Spatial and temporal expression patterns of nicotinic acetylcholine α9 and α10 subunits in the embryonic and early postnatal inner ear.

The expression and function of nicotinic receptor subunits (nAChRs) in the inner ear before the onset of hearing is not well understood. We investigated the mRNA expression of the α9 and α10 nAChR subunits in sensory hair cells of the embryonic and postnatal rat inner ear. We mapped their spatial and temporal expression in cochlear and vestibular hair cells using qPCR, [35S] labeled cRNA in situ hybridization, and α-bungarotoxin (α-Bgt) to label the presumptive membrane-bound receptor on cochlear hair cells. The results suggest that (1) the mRNA expression of the α9 subunit precedes expression of the α10 subunit in both cochlear and vestibular hair cells, (2) the mRNA expression of both the α9 and α10 subunits occurs earlier in the vestibular system than in the cochlea, (3) the mRNA expression of both subunits is required for the assembled receptor complexes, and (4) the presumptive assembled receptor, at least in the cochlea, is associated with synapse formation and the onset of function.

GABAergic neurons in the lateral superior olive of the hamster are distinguished by differential expression of gad isoforms during development.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that is synthesized by two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. Using in situ hybridization and immunocytochemical techniques in hamsters, we investigated the postnatal development of GAD isoforms within the lateral superior olive (LSO) where GABAergic neurons form part of a descending efferent projection to the cochlea. In the neonatal hamster LSO, GAD67 immunoreactivity, GAD67 transcript labeling, and intense GABA immunostaining are at low levels. However, robust GAD65 mRNA expression is found throughout the LSO during the early postnatal period. The neonatal GABAergic expression patterns are in stark contrast to the adult where the LSO has robust GAD67 mRNA expression and weak GAD65 mRNA expression. Cells exhibiting intense GABA immunolabeling were also found in the same LSO locations as robust GAD67 mRNA expression and intense GAD67 immunoreactivity. Additionally, GAD67-positive cells in the LSO were retrogradely labeled from the cochlea confirming that these cells are a part of the lateral olivocochlear system. The late onset of GAD67 expression and intense GABA immunoreactivity in LSO neurons are consistent with the relatively late maturation of the lateral olivocochlear neurons inferred from previous studies. During development, these data lead us to conclude that the GABAergic portion of the lateral olivocochlear system is distinguished by preferential GAD67 expression, intense GABA immunoreactivity, and relatively late postnatal onset.

Expression of alpha and beta parvalbumin is differentially regulated in the rat organ of corti during development.

The expression of two calcium-binding proteins of the parvalbumin (PV) family, the alpha isoform (alphaPV) and the beta isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT-PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while alphaPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, alphaPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished alphaPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but alphaPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of alphaPV immunoreactivity. This study suggests that OM and alphaPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation.

Effect of xerostomic medications on stimulated salivary flow rate in patients with Sjögren's syndrome.

OBJECTIVE: The purpose of this study was to examine the effect of xerostomic medications on the salivary output of patients with Sjögren's syndrome. METHOD AND MATERIALS: Of 62 patients evaluated in this study, 23 were not using medication, and 39 were using between 1 and 6 medications with xerostomic side effect. RESULTS: The mean +/- SEM stimulated parotid output was 0.33 +/- 0.07 mL/min per gland for patients who were not using medication and 0.33 +/- 0.04 mL/min per gland for patients using (1 to 6) medications. Analyses did not reveal a significant difference in salivary output between these groups. The salivary output of patients using various numbers of medications (1 or 2; 3 or 4; 5 or 6) was also compared. Analysis revealed no significant difference in salivary output related to the number of xerostomic medications used. CONCLUSION: The use of xerostomic medications may not necessarily affect stimulated parotid flow rate in patients with Sjögren's syndrome. These results suggested that gustatory stimulation may be adequate to overcome the inhibitory effect induced by xerostomic medications.

Differential development of cholinergic-like neurons in the superior olive: a light microscopic study.

To better understand the development of cholinergic-like neurons within the superior olivary complex, we investigated the onset and distribution of two well-known markers of cholinergic-like neurons in hamsters: choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). From embryonic day (E) 14 through postnatal day (P) 0, olivary cells immunopositive for ChAT were restricted to the rostral periolivary (RPO) area. Between P0 and P3, ChAT-positive cells are found in progressively more caudal and ventral periolivary locations. Although rostral and ventral periolivary cells exhibited an early onset of ChAT expression, stable numbers were not reached until P4. In contrast, ChAT expression within the lateral superior olive (LSO) is not visible until after P0 and higher numbers of ChAT-positive cells are obtained by P5. The AChE expression lags several days but follows roughly the same pattern of onset as for ChAT. Additionally in rostral and ventral periolivary regions as well as in the LSO, there were fewer AChE-labeled cells than ChAT-labeled cells. The observed temporal relationships in cholinergic-like expression within olivary cells suggest that different cholinergic-like populations may be defined on the basis of the onset of neurotransmitter-related enzymes: RPO cells are first, cells in ventral periolivary regions are second, and cells associated with the LSO are last. The differences observed in the onset of ChAT and AChE expression may reflect differences in the timing of target innervation as well as differences in synaptogenesis.

