Localization of immunoreactivity for Deleted in Colorectal Cancer (DCC), the receptor for the guidance factor netrin-1, in ventral tier dopamine projection pathways in adult rodents

Neuroscience 131 (2005) 671– 681

LOCALIZATION OF IMMUNOREACTIVITY FOR DELETED IN COLORECTAL CANCER (DCC), THE RECEPTOR FOR THE GUIDANCE FACTOR NETRIN-1, IN VENTRAL TIER DOPAMINE PROJECTION PATHWAYS IN ADULT RODENTS P. B. OSBORNE,a,b* G. M. HALLIDAY,a H. M. COOPERc AND J. R. KEASTa

Parkinson’s disease (PD) is a progressively disabling, incurable movement disorder caused by the gradual death of dopamine neurons in the substantia nigra pars compacta (Fearnley and Lees, 1991). Recent genetic studies have identified two main cellular systems that appear to be important for this selective degeneration of midbrain dopamine neurons: the ubiquitin proteosome system (GwinnHardy, 2002) and dopamine cell growth and survival systems (Le et al., 2003). Although a majority of genetic mutations identified to date occur in the less common early-onset forms of PD and influence the ubiquitin– proteosome system, the recent identification of mutations in Nur-related factor 1 (Nurr1)—a nuclear receptor critical for the development and maturation of midbrain dopamine neurons—in 10 familial cases of late-onset PD (Le et al., 2003), has focused attention on molecular mechanisms necessary for the development and support of mature dopamine neurons. During development, successive waves of nuclear transcription factors differentiate stem cells into dopamine neurons, and at least two transcription factors contribute to the full maturation of dopamine neurons (Burbach et al., 2003). Nurr1 is one of these, and regulates several genes involved in dopamine synthesis, transport, release, and reuptake. Also expressed in dopamine neurons at the same time as Nurr1 and tyrosine hydroxylase, is the homeobox protein Pitx3. Binding of Pitx3 to the response element of the tyrosine hydroxylase gene results in pronounced up-regulation of its transcription (Cazorla et al., 2000). In contrast to Nurr1, Pitx3 is only found in midbrain dopamine neurons and is reduced in PD (Smidt et al., 1997). Netrin-1 is part of a family of recently identified guidance factors important for directing axons to their targets (Manitt and Kennedy, 2002). Netrin-1 plays an important role in the development and separation of the patch and matrix compartments in the striatum (Hamasaki et al., 2001), as well as in the development of other neural projections (Metin et al., 1997; Braisted et al., 2000; Schwarting et al., 2001). Deleted in colorectal cancer (DCC) is the high affinity cell surface receptor for netrin-1 and is widely expressed in the developing brain but is down-regulated prior to maturation. However, in adult rodents DCC mRNA has been located in the substantia nigra, striatum and cerebellum (Livesey and Hunt, 1997; Volenec et al., 1997). To determine if DCC is colocalized in dopamine neurons or is selectively expressed by particular types of neuronal projections in these areas, we used an antiserum against DCC in conjunction with markers

a Prince of Wales Medical Research Institute, University of New South Wales, Barker Street Randwick, Sydney NSW 2031, Australia b Pain Management Research Institute, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia c

