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Rosenthal and colleagues report
that a constitutively active form
of Smoothened (green) mimics
concentration-dependent actions
of Sonic Hedgehog in the
developing neural tube, including
activation of ventral markers
(such as Nkx2.2, red), suppression editorial
of dorsal markers and induction
2000 Nature America Inc. http://neurosci.nature.com
brief communications
Long-term visual experience recalibrates human orientation perception. . . . . . . . . . . . . . . 13
D Whitaker and PV McGraw
articles
Molecular basis of NMDA receptor-coupled ion channel modulation
by S-nitrosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
YB Choi, L Tenneti, DA Le, J Ortiz, G Bai, HSV Chen and SA Lipton
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contents
Editor's note
Beginning with this issue, Scientific Correspondence will be
renamed Brief Communications, to maintain consistency
with Nature and other Nature titles. The editorial content of
this section will remain unchanged.
editorial
humankind determined its own genetic sequence. Second, they power. This power will most likely come from two emerging tech-
will regard this discovery as significant primarily because of its nologies, single-nucleotide polymorphisms (SNPs) and DNA
implications for understanding the brain. microarrays. SNPs are the most common form of human allelic
Pedants may quibble, but this enormous scientific achievement variation, and are currently being identified in very large num-
will coincide fairly precisely with the millenium. The sequence of bers, with both public and private funding. Some of these poly-
chromosome 22 was announced in December 1999, and a draft morphisms will themselves be causes of phenotypic variation,
sequence of the entire genome (which will be about 90% complete, whereas others will be linked to the causal polymorphisms and
and is likely to be accurate to better than 99%) will be available by will therefore be useful as genetic markers for association studies.
spring 2000. Celera Genomics, a private company, hopes to assem- There are also tremendous efforts being made to develop microar-
ble a full sequence using a shotgun method by 2001. There will be ray methods to the point where whole genomes can be scanned
a justifiable celebration at some point in the next three years or so, rapidly and cheaply for a comprehensive set of SNPs. These tech-
when the public consortium declares the entire sequence opera- nologies are integral to the identification of risk alleles for complex
tionally complete (meaning an error rate of less than 1 in 10,000, diseases, and they should be equally applicable to other complex
minus a few gaps), but the reality is that there will be no clear end traits such as personality and brain function.
point to the project. So the millenium will be good enough. All this will not happen overnight, but the implications for
Although the most immediate practical effects of the genome society seem potentially far-reaching. Plomin, like most thought-
project will probably be new insights into human disease, it does ful scientists, is careful to emphasize that science alone should
not seem far-fetched to suggest that the biggest impact in the long not and cannot determine social policy, which must depend on
term will be in neuroscience. It is, after all, our brains that make judgments of values as well as facts. At present, we can only spec-
us who we are, both as a species and as individuals. The genome ulate on what society will make of this new knowledge. There
project, and the work that will build on it, will provide a new will surely be individuals who will be burning with curiosity to
depth of understanding of how our brains are built and how they know their own genetic makeup and willing to pay for the infor-
differ from those of other species. But perhaps even more impor- mation, for the same reason that they buy books such as Know
tantand more challenging for societywill be the new insights Your Own I.Q. Will people also want to find reproductive part-
it will provide into how individuals differ from each other. ners based on genetic data, and should it be legal to provide a
Some of these issues are discussed1 by Robert Plomin of the service for those who do? Should parents be permitted to have
Institute of Psychiatry in London, writing in a special Nature sup- their children genotyped in order to plan their education? One
plement that is being distributed with this issue of Nature Neu- hopes that genetic testing will never become compulsory, but
roscience. Plomin focuses on intelligence, and more particularly even if the idea is anathema to most western democracies, there
on general cognitive ability (defined by factor analysis and often is no guarantee that all societies will feel the same way.
abbreviated to g), which is one of the most heritable behavioral A deeper understanding of behavioral genetics may also bring
traits. Rather than being determined by a single gene, g is a com- many benefits. It might, for instance, lead to better educational
plex trait that is influenced by many genes, each having a rela- strategies, not just for the most or least gifted, but for everyone.
tively small effect, as well as by the environment. Experts disagree One may also hope that it will help to combat racial prejudice
on the magnitude of the genetic contribution to g, but Plomin lay people are often unaware of the fact that the great majority
argues that it probably around 50%; in other words, that about of human genetic variation cuts across racial categories, and that
half of the variance in g can be attributed to genetic variation there is little evidence for systematic genetic differences between
within the population. Regardless of the exact figure, it seems races, other than in the superficial characteristics by which they
clear that there are genes that affect intelligence, as well as many are normally recognized. On the other hand, genetic informa-
other behavioral traits. tion may give new opportunities for discrimination against indi-
Many of these genes are likely to be identified as a result of the viduals. Clearly, great challenges as well as great uncertainties lie
genome project. Classical population genetics has indicated that ahead. But as Plomin says, the only thing that seems complete-
genetic effects on g are largely additive; that is, individual alleles ly clear is that nothing will be gained by ignoring the issue.
exert effects that are independent of interactions with other alle-
les. This lack of statistical interaction (which is not to be confused 1. Plomin, R. Nature 402, C25C29 (1999).
How should the nonword jat be pro- dure, in which an entire written word is ciations between orthographic and
nounced? Most English speakers would represented as a set of component letters semantic (meaning-based) information,
agree that it should rhyme with bat. Yet or letter clusters, and then these ortho- and then forming associations between
what about the nonword jough? Should graphic subcomponents are associated semantic and phonological information.
it sound like cough, tough, dough or with corresponding sounds or phonolog- Lexical and semantic procedures may help
2000 Nature America Inc. http://neurosci.nature.com
bough? This example illustrates a major ical subcomponents2. These subcompo- maximize reading speed for very common
difficulty of English and other ortho- nents can be used to build up a words, and they may help constrain the
graphically deep languages: mappings representation of an entire word sound. output of the sublexical procedure (for
between how words are spelled (their Sublexical procedures take advantage of instance, to ensure that pint is not pro-
orthography) and how they sound (their the consistent mappings between orthog- nounced so that it rhymes with mint).
phonology) are only partially consistent. raphy and phonology (such as that bad, Paulesu and colleagues1 theorized that lex-
English readers must use the consistency dad, tad, bat, dat, tat, dab and tab ical and semantic procedures are espe-
that is present to support pronunciation are different combinations of the same cially important for English readers,
of unfamiliar words (such as fint), but four letters and sounds) to avoid having because their sublexical procedure gener-
they also must accurately pronounce separate representations of the ortho- ates multiple alternative pronunciations.
words that violate these same generaliza- graphicphonological relationship for (Should fint rhyme with mint or pint?)
tions (for example, pint). Readers of Ital- each word in a language. Paulesu and col- To test their hypothesis that differences
ian and other orthographically shallow leagues theorized that the high degree of in the orthographic consistency of Italian
languages have an easier task because consistency in the Italian language per- versus English may lead subjects to place a
mappings between orthography and mits Italian readers to rely heavily on a different emphasis on sublexical versus
phonology are highly consistent: a partic- sublexical procedure for reading words lexical and semantic procedures (Fig. 1),
ular letter or letter combination is almost aloud. Paulesu and colleagues1 asked Italian and
always associated with the same sound. In Most models of word reading also con- English subjects to read aloud a variety of
this issue, Paulesu and colleagues 1 use tain one or more additional procedures words and nonwords. It took the English
behavioral measures and functional brain that do not depend on sublexical ortho- subjects significantly longer to begin read-
imaging to investigate how the ortho- graphic structure. For instance, in one ing each word. The differences became
graphic consistency of ones native lan- class of models, a lexical procedure is even more pronounced when the stimuli
guage affects reading speed and the degree thought to permit access to stored infor- were nonwords. Control experiments
to which different language-related brain mation about the association between indicated that the English subjects were
areas are used for reading. The work whole word forms and their correspond- not just slow speakers. For instance, Eng-
demonstrates how brain imaging can be ing sounds3. In another class of models4, lish and Italian subjects took the same
used to address basic research questions phonological representations can be amount of time to produce a word for a
about mindbrain relationships and how accessed indirectly, by first forming asso- picture of an object. The faster reading
the results can be applied to questions of
clinical and educational significance.
This study1 was influenced by cogni-
Word sound Word sound
tive models of word reading. Previous (phonology) (phonology)
behavioral work indicates that skilled
readers of all alphabetic languages auto-
Lexical and/or Lexical and/or
matically use multiple procedures to semantic
Sublexical
semantic
Sublexical
translation translation translation
translate between orthography and translation
phonology, although the precise number
and nature of the procedures is hotly Word form Word form
(orthography) (orthography)
debated. Common to most models of
reading is some type of sublexical proce-
ENGLISH SUBJECTS ITALIAN SUBJECTS
Christie Albin
Julie Fiez is in the Department of Psychology, Fig. 1. Italian and English subjects place different emphasis on the two processes used to deter-
605 LRDC, University of Pittsburgh, Pittsburgh, mine how to pronounce written words. English speakers rely more heavily on lexical and/or
Pennsylvania 15260, USA. semantic translation, whereas Italian speakers rely more on sublexical translation (left). These
e-mail: fiez+@pitt.edu strategies lead to differences in the brain activation evoked by reading aloud (right).
times for Italians are consistent with the semantic processing (naming of both should help constrain interpretations of
hypothesis that Italian subjects can rely words and pictures, generating seman- activation differences within the system
almost exclusively on a sublexical proce- tic associations). and theoretical models of reading.
dure, whereas English subjects have to use Although Paulesu and colleagues 1 Indeed, these data1 already provide an
an additional lexical/semantic procedure develop a compelling interpretation for exciting glimpse into the neural basis of
to guide phonological output. their results, it will not be received with- reading. The finding that the left superi-
If the difference in reading speed for out controversy. One reason is that the or temporal, posterior inferior temporal
Italian versus English subjects reflects a existing imaging literature supports mul- and frontal areas are sensitive to the
differential emphasis upon sublexical ver- tiple interpretations. For instance, many orthographic consistency of a subjects
sus lexical/semantic procedures, then the imaging studies show that practice and native language suggests that they are crit-
two groups should have different patterns repetition can also produce relative ically involved in the derivation of
of brain activity during a reading task. To decreases in regional blood flow7. Indeed, phonology from orthography, even if the
test this idea, the authors1 used positron whether a particular region shows an specific contributions that each makes is
emission tomography to measure blood experience-related increase or decrease debatable. There is growing evidence
flow (a measure of neuronal activity) in seems to reflect several factors, such as its from reading researchers and educators
the brains of Italian and English subjects role in the learning process and the that the ability to manipulate and derive
while they read words and nonwords. As amount of practice given8. This suggests phonological representations is a funda-
expected, in both groups, this task acti- a different possible logic: that the more a mental aspect of skilled reading9. By fur-
2000 Nature America Inc. http://neurosci.nature.com
vated a widespread network of brain sublexical, lexical or semantic procedure thering our understanding of the brain
regions previously associated with read- is used, the more efficient the brain regions and cognitive processes involved
ing5. The novel finding is that brain acti- regions that implement the procedure in the analysis of phonology, we should
vation in three areas of this larger might become, with increases in efficien- be in a better position to diagnose and
network depended on subjects native cy detected as smaller increases in activity. eventually treat reading disorders. We can
language. Italians had greater activation Thus, the smaller left frontal and posteri- already form some tantalizing links
in a left superior temporal region during or inferior temporal responses for Italian between normal and impaired perfor-
both word and nonword reading than subjects could reflect their greater skill rel- mance and the areas identified by Paule-
English subjects, who showed greater ative to English subjects at a sublexical su and colleagues. For instance, the left
activation in a left frontal and a posteri- procedure that is particularly important superior temporal region identified in
or inferior temporal region during non- for nonword reading. Similarly, the small- their study is near a region that is acti-
word reading (Fig. 1). er left superior temporal response for vated below normal levels when dyslexic
Paulesu and colleagues1 suggest that English subjects could reflect their greater subjects attempt to read words 10 . A
a sublexical procedure can be localized skill at a lexical or semantic procedure. recently developed technique for exam-
to the superior temporal region, and a This alternative interpretation is also con- ining the connections between brain
lexical/semantic procedure to the left sistent with some imaging results, such as regions indicates that this reduced acti-
frontal and posterior inferior temporal nonword versus word differences in left vation may arise from abnormal connec-
regions. This interpretation is based in frontal cortex activation5. tions between left temporal and frontal
part on the implicit logic that increasing A second issue is that the work of regions in dyslexic subjects (T. Klingberg
the emphasis on a particular procedure Paulesu and colleagues 1 leaves funda- et al. Soc. Neurosci. Abstr. 25, 654.7, 1999.
will produce more activation in regions mental differences in cognitive models of Equally exciting are the implications
that implement the procedure. Thus, word reading unresolved. We may now of this work for our understanding of
because Italians emphasize a sublexical know which brain regions are involved in how experience can shape the organiza-
procedure, the left superior temporal sublexical and lexical/semantic proce- tion of our cognitive systems. Both the
region that shows greater activation in dures, but we still do not know how these Italian and English subjects studied by
Italian subjects is likely to contribute to procedures work. For instance, do we Paulesu and colleagues1 can use sublexi-
this process. The converse is true for the develop rules that codify consistent cal and lexical procedures, but experi-
left frontal and posterior inferior tem- spelling-to-sound relationships in our lan- ence has optimized their use of these
poral regions. Neuroimaging work in guage3, or do we form distributed repre- procedures for the language they read.
other domains provides support for this sentations that capture the statistical Our perceptual systems have a remark-
logic. For instance, piano players have likelihood of both consistent and incon- able ability to self-organize based on
more activation in motor-related regions sistent pronunciations4? Such questions sensory experience 11. Reading proce-
than non-musicians when they perform have remained unresolved because exist- dures may become tuned through simi-
a simple finger-movement task6. To fur- ing behavioral data about the reading per- lar mechanisms, with much of the
ther support their interpretation, Paule- formance of normal and brain-damaged optimization occurring automatically
su and colleagues compare their results subjects is compatible with multiple mod- and continually. An interesting theoret-
to those of previous neuroimaging stud- els of reading. Paulesu and colleagues pro- ical issue with profound practical impli-
ies. They note that the left superior tem- vide a striking demonstration of how cations is how much this tuning may be
poral region is activated by other tasks neuroimaging can be used to provide a influenced by instructional strategy and
that are thought to emphasize sublexi- new type of data, in which the behavior the initial structure of reading materials.
cal phonology (reading nonwords as of individual components of the reading For instance, an early instructional strat-
compared to reading words), whereas system can be teased apart. Further inves- egy (such as phonics) that emphasizes
the left frontal and posterior inferior tigation of how the brain areas involved the consistent features of orthographic-
temporal regions are activated by tasks in reading are affected by experimental to-phonological transformation in Eng-
that are thought to emphasize lexical and and experience-dependent manipulations lish may lead children to emphasize a
2. Patterson, K. & Behrmann, M. J. Exp. Psychol. 7. Raichle, M. E. et al. Cereb. Cortex 4, 826
sublexical transformation procedure that Hum. Percept. Perform. 23, 12171231 (1997). (1994).
involves the superior temporal region, 3. Coltheart, M., Curtis, B., Atkins, P. & Haller, 8. Karni, A. et al. Proc. Natl. Acad. Sci. USA 95,
compared to an early instructional strat- M. Psychol. Rev. 100, 589608 (1993). 861868 (1998).
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focuses on the relationships between & Patterson, K. Psychol. Rev. 103, 56115 Education 1, 157 (1995).
entire word forms and their corre- (1996). 10. Rumsey, J. M. et al. Arch. Neurol. 54, 562573
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Sci. USA 94, 914921 (1998). 11. Recanzone, G. H., Merzenich, M. M., Jenkins,
1. Paulesu, E. et al. Nat. Neurosci. 3, 9196 6. Hund-Georgiadis, M. & von Cramon, D. Y. W. M., Grajski, K. A. & Dinse, H. R.
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bistin I and II, two alternatively spliced A better understanding of the colly- bistin may be involved in the postsynap-
products of the same gene. Analysis of bistins could also shed new light on the tic response to extracellular signaling pro-
their amino-acid sequences revealed two multiple lives of gephyrin. Gephyrin is teins. Such signals might come from the
domains that identify the collybistins as necessary for clustering not only glycine presynaptic terminal, glia or even the
members of the dbl-like family of receptors but also many GABA receptors postsynaptic cell. So far, the only extra-
GDP/GTP exchange factors. This family in vivo12. It has also emerged as a likely cellular signaling molecule known to be
is characterized by dbl domains, which regulator of local mRNA translation13. involved in synapse formation is agrin,
are important for exchange activity, and Most mysteriously, gephyrin is required which is essential for neuromuscular junc-
by pleckstrin homology domains, for the synthesis of a necessary cofactor tion development. Agrin is also expressed
thought to mediate association with the for molybdenum enzymes. Such enzymes in the brain, but its role there is not
plasma membrane. Dbl-like GDP/GTP are expressed in most cell types and are understood. The identity of functionally
exchange factors (GEFs) regulate the involved in the metabolism of sulfites analogous actors at CNS synapses is large-
activity of small GTPases such as rho, rac (among other things). Mutant mice lack- ly unknown, although some candidates
and cdc42 (ref 10). These proteins act as ing gephyrin show severe neurotoxicity are now emerging14.
signaling molecules and can exist in active due to the build-up of these and other Clearly, a complete understanding of
(GTP-bound) or inactive (GDP-bound) metabolites. Finding the mechanisms by synapse formation will require the iden-
forms. Their GTPase activity brings about which collybistins are activated and iden- tification of extracellular, transmembrane,
their inactivation, and they are reactivat- tifying the targets of the collybistins anchoring, cytoskeletal and regulatory
2000 Nature America Inc. http://neurosci.nature.com
ed when the bound GDP is exchanged for should help elucidate gephyrins role in molecules. Once the molecular players
GTP from the cytoplasmic pool. GEFs act these diverse cellular functions. Finally, have been identified, the challenge will be
by promoting this reactivation (Fig. 1). A gephyrin and neural activity are neces- to understand how they collaborate to
major function of GTPases such as rho, sary but not sufficient to cause glycine form and maintain synapses. Among the
rac and cdc42 is to link the activation of receptor clustering on neurons. It will most interesting questions will be how the
cell-surface receptors to reorganization therefore be interesting to find out synapse maintains a structural memory.
of the actin cytoskeleton, especially at whether collybistin is somehow required In other words, how it is that the struc-
membranes11. For example, microinjec- for the activity-dependent regulation of ture of a synapse can remain stable over
tion of rho can induce the formation of synaptic structure. long periods of time, even though the
actin-based stress fibers in many cell More generally, how does this new individual molecules from which it is
types. Thus, the sequence of the colly- information advance our understanding made are constantly turning over? It
bistins, along with their ability to bind of how synaptic architecture is regulated? seems likely that this structural stability,
gephyrin, raises the possibility that they The growing list of molecules that have along with the capacity for use-dependent
may regulate GTPase signaling at been implicated in receptor clustering modification, is a function of feedback
inhibitory synapses. suggests a theme that may be character- loops that integrate networks of structur-
Kins et al.1 tested the biological activity ized as similar stories, different actors. al and regulatory molecules 15 . Thus,
of collybistins by expressing them in vari- Thus, distinct anchoring molecules have although the names may change, com-
ous combinations with gephyrin and now been found for most of the major parative studies can teach us much about
glycine receptors in non-neuronal cells. classes of ligand-gated ion channels, what kinds of molecules are needed to
When expressed individually, both gephyrin including the PSD-95/SAP90 family (for build and regulate the diverse array of
and collybistin are cytoplasmic. However, NMDA receptors)8, GRIP (AMPA-type synapses that hold up the nervous system.
when gephyrin and collybistin II (but not GluRs)3, homer (some mGluRs)9 and rap-
collybistin I) are expressed together, they syn (muscle nAChRs)4, as well as gephyrin 1. Kins, S., Betz, H. & Kirsch, J. Nat. Neurosci. 3,
2229 (2000).
form submembranous aggregates. Impor- (glycine and GABAA receptors). In addi-
2. Chen, H. J., Rojas-Soto, M., Oguni, A. &
tantly, such aggregates can also capture tion to these structural components, reg- Kennedy, M. B. Neuron 20, 895904 (1998).
glycine receptors, thereby targeting them to ulatory molecules have now been linked
3. Kim, J. H. & Huganir, R. L. Curr. Opin. Cell Biol.
clusters within the plasma membrane. to many of these complexes. One that 11, 248254 (1999).
These experiments were performed in seems particularly relevant to under- 4. Sanes, J. R. & Lichtman, J. W. Annu. Rev.
fibroblasts, but it is tempting to think that standing collybistins is SynGap, a GTPase- Neurosci. 22, 389442 (1999).
the same thing may be happening in neu- activating protein (GAP) that is associated 5. Craig, A. M. Neuron 21, 459462 (1998).
rons. A first step toward testing this idea with PSD-95 (refs. 2, 3). GAPs accelerate 6. Prior, P. et al. Neuron 8, 11611170 (1992).
will be to determine the localization of the GTPase activity of small G proteins 7. Feng, G. et al. Science 282, 13211324 (1998).
collybistin proteins in vivo. Collybistin and such as rho, rac and cdc42, thereby pro-
8. Kornau, H. C., Seeburg, P. H. & Kennedy, M. B.
its mRNA are selectively expressed in the moting their auto-inactivation. The effect Curr. Opin. Neurobiol. 7, 368373 (1997).
brain, but the available antibodies were not of GAPs is thus opposite to that of 9. Tu, J. C. et al. Neuron 21, 717726 (1998).
suitable for establishing its cellular local- exchange factors, and so it seems reason-
10. Lin, R., Cerione, R. A. & Manor, D. J. Biol.
ization. It will also be important to able to speculate that collybistin may Chem. 274, 2363323641 (1999).
demonstrate that collybistin does indeed antagonize an as-yet unidentified GAP at 11. Hall, A. Science 279, 509514 (1998).
have GDP/GTP exchange activity, to iden- inhibitory synapses, and conversely that
12. Kneussel, M. et al. J. Neurosci. 19, 92899297
tify its natural substrates, and to determine SynGap may be antagonized by another (1999).
whether the exchange activity is required exchange factor (perhaps related to colly- 13. Sabatini, D. M. et al. Science 284, 11611164
for its biological activity. Because of its bistin) at excitatory synapses. (1999).
interaction with gephyrin, one intriguing Small GTPase signaling pathways are 14. OBrien, R. J. et al. Neuron 23, 309323 (1999).
idea is that gephyrin may be required for often involved in transducing extracellular 15. Lisman, J. E. & Fallon, J. R. Science 283,
activation of its enzymatic activity. signals, raising the possibility that colly- 339340 (1999).
the first half of the 20th century. Resolu- Cajal and Sherrington would have naptic potential when recorded at higher
tion came in the 1950s, and the clear endorsed and Bullock and Eccles did time resolution3. Coupling was specific
answer was both; there are electrical endorse) is that a synapse is a morpholog- and did not occur between inhibitory
synapses and chemical synapses1. At that ically specialized structure mediating inter- neurons and nearby pyramidal cells or
time, many neuroscientists (a term not yet action between neurons or between between pyramidal cells. Moreover, two
widely used) thought electrical transmis- neurons and follower cells. Use of the populations of inhibitory neurons in the
sion would be uncommon in mammals, phrase chemical synapse is implicit same region but with different firing char-
because the first electrical synapses were recognition of this view (as gin martini acteristics were coupled within popula-
found in invertebrates and lower verte- legitimizes martinis made with vodka). tions but hardly at all between them4.
brates. Furthermore, transmission at elec- The major technological advance The extent of coupling remains to be
trical synapses did not show the activity underlying the recent electrophysiologi- determined. How far does the coupling
dependence commonly observed at chem- cal demonstration of electrical synapses3,4 spread over the cortex? Are there small
ical synapses. Indeed, as ultrastructural was infrared differential interference con- domains of coupled cells or does coupling
studies of mammalian CNS became more trast microscopy applied to brain slices7. extend for long distances? Are cell pairs of
refined, almost all synapses proved to have This technique permits visualization of the same type that are not coupled to each
presynaptic vesicles and other specializa- neighboring cells in a brain slice relative- other also not coupled to any other cell?
tions characteristic of chemical transmis- ly far below the cut edge. Slices also per- This coupling does not yet have an
sion, and few synapses showed gap mit placement of two electrodes with anatomical substrate, although the pres-
junctions, the structural underpinning of much less likelihood of mechanical inter- ence of gap junctions is indicated by the
the most common form of electrical actions during the manipulations. Because uncoupling effect of octanol, a common
synapse. (Other modes of excitatory and this approach is not that new, a change in gap junction blocker4. (Octanol has mul-
inhibitory electrical transmission are perceptual set (or equipment budget) may tiple effects on neurons, and alteration of
known1,2.) Now it seems that electrical have contributed to the new observations. other functions, such as repetitive firing,
synapses may be more frequent than most In these studies3,4, the inhibitory neurons by octanol does not indicate gap junction
neuroscientists thought. In a recent issue were identified first visually by shape and involvement.) Dendrodendritic gap junc-
of Nature, two groups reported electrical then by their characteristic firing patterns tions occur between inhibitory neurons8.
coupling of inhibitory neurons in the neo-
cortex 3,4 , and other reports will soon
appear. In addition, a widely expressed, SO WHICH ARE MORE PRIMITIVE, CHEMICAL SYNAPSES OR ELECTRICAL SYNAPSES?
neuron-specific, gap junction protein has Connexins, the family of homologous proteins forming gap junctions in vertebrates,
recently been cloned5,6. are unrelated to innexins, the gap-junction-forming proteins in invertebrates of the
Over the years, the catalog of electrical protostome line16. Innexin gap junctions show an astonishing degree of convergence
synapses in adult mammals, identified with connexin gap junctions in overall morphology, in having four membrane span-
physiologically and/or anatomically, has ning regions with two extracellular loops and in being blocked by the same agents,
grown slowly1,2. However, the incidence of such as hydrogen ions, calcium and octanol. The two types differ not only in sequence,
electrical synapses compared to chemical but also in gap width, interchannel separation and channel diameter. The apparently
synapses in the adult has remained rela- independent, although convergent, evolution of gap-junction-forming proteins is in
tively low. These data would be even more marked contrast to molecules underlying action potential generation and chemically
tilted toward chemical synapses if more mediated transmission, most if not all of which have homologs in vertebrates,
researchers shared the mistaken view that Drosophila and, of course, C. elegans. Thus, it seems that gap-junction-mediated com-
munication evolved after chemical transmission and, from this point of view, is more
Michael Bennett is in the Department of advanced. Later evolutionary development of gap junctions can also be argued from
Neuroscience, Albert Einstein College of unicellular organisms, which secrete and respond to chemical signals but do not com-
Medicine, Bronx, New York 10461, USA. municate via gap junctions.
e-mail: mbennett@aecom.yu.edu
Alternatively, coupling may result from observed in one experiment. Dye coupling studies3,4, electrically coupled neurons
presynaptic fibers that make gap junctions of cortical inhibitory interneurons has could also be connected in one or both
on more than one postsynaptic neuron1. been reported9, but the rats used in these directions by inhibitory chemical synaps-
Neurobiotin injection confirmed the cell experiments may have been younger es. Stimulation of a cell that is connected
type 4 , but tracer coupling was only when dye coupling of cortical neurons is both electrically and chemically produces
more prominent10. Failure to observe dye a biphasic, depolarizinghyperpolarizing
coupling may arise from geometric fac- potential in the postsynaptic cell. Model-
a tors (relationship between junctional con- ing indicates that reciprocal or recurrent
ductance and cell volume and extent of inhibition without electrical coupling can
the coupled network) or from imperme- mediate synchronous firing, given a dis-
ability of junctions to the tracer. tributed excitatory input11. Thus, it is not
What does electrical coupling do for clear that the electrical coupling is
these cells? In both studies, impulses in required for the oscillations observed in
one cell were insufficient to excite anoth- inhibitory neurons (and their follower
er, coupled cell, but if both cells were cells), but it would make the cells fire
depolarized to near threshold, synchro- more synchronously. This synchronizing
10 m nous firing was observed. Thus, a distrib- action is not simply mutual excitation;
uted excitatory input to these cells would whereas a more depolarized cell tends to
2000 Nature America Inc. http://neurosci.nature.com
tend to activate them together, and elec- excite a less depolarized cell, the less depo-
trical coupling would increase the degree larized cell tends to inhibit the more depo-
ba of synchronization. Electrical coupling is larized cell. The synaptic relationship
likely to contribute to the synchronous between them is inhibitory as well as exci-
oscillations that are often observed in tatory, independent of whether there are
pools of inhibitory neurons9,11. In both chemical inhibitory synapses in either or
Cell 1 both directions.
