Functional P2X7 Receptors in the Auditory Nerve of Hearing Rodents Localize Exclusively to Peripheral Glia.

P2X7 receptors (P2X7Rs) are associated with numerous pathophysiological mechanisms, and this promotes them as therapeutic targets for certain neurodegenerative conditions. However, the identity of P2X7R-expressing cells in the nervous system remains contentious. Here, we examined P2X7R functionality in auditory nerve cells from rodents of either sex, and determined their functional and anatomic expression pattern. In whole-cell recordings from rat spiral ganglion cultures, the purinergic agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) activated desensitizing currents in spiral ganglion neurons (SGNs) but non-desensitizing currents in glia that were blocked by P2X7R-specific antagonists. In imaging experiments, BzATP gated sustained Ca2+ entry into glial cells. BzATP-gated uptake of the fluorescent dye YO-PRO-1 was reduced and slowed by P2X7R-specific antagonists. In rats, P2X7Rs were immuno-localized predominantly within satellite glial cells (SGCs) and Schwann cells (SCs). P2X7R expression was not detected in the portion of the auditory nerve within the central nervous system. Mouse models allowed further exploration of the distribution of cochlear P2X7Rs. In GENSAT reporter mice, EGFP expression driven via the P2rx7 promoter was evident in SGCs and SCs but was undetectable in SGNs. A second transgenic model showed a comparable cellular distribution of EGFP-tagged P2X7Rs. In wild-type mice the discrete glial expression was confirmed using a P2X7-specific nanobody construct. Our study shows that P2X7Rs are expressed by peripheral glial cells, rather than by afferent neurons. Description of functional signatures and cellular distributions of these enigmatic proteins in the peripheral nervous system (PNS) will help our understanding of ATP-dependent effects contributing to hearing loss and other sensory neuropathies.SIGNIFICANCE STATEMENT P2X7 receptors (P2X7Rs) have been the subject of much scrutiny in recent years. They have been promoted as therapeutic targets in a number of diseases of the nervous system, yet the specific cellular location of these receptors remains the subject of intense debate. In the auditory nerve, connecting the inner ear to the brainstem, we show these multimodal ATP-gated channels localize exclusively to peripheral glial cells rather than the sensory neurons, and are not evident in central glia. Physiologic responses in the peripheral glia display classical hallmarks of P2X7R activation, including the formation of ion-permeable and also macromolecule-permeable pores. These qualities suggest these proteins could contribute to glial-mediated inflammatory processes in the auditory periphery under pathologic disease states.

Dynamics of the fragile X mental retardation protein correlates with cellular and synaptic properties in primary auditory neurons following afferent deprivation.

Afferent activity dynamically regulates neuronal properties and connectivity in the central nervous system. The Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates cellular and synaptic properties in an activity-dependent manner. Whether and how FMRP level and localization are regulated by afferent input remains sparsely examined and how such regulation is associated with neuronal response to changes in sensory input is unknown. We characterized changes in FMRP level and localization in the chicken nucleus magnocellularis (NM), a primary cochlear nucleus, following afferent deprivation by unilateral cochlea removal. We observed rapid (within 2 hr) aggregation of FMRP immunoreactivity into large granular structures in a subset of deafferented NM neurons. Neurons that exhibited persistent FMRP aggregation at 12-24 hr eventually lost cytoplasmic Nissl substance, indicating cell death. A week later, FMRP expression in surviving neurons regained its homeostasis, with a slightly reduced immunostaining intensity and enhanced heterogeneity. Correlation analyses under the homeostatic status (7-14 days) revealed that neurons expressing relatively more FMRP had a higher capability of maintaining cell body size and ribosomal activity, as well as a better ability to detach inactive presynaptic terminals. Additionally, the intensity of an inhibitory postsynaptic protein, gephyrin, was reduced following deafferentation and was positively correlated with FMRP intensity, implicating an involvement of FMRP in synaptic dynamics in response to reduced afferent inputs. Collectively, this study demonstrates that afferent input regulates FMRP expression and localization in ways associated with multiple types of neuronal responses and synaptic rearrangements.

Combined optogenetic and electrical stimulation of auditory neurons increases effective stimulation frequency-an in vitro study.

