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Neurotransmitters of the cochlea and cochlear nucleus: Immunocytochemical evidence
Am I Otolaryngal 7:100-100, 1980
Neurotransmitters of the Cochlea and Cochlear Nucleus: Immunocytochemical Evidence RICHARDA. ALTSCHULER,PH.D., DOUGLASW. HOFFMAN,PH.D., ANDROBERTJ. WENTHOLD,PH.D. Many neurotransmitter candidates have been identified in the cochlea and cochlear nucleus with the use of immunocytochemical techniques. Choline acetyltransferase immunoreactivity suggests acetylcholine as a transmitter of medial and lateral efferent systems in the cochlea. Immunoreactivities to enkephalins, dynorphins, calcitonin gene-related peptide, and tyrosine hydroxylase (a marker for dopamine) are also found in lateral efferents. Choline acetyltransferase, enkephalin, and dynorphin immunoreactivities are cocontained in neurons of the lateral system. In the anteroventral cochlear nucleus, the inhibitory amino acid transmitters, gamma aminobutyric acid (GABA), and glycine, as well as the presumed excitatory amino acid transmitter of the auditory nerve, have been directly or indirectly localized, immunocytochemically, to discrete populations of terminals on spherical cells with distinct morphologic characteristics.
Transmitters are a basic element of neuronal and sensory function. A major distinction of neurons (and receptor cells such as cochlear hair cells) is their ability to communicate with other cells by means of a n e u r o t r a n s m i t t e r . Knowledge of neurotransmitters enables us to manipulate nervous function pharmacologically. Moreover, neurotransmitters have been implicated in several disorders, such as dopamine in Parkinson's disease. Hawkins once hypothesized that chemical mediators such as neurotransmitters might be involved in aminoglycoside-induced hearing loss, and although this may not be the case, it is very possible that neurotransmitters are involved in aspects of presbycusis, perhaps in inability to extract relevant auditory information embedded in noise. 1 It is likely that increased knowledge of neurotransmitters of the auditory system will help us to
understand, and perhaps treat, auditory dysfunctions, Although the transmitter of cochlear hair cells remains unknown, with some evidence favoring a substance related to an excitatory amino acid, 2 we have been able to identify, by means of imm u n o c y t o c h e m i s t r y , n e u r o t r a n s m i t t e r candidates in cochlear efferents and in the auditory nerve and cochlear nucleus. In the case of efferents, we have been able to co-localize at least three neurotransmitter candidates w i t h i n the same neurons. In the cochlear nucleus, we have localized three neurotransmitter candidates to separate terminals, with distinct synaptic characteristics.
EFFERENTS The olivocochlear efferents, first described by Rasmussen,3.4 have recently been reclassified by Warr et al.~ and Cuinan et al. 6 into lateral and medial systems. The lateral system has its cells of origin in or around the lateral superior olive and sends its axons p r e d o m i n a n t l y or exclusively to the ipsilateral cochlea. In the cochlea, lateral efferent fibers travel in the inner spiral bundle and tunnel spiral bundle and terminate predominantly on afferent dendrites of type I spiral ganglion cells with a few terminating at the bases of inner hair cells (and perhaps a few ending on outer hair cells). The medial system
Received from the KresgeHearingResearch Institute, University of Michigan, Ann Arbor, Michigan (Dr. Altschuler); Department of Psychiatry, Southern Illinois University School of Medicine,Springfield,Illinois (Dr. Hoffman);and the Laboratoryof Neuro-otolaryngology,National Institutes of Health, Bethesda,Maryland (Dr. Wenthold}. Presented at "ContemporaryIssues of Middle and Inner Ear Physiologyand Pathology:A Symposium in Honor of Joseph E. Hawkins, jr., Ph,D,, Sc,D,, and Merle Lawrence, Ph.D.," Ann Arbor, Michigan, October 27, 1985. Accepted for publication at that time, Address reprint requests to Dr, Altschuler:KresgeHearing Research Institute, 1301 E, Ann St., Ann Arbor, MI 48109,
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has its origin in cells in more medially located olivary nuclei and sends its axons mostly to the contralateral cochlea. Some medial efferent fibers may travel for a small distance in inner spiral and tunnel spiral bundles before crossing the tunnel of Corti, whereas most medial efferents immediately cross the tunnel of Corti to terminate predominantly at the bases of outer hair cells (with some also ending on afferent dendrites of type II spiral ganglion cells). There may be axo-axonal connections between the two systems in the inner spiral bundle regionJ Supranuclear and circumnuclear efferent endings on outer hair cells are a minor population and may be part of either or both medial and lateral systems. 8,9 There is considerable evidence suggesting acetylcholine as a transmitter of efferents.,.lo We have found choline acetyltransferase immunereactivity, indicating the presence of acetylcholine, in both lateral and medial systems of olivecochlear efferents (Fig. 1) in the guinea pig,lO and similar findings have been reported in the rat. 1~ This immunoreactivity was found in fibers and terminals in the inner spiral bundle and in the tunnel spiral bundle, corresponding to the lateral system of efferents. Choline acetyltransferase immunoreactive labeling corresponding to the medial system of efferents was seen in upper tunnel crossing fibers and in large puncta corresponding to terminals at outer hair cell bases. In the guinea pig cochlea, nine or more immunoreactive terminals can be seen on first row outer hair cells in basal turns. As one progresses apically along the cochlear spiral, the number of large choline acetyltransferase immunoreactive puncta at outer hair cell bases decreases, beginning in the third row and then decreasing in the second row. By the beginning of the fourth turn, immunoreactive terminals are seen at first row outer hair cells only, and by the middle of the fourth turn, no such terminals are observed on any outer hair cell bases. Choline acetyltransferase immunoreactivity is also seen in supranuclear and circumnuclear efferents, w h i c h have n o t yet been assigned to either system of efferents. These findings add to the evidence that acetylcholine is a transmitter of both medial and lateral systems, although more physiologic and pharmacologic studies are needed to confirm its role in the lateral efferent system. Enkephalins and dynorphins are two separate families of opioid neuropeptides with important neurotransmitter/neuromodulator roles in the nervous system. With the use of most antisera to enkephalins and antisera to dynorphins, im-
ChAT-IR [Acetylcholine] Outer Hair Cells
Inner Hair Cells
Outer Spiralling Fiber
Tunnel Spiral InnerSpiral Bundle Bundle
i
LatSyst eralem System
Medial
Figure 1. Schematic of choline acetyltransferase immunereactivity in the guinea pig organ of Corti. Labeling of both medial and lateral efferent systems is seen.
munoreactive staining is seen in fibers and terminals of the inner spiral bundle and in the tunnel spiral bundle, corresponding to the lateral system of efferents. '~'12,1~-~ Electron microscopic studies have shown enkephalin immunereactivity to be associated with large dense core vesicles in efferent terminals synapsing on afferent dendrites or occasionally on other efferents.16.~8.~9 The presence of three neurotransmitter candidates (acetylcholine, enkephalins, and @norphins) in lateral efferents suggests that there may be more than one neurotransmitter within the same neuron in this system. We have examined this by looking at the cells of origin of the lateral system and using sequential co-localization methods such as the method of Tramu. 2e Our results s h o w that lateral olivocochlear neurons co-contain enkephalins, dynorphins, and choline acetyltransferase (indicating acetylcholine). 27-2g Biochemicai studies have been done that have localized enkephalins and dynorphins, with appropriate receptor-binding characteristics, to olivocochlear efferents, 22-~5 and sound-induced release of an enkephalinlike substance has been reported.no Recent immunocytochemical evidence suggests that the lateral efferent system contains even more neurotransmitter candidates. We have recently found tyrosine hydroxylase immunoreactivity (indicating dopamine as a transmitter) in lateral efferents in the guinea pig cochlea and its ceils of origin in the lateral superior olivary complex. Two different studies have just reported calcitonin gene-related neuropeptide immunoreactivity in lateral efferents. 31,32/t would therefore appear that lateral efferents may act via a complex multitransmitter synapse.
