Histological analysis of laterodorsal tegmental nucleus in the rat brain

Travis Bico
Department of Biological Sciences

(Submitted in partial fulfillment of the requirements

for the degree of Bachelor of Science Honours)

Brock University
St. Catharines, Ontario
April 2013

Abstract:
The laterodorsal tegmental nucleus (LDT), containing Ch6 group of cholinergic neurons,
resides in the upper caudal region of the brainstem. This nucleus is important in various roles
such as, sleep-wake cycle, emotional arousal, alarm calls and alertness. By using stained brain
sections of 15 rats with thionine, neurons of the LDT became observable under light microscopy.
Neurons were observed from the caudal to rostral end with reference to a rat brain atlas and
neuron characteristics were recorded both numerically and schematically. It was discovered that
neurons tended to cluster in the center of the nucleus area and more dorsally in the broader,
medial region of the LDT. Furthermore, larger neurons were found to be located more in the
rostral and medial regions of the nucleus compared to the caudal end, which may be an important
feature for the ascending cholinergic pathway. This study provides fundamental information on
characteristics of the LDT to further strengthen the understanding of the LDT in future studies.

Acknowledgements:
Thank you to Dr. Stefan Brudzynski for his entrustment and guidance in this thesis project as
supervisor, to Dr. Joffre Mercier for his advice and time as a committee member and to Dr.
Cheryl McCormick for the use of her imaging microscope.

Table of Contents
1. Introduction pg. 4
2. Literature Review pg. 5
Connections to and from the LDT pg. 5
Types of neurons found in LDT pg. 7
LDTs response to neurotransmitters/neuromodulators pg. 9
LDTs role in sleep/wakefulness pg. 11
LDT in rat vocalization pg. 13
3. Methods and Materials pg. 15
Rat brain sectioning pg. 15
Nissl staining of brain sections pg. 16
Light microscopy of stained sections pg. 16
Brain section imaging & Statistical analyses pg. 17
4. Results pg. 19
Neuronal characteristics of the LDT pg. 19
Neuronal differences in the LDT between caudal, medial and rostral regions pg. 23
5. Discussion pg. 25
Ascending projection of the LDT pg. 25
Darkly stained neurons pg. 27
Frequent pyramidal neuron morphology pg. 27
6. Conclusion pg. 28
7. Bibliography pg. 29
8. Appendix pg. 32

List of Tables
Table 1: Raw data recordings pg. 32
List of Illustrations
Figure 1: Microscope images of brain sections showcasing used methodology pg. 18
Figure 2: Superimposed shading of neuron density for entire LDT nucleus pg. 19
Figure 3: Histogram of cell shapes encountered throughout the LDT pg. 20
Figure 4: Histogram of neuron density recorded throughout the LDT pg. 20
Figure 5: Correlation graphs for the number of neurons counted in each stereotaxic plane pg.
22
Figure 6: Division of superimposed shading of neuron density into rostral, medial and caudal
regions pg. 24
Figure 7: Bar graph of mean number of large neurons in rostral medial and caudal regions pg.
25

Abbreviations
LDT laterodorsal tegmental nucleus
WGA wheat germ agglutinin
PHA-L Phaseolus vulgaris leucoagglutinin
NTS nucleus of the tractus solitaries
NADPH-d nicotine adenine dinucleotide phosphate diaphorase
Hcrt/Orx hypocretin/orexin
REM rapid eye movement
NREM non-rapid eye movement
DRN dorsal raphe nucleus
LC locus coeruleus
GLM general linear model
PPN pedunculopontine nucleus

Introduction:
The neuronal connections of the mammalian brain allow for a complex array of responses
to environment and unexpected stimuli. Many regions of the brain are organized in fashions that
allow excitation or inhibition of vast areas of the brain. One such region is the laterodorsal
tegmental nucleus (LDT), which is located in the upper caudal region of the brainstem (Honda &
Semba, 1995). Its connections are mainly ascending, activating broad regions of the brain. The
LDT has a large concentration of cholinergic neurons, which release acetylcholine as a
transmitter, intermingled with non-cholinergic neurons which influence the thalamus,
hypothalamus, basal forebrain, pontine reticular formation, septum, basal ganglia and medial
frontal, prefrontal and olfactory cortices (Cornwall, Cooper, & Phillipson, 1990; Luebke,
McCarley, & Greene, 1991; Brudzynski, Kadishevitz, & Fu, 1998; Burlet, Tyler, & Leonard,
2002). LDT neuronal activity can be regulated by the habenula through thalamic relay, as well as
the ventrolateral preoptic nucleus, which contains GABA synthesizing neurons, and the dorsal
raphe, which contain large amounts of serotonergic neurons (Cornwall, Cooper, & Phillipson,
1990; Horner et al., 1997; Saper, Chou, & Scammell, 2001). The LTD nucleus has been shown
to play key roles in wakefulness, REM-sleep, behavioural and defensive state, alertness and
arousal with studies mostly conducted on rats, mice and cats (Mitani, et al., 1988; Brudzynski,
Kadishevitz, & Fu, 1998; Brudzynski, Iku, & Harness, 2011).
Extensive studies have been conducted on the connections of the LDT using
immunohistochemistry and retrograde and anterograde tracing methods to understand where
neurons project (Mesulam et al., 1983; Kayama & Ogawa, 1987; Cornwall, Cooper, &
Phillipson, 1990). Additionally, studies are conducted by injecting selected pharmacological
substances, whether they are neurotransmitters or pharmacological agonists or antagonists, into

