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[D-Ala2]Deltorphin I-Like Immunoreactivity in the Adult Rat Brain

[D-Ala2]Deltorphin I-Like Immunoreactivity in the Adult Rat Brain

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9635-9639, October 1993 [D-Ala2]Deltorphin I-like immunoreactivity in the adult rat : Immunohistochemical localization (amphibian skin peptides/&opioid receptors//calbindin) IKUO TOOYAMA*, HIROMICHI ABE*t, TINDARO RENDAt, VITTORIO ERSPAMER§, AND HIROSHI KIMURA*$ *Institute of Molecular Neurobiology and tDepartment of Ophthalmology, Shiga University of Medical Science, Otsu, 520-21 Japan; and Institutes of tHuman and Medical Pharmacology, University "La Sapienza," 00161 , Contributed by Vittorio Erspamer, July 7, 1993

ABSTRACT Using a specific antiserum recently raised comotor activity, sniffing, rearing, grooming (10), as well as against [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val- facilitation of social contacts (11) and memory consolidation Val-Gly-NH2), a highly selective ligand for 6-opioid receptors, (12). we have previously demonstrated the occurrence of positive We have recently produced a polyclonal anti-DADTI se- immunostaining in several structures of mouse brain. We rum, which specifically recognizes its carboxyl-terminal tet- describe here the neuroanatomical distribution patterns of rapeptide region (13). Immunohistochemical studies of DADTI-immunoreactive neuronal bodies, , and tany- mouse brain have detected DADTI-immunoreactive (IR) cytes in rat brain. Positive neuronal somata were localized neuronal cells in the and positive nerve mainly in the ventral mesencephalon, including the ventral fibers in the accessory and neostriatum. tegmental area and the of the . This study was undertaken to elucidate the neuroanatom- A minor population of positive somata was found in the pars ical distribution patterns of DADTI immunoreactivity in the reticulata and pars lateralis of the substantia nigra, raphe rat brain. nuclei, supramammillary nucleus, and retrorubral reticular nucleus. All these regions, except for the supramammillary nucleus, contain dopamine cell bodies. Intensely stained posi- MATERIALS AND METHODS tive nerve fibers could be traced along the medial Production and Characterization of DADTI Antiserum. The bundle. Dense positive terminals were seen in the neostriatum, DADTI antiserum was raised in the rabbit as described by shell, , septal areas, Abe et al. (13) using as antigen synthetic DADTI conjugated cingulate, and medial prefrontal cortex. Double-immunostain- to poly-L-glutamate with water-soluble carbodiimide. Immu- ing study revealed that, in the substantia nigra, almost all nospot and immunoabsorption assays designed to test spec- (97.8%) DADTI-positive neurons colocalized with tyrosine ificity of the antiserum showed that it reacted specifically hydroxylase (TH), and the doubly stained cells occupied about with the amidated carboxyl-terminal pentapeptide: Phe-Asp- one-third (29.1%) of the total population of TH-positive neu- Val-Val-Gly-NH2. The antiserum, therefore, possessed a rons. Only a few DADTI/TH-positive cells also stained for marked cross-reactivity not only with the [L-Ala2]DADTI 28-kDa calbindin D, although many neurons double-stained for stereoisomer but also with DADTI analogs having the D-ala- 28-kDa calbindin D and TH. In contrast, the supramammillary nine residue substituted by D-leucine, D-aspartate, glycine, nucleus contained a number of DADTI-positive cells, which aminobutyric acid, or sarcosine. The antiserum did not nearly always stained positively for 28-kDa calbindin D but did cross-react with (i) DADTI analogs shortened at the carboxyl not stain for TH. The association of DADTI-like immunore- terminus, (ii) deamidated DADTI, (iii) DADTI analogs hav- activity with certain seems of partic- ing the Asp4 residue substituted by glutamate (as in ular interest. A small population of DADTI-immunostained [D-Ala2]deltorphin II) or D-aspartate, asparagine, or alanine, tanycytes was present in the ventral part of the third ventricle and (iv) Phyllomedusa sauvagei