OBJECTIVE: The performance of neuroprostheses, including cochlear and retinal implants, is currently constrained by the spatial resolution of electrical stimulation. Optogenetics has improved the spatial control of neurons in vivo but lacks the fast-temporal dynamics required for auditory and retinal signalling. The objective of this study is to demonstrate that combining optical and electrical stimulation in vitro could address some of the limitations associated with each of the stimulus modes when used independently. APPROACH: The response of murine auditory neurons expressing ChR2-H134 to combined optical and electrical stimulation was characterised using whole cell patch clamp electrophysiology. MAIN RESULTS: Optogenetic costimulation produces a three-fold increase in peak firing rate compared to optical stimulation alone and allows spikes to be evoked by combined subthreshold optical and electrical inputs. Subthreshold optical depolarisation also facilitated spiking in auditory neurons for periods of up to 30 ms without evidence of wide-scale Na+ inactivation. SIGNIFICANCE: These findings may contribute to the development of spatially and temporally selective optogenetic-based neuroprosthetics and complement recent developments in 'fast opsins'.

Noise Stress Induces an Epidermal Growth Factor Receptor/Xeroderma Pigmentosum-A Response in the Auditory Nerve.

In response to toxic stressors, cancer cells defend themselves by mobilizing one or more epidermal growth factor receptor (EGFR) cascades that employ xeroderma pigmentosum-A (XPA) to repair damaged genes. Recent experiments discovered that neurons within the auditory nerve exhibit basal levels of EGFR+XPA co-expression. This finding implied that auditory neurons in particular or neurons in general have the capacity to mobilize an EGFR+XPA defense. Therefore, the current study tested the hypothesis that noise stress would alter the expression pattern of EGFR/XPA within the auditory nerve. Design-based stereology was used to quantify the proportion of neurons that expressed EGFR, XPA, and EGFR+XPA with and without noise stress. The results revealed an intricate neuronal response that is suggestive of alterations to both co-expression and individual expression of EGFR and XPA. In both the apical and middle cochlear coils, the noise stress depleted EGFR+XPA expression. Furthermore, there was a reduction in the proportion of neurons that expressed XPA-alone in the middle coils. However, the noise stress caused a significant increase in the proportion of neurons that expressed EGFR-alone in the middle coils. The basal cochlear coils failed to mobilize a significant response to the noise stress. These results suggest that EGFR and XPA might be part of the molecular defense repertoire of the auditory nerve.

Cochlear neuropathy in human presbycusis: Confocal analysis of hidden hearing loss in post-mortem tissue.

Recent animal work has suggested that cochlear synapses are more vulnerable than hair cells in both noise-induced and age-related hearing loss. This synaptopathy is invisible in conventional histopathological analysis, because cochlear nerve cell bodies in the spiral ganglion survive for years, and synaptic analysis requires special immunostaining or serial-section electron microscopy. Here, we show that the same quadruple-immunostaining protocols that allow synaptic counts, hair cell counts, neuronal counts and differentiation of afferent and efferent fibers in mouse can be applied to human temporal bones, when harvested within 9 h post-mortem and prepared as dissected whole mounts of the sensory epithelium and osseous spiral lamina. Quantitative analysis of five "normal" ears, aged 54-89 yrs, without any history of otologic disease, suggests that cochlear synaptopathy and the degeneration of cochlear nerve peripheral axons, despite a near-normal hair cell population, may be an important component of human presbycusis. Although primary cochlear nerve degeneration is not expected to affect audiometric thresholds, it may be key to problems with hearing in noise that are characteristic of declining hearing abilities in the aging ear.

Survival, migration and differentiation of mouse tau-GFP embryonic stem cells transplanted into the rat auditory nerve.

Stem cells have been investigated as treatment for a variety of diagnoses such as Parkinson's disease, Alzheimer's disease and spinal cord injuries. Here, we investigated the possibility of using stem cells as a replacement therapy for lesions of the auditory nerve (AN). We transplanted tau-GFP mouse embryonic stem cells into the AN either by the internal auditory meatus or via the modiolus in rats that had been previously deafened by application of ß-bungarotoxin to the round window niche. We investigated the effect of brain derived neurotrophic factor (BDNF) on cell transplant survival and differentiation. Additionally chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core chondroitin sulfate proteoglycans, was used in order to promote possible migration of cells and axons through the transitional zone. A bioactive isoleucine-lysine-valine-alanine-valine (IKVAV) peptide amphiphile (PA) nanofiber gel was applied around the cell injection site. This nanofiber gel has been shown to promote neural differentiation and other similar gels have been used to encapsulate and release proteins. Three weeks after injection, transplanted cells were found in the scala tympani, the modiolus, the AN trunk and the brain stem. As compared to cell transplantation and gel only, BDNF content in the PA gel increased cell survival and neuronal differentiation. In the animals treated with ChABC we observed extensive migration of cells through the transitional zone to or from the CNS.

