Brain Research, 137 (1977) 11-35 © Elsevier/North-Holland Biomedical Press
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AFFERENT CONNECTIONS OF THE NUCLEUS RAPHE DORSALIS IN THE CAT AS VISUALIZED BY THE HORSERADISH PEROXIDASE TECHNIQUE
KAZUYA SAKAI, DENISE SALVERT, MONIQUE TOURET and MICHEL JOUVET Ddpartement de Mddecine Expdrimentale, Facultd de M~decine, Universitd Claude Bernard, 69008 Lyon (France) (Accepted March 10th, 1977)
SUMMARY
Using a retrograde tracer technique with protein horseradish peroxidase (HRP), attempts were made to determine afferent projections to the nucleus raphe dorsalis (NRD). As a control, the injection of the HRP was also made into one of the following structures adjacent to the NRD: (1) mesencephalic periaqueductal gray; (2) nucleus linearis intermedius; and (3) third cranial nucleus. The present results indicate that the NRD, particularly its rostral part, receives direct projections arising from: (1) locus coeruleus complex (locus coeruleus, locus coeruleus a, and locus subcoeruleus); (2) parabrachial nuclei (nucleus parabrachialis lateralis and medialis); (3) nucleus laterodorsalis tegmenti; (4) griseum centrale pontis, particularly the caudal part of the nucleus incertus; (5) substantia nigra; (6) lateral habenular nucleus; (7) hypothalamic areas, particularly dorsal and lateral hypothalamic areas; (8) preoptic areas; (9) an area dorso-lateral to the inferior olivary complex and medial to the lateral reticular nucleus; and (10) raphe nuclei, particularly nucleus linearis intermedius, centralis superior, pontis and magnus. The present findings thus confirm some previous reports on the afferent projections to the NRD described in the cat and rat, and further indicate the richness of afferent connections of the NRD. Some problems of the peroxidase technique have also been discussed.
INTRODUCTION
Evidence from histochemical fluorescence studies indicates that neurons containing a significant concentration of the putative neurotransmitter serotonin (5-HT) have their cell bodies almost exclusively in the brain stem raphe nuclei and their axons projecting extensively throughout the brain, from the spinal cord to the telencephalon 4,23,62,76. Biochemical3°,37,4°,5°,67 and fluorescence 4,7,76 studies further indi-
12 cate that the nucleus raphe dorsalis (NRD) together with the nucleus raphe centralis superior (NCS) constitute a major source of ascending serotonergic systems to various forebrain sites. The widespread efferent projections of the NRD have been also demonstrated recently by anterograde autoradiographic tracer techniques with labeled amino acids s,9,21,2s,74, and some of these connections have been further confirmed by a retrograde tracer technique with the protein horseradish peroxidase (HRp)42,4'5, 46, 48,53,5,5,59,65,66,69 (see also ref. 11). On the other hand, degeneration as well as autoradiographic studies in the rat and cat indicate afferents to the N R D arising from the hypothalamic areas22,57, 79 and lateral habenular nucleus 3. Further, several histochemical and fluorescence studies indicate a direct projection to the N R D from the nucleus locus coeruleus (LC) 2°,47,49. In the present study, using the retrograde tracer technique with the HRP a~, we have attempted to confirm these early observations and to identify further possible afferents to the NRD within the central nervous system. As a comparative study, afferent projections to the adjacent midbrain structures (such as the periaqueductal gray, oculomotor nucleus and nucleus linearis intermedius) have been also examined. The present results should provide some additional anatomical data for the physiological study of the midbrain raphe nuclei and adjacent central tegmental gray, implicated in the regulation of sleep, endocrine activity, emotional behavior, etc. (see refs. 17, 36, 54, 56 for review). MATERIALS AND METHODS The experiments were carried out on 20 adult cats. A single injection of 0.1 #I or 0.15 #1 of a 50 ~o solution (Boehringer Grade I) was made with a Hamilton 5 #1 syringe under stereotaxic guidance. In order to avoid the bony tentorium, the needle was usually inserted into the brain through the cerebellum at a 45 ° angle to the stereotaxic horizontal plane, while, in some cases (M 57, N 57, K 57), the injection was made with the needle inclined 90 ° to the horizontal plane and 3 ° to the sagittal plane. In 8 animals, the HRP was injected into the nucleus raphe dorsalis (NRD) either into the rostral part (A 1.0-AP 0, L 0-0.3, H - - 1.5---2.0) (cases: N 54, M 55, L 56, R 54, W 55, V 56) or into the caudal part of this nucleus (P 1.0-2.0, L 0-0.2, H - - 1 . 