Brain Research, 155 (1978) 103-107 © Elsevier/North-Holland Biomedical Press
103
Short Communications
Avian efferent vestibular neurons identified by axonal transport of pH]adenosine and horseradish peroxidase DIETRICH W. F. SCHWARZ, IRMGARD E. SCHWARZ and R. DAVID TOMLINSON Laboratory of Otoneurology, Medical Sciences Building, Room 7308, Depts. of Otolaryngology and Physiology, University of Toronto, Toronto, Ontario M5S lA8 (Canada)
(Accepted May 25th, 1978)
The sensory epithelia of the vestibular organs in the inner ear are under central nervous influence via efferent axons originating in the brain. The nature of the control provided by these efferents is still enigmatic, despite a considerable number of studies devoted to this problem2-4,10,11,15. Efferent neurons have been identified in mammals (see refs. 2-4, 15) and amphibia lo. Progress has been limited by surgical difficulties encountered when dissecting labyrinths in these species. Vestibular sensory organs of pigeons are relatively easily accessible and we have identified vestibular efferent neurons in this species in preparation for further studies aimed at a functional interpretation. The axonal transport of adenosinea,12,13, ~6-1s proved to be useful for this purpose. In each of 6 pigeons the ampulla of one semicircular canal was opened surgically under general anaesthesia (64 mg/kg ketamine, i.m., followed by N20-halothane inhalation) and sterile precautions in order to expose the corresponding crista ampullaris. A micropipette was inserted into the crista through which 50/~Ci of 2-[3H]adenosine in 1 #1 of distilled water were injected over 30 min. After this operation, the pigeons were allowed to survive for 3 or 4 days. Two experiments were made for each of the 3 canals of the left side. After the survival period the birds were anaesthetized and transcardially perfused with a buffered aldehyde fixative. Serial paraffin sections (10 #m) were cut in a sagittal or coronal plane and prepared for autoradiography in a standard fashionL Neuron somata could be clearly identified as labeled cells at two locations only. (I) The left ganglion of Scarpa was heavily labeled (Fig. 1), which suggests that the direction of the main label trasport is better characterized by the term 'somatopetal' than by 'retrograde'16, is. (2) The labeled somata of vestibular efferent cells were found within a narrow region within the nucleus reticularis pontis caudalis 6 ventral to the medial and ventrolateral vestibular nuclei and just latero-ventro-caudal to the abducens nucleus (Figs. 2, 3 and 5). The maximal extent of this region was 1.6 mm anteroposteriorly, 0.5 mm ventrodorsally and 0.6 mm laterally; the volume involved was always less than 0.5 cu.mm. In all cases the projection was bilateral. Relatively few labeled cell somata were found in each brain stem, the number was estimated to range from 90 to 160 per injection. This estimate was based upon the number of cells counted and section thickness s. No topographic difference could be recognized for injections of different
104
105
Fig. 5. Semidiagrammatic view of efferent vestibular neurons found labeled after [aH]adenosine injection into the left horizontal canal crista. Cells found in this (obliquely cut) section are marked by circles, those found in neighbour sections up to 120/~m rostral or caudal to this section are shown as squares. The region exhibiting anterograde label (cf. Fig. 4) (ipsilateral vestibular nuclei) is stippled. VS, VD, VM, VL, DL, TA nucleus vestibularis superior, descendens, medialis, ventrolateralis, dorsolateralis and tangentialis respectively; TTD descending trigeminal nucleus and tract, OS -- oliva superior; PM, PL nucleus pontis medialis and lateralis; FLM fasciculus longitudinalis medialis; VI nucleus abducens; VII nucleus facialis; GL, ML granular and molecular layer of the lingula. cristae. This a p p a r e n t lack of topographical organization could be a reflection of either a r a n d o m d i s t r i b u t i o n of efferent cells within the nucleus or the presence of a collateralizing supply to different cristae. These possibilities are currently u n d e r investigation in our laboratory. The location of the efferent n e u r o n somata (Fig. 5) was confirmed in one additional pigeon prepared for identification of retrogradely transported horseradish peroxidase (HRP). In this animal 1/A of a 30 ~ H R P (Miles Lab., South Africa) solution in distilled water was injected over one h o u r into the right lateral canal crista. The survival time was two days a n d the histochemical procedure according to Keefer v.
Fig. 1. Ganglion Scarpae, showing labeled ganglion cells at low power, x 110. Fig. 2. Efferent vestibular neurons in caudal pontine reticular nucleus. This distribution of labeled cells between unlabeled elements is characteristic, x 210. Fig. 3. Retrograde label in efferent vestibular neurons in the caudal pontine reticular nucleus ipsilateral to the tangential nucleus shown in Fig. 4. × 530. Fig. 4. Label over neuropil and cell bodies in the tangential nucleus, same brain as in Fig. 3. Unfocussed grains appear relatively pale. × 530.