Walter and Eliza Hall Institute, 1G Royal Parade, Parkville VIC 3050, Australia

Abstract—DCC (deleted in colorectal cancer)—the receptor of the netrin-1 neuronal guidance factor—is expressed and is active in the central nervous system (CNS) during development, but is down-regulated during maturation. The substantia nigra contains the highest level of netrin-1 mRNA in the adult rodent brain, and corresponding mRNA for DCC has also been detected in this region but has not been localized to any particular neuron type. In this study, an antibody raised against DCC was used to determine if the protein was expressed by adult dopamine neurons, and identify their distribution and projections. Significant DCC-immunoreactivity was detected in midbrain, where it was localized to ventrally displaced A9 dopamine neurons in the substantia nigra, and ventromedial A10 dopamine neurons predominantly situated in and around the interfascicular nucleus. Strong immunoreactivity was not detected in dopamine neurons found elsewhere, or in non-dopamine-containing neurons in the midbrain. Terminal fields selectively labeled with DCC antibody corresponded to known nigrostriatal projections to the dorsolateral striatal patches and dorsomedial shell of the accumbens, and were also detected in prefrontal cortex, septum, lateral habenular and ventral pallidum. The unique distribution of DCC-immunoreactivity in adult ventral midbrain dopamine neurons suggests that netrin-1/DCC signaling could function in plasticity and remodeling previously identified in dopamine projection pathways. In particular, a recent report that DCC is regulated through the ubiquitin–proteosome system via Siah/Sina proteins, is consistent with a potential involvement in genetic and sporadic forms of Parkinson’s disease. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: guidance factor, substantia nigra, interfascicular nucleus, striatonigral, mesolimbic, accumbens. *Correspondence to: P. Osborne, Pain Management Research Institute, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia. Tel: ⫹61-2-9926-5539; fax: ⫹61-2-9906-4079. E-mail address: [email protected] (P. Osborne). Abbreviations: DAT, dopamine transporter; DCC, deleted in colorectal cancer; IR, immunoreactivity; MOR, ␮-opioid receptor; Nurr1, Nurrelated factor 1; PB, phosphate buffer; PD, Parkinson’s disease; SNl, substantia nigra pars lateralis; SNv, substantia nigra, ventral tier in pars compacta; SNvd, substantia nigra, ventrally displaced tier in pars reticulata; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.11.043

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of dopamine neurons (tyrosine hydroxylase [TH], calbindin and dopamine transporter [DAT]) and ␮-opioid receptor (MOR), to examine the midbrain and forebrain of adult rats and mice.

EXPERIMENTAL PROCEDURES Tissue removal All experiments on animals were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edn., 2004; http://www.nhmrc.gov.au) and approved by the Animal Ethics Committee of the University of New South Wales. All efforts were made to minimize animal suffering and the number of animals used. Adult male outbred Wistar rats (seven) and C57/Bl6 mice (two) were used in the study. Animals were anesthetized (rats: sodium pentobarbitone 45 mg/kg i.p.; mice: ketamine/xylazine 60 and 10 mg/kg i.p.) and perfused intracardially with freshly made buffered 4% formaldehyde.

Immunofluorescence Frozen sections (50 ␮m) were processed as a 1:4 series for dual-labeling immunofluorescence. Sections were treated with 0.1 M phosphate buffer (PB) containing 5% non-immune horse serum, and then incubated for 48 h at room temperature with combinations of primary antisera diluted in PB containing 2% horse serum and 0.2% Triton X-100. The anti-DCC antibody was raised in rabbit against a C-terminal peptide (antiserum 2473), as characterized previously (Seaman et al., 2001). Dilutions from 1:3000 to 1:5000 were optimal for observing bright DCCimmunoreactivity (IR) and low background staining in sections containing midbrain dopamine neurons. This staining decreased in intensity with an antibody dilution of 1:10,000, and could not be reliably detected using a dilution of 1:30,000. A dilution of 1:1000 revealed numerous very weakly stained neuronal cell bodies that were distributed relatively homogenously throughout the neocortex, forebrain and midbrain. The DCC antiserum was used in combination with antiserum raised against TH (host species sheep; Chemicon International, Temecula, CA, USA AB1542; 1:1500), calbindin (mouse monoclonal; Sigma C-8666; 1:7000), DAT (host species rat; Chemicon MAB369; 1:1000) or MOR (host species guinea-pig; Chemicon AB 1774; 1:2000). Secondary antisera conjugated with Cy3 (rabbit) or FITC (other species; Jackson Immunoresearch Laboratories, West Grove, PA, USA) were then applied for 4 h, before washing and coverslipping. Preadsorption testing of the DCC antiserum was performed by mixing various dilutions (1:5000, 1;10,000 or 1:20,000) with 7.8 –50 ␮M of the C-terminal peptide antigen (SEESHKPTEDPASV: Seaman et al., 2001) for 0.5–24 h prior to incubating midbrain and cerebellum sections. This caused a detectable reduction of weak DCC-IR in neurons in the cerebellum but failed to cause a consistent reduction of the bright DCC-IR in the substantia nigra and ventral tegmental area (VTA).

Microscopy and image documentation Sections were assessed for cellular location of DCC-IR, and expression compared with TH, DAT, calbindin or MOR. Monochrome eight-bit digital images were acquired with a SpotRT camera and were processed only by adjusting saturation and contrast to best resemble the native immunostaining signal. Representative images from key areas have been shown as single or merged pairs (Cy3 and FITC). All observations were made on brain tissue from three to seven rats and two mice. Results from the two species were essentially identical so have been illustrated in detail only for rats.

