Vestibular Periphery Research Articles

Comparison of the vascular innervation of the rat cochlea and vestibular system

In order to gain a better understanding of the neuronal and local control of inner ear blood flow, the vascular innervation to the rat cochlea and vestibular system was examined. Specimens were removed in toto beginning at the basilar artery extending to the anterior inferior cerebellar artery, labyrinthine artery, common cochlear artery, modiolar artery and anterior vestibular artery. When possible the vessels were dissected in continuity through the cribrose area. The vestibular endorgans were also removed. Specimens were examined using immunohistochemical techniques for the presence of vasoactive intestinal peptide, neuronal nitric oxide synthase, neuropeptide-Y, substance P and calcitonin gene related peptide. Results show that the vasculature to the cochlea and vestibular portion of the inner ear receive similar types of nonadrenergic innervation, that within the vestibular endorgans, only CGRP and SP were found in the neuroepithelium or in association with vessels, and that within the vestibular system, the majority of the vascular innervation appears to stop at or near the cribrose area. In the cochlea however, it extends to include the radiating arterioles. These findings suggest that cochlear blood flow is under finer control and that neuronally induced changes in blood flow may have a more global effect in the vestibular periphery.

Topographic distribution of nicotinic acetylcholine receptors in the cristae of a turtle

The neurochemical basis of cholinergic efferent modulation of afferent function in the vestibular periphery remains incompletely understood; however, there is cellular, biochemical and molecular biological evidence for both muscarinic and nicotinic acetylcholine (ACh) receptors (nAChRs) in this system. This study examined the topographic distribution of α-bungarotoxin (α-BTX) nAChRs in the cristae of a turtle species. Cristae were perfusion-fixed, cut at 20 μm on a cryostat and incubated with α-BTX or polyclonal antibodies raised against Torpedo nAChR. Light microscopy showed abundant specific labeling of nAChR in the central zone of each hemicrista on the calyx-bearing afferents surrounding type I hair cells and on the base of the type II hair cells. Within the peripheral zone, dense labeling of type II hair cells near the torus and sparse or no label was observed on type II hair cells near the planum. The α-BTX binding showed a similar pattern within the cristae. The similarity between the topographic distribution of α-BTX binding nAChR and of efferent inhibition of afferents supports the notion that the inhibitory effect of afferents is mediated by nAChR.

Partial cDNAs encoding G-protein α subunits in the rat vestibular periphery

To begin understanding what G-proteins are involved in signal transduction in the vestibular periphery, the expression of G α subunits in rat primary afferent neurons (Scarpa's ganglia) and end-organs was studied. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers corresponding to two conserved regions of the G α protein coding sequence produced partial cDNAs encoding two distinct forms of G α s subunit (G α s2 and anove) G α s2 subunit,GenBank accession number AF1841510); and two forms of G α i2 subunits. A novel truncated form of G α i2 (designated G α i2(vest),Gen Bank accession number AF189020) was detected in the vestibular periphery. G α i2(vest) was also expressed in rat cerebellum and heart. The possible role of the identified G α protein cDNAs in the function of the vestibular periphery is discussed.

Nitric oxide synthase localized in a subpopulation of vestibular efferents with NADPH diaphorase histochemistry and nitric oxide synthase immunohistochemistry.

Efferent innervation of the vestibular labyrinth is known to be cholinergic. More recent studies have also demonstrated the presence of the neuropeptide calcitonin gene-related peptide in this system. Nitric oxide is one of a new class of neurotransmitters, the gaseous transmitters. It acts as a second messenger and neurotransmitter in diverse physiological systems. We decided to investigate the anatomical distribution of the synthetic enzyme for nitric oxide, nitric oxide synthase (NOS), to clarify the role of nitric oxide in the vestibular periphery. NADPH diaphorase histochemical and NOS I immunohistochemical studies were done in the adult chinchilla and rat vestibular brainstem; diaphorase histochemistry was done in the chinchilla periphery. Retrograde tracing studies to verify the presence of NOS in brainstem efferent neurons were performed in young chinchillas. Our light microscopic results show that NOS I, as defined mainly by the presence of NADPH diaphorase, is present in a subpopulation of both brainstem efferent neurons and peripheral vestibular efferent boutons. Our ultrastructural results confirm these findings in the periphery. NADPH diaphorase is also present in a subpopulation of type I hair cells, suggesting that nitric oxide might be produced in and act locally upon these cells and other elements in the sensory epithelium. A hypothesis about how nitric oxide is produced in the vestibular periphery and how it may interact with other elements in the vestibular sensory apparatus is presented in the discussion.

