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The origin of centrifugal fibers to the inner ear in Caiman crocodilus. A horseradish peroxidase study
Neuroscience Letters, 27 (1981) 95-100
95
Elsevier/North-Holland Scientific Publishers Ltd.
T H E ORIGIN OF C E N T R I F U G A L FIBERS TO T H E I N N E R E A R IN C A I M A N CROCODILUS. A HORSERADISH PEROXIDASE STUDY
JORGEN STRUTZ
University o f Freiburg, Department o f Oto-Rhino-Laryngology, Unit f o r Morphological Brain Research, Killianstrafle 5, D-7800 Freiburg i.Br. (F.R.G.) (Received July 31st, 1981; Revised version received September 2nd, 1981; Accepted September 4th, 1981)
Key words: labyrinth - efferent neurons - tracer method - Caiman - horseradish peroxidase
The origin of acoustic and vestibular efferent fibers was investigated in Caiman crocodilus. After injection of horseradish peroxidase (HRP) into the cochlear duct or into the ampullae of the horizontal and anterior semi-circular canals, cells in the medulla oblongata exhibited retrogradely transported H R P reaction product. Efferent vestibular fibers were found in the medial reticular nucleus, at the level of the oliva superior. There were more labeled neurons ipsilateral to the injection site. Efferent acoustic neurons were found close to or inside the rostroventral division of the oliva superior. They spread out into the medial reticular nucleus bilaterally. More labeled efferent acoustic neurons occurred contralateral to the injection site.
Comparative anatomical studies of the reptile ear have demonstrated in species of the order Crocodilia the best developed labyrinth and the most clearly differentiated octavus nuclei [2, 10, 19]. Crocodilia are of particular interest to the comparative morphologist because their brains greatly resemble those of the avain forms [9]. Crocodilia seem to present an important transitional stage in the evolution of the central nervous system. Light and electron microscopical studies of the inner ear in Caiman showed that the basilar papilla is composed of two distinct types of hair cells which are innervated by both afferent and efferent nerve terminals [6, 11]. Boord [4] described the course of a crossed efferent cochlear bundle of the Caiman in a degeneration study; he could not determine, however, the cells of origin of these centrifugal fibers. The present study was undertaken to identify the parent cells of the efferent cochlear and vestibular fibers in the Caiman. In our experimental approach in Caiman crocodilus (snout-vent length 26-30 cm) 4 animals received a injection of 0.15 /~1 of an aqueous solution of 50% horseradish peroxidase (HRP) through the oval window into the cochlear duct. In 3 additional animals the ampullae of the horizontal and anterior semicircular canals were exposed and 0.1 #1 of the HRP solution was injected into each. After 24 h 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Hofland Scientific Publishers Ltd.
96
t( /,
)
Y /
W t\
f~
Fig. 1. a and b: Semi-schematic drawings of transverse sections of the brainstem (every second section) of one animal after HRP application to the left cochlear duct (a) or right ampullae (b). Each dot represents one labeled neuron. Abbreviations: CB, cerebellum; tim, fasciculus longitudinalis medialis; LLv, ventral nucleus of the lemniscus lateralis; NA, nucleus angularis; NL, nucleus laminaris; NM, nucleus magnocellularis; OS, oliva superior; Rm, nucleus reticularis medius; Vds, Vm, nuclei of the trigeminus nerve; VI, abducens nucleus; nVl, nerve of the abducens nucleus, Vlld, Vllm, Vllv, Vllvl, nuclei of the facial nerve; VIlI, nervus octavus.
97
lb
survival the animals were perfused with saline, followed by a mixture of 1.5% glutaraldehyde and 1% paraformaldehyde. Frozen frontal sections of the brain were cut at 40 tzm and treated for the demonstration of H R P according to the tetramethylbenzidine technique (TMB [12]). In all animals the bipolar cells of the ganglion of the octavus nerve ipsilateral to the injection were labeled. In the brainstem, neuronal somata labeled by retrograde axonal transport of H R P from the cochlear duct should be identified bilaterally at two locations: in the medial reticular nucleus (Rm [5]) and in the rostroventral division of the oliva superior (OS). Tracer application to the ampullae effected labeled neurons in the Rm bilaterally.
