Dendrodendritic connections between the cochlear efferent neurons in guinea pig

The outer hair cells of organ of Corti are innervated by the efferent neurons of medial olivocochlear neurons (MOC) of the brainstem which modify the cochlear auditory processing and sensitivity. Most of the MOC neurons are excited by a dominant ear and only a small portion of them is excited by both ears resulting in a binaural facilitation. The functional role of the feedback system between the organ of Corti and the cochlear efferent neurons is the protection of the ear from acoustic injury. The rapid impulse propagation in the bilateral olivocochlear system is suggestive of an electrotonic interaction between the bilateral olivocochlear neurons. The morphological background of the MOC pathway is not yet completely characterized. Therefore, we have labeled the bilateral cochlear nerves with different neuronal tracers in guinea pigs. In the anesthetized animals the cochlear nerves were exposed in the basal part of the modiolus and labeled simultaneously with different retrograde fluorescent tracers. By using confocal laser scanning microscope we could detect close appositions between the dendrites of the neurons of bilateral MOC. The distance between the neighboring profiles suggested close membrane appositions without interposing glial elements. These connections might serve as one of the underlying mechanisms of the binaural facilitation mediated by the olivocochlear system.     

The superior olivary complex of the hamster has multiple periods of cholinergic neuron development

Cholinergic neurons of the superior olivary complex share a common embryological and phylogenetic origin with brainstem motor neurons and serve as the major descending efferent pathway either to the cochlea as part of the olivocochlear system or to the cochlear nucleus. In this study, we investigated the developmental expression patterns of choline acetyltransferase (ChAT) and its co-localization with calcitonin gene-related peptide within the superior olivary complex and neighboring brainstem motor nuclei. At embryonic day 12, neurons in the ventral nucleus of the trapezoid body were first to express ChAT. The temporal expression pattern of both ChAT mRNA and immunoreactivity in this periolivary region mimicked motor neurons in the facial and trigeminal motor nuclei. Just before birth, shell neurons surrounding the lateral superior olive expressed ChAT. Neither ChAT-positive periolivary neurons nor shell neurons co-expressed calcitonin gene-related peptide during development or in the adult. Immediately following birth, intrinsic neurons within the lateral superior olive expressed ChAT but not calcitonin gene-related peptide. However, a transient increase in the number of ChAT-positive neurons in the lateral superior olive coincided with the onset of the calcitonin gene-related peptide co-expression within these neurons. We conclude that ChAT expression appears first in periolivary regions containing medial olivocochlear neurons, precedes the expression of calcitonin gene-related peptide in the superior olivary complex, and is co-expressed with calcitonin gene-related peptide within the lateral superior olive containing lateral olivocochlear neurons. These data suggest that the lateral olivocochlear system co-expresses ChAT and calcitonin gene-related peptide, whereas the medial olivocochlear system does not.     

Gentamicin abolishes all cochlear effects of electrical stimulation of the inferior colliculus

Electrical stimulation of the inferior colliculus (IC) has been shown to result in suppression of cochlear output, due to activation of the medial olivocochlear system. This auditory efferent system originates in the brainstem and terminates on the outer hair cells in the cochlea. Recently, excitatory effects of IC stimulation have also been reported, both on cochlear gross potentials and on primary auditory afferents. It has been hypothesized that this excitation is due to co-activation of the lateral olivocochlear system, which synapses on the primary auditory afferent fibres contacting the inner hair cells. If stimulation of the IC leads to the activation of both the medial and lateral olivocochlear system, resulting in a mixture of inhibitory and excitatory effects in the cochlea, then removal of the inhibitory effects, by blocking the medial system, should lead to more pronounced excitatory effects out in the periphery. To investigate this hypothesis, we recorded the effect of IC stimulation on cochlear gross potentials as well as on single auditory primary afferents in guinea pigs following block of the medial olivocochlear system with gentamicin. We found that administration of gentamicin, whether intraperitoneally or by intracochlear perfusion, blocked all effects of IC stimulation, whether inhibitory or excitatory. These data strongly suggest that all effects observed after IC stimulation, both inhibitory as well as excitatory, are due to the activation of the medial olivocochlear system.     