RESULTS DCC-IR is preferentially localized in dopamine neurons situated in the ventral tier of the rodent substantia nigra To establish if DCC-IR is expressed in mesencephalic dopamine neurons, we performed an immunohistochemical analysis using antibodies raised against TH and a peptide fragment of the C-terminal sequence of DCC (Seaman et al., 2001). Fig. 1 illustrates the pattern of DCC-IR seen in a series of coronal sections of adult rat midbrain. Throughout this region, strong DCC-IR was almost completely restricted to dopamine neurons immunoreactive for TH (Figs. 1, 2 and 3). We analyzed rostralcaudal series of midbrain sections in rat and mouse and determined the localization of these neurons within subregions of the A8, A9 and A10 dopamine cell groups, using the atlas of Paxinos and Watson (1997) and the nomenclature of Gerfen and colleagues (Gerfen, 1985, 1992; Gerfen et al., 1987a,b). In the rostral midbrain, DCC-IR first appeared as a contiguous layer lateral to the midline in A9 dopamine neurons situated in the ventral tier of the substantia nigra pars compacta (SNv) (Fig. 1). DCC-IR was rarely detected in dopamine neurons in the dorsal tier of the SNv, in the substantia nigra pars lateralis (SNl), or in the adjacent retrorubral fields. This basic pattern of localization observed in the rostral midbrain was maintained in the medial and caudal midbrain (Fig. 1c, d), except at these levels, DCC-IR was also localized to ventrally displaced dopamine neurons in the substantia nigra pars reticulata (SNvd). Calbindin-IR was used to further investigate the apparent preferential localization of DCC-IR in ventral tier dopamine neurons (Gerfen et al., 1987a; Gerfen, 1985; McRitchie et al., 1996). This staining revealed that most of the DCC-IR neurons in the pars compacta were situated ventral to the mass of calbindin-IR neurons in the dorsal tier, and ventral to the adjacent dense zone of calbindin-IR fibers in the dorsomedial pars reticulata (Fig. 2e– h). Although co-localization of DCC- and calbindin-IR was rare in the substantia nigra, significant numbers of doubleimmunostained neurons were identified in medial aspects of the pars compacta (Fig. 2d) that corresponded to a region identified as the pars medialis by some reports (McRitchie et al., 1996). In the A10 cell group, DCC-IR was predominantly localized in small dopamine neurons situated in the interfascicular nucleus (Figs. 1b, c and 3a, c). DCC-IR dopamine neurons were rarely observed outside of this subregion of the A10 cell group, and these were mostly situated adjacent to the interfascicular nucleus in the rostral or caudal linear nuclei or along the medial margin of the ventral tegmental area (VTA). As was found in the A9 cell group, calbindin-IR also rarely co-localized with DCC-IR A10 dopamine neurons (Fig. 3d–f). No DCC-IR neurons were present in the extension of the A10 cell group in the supramammillary nucleus, nor were they detected in any of the more rostral dopamine cell groups in the hypothalamus (Fig. 4a, c). However, some DCC-IR

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Fig. 1. DCC-IR is localized in ventral A9 dopamine neurons in the substantia nigra and in A10 dopamine neurons situated in and around the interfascicular nucleus. Shown are image pairs of rat midbrain sections cut in the coronal plane and double-immunostained for TH and DCC, which are shown separately. (A–D) A rostral–caudal series at different midbrain levels containing the A8, A9 and A10 dopamine cell groups. fr, fasciculus retroflexus; IP, interpeduncular nucleus; Li, linear nuclei; ml, medial lemniscus; SNd, substantia nigra, dorsal tier of the pars compacta; 3n, third cranial nerve. Scale bar⫽500 ␮m.

dopamine neurons were detected in the dorsal raphe nucleus, which contains a dorsal, caudal extension of the A10 complex (Hökfelt et al., 1984; Fig. 4d).

