It is well known that adult-like physiological functioning of the mammalian postnatal cochlea occ... more It is well known that adult-like physiological functioning of the mammalian postnatal cochlea occurs coincidentally with the presence of efferent synapses on outer hair cells (OHCs). This study described the cochlear innervation patterns of thick efferent fibers traveling in the vestibular nerve in postnatal hamsters ranging in age from day zero to day 10. At least three kinds of efferent fibers were labeled via an in vitro horseradish peroxidase (HRP) technique: varicose, thin efferents; nonvaricose, thin efferents; and nonvaricose, thick efferents. Nonvaricose thick efferents were reconstructed from the basal third of the cochlea. Reconstructed efferent fibers traversed in the intraganglionic spiral bundle (IGSB) on the peripheral edge of the spiral ganglion and branched profusely in the osseous spiral lamina (OSL). From day zero to day five, large (greater than 1.0 pm) diameter nonvaricose efferent fibers gave rise to branches that either terminated underneath inner hair cells or appeared to end blindly in the OSL. Efferent fibers also had branches that traveled in the inner spiral bundle (ISB) and tunnel spiral bundle (TSB). In cochleae from hamsters six to eight days old, some thin and thick diameter efferent fibers contacted both inner and outer hair cells. By the tenth day, large diameter fibers traveled radially across the tunnel of Corti to terminate on one to five OHCs. As early as day seven, large diameter fibers also appear to terminate preferentially on OHCs in row one. These observations are consistent with the notion that the end of the first postnatal week represents a critical period in the formation of adult-like synapses on the OHCs. The data also suggest a developmental transition period when efferent fibers contact both hair cell types before contacting OHCs separately.
We generated constitutive knockout mouse models for the α9 and α10 nicotinic acetylcholine recept... more We generated constitutive knockout mouse models for the α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits by derivation from conditional knockouts by breeding with CRE deleter mice. We then backcrossed them onto a C57BL/6J genetic background. In this manuscript, we report the generation of the strains and an auditory phenotypic characterization of the constitutive α9 and α10 knockouts and a double α9α10 constitutive knockout. Although the α9 and α10 nAChR subunits are relevant to a number of physiological measures, we chose to characterize the mouse with auditory studies to compare them to existing but different α9 and α10 nAChR knockouts (KOs). Auditory brainstem response (ABR) measurements and distortion product otoacoustic emissions (DPOAEs) showed that all constitutive mouse strains had normal hearing. DPOAEs with contralateral noise (efferent adaptation measurements), however, showed that efferent strength was significantly reduced after deletion of both the α9 and α10 subunits, in comparison to wildtype controls. Animals tested were 3-8 weeks of age and efferent strength was not correlated with age. Confocal studies of single and double constitutive KOs showed that all KOs had abnormal efferent innervation of cochlear hair cells. The morphological results are similar to those obtained in other strains using constitutive deletion of exon 4 of α9 or α10 nAChR. The results of our physiological studies, however, differ from previous auditory studies using a α9 KO generated by deletion of the exon 4 region and backcrossed onto a mixed CBA/CaJ X 129Sv background.
bioRxiv (Cold Spring Harbor Laboratory), Mar 4, 2022
In cochlear outer hair cells (OHCs), a network of Ca 2+ channels, pumps and Ca 2+-binding protein... more In cochlear outer hair cells (OHCs), a network of Ca 2+ channels, pumps and Ca 2+-binding proteins (CaBPs) regulates the localization, spread, and magnitude of free Ca 2+ ions. During early postnatal development, OHCs express three prominent mobile EF-hand CaBPs: oncomodulin (OCM), αparvalbumin (APV) and sorcin. We have previously shown that deletion of Ocm (Ocm-/-) gives rise to progressive cochlear dysfunction in young adult mice. Here, we show that changes in Ca 2+ signaling begin early in postnatal development of Ocm-/mice. While mutant OHCs exhibit normal electrophysiological profiles compared to controls, their intracellular Ca 2+ signaling is altered. The onset of OCM expression at postnatal day 3 (P3) causes a developmental change in KCl-induced Ca 2+ transients in OHCs and leads to slower KCl-induced Ca 2+ transients than those elicited in cells from Ocm-/littermates. We compared OCM buffering kinetics with other CaBPs in animal models and cultured cells. In a double knockout of Ocm and Apv (Ocm-/-;Apv-/-), mutant OHCs show even faster Ca 2+ kinetics, suggesting that APV may also contribute to early postnatal Ca 2+ signaling. In transfected HEK293T cells, OCM slows Ca 2+ kinetics more so than either APV or sorcin. We conclude that OCM controls the intracellular Ca 2+ environment by lowering the amount of freely available [Ca 2+ ]i in OHCs and in transfected HEK293T cells. We propose that OCM plays an important role in shaping the development of early OHC Ca 2+ signals through its inimitable Ca 2+ buffering capacity.
