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Efferent acoustic neurons within the lateral superior olivary nucleus of the guinea pig
174
Brain Research, 299 (1984) 174-177 Elsevier
BRE 20163
Efferent acoustic neurons within the lateral superior olivary nucleus of the guinea pig JORGEN STRUTZ and KATRIN BIELENBERG Department of Oto-Rhino-Laryngology, Unitfor Morphological Brain Research, D-7800 Freiburg i. Br. (F.R.G.) (Accepted January 3rd, 1984) Key words: efferent acoustic neurons - - Fast Blue dye - - horseradish peroxidase - - horseradish peroxidase electron microscopy
The cells of origin of the olivocochlear bundles in the guinea pig were labeled by retrograde axonal transport with either the fluorescent dye 'Fast Blue' or horseradish peroxidase (HRP). Labeled cells were found bilaterally in the superior olivary complex, as described previously. Moreover, an extremely large population of labeled cells occurred within the cell mass of the lateral superior olivary nucleus (LSO) ipsilaterally to the injection site. Preliminary electron microscopical observations confirmed that the labeled LSO cells were distinctly smaller than the unlabeled LSO cells. The labeled cells contained a large nucleus that exhibited extensive invaginations. The crossed and uncrossed olivocochlear bundles (OCB)8 terminate on the outer and inner hair cells of the organ of Corti 6. After the retrograde neuronal tracer horseradish peroxidase (HRP) was injected into the cochlea, the cells of origin of the mammalian OCBs were located bilaterally within the superior olivary complex (SOC)2,1~, 15. 60-70% of the neurons were located on the side ipsilateral to the injection; the majority was found in the dorsal hilus of the lateral superior olivary nucleus (LSO), whereas the nucleus itself remained unlabeled. Labeled efferent neurons also occurred in the ventral and medial nucleus of the trapezoid body (VTB and MTB) respectively, and in the dorsomedial periolivary nucleus (DMPO). Contralaterally to the injected side, the labeled neurons were located mainly in the VTB and D M P O and to a lesser degree in the dorsal hilus of the LSO and in the MTB. A few efferent acoustic cells were determined bilaterally in the ventral nucleus of the lemniscus lateralis (VLL) 12. Preliminary studies with various fluorescent retrograde neuronal tracers, i.e. Fast Blue, Nuclear Yellow and Propidium Jodide, resulted in a large number of strongly fluorescent cells dispersed within the cell mass of the LSO ipsilaterally to the injected side.
The present study was undertaken to substantiate this surprising finding. So far, the LSO is known to receive a massive input from the ipsilateral ventral cochlear nucleus14. The LSO projects to the inferior colliculus and to the nuclei of the lateral lemniscusl,5,11; there is no evidence for a direct projection to the cochlear nucleiS. Five guinea pigs (350-400 g) received, through the round window, a unilateral injection of 0.7 pl of a 4% Fast Blue solution (diamidino compound 253/50, ref. 3) into the scala tympani. After 4 days survival, the animals were perfused transcardially with saline, followed by 8% formaline. Frozen frontal sections of the brains, 4 0 p m thick, were collected in distilled water, mounted and coverslipped. The material was studied with a Leitz microscope equipped with filtermirror system D, which provides excitation light at 390 nm wavelength. An additional 10 guinea pigs received a 0.7-/d injection of a 50% H R P solution (Serva) into the scala tympani. After 24-h survival, the animals were perfused with 1.5% glutaraldehyde and 1% paraformaldehyde. The brains of these specimens were frozen and cut at 40/~m. The sections were treated for the demonstration of H R P according to Mesulam's tetramethylbenzidine technique
Correspondence: J. Strutz, Department of Oto-Rhino-Laryngology, Unit for Morphological Brain Research, Killianstrasse 5, D7800 Freiburg i. Br., F.R.G. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
175
Fig. 1. Fluorescent efferent acoustic neurons within the lateral superior olivary nucleus (LSO) ipsilateral to the injection. Fig. 2. Examples of efferent acoustic neurons within the LSO in a higher magnification. Note fluorescence in the cytoplasm and dendrites. The large nucleus occurs unlabeled. Fig. 3. HRP-labeled efferent acoustic neurons within the LSO ipsilateral to the injection. TMB reaction, neutral red counterstain. Fig. 4. Examples of HRP-labeled efferent acoustic neurons within the LSO in a higher magnification. The labeled neurons are small (10-12 gm) and fusiform in shape. TMB reaction, neutral red counterstain.