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NEUROTRANSMITTERS OF THE COCHLEA Dynorphins-IR E~ Enkephalins-IR Outer Hair Cells
Inner Hair Cells
Outer Spiralling Fiber
Tunnel Spiral Inner Spiral Bundle Bundle
i
m
/(3 Medial System
Figure 2. Schematic of [mmunoreactive labeling a[ the lateral system of efferents in the guinea pig organ of Corti that is seen using antisera to enkephalins and dyuorphins as well a s tyrosine hydroxylase and calcitonin gene-re[ated peptide.
Gamma aminobutyric acid (GABA) immuner e a c t i v i t y , as w e l l as i m m u n o r e a c t i v i t y for GAD (an enzyme marker for GABA), is found in e f f e r e n t f i b e r s a n d t e r m i n a l s i n t h e cochlea,9,12,aa, a4 but it is difficult to ascribe these to medial or lateral systems, as GABA immunereactive label is seen in elements of both systems in the guinea pig cochlea. More GABA immunoreactive fibers and terminals are seen in the apical portions of the cochlea than the basal with the greatest distribution in third and lower f o u r t h t u r n s of t h e g u i n e a pig cochlea. The GABA i m m u n o r e a c t i v i t y appears in efferent terminals u n d e r outer hair cells more apically than we have observed w i t h choline acetyltransferase i m m u n o r e a c t i v e labeling. Apparently, GABA immunoreactive fibers a n d terminals may then constitute a third system of efferents. Physiologic and pharmacologic studies, particularly on the lateral efferent system, will be necessary to d e t e r m i n e w h i c h of these m a n y neurotransmitters f u n c t i o n or ca-function as efferent transmitters and w h a t their actions or coactions m a y be. A U D I T O R Y NERVE AND C O C H L E A R NUCLEUS
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The cochlear nucleus is the first major relay station in the processing of a u d i t o r y information, receiving the p r i m a r y afferent input of the a u d i t o r y nerve. It is d i v i d e d into ventral and dorsal divisions, the former is further divided by the b i f u r c a t i o n of the a u d i t o r y nerve into anterior a n d posterior divisions. The ventral co-
chlear nucleus gets the bulk of the primary afferent i n p u t of the a u d i t o r y nerve, with the dorsal cochlear nucleus getting proportionally less primary afferent input. In the anteroventral cochlear nucleus, there are two major cell types, spherical cells and stellate multipolar cells.~,35-39 Considerable evidence favors an excitatory amino acid as the transmitter of the auditory nerve.l.40-42 Lesion studies of the auditory nerve have been used to identify the morphology and distribution of auditory nerve terminals in the cochlear n u c l e u s . Such p r i m a r y afferent terminals constitute a very large input on spherical cells of the anteroventral cochlear nucleus.l, 35-3U We have i m m u n o c y t o c h e m i c a l l y labeled a u d i t o r y nerve terminals on spherical cells in the anteroventral cochlear nucleus using antisera to enzymes we believe may be involved in the production of excitatory amino acid transmitters (Fig. 3). 43,44 These are often very large t e r m i n a l s and c o n t a i n large r o u n d vesicles, make an asymmetric contact, and are marked by presynaptic invaginations and postsynaptic evaginations. Other terminals using other neurotransmitters are also present in the cochlear nucleus and are presumably involved in the processing of auditory information. These, as well as the primary afferent terminals, give different cells and o u t p u t s of the cochlear nucleus different response properties.l,as,a9 Based on physiologic and pharmacologic evidence, the two major inhibitory transmitters in the cochlear nucleus are GABA and glycine.1,40-42 Using antiserum to GABA, we can identify a large GABAergic population of terminals in the cochlear nucleus.45-47 These are present on all cell types in all divisions of the cochlear nucleus. In the anteroventral cochlear nucleus, GABA immunoreactive terminals were seen making axodendritic synapses in the neuropil and axosomatic synapses on cells with a high density of axosomatic contacts corresponding to spherical bushy cells and on cells with a low density of axosomatic contacts corresponding to stellate cells. These terminals contain oval/pleomorphic vesicles and make symmetric contacts (Fig. 4). Using an antiserum to the glycine receptor, we can identify a separate large population of glycinergic terminals. 4a As in the case of GABA immunereactive terminals, these are seen in all divisions of the cochlear nucleus and appear to be on most major cell types. Terminals apposing i m m u n o r e a c t i v e l y labeled glycine receptors, however, contain flattened vesicles and therefore appear to constitute a separate population
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Figure 3. Aspartate aminotransferase immunoreactivity (shown) and glutaminase immunoreactivity (not shown) label primary afferent (pa) terminals of the auditory nerve on spherical cells in the anteroventral cochlear nucleus of the guinea pig. Other terminals an spherical cells, such as those conraining flattened vesicles (f) are unlabeled with these antisera ( x 24,000).
Figure 4, The GABA immunoreactivity is seen in terminals containing oval/pleomorphic vesicles (ap) on spherical cells in the anteroventral cochlea nucleus of the guinea pig ( x 23,000).
f r o m t h e GABA i m m u n o r e a c t i v e ones. These term i n a l s , i d e n t i f i e d as g l y c i n e r g i c by i m m u n o r e a c t i v e labeling of t h e p o s t s y n a p t i c receptor, are s e e n m a k i n g b o t h a x o s o m a t i c and axodendritic contacts a n d in t h e a n t e r o v e n t r a l cochlear n u c l e u s are s e e n on t h e s o m a s of cells corres p o n d i n g to s p h e r i c a l b u s h y a n d stellate multip o l a r cells (Fig. 5). A l t h o u g h an e x c i t a t o r y a m i n o a c i d is p r o b a b l y the m a j o r excitatory t r a n s m i t t e r of the cochlear
n u c l e u s a n d GABA and glyoine are likely to be the major i n h i b i t o r y t r a n s m i t t e r s , t h e r e are o t h e r i m p o r t a n t n e u r o t r a n s m i t t e r s that h a v e b e e n localized t h r o u g h i m m u n o c y t o c h e m i c a l a n d hist o c h e m i c a l m e t h o d s in t h e c o c h l e a r n u c l e u s . M c D o n a l d and Rasmussen,49 u s i n g a c e t y l c h o l i n esterase s t a i n i n g as a m a r k e r for c h o l i n e r g i c terminals, w e r e able to localize this e n z y m e pred o m i n a n t l y to t e r m i n a l s c o n t a i n i n g s m a l l r o u n d vesicles a n d m a k i n g s y m m e t r i c a l s y n a p s e s . Ace-
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Figure 5. Immunoreactive labeling of the glycine receptor (arrows) apposes terminals with flattened vesicles [J) on spherical cells in the anteroventral cochlear n u c l e u s of the g u i n e a pig
( x 23,0oo).
