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Substance P- and enkephalin-containing projections from the interpeduncular nucleus to the dorsal tegmental region in the rat
Neuroscience Letters, 62 (1985) 311 316 Elsevier Scientific Publishers Ireland Ltd.
31 I
NSL 03672
SUBSTANCE P- AND ENKEPHALIN-CONTAINING P R O J E C T I O N S FROM THE I N T E R P E D U N C U L A R N U C L E U S TO THE D O R S A L TEGMENTAL REGION IN THE RAT
INGE H U I T I N G A , C H R I S T I N E A. VAN DIJK and H E N K J. G R O E N E W E G E N *
D~Tmrtment ~/ Anatomy. Vr(je Universiteit, Van de Boechorststraat 7, 1081 BT Amsterdam (The Netherhinds; (Received July 21st, 1985: Revised version received September 10th, 1985: Accepted September 12th, 1985)
Key words." interpeduncular nucleus tegmental region - rat
fluorescent retrograde tracing
enkephalin
substance P
dorsal
A combination of fluorescent retrograde tracing and immunohistochemical staining for substance P (SP) and Leu-enkephalin (Enk) was used to study the projections from the interpeduncular nucleus (IP) to the dorsal tegmental region, i.e. the dorsal tegrnental nucleus of Gudden, the dorsolateral tegmental nucleus and the caudal extension of the dorsal raphe nucleus. After injections of 'Granular Blue" (GB) in the dorsal tegmental region, followed one or two days later by a colchicine injection near the IP, and subsequently two days later by the immunohistochemical procedure, populations of neurons double labeled for Enk and GB, or SP and GB, cells that only showed GB labeling, and a number of cells stained only for one of the peptides could be identified in the rostral subnucleus of the IP. This study demonstrates the existence of both Enk- and SP-containing projections from the IP to the dorsal tegmental region.
The interpeduncular nucleus (IP) constitutes a distinct, relatively small nuclear mass in the ventral mesencephalon. From a connectional point of view, the IP serves as an integration center through which limbic forebrain structures by way of the habenula can exert their influence on the midbrain raphe nuclei and the dorsal tegmental region [3, 6]. In the latter region, IP efferents are mainly directed to the caudal part of the dorsal raphe nucleus and the dorsolateral tegmental nucleus [3]. These two nuclei give rise to, respectively, serotonergic and cholinergic ascending pathways that reach widespread areas in the forebrain [2, 7, 9, 13]. In functional terms it is of interest to know which neuroactive substances play a role in the regulation of these pathways. Based on cytoarchitectonics, the IP of the rat can be subdivided into 5 paired and 3 unpaired subnuclei [3, 4, 8]. Among the several presumptive neurotransmitters located in the IP, the peptides enkephalin (Enk) and substance P (SP) have been repeatedly documented [3, 5, 6]. Both Enk-positive and SP-positive neurons are
*Author for correspondence and reprint requests. 0304-3940/85/$ 03.30 © 1985 Elsevier Scienti[ic Publishers Ireland Ltd.