Donut-like topology of synaptic vesicles with a central cluster of mitochondria wrapped into membrane protrusions: a novel structure-function module of the adult calyx of Held.

Structural and functional properties of synapses are intricately and reciprocally coupled. To cope with the functional requirements in auditory processing, the calyx of Held developed distinct structural specializations such as a large number of active zones, large size, elaborate morphology, and defined distribution of ion channels. These specializations typically appear during postnatal maturation within the first 3 weeks of life and are accompanied by marked changes in the properties of synaptic transmission. We examined the arrangement of synaptic vesicles at different postnatal stages of maturation by genetically labeling vesicles with the fluorescent fusion protein synaptophysin-enhanced green fluorescent protein. Fluorescence and electron microscopy-based analyses revealed a new anatomical specialization in the mature calyx of Held. Within small, membrane-delimited compartments (swellings), synaptic vesicles formed donut-like assemblies around a central cluster of interconnected mitochondria. Adult calyces contained approximately 100 such structural units, each of them consisting of approximately 800 synaptic vesicles, six to nine mitochondria, and five to nine active zones. A donut of synaptic vesicles measured approximately 1 microm in diameter and was placed in a swelling with a volume of approximately 5 fl. Conspicuously, this structural specialization appears with the onset of hearing and may contribute to maturational changes in presynaptic function.

Ephrin A2 may play a role in axon guidance during hair cell regeneration.

OBJECTIVE: Hair cell regeneration in the avian cochlea is accompanied by frequency specific reinnervation and recovery of physiologic function. The molecular cues that guide ganglion cells to tonotopically appropriate new hair cells have not been identified. We investigated the potential of ephrin A2 in this process. STUDY DESIGN: Ephrin A2 expression was characterized in acoustic ganglion cells of normal and gentamicin-treated early post hatch chicks. METHODS: Ephrin A2 expression was determined by Western analysis of total protein isolated from acoustic ganglia in normal animals. Protein localization was characterized by fluorescence immunohistochemistry in sections of acoustic ganglia of normal and gentamicin treated animals. Patterns of ephrin A2 expression in acoustic ganglia were determined and quantified during hair cell regeneration. RESULTS: Ephrin A2 expression was found in acoustic ganglia by Western analysis. Localization of this protein by immunofluorescence revealed its presence in acoustic ganglion cells in normal chicks. After gentamicin treatment, ephrin A2 expression was lost in a subset of acoustic ganglion cells. The spatial and temporal pattern of ephrin A2 loss coincides with the pattern of hair cell loss and regeneration. CONCLUSIONS: The changes in ephrin A2 immunoreactivity in acoustic ganglion cells during cochlear damage and regeneration suggests that ephrin A2 may be involved in the guidance of ganglion cells to tonotopically appropriate hair cell targets during regeneration. Ephrin A2 in hair cell regeneration.

Pathways controlling trigeminal and auditory nerve-evoked abducens eyeblink reflexes in pond turtles.

An in vitro brain stem preparation from turtles exhibits a neural correlate of eyeblink classical conditioning during pairing of auditory (CS) and trigeminal (US) nerve stimulation while recording from the abducens nerve. The premotor neuronal circuits controlling abducens nerve-mediated eyeblinks in turtles have not been previously described, which is a necessary step for understanding cellular mechanisms of conditioning in this preparation. The purpose of the present study was to neuroanatomically define the premotor pathways that underlie the trigeminal and auditory nerve-evoked abducens eyeblink responses. The results show that the principal sensory trigeminal nucleus forms a disynaptic pathway from both the trigeminal and auditory nerves to the principal and accessory abducens motor nuclei. Additionally, the principal abducens nucleus receives vestibular inputs, whereas the accessory nucleus receives input from the cochlear nucleus. The late R2-like component of abducens nerve responses is mediated by the spinal trigeminal nucleus in the medulla. Both the principal sensory trigeminal nucleus and the abducens motor nuclei receive CS-US convergence and therefore both, or either, might be considered potential sites of synapse modification during in vitro abducens conditioning. Further data are required to determine the role of the principal sensory trigeminal nucleus in in vitro conditioning.

Distribution of anterogradely labeled trigeminal and auditory nerve boutons on abducens motor neurons in turtles: implications for in vitro classical conditioning.