176 (TMB) 7. The other 5 specimens were prepared for electron microscopy. The brain stems were immediately removed and cut with the aid of a Vibratome in 200-pm slices. These slices were processed with TMB for electron microscopy4,13. After Fast Blue injections into the cochlea, retrogradely labeled neurons were found bilaterally in the SOC and VLL, as described in previous H R P studies2A2,15. The labeled neurons displayed a brilliant fluorescent cell body and dendrites with fine silver fluorescent granules. The nucleus, however, showed little fluorescence. Moreover, the ipsilateral cochlear nuclei were labeled by an anterograde transport of the tracer via the octavus nerve. In contrast to the earlier experiments2.12,15, the body of the LSO ipsilateral to the injected cochlea contained an extremely large population of labeled cells (Fig. 1). These labeled LSO neurons were small and fusiform in shape (Fig. 2). More than 1000 labeled cells occurred within this nucleus. All together, as many as 1300 labeled neurons were found in the ipsilateral SOC. In the LSO contralateral to the injected cochlea, only a small number of neurons were labeled (no more than 14). The use of H R P instead of the fluorescent tracer yielded similar results with respect to the distribution and number of labeled neurons. An extremely large population of labeled cells was found again within the body of the ipsilateral LSO (Fig. 3). These neurons were small (10 to 12/~m) and fusiform in shape (Fig. 4). In addition, labeled neurons of similar shape and size appeared in the dorsal hilus of the LSO. Up to 965 labeled cells were found in the ipsilateral LSO, whereas the contralaterai LSO contained only 12 labeled neurons. In order to reduce the possibility of overestimating cell counts due effectively to counting twice those labeled neurons which lie partly in and partly out of the section, a labeled neuron was numbered only if it appeared that most of the pericaryon was contained within the tissue section. In semithin sections (2 ~m), the HRP-labeled cells within the LSO body could be easily identified by their content of brownish T M B - H R P reaction product. About a third of the small neurons were labeled. This proportion, however, differed considerably within the various parts of the nucleus, without any indication of subpopulations of parent cells. In ultrathin sections, the HRP-labeled neurons in
Fig. 5. Electron micrograph of a small labeled LSO neuron. Electron-dense HRP-TMB crystalline structures are accumulated within the pericaryal cytoplasm. Note the nuclear infoldings.
the ipsilateral LSO were readily identified by their contents of electron-dense crystalline structures 4,13. The crystalline H R P - T M B reaction product was accumulated within the pericaryal cytoplasm (Fig. 5). Preliminary electron microscopical observations showed that the labeled LSO cells were distinctly smaller than the unlabeled cells. The labeled neurons exhibited a large nucleus and a relatively thin shell of cytoplasm. In comparison to the unlabeled neurons, the labeled cells contained only a few mitochondria. Characteristically, the nuclei of the labeled cells showed deep nuclear infoldings. Such invaginations were not seen in the nuclei of large unlabeled LSO neurons. Previously, neurons in the dorsal hilus and along the margins of the LSO body (peri-LSO cells 2) have been identified as parent cells of the uncrossed OCB2,12,15. Rasmussen9,10 supposed the main body of the LSO to be the principal source of the uncrossed OCB since he found that the recurrent pathway degenerated after lesions affecting the LSO. Warr 15 and Adams2, however, established the hypothesis that most of the degeneration seen by Rasmussen was due to destruction of periolivary cells or their axons. The present findings indicate strongly that a certain class of small LSO cells contributes to the un-