312
found in the rostral subnucleus (IP-R), whereas Enk-containing cells are also present in the apical subnucleus (IP-A) and the caudal part of the central subnucleus (IP-C) [3, 5, 6]. From the results of recent tracing experiments, it is known that IP-R sends its major output to the raphe nuclei and the dorsal tegmental region [3i. Enk- and SP-positive fibers and terminals have been described in the dorsal tegmental region and the dorsal raphe nucleus [1, I 1, 12, 14]. In order to investigate whether Enk and/ or SP is contained in the descending IP projections, injections of the fluorescent tracer Granular Blue (GB) were placed in the dorsal tegmental region, followed by immunofluorescent staining of Enk and SP. Fourteen female Wistar rats (180-220 g body wt.) were anesthetized with an i.m. injection of a mixture of Ketamine and Rompun (4:3, 1 ml/kg). Injections of 1 /~1 GB (5~ in saline) were placed in the dorsal tegmental region and the dorsal raphe nucleus, after 3-4 days followed by an injection of colchicine (15 ~g in I /4) near the IP. One or two days later, the animals were anesthetized with Nembutal and perfused transcardially with 100 ml saline (4°C), followed by 400 mi 4 ~ paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4 (4~ C). Sections were cut on a freezing microtome (40/~m) or on a cryostat (16/~m). The Leu-enkephalin antiserum (Enk) was kindly provided by Dr. R.J. Miller [10], and the antiserum against SP was purchased from Sera Labs. Two immunohistochemical procedures were used to visualize Enk- or SP-immunoreactivity (IR): the peroxidase-anti-peroxidase (PAP) and the indirect immunofluorescence methods. Incubation with the primary SP and Enk antisera was done overnight at 4'~C, diluted, respectively, 1:40 and 1:500 in 0.05 M Tris-buffered saline (TBS), pH 7.6. For the PAP method the Enk staining was preceded by an incubation in goat anti-rabbit immunoglobulin in TBS containing 0.5~o Triton X-100 (TBS-TX: 1:50), followed by rabbit-PAP in TBS-TX (1:600). The SP sections were incubated with a conjugate of horseradish peroxidase (HRP) with goat anti-rat immunoglobulin (kindly provided by Dr. D. Boorsma) in TBS-TX (1:25). Both Enk and SP staining was ultimately visualized by incubation in diaminobenzidine and H202. For the immunofluorescent method, after the primary antisera, the sections were incubated in fluorescein isothiocyanate (FITC)-conjugated horse anti-rabbit immunoglobulin in TBS-TX (1:30) for Enk staining or in FITC-conjugated rabbit anti-rat immunoglobulin in TBS-TX (1:35) for the visualization of SP. Following unilateral GB injections in the dorsal tegmental region retrogradely labeled cells are found in virtually all subnuclei of the IP. In the rostal (IP-R) and rostral lateral (IP-RL) subnuclei, labeled cells are mainly present at the side of the injection, and to a smaller degree contralaterally. In contrast, the dorsal medial (IP-DM) and dorsal lateral (IP-DL) subnuclei contain GB-labeled neurons predominantly contralaterally. In the caudal aspect of IP, the lateral (IP-L), apical (IP-A) and central (IP-C) subnuclei contain retrograde labeling bilaterally, which is sparse in IP-A and IP-C. In agreement with previous studies [3, 5, 6], SP-positive neurons are found exclusively in IP-R, whereas Enk-containing perikarya are present in IP-R, IP-A and, sporadically, in the caudodorsal part of IP-C. In the present experiments, no significant effect of the incubation procedure was found on the number or the distribution pat-
313
%
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r~ ci,
Fig. 1. Photomicrographs of the rostral subnucleus (IP-R) showing the results of an experiment with retrograde fluorescent tracing of GB and immunohistochemistry on the same tissue section. The injection of GB was placed in the dorsal tegmental region. A and B: section of IP-R stained for Enk immunoreactivity. A: FITC immunofluorescence. B: retrograde labeling with GB. C and D: section of IP-R stained for SP immunoreaclivity. C: FITC immunofluorescence. D: retrograde labeling with GB. Single- and doubleheaded arrows identify double-labeled neurons. Bar = 20/~m.