A conditioned abducens nerve response is generated in in vitro brainstem preparations from turtles by pairing a weak conditioned stimulus (CS) applied to the auditory nerve that immediately precedes an unconditioned stimulus (US) applied to the trigeminal nerve. Tract-tracing studies showed direct projections from auditory and trigeminal nerves to abducens motor neurons. In light of these findings for convergent CS-US inputs, it is hypothesized that auditory and trigeminal nerve synaptic inputs onto abducens motor neurons are in spatial proximity because the CS is a weak input that may be required to be near the US inputs to have an associative effect, and conditioning occurs only when the CS and US are temporally separated by less than 100 ms. This study examined the spatial relationship of 133 anterogradely labeled synaptic boutons conveying CS or US information on retrogradely labeled abducens motor neurons. The results show that trigeminal and auditory nerve terminal fields occupy primarily the soma and proximal dendrites of abducens motor neurons. Quantitative analysis shows that the majority of labeled boutons (76% and 85% from injections of the trigeminal and auditory nerves, respectively) were apposed to somata or were localized to dendritic segments no more than about 30 microm from the nucleus. There were no quantitative differences between trigeminal and auditory nerve boutons in terms of their localization on dendrites or bouton diameter. Finally, triple labeling experiments demonstrated that individual abducens motor neurons receive inputs from both nerves and that these inputs may be in close spatial proximity to one another. This synaptic arrangement allows for the possibility that in vitro abducens conditioning is generated by coincident CS-US detection mediated by NMDA receptors and may utilize a Hebbian-like plasticity mechanism.

Olivocochlear innervation in the mouse: immunocytochemical maps, crossed versus uncrossed contributions, and transmitter colocalization.

To further understand the roles and origins of gamma-aminobutyric acid (GABA) and calcitonin gene-related peptide (CGRP) in the efferent innervation of the cochlea, we first produced in the mouse an immunocytochemical map of the efferent terminals that contain acetylcholine (ACh), CGRP, and GABA. Olivocochlear (OC) terminals in inner and outer hair cell (IHC and OHC) regions were analyzed quantitatively along the cochlear spiral via light-microscopic observation of cochlear wholemounts immunostained with antibodies to glutamic acid decarboxylase (GAD), vesicular acetylcholine transporter (VAT), or the peptide CGRP. Further immunochemical characterization was performed in mice with chronic OC transection at the floor of the fourth ventricle to distinguish crossed from uncrossed contributions and, indirectly, the contributions of lateral versus medial components of the OC system. The results in mouse showed that (1) there are prominent GABAergic, cholinergic, and CGRPergic innervations in the OHC and IHC regions, (2) GABA and CGRP are extensively colocalized with ACh in all OC terminals in the IHC and OHC areas, (3) the longitudinal gradient of OC innervation peaks roughly at the 10-kHz region in the OHC area and is more uniform along the cochlear spiral in the IHC area, (4) in contrast to other mammalian species there is no radial gradient of OC innervation of the OHCs, and (5) all OHC efferent terminals arise from the medial OC system and terminals in the IHC area arise from the lateral OC system.

Synaptophysin in the cochlear nucleus following acoustic trauma.

Chinchillas are notable for a low-frequency hearing range similar to that of humans and a marked sensitivity to loud noise. A single noise exposure that produces cochlear damage may lead to progressive loss of synaptic endings in the cochlear nucleus, followed by new axonal growth. As an index of synaptic regulation during such changes, we have examined the expression of a synaptic vesicle protein, synaptophysin, in the cochlear nucleus following a damaging acoustic stimulus in adult chinchillas. With one ear protected by a plug, following a 3-h exposure to an octave-band noise of 108 dB sound pressure level, centered at 4 kHz, the unprotected cochlea and the cochlear nuclei exhibited degeneration of hair cells and axons over periods of 7, 14, 30, 90, and 150 days. Axonal degeneration, as revealed by a silver degeneration method, was heavy ipsilateral to the cochlear damage, but sparse degeneration also appeared on the contralateral, unexposed side. Synaptophysin immunostaining underwent a major, bilateral decline in the anteroventral and posteroventral cochlear nuclei, interrupted at intervening periods by transient increases in the numbers of stained structures. A distinction in staining between large perisomatic structures and smaller puncta in the neuropil and between the dorsal and the ventral zones of the ventral cochlear nuclei revealed some variations in the response and degree of recovery of synaptophysin staining. These findings could best be explained by degeneration of synaptic endings followed by new growth of terminals and by regulatory changes in the levels of synaptophysin expression and synaptic vesicle accumulation over time.

