[The role of innate immunity in otitis media]. / Rolle des angeborenen Immunsystems bei Otitis media.

Otitis media (OM) belongs to the most common pediatric diseases and causes more medical contacts, surgical interventions, and drug prescriptions than any other infectious disease. Recent findings have identified a critical role of innate immunity in recovery from OM. The middle ear mucosa identifies invading pathogens by sensing pathogen-associated molecule patterns (PAMPs) via pattern recognition receptors such as the Toll-like receptors (TLRs). They generate immediate antimicrobial responses and cytokine release, leading to an inflammatory reaction as seen in acute or chronic OM. Cross-talk between TLRs can enhance or suppress the healing process in the middle ear. In order to prevent over-activation on the one hand and insufficient immune response on the other, the signaling network between different TLRs must be integrated and controlled by positive and negative feedback loops. This guarantees a proper immune response in the middle ear after infection. In this review, we focus on the involvement of the innate immune system and TLRs in OM, as well on their relevance for new vaccination strategies and immunotherapies.

Hair cell stereociliary bundle regeneration by espin gene transduction after aminoglycoside damage and hair cell induction by Notch inhibition.

Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. Although this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin-bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. Although γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 h after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1-43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs.

[The role of the spiral ganglion neurons in cochlear implants. Today and in future regenerative inner ear treatment]. / Die Rolle der Spiralganglienneurone im Kochleaimplantat. Heute und in zukünftigen regenerativen Innenohr-Therapien.

Sensorineural hearing impairment is caused by pathologies within the cochlear portion of the inner ear or the central auditory pathway. Within the last decade, tremendous progress has been made in inner ear biology, thus greatly increasing our understanding of congenital and acquired inner ear pathologies. Moreover, the discovery of hair cell regeneration and the presence of neuronal stem cells in the cochlea has raised hopes of being able to treat the causes of sensorineual hearing impairment in the mid-term future. To do so, the regenerated cells will have to be reinnervated through the peripheral axons of the spiral ganglion neurons (SGNs). So far, most factors with the potential to guide peripheral axons of SGNs have been investigated in the developing cochlea of rodent models but not in humans. Remaining SGNs can already be directly stimulated electrically by cochlear implants, electrode arrays surgically inserted into the cochlea, providing effective treatment for severe cochlear hearing impairment.

Role of inhibitor of apoptosis protein in gentamicin-induced cochlear hair cell damage.

Apoptotic cell death is considered to play a key role in gentamicin-induced cochlear hair cell loss. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis that can prevent activation of effector caspases. This study was designed to investigate the possible involvement of X-linked inhibitor of apoptosis protein (XIAP) in hair cell death due to gentamicin. Basal turn organ of Corti explants from postnatal day (p) p3 or p4 rats were maintained in tissue culture and were exposed to 35 muM gentamicin for up to 48 h. Effects of specific XIAP inhibitors on gentamicin-induced hair cell loss and caspase-3 activation were examined. XIAP inhibitors increased gentamicin-induced hair cell loss but an inactive analog had no effect. Caspase-3 activation was primarily observed at 36 or 48 h in gentamicin-treated hair cells, whereas caspase-3 activation peaked at 24-36 h when explants were treated with gentamicin and an XIAP inhibitor. The inhibitors alone had no effect on hair cells. Finally, a caspase-3 inhibitor decreased caspase-3 activation and hair cell loss induced by gentamicin and an XIAP inhibitor, but caspase-8 and -9 inhibitors did not. The results indicate that XIAP normally acts to decrease caspase-3 activation and hair cell loss during gentamicin ototoxicity, as part of a protective response to potentially damaging stimuli.

Evaluation of an independent prestin mouse model derived from the 129S1 strain.