177 crossed O C B . In fact, these cells a p p e a r to r e p r e s e n t the largest group of p a r e n t cells of the uncrossed OCB. It does not seem plausible that the l a b e l e d L S O neurons are the p r o d u c t of trans-synaptic transport of the tracer. A trans-synaptic t r a n s p o r t could take place in the cochlear nuclei which were l a b e l e d by an anterograde t r a n s p o r t of the tracer via the octavus nerve. H o w e v e r , the survival time of 24 h was too short for trans-synaptic transport. M o r e o v e r , the absence of any L S O p r o j e c t i o n to the cochlear nuclei5 excludes the possibility of labeling L S O neurons by trans-synaptic t r a n s p o r t in the cochlear nuclei. The reason for the failure to label the small L S O cells in our previous study is not entirely clear. Possibly, greater injection volumes and increased sensitivity of 1 Adams, J. C., Ascending projections to the inferior colliculus, J. comp. Neurol., 183 (1979) 519--538. 2 Adams, J. C., Cytology of periolivary cells and the organization of their projections in the cat, J. comp. Neurol., 215 (1983) 275--289. 3 Bentivoglio, M., Kuypers, H. G. J. M., Catsman-Berevoets, C. E., Loewe, H. and Dann, O., Two new fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (1980) 25-30. 4 Carson, K. A. and Mesulam, M.-M., Electron microscopic demonstration of neural connections using horseradishperoxidase: a comparison of the tetramethylbenzidine procedure with seven other histoehemical methods, J. Histochem. Cytochem., 30 (1982) 425-435. 5 Glendenning, K. K. and Masterton, R. B., Acoustic chiasm: efferent projections of the lateral superior olive, J. Neurosci., 3 (1983) 1521-1537. 6 Kimura, R. and Wers~ill, J., Termination of the olivocochlear bundle in relation to the outer hair cells of the organ of Corti in the guinea pig, A cta oto-laryng., 55 (1962) 11-32. 7 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualization neural afferents and efferents, J. Histochern. Cytochem., 26 (1978) 106--117. 8 Rasmussen, G. L., The olivary peduncle and other fiber projections of the superior olivary complex, J. comp. Neu-
the H R P - T M B reaction p r o c e d u r e can account for this. The L S O receives a massive input from the spherical cells region of the ipsilateral ventral cochlear nucleus 14. This may indicate the existence of a short feed-back mechanism to the cochlea, although it is not yet clear whether this p r o j e c t i o n contacts the small L S O cells directly. There could be m o r e physiological studies of the role of the uncrossed O C B . In the guinea pig and cat, for example, the main b o d y of the L S O represents the most p r o m i n e n t nucleus of the SOC, so that this nucleus is of easy access for electrophysiological recordings. This study was s u p p o r t e d by grants from the Deutsche Forschungsgemeinschaft, SFB 70. rol., 84 (1946) 141-219. 9 Rasmussen, G. L., Efferent fibers of the cochlear nerve and cochlear nucleus. In G. L. Rasmussen and W. F. Windle (Eds.), Neural Mechanisms of the Auditory and Vestibular Systems, C. C. Thomas, Springfield, 1960, pp. 105-115. 10 Rasmussen, G. L., Efferent connections of the cochlear nucleus. In A. B. Graham (Ed.), Sensorineural Hearing Process and Disorders, Little, Brown, Boston, 1967, pp. 61-75. 11 Strutz, J., Der Ursprung der afferenten Innervation zum Colliculus inferior beim Meerschweinchen, Arch. Otorhinolaryng., 228 (1980) 285-294. 12 Strutz, J. and Spatz, W. B., Superiorolivary and extraolivary origin of centrifugal innervation of the cochlea in guinea pig. A horseradish peroxidase study, Neurosci. Lett., 17 (1980) 227-230. 13 Stfirmer, C., Bielenberg, K. and Spatz, W. B., Electron microscopical identification of 3,3',5,5'-tetramethylbenzidine-reacted horseradish peroxidase after retrograde axoplasmic transport, Neurosci. Lett., 23 (1981) 1-5. 14 Warr, W. B., Fiber degeneration following lesions in the anterior ventral cochlear nucleus of the cat, Exp. Neurol., 14 (1966) 453-474. 15 Warr, W. B., Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry, J. comp. Neurol., 161 (1975) 159--182.