106 Label in the efferent cells was of the traditional granular type, not o f the Golgi-like appearance seen in Keefer's material. Label resulting f r o m [3H]adenosine injection has been shown to travel in an anterograde as well as in a somatopetal directionS, 12,13,1e-Is. Even transneural t r a n sport within the region of axonal termination fields has been reported 5,12~;3,1~s. It is, therefore, necessary to exclude the possibility that cells in the termination field of primary vestibular afferents might erroneously be interpreted as efferent neurons. In our material there was anterograde labeling of the vestibular nuclei on the left side which was evident by higher than background grain density over neuropil in ~his area (Fig. 4). Occasionally, above b a c k g r o u n d activity was also seen over vestibular nuclei cell somata, the greatest grain density found being shown in Fig. 4. It is uncertain if the corresponding radiation originated from transneurally labeled cell bodies or afferent terminals contacting somata 14. The a m o u n t of this label is, however, much smaller than that seen over neurons in the caudal pontine reticular nucleus (compare Figs. 3 and 4), and it is consequently easy to differentiate somatopetal and transneurat label. Since no transported label was found in the contralateral vestibular nuclei Or the extraocular m o t o r nuclei, our material does not provide any evidence for specific transneural transport of label originating from the [3H]adenosine injections. It can, therefore, be concluded that neurons in the nucleus reticularis pontis caudalis which exhibit label after [all]adenosine injection into semicircular canal cristae are sources of vestibular effercnt fibers. We thank Prof. M. Z i m m e r m a n n for critical comments on the manuscript and Mrs. Linda Szeto for expert histological assistance. This study was supported by the Medical Research Council of Canada.
1 Cowan, W. M.,Gottlieb, D. T.. Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration ofaxonalconnectionsin the centratnervous system, Brain Research. 37(1972) 21-51. 2 Gacek, R. R. and Lyon, M., The localization of vestibular efferent neurons in the kitten with horseradish peroxidase, Acta Otolaryngol., 77 (1974) 92-101. 3 Gacek, R. R., Morphological aspects of the efferent vestibular system, in Handbook of Sensory Physiology, Vol. 6, Part 1, 1974, pp. 213-220. 4 Goldberg, J. M. and Fernandez, C., Efferent vestibular system in the squirrel monkey, Neurosci. Abstr., 111 (1977) 543 (no. 1723). 5 Hunt. S. P. and KiJnzle, H., Bidirectional movement of label and transeuronal transport phenomena after injection of [3H]adenosine into the central nervous system, Brain Research. t 12 (1976) 127-132.
6 Karten. H. J. and Hodos, W.. ,4 Stereotaxic Atlas of the Brain of Pigeon, Johns Hopkins Pl ess, Baltimore, Md.. 1967. 7 Keefer, D. A., Horseradish peroxidase as a retrogradely transported, detailed dendritic marker. Brain Research, 140 (1978) 15-32. 8 Konigsmark, B. W., Methods for the counting of neurons. In W. G. H. Nauta and S. O. E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 3t5-340. 9 Kruger, L. and Saporta, S., Axonal transport of [3H]adenosine in visual and somatosensory pathways, Brain Research, 122 (1977) 132-136. 10 Precht, W., Physiology of the peripheral and central vestibular systems. In R. Llinhs and W. Precht (Eds.), Frog Neurobiology, Springer, Berlin, 1976, pp. 481-512.
107 11 Precht, W., Physiological aspects of the efferent vestibular system, Handbook Sensory Physiology, Vol. 6, Part 1, 1974, pp. 221-238. 12 Schubert, P. and Kreutzberg, G. W., Axonal transport ofadenosine and uridine derivates and transfer to postsynaptic neurons, Brain Research, 76 (1974) 526-530. 13 Schubert, P. and Kreutzberg, G. W., [3H]adenosine, a tracer for neuronal connectivity, Brain Research, 85 (1975) 317 319. 14 Schwarz, D. W. F., Schwarz, I. E. and Fredrickson, J. M., Fine structure of the medial and descending vestibular nuclei in normal rats and after unilateral transection of the vestibular nerve, Acta Otolaryngol., 84 (I 977) 80-90. 15 Warr, W. B., Olivocochlear and vestibular efferent neurons of feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry, J. comp. Neurol., 161 (1974) 159-182. 16 Wise, S. P. and Jones, E. G., Transneural or retrograde transport of [3H]adenosine in the rat somatic sensory system, Brain Research, 107 (1976) 127-131. 17 Wise, S. P. and Jones, E. G., The organization and postnatal development of the commissural projection of the rat somatic sensory system, Brain Research, 168 (1976) 313-344. 18 Wise, S. P., Jones, E. G. and Berman, N., Direction and specificity of axonal and transcellular transport of nucleosides, Brain Research, 139 (1978) 197-217.
