Corticofugal projections from medial primary somatosensory cortex avoid EphA7-expressing neurons in striatum and thalamus

Accepted Manuscript Corticofugal projections from medial primary somatosensory cortex avoid EphA7-expressing neurons in striatum and thalamus Alexander X. Tai, Lawrence F. Kromer PII: DOI: Reference:

S0306-4522(14)00439-4 http://dx.doi.org/10.1016/j.neuroscience.2014.05.039 NSC 15441

To appear in:

Neuroscience

Accepted Date:

21 May 2014

Please cite this article as: A.X. Tai, L.F. Kromer, Corticofugal projections from medial primary somatosensory cortex avoid EphA7-expressing neurons in striatum and thalamus, Neuroscience (2014), doi: http://dx.doi.org/ 10.1016/j.neuroscience.2014.05.039

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Corticofugal projections from medial primary somatosensory cortex avoid EphA7expressing neurons in striatum and thalamus

Alexander X. Taia B.S. and Lawrence F. Kromera,b Ph.D. a

Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road

NW, Washington, DC 20007, USA. bInterdisciplinary Program in Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007, USA

Abbreviated title: EphA7 guides corticofugal projections Key words: matrix, matrisome, anatomy, ephrin, corticostriatal, corticothalamic

Correspondence to:

Lawrence F. Kromer, Ph.D. Department of Neuroscience, Georgetown University Medical Center Rm EG11, The Research Bldg., 3970 Reservoir Road, NW Washington, DC 20057, USA Tel: 202-687-1827 Fax: 202-687-0617 Email: [email protected]

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Abbreviations Biotinylated dextran amine Cytochrome oxidase Diaminobenzidine External capsule Medial posterior complex of the thalamus Medium spiny neurons Postnatal day Primary somatosensory cortex Ventrobasal complex of the thalamus

BDA CO DAB ec POm MSNs P S1 VB

Highlights Corticostriatal projections from the medial somatosensory cortex avoid EphA7 expressing neurons in the striatum Corticothalamic projections from the somatosensory cortex avoid EphA7 expressing neurons in the thalamus EphA7-ephrinA interactions mediate corticofugal terminal topography from medial S1 cortex

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Abstract Within the first two postnatal weeks, corticostriatal axons from the primary somatosensory cortex (S1) form topographic projections that organize into characteristic bands of axon terminals in the dorsolateral striatum. Molecules regulating the development of these topographically organized projections are currently unknown.

Thus the present study

investigated whether EphA receptor tyrosine kinases, which regulate axonal guidance in the visual system via axon repulsion, could participate in the formation of corticostriatal connections during development. Prior studies indicate that EphA7 expressing striatal neurons are organized into banded compartments resembling the matrisome innervation pattern formed by cortical afferents from S1 cortex and that ephrin-A5, a known EphA7 ligand, is expressed in a medial (high) to lateral (low) gradient in S1. Thus, we hypothesized that the organization of EphA7 expressing striatal neurons into banded domains provides a repulsive barrier preventing corticostriatal axons containing EphA7-ligands from innervating inappropriate regions of the striatum. To evaluate this, we injected the anterograde tracer, BDA, into two locations in medial areas of S1 (the anterior and posterior whisker fields), which are reported to express high levels of ephrin-A5 during development. Injections were made in mouse pups on postnatal day 9 (P9) and animals were processed for immunohistochemistry on P12. Our data demonstrate that projections from both the forelimb/anterior whisker field and the posterior whisker field avoid EphA7-expressing neurons and terminate in a banded pattern in regions with very low EphA7expression. We also determined that corticothalamic projections from medial S1 also exhibit a restricted distribution in the thalamus and avoid neurons expressing EphA7. Thus, our results support the hypothesis that the anatomical organization of striatal and thalamic neurons

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expressing EphA7 receptors restricts the topographic distribution of cortical afferents from medial regions of S1 which express high levels of ephrin-A5.

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1. Introduction As the primary input nucleus of the basal ganglia, the striatum plays a central role in regulating cognitive, sensory, motor and limbic modalities. Its primary function is to receive and process basal ganglia-directed activity (a major component of which is from the cortex) and relay it to basal ganglia output nuclei: the globus pallidus and substantia nigra. These nuclei then modulate various modalities directly through inhibitory projections to thalamic and brainstem nuclei (Bolam et al., 2000). Medium spiny neurons (MSNs), the output neurons of the striatum and the main targets for glutaminergic input from the cortex (Gerfen, 2004), are organized into distinct biochemical compartments: the island-like striosomes, which express µ-opioid receptors (Graybiel and Ragsdale, 1978, Herkenham and Pert, 1981), and the surrounding matrix, which expresses EphA4 and EphB1 (Martone et al., 1997, Janis et al., 1999, Richards et al., 2007, Tai et al., 2013). Data indicate that different populations of cortical neurons target the striosomes versus the matrix suggesting the striosomes and matrix possess different molecular signatures (Gerfen, 1984, 1989, Crittenden and Graybiel, 2011). Additional studies also indicate that the matrix compartment itself is comprised of several topographically organized subpopulations of neurons that receive unique cortical inputs (Alloway et al., 1998, Brown et al., 1998, Alloway et al., 1999, Hoffer and Alloway, 2001, Hoffer et al., 2005). Since striatal function is highly dependent upon the topographical organization of its neurons and afferents (Voorn et al., 2004), the formation of these complex and selective corticostriatal connections is crucial to proper striatal function. Corticostriatal projections arrive in the striatum shortly after birth but do not achieve their mature topographic organization until the end of the second postnatal week (Iniguez et al., 1990, Christensen et al., 1999, Jain et al., 2001, Sohur et al., 2014). Several molecules, such as netrin-

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1 and semaphorins, are reported to regulate the axonal outgrowth of corticofugal axons and their guidance within the internal capsule to their target brain regions in the ventral forebrain and thalamus (Grant et al, 2012). However, the molecules that regulate the topographic organization of corticostriatal terminal fields within the striatum have not been identified. One family of axon guidance molecules that could possible regulate this process are the Eph family of receptor tyrosine kinases and their membrane bound ligands, ephrins.