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tylcholinesterase is not a completely reliable marker for cholinergic terminals, however, and further studies using antisera to choline acetyltransferase as a marker are called for. Smaller populations and catecholaminergic and peptidergic terminals have also been identified. M o n o a m i n e - a n d c a t e c h o l a m i n e - c o n taining fibers and terminals in the cochlear nucleus have b e e n identified by histofluorescence,~U,sl and recently we have localized these immunocytochemically using antibodies to tyrosine hydroxylase and dopamine beta hydroxylose. Neuropeptides that have been immunocytochemically identified in the cochlear nucleus include enkephalins, dynorphins, calcitonin gene-related n e u r o p e p t i d e , substance P, somatostatin, and CCK.a2.s2-s6 These have yet to be localized to specific terminals in electron microscopic studies and m a y be co-localized with each other or with other previously mentioned neurotransmitters. There is evidence that enkephalins and GABA may be co-localized. 4a With these i m m u n o c y t o c h e m i c a l studies adding to previous biochemical, physiologic/ pharmacologic, and anatomic s t u d i e s , 1'as-42,57-59 it is now possible in the anteroventral cochlear nucleus to propose a neurotransmitter candidate for each of the major classes of terminals on spherical bushy cells that have previously been identified in anatomical studies. 1,3s-39 We have done so in Figure 6, assigning an excitatory amino acid (aspartate or glutamate being the most likely candidates) to primary afferent terminals of the auditory nerve. We assign GABA to the class of terminals containing oval/pleo-
morphic vesicles which make symmetrical contacts with spherical bushy cells and glycine to the terminals containing flattened vesicles, which also make symmetrical contacts with these cells. Acetylcholine can be assigned to the class of terminals with small round vesicles.
CONCLUSIONS Although our knowledge of auditory neurotransmitters is still incomplete, many of the neurotransmitters that may be involved in the processing of auditory information have been identified. Either by localizing these to discrete populations of neurons and terminals or by finding out how they co-exist, we can help to learn the anatomic and neurochemical basis of
(-) (-)
~+11-1 NEUROPEPTIDES CATECEHRO T%MIINES
Figure 6. Schematic of the different transmitter inputs to spherical cells in the allteroventral cochlear nucleus. Acetylcholine (ACh) is in terminals containing small round vesicles (sr], glycine (GLY) is in terminals containing fiattened vesicles (fl], GABA is ifl terminals containing oval/ pleamorphic vesicles (o/p), and aspartafe (ASP) and/or glutamate (GLUT) is in primary afferent terminals of the auditory nerve (PA) containing large round vesicles [lr), Several neuropeptides, monoamines (serotonin], and catecholamines may be transmitters onto spherical ceils hut have not yet been ascribed topartiCular terminal types.
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auditory function. Once neurotransmitters have been identified and their distributions quantified, we can examine how different inputs and their transmitters change with development, aging, lesions, auditory enrichment, or deprivation, as well as with diseases. This knowledge will likely contribute to our knowledge of presbycusis, as well as other auditory dysfunctions.
Acknowledgments. T h e authors a c k n o w l e d g e the i n d i s p e n s a b l e c o n t r i b u t i o n of Dr, JOrgen F e x to the s t u d i e s described in this article and t h a n k M a r i a n n e P a r a k k a ] and Karen Reeks for t h e i r e x c e l l e n t technical assistance. The authors also thank Drs. Betz, C u r t h o y s , Eckenstein, Haser, and W e b e r for their gifts of antisera.
References 1. Caspary DM: Cochlear nuclei: functional neuropharmacology of the principal cell types, in Altschuler RA, Hoffman DW, Bobbin RP (ads): N~urobiology of Hearing: The Cochlea. New York, Raven Press, in press 2. Bobbin RP, Bledsoe SC, lenisen GL: Neurotransmitters of the cochlea and lateral line organ, in Berlin CI (ed): Hearing Science: Recent Advances. San Diego, College-Hill Press, 1984, pp 159-180 3. Rasmussen GL: The olivary peduncle and other projections of the superior olivary complex. I Comp Neurol 84:141-219, 1946 4. Rasmussen GL: Efferent fibers of the cochlear nerve and cochlear nucleus, in Rasmussen GL, Windle WF (eds): Neural Mechanisms of the Auditory and Vestibular System. Springfield, Illinois, Charles C Thomas, 1960, pp 105-115 5. Warr WB, Guinan H, White JS: Organization of the efferent fibers: the lateral and medial olivocochlear systems, in Altschuler RA, Hoffman DW, Bobbin RP (ads): Neurobiology of Hearing: The Cochlea. New York, Raven Press, in press 6. Guinan JJ, Warr WB, Norris BE: Differential olivocoehiear projections from lateral versus medial zones of t h e s u p e r i o r o l i v a r y c o m p l e x . J Camp Neurol 221:358-370, 1983 7. Liberman MC: Efferent synapses in the inner hair cell area of the cat cochlea: an electron microscopic study of serial sections. Hearing Res 3:189-204, 1980 8. Brown MC: Peripheral projections of labeled efferent nerve fibers in the guinea pig cochlea: an anatomical study. Abstracts of the Eighth Midwinter Research Meeting, Association for Research in Otolaryngology, 1985, p 9 9. Altschuler RA, Fex J: Efferent neurotransmitters, in Altschuler RA, Hoffman DW, Bobbin RP {ads]: Neurcbiology of Hearing: The Cochlea. New York, Raven Press, in press 10. Altschuler RA, Kachar B, Rubio JA, et ah Immunecytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea. Brain Res 338:1-11, 1985 11. Eybalin M, Pujol R: Ultrastructural localization of choline acetyltransferase-like immunoreactivity in the organ of Corti. Neuroscienca Letters (suppl] 18:5244, 1984 12. Fox J, Altschuler RA: Immunohistochemistry of the m a m m a l i a n cochlea: results and expectations, in Drescher DG (ed): Auditory Biochemistry, Springfield, Illinois, Charles C Thomas, 1985
13. Godfrey DA, Wiet GJ, Ross CD: Quantitative histochemistry of the cochlea, in Altschu]er RA, Hoffman DW, Bobbin RP (ads): Neurobiology of Hearing: The Cochlea. New York, Raven Press, in press 14, Guth P, Melamed B: Neurotransmission in the auditory system: a primer for pharmacologists. Ann Rev Pharmace[ Toxicol 22:383-412, 1982 15. Klinke R: Neurotransmitters in the cochlea and the cochlea nucleus. Acta Otolaryngol 91:541-554, 1981 16. Altschuler RA, Parakkal MH, Ruhio ]A, et al" Enkepholin-like immunoreactivity in the guinea pig organ of Corti: ultrastructural and lesion studies. Hearing Res 18:17-31, 1984 17. Altschuler RA, Hoffman DW, Reeks KA, et ah Localization of dynorphin B-like and alpha-:neoendorphin-like immunoreactivities in the guinea pig. Hearfng Res 17',249-258, 1985 18. Eybalin M, Cups A, Pujol R: Localisation ultrastructale des immunoreactians a u n anticorps met-enkephaline dans l'organe de Certi. C R A c a d Sci 296'.1125-1128, 1983 19. Eybalin M, Cupo AA, Pujol R: Met-enkephalin characterization in the cochlea: high performance liquid chromatography and immunoelectran microscopy, Brain Res 305;313-322, 1984 20. Eybalin M, Pujol R: Immunofluorescence with met-enkephalin and leu-enkephalin antibodies in the guinea pig cochlea. Hearing Res 12:135-140, 1984 21. Fox J, Altschuler RA: Enkephalin-like immunoreactivity of olivocochlear nerve fibers in cochlea of guinea pig and cat. Proc Natl Acad Sci USA 78:1255-1259, 1981 22. Hoffman DW, Altschuler RA, Fox J: High performance liquid chromatographic identification of enkephalinlike peptides in the cochlea. Hearing Res 9:71-79, 1983 23, Hoffman DW, Ruble JA, Altschuler RA, et a[: Several distinct receptor binding enkephalins in olivocachlear fibers and terminals in the organ of Carti. Brain Res 322:59-65, 1984 24. Hoffman DW, Zamir N, Ruble JA, et ah Proenkephalin and prc3dynorphin-related neuropeptides in the cochlea. Hearing Res 17:47-50, 1985 25. Hoffman DW: Opioid mechanisms in the cochlea, in Altschuler RA, Hoffman DW, Bobbin RP (ads): Neurobiology of Hearing: The Cochlea. New York, Raven Press, in press 26. Tramu G, Pillez A, Leonardelli J: An efficient method for antibody elution far the successive or simultaneous l o c a l i z a t i o n of two a n t i b o d i e s by i m m u n a c y t a chemistry. 