the LDT to activate its neuronal pathways (Luebke, McCarley, & Greene, 1991; Horner et al.,
1997; Portas et al., 1997; Brudzynski, Kadishevitz, & Fu, 1998). These studies contribute to
explaining the role of the LDT in the brain. However, a summary of these findings with
reference to the location of neurons involved directly in these pathways would be beneficial to
further understanding the function of the LDT and its connections.
Literature Review:
Connections to and from LDT:
To understand the functionality of the LDT, it is necessary to review our knowledge of
where the neurons of this nucleus connect to and how this knowledge was obtained. In a
comprehensive study by Cornwall, Cooper & Phillipson (1990), the efferent and afferent
connections were explored in rats using retrograde and anterograde tracers. For retrograde
tracing, wheat germ agglutinin (WGA) was microinjected iontophorectically into sites of
interest. Anterograde tracing uses the same method, but instead of WGA, Phaseolus vulgaris
leucoagglutinin (PHA-L) was utilized.
Connections made to the LDT and found through retrograde tracing were divided into
five main groups based on the region or regions from which the input orginated. These regions
are the cortex, basal forebrain/hypothalamus, habenula and interpeduncular nucleus, midbrain
and lower brainstem. Large inputs were found to the LDT from the orbital cortex, while minor
inputs derive from the infralimbic cortex. Inputs from the basal forebrain/hypothalamic inputs
were found to be from the diagonal band, substantia innominata, medial preoptic area and lateral
hypothalamus. As for the habenula and interpeduncular nucleus (IPN), the connections from the
habenula to the LDT were localized in the lateral habenula, suggesting that it plays a role in

motor and limbic activity. Like the habenula, the inputs of the IPN were localized in the
rostrolateral and dorsolateral subnuclei. The midbrain displayed a high number of connections
from the substania nigra, ventral tegmental area, medial terminal nucleus and medial pretectum,
which further strengthens the LDTs role in the visual-motor system. Further connections from
the midbrain were found from the supraoculomotor nucleus, medial longitudinal fasciculus, and
Edinger-Westpaphl and Darkschewitsch nuclei. Lastly, connections from the lower brainstem
included the paramedial portions of the pontine reticular formation and parabrachial nucleus, and
the nucleus of the tractus solitarius (NTS) (Cornwall, Cooper, & Phillipson, 1990). Interestingly,
researchers suggest that the NTS-LDT pathway may be adrenergic as adrenaline synthesizing
neurons have been found in this connection.
Outputs from the LDT found through anterograde tracing were divided into six groups:
the cortex, septal region and basal forebrain, thalamus, epithalamus, hypothalamus and
midbrain/pons/medulla (Cornwall, Cooper, & Phillipson, 1990). Outputs were mainly to the
medial bank of the prefrontal cortex with minor projections to the hippocampal cortex from the
rostral end of the LDT. Abundant projections in the septal region were attributed to the lateral
septal nuclei and posterior septum, suggesting relationships to the hippocampus. In the basal
forebrain, innervation was found in the horizontal limb of the diagonal band, and magnocellular
and medial preoptic area. To the thalamus, projections were heavy in the ventral midline
thalamic group as well to the anterior and mediodorsal nuclei, which the researchers suggested
play a role in relaying information to the cingulate cortex. The epithalamus had innervation from
the LDT via the lateral habenular nucleus. A large portion of the LDT output was found to be to
the hypothalamus, specifically to the lateral hypothalamus and lateral mammillary nucleus. In the
midbrain, the main output of the LDT was to the medial substantia nigra pars compacta and

interpeduncular nucleus. Finally, projections from the LDT to the pons and medulla were
described roughly as sites known to have visual function and more specific connections to the
superior colliculus (Cornwall, Cooper, & Phillipson, 1990). These findings demonstrate the
complex array of connections from and to the LDT, as well as its connection to the visual system
and its ability to affect distant regions such as the cortex.
Types of neurons found in LDT:
Although it was known at the time of the study by Cornwall et al. (1990) that neurons in
the LDT were cholinergic, this was not the main focus of their study. An early study of the
classification of LDT neurons was conducted by Mesulam et al. (1983). This study was
conducted on 23 rats using choline acetyltransferase (ChAT) immunohistochemistry to identify
the cholinergic neurons. In their study they classified large sectors of cells throughout the brain
that were found to be cholinergic. The authors have developed their own nomenclature for
differentiating between cholinergic cell groups. Cell groups were denoted as Ch1-Ch6, where
Ch6 referred exclusively to cholinergic cells found in the LDT.
Further studies conducted on the LDT discovered the makeup of the non-cholinergic
neurons that reside in the LDT. Clements & Grant (1990) examined the possibility of glutamate
synthesizing neurons in the LDT, since glutamate is an abundant neurotransmitter. In their study,
8 rats were tested using glutamate immunohistochemistry, as well as nicotine adenine
dinucleotide phosphate diaphorase (NADPH-d) histochemistry to identify cholinergic neurons.
They discovered that glutamatergic neurons exist within the LDT and postulated that noncholinergic projections from the LDT may also contribute to the previously described functions
of the nucleus.

Ford et al. (1995) decided to explore the possibility of GABA-synthesizing neurons being
present in the LDT. Their tests were conducted on rats, using immunohistochemistry to detect
glutamic acid decarboxylase (GAD), an essential enzyme for GABA synthesis. Also, ChAT
immunochemistry was utilized to compare their findings for GABAergic neurons with
cholinergic neurons. They observed that GABAergic neurons were intermingled throughout the
LDT and outnumbered the cholinergic neurons by a factor of two. The researchers hypothesized,
because of the connections of the GABAergic neurons to surrounding areas that are both heavily
and not heavily innervated by cholinergic neurons, that the GABAergic neurons may work in a
relay system to modulate cholinergic activity or play an antagonist role in cholinergic pathways.
Up until 2009, it was not entirely known where the distribution of neurons in the LDT
was situated. Also, at this time it was thought that cholinergic cells might be co-releasing
acetylcholine with GABA and/or glutamate. In a study by Wang & Morales (2009), using in situ
hybridization, the composition of neurons within the LDT was found to be comprised of
cholinergic, glutamateric and GABAergic neurons. It was also discovered that the majority of
neurons lacked the capability of co-releasing acetylcholine, GABA and/or glutamate. In their
study they used in situ hybridization on seven rats to detect ChAT, glutamic acid decarboxylase
(GAD), or glutamate vesicle transporters (vGluT1, vGluT2 and vGluT3). As expected, ChAT
expression was seen, as well as GAD. Interestingly, vGluT1 and vGluT3 expression was not
seen in the LDT; however, vGluT2 was observed. To test the hypothesis of co-release they
utilized a double in situ hybridization method and found that with ChAT expression, vGluT2 or
GAD expression was not found in 95% of cases, suggesting that the hypothesis of co-release of
neurotransmitter as a major factor is not likely. After this finding the researchers looked at the
subpopulations of cholinergic, GABAergic and glutamatergic neurons to find their distribution