During development these

molecules guide axons and regulate the formation of topographic projections in multiple brain regions, primarily through repulsive interactions (Flanagan and Vanderhaeghen, 1998, O'Leary and Wilkinson, 1999, Pasquale, 2005, Boyd et al., 2014). These repulsive mechanisms can be mediated either via “forward” signaling through the EphA receptor or via “reverse” signaling through the corresponding ephrin (Holland et al., 1996, Feldheim et al., 1998, Chilton, 2006, Feldheim and O'Leary, 2010). Recent studies indicate that cortical afferents enter the striatum postnatally (Sohur et al., 2014), after EphA7 expressing matrisome neurons have organized into their characteristic banded distribution within the matrix compartment of the striatum (Janis et al., 1999, Tai et al., 2013). Interestingly, the banded domains of EphA7+ striatal neurons resemble the reported topographic distribution of corticostriatal axons from the primary somatosensory cortex (S1), which also terminate in banded domains within the dorsolateral striatum (Brown et al., 1998, Alloway et al., 1999, Hoffer and Alloway, 2001, Alloway et al., 2009). Molecular studies indicate that corticostriatal and corticothalamic neurons located in layers V and VI of the medial S1 barrel cortex express high levels of ephrin-A5 perinatally, and that these neurons are segregated from adjacent cortical areas containing EphA7-expressing neurons (Vanderhaeghen et al., 2000, Miller et al., 2006, Dye et al., 2011a, b). Ephrin-A5 exhibits a high binding affinity to EphA7 (Himanen et al., 2004) and this ligand-receptor pair

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was recently implicated in the guidance of corticothalamic projections to various thalamic nuclei via a repulsive mechanism (Torii and Levitt, 2005, Torii et al., 2013). Thus, we hypothesize that repulsive interactions between ephrin-As, such as ephrin-A5, on cortical axons and EphA7 receptors present on a subset of striatal neurons also may serve to guide the topographic distribution of corticostriatal axons. To evaluate this hypothesis, we injected the anterograde axonal tracer, biotinylated dextran amine (BDA) into medial regions of the S1 cortex where neurons in layer V and VI express high levels of ephrin-A5 perinatally (Vanderhaeghen et al., 2000, Miller et al., 2006, Dye et al., 2011a, b) and evaluated the organization of the labeled corticostriatal terminal arbors within the striatum. In addition, we also evaluated whether corticothalamic afferents from the medial S1 barrel cortex avoid regions of the ventrobasal (VB) and medial posterior complex (POm) of the thalamus which contain neurons expressing EphA7 receptors.

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2. Experimental Procedures 2.1 Animals All animal experiments strictly conformed to guidelines set forth by the National Institutes of Health (NIH) and the Georgetown University Institutional Animal Care and Use Committee.

Mice for these studies were on a mixed Swiss-Webster:C57BL-6:129:FVB/N

background and were bred at Georgetown University as part of a transgenic mouse colony. Founder mice for this colony were obtained from the MMRRC and Dr. David Feldheim (UC, Santa Cruz).

2.2 BDA injections Mice were postnatal day 9 (P9) at the time of surgery. Prior to surgery, each mouse pup was anesthetized with intraperitoneal injections of a cocktail containing ketamine (10 mg/ml), acepromazine (1 mg/ml) and xylazine (10 mg/ml) in sterile saline with dosing based on their weight (0.01 ml/1gm). After anesthesia induction, the skin over the cranium was cleaned with antiseptic scrub and resected, and the animal was placed in a mouse stereotaxic instrument. Approximately 50 nl of 10% BDA solution (Molecular Probes, D-7135, Eugene, OR) was injected into anterior or posterior regions of the right medial S1 barrel cortex with a glass pipette coupled to a Hamilton microsyringe (Reno, NV). Injection coordinates were based on the Atlas of the Developing Mouse Brain by Paxinos & Franklin (Paxinos and Franklin, 2001). Following tracer injections, the wound margin was closed with LiquiVet (Oasis Medical, Mettawa, IL). After recovery from anesthesia, the pups were returned to their mothers and survived for 3 days postinjection to allow transport of the injected BDA to axonal terminals before the animals were prepared for histology.