deltorphin (Tyr-D-Met-Phe- wall. His-Leu-Met-Asp-NH2). Similarly, no cross-reaction was observed with dermorphins or with any of the classical [D-Ala2]Deltorphin I (DADTI), an opioid peptide recently mammalian opioid peptides or other common brain neu- isolated from the skin of the South American frog Phyllo- ropeptides. The DADTI sequence has little homology with medusa bicolor, displays high affinity and selectivity for known peptides or proteins other than those of the dermor- 8-opioid receptors (1, 2). cDNAs encoding deltorphin pre- phin/deltorphin family. cursors have been cloned from the skin of the same amphib- Tissue Preparation. Adult male Wistar rats, weighing 200- ian species (3). The precursors contained three DADTI 250 g, were used for the study. Under deep anesthesia with sequences and only a single [D-Ala2]deltorphin II sequence. Nembutal (50 mg/kg, i.p., Abbott), each animal was perfused Because its L-isomer is pharmacologically inactive (1), D-ala- via the left ventricle with 0.01 M phosphate-buffered saline nine or at least a D-amino acid in the second position appears (pH 7.4), followed by an ice-cold fixative containing 4% essential to its opiate-like activity. In turn, the carboxyl- (wt/vol) paraformaldehyde, 0.2% picric acid, and 0.35% terminal region of deltorphins seems crucial for addressing glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brain the molecules toward the 6-type receptor (4-8). Recently, was quickly removed from the skull and sliced into 5- to 6-mm DADTI-binding sites have been demonstrated by autoradi- coronal blocks, which were postfixed for 2 days in a post- ography with either 125I-labeled DADTI in mouse brain (9) or fixative containing 4% paraformaldehyde and 0.2% picric [3H]DADTI in rat brain (unpublished work). DADTI or acid in 0.1 M phosphate buffer at 4°C. After cryoprotection [D-Ala2]deltorphin II injected into the rat cerebral ventricle by placing the tissue for 2 days in 0.1 M phosphate buffer/ induces several central effects, including stimulation of lo- 15% sucrose/0.1 M sodium azide at 4°C, each block was cut

The publication costs of this article were defrayed in part by page charge Abbreviations: DADTI, [D-Ala2]deltorphin I; TH, tyrosine hydroxy- payment. This article must therefore be hereby marked "advertisement" lase; CaBP, 28-kDa calbindin D; IR, immunoreactive. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. 9635 Downloaded by guest on September 23, 2021 9636 Pharmacology: Tooyama et al. Proc. Natl. Acad. Sci. USA 90 (1993) into 20-,um-thick coronal sections in a cryostat (Yamato, for 1-2 days at 4°C. The antibody binding was detected Tochigi, Japan). The sections were rinsed for at least 4 days similarly to the first cycle, except that nickel ammonium with several changes of 0.1 M phosphate-buffered saline/ sulfate was eliminated from the 3,3'-diaminobenzidine solu- 0.3% Triton-X 100 (PBST). To improve antibody penetration, tion, yielding a yellow-brown reaction product. some sections were treated with protease papain (0.1 inter- national unit per ml of PBST, 10 min at 37°C). Immunohistochemical Procedure. Free-floating sections RESULTS were incubated for 2-3 days at 4°C with the DADTI antiserum (diluted 1:5000-10,000), for 1 hr at room temperature with DADTI immunoreactivity was observed in neurons and biotinylated anti-rabbit IgG (diluted 1:1000, Vector), and for tanycytes. Fig. 1 presents maps of DADTI-IR neuronal 1 hr at room temperature with avidin-biotin-peroxidase somata and their processes in seven schematic drawings of complex (diluted 1:4000, ABC Elite, Vector). All sera were coronal sections, with reference to the atlas of Paxinos and diluted with PBST, and sections were always rinsed in PBST Watson (15). Typical examples are shown with micrographs after each step. Peroxidase activity was revealed by 0.02% in Figs. 2 and 3. 