Brain Research, 178 (1979) 179-184 © Elsevier/North-Holland Biomedical Press
179
The mesencephalic exterolateral posterior nucleus of the, mormyrid fish Bryenomyrus niger: efferent connections studied by the HRP method
F. HAUGEDE-CARRE C.N.R.S., Laboratoire de Physiologie Nerveuse, Department de Neurophysiologie Sensorielle, F-91190 Gif Sur Yvette (France)
(Accepted August 16th, 1979) Key words: teleost - - fish - - mesencephalon - - torus semicircularis - - brain stem - -
postoptic commissure - - H R P
The torus semicircularis of teleosts has been described by several authors as a single nucleus, the nucleus lateralis mesencephalP,5,8,10, the symmetrical structures of which appear separated in the midline by a relatively large valvula cerebelli. In catfish, the torus semicircularis was recently shown both physiologically and anatomically 11, to be divided into two distinct nuclei, the nuclei lateralis and centralis. In mormyrids, the torus semicircularis was also considered earlier a to be a single structure, the ganglion mesencephali laterale, but was later subdivided by StendelP 2 into two nuclei, the ganglion mesencephali laterale (sensu stricto) and the ganglion mesencephali exterolaterale lz, both being the termination sites of the major part of the tractus octavo-lateralis mesencephalicus, i.e. lemniscus lateralis. Recent personal observations (Szabo, Ravaille, Libouban and Enger, personal communications) suggest that the ganglion, i.e. nucleus mesencephali lateralis is an even more complex structure, containing at least 5 different nuclei: the lateral mesencephalic nucleus (nL), which constitutes the principal mass of the torus semicircularis, the medio-dorsal nucleus, the medio-ventral nucleus (nMV), the dorsal nucleus and the ventro-posterior nucleus (nVP). Also the ganglion or nucleus exterolateralis can actually be divided into two nuclei exhibiting distinct structures and electrical activities2,a: the anterior exterolateral nucleus (nELa), the connections of which have been established recently 13, and the posterior exterolateral nucleus (nELp), which is the subject of the present communication. For this study 8 Bryenomyrus niger (7-9 cm in length) and 2 Gnathonernus petersii (10-15 cm in length) were used. Under MS 22 (Sandoz) anaesthesia, concentrated H R P (45 ~ ) of Sigma VI type was injected into the posterior exterolateral nucleus. The animals were allowed to survive 4-6 days and were then perfused through the aortic bulb with a mixture of 3 0 ~ formaldehyde (2~o)/25 ~ glutaraldehyde ( 8 ~ ) in a phosphate buffer 0.1 M (see ref. 9). After 3 h in a phosphatesucrose buffer, the brains were cut with a freezing microtome.
180
8
181 The sections (40 # m ) were incubated for 20 min in a solution o f 3, 3' diaminobenzidine tetrahydrochloride in Tris.HC1 (pH 7.6). The enzyme was then revealed during 3 rain by addition of hydrogen peroxide in the incubation bath. After rinsing in a phosphate buffer (pH 7.2), the brain sections were m o u n t e d in gelatin (2 ~o) then slightly stained with toluidine blue (1 ~). The posterior mesencephalic exterolaterat nucleus (nELp) can be distinguished f r o m the anterior exterolateral nucleus (nELa) by its cellular composition; the former displays a particularly large n u m b e r o f intermediate-sized and large cells (6-14/~m); whereas in the nELa, the small cells (3-4 # m ) prevail, a m o n g which relatively few large cells (12-16 # m ) are scattered. After H R P injection in the n E L p , a labelled tract o f fibres was observed which passes t h r o u g h the posterior part o f the mesencephalic lateral nucleus (nL) towards the brain stem (Fig. 4). This tract is composed of m a n y fine fibres, intermingled with few thick fibres. W h e n they arrive at the level of ' T e c t u m Marklager' (TM) 1~ which represents the ventral limit of torus semicircularis, the fine fibres divide into two bundles taking opposite directions (Fig. 4): the first one (a) runs caudally and terminates ipsilaterally, whereas the second (b) takes a cranial direction and ends contralaterally. The caudal bundle follows the lateral part o f the T M (Fig. 4, double arrow) and penetrates into a small nucleus, which lies ventrally to the nucleus preeminentialis (nP) and is therefore called the nucleus subpreeminentialis (nSP) (Fig. 6, arrow). Comprised o f cells smaller than those of the nP, the nSP is situated beneath the rostral half o f the nP. A small part o f the caudal bundle runs beyond the nSP towards the medulla (Fig. 6, double arrow) and distributes endings in the region o f the inferior olive. The cranial bundle forms a b o u n d a r y on the ventrolateral mesencephalic and diencephalic edge of the brain stem (Fig. 4, arrow); together with its contralateral Figs. 1-4 and 6. Photomicrographs of transverse sections through the brain stem of Bryenomyrus niger showing the different labelled tracts and nuclei after injection in the nucleus exterolateralis posterior, hELp, and (Figs. 5 and 7), after injection into the valvula cerebelli. Figs. 1 and 2. The postchiasmatic commissure; its labelled ventral part (double-headed arrows). Fig. 1 : vertical dashed line: midline plane, x 43. Fig. 2 is a detail of Fig. 1 indicated by a square, x 110. Darkfield. Fig. 3. Labelled endings of thick fibres in the ipsilateral ganglion isthmi, GI; x 110; inset: labelled ending in the GI at higher magnification showing several 'boutons terminaux' (arrows); x 1100. Fig. 4. Course of the labelled tract (encircled) originating from the hELp. a: ipsilateral bundle ending in the medio-ventral nucleus nMV; b: ipsilateral caudal bundle in the 'Tectum Marklager', TM, providing fibres for nucleus subpreeminentialis nSP, GI and the region of the inferior olive; c: cranial bundle running towards the contralateral side through the postchiasmatic commissure (see also Figs. 1 and 3). x 43. D~.rkfield. Fig. 5. HRP-labelled terminal ramifications in the nucleus subpreeminentialis (nSP) at high magnification (x 410); arrow shows a point of bifurcation. Fig. 6. Course of fine fibres passing through the GI and ending in the nCP (arrowhead). Double arrow points to the small bundle descending towards the inferior olive, x 43. Darkfield. Fig. 7. Preterrninal area of thick fibres in the medio-ventral nucleus (nMV), see Fig. 4. Ramifications are oriented preferably in a frontal plane of preterminal fibres: arrow indicates a point of bifurcation. x 250. Darkfield. Fig. 8. Small descending bundle of fine fibres (encircled) towards the inferior olive region (arrow), x 100. Darkfield.
182
Fig. 9. Schematic drawing of efferent connections of the nucleus exterolateralis posterior mesencephali (hELp) in the mormyrid fish Bryenomyrusnigerrepresented in transverse sections. Thick black line indicates the course of thick and thin efferent fibres originating in the left nELp. Cc, corpus cerebelli; EG, eminentia granularis; GI, ganglion isthmi; l.inf., lobus inferior; 11, lemniscus lateralis; nELa, nucleus exterolateralis anterior; nELp, nucleus exterolateralis posterior; nL, nucleus lateralis; nMV, nucleus medio-ventralis; nP, nucleus preeminentialis; nSP, nucleus sub-preeminentialis; nVP, nucleus ventro-posterior; p.ch.comm., postchiasmatic commissure; tect., tectum; TM, Tectum Marklager; v, ventricle; valv., valvula. A-P arrow indicates antero-posterior direction.
homologue, it constitutes the ventral part of the postoptic commissure (Figs. 1 and 2), i.e. the commissura transversa 10 or the postchiasmatic commissurO 2, so-called because of its situation immediately posterior to the optic chiasma. F r o m this commissure this bundle of fine fibres runs caudalwards symmetrically on the contralateral side and enters into the T M at the level of the nELp and finally ends in the contralateral nSP (see Fig. 8). The course of the thick fibres is almost the same as that of the fine fibres except for the termination sites: the ipsilateral fibres end in the medio-ventral nucleus (Fig. 4, a), whereas the remaining fibres lead ventrally towards the T M where they divide into two fascicles running in opposite directions: the caudal one runs through the T M (Fig. 4, b, double arrow) and ends in the ipsilateral ganglion isthmi (GI) lz situated laterally to the nP (Figs. 3 and 6), whereas the cranially directed fascicle passes through the postchiasmatic commissure (Figs. 1 and 2), then turns caudally and enters into the contralateral T M and terminates in the contralateral G I (Fig. 8).