Electrical transmission is commonly
thought to be faster than chemical trans-
Fig. 1. Electrical transmission in practice and mission, and this is true in respect to the
in theory. (a) IR-DIC micrograph of two corti-
beginning of charge transfer across a gap
cal inhibitory neurons just before recording4.
The electrodes enter the image from either junction during the rising phase of the
side. (b) Depolarizing and hyperpolarizing presynaptic impulse. However, because
Cell 2 pulses applied in cell 1 (superimposed records, there is a threshold for detection, the mea-
currents on middle trace) spread to cell 2 (ref. sured postsynaptic potential is delayed
4). Impulses in cell 1 caused brief depolariza- with respect to its actual onset, and the
tions in cell 2; relative hyperpolarizations time required to charge the postsynaptic
between these depolarizations may have capacitance increases this delay. The delay
included a contribution from inhibitory can be a significant fraction of the rise time
26 mV
2 chemical synapses. Synaptic noise observed
of the presynaptic action potential
50 ms during hyperpolarization was asynchronous
in the two cells, indi
(Fig. 1c) and easily longer than the mini-
c cating that they had mum delay of chemical transmission at
separate synaptic inputs. mammalian body temperature (0.2 ms).
Vpre gj Vpost (c) Equivalent circuit There are few sites in the mammalian ner-
of transmission at an vous system where the time saved in elec-
electrical synapse1. The trical transmission is important. The
C gpost
gap junction has conduc- selection advantages of electrical synapses
tance, gj; the postsynap- seem to lie in reciprocity and transmission
tic cell has conductance, of subthreshold potentials. Where activi-
gpost, and capacitance, C.
ty of similar cells is synchronized, the syn-
The postsynaptic poten-
Voltage
transmission almost irrelevant (but we ately, phrases such as gap junction-like. ascribed to reciprocity and transmission
should not forget our roots, and new The freeze-fracture technique displays of subthreshold potentials that facilitate
chemical transmitters keep appearing). All large areas of membrane surface and can synchronization. This general physiolog-
neurons may express machinery for reveal junctions smaller in diameter than ical outcome may be achievable in other
chemical transmission, but many of them the usual 70 nm of thin sections, and ways, but electrical transmission is defi-
also transmit electrically. Identification of these junctions would be difficult to detect nitely in the common neuronal repertoire.
connexins, the family of gap junction pro- by that technique. In freeze fracture, cell
teins, should be nearing completion for type may be hard to identify, and because 1. Bennett, M. V. L. in Cellular Biology of
mammals. The sequence information per- there are many CNS gap junctions Neurons Vol. I, Sec. I, Handbook of
Physiology. The Nervous System (ed., Kandel,
mits identification by in situ hybridization between glia, neuronal localization of a E. R.) 357416 (Williams and Wilkins,
and northern analysis as well as RT-PCR gap junction must be demonstrated. The Baltimore, 1977).
from single cells. Antibodies to connexin- situation can be improved by a new and 2. Jefferys, J. G. R. Physiol. Rev. 75, 689723 (1995).
specific peptides are being used at the light laborious approach, where there is the 3. Galarreta M. & Hestrin, S. Nature 402, 7275
and electron microscope levels. A major interest and manpower to apply it15. Slices (1999).
breakthrough for mammalian electrical of CNS tissue are frozen, fractured and 4. Gibson, J. R., Beierlein, M. & Connors, B. W.
synapses is the cloning of Cx36, a (near- shadowed, and the remaining tissue and Nature 402, 7579 (1999).
ly) neuron-specific connexin5,6. In situ replica are mounted, replica down, on an 5. Condorelli, D. F. et al. Eur. J. Neurosci. 10,
hybridization and immunocytochemistry EM grid. After confocal microscopy to 12021208 (1998).
2000 Nature America Inc. http://neurosci.nature.com
show much broader distribution of this identify cells at specific sites, the tissue is 6. Shl, G., Degen, J., Teubner, B. & Willecke, K.
FEBS Lett. 428, 2731 (1998).
connexin than was previously appreciat- dissolved away and the replica examined
7. Dodt, H. U. & Zieglgnsberger, W. Brain Res.
ed for electrical synapses (J.E. Rash, T. with knowledge of what cells were at the 537, 333336 (1990).
Yasumura, W.A. Staines, D. Patel & J.I. fracture surface. This approach has
8. Katsumaru, H., Kosada, T., Heizmann, C. W.
Nagy, Mol. Biol. Cell. 10, 404a, 1999). demonstrated a high incidence of mor- & Hama, K. Exp. Brain Res. 72, 363370
Cx36 appears abundant not only in areas phologically mixed synapses, that is, with (1988).
where many electrical synapses are known both gap junctions and transmitter release 9. Benardo, L. S. J. Neurophysiol. 77, 31343144
(retina, inferior olive, olfactory bulb, stria- sites, in the rat spinal cord. (1997).
tum and now neocortex) but also in areas Clearly, tissue slices and IR microscopy 10. Peinado, A., Yuste, R. &, Katz, L. C. Neuron 10,
where electrical coupling or gap junctions coupled with whole-cell patch clamping 103114 (1993).
are less clearly demonstrated. make feasible the characterization of the 11. Jefferys, J. G. R., Traub, R. D. & Whittington,
M. A. Trends Neurosci. 19, 202208 (1996).
Where have all these putative gap junc- microcircuitry of neighboring neurons.
tions been hiding? Very likely some of Cells can be visualized, activated directly 12. Ishimatsu, M. & Williams, J. T. J. Neurosci. 16,
51965204 (1996).
them have been seen, but adequate fixa- or synaptically, and filled with tracer for
13. Hatton, G. I. & Li, Z. Adv. Exp. Med. Biol. 449,
tion of the mammalian CNS for electron Golgi-like analysis. Immunocytochem- 7995 (1998).
microscopy is difficult. Visualization of the istry of physiologically characterized neu-
14. Nolan, M. F., Logan, S. D. & Spanswick, D. J.
diagnostic seven layers of a gap junction rons is in the offing. Neurons can express Physiol. (Lond.) 519, 753764 (1999).
in cross section requires optimum prepa- specific connexins, and it is clearer than 15. Rash, J. E. et al. Proc. Natl. Acad. Sci. USA 93,
rations, and junction diameter must be before that electrical transmission is like- 42354239 (1996).
large enough to extend through the entire ly to be found wherever it is useful. At 16. Phelan, P. et al. Trends Genet. 14, 348349
section. The literature contains, appropri- many sites, the selective advantage can be (1998).
Targeting visual motion into how the brain analyzes the motion
of complex visual patterns.
Jochen Braun An early step in understanding motion
processing was the moving plaid stimu-
A new stimulus display reveals that humans summate the lus4, which is a superposition of two sinu-
soidal gratings moving in different
motion energies of all components consistent with a single directions. Depending on the details of the
velocity, rather than optimizing sensitivity by ignoring noise. stimulus, this display can be perceived
either as two sets of gratings sliding past
In vision research, decisive advances are targeted displays is to remove all but one each other, or as a single coherent plaid
often the result of cleverly targeted stim- type of visual information, so that the pattern moving in a third direction
ulus displays. Two classic examples are stimulus activates only a very restricted (Fig. 1a). Where in the brain do these per-
Bela Julesz demonstrations that the visu- class of perceptual mechanisms. This ceptions arise? In visual cortical area V1,
al system can process both binocular dis- allows the mechanisms in question to be which represents an early stage in visual
parity1 and differences in visual textures2 studied in quasi-isolation, and in many processing, motion-sensitive neurons
at a surprisingly early level. The aim of cases it can lead to the identification of respond merely to the separate compo-
the underlying neural substrates of per- nent motions of the two gratings5. This is
Jochen Braun is at the Institute of ception within the visual cortex. On page because neurons at this level are sensitive
Neuroinformatics, Uni/ETH Zrich, 64 of this issue, Schrater et al.3 present a only to contours at a particular visual loca-
Winterthurerstr. 190, 8057 Zrich, Switzerland. new type of motion stimulus that tion and orientation, and can only signal
e-mail: achim@ini.unizh.ch promises to provide important insights the motion component that is orthogonal
to their preferred orientation (the so-called tains information at many different spatial plaids, but also from translating patterns
aperture problem). The perception of scales. Because velocity equals temporal fre- of random dots10, which have also been
coherent motion, which requires the sep- quency divided by spatial frequency, a multi- popular for studying motion. Whereas
arate motion components to be combined, scale pattern that is moving with a given Schrater et al. can target particular popu-
seems to arise in MT, a higher area that is velocity contains motion energy at many lations of V1 neurons, random dot dis-
known to be involved in motion percep- different combinations of spatial and tem- plays give rise to motion energy that is
tion, and where a proportion of neurons poral frequencies. Moreover, because of the distributed over the entire velocity plane.
responds to the coherent pattern motion aperture problem, the amount of energy at Random dot displays thus suffer from an
that is actually perceived6,7. The moving a particular combination of spatial and tem- important but rarely recognized limita-
plaid is of course a highly simplified stim- poral frequency depends also on the orien- tion: they make it difficult to study inter-
ulus, and real-world objects are typically tation preference of the detector. For this actions between the respective neural
defined by outlines composed of contours reason, motion components along the x and representations of distinct but consistent
with many different orientations. The var- y axes must be considered separately. component motions, because they do not
ious component motions from these con- Movement in a given velocity thus cre- stimulate these representations differen-
tours are expected to be represented in ates motion energy over a range of tem- tially. In other words, random dot displays
area V1, but underlying motion of the poral frequencies and spatial frequencies turn out to be a poor choice for studying
object as a whole is thought to be recov- along the x and y axes. The set of all com- the integration of component motions.
ered only in area MT (Fig. 1b). A major binations that are consistent with a par- Armed with their new displays,
2000 Nature America Inc. http://neurosci.nature.com
open issue is how this recovery is accom- ticular velocity (speed and direction of Schrater et al. revisit the question of how
plished; that is, how do neurons in area movement) lie on a tilted plane; specifi- component motions are integrated to
MT integrate and combine the informa- cally, the tilt angle (angle between the recover pattern motion. To this end, they
tion they receive through projections from plane and the t-axis) reflects the speed, combine one or more component
neurons in area V1? and the direction of the tilt (the angle in motions and, by varying the contrast in
When moving plaid displays were first the x,y-plane) reflects the direction of their stimuli, measure how readily an
introduced, there were high hopes that they the velocity vector. observer can detect the presence of
would reveal how component motions are Figure 2 illustrates the energy distribu- motion in each case. Statistical decision
combined into pattern motion. Unfortu- tion for each of the five variants of the dis- theory predicts how an ideal observer
nately, these hopes have not been fulfilled. play used by Schrater and colleagues. The would perform in this situation: the con-
The problem is that the moving plaid dis- simplest variant is the component display, trast necessary to detect motion of n com-
play is not sufficiently targeted, in that it which contains only one component ponents decreases in proportion to 1/n.
still contains several kinds of motion infor- motion and targets one population of area By definition, an ideal observer summates
mation. Specifically, the intersections V1 neurons. A slightly more complex vari- motion energy from all relevant parts of
between the two gratings create moving ant is equivalent to a plaid display, in x,y,t space (that is, parts stimulated
discontinuities, or nodes, and these nodes which two component motions target two by a component motion) and ignores
(rather than the component gratings) can separate populations of area V1 neurons motion energy from all irrelevant parts.
govern the perception of coherent (see also Fig. 1c). The authors show that human observers
motion7,8. For this reason, moving plaids The displays of Schrater and col- generally do not conform to this prediction,
may tell us more about visual sensitivity to leagues 3 differ not only from moving which implies that they are unable to mon-
moving nodes than about the integration
of component motions. Schrater et al. now
introduce a new type of visual display that
finally overcomes this limitation3 (Fig. 1c). a
A bB c
C
To generate this display, the authors filter
dynamic random noise in a way that con-
centrates motion energy in several distinct
subregions of spatio-temporal frequency
space, each corresponding to one compo- pattern component
nent motion (Fig. 2). The stimulus is per-
ceived as an amorphous pattern that y pattern y
appears to drift in one or more directions,
but which (unlike the moving plaid dis- component
play) lacks any persistent features that t t
could serve as the basis for a feature-track-
x x Amy Center
ing mechanism of motion detection. Cru-
cially, the energy distribution within each Fig. 1. Computation of motion from its components. (a) Real-world objects contain contour seg-
subregion matches the sensitivity of V1 ments of many orientations. Object motion causes each contour segment to move orthogonally
neurons, being concentrated at one partic- to its orientation. These component motions must be pooled to recover the pattern motion of
the object as a whole. (b) Moving plaid stimulus, consisting of two gratings drifting, respectively,
ular combination of spatial and temporal
toward the upper right and the lower right of the xy plane. The pattern as a whole is perceived as
frequencies, x, y, and t5,9. moving directly rightward. The top and side surfaces show how the pattern changes over time (xt
To understand this, recall that V1 neu- plane and yt plane. (c) Stimulus of the type used by Schrater et al. This stimulus is equivalent to
rons are tuned not only for orientation but the moving plaid in (b), in that it contains motion energy in the same two directions and is per-
also for particular spatial and temporal fre- ceived as moving directly rightward. Note, however, the lack of specific features that can be
quencies. A typical real-world pattern con- tracked over longer times (compare top surfaces in b and c).
brief communications
Long-term visual experience uli were presented for 250 milliseconds twiceonce with either a
clockwise or counterclockwise reference tilt, and once with one of
recalibrates human seven possible inverted comparison tilts. Both reference-tilt direc-
tion and the presentation order of reference and comparison were
orientation perception randomly interleaved. Observers were required to indicate which
of the two trials produced the greatest impression of tilt. Reference
tilts between 5 and 20 were investigated. As a control, the same
David Whitaker and Paul V. McGraw observers were presented with non-alphanumeric stimuli using the
same procedure. These consisted of three circular Gabor patches
Department of Optometry, University of Bradford, Bradford BD7 1DP, UK
Correspondence should be addressed to D.W. (d.j.whitaker@bradford.ac.uk)
within which the sinusoidal luminance modulation was tilted either
clockwise or counterclockwise. These stimuli were chosen because
Sensory neural reorganization occurs as a result of visual experi- they are ideally suited to analysis by orientation- and frequency-
ence in the adult as well as early in life. Here we report and quanti- selective mechanisms early in the visual cortex.
fy a misperception of visual orientation: when alphanumeric From responses averaged across six observers, we calculated the
characters that are commonly tilted clockwise were presented with counterclockwise tilt that appeared perceptually equivalent to any given
a clockwise tilt, they were perceived as less tilted than the same stim- clockwise digit tilt, and vice versa (Fig. 1c). The diagonal dashed line
ulus horizontally inverted. In contrast, subjective perception of tilt represents the prediction that the matching tilt is exactly equal and
magnitude for horizontally inverted non-alphanumeric stimuli was opposite to the reference tilt. The control data for non-alphanumeric
2000 Nature America Inc. http://neurosci.nature.com
similar to that for non-inverted stimuli. Therefore, misassessment stimuli lie almost exactly on this diagonal line. In contrast, a best-fit-
of tilt of alphanumeric characters probably reflects a persistent sen- ting linear regression to the alphanumeric data has a gradient almost
sory recalibration of orientation perception as a result of previous identical to that of the predicted function, but displays a constant error
long-term visual experience. of approximately 2.4. Thus, counterclockwise tilts are consistently per-
Considerable evidence suggests that specific visual experience early ceived as more tilted than their clockwise counterparts, providing
in life can result in a reorganization of orientation selectivity among empirical support for the perceptual observations of Fig. 1a and b.
cortical neurons; for example, the rearing of animals in an environ- It is well established that perceived orientation can be influenced
ment containing a restricted range of orientations leads to a prepon- by previous adaptation to a tilted stimulus7. This is classically known
derance of cortical neurons tuned to that orientation1,2. In addition, as the tilt aftereffect, an illusion that decays rapidly over time8. The
sensory neural reorganization can also occur throughout adulthood36. present findings, however, reflect a relatively persistent form of adap-
Alphanumeric characters such as those used in digital displays and tation within the visual system, a phenomenon probably conditioned
italicized text are commonly tilted clockwise by around 510; there- by extensive previous visual experience of alphanumeric characters
fore we asked whether long-term visual exposure to clockwise-tilted whose tilt, if present, is almost always in a clockwise direction. Restric-
alphanumeric characters influences our visual perception of tilt. tion of this misperception to alphanumeric characters implicates rel-
We found that the tilt of clockwise-slanted characters appeared atively high-level processes involved in character recognition. This
to be of much smaller magnitude than when such characters were kind of top-down regulation of perceived orientation receives con-
horizontally inverted (as when viewed in a mirror; Fig. 1a and b). To siderable physiological support from dynamic changes in the orien-
quantify this effect, we presented observers with stimuli consisting tation tuning of visual neurons via intracortical feedback
of three digits arranged side by side (888) and surrounded by a rec- mechanisms9. Our findings support the view that, in the sense that
tangular reference frame defining true vertical and horizontal. Stim- our everyday perceptions can be markedly influenced by unique prior
visual experience, we learn to perceive. A robust test of this interpre-
tation would be to establish whether the misperceptions we describe
a b exist in cultures that provide little or no exposure to italicized Latin
text or tilted digital numerals10.
Original display
ACKNOWLEDGEMENTS
P.V.M. was supported by a Vision Research Training Fellowship from the Wellcome Trust.
10 Fig. 1. Normal and horizontally inverted versions of a digital display (a) and
y = x italicized text (b). The horizontally inverted versions produce a greater impres-
Counter clockwise sion of tilt. This effect is quantified in the experimental data (c). For each one of
20 several clockwise (positive) and counterclockwise (negative) reference tilts, we
plotted the mirror-image tilt that appears perceptually equivalent. Solid sym-
20 15 10 5 0 5 10 15 20 bols, data for the alphanumeric characters; open symbols, data for Gabor
Reference tilt patches (spatial frequency, 8 cycles per degree). Error bars represent s.e. The
linear regression that best fits the data has the form y = 2.385 0.989x.
articles
1 Cerebrovascular and Neuroscience Research Institute, Brigham and Womens Hospital, and Program in Neuroscience, Harvard Medical School, 221 Longwood
Avenue, LMRC First Floor, Boston, Massachusetts 02115, USA
2 Present address: Del E. Webb Center for Neuroscience and Aging Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA
Correspondence should be addressed to S.A.L. (slipton@burnham-inst.org)
2000 Nature America Inc. http://neurosci.nature.com
Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular
basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion
channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed
mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation
(NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing
NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the
effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.
The N-methyl-D-aspartate class of glutamate receptor (NMDAR) increases during neuropathological conditions including
is critical for development, learning and memory in the central ischemia, trauma and HIV-associated dementia, and may be
nervous system1,2. Excessive activation of NMDARs, however, responsible at least in part for neuronal apoptosis and necrosis
contributes to the pathological processes of stroke, epilepsy and in such neurodegenerative conditions10,2831. Second, NMDAR
several neurodegenerative disorders36. Hence, precise regulation activation stimulates neuronal nitric oxide synthase (nNOS)32,33,
of NMDAR activity is critical to normal brain function as well and endogenous NO increases in brain tissue are detected with an
as the avoidance of neuronal damage and death; accordingly, electrochemical probe34. Third, putative inhibition of NMDAR
there are several endogenous mechanisms for receptor modula- function by endogenous NO/NO-related species should serve as
tion1,2. One mechanism of control of NMDAR-associated chan- negative feedback for excessive NMDAR-associated channel activ-
nels715 and other membrane ion channels1625 involves direct ity. Here, using site-directed mutagenesis of recombinant
modulation by nitric oxide (NO) via an unknown, cyclic GMP- NMDAR subunits, we demonstrate regulation of a channel by
independent molecular mechanism. Here we show that the endogenous NO via S-nitrosylation.
NMDAR-associated ion channel was modulated by both endoge-
nous and exogenous NO. We used site-directed mutagenesis on RESULTS
the NR2A subunit to identify a cysteine residue (Cys 399) critical Initially, we examined inhibition of recombinant NR1/NR2A
to this modulation, which is S-nitrosylated (NO+ is transferred receptor responses by exogenous S-nitrosothiols (SNOs) in the
to cysteine thiol) under physiological conditions. In cells express- Xenopus laevis oocyte, as this NMDAR subunit combination
ing NMDARs with NR2A subunits in which this cysteine was shows greatest modulation by these NO-generating com-
mutated, inhibition of NMDA-evoked responses by endogenous pounds13,14. Application of S-nitrosocysteine (SNOC) decreased
NO was abolished. Moreover, reversed-phase high-performance NMDA-evoked currents by 24 2% (mean s.e., n = 11;
liquid chromatography (RP-HPLC) demonstrated S-nitrosyla- Fig. 1a). The decrease in NMDA-evoked currents persisted with-
tion of Cys 399 in an NR2A peptide after NO+ transfer. Taken out significant spontaneous recovery for over ten minutes, but
together, these results suggest that S-nitrosylation can modulate was rapidly reversed by the chemical reducing agent dithiothre-
ion channel activity via endogenous NO-thiol reactions in intact itol (DTT), suggesting covalent modification of cysteine
cell preparations. Evidence both in vitro and in vivo indicates that residue(s) on the receptor.
nitric oxide synthase (NOS) activity can affect NMDA-evoked Next, we tested methanethiosulfonate derivatives, which react
responses of primary neurons11,26, suggesting that endogenous specifically and rapidly with thiol groups to form mixed disul-
form(s) of NO-related species can inhibit NMDAR function. In fides35, for ability to mask the action of NO-related species on
single-channel recordings from primary cortical neurons, NO recombinant NMDARs. Similar to SNOC, a two-minute appli-
decreases opening frequency of the NMDAR-associated ion chan- cation of 2-aminoethylmethanethiosulfonate (MTSEA; 500 M)
nel27. We used recombinant systems to describe the molecular led to inhibition of NMDA-evoked currents in oocytes express-
basis of this effect on NMDAR responses. ing NR1/NR2A receptors; MTSEA also occluded further inhibi-
Interactions between NO and NMDARs are particularly inter- tion by subsequent SNOC application (Fig. 1b). This result
esting for three reasons. First, similar to NMDAR activity, NO suggests that MTSEA interacts with cysteine residues and blocks
articles
ISNOC/Icontrol (%)
possible involvement of cysteine residues in
this phenomenon. Using the reported con-
c sensus motif for S-nitrosylation15, we inspect-
ed NR1 and NR2A subunits for candidate
nitrosylation sites. From the candidate
cysteine residues, we chose to mutate to Ala
Cys 399, which is unique to NR2A among
NMDAR subunits and, according to pro
posed NMDAR-subunit transmembrane
No MTSEA Post MTSEA topology 3739, located in the extracellular N
treatment treatment
terminus of NR2A. We then tested the effect
Fig. 1. NO species generated by S-nitrosocysteine (SNOC) inhibit NMDA-evoked currents in of SNOC on NR1/NR2A(C399A) mutant
2000 Nature America Inc. http://neurosci.nature.com
NR1/NR2A receptors, and the cysteine modifying agent (2-aminoethyl) methanethiosulfonate receptors. Inhibition of NMDA-evoked cur-
(MTSEA) occludes NO inhibition. (a) Inhibition of NMDA-evoked currents (200 M NMDA rents by SNOC-generated NO was virtually
and 100 M glycine) after SNOC application for 3 min in Xenopus oocytes expressing abolished in NR1/NR2A(C399A) receptors
NR1/NR2A receptors. Holding potential, 80 mV; pH 7.5. Following washout of SNOC, the (Fig. 3a and b). Inhibition of NMDA-evoked
decrease in NMDA-evoked currents persisted for 10 min. (b) Modifying cysteine residues currents by methanethiosulfonate agents was
with MTSEA (0.5 M) for 2 min inhibited NMDA-evoked currents in oocytes expressing
also virtually abolished in NR1/NR2A(C399A)
NR1/NR2A receptors. Exposure to MTSEA occluded further inhibition of NMDA-evoked
currents by SNOC. (c) SNOC inhibition of NR1/NR2A receptors expressed as a percentage
receptors (Fig. 3c).
of the response to NMDA before SNOC application (mean s.e., n = 4 in each case). SNOC Inspection of NR1 and NR2 subunits revealed
inhibition after MTSEA treatment was significantly different from that before treatment (*p < an additional four cysteine residues resembling
0.01; Students t-test). the consensus motif for S-nitrosylation. In addi-
tion to NR2A Cys 399, only Cys 744 and Cys 798
(in the M3M4 extracellular linker of NR1) and
Cys 87 and Cys 320 (in the extracellular N ter-
their interaction with the NO-related species generated by SNOC. minus of NR2A) fit the consensus motif. Therefore, we also mutat-
Within one batch of oocytes, inhibition of NMDA-evoked cur- ed these cysteines to alanines, yielding the heteromeric receptor
rents by SNOC in the absence of MTSEA (21 3%, n = 4) sig- NR1(C744A,C798A)/NR2A(C87A, C320A,C399A). The crystal
nificantly differed from inhibition after MTSEA modification structure of the GluR2 ligand-binding domain reveals a disulfide
(5 3%, n = 4; Fig. 1c). Additionally, a larger, charged agent, 2- bond between cysteine residues homologous to NR1 C744 and
(trimethylammonium)ethyl methanethiosulfonate (MTSET) had C798; this may also be true for NR2A C87 and C320 (ref. 40). In
an effect similar to that of MTSEA and also occluded SNOC inhi- the form of disulfide, these additional cysteine residues would not
bition of NMDA-evoked currents. Prevention of NOs effect by possess free thiol groups and would therefore not be expected to
bath application of this permanently charged, membrane-imper- react with NO. Indeed, under physiological conditions, we could
meant compound suggested that the critical thiol group(s)
were located extracellularly.
We also considered if the modulation of NMDAR cur-
rents by NO was voltage dependent. If the critical cysteine
residue were extracellular, we would predict that the effect
of NO would be voltage independent. We found that the
currentvoltage relationship for wild-type NR1/NR2A was
approximately linear before and after exposure to SNOC
(Fig. 2), indicating that inhibition of NMDA-evoked cur-
rents by NO was not significantly voltage dependent.
Results with methanethiosulfonate agents, coupled
with our previous finding in primary rat cortical cul-
tures that N-ethylmaleimide prevents the inhibitory
effect of NO donors such as SNOC and nitroglycerin
articles
ISNOC/Icontrol (%)
results were obtained in n = 11 oocytes.
(b) Inhibition of NMDA-evoked currents by
SNOC in wild-type or mutant NR1/NR2A
receptors expressed as a percentage of the
response to NMDA before SNOC applica-
tion. Values are mean s.e.; n = 511 oocytes
for each group. SNOC inhibition of receptors c
containing the NR2A(C399A) subunit was
significantly different from that of wild-type
NR1/NR2A receptors (*p < 0.01, ANOVA
followed by Fishers PLSD test). (c) Current
tracings showing that inhibition of NMDA-
evoked currents by MTSEA (0.5 mM) was
nearly abolished in NR1/NR2A(C399A)
2000 Nature America Inc. http://neurosci.nature.com
not detect a statistically significant difference in SNOC-induced (Fig. 3e). Additionally, voltage-dependent blockade of NMDA-
inhibition of NMDA-evoked currents between NR1/NR2A(C399A) evoked currents from NR1/NR2A(C399A) receptors by 100 M
and NR1(C744A,C798A)/NR2A(C87A,C320A,C399A) receptors Mg2+ was indistinguishable from that of wild-type receptors.
