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A subpopulation of dopaminergic neurons in rat ventral mesencephalon contains both neurotensin and cholecystokinin
Brain Research, 455 (1988) 88-98 Elsevier
88 BRE 13753
A subpopulation of dopaminergic neurons in rat ventral mesencephalon contains both neurotensin and cholecystokinin K. Seroogy 1. , S. Ceccatelli 1, M. Schalling 1, T. H6kfelt 1, P. Frey 2, J. Walsh 3, G. Dockray 4, J. Brown 5, A. B u c h a n 5 a n d M. G o l d s t e i n 6 t Department of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden), 2Sandoz Research Institute, Bern (Switzerland), 3Centerfor Ulcer Research and Education, VA Medical Center-Wadsworth, Los Angeles, CA (U.S.A.), 4The Physiological Laboratory, University of Liverpool, Liverpool (U. K.), 5Medical Research Council of Canada, Regulatory Peptide Group, University of British Columbia, Vancouver, BC (Canada) and 6Department of Psychiatry, New York University Medical Center, New York, NY (U.S.A.) (Accepted 2 February 1988)
Key words: Neuropeptide; Tyrosine hydroxylase; Colocalization; Ventral tegmental area; Immunohistochemistry
The coexistence of the neuropeptides neurotensin and cholecystokinin and the catecholamine-synthesizing enzyme tyrosine hydroxylase within neurons of the ventral mesencephalon was analyzed using an immunofluorescence triple-labeling technique. Virtually all of the neurotensin-positive cell bodies in the ventral tegmental area, medial substantia nigra pars compacta, retrorubral field, and rostral and caudal linear raphe nuclei were found to contain both cholecystokinin and tyrosine hydroxylase immunoreactivities. The degree of colocalization was lower and more variable in other regions including the ventral and central periaqueductal grey matter and dorsal raphe nucleus. It appeared that immunoreactivities for these 3 neuroactive substances were not contained within the same axonal-like fibers and terminals in the ventral midbrain. These results demonstrate that a subpoputation of dopaminergic neurons, which presumably comprise part of the ascending mesotelencephalic system, contains the two peptides neurotensin and cholecystokinin. Thus, the data suggest a morphological basis for some of the reported functional interactions of these 3 putative neurotransmitters/neuromodulators within this system.
INTRODUCTION In addition to the well-known distribution of dopaminergic neurons throughout the ventral mesencephalon 1'3'6'12, the neuropeptides cholecystokinin (CCK) and neurotensin (NT) have been localized within neuronal perikarya in this region 10"16"18"43'44. These studies have shown that CCK-containing somata are widely distributed throughout the ventral tegmental area, substantia nigra pars compacta and pars lateralis and several midline nuclei. N T cell bodies encompass a relatively smaller population of neurons restricted predominantly to the ventral tegmental area, although a few cells are found in the substantia nigra pars compacta and various raphe nuclei. It has been determined with immunocytochem-
ical 1°'33 and biochemical 9'21'39'4°'47 techniques that many of the CCK cells of the ventral midbrain comprise a subpopulation of the dopaminergic neurons. Virtually all of the N T neurons throughout this region have been shown to contain immunoreactivity for tyrosine hydroxylase 11, the synthesizing enzyme for catecholamines. By using a double-labeling indirect immunofluorescence procedure, we have recently demonstrated 3t the coexistence of CCK and N T within neurons of the rat ventral mesencephalon. It was found that more than 90% of the NT-positive neurons (primarily located in the ventral tegmental area) also contained CCK immunoreactivity. Based on these results, it was postulated that neurons in the ventral midbrain containing both peptides may also contain
* Present address: Department of Physiology, University of North Carolina, Chapel Hill, NC 27599, U.S.A. Correspondence: T. H6kfelt, Department of Histology and Neurobiology, Karolinska Institute, S-104 01 Stockholm, Sweden. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
89 the monoamine dopamine. The present study was conducted to test this hypothesis and thereby provide an anatomical basis for some of the reported functional interactions 3"29 of these 3 neuroactive substances in the ventral mesencephalon. In addition, a more extensive analysis of the extent of coexistence of these compounds in periaqueductal and raphe regions was undertaken. MATERIALS AND METHODS Adult male albino rats (200 g; ALAB, Stockholm, Sweden) were used in these experiments. The animals were anesthetized with chloral hydrate and stereotaxically injected with colchicine into a lateral ventricle (120/tg in 20/~10.9% NaC1) or both intracerebroventricularly and locally (1.8 ~g in 0.3/A NaCI), approximately 2 mm dorsal to the substantia nigraventral tegmental area complex. After 24-48 h, the rats were re-anesthetized and perfused via the ascending aorta with 50 ml warm (37 °C) calcium-free Tyrode's solution, then 50 ml warm 0.3% picric acidformalin 4s in 0.1 M phosphate buffer (PB), followed by the same ice-cold fixative for 6 min. The brains were removed, immersed in ice-cold fixative for 90 min, and placed in 10% sucrose in PB overnight at 4 °C. Coronal sections (14/~m) through the ventral mesencephalon were cut in a cryostat (Dittes, Heidelberg, F.R.G.), thaw-mounted onto gelatin-coated glass slides, and rinsed in phosphate buffered saline (PBS). The sections were then processed for the simultaneous detection of NT and CCK using a modification of the indirect immunofluorescence technique 5. Briefly, the sections were incubated with a mixture of rabbit anti-NT antiserum (diluted 1:400) and mouse monoclonal anti-CCK antibody (1:400) or, alternatively, with a mixture of rabbit anti-CCK antisera (1:400) and mouse monoclonal anti-NT antibody (1:100) 30a. Following an incubation period of 18-24 h at 4 °C in a humid chamber, the sections were rinsed in PBS and then incubated with a mixture containing tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit immunoglobulins (IgG) (1:40; Boehringer Mannheim Scandinavia, Stockholm, Sweden) and either fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or sheep anti-mouse IgG (both 1:40; Boehringer Mann-
heim and Amersham, Amersham, U.K., respectively) for 30 min at 37 °C. The sections were then rinsed in PBS, coverslipped in a 3:1 solution of glycerol: PBS containing 0.1% p-phenylenediamine 17.28, and examined with a Zeiss standard fluorescence microscope equipped with an oil darkfield condensor and proper filter combinations. After photography, the sections were processed according to the elution/restraining technique 42 as described in detail elsewhere 1°. Briefly, the antibodies were eluted with a solution of potassium permanganate and hydrogen sulfate, and the sections then reincubated in the secondary antisera cocktail described above to check for absence of immunofluorescence and thus assure complete elution of the antibodies. Sections were then processed for tyrosine hydroxylase (TH) immunoreactivity using a rabbit antiTH antiserum (1:400) 20 and FITC- or TRITClabeled goat anti-rabbit IgG secondary antisera. Photographs of the NT/CCK double-labeling were compared with those of the TH immunostaining in the appropriate areas to examine for coexistence. In control experiments, preabsorption of the CCK/NT primary antisera mixture with 10/~M CCK peptide eliminated the staining for CCK but did not affect the anti-NT staining. Similarly; preabsorption with 10/~M NT resulted in absence of immunofluorescence for NT but did not block the CCK immunostaining. Controls for secondary antisera species specificity and possible cross-reactivity, as described in detail previously31, consisted of incubation of tissue with each of the primary antisera separately, followed by the mixture of both secondary antisera. In each instance, only immunofluorescence from the appropriate secondary antiserum was observed. Replacement of the TH antiserum with normal rabbit serum resulted in the absence of all T H immunostaining. Omission of both primary antisera from the initial incubation step followed by processing with the combined secondary antisera mixture resulted in the complete absence of all immunofluorescence under both FITC and TRITC filters. RESULTS Numerous CCK-like immunoreactive (CCK-I) cell bodies were observed throughout the rostrocaudal extent of the ventral mesencephalon, as de-
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Fig. 1. Low-magnification immunofluorescence photomontages of the same section through the ventral mesencephalon illustrating the colocalization of NT-I (a, FITC filter), CCK-I (b, TRITC filter) and TH-I (c, FITC filter after elution/restaining procedure). Examples of neurons triple-labeled with all 3 substances are indicated by arrows. Note that virtually all of the NT-I cells contain both CCK-I and TH-I and the higher degree of CCK-I/TH-I coexistence. The boxed-in region of the ventral tegmental area in (a) and the corresponding areas in (b) and (c) are shown at higher magnification in Fig. 2. Note the differential distribution of especially NT and CCK immunoreactive terminals in the interpeduncular nucleus (IPN). Bar = 100/~m.