Studies using the prestin knockout mouse indicate that removal of the outer hair cell (OHC) motor protein is associated with loss of sensitivity, frequency selectivity and somatic electromotility. Here we provide data obtained from another prestin mouse model that was produced commercially. In vivo electrical recordings from the round window indicate that the phenotype is similar to that of the original knockout generated by the Zuo group at St. Jude Children's Research Hospital. Hence, compound action potential (CAP) thresholds are shifted in a frequency-dependent manner and CAP tuning curves at 12 kHz are flat for masker frequencies between 3 and 18 kHz. Although CAP input-output functions at 6 kHz show a shift in sensitivity at low levels, responses approach wild-type magnitudes at high levels where the cochlear amplifier has less influence. In order to confirm that the loss of sensitivity and frequency selectivity is due to loss of prestin, we performed immunohistochemistry using a prestin antibody. Cochlear segments from homozygous mutant mice showed no fluorescence, while wild-type mice displayed a fluorescent signal targeted to the OHC's lateral membrane. Absence of prestin protein was confirmed using LDS-PAGE/Western blot analysis. These results indicate that the loss of function phenotype is associated with loss of prestin protein. Lack of prestin protein also results in a shortening of OHC length to approximately 60% of wild-type, similar to that reported previously by Liberman's group. The linkage shown between the loss of prestin protein and abnormal cochlear function validates the original knockout and attests to the importance of OHC motor function in the auditory periphery.

Expression of the P2X(2) receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission.

Extracellular ATP has multimodal actions in the cochlea affecting hearing sensitivity. ATP-gated ion channels involved in this process were characterized in the guinea pig cochlea. Voltage-clamped hair cells exhibited a P2 receptor pharmacology compatible with the assembly of ATP-gated ion channels from P2X(2) receptor subunits. Reverse transcription-PCR experiments confirmed expression of the P2X(2-1) receptor subunit mRNA isoform in the sensory epithelium (organ of Corti); a splice variant that confers desensitization, P2X(2-2), was the predominant subunit isoform expressed by primary auditory neurons. Expression of the ATP-gated ion channel protein was localized using a P2X(2) receptor subunit-specific antiserum. The highest density of P2X(2) subunit-like immunoreactivity in the cochlea occurred on the hair cell stereocilia, which faces the endolymph. Tissues lining this compartment exhibited significant P2X(2) receptor subunit expression, with the exception of the stria vascularis. Expression of ATP-gated ion channels at these sites provides a pathway for the observed ATP-induced reduction in endocochlear potential and likely serves a protective role, decoupling the "cochlear amplifier" in response to stressors, such as noise and ischemia. Within the perilymphatic compartment, immunolabeling on Deiters' cells is compatible with purinergic modulation of cochlear micromechanics. P2X(2) receptor subunit expression was also detected in spiral ganglion primary afferent neurons, and immunoelectron microscopy localized these subunits to postsynaptic junctions at both inner and outer hair cells. The former supports a cotransmitter role for ATP in a subset of type I spiral ganglion neurons, and latter represents the first characterization of a receptor for a fast neurotransmitter associated with the type II spiral ganglion neurons.

Identification of a novel myosin heavy chain gene expressed in the rat larynx.

Based on reactivity to antibodies against known myosin heavy chains, expression of a novel fast myosin heavy chain (MHC) gene was suspected in the thyroarytenoid (TA) muscle of the rat larynx. The 3' ends of MHC transcripts in the TA were amplified by RT-PCR using a primer to a highly conserved MHC sequence and to the poly(A) tail. The resultant products were cloned and fourteen PCR products were screened by dot-blotting with oligonucleotides specific for known skeletal muscle MHC genes. A clone that reacted weakly to the 2B oligo was sequenced and found to encode a novel fast MHC transcript, termed 2L, that appears to represent an eighth vertebrate skeletal muscle MHC gene. By homology analysis, the 2L sequence is most similar to the extraocular MHC, suggesting a possible evolutionary relationship between MHCs associated with the branchial arches.

Amino acid labeling patterns in the efferent innervation of the cochlea: an electron microscopic autoradiographic study.