183 The GI is composed of densely-packed round small ceils similar to cerebellar granular cells but somewhat larger in diameter (6-9 #m). This nucleus which was identified as the ganglion isthmi 12 belongs to a large group of granular-like cells (Libouban and Szabo, personal communication, Hauged6-Carr6 and Szabo, personal observations) which occupy an important part of the mesencephalic tegmentum and are dispersed around the various white matter structures of the brain stem. The labelled endings observed in this nucleus after injection into the nELp are quite particular: the fibre splits in several preterminal branches ending in 5-10 'boutons terminaux' which surround the same cell in the form of a basket (Fig. 3, inset). In conclusion (see Fig. 8), the hELp projects bilaterally onto the nSP and GI through the TM and the postchiasmatic commissure. Its ipsilateral connections with the nMV and the region of the inferior olive are established. The efferent connections of the nELp with the nSP and the inferior olive are made through the fine fibres, whereas those with the GI and nMV are made by means of thick fibres. The projections of nELp onto the GI are a matter of interest because injections made in the valvula cerebelli (personal observations) result in retrograde labelling of cells in the ipsilateral GI. Consequently, the latter should be considered as a site of transmission for impulses from the nELp towards the valvula. Furthermore, the presence of small labelled cells in the nELa after injection in the nELp (personal observations, see Fig. 8) suggests that the nELp receives fibres from the nELa which is the arrival site of the rapid electrosensory pathway 13. Thus the connections between tuberous electroreceptors (Knollenorgan) and the valvula seem to be established over the nELp 14.
1 Edinger, L., Vorlesungen iiber den Bau der nervOsen Centralorgane des Menschen und der Tiere, F. C. W. Vogel, Leipzig, 1908, 7. Auflage. 2 Enger, P. S., Libouban, S, and Szabo, T., Fast conducting electrosensory pathway in the Mormyrid fish Gnathonemus petersii, Neurosci. Lett., 2 (1976a) 127-133. 3 Enger, P. S., Szabo, T. and Libouban, S., Rhombo-mesencephalic connections in the fast conducting electrosensory system of the mormyrid fish, Gnathonemus petersii. An HRP study, Neurosei. Lett., 3 (1976) 239-243. 4 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system., Brain Research, 4 (1967) 369-374. 5 Franz, V., Das Mormyridenhirn, Zool. J., 32 (1911) 465-492. 6 Franz, V., Zur mikroskopischen Anatomic der Mormyriden, Zool. J., 42 (1920) 91-148. 7 Graham, R. C. and Karnowsky, M. F., The early stages of absorption of injected HRP in the proximal tubule of mouse kidney: ultrastruetural cytochemistryby a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 8 Ito, H., Fine structure of the torus semicircularis of some teleosts, Y. MorphoL, 142 (1974) 137-152. 9 Kaisermann-Abramoff, I. P. and Palay, S. L., Fine structural studies of the cerebellar cortex in a Mormyrid fish. In R. Llinas (Ed.), Neurobiology of Cerebellar Evohaion and Evolution, Amer. Med. Ass. Educ. and Res. Fdn., Chicago, 1969, 171-205. 10 Kappers, A. C. U., Huber, G. C. and Crosby, E. C., The Comparative Anatomy of the Nervous System of Vertebrates Including Man, VoL 1, MacMillan, New York, 1936, pp. 1239. 11 Knudsen, E. I., Distinct auditory and lateral line nuclei in the midbrain of catfish, J. comp. NeuroL, 173 (1977) 417432. 12 StendelI, W., Die Faseranatomie des Mormyridengehirns, Abh. Senekenberg. Forsch. Ges., 36 (1914) 3-40.
184 13 Szabo, T., Enger, P. S., Libouban, S., Electrosensory systems in the Mormyrid fish, Gnathonemus petersii: special emphasis on the fast conducting pathway, J. Physiol. (Paris), in press. 14 Zipser, B. and Bennett, M. V. L., Interaction of electrosensory and electromotor signals in the lateral line lobe of a mormyrid fish, J. NeurophysioL, 39 (1976) 713-721.