91 scribed in detail in previous reports 1°'32. Briefly, high numbers of CCK-I perikarya were found in the ventral tegmental area (A10 cell group region according to the terminology of Dahlstr6m and Fuxe6), rostral and intermediate levels of the substantia nigra pars compacta (A9), interfascicular nucleus, and ventral periaqueductal grey matter. Relatively fewer immunoreactive neurons were present in the rostral and caudal linear raphe nuclei, central periaqueductal grey matter, dorsal raphe nucleus, substantia nigra pars lateralis, substantia nigra pars compacta at caudal levels, retrorubral field (A8), and the peripeduncular region. NT-like immunoreactive (NT-I) perikarya were present in much fewer numbers in several of the same regions throughout the ventral mesencephalon including the ventral tegmental area, medial substantia nigra pars compacta (at rostral levels), rostral and caudal linear nuclei, central and ventral periaqueductal grey matter, retrorubral region, lateral aspects of the substantia nigra pars compacta, and in peripeduncular region. Double-labeling analysis revealed that the vast majority of the NT-I neurons (located for the most part in the ventral tegmental area) also contained immunoreactivity for CCK (Figs. la,b; 2a,b). The results were the same regardless of which combination of primary antisera mixture was used. These NT-I/CCK-I neurons were situated in both the parabrachialis pigmentosus and paranigralis subdivisions of the ventral tegmental area, although more frequently in the former. At more rostral levels of the ventral mesencephalon, these double-labeled cell bodies occupied a more dorsal position in the ventral tegmental area, whereas more caudally they were characteristically observed intermingled among the rootlets of the oculomotor nerve (III). Following the elution/restraining procedure, it was found that virtually all of the perikarya in this region containing both NT and CCK immunoreactivities also exhibited immunostaining for TH (Figs. l a - c ; 2a-c). Much greater numbers of neurons contained immunoreactivities for both CCK and T H but not NT. Very infrequently, a neuron displaying NT-I and TH-I but not CCK-I could be found. Somata in other regions of the ventral midbrain were also observed to contain immunoreactivities for both of the peptides as well as for the catecholamine-
Fig. 2. High-power magnifications of the boxed-in region in Fig. la and the corresponding areas in lb and lc demonstrating the perikaryal colocalization of NT-I (a), CCK-I (b) and TH-I (c) in the ventral tegmental area. Arrows indicate examples of the triple-labeled neurons. Bar = 50 pm.
92 synthesizing enzyme. A l t h o u g h the NT-I s o m a t a located in or usually above the lateral ventral tegmental area and medial substantia nigra pars compacta at rostral levels of the ventral m e s e n c e p h a l o n typically did not contain C C K - I or T H - I , p e r i k a r y a were occasionally found in these areas i m m u n o s t a i n e d for all 3 substances (Fig. 3 d - f ) . In the lateral part of the substantia nigra pars c o m p a c t a , infrequent triplelabeled neurons were observed. H o w e v e r , a small population of NT-I cell bodies located a b o v e this re-
gion, as well as above the substantia nigra pars lateralis and in the p e r i p e d u n c u l a r region, typically did not display staining for either C C K or T H . The few NT-I p e r i k a r y a found in the retrorubral field did exhibit both CCK-I and TH-I. Although a few of the large neurons of the ventral periaqueductal grey m a t t e r (including part of the E d i n g e r - W e s t p h a l nucleus) were d o u b l e - l a b e l e d with NT-1 and CCK-I, these cells were never observed to contain T H - I (Fig. 3 a - c ) . Virtually all of
Fig. 3. (a-c) Immunofluorescence photomicrographs of the same section through the ventral periaqueductal grey matter showing large neurons (including Edinger-Westphal nucleus) displaying both NT-I (a) and CCK-I (b) (arrowheads) but not TH-I (c) in this midline group. Star denotes ventral edge of aqueduct. (d-f) Immunofluorescence photomicrographs of the same section through the substantia nigra pars compacta illustrating the colocalization of NT-I (d), CCK-I (e) and TH-I (f) within perikarya in both dorsal and ventral aspects at the rostral level of this region. Arrowheads indicate some of the triple-labeledneurons. Bar in a = 50/~m for a-c; in d = 50pm for d-f.
93 the small N T - I s o m a t a l o c a t e d in t h e m i d l i n e rostral
cular nucleus, m o r e differential p a t t e r n s o f coexis-
linear nucleus w e r e also i m m u n o s t a i n e d for b o t h
t e n c e versus n o n - c o e x i s t e n c e of N T - , C C K - , and
C C K and T H . A t levels b e g i n n i n g just c a u d a l to the i n t e r p e d u n -
T H - I w e r e o b s e r v e d . In the caudal linear nucleus, the vast m a j o r i t y of the N T - I n e u r o n s w e r e also im-
,j
Fig. 4. Immunofluorescence photomontages of the same section illustrating the extent of colocalization of NT-I (a), CCK-I (b) and TH-I (c) in juxta-aqueductal, dorsal raphe and caudal linear raphe regions. Note that, at this level, few NT-I and TH-I cells are situated in the dorsal raphe nucleus, whereas many CCK-I somata are present. Also, almost all of the NT-I perikarya in the caudal linear nucleus (a) contain both CCK-I (b) and TH-I (c). Arrows indicate examples of triple-labeled neurons. Examples of neurons doublelabeled only with CCK-I (b) and TH-I (c) but not NT-I are indicated by arrowheads. Open arrows identify examples of cell bodies single-labeled for each substance in their respective panels. Star denotes aqueduct. Bar = 50 #m.