3,3'-diaminobenzidine (Wakenyaku, Kyoto, Japan) in 0.05 M Localization ofDADTI-IR Cell Bodies. DADTI-IR neuronal Tris HCl buffer, pH 7.6/0.005% H202/0.3% nickel ammo- somata were localized mainly in the ventral part of the nium sulfate. The stained sections were mounted on glass , including the slides, dehydrated, and cleared; coverslips were applied with and the pars compacta of the substantia nigra (Figs. 1 and 2 Entellan. Control experiments included the omission of the A and B). In the pars compacta, positive somata were primary antiserum or its substitution with either buffer or distributed throughout the rostrocaudal extent of the struc- preimmune antiserum or preabsorbed serum (13). None of ture, but in a coronal plane they were almost always confined the controls showed specific staining. to the ventral tier (Fig. 2A). Positive cell bodies were also Sections were double-immunostained for DADTI and ty- scattered in the supramammillary nucleus (Fig. 1E), the rosine hydroxylase (TH) or 28-kDa calbindin D (CaBP), retrorubral reticular nucleus (so-called A8 region, Fig. 1G), according to Akiyama et al. (14). In brief, after completion of the caudal linear nucleus ofraphe (Fig. 1G), the rostroventral the first 3,3'-diaminobenzidine/nickel reaction for DADTI, part of the raphe dorsalis (Fig. .G), and both the pars yielding a blue-purple reaction product, sections were treated reticulata and the pars lateralis of the substantia nigra (Fig. with 0.5% H202/PBST for 30 min to destroy any residual 2A). Around these positive cells, many dendritic or axonal peroxidase activity. Sections were then incubated for the processes were clearly observed branching into the pars second cycle with mouse monoclonal antibodies to TH reticularis (Fig. 2B). Most positive axons projected medially (diluted 1:40,000, Chemicon) or CaBP (diluted 1:5000, Sigma) to join the medial forebrain bundle (Fig. 1 C and D). A BC Co

FIG. 1. Schematic mapping of DADTI-IR cells (0) and fibers (-). Levels ofbregma correspond to those in ref. 15. AcC, nucleus accumbens core; AcS, nucleus accumbens shell; A8, A8 region (retrorubral reticular nucleus); Cg, ; Cl, claustrum; CLi, caudal linear raphe nucleus; CP, caudate putamen; DR, ; f, medial forebrain bundle; IL, infralimbic cortex; LHb, lateral habenular nucleus; LSI, lateral septal nucleus, intermediate; PF, medial prefrontal cortex; SNc, substantia nigra, pars compacta; SNr, substantia nigra, pars reticularis; Su, supramammillary nucleus; Tu, olfactory tubercle; tv, third ventricle, tanycytes; VTA, ventral tegmental area. Downloaded by guest on September 23, 2021 Pharmacology: Tooyama et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9637

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FIG. 2. Photomicrographs of DADTI-IR cells in ventral midbrain tegmentum. (A) DADTI-IR neurons in pars compacta of substantia nigra and ventral tegmental area. (B) Higher magnification ofboxed area in A. (C) Double immunostaining for DADTI (blue) and TH (yellow-brown) in pars compacta of substantia nigra. (D) Double immunostaining for DADTI (blue) and CaBP (yellow-brown) in pars compacta of substantia nigra. (E) Double immunostaining for DADTI (blue) and TH (yellow-brown) in supramammillary nucleus. (F) Double immunostaining for DADTI (blue) and CaBP (yellow-brown) in supramammillary nucleus. SNc, substantia nigra, pars compacta; SNr, substantia nigra, pars reticularis; Su, supramammillary nucleus; MM, medial mammillary nucleus. (Bars = 50 gm.) Double immunostaining in the substantia nigra showed that prefrontal (Figs. 1A and 3G), and anterior cingulate (Fig. 1B) almost all DADTI-IR neurons colocalized with TH (Fig. 2C). cortical areas. In the anterior cingulate cortex the fiber These double-stained cells, always located near the ventral density decreased caudally, and positive staining did not edge of the pars compacta, were readily distinguishable from extend to the posterior cingulate cortex (Fig. 1 D and E). The the more dorsally situated cells, which stained singly for TH. claustrum, situated near the frontal cortex, contained only a On the other hand, very few neurons double-stained for few (Fig. 1 A-C). Other cortical regions including the sulcal DADTI and CaBP. Cells singly labeled for DADTI were cortex surrounding the sulcus rhinalis contained no positive situated exclusively