The antibody dilution (1:3000) that produced optimal DCC immunostaining of mesencephalic dopamine neurons did not reveal similar staining in neurons in any

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Fig. 2. Localization of DCC-IR in the ventral tier dopamine neurons of the substantia nigra. Shown are merged images of rat midbrain sections containing the SN and VTA, which in the left column are of sections double-immunostained for TH (green) and DCC (red) and in the right column are of sections double-immunostained for calbindin (green) and DCC (red). (A–D) DCC-IR dopamine neurons (yellow) were distributed in the SNv in the pars compacta and in the SNvd in the pars reticulata but were rarely observed in the SNd. (E–H) DCC-positive neurons (red) were mostly situated between the layer of calbindin-IR neurons corresponding to the SNd, and the dense calbindin-IR terminal fields in the dorsal SNr. Small numbers of neurons in which DCC co-localized with calbindin were observed in the medial SNc (arrowheads). fr, fasciculus retroflexus; IF, interfascicular nucleus; ml, medial lemniscus; SNd, substantia nigra, dorsal tier of the pars compacta; SNr, substantia nigra pars reticulata. Scale bars⫽500 ␮m (A, E), 200 ␮m (B–D, F–H).

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Fig. 3. DCC-IR A10 dopamine neurons were distributed in the IF but were rare in the VTA. Shown are merged image pairs of sections double-immunostained for TH (green) and DCC (red) in the left column, and calbindin (green) and DCC (red) in the right column. (A–C) A majority of A10 neurons that contained DCC co-localized with TH (yellow) were situated in the IF and were only present in the VTA in significant but low numbers at the level of the fr or ventrally in the region adjacent to IP. (D–F) DCC-IR in A10 neurons rarely co-localized with calbindin-IR in the VTA or IF. fr, fasciculus retroflexus; IF, interfascicular nucleus; IP, interpeduncular nucleus; ml, medial lemniscus; SNd, substantia nigra, dorsal tier of the pars compacta; SNr, substantia nigra pars reticulata; 3n, third nerve. Scale bars⫽400 ␮m (A, B, D–F), 200 ␮m (C).

other midbrain, forebrain or cortical structure. Only weakly immunoreactive neuronal cell bodies were identified in the hippocampus and cerebellum, structures in which DCC mRNA has been detected by in situ hybridization (Livesey and Hunt, 1997; Volenec et al., 1997). Strong DCC-IR was detected in a restricted region of the ventromedial wall of the third ventricle adjacent to the medial eminence and extending into the arcuate nucleus and periventricular hypothalamus (Fig. 4c). However, the distribution and appearance of these DCC-IR cells was identical with previous descriptions of non-neuronal tanycytes, which are a specialized type of glial cell (e.g. Chauvet et al., 1998).

Localization of DCC in nigrostriatal and mesolimbic dopamine projections The striatum is the major projection target of mesencephalic dopamine neurons. We predicted that DCC-IR terminals would be enriched in striatal patches, as most of the projections of the ventral dopamine neurons are located in these structures (reviewed by Joel and Weiner, 2000). Fig. 5a illustrates that DCC-IR terminals were most evident in the dorsal striatum where they concentrated in patches, and an extended zone along the lateral margin of the striatum adjacent to the external capsule. These DCC-enriched areas were also immunoreactive for MOR, which is a marker of the

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Fig. 4. DCC-IR is not localized in hypothalamic dopamine cell groups. Images of rat brain sections at the level of the arcuate nucleus. (A, B) DCC-IR is co-localized with TH-positive fibers of passage traveling in the ventral parts of the medial forebrain bundle. (C) DCC-IR was absent in TH-positive cell groups situated at the level of the arcuate nucleus, but non-neuronal DCC-IR cells that were TH-negative lined the ventral half of the third ventricle. (D) DCC-IR is localized in a small number of dorsal A10 dopamine neurons in the dorsal raphe nucleus. Images of rat caudal midbrain sections double-immunolabeled for TH and DCC. Arc, arcuate nucleus; DMD, dorsomedial hypothalamic area, dorsal part; mfb, medial forebrain bundle; Pe, periventricular nucleus; 3V, third ventricle. Scale bars⫽500 ␮m (A, D), 200 ␮m (B), 50 ␮m (C), 100 ␮m (D).

striatal patch compartment (Fig. 5a), although the density of DCC terminals appeared to decline in patches located more ventrally. Inspection of the terminal fields under high magnification established that a majority of DCC-IR varicosities in

patches also appeared to be co-localized with TH (Fig. 5b) and DAT (data not shown). The sources of the dopamine projection to the accumbens in the ventral striatum are also segregated, but in