The human olivocochlear system: organization and development.

The goals of the present study were to identify olivocochlear neurons in the human brainstem, to establish the time course of their early development and to compare the organization of the human olivocochlear system to that of other mammals. To accomplish these goals, we used immunohistochemistry for choline acetyltransferase (ChAT) and calcitonin gene-related peptide (CGRP) in postmortem brainstems of human subjects ranging in age from 16 fetal weeks to 17 years. By immunostaining, we identified two classes of cells in the superior olivary complex: both classes were seen to be present from the twenty-first fetal week to the seventeenth year. Neurons which are immunostained only for ChAT are located primarily in the dorsomedial, ventral and ventrolateral sectors of the periolivary region. These neurons are predominantly bipolar or multipolar cells, and are probably homologous to medial olivocochlear neurons in other species. A second population of cells is immunoreactive for both ChAT and CGRP. This population includes a cluster of mostly small oval neurons, located on the dorsal edge of the olivary complex, and a variable number of cells found along the margin of the lateral olivary nucleus. These ChAT- and CGRP-immunoreactive cells are likely to be homologous to the lateral olivocochlear system in other mammals. With increasing age, the dorsal cluster of small cells shifts from its original cap-like position over the lateral olivary nucleus to become an extended column of cells lying among the fibers of the olivocochlear bundle.

NADPH-diaphorase histochemistry reveals an autonomic-like innervation in the postnatal hamster cochlea.

Previous studies used nicotinamide adenine diphosphate (NADPH)-diaphorase histochemistry as an indicator of nitric oxide synthase (NOS) expression in the adult mammalian cochlea. In this study, we investigated the early postnatal expression of diaphorase activity in the hamster cochlea. Two types of extrinsic fibers were intensely labeled as early as postnatal day 3 (P3) in the portion of the cochlear nerve that innervates the base of the modiolus. By P10, these fibers had reached the spiral ganglion and were projecting toward the organ of Corti. The perivascular type of fiber did not project into the organ of Corti; however, the nonperivascular type could be traced among the supporting cells below the outer hair cells. Spiral ganglion cell somata were also labeled as early as P3. The onset of diaphorase expression in the spiral ganglion cells corresponds to a critical period of synaptogenesis for these sensorineural cells. If NADPH-diaphorase activity is an indicator of NOS, then our results suggest that NO may play a role during postnatal cochlear development.

Choline acetyltransferase expression during a putative developmental waiting period.

The relationship between the cholinergic expression, morphological development, and target cell innervation of olivocochlear (OC) efferent neurons was investigated in the postnatal hamster. Similar to what was found in previous studies, tracer injections into the contralateral cochlea labeled cells bodies retrogradely in periolivary regions and labeled cell bodies only rarely in the lateral superior olive (LSO). Few morphological differences were found among cell bodies labeled between postnatal day 1 (P1) and P30. Tracer injections into the crossed OC bundles within the brainstem anterogradely labeled terminals below the inner hair cells of the cochlea prior to P5 and labeled terminals below outer hair cells after P5, consistent with a period of transient innervation, as hypothesized previously. Within the superior olive, choline acetyltransferase (ChAT) was expressed differentially. In periolivary regions, ChAT was expressed as early as P0. ChAT-immunoreactive cell bodies in periolivary regions were similar morphologically to retrogradely labeled OC neurons. In contrast, within the LSO, ChAT was not expressed until after P2. Consistent with a medical OC projection to the cochlea at early postnatal ages, ChAT immunoreactivity was detected below inner hair cells as early as P2 but was not detected below outer hair cells until after P6. Our results suggest that medial OC neurons not only provide transient connections to inner hair cells but also may express ChAT when they are below inner hair cells. Furthermore, these results raise the possibility that OC neurons may be capable of acetylcholine synthesis and release prior to or simultaneous with their innervation of the cochlea.

Differential expression of the alpha 9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells.