in the ventral tier of the pars compacta, fibers. whereas CaBP-IR cells were distributed mostly in the dorsal The neostriatum exhibited a unique pattern of fiber distri- tier of the pars compacta (Fig. 2D). bution. Just beneath the corpus callosum or the external In a semiquantitative analysis, TH-IR cells were over three DADTI-IR were in the times more numerous than DADTI-IR cells (2771 vs. 825), capsule, very dense fibers present and as many as 97.8% (807) of DADTI-IR cells colocalized dorsolateral rim of the (Figs. 1 A-C and 3A). The with TH. In contrast, 92.3% (798) ofDADTI-IR neurons were positive fibers were distributed more densely in the rostro- CaBP negative, even though CaBP-IR cells were -60% dorsal than in the caudoventral parts of the striatum (Fig. 1 (1274) more numerous than DADTI-IR cells. A-C). At higher magnifications, the fiber networks in the In the supramammillary nucleus the opposite occurred: rostral striatum appeared to be composed of numerous fine almost all DADTI-IR neurons colocalized with CaBP (Fig. axons and terminal-like dots, intermingled with a few larger 2F), but very few co-stored TH (Fig. 2E). terminal boutons (Fig. 3B). In the ventral parts of the Localization of DADTI-IR Nerve Fibers. Positive fibers striatum, many DADTI-IR axons passed through the white were moderately dense in the infralimbic (Fig. 1A), medial matter bundles, probably indicating the route ofmesocortical Downloaded by guest on September 23, 2021 9638 Pharmacology: Tooyama et al. Proc. Natl. Acad. Sci. USA 90 (1993)

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FIG. 3. Photomicrographs of DADTI-IR fibers and terminals in neostriatum (A and B), nucleus accumbens shell (A and C), septum (A and D), rostral (E) and caudal (F) part of the olfactory tubercle, medial prefrontal cortex (G), and lateral nucleus (H). B, C, and D are higher magnifications of areas indicated in A by respective arrows. LHb, lateral habenular nucleus; MHb, medial habenular nucleus. (Bars = 500 ,tm in A, 25 ,um in B-D, and 100 ,um in E-H.) projections (Fig. 3A). In the nucleus accumbens, the rostro- observed in the and the , which dorsal shell of the nucleus was packed with extremely dense are structures having afferent inputs from the ventral mes- DADTI-IR structures consisting mainly of terminal-like tiny encephalon. dots (Figs. 1 A and B and 3 A and C), whereas the core of the Tanycytes. The ventral part of the third ventricle wall, near nucleus contained only a few fine positive fibers and termi- the infundibulum, contained intensely stained DADTI-IR nals (Fig. 3A). somata of tanycytes (Fig. 1D). From the somata thick pro- Although the medial septum had few DADTI-IR struc- cesses spread ventrolaterally to reach the brain surface. tures, a number ofpositive fibers were scattered in the lateral Although some of these processes traversed the arcuate septal nucleus. Particularly in the intermediate part of the nucleus, no positive neurons were seen within the nucleus. nucleus, terminal-like positive dots were situated close to immunonegative neuronal somata, implying the presence of DISCUSSION axosomatic contacts (Fig. 3D). In the different subdivisions of the olfactory tubercle, Our results do not show that the genuine frog skin peptide DADTI-IR fibers were distributed with sharply different [D-Ala2]deltorphin I actually exists in the central nervous densities. The rostral tubercle exhibited plenty of positive system ofmammals. But they do show that DADTI antiserum fibers and terminals (Fig. 3E), whereas the caudal tubercle clearly stains neuronal somata and fibers in precise areas of contained almost none (Fig. 3F). A moderate density of the rat central nervous system. positive fine fibers and terminals was observed in the lateral It is certainly possible that the DADTI antiserum recog- but was not seen in the medial, habenular nucleus (Figs. 1 C nizes not only the amidated active deltorphin heptapeptide and D and 3H). Finally, no or few DADTI-IR fibers were but also peptides deriving from the processing of a precursor Downloaded by guest on September 23, 2021 