Ephrin B2 based hierarchy determines midbrain dopaminergic projection targeting

Abstract
The mesocortical dopamine projections are involved in several important behaviors, like
motor function and reward, as well as pathologies, including Parkinsons disease and
Schizophrenia. The Ephrin-B1/EphB2 ligand-receptor pair has been implicated as one of the
molecular mechanisms that give rise to the segregation of neurons from two of these pathways,
the nigrostriatal and the mesolimbic pathways. This experiment further substantiates the role of
Ephrin-B1/EphB2 in the determination of those two pathways by verifying that segregation and
expression of the ligand receptor pair occur contemporaneously as well as demonstrating
necessity of the system. The main goal of this experiment is to demonstrate that the same
mechanisms involved in the differentiation of the mesolimbic and nigrostriatal projections lead
to the differentiation of the third important pathway, the mesocortical projection.
Importance of Midbrain Dopaminergic Projections
Dopamine is one of major neurotransmitters in the brain, and dopaminergic neuron
groups have been implicated in several higher cognitive abilities and behaviors. While there is
dopamine-based signaling in many different areas of the brain, including the olfactory system
and in vision, the largest and most interesting group of dopaminergic fibers emanates from the
ventral mesencephalon. Three afferent projections originate from one of two adjacent subregions
of the ventral mesencephalon: either the substantia nigra (SN) or the ventral temgental area
(VTA)1. Each pathway subserves a different set of functions and is consequentially involved in

distinct neuropathologies. The molecular mechanisms of the differentiation of these pathways

are currently under investigation and will be the primary focus of this paper.
The nigrostriatal system runs from the substantia nigra to the basal ganglia terminating
specifically in the caudate putamen (CP). This pathway is central for the control of voluntary
movement is implicated in several movement disorders. Patients afflicted with Parkinsons
disease exhibit extensive cell death within this pathway, which gives rise to characteristic
movement symptoms2. Patients show limited spontaneous movement as well as a tremor while
resting. The elucidation of pathway determinants may give rise to cell replacement therapies in
the treatment of Parkinsons disease.
The mesolimbic system projects from
the VTA to the nucleus accumbens (NA) of the
limbic system. Dopaminergic neurons in the
mesolimbic system have been implicated in
schizophrenia, obsessive-compulsive disorder,
and substance abuse3. This pathway is also
thought to be a key component in the reward
system whereby certain behaviors are
reinforced through dopamine release by these

Figure 1: Diagram of the dopaminergic

projection in the embryonic mouse brain. VTA,
Ventral Tegmental Area; SN, Substantia Nigra;
MFB, Medial Forbrain Bundle; St, Striatum
(Caudate Putamen); NA, Nucleus Accumbins;
PFC, prefrontal cortex. Image from reference 1.

neurons. This pathway is intimately related to the mesocortical pathway.

Similar to the mesolimbic system, the mesocortical pathway originates in the VTA but it
terminates in cerebral cortex, predominantly the fifth layer of the medial prefrontal cortex
(mPFC)3. The mPFC is involved in several higher cognitive functions such as working memory
and goal maintenance; consequentially, the mesocortical pathway is central in models of

attentional deficit and hyperactivity disorder4. Current theories advanced by Miller & Cohen have
suggested that the mesocortical projection to the mPFC facilitates reward driven updating or
shifting of goal representations5.
An inhibitory connection from the prefrontal cortex to the VTA and limbic system forms
a circuit including both the mesolimbic and mesocortical projections. Schizophrenia is thought
to result from abnormalities in this larger circuit, with vegetative symptoms arising due to
reduced activity in the mesocortical circuit and hallucinations due to hyperactivity of the
mesolimbic circuit6. Based on this model, it would be important to determine the molecules that
guide the development of the mesocortical projection, both to find candidate genes for
susceptibility to schizophrenia as well as potential future interventions for treatment of refractory
cases of schizophrenia.
Pathway Development
The cellular development and axon pathfinding of these pathways is an active field of
research. The neurons from all of these fibers follow the same rostral path from the midbrain to
higher cortical areas. As can be seen in Figure 1 all three projections are a part of the medial
forebrain bundle (MFB) as they leave the VTA and SN, and they do not diverge until they
approach their target areas. A study by Nakamura et al. demonstrated that local cues, as opposed
to secreted chemicals, guide the initial pathfinding of these neurons7 toward the cortex, NA and
CP. A separate study located the factor that causes rostral growth to type II astrocytes 8. While the
specific guidance molecules that lead to the rostral growth impetus of dopaminergic axons have
not been identified, other common guidance molecules have been implicated in different stages
of dopaminergic axon growth.