The expression of the alpha9 nicotinic acetylcholine receptor (nAChR) subunit was investigated in perinatal and adult rat cochleae using [35S] labeled cRNA in situ hybridization techniques. In the adult, alpha9 expression showed both longitudinal and radial gradients. The highest expression occurs over outer hair cells (OHCs) in basal regions, and particularly, OHCs in row 1. In contrast, expression over IHCs is lowest in basal regions and highest in apical regions. During embryonic and postnatal ages, the pattern of alpha9 expression differs. Expression of alpha9 was nearly equivalent over IHCs and OHCs. Additionally, the greater epithelial ridge, which is adjacent to IHCs before birth, shows a high level of alpha9 expression. These data are consistent with current models of efferent synaptogenesis and suggest that the expression of the alpha9 nAChR may be influenced by the arrival of efferent axons.

Developmentally regulated expression of calcitonin gene-related peptide in the superior olive.

During postnatal development, the expression of calcitonin gene-related peptide (CGRP) and its mRNA was investigated in the superior olivary complex of the hamster in order to better understand its role in the development of the efferent olivocochlear (OC) pathway. Although both the peptide and its mRNA were expressed at birth in a few periolivary cells, neither CGRP mRNA nor any immunoreactivity could be detected in the lateral superior olive until after postnatal day (P) 5. By P9, CGRP expression had significantly increased and was mostly contained within the lateral superior olive. Between P7 and P18, there appears to be a transient increase in the transcript expression both in periolivary regions and in the lateral superior olive. Notably, both peptide and mRNA expression decreased precipitously throughout the superior olive after P18. In comparison, although both the facial and trigeminal motor nuclei had significant CGRP expression at birth, the facial motor nucleus demonstrated a decrease in the level of CGRP expression between P1 and P6, while the trigeminal motor nucleus reached a maximal level of expression around P18. If CGRP expression is related to synaptogenesis in OC neurons, as has been suggested for certain motor neurons, then we predict that the ephemeral increases in transcript expression in OC neurons are related to synaptogenetic mechanisms in the cochlear periphery. Importantly, the time course for CGRP expression in lateral OC neurons indicates that their OC terminals in the cochlear periphery may not begin forming synapses until near the end of the 1st postnatal week.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study.

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons form part of a later, secondary innervation to the cochlea.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: evidence for a waiting period.

Reconstructions of the efferent innervation of the hamster (Mesocricetus auratus) cochlea were done during postnatal development. Efferent neurons were labeled via injections of biocytin and horseradish peroxidase into the crossed olivocochlear (OC) bundles using an in vitro brainstem technique. Such injections retrogradely labeled cell bodies in ventral periolivary regions of the superior olive consistent with their being medial OC neurons. Anterogradely labeled axons were traced to the cochlea, where they terminated on or below inner hair cells (IHCs) prior to postnatal day 5 (P5). After P5, labeled axons terminated on IHCs and outer hair cells (OHCs) and after P10, the majority of labeled axons terminated on the OHCs. In the electron microscope, small labeled terminals containing densely packed synaptic vesicles were found both adjacent to IHCs (axosomatic) as well as apposed to afferent and efferent fibers below IHCs prior to P5. By P10, large labeled terminals were axosomatic to OHCs and no longer found on IHCs. Consistent with previous reports, these data suggest that medial OC axons form part of an early primary innervation on and below IHCs before terminating on OHCs. This raises the possibility that OC neurons demonstrate a period of waiting below an intermediate target similar to that described in the development of thalamocortical projections.

Morphological gradients in sensory hair cells of the amphibian papilla of the frog, Rana pipiens pipiens.

The sensory hair cells of the amphibian papilla (AP) of the northern leopard frog were examined in a light-microscopic analysis. Hair cell length and cross-sectional area were found to vary systematically along the rostro-caudal axis of the endorgan. The AP was readily divided into three morphological regions. Rostrally-located hair cells are tall, cylindrically-shaped cells with large cross-sectional areas and long stereocilia; caudally-located hair cells are short, goblet-shaped cells with small cross-sectional areas and short stereocilia. In the middle region, hair cells exhibit features intermediate to those of hair cells located at the AP extremes. The detailed pattern of changes in hair cell morphology along the endorgan correlates well with its observed tonotopy and may have implications for the intrinsic tuning of the AP.

Acute effects of capsaicin on the postnatal spiral ganglion.

Capsaicin exerts both excitatory and desensitizing effects on a subset of sensory neurons during development in the dorsal root ganglion, but it is not known how widespread these effects are in other sensory ganglia especially those not known to have any pain sensitivity. The capsaicin sensitivity of auditory neurons in the spiral ganglion was investigated in acute preparations from three postnatal age groups in the hamster: postnatal days 6-8 (group 1), postnatal days 13-15 (group 2) and postnatal days 20-22 (group 3). Morphologically, capsaicin altered the shape, size, and staining characteristics of spiral ganglion cells (SGCs) across all age groups and this effect was apparently not selective for any identifiable subpopulation. However, SGCs from the youngest age groups were particularly sensitive to capsaicin neurotoxicity. When incubated in media that contained cobalt, capsaicin stimulated cobalt uptake within SGCs. KCl depolarization was able to produce the same level of cobalt uptake as capsaicin treatment. Our data suggest that the extent of capsaicin neurotoxicity in the spiral ganglion is strongly age dependent, involves the entry of calcium ions and is relatively nonspecific.