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2.3 Histochemistry For immunohistochemical staining procedures, P12 mouse pups were deeply anesthetized with a cocktail containing ketamine, acepromazine, and xylazine, perfused transcardially with 0.1M phosphate buffer (pH 7.2–7.4), followed with 4% paraformaldehyde in 0.1M phosphate buffer. After perfusion, the brain was removed, postfixed for 3 hours in 4% buffered paraformaldehyde (4°C), and then cryoprotected in 20% (w/v) sucrose in 0.1M phosphate buffer for 24–72 hours at 4°C. The brains were then frozen in Tissue Tek (Sakura Finetek, Torrance, CA), stored at -80°C until sections (20 µm) were cut on a cryostat and mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Mounted sections were stored at -20°C to -80°C until they were processed for histology. 2.3.1 EphA7 immunohistochemistry Antigen retrieval was used to obtain optimal detection of EphA7 receptor protein expression in tissue sections. For this procedure mounted brain sections were autoclaved for 2 minutes in 0.01M citrate buffer (pH 6.0) at 120°C to unmask antigenic sites (Wang et al., 2007). After allowing sections to cool, they were washed in 0.1M PBS (pH 7.4) and treated with 0.1% H2O2 in methanol to quench endogenous peroxidase activity. Sections were then preincubated in blocking solution containing 0.1M PBS, 10% donkey serum, and 0.2% Tween 20 for 1 hour at room temperature. Tissue sections from specimens injected with BDA were treated with Vector Avidin/Biotin blocking kit to prevent cross-reactivity of the injected BDA with subsequent steps requiring antibody detection of EphA7. EphA7 was detected by incubating tissue sections with a goat anti-EphA7 polyclonal primary antibody (1:200, R&D Systems, Cat. No. AF608, Lot no. CCG036031) in 0.1M PBS containing 10% blocking solution and 0.2% Tween 20 for 48 hours at 4°C. Subsequently, sections were rinsed in 0.1M PBS and incubated in biotinylated donkey anti-

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goat secondary antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.1M PBS containing 10% blocking solution and 0.2% Tween 20 for 2 hours at room temperature.

Localization of antibody binding on tissue sections was visualized with the

Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA) per the manufacturer’s instructions by incubating sections in a reaction solution containing 0.05% diaminobenzidine (DAB), 0.08% NiCl, and 0.01% H2O2 in 0.1M Tris HCl (pH 7.6) to produce a blue-black reaction product. Sections were then dehydrated in ethanol, cleared in Histoclear (National diagnostics, Atlanta, GA) and coverslipped with Permount.

2.3.2 BDA histochemistry Mounted sections were rinsed in 0.1M PBS (pH 7.4) and treated with 0.1% H2O2 in methanol to quench endogenous peroxidase activity. Antigen retrieval was then performed in 1% sodium borohydride in 0.1M PBS. Localization of BDA tracer on tissue sections was visualized with the Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA) per the manufacturer’s instructions by incubating sections in a reaction solution containing 0.05% DAB, 0.08% NiCl, and 0.01% H2O2 in 0.1M Tris HCl (pH 7.6) to produce a blue-black reaction product. Sections were then dehydrated in ethanol, cleared in Histoclear and coverslipped. 2.3.3 Cytochrome oxidase activity Mounted sections were rinsed in 0.1M PBS (pH 7.4) and treated with 0.1% H2O2 in 0.1M PBS to quench endogenous peroxidase activity. Sections were then pretreated in 10% sucrose in 0.1M PBS for 10 min at 37°C and subsequently reacted in cytochrome oxidase reaction solution (10% sucrose, 0.08% NiCl, 0.03% DAB, 0.002% cytochrome C and 0.002% catalase in 0.1M PBS) for 8-14 hrs in 37°C. Reacted sections were passed through increasingly dilute sucrose

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solutions (10%, 5%, 0% sucrose in 0.1M), dehydrated in ethanol, cleared in Histoclear and coverslipped. 2.4 Figure preparation Histological sections were evaluated and microscope images photographed using an Olympus BX51 microscope equipped with an Olympus DL-72 digital camera. Image quality (contrast, brightness) was optimized, figures were assembled and labeled, and overlays were generated using Adobe Photoshop (San Jose, CA).

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3. Results 3.1 Postnatal expression of EphA7 in the striatum and cortex Using antibodies directed against the EphA7 receptor, we documented the postnatal expression pattern of EphA7 in the developing mouse striatum (Fig. 1). By postnatal day 6 (P6), neurons expressing high levels of EphA7 are arranged in distinct banded compartments in the anterior and posterior striatum (arrows in Fig. 1A & D). These compartments are oriented along a diagonal dorsomedial to ventrolateral axis with a prominent band abutting the dorsolateral border of the striatum (arrowheads in Fig. 1A-F). These compartments closely resemble the morphology and anatomical organization of the EphA7-expressing matrisome compartments described in the postnatal rat striatum (Tai et al., 2013). The matrisome pattern of EphA7+ neurons remains static and visible well into the second postnatal week (Fig. 1B, C, E, & F). However, after the second postnatal week, expression of EphA7 receptors on striatal neurons declines rapidly to levels not detectable on tissue sections by our anti-EphA7 antibody. At P6 we also detected low levels of EphA7 immunostaining in S1 barrel cortex, but little detectable EphA7 staining was observed at P12. However, EphA7 was still clearly present on neurons located in specific layers of the cingulate cortex at this postnatal age (Fig. 2C & F).

3.2 Corticostriatal S1 projections avoid EphA7-expressing striatal compartments BDA tracer was injected into the medial S1 barrel cortex on P9 and the distribution of labeled axons analyzed on P12. At this age the overall topographic distribution of corticostriatal afferents has stabilized and the extent of their terminal axonal arborization is nearly mature (Iniguez et al., 1990, Jain et al., 2001). We chose this time point for data analysis since EphA7expression is still detectable by immunohistochemistry at P12, which permits co-localization of EphA7+ striatal neurons with the distribution of BDA-labeled corticofugal axons and terminals.