94 munostained for CCK-I and TH-I (Fig. 4). However, a rare single-labeled NT-I perikaryon could be found (see Fig. 4a). The remaining CCK-I and TH-I somata displayed a relatively high degree of coexistence (Fig. 4b,c) with additional neurons single-labeled for each substance also present (see Fig. 4a-c). At rostral levels of the dorsal raphe nucleus, separate populations of NT-, CCK-, and T H - I cell bodies existed, although a few triple-labeled neurons were observed. In the fountain of Sheehan and in more caudal aspects of the dorsal raphe nucleus, most of the somata immunoreactive for NT also contained CCKand TH-I (Fig. 5). Again, the numbers of neurons double-labeled for both CCK-I and TH-I exceeded that of the triple-labeled cells. Immunoreactive neurons located mainly ventrally (but occasionally laterally) adjacent to the cerebral aqueduct in the central periaqueductal grey matter also displayed a differential pattern of coexistence based upon their rostrocaudal distribution throughout the mesencephalon. At rostral and intermediate levels, the few NT-I somata present were not found to be immunoreactive for either CCK or TH, whereas immunofluorescence for the latter two compounds
was often observed within the same perikarya (see Fig. 4b,c). At caudal aspects, the small immunoreactive NT cells were found to contain both CCK- and TH-I, only TH-I, or neither of these two substances (Fig. 5). It could often be observed that, in the same section, most of the larger and more numerous NT-I perikarya situated slightly more ventrally within the dorsal raphe nucleus were immunostained for both CCK and TH (Fig. 5). At these more caudal levels, virtually all of the relatively more numerous CCK-I cells exhibited T H immunofluorescence. Throughout the entire ventral mesencephalon, and especially in the ventral tegmental area, a dense to moderately dense plexus of terminal-like immunoreactivity for NT was present (see Figs. la; 2a). This punctate NT-I was usually more dense than the CCK-immunoreactive puncta observed in these regions. Although, as described above, almost all of the NT-I somata in the ventral tegmental area also contained CCK-I, immunofluorescence for both peptides could not be observed within the same puncta. However, the fact that colchicine was used may account for the apparent lack of observable CCK-I within axonal-like fibers and terminals throughout
Fig. 5. Immunofluorescence photomicrographs of the same section through caudal regions of the dorsal raphe nucleus and ventral periaqueductal regions showing the perikaryal colocalization of NT-I (a), CCK-I (b) and TH-I (c). At this level, virtually all of the NTI cells (a) within the dorsal raphe nucleus contain both CCK-I (b) and TH-I (c) as indicated by the arrows. Arrowheads in (a) and (c) identify a neuron containing NT-I (a) and TH-I (c) but lacking CCK-I (b), while examples of those exhibiting only CCK-I (b) and TH-I (c) are indicated by curved arrows in (b) and (c). Open arrow in (c) identifies a single-labeled TH-I cell. Asterisk denotes aqueduct. Bar = 50~m.
95 the ventral midbrain, and thus, a definite statement concerning axonal or terminal peptide coexistence cannot be made. Nevertheless, where terminal-like immunoreactivity for both substances was present, there appeared to be no coexistence as well as a differential or sometimes complementary pattern of immunostaining. Examples of this were best observed in the paranigralis subdivision of the ventral tegmental area and within the interpeduncular nucleus (see Fig. la,b). It should be noted, however, that at more caudal levels of the ventral midbrain (for example in the caudal linear raphe nucleus or in the fountain of Sheehan), single examples of fibers were detected containing immunoreactivity for both peptides. The pattern of TH-I fibers and puncta throughout all of the above regions generally resembled that of the NT-I staining (cf. Fig. la to lc), although, again, unequivocable demonstration of the colocalization of these two substances within puncta could not be made. DISCUSSION The present demonstration of NT/CCK coexistence within neurons of the ventral mesencephalon confirms and extends our recent findings 31. In addition, we have provided direct evidence that virtually all of the NT/CCK perikarya also contain the enzyme TH, the first enzyme in dopamine synthesis in these cells (see refs. 3, 12, 13). The results are also in good agreement with the previous immunocytochemical reports of CCK/TH 1°'33 and NT/TH 11 coexistence in rat ventral midbrain. Moreover, it has recently been shown that a very small population of dopamine neurons in the ventral tegmental area exhibit vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-immunoreactivities 34. Thus, there is now strong evidence that several heterogeneous subpopulations of dopaminergic neurons exist in the ventral mesencephalon: those containing (1) both CCK and NT, (2) CCK alone, (3) NT alone (rare), (4) both CCK and VIP/PHI (rare), (5) VIP/PHI alone (rare), or (6) none of the peptides. With the use of combined retrograde tracing and double-labeling immunocytochemistry, some of the connections of the NT/CCK neurons have been previously determined. Perikarya containing both peptides located preferentially in the ventral tegmental
area were found to innervate the nucleus accumbens, prefrontal cortex, and amygdala 31. Dopaminergic projections arising from the ventral tegmental area to the above forebrain sites are well-established (see refs. 1, 3, 41, 45). Based on the present results, it can now be firmly stated that the NT/CCK pathways are dopaminergic. Therefore, a component of the ascending mesotelencephalic system innervating at least some limbic and cortical areas contains the two peptides NT and CCK, as well as the catecholamine dopamine. Whether or not the NT/CCK/dopamine cells extend projections to additional forebrain regions, as has been shown for the CCK- or CCK/THpositive neurons of the ventral midbrain 32'33, remains to be determined. The presence of all 3 compounds within a few neurons of the rostral substantia nigra pars compacta suggests a minor NT/CCK/dopaminergic nigrostriatal pathway. In contrast to the situation in the ventral tegmental area, a differential pattern of coexistence was found in the ventral and central periaqueductal grey matter (PAG) and in the dorsal raphe nucleus at caudal levels of the midbrain. NT immunoreactivity was infrequently found in the large neurons of the midline ventral PAG (also identified as the Edinger-Westphal nucleus by Innis and Aghajanian15). When present, however, it was always colocalized with CCK. TH immunostaining was never observed in these doublelabeled cells nor in any of the larger population of intensely immunofluorescent CCK cell bodies. However, the peptide substance P has been colocalized with CCK in these neurons 37. Moreover, these CCK/substance P-containing neurons have been shown to project to the spinal cord 19'26'38. It is possible that a minor portion of this efferent pathway also contains NT. The distribution of NT somata observed in this study within the central P A G is generally in good agreement with earlier reports 2'16'35. The present resuits demonstrate that the NT perikarya located in the ventrolateral and ventromedial parts of the P A G adjacent to the aqueduct typically did not contain either CCK or TH. In contrast, almost all of the CCK cell bodies present in these same areas also exhibited TH (see ref. 33). In the region of the dorsal raphe nucleus, a rostrocaudal gradient appeared to exist, in which a higher degree of coexistence among the 3 substances was observed at more caudal levels. Thus,
96 the extent of NT/CCK/dopamine coexistence at the caudal levels is much more variable than at more rostral levels, i.e. in the ventral tegmental area. Minor CCK-containing projections from the dorsal raphe nucleus to such forebrain regions as the caudate-putamen and amygdala have been reported32; therefore, NT may constitute some portions of these pathways. On the other hand, descending projections from central P A G regions to hindbrain raphe nuclei have been shown to be NT- but not CCK-positive 2. Despite the presence of all 3 compounds within cell bodies of the ventral tegmental area and medial substantia nigra pars compacta, their colocalization within individual axonal-like fibers and terminals in these regions could not reliably be observed. Allowing for the inherent difficulty of identifying t h e same individual puncta in relatively dense fields when observed under different filters of the fluorescent microscope or even upon comparison of photographs, and for effects of colchicine treatment, it was apparent that there were often complementary patterns of especially the NT and CCK immunoreactivities. This was most evident in the interpeduncular nucleus, medial substantia nigra, ventral tegmental area, and even in ventral and central P A G regions. In fact, it appeared that the overall pattern of NT terminal-like immunostaining was more similar to that of TH than C C K . These data suggest that although some of the efferents of the ventral mesencephalon contain all 3 molecules, the majority of the NT-, CCK-, and TH-positive afferents to this area (see ref. 25 for review of ventral midbrain connections) arise from separate populations of neurons. The effects of NT or CCK upon various dopamine-
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mediated functions have been extensively reviewed elsewhere 13'22'24'31'5°.Generally, in those studies pertaining to the mesotelencephalic system, the actions of both peptides appear to be similar (see refs. 7, 8, 23, 24, 27, 30, 36, 46, 49), although differences have been noted (see ref. 31). In more recent investigations which have directly compared the effects of NT and CCK upon certain dopamine- or TH-associated activities in the same experiments and paradigms, both peptides were found to potentiate apomorphine-induced stereotypy 4, to reduce striatal T H activity TM, or to enhance the release of dopamine from the nucleus accumbens 29. Thus, these and the many previous functional studies indicate that the peptides NT and CCK modulate dopaminergic function in the ascending mesotelencephalic system. The present demonstration of the coexistence of both peptides within dopaminergic neurons which are presumed to contribute to this ascending system, provides evidence for an anatomical substrate for some of the complex interactions of these 3 neuroactive substances.