(Fig. 3b). However, if we pretreated NR1/NR2A(C399A) with DTT These results indicate that the agonist sites and channel pore of
to reduce the disulfide bonds to free thiol groups, we then observed the NMDA receptor-channel complex were largely unchanged
an additional small but statistically significant inhibition by NO by the NR2A(C399A) mutation.
donors (11 4%, n = 10; p < 0.05 by ANOVA). Reminiscent of Next, we sought to detect inhibition of NMDAR responses
polynitrosylation of the ryanodine receptor25, these results suggest by endogenous NO and to determine the underlying molecular
that multiple cysteine residues may react with NO depending on mechanism for this effect. For this purpose, HEK293 cells stably
the redox state of the NMDAR; this may explain divergent find- transfected with nNOS42 were transiently transfected with wild-
ings 27,41. Our results indicate, however, that even though type NR1/NR2A or mutant NR1/NR2A(C399A) receptors.
NR1(C744,C798) and NR2A(C87,C320) can interact with NO, NMDA responses were monitored by digital Ca2+ imaging with
the effect is quite small and occurs under nonphysiological reduc- fura-2. Production of endogenous NO by these HEK-nNOS cells
ing conditions. Therefore, we characterized effects of endogenous was measured with diaminofluorescein (DAF-2), a new fluores-
NO on NMDAR responses by studying NR2A(C399) under phys- cent NO indicator43. In these cells, endogenous NO increased in
iological conditions (without previous exposure to reducing or response to NMDA in the presence of L-arginine, the substrate
oxidizing agents). for NOS. NO similarly increased in cells expressing either wild-
Before considering possible differences in physiological effects type NR1/NR2A or mutant NR1/NR2A(C399A) receptors
of endogenous NO on NR1/NR2A(C399A) or wild-type (Fig. 4b and c). Quantification of DAF-2 fluorescence in three
NR1/NR2A receptors, we had to determine whether this muta- experiments showed that NO level did not statistically differ
tion produced extensive structural changes affecting other between the wild-type and mutant receptors (wild-type, 84 11;
NMDAR properties. We constructed NMDA and glycine mutant, 102 16; mean arbitrary fluorescence units s.e.,
doseresponse curves for the wild-type and mutant receptors. n = 757 cells; p > 0.4 by Students t-test). In contrast, little or no
The NMDA EC 50 for NR1/NR2A(C399A) receptors was NO was detected in cells pretreated with L-nitro-arginine, a
54.8 1.6 M, compared with 37.5 1.7 M for wild-type recep- well -known NOS inhibitor (Fig. 4df). In the presence of L-argi-
tors (Fig. 3d). The glycine EC50 for NR1/NR2A(C399A) recep- nine, repeated pulses of NMDA, which led to progressive endoge-
tors was 2.1 0.2 M, compared with 2.3 0.2 M for wild-type nous production of NO, progressively reduced the magnitude of
articles
wild-type NR1/NR2A receptor Ca2+ responses (Fig. 4g). In con- tide showed a retention time of 12.5 min, irrespective of expo-
trast, responses of mutant NR1/NR2A(C399A) receptors were sure to the NO donor. These findings strongly suggest formation
not inhibited with serial NMDA application. Moreover, pre- of S-nitrosothiol (SNO) at cysteine 399 (ref. 44). We used this
treatment with L-nitro-arginine not only prevented NMDAs inhi- chemical approach to distinguish between different possible
bition of responses elicited from wild-type receptors, but also chemical modifications of the peptide; results implicated S-nitro-
actually increased response amplitudes (Fig. 4h). This result sug- sylation rather than formation of sulfenic acid or another deriv-
gests that endogenous NO already inhibited NMDAR responses ative. This finding is consistent with the notion that NO donors
at the time of application of L-nitro-arginine, but, because gen- induce S-nitrosylation of cysteine 399 rather than some other
eration of additional NO was blocked by this agent, the inhibi- chemical modification. Although relatively stable, the reaction
tion was relieved in 1020 minutes. As expected, L-nitro-arginine may spontaneously break down over tens of minutes; we observed
had essentially no effect on mutant NR1/NR2A(C399A) respons- this reversal of NOs effect on NR2A(C399) in both chemical and
es to NMDA. Taken together, these results can be best explained physiological experiments.
by physiological reaction of endogenous NO-related species with
the cysteine residue at position 399, in the extracellular domain of DISCUSSION
the NR2A subunit, to inhibit NR1/NR2A receptor responses. This study reports a mechanism of regulation of ion channel
To characterize the chemical reaction between NO and cys- activity by S-nitrosylation resulting from endogenous NOS activ-
teine 399, we synthesized a decapeptide containing the sequence ity in an intact cell system. S-nitrosylation of the NMDAR-asso-
surrounding this residue (KSFSDCEPDD) and a mutated pep- ciated ion channel is of particular interest because the action of
tide containing an alanine residue in place of the cysteine. We NO in the central nervous system is closely linked to the
then reacted these decapeptides with an NO donor and charac- NMDAR. NO is produced in certain neurons following NMDAR
terized the products by RP-HPLC with UV detection. Resulting activation and, in turn, triggers an increase in cGMP32; further-
chromatographs of the wild-type peptide revealed a shift in the more, entry of Ca2+ through NMDAR-associated channels stim-
major peak from a retention time of 13.5 minutes to 15.0 min- ulates nNOS activity42. The NMDAR NR1 subunit is physically
utes after reaction with the NO donor (Fig. 5). The mutated pep- coupled to nNOS by an intermediary adaptor protein, PSD95,
articles
articles
NO43. DAF-2 was used to measure nitric oxide formation in HEK-nNOS 8. Lei, S. Z. et al. Effect of nitric oxide production on the redox modulatory site
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9. Hoyt, K. R., Tang, L.-H., Aizenman, E. & Reynolds, I. J. Nitric oxide
NMDAR subunits and just before the experiment, the culture medium modulates NMDA-induced increases in intracellular Ca2+ in cultured rat
was exchanged for a physiological saline based on the Hanks balanced forebrain neurons. Brain Res. 592, 310316 (1992).
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2 M DAF-2 DA in HBSS at room temperature for 60 min and rinsed. ions in the nitric oxide-induced blockade of N-methyl-D-aspartate receptors
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13. Omerovic, A., Chen, S.-J., Leonard, J. P. & Kelso, S. R. Subunit-specific redox
presence of 1 mM L-Arg or 1 mM L-Arg plus 1 mM L-nitro-Arg. DAF-2 modulation of NMDA receptors expressed in Xenopus oocytes. J. Recept.
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with 10 M fura-2 acetoxymethylester (fura-2/AM) instead of DAF-2 from genes to channels. Trends Pharmacol. Sci. 17, 348355 (1996).
15. Stamler, J. S., Toone, E. J., Lipton, S. A. & Sucher, N. J. (S)NO signals:
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between 350 10 nm and 380 10 nm, with emission at 500 nm. Images oxide directly activates calcium-dependent potassium channels in vascular
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suggests a three transmembrane domain topology for the glutamate receptor Feelisch, M. & Stamler, J. S.) 521539 (Wiley, Chichester, 1996).
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2000 Nature America Inc. http://neurosci.nature.com
articles
1 Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstr. 46, D-60528 Frankfurt, Germany
2 Present address: Department of Psychiatric Research, University of Zrich, August-Forel-Str. 7, CH-8008 Zrich, Switzerland
3 Department of Anatomy and Cellular Neurobiology, University of Ulm, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
Correspondence should be addressed to H.B. (neurochemie@mpih-frankfurt.mpg.de)
2000 Nature America Inc. http://neurosci.nature.com
The formation of postsynaptic GABAA and glycine receptor clusters requires the receptor-associated
peripheral membrane protein gephyrin. Here we describe two splice variants of a novel gephyrin-binding
protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors
(GEFs). Co-expression of collybistin II with gephyrin induced the formation of submembrane gephyrin
aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regu-
lates the membrane deposition of gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this
protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.
Information processing in the CNS depends on fast neurotrans- A high-affinity gephyrin-binding motif in the cytoplasmic
mission, which is mediated by receptor proteins that are selec- loop region of the GlyR subunit1618 suggests that heterologously
tively concentrated at distinct postsynaptic membrane expressed gephyrin is not localized in submembrane areas, but
specializations. Mechanisms that establish these specialized mem- forms large intracytoplasmic aggregates16. This suggests that
brane domains during synaptogenesis and determine the posi- important regulatory elements of gephyrin targeting to synaptic
tions, size and packing densities of postsynaptic neurotransmitter sites are still unknown. Here we used the yeast two-hybrid sys-
receptor clusters are poorly understood. Receptor-associated pro- tem19 to search for gephyrin-binding proteins that may be involved
teins are assumed to be important in these processes13. in regulating the subcellular distribution of gephyrin. We describe
Postsynaptic clustering of the inhibitory glycine receptor (GlyR)4 a new dbl-like GDP/GTP exchange factor that induced the for-
involves a multi-step process requiring the local release of neuro- mation of submembrane gephyrin-rich microdomains. These
transmitter from apposed nerve terminals followed by activation submembrane gephyrin clusters were capable of accumulating
of GlyRs and depolarization of the postsynaptic membrane5,6. The hetero-oligomeric GlyRs. Our findings suggest that the novel GEF,
latter is thought to trigger Ca2+ influx through voltage-gated L-type for which we propose the name collybistin (from Greek
calcium channels; this influx then induces submembrane accumu- : to exchange), is an important determinant of gephyrin
lation of the GlyR-associated protein gephyrin5. The primordial localization in neurons, and thus of both GlyR and GABAAR clus-
gephyrin clusters are proposed to trap GlyRs that diffuse laterally tering at developing postsynaptic sites.
in the neuronal plasma membrane, thereby concentrating recep-
tors beneath active presynaptic terminals5. RESULTS
Studies on cultured spinal neurons7 and genetic approaches8 Collybistins I and II, two dbl-like GEFs
underscore the importance of gephyrin in GlyR clustering. Both To identify proteins that interact with gephyrin, we screened a yeast
attenuation of gephyrin expression by antisense oligonucleotides7 two-hybrid cDNA library from newborn rat brain using the
and the targeted disruption of the gephyrin gene8 prevent forma- gephyrin clone p1 (ref. 20) as bait. This resulted in the isolation of
tion of postsynaptic GlyR clusters. Moreover, immunohistochem- five cDNAs representing two alternatively spliced mRNAs of 3786
istry9,10, experiments with cultured neurons11 and analysis of bp and 3068 bp (Fig. 1a). The corresponding open reading frames
gephyrin-knockout mice12 also implicate gephyrin in the synaptic encode proteins of 493 and 413 amino acids with calculated mole-
localization of -aminobutyric acid type A receptors (GABAARs). cular weights of 58.2 kDa and 48.6 kDa. In the 5 UTR, several stop
In addition, gephyrin has non-synaptic functions as a coenzyme- codons were found in frame with the putative start codon, indi-
synthesizing protein in different peripheral tissues8 and species13. cating that full-length cDNAs were identified. A BLAST search of
Overlay and copolymerization assays demonstrate high-affin- the GenBank database revealed high homology to the human com-
ity binding of gephyrin to polymerized tubulin14. Furthermore, plete cDNA clone KIAA0424, with 93% identity throughout the
the morphology of postsynaptic gephyrin/GlyR clusters is regu- coding region and 6070% identity in the 3 UTR, but no significant
lated by cytoskeletal elements15. These findings are consistent homology in the 5 UTR. The homology between the rat and
with a model in which gephyrin serves as both an anchor and an human cDNAs starts abruptly 27 bp downstream from the first
organizing molecule linking postsynaptic receptors to the sub- potential start codon at the second methionine codon, suggesting
membrane cytoskeleton15. that the latter may serve as translational start signal. The predict-
articles
rat 240
DH moiety (Fig. 2c) mediates the GDP/GTP human:
exchange activity of dbl-like oncoproteins21. rat 280
The role of the poorly conserved PH domain human:
rat 320
(Fig. 2d) is unclear and might involve the human:
proper subcellular localization of GEFs22 and rat 360
binding of the second messenger phos- human:
phatidylinositol 4,5-bisphosphate (PIP2)23. rat 400
Because these homologies suggest that our human:
cDNAs encode a GEF for small GTPases of the rat 440
human:
Rho/Rac-family, we named the two splice vari-
rat 480
ants collybistin I and II. human:
The N-terminal region of the longer colly- 520
bistin splice variant contains an additional Src rat
human:
homology 3 (SH3) domain24. The primary 560
human:
structures of SH3 domains, known to mediate
interactions with proline-rich regions of other Fig. 1. Primary structures of rat and human collybistin. (a) Schematic representation of colly-
bistin I and II cDNAs. Nucleotide sequences encoding src homology 3 (SH3, gray box), Dbl
polypeptides24, are highly variable among dif-
homology (DH, black box), Pleckstrin homology (PH, black box) and the predicted coiled-coil
ferent protein families (Fig. 2b). The SH3 domain (CC, hatched box) are indicated. The cDNAs of collybistin I and II differ only by
domain identified here displays a higher nucleotide sequences encoding SH3 and coiled-coil domains, absent from the collybistin II
homology to SH3 domains of cytoskeletal cDNA. (b) Comparison of the primary structures of rat and human collybistin. Identical amino
proteins (Fig. 2b) than to those of the PAK- acids are marked by an asterisk, nonidentical residues with (). The homology domains shown in
interacting exchange factors (PIXs)25. A sec- (a) are boxed, and the predicted coiled-coil domain is underlined. Collybistin II has a unique C
ond region of variation is found in the terminus (VTQRKWHY*), starting with residue 462 of the collybistin I sequence.
C-terminal region of the longer variant, which
contains a segment predicted to form a coiled
coil, a structure known to mediate protein
interactions (Fig. 1a). the adult rat brain (Fig. 4a and b). Hybridization signals were
localized to neuronal somata (not shown). Distribution patterns
Collybistin transcripts expressed predominantly in brain of collybistin transcripts resembled those of gephyrin mRNAs
Northern hybridizations of polyA+ mRNA from several rat tissues (Fig. 4c), although hybridization intensities differed in various
with oligonucleotides specific for either the collybistin I or both the brain regions. Most prominently, collybistin expression was near-
collybistin I and II mRNAs (Fig. 3a) and with a random-primed ly equal in the granular layer of the cerebellum and in the dien-
collybistin cDNA revealed predominant expression of 5.5 kb and cephalon, whereas gephyrin expression was considerably weaker
6.0 kb transcripts in brain (Fig. 3b). In heart and skeletal muscle, in diencephalon than in cerebellum.
weak signals were detected at 6.0 kb and 6.5 kb and at 6.5 kb and To express collybistin proteins, we generated collybistin I and
6.8 kb respectively upon prolonged exposure (not shown); none of II cDNAs that encoded a hemagglutinin- (HA) epitope tag at
the other tissues examined showed significant hybridization their C-terminal ends and subcloned both constructs in a mam-
(Fig. 3b). The oligonucleotide probes used and the random-primed malian expression vector. Expression of the recombinant pro-
collybistin cDNA produced identical results, indicating similar dis- teins in human embryonic kidney (HEK) 293 cells transfected
tribution and abundance of collybistin I and II mRNAs. These data with these constructs was demonstrated using a polyclonal anti-
show that the collybistin gene is predominantly transcribed in brain HA antibody and an antibody generated against a peptide posi-
and, to a rather low extent, in heart and skeletal muscle. tioned amino-terminal to the DH domain and common to both
In-situ hybridization using either antisense oligonucleotide collybistin splice variants (Fig. 3a). In western blots of lysates
on horizontal and parasagittal sections revealed widespread from cells transfected with the HA-tagged collybistin I or II
expression of the collybistin gene throughout the gray matter of cDNAs, proteins migrating at 62 or 55 kDa, respectively, were
articles
aa
bb
Collybistin I
Stac
Myosin
Spectrin
Vav
DrhoGEF
rat PIX
2000 Nature America Inc. http://neurosci.nature.com
c
Collybistin I
Dbl
Tiam 1
PIX
Vav
Collybistin
Dbl
Tiam 1
PIX
Vav
d
Collybistin
Dbl
Tiam 1
PIX
Vav
Fig. 2. Domain comparison of collybistin and other members of the GEF family. (a) Comparison of the modular organization of collybistin I, Dbl
(P10911), Tiam 1 (Q60610), DrhoGEF (E1264107), rat PIX (D25304), Dbs (Q63406) and Vav (P54100). Accession numbers of the respective pro-
tein sequences are given in brackets. The domains are drawn roughly to scale. In addition to the DH, PH, SH3 and coiled-coil (slashed box) domains,
myosin-like motifs and, in Vav, a helix-loop-helix motif (HLH), a putative diacylglycerol-binding motif (DAG) and a SH2 domain are indicated.
(bd) Multiple sequence alignments of the SH3 (b), DH (c) and PH (d) domains of collybistin and related proteins. Residues present in at least 50%
of the sequences are highlighted by bold letters. Only amino-acid sequences displaying comparatively high homology to the respective domains of
collybistin are shown. In addition, the SH3 domains of myosin and spectrin are presented in (b); note that the SH3 domains of these cytoskeletal pro-
teins are more homologous to the SH3 domain of collybistin than those of other GEF family members.
specifically stained with either the anti-HA or the anti-collybistin- Collybistin associates with gephyrin in HEK 293 cells
peptide antibody (Fig. 5a). These findings are consistent with the To detect interaction of collybistin with gephyrin in intact mam-
predicted molecular weights of 58 kDa and 49 kDa, respective- malian cells, we used an assay previously employed to character-
ly. However, no specific immunolabeling was observed in brain ize the interaction of the GlyR subunit with gephyrin16,17. This
sections and cultured spinal neurons with this or a second anti- assay is based on the observation that recombinant gephyrin forms
peptide antibody (data not shown). Furthermore, an anti-HA large intracytoplasmic aggregates that trap hetero-oligomeric
antibody immunoprecipitated a complex containing gephyrin GlyRs16 as well as mutant GABAARs17 and NMDA receptors27 or
and collybistin I from extracts of HEK 293 cells co-expressing green fluorescent protein carrying a gephyrin-binding motif18.
these proteins (Fig. 5b) but not of cells transfected with cDNAs of Immunostaining of HA-tagged collybistins I and II expressed in
collybistin II (not shown). These data confirm the interaction of transfected HEK 293 cells localized both collybistin isoforms to
collybistin with gephyrin detected in our two-hybrid screen. We the cytoplasm (Fig. 6c and data not shown). However, when co-
also tried to co-immunoprecipitate collybistin solubilized from expressed with gephyrin, collybistin I was redistributed to
brain and spinal cord homogenates using anti-gephyrin anti- gephyrin-rich cytoplasmic regions as previously observed for
bodies. However, no specific band immunoreactive with the anti- polypeptides carrying a gephyrin-binding motif (Fig. 6d), again
collybistin peptide antibodies could be detected (not shown). demonstrating binding of the two proteins. By contrast, two addi-
This may reflect the poor reactivity of anti-gephyrin antibodies tional polypeptides identified as putative gephyrin-binding pro-
with detergent-solubilized gephyrin26. teins during the two-hybrid screen, BS69, a suppressor of
articles
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ulates postsynaptic GlyR-cluster formation at a step crucial for domains and thereby initiating GlyR and GABAAR clustering. In
2000 Nature America Inc. http://neurosci.nature.com
gephyrin deposition at presumptive postsynaptic sites. addition, it may localize subsynaptic signaling cascades to post-
synaptic sites34 by GEF-dependent activation of Rho-family
Collybistin is a GEF expressed in brain GTPases. Additional domains found in many GEFs include,
GEFs activate GTPases of the Rho/Rac family by rapidly exchang- among others, one or several SH2 or SH3 domains and regions
ing GDP for GTP21. Nucleotide exchange is mediated by the DH predicted to form coiled-coil structures22. In line with this mod-
moiety of the DH/PH tandem domain present in all GEFs ular-building principle, collybistin I harbors a SH3 domain in
known22. From sequence comparison with the DH domains of the aminoterminal region and a putative coiled-coil domain at
other GEFs, Rac and Cdc 42 seem the most likely candidates for the carboxyterminal end in addition to the central DH/PH
activation by collybistin. The PH region is thought to be required domain. The SH3 domain of collybistin I displays highest homol-
for the intracellular localization of GEFs33 and binds PIP2 (ref. ogy to SH3 domains of cytoskeletal proteins (Fig. 2b) and not to
23). The latter property may be important for recruiting colly- those of the PIX family25; therefore, signaling through PAK kinas-
bistin/gephyrin aggregates to PIP 2-rich plasma-membrane es seems unlikely. Both SH3 and coiled-coil domains mediate
interactions with other polypeptides, but there is no evidence for
proteins binding to the corresponding domains of collybistin I.
Notably, submembrane collybistin/gephyrin microclusters do
a b not form when collybistin II is replaced with collybistin I, which
differs only by the presence of the SH3 and coiled-coil domains.
This suggests that proteins recognizing these collybistin I-spe-
cific domains might regulate GEF function and/or interaction
with gephyrin. Conversely, the absence of such domains in colly-
bistin II indicates that gephyrin binding must be mediated by
segments adjacent to or within the DH/PH domains.
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and 240 bp were subcloned into pBluescript SK- (Stratagene) and RECEIVED 3 SEPTEMBER; ACCEPTED 3 NOVEMBER 1999
sequenced. Three independent clones were analyzed. The protein and
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EMBL/GeneBank data library under accession number AJ250425. Neurosci. 16, 347368 (1993).
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Antibodies. Monoclonal antibodies mAb 5a against gephyrin and mAb of glutamate receptors. Curr. Opin. Cell Biol. 8, 484489 (1996).
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2000 Nature America Inc. http://neurosci.nature.com
articles
1 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
2 Department of Biological Chemistry, University of California Los Angeles School of Medicine, Los Angeles, California 90095, USA
Correspondence should be addressed to A.G. (aghosh@jhmi.edu)
To understand the function of Notch in the mammalian brain, we examined Notch1 signaling and its
2000 Nature America Inc. http://neurosci.nature.com
cellular consequences in developing cortical neurons. We found that the cytoplasmic domain of
endogenous Notch1 translocated to the nucleus during neuronal differentiation. Notch1 cytoplasmic-
domain constructs transfected into cortical neurons were present in multiple phosphorylated forms,
localized to the nucleus and could induce CBF1-mediated transactivation. Molecular perturbation
experiments suggested that Notch1 signaling in cortical neurons promoted dendritic branching and
inhibited dendritic growth. These observations show that Notch1 signaling to the nucleus exerts an
important regulatory influence on the specification of dendritic morphology in neurons.
Although the role of cellcell interactions in the regulation of Four mammalian homologs of Notch (Notch1, Notch2, Notch3
mammalian neural development is not well understood, it has and Notch4) are expressed in the developing nervous sys-
become increasingly apparent from studies in invertebrates that tem2529. Mammalian homologs of the Notch ligands Delta and
such interactions can exert an important regulatory influence on Serrate are expressed in the developing brain and spinal
differentiation. The role of cellcell interactions has been exten- cord3033. Mice lacking Notch1 die embryonically, before the
sively studied in the context of lateral specification in inverte- period of neuronal differentiation, and show a high occurrence
brates. Such interactions among developmentally equivalent cells of cell death in the central nervous system34,35. In addition,
allow a single cell within the group to adopt a cell fate distinct transgenic mice overexpressing Notch3 have neural tube
from the neighboring cells and have an important role in neu- defects36. These observations suggest that, as in other verte-
roblast specification1,2. Molecular and genetic studies indicate brates, mammalian Notch homologs regulate early neural
that the cell-surface protein Notch is a central mediator of later- development.
al specification in invertebrates35. Protein localization studies also suggest a role for mammalian
Notch is a type I cell-surface protein, approximately 300 kDa Notch in neuronal differentiation. In the ventricular zone of devel-
in size, which functions as a receptor. Proteolytic processing of oping ferret cortex, Notch1 is localized to the basal pole of divid-
full-length Notch generates two fragments that seem to associ- ing progenitor cells37. Because the basal daughter is thought to
ate at the membrane to form a receptor complex69. One of the become postmitotic in asymmetric cell divisions, this observa-
fragments (p180) contains most of the extracellular domain, tion suggests that Notch1 is preferentially inherited by the post-
and the other fragment (p120) contains the transmembrane mitotic cell. This study suggests that Notch may continue to
and cytoplasmic domains. An important intracellular target of function in differentiated neurons after cell fate has been decided.
Notch signaling in Drosophila is the transcription factor, Sup- To explore the role of Notch in mammalian neural develop-
pressor of Hairless [Su(H)]10,11. There is also considerable evi- ment, we examined the molecular and cellular consequences of
dence that the mammalian homolog of Su(H), called CBF1, is a Notch signaling in developing cortical neurons. We report that
target of Notch signaling in mammalian cells1215. Although the neuronal Notch1 signals to the nucleus during differentiation and
mechanisms by which Notch signaling leads to transactivation regulates the dendritic development of cortical neurons.
via Su(H)/CBF1 are poorly understood, Notch-receptor acti-
vation seems to involve cleavage and nuclear translocation of RESULTS
the cytoplasmic domain of the receptor1619. Developmental regulation of Notch1 localization
Several observations suggest that vertebrate Notch proteins To characterize expression of Notch1 in cortical tissues, we used
are involved in regulating neural development in nonmam- affinity-purified antibodies generated against the intracellular
malian vertebrates. For example, overexpression of the Notch domain of Notch1 (-Notch.IC). These antibodies recognize a
ligand Delta or a constitutively active form of Notch inhibits truncated Notch1 construct when expressed in COS cells, and
primary neurogenesis in Xenopus2022. Similarly, Notch-medi- this interaction can be specifically blocked by preincubation of
ated interactions seem to negatively regulate neuronal differ- the affinity-purified antibodies with recombinant Notch1 pro-
entiation in Xenopus and chick retina23,24. It is likely that Notch tein (Fig. 1a). In addition, these antibodies recognize full-length
is also involved in regulating neural development in mammals. Notch1 but not Notch2, Notch3 or Notch4 in western blots of
articles
recombinant proteins (Fig. 1b). Western blots showed that the To determine Notch1 protein localization in developing cor-
two previously described forms of Notch1, full-length p300 and tex, we performed immunohistochemical staining with
cleaved p120, were present in cortical neurons at all ages -Notch.IC on cortical sections from late embryonic and early
(Fig. 1c). Proteins migrating at about 140 kDa and 100 kDa were postnatal rats. In agreement with previous studies25,30,37, we
found at embryonic and early postnatal ages, but not at later ages. detected Notch1 protein in cells of the ventricular zone of E18
This pattern suggests that Notch1 might regulate developmental cortex (Fig. 1e). In addition, at both embryonic and postnatal
events both during and after neurogenesis. ages, we detected significant levels of Notch1 immunoreactiv-
To define the cell-surface form of Notch1 in developing cor- ity in the cortical plate, suggesting that Notch1 may regulate
tical neurons, we did surface biotinylation experiments in E18 the development of postmitotic neurons.
cortical neurons in culture. After surface biotin labeling, We performed confocal microscopy on immunofluorescent-
biotinylated proteins were precipitated using streptavidin- ly labeled sections through the developing cortex to determine
agarose and separated by SDS-PAGE. Western blots using anti- the subcellular localization of Notch1 in cortical cells. In the ven-
bodies to the intracellular and extracellular domains of Notch1 tricular zone, Notch1 was concentrated near the cell membranes
indicated that the p120 form of the Notch1 protein was the and seemed largely excluded from the nucleus (Fig. 2ac). By
dominant cell-surface form of the receptor recognized by the contrast, Notch1 was localized predominantly in the nucleus in
intracellular-domain antibody, and the p180 form of the pro- cortical plate neurons (Fig. 2 di). Because cells migrate from
tein was the dominant cell-surface form recognized by the extra- the ventricular zone to the cortical plate once they become post-
cellular-domain antibody (Fig. 1d). This is consistent with a mitotic, these observations suggest that neuronal differentiation
model in which the cell-surface Notch1 receptor is a het- is accompanied by a translocation of Notch1 to the nucleus.
erodimer of p120 and p180 forms. We also found a high-mole- To examine the relationship between Notch 1 localization and
cular-weight form (Fig. 1d) that may represent uncleaved, neuronal differentiation more carefully, we carried out experiments
full-length Notch1 at the cell surface. in E18 dissociated cortical cell cultures, in which the transition from
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postmitotic neurons.
articles
a b
c d e
2000 Nature America Inc. http://neurosci.nature.com
Fig. 5. Characterization of nuclear forms of Notch1. (a) The distribution of Notch1 in whole tissue homogenates (H), the cytosolic fraction (C) and
the nuclear fraction (N) in extracts prepared from P3 and adult cortex as determined by western blotting using -Notch.IC. The nuclear extract con-
tains two Notch1-immunoreactive proteins that migrate at 120 kDa and 100 kDa (arrows). Probing the same blot with an antibody to a membrane
protein (Na+/K+ ATPase) does not give a signal in the nuclear fraction, indicating that the nuclear Notch1 signal does not represent membrane-asso-
ciated Notch1. (b) Western blot of nuclear fraction from P3 cortex loaded alongside immunoprecipitates of HA-tagged truncated Notch1 constructs
and probed with -Notch.IC. The upper and lower bands present in P3 nuclear fraction comigrate with the bands found with ZEDN1.HA and
FCDN1.HA. CDN1.HA lacks sequences in FCDN1 and migrates lower than the two bands in the P3 nuclear fraction. (c) Phosphatase treatment of
ZEDN1.HA and FCDN1.HA immunoprecipitates indicates that the p120 form of these proteins is phosphorylated. E18 cortical neurons were trans-
fected with either ZEDN1.HA or FCDN1.HA, immunoprecipitated with anti-HA, treated with PPase or 10 mM Na3VO4 and separated by PAGE.