Brain Research, 299 (1984) 174-177 Elsevier
BRE 20163
Efferent acoustic neurons within the lateral superior olivary nucleus of the guinea pig JORGEN STRUTZ and KATRIN BIELENBERG Department of Oto-Rhino-Laryngology, Unitfor Morphological Brain Research, D-7800 Freiburg i. Br. (F.R.G.) (Accepted January 3rd, 1984) Key words: efferent acoustic neurons - - Fast Blue dye - - horseradish peroxidase - - horseradish peroxidase electron microscopy
The cells of origin of the olivocochlear bundles in the guinea pig were labeled by retrograde axonal transport with either the fluorescent dye 'Fast Blue' or horseradish peroxidase (HRP). Labeled cells were found bilaterally in the superior olivary complex, as described previously. Moreover, an extremely large population of labeled cells occurred within the cell mass of the lateral superior olivary nucleus (LSO) ipsilaterally to the injection site. Preliminary electron microscopical observations confirmed that the labeled LSO cells were distinctly smaller than the unlabeled LSO cells. The labeled cells contained a large nucleus that exhibited extensive invaginations. The crossed and uncrossed olivocochlear bundles (OCB)8 terminate on the outer and inner hair cells of the organ of Corti 6. After the retrograde neuronal tracer horseradish peroxidase (HRP) was injected into the cochlea, the cells of origin of the mammalian OCBs were located bilaterally within the superior olivary complex (SOC)2,1~, 15. 60-70% of the neurons were located on the side ipsilateral to the injection; the majority was found in the dorsal hilus of the lateral superior olivary nucleus (LSO), whereas the nucleus itself remained unlabeled. Labeled efferent neurons also occurred in the ventral and medial nucleus of the trapezoid body (VTB and MTB) respectively, and in the dorsomedial periolivary nucleus (DMPO). Contralaterally to the injected side, the labeled neurons were located mainly in the VTB and D M P O and to a lesser degree in the dorsal hilus of the LSO and in the MTB. A few efferent acoustic cells were determined bilaterally in the ventral nucleus of the lemniscus lateralis (VLL) 12. Preliminary studies with various fluorescent retrograde neuronal tracers, i.e. Fast Blue, Nuclear Yellow and Propidium Jodide, resulted in a large number of strongly fluorescent cells dispersed within the cell mass of the LSO ipsilaterally to the injected side.
The present study was undertaken to substantiate this surprising finding. So far, the LSO is known to receive a massive input from the ipsilateral ventral cochlear nucleus14. The LSO projects to the inferior colliculus and to the nuclei of the lateral lemniscusl,5,11; there is no evidence for a direct projection to the cochlear nucleiS. Five guinea pigs (350-400 g) received, through the round window, a unilateral injection of 0.7 pl of a 4% Fast Blue solution (diamidino compound 253/50, ref. 3) into the scala tympani. After 4 days survival, the animals were perfused transcardially with saline, followed by 8% formaline. Frozen frontal sections of the brains, 4 0 p m thick, were collected in distilled water, mounted and coverslipped. The material was studied with a Leitz microscope equipped with filtermirror system D, which provides excitation light at 390 nm wavelength. An additional 10 guinea pigs received a 0.7-/d injection of a 50% H R P solution (Serva) into the scala tympani. After 24-h survival, the animals were perfused with 1.5% glutaraldehyde and 1% paraformaldehyde. The brains of these specimens were frozen and cut at 40/~m. The sections were treated for the demonstration of H R P according to Mesulam's tetramethylbenzidine technique
Correspondence: J. Strutz, Department of Oto-Rhino-Laryngology, Unit for Morphological Brain Research, Killianstrasse 5, D7800 Freiburg i. Br., F.R.G. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
175
Fig. 1. Fluorescent efferent acoustic neurons within the lateral superior olivary nucleus (LSO) ipsilateral to the injection. Fig. 2. Examples of efferent acoustic neurons within the LSO in a higher magnification. Note fluorescence in the cytoplasm and dendrites. The large nucleus occurs unlabeled. Fig. 3. HRP-labeled efferent acoustic neurons within the LSO ipsilateral to the injection. TMB reaction, neutral red counterstain. Fig. 4. Examples of HRP-labeled efferent acoustic neurons within the LSO in a higher magnification. The labeled neurons are small (10-12 gm) and fusiform in shape. TMB reaction, neutral red counterstain.