Origin of the noradrenergic innervation of the superior olivary complex in the rat

In the rat, the superior olivary complex contains lateral and medial olivocochlear neurones, which respectively innervate two separate targets within the cochlea; the auditory afferents contacting the inner hair cells and the outer hair cells themselves. Previous double label immunohistochemical studies have shown that both lateral and medial olivocochlear neurones are contacted by noradrenergic nerve endings, and electrophysiological studies on in-vitro rat brain slices have demonstrated that noradrenaline exerts a direct, predominantly excitatory effect on medial olivocochlear neurones. In this paper, we have investigated the origin of the noradrenergic input to the superior olivary complex (SOC). A retrograde tracer, Fluorogold, was used to map the inputs to the SOC, and this was combined with immunofluorescent staining for dopamine-beta-hydroxylase (DβH) to identify which of the afferent inputs was noradrenergic. These experiments showed small numbers of neurones double-stained for both Fluorogold and DβH in the A6 cell group (the locus coeruleus). In the A7 cell group, within and medial to the lateral lemniscus, numerous Fluorogold labelled and DβH positive neurones were found, but no neurones were seen that were double-labelled. In none of the other major noradrenergic cell groups were labelled Fluorogold neurones ever detected. To confirm the results obtained by retrograde tracer injections, anterograde tracer injections with biotinylated dextran amine were made in the locus coeruleus. This resulted in labelled fibres within all subdivisions of the superior olivary complex. These experiments indicate that the noradrenergic input to the olivocochlear neurones originates solely from the locus coeruleus. The small numbers of double-labelled neurones found in the locus coeruleus indicate a very divergent non-selective noradrenergic input to the SOC.     

The presence of a calcitonin gene-related peptide in the olivocochlear bundle in rat

Summary The origins of calcitonin gene-related peptide immunoreactive (CGRPI) fibers in the cochlea were examined in rats. Parasagittal transection of the brain just medial to the principal sensory trigeminal nucleus resulted in the ipsilateral disappearance of CGRPI fibers in the cochlea, indicating that the origins of these fibers lie in the central nervous system. Next, we used a highly sensitive method combining retrograde tracing and immunohistochemistry to identify the origins of the CGRPI fibers in the cochlea. After injection of biotin-wheat germ agglutinin (b-WGA) into the cochlea, CGRPI neurons in the ipsilateral lateral superior olivary nucleus also contained b-WGA granules. These findings indicated tht CGRPI efferent fibers are major components of the olivocochlear bundle.     

Environmental enrichment to sound activates dopaminergic pathways in the auditory system

Environmental enrichment to sound stimulation, in the adult, can promote physiological changes and protection against trauma in the auditory peripheral and central nervous system. Sound enrichment, or sound conditioning is a method that utilizes a low-level, non-damaging acoustic stimulus as a protective agent. Pre-treating subjects to a moderate or low-level acoustic stimulus reduces the damaging effects of a subsequent traumatic stimulus. The intention of this review is to describe how environmental enrichment to sound affords protection against a subsequent trauma, and the role that the dopaminergic pathways in the cochlea and the auditory brainstem play in this protection. Dopamine is released from the lateral efferents and exerts a tonic inhibition of auditory nerve activity thus preserving auditory sensitivity and protecting against excitotoxicity. Sound conditioning up-regulated tyrosine hydroxylase in the lateral efferents under the inner hair cells and acoustic trauma reduced these levels. Thus, sound conditioning triggers an up-regulation of tyrosine hydroxylase both in the lateral efferent of cochlea and in the lateral superior olivary complex. These findings expand our understanding of the neurochemical balance and regulation between the lateral olivocochlear neurons and the lateral efferent terminals in the cochlea during sound stimulation.     