179
The mesencephalic exterolateral posterior nucleus of the, mormyrid fish Bryenomyrus niger: efferent connections studied by the HRP method
F. HAUGEDE-CARRE C.N.R.S., Laboratoire de Physiologie Nerveuse, Department de Neurophysiologie Sensorielle, F-91190 Gif Sur Yvette (France)
(Accepted August 16th, 1979) Key words: teleost - - fish - - mesencephalon - - torus semicircularis - - brain stem - -
postoptic commissure - - H R P
The torus semicircularis of teleosts has been described by several authors as a single nucleus, the nucleus lateralis mesencephalP,5,8,10, the symmetrical structures of which appear separated in the midline by a relatively large valvula cerebelli. In catfish, the torus semicircularis was recently shown both physiologically and anatomically 11, to be divided into two distinct nuclei, the nuclei lateralis and centralis. In mormyrids, the torus semicircularis was also considered earlier a to be a single structure, the ganglion mesencephali laterale, but was later subdivided by StendelP 2 into two nuclei, the ganglion mesencephali laterale (sensu stricto) and the ganglion mesencephali exterolaterale lz, both being the termination sites of the major part of the tractus octavo-lateralis mesencephalicus, i.e. lemniscus lateralis. Recent personal observations (Szabo, Ravaille, Libouban and Enger, personal communications) suggest that the ganglion, i.e. nucleus mesencephali lateralis is an even more complex structure, containing at least 5 different nuclei: the lateral mesencephalic nucleus (nL), which constitutes the principal mass of the torus semicircularis, the medio-dorsal nucleus, the medio-ventral nucleus (nMV), the dorsal nucleus and the ventro-posterior nucleus (nVP). Also the ganglion or nucleus exterolateralis can actually be divided into two nuclei exhibiting distinct structures and electrical activities2,a: the anterior exterolateral nucleus (nELa), the connections of which have been established recently 13, and the posterior exterolateral nucleus (nELp), which is the subject of the present communication. For this study 8 Bryenomyrus niger (7-9 cm in length) and 2 Gnathonernus petersii (10-15 cm in length) were used. Under MS 22 (Sandoz) anaesthesia, concentrated H R P (45 ~ ) of Sigma VI type was injected into the posterior exterolateral nucleus. The animals were allowed to survive 4-6 days and were then perfused through the aortic bulb with a mixture of 3 0 ~ formaldehyde (2~o)/25 ~ glutaraldehyde ( 8 ~ ) in a phosphate buffer 0.1 M (see ref. 9). After 3 h in a phosphatesucrose buffer, the brains were cut with a freezing microtome.
180
8
181 The sections (40 # m ) were incubated for 20 min in a solution o f 3, 3' diaminobenzidine tetrahydrochloride in Tris.HC1 (pH 7.6). The enzyme was then revealed during 3 rain by addition of hydrogen peroxide in the incubation bath. After rinsing in a phosphate buffer (pH 7.2), the brain sections were m o u n t e d in gelatin (2 ~o) then slightly stained with toluidine blue (1 ~). The posterior mesencephalic exterolaterat nucleus (nELp) can be distinguished f r o m the anterior exterolateral nucleus (nELa) by its cellular composition; the former displays a particularly large n u m b e r o f intermediate-sized and large cells (6-14/~m); whereas in the nELa, the small cells (3-4 # m ) prevail, a m o n g which relatively few large cells (12-16 # m ) are scattered. After H R P injection in the n E L p , a labelled tract o f fibres was observed which passes t h r o u g h the posterior part o f the mesencephalic lateral nucleus (nL) towards the brain stem (Fig. 4). This tract is composed of m a n y fine fibres, intermingled with few thick fibres. W h e n they arrive at the level of ' T e c t u m Marklager' (TM) 1~ which represents the ventral limit of torus semicircularis, the fine fibres divide into two bundles taking opposite directions (Fig. 4): the first one (a) runs caudally and terminates ipsilaterally, whereas the second (b) takes a cranial direction and ends contralaterally. The caudal bundle follows the lateral part o f the T M (Fig. 4, double arrow) and penetrates into a small nucleus, which lies ventrally to the nucleus preeminentialis (nP) and is therefore called the