103
Short Communications
Avian efferent vestibular neurons identified by axonal transport of pH]adenosine and horseradish peroxidase DIETRICH W. F. SCHWARZ, IRMGARD E. SCHWARZ and R. DAVID TOMLINSON Laboratory of Otoneurology, Medical Sciences Building, Room 7308, Depts. of Otolaryngology and Physiology, University of Toronto, Toronto, Ontario M5S lA8 (Canada)
(Accepted May 25th, 1978)
The sensory epithelia of the vestibular organs in the inner ear are under central nervous influence via efferent axons originating in the brain. The nature of the control provided by these efferents is still enigmatic, despite a considerable number of studies devoted to this problem2-4,10,11,15. Efferent neurons have been identified in mammals (see refs. 2-4, 15) and amphibia lo. Progress has been limited by surgical difficulties encountered when dissecting labyrinths in these species. Vestibular sensory organs of pigeons are relatively easily accessible and we have identified vestibular efferent neurons in this species in preparation for further studies aimed at a functional interpretation. The axonal transport of adenosinea,12,13, ~6-1s proved to be useful for this purpose. In each of 6 pigeons the ampulla of one semicircular canal was opened surgically under general anaesthesia (64 mg/kg ketamine, i.m., followed by N20-halothane inhalation) and sterile precautions in order to expose the corresponding crista ampullaris. A micropipette was inserted into the crista through which 50/~Ci of 2-[3H]adenosine in 1 #1 of distilled water were injected over 30 min. After this operation, the pigeons were allowed to survive for 3 or 4 days. Two experiments were made for each of the 3 canals of the left side. After the survival period the birds were anaesthetized and transcardially perfused with a buffered aldehyde fixative. Serial paraffin sections (10 #m) were cut in a sagittal or coronal plane and prepared for autoradiography in a standard fashionL Neuron somata could be clearly identified as labeled cells at two locations only. (I) The left ganglion of Scarpa was heavily labeled (Fig. 1), which suggests that the direction of the main label trasport is better characterized by the term 'somatopetal' than by 'retrograde'16, is. (2) The labeled somata of vestibular efferent cells were found within a narrow region within the nucleus reticularis pontis caudalis 6 ventral to the medial and ventrolateral vestibular nuclei and just latero-ventro-caudal to the abducens nucleus (Figs. 2, 3 and 5). The maximal extent of this region was 1.6 mm anteroposteriorly, 0.5 mm ventrodorsally and 0.6 mm laterally; the volume involved was always less than 0.5 cu.mm. In all cases the projection was bilateral. Relatively few labeled cell somata were found in each brain stem, the number was estimated to range from 90 to 160 per injection. This estimate was based upon the number of cells counted and section thickness s. No topographic difference could be recognized for injections of different
104
105
Fig. 5. Semidiagrammatic view of efferent vestibular neurons found labeled after [aH]adenosine injection into the left horizontal canal crista. Cells found in this (obliquely cut) section are marked by circles, those found in neighbour sections up to 120/~m rostral or caudal to this section are shown as squares. The region exhibiting anterograde label (cf. Fig. 4) (ipsilateral vestibular nuclei) is stippled. VS, VD, VM, VL, DL, TA nucleus vestibularis superior, descendens, medialis, ventrolateralis, dorsolateralis and tangentialis respectively; TTD descending trigeminal nucleus and tract, OS -- oliva superior; PM, PL nucleus pontis medialis and lateralis; FLM fasciculus longitudinalis medialis; VI nucleus abducens; VII nucleus facialis; GL, ML granular and molecular layer of the lingula. cristae. This a p p a r e n t lack of topographical organization could be a reflection of either a r a n d o m d i s t r i b u t i o n of efferent cells within the nucleus or the presence of a collateralizing supply to different cristae. These possibilities are currently u n d e r investigation in our laboratory. The location of the efferent n e u r o n somata (Fig. 5) was confirmed in one additional pigeon prepared for identification of retrogradely transported horseradish peroxidase (HRP). In this animal 1/A of a 30 ~ H R P (Miles Lab., South Africa) solution in distilled water was injected over one h o u r into the right lateral canal crista. The survival time was two days a n d the histochemical procedure according to Keefer v.
Fig. 1. Ganglion Scarpae, showing labeled ganglion cells at low power, x 110. Fig. 2. Efferent vestibular neurons in caudal pontine reticular nucleus. This distribution of labeled cells between unlabeled elements is characteristic, x 210. Fig. 3. Retrograde label in efferent vestibular neurons in the caudal pontine reticular nucleus ipsilateral to the tangential nucleus shown in Fig. 4. × 530. Fig. 4. Label over neuropil and cell bodies in the tangential nucleus, same brain as in Fig. 3. Unfocussed grains appear relatively pale. × 530.