The lobulus petrosus of the paraflocculus relays cortical visual inputs to the posterior interposed and lateral cerebellar nuclei: an anterograde and retrograde tracing study in the monkey

The afferent and efferent projections of the lobulus petrosus (LP) of the paraflocculus were examined by retrograde tracing with cholera toxin subunit B (CTB) and anterograde tracing with CTB or biotinylated dextran (BD) in the macaque. Following injections of the tracers into the LP, labeled neurons were seen mainly in the lateral, dorsal, paramedian and dorsolateral parts of the contralateral pontine nuclei (Pn) and in the ventromedial part of the principal olivary nucleus and the lateral pole of the medial accessory olivary nucleus at the rostral levels. Labeled terminals were seen ipsilaterally in the ventrolateral part of the posterior interposed nucleus and the basal interstitial nucleus of the cerebellum at their rostral levels, and in the ventromedial part of the lateral cerebellar nucleus. To reveal the connections between the LP-projecting pontine neurons and the corticopontine axons, BD was injected into the left prearcuate region or extrastriate visual areas, and CTB into the right LP. Labeled terminals from the prearcuate region were seen mainly in the left dorsal, medial and paramedian Pn, whereas those from the extrastriate visual areas were seen in the lateral and dorsolateral Pn. The distribution of the LP-projecting neurons overlapped that of the terminals from the extrastriate visual areas but did not overlap that of the terminals from the prearcuate region. The present study demonstrates that the LP of the monkey receives cortical visual inputs and projects to the posterior interposed and lateral cerebellar nuclei, suggesting that the LP may be involved in the control of voluntary eye movements. Electronic Publication     

Brainstem organization of efferent projections to the guinea pig cochlea studied using the fluorescent tracers fast blue and diamidino yellow

Summary Intracochlear injection of the fluorescent retrograde neuronal tracers fast blue and diamidino yellow was used to investigate the distribution within the brainstem of neurones projecting to the cochlea in the guinea pig. The overall pattern of distribution of cells within the brainstem auditory nuclei was the same for both tracers and was also in broad agreement with recent studies in this species using horse-radish peroxidase as the neuronal tracer. However, the total number of neurones found (mean of 1234 projecting to each cochlea) was significantly greater than that reported using horseradish peroxidase, largely as a result of more small labelled neurones being detected within the lateral superior olivary nucleus ipsilateral to the injected cochlea. The yield of labelled cells was greatest in animals in which care was taken to perfuse the whole length of the cochlear epithelium. After bilateral injections of both tracers, double-labelled cells were found in small numbers within all the large neurone medial system nuclei and the ventral nucleus of the lateral lemniscus. It was concluded that between 1–5% of the medial system neurones project to both cochleas.     

Enkephalin mRNA production by cochlear and vestibular efferent neurons in the gerbil brainstem

Summary Preproenkephalin mRNA production by efferent neurons projecting to the gerbil inner ear was assessed using combined in situ hybridization and retrograde labeling with florescent tracers. Virtually all vestibular efferent neurons were positive for preproenkephalin mRNA. Of the cochlear efferents, one-half of the medial olivocochlear neurons were positive for enkephalin. All lateral olivocochlear neurons were negative for enkephalin. The results suggest that there are two, biochemically distinct subpopulations of medial olivocochlear efferents in the gerbil. Offprint requests to: Division of Otolaryngology, ENT, V-112C     

Diversity of axonal ramifications belonging to single lateral and medial olivocochlear neurons

A classification of olivocochlear (OC) neurons based on their location in either the lateral or medial region of the superior olivary complex provides a powerful tool for predicting their terminations beneath the inner (IHC) or outer hair cells (OHC) of the cochlea, respectively. Yet the morphology of axonal terminations belonging to single lateral OC (LOC) and medial OC (MOC) neurons, which can provide clues about the functional capabilities of individual efferent neurons, has been relatively unexplored. Following injections of biotinylated dextran amine into regions containing OC neurons in the rat, we reconstructed 19 LOC and 15 MOC axons in surface preparations of the cochlea. Confirming previous studies, LOC axons could be classified as either intrinsic or shell based on the length (short versus long) of their terminal ramifications beneath the IHC. However, intrinsic LOC axons were of two types, those that traveled to the organ of Corti without branching (simple intrinsic) and those that had three or more branches that converged on the same discrete patch of IHCs (converging intrinsic). Regarding shell neurons, we found that they may have as many as four intraganglionic branches that could innervate as much as 41% of cochlear length. Lastly, we found that MOC neurons were extremely diverse, not only in the number of their tunnel-crossing fibers (1-15), but also in both the number of boutons they formed (1-48) and in their basal-apical spans (1-45%). Analysis revealed that the number of tunnel-crossing fibers formed by a given axon was closely related to the total number of its terminal boutons, but not to its cochlear span. Analysis further suggested the existence of two distinct subtypes of MOC neurons on the basis of the number of tunnel-crossing fibers and boutons each possessed: the more common sparsely-branched and the quite rare highly-branched MOC neurons. In conclusion, the variety of axonal ramifications of individual LOC and MOC neurons has functional implications and raises the question of whether the various types of efferent neurons might be subject to selective control by ascending and descending central auditory and possibly non-auditory pathways.     