10

throughout the LDT. They divided their preparations into three sections, medial, caudal and
rostral. In the caudal portion of the LDT, GABAergic neurons were the most concentrated at
58% of all neurons in this portion, while cholingergic and glutamatergic were 14% and 28%,
respectively. In the medial region, GABAergic neurons were most dense again at 36% of all
neurons, while cholinergic and glutamatergic were 32% each. Lastly, in the rostral region
glutamatergic neurons were most densely populated at 54% of all neurons, with cholinergic
neurons being 19% and GABAergic 27%. In conclusion, the LDT is comprised of three
subpopulations of neurons unevenly distributed throughout the nucleus, with cholinergic cells
being the least populous and GABAergic cells being the most populous. Now that the
connections of the LDT and the neurons that compose the nucleus are better understood, the
LDTs role in the brain can be explored.
LDTs response to neurotransmitters/neuromodulators:
In order to understand how LDT neurons act physiologically, their response to
neurotransmitters/neuromodulators from various inputs had to be studied. The most common
way to achieve this is by injecting the substance of interest into the LDT and observing the
response. Since it is known the dorsal ralpe nucleus contain a large population of serotonergic
neurons that innervate the LDT, the effect of serotonin was explored by Horner et al. (1997). In
their study they used ten freely moving adult rats and monitored their responses to
microinjections of serotonin. Since it is known that the LDT plays a role in rapid eye movement
(REM) sleep, they looked for changes in REM sleep time by monitoring changes in the
electroencephalogram, electromyogram and ponto-geniculo-occipital wave activity as well as
visual cues. They discovered that serotonin can inhibit the LDTs activity, as episodes of REM
sleep, which are normally stimulated by LDT activity, were significantly reduced upon injection.

11

This was found to be dose-dependent, as the smaller injections, 1 mM versus 1.5 mM serotonin,
did not produce a significant decrease in REM sleep time compared to the control in the first 20
minutes after injection. However, after this period there was a significant decrease, suggesting
that serotonin does not act to abolish REM sleep, but its ability to inhibit the LDT activity acts
over time altering the maintenance of the REM sleep state.
In contrast, Portas et al. (1997) observed that perfusion of 300 M adenosine into the
LDT increased the duration REM sleep state. This study was performed on 12 adult cats, and
adenosine was microdialysed into the LDT region after the first waking of the sleep cycle. They
found a 52% decrease in waking and a 46% increase in time spent in REM sleep, suggesting that
adenosine reinforces the sleep state when given to the LDT. How this occurs was not explained;
however, in a later review it is explained that the increased levels of adenosine may be a result of
its accumulation over time from mainly in the basal forebrain and cortex (Strecker et al. 2000).
As these levels increase during wakefulness, they begin to promote the sleep state and eventually
REM sleep as adenosine acts on the LDT.
Additional responses in the LDT were tested by Luebke et al. (1991), who explored the
possibility of acetylcholine acting in a feedback loop since the concentration of cholingergic
cells within and around the LDT are high. They dissected out neurons in the LDT from male and
female rat pups and separated cholinergic from noncholinergic neurons using NADPHdiaphorase. To test the neurons response to acetylcholine, they used the acetylcholine analogue,
carbachol, and the muscarinic agonist, methacholine, to specifically look at the LDTs response
to muscarinic acetylcholine receptor activation. Furthermore, they demonstrated that non-M1
receptors were responsible for cholinergic neuron inhibition by using pirenzepine, an M-1
antagonist. In their results they found that activation of the non-M1 receptors increased the

12

potassium conductance of the neurons, promoting a hyperpolarized and less active state. This
suggests that acetylcholine from the LDT or surrounding cholinergic cells can act in a negative
feedback loop to inhibit their activity.
A more recent study by Burlet et al. (2002), explored the effect of hypocretin/orexin
(Hcrt/Orx) peptides on LDT brain slices of mice, as these peptides have been linked with
narcolepsy and REM sleep. To monitor the effect of Hcrt/Orx on LDT neurons they recorded
excitatory postsynaptic currents (EPSC) using whole cell recordings and observed a significant
increase in amplitude after 1 M of Hcrt/Oxn was introduced into the bathing solution. They also
observed an increase in neuron firing in loose-patch extracellular recordings with the addiction
of 300 nM into the extracellular solution. Since Hcrt/Orx peptide secreting neurons project from
the hypothalamus to the LDT, it is believed these could excite specific regions of the LDT to
modulate LDT output (Burlet et al., 2002).
LDTs role in sleep/wakefulness:
The role of the LDT in REM sleep was examined by Webster & Jones (1988) in a lesion
study of the LDT area using kainic acid. Their study was performed on fifteen cats which were
injected bilaterally with 1.2 L of kainic acid then monitored for a month before being
euthanized to observe the brain for the loss of cholinergic neurons. It was found that 25-85% of
the cholinergic cells were destroyed using this method. They also discovered in the first three
weeks that REM sleep was nearly abolished. Increased episodes of wakefulness were also
observed. These results reinforce the importance of the LDT in REM sleep.
In REM sleep and during wakefulness, neurons of the LDT display similar rates of
impulse firing, while decreased rates of firing are observed in non-REM sleep (NREM) (Saper,