ACKNOWLEDGEMENTS This study was supported by the Swedish M R C (04X-2887), Alice och Knut Wallenberg Stiftelse, Petrus och Augusta Hedlunds Stiftelse, Fredrik och Ingrid Thurings Stiftelse, and the National Institute of Mental Health (MH 43230). K.S. was supported by a N A T O Postdoctoral Fellowship in Science and S.C. was supported by grants from the Swedish Institute and the Wenner-Gren Foundation.
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97 8 Ervin, G.N., Birkemo, L.S., Nemeroff, C.B. and Prange, Jr., A.J., Neurotensin blocks certain amphetamine-induced behaviors, Nature (Lond.), 291 (1981) 73-76. 9 Gilles, C., Lotstra, F. and Vanderhaeghen, J.-J., CCK nerve terminals in rat striatum and limbic areas originate partly in the brain stem and partly in telencephalic areas, Life Sci., 32 (1983) 1683-1690. 10 H6kfelt, T., Skirboll, L., Rehfeld, J.H., Goldstein, M . , Markey, K. and Dann, O., A subpopulation of mesencephalic dopamine neurons projecting to limbic areas contains a cholecystokinin-like peptide: evidence from immunohistochemistry combined with retrograde tracing, Neuroscience, 5 (1980) 2093-2124. 11 H6kfelt, T., Everitt, B.J., Theodorsson-Norheim, E. and Goldstein, M., Occurrence of neurotensin like immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons, J. Comp. Neurol., 222 (1984) 543-559. 12 H6kfelt, T., M~rtensson, R., Bj6rklund, A., Kleinau, S. and Goidstein, M., Distributional maps of tyrosine hydroxylase-immunoreactive neurons in the rat brain. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2: Classical Transmitters in the CNS, Part 1, Elsevier, Amsterdam, 1984, pp. 277-379. 13 H6kfelt, T., Holets, V.R., Staines, W., Meister, B., Melander, T., Schalling, M., Schultzberg, M., Freedam, J., BjOrklund, H., Olson, L., Lindh, B., Elvin, L.G., Lundberg, J.M., Lindgren, J., ~., Samuelsson, B., Pernow, B., Terenius, L., Post, C., Everitt, B. and Goldstein, M., Coexistence of neuronal messengers: an overview. In T. H6kfelt, K. Fuxe and B. Pernow (Eds.), Progress in Brain Research, Vol. 68, Elsevier, Amsterdam, 1986, pp. 33-70. 14 Hole, K., Russo, P.V. and Mandell, A.J., The influence of neuropeptides on amine synthesis: cholecystokinin-8 and neurotensin reduce striatal tyrosine hydroxylase activity, Neurochem. Int., 9 (1986) 493-498. 15 Innis, R.B. and Aghajanian, G.K., Cholecystokinin-containing and nociceptive neurons in rat Edinger-Westphal nucleus, Brain Research, 363 (1986) 230-238. 16 Jennes, L., Stumpf, W.E. and Kalivas, P.W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. Comp. Neurol., 210 (1982) 211-224. 17 Johnson, D.G. and Nogueira Araujo, G.M., A simple method of reducing the fading of immunofluorescence during microscopy, J. Immunol. Meth., 43 (1981) 349-350. . 18 Kalivas, P.W., Neurotensin in the ventromedial mesencephalon of the rat: anatomical and functional considerations, J. Comp. Neurol. , 226 (1984)495-507. 19 Maciewicz, R., Phipps, B.S., Grenier, J. and Poletti, C.E., Edinger-Westphal nucleus: cholecystokinin immunocytochemistry and projections to spinal cord and trigeminal nucleus in the rat, Brain Research, 299 (1986) 139-145. 20 Markey, K.A., Kondo, S., Shenkman, L. and Goldstein, M., Purification and characterization of tyrosine hydroxylase from a clonal pheochromocytoma cell line, Mol. Pharmacol., 17 (1980) 79-85. 21 Marley, P.O., Emson, P.C. and Rehfeld, J.H., Effect of 6hydroxydopamine lesions of the medial forebrain bundle on the distribution of cholecystokinin in rat forebrain, Brain Research, 252 (1982) 382-385. 22 Nemeroff, C.B., Hernandez, D.E., Luttinger, D., Kalivas, P.W. and Prange, Jr., A.J., Interactions of neurotensin with brain dopamine systems, Ann. N.Y. Acad. Sci., 400 (1982) 330-344.