1 Histochem Cytochem 26:322, 1978 27. Altschuler RA, Fox J, Parakkal MH, et ah Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig. J Histochem Cytochem 32:839-843, 1984 28. Altschuler RA, Parakkal MH, Fex J: Localization of enkephalin-like immunoreactivity in acetylchelinesterase positive cells in the guinea pig lateral superior olivary complex that project to the cochlea. Neuroscience 9:621-630, 1983 29, Altschuler RA, Hoffman DW, Reeks KA, et ah Colocalization of immunoreactivities for dynorphins and enkephalins in lateral olivocochlear neurons in the guinea pig. Abstracts of the Eighth Midwinter Research Meeting, Association for Research in Otolaryngology, 1985, p 139 30. Drescher M], Drescher DG, Medina JE: Effects of sound stimulation at several levels of concentrations of primary amines, including neurotransmitter candidates, in perilymph of the guinea pig inner ear. J Neurochem 41:309-320, 1983 31. Kitajiri M, Yamashita T, Amano H, et ah Localization of caleitonin gone-related paptide in the organ of Corti of the rat: an immunohistoehemical study. Brain Res, in press
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NEUROTRANSMITTERS OF THE COCHLEA 32. S c h w e i t z e r LF, Lu SM, Dawbarn D, et ah CaIciton gonerelated peptide in the superior olivary complex of cat and rat: a specific label for the lateral olivocochlear system. Neuroscience Abet, 1985 33. Fex 1, Altschnler RA: Clutamic acid decarboxylase immunoreactivity of olivocochlear neurons in the organ of Gorti of guinea pig and rat. Hearing Res 15:123131, 19114 34. Fox J, Altschuler RA, Kachar B, etah CABA visualized by immunecytochemistry in olivocochlear neurons in the guinea pig cochlea. Brain Res, in press 35. Cant NB, Merest DK: The bushy cells in the anterovem tral cochlear nucleus of the cat: a study with the electron microscope. Neuroscience 4:1925-1945, 1979 36. Cant NB, Merest DK: The structural basis for stimulus coding in the cochlear nucleus of the cat, in Berlin CI (ed]: Hearing Science'. Recent Advances. San Diego, College-Hill Press, 1984, pp 271-421 37. Culley RL, Landis DMD, Reese TS: Internal organization of the end bulbs of Held in the anteroventral cochlear nucleus. ] Comp Neurol 180:707-742, 1978 38. Schwartz AM, Galley RL: Non-primary afferents to the principal cells of the rostral anteroventral cochlear nucleus of the guinea pig. I Anat 153:489-508, 1978 39. Moore IK: Cochlear nuclei: relationship of neurons to the auditory nerve, in Altschuler RA, Hoffman DW, Bobbin RP (eds): Neurobiology of Hearing: The Cochlea, New York, Raven Press, in press 40. Caspary DM, Rybak LP. Faingold CL: The effects of inhibitory and excitatory amino-acid neurotransmitters on the response p r o p e r t i e s of brainstem auditory neurons, in Drescher DG (ed): Auditory Biochemistry. Springfield, Illinois, Charles C Thomas, 1985, pp. 198-226 41, Wenthold RJ, Martin MR: Neurotransmitters of the auditory nerve and central auditory system, in Berlin Cl (ed): Hearing Science: Recent Advances, San Diego, College-Hill Press, 1984, pp. 321-389. 42, Wenthold RJ: Glutamate and aspartate as neurotransmitters of the auditory nerve, in Drescher D (ed): Auditory Biochemistry. Springfield, Illinois, Charles C Thomas, 1985, pp 125-140 45. Altsehuler RA, Neises GR, Marmison GG, etal: Immunocytochemical localization of aspartate aminotransferase immunoreactivity in cochlear nucleus of the guinea pig. Proc Nail Acad Sci USA 78:6553-6557, 1981 44. Altschuler RA, Wenthold R;, Schwartz AM, e t a h hnmunooytochemical localization of glutaminase-like immunoreactivity in the auditory nerve. Brain Res 291:173-178, 1984
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45. Wenthold RJ, Zempel JM, Parakkal MH etah Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig. Brain Res, in press 46. Adams JC, Mugnaini E: GAD-like immunoreactivity in the ventral cochlear nucleus. Neuroscience Abet, 393, 1984 47. Moore JK, Moore RY: GAD-like immunoreactivity in the cochlear nuclei and superior olivary complex. Soc Neurosci Abet 10:843, 1984 48. Altschuler RA, Betz H, Parakkal MH, etah Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res, in press 49. McDonald DM, Rasmussen GL: Ultrastructural characteristics of synaptic endings in the cochlear nucleus having acetylcholinesterase activity. Brain Res 28:1-18, 1971 50. Fuxe K: Evidence for the existence of monoamine neurons in the central nervous system: IV. Distribution of monoamine terminals in the central nervous system. Acta Physio} 64 (suppl 247}:39-85, 1965 51. Kromer LF, Moore RY: Cochlear nucleus innervation by central norepinephrine neurons in the rat. Brain Res 118:531-537, 1976 52. Altschuler RA: Met-enkephalin positivity in the small cells of the deep dorsal cochlear nucleus and posteroventral cochlear nucleus of the rat. Neurosci Abet 5:15, 1979 53. Tachibana M, Rothman JM, Cuth PS: Somatostatin along the auditory pathway. Hearing Res 1:365-368, 1979 54. Adams ]C, Mugnaini E: Patterns of immunostaining with antisera to peptides in the auditory brainstem of cat. Neurosci Abet 11:32, 1985 55. Spangler KM, Morley BJ: Somatostatin-like immunereactivity in the midbrain of the cat. Neurosci Abet 11:803, 1985 56. yon Krosigk M, Vincent SR, Brown ]C, e t a h Somatostatin immunoreactivity in second-order auditory neurons of the rat. Neurosci Abet 11:352,1985 57. Godfrey DA, Carter IA, Lowry OH, et ah Distribution of gamma aminobutyric acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat. ] Histochain Cytochem 26:118-126, 1978 58. Potashner SJ, Lindberg N, Merest DK: Uptake and release of GABA in the guinea pig cochlear nucleus after axotomy of cochlear and centrifugal fibers. I Neurochem, in press 59. Schwartz IR: The differential distribution of label following uptake of 3H-labeled amino acids in the dorsal cochlear nucleus of the cat. Exp Neurol 73'.601-617, 1981
Neurotransmitters of the Cochlea and Cochlear Nucleus: Immunocytochemical Evidence RICHARDA. ALTSCHULER,PH.D., DOUGLASW. HOFFMAN,PH.D., ANDROBERTJ. WENTHOLD,PH.D. Many neurotransmitter candidates have been identified in the cochlea and cochlear nucleus with the use of immunocytochemical techniques. Choline acetyltransferase immunoreactivity suggests acetylcholine as a transmitter of medial and lateral efferent systems in the cochlea. Immunoreactivities to enkephalins, dynorphins, calcitonin gene-related peptide, and tyrosine hydroxylase (a marker for dopamine) are also found in lateral efferents. Choline acetyltransferase, enkephalin, and dynorphin immunoreactivities are cocontained in neurons of the lateral system. In the anteroventral cochlear nucleus, the inhibitory amino acid transmitters, gamma aminobutyric acid (GABA), and glycine, as well as the presumed excitatory amino acid transmitter of the auditory nerve, have been directly or indirectly localized, immunocytochemically, to discrete populations of terminals on spherical cells with distinct morphologic characteristics.