106 Label in the efferent cells was of the traditional granular type, not o f the Golgi-like appearance seen in Keefer's material. Label resulting f r o m [3H]adenosine injection has been shown to travel in an anterograde as well as in a somatopetal directionS, 12,13,1e-Is. Even transneural t r a n sport within the region of axonal termination fields has been reported 5,12~;3,1~s. It is, therefore, necessary to exclude the possibility that cells in the termination field of primary vestibular afferents might erroneously be interpreted as efferent neurons. In our material there was anterograde labeling of the vestibular nuclei on the left side which was evident by higher than background grain density over neuropil in ~his area (Fig. 4). Occasionally, above b a c k g r o u n d activity was also seen over vestibular nuclei cell somata, the greatest grain density found being shown in Fig. 4. It is uncertain if the corresponding radiation originated from transneurally labeled cell bodies or afferent terminals contacting somata 14. The a m o u n t of this label is, however, much smaller than that seen over neurons in the caudal pontine reticular nucleus (compare Figs. 3 and 4), and it is consequently easy to differentiate somatopetal and transneurat label. Since no transported label was found in the contralateral vestibular nuclei Or the extraocular m o t o r nuclei, our material does not provide any evidence for specific transneural transport of label originating from the [3H]adenosine injections. It can, therefore, be concluded that neurons in the nucleus reticularis pontis caudalis which exhibit label after [all]adenosine injection into semicircular canal cristae are sources of vestibular effercnt fibers. We thank Prof. M. Z i m m e r m a n n for critical comments on the manuscript and Mrs. Linda Szeto for expert histological assistance. This study was supported by the Medical Research Council of Canada.
1 Cowan, W. M.,Gottlieb, D. T.. Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration ofaxonalconnectionsin the centratnervous system, Brain Research. 37(1972) 21-51. 2 Gacek, R. R. and Lyon, M., The localization of vestibular efferent neurons in the kitten with horseradish peroxidase, Acta Otolaryngol., 77 (1974) 92-101. 3 Gacek, R. R., Morphological aspects of the efferent vestibular system, in Handbook of Sensory Physiology, Vol. 6, Part 1, 1974, pp. 213-220. 4 Goldberg, J. M. and Fernandez, C., Efferent vestibular system in the squirrel monkey, Neurosci. Abstr., 111 (1977) 543 (no. 1723). 5 Hunt. S. P. and KiJnzle, H., Bidirectional movement of label and transeuronal transport phenomena after injection of [3H]adenosine into the central nervous system, Brain Research. t 12 (1976) 127-132.
6 Karten. H. J. and Hodos, W.. ,4 Stereotaxic Atlas of the Brain of Pigeon, Johns Hopkins Pl ess, Baltimore, Md.. 1967. 7 Keefer, D. A., Horseradish peroxidase as a retrogradely transported, detailed dendritic marker. Brain Research, 140 (1978) 15-32. 8 Konigsmark, B. W., Methods for the counting of neurons. In W. G. H. Nauta and S. O. E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 3t5-340. 9 Kruger, L. and Saporta, S., Axonal transport of [3H]adenosine in visual and somatosensory pathways, Brain Research, 122 (1977) 132-136. 10 Precht, W., Physiology of the peripheral and central vestibular systems. In R. Llinhs and W. Precht (Eds.), Frog Neurobiology, Springer, Berlin, 1976, pp. 481-512.
107 11 Precht, W., Physiological aspects of the efferent vestibular system, Handbook Sensory Physiology, Vol. 6, Part 1, 1974, pp. 221-238. 12 Schubert, P. and Kreutzberg, G. W., Axonal transport ofadenosine and uridine derivates and transfer to postsynaptic neurons, Brain Research, 76 (1974) 526-530. 13 Schubert, P. and Kreutzberg, G. W., [3H]adenosine, a tracer for neuronal connectivity, Brain Research, 85 (1975) 317 319. 14 Schwarz, D. W. F., Schwarz, I. E. and Fredrickson, J. M., Fine structure of the medial and descending vestibular nuclei in normal rats and after unilateral transection of the vestibular nerve, Acta Otolaryngol., 84 (I 977) 80-90. 15 Warr, W. B., Olivocochlear and vestibular efferent neurons of feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry, J. comp. Neurol., 161 (1974) 159-182. 16 Wise, S. P. and Jones, E. G., Transneural or retrograde transport of [3H]adenosine in the rat somatic sensory system, Brain Research, 107 (1976) 127-131. 17 Wise, S. P. and Jones, E. G., The organization and postnatal development of the commissural projection of the rat somatic sensory system, Brain Research, 168 (1976) 313-344. 18 Wise, S. P., Jones, E. G. and Berman, N., Direction and specificity of axonal and transcellular transport of nucleosides, Brain Research, 139 (1978) 197-217.