5 - - - 2 . 5 ) (cases: S 55, D 57). in other experimental animals, the HRP was injected into one of the following structures adjacent to the NRD: (i) periaqueductal gray (Gc) (cases: X 56, A 57, N 57); (2) nucleus linearis intermedius (Li) (cases: T 55, Z 56, K 57); and (3) third cranial nucleus (N II|) (cases: Y 55, M 56, M 57). In three control animals, the HRP was injected into the cerebral aqueduct and the animals were subjected to the same perfusion and incubation procedures as the experimental animals. Following survival times of 19 or 24 h, the brain was perfused with 3% paraformaldehyde-1%o glutaraldehyde in 0.1 M phosphate buffer (pH 7.2-7.4). The brain was removed, placed in fresh fixative at 4 °C for 16-24 h, and then rinsed for about 24 h at 4 °C in 0.1 M phosphate buffer containing 5 ~/o sucrose. Frontal sections 50 #m
13 thick were cut on a freezing microtome usually from Horsley-Clarke (HC) plane A 15-P 15. The sections were soaked for 30 min at 37 °C in a preincubation solution containing 0.05 ~o 3,3'-diaminobenzidine (DAB), and then incubated for 30 min in a 0.05 ~o DAB solution containing 0.03 ~ hydrogen peroxide, washed and mounted 26,4a, 65. Some sections were lightly counterstained with cresylecht violet. Examination for the HRP-containing neurons was carried out under both light and dark-field illumination, and care was taken to differentiate the retrograde HRP-labeled neurons from HRP-labeled endothelial or vascular cells, according to the criteria described by Nauta et al. 59. RESULTS L Results obtained from control animals HRP-labeled cells were not found in any studied brain structures (from the preoptic area to the medulla oblongata), when the HRP had been injected into the cerebral aqueduct. This negative result ruled out, at least in our study: (1) a possible uptake of HRP by brain parenchyma adjacent to the ventricle and subsequent retrograde transport to the somata of the axons2,1°; and (2) any possible confusion between retrograde exogenous HRP granules and granules endogenously present in the brain such as lipofuscin, neuromelanin, heme-enzymes and other hemo-proteins 43,52,~9,s°. 1I. Results obtained from experimental animals As described by Taber 73, Taber et al. 72 and other authors 6,77, the NRD is situated in and ventral to the central periaqueductal gray. The ventral part of the nucleus is located between the two medial longitudinal fasciculi (MLF) and adjacent to the commissure of Probst 5,77 and decussating fibers of the brachium conjunctivum (BC). Most rostrally, the nucleus adjoins the oculomotor nucleus (N III) and merges ventrally with the nucleus linearis intermedius (Li). At the level of the trochlear nucleus (N IV), it has a laterally extending wing on either side (lateral division of the NRD). The lateral part of the NRD thus adjoins ventrally the N IV and nucleus annularis (An), laterally the nucleus laterodorsalis tegmenti (Ldt), and ventro-laterally the ascending dorsal catecholaminergic (CA) bundle 51. The caudal part of the nucleus adjoins laterally the dorsal tegmental nucleus of Gudden (TD) and nucleus incertus (INC), and fuses ventrally with nucleus centralis superior (NCS) (cf. Figs. 2 and 4). Considering the complexity of these surrounding structures, it is of great importance, for the mapping of the specific projection to the NRD, to determine the precise location of the needle tip and the extent of the diffusion of HRP, particularly since: (1) although numerous recent HRP studies have pointed out that the effective zone of uptake tends to be limited to the tissue immediately surrounding the needle tip 15,34,45,59,65, evidence against it has also been reported44; and (2) the HRP can be taken up by axons of passage (particularly those damaged by the injection) and transported in a retrograde direction to the parent cell bodies 15,as,as,44. In order to control these possibilities of uptake by diffusion and by damaged axons of passage (and subsequent retrograde transport), we have attempted to inject a