tern o f r e t r o g r a d e l y labeled cells in the IP. F u r t h e r m o r e , no essential differences were n o t e d in the n u m b e r a n d d i s t r i b u t i o n o f i m m u n o r e a c t i v e n e u r o n s in e x p e r i m e n t s with or w i t h o u t a G B injection in the d o r s a l t e g m e n t a l region, i n d i c a t i n g that the retrograde tracer does n o t n o t i c e a b l y decrease the antigenicity o f Enk o r SP in p e r i k a r y a o f IP. F o l l o w i n g injections o f G B in the d o r s a l t e g m e n t a l region, in which sections t h r o u g h the IP were a l t e r n a t e l y stained for E n k - I R a n d S P - I R , 5 distinct p o p u l a t i o n s o f n e u r o n s could be identified in the IP. These p o p u l a t i o n s include cells that only c o n t a i n e d the r e t r o g r a d e tracer G B , n e u r o n s that were i m m u n o r e a c t i v e for Enk or SP b u t did n o t show the r e t r o g r a d e tracer, a n d p e r i k a r y a c o n t a i n i n g either E n k - I R o r S P - I R t o g e t h e r with the r e t r o g r a d e t r a c e r (Fig. 1). These latter d o u b l e - l a b e l e d cells in the IP are virtually only present in the rostral subnucleus I P - R , b o t h in case o f Enk a n d SP, b u t very o c c a s i o n a l y an E n k - n e u r o n in the I P - A was also r e t r o g r a d e l y filled with GB. F o l l o w i n g small unilateral injections o f G B in the d o r s a l t e g m e n t a l region, relatively few d o u b l e - l a b e l e d n e u r o n s were present in the I P - R . In 3 b r a i n s o f which in total 18 E n k - s t a i n e d a n d 12 SP-stained frozen sections (40/zm) t h r o u g h
314
the IP-R were examined, 500 Enk- and 419 SP-positive perikarya were counted, of which, respectively, 37 (7.4~) and 40 (9.6~) also exhibited GB fluorescence. A larger
BILATERAL GRANULAR BLUE INJECTIONS
ENKEPHALIN
SUBSTANCE P
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bilateral injection of GB in the dorsal tegmental region, in an experiment in which cryostat sections (16 ~m) of the IP were cut, gave rise to a larger number of retrogradely labeled neurons in the IP and a higher proportion of double-labeled cells in the IP-R (Fig. 2). In this case, 15 Enk-immunoreacted sections contained 262 Enk-posirive cells, of which 47.9~o were double-labeled. In 12 SP-stained sections. 46.9°i, of the 130 SF,-positive neurons also contained the retrograde tracer GB. The present results indicate the existence in the IP-R of a population of Enk-positive neurons and a group of SP-containing cells that project to the dorsal tegmental rcgiol". In the target area, Enk-positive fibers and terminals are found in highest concentration in the periphery of the dorsal raphe nucleus, and dorsally and medially to the dorsal tegmental nucleus of Gudden near the midline and in the periventricular zone [10, 11, 14]. An almost similar distribution within the dorsal tegmental region has been described for SP fibers and terminals [!, 12]. Likewise, the most dense termination areas of IF, fibers, as described with the anterograde autoradiographic tracing method, are located in the midline, the periphery of the dorsal tegmental nucleus of Gudden and in the dorsolateral tegmental nucleus [3]. Depending on the size of the injection, up to about one-half of the Enk and SP neuronal populations in the IP-R can be double labeled following injections of G B in the dorsal tegmental region. Moreover, in all experiments there is a large group of neurons which contains only SP-IR or Enk-IR, without being retrogradely labeled. Whether these neurons project to other mesencephalic areas, such as the rostral parts of the dorsal raphe nucleus and the median raphe nucleus, or constitute intranuclear connections within the IF', cannot be concluded from the present results. In recent tracing experiments, it was established that IP-R not only projects to the raphe nuclei and the dorsal tegmental region, but also to the caudal IF, subnuclei 1PA and IP-L [3]. In the IF,-A both Enk- and SF,-positive fibers are present, and IP-L contains a dense SP-fiber plexus [3, 5, 6]. It must further be emphasized that the presence of large numbers of neurons in the 1P, retrogradely filled from the dorsal tegmental region without showing Enk-IR or SF,-IR, indicates that the interpedunculotegmental projections also may contain other neurotransmitters. Potential candidates are 7-aminobutyric acid, vasoactive intestinal polypeptide, somatostatin and serotonin, as all of these substances or one of their synthesizing enzymes has been demonstrated immunocytochemically to be contained in IF, neurons [5, 6]. In conclusion, the results of the present experiments have demonstrated the existence of peptidergic pathways, containing Enk and SF,, from the IF' to the dorsal tegmental region. What functional role these connections play remains to be established, but the present data suggest that the limbic regulation of the ascending serotonergic Fig. 2. Chartings of 4 pairs of adjacent coronal cryostat sections ( 16/~m) of the rostral half of the IP from an experiment in which retrograde tracing and immunohistochemistry were applied to the samc section. Adjacent sections were stained either for Enk (left column) or SP (right column). The location of the two GB deposits in the dorsal tegmental region and dorsal raphe nucleus is shown at the top. ()pen circles indicate single immunoreactive Enk or SP neurons, and stars identify immunoreactive neurons also containing GB. D L T N , dorsolateral tegmental nucleus; D R N , dorsal raphe nucleus: DTN, dorsal tegmental nucleus of Gudden: tim, fasciculus longitudinalis medialis; hp, habenulo-interpeduncular tract: 1P-I, intermediate subnucleus: LC, locus coeruleus; VTA, ventral tegmental area.