A role of glucocorticoid receptors in the guinea pig vestibular system

To investigate glucocorticoid receptor (GR) function in the vestibular periphery, GR antagonist RU38486 was administered to the guinea pig inner ear by osmotic pump, and we observed post-rotatory nystagmus (PRN) changes as a marker of vestibular function. Ten days after treatment, RU38486 (1 mM) resulted in ipsilateral vestibule hyperexcitability in response to rotation stimulation. This effect was dose-dependent. These data indicate that steroid hormones may play an important role in maintaining vestibular function.

Co-localization of glutamate, choline acetyltransferase and glycine in the mammalian vestibular ganglion and periphery.

Glutamate (Glu) is considered to be the main transmitter at the central synapses of primary vestibular afferents (PVA) and glycine (Gly) is assumed to play a modulatory role. In the vestibular periphery a transmitter role for acetylcholine (ACh) has been attributed chiefly to vestibular efferents (VE), however only a subset of VE neurons displays immunoreactivity (ir) for choline acetyltransferase (ChAT) and acetylcholine esterase (AChE). Controversial results exist on the presence of these two enzymes in PVA. In this study the presence of Glu, ChAT, Gly and their co-localization in the vestibular ganglia (VG) and end organs of mouse, rat, guinea pig and squirrel monkey were investigated. In the VG all bipolar neurons display strong Glu-ir and the majority of cells show a graded ChAT-ir and Gly-ir in all species examined. ChAT and Gly are present in highly overlapping neuronal populations and with a similar gradation. In the end organs ChAT and Gly are again co-localized in the same sets of fibers and endings. In conclusion, in the vestibular ganglion and end organs ChAT appears also to be present in primary afferents rather than being restricted to efferent processes. ChAT in primary afferents might indicate a modulatory or co-transmitter function of acetylcholine.

Vestibular hair cells of the chick express the nicotinic acetylcholine receptor subunit α9

The efferent cholinergic pathways to the vestibular periphery have yet to be fully characterized. While the nicotinic acetylcholine receptor subunit (nAChR) α9 is now regarded as the principle receptor for efferent cholinergic signaling to the organ of Corti, there is still uncertainty over how the more complex efferent effects of the labyrinth are produced. Recent experimental work has demonstrated that the nAChR α9 is present in the vestibular end-organs of the rat and mouse, suggesting that α9 may be one of the mediators of efferent cholinergic signaling in the vestibular periphery as well. In this experiment, we sought to determine whether α9 was also present in the vestibular end-organs of the chick. A homologue of α9 has been cloned recently from the chick cochlea. Using reverse transcription polymerase chain reaction (RT-PCR), individual vestibular end-organ preparations, including posterior ampulla, combined horizontal and superior ampulla, saccule, utricle, and the vestibular ganglion were screened for α9 messenger RNA expression. In each end-organ and the vestibular ganglion, a cDNA of the expected size was obtained by RT-PCR and was confirmed to be α9 by sequence analysis. Further, α9 mRNA was identified by RT-PCR from individually isolated type I and type II vestibular hair cells (single-cell RT-PCR). Lastly, insitu hybridization using digoxigenin-labeled α9 riboprobes confirmed the presence of α9 in type I and type II hair cells throughout the vestibular periphery. These results demonstrate the expression of α9 in the vestibular end-organs of the chick, and lend further support for the role of α9 as a mediator of efferent cholinergic signaling in vestibular hair cells.

Protein kinase C inhibition blocks the early appearance of vestibular compensation