|1
AFFERENT CONNECTIONS OF THE NUCLEUS RAPHE DORSALIS IN THE CAT AS VISUALIZED BY THE HORSERADISH PEROXIDASE TECHNIQUE
KAZUYA SAKAI, DENISE SALVERT, MONIQUE TOURET and MICHEL JOUVET Ddpartement de Mddecine Expdrimentale, Facultd de M~decine, Universitd Claude Bernard, 69008 Lyon (France) (Accepted March 10th, 1977)
SUMMARY
Using a retrograde tracer technique with protein horseradish peroxidase (HRP), attempts were made to determine afferent projections to the nucleus raphe dorsalis (NRD). As a control, the injection of the HRP was also made into one of the following structures adjacent to the NRD: (1) mesencephalic periaqueductal gray; (2) nucleus linearis intermedius; and (3) third cranial nucleus. The present results indicate that the NRD, particularly its rostral part, receives direct projections arising from: (1) locus coeruleus complex (locus coeruleus, locus coeruleus a, and locus subcoeruleus); (2) parabrachial nuclei (nucleus parabrachialis lateralis and medialis); (3) nucleus laterodorsalis tegmenti; (4) griseum centrale pontis, particularly the caudal part of the nucleus incertus; (5) substantia nigra; (6) lateral habenular nucleus; (7) hypothalamic areas, particularly dorsal and lateral hypothalamic areas; (8) preoptic areas; (9) an area dorso-lateral to the inferior olivary complex and medial to the lateral reticular nucleus; and (10) raphe nuclei, particularly nucleus linearis intermedius, centralis superior, pontis and magnus. The present findings thus confirm some previous reports on the afferent projections to the NRD described in the cat and rat, and further indicate the richness of afferent connections of the NRD. Some problems of the peroxidase technique have also been discussed.
INTRODUCTION
Evidence from histochemical fluorescence studies indicates that neurons containing a significant concentration of the putative neurotransmitter serotonin (5-HT) have their cell bodies almost exclusively in the brain stem raphe nuclei and their axons projecting extensively throughout the brain, from the spinal cord to the telencephalon 4,23,62,76. Biochemical3°,37,4°,5°,67 and fluorescence 4,7,76 studies further indi-
12 cate that the nucleus raphe dorsalis (NRD) together with the nucleus raphe centralis superior (NCS) constitute a major source of ascending serotonergic systems to various forebrain sites. The widespread efferent projections of the NRD have been also demonstrated recently by anterograde autoradiographic tracer techniques with labeled amino acids s,9,21,2s,74, and some of these connections have been further confirmed by a retrograde tracer technique with the protein horseradish peroxidase (HRp)42,4'5, 46, 48,53,5,5,59,65,66,69 (see also ref. 11). On the other hand, degeneration as well as autoradiographic studies in the rat and cat indicate afferents to the N R D arising from the hypothalamic areas22,57, 79 and lateral habenular nucleus 3. Further, several histochemical and fluorescence studies indicate a direct projection to the N R D from the nucleus locus coeruleus (LC) 2°,47,49. In the present study, using the retrograde tracer technique with the HRP a~, we have attempted to confirm these early observations and to identify further possible afferents to the NRD within the central nervous system. As a comparative study, afferent projections to the adjacent midbrain structures (such as the periaqueductal gray, oculomotor nucleus and nucleus linearis intermedius) have been also examined. The present results should provide some additional anatomical data for the physiological study of the midbrain raphe nuclei and adjacent central tegmental gray, implicated in the regulation of sleep, endocrine activity, emotional behavior, etc. (see refs. 17, 36, 54, 56 for review). MATERIALS AND METHODS The experiments were carried out on 20 adult cats. A single injection of 0.1 #I or 0.15 #1 of a 50 ~o solution (Boehringer Grade I) was made with a Hamilton 5 #1 syringe under stereotaxic guidance. In order to avoid the bony tentorium, the needle was usually inserted into the brain through the cerebellum at a 45 ° angle to the stereotaxic horizontal plane, while, in some cases (M 57, N 57, K 57), the injection was made with the needle inclined 90 ° to the horizontal plane and 3 ° to the sagittal plane. In 8 animals, the HRP was injected into the nucleus raphe dorsalis (NRD) either into the rostral part (A 1.0-AP 0, L 0-0.3, H - - 1.5---2.0) (cases: N 54, M 55, L 56, R 54, W 55, V 56) or into the caudal part of this nucleus (P 1.0-2.0, L 0-0.2, H - - 1 . 