316 [7] a n d c h o l i n e r g i c [9, 13] s y s t e m s t h r o u g h t h e I P m a y in p a r t b e m e d i a t e d b y p e p t i dergic substances, The authors thank Drs. Pieter Voorn, Fokje Russchen and Tony Lohman tbr their suggestions and reading of the manuscript. The expert technical assistance of John Bol, D i r k d e J o n g , N a n e t t e K a a t a n d R i t a S t o e v e l a a r is g r a t e f u l l y a c k n o w l e d g e d . 1 Cuello, A.C. and Kanazawa, I., The distribution of substance P immunoreactive fibers in the rat central nervous system. J. Comp, Neurol., 178 (1978) 129 156. 2 Groenewegen, H.J. and Van Dijk, C.A., Efferent connections of the dorsal tegmental region in the rat, studied by means of anterograde transport of the lectin Phaseolus vulgaris-leucoagglutinin (PHAL), Brain Res., 304 (1984) 367-371. 3 Groenewegen, H.J., Ahlenius, S., Haber, S.N., Kowall, N.W. and Nauta, W.J.I-I., Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat, J. Comp. Neurol., in press. 4 Hamill, G.S. and Lenn, N.J., The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopic study, J. Comp. Neurol., 222 (1984) 396~408. 5 Hamill, G.S., Olschowska, J.A., Lenn, N.J. and Jacobowitz, D.M., The subnuclear distribution of substance P. vasoactive intestinal polypeptide, somatostatin, Leu-enkephalin, dopamine-//-hydroxylasc, and serotonin in the rat interpeduncular nucleus, J. Comp. Neurol., 226 (1984) 580-596. 6 Hemmendinger, L.M. and Moore, R.Y., lnterpeduncular nucleus organization in the rat: cytoarchitecture and histochemical analysis, Brain Res. Bull., 13 (1984) 163 179. 7 K6hter, C. and Steinbush, H.W.M., Identification of serotonin and non-serotonin containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent tracing study in the rat brain, Neuroscience, 7 (1982) 951-975. 8 Lenn, N.J. and Hamill, G.S., Subdivisions of the interpeduncular nucleus: a proposed nomenclature, Brain Res. Bull, 13 (1984) 203-204. 9 Mesulam, M.-M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch 1 Ch6), Neuroscience, I 0 (1983) 1185-1201. 10 Miller, R.J. and Pickel, V.M., The distribution and functions of the enkephalins, J. Histochem. Cytochem., 28 (1980) 903 917. 11 Moss, M.S., Glazer, E.J. and Basbaum, A.I., The peptidergic organization of the cat periaqueductal gray. I. The distribution of immunoreactive enkephalin-containing neurons and terminals, J. Neurosci., 3 (1983) 603 616. 12 Moss, M.S. and Basbaum, A.I., The peptidergic organization of the cat periaqueductal gray. 1I. The distribution of immunoreactive substance P and vasoactive intestinal polypeptide, J. Neurosci., 3 (1983) 1437-.1449. 13 Sofroniew, M.V., Priestley, J.V., Consolazione, A., Eckenstein, F. and Cuello, A.C., Cholinergic projcctions from the midbrain and pons to the thalamus in the rat, identified by combined retrograde t racing and choline acetyltransferase immunocytochemistry, Brain Res., 329 (1985) 213-223. 14 Uhl, G.R., Goodman, R.R., Kuhar, M.J., Childers, S.R. and Snyder, S.H., lmmunohistoehemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Res., 166 (1979) 75-94.