The western blot was then probed with -Notch.IC. Treatment with PPase causes a reduction in the p120 band compared to the p100 band. The
residual p120 signal in the ZEDN1.HA-transfected cells may correspond to the p120 transmembrane form of Notch1 that has not yet undergone
cleavage in the intracellular domain. (d) Metabolic labeling of FCDN1.HA-transfected neurons with [32P]orthophosphate followed by immunoprecip-
itation with HA antibodies identifies the p120 form of FCDN1.HA as a phosphorylated protein. Immunoprecipitates from [32P]-labeled FCDN1.HA
(+) and control () transfected neurons were separated by PAGE, transferred to nitrocellulose and exposed to film to detect [32P] radioactive signal
(right). Subsequently, the blot was probed with -Notch.IC to reveal the p100 and p120 forms of FCDN1 (left). Lanes 3 and 4 in the western blot
(left) are the same two lanes shown in the film exposure (right). Comparison of the two indicates that the p120 form of FCDN1 is phosphorylated
(arrow). (e) Relative mobility of nuclear Notch1 bands (P3 nuclear) and the biotinylated transmembrane p120 form of Notch1 (E18 + 5DIV biotiny-
lated). The upper (phosphorylated) nuclear band comigrates with the transmembrane p120 form of Notch1.
To determine if the phosphorylated form of nuclear Notch1 in postmitotic cortical neurons, we transfected a series of Notch1-
was distinguishable from the truncated transmembrane form by deletion constructs into cortical neurons (Fig. 6a) together with a
size, we ran cortical nuclear extracts next to biotinyated cell-sur- reporter driven by CBF1 binding sites14 (CBF1-CAT). By immuno-
face proteins and probed the blot with -Notch.IC. The upper fluorescence, transfected full-length Notch1 was excluded from
nuclear Notch1 band comigrated with p120 transmembrane the nucleus and was present mainly in intracellular organelles (and
Notch1 (Fig. 5d). Thus, neurons had two processed forms of perhaps at the cell surface, to some extent). Transfected 0CDN1
Notch1 that migrated at about 120 kDa: the truncated trans- was present mainly on the cell surface and in the cytoplasm. In
membrane receptor and the phosphorylated intracellular domain contrast, CDN1 and CDCN1T were present both in the cytoplasm
of Notch1. These two forms could not be distinguished by west- and in the nucleus, and ZEDN1 and FCDN1 were present mainly
ern blots of whole-cell lysates, and their identification required in the nucleus (Fig. 3 and data not shown).
surface biotinylation and cell-fraction experiments. In neurons transfected with reporter together with the par-
ent vector (pBos), there was a low basal level of reporter activ-
Regulation of transcription by nuclear Notch1 ity (Fig. 6b and c). Transfection with the various Notch1
In non-neuronal cell lines, the cytoplasmic domain of Notch1 can constructs led to markedly different levels of transactivation
translocate to the nucleus and induce CBF1-mediated transacti- (Fig. 6b and c). Whereas transfection with full-length Notch1
vation. To determine whether this signaling pathway is functional led to a weak but significant activation of the reporter, trans-
articles
Fold induction
(pBos) or full-length
Notch1 (N1) together
2000 Nature America Inc. http://neurosci.nature.com
w i t h C B F 1 - C AT a n d
increasing amounts of
0CDN1. 0CDN1 attenu-
ates Notch1 activation of
CBF1-CAT.
by CDN1, a construct
lacking the RAM
domain, a region
between the trans-
membrane domain and
the ankyrin repeats.
Thus the cytoplasmic
domain of Notch1 can
strongly activate CBF1-
mediated transcription
in cortical neurons,
and Notch1 activation
of CBF1-mediated
transcription requires
the RAM domain.
A form of Notch lacking the intracellular domain (0CDN1) (Fig. 7). In this experiment, Notch1 was likely activated by lig-
can act as a dominant-negative receptor39. When expressed in ands expressed by the cultured cortical neurons. In contrast to
cortical neurons, 0CDN1 did not activate the CBF1-CAT the CBF1-CAT reporter, which is strongly activated by ZEDN1
reporter and inhibited activation of CBF1-CAT by full-length and FCDN1, the NSE-CAT reporter was not activated by trun-
Notch1, indicating that it could inhibit Notch1 signaling to cated Notch1 constructs. The failure of truncated Notch1 con-
CBF1 (Fig. 6d and e). structs to activate NSE.CAT suggests that Notch1 signaling to
We next asked if Notch1 signaling could regulate neuron-spe- NSE-CAT may be CBF1 independent. This is not entirely unex-
cific gene expression. The neuron-specific enolase (NSE) gene is pected, as alternate Notch1 signaling pathways have been
expressed exclusively in terminally differentiated neurons and described both in Drosophila development and in myogenesis42.
neuroendocrine cells40, and an 1800-bp fragment of the promoter
can drive neuron-specific expression of a LacZ reporter in trans- Notch1 signaling regulates dendritic morphology
genic mice41. A CAT-reporter construct driven by this 1800-bp The Notch1 localization and signaling experiments suggested
fragment (NSE-CAT) transfected into cortical neurons gave a that Notch1 might regulate aspects of neuronal differentiation.
low level of reporter activity (Fig. 7). Cotransfection of full-length We thus examined the effects of inhibiting Notch1 signaling on
Notch1 led to increased reporter expression, indicating that one of the clearest manifestations of neuronal differentiation,
Notch1 signaling can lead to activation of the NSE promoter the development of dendrites. The dendritic morphology of cul-
articles
Fold induction
treatments, we rescued the effects by over-
expressing an activated Notch1 construct
(FCDN1). The effects of antisense treat-
ment on various dendritic parameters were
partially or completely reversed by overex-
pressing FCDN1 (Fig. 9gj). These obser-
vations support the results of the
transfection experiments, and suggest that
Notch1 signaling exerts a positive effect on
dendritic branching and a negative effect
on dendritic growth.
DISCUSSION
2000 Nature America Inc. http://neurosci.nature.com
articles
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Branching index
sense-(CDC.AS) treated neurons.
Branching index
articles
pBos and either 0CDN1 or pBos at molar ratios of 1:1:1 and 1:1:3. The trol and FCDN1.HA-transfected cultures with 200 Ci per 60-mm dish
final amount of DNA transfected was equalized with pBluescript for each of [32P]orthophosphate (H3PO4; ICN) in phosphate-free DMEM (Gibco)
condition. For morphological analysis, E18 cortical cultures were trans- for 4 h at 37C. Cells were lysed, immunoprecipitated with anti-HA and
fected at 2 DIV with RSV--galactosidase together with the parent expres- transferred to nitrocellulose as above. The blot was first exposed to film
sion vector (control), the 0CDN1 expression plasmid or the ZEDN1 to detect radioactivity and then immunoprecipitated. FCDN1.HA was
expression plasmid in a molar ratio of 1:2 (gal construct) and were fixed detected with -Notch.IC as described above.
and processed for -galactosidase immunocytochemistry at 5 DIV before For the biotinylation experiments, E18 cortical cultures in 60-mm
morphology of transfected neurons was analyzed (see Analysis below). dishes were washed with dextrose (1 mg per ml) in PBS, then incubated
at 4C in 2 mM EZ link Sulfo-NHS-LC-Biotin (Pierce) in dextrose (1 mg
Western blots and cell fractionation. We used 10 g of Notch1, Notch2, per ml) in PBS for 30 min. Cells were rinsed multiple times with dex-
Notch3-HA, Notch4-HA or parent vector (pBos) to transfect 293 cells trose (1 mg per ml) in PBS and lysed as above for immunoprecipitation.
using a calcium phosphate procedure. Cells collected from the same trans- Lysis supernatants were incubated with immobilized Streptavidin (Pierce)
fection were lysed in RIPA buffer, pooled and then split for immunopre- overnight at 4C, washed and separated by PAGE. Proteins were trans-
cipitation. Lysates were immunoprecipitated with 1:100 of rabbit ferred to nitrocellulose and incubated with -Notch.IC (93-4) or an
polyclonal antibodies to either Notch1 (93-4) or Notch2 (93-3) or with extracellular-domain antibody (5261) and detected as above.
monoclonal antibody to HA (12CA5) and captured with Protein-A
agarose. Immunoprecipitates were analyzed by western blot using 93-4 CAT assays. Cells were harvested 1 (NSE-CAT) or 2 (CBF1-CAT) days
(1:5000) to detect Notch1, 93-3 (1:3000) to detect Notch2 and 12CA5 after transfection in isotonic TNE (10 mM Tris, 150 mM NaCl and 1 mM
(1:1000) monoclonal to detect the HA tag present on Notch3 and Notch4. EDTA at pH 7.8). Cells were spun down gently and subjected to three
Antibodyprotein interactions were detected by ECL. cycles of freeze-thaw lysis. Lysis supernatants were incubated with 0.5
2000 Nature America Inc. http://neurosci.nature.com
For tissue westerns, cortices were dissected from embryonic (E13 and Ci 14C-labeled chloramphenicol (Amersham) and 0.8 mmol acetyl CoA
E18) and postnatal rats (P0, P7, P14 and adult). Tissues were homogenized (Boehringer Mannheim) at pH 7.8 and at 37C for 1 h. Reaction mix-
with a dounce homogenizer in lysis buffer (137 mM NaCl, 20 mM Tris, 1% tures were extracted with ethyl acetate, speed-vacuumed, resuspended
NP40, 10% glycerol, 1 mM PMSF, 10 g per ml aprotinin, 1 g per ml pep- in chloroform, spotted onto thin-layer chromatography (TLC) plates
statin A and 1g per ml leupeptin at pH 8.0). To disrupt nuclei, (J.T. Baker) and separated by ascending chromatography for 2 h (95%
homogenates were sonicated with 2 bursts (15 s), homogenized with pestel chloroform, 5% methanol). To normalize for transfection efficiency,
B and mixed for 1 h at 4C. E18 cultured cells were rinsed in TBS, harvest- equal amounts of a RSV--galactosidase plasmid were cotransfected in
ed and homogenized as for whole tissue. BCA Protein Assay (Pierce, Rock- all reporter experiments, and -galactosidase activity was used for nor-
ford, Illinois) was used to determine protein concentration, and 10 g of malization. Data from any single histogram were quantified from exper-
each homogenate was separated by PAGE. Proteins were transferred to nitro- iments on cells that were simultaneously cultured, transfected and assayed
cellulose using a Genie Electroblotter system (Idea Scientific, Minneapolis, to minimize variability due to subtle differences in experimental proce-
Minnesota). Blots were blocked with 4% BSA in TBST and incubated with dure. Fold induction was calculated relative to control transfected cul-
-Notch.IC (93-4) diluted 1:10,000 in blocking solution overnight at 4C. tures. Shown are representative examples of experiments performed three
In control experiments, -Notch.IC was incubated at 1:15,000 with an excess times. For measurements of relative CAT activity, levels of [14C] emis-
of Notch1 fusion protein (20 g per ml) overnight at 4C before incuba- sions on TLC plates were quantified by phosphorimager scans. Statisti-
tion with blots of FCDN1.HA immunoprecipitates. Blots were incubated cally significant differences (Students t-test, p < 0.05) in reporter activity
with peroxidase-conjugated anti-rabbit secondary antibody (Amersham) are indicated by an asterisk.
diluted 1:20,000 in blocking solution and visualized using a chemilumi-
nescent detection system (SuperSignal Substrate, Pierce). Antisense oligonucleotides. Oligonucleotides were designed against two
Cell fractionation and isolation of nuclear proteins was carried out distinct regions of rat Notch1 as reported24, a portion of the 5 intra-
as described50. Cortices from P3 and adult rats were minced and homog- cellular cdc10/ankyrin repeat region (CDC) and the extracellular
enized by two-component dounce homogenization, and nuclei were lin12/Notch repeat region (LN). The CDC antisense sequence, 5-CCTC-
separated on a sucrose density gradient. Isolated nuclei were resuspended CGCTGCAGGAGGCAATCAT-3, and the LN antisense sequence,
in lysis buffer and disrupted by sonication. Protein concentrations were 5-CCAGCACTGCAGGGACTGAGTGC-3, were used. For each anti-
determined by BCA protein assay, and 10 g of each fraction sense oligonucleotide, corresponding sense oligonucleotides were syn-
homogenate, cytoplasmic and nuclearwere separated by PAGE and thesized and used in parallel in each experiment. All oligonucleotides
transferred to nitrocellulose. Notch1 was detected using -Notch.IC as were 23 bp in length, with phosphothioate linkages added between all
described above. Parallel blots were blocked with 3% nonfat dry milk bases. Oligonucleotides were synthesized at the Johns Hopkins School
in PBS and incubated overnight with an antibody to the -1 subunit of of Hygiene and Public Health DNA Synthesis Core Facility. Optical den-
the Na+/K+ ATPase (Upstate Biotechnology) at 1:1000 in blocking solu- sity (OD260) readings were taken to determine concentration. For anti-
tion at 4C. Blots were incubated with peroxidase-conjugated anti-rab- sense treatment experiments, E18 cortical neurons were cultured as
bit secondary antibody (Amersham) diluted 1:10,000 in blocking described on 12-well tissue culture plates. Cells were transfected with
solution and were visualized by chemiluminescence, as above. RSV--galactosidase at 2 DIV. The day after transfection, sense and anti-
sense oligonucleotides were added to a final concentration of 2 M and
Immunoprecipitation, metabolic labeling and cell-surface biotinylation. maintained for an additional two days before fixation. The morphology
E18 cortical cultures in 60-mm dishes were transfected with 6 g per of the transfected neurons was revealed by -galactosidase immunocy-
dish of ZEDN1.HA, FCDN1.HA or CDN1.HA. Three days after trans- tochemistry and analyzed as described below.
fection, cells were lysed in lysis buffer. Lysed cells were homogenized with
pestel B of a dounce homogenizer. Lysis supernatants were separated Analysis. Transfection experiments were carried out in duplicate wells,
from fragmented cells by centrifugation, incubated with anti-HA anti- and all of the experiments described here were repeated multiple times in
bodies and Gamma Bind G Sepharose (Pharmacia), washed and sepa- several independent experiments with comparable results. To analyze the
rated by PAGE. For the analysis of protein phosphorylation, cells from effects of antisense treatment, ZEDN1 and 0CDN1 expression on dendritic
the same transfection condition were lysed as above, pooled and then morphology, images of -galactosidase-transfected neurons were captured
split for immunoprecipitation. After binding to Gamma Bind G using a digital CCD camera (Dage, MTI, Michigan City, Indiana) attached
Sepharose, immunoprecipitates were incubated for 1 hour at 30C with to the side port of a Nikon Eclipse TE300 inverted microscope driven by
50 U Phosphatase (NEB) or 10 mM sodium orthovanadate (Na3VO4) in IP Lab Spectrum 3.1.1 image-acquisition software (Scanalytics, Fairfax,
50 mM TrisHCl, 5 mM DTT, 2 mM MnCl2, 100 g per ml BSA and pro- Virginia). Captured cells were then traced using ClarisDraw software. To
tease inhibitors as above at pH 7.8 and washed and separated by PAGE. assess the effects of ZEDN1 and 0CDN1 expression on dendritic mor-
After separation by PAGE, proteins were transferred to nitrocellulose and phology, the dendritic trees of at least 50 Bos-(control), ZEDN1- and
detected as above. Metabolic labeling was performed by incubating con- 0CDN1-transfected neurons were reconstructed and scored for number
articles
of primary processes, dendritic branch points, average dendritic length 20. Coffman, C., Harris, W. & Kintner, C. Xotch, the Xenopus homolog of
and branching index (number of branch points divided by average den- Drosophila notch. Science 249, 14381441 (1990).
21. Coffman, C. R., Skoglund, P., Harris, W. A. & Kintner, C. R. Expression of an
dritic length, expressed as percent of control). For quantitative analysis of extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73,
the effects of oligonucleotide treatments on dendritic morphology, the 659671 (1993).
dendritic trees of at least 100 transfected neurons from each treatment 22. Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. & Kintner, C. Primary
condition per experiment were reconstructed and scored for various den- neurogenesis in Xenopus embryos regulated by a homologue of the
dritic parameters. Independent blind analysis of dendritic morphology Drosophila neurogenic gene Delta. Nature 375, 761766 (1995).
23. Dorsky, R. I., Rapaport, D. H. & Harris, W. A. Xotch inhibits cell
under various experimental conditions showed similar results. Data are differentiation in the Xenopus retina. Neuron 14, 487496 (1995).
shown as mean standard error. Statistically significant differences (Stu- 24. Austin, C. P., Feldman, D. E., Ida, J. A. Jr. & Cepko, C. L. Vertebrate retinal
dents t-test; p < 0.05) are indicated by an asterisk. To analyze the relative ganglion cells are selected from competent progenitors by the action of
amounts of intracellular domain Notch1 in the nucleus and cytoplasm of Notch. Development 121, 36373650 (1995).
25. Weinmaster, G., Roberts, V. J. & Lemke, G. A homolog of Drosophila Notch
double-labeled cells (Fig. 4m), we captured images of Notch1-immunos- expressed during mammalian development. Development 113, 199205 (1991).
tained neurons as above. The intensity of Notch1 immunofluorescence 26. del Amo, F. F. et al. Expression pattern of Motch, a mouse homologue of
was then measured in identical volumes of the nucleus and cytoplasm from Drosophila Notch, suggests an important role in early postimplantation
the same cell using IP Lab Spectrum 3.1.1 image software. mouse development. Development 115, 737744 (1992).
27. Reaume, A. G., Conlon, R. A., Zirngibl, R., Yamaguchi, T. P. & Rossant, J.
Expression analysis of a Notch homologue in the mouse embryo. Dev. Biol.
ACKNOWLEDGEMENTS 154, 377387 (1992).
We thank Diane Hayward for providing us with CBF1 reporter constructs, 28. Weinmaster, G., Roberts, V. J. & Lemke, G. Notch2: a second mammalian
Carrie Shawber and Libby Walker for the pCDNA3.FCDN1 plasmid, Donna Notch gene. Development 116, 931941 (1992).
29. Lardelli, M., Dahlstrand, J. & Lendahl, U. The novel Notch homologue
Nofziger for affinity purification of the 93-4 antisera, Greg Sutcliffe for the NSE-
2000 Nature America Inc. http://neurosci.nature.com
articles
1 Departments of Neuroscience and 2Molecular Oncology, Genentech, Inc., South San Francisco, California 94080, USA
Correspondence should be addressed to M.H. (mah@gene.com) and A.R. (ar@gene.com)
Sonic Hedgehog (Shh) is a secreted protein that controls cell fate and mitogenesis in the develop-
2000 Nature America Inc. http://neurosci.nature.com
ing nervous system. Here we show that a constitutively active form of Smoothened (Smo-M2)
mimics concentration-dependent actions of Shh in the developing neural tube, including
activation of ventral marker genes (HNF3, patched, Nkx2.2, netrin-1), suppression of dorsal
markers (Pax-3, Gli-3, Ephrin A5) and induction of ventral neurons (dopaminergic, serotonergic)
and ventrolateral motor neurons (Islet-1+, Islet-2+, HB9+) and interneurons (Engrailed-1+,
CHX10+). Furthermore, Smo-M2s patterning activities were cell autonomous, occurring
exclusively in cells expressing Smo-M2. These findings suggest that Smo is a key signaling
component in the Hh receptor and that Shh patterns the vertebrate nervous system as a
morphogen, rather than through secondary relay signals.
Sonic hedgehog (Shh) is a vertebrate homolog of the secreted and basal cell carcinomas21. In contrast, Smo acquires activating
segment-polarity gene, Hedgehog (Hh), which is produced by mutations in sporadic forms of this disorder22.
the embryonic notochord and floor plate. Genetic studies in Taken together, these findings lead to the hypothesis that the
human1,2 and mouse3 as well as studies with cultured chick and receptor for Shh is composed of both Ptc and Smo15,20,23. How-
rodent neural-tube explants4 revealed that Shh controls multiple ever, although Smo mediates simple mitogenic signals of Shh in
cell-patterning events along the ventral aspect of the neural tube. vertebrate skin22, the question of whether it is involved in the
An early activity of Shh is to restrict the expression of multiple coordination of the complex patterning actions of Shh in the
transcription factors (for example, Pax3, Pax7, Msx1 and Msx2)4 neural tube remains open. To directly examine this issue, we
and secreted proteins (for instance, Ephrin-A5)5 to the dorsal expressed a constitutively active form of Smo (Smo-M2) in the
aspect of the neural tube. Concomitant with the exclusion of neural tubes of mice and chick embryos. We show that ectopic
future dorsal cell markers, Shh upregulates the expression of expression of Smo-M2 resulted in downregulation of dorsal cell
ventral transcription factors [for example, HNF3, Nkx 2.2 (ref. markers, induction of ventral cell markers and the ectopic for-
6) Pax 6 (ref. 7) and Gli-1 (ref. 5)] as well as cell surface (Ptc)8 mation of ventral neurons.
and secreted (for example, Netrin-1)9,10 proteins in the ventral Having obtained evidence that Smo-M2 is a key signaling com-
neural tube, thus committing the affected neural precursors to a ponent of the Shh receptor, we asked whether Shh induces distinct
general ventral cell fate. Shh further induces the specification of cell types in the neural tube directly or through a second wave of
these generic ventral progenitors to distinct neuronal and non- secreted inducers. We assumed that if Shh specified neural prog-
neuronal cell types that typify the ventral aspect of the mature enitors directly, its activated receptor would suppress dorsal cell
vertebrate nervous system. These include floor plate cells, Islet- markers and upregulate ventral cell markers only in the cells in
1, 2 and HB9-positive motor neurons, Chx10+ and Engrailed-1+ which it was expressed. On the other hand, if a relay signal were
(En-1) interneurons4, serotonergic (5HT)5,11, dopaminergic involved, cells that did not express Smo-M2 would also be affect-
(DA)12,13 and multiple forebrain6 neurons. ed, as they would respond to secreted relay signals produced by
The mechanism of Shh signal transduction is not fully Smo-M2-expressing cells. Studies in non-neuronal tissues in
understood. However, biochemical studies demonstrate that Drosophila and vertebrates suggest that, depending on the partic-
Shh binds the 12-transmembrane (TM) protein Ptc14,15, and ular system, Hh can mediate its activities directly or via a relay
that Ptc in turn forms a physical complex with Smo15. Gene mechanism. For example, the mitogenic and patterning effects of
ablation and overexpression experiments in Drosophila as well Shh in the vertebrate limb are executed via members of both the
as gene ablation of Ptc in mice further suggest that Smo is fibroblast growth factor (FGF) and the bone morphogenic factors
required for Hh signal transduction16,17. In contrast, Ptc seems (BMP) protein families24,25, which act as relay signals. Likewise,
to be a negative regulator in this signaling system1820. Consistent the cell-patterning effects of Hh in the anterior compartment of
with these suggestions, Ptc is inactivated in hereditary basal cell the Drosophila leg imaginal disc are thought to be mediated indi-
nevus syndrome (BCNS), a disease associated with develop- rectly via the induction of BMP, dorsally, and wingless, ventrally26.
mental abnormalities and high incidence of medulloblastomas In addition, Hh seems to influence cell polarity in the Drosophila
articles
RESULTS
Smo-M2 alters cell pattern in the neural tube
We expressed cDNAs encoding wild-type (WT) Smo15 or a con-
2000 Nature America Inc. http://neurosci.nature.com
articles
articles
articles
Fig. 5. Smo-M2 induces ventral and ventrolateral markers and Ventral markers SmoM2/GFP Overlap
cell types in the spinal cord. (ao) Smo-M2 induces the marker
for ventral spinal cord and d-MN progenitor, Nkx2.2 (ac), the
v-MN motor neuron markers HB9 (df) and islet-2 (gi), and
the progressively more dorsal V1 and V2 interneuron markers
Chx10 (jl) and En-1 (mo). Each of these markers and cell
types is induced in a cell-autonomous manner in the electropo-
rated (right) but not control (left) side of the neural tube
(examples marked by arrowheads). Scale bar, ~0.5 mm (ao).
articles
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for the HNF-3 antiserum. We also thank E. Berry and A. Bruce for help with prepa- of the homeobox containing gene en-2 delineates a specific region in the
ration of the manuscript and A. Shih and K. Poulsen for technical assistance. developing mouse brain. Genes Dev. 2, 361371 (1988).
35. Tremblay, P., Pituello, F. & Gruss, P. Inhibition of floor plate differentiation by
pax3: evidence from ectopic expression in transgenic mice. Development 122,
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36. Winslow, J. W. et al. Cloning of AL-1, a ligand for an Eph-related tyrosine kinase
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articles
1 Department of Physiology, University of California, San Francisco, San Francisco, California 94143-0444, USA
2 Department of Anatomy, Box 0452, University of California, San Francisco, San Francisco, California 94143-0452, USA
3 Department of Neurology, Box 0453, University of California, San Francisco, San Francisco, California 94143-0453, USA
Correspondence should be addressed to H.L.F. (hlf@itsa.ucsf.edu)
Repeated administration of an opioid in the presence of specific environmental cues can induce
tolerance specific to that setting (associative tolerance). Prolonged or repeated administration of an
2000 Nature America Inc. http://neurosci.nature.com
opioid without consistent contextual pairing yields non-associative tolerance. Here we demonstrate
that cholecystokinin acting at the cholecystokinin-B receptor is required for associative but not non-
associative morphine tolerance. Morphine given in the morphine-associated context increased Fos-
like immunoreactivity in the lateral amygdala and hippocampal area CA1. Microinjection of the
cholecystokinin B antagonist L-365,260 into the amygdala blocked associative tolerance. These
results indicate that cholecystokinin acting in the amygdala is necessary for associative tolerance to
morphines analgesic effect.