Transmitters are a basic element of neuronal and sensory function. A major distinction of neurons (and receptor cells such as cochlear hair cells) is their ability to communicate with other cells by means of a n e u r o t r a n s m i t t e r . Knowledge of neurotransmitters enables us to manipulate nervous function pharmacologically. Moreover, neurotransmitters have been implicated in several disorders, such as dopamine in Parkinson's disease. Hawkins once hypothesized that chemical mediators such as neurotransmitters might be involved in aminoglycoside-induced hearing loss, and although this may not be the case, it is very possible that neurotransmitters are involved in aspects of presbycusis, perhaps in inability to extract relevant auditory information embedded in noise. 1 It is likely that increased knowledge of neurotransmitters of the auditory system will help us to
understand, and perhaps treat, auditory dysfunctions, Although the transmitter of cochlear hair cells remains unknown, with some evidence favoring a substance related to an excitatory amino acid, 2 we have been able to identify, by means of imm u n o c y t o c h e m i s t r y , n e u r o t r a n s m i t t e r candidates in cochlear efferents and in the auditory nerve and cochlear nucleus. In the case of efferents, we have been able to co-localize at least three neurotransmitter candidates w i t h i n the same neurons. In the cochlear nucleus, we have localized three neurotransmitter candidates to separate terminals, with distinct synaptic characteristics.
EFFERENTS The olivocochlear efferents, first described by Rasmussen,3.4 have recently been reclassified by Warr et al.~ and Cuinan et al. 6 into lateral and medial systems. The lateral system has its cells of origin in or around the lateral superior olive and sends its axons p r e d o m i n a n t l y or exclusively to the ipsilateral cochlea. In the cochlea, lateral efferent fibers travel in the inner spiral bundle and tunnel spiral bundle and terminate predominantly on afferent dendrites of type I spiral ganglion cells with a few terminating at the bases of inner hair cells (and perhaps a few ending on outer hair cells). The medial system
Received from the KresgeHearingResearch Institute, University of Michigan, Ann Arbor, Michigan (Dr. Altschuler); Department of Psychiatry, Southern Illinois University School of Medicine,Springfield,Illinois (Dr. Hoffman);and the Laboratoryof Neuro-otolaryngology,National Institutes of Health, Bethesda,Maryland (Dr. Wenthold}. Presented at "ContemporaryIssues of Middle and Inner Ear Physiologyand Pathology:A Symposium in Honor of Joseph E. Hawkins, jr., Ph,D,, Sc,D,, and Merle Lawrence, Ph.D.," Ann Arbor, Michigan, October 27, 1985. Accepted for publication at that time, Address reprint requests to Dr, Altschuler:KresgeHearing Research Institute, 1301 E, Ann St., Ann Arbor, MI 48109,
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has its origin in cells in more medially located olivary nuclei and sends its axons mostly to the contralateral cochlea. Some medial efferent fibers may travel for a small distance in inner spiral and tunnel spiral bundles before crossing the tunnel of Corti, whereas most medial efferents immediately cross the tunnel of Corti to terminate predominantly at the bases of outer hair cells (with some also ending on afferent dendrites of type II spiral ganglion cells). There may be axo-axonal connections between the two systems in the inner spiral bundle regionJ Supranuclear and circumnuclear efferent endings on outer hair cells are a minor population and may be part of either or both medial and lateral systems. 8,9 There is considerable evidence suggesting acetylcholine as a transmitter of efferents.,.lo We have found choline acetyltransferase immunereactivity, indicating the presence of acetylcholine, in both lateral and medial systems of olivecochlear efferents (Fig. 1) in the guinea pig,lO and similar findings have been reported in the rat. 1~ This immunoreactivity was found in fibers and terminals in the inner spiral bundle and in the tunnel spiral bundle, corresponding to the lateral system of efferents. Choline acetyltransferase immunoreactive labeling corresponding to the medial system of efferents was seen in upper tunnel crossing fibers and in large puncta corresponding to terminals at outer hair cell bases. In the guinea pig cochlea, nine or more immunoreactive terminals can be seen on first row outer hair cells in basal turns. As one progresses apically along the cochlear spiral, the number of large choline acetyltransferase immunoreactive puncta at outer hair cell bases decreases, beginning in the third row and then decreasing in the second row. By the beginning of the fourth turn, immunoreactive terminals are seen at first row outer hair cells only, and by the middle of the fourth turn, no such terminals are observed on any outer hair cell bases. Choline acetyltransferase immunoreactivity is also seen in supranuclear and circumnuclear efferents, w h i c h have n o t yet been assigned to either system of efferents. These findings add to the evidence that acetylcholine is a transmitter of both medial and lateral systems, although more physiologic and pharmacologic studies are needed to confirm its role in the lateral efferent system. Enkephalins and dynorphins are two separate families of opioid neuropeptides with important neurotransmitter/neuromodulator roles in the nervous system. With the use of most antisera to enkephalins and antisera to dynorphins, im-
ChAT-IR [Acetylcholine] Outer Hair Cells
Inner Hair Cells
Outer Spiralling Fiber
Tunnel Spiral InnerSpiral Bundle Bundle
i
LatSyst eralem System
Medial
Figure 1. Schematic of choline acetyltransferase immunereactivity in the guinea pig organ of Corti. Labeling of both medial and lateral efferent systems is seen.
munoreactive staining is seen in fibers and terminals of the inner spiral bundle and in the tunnel spiral bundle, corresponding to the lateral system of efferents. '~'12,1~-~ Electron microscopic studies have shown enkephalin immunereactivity to be associated with large dense core vesicles in efferent terminals synapsing on afferent dendrites or occasionally on other efferents.16.~8.~9 The presence of three neurotransmitter candidates (acetylcholine, enkephalins, and @norphins) in lateral efferents suggests that there may be more than one neurotransmitter within the same neuron in this system. We have examined this by looking at the cells of origin of the lateral system and using sequential co-localization methods such as the method of Tramu. 2e Our results s h o w that lateral olivocochlear neurons co-contain enkephalins, dynorphins, and choline acetyltransferase (indicating acetylcholine). 27-2g Biochemicai studies have been done that have localized enkephalins and dynorphins, with appropriate receptor-binding characteristics, to olivocochlear efferents, 22-~5 and sound-induced release of an enkephalinlike substance has been reported.no Recent immunocytochemical evidence suggests that the lateral efferent system contains even more neurotransmitter candidates. We have recently found tyrosine hydroxylase immunoreactivity (indicating dopamine as a transmitter) in lateral efferents in the guinea pig cochlea and its ceils of origin in the lateral superior olivary complex. Two different studies have just reported calcitonin gene-related neuropeptide immunoreactivity in lateral efferents. 31,32/t would therefore appear that lateral efferents may act via a complex multitransmitter synapse.