31 I
NSL 03672
SUBSTANCE P- AND ENKEPHALIN-CONTAINING P R O J E C T I O N S FROM THE I N T E R P E D U N C U L A R N U C L E U S TO THE D O R S A L TEGMENTAL REGION IN THE RAT
INGE H U I T I N G A , C H R I S T I N E A. VAN DIJK and H E N K J. G R O E N E W E G E N *
D~Tmrtment ~/ Anatomy. Vr(je Universiteit, Van de Boechorststraat 7, 1081 BT Amsterdam (The Netherhinds; (Received July 21st, 1985: Revised version received September 10th, 1985: Accepted September 12th, 1985)
Key words." interpeduncular nucleus tegmental region - rat
fluorescent retrograde tracing
enkephalin
substance P
dorsal
A combination of fluorescent retrograde tracing and immunohistochemical staining for substance P (SP) and Leu-enkephalin (Enk) was used to study the projections from the interpeduncular nucleus (IP) to the dorsal tegmental region, i.e. the dorsal tegrnental nucleus of Gudden, the dorsolateral tegmental nucleus and the caudal extension of the dorsal raphe nucleus. After injections of 'Granular Blue" (GB) in the dorsal tegmental region, followed one or two days later by a colchicine injection near the IP, and subsequently two days later by the immunohistochemical procedure, populations of neurons double labeled for Enk and GB, or SP and GB, cells that only showed GB labeling, and a number of cells stained only for one of the peptides could be identified in the rostral subnucleus of the IP. This study demonstrates the existence of both Enk- and SP-containing projections from the IP to the dorsal tegmental region.
The interpeduncular nucleus (IP) constitutes a distinct, relatively small nuclear mass in the ventral mesencephalon. From a connectional point of view, the IP serves as an integration center through which limbic forebrain structures by way of the habenula can exert their influence on the midbrain raphe nuclei and the dorsal tegmental region [3, 6]. In the latter region, IP efferents are mainly directed to the caudal part of the dorsal raphe nucleus and the dorsolateral tegmental nucleus [3]. These two nuclei give rise to, respectively, serotonergic and cholinergic ascending pathways that reach widespread areas in the forebrain [2, 7, 9, 13]. In functional terms it is of interest to know which neuroactive substances play a role in the regulation of these pathways. Based on cytoarchitectonics, the IP of the rat can be subdivided into 5 paired and 3 unpaired subnuclei [3, 4, 8]. Among the several presumptive neurotransmitters located in the IP, the peptides enkephalin (Enk) and substance P (SP) have been repeatedly documented [3, 5, 6]. Both Enk-positive and SP-positive neurons are
*Author for correspondence and reprint requests. 0304-3940/85/$ 03.30 © 1985 Elsevier Scienti[ic Publishers Ireland Ltd.