This study tests the hypothesis that activation of protein kinase C (PKC) is a critical step for early recovery from spontaneous nystagmus after unilateral ablation of the vestibular periphery. Halothane–NO 2–O 2-anesthetized Long–Evans rats received a 5-μl intracerebroventricular bolus of vehicle (distilled water, six rats), PKC inhibitor [Iso-H-7 (10 mM, four rats; 50 mM, five rats) or bisindolemaleimide I (Bis-I, 10 μM, six rats)], PKG and PKA inhibitor (A-3, 1 mM, six rats), or the serine–threonine protein kinase inhibitor H-7 (1 mM, five rats; 10 mM, five rats). Surgical unilateral labyrinthectomy (UL) was completed within 15 min. Sham control groups showed no nystagmus. Bis-I and Iso-H-7 significantly retarded the disappearance of spontaneous nystagmus quick phases for 8 h after UL ( p<0.05). The effects of Iso-H-7 were dose-dependent: more nystagmus quick phases ( p<0.05) were present in the 50 mM than the 10 mM group at 7 and 8 h post-UL. The rats given A-3 showed a delayed retardation of nystagmus loss, which differed significantly ( p<0.05) from controls at 4–8 h after labyrinthectomy. The number of nystagmus quick phases was significantly greater than controls ( p<0.05) in the 10 mM H-7 group at 4, 5, 6 and 48 h post-UL, but only at 6 and 24 h post-UL in the 1 mM H-7 group. Thus, PKC activation is an important early requirement for vestibular compensation during the acute post-labyrinthectomy period, while cyclic-nucleotide dependent kinases may be important in a later time frame.

Immune cytokines and dexamethasone influence sensory regeneration in the avian vestibular periphery.

Prior studies have shown that macrophages are recruited to sites of hair cell lesions in the avian inner ear in vitro (Warchol, 1997) and in vivo (Bhave et al., 1998). Although the avian ear has a high capacity for sensory regeneration (Oberholtzer & Corwin, 1997; Stone et al., 1998), the role of macrophages in the regenerative process is uncertain. The present study examined the possible influence of macrophages and immune cytokines on regenerative proliferation in the avian utricle, one of the sensory endorgans of the vestibular system. Utricles from post-hatch chicks were placed in organ culture and hair cell lesions were created by incubation in neomycin. The cultures were then maintained for an additional 24-48 hours in vitro, and some cultures were treated with dexamethasone, which inhibits macrophage activation and cytokine production. Following fixation, resident macrophages were identified by immunoreactivity to CD68. Labeled macrophages were present in all specimens and increased numbers of macrophages were observed following neomycin treatment. Regenerative proliferation in dexamethasone-treated specimens was reduced by about 50%, relative to untreated controls. Additional experiments showed that two macrophage secretory products-TGF-alpha and TNF-alpha-enhanced the proliferation of utricular supporting cells. The results are consistent with a role for macrophages in hair cell regeneration.

Evidence for three additional P2X 2 purinoceptor isoforms produced by alternative splicing in the adult rat vestibular end-organs

P2X 2 receptors are ligand-gated ion channels that are activated by extracellular ATP. To characterize the expression of P2X 2 purinoceptor in the adult rat vestibular periphery, reverse transcription-polymerase chain reaction (RT-PCR) was used. No transcript for P2X 2 receptor was found in the vestibular primary afferent neurons (Scarpa's ganglia); however, partial cDNAs encoding four splice variants of the P2X 2 receptor were isolated from vestibular end-organs. In all four cDNAs, the deletions were of different lengths but started at the same position on the P2X 2 gene (Val-370 codon) located toward the intracellular carboxyl terminus. One of these receptor isoforms was identical in sequence to the recently published P2X 2(b) receptor (Simon et al., 1997, Mol. Pharmacol. 52, 237–248) (also known as P2X 2-2, in the nomenclature of Brändle et al., 1997, FEBS Lett. 404, 294–298). The remaining three novel splice variants of the P2X 2 receptor were designated P2X 2(e), P2X 2(f) and P2X 2(g) (GenBank accession numbers AF028603, AF028604 and AF028605, respectively). The functional significance of these three splice variants remains to be determined. Pituitary and cerebellum were used as survey tissues and only the P2X 2(b) receptor cDNA was found.

The role of N-methyl- d-asparate receptor in the guinea pig inner ear after unilateral labyrinthectomy

To investigate the role of the N-methyl- d-aspartate (NMDA) receptor in the vestibular periphery, vestibular compensation in the guinea pig was studied following chemical unilateral labyrinthectomy by osmotic pump administration of streptomycin sulfate (SM) with or without d-2-amino-5-phosphonovalerate ( d-APV), one of the NMDA receptor antagonists. All animals administrated SM (SM group) or SM and d-APV (SM+ d-APV group) showed spontaneous nystagmus and head tilt. The maximum degree of yaw head tilt in the SM+ d-APV group was statistically smaller than that in the SM group. Moreover the time constant for head tilt in the SM+ d-APV group was statistically shorter than that in the SM group. These results indicate that the NMDA receptor in the vestibular periphery influences vestibular compensation after unilateral labyrinthectomy.