98
2
~ ,50 ,,tim i
3
tsoprn
'
Fig. 2. Transverse section of the rostroventral division of the oliva superior with 8 labeled efferent cochlear neurons, contralateral to the injection site. TMB reaction, neutral red counterstain. Fig. 3. Transverse section of the Rm, ipsilateral to the HRP injection of the ampullae. Fusiform neurons are labeled. TMB reaction, neutral red counterstain.
The most prominent accumulation o f labeled acoustic neurons was f o u n d inside or a r o u n d the rostroventral division o f the OS (Fig. la). A detailed description o f the OS in the alligator has been presented by Ariens-Kappers et al. [l] and since these structures are similar in Caiman we used this description. The small-celled complex consists o f two parts: the caudal and m o r e dorsal portion is wedge-shaped, while the second part is recognizable more ventrally, medial to the first subdivision. The ventral part extends farther rostraUy in the pontine tegmentum and turns progressively to a more lateral position. The labeled cells f o u n d inside or close to the latter rostroventral division o f the OS were small, round, or polygonal neurons (Fig. 2). Dorsally, labeled neurons shift to the adjacent R m bilaterally. There was no clear b o u n d a r y between labeled neurons in the rostroventral division o f the OS and those in the Rm. However, labeled R m neurons were fusiform in shape with two or three dendrites. Up to 263 labeled cells were f o u n d in these regions after tracer application to the cochlear duct. More labeled neurons appeared contralateral to the injection site with a distribution o f 167 to 96 neurons ipsilateral. Labeled efferent vestibular cells were discernible bilaterally in the Rm (Fig. lb). Labeled Rm neurons were fusiform in shape with two or three dendrites (Fig. 3). A few o f these medium-sized neurons were f o u n d dorsal to the abducens nucleus.
99
Labeled neurons were not found either in the rostroventral division of the oliva superior or medial to the intramedullary portion of the abducens nerve. More labeled cells were located ipsilateral to the injection site (up to 116) than contralateral (up to 80). Compared with Rm-labeled neurons after injection into the cochlear duct, no demonstrable difference in distribution, size, location and label were found. The fact that there were labeled cells within the Rm following HRP application to the cochlear duct can depend on involvement of the maculae lagenae, which is an organ of balance. In lower vertebrates the reticular formation has been shown repeatedly to be the origin of efferent vestibular fibers. In the goldfish [16] efferent labyrinthine fibers originate in the nucleus motorius tegmenti (NMT [3] ) on the ipsilateral side. In the frog parent cells of the centrifugal fibers to the inner ear originate in then nucleus reticularis medius (Rm [13, 17]). In birds these efferent fibers to the vestibular apparatus arise from the nucleus reticularis pontis caudalis (RP [14, 15]) bilaterally. The NMT in the fish, the Rm in the frog and Caiman, and the RP in birds and mammals [8], seem to be homologous reticular nuclei. In addition, efferent vestibular neurons in mammals originate in a dorsal position lateral to the abducens nucleus [7, 8, 18]. This study in a reptile completes the data of the origin of efferent labyrinthine fibers in vertebrates. A separation of acoustic from vestibular efferent cells is first seen in Caiman. A similar distribution of efferent acoustic and vestibular neurons is described in birds [14, 15]. These data provide evidence that the brain of Crocodilia greatly resembles that of avian forms. Supported by grants of the Deutsche Forschungsgemeinschaft, SFB 70.
1 Ariens Kappers, C.U., Huber, G.C. and E.C. Crosby, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Hafner, New York, 1936. 2 Baird, I.L., The anatomy of the reptilian ear. In C. Gans and Parson (Eds.), Biology of the Reptilia, Vol. 2, Academic Press, New York, 1970, pp. 193-275. 3 Bartelmez, G.W., Mauthner's cell and the nucleus motorius tegmenti, J. comp. Neurol., 25 (1915) 87-126. 4 Boord, R.L., The efferent cochlear bundle in the Caiman and pigeon, Exp. Neurol., 3 (1961) 225-239. 5 Donkelaar, H.J. ten, and Nieuwenhuys, R., in C. Gans (Ed.), Biology of the Reptilia, Vol. I0, Academic Press, New York, 1979, pp. 160-200. 6 Daring, M. von, Karduck, A. and Richter, H.G., The fine structure of the inner ear in Caiman crocodilus, Z. Anat. Entwickl.-Gesch., 145 (1974)41-65. 7 Gacek, R.R. and Lyon, M., The localization of vestibular efferent neurons in the kitten with horseradish peroxidase, Acta otolaryng. (Stockh.), 77 (1974) 92-101. 8 Goldberg, J.M. and Fernandez, C., Efferent vestibular system in the squirrel monkey: anatomical location and influence on afferent activity, J. Neurophysiol., 43 (1980) 986-1025.