13

Chou, & Scammell, 2001). The difference in states is attributed to the interaction of the LDT
with other regions that can inhibit or enhance the activity of the LDT. In a review by McCarley
et al. (1995) a possible schematic for the LDTs role in sleep is presented. The cycle begins in
NREM sleep where the dorsal raphe nucleus (DRN), releasing serotonin, and locus coeruleus
(LC), releasing norepinephrine, gradually lose their ability to inhibit the LDT. Eventually, the
inhibition of LDT neurons is lifted, allowing them to excite the reticular formation which
initiates REM sleep state. This state is reinforced by a positive feedback loop from the pontine
reticular formation which connects back to the LDT that they suggest releases an excitatory
amino acid to further excite LDT neurons. At this time it was not fully understood how the DRN
and LC lose their ability to inhibit the LDT. Two possibilities were proposed, the first being that
the DRN and LC slowly inhibit themselves as they release their inhibitory neurotransmitters
acting both on their own receptors as well as the LDT. The second possibility proposed was that
GABA from the reticular formation may be inhibiting DRN and LC, preventing them from
continuing to inhibit the LDT. An additional question remained regarding the termination of the
REM sleep state. The authors hypothesized that the cholinergic connections of the LDT to the
DRN and LC may gradually excite the DRN and LC enough to inhibit the LDT. As a result, the
LDT projections to the reticular formation can become inhibited and the REM sleep state cannot
be maintained (McCarley et al., 1995).
In a more recent review, Saper et al. (2001) suggest that the ventrolateral preoptic
nucleus (VLPO) plays a pivotal role in transitioning between sleep and wakefulness. They also
add that the DRN, LC and tubermammillary nucleus (TMN) are most active in wakefulness,
slow down in NREM sleep and then are almost completely inactive in REM sleep. These
connections are important as they activate the thalamocortical system to stimulate the whole

14

cerebral hemisphere. As previously stated, the alleviation of inhibition in the LDT was not
known, and it was hypothesized that GABA from the reticular formation prevented the DRN and
LC from inhibiting the LDT. The scheme in this article differs from that of McCarley et al.
(1995), as the VLPO is responsible for releasing GABA and galanin into the DRN, LC and TMN
to inactivate them, allowing the LDT to activate the reticular formation and generate REM sleep.
As for the LDTs activity in wakefulness, the projections to the thalamus and
hypothalamus play a large role in exciting or activating their neurons; however, they are not
essential (Mansari et al., 1989). In the lesion experiments previously mentioned by Webster &
Jones (1988), there were no significant changes in the cats behaviour while awake that the
researchers observed. This was followed by another lesion study by Inglis & Semba (1997), who
attempted to lesion cholinergic LDT neurons specifically, since they have the most extensive
connections to outside regions. Their study was conducted on 55 male rats using AMPA,
NMDA, ibotenic acid and quinolinic acid injections to lesion the LDT unilaterally. Injections of
ibotenic acid caused the largest lesion in the LDT, 80-90% cholinergic cell loss, which was
estimated by identifying cholinergic cells after the lesion with NADPH-diaphorase. These rats
also experienced the greatest behavioral change, rotating away from the side of the lesion. Upon
injections with a higher concentration of ibotenic acid, Cheyne-Stokes breathing patterns were
observed followed by death. These results show that the LDT does not contribute greatly to
stimulating the wakeful state, but regulates behaviours during wakefulness.
LDT in rat vocalization:
Extensive work has been compiled on the vocalization of rats in response to LDT
stimulation. Early work by Brudzynski & Barnabi (1996) distinguished the LDT as the principle

15

inducer of 22 kHz calls, which correspond to defensive or alarm calls in order to alert other rats
to possible threats. This study took 30 rats, implanted them with cannula in the LDT and
hypothalamic preoptic region. Rat vocalizations were then recorded immediately after various
combinations of injections to the LDT and/or hypothalamic preoptic region. In these injections,
the LDT was either injected with L-glutamate or saline, or was left uninjected. It was discovered
that in the medial caudal region of the LDT, vocalizations induced by L-glutamate were nearly
identical to vocalizations induced by carbachol in the hypothalamic preoptic region. Injection of
scopolamine, a muscarinic antagonist, into the hypothalamic preoptic area prior to L-glutamate
injection into the LDT showed a significant decrease in response to L-glutamate. In conjunction
with retrograde studies linking the LDT to the hypothalamus, the LDT exerts a major influence
on 22 kHz calls in rats (Jones et al., 1986; Cornwall et al., 1990). It was later discovered by
Brudzynski et al. (1998) that the cholinergic output to the hypothamic preoptic region is
predominantly inhibitory.
Further research by Brudzynski et al. (2011) analyzed the activity of the LDT cholinergic
neurons in rats when 22 kHz calls were evoked with an air puff. At this time, LDT projections
eliciting this response were refined to the basal forebrain, diagonal band, septum and
hypothalamus, collectively termed the medial cholinoceptive vocalization strip. To study
changes in cholinergic activity during stimulation to air puffs, c-fos was employed. Also,
injections of carbachol in the medial cholinoceptive vocalization strip were used. Since some rats
have been shown not to respond to air puffs, rats were first divided into four groups based on
their responses: nave, air puffed but not vocalizing, air puffed and short-vocalizing, and air
puffed and long vocalizing. It was discovered that longer vocalizing rats showed significantly
higher c-fos expression in some cholinergic neurons compared to the other groups, while non-

16

vocalizing rats showed little to no increase in cholinergic activity. Since vocalizing rats did show
an increase, it is clear that LDT activation from stressful stimuli can act on the medial
cholinoceptive vocalization strip to produce alarm calls.
Given this information, much of the literature surrounding the LDT focusses on using
methods to identify neurons of interest and connections without creating a picture of how the
neurons are distributed throughout the whole LDT nucleus. The aim of this study is to observe
the neurons of the LDT using light microscopy to map how the densities of neurons change from
the rostral to the caudal end of the nucleus. This will be completed by summarizing findings
from 15 rat brains which were sectioned using a freezing microtome and stained with thionine. It
is likely that many large neurons will be found throughout the entire nucleus. Finally, the vast
outputs of the LDT may include different types of neurons cell bodies with to support axon
projections to multiple areas. Thus, this project will attempt to study the distribution of different
neuronal shapes and sizes within the LDT.
Methods and Materials:
Rat brain sectioning:
Fifteen adult Long-Evens rat brains preserved in 4% formalin were used to produce
transverse brains sections within the LDT region. Sectioning took place on a BFS Series freezing
microtome from Hacker instruments, using Tissue-Tek O.C.T. compound to glue the rat brains to
the stage. A 3 ampere current was run continually to allow brains to freeze using the Peltier
effect. Before the brains were completely frozen, sectioning took place using a radial microtome
producing 40-50 m transverse sections through 2 mm of tissue around the LDT. Brain sections
were then transferred to gelatinized microscope slides and allowed to dry overnight.