Ultrastructural localization of GABA-like immunoreactivity in the human utricular macula

In the vertebrate vestibular periphery, γ-aminobutyric acid (GABA) has long been presumed to be a neurotransmitter candidate. However, experimental reports about the localization and function of GABA in the vestibular systems of vertebrates are contradictory. In addition, there is no information in the literature concerning the localization of GABA in the human vestibular periphery. The present study investigates the ultrastructural localization of GABA-like immunoreactivity in the human utricular macula. A modified pre-embedding immunostaining electron microscopy technique was applied using two different commercially available polyclonal antibodies to GABA. GABA-like immunoreactivity is confined to the vesiculated nerve fibers and terminals of the human vestibular neurosensory epithelia. The GABA-containing nerve terminals make asymmetrical axo-dendritic synapses with the afferent chalices surrounding the type I sensory hair cells. Type I and type II hair cells as well as afferent chalices are devoid of GABA-like immunoreactive staining. The present study demonstrates that GABA exists in the human vestibular periphery, and that GABA is a neurotransmitter candidate of the human efferent vestibular system.

Ultrastructural localization of ChAT-like immunoreactivity in the human vestibular periphery

Acetylcholine (ACh) has long been considered a neurotransmitter candidate in the efferent vestibular system of mammals. Recently, choline acetyltransferase (ChAT), the synthesizing enzyme for ACh, was immunocytochemically localized in all five end-organs of the rat vestibule (Kong et al. (1994) Hear. Res. 75, 192–200). However, there is little information in the literature concerning the cholinergic innervation in the vestibular periphery of man. In the present study the ultrastructural localization of the ChAT-like immunoreactivity in the human vestibular periphery was investigated in order to reveal the cholinergic innervation in the human vestibular end-organs. A modified method of pre-embedding immunoelectron microscopy was applied. It was found that the ChAT-like immunoreactivity was located in the bouton-type vesiculated nerve terminals in the vestibular neurosensory epithelia of man. These ChAT-like immunostained nerve terminals make synaptic contacts either with afferent chalices surrounding type I vestibular sensory hair cells, or with type II vestibular sensory hair cells. These results show that the ChAT-like immunoreactivity in the human vestibular periphery is confined to the efferent vestibular system. The ChAT-containing efferents innervate both type I hair cells and type II hair cells, making postsynaptic and presynaptic contacts, respectively. This study presents evidence that ACh is a neurotransmitter candidate in the efferent vestibular system of man.

Immunohistochemical investigation of enkephalins in the human inner ear

Enkephalins are generally considered as neuropeptides in the central and peripheral nervous system of mammals bound to three large precursor molecules. Several animal studies demonstrated the distribution of met- and leu-enkephalin-like immunoreactivities in neurons and terminals of the lateral olivocochlear system. The immunostainings in the medial system are more controversial. No data about the presence of different enkephalin sequences in the vestibular efferent terminals are known. In the present study, the ultrastructural localization and distribution of immunoreactivities for six different antibodies against met- and leu-enkephalins in the human cochlear and vestibular periphery were investigated. A modified method of pre-embedding immunoelectronmicroscopy was applied. Met- and leu-enkephalin-like immunoreactivities were observed in the efferent terminals of the human outer and inner hair cell region. Using different met- and leu-enkephalin antibodies, the distribution of immunoreactivities remained similar. In the five human vestibular endorgans, enkephalin-like immunostaining was absent.

Ultrastructural Localization of GABA-like Immunoreactivity in the Vestibular Periphery of the Rat

Gamma-aminobutyric acid (GABA) is presumed to be a neurotransmitter candidate in the vestibular periphery of mammals. However, experimental reports about the localization of GABA in afferents or efferents of the vestibular systems are contradictory. It is an open question whether there are species differences in the amammalian vestibular system. The present study was designed to investigate the ultrastructural localization of GABA-like immunoreactivity in the vestibular periphery of the rat. A modified preembedding immunoelectron microscopy technique was applied using a polyclonal antibody to GABA as a marker. GABA-like immunoreactivity was revealed in the vestibular periphery of the rat, confined to the vesiculated nerve fibers and terminals of the rat vestibular neurosensory epithelia. Type I hair cells and type II hair cells as well as efferent chalices are devoid of GABA-like immunoreactive staining. These findings indicate that GABA is a neurotransmitter candidate of the efferent vestibular system of the rat.