23 Nemeroff, C.B., Luttinger, D., Hernandez, D.E., Mailman, R.B., Mason, G.A., Davis, S.D., WiderlOv, E., Freye, G.D., Kilts, C.A., Beaumont, K., Breese, G.R. and Prange, Jr., A.J., Interactions of neurotensin with brain dopamine systems: biochemical and behavioral studies, J. Pharmacol. Exp. Ther., 225 (1983) 337-345. 24 Nemeroff, C.B., The interaction of neurotensin with dopaminergic pathways in the central nervous system: basic neurobiology and implications for the pathogenesis and treatment of schizophrenia, Psychoneuroendocrinology, 11 (1986) 15-37. 25 Oades, R.D. and Halliday, F.M., Ventral tegmental (A10) system: neurobiology. I. Anatomy and connectivity, Brain Res. Rev., 12 (1987) 117-165. 26 Phipps, B.S., Maciewicz, R., Sandrew, B.B., Poletti, C.E. and Foote, W.E., Edinger-Westphal neurons that project to spinal cord contain substance P, Neurosci. Lett., 36 (1983) 125-131. 27 Pinnock, R.D., Neurotensin depolarizes substantia nigra dopamine neurones, Brain Research, 338 (1985) 151-154. 28 Platt, J.L. and Michael, A.F., Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine, J. Histochem. Cytochem., 31 (1983) 840-842. 29 Ruggeri, M., Ungerstedt, U., Agnati, L.F., Mutt, V., H~irfstrand, A. and Fuxe, K., Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat, Neurochem. Int., 10 (1987) 509-520. 30 Schneider, L.H., Alpert, J.E. and Iversen, S.D., CCK-8 modulation of mesolimbic dopamine: antagonism of amphetamine-stimulated behaviors, Peptides, 4 (1983) 749-753. 303 Seal, A., Liu, E., Buchan, A. and Brown, J., Immunoneutralization of somatostatin and neurotensin: effect on gastric acid secretion, Am. J. Physiol., in press. 31 Seroogy, K.B., Mehta, A. and Fallon J.H., Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: projections to forebrain, Exp. Brain Res., 68 (1987) 277-289. 32 Seroogy, K.B. and Fallon, J.H., Forebrain projections from cholecystokinin-like immunoreactive neurons in the rat midbrain, J. Comp. Neurol., in press. 33 Seroogy, K.B., Dangaran, K., Lim, S., Haycock, J. and Fallon, J.H., Ventral mesencephalic neurons containing both cholecystokinin- and tyrosine hydroxylase-like immunoreactivities project to forebrain regions, J. Comp. Neurol., in press. 34 Seroogy, K., Tsuruo, Y., H6kfelt, T., Walsh, J., Fahrenkrug, J., Emson, P.C. and Goldstein, M., Further analysis of presence of peptides within dopamine neurons: cholecystokinin, peptide histidine isoleucine/vasoactive intestinal polypeptide and substance P in rat supramammillary region and mesencephalon, Exp. Brain Res., in press. 35 Shipley, M.T., McLean, J.H. and Behbehani, M.M., Heterogeneous distribution of neurotensin-like immunoreactire neurons and fibers in the midbrain periaqueductal gray of the rat, J. Neurosci., 7 (1987) 2025-2034. 36 Skirboll, L.R., Grace, A.A., Hommer, D.W., Rehfeld, J., Goldstein, M., H6kfelt, T. and Bunney, B.S., Peptidemonoamine coexistence: studies of the actions of cholecystokinin-like peptide on the electrical activity of midbrain dopamine neurons, Neuroscience, 6 (1981) 2111-2124.
98 37 Skirboll, L., HOkfelt, T., Rehfeld, J., Cuello, A.C. and Dockray, G., Coexistence of substance P- and cholecystokinin-like immunoreactivity in neurons of the mesencephalic periaqueductal central gray, Neurosci. Lett., 28 (1982) 35-39. 38 Skirboll, L., H6kfelt, T., Dockray, G., Rehfeld, J., Brownstein, M. and Cuello, A.C., Evidence for periaqueductal cholecystokinin-substance P neurons projecting to the spinal cord, J. Neurosci., 3 (1983) 1151-1157. 39 Studler, J.M., Simon, H., Cesselin, F., Legrand, J.C., Glowinski, J. and Tassin, J.P., Biochemical investigation on the localization of the cholecystokinin octapeptide in dopaminergic neurons originating from the ventral tegmental area, Neuropeptides, 2 (1981) 131-139. 40 Studler, J.M., Reibaud, M., Tramu, G., Blanc, G., Glowinski, J. and Tassin, J.P., Pharmacological study on the mixed CCK8/DA meso-nucleus accumbens pathway: evidence for the existence of storage sites containing the two transmitters, Brain Research, 298 (1984) 91-97. 41 Swanson, L.W., The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat, Brain Res. Bull., 9 (1982) 321-353. 42 Tramu, G., Pillez, A. and Leonardelli, J., An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry, J. Histochem. Cytochem., 26 (1978) 322-324. 43 Uhl, G.R., Goodman, R.R. and Snyder, S.H., Neuroten-
sin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: immunohistochemical mapping, Brain Research, 167 (1979) 77-91. 44 Vanderhaeghen, J.-J., Lotstra, F., De Mey, J. and Gilles, C., Immunochemical localization of cholecystokinin- and gastrin-like peptides in the brain and hypophysis of the rat, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1190-1194. 45 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta Physiol. Scand., 367 (Suppl.) (1971) 1-48. 46 Van Ree, J.M., Gaffori, O. and De Wied, D., In rats, the behavioral profile of CCK-8-related peptides resembles that of antipsychotic agents, Eur. J. Pharmacol., 93 (1983) 63-78. 47 Williams, R.G., Gayton, R.J., Zhu, W.-Y. and Dockray, G.J., Changes in brain cholecystokinin octapeptide following lesions of the medial forebrain bundle, Brain Research, 213 (1981) 227-230. 48 Zamboni, L. and De Martino, C., Buffered picric acid formaldehyde: a new rapid fixation for electron microscopy, J. Cell. Biol., 35 (1967) 148A. 49 Zetler, G., Neuroleptic-like effects of ceruletide and cholecystokinin octapeptide: interactions with apomorphine, methylphenidate and picrotoxin, Eur. J. Pharmacol., 94 (1983) 261-270. 50 Zetler, G., Neuropharmacological profile of cholecystokinin-like peptides, Ann. N.Y. Acid. Sci., 448 (1985) 448-469.
88 BRE 13753
A subpopulation of dopaminergic neurons in rat ventral mesencephalon contains both neurotensin and cholecystokinin K. Seroogy 1. , S. Ceccatelli 1, M. Schalling 1, T. H6kfelt 1, P. Frey 2, J. Walsh 3, G. Dockray 4, J. Brown 5, A. B u c h a n 5 a n d M. G o l d s t e i n 6 t Department of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden), 2Sandoz Research Institute, Bern (Switzerland), 3Centerfor Ulcer Research and Education, VA Medical Center-Wadsworth, Los Angeles, CA (U.S.A.), 4The Physiological Laboratory, University of Liverpool, Liverpool (U. K.), 5Medical Research Council of Canada, Regulatory Peptide Group, University of British Columbia, Vancouver, BC (Canada) and 6Department of Psychiatry, New York University Medical Center, New York, NY (U.S.A.) (Accepted 2 February 1988)
Key words: Neuropeptide; Tyrosine hydroxylase; Colocalization; Ventral tegmental area; Immunohistochemistry
The coexistence of the neuropeptides neurotensin and cholecystokinin and the catecholamine-synthesizing enzyme tyrosine hydroxylase within neurons of the ventral mesencephalon was analyzed using an immunofluorescence triple-labeling technique. Virtually all of the neurotensin-positive cell bodies in the ventral tegmental area, medial substantia nigra pars compacta, retrorubral field, and rostral and caudal linear raphe nuclei were found to contain both cholecystokinin and tyrosine hydroxylase immunoreactivities. The degree of colocalization was lower and more variable in other regions including the ventral and central periaqueductal grey matter and dorsal raphe nucleus. It appeared that immunoreactivities for these 3 neuroactive substances were not contained within the same axonal-like fibers and terminals in the ventral midbrain. These results demonstrate that a subpoputation of dopaminergic neurons, which presumably comprise part of the ascending mesotelencephalic system, contains the two peptides neurotensin and cholecystokinin. Thus, the data suggest a morphological basis for some of the reported functional interactions of these 3 putative neurotransmitters/neuromodulators within this system.