The role of cholecystokinin (CCK) as an anti-opioid peptide is erance (group A), we used a balanced crossover design in which all
firmly established. CCK suppresses the analgesic effect of mor- rats received i.v. morphine in one of two distinct environments
phine and other -opioid receptor agonists1,2. CCK antagonists and, on alternate days, i.v. saline in the other environment. After
potentiate the analgesic effects of exogenous and endogenous several administrations, morphine was ineffective when given in
-opioid receptor agonists38 and enhance the inhibitory effect the environment in which the rat had previously received mor-
of morphine on nociceptive dorsal horn neurons9,10. Addition- phine (Fig. 1a, black circles). However, the same dose of mor-
ally, CCK antagonists impede the acquisition of morphine toler- phine was fully effective when administered in the context
ance yet have no effect on morphine dependence or previously paired with saline (Fig. 1b). Thus, using this behav-
withdrawal3,1116. Although CCK antagonists potentiate the effects ioral protocol and method of drug administration, the analgesic
of opioids, they do not alter nociceptive threshold levels when tolerance to morphine could be accounted for by learning factors
administered independently8,16. There are two distinct CCK and therefore was associative. To produce non-associative toler-
receptors (CCK-A and CCK-B) in the CNS1720. ance (group NA), morphine pellets were implanted in rats that
Learning can contribute to drug tolerance2127. When mor- then received i.v. saline in either environment used to condition
phine administration is paired with specific environmental cues, rats in the associative tolerance protocol. Group NA animals were
these cues can act as conditioned stimuli that elicit a conditioned tolerant to morphine in all experimental environments (data not
response opposing the agonist effect of morphine (associative shown), indicating that the morphine tolerance observed in these
tolerance). Although associative analgesic tolerance following animals was largely independent of context (non-associative).
repeated opioid administration is well established, the underlying
neurobiological mechanisms are unknown. RESULTS
The -opioid receptor is necessary and sufficient for mor- Systemic administration of the CCK-B antagonist L-365,260,
phine analgesia28. Animals that become non-associatively tolerant but not the CCK-A antagonist MK-329, blocked the expression
to morphine are selectively tolerant to other -opioid receptor of associative tolerance (Fig. 1c and d). Neither L-365,260 nor
agonists. In contrast, associative morphine tolerance generalizes MK-329 affected the expression of non-associative tolerance
such that the effects of agonists acting at other opioid receptors (Fig. 1e and f).
are also reduced29. This suggests that these two types of morphine Although the same dose of morphine and same baseline tail-
tolerance involve different CNS circuitry. However, direct evi- flick latency were used to assess analgesic tolerance, it is possible
dence for such circuitry differences has yet to be demonstrated. that the non-associative tolerance was of greater magnitude and
Because of its role as an anti-opioid peptide, we tested the thus obscured a possible morphine-potentiating effect of
hypothesis that CCK is required for associative but not non-asso- L-365,260. To address this possibility, we studied a second group
ciative morphine tolerance. In addition, we investigated poten- of non-associatively tolerant rats following three instead of five
tial differences in the CNS circuitry underlying these two types days of morphine pelleting. These rats were only partially toler-
of morphine tolerance. ant to morphine, so any potentiating effect of L-365,260 on mor-
CCK-A and CCK-B receptor antagonists were administered phine analgesia should have been easy to demonstrate. However,
i.v. following the acquisition of either associative or non-associa- L-365,260 had no effect on morphine analgesia in these partial-
tive analgesic tolerance to morphine. To produce associative tol- ly but non-associatively morphine-tolerant rats (Fig. 1g and h).
articles
7 7
6 6
5 5
articles
VTA CA1
p < 0.05, respectively). In contrast, no
Number Fos-positive cells
*
core, NAcc shell and Ce amygdala;
15 210
Fig. 2). These results indicate that
7 105 activity of neurons in both lateral and
basolateral amygdala and in CA1 of the
hippocampus was elevated in associa-
AS AM NAM NAS AS AM NAM NAS
tively tolerant animals receiving mor-
phine in a morphine-associated
context as compared with activity
CeA BLA under conditions of non-associative
Number Fos-positive cells
120
overall statistical analysis, there was a
** significant main effect of treatment, but
90
only within the lateral amygdala
60 (p < 0.0001). Post-hoc tests revealed sig-
nificant differences between animals
30 receiving morphine in the morphine
context and those receiving either saline
in the morphine context (p < 0.01) or
AS AM NAM NAS
morphine in the saline context
Associative Non-associative (p < 0.01). Specifically, when given
saline in the morphine context, animals
had 12.6 12.2 (mean s.e.) cells in
Associatively tolerant rats receiving morphine in the mor- the lateral amygdala, whereas those given morphine in the saline
phine-paired environment had significantly more Fos-positive context had 7.5 4.9 cells. In contrast, rats given morphine in the
cells in the lateral amygdala (LA) than did animals in the three morphine context had 76.4 24.6 cells in the lateral amygdala.
other groups (Figs. 2 and 3; p < 0.01). These same rats also had a Thus, the appropriate morphinecontext pairing was necessary
significantly greater number of Fos-positive cells in the basolat- for fos activation within the lateral amygdala, and the activation
eral amygdala than either group of non-associatively tolerant ani- was not a nonspecific effect of morphine administration.
mals (p < 0.05). Associatively tolerant animals receiving either To determine if the reversal of associative tolerance fol-
morphine in the morphine-paired context (AM) or saline in the lowing the systemic administration of the CCK-B antagonist
saline-paired context (AS) had significantly more Fos-positive L-365,260 was due to blockade of an action of CCK in either
cells in area CA1 than did either group of non-associatively tol- the amygdala or hippocampus, L-365,260 was microinjected
erant animals (NAM and NAS groups; p < 0.01 and into L/BL amygdala and CA1 following the acquisition of asso-
articles
articles
METHODS
a
A Subjects. Male Sprague-Dawley rats (Charles River, Wilmington, Mass-
achusetts) weighing 300325 g were individually housed. The colony
12
room was kept constant at 21C and followed a standard 12-h light/dark
**
** cycle with food and water available ad libitum. Animals were tested at
Tail-flick latency (s)
10
the same time during their light cycle each day.
8
*
Drugs. Morphine sulfate powder (supplied by the National Institute on
6 Drug Abuse) was dissolved in physiological saline for micro- and i.v.
injections. Subcutaneous morphine pellets (75 mg morphine base) were
4
obtained from the National Institute on Drug Abuse. MK-329 was
2
obtained from Merck Pharmaceuticals (Terlings Park, UK) and was dis-
solved in distilled water. The CCK-B antagonist L-365,260 (a gift from
0 Les Iversen, Panos Pharmaceuticals) was dissolved in 90% ethanol.
SYST L/BL Ce A VTA Core CA1 Surgery. Animals were anesthetized with 50 mg per kg Nembutal,
implanted with jugular catheters (PE 50), and allowed a one week recov-
ery period. Group A animals in the microinjection study were implant-
ed bilaterally with stainless steel 26 gauge chronic guide cannula (Plastics
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articles
site) in the saline-paired environment while the other half received vol- and 0.3% TritonX before being incubated for 1 h at room temperature
ume-matched microinjections of vehicle (90% ethanol) in the saline in a blocking solution of 0.05 M Tris-phosphate-buffered saline with 3%
paired environment. Tail-flick latency was then measured as before. normal goat serum and 0.3% TritonX. Primary antibody (rabbit poly-
clonal, provided by Dennis Slamon, University of California, Los Ange-
Group NA (non-associative tolerance). On the first experimental day, les) was then applied to the tissue. Tissue was incubated overnight at
animals received 0.05 ml saline i.v. followed by a 0.2-ml heparinized saline room temperature. Sections were thoroughly washed before the appli-
flush. Animals were allowed a 15-min habituation period in one of the cation of secondary antibody (goat anti-rabbit IgG and avidin-biotin-
two experimental environments used to condition rats in the associative peroxidase complex, Vector Labs, Burlingame, California). Secondary
tolerance study. Tail-flick latency was then measured every 10 min for antibody was left on the tissue for a period of 2 h. A nickel/diaminoben-
60 min. Animals were then returned to their home cages. Sixty min later, zidine (DAB) reaction was used to visualize Fos immunoreactivity50. Tis-
animals were implanted with two subcutaneous morphine pellets and sue was processed simultaneously for Group A and NA animals.
returned to their home cages. Thus all non-associatively tolerant animals
were exposed to high levels of morphine in their home cages as well as Fos quantification. Sections were examined under dark-field illumination
in the two contexts used for saline and morphine conditioning (for the to identify the boundaries of each brain region of interest. To simplify quan-
associatively tolerant rats). titation, regional boundaries were defined (NAcc, AP 2.2 to AP 0.48; VTA,
AP 5.2 to AP 6.8; CA1, AP 2.3 to AP 4.16; CeA, AP 1.8 to AP 3.0;
Systemic study. Animals were pellet-implanted as above. On the last BLA, AP 1.8 to AP 3.3; LA, AP 2.5 to AP 3.3). Fos-positive cells were
experimental day, animals were divided into 2 groups. Seven min before quantified at 20 magnification using light-field illumination. An observ-
i.v. administration of 2.5 mg per kg morphine, group 1 received either er blind to the experimental treatment counted Fos-positive cells. Once
MK-329 or L-365,260. Forty-five min following the i.v. administration quantitation was completed, the total number of cells for each animal in
2000 Nature America Inc. http://neurosci.nature.com
of 2.5 mg per kg morphine, group 2 received either MK-329 or L-365,260. each brain region was divided by the number of section per region, ren-
Following morphine administration, group 1 animals were placed in one dering a mean number of Fos cells for each animal for each brain nucleus.
of the experimental environments and allowed a 15-min habituation
period. Tail-flick latency was measured every 10 min for 60 min. Fol- Statistical analysis: Fos study. A one-way randomized ANOVA was used
lowing morphine administration, group 2 animals were placed in one of to analyze the data collected from each brain region. Tukey-Kramer post-
the experimental environments and allowed a 15-min habituation peri- hoc tests were conducted for pairwise comparisons.
od before tail-flick latency was measured every 5 min for 30 min. Group
2 animals were then removed from the experimental environment, Statistical analysis: systemic and microinjection studies. The mean of
administered either MK-329 or L-365,260 and then placed back in the the six tail-flick latencies measured for each animal was calculated for
experimental environment. Animals were allowed a 15-min habituation each test day. As the data did not follow a standard distribution (due to
period. Tail-flick latency was measured every 5 min for 30 min. Care was the use of a 12-s cut-off) non-parametric statistics were used for data
taken to treat NA animals in such a way as to prevent the association of analysis. Wilcoxon two-group signed-rank tests were used to compare
morphine administration with any environmental cues. the mean tail-flick latencies on the different test days. Statistical signifi-
cance was set at p < 0.05. All statistical tests were conducted using GB-
Microinjections. All injection sites were determined based on the atlas STAT, version 6.5. All figures were created using StatView, version 4.0,
of Paxinos and Watson (1986). Bilateral microinjections were made into (SAS Institute, Cory, North Carolina) and Adobe Illustrator, version 5.5.
the following brain structures: VTA (n = 7; AP, 5.8; ML, 0.5; DV, 8.5),
NAcc core (n = 10; AP, 2.5; ML, 1.2; DV, 6.5), CA1 (n = 12; AP, 3.6;
ML, 1.5; DV, 3.0), CeA (n = 7, AP, 2.5; ML, 4.3; DV, 8.3), L/BLA ACKNOWLEDGEMENTS
(n = 12; AP, 2.8; ML, 5.2; DV, 8.5). Each injection was made using a We wish to thank our colleagues Mary Dallman, Michael Silver and Larry Tecott
1 l syringe (Hamilton, Reno, Nevada) attached to 10 cm of PE 50 tub- for reading this manuscript. This work was supported by NINDS grant # 21445,
ing connected to a 33-gauge injection cannula (Plastics One). Microin- NIDA grant # 01949, the Wheeler Center for the Neurobiology of Addiction and
jection was conducted at a rate of 0.5 l per min. The injection cannula
by an NSF student fellowship.
extended 2 mm past the guide cannula and was left in place for 1 min
following microinjection to minimize the backflow of drug solution.
At the conclusion of the experiment, animals were anesthetized with RECEIVED 27 SEPTEMBER; ACCEPTED 3 NOVEMBER 1999
pentobarbital, injected with 0.5 l pontamine sky blue per brain site and
perfused intracardially through the ascending aorta. Brains were blocked
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articles
1 Neuroscience Program, University of Southern California, Los Angeles, California 90089-2520, USA
2 Department of Biomedical Engineering, University of Southern California, Los Angeles, California 90089-1451, USA
Correspondence should be addressed to K.A.A. (karchie@lnc.usc.edu)
Many complex cells in mammalian primary visual cortex are finely tuned to binocular disparity. In
the prevailing model, several binocular simple cells drive each disparity-tuned complex cell.
However, some cat complex cells receive direct LGN input, and binocular simple cells are rare in
2000 Nature America Inc. http://neurosci.nature.com
macaque. In our biophysically detailed compartmental model, active dendrites of a single neuron
perform the multiple simple-cell-like subunit computations that underlie both orientation and
disparity tuning. The responses of our detailed model could be predicted by a simple algebraic
formula closely related to an energy model. Adding inhibitory synapses led to sharper, more
contrast-invariant tuning curves. Thus active dendrites could contribute to binocular-disparity
tuning in complex cells.
Neurons in mammalian primary visual cortex are grouped into computations19,2227. Also, dendrites of neocortical pyramidal cells
two broad categories: simple cells, sensitive to stimulus orienta- contain voltage-dependent channels that could profoundly influ-
tion and position (or phase), and complex cells, relatively insen- ence their integrative behavior, including NMDA channels and
sitive to the spatial phase of an oriented stimulus. Many complex voltage-dependent Na+ and Ca2+ conductances that can amplify
cells receive input from both eyes and are tuned to binocular dis- synaptic inputs and generate both fast and slow dendritic spikes28,29.
parity, the horizontal offset of features between the two eyes that Based on this evidence, two modeling studies show that a sin-
conveys stimulus depth1,2. Whereas simple cells are often approx- gle neuron with active dendrites can reproduce the defining spatial
imated as linear3, complex cells are fundamentally nonlinear: nonlinearities of complex cells, including position-invariant ori-
their responses to combinations of stimuli cannot be predicted entation tuning17 and binocular-disparity tuning30. The morpho-
by summing the responses to individual stimuli. The form of the logically realistic pyramidal cell and the random subunit allocation
response nonlinearity suggests a hierarchy in which outputs of used in these studies, however, make it difficult to quantify con-
multiple simple-cell-like subunits are pooled by complex cells4,5. tributions of active dendritic currents, or to compare the behav-
In a disparity-tuned complex cell, those subunits seem to be ior of the biophysical model to existing mathematical models.
binocular and oriented6,7. Here we developed a simplified model, patterned after an
An early proposal held that simple cells receive input from existing energy model for disparity tuning6,7, in which four
unoriented cells in the lateral geniculate nucleus (LGN) in cat or binocular simple-cell-like subunits were mapped onto four basal
cortical layer 4C in monkey, and complex cells pool over the ori- branches of a dendritic tree. In addition, although it was previ-
ented simple-cell outputs4,8. In similar models for disparity tuning, ously shown that excitatory inputs onto active dendrites can pro-
a set of binocular simple cells performs the requisite subunit com- duce nonlinear boosting interactions, we asked whether observed
putations6,7,9,10. However, evidence rules out such a strict hierarchy. suppressive interactions between stimuli57,15 could likewise result
In cat, some first order complex cells receive direct LGN input, from intradendritic computations, by assuming more realistic
circumventing the simple-cell stage11,12. In macaque, the binocu- (nonzero) background firing rates of inputs, or by including
lar simple cells required by conventional models are rare or nonex- synaptic inhibition16,19.
istent. Although many disparity-tuned neurons are found in
macaque V1, a recent study found no binocular simple cells: a few RESULTS
had spatially offset light and dark subregions (accounting for the We used compartmental simulations to study the response of a
small proportion of binocular cells classified as simple in earlier simplified pyramidal neuron with active dendrites (Fig. 1). Low
studies13,14), but all binocular cells showed position-invariant dis- input resistance at the soma (87 M) meant that voltage signals
parity tuning15. generated within the thin basal dendrites were strongly attenuated
If the simple-cell stage is bypassed, where might the required when recorded at the cell body, electrically isolating the branch-
subunit computations be carried out? It has been suggested that es from one another such that a synaptic input changed voltage
simple-cell-like subunit computations might be performed direct- within its associated branch much more than in the other three.
ly within the dendrites of complex cells8,16,17. In theoretical stud- Nonlinear interactions between synaptic inputs were thus lim-
ies, spatially extended dendrites electrotonically compartmentalize ited primarily to those among synapses on the same branch and
synaptic potentials18,1921, potentially allowing different dendrites of mediated by dendritic Na+ and K+ currents and voltage-depen-
a single neuron to perform quasi-independent nonlinear subunit dent NMDA-type synaptic currents.
articles
OFF-center cell
total input to the cell. This modulation was distinct from that
L L seen in responses of typical simple cells in that the peaks and
R R valleys of the modulation occurred at identical locations for
2000 Nature America Inc. http://neurosci.nature.com
articles
20 20
contrast response from the mean same-contrast
16
0
16
0
response (Fig. 5a). The diagonally elongated
12 12
8 12
16
8 12
16 structure with alternating excitatory and
4
8
4
8 inhibitory lobes closely resembled kernels gen-
n1 4
n n1 4
n erated from binocular complex cells in cat visu-
0 0 2 0 0 2
al cortex (Fig. 5b), which are often well fit by an
Fig. 2. Interaction between synaptic inputs on two branches. Each frame shows a cells mean
energy model consisting of four simple-cell-like
firing rate (averaged over 64 simulations, 1 s each) as the number of fully active (100 Hz)
synapses was increased on the 2 branches. Within each branch, synapse locations were ran-
subunits (negative and positive parts of even-
7
dom. The other 2 branches each contained 16 synapses firing at a background rate of 15 Hz. and odd-symmetric Gabor filters) . We attempt-
Cell parameters were as in Table 1. (a) Response of the model cell. Solid and dashed lines ed to predict the binocular kernels of our bio-
show two iso-synapse-count contours (see text; solid, 12 total synapses, dashed, 16 total physically modeled cell using a simple algebraic
synapses). Dips in these contours reflect compartmentalization of cell. (b) Response of an formula inspired by the structure of the energy
idealized cell with single-branch nonlinearity as in (a), but with perfect independence of the model. To do this, we calculated the linear stage
two branches. Each branch was assumed to produce an independent nonlinear response f(n), of each of the four subunits by adding the mean
where n was the number of active synapses on that branch. f(n) was taken as the average of firing rates of the 16 afferent inputs to each
the values on the branch 1 and branch 2 axes in (a), which differed slightly because of the ran- branch. Each of the subtotals was then squared,
domization of spike train inputs used in these simulations. The idealized cell output was the
sum of the individual branch responses, f(n1) + f(n2). (c) Difference between (a) and (b).
and the four resulting values were added to pre-
Raised portions represent cell responses above linear prediction. (d) Model cell whose out- dict the cells overall response. The resulting
put is proportional to n12 + n22. The result is scaled for comparison with other frames. Iso- binocular disparity kernel (Fig. 5c) resembled
synapse-count contours are similar to those shown in (a). (e) Response of a cell with branch corresponding plots generated by the compart-
nonlinearity f(n) as in (a), but with no subunit isolation, giving a response f(n1 + n2). All iso- mental simulations (Fig. 5a) as well as experi-
synapse-count contours are flat, indicating that the cell is insensitive to the arrangement of mental data (Fig. 5b).
synapses across the two branches. To assess the importance of the squaring
nonlinearity in determining the basic form of
the binocular kernel, we applied three other
expansive nonlinear functions to the branch-
We tested the binocular-disparity tuning of the cell using bars subunit outputs in the algebraic model: x1.05 (Fig. 5d), x3(Fig. 5e)
at its optimal orientation. Following experimental protocols used and ex/10(Fig. 5f). Binocular kernels produced by all cases were
in cat7 and monkey15, we ran four stimulus conditions consist- similar, and on this basis would be difficult to distinguish exper-
ing of light and dark bars presented simultaneously to the left imentally. However, the magnitude of the nonlinear response
and right eyes in each of the four possible combinations (Fig. 4). components that make up the kernel depended strongly on the
For each condition, we presented the two bars at 64 horizontal subunit exponent: the output of the x1.05 model, for instance,
locations in each of the two eyes to generate a two-dimensional contained a nonlinear component that reached only 3% of the
binocular-response plot (Fig. 4a). In the same-contrast condi- peak cell response, whereas the contribution rose to 38% in the
tions (both light or both dark), the cell responded best when the quadratic model. For comparison, the magnitude of the nonlin-
articles
40 active, ordered 40
30 30 the cell body. In the distal condition,
passive, ordered inhibitory contacts were distributed uni-
20 20 formly over the distal 180 m of the
active, scrambled branch, avoiding the 20 proximal m.
10 10
Counter to expectations, proximal inhi-
0 0 bition weakened orientation tuning relative
90 45 0 45 90 0 120 240 360 to that seen in the excitation-only condition
Grating orientation Grating phase (compare Fig. 6a with Fig. 3c). Blunting of
orientation tuning was particularly evident
Fig. 3. Response of the model cell to sinusoidal gratings. (a) Presynaptic firing rates are indicated at higher contrasts, and was due to both an
by the size of the synapse marker; synapses with firing rate of 0 Hz are not shown. An input at the
elevation of responses at null orientations
preferred (vertical) orientation leads to grouping of the most active synapses on a single branch.
This grouped input leads to robust firing at the cell body (inset voltage trace). The visual stimulus is and an anomalous suppression of respons-
shown above the voltage trace, with the grid of LGN cell centers indicated by black dots. One LGN es to optimal stimuli. Binocular-response
receptive field is shown for scale. (b) An input at the null orientation leads to dispersal of the most plots (Fig. 6b and c) were qualitatively sim-
active synapses throughout the basal dendrites; the cell responds only weakly to this diffuse pattern ilar to those in the excitation-only condi-
of input. (c) Firing rate as a function of grating orientation shows clear orientation tuning (dia- tion, though monocular responses were
monds) that is abolished when afferents were randomly reassigned to the four branches (squares) very weak. The binocular interaction plots
or when the NMDA and dendritic Na+ channels were blocked (crosses). Higher synaptic conduc- differed from the excitation-only case in that
tances were used in the latter case to boost firing rates. Each data point is an average over 1-s runs they consisted almost entirely of boosting,
at each of 18 different phases. (d) Firing rate for an optimally oriented grating as a function of grat-
but not suppresive nonlinearities (Fig. 6d
ing phase (diamonds). Modulation is due to spatial variation in total LGN input to cell (see text).
Each data point represents a single 1-s run. Two lower curves are for conditions as in part (c).
and e). Although suppressive binocular
interactions were almost absent in this cell,
the binocular disparity kernel appeared
normal in form, containing both positive
and negative lobes (Fig. 6f).
ear component of the biophysically modeled cell response was In contrast to proximal inhibition, distal inhibition produced
in the range of 2560% of the peak response of the cell (1015 both sharper orientation tuning than was seen with excitation
Hz boost over a 40-Hz peak above background in the same-con- alone, and approximately multiplicative scaling of the tuning
trast conditions; 20-Hz suppression relative to peak of 35 Hz in curve with increased contrast (Fig. 6g). The cell lacked monocu-
different-contrast conditions), values comparable to those report- lar responses almost entirely, but showed strong, disparity-tuned
ed for a disparity-tuned complex cell in monkey V1 (ref. 15). binocular responses (Fig. 6h and i). Binocular interactions were
We added synaptic inhibition to the model to investigate again exclusively boosting (Fig. 6j and k), and binocular kernels
whether it could also contribute to the dendritic subunit com- seemed normal for a disparity-tuned complex cell, with pro-
putations underlying disparity tuning. Through an inhibitory nounced positive and negative lobes (Fig. 6l).
interneuron, each excitatory LGN afferent was assumed to drive To identify the source of differences between cases with and
a single GABAA-type synapse placed on one of the four dendrit- without inhibition, we recorded simultaneously from each of the
ic compartments. This shunting inhibitory synapse was mapped four dendritic branches and the soma while presenting an opti-
onto a pyramidal cell dendrite at random and balanced so that mally oriented grating (Fig. 7a). Somatic action potentials were
every branch received an identical number of inhibitory contacts most often triggered when two or more dendritic branches fired
and direct excitatory and indirect inhibitory inputs driven by any in rapid succession, one spike riding piggyback on the other.
given LGN afferent never fell on the same branch. This wiring For an optimally oriented stimulus, the initial spike was most
constraint reflected the assumption that a Hebb-type rule gov- often generated within the primary subunit, followed quickly by
erning synapse survival would discourage stabilization of corre- a secondary spike in another branch. Isolated dendritic spikes
lated excitatory and inhibitory synapses within the same that did not trigger a somatic spike led to EPSP-like somatic depo-
articles
same contrast opposite contrast one subunit far more than the
L R L R L R L R other three (Fig. 3a). In this con-
figuration, spikes continued to be
generated frequently within the pri-
mary subunit, but weak activation
of the other branches reduced the
a incidence of secondary spikes in
2 45 Hz these branches and increased spike
failures at the cell body.
In contrast to proximal inhibi-
0 tion, an identical set of inhibitory
Right stimulus position
articles
articles
+25 Hz
dd e
e jj kk neurons in macaque V1 have spa-
interactions
articles
articles
outputs of these nonlinear subunits. Note that the basic struc- strained by available data. The values of these parameters were
ture of this nonlinear computationsometimes expressed as a thus set within reasonable ranges, then adjusted until the inhibi-
sum of product termsoccurs frequently in analyses of both tion produced strong, but not total, suppression of cell firing rates.
classical and extraclassical receptive-field properties in visual cor- Stimulus contrasts were then adjusted to drive the cell through a
tex41. For example, beyond our illustration of phase-invariant reasonable range of output rates.
orientation and disparity tuning, similar computations are per- The cell began each simulation uniformly polarized at a rest-
formed by cells whose responses are facilitated by stimuli pre- ing potential near 70 mV. The firing rate typically stabilized after
sented in the extraclassical receptive field42, by cells that respond a brief initial period of depolarization from rest. Each stimulus
to illusory contours43 and by cells that show multiplicative boost- was presented for 1.05 s, with all response rates calculated over a
ing under the influence of focal visual attention44. This com- 1-s interval following the initial 50 ms simulation transient.
monality could indicate that active dendritic processing
contributes widely to the information-processing functions of ACKNOWLEDGEMENTS
cortical neurons. This work was supported by the National Science Foundation. We thank Dan
Ruderman for contributions to early stages of this work, and Margaret
METHODS Livingstone and Gary Holt for comments.
Responses of a single cortical neuron were modeled using the
NEURON simulation environment45. The cell morphology is RECEIVED 29 JUNE; ACCEPTED 5 NOVEMBER 1999
described in Fig. 1; dendritic spines were not modeled, but pre-
2000 Nature America Inc. http://neurosci.nature.com
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articles
1 Department of Psychology, University of Minnesota, N218 Elliott Hall, 75 E. River Dr., Minneapolis, Minnesota 55455, USA
2 Department of Psychology, University of Pennsylvania, 3815 Walnut St., Philadelphia, Pennsylvania 19104, USA
3 Center for Neural Science, New York University, 4 Washington Place, New York, New York 10003, USA
Correspondence should be addressed to P.R.S. (schrater@eye.psych.umn.edu)
Visual motion is processed by neurons in primary visual cortex that are sensitive to spatial
orientation and speed. Many models of local velocity computation are based on a second stage that
2000 Nature America Inc. http://neurosci.nature.com
pools the outputs of first-stage neurons selective for different orientations, but the nature of this
pooling remains controversial. In a human psychophysical detection experiment, we found
near-perfect summation of image energy when it was distributed uniformly across all orientations,
but poor summation when it was concentrated in specific orientation bands. The data are consistent
with a model that integrates uniformly over all orientations, even when this strategy is sub-optimal.