Pharmacology: Tooyama et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9639 that could still contain the L-alanine residue instead of the derive from the positive cells situated near the midline ofthe D-alanine residue. This hypothesis could explain our failure ventral tegmental area. in preliminary experiments to detect deltorphin-like activity Recently, the heterogeneity ofmidbrain dopaminergic neu- in mammalian brain extracts by bioassay. The final, unequiv- rons has been defined immunocytochemically with antibod- ocal demonstration of the occurrence of deltorphins (and the ies against, for example, cholecystokinin (23), neurotensin same is true for the dermorphins) in mammalian nervous (24), and 27-kDa basic fibroblast growth factor-like protein system will, of course, be afforded by the demonstration of (25). The colocalization pattern of dopamine, in various the existence and expression of the related gene, as in combinations, with these peptides or proteins may help to skin. characterize specific pathways in the mesencephalic dopa- amphibian minergic system. Except in the supramammillary nucleus, With this reservation, this study presents a map of the the DADTI-like molecule colocalized in a subpopulation of distribution patterns of DADTI-IR cell components in the TH-positive but CaBP-negative dopaminergic neurons ofthe adult rat brain. Except for a few tanycytes, the major positive midbrain. Interestingly, CaBP-negative dopaminergic neu- cellular components are neurons. Positive neuronal somata rons are now known to be particularly vulnerable in - are confined to a limited region of the midbrain, and their son disease (26). axons give rise to trajectories that project toward certain The significance of DADTI-like immunostaining in a small forebrain regions. Despite being less abundant, the overall population of tanycytes is obscure. anatomical distribution of the DADTI-IR neuronal system For the sake of completeness we add the information that closely resembles that of the mesencephalic dopaminergic intense DADTI-like immunoreactivity has also been ob- system. In other words, DADTI-IR neurons appear to par- served in a subpopulation of amacrine cells of the rat retina ticipate in the composition of all four subgroups of dopami- and in the mouse vomeronasal/accessory nergic pathways represented by the nigrostriatal, mesolim- (unpublished results). bic, mesocortical, and mesothalamic projections (16-18). The patchy distribution of DADTI-IR nerve fibers in the This paper was partially supported by the Italian Research Council striatum coincides well with that of dopaminergic striosomes (Consiglio Nazionale delle Ricerche, bilateral project T.R./H.K.), Uehara Medical Foundation (I.T.), Sasagawa Medical Research or neostriatal patches. The dopaminergic fibers belonging to Foundation (I.T.), Shiga International Cooperation for Medical the nigrostriatal pathways originate from neuronal somata Research (I.T.), and Grant in Aid by the Ministry of Education, situated in a ventral tier ofthe pars compacta ofthe substantia Science and Culture of Japan (H.K.). nigra (18-21). The results of our double-immunostaining studies clearly demonstrated that virtually all the DADTI-IR 1. Erspamer, V., Melchiorri, P., Falconieri-Erspamer, G., Negri, L., Corsi, R., Severini, C., Barra, D., Simmaco, M. & Kreil, G. (1989) Proc. Natl. somata located in the nigral ventral tier possess the dopami- Acad. Sci. USA 86, 5188-5192. nergic index TH. Thus, DADTI-like molecule(s) may be an 2. Erspamer, V. (1992) Int. J. Dev. Neurosci. 10, 3-30. intimate partner of dopamine in the striosome-forming ni- 3. Richter, K., Egger, R., Negri, L., Corsi, R., Severini, C. & Kreil, G. grostriatal pathway. In the striatum, most DADTI-IR fibers (1990) Proc. Natl. Acad. Sci. USA 87, 4836-4839. 4. Lazarus, L. H., Salvadori, S., Tomatis, R. & Wilson, W. E. (1991) are fine; only a few display large terminal boutons. Because Biochem. Biophys. Res. Commun. 178, 110-115. dopaminergic large terminals make asymmetric synapses 5. Lazarus, L. H., Salvadori, S., Santagada, V., Tomatis, R. & Wilson, mainly on spines of striatal cells (22), a minor population of W. E. (1991) J. Med. Chem. 34, 1350-1355. 