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BDA was injected at two sites in the somatosensory cortex: an anterior site within the medial S1 cortex corresponding to the forelimb and anterior whisker bed (Fig. 2A-C) and a posterior site at the posterior border of the medial S1 cortex corresponding to the posterior whisker bed (Fig. 2D-F). These sites were located the same distance from the midline, but were separated by four millimeters in the anterior-posterior axis. In all specimens, the BDA injections were restricted primarily to layer V and superficial layer VI of the cortex. In some specimens the injection site extended into deep layer VI but did not extend into the external capsule. The location of each injection was confirmed by comparing the sites of BDA staining to cytochrome oxidase (CO) histochemical staining on adjacent sections (Fig. 2A & D). Since CO is a wellknown marker for the barrels in layer IV of S1, this approach permitted us to correlate the location of the BDA injection site relative to the barrel cortex and map this to figures in a postnatal mouse brain atlas (Paxinos et al., 2007). This approach also allowed us to determine that our injections clearly labeled cortical layer V and extended into layer VI since our injections labeled areas deep to layer IV but superficial to the external capsule. Comparison of areas exhibiting EphA7 and CO staining indicated that P12 the whisker barrel fields exhibited low levels of EphA7 staining compared to the cingulate cortex (Fig. 2C & F). At P12, BDA-labeled afferents were readily visible entering into the ipsilateral internal capsule and extending into the striatum and thalamus. BDA-labeled interhemispheric cortical fibers also were visible entering into the corpus callosum. Corticostriatal afferents from the anterior injection site innervated ipsilateral anterior and posterior areas of the dorsolateral striatum (Fig. 3B, E & H). The terminal arborization of these afferents was organized into elongated bands in the anterior dorsolateral striatum (Fig. 3B), but form denser patches in the posterior striatum (Fig. 3E & H). When we compared adjacent striatal sections labeled for BDA

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(Fig. 3B, E & H) and EphA7 receptors (Fig. 3A, D & G), and created overlay images (Fig. 3C, F & I), it was possible to determine that the BDA-labeled terminal fields avoid areas with high levels of EphA7 expression and densely innervate areas of low EphA7 immunostaining. This pattern is apparent in both anterior and posterior striatal regions.

Examination of higher

magnification views of terminal axon arbors in the overlay images (Fig. 3C, F & I) confirmed that the BDA-labeled afferents consistently form a complementary pattern of terminal fields in striatal areas located between and immediately adjacent to regions with high levels of EphA7 staining. Evaluation of overlay images indicated that bundles of BDA-labeled cortical axons coursing into or through the striatum in the internal capsule did not appear to respect the boundaries of high EphA7 expression as stringently as their terminals and occasionally appeared to pass through areas of high EphA7 expression (white arrow in Fig. 3C). However, given the thickness of the tissue sections and the loss of depth of field in the overlay images, it is possible that the axonal bundles in the internal capsule were surrounded by the EphA7+ cellular areas, which could form boundaries restricting the spread of these fasciculated axon bundles. Corticostriatal afferents from the posterior injection site also innervated anterior and posterior areas of the dorsolateral striatum, though afferents from this site appear to innervate the posterior striatum to a greater extent (Fig. 3K, N & Q). The terminal arbors of these afferents were less expansive than their counterparts from the anterior site. This observation is likely a result of greater targeting of the posterior striatum by afferents from the posterior site and that corticostriatal axon terminals coalesce in the posterior striatum more than they do in the anterior striatum. These afferents clearly innervated areas with low EphA7 staining and avoided areas with high EphA7 expression. This complementary spatial relationship between BDA-labeled afferents and EphA7+ neurons was apparent at low (Fig. 3M-0) and high magnifications (Fig.

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3P-R). Bundles of labeled axons from posterior S1 also occasionally traveled through areas with high levels of EphA7 expression to reach their terminal fields similar to their anterior counterparts (white arrow in Fig. 3L). Together, these data indicate that the terminal arbors of corticostriatal afferents from medial regions of the anterior and posterior S1 barrel cortex occupy striatal regions containing low levels of EphA7 expression and avoid innervating striatal regions containing neurons expressing high levels of EphA7 receptor. 3.3 Corticothalamic S1 projections avoid EphA7-expressing regions in the thalamus At P6, several thalamic nuclei express high levels of the EphA7 receptor. In particular, the ventrobasal (VB) and medial posterior nuclear complex (POm) express high levels of EphA7 along their mutual border (arrows in Fig. 4A).

Within the VB, EphA7 is expressed in a

dorsolateral (high) to ventromedial (low) gradient with individual thalamic barreloids located in the dorsolateral region clearly labeled by EphA7 immunostaining (arrowheads in Fig. 4A). EphA7 expression levels decrease rapidly after the first postnatal week and it is difficult to identify EphA7+ barreloids by P9 (Fig. 4B). At both P9 and P12 there is little to no EphA7 immunostaining visible in the dorsolateral VB (arrowheads in Fig. 4B-C) and only sparse immunostaining along the VB/POm border (arrows in Fig. 4B-C). At P12, BDA-labeled corticothalamic afferents from the anterior and posterior medial S1 barrel cortex injection sites are clearly visible coursing through the internal capsule. After entering the thalamus these fibers form dense terminal fields within specific areas of VB and POm (Fig. 4E-F & I-J). Afferents from the anterior S1 injection site innervate ventromedial regions of VB and dorsomedial regions of POm (Fig.4E-F). BDA-labeled axons from the posterior S1 injection site innervate thalamic regions immediately lateral to their anterior counterparts in VB and POm (Fig. 4I-J).

Comparison of these terminal arbors to EphA7

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immunostaining in the VB and POm reveal that they avoid the VB/POm border where EphA7 staining (arrows) is still detected (Fig. 4G-H & K-L). Although we did not trace corticothalamic afferents at P6, BDA-labeled afferents observed at P12 clearly innervate regions in VB and POm that express low level of EphA7 expression at P6 (magenta shaded area in Fig. 4D). Thus, these results indicate that the terminal arbors of corticothalamic and corticostriatal afferents from medial S1 barrel cortex avoid thalamic and striatal regions expressing high levels of EphA7 during postnatal development.