176 (TMB) 7. The other 5 specimens were prepared for electron microscopy. The brain stems were immediately removed and cut with the aid of a Vibratome in 200-pm slices. These slices were processed with TMB for electron microscopy4,13. After Fast Blue injections into the cochlea, retrogradely labeled neurons were found bilaterally in the SOC and VLL, as described in previous H R P studies2A2,15. The labeled neurons displayed a brilliant fluorescent cell body and dendrites with fine silver fluorescent granules. The nucleus, however, showed little fluorescence. Moreover, the ipsilateral cochlear nuclei were labeled by an anterograde transport of the tracer via the octavus nerve. In contrast to the earlier experiments2.12,15, the body of the LSO ipsilateral to the injected cochlea contained an extremely large population of labeled cells (Fig. 1). These labeled LSO neurons were small and fusiform in shape (Fig. 2). More than 1000 labeled cells occurred within this nucleus. All together, as many as 1300 labeled neurons were found in the ipsilateral SOC. In the LSO contralateral to the injected cochlea, only a small number of neurons were labeled (no more than 14). The use of H R P instead of the fluorescent tracer yielded similar results with respect to the distribution and number of labeled neurons. An extremely large population of labeled cells was found again within the body of the ipsilateral LSO (Fig. 3). These neurons were small (10 to 12/~m) and fusiform in shape (Fig. 4). In addition, labeled neurons of similar shape and size appeared in the dorsal hilus of the LSO. Up to 965 labeled cells were found in the ipsilateral LSO, whereas the contralaterai LSO contained only 12 labeled neurons. In order to reduce the possibility of overestimating cell counts due effectively to counting twice those labeled neurons which lie partly in and partly out of the section, a labeled neuron was numbered only if it appeared that most of the pericaryon was contained within the tissue section. In semithin sections (2 ~m), the HRP-labeled cells within the LSO body could be easily identified by their content of brownish T M B - H R P reaction product. About a third of the small neurons were labeled. This proportion, however, differed considerably within the various parts of the nucleus, without any indication of subpopulations of parent cells. In ultrathin sections, the HRP-labeled neurons in
Fig. 5. Electron micrograph of a small labeled LSO neuron. Electron-dense HRP-TMB crystalline structures are accumulated within the pericaryal cytoplasm. Note the nuclear infoldings.
the ipsilateral LSO were readily identified by their contents of electron-dense crystalline structures 4,13. The crystalline H R P - T M B reaction product was accumulated within the pericaryal cytoplasm (Fig. 5). Preliminary electron microscopical observations showed that the labeled LSO cells were distinctly smaller than the unlabeled cells. The labeled neurons exhibited a large nucleus and a relatively thin shell of cytoplasm. In comparison to the unlabeled neurons, the labeled cells contained only a few mitochondria. Characteristically, the nuclei of the labeled cells showed deep nuclear infoldings. Such invaginations were not seen in the nuclei of large unlabeled LSO neurons. Previously, neurons in the dorsal hilus and along the margins of the LSO body (peri-LSO cells 2) have been identified as parent cells of the uncrossed OCB2,12,15. Rasmussen9,10 supposed the main body of the LSO to be the principal source of the uncrossed OCB since he found that the recurrent pathway degenerated after lesions affecting the LSO. Warr 15 and Adams2, however, established the hypothesis that most of the degeneration seen by Rasmussen was due to destruction of periolivary cells or their axons. The present findings indicate strongly that a certain class of small LSO cells contributes to the un-