INTRODUCTION In addition to the well-known distribution of dopaminergic neurons throughout the ventral mesencephalon 1'3'6'12, the neuropeptides cholecystokinin (CCK) and neurotensin (NT) have been localized within neuronal perikarya in this region 10"16"18"43'44. These studies have shown that CCK-containing somata are widely distributed throughout the ventral tegmental area, substantia nigra pars compacta and pars lateralis and several midline nuclei. N T cell bodies encompass a relatively smaller population of neurons restricted predominantly to the ventral tegmental area, although a few cells are found in the substantia nigra pars compacta and various raphe nuclei. It has been determined with immunocytochem-
ical 1°'33 and biochemical 9'21'39'4°'47 techniques that many of the CCK cells of the ventral midbrain comprise a subpopulation of the dopaminergic neurons. Virtually all of the N T neurons throughout this region have been shown to contain immunoreactivity for tyrosine hydroxylase 11, the synthesizing enzyme for catecholamines. By using a double-labeling indirect immunofluorescence procedure, we have recently demonstrated 3t the coexistence of CCK and N T within neurons of the rat ventral mesencephalon. It was found that more than 90% of the NT-positive neurons (primarily located in the ventral tegmental area) also contained CCK immunoreactivity. Based on these results, it was postulated that neurons in the ventral midbrain containing both peptides may also contain
* Present address: Department of Physiology, University of North Carolina, Chapel Hill, NC 27599, U.S.A. Correspondence: T. H6kfelt, Department of Histology and Neurobiology, Karolinska Institute, S-104 01 Stockholm, Sweden. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
89 the monoamine dopamine. The present study was conducted to test this hypothesis and thereby provide an anatomical basis for some of the reported functional interactions 3"29 of these 3 neuroactive substances in the ventral mesencephalon. In addition, a more extensive analysis of the extent of coexistence of these compounds in periaqueductal and raphe regions was undertaken. MATERIALS AND METHODS Adult male albino rats (200 g; ALAB, Stockholm, Sweden) were used in these experiments. The animals were anesthetized with chloral hydrate and stereotaxically injected with colchicine into a lateral ventricle (120/tg in 20/~10.9% NaC1) or both intracerebroventricularly and locally (1.8 ~g in 0.3/A NaCI), approximately 2 mm dorsal to the substantia nigraventral tegmental area complex. After 24-48 h, the rats were re-anesthetized and perfused via the ascending aorta with 50 ml warm (37 °C) calcium-free Tyrode's solution, then 50 ml warm 0.3% picric acidformalin 4s in 0.1 M phosphate buffer (PB), followed by the same ice-cold fixative for 6 min. The brains were removed, immersed in ice-cold fixative for 90 min, and placed in 10% sucrose in PB overnight at 4 °C. Coronal sections (14/~m) through the ventral mesencephalon were cut in a cryostat (Dittes, Heidelberg, F.R.G.), thaw-mounted onto gelatin-coated glass slides, and rinsed in phosphate buffered saline (PBS). The sections were then processed for the simultaneous detection of NT and CCK using a modification of the indirect immunofluorescence technique 5. Briefly, the sections were incubated with a mixture of rabbit anti-NT antiserum (diluted 1:400) and mouse monoclonal anti-CCK antibody (1:400) or, alternatively, with a mixture of rabbit anti-CCK antisera (1:400) and mouse monoclonal anti-NT antibody (1:100) 30a. Following an incubation period of 18-24 h at 4 °C in a humid chamber, the sections were rinsed in PBS and then incubated with a mixture containing tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit immunoglobulins (IgG) (1:40; Boehringer Mannheim Scandinavia, Stockholm, Sweden) and either fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or sheep anti-mouse IgG (both 1:40; Boehringer Mann-
heim and Amersham, Amersham, U.K., respectively) for 30 min at 37 °C. The sections were then rinsed in PBS, coverslipped in a 3:1 solution of glycerol: PBS containing 0.1% p-phenylenediamine 17.28, and examined with a Zeiss standard fluorescence microscope equipped with an oil darkfield condensor and proper filter combinations. After photography, the sections were processed according to the elution/restraining technique 42 as described in detail elsewhere 1°. Briefly, the antibodies were eluted with a solution of potassium permanganate and hydrogen sulfate, and the sections then reincubated in the secondary antisera cocktail described above to check for absence of immunofluorescence and thus assure complete elution of the antibodies. Sections were then processed for tyrosine hydroxylase (TH) immunoreactivity using a rabbit antiTH antiserum (1:400) 20 and FITC- or TRITClabeled goat anti-rabbit IgG secondary antisera. Photographs of the NT/CCK double-labeling were compared with those of the TH immunostaining in the appropriate areas to examine for coexistence. In control experiments, preabsorption of the CCK/NT primary antisera mixture with 10/~M CCK peptide eliminated the staining for CCK but did not affect the anti-NT staining. Similarly; preabsorption with 10/~M NT resulted in absence of immunofluorescence for NT but did not block the CCK immunostaining. Controls for secondary antisera species specificity and possible cross-reactivity, as described in detail previously31, consisted of incubation of tissue with each of the primary antisera separately, followed by the mixture of both secondary antisera. In each instance, only immunofluorescence from the appropriate secondary antiserum was observed. Replacement of the TH antiserum with normal rabbit serum resulted in the absence of all T H immunostaining. Omission of both primary antisera from the initial incubation step followed by processing with the combined secondary antisera mixture resulted in the complete absence of all immunofluorescence under both FITC and TRITC filters. RESULTS Numerous CCK-like immunoreactive (CCK-I) cell bodies were observed throughout the rostrocaudal extent of the ventral mesencephalon, as de-
90
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I
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Fig. 1. Low-magnification immunofluorescence photomontages of the same section through the ventral mesencephalon illustrating the colocalization of NT-I (a, FITC filter), CCK-I (b, TRITC filter) and TH-I (c, FITC filter after elution/restaining procedure). Examples of neurons triple-labeled with all 3 substances are indicated by arrows. Note that virtually all of the NT-I cells contain both CCK-I and TH-I and the higher degree of CCK-I/TH-I coexistence. The boxed-in region of the ventral tegmental area in (a) and the corresponding areas in (b) and (c) are shown at higher magnification in Fig. 2. Note the differential distribution of especially NT and CCK immunoreactive terminals in the interpeduncular nucleus (IPN). Bar = 100/~m.