nucleus subpreeminentialis (nSP) (Fig. 6, arrow). Comprised o f cells smaller than those of the nP, the nSP is situated beneath the rostral half o f the nP. A small part o f the caudal bundle runs beyond the nSP towards the medulla (Fig. 6, double arrow) and distributes endings in the region o f the inferior olive. The cranial bundle forms a b o u n d a r y on the ventrolateral mesencephalic and diencephalic edge of the brain stem (Fig. 4, arrow); together with its contralateral Figs. 1-4 and 6. Photomicrographs of transverse sections through the brain stem of Bryenomyrus niger showing the different labelled tracts and nuclei after injection in the nucleus exterolateralis posterior, hELp, and (Figs. 5 and 7), after injection into the valvula cerebelli. Figs. 1 and 2. The postchiasmatic commissure; its labelled ventral part (double-headed arrows). Fig. 1 : vertical dashed line: midline plane, x 43. Fig. 2 is a detail of Fig. 1 indicated by a square, x 110. Darkfield. Fig. 3. Labelled endings of thick fibres in the ipsilateral ganglion isthmi, GI; x 110; inset: labelled ending in the GI at higher magnification showing several 'boutons terminaux' (arrows); x 1100. Fig. 4. Course of the labelled tract (encircled) originating from the hELp. a: ipsilateral bundle ending in the medio-ventral nucleus nMV; b: ipsilateral caudal bundle in the 'Tectum Marklager', TM, providing fibres for nucleus subpreeminentialis nSP, GI and the region of the inferior olive; c: cranial bundle running towards the contralateral side through the postchiasmatic commissure (see also Figs. 1 and 3). x 43. D~.rkfield. Fig. 5. HRP-labelled terminal ramifications in the nucleus subpreeminentialis (nSP) at high magnification (x 410); arrow shows a point of bifurcation. Fig. 6. Course of fine fibres passing through the GI and ending in the nCP (arrowhead). Double arrow points to the small bundle descending towards the inferior olive, x 43. Darkfield. Fig. 7. Preterrninal area of thick fibres in the medio-ventral nucleus (nMV), see Fig. 4. Ramifications are oriented preferably in a frontal plane of preterminal fibres: arrow indicates a point of bifurcation. x 250. Darkfield. Fig. 8. Small descending bundle of fine fibres (encircled) towards the inferior olive region (arrow), x 100. Darkfield.
182
Fig. 9. Schematic drawing of efferent connections of the nucleus exterolateralis posterior mesencephali (hELp) in the mormyrid fish Bryenomyrusnigerrepresented in transverse sections. Thick black line indicates the course of thick and thin efferent fibres originating in the left nELp. Cc, corpus cerebelli; EG, eminentia granularis; GI, ganglion isthmi; l.inf., lobus inferior; 11, lemniscus lateralis; nELa, nucleus exterolateralis anterior; nELp, nucleus exterolateralis posterior; nL, nucleus lateralis; nMV, nucleus medio-ventralis; nP, nucleus preeminentialis; nSP, nucleus sub-preeminentialis; nVP, nucleus ventro-posterior; p.ch.comm., postchiasmatic commissure; tect., tectum; TM, Tectum Marklager; v, ventricle; valv., valvula. A-P arrow indicates antero-posterior direction.
homologue, it constitutes the ventral part of the postoptic commissure (Figs. 1 and 2), i.e. the commissura transversa 10 or the postchiasmatic commissurO 2, so-called because of its situation immediately posterior to the optic chiasma. F r o m this commissure this bundle of fine fibres runs caudalwards symmetrically on the contralateral side and enters into the T M at the level of the nELp and finally ends in the contralateral nSP (see Fig. 8). The course of the thick fibres is almost the same as that of the fine fibres except for the termination sites: the ipsilateral fibres end in the medio-ventral nucleus (Fig. 4, a), whereas the remaining fibres lead ventrally towards the T M where they divide into two fascicles running in opposite directions: the caudal one runs through the T M (Fig. 4, b, double arrow) and ends in the ipsilateral ganglion isthmi (GI) lz situated laterally to the nP (Figs. 3 and 6), whereas the cranially directed fascicle passes through the postchiasmatic commissure (Figs. 1 and 2), then turns caudally and enters into the contralateral T M and terminates in the contralateral G I (Fig. 8).