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4. Discussion Several studies have described the mature topographic distribution of corticostriatal (Alloway et al., 1998, Brown et al., 1998, Alloway et al., 1999, Alloway et al., 2000, Hoffer and Alloway, 2001, Hoffer et al., 2005, Alloway et al., 2006, Alloway et al., 2009) and corticothalamic projections (Alloway et al., 2003), as well as the developmental time course for the arrival of cortical projections to the striatum and thalamus in mice and rats (Iniguez et al., 1990, Christensen et al., 1999, Jacobs et al., 2007, Grant et al., 2012, Torii et al., 2013, Sohur et al., 2014). However, the molecular mechanisms underlying the formation of these complex topographic connections remain unclear. In a previous study in rats, we used mRNA in situ hybridization

and

immunohistochemistry

to

characterize

a

unique

striatal

matrix

subcompartment containing neurons expressing the EphA7 receptor, which we termed the matrisome subcompartment (Tai et al., 2013), since it closely resembled the topographic “matrisome” distribution of S1 corticostriatal afferents in the dorsolateral striatum (Flaherty and Graybiel, 1991, Graybiel et al., 1991, Hoffer et al., 2005, Alloway et al., 2009). That study also demonstrated that EphA7+ striatal neurons bind ephrin-A5, a known high affinity ligand for EphA7 (Himanen et al., 2004). In the present study, we report that the organization of the EphA7+ matrisome compartment in mice is identical to the matrisome organization in rats. We also demonstrate that the distribution of corticostriatal terminals from medial areas of S1 cortex, a region that expresses high levels of ephrin-A5 perinatally, avoid the EphA7-expressing matrisome subcompartment and innervate adjacent regions containing little to no EphA7 expression. In addition, our data demonstrate that corticothalamic projections from areas of medial S1 known to express high levels of ephrin-A5 also avoid thalamic regions with high postnatal EphA7 expression.

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4.1 S1 barrel cortex projections To identify the topographic distribution of corticostriatal terminals from the forelimb and whisker regions of medial S1 cortex, we injected BDA into cortical layers V and VI. We observed that the distribution of the BDA-labeled cortical terminals in the ipsilateral striatum in P12 mice closely matches the topographic organization of corticostriatal terminal arbors from the forelimb and whisker regions of S1 that were described in anatomical tracing studies in adult rats (Alloway et al., 1999, Hoffer et al., 2005). Both species of rodents possess topographically organized, curved bands of corticostriatal terminal fields that roughly parallel the curvature of the external capsule when viewed in coronal sections.

The distribution of corticothalamic

afferents labeled by our injections also resembles those labeled by medial S1 cortex injections in adult rats (Alloway et al., 2003). Though the rodents in these studies differ in age and species, the similarity between our findings in postnatal mice and the distribution of corticostriatal and corticothalamic afferents from the forelimb/medial whisker barrel cortex in adult rats demonstrates the consistency in the topographic distribution of corticofugal projections across rodent species and indicates that the adult pattern of corticostriatal and corticothalamic projections is achieved by the end of the second postnatal week in mice.

4.2 Ephrin-A5 as a potential binding partner for EphA7 receptors Several studies have reported that there is a complementary expression pattern for ephrinA5 and EphA7 within the somatosensory cortex (Torii and Levitt, 2005, Miller et al., 2006, Dye et al., 2011a, b, Torii et al., 2013). For example, ephrin-A5 expression is highest in medial S1 and lowest in lateral regions; whereas EphA7 expression is highest in the lateral S1 and low medially. This complementary expression is most evident postnatally during the time when

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corticofugal axons are innervating their targets within the striatum and thalamus (Vanderhaeghen et al., 2000, Miller et al., 2006, Dye et al., 2011a, b). Along with the cortex, thalamic nuclei are also reported to express EphA7 and ephrin-A5 in complementary gradients (Torii and Levitt, 2005, Lehigh et al., 2013) and striatal regions may exhibit a similar relationship since ephrin-A5 is known to be expressed on select striatal neurons as well (Passante et al., 2008). However, this has yet to be tested. Several studies indicate that ephrin-A5 is a binding partner for EphA7 in the developing cortex (Miller et al., 2006), striatum (Janis et al., 1999, Tai et al., 2013) and thalamus (Lehigh et al., 2013) where it may play a role in axonal guidance, cell segregation, or both. Thus, ephrin-A5 appears to be a strong candidate to be the primary binding partner for EphA7 in the cortex, striatum, and thalamus.

Support for this is provided by studies indicating that

experimental manipulations that alter the levels of EphA7 receptors in the somatosensory cortex result in an abnormal topographic distribution of corticothalamic afferents to thalamic regions expressing ephrin-A5 (Torii and Levitt, 2005, Torii et al., 2013). These results, in conjunction with our data regarding the distribution of EphA7 receptors in the postnatal striatum and thalamus, indicate that EphA7 (and ephrin-A5) can be present either on postsynaptic striatal and thalamic neurons or presynaptic cortical axons.