Kainic acid stimulates uptake of divalent cations in postnatal cochlear neurons.

Excitatory amino acids were tested for their ability to induce cobalt uptake in cochlear explants of spiral ganglion neurons. Explants were taken from postnatal day 5 hamsters. Compared with NMDA and quisqualate, kainic acid (KA) stimulated the greatest amount of Co2+ uptake within spiral ganglion neurons as indicated by precipitation of the Co2+ accumulation and relative optical density measurements. Both L-glutamate and KA stimulation were blocked by non-NMDA receptor antagonists kynurenate and DNQX. Nifedipine, a dihydropyridine-sensitive calcium channel blocker, also partially inhibited Co2+ uptake. These data suggest that a mixed population of NMDA and non-NMDA receptors are present. Additionally non-NMDA receptors may have a role in cochlear synaptic maturation.

A transient afferent innervation of outer hair cells in the postnatal cochlea.

In vitro retrograde labeling techniques were used to reconstruct the peripheral terminations of afferent fibers to the outer hair cells in postnatal hamsters. Between postnatal days 4 and 6, both radial and spiral afferent fibers terminated on outer hair cells. The cell bodies which gave rise to such fibers were not distinguishable. These outer radial-fiber terminations may be a transient phenomenon since they were not observed either before or after this period.

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 cholinergic 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.

Innervation of the amphibian and basilar papillae in the leopard frog: reconstructions of single labeled fibers.

Amphibians have two auditory organs specialized for reception of airborne sounds: the amphibian papilla and the basilar papilla. In this report we examine the morphology of the ganglion cells and the afferent innervation of the sensory epithelium in both auditory organs of the leopard frog, Rana pipiens pipiens. Extracellular injections of either biocytin or horseradish peroxidase (HRP) were made into the VIII nerve; they labeled ganglion cells, their axons, and their terminal fibers within the papillae. Ganglion cells that projected to either the amphibian papilla or basilar papilla had cell bodies that were morphologically distinct from other labeled cells. In the amphibian papilla thick fibers terminated in the rostral portion and thin fibers terminated in the caudal portion. Labeled fibers in the rostral portion traveled short distances before making contacts with up to nine hair cells whereas labeled fibers in the caudal portion traveled longer distances and contacted no more than five hair cells. In the basilar papilla labeled fibers were thick (around 4 microns) and terminated on as many as nine hair cells. Consistent with studies from the bullfrog, Rana catesbeiana, our results suggest that the amphibian papilla of R. pipiens pipiens has a convergent innervation (i.e., multiple hair cells provide input to a single ganglion cell) and is topographically organized. However, in contrast to reports in other ranid species, a highly convergent innervation like that found in the amphibian papilla is also found in the basilar papilla.

Postnatal maturation of spiral ganglion neurons: a horseradish peroxidase study.

Using an in vitro cochlear preparation from postnatal hamsters, spiral ganglion cells (SGCs) were labeled retrogradely following extracellular injections of HRP into the cochlear nerve. In 24 cochleae from hamsters between postnatal days (P) 0 and 10, the neuronal morphology of 201 SGCs and their peripheral axons were analyzed. From P 0 to 3, labeled SGCs had few distinguishable features. Although SGCs could be traced separately to inner hair cells (IHCs) and outer hair cells (OHCs), they all had roughly bipolar-shaped cell bodies. Approximately half of the labeled SGCs had peripheral axons that spiraled some distance before entering radial fiber bundles. From P 3 to 7, SGCs increased in size by nearly 30% and the number of SGCs with spiraling peripheral axons decreased to near zero. At P 10, the central axon diameter to peripheral axon diameter ratios distinguished two populations of SGCs. The hair-cell innervation patterns of SGCs also changed morphologically as a function of postnatal age. At P 0, radial fiber (RF) terminals of peripheral axons contacted as many as 8 IHCs; by P 3, RFs contacted typically one or two IHCs. The terminal portions of peripheral axons contacting OHCs did not show any appreciable spiral until P 2. By P 5, individual outer spiral fibers (OSFs) had greater spiral lengths underneath row-3 OHCs and the number of OHC contacts was also greatest for row-3 OSFs. These data suggest that SGCs undergo a systematic maturational process. Furthermore, the morphological differentiation of SGCs occurs after they have established separate inner and outer hair cell innervations.