Catecholaminergic innervation of guinea pig superior olivary complex

In mammals, olivocochlear neurons in the superior olivary complex project to the cochlea, providing input to outer hair cells and auditory afferents contacting inner hair cells. In the rat it has been demonstrated that olivocochlear neurons receive noradrenergic input, arising from the locus coeruleus and it has been demonstrated in this species using in vitro brain slices that noradrenaline exerts a direct, mostly excitatory effect on an olivocochlear subpopulation. The guinea pig is a more commonly used animal in auditory physiology than the rat and anatomical data on noradrenaline in the auditory brainstem in this species are lacking. Because it has been shown that a compact locus coeruleus is not present in the guinea pig, subtle species differences might be expected. Therefore, using immunohistochemical and tracing techniques we have investigated in the guinea pig (1) the noradrenergic and dopaminergic innervation of the superior olivary complex, (2) the anatomical relationship between noradrenergic fibres and olivocochlear neurons and (3) the origin of the noradrenergic input to this brainstem region. The results show that the guinea pig superior olivary complex receives moderately dense noradrenergic innervation and no dopaminergic innervation. In addition, noradrenergic fibres and varicosities were observed in close contact with both somata and dendrites of olivocochlear neurons, strongly suggestive of synaptic contacts. Finally the results show that a significant component of the noradrenergic innervation of the guinea pig superior olivary complex arises in the locus subcoeruleus, which is a structure likely to be the homologue of the locus coeruleus in rats and other species.     

Neurochemistry of Olivocochlear Neurons in the Hamster

The present study was conducted to characterize the superior olivary complex (SOC) of the lower brain stem in the pigmented Djungarian hamster Phodopus sungorus. Using Nissl‐stained serial cryostat sections from fresh‐frozen brains, we determined the borders of the SOC nuclei. We also identified olivocochlear (OC) neurons by retrograde neuronal tracing upon injection of Fluoro‐Gold into the scala tympani. To evaluate the SOC as a putative source of neuronal nitric oxide synthase (nNOS), arginine‐vasopressin (AVP), oxytocin (OT), vasoactive intestinal polypeptide (VIP), or pituitary adenylate cyclase‐activating polypeptide (PACAP) that were all found in the cochlea, we conducted immunohistochemistry on sections exhibiting retrogradely labeled neurons. We did not observe AVP‐, OT‐, or VIP‐immunoreactivity, neither in OC neurons nor in the SOC at all, revealing that cochlear AVP, OT, and VIP are of nonolivary origin. However, we found nNOS, the enzyme responsible for nitric oxide synthesis in neurons, and PACAP in neuronal perikarya of the SOC. Retrogradely labeled neurons of the lateral olivocochlear (LOC) system in the lateral superior olive did not contain PACAP and were only infrequently nNOS‐immunoreactive. In contrast, some shell neurons and some of the medial OC (MOC) system exhibited immunofluorescence for either substance. Our data obtained from the dwarf hamster Phodopus sungorus confirm previous observations that a part of the LOC system is nitrergic. They further demonstrate that the medial olivocochlear system is partly nitrergic and use PACAP as neurotransmitter or modulator. Anat Rec, 292:461–471, 2009. © 2009 Wiley‐Liss, Inc.     