91 scribed in detail in previous reports 1°'32. Briefly, high numbers of CCK-I perikarya were found in the ventral tegmental area (A10 cell group region according to the terminology of Dahlstr6m and Fuxe6), rostral and intermediate levels of the substantia nigra pars compacta (A9), interfascicular nucleus, and ventral periaqueductal grey matter. Relatively fewer immunoreactive neurons were present in the rostral and caudal linear raphe nuclei, central periaqueductal grey matter, dorsal raphe nucleus, substantia nigra pars lateralis, substantia nigra pars compacta at caudal levels, retrorubral field (A8), and the peripeduncular region. NT-like immunoreactive (NT-I) perikarya were present in much fewer numbers in several of the same regions throughout the ventral mesencephalon including the ventral tegmental area, medial substantia nigra pars compacta (at rostral levels), rostral and caudal linear nuclei, central and ventral periaqueductal grey matter, retrorubral region, lateral aspects of the substantia nigra pars compacta, and in peripeduncular region. Double-labeling analysis revealed that the vast majority of the NT-I neurons (located for the most part in the ventral tegmental area) also contained immunoreactivity for CCK (Figs. la,b; 2a,b). The results were the same regardless of which combination of primary antisera mixture was used. These NT-I/CCK-I neurons were situated in both the parabrachialis pigmentosus and paranigralis subdivisions of the ventral tegmental area, although more frequently in the former. At more rostral levels of the ventral mesencephalon, these double-labeled cell bodies occupied a more dorsal position in the ventral tegmental area, whereas more caudally they were characteristically observed intermingled among the rootlets of the oculomotor nerve (III). Following the elution/restraining procedure, it was found that virtually all of the perikarya in this region containing both NT and CCK immunoreactivities also exhibited immunostaining for TH (Figs. l a - c ; 2a-c). Much greater numbers of neurons contained immunoreactivities for both CCK and T H but not NT. Very infrequently, a neuron displaying NT-I and TH-I but not CCK-I could be found. Somata in other regions of the ventral midbrain were also observed to contain immunoreactivities for both of the peptides as well as for the catecholamine-
Fig. 2. High-power magnifications of the boxed-in region in Fig. la and the corresponding areas in lb and lc demonstrating the perikaryal colocalization of NT-I (a), CCK-I (b) and TH-I (c) in the ventral tegmental area. Arrows indicate examples of the triple-labeled neurons. Bar = 50 pm.
92 synthesizing enzyme. A l t h o u g h the NT-I s o m a t a located in or usually above the lateral ventral tegmental area and medial substantia nigra pars compacta at rostral levels of the ventral m e s e n c e p h a l o n typically did not contain C C K - I or T H - I , p e r i k a r y a were occasionally found in these areas i m m u n o s t a i n e d for all 3 substances (Fig. 3 d - f ) . In the lateral part of the substantia nigra pars c o m p a c t a , infrequent triplelabeled neurons were observed. H o w e v e r , a small population of NT-I cell bodies located a b o v e this re-
gion, as well as above the substantia nigra pars lateralis and in the p e r i p e d u n c u l a r region, typically did not display staining for either C C K or T H . The few NT-I p e r i k a r y a found in the retrorubral field did exhibit both CCK-I and TH-I. Although a few of the large neurons of the ventral periaqueductal grey m a t t e r (including part of the E d i n g e r - W e s t p h a l nucleus) were d o u b l e - l a b e l e d with NT-1 and CCK-I, these cells were never observed to contain T H - I (Fig. 3 a - c ) . Virtually all of
Fig. 3. (a-c) Immunofluorescence photomicrographs of the same section through the ventral periaqueductal grey matter showing large neurons (including Edinger-Westphal nucleus) displaying both NT-I (a) and CCK-I (b) (arrowheads) but not TH-I (c) in this midline group. Star denotes ventral edge of aqueduct. (d-f) Immunofluorescence photomicrographs of the same section through the substantia nigra pars compacta illustrating the colocalization of NT-I (d), CCK-I (e) and TH-I (f) within perikarya in both dorsal and ventral aspects at the rostral level of this region. Arrowheads indicate some of the triple-labeledneurons. Bar in a = 50/~m for a-c; in d = 50pm for d-f.
93 the small N T - I s o m a t a l o c a t e d in t h e m i d l i n e rostral
cular nucleus, m o r e differential p a t t e r n s o f coexis-
linear nucleus w e r e also i m m u n o s t a i n e d for b o t h
t e n c e versus n o n - c o e x i s t e n c e of N T - , C C K - , and
C C K and T H . A t levels b e g i n n i n g just c a u d a l to the i n t e r p e d u n -
T H - I w e r e o b s e r v e d . In the caudal linear nucleus, the vast m a j o r i t y of the N T - I n e u r o n s w e r e also im-
,j
Fig. 4. Immunofluorescence photomontages of the same section illustrating the extent of colocalization of NT-I (a), CCK-I (b) and TH-I (c) in juxta-aqueductal, dorsal raphe and caudal linear raphe regions. Note that, at this level, few NT-I and TH-I cells are situated in the dorsal raphe nucleus, whereas many CCK-I somata are present. Also, almost all of the NT-I perikarya in the caudal linear nucleus (a) contain both CCK-I (b) and TH-I (c). Arrows indicate examples of triple-labeled neurons. Examples of neurons doublelabeled only with CCK-I (b) and TH-I (c) but not NT-I are indicated by arrowheads. Open arrows identify examples of cell bodies single-labeled for each substance in their respective panels. Star denotes aqueduct. Bar = 50 #m.
94 munostained for CCK-I and TH-I (Fig. 4). However, a rare single-labeled NT-I perikaryon could be found (see Fig. 4a). The remaining CCK-I and TH-I somata displayed a relatively high degree of coexistence (Fig. 4b,c) with additional neurons single-labeled for each substance also present (see Fig. 4a-c). At rostral levels of the dorsal raphe nucleus, separate populations of NT-, CCK-, and T H - I cell bodies existed, although a few triple-labeled neurons were observed. In the fountain of Sheehan and in more caudal aspects of the dorsal raphe nucleus, most of the somata immunoreactive for NT also contained CCKand TH-I (Fig. 5). Again, the numbers of neurons double-labeled for both CCK-I and TH-I exceeded that of the triple-labeled cells. Immunoreactive neurons located mainly ventrally (but occasionally laterally) adjacent to the cerebral aqueduct in the central periaqueductal grey matter also displayed a differential pattern of coexistence based upon their rostrocaudal distribution throughout the mesencephalon. At rostral and intermediate levels, the few NT-I somata present were not found to be immunoreactive for either CCK or TH, whereas immunofluorescence for the latter two compounds
was often observed within the same perikarya (see Fig. 4b,c). At caudal aspects, the small immunoreactive NT cells were found to contain both CCK- and TH-I, only TH-I, or neither of these two substances (Fig. 5). It could often be observed that, in the same section, most of the larger and more numerous NT-I perikarya situated slightly more ventrally within the dorsal raphe nucleus were immunostained for both CCK and TH (Fig. 5). At these more caudal levels, virtually all of the relatively more numerous CCK-I cells exhibited T H immunofluorescence. Throughout the entire ventral mesencephalon, and especially in the ventral tegmental area, a dense to moderately dense plexus of terminal-like immunoreactivity for NT was present (see Figs. la; 2a). This punctate NT-I was usually more dense than the CCK-immunoreactive puncta observed in these regions. Although, as described above, almost all of the NT-I somata in the ventral tegmental area also contained CCK-I, immunofluorescence for both peptides could not be observed within the same puncta. However, the fact that colchicine was used may account for the apparent lack of observable CCK-I within axonal-like fibers and terminals throughout
Fig. 5. Immunofluorescence photomicrographs of the same section through caudal regions of the dorsal raphe nucleus and ventral periaqueductal regions showing the perikaryal colocalization of NT-I (a), CCK-I (b) and TH-I (c). At this level, virtually all of the NTI cells (a) within the dorsal raphe nucleus contain both CCK-I (b) and TH-I (c) as indicated by the arrows. Arrowheads in (a) and (c) identify a neuron containing NT-I (a) and TH-I (c) but lacking CCK-I (b), while examples of those exhibiting only CCK-I (b) and TH-I (c) are indicated by curved arrows in (b) and (c). Open arrow in (c) identifies a single-labeled TH-I cell. Asterisk denotes aqueduct. Bar = 50~m.