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NEUROTRANSMITTERS OF THE COCHLEA Dynorphins-IR E~ Enkephalins-IR Outer Hair Cells
Inner Hair Cells
Outer Spiralling Fiber
Tunnel Spiral Inner Spiral Bundle Bundle
i
m
/(3 Medial System
Figure 2. Schematic of [mmunoreactive labeling a[ the lateral system of efferents in the guinea pig organ of Corti that is seen using antisera to enkephalins and dyuorphins as well a s tyrosine hydroxylase and calcitonin gene-re[ated peptide.
Gamma aminobutyric acid (GABA) immuner e a c t i v i t y , as w e l l as i m m u n o r e a c t i v i t y for GAD (an enzyme marker for GABA), is found in e f f e r e n t f i b e r s a n d t e r m i n a l s i n t h e cochlea,9,12,aa, a4 but it is difficult to ascribe these to medial or lateral systems, as GABA immunereactive label is seen in elements of both systems in the guinea pig cochlea. More GABA immunoreactive fibers and terminals are seen in the apical portions of the cochlea than the basal with the greatest distribution in third and lower f o u r t h t u r n s of t h e g u i n e a pig cochlea. The GABA i m m u n o r e a c t i v i t y appears in efferent terminals u n d e r outer hair cells more apically than we have observed w i t h choline acetyltransferase i m m u n o r e a c t i v e labeling. Apparently, GABA immunoreactive fibers a n d terminals may then constitute a third system of efferents. Physiologic and pharmacologic studies, particularly on the lateral efferent system, will be necessary to d e t e r m i n e w h i c h of these m a n y neurotransmitters f u n c t i o n or ca-function as efferent transmitters and w h a t their actions or coactions m a y be. A U D I T O R Y NERVE AND C O C H L E A R NUCLEUS
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The cochlear nucleus is the first major relay station in the processing of a u d i t o r y information, receiving the p r i m a r y afferent input of the a u d i t o r y nerve. It is d i v i d e d into ventral and dorsal divisions, the former is further divided by the b i f u r c a t i o n of the a u d i t o r y nerve into anterior a n d posterior divisions. The ventral co-
chlear nucleus gets the bulk of the primary afferent i n p u t of the a u d i t o r y nerve, with the dorsal cochlear nucleus getting proportionally less primary afferent input. In the anteroventral cochlear nucleus, there are two major cell types, spherical cells and stellate multipolar cells.~,35-39 Considerable evidence favors an excitatory amino acid as the transmitter of the auditory nerve.l.40-42 Lesion studies of the auditory nerve have been used to identify the morphology and distribution of auditory nerve terminals in the cochlear n u c l e u s . Such p r i m a r y afferent terminals constitute a very large input on spherical cells of the anteroventral cochlear nucleus.l, 35-3U We have i m m u n o c y t o c h e m i c a l l y labeled a u d i t o r y nerve terminals on spherical cells in the anteroventral cochlear nucleus using antisera to enzymes we believe may be involved in the production of excitatory amino acid transmitters (Fig. 3). 43,44 These are often very large t e r m i n a l s and c o n t a i n large r o u n d vesicles, make an asymmetric contact, and are marked by presynaptic invaginations and postsynaptic evaginations. Other terminals using other neurotransmitters are also present in the cochlear nucleus and are presumably involved in the processing of auditory information. These, as well as the primary afferent terminals, give different cells and o u t p u t s of the cochlear nucleus different response properties.l,as,a9 Based on physiologic and pharmacologic evidence, the two major inhibitory transmitters in the cochlear nucleus are GABA and glycine.1,40-42 Using antiserum to GABA, we can identify a large GABAergic population of terminals in the cochlear nucleus.45-47 These are present on all cell types in all divisions of the cochlear nucleus. In the anteroventral cochlear nucleus, GABA immunoreactive terminals were seen making axodendritic synapses in the neuropil and axosomatic synapses on cells with a high density of axosomatic contacts corresponding to spherical bushy cells and on cells with a low density of axosomatic contacts corresponding to stellate cells. These terminals contain oval/pleomorphic vesicles and make symmetric contacts (Fig. 4). Using an antiserum to the glycine receptor, we can identify a separate large population of glycinergic terminals. 4a As in the case of GABA immunereactive terminals, these are seen in all divisions of the cochlear nucleus and appear to be on most major cell types. Terminals apposing i m m u n o r e a c t i v e l y labeled glycine receptors, however, contain flattened vesicles and therefore appear to constitute a separate population
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Figure 3. Aspartate aminotransferase immunoreactivity (shown) and glutaminase immunoreactivity (not shown) label primary afferent (pa) terminals of the auditory nerve on spherical cells in the anteroventral cochlear nucleus of the guinea pig. Other terminals an spherical cells, such as those conraining flattened vesicles (f) are unlabeled with these antisera ( x 24,000).
Figure 4, The GABA immunoreactivity is seen in terminals containing oval/pleomorphic vesicles (ap) on spherical cells in the anteroventral cochlea nucleus of the guinea pig ( x 23,000).
f r o m t h e GABA i m m u n o r e a c t i v e ones. These term i n a l s , i d e n t i f i e d as g l y c i n e r g i c by i m m u n o r e a c t i v e labeling of t h e p o s t s y n a p t i c receptor, are s e e n m a k i n g b o t h a x o s o m a t i c and axodendritic contacts a n d in t h e a n t e r o v e n t r a l cochlear n u c l e u s are s e e n on t h e s o m a s of cells corres p o n d i n g to s p h e r i c a l b u s h y a n d stellate multip o l a r cells (Fig. 5). A l t h o u g h an e x c i t a t o r y a m i n o a c i d is p r o b a b l y the m a j o r excitatory t r a n s m i t t e r of the cochlear
n u c l e u s a n d GABA and glyoine are likely to be the major i n h i b i t o r y t r a n s m i t t e r s , t h e r e are o t h e r i m p o r t a n t n e u r o t r a n s m i t t e r s that h a v e b e e n localized t h r o u g h i m m u n o c y t o c h e m i c a l a n d hist o c h e m i c a l m e t h o d s in t h e c o c h l e a r n u c l e u s . M c D o n a l d and Rasmussen,49 u s i n g a c e t y l c h o l i n esterase s t a i n i n g as a m a r k e r for c h o l i n e r g i c terminals, w e r e able to localize this e n z y m e pred o m i n a n t l y to t e r m i n a l s c o n t a i n i n g s m a l l r o u n d vesicles a n d m a k i n g s y m m e t r i c a l s y n a p s e s . Ace-
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NEUROTRANSM[TTERS OF THE COCHLEA
Figure 5. Immunoreactive labeling of the glycine receptor (arrows) apposes terminals with flattened vesicles [J) on spherical cells in the anteroventral cochlear n u c l e u s of the g u i n e a pig
( x 23,0oo).