17

Nissl staining of brain sections:

After brain sections dried overnight, sections were stained with thionine using a modified
Nissl staining technique. Microscope slides containing brain sections were delipidized by being
placed in 95% then 70% ethanol for 1 minute each before being placed in distilled water for 2
minutes. Slides were then set in a 0.5% thionine solution for 1 minute before being briefly
washed with distilled water. In order to dehydrate the preparations, they were then placed in 70%
ethanol for 2 minutes, then two more washes in 95% and absolute ethanol for 2 minutes each,
before slides were placed in a 1:1 solution of ethanol/isopropanol. Finally, slides were placed in
xylene for 5 minutes. Before the xylene evaporated from the slides, Permount medium was
dipped onto the slide to allow for covering with glass coverslips.
Light microscopy of stained sections
Using a standard light microscope, sections were observed at 40x magnification to
determine the approximate stereotaxic plane and area of the LDT using a rat brain atlas by
Paxinos & Watson (2009) as a guide. How this was determined can be seen in Fig. 1A. Under
40x magnification, neurons were counted, unilaterally in a 100 m by 100 m square at the
highest density region for that area. Neurons were classified and counted as large neurons (>20
m), medium-sized neurons (8-19 m), and darkly stained neurons, which were simply neurons
within the counting square that were more darkly stained relative to the surrounding neurons in
the section. Neurons less than 8 m were too numerous and only included when counting darkly
stained neurons. The dominant neuron shape was recorded at 100x magnification throughout the
stereotaxic plane. Shapes were recorded as spherical, bipolar, pyramidal and multipolar or any
combination of the four if two shapes appeared to be dominant. Examples of neuron shapes can

18

be seen in Fig. 1B. The density of neurons within the nucleus was recorded as low, medium or
high. Quantitative density was not recorded due to the fact that neurons were observed in clusters
which an overall density number would fail to express. In order to record the neuron cluster
locations, regions within the LDT that were more densely packed with neurons were recorded
schematically by shading throughout entire LDT sections. Shaded LDT sections were then
digitalized and superimposed using Microsoft Paint.
Brain section imaging
Images of stained brain sections were taken with a Nikon 80i Eclipse imaging
microscope to demonstrate the appearance of the LDT nucleus under the microscope. Act- 1
software was utilized to digitalize microscopy images. The images act as representations of how
the cell shapes and the outline of the nucleus area was determined.
Statistical analyses
SPSS was used to perform statistical tests at an of P < 0.05 level of significance. Linear
regressions and post-hoc Tukey tests were utilized for data analysis. General linear model
(GLM) test was also used for comparisons since data were not normally distributed (p < 0.0001
for all data sets).

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3.
3.
1.

2.
4.

2.

1.
Spherical

3.
Bipolar

Pyramidal

4.
Multipolar

Figure 1: A) -8.88 mm (bregma) brain section image of stained neurons at 40x magnification.
Large red outline indicates the area of the LDT nucleus, while the red square is an example of a
counting square area used to count neurons. B) -9.00 mm (bregma) brain section image of
stained neurons at 100x magnification. Numbers correspond to each cell shape. Red outlines
display different cell shapes used to determine dominant cell shape of sections.

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Results:
Neuronal Characteristics of the LDT
After superimposing shading of the LDT from raw observations for the entire LDT
nucleus, between 15 rats, a composite figure was constructed (Fig. 2). Areas of shading indicate
area in which the density of neurons was larger compared to the rest of the nucleus in that
section. Since the area, in which neuron counting occurred, was in the densest region of the
observed section, the square area used for neuron counting was also included in the shading. The
plane -9.48 mm from bregma, due to its size, has the darkest shading throughout the entire area
because the counting square covered its entire area. The darker regions appeared to be in the
center of the nucleus, indicating that more neurons are likely to be located in the center of the
nucleus as opposed to the periphery. In sections -8.52 mm, -8.64 mm, -8.88 mm, and -9.00 mm
from bregma, neurons appeared to be more densely clustered in the dorsal half of the nucleus.
There was an exception, -8.76 mm from bregma, in which the neuron density was large
throughout the center of the nucleus dorsal-ventrally. Sections -8.40 mm, -9.00 mm and -9.12
mm from bregma appeared to have dense clusters of neurons in the ventral perimeter of the
nucleus. Cell shapes appeared to favour pyramidal cell types in most sections, with bipolar cells
being a distant second and multipolar being the least common shape (Fig. 3). Medium neuron
density was most common in sections followed by high, and low neuron density was the least
frequent (Fig. 4).

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Rostral

-8.16mm (11)
-8.28mm (12)

Density of neurons
10

-8.40mm (13)

9
8

Caudal

7
6

-8.52mm (10)

5
4
3
2

-8.64mm (12)

-8.76mm (12)

-8.88mm (11)

-9.00mm (12)

-9.12mm (13)

-9.24mm (10)
Dorsal

-9.36mm (13)
Lateral

Medial

-9.48mm (10)
Ventral

Figure 2: Shaded areas of dense groups of neurons collected from 15 rats, superimposed with
increasing shades of grey to represent the number of overlaps between rats in the stereotaxic
plane. Stereotaxic coordinates given in bregma. Number of rats per section in brackets.