5 - - - 2 . 5 ) (cases: S 55, D 57). in other experimental animals, the HRP was injected into one of the following structures adjacent to the NRD: (i) periaqueductal gray (Gc) (cases: X 56, A 57, N 57); (2) nucleus linearis intermedius (Li) (cases: T 55, Z 56, K 57); and (3) third cranial nucleus (N II|) (cases: Y 55, M 56, M 57). In three control animals, the HRP was injected into the cerebral aqueduct and the animals were subjected to the same perfusion and incubation procedures as the experimental animals. Following survival times of 19 or 24 h, the brain was perfused with 3% paraformaldehyde-1%o glutaraldehyde in 0.1 M phosphate buffer (pH 7.2-7.4). The brain was removed, placed in fresh fixative at 4 °C for 16-24 h, and then rinsed for about 24 h at 4 °C in 0.1 M phosphate buffer containing 5 ~/o sucrose. Frontal sections 50 #m
13 thick were cut on a freezing microtome usually from Horsley-Clarke (HC) plane A 15-P 15. The sections were soaked for 30 min at 37 °C in a preincubation solution containing 0.05 ~o 3,3'-diaminobenzidine (DAB), and then incubated for 30 min in a 0.05 ~o DAB solution containing 0.03 ~ hydrogen peroxide, washed and mounted 26,4a, 65. Some sections were lightly counterstained with cresylecht violet. Examination for the HRP-containing neurons was carried out under both light and dark-field illumination, and care was taken to differentiate the retrograde HRP-labeled neurons from HRP-labeled endothelial or vascular cells, according to the criteria described by Nauta et al. 59. RESULTS L Results obtained from control animals HRP-labeled cells were not found in any studied brain structures (from the preoptic area to the medulla oblongata), when the HRP had been injected into the cerebral aqueduct. This negative result ruled out, at least in our study: (1) a possible uptake of HRP by brain parenchyma adjacent to the ventricle and subsequent retrograde transport to the somata of the axons2,1°; and (2) any possible confusion between retrograde exogenous HRP granules and granules endogenously present in the brain such as lipofuscin, neuromelanin, heme-enzymes and other hemo-proteins 43,52,~9,s°. 1I. Results obtained from experimental animals As described by Taber 73, Taber et al. 72 and other authors 6,77, the NRD is situated in and ventral to the central periaqueductal gray. The ventral part of the nucleus is located between the two medial longitudinal fasciculi (MLF) and adjacent to the commissure of Probst 5,77 and decussating fibers of the brachium conjunctivum (BC). Most rostrally, the nucleus adjoins the oculomotor nucleus (N III) and merges ventrally with the nucleus linearis intermedius (Li). At the level of the trochlear nucleus (N IV), it has a laterally extending wing on either side (lateral division of the NRD). The lateral part of the NRD thus adjoins ventrally the N IV and nucleus annularis (An), laterally the nucleus laterodorsalis tegmenti (Ldt), and ventro-laterally the ascending dorsal catecholaminergic (CA) bundle 51. The caudal part of the nucleus adjoins laterally the dorsal tegmental nucleus of Gudden (TD) and nucleus incertus (INC), and fuses ventrally with nucleus centralis superior (NCS) (cf. Figs. 2 and 4). Considering the complexity of these surrounding structures, it is of great importance, for the mapping of the specific projection to the NRD, to determine the precise location of the needle tip and the extent of the diffusion of HRP, particularly since: (1) although numerous recent HRP studies have pointed out that the effective zone of uptake tends to be limited to the tissue immediately surrounding the needle tip 15,34,45,59,65, evidence against it has also been reported44; and (2) the HRP can be taken up by axons of passage (particularly those damaged by the injection) and transported in a retrograde direction to the parent cell bodies 15,as,as,44. In order to control these possibilities of uptake by diffusion and by damaged axons of passage (and subsequent retrograde transport), we have attempted to inject a