100 9 Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of the Pigeon (Columbia livia), John Hopkins Press, Baltimore, MD, 1967. 10 Leake, P.A., Central projections of the statoacoustic nerve in Caiman crocodilus, Brain Behav. Evol., 10 (1974) 170--196. 11 Leake, P.A., Scanning Electron Microscopic Observations of Labyrinthine Sense Organs and Fiber Degeneration Studies of Secondary Vestibular and Auditory Pathways in Caiman crocodilus, Dissertation, Univ. California, San Fransicso, CA, 1976. 12 Mesulam, M.M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non carcinogenic blue reaction product with superior sensitivity for visualization neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106 ~117. 13 Opdam, P., Kemali, M. and Nieuwenhuys, R., Topological analysis of the brain stem of the frogs Rana esculenta and Rana catesbeiana, J. comp. Neurol., 165 (1976) 307-332. 14 Schwarz, I.E., Schwarz, D.W.F., Fredrickson, J.M. and Landolt, J.P., Efferent vestibular neurons: a study employing retrograde tracer methods in the pigeon (Columbia livia), J. comp. Neurol., 196 (1981) 1-12. 15 Strutz, J. and C.L. Schmidt, Acoustic and vestibular efferent neurons in the chicken (Gallus domesticus). A horseradish peroxidase study, Acta oto-laryng. (Stockh.), in press. 16 Strutz, J., Schmidt, C i . and Stt~rmer, C., Origin of efferent fibers of the vestibular apparatus in goldfish. A horseradish peroxidase study, Neurosci. Lett., 18 (1980) 5-9. 17 Strutz, 3., Spatz, W.B., Schmidt, C.L., St~rmer, C., Origin of centrifugal fibers to the labyrinth in the frog (Rana esculenta). A study with the fluorescent retrograde neuronal tracer 'Fast Blue', Brain Res., 215 (1981) 323-328. 18 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. Neuro[., 161 (1975) 159-182. 19 Wever, E.G., The Reptile Ear, Princeton University Press, N J, 1978, pp. 923-964.
95
Elsevier/North-Holland Scientific Publishers Ltd.
T H E ORIGIN OF C E N T R I F U G A L FIBERS TO T H E I N N E R E A R IN C A I M A N CROCODILUS. A HORSERADISH PEROXIDASE STUDY
JORGEN STRUTZ
University o f Freiburg, Department o f Oto-Rhino-Laryngology, Unit f o r Morphological Brain Research, Killianstrafle 5, D-7800 Freiburg i.Br. (F.R.G.) (Received July 31st, 1981; Revised version received September 2nd, 1981; Accepted September 4th, 1981)
Key words: labyrinth - efferent neurons - tracer method - Caiman - horseradish peroxidase
The origin of acoustic and vestibular efferent fibers was investigated in Caiman crocodilus. After injection of horseradish peroxidase (HRP) into the cochlear duct or into the ampullae of the horizontal and anterior semi-circular canals, cells in the medulla oblongata exhibited retrogradely transported H R P reaction product. Efferent vestibular fibers were found in the medial reticular nucleus, at the level of the oliva superior. There were more labeled neurons ipsilateral to the injection site. Efferent acoustic neurons were found close to or inside the rostroventral division of the oliva superior. They spread out into the medial reticular nucleus bilaterally. More labeled efferent acoustic neurons occurred contralateral to the injection site.
Comparative anatomical studies of the reptile ear have demonstrated in species of the order Crocodilia the best developed labyrinth and the most clearly differentiated octavus nuclei [2, 10, 19]. Crocodilia are of particular interest to the comparative morphologist because their brains greatly resemble those of the avain forms [9]. Crocodilia seem to present an important transitional stage in the evolution of the central nervous system. Light and electron microscopical studies of the inner ear in Caiman showed that the basilar papilla is composed of two distinct types of hair cells which are innervated by both afferent and efferent nerve terminals [6, 11]. Boord [4] described the course of a crossed efferent cochlear bundle of the Caiman in a degeneration study; he could not determine, however, the cells of origin of these centrifugal fibers. The present study was undertaken to identify the parent cells of the efferent cochlear and vestibular fibers in the Caiman. In our experimental approach in Caiman crocodilus (snout-vent length 26-30 cm) 4 animals received a injection of 0.15 /~1 of an aqueous solution of 50% horseradish peroxidase (HRP) through the oval window into the cochlear duct. In 3 additional animals the ampullae of the horizontal and anterior semicircular canals were exposed and 0.1 #1 of the HRP solution was injected into each. After 24 h 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Hofland Scientific Publishers Ltd.