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Fig. 5. DCC-IR is localized in the terminal fields of nigrostriatal and mesolimbic dopamine neurons. Shown are forebrain sections doubleimmunostained for TH and DCC in the image pairs in the left columns and MOR and DCC in the image pairs in the right hand columns. (A) A low-magnification image pair of the striatum shows DCC-IR terminals predominantly localized in the dorsal striatum where it was enriched in patches and the lateral subcallosal streak (arrowheads). (B) High-magnification image of a DCC-IR patch (arrowheads) showing co-localization of DCC-IR in TH-positive varicosities. (C) Low-magnification view of the ventral striatum showing the registration of TH and DCC immunostaining in a region of the dorsomedial accumbens shell. (D) High-magnification view of dorsal striatum adjacent to the external capsule where DCC-IR terminals were enriched in patches identified by MOR-IR (arrowheads) but also distributed in significant numbers in matrix. (E) DCC-IR terminals could not be readily discerned outside of MOR patches (arrowheads) more ventrally in the striatum. (F) DCC-IR patches in the dorsomedial accumbens were weakly immunoreactive for MOR. ac, anterior commisure; AcbC, accumbens core; AcbS, accumbens shell; ec, external capsule; LSV, lateral septum ventral; LV, lateral ventricle; Str, striatum. Scale bars⫽500 ␮m (A, C), 50 ␮m (B, D, E), 200 ␮m (F).

comparison to the striatum, the topographic organization is much less well characterized. Fig. 5c, f illustrates the striking concentration of DCC-IR terminals in the accumbens, which was largely restricted to the dorsal part of the medial shell. The accumbens is not organized into patch and matrix but shows a more complex arrangement of neuronal ensembles (Pennartz et al., 1994). As was the case in dorsal striatum, DCC-IR viewed with high magnification fluorescence microscopy was co-localized in TH-IR terminals. In contrast to the striatum, MOR IR was enriched in areas of the dorsomedial shell largely adjacent to DCClabeled terminals (Fig. 5f). DCC-IR terminals were also present in other regions that receive projections from the mesencephalic dopamine system (Björklund and Lindvall, 1984). In the prefrontal cortex, the prelimbic (Fig. 6) and cingulate cortices were sparsely innervated and whereas all of the DCC-IR terminals in these regions appeared to be immunoreactive for TH, not all of them were immunoreactive for DAT (data not shown). DCC-IR terminals appeared to be absent in other parts of the cortex and in the amygdala but were present in the lateral septal nucleus (Fig. 6) and ventral pallidum. In the

hypothalamus, the ventral part of the medial forebrain bundle contained a high density of DCC-IR fibers of passage, virtually all of which appeared to co-localize TH (Fig. 4b) or DAT (data not shown). In the lateral habenula (Fig. 6), DCC-IR appeared to be localized in a subset of large caliber TH-immunoreactive terminals. More dorsal in the medial habenular, similar large caliber DCC- and TH-positive terminals surrounded a patch of smaller-caliber DCC-IR terminals that were TH-negative.

DISCUSSION The major finding of this study was that significant DCC-IR was expressed in a very restricted population of dopamine neurons—mainly those vulnerable to the neurodegeneration of PD (Fearnley and Lees, 1991). A majority of DCC-IR neurons were ventral A9 dopamine neurons situated in the pars compacta and pars reticulata of the substantia nigra. DCC-IR was not detected in the rostral hypothalamic dopamine cell groups nor the A8 retrorubral field complex, but was present in some A10 dopamine neurons, which were concentrated around the interfascicular nucleus. Livesey and

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Fig. 6. Localization of DCC-IR in TH-positive terminals outside of the striatum. Low-magnification views of TH-immunostained sections containing prelimbic cortex (A), lateral septum (B) and habenula (C). The corresponding image pairs in each row (A=–C=) show high-magnification views of the relative distribution of TH- and DCC-IR terminals. Terminals containing co-localized TH and DCC-IR (e.g. arrowheads) were surrounded by numerous fibers positive for TH only, but relatively few fibers that were positive for DCC only. ac, anterior commissure; cc, corpus callosum; Hb, habenula; LS, lateral septum; PrL, prelimbic cortex; Str, striatum. Scale bars⫽500 ␮m (A, B), 200 ␮m (C) and 50 ␮m (A=–C=).