Light microscopic autoradiography and electron microscopic autoradiography were used to study the distribution of label in the cochlear efferents following in vivo incubation with tritiated amino acids. Two basic patterns of labeling were observed. These patterns correspond closely to the lateral and medial superior olivary complex (SOC) olivocochlear systems identified by Warr and Guinan ('79, Brain Res. 173:152-155). Our electron microscopic observations suggest that, at least in the gerbil, the complete separation of outer hair cell (OHC) versus inner hair cell (IHC) efferent innervation proposed by these investigators based upon light microscopic data does not occur. Rather, our data suggest that while the lateral SOC system supplies endings only to the region under the IHC, the medial SOC system may supply endings beneath both the IHCs and OHCs.

Regional (14C) 2-deoxyglucose uptake during forelimb movements evoked by rat motor cortex stimulation: pons, cerebellum, medulla, spinal cord, muscle.

Electrical stimulation of the right forelimb motor (MI) sensory (SI) cortex in normal, adult rats produced repetitive left forelimb movements. Regions of increased (14C) 2-deoxyglucose (2DG) uptake were mapped auto-radiographically during these movements. MI stimulation activated the ipsilateral reticular tegmental pontine nucleus (RTP) and the middle (rostral-caudal) third of the pontine nuclei including pyramidal (P), medial (POM), ventral (POV), and lateral (POL) pontine nuclei. The ipsilateral inferior olivary complex was activated including dorsal accessory olive (DAO), principal olive (PO), and medial accessory olive (MAO). The contralateral lateral reticular (LR) nucleus and nucleus cuneatus (CU) were activated. Lateral vermal, paravermal, and hemispheric portions of the contralateral cerebellum were also activated. Parts of vermian lobules IV, V, VI, VII, and VIII, and lobulus simplex, crus I, crus II, paramedian lobule, and copula pyramidis were activated. Granule cell layers were activated much more than molecular layers. Discrete microzones of high granule cell 2DG uptake alternated with zones of low uptake in left paramedian lobule and copula pyramidis and may correlate with the fractured cerebellar somatotopy described physiologically by Welker and his associates. Portions of the left lateral and interpositus nuclei were metabolically activated. Medial portions of laminae I-VI were activated in the dorsal horn of cervical spinal cord. The 2DG uptake was either unchanged or decreased in the ventral horn. Thoracic and lumbar spinal cord were not activated. Monsynaptic MI and SI connections to P, POM, POV, POL, RTP, DAO, PO, MAO, LR, CU, and spinal cord could account for activation of those structures. However, there are no direct MI or SI connections to the deep cerebellar nuclei, the cerebellar hemisphere, or the muscles. Activation of these structures must be due to activation of polysynaptic pathways, sensory feedback from the moving forelimb, or both. The present experiments cannot distinguish these possibilities. Comparison of the regions activated during forelimb MI stimulation (FLMIS) to those activated during vibrissae MI stimulation (VMIS) suggests that the pontine nuclei, cerebellar hemisphere, and possibly the deep cerebellar nuclei are somatotopically organized. RTP, LR, CU, and spinal cord were activated during FLMIS but were not activated during VMIS. The failure to activate the ventral horn of cervical spinal cord may be due to known inhibition of alpha-motor neurons during motor cortex stimulation.

Distribution of non-NMDA glutamate receptor mRNAs in the developing rat cochlea.

In situ hybridization was used to document the distribution of mRNA encoding six subunit isoforms of non-N-methyl D-aspartic acid (NMDA) glutamate receptors (GluR1, GluR2, GluR3, GluR4, GluR5 and GluR6) in the inner ears of embryonic, postnatal and adult rats. GluR2 and GluR3 expression in the spiral ganglion appeared well before birth, and reached adult levels several days before the onset of function in the cochlea. In the spiral limbus, expression of GluR2 and GluR3 mRNA reached very high levels at around the time of birth, then declined after a few days. Low levels of GluR1, GluR4 and GluR6 expression were detected in various tissues of the cochlea during development. In the adult cochlea, GluR expression was limited to GluR2 and GluR3 mRNAs in the spiral ganglion neurons and GluR2 mRNA in fibrocytes of the spiral limbus, a non-neural tissue. The ontogenetic expression of additional GluR subunit genes and their appearance in different cochlear tissues could reflect different roles for these genes during development, or less precise regulation of gene expression within the GluR family. In particular, the very high levels of GluR gene expression in the spiral limbus during the perinatal period support a non-neural function, perhaps as cell surface receptors during tissue differentiation.