4.3 EphA7 mediates the topographic organization of corticofugal projections from medial S1 cortex In the present study, we demonstrate that striatal and thalamic neurons express EphA7 protein in a spatially and temporally regulated manner that coincides with the postnatal period when the topographic organization of corticostriatal and corticothalamic terminal fields develops. During this postnatal period neurons located in cortical layers V and VI of the medial S1 cortex express high levels of ephrin-A5 and low-levels of EphA7 mRNA (Torii and Levitt,

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2005, Dye et al., 2011a, b). As cortical axons begin extending into the mouse striatum at P4 (Sohur et al., 2014), striatal neurons expressing EphA7 mRNA already are organized into their adult striosome distribution pattern (Dye et al., 2011a), which is consistent with the EphA7 immunohistochemical data in the present study (Fig. 1). Moreover, our data indicate that by P12 BDA-labeled cortical axons arising from neurons in medial S1 avoid the EphA7+ matrisome compartment (Fig. 3) and exhibit the characteristic mature banded innervation pattern present in the adult striatum. Our data also indicate that BDA-labeled corticothalamic axons from medial S1 cortex avoid regions in VB and POm that express EphA7 postnatally (Fig. 6). Although corticothalamic axons begin innervating the thalamus by P0 (Jacobs et al., 2007, Grant et al., 2012), there already is a topographically organized distribution of thalamic neurons expressing EphA7 mRNA in VB and POm (Dye et al., 2011a) that is similar to the distribution of thalamic neurons that we observed to be immunostained for EphA7 at P6.

Together, the above

observations support the hypothesis that EphA7 receptors on striatal and thalamic neurons likely function to mediate the topographic organization of corticostriatal afferents expressing ephrin-A proteins via repulsive interactions. This EphA7−ephrin-A mediated repulsive interaction between the postsynaptic neurons and presynaptic axons can occur either via forward signaling through the EphA tyrosine kinase receptor or by reverse signaling through ephrins (Holland et al., 1996, Pasquale, 2005, Feldheim and O'Leary, 2010).

Since striatal neurons expressing EphA7 are distributed in a mature

matrisome organization and EphA7+ thalamic neurons are present in the dorsal VB and along the POm-VB border prior to the arrival of cortical axons, we propose that reverse signaling through ephrin-A ligands on cortical axon projections from medial S1 is the predominant mechanism by which EphA7 mediates the distribution of cortical axon terminals from medial S1.

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It is important to note that there is a high medial to low lateral gradient in the perinatal expression of ephrin-A5 in neurons located in cortical layers V and VI of the somatosensory cortex. Thus, neurons located in layers V and VI of the lateral somatosensory cortex express low levels of ephrin-A5 but high levels of EphA7 receptors perinatally (Torii and Levitt, 2005). Thus, forward signaling through EphA7 receptors on corticofugal axons originating from neurons in lateral regions of S1 may be primarily responsible for regulating repulsive interactions that restrict the distribution of lateral S1 axon terminals in the thalamus and striatum. Though our study supports a role for EphA7 receptors on striatal and thalamic neurons in restricting the terminal distribution of corticostriatal and corticothalamic projections from medial regions of S1 and implicates ephrin-A5 as the possible binding partner on medial S1 axons, our results do not specifically identify which ephrin-A ligand binding partner is actually present on these cortical axons. In addition to ephrin-A5, ephrin-A2 and ephrin-A3 both bind to striatal regions containing neurons expressing EphA7 (Janis et al., 1999, Tai et al., 2013). However, ephrin-A5 is a strong candidate to be the primary binding partner on both corticostriatal and corticothalamic projections from medial regions of S1 since layer V and VI neurons in this region express high levels of ephrin-A5 mRNA during postnatal development and ephrin-A5 is reported to exhibit high binding affinity to EphA7 receptors (Himanen et al., 2004). Although EphA7−ephrin-A5 interactions likely participate in the formation of corticostriatal connections as they do for corticothalamic connections, the degree to which the formation of corticostriatal connections relies solely on EphA7−ephrin-5 interactions remains to be determined.

Future studies examining the topographic distribution of corticostriatal

projections in mice with single or multiple deletions of specific A-ephrins or EphA7 receptors,

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and/or animal studies in which there is experimental overexpression of these molecules, will be necessary to clarify this issue.

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5. References Alloway KD, Crist J, Mutic JJ, Roy SA (1999) Corticostriatal projections from rat barrel cortex have an anisotropic organization that correlates with vibrissal whisking behavior. J Neurosci 19:10908-10922. Alloway KD, Hoffer ZS, Hoover JE (2003) Quantitative comparisons of corticothalamic topography within the ventrobasal complex and the posterior nucleus of the rodent thalamus. Brain Res 968:54-68. Alloway KD, Lou L, Nwabueze-Ogbo F, Chakrabarti S (2006) Topography of cortical projections to the dorsolateral neostriatum in rats: multiple overlapping sensorimotor pathways. J Comp Neurol 499:33-48. Alloway KD, Mutic JJ, Hoffer ZS, Hoover JE (2000) Overlapping corticostriatal projections from the rodent vibrissal representations in primary and secondary somatosensory cortex. J Comp Neurol 428:51-67. Alloway KD, Mutic JJ, Hoover JE (1998) Divergent corticostriatal projections from a single cortical column in the somatosensory cortex of rats. Brain Res 785:341-346. Alloway KD, Smith JB, Beauchemin KJ, Olson ML (2009) Bilateral projections from rat MI whisker cortex to the neostriatum, thalamus, and claustrum: forebrain circuits for modulating whisking behavior. J Comp Neurol 515:548-564. Bolam JP, Hanley JJ, Booth PA, Bevan MD (2000) Synaptic organisation of the basal ganglia. Journal of anatomy 196 ( Pt 4):527-542. Boyd AW, Bartlett PF, Lackmann M (2014) Therapeutic targeting of EPH receptors and their ligands. Nature reviews Drug discovery 13:39-62.