Hunt (1997) had previously reported that the substantia nigra contains the highest levels of netrin-1 mRNA expression in the adult CNS and had detected DCC mRNA in this region, but the transmitter phenotype of the neurons expressing this message was not determined by this earlier study. Technical considerations The rabbit polyclonal antiserum (2473) used in this study was raised against a C-terminal peptide (SEESHKPTEDPASV) corresponding to amino acids 1406 –1419 of the mouse DCC protein (Seaman et al., 2001). In a previous report, DCC-IR revealed in the peripheral and enteric ner-

vous system of embryonic mice using a 1:200 dilution of this antibody was lost when the antisera was pre-incubated with the peptide antigen. In the present study, an optimal dilution of 1:3000 revealed the bright immunostaining obtained with this antiserum in mesencephalic dopamine neurons and their terminal fields, as well as non-neuronal tanycytes in the arcuate nucleus adjacent to the third ventricle. The strong IR seen in these structures contrasted with weakly immunoreactive cell bodies in the cerebellum and hippocampus, which have also previously been reported to contain DCC mRNA detected by in situ hybridization (Livesey and Hunt, 1997; Volenec et al., 1997).

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However, it should be noted that pre-incubation with the peptide antigen reduced the weak immunostaining of neurons in the cerebellum but failed to cause a consistent detectable reduction of strong immunostaining in dopamine neurons in the mesencephalon. On this basis, there is a possibility that a different unknown molecule was recognized by the antibody. However, the presence of DCC-IR dopamine neurons in the substantia nigra and VTA is consistent with previous localization of DCC mRNA in these areas, together with the highest levels of netrin-1 in adult brain (Livesey and Hunt, 1997). DCC-IR is a marker of ventral tier dopamine neurons in the rat substantia nigra To our knowledge, the Pitx3 homeobox gene is the only marker previously identified in midbrain dopamine neurons that is expressed in a pattern similar to the DCC-IR neurons described here (Hwang et al., 2003; van den Munckhof et al., 2003; Nunes et al., 2003). It has been reported in adult mice that Pitx3-IR is colocalized in most of the TH-positive neurons in the ventral SNc and in about half of the neurons in the VTA (van den Munckhof et al., 2003). Furthermore, in Pitx3deficient aphakia mice TH-positive neurons are reduced by 70% in the SN and by 52% in the VTA. This is matched by a marked loss of striatal TH-IR fibers in the dorsolateral striatum, but relative sparing in the ventral striatum. It is notable however, in a previous report that some loss of TH-IR fibers is apparent in the dorsomedial shell (see Fig. 3; van den Munckhof et al., 2003). Parallels have been drawn between the cardinal features of PD and aphakia mice, which show preferential loss of ventral tier SN dopamine neurons, severe depletion of the dopamine projections to the dorsal striatum, and display akinesia. Pitx3 is a bicoid-related homeodomaincontaining transcription factor required for the specification and/or survival of specific midbrain dopamine neurons during development and is only expressed by these neurons in adult brain. As Pitx3-deficient mice have the same pattern of midbrain dopamine neuronal losses to that observed in PD (Hwang et al., 2003; van den Munckhof et al., 2003), the similarity of the anatomical relationship is consistent with a potential role for netrin-1 and DCC in the neurodegeneration associated with PD. This concept is further strengthened by the knowledge that DCC is regulated intracellularly via the Siah/Sina family of proteins through the ubiquitin– proteosome pathway (Hu et al., 1997). Dysfunction in the cellular degradation of proteins through the ubiquitin–proteosome system provides a common intracellular mechanism for the genetic and sporadic forms of PD (Gwinn-Hardy, 2002). The unique distribution in adult ventral midbrain dopamine neurons and regulation by an intracellular mechanism known to be dysfunctional in PD, identify DCC as a potentially important effector mechanism for this neurodegenerative disease, possibly due to its role in the plasticity and remodeling of selected dopamine pathways. Netrin-1 mRNA can be detected in the developing substantia nigra as early as E13 and continues to be expressed in adults (Livesey and Hunt, 1997). The corresponding expression of the receptor indicates the potential for functional netrin-DCC signaling in the cell bodies or