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tylcholinesterase is not a completely reliable marker for cholinergic terminals, however, and further studies using antisera to choline acetyltransferase as a marker are called for. Smaller populations and catecholaminergic and peptidergic terminals have also been identified. M o n o a m i n e - a n d c a t e c h o l a m i n e - c o n taining fibers and terminals in the cochlear nucleus have b e e n identified by histofluorescence,~U,sl and recently we have localized these immunocytochemically using antibodies to tyrosine hydroxylase and dopamine beta hydroxylose. Neuropeptides that have been immunocytochemically identified in the cochlear nucleus include enkephalins, dynorphins, calcitonin gene-related n e u r o p e p t i d e , substance P, somatostatin, and CCK.a2.s2-s6 These have yet to be localized to specific terminals in electron microscopic studies and m a y be co-localized with each other or with other previously mentioned neurotransmitters. There is evidence that enkephalins and GABA may be co-localized. 4a With these i m m u n o c y t o c h e m i c a l studies adding to previous biochemical, physiologic/ pharmacologic, and anatomic s t u d i e s , 1'as-42,57-59 it is now possible in the anteroventral cochlear nucleus to propose a neurotransmitter candidate for each of the major classes of terminals on spherical bushy cells that have previously been identified in anatomical studies. 1,3s-39 We have done so in Figure 6, assigning an excitatory amino acid (aspartate or glutamate being the most likely candidates) to primary afferent terminals of the auditory nerve. We assign GABA to the class of terminals containing oval/pleo-
morphic vesicles which make symmetrical contacts with spherical bushy cells and glycine to the terminals containing flattened vesicles, which also make symmetrical contacts with these cells. Acetylcholine can be assigned to the class of terminals with small round vesicles.
CONCLUSIONS Although our knowledge of auditory neurotransmitters is still incomplete, many of the neurotransmitters that may be involved in the processing of auditory information have been identified. Either by localizing these to discrete populations of neurons and terminals or by finding out how they co-exist, we can help to learn the anatomic and neurochemical basis of
(-) (-)
~+11-1 NEUROPEPTIDES CATECEHRO T%MIINES
Figure 6. Schematic of the different transmitter inputs to spherical cells in the allteroventral cochlear nucleus. Acetylcholine (ACh) is in terminals containing small round vesicles (sr], glycine (GLY) is in terminals containing fiattened vesicles (fl], GABA is ifl terminals containing oval/ pleamorphic vesicles (o/p), and aspartafe (ASP) and/or glutamate (GLUT) is in primary afferent terminals of the auditory nerve (PA) containing large round vesicles [lr), Several neuropeptides, monoamines (serotonin], and catecholamines may be transmitters onto spherical ceils hut have not yet been ascribed topartiCular terminal types.
ALTSCHULER ET AL.
auditory function. Once neurotransmitters have been identified and their distributions quantified, we can examine how different inputs and their transmitters change with development, aging, lesions, auditory enrichment, or deprivation, as well as with diseases. This knowledge will likely contribute to our knowledge of presbycusis, as well as other auditory dysfunctions.
Acknowledgments. T h e authors a c k n o w l e d g e the i n d i s p e n s a b l e c o n t r i b u t i o n of Dr, JOrgen F e x to the s t u d i e s described in this article and t h a n k M a r i a n n e P a r a k k a ] and Karen Reeks for t h e i r e x c e l l e n t technical assistance. The authors also thank Drs. Betz, C u r t h o y s , Eckenstein, Haser, and W e b e r for their gifts of antisera.
References 1. Caspary DM: Cochlear nuclei: functional neuropharmacology of the principal cell types, in Altschuler RA, Hoffman DW, Bobbin RP (ads): N~urobiology of Hearing: The Cochlea. New York, Raven Press, in press 2. Bobbin RP, Bledsoe SC, lenisen GL: Neurotransmitters of the cochlea and lateral line organ, in Berlin CI (ed): Hearing Science: Recent Advances. San Diego, College-Hill Press, 1984, pp 159-180 3. Rasmussen GL: The olivary peduncle and other projections of the superior olivary complex. I Comp Neurol 84:141-219, 1946 4. Rasmussen GL: Efferent fibers of the cochlear nerve and cochlear nucleus, in Rasmussen GL, Windle WF (eds): Neural Mechanisms of the Auditory and Vestibular System. Springfield, Illinois, Charles C Thomas, 1960, pp 105-115 5. Warr WB, Guinan H, White JS: Organization of the efferent fibers: the lateral and medial olivocochlear systems, in Altschuler RA, Hoffman DW, Bobbin RP (ads): Neurobiology of Hearing: The Cochlea. New York, Raven Press, in press 6. Guinan JJ, Warr WB, Norris BE: Differential olivocoehiear projections from lateral versus medial zones of t h e s u p e r i o r o l i v a r y c o m p l e x . J Camp Neurol 221:358-370, 1983 7. Liberman MC: Efferent synapses in the inner hair cell area of the cat cochlea: an electron microscopic study of serial sections. Hearing Res 3:189-204, 1980 8. Brown MC: Peripheral projections of labeled efferent nerve fibers in the guinea pig cochlea: an anatomical study. Abstracts of the Eighth Midwinter Research Meeting, Association for Research in Otolaryngology, 1985, p 9 9. Altschuler RA, Fex J: Efferent neurotransmitters, in Altschuler RA, Hoffman DW, Bobbin RP {ads]: Neurcbiology of Hearing: The Cochlea. New York, Raven Press, in press 10. Altschuler RA, Kachar B, Rubio JA, et ah Immunecytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea. Brain Res 338:1-11, 1985 11. Eybalin M, Pujol R: Ultrastructural localization of choline acetyltransferase-like immunoreactivity in the organ of Corti. Neuroscienca Letters (suppl] 18:5244, 1984 12. Fox J, Altschuler RA: Immunohistochemistry of the m a m m a l i a n cochlea: results and expectations, in Drescher DG (ed): Auditory Biochemistry, Springfield, Illinois, Charles C Thomas, 1985