Localization of mRNA encoding the P2X2 receptor subunit of the adenosine 5'-triphosphate-gated ion channel in the adult and developing rat inner ear by in situ hybridization.

Localization of expression of the adenosine 5'-triphosphate (ATP)-gated ion channel P2X2 receptor subunit (P2X2R) in the rat inner ear at different stages of development was achieved by using in situ mRNA hybridization. In the adult, P2X2R mRNA was strongly expressed in many of the cells bordering the cochlear endolymphatic compartment. This included the interdental cells of the spiral limbus, all cells of the inner sulcus and organ of Corti, and cells of the spiral prominence. In the vestibular labyrinth, strong expression was noted in the transitional cells at the base of the crista ampullaris and in the sensory epithelium of the crista and maculae. During development, P2X2R mRNA expression was evident in the precursors of these structures at the earliest period studied, embryonic day 12 (E12). Expression increased during the ontogeny in both the cochlear and the vestibular end organs. In addition, both the spiral and vestibular ganglia showed developmental expression. In contrast to the supporting cells of the organ of Corti, both inner and outer hair cells exhibited P2X2R mRNA only after postnatal day 10 (P10) through P12, concomitant with the onset of hearing. P2X2R expression levels in all cells fell from a maximum at P12-P18 to lower levels in the adult. In the adult, P2X2R mRNA levels were modest in outer hair cells in the basal (high-frequency) encoding region of the cochlea, and inner hair cell labeling was low throughout the cochlea. Reissner's membrane, which maintains an electrochemical barrier between scala vestibuli and scala media, showed considerable expression of P2X2R mRNA in early postnatal development, and expression was maintained at moderate levels in the adult cochlea. These data are consistent with a role for the P2X2R subunit in the processes of labyrinthine development and the regulation of the electrochemical gradients supporting auditory and vestibular sensory transduction.

Tonotopic organization in the central auditory pathway of the Mongolian gerbil: a 2-deoxyglucose study.

The uptake of 2-deoxyglucose (2-DG) was employed to map functional activation of the central auditory pathway in the mongolian gerbil, during 85 dB SPL stimulation with pure tonal stimuli at frequencies of 0.75, 3.0, or 12.0 kHz. Pure tones produced foci of very high 2-DG uptake, when compared to adjacent tissue, in the cochlear nucleus, superior olivary complex, and inferior colliculus. Less distinct areas of elevated 2-DG uptake were seen in the dorsal and ventral nuclei of the lateral lemniscus, medial geniculate nucleus, and auditory cortex. Little or no change in the distribution of 2-DG uptake was noted in the nucleus of the trapezoid body. The location of discrete regions of relatively high 2-DG uptake varied systematically with stimulus frequency. The tonotopic organization demonstrated by 2-DG mapping agreed well with the results of previous electrophysiological studies for most structures. However, in the inferior colliculus, stimulus-evoked increases in 2-DG uptake were found to occur in a fixed pattern of three to four bands across the central nucleus, which did not correspond to any previously reported anatomical or physiological organization. Pure tonal stimuli activated discrete portions of this banding pattern. Also, a small area at the ventromedial edge of the colliculus was more broadly tuned than other regions of the nucleus. It is concluded that 2-DG uptake is well suited to the investigation of tonotopic organization. This technique reveals patterns of activation which have not been observed with other methodologies.