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Brown LL, Smith DM, Goldbloom LM (1998) Organizing principles of cortical integration in the rat neostriatum: corticostriate map of the body surface is an ordered lattice of curved laminae and radial points. J Comp Neurol 392:468-488. Chilton JK (2006) Molecular mechanisms of axon guidance. Developmental biology 292:13-24. Christensen J, Sorensen JC, Ostergaard K, Zimmer J (1999) Early postnatal development of the rat corticostriatal pathway: an anterograde axonal tracing study using biocytin pellets. Anatomy and embryology 200:73-80. Crittenden JR, Graybiel AM (2011) Basal Ganglia disorders associated with imbalances in the striatal striosome and matrix compartments. Frontiers in neuroanatomy 5:59. Dye CA, El Shawa H, Huffman KJ (2011a) A lifespan analysis of intraneocortical connections and gene expression in the mouse I. Cerebral cortex 21:1311-1330. Dye CA, El Shawa H, Huffman KJ (2011b) A lifespan analysis of intraneocortical connections and gene expression in the mouse II. Cerebral cortex 21:1331-1350. Feldheim DA, O'Leary DD (2010) Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition. Cold Spring Harb Perspect Biol 2:a001768. Feldheim DA, Vanderhaeghen P, Hansen MJ, Frisen J, Lu Q, Barbacid M, Flanagan JG (1998) Topographic guidance labels in a sensory projection to the forebrain. Neuron 21:1303-1313. Flaherty AW, Graybiel AM (1991) Corticostriatal transformations in the primate somatosensory system. Projections from physiologically mapped body-part representations. Journal of neurophysiology 66:1249-1263. Flanagan JG, Vanderhaeghen P (1998) The ephrins and Eph receptors in neural development. Annual review of neuroscience 21:309-345.

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Gerfen CR (1984) The neostriatal mosaic: compartmentalization of corticostriatal input and striatonigral output systems. Nature 311:461-464. Gerfen CR (1989) The neostriatal mosaic: striatal patch-matrix organization is related to cortical lamination. Science 246:385-388. Gerfen CR (2004) Basal Ganglia. In: The Rat Nervous System (Paxinos, G., ed), pp 455-508 San Diego, CA: Elsevier Academic Press. Grant E, Hoerder-Suabedissen A, Molnar Z (2012) Development of the corticothalamic projections. Frontiers in neuroscience 6:53. Graybiel AM, Flaherty AW, Gimenez-Amaya J-M (1991) The basal ganglia III. New York: Plenum Press. Graybiel AM, Ragsdale CW, Jr. (1978) Histochemically distinct compartments in the striatum of human, monkeys, and cat demonstrated by acetylthiocholinesterase staining. Proceedings of the National Academy of Sciences of the United States of America 75:5723-5726. Herkenham M, Pert CB (1981) Mosaic distribution of opiate receptors, parafascicular projections and acetylcholinesterase in rat striatum. Nature 291:415-418. Himanen JP, Chumley MJ, Lackmann M, Li C, Barton WA, Jeffrey PD, Vearing C, Geleick D, Feldheim DA, Boyd AW, Henkemeyer M, Nikolov DB (2004) Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling. Nature neuroscience 7:501-509. Hoffer ZS, Alloway KD (2001) Organization of corticostriatal projections from the vibrissal representations in the primary motor and somatosensory cortical areas of rodents. J Comp Neurol 439:87-103.

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Hoffer ZS, Arantes HB, Roth RL, Alloway KD (2005) Functional circuits mediating sensorimotor integration: quantitative comparisons of projections from rodent barrel cortex to primary motor cortex, neostriatum, superior colliculus, and the pons. J Comp Neurol 488:82-100. Holland SJ, Gale NW, Mbamalu G, Yancopoulos GD, Henkemeyer M, Pawson T (1996) Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands. Nature 383:722-725. Iniguez C, De Juan J, al-Majdalawi A, Gayoso MJ (1990) Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase. Brain research Developmental brain research 57:43-53. Jacobs EC, Campagnoni C, Kampf K, Reyes SD, Kalra V, Handley V, Xie YY, Hong-Hu Y, Spreur V, Fisher RS, Campagnoni AT (2007) Visualization of corticofugal projections during early cortical development in a tau-GFP-transgenic mouse. The European journal of neuroscience 25:17-30. Jain M, Armstrong RJ, Barker RA, Rosser AE (2001) Cellular and molecular aspects of striatal development. Brain Res Bull 55:533-540. Janis LS, Cassidy RM, Kromer LF (1999) Ephrin-A binding and EphA receptor expression delineate the matrix compartment of the striatum. J Neurosci 19:4962-4971. Lehigh KM, Leonard CE, Baranoski J, Donoghue MJ (2013) Parcellation of the thalamus into distinct nuclei reflects EphA expression and function. Gene Expr Patterns 13:454-463. Martone ME, Holash JA, Bayardo A, Pasquale EB, Ellisman MH (1997) Immunolocalization of the receptor tyrosine kinase EphA4 in the adult rat central nervous system. Brain Res 771:238-250.

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Torii M, Levitt P (2005) Dissociation of corticothalamic and thalamocortical axon targeting by an EphA7-mediated mechanism. Neuron 48:563-575. Torii M, Rakic P, Levitt P (2013) Role of EphA/ephrin--a signaling in the development of topographic maps in mouse corticothalamic projections. J Comp Neurol 521:626-637. Vanderhaeghen P, Lu Q, Prakash N, Frisen J, Walsh CA, Frostig RD, Flanagan JG (2000) A mapping label required for normal scale of body representation in the cortex. Nature neuroscience 3:358-365. Voorn P, Vanderschuren LJ, Groenewegen HJ, Robbins TW, Pennartz CM (2004) Putting a spin on the dorsal-ventral divide of the striatum. Trends in neurosciences 27:468-474. Wang HB, Deng YP, Reiner A (2007) In situ hybridization histochemical and immunohistochemical evidence that striatal projection neurons co-containing substance P and enkephalin are overrepresented in the striosomal compartment of striatum in rats. Neuroscience letters 425:195-199.