When a viewer moves relative to the environment, the visual ral frequency bands that lie on a common plane (Fig. 1c)810. The
image projected onto the retina changes accordingly. Within small population response of a family of detectors built in this way can
regions of the retina and for short durations, this motion is com- determine the presence and velocity of local translating patterns,
monly approximated as a two-dimensional translation. The field and the responses of individual detectors are well matched to the
of velocity vectors associated with each such region is referred to behaviors of a subset of neurons in visual area MT10.
as optic flow. Physiological and psychophysical experiments Alternatively, the visual system could use an adaptive integra-
demonstrate that mammalian cortex uses mechanisms sensitive tion rule, selectively combining only those first-stage detectors
to local image motion1. These mechanisms are generally consid- that are tuned to the spatial structure of the image. For example,
ered to form the neural substrate for representing optic flow. one type of model makes initial robust estimates of the compo-
Neurons in primary visual cortex perform the first stage of cor- nents of a pattern by selecting those detectors responding to the
tical motion processing. They are selective for both the spatial ori- stimulus but ignoring those responding to noise11. These one-
entation and speed of the visual input within a spatially localized dimensional estimates are then combined to produce an estimate
region. Because of their orientation specificity, however, these neu- of the two-dimensional pattern velocity that is most consistent
rons are impaired by an ambiguity commonly known as the aper- with the measured one-dimensional velocity components. In gen-
ture problem: each neuron can signal the speed only of motion eral, such adaptive pooling rules produce more efficient motion
perpendicular to the orientation to which it is tuned2; thus, it is detectors than fixed pooling rules because the detector is better
insensitive to the velocity component parallel to this orientation. It matched to the signal. Human observers adapt their spatial pool-
is suggested that a second stage of processing, commonly associ- ing to improve the detection of static images12. Thus, it is plausi-
ated with visual area MT, computes an unambiguous representa- ble that they may do the same for moving images.
tion of local pattern velocity by selectively combining the outputs We designed a set of psychophysical detection experiments to
of the first-stage detectors25. Although such an integration stage rigorously test the predictions of three models for pooling of spec-
seems largely consistent with psychophysical and physiological tral energy: (1) a planar power detector that sums the signal ener-
data, the precise form of the combination rule remains unclear. gy over all orientations in a fixed planar region of spatiotemporal
Here we describe psychophysical experiments designed to test the frequency, (2) an adaptive planar power detector that sums ener-
predictions of several general combination rules. gy only over those regions of a plane containing the signal and
The simplest combination strategy is to integrate responses of (3) an adaptive unrestricted power detector that can sum energy
all those first-stage mechanisms consistent with a particular two- over arbitrary regions of spatiotemporal frequency. Experimental
dimensional velocity2. It is convenient to describe this construction stimuli were constructed by summing dynamic random noise
in the three-dimensional spatiotemporal frequency domain, where patterns that were band-limited using spatiotemporally oriented
the spectral energy of a rigidly translating image is concentrated on filters analogous to those used to describe first-stage motion
an oblique plane (Fig. 1a and b). The orientation of this plane mechanisms. Each of the three models makes distinct subthresh-
uniquely specifies the translational velocity (both speed and direc- old summation predictions for the detection of such stochastic
tion) of the luminance pattern6. First-stage motion detectors in the signals embedded in white noise. The planar power detector pre-
mammalian visual system can be described as computing the spec- dicts optimal summation performance when signal energy is dis-
tral energy within limited bands of spatiotemporal frequency7. Thus, tributed uniformly over all spatial orientations in a plane.
a pattern-velocity detector may be constructed by summing the However, this model predicts suboptimal performance when the
weighted outputs of first-stage mechanisms tuned to spatiotempo- signal energy is concentrated in a subset of planar orientation
articles
x
x x
val contained the signal. Thresh-
olds were estimated by fitting a
psychometric function to the
Fig. 1. A translational motion detector. (a) Space-time luminance pattern of an image translating to the right. This is a data and selecting the contrast
representation of the intensity information in the retinal image (the xy plane) over time (t). The rightward motion can
energy producing 81.1%
be inferred from the oriented pattern on the xt face. (b) The Fourier amplitude spectrum of the luminance pattern,
represented by the intensity of points in a three-dimensional spatiotemporal frequency domain. Non-zero Fourier detectability. Different stimulus
amplitudes are constrained to lie on a plane through the origin. The orientation of this plane uniquely specifies the types were presented in distinct
direction and speed of translation. (c) Construction of a translation detector10, illustrated in the Fourier domain. Pairs experimental sessions, and sub-
2000 Nature America Inc. http://neurosci.nature.com
of balls symmetric about the origin indicate the Fourier amplitude spectra of band-pass filters whose peak frequencies jects were told which stimulus
lie in the plane. A translation detector can be constructed by summing the squared outputs of such filters. type was being used. Before data
collection, subjects were given
several hours practice for each
stimulus type over the course of
bands or distributed over non-coplanar regions of spatiotempo- several days, until performance saturated (see Methods).
ral frequency, because the model sums both signal and noise in To compare detection performance across stimuli, we assumed
these cases. Similarly, the adaptive planar power detector predicts that performance for the component stimulus reflects the detec-
optimal performance for any signal whose energy is concentrated tion efficiency of a band-limited, first-stage motion-energy detector
in a plane, regardless of orientation content, but sub-optimal per- mechanism. (The component signal has a bandwidth similar to
formance for stimuli composed of non-coplanar distributions of psychophysically optimal bandwidths13). Specifically, we assumed a
signal energy. Finally, the adaptive unrestricted power detector model in which subjects made detection judgments for the com-
predicts optimal performance for any distribution of signal ener- ponent stimulus based on output of a filter matched to the com-
gy, with no improvement in performance for planar distributions. ponent signal; this output was then corrupted by additive internal
noise. The internal noise represented the added uncertainty creat-
RESULTS ed by neural noise, filter/signal mismatch and central-decision noise.
In the first experiment, we tested these predictions by measuring We estimated internal noise by comparing subjects thresholds with
detection thresholds for three types of spatially localized stochastic that of an ideal observer for the component stimulus. Assuming
signals embedded in white noise. Signals were generated by filter- approximately constant internal noise across all stimulus condi-
ing spatiotemporally white noise with different sets of band-pass fil- tions, we derived optimal summation predictions for the plaid and
ters. Component stimuli (Fig. 2a) had spectral energy confined to planar stimuli. These predictions reflected the performance of ideal
a single pass-band. Plaid stimuli (Fig. 2b) had two component observers that sum energy over only the spatiotemporal frequency
bands on the same plane. Planar stimuli (Fig. 2c) were constructed bands containing the signal in a stimulus and were corrupted by a
using a set of component filters arranged in a ring on an oblique constant level of internal noise. (Such a model gives the common-
plane (so that signal energy was uniformly distributed over an annu- ly cited square-root law for contrast summation.)
articles
2 0.16
Fig. 3. Detection thresholds. (a) Per
Component Component formance of three subjects for the three
Plaid Plaid stochastic signals of Fig. 2. Threshold
Planar 0.14 Planar
signal-to-noise ratio (SNR) for 81.1%
detectability. SNR is calculated as the
1.5 0.12 ratio of the signal power to the back-
ground-noise power. Heavy black lines
Threshold SNR
Efficiency
tion, derived from the component con-
1 0.08 dition thresholds. (b) Detection
efficiencies for the three stimulus types.
0.06 Efficiencies are plotted in proportions,
with 1.0 reflecting perfect performance
0.5 0.04 (matching that of an ideal observer
tuned to the signal structure for that
particular stimulus). The differences
0.02
between the efficiencies of the pattern
stimuli (plaid and planar stimuli) and the
0 0 component stimulus provide a quantita-
a
(a) PS ML AS (b
b) PS ML AS tive measure of summation of the pat-
2000 Nature America Inc. http://neurosci.nature.com
Figure 3a shows subjects detection thresholds for the compo- ence between those for plaid and non-planar-triplet stimuli
nent, plaid and planar stimuli. The heavy black lines on the plaid (Fig. 4b). Figure 4c shows subjects efficiencies for detecting each
and planar plots represent predictions of ideal summation derived of the three signals used in the experiment. Detection efficiency
from the component data. The plaid data show little or no sum- was significantly better for the planar triplet than for the plaid,
mation, whereas thresholds for the planar stimulus fell well with- implying improved summation for the triplet stimulus (Fig. 4c).
in the range predicted by perfect summation. Figure 3b shows an Whereas detection efficiencies for the planar triplet were sub-
alternative characterization of the results. We plotted subjects stantially better than for the plaid stimulus, subjects efficiencies
detection efficiencies for each of the three types of stimuli. Detec- for the planar triplet remained lower than for the planar stimu-
tion efficiencies were computed by comparing subjects detection lus in experiment 1 (Fig. 3). Again, this is consistent with the pla-
thresholds with those of ideal observers optimally tuned to the nar power-detector model, which predicts progressively better
signals contained in the stimuli. (Note that the ideal observers are summation as signal energy is distributed more broadly over a
different for each of the three stimulus types.) Efficiency provides plane in spatiotemporal frequency, attaining ideal summation
a measure of the fraction of stimulus information effectively used only when the energy is distributed uniformly over the plane
by subjects in performing the detection task. Because perfect sum- a stimulus that best matches the putative detector.
mation is ideal for these experiments, efficiency is also a measure
of summation. The significantly lower detection efficiencies for DISCUSSION
the plaid stimulus than for the component stimulus reflect poor We can summarize the qualitative results of experiments 1 and
summation for the plaid. Conversely, equal detection efficiencies 2 as follows: subthreshold summation of signal-contrast energy
for the planar stimulus and the component stimulus reflect near- improved as the energy was distributed more and more broadly
ly optimal summation for the planar stimulus. around different orientations in a plane in spatiotemporal fre-
Of the three detection models described above, the data are quency, but did not improve when the energy was distributed
clearly most consistent with the planar power-detector model. into non-planar regions of spatiotemporal frequency. The lack
The experiment, however, only compared summation perfor- of summation for non-planar regions agrees with previous stud-
mance for two types of pattern stimuli. Motivated by the obser- ies suggesting a lack of generic summation for components mov-
vation that the planar power-detector model predicts ing in opposite directions1416. Stated more plainly, detection
progressively improved summation when signal energy is dis- efficiencies improve as more orientations are added to a moving
tributed more broadly across spatial orientations in a plane, but pattern, as long as the motion of those orientation components
not when the energy is distributed out of a plane, we ran a second are consistent with the velocity of the pattern. Of the three detec-
experiment. We generated a new set of stochastic signals to test tion models proposed in the introduction, these results are most
this prediction. Signals for the stimuli were created by passing consistent with the planar power-detector model.
spatiotemporal white noise through three configurations of fil- We extended the analysis one step further by comparing per-
ters (Fig. 4a). The first was a plaid signal, similar to the plaid used formance of a particular planar power detector on the five dif-
in experiment 1. The second was a planar triplet, created by ferent pattern stimuli used in the experiments with that of the
adding a component band to the plaid, in the same plane as the subjects. The detector optimally summed energy over the band of
plaid. The third was a non-planar triplet, created by adding a frequencies contained in the planar stimulus from experiment 1
component band out of the plane of the plaid. Detection thresh- (using a matched power filtersee Methods). We assumed this
olds were measured using the same method described for exper- detectors output to be corrupted by levels of internal noise esti-
iment 1. The planar power-detector model predicts improved mated from subjects detection thresholds for the component
summation for the planar triplet relative to the plaid, but no stimulus in experiment 1. Figure 5 shows the model predictions
improved summation for the non-planar triplet. for the five pattern stimuli used. Given the assumptions built
Detection thresholds for the planar triplet were, as predicted, into the model concerning the exact spatiotemporal frequency
lower than for the plaid, whereas there was no significant differ- band covered by the planar power detector, the match is sur-
articles
Efficiency
As with all psychophysical studies, we had
0.3 0.06
to make certain assumptions to draw con-
x y clusions about the underlying neural mech-
0.2 0.04
anisms. In particular, we assumed that
In plane
t 0.1 0.02
detection of single component signals is
mediated by responses of first-stage energy
0 0 mechanisms, but that detection of compound
PS ML PS ML signals is mediated by second-stage respons-
y Subject Subject es. In addition, we assume that observers can
2000 Nature America Inc. http://neurosci.nature.com
0.1
Fig. 5. Threshold SNRs for detecting the five types of pattern stimuli
replotted from experiments 1 and 2, where Plaid 1 in the legend denotes
0 the plaid from the first experiment and Plaid 2 from the second. Plaid 1 dif-
PS ML fers from Plaid 2 in that its energy is more diffusely spread over frequency.
Black bands indicate the predictions of a planar filter, based on subjects
Subject detection thresholds for the component stimulus used in experiment 1.
articles
the Hessian of the likelihood function of the fit and using a Monte bers, Es En is Gaussian distributed, and hence can be charac-
Carlo sampling method. terized by its mean and variance. The mean and variance of
The component filter has an amplitude spectrum given by Es En are simply the sums of the means and variances of the
C(r, , ) = R(r) |cos()|9 |cos( )|9 in spherical fre- 2(2) variables at each frequency. Computing the means and vari-
quency coordinates. The frequency radius r is given by ances of the 2(2) variables that have been scaled by |F(x, y,
0
r = x2 + y2 + (t/2.1)2 (cycles per degree, cycles per degree, t)|2 (s2 |F(x, y, t)|2 + |N(x, y, t)|2) on the signal interval
cycles per s). The elevation shift = 36.9 acts to rotate the filter and |F(x, y, t)|2 (|N(x, y, t)|2) on the noise interval, and
letting FmSn = |F(x, y, t)|m |N(x, y, t)|n, then with a lit-
0
out of the spatial frequency plane into a plane corresponding to a
1.93 degrees per s translation downward. The radial frequency func- tle algebra, the mean and variance E s E n are given by
tion R is a smooth box function whose transitions from 0 to 1 are id = 2s2F2F2 and 2id = 8(s4F4F4 + 2 s2F4F2 N + 2 F4N2), respec-
given by 1/2 cycle of a cosine function. This function has low/high tively. Then p(Es En > 0) = 1 (0, id, 2id), where is the
cutoffs of (0.49, 7.6) cycles per degree and the cosine transition cumulative Gaussian distribution.
regions have widths of 1.45 cycles per degree. The plaid signal was To compute summation predictions, we modeled component
constructed by adding two component filters, rotated by +70 and performance as ideal but with the variance given by 2id + 2internal,
70 within the common plane. The planar signal was construct- so that p(E s E n > 0) = 1 (0, id, 2id + 2internal). Setting
ed using a sum of 10 component filters, rotated by multiples of 18 p(Es En > 0) = 81.1 and plugging in the subjects threshold into id
and constrained to lie in a common plane. For the second experi- and 2id, we solved for 2internal. To generate predictions, the
ment, the filters were modified to decrease the spectral spread. They 2internal was then added to the ideal variance for each of the other
2000 Nature America Inc. http://neurosci.nature.com
have the form BP(r, , ) = Wr(r)W()W(), where Wx is signal types, and a predicted threshold for 81.1% was computed.
a smooth box function on the variable x. Wr had a transition region Predictions for the planar model were generated in a similar man-
width of 1.45 and low/high frequency cutoffs of (0.49, 7.6) cycles ner. If we represent the planar filter amplitude spectrum as
per degree visual angle. W and W had transition widths of 8 and P( x, y, t) then the planar model computes the energies
high/low cutoffs that spanned 36. These filters were rotated to lie in Epl = |P(x, y, t)|2 |S(x, y, t)|2 on both intervals. The deci-
the same positions as the component and plaid filters for the plaid sion variable mean and variance are then given by id = 2s2P2F2
and planar triplet. The off-plane non-planar triplet component, and 2id = 8(s4P4F4 + 2s2P4F2 N + 2P4N2). Threshold predictions
however, had an elevation shift corresponding to a slower 0.26 were generated from these expressions using the internal noise
degrees per s translation downward. estimated from the observers component stimulus performance.
Ideal observers for the task are matched filter power detec-
tors17. Let F(x, y, t) denote the spectrum of the signal nor- ACKNOWLEDGEMENTS
malized to one, and N(x, y, t) the (complex) spectrum of the P.S. was supported by an NIH training grant, D.C.K. was supported by a grant
noise. Let |X(x, y, t)|2 = X(x, y, t) . X(x, y, t)* denote from the NIH and E.P.S. was supported by a Sloan Fellowship, an NSF CAREER
the inner product of the complex function with its complex con- grant and the Sloan Program in Theoretical Neurobiology at New York University.
jugate. Then the spectrum of the received signal |S(x, y, t)|2 is
s2 |F(x, y, t)|2 + |N(x, y, t)|2 on the signal plus noise inter- RECEIVED 11 AUGUST; ACCEPTED 11 NOVEMBER 1999
val and |N(x, y, t)|2 = N2 on the noise-alone interval. The best
possible performance can be achieved by computing the energy 1. Nakayama, K. Biological image motion processing: a review. Vision Res. 25,
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1.3 105 frequency samples. 4. Movshon, J. A., Adelson, E. H., Gizzi, M. S. & Newsome, W. T. in Experimental
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either s2 |F(x, y, t)|2 + |N(x, y, t)|2 on the signal interval from the perceived motions of their components. Vision Res. 31, 139149
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the squared amplitude spectrum of the signal. Because the ener- selective for direction of movement. J. Physiol. (Lond.) 250, 347366 (1975).
15. Watson, A. B., Thompson, P. G., Murphy, B. J. & Nachmias, J. Summation and
gy is the sum of an extremely large number of 2(2) random vari- discrimination of gratings moving in opposite directions. Vision Res. 20,
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articles
The visual system uses several signals to deduce the three-dimensional structure of the environment,
including binocular disparity, texture gradients, shading and motion parallax. Although each of
these sources of information is independently insufficient to yield reliable three-dimensional
2000 Nature America Inc. http://neurosci.nature.com
structure from everyday scenes, the visual system combines them by weighting the available
information; altering the weights would therefore change the perceived structure. We report that
haptic feedback (active touch) increases the weight of a consistent surface-slant signal relative to
inconsistent signals. Thus, appearance of a subsequently viewed surface is changed: the surface
appears slanted in the direction specified by the haptically reinforced signal.
Combining different sources of three-dimensional information These weights affect the appearance of the visual world and
helps the interpretation of ambiguous signals and reduces the the manner in which we interact with it. Here we asked how the
effects of measurement noise. The method of combination has nervous system determines weights applied to different estima-
been successfully examined by using cue-conflict protocols in tors. There are at least three ways in which the weights could be
which signals are manipulated independently15. In one study, determined. First, weights could be fixed for a given viewing sit-
for example, subjects adjusted the three-dimensional shape of uation and individual and not subject to change through feed-
convex surfaces with elliptical cross-sections until they appeared back (although they may have been changeable during infancy
cylindrical (circular cross section)4. The shape was specified by and childhood to compensate for anatomical changes in the sen-
conflicting disparity (three-dimensional information provided sory apparatus, for example). Second, weights could be adjust-
by the differences in images between the two eyes) and texture ed by comparing a given estimators output with those of other
gradients (three-dimensional information given by projection of estimators and with feedback from motor behavior. Third,
a surface with statistically regular markings onto the retina). weights could be determined directly from statistical measures
Affected by both the disparity and texture signals, the shape set- of estimator outputs. For example, if the output of one estimator
tings were well described by a linear weight combination rule: for a given viewing situation fluctuated less over time than that of
S = wtSt + wdSd (1) another estimator, the formers weight could be increased rela-
St and Sd are the outputs of shape estimators with weights wt and tive to the latters. We examined the first two of these possibilities.
wd that use texture and disparity signals, respectively. Each shape Specifically, we asked whether the weights assigned to different
estimator may use a variety of input signals. For example, the estimators can be changed by haptic feedback (sensation of touch
disparity-based estimator uses inputs of horizontal disparity, ver- generated by active, exploratory hand and finger movements)
tical disparity and their gradients1. One cannot distinguish a that is consistent with one estimator and not another.
change in weight from a change in estimator gain, so for our pur- In numerous reported visualhaptic interactions, visual appear-
poses a weight change will refer to both possibilities. Equation 1 ance is unaffected by haptic feedback. For example, subjects grasp-
is a maximum-likelihood estimator if the estimators, St and Sd, ing a square viewed through an optical device that distorts the
are Gaussian distributed and statistically independent and the image to make it appear rectangular see and feel the square as a
weights, wt and wd, are equal to the estimators inverse variances, rectangle13. In this case, perception is determined entirely by the
normalized to add up to one68. visual information; thus it is an example of visual capture1317.
This linear weighting scheme suffices for understanding many Perhaps this finding is not surprising, because the visual stimulus
phenomena in visual perception15,911. A statistically sensible clearly specifies perceived shape. Consistent with this idea, touch
method for estimation would give high weight to more informative can affect appearance of an impoverished visual stimulus18. These
estimators and low weight to uninformative ones5, because such visualhaptic studies sought concurrent perceptual effects, but we
weighting should yield more stable percepts58,12. The weights must were primarily concerned with persistent effects of haptic feed-
depend on viewing conditions because, for example, the informa- back on visual perception. To increase the probability of observ-
tion content of the disparity signal decreases as a function of dis- ing a positive influence of haptic feedback, we presented visual
tance4,12. Furthermore, experiments show that the weights vary stimuli with a range of possible interpretations (Fig. 1).
from one individual to another for a given viewing situation. For Many studies of visuomotor adaptation show that humans can
example, some subjects consistently give more weight to disparity, adapt behaviorally to changes in the mapping between the envi-
whereas others preferentially weight texture gradients4,12. ronment and sensory signals15,1921. Helmholtz first demonstrat-
articles
articles
Conflict angle ()
articles
When haptic feedback was consistent with the disparity-specified for the particular stimulus properties under examination. When
slant (middle column), the average weight assigned to the disparity- the attended property is size, length, curvature or angular separa-
based estimator increased from 0.60 to 0.66 (middle panel); 7 of the tion, vision dominates the percept, even when touch is in con-
10 subjects showed an increase in this weight and 2 showed a small flict1317, demonstrating visual capture. The visual system is well
decrease (lower middle). designed for making fine discriminations of size, length, curvature
A statistical test on the pre- and post-test weights revealed a and angular separation, so the reliability of visual estimates of those
significant interaction: the increase in texture weight was greater properties is high. The haptic system is not capable of such fine
after texture-reinforced training than after disparity-reinforced discriminations23, so its reliability is lower than that of the visual
training (F1,9 = 10.597, p < 0.01). These results show that 3045 estimates. A sensible estimation method would give greater weight
minutes of haptic feedback cause an upweighting of the reinforced to the more reliable estimate; that is, the final estimate would be
estimator and a corresponding change in visual appearance. The dominated by vision and barely affected by conflicting haptic
weight change was small and variable across subjects. information. However, when the stimulus property under exami-
The average weight assigned to the disparity-based estimator in nation is coarseness of texture, vision and touch influence the per-
the no-feedback control experiment (Fig. 4, right column) revealed cept 18. Visual and haptic discrimination of coarseness
an average disparity-based estimator weight of 0.56 in the pre-test (just-discriminable percentage change) are comparable18, so the
and 0.57 in the post-test (middle right), values that are statistically reliabilities of visual and haptic estimates are roughly equivalent.
indistinguishable (F1,9 = 0.142, p = 0.715). Thus, haptic feedback Both estimates should, therefore, be able to influence the final esti-
is indeed necessary for the weight change to occur. mate20. In the experiment reported here, the visual information
2000 Nature America Inc. http://neurosci.nature.com
We conducted a second control experiment (data not shown), was by design ambiguous (texture and disparity specified differ-
in which the texture- and disparity-specified slants were congruent ent slants), so it is reasonable to assume that additional haptic
during the training phase. Haptic feedback was consistent with information consistent with one of the slants would be used.
both visual signals, and the weights did not change. Thus, weight In summary, we have shown that haptic feedback provides
changes occur only when haptic feedback is consistent with one one means for adjusting the weights given different sources of
signal and not the other. visual information. An interesting byproduct is that haptic feed-
back consistent with one source of information can cause a
DISCUSSION change in subsequent visual appearance: a surface with conflict-
We showed that haptic feedback can change the subsequent visu- ing disparity and texture signals will look more like the haptical-
al perception of surface slant. Specifically, when subjects are given ly reinforced slant than it did before.
haptic stimulation consistent with the texture-specified slant of a
visual stimulus, their subsequent visual percepts are closer to the
METHODS
texture slant than they were before training. The opposite occurs Nine naive subjects and M.O.E. completed the three conditions of the
when they are given haptic stimulation consistent with the dis- experiment. All had normal or corrected-to-normal vision and good
parity-specified slant. We interpreted the effect as a change in the stereo vision. Informed consent was obtained.
weights given to different slant estimators, in this case, to texture- The visual stimulus was a textured plane viewed binocularly. The tex-
based and disparity-based estimators. ture was a regular grid mapped onto the plane and then displayed on a
The weight changes were small. When the texture-specified CRT using OpenGL routines. The stimulus was viewed stereoscopically
slant was reinforced, the texture weight increased from pre- to with liquid-crystal shutter glasses. The apparent position of the plane
post-test by an average of 40%. When the disparity slant was rein- was below the mirror (Fig. 3). The surroundings were dark. At the begin-
ning of each stimulus presentation, a bright white field was presented to
forced, the texture weight decreased by 15%. There are two obvi-
maintain light adaptation and make the CRT frame less visible.
ous reasons for the small magnitudes. First, although we The stimulus planes slant was specified by binocular disparity and a
separately manipulated the texture- and disparity-specified slants texture gradient (Fig. 1). In the pre- and post-tests, the disparity- and tex-
during training, the values were correlated (r = 0.59). Second, ture-specified slants differed (conflict angle = 0, 10, 20 or 30, tilt = 0).
calibration of a sensory system is probably best served by slow Despite the differing slant specifications, the stimulus always looked like
changes in response to large amounts of data. If brief exposures to a single plane with a well-defined slant. Each stimulus was presented for
new correlations among information sources resulted in large 500 ms, and subjects reported whether the planes left or right side appeared
changes in calibration, a sensory system could become an unsta- closer. The slant was varied (holding constant) according to an adaptive
ble estimator of environmental information because its estimates staircase procedure (seven reversals) to estimate the surface slant that
would be subject to the vagaries of the particular sequence of appeared frontoparallel (). The estimates were determined from an aver-
age of the last four reversals. From these estimates, we calculated the weights
recent events. Thus, the 1540% change we observed is a rea-
assigned to the disparity- and texture-based slant estimators (Fig. 4).
sonable response to 3045 minutes of altered experience. The haptic-training phase occurred between the pre- and post-tests. Again,
The perceptual effect we observed is persistent: remnants of the disparity- and texture-specified slants were manipulated separately. Dur-
the effect last at least 24 hours, as subjects weights had not ing training, haptic stimulation was provided along with the visual displays.
returned to their initial values by the second day of testing. The The haptic stimulus was created by a haptic force-feedback device (PHAN-
evidence for this is the difference in the pre-test weights in the ToM Model 1.5, http://www.sensable.com). The virtual haptic stimulus
texture- and disparity-feedback conditions (Fig. 4). This persis- consisted of the plane and a small cube lying flat on the plane. The right
tence is striking, given that subjects presumably received sub- index finger was placed in a thimble-like holder attached to the device. To
stantial haptic feedback from normal interaction with their create the haptic sensation, the three-dimensional position of the right index
finger was monitored in real time. When the fingertip reached the simulat-
environment during the intervening period.
ed haptic plane or cube, the device applied an appropriate force on the fin-
It is interesting to consider our observations in light of known gertip, creating a compelling sensation of touching solid objects (the
visualhaptic interactions. As stated earlier, the majority of this stationary plane and movable cube)23. By these means of creating stimuli,
work failed to observe an effect of touch on visual appearance. we could independently manipulate visual and haptic stimulation.
Why did we succeed where others had not? A sensible answer During haptic training, a subject used the index finger to move the cube
comes from analyzing information provided by vision and touch along the plane to the target by pressing down on top of the cube and mov-
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articles
Center for Neural Science, New York University, New York, New York 10003, USA
Correspondence should be addressed to K.M. (karim@cns.nyu.edu)
Fear-arousing stimuli elicit innate reactions and can reinforce acquisition of new responses. We tested
whether mechanisms mediating these conditioned stimulus (CS) properties were isomorphic or disso-
2000 Nature America Inc. http://neurosci.nature.com
ciable within the amygdala. Rats trained on a fear-conditioning task (CS paired with footshock) were
then trained on an escape-from-fear task (EFF) in which the CS reinforced a locomotor response
terminating the CS. Lateral nucleus (LA) lesions blocked acquisition of both conditioned freezing
responses and the CSs reinforcement of a new response in the EFF task. Central nucleus (CE) lesions
blocked conditioned freezing but not the EFF, whereas basal nucleus (B) lesions blocked the EFF but
not conditioned freezing. Thus, activation of the LA by a CS seems to trigger conditioned reactions
via CE and conditioned aversion via B activation, reduction of which reinforces new actions .