Ephrin is a membrane bound ligand that has been shown to play an important role in the
specification of neuron targeting in the central nervous system. It interacts with the Eph
Receptor in a contact dependant manner. Studies by Feldheim and colleagues indicated that these
molecules result in repulsion of cells expressing the Eph receptor9. Subsequent work has
demonstrated not only that different members of the Ephrin/Eph families can function as contact
dependant attractants, but that the same protein may behave as either an attractant or a repellent
in different cells. In vitro studies have established that Ephrin has an inhibitory effect on neurite
growth in dopaminergic neurons. This work has been elaborated with work in the midbrain
pathways specifically.
In 1999, Yue and colleagues provided evidence that the Ephrin/Eph system specifies the
differentiation of the nigrostriatal system from the mesolimbic system10. Using in situ
hybridization for the mRNA of various Eph receptors they found high levels of expression of
EphB1 in neurons originating from the SN, while nerves from the VTA had low levels of the
EphB1 receptor. The researchers then tested for Ephrin-B2 ligand expression in the target areas
of SN and VTA neurons again using in situ hybridization. High levels of Ephrin-B2 mRNA were
detected in the NA and significantly lower levels were observed in both the medial CP and the
lateral CP. Researchers detected these differences in expression from embryonic day eighteen,
their earliest data point, through adulthood. Because SN neurons are known to project to the CP
and not the NA the researchers concluded that the Ephrin-B2/EphB1 system functions as a
repellant of dopaminergic cells; however, because they did not manipulate Ephrin-B2 or EphB1
in their study they did not demonstrate sufficiency or necessity.
Building on Yues work, Zhaoliang Hu and colleagues conducted a study to determine
how discrete innervations of the CP and NA arise11. They used different retrograde tracers

applied to CP or NA to assay the segregation of the nigrostriatal and mesolimbic pathways. They
repeated their procedure several times during mouse development. During early stages of
embryonic development distinct retroactive tracers placed in CP and NA co-labeled cells in both
the SN and VTA. Beginning with embryonic day seventeen they saw emerging specificity; some
cells only expressed one of the labels while others continued to express both. By birth, the
characteristic segregation of the nigrostriatal and mesolimbic pathways was observed, with cells
in the SN expressing only the label placed in the CP and VTA neurons expressing the other
tracer. This evidence can be interpreted to say that neurons initially project to both the NA and
CP, but a contact dependant signal like Ephrin/Eph leads to collateral pruning beginning around
embryonic day seventeen. A problem emerges from the juxtaposition of this study with the Yue
study: according to Hu, pruning and specificity generation occurs prior to the earliest point that
Yue tested for the differential expression of Ephrin B2 and EphB1.
In addition to bolstering the case for Ephrin B2 and EphB1 in the establishment of the
mesocortical and nigrostriatal pathways I hope to address mechanisms by which the mesocortical
projection is separated from the either two. This proposed study tests the hypothesis that the
same Ephrin B2/EphB1 system that leads to differentiation of the nigrostriatal system gives rise
to the segregation of the mesolimbic and mesocortical projections by pruning inappropriate
collaterals in the mPFC from cells that also project to the NA.
Experimental Proposal
While many psychological studies emphasize the mesocortical projection, most
molecular and cellular studies of the midbrain dopamine system to date have not distinguished
between the mesocortical projection and mesolimbic projection or have ignored the mesocortical
projection altogether. This may be due to the involvement of these two pathways in the same