Fibronectin-like immunoreactivity of the basilar membrane of young and aged rats.

Dysfunction of cochlear mechanics has been hypothesized to be a source of age-related hearing loss and the basilar membrane mass and stiffness contribute to normal cochlear mechanics. Fibronectin, a large, extracellular matrix protein and a major component of the basilar membrane, may contribute to both the mass and stiffness of the membrane. Mesothelial cells underlying the basilar membrane may produce the fibronectin and also contribute to the mass of the membrane. Changes in either the fibronectin or the mesothelial cells might, therefore, have an effect on cochlear mechanics. In order to assess basilar membrane changes in aged animals, young adult (2-4 months) and aged (24-26 months) Sprague-Dawley rats were evaluated for the presence of fibronectin-like protein and mesothelial cells. The basilar membrane in the young animals had strong fibronectin-like immunoreactivity throughout its length. The old animals, on the other hand, showed normal fibronectin immunoreactivity in the basilar membrane of the basal turn, but little or no reactivity in the apical cochlear turn. The number of mesothelial cells was reduced throughout the length of the membrane in aged animals, with the greatest loss in the basal turn (60% fewer cells). These two degenerative changes, which appear to be independent of each other, may contribute to the observed threshold shifts in aged cochleas.

Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear.

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.

Effects of stimulus frequency and intensity on c-fos mRNA expression in the adult rat auditory brainstem.

Induction of the cellular fos gene (c-fos) is one of the earliest transcriptional changes observed following neuronal excitation. Although not an activity marker in the strict electrophysiological sense, many neurons in the central nervous system increase their c-fos expression after periods of sustained stimulation at physiological levels of intensity. In the present study, induction of c-fos mRNA expression was examined in the auditory brainstem after 1 hour of continuous free-field acoustic stimulation. Sprague-Dawley rats were exposed to pure tones of 2, 8, 16, or 32 kHz or half-octave noise bands centered on 2, 8, or 32 kHz at 80-120 dB SPL. Stimulation-induced c-fos mRNA expression was evident at all levels of the auditory brainstem, and this expression was intensity dependent. In some brain areas, induced expression manifested a clear tonotopic organization, i.e., in dorsal, posteroventral, and anteroventral cochlear nuclei, and in the medial nucleus of the trapezoid body. The inferior colliculus exhibited multiple tonotopic representations. The dorsal nucleus of the lateral lemniscus had a crude tonotopy. Although expression was present, tonotopy was not evident in periolivary nuclei or in the ventral or intermediate nuclei of the lateral lemniscus. Free-field diotic stimulation did not induce c-fos mRNA expression in the medial or lateral superior olivary nuclei. Expression was induced in the lateral superior olive by dichotic stimulation (after a unilateral cochlear ablation), and that expression was tonotopically organized. The results suggest that stimulation-induced c-fos mRNA expression can be an effective way of mapping neuronal activity in the central auditory system under both normal and pathological conditions.

Cochlear ablation alters acoustically induced c-fos mRNA expression in the adult rat auditory brainstem.

Expression of c-fos mRNA was studied in the adult rat brain following cochlear ablations by using in situ hybridization. In normal animals, expression was produced by acoustic stimulation and was found to be tonotopically distributed in many auditory nuclei. Following unilateral cochlear ablation, acoustically driven expression was eliminated or decreased in areas normally activated by the ablated ear, e.g., the ipsilateral dorsal and ventral cochlear nuclei, dorsal periolivary nuclei, and lateral nucleus of the trapezoid body and the contralateral medial and ventral nuclei of the trapezoid body, lateral lemniscal nuclei, and inferior colliculus. These deficits did not recover, even after long survivals up to 6 months. Results also indicated that neurons in the dorsal cochlear nucleus could be activated by contralateral stimulation in the absence of ipsilateral cochlear input and that the influence of the contralateral ear was tonotopically organized. Results also indicated that c-fos expression rose rapidly and persisted for up to 6 months in neurons in the rostral part of the contralateral medial nucleus of the trapezoid body following a cochlear ablation, even in the absence of acoustic stimulation. This response may reflect a release of constitutive excitatory inputs normally suppressed by missing afferent input or changes in homeostatic gene expression related to sensory deprivation. Instances of transient, surgery-dependent increases in c-fos mRNA expression in the absence of acoustic stimulation were observed in the superficial dorsal cochlear nucleus and the cochlear nerve root on the ablated side.