Frequency

22

80
70
60
50
40
30
20
10
0
Spherical

Bipolar

Pyramidal

Multipolar

Spherical &
Bipolar

Spherical &
Pyramidal

Bipolar &
Pyramidal

Cell Shape

Figure 3: Histogram of dominant cell shapes recorded throughout entire LDT nucleus among
each section. (n = 139). Last three categories on the right for cell shape indicate regions in which
two cell shapes were both dominant.
120

Frequency

100
80
60
40
20
0
Low

Medium

High

Neuron Density

Figure 4: Histogram of neuron density from each section of the entire LDT nucleus. (n = 139).
Linear regressions were performed to compare the stereotaxic plane to the numbers of
large, medium and darkly stained neurons, as well as cell density and cell shape (n = 139; Fig. 5).
Both medium neurons and darkly stained neurons were found to have no relation to the
stereotaxic planes, suggesting that their distribution is uniform throughout the nucleus (R =
0.078, p = 0.364; R = 0.029, p = 0.737, respectively). Since darkly stained cells were observed to

23

be medium cells largely in this study, the possibility of a significant relation was explored and
found, suggesting that darkly stained neurons are likely to be medium-sized neurons (R = 0.47, r2
= 0.215, p < 0.0001) (Fig. 5).

35

25
20

15
10
5
0

-10

-9.5

-9

-8.5

Number of Darkly Stained

Neurons

Number of Medium Sized

Neurons

30

30
25
20
15
10
5

-8

0
-10

Stereotaxic Plane (mm bregma)

-9.5
-9
-8.5
Stereotaxic Plane (mm bregma)

-8

C
Number of Darkly Stained Neurons

35

30
25
20
15

10
5
0
0

10

15

20

25

30

Number of Medium Sized Neurons

Figure 5: A and B) Number of medium sized neurons and darkly stained neurons throughout
LDT sections (n = 139 for each) C) Number of medium sized neurons correlated with the
number of darkly stained neurons from all 15 rats displaying a positive relationship. (n = 278).

24

Cell shapes were also compared to the stereotaxic plane, and no relation was found (R =
0.040, p = 0.640). A weak significant relation between large neurons and stereotaxic plane was
observed, suggesting that the number of large neurons may differ depending on the stereotaxic
plane (R = 0.21, r2 = 0.047, p = 0.011). A GLM test showed that there was a significant
difference between large neurons and stereotaxic planes (p = 0.005). Which stereotaxic planes
display different numbers of large neurons was found using a post-hoc Tukey test, finding a
significant difference only between -9.48 mm and -8.88 mm (bregma) planes (p = 0.039).
Finally, neuron densities between stereotaxic planes were found to not have any significant
relation (R = 0.144, p = 0.091).
Neuronal differences in the LDT between caudal, medial and rostral regions
To investigate difference in neuronal characteristics the LDT more thoroughly, the LDT
was divided into three regions, rostral (n = 46), medial (n = 47) and caudal (n= 46) (Fig. 6).
Linear regressions were utilized again to identify any similarities and differences between
regions. Once again, the number of medium and darkly stained neurons was found to have no
significant relationship between regions, suggesting that they are found in similar numbers
throughout each region (R = 0.061, p = 0.477; R = 0.037, p = 0.665, respectively). Both cell
shape and density were also found to have no significant relationship between the three regions,
suggesting that densities and cell shapes are similar between each region (R= 0.036, p = 0.676; R
= 0.133, p = 0.118, respectively). However, neuron density was found to be significantly higher
in the medial region compared to the caudal and rostral regions from a GLM and post-hoc Tukey
test (p < 0.0001; p = 0.026, respectively). Large neurons were found to have a higher density in
the medial region when compared in this manner (R = 0.238, r2 = 0.057, p = 0.005). To examine
if there is a difference in large neurons between the regions a GLM test was used and a

25

significant difference was found (p < 0.0001). Using a post-hoc Tukey test there were significant
differences in the number of large neurons between the regions was found to be between the
rostral and caudal and the medial and caudal regions (p = 0.009; p < 0.0001, respectively),
whereas no significant difference was found between rostral and medial regions (p = 0.211).
These results suggest the rostral and medial regions have more large neurons than the caudal
region since the mean number of neurons is also larger in rostral and medial regions compared to
the caudal region (Fig. 7).
Medial

Rostral
-8.16mm (11)

Caudal

-8.64mm (12)

Density of Neurons
10

-9.12mm (13)

-8.28mm (12)

9
8

-9.24mm (10)
-8.40mm (13)

7
6

-8.76mm (12)

-9.36mm (13)

-8.52mm (10)

5
4

-9.48mm (10)

3
2
1

-8.88mm (11)

-9.00mm (12)

Figure 6: Shaded areas of dense groups of neurons collected from 15 rats, superimposed with
increasing shades of grey to represent overlap between separate rats. The data are the source of

26

Fig. 2, but sections divided into rostral, medial and caudal regions. Stereotaxic coordinates given
from bregma. Number of rats per section in brackets.
Mean Number of Large Neurons

5
4
4
3
3
2
2
1
1
0
Rostral

Medial

Caudal

Region of LDT nucleus

Figure 7: Mean number of large neurons observed within each region of the LDT, corresponding
to the rostral, medial and caudal sections illustrated in Fig. 6. SE = 0.206, n = 139.
Discussion:
Using Nissl staining on rat brain sections, neuronal characteristics of the LDT were
studied. The main finding of this study is that neurons of the LDT are largely situated in the
center of the nucleus. In the broadest cross-sections of the LDT, neurons were observed more
densely in the dorsal half. Large neurons were found to have an unequal distribution throughout
the entirety of the nucleus, being more common in the rostral and medial regions as opposed to
the caudal region. Of four possible neuron shapes, pyramidal shaped cells were the most
frequent. Low and high densities of neurons were found to be less frequent than medium density
throughout the LDT.
Ascending projections of the LDT
The importance of ascending projections from the LDT has been well documented (Tago
et al., 1989; Oakman et al., 1995; Brudzynski, Kadishevitz, & Fu, 1998). These projections can