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dendritic fields of adult ventral tier dopamine neurons. However, netrin-1 is also expressed in the adult striatum where it could affect the terminal fields of the nigrostriatal dopamine projections. In the striatum, the localization of immunohistochemical staining for DCC conformed to the pattern predicted by the distribution of DCC-IR in ventral dopamine neurons (Fallon and Moore, 1978; Gerfen, 1985, 1992; Gerfen et al., 1987a,b; Jimenez-Castellanos and Graybiel, 1987; Langer and Graybiel, 1989; Prensa and Parent, 2001). Reconstruction of single axons projecting from cell bodies filled with biotinylated dextran amine has shown that most ventral dopamine neurons arborize profusely in the patch compartment and subcallosal streak (Prensa and Parent, 2001). We showed that DCC-IR terminals were enriched in these structures, which were identified by immunolabeling for MOR. Both nigral and striatal neurons are reported to express DCC and netrin-1 mRNAs from the earliest point in their development, and throughout the postnatal period into adulthood (Livesey and Hunt, 1997). During development, patch neurons are generated first to form a striatal primordium, an event that precedes a massive migration of matrix neurons. At this stage netrin-1 exerts a repulsive effect on these migrating neurons, which can be blocked using DCC antibodies (Hamasaki et al., 2003). However, netrin-DCC signaling can also function in attractive guidance and has been suggested to be important in the maintenance of striatonigral connections in the embryo (Livesey and Hunt, 1997). The present finding that DCC is localized in adult ventral tier dopamine neurons could reflect the expression of netrin-1 in developing patches as nigrostriatal dopamine terminals distribute in patches early in development, with the matrix being innervated subsequently (Olson et al., 1972). In adults, it has been repeatedly demonstrated that grafted dopamine neurons can form abundant and appropriate synaptic connections when inserted into the striatum. However, only a relatively small proportion of the total dopamine cell population makes these connections (Isacson et al., 2003). This suggests the continued activity of guidance factors could be important in maintaining the specificity of dopamine synaptic connections with the correct target. As we observed in the striatonigral dopamine projection, DCC-IR appeared to define specific mesolimbic dopamine projection pathways. These originate from A10 dopamine neurons, which show an inconsistent and crude topographic organization, with an inverted dorsal-to-ventral relationship between source and targets, as well as non-inverted anteriorto-posterior and medial-to-lateral topographic gradients (Björklund and Lindvall, 1984; Oades and Halliday, 1987). Notably DCC-IR was localized to dopamine terminals in the lateral habenula, which predominantly originate from the interfascicular and linear nuclei (Phillipson, 1979; Phillipson and Griffiths, 1985). Neurons in these regions also project to the accumbens, where the present study found that DCC-IR terminals were predominantly localized to the caudal dorsomedial shell—a limbic area that receives overlapping input from the deep layers of the ventral prelimbic cortex and the caudal parvicellular basal amygdala (Wright and Groenewe-

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gen, 1995; Wright et al., 1996). There is mounting evidence that drug-induced plasticity and other behavioral modifications can lead to extensive structural remodeling in striatal circuits (Parish et al., 2002; Kolb et al., 2003; Li et al., 2003). Dopamine terminals in the dorsomedial shell target GABA projection neurons containing cocaine- and amphetamineregulated transcript (Smith et al., 1999), and have been reported to be increased following repeated injections of cocaine that induce behavioral sensitization (Todtenkopf and Stellar, 2000). In conclusion, this study has determined that DCC mRNA previously detected in the adult rodent substantia nigra is mostly likely localized in ventral tier dopamine neurons and a subpopulation of calbindin-negative A10 dopamine neurons. In the developing brain, DCC and the preferred ligand netrin-1 are widely distributed and function in targeting developing axons and neurons, but in adult brain both DCC and netrin-1 have a limited distribution, the functional significance of which is not yet known. The presence of DCC in populations of adult dopamine neurons that express axonal degeneration and/or remodeling suggests further investigation of the possible function of netrin-1/DCC signaling in these neurons is warranted. Acknowledgments—This work was supported by NHMRC project grants 157158 (P.B.O) and 102447 (J.R.K); NHMRC fellowships 157213 (J.R.K) and 157212 (G.M.H), NHMRC block grant (WEHI; H.C.) and a grant from Spinalcure Australia (Roads and Traffic Authority, NSW; J.R.K). We thank Mr. Craig Thomas for expert technical assistance.

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(Accepted 4 November 2004)