95 the ventral midbrain, and thus, a definite statement concerning axonal or terminal peptide coexistence cannot be made. Nevertheless, where terminal-like immunoreactivity for both substances was present, there appeared to be no coexistence as well as a differential or sometimes complementary pattern of immunostaining. Examples of this were best observed in the paranigralis subdivision of the ventral tegmental area and within the interpeduncular nucleus (see Fig. la,b). It should be noted, however, that at more caudal levels of the ventral midbrain (for example in the caudal linear raphe nucleus or in the fountain of Sheehan), single examples of fibers were detected containing immunoreactivity for both peptides. The pattern of TH-I fibers and puncta throughout all of the above regions generally resembled that of the NT-I staining (cf. Fig. la to lc), although, again, unequivocable demonstration of the colocalization of these two substances within puncta could not be made. DISCUSSION The present demonstration of NT/CCK coexistence within neurons of the ventral mesencephalon confirms and extends our recent findings 31. In addition, we have provided direct evidence that virtually all of the NT/CCK perikarya also contain the enzyme TH, the first enzyme in dopamine synthesis in these cells (see refs. 3, 12, 13). The results are also in good agreement with the previous immunocytochemical reports of CCK/TH 1°'33 and NT/TH 11 coexistence in rat ventral midbrain. Moreover, it has recently been shown that a very small population of dopamine neurons in the ventral tegmental area exhibit vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-immunoreactivities 34. Thus, there is now strong evidence that several heterogeneous subpopulations of dopaminergic neurons exist in the ventral mesencephalon: those containing (1) both CCK and NT, (2) CCK alone, (3) NT alone (rare), (4) both CCK and VIP/PHI (rare), (5) VIP/PHI alone (rare), or (6) none of the peptides. With the use of combined retrograde tracing and double-labeling immunocytochemistry, some of the connections of the NT/CCK neurons have been previously determined. Perikarya containing both peptides located preferentially in the ventral tegmental
area were found to innervate the nucleus accumbens, prefrontal cortex, and amygdala 31. Dopaminergic projections arising from the ventral tegmental area to the above forebrain sites are well-established (see refs. 1, 3, 41, 45). Based on the present results, it can now be firmly stated that the NT/CCK pathways are dopaminergic. Therefore, a component of the ascending mesotelencephalic system innervating at least some limbic and cortical areas contains the two peptides NT and CCK, as well as the catecholamine dopamine. Whether or not the NT/CCK/dopamine cells extend projections to additional forebrain regions, as has been shown for the CCK- or CCK/THpositive neurons of the ventral midbrain 32'33, remains to be determined. The presence of all 3 compounds within a few neurons of the rostral substantia nigra pars compacta suggests a minor NT/CCK/dopaminergic nigrostriatal pathway. In contrast to the situation in the ventral tegmental area, a differential pattern of coexistence was found in the ventral and central periaqueductal grey matter (PAG) and in the dorsal raphe nucleus at caudal levels of the midbrain. NT immunoreactivity was infrequently found in the large neurons of the midline ventral PAG (also identified as the Edinger-Westphal nucleus by Innis and Aghajanian15). When present, however, it was always colocalized with CCK. TH immunostaining was never observed in these doublelabeled cells nor in any of the larger population of intensely immunofluorescent CCK cell bodies. However, the peptide substance P has been colocalized with CCK in these neurons 37. Moreover, these CCK/substance P-containing neurons have been shown to project to the spinal cord 19'26'38. It is possible that a minor portion of this efferent pathway also contains NT. The distribution of NT somata observed in this study within the central P A G is generally in good agreement with earlier reports 2'16'35. The present resuits demonstrate that the NT perikarya located in the ventrolateral and ventromedial parts of the P A G adjacent to the aqueduct typically did not contain either CCK or TH. In contrast, almost all of the CCK cell bodies present in these same areas also exhibited TH (see ref. 33). In the region of the dorsal raphe nucleus, a rostrocaudal gradient appeared to exist, in which a higher degree of coexistence among the 3 substances was observed at more caudal levels. Thus,
96 the extent of NT/CCK/dopamine coexistence at the caudal levels is much more variable than at more rostral levels, i.e. in the ventral tegmental area. Minor CCK-containing projections from the dorsal raphe nucleus to such forebrain regions as the caudate-putamen and amygdala have been reported32; therefore, NT may constitute some portions of these pathways. On the other hand, descending projections from central P A G regions to hindbrain raphe nuclei have been shown to be NT- but not CCK-positive 2. Despite the presence of all 3 compounds within cell bodies of the ventral tegmental area and medial substantia nigra pars compacta, their colocalization within individual axonal-like fibers and terminals in these regions could not reliably be observed. Allowing for the inherent difficulty of identifying t h e same individual puncta in relatively dense fields when observed under different filters of the fluorescent microscope or even upon comparison of photographs, and for effects of colchicine treatment, it was apparent that there were often complementary patterns of especially the NT and CCK immunoreactivities. This was most evident in the interpeduncular nucleus, medial substantia nigra, ventral tegmental area, and even in ventral and central P A G regions. In fact, it appeared that the overall pattern of NT terminal-like immunostaining was more similar to that of TH than C C K . These data suggest that although some of the efferents of the ventral mesencephalon contain all 3 molecules, the majority of the NT-, CCK-, and TH-positive afferents to this area (see ref. 25 for review of ventral midbrain connections) arise from separate populations of neurons. The effects of NT or CCK upon various dopamine-
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ACKNOWLEDGEMENTS This study was supported by the Swedish M R C (04X-2887), Alice och Knut Wallenberg Stiftelse, Petrus och Augusta Hedlunds Stiftelse, Fredrik och Ingrid Thurings Stiftelse, and the National Institute of Mental Health (MH 43230). K.S. was supported by a N A T O Postdoctoral Fellowship in Science and S.C. was supported by grants from the Swedish Institute and the Wenner-Gren Foundation.
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