Muscarinic receptor subtypes are differentially distributed in the rat cochlea

Five different genes encode the muscarinic acetylcholine receptors. The muscarinic receptor subtypes M1, M3, and M5 are typically coupled to activation of the Galpha(q/11)-phosphatidyl inositol pathway, whereas the M2 and M4 subtypes are typically linked to Galpha(i) and adenylyl cyclase inhibition. In order to localize muscarinic receptors in the rat cochlea, we applied polyclonal antibodies for subtypes M1, M2, M3, and M5, and monoclonal antibody for subtype M4 to paraffin sections. In the organ of Corti, outer hair cells exhibited strong immunoreactivity for M3 and weak immunoreactivity for M1. Deiters' cells were strongly immunoreactive to antibodies for the M1 and M2 subtypes, with weak staining observed for M3, and weaker yet for M5. Inner hair cells showed moderate immunoreactivity for the M1 subtype, weaker staining for the M5 subtype, and slight staining for the M3 subtype. Among the spiral ganglion neurons, weak to moderate immunoreactivity was detected for M3 and M5 subtypes and weak staining was observed for the M1 subtype. The efferent fibers of the intraganglionic spiral bundle were positive for M2 and M5. In the lateral wall, weak to moderate staining was detected for M5 in the stria vascularis corresponding in position to the basolateral extensions of marginal cells. Staining for M3 was observed associated with capillaries. Fibrocytes of the spiral ligament exhibited limited but selective subtype immunoreactivity. No immunoreactivity was detected in the cochlea for the M4 subtype.From the present findings we suggest that M3 is the primary muscarinic receptor subtype in outer hair cells mediating a postsynaptic response to the medial olivocochlear cholinergic efferent input. The muscarinic receptor subtypes M1, M3, and M5 appear to subserve the action of cholinergic lateral olivocochlear efferent stimulation on postsynaptic responses in type I afferents. Whether M1, M3, and M5 protein in inner hair cells indicates constitutive or vestigial expression remaining from development is unknown. M2 and M5 muscarinic receptors expressed presynaptically may modulate the efferent signal. Finally, expression by Deiters' cells of several muscarinic subtypes raises the possibility that cholinergic efferents couple to these non-sensory cells through muscarinic receptors.     

Postnatal calcitonin gene-related peptide in the superior olivary complex

The purpose of this study was to investigate quantitatively the distribution of calcitonin gene-related peptide (CGRP)-positive neurons within the superior olivary complex (SOC) at various postnatal ages. In the lateral superior olive (LSO), most if not all, CGRP-positive cells correspond to the cholingeric portion of the lateral olivocochlear system which innervates the cochlea. Brains from 1-day old (P1) through juvenile (P30) hamsters were used. At all ages, CGRP-positive neurons were seen throughout the various nuclei of the SOC. There was a dramatic shift in the distribution of CGRP-positive neurons from being predominantly periolivary to being predominantly confined within the LSO. The number of CGRP-positive neurons clearly increased as a function of increasing age. At the earliest postnatal ages, the LSO contained few if any immunostained cells, whereas at later ages the LSO contained the majority (greater than 70%) of the immunostained cells. Assuming that these CGRP-positive cells within the LSO correspond to olivocochlear neurons, these data suggest that in hamsters the lateral olivocochlear system may be immature at birth up until the second postnatal week.     

Hypothalamic and zona incerta neurons expressing hypocretin, but not melanin concentrating hormone, project to the hamster intergeniculate leaflet