177 crossed O C B . In fact, these cells a p p e a r to r e p r e s e n t the largest group of p a r e n t cells of the uncrossed OCB. It does not seem plausible that the l a b e l e d L S O neurons are the p r o d u c t of trans-synaptic transport of the tracer. A trans-synaptic t r a n s p o r t could take place in the cochlear nuclei which were l a b e l e d by an anterograde t r a n s p o r t of the tracer via the octavus nerve. H o w e v e r , the survival time of 24 h was too short for trans-synaptic transport. M o r e o v e r , the absence of any L S O p r o j e c t i o n to the cochlear nuclei5 excludes the possibility of labeling L S O neurons by trans-synaptic t r a n s p o r t in the cochlear nuclei. The reason for the failure to label the small L S O cells in our previous study is not entirely clear. Possibly, greater injection volumes and increased sensitivity of 1 Adams, J. C., Ascending projections to the inferior colliculus, J. comp. Neurol., 183 (1979) 519--538. 2 Adams, J. C., Cytology of periolivary cells and the organization of their projections in the cat, J. comp. Neurol., 215 (1983) 275--289. 3 Bentivoglio, M., Kuypers, H. G. J. M., Catsman-Berevoets, C. E., Loewe, H. and Dann, O., Two new fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (1980) 25-30. 4 Carson, K. A. and Mesulam, M.-M., Electron microscopic demonstration of neural connections using horseradishperoxidase: a comparison of the tetramethylbenzidine procedure with seven other histoehemical methods, J. Histochem. Cytochem., 30 (1982) 425-435. 5 Glendenning, K. K. and Masterton, R. B., Acoustic chiasm: efferent projections of the lateral superior olive, J. Neurosci., 3 (1983) 1521-1537. 6 Kimura, R. and Wers~ill, J., Termination of the olivocochlear bundle in relation to the outer hair cells of the organ of Corti in the guinea pig, A cta oto-laryng., 55 (1962) 11-32. 7 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualization neural afferents and efferents, J. Histochern. Cytochem., 26 (1978) 106--117. 8 Rasmussen, G. L., The olivary peduncle and other fiber projections of the superior olivary complex, J. comp. Neu-
the H R P - T M B reaction p r o c e d u r e can account for this. The L S O receives a massive input from the spherical cells region of the ipsilateral ventral cochlear nucleus 14. This may indicate the existence of a short feed-back mechanism to the cochlea, although it is not yet clear whether this p r o j e c t i o n contacts the small L S O cells directly. There could be m o r e physiological studies of the role of the uncrossed O C B . In the guinea pig and cat, for example, the main b o d y of the L S O represents the most p r o m i n e n t nucleus of the SOC, so that this nucleus is of easy access for electrophysiological recordings. This study was s u p p o r t e d by grants from the Deutsche Forschungsgemeinschaft, SFB 70. rol., 84 (1946) 141-219. 9 Rasmussen, G. L., Efferent fibers of the cochlear nerve and cochlear nucleus. In G. L. Rasmussen and W. F. Windle (Eds.), Neural Mechanisms of the Auditory and Vestibular Systems, C. C. Thomas, Springfield, 1960, pp. 105-115. 10 Rasmussen, G. L., Efferent connections of the cochlear nucleus. In A. B. Graham (Ed.), Sensorineural Hearing Process and Disorders, Little, Brown, Boston, 1967, pp. 61-75. 11 Strutz, J., Der Ursprung der afferenten Innervation zum Colliculus inferior beim Meerschweinchen, Arch. Otorhinolaryng., 228 (1980) 285-294. 12 Strutz, J. and Spatz, W. B., Superiorolivary and extraolivary origin of centrifugal innervation of the cochlea in guinea pig. A horseradish peroxidase study, Neurosci. Lett., 17 (1980) 227-230. 13 Stfirmer, C., Bielenberg, K. and Spatz, W. B., Electron microscopical identification of 3,3',5,5'-tetramethylbenzidine-reacted horseradish peroxidase after retrograde axoplasmic transport, Neurosci. Lett., 23 (1981) 1-5. 14 Warr, W. B., Fiber degeneration following lesions in the anterior ventral cochlear nucleus of the cat, Exp. Neurol., 14 (1966) 453-474. 15 Warr, W. B., Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry, J. comp. Neurol., 161 (1975) 159--182.