96
t( /,
)
Y /
W t\
f~
Fig. 1. a and b: Semi-schematic drawings of transverse sections of the brainstem (every second section) of one animal after HRP application to the left cochlear duct (a) or right ampullae (b). Each dot represents one labeled neuron. Abbreviations: CB, cerebellum; tim, fasciculus longitudinalis medialis; LLv, ventral nucleus of the lemniscus lateralis; NA, nucleus angularis; NL, nucleus laminaris; NM, nucleus magnocellularis; OS, oliva superior; Rm, nucleus reticularis medius; Vds, Vm, nuclei of the trigeminus nerve; VI, abducens nucleus; nVl, nerve of the abducens nucleus, Vlld, Vllm, Vllv, Vllvl, nuclei of the facial nerve; VIlI, nervus octavus.
97
lb
survival the animals were perfused with saline, followed by a mixture of 1.5% glutaraldehyde and 1% paraformaldehyde. Frozen frontal sections of the brain were cut at 40 tzm and treated for the demonstration of H R P according to the tetramethylbenzidine technique (TMB [12]). In all animals the bipolar cells of the ganglion of the octavus nerve ipsilateral to the injection were labeled. In the brainstem, neuronal somata labeled by retrograde axonal transport of H R P from the cochlear duct should be identified bilaterally at two locations: in the medial reticular nucleus (Rm [5]) and in the rostroventral division of the oliva superior (OS). Tracer application to the ampullae effected labeled neurons in the Rm bilaterally.
98
2
~ ,50 ,,tim i
3
tsoprn
'
Fig. 2. Transverse section of the rostroventral division of the oliva superior with 8 labeled efferent cochlear neurons, contralateral to the injection site. TMB reaction, neutral red counterstain. Fig. 3. Transverse section of the Rm, ipsilateral to the HRP injection of the ampullae. Fusiform neurons are labeled. TMB reaction, neutral red counterstain.
The most prominent accumulation o f labeled acoustic neurons was f o u n d inside or a r o u n d the rostroventral division o f the OS (Fig. la). A detailed description o f the OS in the alligator has been presented by Ariens-Kappers et al. [l] and since these structures are similar in Caiman we used this description. The small-celled complex consists o f two parts: the caudal and m o r e dorsal portion is wedge-shaped, while the second part is recognizable more ventrally, medial to the first subdivision. The ventral part extends farther rostraUy in the pontine tegmentum and turns progressively to a more lateral position. The labeled cells f o u n d inside or close to the latter rostroventral division o f the OS were small, round, or polygonal neurons (Fig. 2). Dorsally, labeled neurons shift to the adjacent R m bilaterally. There was no clear b o u n d a r y between labeled neurons in the rostroventral division o f the OS and those in the Rm. However, labeled R m neurons were fusiform in shape with two or three dendrites. Up to 263 labeled cells were f o u n d in these regions after tracer application to the cochlear duct. More labeled neurons appeared contralateral to the injection site with a distribution o f 167 to 96 neurons ipsilateral. Labeled efferent vestibular cells were discernible bilaterally in the Rm (Fig. lb). Labeled Rm neurons were fusiform in shape with two or three dendrites (Fig. 3). A few o f these medium-sized neurons were f o u n d dorsal to the abducens nucleus.