The hypocretins (Hcrt; also known as orexins) and melanin-concentrating hormone comprise distinct families of neuropeptides synthesized in cells located in the lateral hypothalamus and adjacent areas. The Hcrts are thought to modulate food intake and sleep/wake patterns in mammals. Melanin-concentrating hormone has a well-documented role in energy metabolism. A moderate plexus of Hcrt immunoreactive terminals has been described in the hamster intergeniculate leaflet, part of the circadian rhythm system. This study investigated the origin of Hcrt-immunoreactive terminals in the intergeniculate leaflet and determined whether melanin-concentrating hormone neurons also project to the intergeniculate leaflet. The tracer, cholera toxin beta-subunit, was injected into the intergeniculate leaflet of the golden hamster. Double-label fluorescent immunohistochemistry for cholera toxin beta-subunit and Hcrt or melanin-concentrating hormone was then performed to identify retrogradely labeled cells also containing immunoreactive peptide. Most cholera toxin beta-subunit-labeled cells were detected in the medial zona incerta and sub-incertal zone, with few observed in the lateral hypothalamus. Hcrt-immunoreactive cells were abundant and found largely in the lateral hypothalamus and adjacent nuclei. Melanin-concentrating hormone cells were also abundant in the medial zona incerta, in close proximity to cholera toxin beta-subunit-labeled cells, but ventral to them. Cells containing both cholera toxin beta-subunit and Hcrt-immunoreactive, were present in the dorsal aspect of the lateral hypothalamus. The number observed was small, < or = 1% of the total number of Hcrt cells counted in the hamster. No cholera toxin beta-subunit-immunoreactive cells also contained melanin-concentrating hormone and no melanin-concentrating hormone-immunoreactive processes were evident in the intergeniculate leaflet. The results show that a small number of lateral hypothalamus cells containing Hcrt-immunoreactivity project to the intergeniculate leaflet, but they are scattered rather than collected into a discrete group. At the present time there is no information regarding the function of these cells, although they may contribute to the regulation of sleep/arousal, circadian rhythmicity, or vestibulo-oculomotor function.     

Mossy and climbing fiber collateral inputs in monkey cerebellar paraflocculus lobulus petrosus and hemispheric lobule VII and their relevance to oculomotor functions

Recent lesion studies on monkeys suggest that the cerebellar lobulus petrosus of the paraflocculus (LP) and crura I and II of hemispheric lobule VII (H-7) are involved in smooth pursuit eye movement control. To reveal the relationship between the LP and H-7, we studied mossy and climbing fiber collateral inputs to these areas in four cynamolgus monkeys. After unilateral injections of retrograde tracers into the LP, labeled mossy fibers were seen ipsilaterally in the crura I and II of H-7. A very small number of labeled mossy fiber collaterals were also seen in the dorsal paraflocculus (DP). Labeled climbing fibers were seen exclusively in the ipsilateral crus I. No labeled mossy/climbing fibers were seen in the flocculus, ventral paraflocculus and other cortical areas. Combined injections of fast blue in the LP and cholera toxin subunit B in the posterior crus I and crus II of H-7 resulted in a small number of the double-labeled pontine and principal olivary neurons. Combined injections in the LP and DP induced only a few double-labeled neurons in the pontine nuclei, and no double-labeled neurons in the olivary nuclei. These results suggest that the LP and crura I and II of H-7 may share some of their mossy and climbing fiber inputs and mediate similar functional roles involving smooth pursuit eye movement control.     

Distribution of cochlear efferents and olivo-collicular neurons in the brainstem of rat and guinea pig

Summary In rat and guinea pig, cochlear efferents to the two ears were labeled simultaneously with different fluorescent tracers. It was found that in both species only few (1–3%) olivo-cochlear neurons were double-labeled and project to both cochleae. In most periolivary regions large olivocochlear neurons (OCN) projecting to the ipsilateral and contralateral side are intermingled and form a continuous cell column between the facial nucleus and lateral lemniscus. In a second series of experiments in rat, cochlear efferents and ascending olivo-collicular neurons were labeled. Olivo-cochlear and olivo-collicular neurons are intermingled in the lateral superior olive (LSO) and in the ventromedial periolivary region. No double-labeled neurons were found that project to the cochlea and the inferior colliculus.     

Localization of dopamine receptor subtypes in the rat spiral ganglion

Although dopaminergic neurons are thought to exist in the lateral olivocochlear efferent system and modulate the afferent nerve activity, the distribution of dopamine (DA) receptor subtypes is still obscure. In the present study, we investigated the localization of five subtypes of DA receptor (D1-5) by immunocytochemical analysis and the gene expression of D1-5 using RT-PCR procedure in the rat cochlea. Most, but not all, spiral ganglion neurons were immunolabeled with all the anti-DA receptor subunit antibodies and faint punctuate immunoreactivities were observed in inner hair cell regions. Gene expression for all receptors was detected. These results suggest that all DA receptor subtypes are present in spiral ganglion cells, and potentially regulate afferent neurotransmission.