312
found in the rostral subnucleus (IP-R), whereas Enk-containing cells are also present in the apical subnucleus (IP-A) and the caudal part of the central subnucleus (IP-C) [3, 5, 6]. From the results of recent tracing experiments, it is known that IP-R sends its major output to the raphe nuclei and the dorsal tegmental region [3i. Enk- and SP-positive fibers and terminals have been described in the dorsal tegmental region and the dorsal raphe nucleus [1, I 1, 12, 14]. In order to investigate whether Enk and/ or SP is contained in the descending IP projections, injections of the fluorescent tracer Granular Blue (GB) were placed in the dorsal tegmental region, followed by immunofluorescent staining of Enk and SP. Fourteen female Wistar rats (180-220 g body wt.) were anesthetized with an i.m. injection of a mixture of Ketamine and Rompun (4:3, 1 ml/kg). Injections of 1 /~1 GB (5~ in saline) were placed in the dorsal tegmental region and the dorsal raphe nucleus, after 3-4 days followed by an injection of colchicine (15 ~g in I /4) near the IP. One or two days later, the animals were anesthetized with Nembutal and perfused transcardially with 100 ml saline (4°C), followed by 400 mi 4 ~ paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4 (4~ C). Sections were cut on a freezing microtome (40/~m) or on a cryostat (16/~m). The Leu-enkephalin antiserum (Enk) was kindly provided by Dr. R.J. Miller [10], and the antiserum against SP was purchased from Sera Labs. Two immunohistochemical procedures were used to visualize Enk- or SP-immunoreactivity (IR): the peroxidase-anti-peroxidase (PAP) and the indirect immunofluorescence methods. Incubation with the primary SP and Enk antisera was done overnight at 4'~C, diluted, respectively, 1:40 and 1:500 in 0.05 M Tris-buffered saline (TBS), pH 7.6. For the PAP method the Enk staining was preceded by an incubation in goat anti-rabbit immunoglobulin in TBS containing 0.5~o Triton X-100 (TBS-TX: 1:50), followed by rabbit-PAP in TBS-TX (1:600). The SP sections were incubated with a conjugate of horseradish peroxidase (HRP) with goat anti-rat immunoglobulin (kindly provided by Dr. D. Boorsma) in TBS-TX (1:25). Both Enk and SP staining was ultimately visualized by incubation in diaminobenzidine and H202. For the immunofluorescent method, after the primary antisera, the sections were incubated in fluorescein isothiocyanate (FITC)-conjugated horse anti-rabbit immunoglobulin in TBS-TX (1:30) for Enk staining or in FITC-conjugated rabbit anti-rat immunoglobulin in TBS-TX (1:35) for the visualization of SP. Following unilateral GB injections in the dorsal tegmental region retrogradely labeled cells are found in virtually all subnuclei of the IP. In the rostal (IP-R) and rostral lateral (IP-RL) subnuclei, labeled cells are mainly present at the side of the injection, and to a smaller degree contralaterally. In contrast, the dorsal medial (IP-DM) and dorsal lateral (IP-DL) subnuclei contain GB-labeled neurons predominantly contralaterally. In the caudal aspect of IP, the lateral (IP-L), apical (IP-A) and central (IP-C) subnuclei contain retrograde labeling bilaterally, which is sparse in IP-A and IP-C. In agreement with previous studies [3, 5, 6], SP-positive neurons are found exclusively in IP-R, whereas Enk-containing perikarya are present in IP-R, IP-A and, sporadically, in the caudodorsal part of IP-C. In the present experiments, no significant effect of the incubation procedure was found on the number or the distribution pat-
313
%
A
r~ ci,
Fig. 1. Photomicrographs of the rostral subnucleus (IP-R) showing the results of an experiment with retrograde fluorescent tracing of GB and immunohistochemistry on the same tissue section. The injection of GB was placed in the dorsal tegmental region. A and B: section of IP-R stained for Enk immunoreactivity. A: FITC immunofluorescence. B: retrograde labeling with GB. C and D: section of IP-R stained for SP immunoreaclivity. C: FITC immunofluorescence. D: retrograde labeling with GB. Single- and doubleheaded arrows identify double-labeled neurons. Bar = 20/~m.