Stimuli in the presence of painful or threatening stimuli acquire tions813. In contrast, several lines of investigation in which LA
aversive properties and, as a result, later elicit fear reactions. This and basal nucleus of the amygdala were lesioned together sug-
form of learning is extensively studied using a Pavlovian fear- gest that the LA or B is involved in the ability of a CS to serve as
conditioning protocol. In a typical experiment, a tone (condi- a conditioned reinforcer14,15, but it is not clear whether both are
tioned stimulus) is paired with an unconditioned stimulus (US), involved. Further, given that LA projects to CE directly and by
a footshock. Subsequently, the CS elicits unlearned species-typ- way of B16,17, it is important to determine whether the Pavlovian
ical defense reactions in the absence of the US1,2. Thus, fear con- conditioning of fear reactions is mediated by the direct projec-
ditioning does not create fear responses, but instead establishes tion from LA to CE or by way of the projection from LA to B and
the environmental conditions under which innate fear respons- from there to CE. Therefore, we examined the effects of lesions of
es will be expressed. LA, B or CE on the acquisition of both a Pavlovian and an instru-
Although innate fear reactions may be adaptive, ability to take mental conditioned response, with the stimulus that served as
novel actions in threatening situations may also be advantageous. the CS in the Pavlovian task also serving as the conditioned rein-
In the present study we used a modified escape-from-fear3 task forcer in the instrumental task.
to demonstrate mediation of these two kinds of responses by dif-
ferent neural pathways. The task involved two phases. In the first, RESULTS
fear reactions were conditioned to a CS by pairing it with shock. Histology
Subsequently, the animals were placed in a new chamber, where The LA was targeted in 20 rats. Fourteen rats were excluded
the CS was presented. They then learned that an arbitrary because of either insufficient tissue damage to the LA or dam-
response, stepping into the adjoining identical chamber, termi- age that grossly infringed on the B and/or CE. The final LA group
nated the CS. Termination of the CS reinforced the novel action, consisted of six rats. These animals had lesions destroying most
presumably because it decreased the conditioned fear elicited by of the dorsal LA and approximately 75% of the ventral LA
the CS. The CS from the conditioned fear task thus functioned as (Fig. 1b and 2a). The lesions infringed slightly on the dorsal
a conditioned negative reinforcer in the EFF task4. endopiriform nucleus laterally, but spared both B and CE. Fif-
The neural basis for the acquisition of Pavlovian fear condi- teen animals received lesions of CE. Nine of these animals were
tioning is well studied57, but the manner in which it fits into a excluded either because CE was spared or because lesions dam-
broader network of mental and behavioral systems is poorly under- aged LA and/or B. The remaining six animals included in the
stood. Here we begin to integrate the anatomy of fear conditioning analysis had lesions destroying most of both the medial and lat-
with other systems involved in more complex aspects of behavior. eral subnuclei of CE but sparing both LA and B (Fig. 1c and 2b).
Fear conditioning is believed to involve the relay of sensory Of the 16 rats that underwent B lesions, 8 were excluded from
information about the CS first to the lateral nucleus of the amyg- behavioral analyses because the lesions spared most of B or
dala and from there to the central nucleus of the amygdala57. infringed on LA and/or CE. Acceptable lesions in eight animals
The conclusion that these circuits are involved in fear condi- damaged much of B and infringed on the ventral portion of LA
tioning is based on anatomical tracing, lesion, pharmacological as well as the accessory basal nucleus (Fig. 1d and 2c). The dam-
and unit-recording studies. Particularly relevant here, lesions of age to the accessory basal was not consistent and was not evident
LA and CE interfere with the Pavlovian conditioning of fear reac- in all the animals included in this experimental group.
articles
a Sham lesion b LA lesion indicated that the B and sham groups had comparable freezing
scores (p > 0.05). Rats in groups CE and LA had similar scores
(p > 0.05). Freezing scores in both the sham and B-lesioned groups
were significantly different from scores in groups LA and CE
(p < 0.05 for all). These results demonstrate that the LA and CE are
necessary for the acquisition of freezing behavior, but the B is not.
The findings from the instrumental learning task (EFF) over-
lapped and diverged with those from the Pavlovian task. Lesions of
the LA and B blocked acquisition of the EFF task (Fig. 4b), but
c CE lesion d B lesion lesions of the CE had no effect on this task. An ANOVA compar-
ing the groups (sham, B, CE, LA) with the number of escape
responses over 4 blocks of 5 trials revealed a significant interaction
(F9,87 = 4.3, p < 0.05). Furthermore, there was a main effect of both
group (F3,29 = 5.6, p < 0.05) and blocks (F3,87 = 3.1, p < 0.05). New-
man-Keuls post-hoc analyses with group and block as variables
revealed that all groups demonstrated comparable scores on
block 1 (p > 0.05). Furthermore, only rats with either sham or CE
lesions acquired the EFF task. Specifically, the scores for sham and
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Fig. 1. Coronal unilateral images of representative lesions of amygdala subdi- CE rats from block 4 were significantly different from their respec-
visions. (a) Sham; (b) lateral amygdala; (c) central amygdala; (d) basal amygdala. tive scores on block 1 (p < 0.05). Conversely, rats with lesions of the
B or LA did not differ between blocks 1 and 4 (or any of the other
blocks; p > 0.05). These data demonstrate that the LA and B are
Behavioral results necessary for the acquisition of the EFF task, but that the CE is not.
Unlesioned animals receiving paired CSUS presentations froze
significantly more than rats presented with unpaired stimuli DISCUSSION
(F1,11 = 19.1, p < 0.05; Fig. 3a). Behavior of the animals also dif- LA is believed to be the sensory interface, the locus where CS
fered with regard to the acquisition of the EFF (Fig. 3b). A one- information enters the amygdala8,1621. Damage to LA should
way analysis of variance (ANOVA) comparing the number of therefore disrupt amygdala-dependent responses elicited by sen-
escape responses over five trial blocks as a repeated measure with sory stimuli. Indeed, here and in other studies, damage to LA pre-
the CSUS relationship (paired versus unpaired) revealed a sig- vents conditioning of fear reactions to a CS, as well as expression
nificant interaction between these two variables (F3,33 = 3.2, of previously conditioned fear reactions9,11. We also found that
p < 0.05). There was no main effect, however, of either block of lesions of LA disrupted the ability of the same CS to serve as a
trials or CSUS relationship (p > 0.05). Post-hoc Newman-Keuls conditioned reinforcer of a novel instrumental response, a con-
analyses with group and block as variables revealed that the escape ditioned fear-motivated action. Furthermore, we have demon-
scores for paired animals were significantly higher on block 4 than strated that these two properties of a CS are differentially affected
on block 1 (p < 0.05). Recipients of unpaired stimuli, however, by lesions placed in different targets of LA within the amygdala.
demonstrated comparable escape responses on the first and last Specifically, lesions of the CE blocked the acquisition of the freez-
training blocks (p > 0.05). Thus, the acquisition of both the freez- ing response elicited by the CS in the Pavlovian task, but had no
ing and EFF were contingent on association of the CS with the US. effect on the ability of the CS to reinforce acquisition of the instru-
mental response in the EFF task. Conversely, lesions of the B
Effects of amygdala lesions blocked reinforcing effects of the CS in the EFF task, but had no
Lesions of the LA and CE blocked the acquisition of Pavlovian fear effect on acquisition of freezing to the CS in the Pavlovian task.
conditioning, as measured by freezing to the CS (Fig. 4a). Howev- Different outputs of LA thus seem to mediate the ability of the CS
er, lesions confined to the B had no effect on freezing. An ANOVA to elicit fear reactions and to reinforce novel actions.
comparing the freezing scores with groups revealed a significant The effects of CE lesions on the Pavlovian conditioned freezing
effect of group (F3,29 = 10.7, p < 0.05). Newman-Keuls post-hoc tests response is consistent with previous reports that similar lesions
block freezing as well as other
Table 1. Coordinates relative to the skull surface at bregma (mm) and current duration. conditioned fear responses, such
Posterior Medial/lateral Ventral Current duration (s) as fear-potentiated startle, auto-
LA 2.3 5.1 8 9 nomic and endocrine changes
3.2 5.3 8.1 10
and alterations in pain reactivi-
ty913,22. Thus, with regard to fear
4.0 5.5 8.1 11
conditioning, the CE seems to be
the motor output for the expres-
B 2.1 4.9 9.1 12 sion of various hardwired reac-
2.8 4.9 9.3 15 tions elicited by the Pavlovian
3.3 5.3 9.2 15 CS57. The failure of damage to B
4.2 5.3 9.3 15 to affect conditioned freezing
suggests that, although LA pro-
CE 1.8 4.4 8.4 12 jects to CE directly and by way of
2.3 4.4 8.4 12 B16,17, the direct projection is suf-
ficient to mediate Pavlovian fear
2.8 4.4 8.4 12
conditioning.
articles
a
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Fig. 2. Camera lucida drawings of the extent of (a) lateral (b) central and (c) basal amygdala lesions over three rostrocaudal planes. The stippled area
represents the area of neuronal loss. LA, lateral nucleus; B, basal nucleus; Bi, intermediate division; Bpc, parvicellular division; AB, accessory basal
nucleus; ABmc, magnocellular division; ABpc, parvicellular division; BAOT, bed nucleus of the accessory olfactory tract; CO, cortical nucleus; M, medial
nucleus; PAC, periamygdaloid cortex; Ce, central nucleus; AHA, amygdalohippocampal area; AHAl, lateral division; I, intercalated nuclei; Pir, piriform
cortex; DEn, dorsal endopiriform nucleus; opt, optic tract; cp, cerebral peduncle.
The ability of a CS paired with a US to reinforce acquisition of age to the CE on acquisition of conditioned reinforcement shows
a new task defines the CS as a conditioned reinforcer23. Lesions of that CE, required for the expression of Pavlovian fear conditioning,
B blocked the conditioned reinforcing properties of an aversively does not mediate all of the effects of the fear-conditioned CS.
conditioned CS. Past studies using appetitive USs found that com- Although intra-amygdala pathways have been anatomically
bined lesions of B and LA interfere with the ability of an appetitive- mapped in great detail16,17,26, the paucity of studies explicitly exam-
ly conditioned CS to support acquisition of a new task14. ining contributions of component nuclei within the amygdala to
Furthermore, combined lesions of LA and B block the acquisition fear conditioning prevents us from elaborating further on the
of appetitive second-order conditioning24. Although B is required intra-amygdala circuitry over which information is relayed. Most
for the establishment of the new response by the conditioned rein- studies focus on lesions of LA, B or CE, so the effects of lesions
forcer, B is not necessarily the locus of motor control nor the locus of of other areas of the amygdala are generally not known. Howev-
plasticity underlying the association of the stimulus and response. er, preliminary studies aimed at addressing this question suggest
B is instead more likely the source of the conditioned reinforcement. that, whereas damage to LA and CE disrupt freezing, damage to
By way of anatomical interactions between the B and striatal response other areas (medial, cortical, B and accessory amygdala nuclei)
control circuits, conditioned reinforcement established in the amyg- have no effect on auditory fear conditioning as measured by freez-
dala may reinforce novel motor responses14,25. Taken together, the ing (P. Majidishad, D. G. Pelli, & J. E. L., personal communica-
various results suggest some overlap in the mechanisms that enable tion). The contribution of these and other amygdala areas to
a CS to reinforce new learning after being conditioned with either contextual fear conditioning and to conditioned reinforcement
an appetitive or an aversive US. In addition, the lack of effect of dam- is not yet known.
articles
Freezing (s)
The behavioral effects of B
and CE lesions are not due to
general sensory, motor or
learning impairments. Thus, as
animals with lesions of the B
showed normal freezing behav-
ior in the presence of the CS, Paired Unpaired
Blocks of five trials
they were competent to per-
ceive the CS, form an association and perform a conditioned ing scores) and also to those of the sham control group that received
response. By the same logic, the ability of rats with lesions of the CE paired training. This demonstrates that the acquisition of the active
to acquire the EFF task demonstrates that they could perceive the response (stepping into the alternate environment) was not the
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CS, form an association and perform an appropriate motor response. default behavior of the rats in this situation and was instead gradu-
To damage LA, B and CE, we used electrolytic lesions, which ally acquired over the training session.
affect both cells in the region and fibers of passage. As a result, it is It has been suggested that avoidance responses, such as running
difficult to conclude whether the effect was due to damage to the away in an active-avoidance protocol, are actually Pavlovian in
cells within the lesion or to fibers of cells in other parts of the brain; nature and not instrumental2, and that the particular response of
we chose this method because it was the only one practical for an animal in a given task depends on the situation28. Thus, it is pos-
inducing damage confined to the LA or B, with little or no involve- sible that the EFF task measured a hard-wired reactive response
ment of the other. However, the dissociation we observed between (running) that is only expressed when an exit is available. This is
effects of electrolytic lesions of LA and B, combined with studies unlikely for a number of reasons. First, running is thought to be an
using fiber-sparing excitotoxic lesions covering both the LA and unconditioned reaction to shock, and no shock was delivered in
B11,15,27, allow us to conclude that cells in LA mediate the acquisi- the EFF protocol29. Second, even when animals approach an exit,
tion of conditioned fear reactions to the CS, and cells in B the acqui- presentation of a previously fear-conditioned stimulus elicits freez-
sition of the conditioned reinforcing properties of the CS. That is, ing and not running away30. Third, if performance in the EFF task
given that excitotoxic lesions of LA together with B prevent the reflected Pavlovian rather than instrumental conditioning, then CS
acquisition of both conditioned fear and conditioned reinforce- presentation should have elicited maximal escape responses on the
ment, the dissociation produced by electrolytic lesions of the indi- first block of trials. Instead, they showed a gradual acquisition curve
vidual nuclei shows the necessity of cells within these structures. over trials, typical of what is observed in instrumental training.
An alternative interpretation of our findings is that the acquisition Thus, the EFF is unlikely to be sampling a Pavlovian response.
of the EFF task depends on the absence of freezing. That is, rats can- Our distinction between the B mediating active responses and
not perform the active response as long as they are freezing, so elim- the CE mediating reactive responses is similar to distinctions made
ination of freezing by damaging some area (like CE) allows the rat to by others 15, though important differences also exist. These
step into the other chamber. If freezing were simply competing with researchers found that combined damage of LA and B interferes
stepping into the adjacent chamber, then all groups with minimal with the ability of an aversive CS to reinforce a new response, where-
freezing should have demonstrated high escape responses on the as lesions of CE, but not combined LA/B lesion, interfere with the
very first block. This was not observed. Animals with lesions of the conditioned reactive responses, and suggested that the amygdala
CE or LA both had reduced freezing responses to the CS in the con- has two learning systems, the LA/B (for conditioned reinforcement)
ditioned fear task. At the same time, in the EFF task, escape scores and the CE (for conditioned reactions)15. However, as noted above,
of both the LA- and CE-lesioned groups to the CS on block 1 were our finding that lesions restricted to the LA block Pavlovian con-
comparable to those of rats with B lesions (which had normal freez- ditioned reactions replicates several past studies8,11. At present, the
reason for the discrepant find-
a b Central
ings is not apparent, although a
Sham number of possibilities have
Number of escape responses
articles
actions, to learn the action at the time through trial and error or
Reactive
to devise a plan of action on the spot. By studying how different
responses actions develop in response to a single eliciting stimulus, we will
CS information
be able to further explore aspects of these cognitiveemotional
2000 Nature America Inc. http://neurosci.nature.com
articles
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grants MH46516, K02 MH00956 and R37 MH38774 to J.E.L. and a HFS
deficits produced by neurotoxic lesions of the basolateral amygdala.
Fellowship to K.N. The work was also supported by a grant from the W.M. Keck J. Neurosci. 18, 30883097 (1998).
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articles
Section on Functional Brain Imaging, Laboratory of Brain and Cognition, National Institute of Mental Health, Building 10, Room 4C104, Bethesda, Maryland
20892-1366, USA
Correspondence should be addressed to E.A.H. (elizabeth_hoffman@nih.gov)
Face perception requires representation of invariant aspects that underlie identity recognition as well as
2000 Nature America Inc. http://neurosci.nature.com
representation of changeable aspects, such as eye gaze and expression, that facilitate social
communication. Using functional magnetic resonance imaging (fMRI), we investigated the perception of
face identity and eye gaze in the human brain. Perception of face identity was mediated more by regions
in the inferior occipital and fusiform gyri, and perception of eye gaze was mediated more by regions in
the superior temporal sulci. Eye-gaze perception also seemed to recruit the spatial cognition system in
the intraparietal sulcus to encode the direction of anothers gaze and to focus attention in that direction.
Whereas the invariant aspects of a face allow one to recognize ity in the STS, then selective attention to gaze should elicit a
who someone is, the changeable aspects of a face can be used to stronger response in that region than attention to identity. In our
infer information about that persons state of mind. Most face second experiment, we examined the strength of responses elicit-
viewing occurs in the context of social communication after iden- ed by passive viewing of faces in which gazes were averted as com-
tity has been established. One example of information gleaned pared with viewing of faces in which gazes were direct (Fig. 1,
from a face during social interaction is the direction of anothers lower panel). Given that the perception of averted gaze causes a
gaze, which can indicate where that persons attention is directed seemingly reflexive attention shift15, passive viewing of faces in
and can be used to similarly direct ones own attention. which gazes are averted should elicit stronger responses in the
Impaired face recognition (prosopagnosia) is associated with IPS, a brain region associated with covert shifts of spatial atten-
ventral temporal lesions13. Within ventral temporal cortex, neu- tion1618, than viewing of faces in which gazes are direct.
roimaging studies of face perception identify a region in the lat-
eral fusiform gyrus (LFG) that responds more to faces than to RESULTS
other objects47. It is unclear, however, whether the perceptual Accuracy and response times were similar for selective attention to
analysis of all aspects of faces is mediated by this region. Other identity and eye gaze (93 6.0%, mean s.d., versus 92 5.0%,
regions that respond preferentially, but less consistently, to faces n.s.; 686 ms 129 ms versus 722 ms 147 ms, n.s.), indicating that
are identified in the lateral inferior occipital gyri (IOG) and in the tasks were well matched on difficulty and attentional demand.
the posterior superior temporal sulcus (STS)4,6,8. The STS region Four bilateral regions that responded more to faces than to
is also associated with the perception of eye and mouth move- control stimuli (scrambled pictures) were identified, in the LFG,
ment9,10 and may be homologous to a region in the superior bank STS, IOG and IPS (Table 1 and Fig. 2). Of the seven subjects who
of the STS in the monkey in which cells respond preferentially showed significant activations in the regions that were the subject
to faces, eye-gaze direction and face expression1114. We hypoth- of our experimental hypotheses, namely the LFG and the STS,
esized that the human face-responsive region in the STS may also bilateral LFG and IOG regions were identified in all subjects, STS
be more involved in the perception of changeable aspects of faces regions were identified in all subjects on the right and in four sub-
and that the human face-responsive region in the LFG may be jects on the left, and IPS regions were identified in all subjects on
more involved in the perception of face identity. We hypothe- the left and in five subjects on the right. As we had hypothesized,
sized further that perception of the direction of eye gaze would selective attention to face identity and eye gaze had opposite effects
elicit activity in regions associated with spatial perception and in the LFG and STS (region attention interaction, p < 0.001 on
spatially directed attention, namely the intraparietal sulcus (IPS). both the right and left), demonstrating that these regions partici-
We conducted two experiments to test these hypotheses. In pate differentially in the representation of the invariant and change-
our first experiment, we tested whether selective attention to able aspects of a face. In the LFG, attention to identity elicited a
identity and eye-gaze direction modulated activity differently in stronger response than did attention to gaze (1.21% versus 0.90%,
these brain regions (Fig. 1, upper panel). If the representation of n = 7, p < 0.001, on the right; 1.23% versus 0.95%, n = 7, p < 0.001,
identity were more dependent on activity in the LFG, then selec- left). By contrast, in the STS, attention to gaze elicited a stronger
tive attention to identity should elicit a stronger response in that response than did attention to identity on the left (0.74% versus
region than attention to eye-gaze direction. Similarly, if the rep- 0.47%, n = 4, p < 0.001), with no significant difference in the same
resentation of eye-gaze direction were more dependent on activ- direction in the right STS (0.79% versus 0.72%, n = 7, n.s.). The
articles
Selective attention
left IPS than did attention to identity (0.99% versus 0.80%, n = 7,
p < 0.001), with a nonsignificant difference in the same direction
in the right IPS (0.88% versus 0.85%, n = 5, n.s.). Similar to the
findings in the STS, the effects of task on responses in the right
Task cue and left IPS did not differ significantly (p > 0.1).
The enhanced response in the IPS while attending to eye gaze
suggested recruitment of the spatial cognition system. The direc-
tion of anothers eye gaze is a potent cue for directing ones own
spatial attention. Shifts of attention in response to the percep-
tion of averted gaze are observed in monkeys, apes and young
Task cue infants1923, and are elicited in adults while fixating on a face, even
when the direction of gaze is task-irrelevant15, suggesting that
our subjects also made covert, reflexive shifts of attention when
the perceived gaze was averted.
It was also possible that the differential response in the IPS
Passive viewing could be attributed to differences in the aperture of spatial atten-
tion. Presumably, the aperture of attention is narrower when
Averted attending to eye gaze than when attending to identity. To rule out
2000 Nature America Inc. http://neurosci.nature.com
gaze
this alternative explanation, we conducted a second experiment
using two passive viewing conditions in which subjects were not
instructed to attend to a specific facial feature. Subjects passive-
ly viewed faces that had averted gazes in one condition and pas-
sively viewed faces that had direct gazes in the other (Fig. 1, lower
Direct
gaze
panel). The faces in each condition never repeated, so that the
entire stimulus, not just the eye region in the averted gaze con-
dition, varied from one trial to the next. Passive viewing of faces
with averted gazes elicited significantly stronger responses than
did passive viewing of faces with direct gazes in the IPS bilaterally
Fig. 1. Face-perception tasks. The one-back repetition detection tasks (on the right, 0.35% versus 0.16%, n = 5, p < 0.001; on the left,
in experiment 1 are displayed in the upper panel. Subjects attended 0.17% versus 0.06%, n = 7, p < 0.05) and in the left STS (0.39%
selectively to the direction of eye gaze or the identity of each face. In the versus 0.27%, n = 4, p < 0.01). By contrast, direction of gaze had
passive-viewing conditions in experiment 2 (lower panel), subjects no effect on the response to faces in the right STS or bilaterally
viewed series of faces that either had the eyes all directed away from the in the IOG or LFG. The difference in the right STS, however, was
viewer or all directed at the viewer.
in the same direction as in the left STS, and the sizes of this effect
in the right and left did not differ significantly (p > 0.1).
DISCUSSION
effects of task on responses in the right and left STS did not differ The results of these experiments indicate that face identity and
significantly (p > 0.1). Additionally, we found that, as in the LFG, eye gaze have distinct representations within the distributed
attention to identity elicited a stronger bilateral response in the human neural system for face perception. This distributed sys-
IOG than did attention to gaze (on the right, 0.99% versus 0.85%, tem includes bilateral regions in the IOG, LFG and STS, all of
n = 7, p < 0.005; on the left, 1.10% versus 0.85%, n = 7, p < 0.001). which show a greater response to faces than to other objects4,6,8,24.
As in the STS, attention to gaze elicited a stronger response in the Additionally, a region in the IPS was activated by our tasks,
Table 1. Volumes and stereotaxic brain atlas coordinates45 for the brain regions activated by viewing faces as compared
to viewing scrambled pictures (mean s.d.).
Talairach coordinates (mm)
Region Hemisphere n Volume (cm3) x y z
Fusiform gyrus left 7 2.5 0.7 37 1 60 1 22 4
right 7 2.7 0.6 39 2 55 2 22 3
articles
Experiment 1 Experiment 2
Selective attention Passive viewing of faces
to gaze and identity with averted and direct gazes
Lateral view
Ventral view
Superior
temporal
sulci
percent change
Flattened cortex
Lateral
fusiform
2000 Nature America Inc. http://neurosci.nature.com
gyri
Intraparietal
sulcus
percent change
Inferior Inferior
occipital occipital
Superior temporal gyri
gyri
sulcus
Fusiform gyrus
Attention Attention Averted Direct
to gaze to identity gaze gaze
Right hemisphere
Left hemisphere
Fig. 2. fMRI results. The left panel shows regions activated by the face-perception tasks (z > 4.0) in the right hemisphere of one subject. Regions are
shown on the folded surface, presented in lateral and ventral views (upper left figures) and on the cortical surface, inflated to show the extent of acti-
vated regions in the sulci. The lower figure shows the cortical surface for the entire right hemisphere presented as a flat, two-dimensional surface.
Sulcal cortex obscured in the folded surface is shown with a darker shade of gray on the inflated and flattened surfaces. On the flattened cortex,
occipital cortex is on the left and frontal cortex is on the right. The right panel shows mean time series in regions of interest, averaged across voxels
in the regions, repetitions of task blocks and subjects. Gray bars indicate the presentation of task blocks. White spaces following task blocks indicate
control task blocks that follow each task. See text for statistical comparisons.
although this region does not typically show selectivity for faces The representation of eye gaze, a changeable aspect of the face,
but, rather, is more typically associated with spatial perception depends more on activity in the STS than on activity in the IOG
and spatial attention1618,25. and LFG. Our results show that selective attention to gaze direc-
The representation of face identity, which is based on aspects of tion elicits a stronger response in the left STS than does attention
facial structure that are invariant across changes in eye gaze or to identity. An earlier neuroimaging study showed that perception
expression, is more dependent on activity in the IOG and LFG than of eye and mouth movement selectively activates the STS bilater-
on activity in the STS. In the monkey, neurons in the convexity of ally9. An event-related potential (ERP) study with scalp electrodes
the inferior temporal (IT) gyrus show greater selectivity for differ- using the same moving stimuli that evoked activity in the STS
ent individual faces than do neurons in the STS11, although some revealed that perception of averted gaze evokes a stronger N170
STS neurons also respond differentially to individual faces1214. The response than does perception of direct gaze30, consistent with our
human ventral temporal regions that are face-responsive may be results. Moreover, N200 responses measured with subdural elec-
homologous to the monkey IT region that is tuned to face identity. trodes placed on ventral face-specific sites do not differ signifi-
This conclusion is consistent with the literature on lesions that cause cantly for perception of averted and direct gaze31, consistent with
prosopagnosia13,26. Neuroimaging research on the role of the LFG our findings in the LFG in experiment 2. A positron emission
has been ambiguous27,28. An early study showed that the fusiform tomography study showed STS activation during perception of
gyrus was activated more when subjects attended to face identity averted and direct gaze10, but no difference between these condi-
than when they attended to gender27. Studies of the effect of face tions, in contrast with our fMRI results and the scalp ERP results30.
inversion on the activity in the LFG, however, generate doubt as to Our results indicate that the STS has a more general role in the
whether this region encodes face identity or simply the generic facial perception of changeable aspects of faces, even when viewing sta-
configuration4,5,29. Inversion impedes recognition of identity but tic images. This conclusion is consistent with electrophysiological
has only a small and nonspecific effect on LFG activity. Our results and lesion studies in monkeys. Monkey STS contains cells that
clearly implicate the LFG in the perception of identity. The effect respond differentially to gaze directions and facial expressions in
of face inversion suggests that LFG activity may reflect the attempt static pictures1114. These findings have led to the proposal that
to perceive identity, not the successful generation of a distinct rep- within face-responsive regions there are independent cell popula-
resentation of an individuals face. tions that perceive social signals from the face1214, and that these
articles
cells are more prevalent in the STS than in IT cortex11. Lesions of the transfer of information about social signals gleaned from the
monkey STS are associated with impaired perception of eye gaze face, particularly those concerning direction of spatial attention38.
with preservation of the perception of face identity32,33. Dissociation Such connections in the human brain could mediate the recruit-
of impairments of face-identity recognition, on the one hand, from ment of the IPS when the STS detects an averted gaze. One fMRI
impairments of the perception of eye-gaze direction or facial study showed that perception of lateral eye movement also acti-
expression, on the other, is reported in human lesion studies, but vates the IPS9, consistent with our contention that the IPS activ-
the anatomical locations of lesions that can cause selective impair- ity is specifically associated with the spatial aspects of perceived
ment of eye-gaze perception are unclear26,32. eye gaze and its role in directing attention.