99
Labeled neurons were not found either in the rostroventral division of the oliva superior or medial to the intramedullary portion of the abducens nerve. More labeled cells were located ipsilateral to the injection site (up to 116) than contralateral (up to 80). Compared with Rm-labeled neurons after injection into the cochlear duct, no demonstrable difference in distribution, size, location and label were found. The fact that there were labeled cells within the Rm following HRP application to the cochlear duct can depend on involvement of the maculae lagenae, which is an organ of balance. In lower vertebrates the reticular formation has been shown repeatedly to be the origin of efferent vestibular fibers. In the goldfish [16] efferent labyrinthine fibers originate in the nucleus motorius tegmenti (NMT [3] ) on the ipsilateral side. In the frog parent cells of the centrifugal fibers to the inner ear originate in then nucleus reticularis medius (Rm [13, 17]). In birds these efferent fibers to the vestibular apparatus arise from the nucleus reticularis pontis caudalis (RP [14, 15]) bilaterally. The NMT in the fish, the Rm in the frog and Caiman, and the RP in birds and mammals [8], seem to be homologous reticular nuclei. In addition, efferent vestibular neurons in mammals originate in a dorsal position lateral to the abducens nucleus [7, 8, 18]. This study in a reptile completes the data of the origin of efferent labyrinthine fibers in vertebrates. A separation of acoustic from vestibular efferent cells is first seen in Caiman. A similar distribution of efferent acoustic and vestibular neurons is described in birds [14, 15]. These data provide evidence that the brain of Crocodilia greatly resembles that of avian forms. Supported by grants of the Deutsche Forschungsgemeinschaft, SFB 70.
1 Ariens Kappers, C.U., Huber, G.C. and E.C. Crosby, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Hafner, New York, 1936. 2 Baird, I.L., The anatomy of the reptilian ear. In C. Gans and Parson (Eds.), Biology of the Reptilia, Vol. 2, Academic Press, New York, 1970, pp. 193-275. 3 Bartelmez, G.W., Mauthner's cell and the nucleus motorius tegmenti, J. comp. Neurol., 25 (1915) 87-126. 4 Boord, R.L., The efferent cochlear bundle in the Caiman and pigeon, Exp. Neurol., 3 (1961) 225-239. 5 Donkelaar, H.J. ten, and Nieuwenhuys, R., in C. Gans (Ed.), Biology of the Reptilia, Vol. I0, Academic Press, New York, 1979, pp. 160-200. 6 Daring, M. von, Karduck, A. and Richter, H.G., The fine structure of the inner ear in Caiman crocodilus, Z. Anat. Entwickl.-Gesch., 145 (1974)41-65. 7 Gacek, R.R. and Lyon, M., The localization of vestibular efferent neurons in the kitten with horseradish peroxidase, Acta otolaryng. (Stockh.), 77 (1974) 92-101. 8 Goldberg, J.M. and Fernandez, C., Efferent vestibular system in the squirrel monkey: anatomical location and influence on afferent activity, J. Neurophysiol., 43 (1980) 986-1025.
100 9 Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of the Pigeon (Columbia livia), John Hopkins Press, Baltimore, MD, 1967. 10 Leake, P.A., Central projections of the statoacoustic nerve in Caiman crocodilus, Brain Behav. Evol., 10 (1974) 170--196. 11 Leake, P.A., Scanning Electron Microscopic Observations of Labyrinthine Sense Organs and Fiber Degeneration Studies of Secondary Vestibular and Auditory Pathways in Caiman crocodilus, Dissertation, Univ. California, San Fransicso, CA, 1976. 12 Mesulam, M.M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non carcinogenic blue reaction product with superior sensitivity for visualization neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106 ~117. 13 Opdam, P., Kemali, M. and Nieuwenhuys, R., Topological analysis of the brain stem of the frogs Rana esculenta and Rana catesbeiana, J. comp. Neurol., 165 (1976) 307-332. 14 Schwarz, I.E., Schwarz, D.W.F., Fredrickson, J.M. and Landolt, J.P., Efferent vestibular neurons: a study employing retrograde tracer methods in the pigeon (Columbia livia), J. comp. Neurol., 196 (1981) 1-12. 15 Strutz, J. and C.L. Schmidt, Acoustic and vestibular efferent neurons in the chicken (Gallus domesticus). A horseradish peroxidase study, Acta oto-laryng. (Stockh.), in press. 16 Strutz, J., Schmidt, C i . and Stt~rmer, C., Origin of efferent fibers of the vestibular apparatus in goldfish. A horseradish peroxidase study, Neurosci. Lett., 18 (1980) 5-9. 17 Strutz, 3., Spatz, W.B., Schmidt, C.L., St~rmer, C., Origin of centrifugal fibers to the labyrinth in the frog (Rana esculenta). A study with the fluorescent retrograde neuronal tracer 'Fast Blue', Brain Res., 215 (1981) 323-328. 18 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. Neuro[., 161 (1975) 159-182. 19 Wever, E.G., The Reptile Ear, Princeton University Press, N J, 1978, pp. 923-964.