Differential expression of α2–7, α9 and β2–4 nicotinic acetylcholine receptor subunit mRNA in the vestibular end-organs and Scarpa's ganglia of the rat

To further characterize the pattern of expression of the nicotinic acetylcholine receptor (nAChR) subunits in the peripheral vestibular system, we conducted RT-PCR of all known mammalian nAChR α and β subunits in mRNA extracted from adult rat vestibular primary afferent neurons (Scarpa's ganglia) and vestibular end-organs. Transcripts encoding the α2–7 and β2–4 nAChR subunits were found in the vestibular ganglia, while α3, α5–7, α9 and β2–4 nAChR subunits were expressed in the vestibular end-organs. These results support previous electrophysiological, immunocytochemical and molecular biological data, and also provide a more complete understanding of the role of nAChRs in the neurochemical transmission subserving the efferent–afferent interaction in the vestibular periphery.

Innervation of cholinergic vestibular efferent system in vestibular periphery of rats.

The innervation of cholinergic efferent fibers in the vestibular endorgans of the rats was investigated using a modified preembedding immunostaining technique of immunoelectron microscopy. A monoclonal antibody to choline acetyltransferase (ChAT) was used as a marker of cholinergic fibers. It was found that there were four types of cholinergic innervation in the vestibular endorgans of the rat: (1) cholinergic nerve endings formed axo-dendritic synapses with afferent chalice surrounding the type I sensory hair cells; (2) cholinergic nerve endings formed axo-somatic synapses with type II hair cells; (3) cholinergic fibers synapse with afferent nerve fibers and (4) a synaptic contact developed between cholinergic nerve endings. The results demonstrated that a multiform innervation of the cholinergic efferents exists in the rats vestibular periphery.

Expression of α4 and β2 nicotinic acetylcholine receptor subunit mRNA and localization of α-bungarotoxin binding proteins in the rat vestibular periphery

In situ hybridization histochemistry was used to map the distribution of α2, α3, α4, and β2 nAChR subunit mRNAs throughout the peripheral vestibular system of the rat. The α4 and β2 nAChR subunit genes were co-expressed by populations of primary afferent neurons within Scarpa's ganglion, while there was no expression of the α2, α3, α4, or β2 nAChR subunit genes by type I or type II vestibular hair cells. α-bungarotoxin binding to nAChRs in the vestibular end-organs was primarily limited to the afferent chalices surrounding type I hair cells and the basal aspect of type II hair cells. These data suggest that nAChRs composed of α4 and β2 subunits are localized on afferent chalices innervating the type I vestibular hair cells and that the direct cholinergic efferent innervation of the type II vestibular hair cells utilizes nAChR composed of other subunits.

Expression of Substance P, CGRP, and GABA in the Vestibular Periphery, With Special Reference to Species Differences

The present study was conducted to elucidate species differences in the distribution of neuroactive substances, including substance P (SP), calcitonin gene-related peptide (CGRP) and gamma-aminobutyric acid (GABA), in the vestibular periphery of various animals (chicken, pigeon, rat, guinea pig and squirrel monkey). SP-like immunoreactivity was found in a subset of the primary vestibular afferents with no marked species differences in the staining pattern. In contrast, the localization of CGRP- and GABA-like immunoreactivities in the efferent nerve fibers varied according to species. This difference in the distribution pattern of neuroactive substances, found in the efferent system, may indicate that each species has a chemically (and probably functionally) distinct efferent system which is related to its specific environment and/or evolution.

Neuroactive substances in the human vestibular end organs.

In order to evaluate the involvement of neuroactive substances in the human vestibular periphery, the immunocytochemical distribution of substance P (SP), calcitonin gene-related peptide (CGRP), and choline acetyltransferase (ChAT) was examined. SP-like immunoreactivity (LI) was present around and beneath sensory hair cells, probably corresponding to their afferent nerve endings. SP-LI was found predominantly in subpopulations of the primary afferents distributed in the peripheral region of the end organs. ChAT-LI and CGRP-LI were found throughout as small puncta below the hair cell layer, probably corresponding to efferent endings. The present results indicate that these neuroactive substances, previously described in animals, are also distributed in the human vestibular periphery, and almost certainly contribute to human vestibular function.