13. Godfrey DA, Wiet GJ, Ross CD: Quantitative histochemistry of the cochlea, in Altschu]er RA, Hoffman DW, Bobbin RP (ads): Neurobiology of Hearing: The Cochlea. New York, Raven Press, in press 14, Guth P, Melamed B: Neurotransmission in the auditory system: a primer for pharmacologists. Ann Rev Pharmace[ Toxicol 22:383-412, 1982 15. Klinke R: Neurotransmitters in the cochlea and the cochlea nucleus. Acta Otolaryngol 91:541-554, 1981 16. Altschuler RA, Parakkal MH, Ruhio ]A, et al" Enkepholin-like immunoreactivity in the guinea pig organ of Corti: ultrastructural and lesion studies. Hearing Res 18:17-31, 1984 17. Altschuler RA, Hoffman DW, Reeks KA, et ah Localization of dynorphin B-like and alpha-:neoendorphin-like immunoreactivities in the guinea pig. Hearfng Res 17',249-258, 1985 18. Eybalin M, Cups A, Pujol R: Localisation ultrastructale des immunoreactians a u n anticorps met-enkephaline dans l'organe de Certi. C R A c a d Sci 296'.1125-1128, 1983 19. Eybalin M, Cupo AA, Pujol R: Met-enkephalin characterization in the cochlea: high performance liquid chromatography and immunoelectran microscopy, Brain Res 305;313-322, 1984 20. Eybalin M, Pujol R: Immunofluorescence with met-enkephalin and leu-enkephalin antibodies in the guinea pig cochlea. Hearing Res 12:135-140, 1984 21. Fox J, Altschuler RA: Enkephalin-like immunoreactivity of olivocochlear nerve fibers in cochlea of guinea pig and cat. Proc Natl Acad Sci USA 78:1255-1259, 1981 22. Hoffman DW, Altschuler RA, Fox J: High performance liquid chromatographic identification of enkephalinlike peptides in the cochlea. Hearing Res 9:71-79, 1983 23, Hoffman DW, Ruble JA, Altschuler RA, et a[: Several distinct receptor binding enkephalins in olivocachlear fibers and terminals in the organ of Carti. Brain Res 322:59-65, 1984 24. Hoffman DW, Zamir N, Ruble JA, et ah Proenkephalin and prc3dynorphin-related neuropeptides in the cochlea. Hearing Res 17:47-50, 1985 25. Hoffman DW: Opioid mechanisms in the cochlea, in Altschuler RA, Hoffman DW, Bobbin RP (ads): Neurobiology of Hearing: The Cochlea. New York, Raven Press, in press 26. Tramu G, Pillez A, Leonardelli J: An efficient method for antibody elution far the successive or simultaneous l o c a l i z a t i o n of two a n t i b o d i e s by i m m u n a c y t a chemistry. 1 Histochem Cytochem 26:322, 1978 27. Altschuler RA, Fox J, Parakkal MH, et ah Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig. J Histochem Cytochem 32:839-843, 1984 28. Altschuler RA, Parakkal MH, Fex J: Localization of enkephalin-like immunoreactivity in acetylchelinesterase positive cells in the guinea pig lateral superior olivary complex that project to the cochlea. Neuroscience 9:621-630, 1983 29, Altschuler RA, Hoffman DW, Reeks KA, et ah Colocalization of immunoreactivities for dynorphins and enkephalins in lateral olivocochlear neurons in the guinea pig. Abstracts of the Eighth Midwinter Research Meeting, Association for Research in Otolaryngology, 1985, p 139 30. Drescher M], Drescher DG, Medina JE: Effects of sound stimulation at several levels of concentrations of primary amines, including neurotransmitter candidates, in perilymph of the guinea pig inner ear. J Neurochem 41:309-320, 1983 31. Kitajiri M, Yamashita T, Amano H, et ah Localization of caleitonin gone-related paptide in the organ of Corti of the rat: an immunohistoehemical study. Brain Res, in press
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NEUROTRANSMITTERS OF THE COCHLEA 32. S c h w e i t z e r LF, Lu SM, Dawbarn D, et ah CaIciton gonerelated peptide in the superior olivary complex of cat and rat: a specific label for the lateral olivocochlear system. Neuroscience Abet, 1985 33. Fex 1, Altschnler RA: Clutamic acid decarboxylase immunoreactivity of olivocochlear neurons in the organ of Gorti of guinea pig and rat. Hearing Res 15:123131, 19114 34. Fox J, Altschuler RA, Kachar B, etah CABA visualized by immunecytochemistry in olivocochlear neurons in the guinea pig cochlea. Brain Res, in press 35. Cant NB, Merest DK: The bushy cells in the anterovem tral cochlear nucleus of the cat: a study with the electron microscope. Neuroscience 4:1925-1945, 1979 36. Cant NB, Merest DK: The structural basis for stimulus coding in the cochlear nucleus of the cat, in Berlin CI (ed]: Hearing Science'. Recent Advances. San Diego, College-Hill Press, 1984, pp 271-421 37. Culley RL, Landis DMD, Reese TS: Internal organization of the end bulbs of Held in the anteroventral cochlear nucleus. ] Comp Neurol 180:707-742, 1978 38. Schwartz AM, Galley RL: Non-primary afferents to the principal cells of the rostral anteroventral cochlear nucleus of the guinea pig. I Anat 153:489-508, 1978 39. Moore IK: Cochlear nuclei: relationship of neurons to the auditory nerve, in Altschuler RA, Hoffman DW, Bobbin RP (eds): Neurobiology of Hearing: The Cochlea, New York, Raven Press, in press 40. Caspary DM, Rybak LP. Faingold CL: The effects of inhibitory and excitatory amino-acid neurotransmitters on the response p r o p e r t i e s of brainstem auditory neurons, in Drescher DG (ed): Auditory Biochemistry. Springfield, Illinois, Charles C Thomas, 1985, pp. 198-226 41, Wenthold RJ, Martin MR: Neurotransmitters of the auditory nerve and central auditory system, in Berlin Cl (ed): Hearing Science: Recent Advances, San Diego, College-Hill Press, 1984, pp. 321-389. 42, Wenthold RJ: Glutamate and aspartate as neurotransmitters of the auditory nerve, in Drescher D (ed): Auditory Biochemistry. Springfield, Illinois, Charles C Thomas, 1985, pp 125-140 45. Altsehuler RA, Neises GR, Marmison GG, etal: Immunocytochemical localization of aspartate aminotransferase immunoreactivity in cochlear nucleus of the guinea pig. Proc Nail Acad Sci USA 78:6553-6557, 1981 44. Altschuler RA, Wenthold R;, Schwartz AM, e t a h hnmunooytochemical localization of glutaminase-like immunoreactivity in the auditory nerve. Brain Res 291:173-178, 1984
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45. Wenthold RJ, Zempel JM, Parakkal MH etah Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig. Brain Res, in press 46. Adams JC, Mugnaini E: GAD-like immunoreactivity in the ventral cochlear nucleus. Neuroscience Abet, 393, 1984 47. Moore JK, Moore RY: GAD-like immunoreactivity in the cochlear nuclei and superior olivary complex. Soc Neurosci Abet 10:843, 1984 48. Altschuler RA, Betz H, Parakkal MH, etah Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res, in press 49. McDonald DM, Rasmussen GL: Ultrastructural characteristics of synaptic endings in the cochlear nucleus having acetylcholinesterase activity. Brain Res 28:1-18, 1971 50. Fuxe K: Evidence for the existence of monoamine neurons in the central nervous system: IV. Distribution of monoamine terminals in the central nervous system. Acta Physio} 64 (suppl 247}:39-85, 1965 51. Kromer LF, Moore RY: Cochlear nucleus innervation by central norepinephrine neurons in the rat. Brain Res 118:531-537, 1976 52. Altschuler RA: Met-enkephalin positivity in the small cells of the deep dorsal cochlear nucleus and posteroventral cochlear nucleus of the rat. Neurosci Abet 5:15, 1979 53. Tachibana M, Rothman JM, Cuth PS: Somatostatin along the auditory pathway. Hearing Res 1:365-368, 1979 54. Adams ]C, Mugnaini E: Patterns of immunostaining with antisera to peptides in the auditory brainstem of cat. Neurosci Abet 11:32, 1985 55. Spangler KM, Morley BJ: Somatostatin-like immunereactivity in the midbrain of the cat. Neurosci Abet 11:803, 1985 56. yon Krosigk M, Vincent SR, Brown ]C, e t a h Somatostatin immunoreactivity in second-order auditory neurons of the rat. Neurosci Abet 11:352,1985 57. Godfrey DA, Carter IA, Lowry OH, et ah Distribution of gamma aminobutyric acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat. ] Histochain Cytochem 26:118-126, 1978 58. Potashner SJ, Lindberg N, Merest DK: Uptake and release of GABA in the guinea pig cochlear nucleus after axotomy of cochlear and centrifugal fibers. I Neurochem, in press 59. Schwartz IR: The differential distribution of label following uptake of 3H-labeled amino acids in the dorsal cochlear nucleus of the cat. Exp Neurol 73'.601-617, 1981