tern o f r e t r o g r a d e l y labeled cells in the IP. F u r t h e r m o r e , no essential differences were n o t e d in the n u m b e r a n d d i s t r i b u t i o n o f i m m u n o r e a c t i v e n e u r o n s in e x p e r i m e n t s with or w i t h o u t a G B injection in the d o r s a l t e g m e n t a l region, i n d i c a t i n g that the retrograde tracer does n o t n o t i c e a b l y decrease the antigenicity o f Enk o r SP in p e r i k a r y a o f IP. F o l l o w i n g injections o f G B in the d o r s a l t e g m e n t a l region, in which sections t h r o u g h the IP were a l t e r n a t e l y stained for E n k - I R a n d S P - I R , 5 distinct p o p u l a t i o n s o f n e u r o n s could be identified in the IP. These p o p u l a t i o n s include cells that only c o n t a i n e d the r e t r o g r a d e tracer G B , n e u r o n s that were i m m u n o r e a c t i v e for Enk or SP b u t did n o t show the r e t r o g r a d e tracer, a n d p e r i k a r y a c o n t a i n i n g either E n k - I R o r S P - I R t o g e t h e r with the r e t r o g r a d e t r a c e r (Fig. 1). These latter d o u b l e - l a b e l e d cells in the IP are virtually only present in the rostral subnucleus I P - R , b o t h in case o f Enk a n d SP, b u t very o c c a s i o n a l y an E n k - n e u r o n in the I P - A was also r e t r o g r a d e l y filled with GB. F o l l o w i n g small unilateral injections o f G B in the d o r s a l t e g m e n t a l region, relatively few d o u b l e - l a b e l e d n e u r o n s were present in the I P - R . In 3 b r a i n s o f which in total 18 E n k - s t a i n e d a n d 12 SP-stained frozen sections (40/zm) t h r o u g h
314
the IP-R were examined, 500 Enk- and 419 SP-positive perikarya were counted, of which, respectively, 37 (7.4~) and 40 (9.6~) also exhibited GB fluorescence. A larger
BILATERAL GRANULAR BLUE INJECTIONS
ENKEPHALIN
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315
bilateral injection of GB in the dorsal tegmental region, in an experiment in which cryostat sections (16 ~m) of the IP were cut, gave rise to a larger number of retrogradely labeled neurons in the IP and a higher proportion of double-labeled cells in the IP-R (Fig. 2). In this case, 15 Enk-immunoreacted sections contained 262 Enk-posirive cells, of which 47.9~o were double-labeled. In 12 SP-stained sections. 46.9°i, of the 130 SF,-positive neurons also contained the retrograde tracer GB. The present results indicate the existence in the IP-R of a population of Enk-positive neurons and a group of SP-containing cells that project to the dorsal tegmental rcgiol". In the target area, Enk-positive fibers and terminals are found in highest concentration in the periphery of the dorsal raphe nucleus, and dorsally and medially to the dorsal tegmental nucleus of Gudden near the midline and in the periventricular zone [10, 11, 14]. An almost similar distribution within the dorsal tegmental region has been described for SP fibers and terminals [!, 12]. Likewise, the most dense termination areas of IF, fibers, as described with the anterograde autoradiographic tracing method, are located in the midline, the periphery of the dorsal tegmental nucleus of Gudden and in the dorsolateral tegmental nucleus [3]. Depending on the size of the injection, up to about one-half of the Enk and SP neuronal populations in the IP-R can be double labeled following injections of G B in the dorsal tegmental region. Moreover, in all experiments there is a large group of neurons which contains only SP-IR or Enk-IR, without being retrogradely labeled. Whether these neurons project to other mesencephalic areas, such as the rostral parts of the dorsal raphe nucleus and the median raphe nucleus, or constitute intranuclear connections within the IF', cannot be concluded from the present results. In recent tracing experiments, it was established that IP-R not only projects to the raphe nuclei and the dorsal tegmental region, but also to the caudal IF, subnuclei 1PA and IP-L [3]. In the IF,-A both Enk- and SF,-positive fibers are present, and IP-L contains a dense SP-fiber plexus [3, 5, 6]. It must further be emphasized that the presence of large numbers of neurons in the 1P, retrogradely filled from the dorsal tegmental region without showing Enk-IR or SF,-IR, indicates that the interpedunculotegmental projections also may contain other neurotransmitters. Potential candidates are 7-aminobutyric acid, vasoactive intestinal polypeptide, somatostatin and serotonin, as all of these substances or one of their synthesizing enzymes has been demonstrated immunocytochemically to be contained in IF, neurons [5, 6]. In conclusion, the results of the present experiments have demonstrated the existence of peptidergic pathways, containing Enk and SF,, from the IF' to the dorsal tegmental region. What functional role these connections play remains to be established, but the present data suggest that the limbic regulation of the ascending serotonergic Fig. 2. Chartings of 4 pairs of adjacent coronal cryostat sections ( 16/~m) of the rostral half of the IP from an experiment in which retrograde tracing and immunohistochemistry were applied to the samc section. Adjacent sections were stained either for Enk (left column) or SP (right column). The location of the two GB deposits in the dorsal tegmental region and dorsal raphe nucleus is shown at the top. ()pen circles indicate single immunoreactive Enk or SP neurons, and stars identify immunoreactive neurons also containing GB. D L T N , dorsolateral tegmental nucleus; D R N , dorsal raphe nucleus: DTN, dorsal tegmental nucleus of Gudden: tim, fasciculus longitudinalis medialis; hp, habenulo-interpeduncular tract: 1P-I, intermediate subnucleus: LC, locus coeruleus; VTA, ventral tegmental area.