Neuroimaging studies of other changeable aspects of faces Another fMRI study found that perception of direct gaze, but
also implicate the STS. In one study, perception of facial expres- not averted gaze, elicits activity in the amygdala39, but detected no
sion elicited a response in regions with coordinates close to our face-responsive region in the STS, perhaps because the control task
STS region34. In another study, lip reading also elicited a response (opening and closing of the eyes) also involves perception of facial
in similar regions35. Together with our results, these findings and movement. The amygdala was not included in the volume we
studies of monkey STS suggest that the STS may play a more gen- scanned. An amygdalar response may reflect the emotional signif-
eral role in the representation of changeable aspects of the face. icance of direct gaze, which is perceived as more socially engaging
Studies of the perception of face expression and lip-reading or potentially threatening than is averted gaze 40,41. Monkeys
suggest that these operations also elicit activity in additional sys- respond more emotionally and make more appeasement gestures
tems that process the significance of information gleaned from when gaze is directed at them than when gaze is directed away42,43.
2000 Nature America Inc. http://neurosci.nature.com
the face. Perception of fearful and disgusted facial expressions Connections between the STS and the amygdala may mediate pro-
elicit further activity in limbic regions associated with process- cessing of the emotional content of direct gaze.
ing emotion34,36,37. Lip-reading elicits further activity in regions Face perception can provide a wealth of information that facil-
associated with auditory processing of speech sounds35. itates social communication. There are two classes of face-percep-
Our results show that the perception of averted eye gaze elic- tion operations that require independent representations. One
its further activity in the spatial cognition system in the IPS. The class involves the perception of the changeable aspects of the face,
IPS is activated during tasks involving spatial perception and such as expression and eye-gaze direction, whereas the other
covert shifts of spatial attention1618,25. Presumably, the IPS was involves the perception of aspects of facial structure that are invari-
recruited in our tasks to encode the spatial direction of anoth- ant across these changes. Perception of changeable aspects pro-
ers gaze and, additionally, perhaps, to mediate covert, reflexive vides information about another persons current state of mind44.
shifts of spatial attention in that direction. The role played by the Eye gaze, in particular, is a powerful social signal that can guide
IPS in mediating covert shifts of spatial attention is presumably our attention and can inform us about the intentions and interest
the same whether that shift is elicited by perceived eye gaze, as of another person. Perception of invariant aspects of facial structure
in our experiment, or by some other spatial cue. underlies the recognition of identity. Our results indicate that face
An alternative account of the differential IPS response is that perception is mediated in humans by a distributed system that
perception of averted gaze elicited eye movements that resulted in comprises multiple regions, and that changeable and invariant
enhanced IPS activity. To rule out this explanation, we recorded aspects of faces have distinct representations within this system.
eye movements using the ISCAN eye tracking system (Burlington,
Massachusetts) while four different subjects performed the selec- METHODS
tive attention and passive viewing tasks outside the scanner. The Tasks. In experiment 1, subjects performed repetition-detection tasks
that directed attention to identity or eye gaze. In each block of trials, nine
mean number of saccadic eye movements was similar for selective
faces were presented sequentially in the center of a screen for 0.5 s with an
attention to identity and eye gaze (18-s stimulus block; 29 5 s, interstimulus interval of 1.5 s. At the beginning of each block of trials, a
mean s.d., versus 28 7 s, n.s.) and for passive viewing of direct cue word (identity or gaze) was displayed for 1 s to inform the subject
and averted gaze (20 7 s versus 18 7 s, n.s.). Moreover, the hor- as to which task to perform. Subjects indicated whether the selectively
izontal and vertical amplitudes of eye movements did not differ attended aspect of each face matched that of the preceding face by press-
for selective attention to identity versus eye gaze (horizontal, ing a button with the right (match) or left (nonmatch) thumb. In a con-
1.6 0.4 versus 1.7 0.3, n.s.; vertical, 0.6 0.3 versus 0.4 0.2, trol task, scrambled, nonsense, color images were presented at the same
n.s.) or for passive viewing of direct versus averted gaze (horizon- rate and in the same format as the stimuli in the repetition-detection
tal, 1.5 0.2 versus 1.3 0.3, n.s.; vertical, 0.6 0.5 versus tasks. In these trials, subjects pressed both the right and left buttons
0.5 0.4, n.s.). Given these results, it is unlikely that the enhanced simultaneously when each stimulus appeared. Blocks of control trials
alternated with repetition-detection blocks. In experiment 2, subjects
IPS activation in experiments 1 and 2 was due to greater eye move-
passively viewed color faces that were blocked by gaze direction (lateral-
ments during selective attention to gaze or passive viewing of avert- ly averted or direct). Subjects were instructed simply to look directly at
ed gaze. These results also suggest that attention to identity, as each picture. Stimuli were presented sequentially at a rate of 2 per s in
compared to attention to eye gaze, did not result in significantly the center of the screen. Each block consisted of 36 stimuli. Face blocks
more scanning of the face or more eye movements with a vertical alternated with control blocks during which nonsense stimuli were pre-
component. Therefore, it is unlikely that differential responses sented at the same rate and in the same format as were stimuli in the face
when attending to gaze and identity can be attributed to differ- blocks. The order of blocks in both experiments was counterbalanced
ences in the parts of the faces that were foveated during these tasks. across subjects and time series. Ten time series, each consisting of eight
Behavioral studies indicate that monkeys and apes use gazes of face blocks and nine control blocks, were obtained for each subject (six in
experiment 1 and four in experiment 2).
others as cues to direct attention19,23. Human infants as young as
three months shift attention in the direction of perceived gaze21,22. Imaging. We scanned 9 healthy volunteers (3 male, 6 female, mean age,
One study with adult subjects suggests that these shifts may be 24 2.5 years). Each subject gave written informed consent and was com-
reflexive, occurring even when the direction of perceived gaze is pensated for participation. Our experimental protocol was approved by
task-irrelevant15. Reciprocal connections between cell popula- the institutional review board of the National Institute of Mental Health.
tions in the superior bank of the STS and the IPS38 could mediate Twenty contiguous, coronal, 5-mm thick slices were obtained in 10 time
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articles
1 Program in Neurosciences and 2Dept. of Psychology, Jordan Hall, Bldg. 420, Stanford University, Stanford, California 94305, USA
Correspondence should be addressed to V.P. (vivekpr@leland.stanford.edu)
Ability to integrate diverse forms of information in current thought, or working memory, is essential
for human reasoning and problem solving. We used functional imaging to identify brain regions
2000 Nature America Inc. http://neurosci.nature.com
Working memory involves the short-term maintenance of infor- respectively, had appeared in the previous display. For the ver-
mation relevant to current goals. Neuroimaging studies show bal scans, the letters were shown at study and test in different
that working memory is mediated by frontal and posterior cor- cases, so that subjects coded the verbal identity rather than the
tical regions differing in the types of information maintained (for visual appearance of letters.
instance, verbal, spatial or object) and in the kinds of contribu- In two other scans, subjects were asked to maintain both spa-
tions made to working memory (for instance, directing rehearsal tial and verbal information either in an integrated or in an unin-
versus storing information per se)126. Whereas posterior corti- tegrated fashion. In both scans, subjects saw a target display of
cal regions seem to specialize in the type of information held in four letters and four spatial locations appeared for two seconds
working memory5,10,17,22,23,2729, several findings suggest that pre- and then disappeared (Fig. 2). In the integrated scan, the four
frontal areas have a special role in integrating different types of letters to be remembered were displayed in the four locations to
information in working memory. Electrophysiological studies in be remembered; thus verbal information and spatial informa-
nonhuman primates reveal frontal lobe cells that maintain both tion were bound together. In the unintegrated scan, the four let-
spatial and object information in working memory3032. Neu- ters were presented centrally and separately from the four
roanatomical tracing in nonhuman primates suggests prefrontal indicated locations; thus, verbal information and spatial infor-
cortex as a region of polymodal sensory convergence from pos- mation were separate. Subjects then had to maintain those let-
terior cortical areas (parietal, temporal and visual regions)33,34. ters and locations in working memory over a retention interval.
Neuroimaging studies in humans have shown proximal foci of Subsequently, they saw a single probe letter in a single probe loca-
activation in frontal cortex for maintaining different types of tion and had to judge whether both the letter and the location
information (for example, letters, objects, locations or faces) in had been shown in the previous display (regardless of whether
working memory. The present study aimed to test directly the that particular letter had been in that particular location). Posi-
hypothesis that human frontal cortex has a specialized role in tive-probe trials presented a letter and a location that had been in
maintaining integrated information in working memory35,36. the target display; negative probe trials presented either a differ-
Six subjects (right-handed; mean age, 24.5 years) were ent letter or a different location or both.
scanned via functional magnetic resonance imaging (fMRI). Scans using a 5-second retention interval between the target
Each subject performed four different tasks in four successive and probe displays were compared to a baseline identical in all
scans. Subjects performed two scans in which they were asked to regards except the retention interval between the target and probe
maintain either spatial information or verbal information displays was only 250 ms, so that information did not have to be
(Fig. 1). In the spatial scan, subjects saw a target display of four maintained in working memory. This allowed for isolation of
spatial locations appear for two seconds and then disappear. In areas involved in maintaining different types of information (spa-
the verbal scan, subjects saw a target display of four letters tial, verbal, verbal and spatial/integrated, verbal and spatial/unin-
appear for two seconds and then disappear. Over a retention tegrated) in working memory. As processes involved in encoding
interval, subjects then had to maintain those letters or locations the target information and in responding to the probe were
in working memory. Subsequently, they saw a single lower-case equivalent for the delay (5-second interval) and no-delay (250-ms
probe letter in the verbal scan or a single probe location in the interval) trials, our scans did not allow identification of areas
spatial scan, and decided whether the letter or the location, involved in these processes.
articles
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p = 0.0005
p = 0.025
2000 Nature America Inc. http://neurosci.nature.com
Fig. 5. Activation in the right frontal cortex in six subjects. For each subject, a minimum of two slices depicting differential activation levels in the right frontal
cortex were chosen. The right frontal cortex was identified anatomically as a region of interest (ROI) in these slices. Statistical analyses were confined to that
ROI and superimposed on each subjects anatomical MR image. The upper row shows robust right frontal activation for maintaining bound information for
5 s versus 250 ms; the lower row shows that this region was minimally activated while maintaining separate information for 5 s versus 250 ms.
was evident not only in the group average, but also in each mation alone (Fig. 6). Comparison of separate versus spatial and
individual subject (Fig. 5). Maintenance of unintegrated, rel- verbal scans revealed posterior regions that were more involved in
ative to integrated, information resulted in greater activity in maintaining unintegrated verbal and spatial information than in
multiple posterior brain areas, including bilateral parietal, maintaining either spatial or verbal information alone. There was
temporal and cerebellar regions. not, however, any difference in the right prefrontal cortex for
In a second analysis, bound and separate conditions were maintaining unintegrated verbal and spatial information. Thus,
compared to the combination of the purely spatial and verbal both analyses provide convergent evidence for a dissociation of
conditions. Here also, the bound versus spatial and verbal com- the right frontal region preferentially involved in the maintenance
parison revealed substantially greater involvement of the right of integrated working-memory representations, and multiple
prefrontal cortex in maintaining integrated spatial and verbal posterior brain regions preferentially involved in the maintenance
information than in maintaining either spatial or verbal infor- of non-integrated working-memory representations.
articles
scan consisted of six cycles, with each cycle comprising blocks of 5-s main-
DISCUSSION
tenance trials alternated with blocks of 250-ms maintenance trials. There
The right prefrontal activation associated with the integration of were 4 trials per block with a total of 24 (12 negative-probe trials and 12
verbal and spatial information is in agreement with meta-analy- positive-probe trials) 5-s maintenance trials and 24 (12 negative-probe
ses showing right prefrontal involvement in both spatial and non- trials and 12 positive-probe trials) 250-ms maintenance trials in each scan.
spatial working memory9,35,36. In contrast, left prefrontal regions In the bound scan, the positive-probe trials were equally divided into con-
show involvement only in nonspatial working memory. Thus, gruent and incongruent trials. The scan order was counterbalanced across
right prefrontal regions seem to have a flexible representational the subjects. Stimuli were generated from a computer and back-project-
architecture that processes both spatial and nonspatial informa- ed onto a screen located above the subjects neck via a magnet-compati-
tion. Posterior activations have been more material specific, with ble projector. Visual images were viewed from a mirror mounted above
specific areas in parietal and temporal regions involved either in the subjects head. The sequence of the presentations of the stimuli were
synchronized with the imaging sequence of the scanner.
verbal or spatial working memory5,10,17,22,23.
Many studies have shown greater activation in prefrontal cor- fMRI methodology. Imaging was performed with a 1.5T whole-body MRI
tex due to increased demands of load, duration or manipulation scanner (General Electric Medical Systems Signa, Rev. 5.5, Waukeshau,
on working memory123. In contrast, greater prefrontal activation Wisconsin). A T2* sensitive gradient echo spiral sequence46 was used
in the present study occurred in the easier condition: subjects were for functional imaging with parameters of TR = 1440 ms,
more accurate and faster in the bound than in the separate condi- TE = 40 ms, flip angle = 83, FOV = 20 cm, inplane resolution = 1.56 mm2,
tion. The prefrontal activation, therefore, reflected the nature of sampling interval = 2.88 s and number of temporal frames or image vol-
the working-memory representation, rather than working-mem- umes = 160. Sixteen 7-mm thick slices with a 0-mm inter-slice interval
2000 Nature America Inc. http://neurosci.nature.com
ory load or duration, which were equal in the two conditions. and covering the whole brain were acquired in the horizontal plane of the
Talairach and Tournoux atlas47.
Baddeley has proposed separate slave system buffers that allow
for temporary retention of discrete information in working mem- fMRI analysis. Functional images were motion-corrected and normal-
ory, including a visuospatial buffer and a phonological buffer37. ized using SPM96, interpolated to 2 2 4 mm3 voxels and spatially
Logie and others have further decomposed the visuospatial buffer smoothed with a Gaussian filter (full width at half maximum, 8 mm).
into separate visual and spatial buffers38. The present fMRI results Low-frequency noise and differences in global signal were removed. Sin-
provide evidence for another type of buffer, namely, one that gle subject data were analyzed with a fixed-effects model. Group data
allows for temporary retention of integrated information. were analyzed using a random-effects model. For the group analysis,
The capacity to integrate information in working memory images were averaged to create one image of mean activity per condi-
may enhance the efficiency of working memory in several ways. tion and subject. These average images were used to create a series of
For example, a study examining the capacity of short-term mem- SPM{Z} maps depicting differences in brain activity between task con-
ditions. Activation maps for both fixed-effects model and random-effects
ory for visuospatial information found that subjects show equiv-
model analysis were created with SPM96 software
alent accuracy in maintaining the memory of a single feature of (http://www.fil.ion.ucl.ac.uk/spm)48 with an intensity threshold of
objects (for instance, color of an object) as in maintaining four p < 0.025 and spatial-extent threshold of p < 0.05.
features of objects (for instance, color, size, orientation and
shape)39. However, performance decreases as the number of
objects that need to be remembered increases. In other words, ACKNOWLEDGEMENTS
more information could be maintained for visuospatial infor- This work was supported by grants from the National Institute on Aging and the
mation in a bound rather than separate display. In our study, National Center for Research Resources. V.P. is supported by a NRSA training
the finding subjects were significantly more accurate and tend- grant awarded by the National Institutes of Health. The authors thank Mark
ed to be faster in the bound than in the separate condition sug- DEsposito for comments on earlier drafts of this manuscript.
gests more efficient access to integrated relative to unintegrated
information. Further, the mean volume of brain area preferen- RECEIVED 3 SEPTEMBER; ACCEPTED 3 NOVEMBER 1999
tially activated by the unintegrated information (11,483 mm3)
was more than twice that activated preferentially by the inte-
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articles
We present behavioral and anatomical evidence for a multi-component reading system in which
different components are differentially weighted depending on culture-specific demands of orthogra-
phy. Italian orthography is consistent, enabling reliable conversion of graphemes to phonemes to
yield correct pronunciation of the word. English orthography is inconsistent, complicating mapping
of letters to word sounds. In behavioral studies, Italian students showed faster word and non-word
reading than English students. In two PET studies, Italians showed greater activation in left superior
temporal regions associated with phoneme processing. In contrast, English readers showed greater
activations, particularly for non-words, in left posterior inferior temporal gyrus and anterior inferior
frontal gyrus, areas associated with word retrieval during both reading and naming tasks.
In English there are 1120 ways of representing 40 sounds (phonemes) We conducted two PET scan studies, again with university stu-
by different letters or letter combinations (graphemes)1. The map- dents. The first study addressed explicit reading. Here participants
pings between graphemes, phonemes and whole word sound are had to read words and non-words aloud. A second study addressed
essentially ambiguous, as illustrated by pairs such as pint/mint, implicit reading, where we assessed physiological responses induced
cough/bough, clove/love. By contrast, in Italian, 33 graphemes are by the mere presence of print in the visual field. Participants were
sufficient to represent the 25 phonemes of the language2, and the not asked to read the stimuli, but to perform a visual feature-detec-
mappings from graphemes to phonemes are unequivocal. Young tion task on words, non-words and false-font stimuli7.
Italian readers can achieve 92% accuracy on word reading tests after Results from both experiments were combined to show only
only 6 months of schooling3, whereas learning to read in English those effects that were reliable in both studies (Fig. 2 and Table 2).
takes much longer. Compared to German, another consistent We identified a common brain system that was active during read-
orthography, accuracy levels in English are lower and reading speed ing, explicitly and implicitly, across the two languages. This sys-
is slower even after three years of schooling4,5. Adult English read- tem included inferior frontal and premotor cortex, superior,
ers are slower at reading non-words than readers of the consistent middle and inferior temporal gyri and fusiform gyrus on the left,
Serbocroat orthography6. We investigated reading in Italian and and superior temporal gyrus on the right. The majority of the left-
English university students. These students read high-frequency reg- hemisphere areas were more active for non-words than for words,
ular words in their respective languages, a set of international words although no single region showed reliably greater activation for
and two sets of non-words derived from both languages. We also words. Interaction effects showed a language-related difference:
investigated the neurophysiological basis of reading in Italian and English readers, particularly when reading non-words, showed
English using positron emission tomography (PET). greater activations in the left posterior inferior temporal region
and in the anteriormost part of the inferior frontal gyrus. When
RESULTS reading words or non-words, Italian readers showed greater acti-
Italian students were faster at both word and non-word reading, vations at the junction between left superior temporal gyrus and
even when the non-words were derived from English words. Con- inferior parietal cortex, a region known as planum temporale.
trol tasks showed that this advantage could not be attributed to
faster reaction times, articulation speed, naming speed or verbal DISCUSSION
fluency (Table 1 and Fig. 1). Both groups were slower at reading In spite of the large amount of empirical data on normal and
non-words as compared with words. This difference was signifi- abnormal reading, it is still a matter of debate as to how word and
cantly greater for English readers. The Italian students read non- non-word reading is achieved, particularly in a deep orthography
words and international words derived from Italian faster than such as English812. Our behavioral results showed that even with
those derived from English. The English students were unaffected simple and regularly spelled stimuli, background effects of the
by the source of the words. complex English orthography incur a cost in terms of reading
articles
words faster than non-words (F2,140 = 114.90, p < 0.0001), but this effect
was more marked for English (group by task interaction; F2,140 = 15,53;
p < 0.0001). In a post-hoc analysis of non-word reading, English students
were equally slow when reading non-words derived from Italian or from
English words. In contrast, Italian students, although significantly faster
than English students for both kinds of non-words (group main effect;
F1,70 = 13.6; p < 0.0005), were faster still with non-words derived from
Italian words (group task interaction effect: F1,70 = 42,51; p < 0.0001).
In reading familiar international words, analysis of variance showed no
overall group effect (F1,70 = 0.97; n.s.), but a group by task interaction
Words
Non-words Non-words International International (F1,70 = 7.9; p < 0.007). This was because Italian subjects were faster at
from Italian from English words words reading international words conforming to their own orthography.
conforming to conforming to
Italian English
2000 Nature America Inc. http://neurosci.nature.com
speed. According to a dual-route perspective, two processes are spelling patterns. Thus, for English readers, Italian non-words had
necessary in reading, letter-to-sound conversion and access to a the same bigram frequency as English non-words and did not slow
lexicon of orthographic patterns to resolve ambiguities in pro- them down. In contrast, Italians read the English non-words with
nunciation8,9,11. According to a single-route connectionist per- their less-familiar spelling patterns more slowly, as expected from
spective, one process is needed, which involves conversion from their lower bigram frequency in Italian.
orthography to phonology, and in a deep orthography such as Eng- Our PET scan data provide the first cross-cultural anatomi-
lish, an extensive translation from orthography to semantics to cal information about a common reading system for different
phonology10,12. Our data can be interpreted within either of these alphabetic orthographies. Reading in both complex and trans-
models. The complexity of English orthography derives partly from parent orthographies depends on a distributed network of pri-
the historical influence of other orthographies, including Italian marily left-sided language areas. Within the common network,
Table 1. Performance in reading tasks and in control tasks in 36 university students from London and 36 from Milan.
Reading tasks
Words Non-words Non-words International words International
derived from derived from conforming to words conforming
( s.d.) Italian words English words Italian (IW1) to English (IW2)
(ms) (NW1) (ms) (NW2) (ms) (ms) (ms)
English subjects 442.6 47.4 526.4 93.9 528.8 101.5* 450.9 48.2 448.0 57.4**
(n = 36)
Italian subjects 410.7 33.1 437.3 39.0 485.9 56.8+ 424.8 36.5 452.8 53.3++
(n = 36)
Mean difference 31.9 89 42.8 26.1 4.8
Two-tailed t value 3.3 5.2 2.2 2.6 0.37
p value < 0.0015 < 0.0001 < 0.03 < 0.012 0.7
Control tasks
Simple vocal Articulation Semantic verbal Letter verbal Picture-
reaction time speed for pairs of fluency fluency (words naming
words (words in (animal names in starting with m latency
(ms) 15 min) 1 min) in 1 min) (ms)
English subjects 310.5 37.8 40.4 3.5 25.4 6.7 16.5 6.7 546.7 41.8
(n = 36)
Italian subjects 313.1 28.8 41.9 6.1 27.5 6.4 16.6 4.7 561.0 47.5
(n = 36)
Mean difference 2.6 1.5 2.1 0.1 14.4
Two-tailed t value 0.33 1.3 1.3 0.08 1.4
p value 0.73 0.19 0.18 0.93 0.17
The results of the control tasks show that differences in reading speed were not due to differences in the sample population or to more general factors such
as sensorimotor speed, articulation speed or verbal fluency. Other statistical comparisons: *Comparison of NW1 and NW2 for English subjects; mean differ-
ence, 2.4 ms; paired t35 = 0.5; n.s. +Comparison of NW1 and NW2 for Italian subjects; mean difference, 48.6 ms; paired t35 = 9.6; p < 0.0001.
**Comparison of IW1 and IW2 for English subjects; mean difference, 2.9 ms; paired t35 = 0.4; n.s. ++Comparison of IW1 and IW2 for Italian subjects; mean
difference, 28.0 ms; paired t35 = 4.8; p < 0.0001.
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Table 2. Functional commonalities and differences between English and Italian reading systems and meta-analysis of
previous neuroimaging findings during naming/semantic tasks and phonological tasks.
Brain region Left hemisphere Right hemisphere
x y z z score x y z z score
(a) Main effect of reading
Inferior frontal gyrus (BA 44) 44 4 20 4.6
Inferior frontal gyrus / insula 38 22 12 4.4
Precentral gyrus (BA 6/4) 48 8 32 4
Insula 22 2 16 4.4 32 26 6 3.4
Temporo/parietal junction (BA 22/40) 38 36 22 3.6 70 40 24 3.5
Superior temporal gyrus (BA 22) 64 44 12 5.3 54 24 6 5.2
Middle temporal gyrus (BA 21) 60 50 8 5
Inferior temporal gyrus (BA 20) 50 58 22 5.6
Fusiform gyrus (BA 37) 40 52 24 7.4
Caudate nucleus 18 10 22 3.5 26 6 20 3.4
2000 Nature America Inc. http://neurosci.nature.com
Thalamus 8 30 0 5.2 24 12 14 4
Cerebellum 22 68 26 5
(c) Greater activations for non-words in English readers as compared with Italian readers
Anterior inferior frontal gyrus (BA 45) 46 18 20 2.7
Inferior temporal gyrus (BA 21/37) 58 58 14 2.9
(d) Greater activations for words and non-words in Italian readers as compared with English readers
Superior temporal gyrus (BA 22/42) 48 34 16 2.6
(e) Other tasks activating left basal temporal region for whole-word processing
Naming33 37 46 20
Semantics; words and pictures13 46 46 20
Conjunction word and object naming34 44 62 16
Stress assignment+ 48 58 20
(f) Other tasks activating left anterior inferior frontal gyrus for whole-word processing
Semantic verbal fluency16 36 24 16
Meta-analysis of semantic tasks15* 37 27 14
(g) Other tasks activating left perisylvian temporal region: orthographic translation and sub-lexical phonological processing
Wordspictures35 42 40 20
Wordspictures36 58 46 28
Nonwordword32 50 36 32
Phonological short-term memory22 44 32 24
Localization is based on stereotactic coordinates. These coordinates refer to the location of maximal activation indicated by the highest z score in a particular
anatomical structure. Distances are relative to the intercommissural (ACPC) line in the horizontal (x), antero-posterior (y) and vertical (z) directions. Z
scores indicate the magnitude of the statistical significance. *Average stereotactic coordinates derived by a published meta-analysis (semantic decision)15.
+Stereotactic coordinate of activation associated with stress-assignment task for visually presented trisyllabic Italian words (E.P. et al., unpublished results).
articles
The bigram frequency of the stimuli was analyzed in terms of the number permitting a mixed-effects analysis appropriate for population infer-
of occurrences of a given bigram in a corpus of the 7500 most frequent ence30. The analysis was based on a 2 (English, Italian subjects) 2
words in each language (DeMauro Vocabulario di Base28; CELEX English (implicit, explicit reading) 3 (words, non-words, baseline) factorial
database, http://www.kun.nl/celex). The absolute bigram frequency of the design. We first calculated the main effect of the activation patterns asso-
stimuli across languages was different, as expected by their different ortho- ciated with reading as the conjunction of the four main effects of reading
graphic and phonological structure (English words versus non-words, (reading minus baseline) in each of the four groups (statistical thresh-
266.2 versus 246.4; Italian words versus non-words, 455.2 versus 403.8). old, p < 0.001 corrected for spatial extent)31. We then calculated the main
Within each set of stimuli, there was a nonsignificant trend for a lower effect of non-word minus word reading and vice versa, and the differ-
bigram frequency of the non-words, and there was no significant interac- ences between groups as group task interaction effects. All interaction
tion with language. In a separate task, subjects read 12 familiar interna-
effects were computed on the voxels identified by the linear contrast of the
tional words with the same meaning in both languages: tennis, boiler,
basket, corner, partner, bitter, coma, taxi, panda, bravo, villa and pasta. relevant main effects. For these latter, more subtle comparisons, a thresh-
Only the last six conform to Italian orthography. old of p < 0.01 was adopted32.
We used several control tasks. Simple vocal-reaction time was mea-
sured by asking subjects to say go as quickly as possible every time a ACKNOWLEDGEMENTS
small dot appeared on a computer screen at random intervals. Articula-
The studies were funded by the EEC-BIOMED II grant (contract BMH4-CT96-
tion speed was measured by asking subjects to repeat aloud as quickly as
0274) and by the Gatsby Charitable Foundation. We are grateful to Andrew Holmes
possible, for 15 seconds, pairs of words common to both vocabularies
(gorilla/banana; tennis/polo). Two tasks measured ease of word retrieval. for statistical advice and Caroline Moore for help with preparing the bibliography.
In the verbal-fluency tasks, subjects generated in one minute as many
words as possible starting with the letter m (letter fluency) or belong-
2000 Nature America Inc. http://neurosci.nature.com
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