Selective retrograde labeling of lateral olivocochlear neurons in the brainstem based on preferential uptake of 3H-D-aspartic acid in the cochlea.

We have previously shown that perfusion of the gerbil cochlea with probe concentrations of 3H-D-aspartic acid (D-ASP) results in immediate, selective labeling of 50-60% of the efferent terminals under the inner hair cells, presumably by high-affinity uptake. The present study was undertaken to determine the origin of these endings. Twenty-four hours after cochlear perfusion with D-ASP, labeled neurons were observed in the ipsilateral, and to a much lesser extent in the contralateral, lateral superior olivary nucleus (LSO). The cells were small, primarily fusiform, and showed fewer synaptic contacts than other LSO cells. Combined transport of D-ASP and horseradish peroxidase indicated that all olivocochlear neurons within the LSO that projected to the injected cochlea were labeled by D-ASP. Labeled fibers coursed dorsally from the LSO, joined contralateral fibers that had passed under the floor of the fourth ventricle, and entered the VIIIth nerve root at its ventromedial edge. Adjacent to the ventral cochlear nucleus (VCN), densely labeled collateral fibers crossed the nerve root to enter the VCN. Labeled fibers and terminals were prominent in the central VCN. Neither retrograde transport of D-ASP by medial olivocochlear and vestibular efferents nor anterograde transport by VIIIth nerve afferents was observed. The D-ASP-labeled cells and fibers are clearly lateral olivocochlear efferents. Retrograde transport of D-ASP thus allows the cells, axons, and collaterals of the lateral olivocochlear system to be studied, morphologically, in isolation from other cells that project to the cochlea. Since the olivocochlear neurons are almost certainly cholinergic, retrograde amino acid transport does not necessarily identify the primary neurotransmitter of a neuron. Rather, it indicates the presence of selective uptake by the processes of that neuron at the site of amino acid injection. Retrograde labeling appears to be markedly enhanced by the use of metabolically inert compounds such as d-isomer amino acids.

Selective retrograde transport of nipecotic acid, a GABA analog, labels a subpopulation of gerbil olivocochlear neurons.

Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.

Collaterals from lateral and medial olivocochlear efferent neurons innervate different regions of the cochlear nucleus and adjacent brainstem.

Two populations of superior olivary neurons which project to different sensory cell regions in the cochlea also give off collateral projections to the ventral cochlear nucleus (VCN) and adjacent brainstem. To determine whether these VCN projections also have different targets they were characterized by selective retrograde amino acid transport. Retrograde transport of 3H-d-aspartate (D-ASP) selectively labeled the unmyelinated fibers and neurons of the lateral olivocochlear (OC) system including a dense collateral projection to the central VCN. Retrograde transport of 3H-nipecotic acid (NIP) labeled the myelinated fibers and neurons of the medial OC system, including collateral projections to the peripheral VCN, subpeduncular granule cells, and nucleus Y. Medial and lateral OC efferent collaterals thus innervate different regions of the CN. Lateral system collaterals overlap extensively with Type I spiral ganglion cell afferent input. They are well positioned to play a role in modulating afferent input to the central auditory system, as is the primary projection of these efferents to the cochlea. The medial system collaterals project near the recently described afferent projections of Type II spiral ganglion cells. The medial system collaterals may therefore be related to the function of outer hair cells, as the medial system primary axons appear to be in the cochlea.

Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system.

The distribution of the P2X2 receptor subunit of the adenosine 5'-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor-specific antisera and riboprobe-based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.