27

innervate vast regions of the brain, contributing to diverse behavioural responses. In this study,
the areas of higher neuron density appeared mostly in the dorsal or medial areas of the nucleus.
Furthermore, larger neurons were more frequent in the medial and rostral regions of the nucleus
which may be a structural characteristic of the LDT nucleus to provide greater cholinergic input
into the ascending cholinergic pathway. It may be assumed that the large neurons are cholinergic
neurons sending their axons over large distances (even as far as the frontal cortex). Their axons
are very long, hence, the cell bodies supporting these axons should also be larger in size. The
large neurons cannot contribute to descending pathways because Woolf & Butcher (1989) have
not found descending pathways projecting from the LDT.
In a pharmacological-behavioural study, Brudzynski & Barnabi (1996) reported that the
strongest activation of the ascending cholinergic pathway to the anterior hypothalamic-preoptic
region was from the caudal region of the LDT. Since there is a moderate distance from the LDT
to the hypothalamic-preoptic region, one may conclude that the cholinergic neurons providing
innervations to this structure are rather medium in size. The occurrence of small dense areas of
neurons in the ventral portion of sections -8.40 mm, -8.88 mm, -9.00 mm and -9.12 mm from
bregma, may act to relay information to the more neuron-rich dorsal half of the LDT since
descending pathways are not present in the LDT. Since the locus coeruleus projects to the LDT,
the ventral area of the LDT may relay information from the locus coeruleus to the rest of the
LDT (Webster & Jones, 1988).
Brudzynski et al. (1998) recorded inhibitory responses in the anterior hypothalamicmedial preoptic region after electrical stimulation of the LDT nucleus. The release of
acetylcholine in the hypothalamic-preoptic region is important for emotional responses and
thermoregulation (Boulant, 2000; Brudzynski, 2001). There are many studies reporting

28

innervation from the LDT nucleus by reterograde and anterograde tracing studies (Cornwall,
Cooper, & Phillipson, 1990; Omelchenko & Sesack, 2005).
Darkly stained neurons
Nissl staining technique is useful for visualizing neurons and their physiological state.
Darkly stained neurons by Nissl methods are regarded as healthy neurons. Inflammation or
neuronal damage would cause disintegration of Nissl substance and light staining. In this study,
darkly stained neurons were found to be uniformly distributed throughout the nucleus,
confirming that the neurons of the studied brains were free of pathologies. The darkly stained
neurons were closely related to medium size neurons, and many of them could also be
cholinergic in nature. As shown by Spann & Grofova (1992) in the pedunculopontine nucleus,
another heavily cholinergic nucleus, cholinergic and non-cholinergic cells can both vary
considerably in size. Furthermore, a study by Mesulem et al. (1989) indicated that cholinergic
and non-cholinergic neurons may not be distinguishable based on their shape and size.
Therefore, the darkly stained cells in this study may represent both populations of neurons, if the
LDT shows similar characteristics to the pedunculopontine nucleus (PPN). Additional studies
would have to be conducted to explore if larger neurons are mainly cholinergic, or if neuron size
and function are related.
Frequent pyramidal shape neuron morphology
Although the morphology of neurons in the LDT has not been thoroughly studied in
terms of cell shape, this study found that pyramidal cell bodies dominated compared to any other
shape. Possible electrophysiological functions may be attributed to the cell shape. Cell size and
shape was observed to have some distinguishable electrophysiological characteristics by

29

Takakisaki et al. (1997) from two different types of neurons identified in the PPN. Since the PPN
is also largely cholinergic, similar characteristics may be observed in the LDT. Cell shape and
neuron function need to be studied in more detail. As shown by Wang & Morales (2009),
glutamatergic, cholinergic and GABAergic neurons exist in the LDT. By comparing
electrophysiological characteristics of these neurons with physical characteristics, as cell shape, a
better functional understanding can be gained. Current technology is helping to make this
possible as well, with the creation of imaging software such as SynD, allowing for rapid
identification of neuron shapes and characteristics, such as synaptic locations (Schmitz et al.,
2011).
Conclusion:
The occurrence of dense areas of neurons in the LDT favoured a central location
throughout the entirety of the nucleus after data from 15 rats were superimposed on each other.
No relationship between medium sized neurons or darkly stained neurons was found within the
regions of the LDT; however, large neurons were found to be located more in the medial and
rostral regions of the nucleus. Interestingly, pyramidal shaped neurons were the most frequent
shape, although the functional significance of this is not known. In conclusion, the LDT nucleus
continues to be an area of interest due to its widespread effect on various behavioral and
physiological changes within mammalian species.

30

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33

Appendix
Table 1: Characteristics of LDT nucleus from raw observations of 15 rat brains.
Rat
Number

Stereotaxi
c Plane
(Bregma)

Number
of
Medium
-sized
Neurons

Number
of
Largesized
Neurons

Number
of
Darkly
Stained
Neurons

Neuron
Density

Cell Shape

1
1
1
1
1
1
1
1
2

-9.36
-9.24
-9.12
-9
-8.88
-8.64
-8.28
-8.16
-9.48

19
21
12
16
9
8
14
10
6

0
0
0
0
0
0
0
3
0

16
25
17
19
5
18
30
20
10

medium
medium
medium
medium
low
high
medium
medium
low

2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3

-9.36
-9.24
-9.12
-9
-8.88
-8.76
-8.64
-8.52
-8.4
-8.28
-8.16
-9.48
-9.36
-9.24
-9.12
-9
-8.88
-8.64

12
8
10
10
9
11
17
17
12
12
16
18
27
15
10
14
15
7

0
2
0
1
2
1
1
0
2
1
1
3
0
2
3
6
3
0

21
20
14
17
13
20
11
17
21
15
18
21
32
22
18
21
18
18

medium
medium
high
high
medium
high
medium
medium
high
low
medium
medium
high
medium
medium
high
medium
medium

3
3
3

-8.4
-8.28
-8.16

14
11
13

1
2
3

17 high
15 medium
16 medium

spherical+bipolar
spherical+bipolar
bipolar
bipolar
bipolar
bipolar
spherical+bipolar
bipolar
spherical+pyramid
al
bipolar
bipolar+pyramidal
spherical
spherical
pyramidal
bipolar
bipolar
spherical+bipolar
pyramidal
spherical
spherical
bipolar
bipolar
pyramidal
spherical
bipolar
pyramidal
spherical+pyramid
al
bipolar
spherical
spherical

LDT
Region

caudal
caudal
caudal
medial
medial
medial
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
medial
medial
rostral
rostral
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
medial
rostral
rostral
rostral