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6. Figure Legends 6.1 Figure 1. Postnatal EphA7 receptor expression is stable from P6 through the second postnatal week. EphA7 immunostaining pattern in the mouse striatum during mid-postnatal development. Similar rostral (A-C) and caudal (D-E) levels are illustrated for coronal sections through the dorsal striatum at postnatal ages P6 (A, D), P9 (B, E) and P12 (C,F). At all ages, EphA7-expressing neurons are organized into curved bands (arrows in A and D) along a dorsomedial to ventrolateral axis, with a prominent band located immediately ventral to the external capsule (ec; arrowheads in A-F). Dotted line approximates the striatopallidal border in D-F. M, medial; L, lateral; D, dorsal; V, ventral; GP, globus pallidus. Scale bar = 200 µm for all panels

6.2 Figure 2. BDA tracer injections into the anterior and posterior medial S1 barrel cortex. Adjacent coronal sections illustrating cytochrome oxidase (CO) activity (A, D), BDA tracer injection sites (B, E), and EphA7 immunolabeling (C, F) in the cortex at P12. Cross-correlation of the BDA injection sites (arrows) with the detection of CO activity as a marker for the barrel cortex confirmed that the tracer injections were localized to layers V and VI of the anterior and posterior S1 barrel cortex. At P12, low levels of EphA7 immunostaining (C, F) are observed in the barrel cortex, although a prominent laminar pattern is still present in the cingulate cortex (arrowheads). The approximate location of the barrel cortex is indicated by short lines in B, C, E, and F and the approximate location of layers 4, 5 and 6 are indicted by IV, V and VI. Cg, cingulate cortex; HPC; hippocampus; ec, external capsule; STR, striatum. Scale bar = 650 µm for all panels.

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6.3 Figure 3. BDA-labeled afferents from the medial S1 barrel cortex avoid EphA7-expressing striatal neurons. EphA7 immunolabeling of coronal sections through the anterior (A, D, G) and posterior (J, M, P) dorsolateral striatum at P12 compared to adjacent sections immunostained for BDA-labeled afferents originating from either an anterior (B, E, H) or posterior (K, N, Q) site in medial S1 barrel cortex.

Merged images of EphA7 immunolabeling and BDA-labeled

corticostriatal afferents (C, F, I, L, O, R) demonstrate that afferents from the medial S1 barrel cortex terminate in domains that avoid EphA7-expressing striatal neurons. White arrows in C and L highlight bundles of BDA-labeled afferents from the medial S1 barrel cortex piercing the band of EphA7-expressing striatal neurons immediately ventral to the external capsule. G-I and P-R. High magnification images of the boxes in D-F and M-O demonstrate the complementary distribution of EphA7 immunostaining (green) in the striatum and the distribution of corticostriatal afferents (magenta) from medial S1 barrel cortex. Ant. STR, anterior striatum; Post. STR, posterior striatum; High Mag, high magnification; M, medial; L, lateral; D, dorsal; V, ventral. Scale bar in A and J = 200 µm and applies to A-F and J-O, respectively. Scale bar in G and P = 50 µm and applies to G-I and P-R, respectively.

6.4 Figure 4. BDA-labeled afferents from the medial S1 barrel cortex innervate thalamic regions exhibiting low postnatal EphA7 expression. A-D. EphA7 immunolabeling of coronal sections through the ventrobasal (VB) and medial posterior complex (POm) of the thalamus at P6, P9 and P12. At P6 (A) individual thalamic barreloids in the dorsolateral VB exhibit EphA7 staining (arrowheads in A) and high levels of EphA7 are observed along the VB/POm border (arrows in A). By P9 (B) and P12 (C), EphA7 expression is down-regulated in the dorsolateral VB (arrowheads in B and C) and individual EphA7+ barreloids cannot be discerned. However,

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EphA7 is still detected along the VB/POm border (arrows in B and C). E-L. Coronal sections stained for cytochrome oxidase (CO), BDA, and EphA7. BDA-labeled afferents from medial S1 barrel cortex (F, J) innervate restricted regions of the VB and POm, which are identified by their unique pattern of CO activity, as illustrated in the composite image of adjacent sections (E, I) stained for CO and BDA. Although EphA7 staining is decreased in the thalamus at P12 (G, K), overlay images (H, L) of BDA-labeled afferents and EphA7 immunolabeling in adjacent sections demonstrate that BDA-labeled corticothalamic afferents avoid thalamic regions that still express EphA7. Arrows in E and I indicate BDA-labeled axons in the internal capsule. D. Schematic representation of the distribution of corticothalamic afferents from medial S1 barrel cortex at P12 superimposed on EphA7 immunostaining at P6 to illustrate that the terminal arbors of corticothalamic axons avoid thalamic regions with high levels of EphA7 immunostaining. Scale bar in A = 100 µm and applies to A-D. Scale bar in E = 200 µm and applies to E-L.

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Highlights Corticostriatal axons from medial S1 cortex avoid EphA7 expressing striatal neurons Corticothalamic axons from medial S1 cortex avoid EphA7 expressing thalamic neurons EphA7-ephrinA interactions mediate organization of corticofugal terminals from S1 cortex

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