316 [7] a n d c h o l i n e r g i c [9, 13] s y s t e m s t h r o u g h t h e I P m a y in p a r t b e m e d i a t e d b y p e p t i dergic substances, The authors thank Drs. Pieter Voorn, Fokje Russchen and Tony Lohman tbr their suggestions and reading of the manuscript. The expert technical assistance of John Bol, D i r k d e J o n g , N a n e t t e K a a t a n d R i t a S t o e v e l a a r is g r a t e f u l l y a c k n o w l e d g e d . 1 Cuello, A.C. and Kanazawa, I., The distribution of substance P immunoreactive fibers in the rat central nervous system. J. Comp, Neurol., 178 (1978) 129 156. 2 Groenewegen, H.J. and Van Dijk, C.A., Efferent connections of the dorsal tegmental region in the rat, studied by means of anterograde transport of the lectin Phaseolus vulgaris-leucoagglutinin (PHAL), Brain Res., 304 (1984) 367-371. 3 Groenewegen, H.J., Ahlenius, S., Haber, S.N., Kowall, N.W. and Nauta, W.J.I-I., Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat, J. Comp. Neurol., in press. 4 Hamill, G.S. and Lenn, N.J., The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopic study, J. Comp. Neurol., 222 (1984) 396~408. 5 Hamill, G.S., Olschowska, J.A., Lenn, N.J. and Jacobowitz, D.M., The subnuclear distribution of substance P. vasoactive intestinal polypeptide, somatostatin, Leu-enkephalin, dopamine-//-hydroxylasc, and serotonin in the rat interpeduncular nucleus, J. Comp. Neurol., 226 (1984) 580-596. 6 Hemmendinger, L.M. and Moore, R.Y., lnterpeduncular nucleus organization in the rat: cytoarchitecture and histochemical analysis, Brain Res. Bull., 13 (1984) 163 179. 7 K6hter, C. and Steinbush, H.W.M., Identification of serotonin and non-serotonin containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent tracing study in the rat brain, Neuroscience, 7 (1982) 951-975. 8 Lenn, N.J. and Hamill, G.S., Subdivisions of the interpeduncular nucleus: a proposed nomenclature, Brain Res. Bull, 13 (1984) 203-204. 9 Mesulam, M.-M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch 1 Ch6), Neuroscience, I 0 (1983) 1185-1201. 10 Miller, R.J. and Pickel, V.M., The distribution and functions of the enkephalins, J. Histochem. Cytochem., 28 (1980) 903 917. 11 Moss, M.S., Glazer, E.J. and Basbaum, A.I., The peptidergic organization of the cat periaqueductal gray. I. The distribution of immunoreactive enkephalin-containing neurons and terminals, J. Neurosci., 3 (1983) 603 616. 12 Moss, M.S. and Basbaum, A.I., The peptidergic organization of the cat periaqueductal gray. 1I. The distribution of immunoreactive substance P and vasoactive intestinal polypeptide, J. Neurosci., 3 (1983) 1437-.1449. 13 Sofroniew, M.V., Priestley, J.V., Consolazione, A., Eckenstein, F. and Cuello, A.C., Cholinergic projcctions from the midbrain and pons to the thalamus in the rat, identified by combined retrograde t racing and choline acetyltransferase immunocytochemistry, Brain Res., 329 (1985) 213-223. 14 Uhl, G.R., Goodman, R.R., Kuhar, M.J., Childers, S.R. and Snyder, S.H., lmmunohistoehemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Res., 166 (1979) 75-94.