cognitive abilities or their common anatomic origin. However, it is important to study the
differences between these pathways because they have opposing roles in a balanced circuit;
elucidating the mechanisms that give rise to those roles may aid in the treatment of diseases like
schizophrenia and substance abuse where that circuit is out of balance.
I propose that the three mesocortical dopamine pathways are all targeted using the
Ephrin-B1/EphB2 ligand-receptor system. High levels of Ephrin-B1 exclude mesolimbic and
nigrostriatal neurons from the mPFC and intermediate levels exclude nigrostriatal neurons from
the NA. In order to test this hypothesis several steps must be taken. First, the timing of
segregation of the mesolimbic and mesocortical pathways will be established. Second, levels of
expression of guidance molecules will be assessed. Finally, necessity and sufficiency of Ephrin
ligand for specificity will be demonstrated.
Specific Aim 1: Determine the timing of pathway segregation.
Determining the developmental time at which these pathways segregate is important for
two reasons. First, if all three pathways differentiate simultaneously it would suggest that they
share a common mechanism and provide evidence for the Ephrin B2/EphB1 system for the
specification of the mesocortical pathway. Second, it provides a time point for testing
differential expression of the Ephrin B2 ligand in the target regions, because the Ephrin
B2/EphB1 pairing cannot determine specificity if it is not expressed during the period of
segregation. This is important both as a confirmatory measure for the mechanism of separation
of the nigrostriatal pathway from the mesolimbic as well as essential if the same system will
specify the distinction of the mesocortical pathway.
The timing of mesolimbic/mesocortical pathway segregation will be determined in a
similar way that the Hu established the timing of the other two pathways. Two distinct

retrograde labels will be injected into the different target regions of the mesolimbic and
mesocortical pathways, the NA and the mPFC, respectively. The VTA will be subsequently
visualized and cells and axons in the VTA will be checked for whether they are co-labeled or
labeled with only one of the retrograde labels.
Regardless of whether or not the processes are pruned in parallel the timing of collateral
pruning from the mPFC will be used as the primary stage of testing for the second stage of the
experiment. Should the pathways differentiate at different times it does not rule out Ephrin
based signaling as the primary mechanism, but it may mean that expression of Ephrin is timed
differently in the mPFC, which would be explored further.
Specific Aim 2: Characterize which chemical cues guide mesocortical specificity.
The primary motivation for this test is to implicate the Ephrin-B2/EphB1 system in the
repulsion of collaterals from cells that also project to the NA. A secondary goal for this
experiment is to demonstrate that the pattern of Ephrin-B2/EphB1 expression observed by Yue et
al. is present at embryonic day seventeen when pathway segregation begins.
Immunohistochemistry, using a labeled antibody specific for Ephrin-B2, will be used to
quantify levels of Ephrin-B2 expression on cells within the CP, NA, and mPFC at the onset of
segregation as indicated by the first experiment. A hierarchy of Ephrin-B2 expression is
expected with CP having low levels, NA having intermediate levels, and mPFC showing the
highest levels. As an additional validation of the Yue study, in situ hybridization for the EphB1
receptor will be conducted on VTA and SN neurons, using an anti-sense control. This will be
relatively uninformative for the primary goal of separating the mesocortical projection from the
mesolimbic, but is necessary to validate the Yue results.