34

4
4
4
4
4
4
5
5
5
5
5
5
5
5

-9.48
-9.36
-8.88
-8.64
-8.4
-8.16
-9.48
-9.36
-9.24
-9.12
-9
-8.88
-8.76
-8.64

12
9
8
7
7
6
4
8
5
7
11
6
10
5

2
4
7
13
6
4
0
2
1
5
8
7
4
5

16
15
18
23
15
11
7
12
16
14
9
14
9
15

medium
high
high
medium
medium
medium
medium
medium
medium
medium
medium
medium
high
medium

5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8

-8.52
-8.4
-8.28
-8.16
-9.48
-9.36
-9.24
-9.12
-9
-8.88
-8.76
-8.64
-8.52
-8.4
-8.28
-8.16
-9.48
-9.36
-9.24
-9.12
-9
-8.88
-8.76
-8.64
-8.52
-8.4
-9.36

11
8
4
6
5
5
10
18
10
6
6
11
11
7
8
8
4
11
7
14
8
8
9
13
8
12
17

5
4
2
3
0
2
1
1
5
9
9
4
4
5
2
0
0
0
2
2
7
3
2
3
3
0
0

15
11
6
10
8
6
10
12
14
12
10
11
3
9
4
7
9
15
10
15
8
3
9
9
10
12
5

medium
medium
medium
medium
medium
low
medium
medium
medium
medium
medium
medium
medium
high
low
low
low
medium
medium
medium
medium
medium
medium
high
medium
medium
low

bipolar
bipolar+pyramidal
bipolar
pyramidal
bipolar+pyramidal
pyramidal
spherical
spherical
spherical
bipolar
pyramidal
pyramidal
bipolar+pyramidal
spherical+pyramid
al
pyramidal
spherical
pyramidal
pyramidal
pyramidal
bipolar+pyramidal
pyramidal
spherical
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
bipolar
pyramidal
bipolar
bipolar+pyramidal
pyramidal
pyramidal
bipolar+pyramidal
bipolar
pyramidal

caudal
caudal
medial
medial
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
medial
medial
rostral
rostral
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
medial
medial
rostral
rostral
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
medial
medial
rostral
rostral
caudal

35

8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
10
10
10
10

-9.24
-9.12
-9
-8.76
-8.64
-8.52
-8.4
-8.28
-8.16
-9.48
-9.36
-9.24
-9.12
-8.76
-8.64
-8.4
-8.28
-8.16
-9.48
-9.36
-9.24
-9.12

13
14
10
5
9
8
8
13
8
9
6
10
7
8
3
14
6
7
14
6
5
5

0
1
0
4
5
3
6
5
3
0
1
1
0
2
7
1
0
0
1
1
4
3

11
15
6
10
15
14
11
8
11
8
5
8
11
10
10
10
14
7
12
12
8
7

medium
medium
medium
high
medium
medium
medium
medium
medium
low
low
medium
medium
medium
medium
medium
medium
medium
medium
medium
medium
medium

10
10
10
10
10
10
10
10
11
11
11
11
11

-9
-8.88
-8.76
-8.64
-8.52
-8.4
-8.28
-8.16
-9.48
-9.36
-9.12
-9
-8.88

9
10
13
11
13
11
7
7
10
9
7
11
11

6
6
1
1
0
3
1
3
1
0
2
1
2

7
9
6
10
10
11
7
4
11
14
11
13
12

medium
medium
medium
medium
medium
medium
medium
medium
low
medium
medium
medium
medium

11
11
11
11
11

-8.76
-8.64
-8.52
-8.4
-8.28

7
14
15
12
15

4
6
2
4
1

9
11
14
12
16

high
high
medium
medium
high

pyramidal
bipolar+pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
multipolar
pyramidal
spherical
bipolar
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
bipolar
spherical+pyramid
al
bipolar+pyramidal
pyramidal
spherical+bipolar
pyramidal
pyramidal
pyramidal
pyramidal
bipolar+pyramidal
bipolar+pyramidal
spherical
bipolar
pyramidal
spherical+pyramid
al
bipolar+pyramidal
pyramidal
bipolar
pyramidal
pyramidal

caudal
caudal
medial
medial
medial
rostral
rostral
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
rostral
rostral
rostral
caudal
caudal
caudal
caudal
medial
medial
medial
medial
rostral
rostral
rostral
rostral
caudal
caudal
caudal
medial
medial
medial
medial
rostral
rostral
rostral

36

11
12
12
12
12
12
12
13
13
13
13
13
13
13
14

-8.16
-9.12
-9
-8.76
-8.64
-8.4
-8.28
-9.48
-9.36
-9.12
-9
-8.88
-8.76
-8.52
-9

14
13
14
13
11
13
7
9
7
10
10
11
10
9
14

0
3
1
2
5
5
10
1
8
3
6
1
2
3
1

15
7
14
10
11
11
11
5
14
7
9
8
14
13
17

medium
medium
high
high
high
medium
medium
medium
medium
medium
high
medium
high
high
high

14

-8.88

11

18 high

14

-8.76

8 high

14
14
14
14
15
15
15

-8.52
-8.4
-8.28
-8.16
-9.36
-9.24
-9.12

6
6
11
8
12
8
13

7
5
2
1
0
1
0

11
14
12
8
8
8
12

15

-8.76

11

10 high

15
15
15

-8.52
-8.4
-8.28

8
7
7

8
2
7

16 medium
6 high
11 medium

medium
high
high
medium
medium
high
medium

pyramidal
bipolar+pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
bipolar
pyramidal
bipolar+pyramidal
spherical+pyramid
al
spherical+pyramid
al
spherical+pyramid
al
spherical
pyramidal
pyramidal
pyramidal
pyramidal
pyramidal
spherical+pyramid
al
spherical+pyramid
al
spherical
pyramidal
spherical+pyramid
al

rostral
caudal
medial
medial
medial
rostral
rostral
caudal
caudal
caudal
medial
medial
medial
rostral
medial
medial
medial
rostral
rostral
rostral
rostral
caudal
caudal
caudal
medial
rostral
rostral
rostral