183 The GI is composed of densely-packed round small ceils similar to cerebellar granular cells but somewhat larger in diameter (6-9 #m). This nucleus which was identified as the ganglion isthmi 12 belongs to a large group of granular-like cells (Libouban and Szabo, personal communication, Hauged6-Carr6 and Szabo, personal observations) which occupy an important part of the mesencephalic tegmentum and are dispersed around the various white matter structures of the brain stem. The labelled endings observed in this nucleus after injection into the nELp are quite particular: the fibre splits in several preterminal branches ending in 5-10 'boutons terminaux' which surround the same cell in the form of a basket (Fig. 3, inset). In conclusion (see Fig. 8), the hELp projects bilaterally onto the nSP and GI through the TM and the postchiasmatic commissure. Its ipsilateral connections with the nMV and the region of the inferior olive are established. The efferent connections of the nELp with the nSP and the inferior olive are made through the fine fibres, whereas those with the GI and nMV are made by means of thick fibres. The projections of nELp onto the GI are a matter of interest because injections made in the valvula cerebelli (personal observations) result in retrograde labelling of cells in the ipsilateral GI. Consequently, the latter should be considered as a site of transmission for impulses from the nELp towards the valvula. Furthermore, the presence of small labelled cells in the nELa after injection in the nELp (personal observations, see Fig. 8) suggests that the nELp receives fibres from the nELa which is the arrival site of the rapid electrosensory pathway 13. Thus the connections between tuberous electroreceptors (Knollenorgan) and the valvula seem to be established over the nELp 14.
1 Edinger, L., Vorlesungen iiber den Bau der nervOsen Centralorgane des Menschen und der Tiere, F. C. W. Vogel, Leipzig, 1908, 7. Auflage. 2 Enger, P. S., Libouban, S, and Szabo, T., Fast conducting electrosensory pathway in the Mormyrid fish Gnathonemus petersii, Neurosci. Lett., 2 (1976a) 127-133. 3 Enger, P. S., Szabo, T. and Libouban, S., Rhombo-mesencephalic connections in the fast conducting electrosensory system of the mormyrid fish, Gnathonemus petersii. An HRP study, Neurosei. Lett., 3 (1976) 239-243. 4 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system., Brain Research, 4 (1967) 369-374. 5 Franz, V., Das Mormyridenhirn, Zool. J., 32 (1911) 465-492. 6 Franz, V., Zur mikroskopischen Anatomic der Mormyriden, Zool. J., 42 (1920) 91-148. 7 Graham, R. C. and Karnowsky, M. F., The early stages of absorption of injected HRP in the proximal tubule of mouse kidney: ultrastruetural cytochemistryby a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 8 Ito, H., Fine structure of the torus semicircularis of some teleosts, Y. MorphoL, 142 (1974) 137-152. 9 Kaisermann-Abramoff, I. P. and Palay, S. L., Fine structural studies of the cerebellar cortex in a Mormyrid fish. In R. Llinas (Ed.), Neurobiology of Cerebellar Evohaion and Evolution, Amer. Med. Ass. Educ. and Res. Fdn., Chicago, 1969, 171-205. 10 Kappers, A. C. U., Huber, G. C. and Crosby, E. C., The Comparative Anatomy of the Nervous System of Vertebrates Including Man, VoL 1, MacMillan, New York, 1936, pp. 1239. 11 Knudsen, E. I., Distinct auditory and lateral line nuclei in the midbrain of catfish, J. comp. NeuroL, 173 (1977) 417432. 12 StendelI, W., Die Faseranatomie des Mormyridengehirns, Abh. Senekenberg. Forsch. Ges., 36 (1914) 3-40.
184 13 Szabo, T., Enger, P. S., Libouban, S., Electrosensory systems in the Mormyrid fish, Gnathonemus petersii: special emphasis on the fast conducting pathway, J. Physiol. (Paris), in press. 14 Zipser, B. and Bennett, M. V. L., Interaction of electrosensory and electromotor signals in the lateral line lobe of a mormyrid fish, J. NeurophysioL, 39 (1976) 713-721.