Using immunohistochemistry for this assay is an improvement from the Yue study, which
used in situ hybridization sensitive for mRNA not protein expression. Unfortunately, in situ
hybridization must be used for EphB1 detection because it will lead to labeling of the cell body
as opposed to the axons, which would be uninformative. If Ephrin-B2 expression in the mPFC is
the same as or lower than in the NA then a different molecule is likely responsible for pathway
differentiation and a microarray analysis of mRNA expression would help elucidate what it is. A
major shortcoming of both this experiment and the Yue et al. study is that it does not show
necessity or sufficiency. It merely demonstrates the presence of a ligand-receptor pair that has
been shown to deter axonal growth in different conditions.
Specific Aim 3: Establish necessity and sufficiency of Ephrin-B2 for target specificity
The hallmark of necessity studies is the mouse knockout; however, Ephrin-B2 has been
implicated in numerous processes throughout the body, and also plays a significant role in the
development of many neural structures. Consequently, a pure knockout of Ephrin-B2 would
likely lead to a severely deformed phenotype. To circumvent this problem I will utilize a
tetracycline-controlled transcriptional activation system12. This system, developed by Gossen
and Bujard, allows for temporal control of gene expression, in this case Ephrin-B2, through the
administration of a tetracycline antibiotic. In this system, the gene is expressed normally until
the drug is administered, at which point transcription is inhibited; however, transcription returns
to normal when the antibiotic is no longer present. For this part of my study I will generate a
transgenic mouse line with tetracycline-controlled transcription of Ephrin-B1. Mice will be
treated with antibiotic on embryonic day fifteen, before pathway segregation. The primary
manipulation in this experiment will be the duration of antibiotic administration. There will be
two experimental groups plus two control groups.

The first group will receive antibiotic until birth, eliminating Ephrin-B1 expression for
the entire course of specificity development, at which point pathway specificity would be
assayed using differential retrograde labeling of the mPFC and the NA. If neurons innervate
both regions at birth this would prove that Ephrin-B1 is necessary for specificity formation. If
not Ephrin-B1 must not be involved in pruning and the involvement of different molecules
would need to be explored using a microarray analysis.
The second group would have the antibiotic administered from embryonic day fifteen to
seventeen, a midpoint in specificity development. Half of the animals in this condition would be
tested for pathway specificity at embryonic day seventeen. The other half would be allowed to
develop until birth without the antibiotic, Ephrin-B1 expression will resume after embryonic day
seventeen, before connection specificity is assayed. To demonstrate that the reinstatement of
Ephrin-B1 can rescue the phenotype there would have to be no pathway specificity at day
seventeen and at least partial pathway specificity at birth. Pathway specificity at birth may not
be observed if the process of specificity generation is temporally sensitive.
Two controls groups will be used for this experiment, the first will consist of transgenic
mice that never receive antibiotics to rule out a disruptive function of the added gene construct.
The second will consist of wild type mice in two subgroups administered with antibiotics
matching each of the test groups. This will serve to control for the effect of embryonic
administration of antibiotics.
There are several shortcomings with this approach. The first is the difficulty of creating a
transgenic mouse expressing with the construct. Secondly, the assay depends on the success of
the earlier tests; however, this could also be conducted on the nigrostriatal and mesolimbic
systems to establish necessity in that context if the prior studies yield unexpected results.

Finally, this does not establish true sufficiency as the re-expression of Ephrin-B1 is in the same
environment as it is normally expressed and it may interact with other ligands beyond the EphB2
receptor to cause repulsion. Sufficiency may be better explored using ectopic or overexpression
of Ephrin-B1.
Conclusion
Little attention has been given to the characterization of the molecular mechanisms
involved in development of the mesocortical projection. This is surprising because this pathway
receives a lot of attention in psychology and has been implicated both in reward and in pervasive
mental disorders like schizophrenia and substance abuse. Because of evidence from earlier
studies, this experiment investigates the Ephrin-B1/EphB2 system as a major determinant of
mesocortical pathway specificity. Ephrin-B1 is established as a necessary component of
pathway separation by this study. A major shortcoming of the model proposed by this paper is
that viewing Ephrin-B1/EphB2 as the only repellant signal does not explain the mechanism that
drives neuron pruning from the CP and excludes mesocortical neurons from the NA. Local
chemical cues that guide axons rostrally could potentially serve this function, but more work is
need to specifically identify this chemical. In sum, progress has been made in the elucidation of
the molecular cues that determine the specificity of the midbrain dopamine pathways, but a lot of
work remains before this knowledge can be applied clinically.

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