Central and peripheral myelin in the rat cochlear and vestibular nerves.

In the bipolar neurons of vertebrate cochlear and vestibular nerves, the myelin envelopes without interruption the axon, the perikaryon and the dendrite. The perikaryal myelin is thin and partially loose, whereas axon and dendrite are enveloped by compacted myelin. The expression of protein 0 and myelin basic protein, constituents of peripheral and central myelin respectively, has been investigated in the rat by immunohistochemical study at the light microscopic level. Our data indicate that both in the cochlear and vestibular nerves the myelin of the perikaryon and dendrite is composed by specific peripheral myelin proteins. The axon segment between the perikaryon and the transitional zone expresses peripheral myelin proteins in the cochlear nerve, while both types of myelin proteins are present in the vestibular nerve. Between the transitional zone and the brainstem the myelin of the axon is exclusively of the central type. The peripheral-central myelin transitional zone is in close proximity to the axonal pole in the vestibular ganglion cells, while in the cochlear nerve it is near the spiral foramina, at variable distance from the axonal pole of ganglion cells.

Expression of Na, K-ATPase alpha and beta isoforms in the neonatal rat cochlea.

The postnatal expression of five Na, K-ATPase alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) subunit isoforms in the rat cochlea was investigated by immunocytochemistry. High levels of expression of the alpha 1 and beta 2 isoforms were observed in stria vascularis (SV) at all developmental stages. alpha 1 and beta 1 isoforms showed a distinct time-dependent developmental expression pattern in tissues of the spiral ligament (SL) and spiral limbus (SLi). Limited, temporary expression of alpha 2 and alpha 3 subunit isoforms were found in SV and SL. Expression of each isoform was also seen in organ of Corti (OC), spiral ganglion (SG), cochlear nerve (CN) and Kölliker's Organ (KO). These observations suggest that individual isoforms may exert specific actions postnatally during final cochlear maturation.

Cochlear polyamines: markers of otitis media-induced cochlear damage.

High-performance liquid chromatography (HPLC) set to the femtomole [corrected] sensitivity level was used to identify and quantify the polyamines spermidine and spermine as well as the diamine putrescine in the different tissues of the inner ears of guinea pigs with experimentally induced otitis media. The tissues examined were the lateral wall (stria vascularis and the spiral ligament), the organ of Corti, and the cochlear nerve. The difference in polyamine profile in the different tissues of the control noninfected guinea pigs suggests a relation to the particular function of each of these tissues [see erratum notice re: preceding sentence]. The difference in polyamine profile in infected different inner ear tissues compared to controls encourages the assumption that the polyamines may be involved in a repair process of the inner ear after injury and that they may be considered as biochemical markers for inner ear damage secondary to acute otitis media.

Post-embedding tem signal-to-noise ratio of S-100.

We assessed the reactivity of purified S-100 antiserum in immuno-electron microscopy by counting the number of gold particles per microns 2 over inner ear tissues embedded in different media. Sections containing predominantly Schwann's cell cytoplasm and nucleus, afferent fiber axoplasm and myelin sheath of chick cochleae were reacted with anti-S-100 IgG, an antibody to a calcium binding protein of neuronal tissues, then labeled with anti-IgG-gold conjugate. This investigation was conducted because previously published procedures, unmodified, did not yield acceptable results. Preparation of all specimens was identical. Only the medium (PolyBed 812, Araldite or Spurr epoxies; and LR White, LR Gold or Lowicryl plastics) was changed. The medium was made the changing variable because antigens available in post-embedding immuno-electron microscopy are decreased by heat, either used and/or released during polymerization of the embedding medium. The results indicate that: (a) none of the embedding media above provided optimal signal-to-noise ratio for all parts of the nerve stained in the same section; (b) aggregation of gold particles over cells was highest in embedding media with high background labeling over areas devoid of tissue (noise); (c) aggregation occurred randomly throughout both cellular and acellular regions; and (d) particles aggregated less and were distributed more evenly in tissues from media yielding good ultrastructural integrity.

Transient appearance of calbindin-D28k-positive neurons in the superior olivary complex of developing rats.