6. Lazarus, L. H., Salvadori, S., Balboni, G., Tomatis, R. & Wilson, W. E. the DADTI-IR fibers may exert an excitatory action on (1992) J. Med. Chem. 35, 1222-1227. striatal neurons via the large boutons with asymmetric syn- 7. Melchiorri, P., Negri, L., Falconieri-Erspamer, G., Severini, C., Corsi, R., Soaje, M., Erspamer, V. & Barra, D. (1991) Eur. J. Pharmacol. 195, apses. Further studies by immunoelectron microscopy are 201-207. required for a more precise understanding ofthe synaptology 8. Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Kramer, T. H. of DADTI-IR terminals. & Yam Hruby, V. J. (1992) Life Sci. 51, 1025-1032. In most terminal fields known as the dopaminergic me- 9. Dupin, S., Tafani, J. A., Marzaguil, H. & Zajac, J. M. (1991) Peptides 12, solimbic system, the distribution of DADTI-IR fibers and 825-830. 10. Longoni, R., Spina, L., Mulas, A., Carboni, E., Garau, L., Melchiorri, terminals resembles that of the dopamine-containing fibers. P. & Di Chiara, G. (1991) J. Neurosci. 11, 1565-1576. These fields include the nucleus accumbens shell, the lateral 11. Negri, L., Noviello, V. & Angelucci, F. (1991) Eur. J. Pharmacol. 209, , and the rostral part of the olfactory tubercle. The 163-168. 12. Pavone, F., Populin, R., Castellano, C., Kreil, G. & Melchiorri, P. (1990) pattern of distribution of DADTI-IR fibers also matches well Peptides 11, 591-594. that of 8-opioid-binding sites in mouse (9) and rat (unpub- 13. Abe, H., Tooyama, I., Renda, T., Erspamer, V. & Kimura, H. (1992) lished work). In the nucleus accumbens, the motor stimulant NeuroReport 3, 669-672. 14. Akiyama, H., Itagaki, S. & McGeer, P. L. (1988) J. Neurosci. Res. 20, effect evoked by intraaccumbens injection of [D-Ala2]del- 147-157. torphin II was antagonized by the Dl-receptor blocker SCH 15. Paxinos, G. & Watson, C. (1986) The Rat Brain in Stereotaxic Coordi- 23390 (10). In the mesolimbic system of mammals, DADTI- nates (Academic, San Diego). like molecule(s) may, therefore, cooperate with dopamine 16. Fallon, J. H. & Moore, R. Y. (1978) J. Comp. Neurol. 180, 533-544. through mechanisms involving S-opioid receptors. 17. Fallon, J. H. & Moore, R. Y. (1978) J. Comp. Neurol. 180, 545-580. 18. Fuxe, K., Agnati, L. F., Kalia, M., Goldstein, M., Andersson, K. & Among the cortical regions reported to receive dopami- Harfstrand, A. (1985) The Dopaminergic System (Springer, Berlin), pp. nergic inputs, the prefrontal, infralimbic, and anterior cingu- 11-25. late cortices possess DADTI-IR fibers, whereas the supra- 19. Fallon, J. H., Riley, J. N. & Moore, R. Y. (1978) Neurosci. Lett. 7, rhinal cortex, amygdala, and hippocampus do not. This 157-162. 20. Gerfen, C. R., Herkenham, M. & Thibault, J. (1987) J. Neurosci. 7, absence may be explained by the observation that no 3915-3934. DADTI-IR somata occur in the dorsal tier of the substantia 21. Gerfen, C. R., Baimbridge, K. G. & Thibault, J. (1987) J. Neurosci. 7, nigra or the ventral tegmental area from which mesocortical 3935-3944. afferents to the latter regions originate (18, 19). 22. Kaiya, H. & Namba, M. (1981) Neurosci. Lett. 25, 251-256. 23. Hokfelt, T., Skirboll, L., Rehfeld, J. F., Goldstein, M., Markey, K. & The mesothalamic dopaminergic projection has been di- Dann, 0. (1980) Neuroscience 5, 2093-2124. vided into two pathways: one running from the intrafascicular 24. Hokfelt, T., Everitt, B. J., Theodorsson-Norheim, E. & Goldstein, M. nucleus to the medial habenula and the other running from the (1984) J. Comp. Neurol. 222, 543-559. 25. Tooyama, I., Walker, D., Yamada, T., Hanai, K., Kimura, H., McGeer, medial paranigral nucleus to the lateral habenular nucleus E. G. & McGeer, P. L. (1992) Brain Res. 593, 274-280. (18). We found DADTI-IR fibers only in the lateral, and not 26. Yamada, T., McGeer, P. L., Baimbridge, K. G. & McGeer, E. G. (1990) in the medial, habenular nucleus. These fibers appear to Brain Res. 526, 303-307. Downloaded by guest on September 23, 2021

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