Calbindin-D28k (CaBP) is a calcium-binding protein that is prominent in various parts of the mammalian auditory system. In order to shed some light on the possible role of CaBP during ontogeny, when calcium ions play key roles in several processes, the location of CaBP was examined immunocytochemically in the auditory system of the developing rat. This study focuses on the principal nuclei of the superior olivary complex, which show distinct CaBP labeling in the adult. Consistent with previous reports in the rat and other mammals, CaBP immunoreactivity in adults was intense in somata of the medial nucleus of the trapezoid body (MNTB) and in the neuropil (presumably in axons) of the lateral superior olive (LSO), the superior paraolivary nucleus (SPN), and the medial superior olive (MSO). In fetal and neonatal animals, however, the labeling pattern was strikingly different. Around birth, MNTB neurons are immunonegative for CaBP, whereas somata and processes in the LSO, probably neuronal, are heavily labeled at that age. This labeling pattern persists throughout the first week of postnatal life and begins to change at P8, when MNTB neurons become immunopositive for CaBP. During the next 10 days labeling intensity in MNTB neurons increases considerably, and the increase is paralleled by an increase in labeling intensity of the neuropil in the LSO, SPN, and MSO, indicating that the labeled processes in these nuclei may be axons originating from MNTB neurons. Immunoreactivity in LSO cells begins to decline around P8, decays rapidly between P10 and P18, and reaches its adult level around P28, when the CaBP labeling pattern in the whole superior olivary complex is indistinguishable from that in the adult. The present results show that the development of CaBP immunoreactivity in the rat superior olivary complex is characterized by two reciprocally related processes: as immunoreactivity within MNTB somata and fibers in the SPN, and LSO, and the MSO increases between P8 and about P21, the immunoreactivity in LSO neurons declines. CaBP immunoreactivity in LSO neurons is only transiently present, suggesting a critical period in development during which the control of Ca2+ homeostasis in LSO neurons may be of particular importance.

GAD- and GABA-immunoreactivity in the ascending auditory pathway of horseshoe and mustached bats.

A comparative study of the immunostain to antibodies directed against glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the ascending auditory pathway was carried out in horseshoe bats (Rhinolophus rouxi) and mustached bats (Pteronotus parnellii). In both species GAD/GABA-positive puncta (presumed axonal boutons) and GAD/GABA-positive cells were found in the cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus the inferior colliculus, and the medial geniculate body. General features of the immunostaining pattern in the auditory pathway agree with observations in other mammals. Quantitative analysis of puncta distribution shows that many auditory centers are characterized by subregional differences in puncta density and distribution. This indicates local differences in putatively inhibitory input related to connectivity and tonotopic organization. The following species characteristic features were found: 1) The dorsal non-laminated portion of the dorsal cochlear nucleus in horseshoe bats lacks the GAD/GABA-immunoreactive cells typical for the ventral laminated portion and the dorsal cochlear nucleus of other species. Clearly, a cytoarchitectonic specialization is accompanied by a loss of putatively GABAergic local inhibitory circuits. 2) The ventral division of the medial geniculate body of the mustached bat lacks GAD/GABA-immunopositive cells. Such cells are present in the horseshoe bat and other mammals. This finding implies functional differences in the organization of the medial geniculate body within the same mammalian order.

An atlas of glycine- and GABA-like immunoreactivity and colocalization in the cochlear nuclear complex of the guinea pig.

The distribution and colocalization of gamma-aminobutyric acid (GABA)- and glycine-like immunoreactivity in the cochlear nuclear complex of the guinea pig have been studied to produce a light microscopic atlas. The method used was based on post-embedding immunocytochemistry in pairs of 0.5-micron-thick plastic sections treated with polyclonal antibodies against conjugated GABA and glycine respectively. Immunoreactive cells, presumably short axon neurones, predominated in the dorsal cochlear nucleus, with mostly single-GABA-labelled cells in the superficial layer, double-labelled in the middle, and single-glycine-labelled in the deep layers. A few large single-glycine-labelled cells, interpreted as commissural neurons, occurred in the ventral nucleus. Scattered double-labelled cells, probably Golgi cells, were seen in the granule cell domain. Immunolabeled puncta of all three staining categories occurred in large numbers throughout the complex, apposed to somata and in the neuropil, showing a differential distribution onto different types of neuron. Three immunolabeled tracts were noted: the tuberculoventral tract, the commissural acoustic stria, and the trapezoidal descending fibres. Most of the fibres in these tracts were single-labelled for glycine, although in the last mentioned tract single-GABA- and double-labelled fibres were also found. Some of the immunolabeled cell types described here are proposed as the origins of the similarly labelled puncta and fibres on the basis of known intrinsic connections.