Changes in the expression of several neurochemical markers associated with either axonal growth (... more Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the choliaergic 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 ~aeurons form part of a later, secondary innervation to the cochlea.
Cochlear outer hair cells (OHCs) are responsible for the exquisite frequency selectivity and sens... more Cochlear outer hair cells (OHCs) are responsible for the exquisite frequency selectivity and sensitivity of mammalian hearing. During development, the maturation of OHC afferent connectivity is refined by coordinated spontaneous Ca2+activity in both sensory and non-sensory cells. Calcium signaling in neonatal OHCs can be modulated by Oncomodulin (OCM, β-parvalbumin), an EF-hand calcium-binding protein. Here, we investigated whether OCM regulates OHC spontaneous Ca2+activity and afferent connectivity during development. Using a genetically encoded Ca2+sensor (GCaMP6s) expressed in OHCs in wild-type (Ocm+/+) and Ocm knockout (Ocm-/-) littermates, we found increased spontaneous Ca2+activity and upregulation of purinergic receptors in OHCs from GCaMP6s Ocm-/-cochlea immediately following birth. The afferent synaptic maturation of OHCs was delayed in the absence of OCM, leading to an increased number of ribbon synapses and afferent fibers on GCaMP6s Ocm-/-OHCs before hearing onset. We pr...
In cochlear outer hair cells (OHCs), a network of Ca2+ channels, pumps and Ca2+-binding proteins ... more In cochlear outer hair cells (OHCs), a network of Ca2+ channels, pumps and Ca2+-binding proteins (CaBPs) regulates the localization, spread, and magnitude of free Ca2+ ions. During early postnatal development, OHCs express three prominent mobile EF-hand CaBPs: oncomodulin (OCM), α-parvalbumin (APV) and sorcin. We have previously shown that deletion of Ocm (Ocm-/-) gives rise to progressive cochlear dysfunction in young adult mice. Here, we show that changes in Ca2+ signaling begin early in postnatal development of Ocm-/- mice. While mutant OHCs exhibit normal electrophysiological profiles compared to controls, their intracellular Ca2+ signaling is altered. The onset of OCM expression at postnatal day 3 (P3) causes a developmental change in KCl-induced Ca2+ transients in OHCs and leads to slower KCl-induced Ca2+ transients than those elicited in cells from Ocm-/- littermates. We compared OCM buffering kinetics with other CaBPs in animal models and cultured cells. In a double knockout...
Using an in vitro cochlear preparation from postnatal hamsters, spiral ganglion cells WXsl were l... more Using an in vitro cochlear preparation from postnatal hamsters, spiral ganglion cells WXsl were labeled retrogradely following extracellular injections of HRP into the cochlear nerve. In 24 cochleae from hamsters between postnatal days iP) 0 and IO, 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 ~pulations of SGCs. The hair-cell inne~ation 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 1HCs; by P 3, RFs contacted typically one or two IHC's. 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. Development; Cochlea; Organ of Corti; Neuronal morphology; Branching patterns introduction Recent studies of cochlear innervation in adult animals suggest there are significant differences between the neuroanatomy of the neonate and adult. In adult mammals, two fundamentally different types of spiral ganglion neurons contact separately the two types of hair cells. Type-I neurons send peripheral axons to terminate on inner hair cells (IHCs) via large radial fibers (RFs) and type-II neurons send their peripheral axons to terminate on outer hair cells (OHCs) via thin outer spiral fibers (OSFs). The type-1 neurons constitute between 90-95% of the afferent neuron population, whereas type-II neurons comprise the remainder of the afferent neuron population (Spoendlin, 1972; Spoendlin, 1974). The cochlear connections and response properties of type-1 neurons are well described Wang et al., 1965; Liberman and Oliver, 1984): most type-1 neurons contact only a single IHC. On the other hand, there is only a single report that gives a possible type of a response property for type-II neurons (Robertson, 1984). Recent anatomical studies show type-II neurons with very complicated cochlear connections:
Regeneration can occur in peripheral neurons after injury, but the mechanisms involved are not fu... more Regeneration can occur in peripheral neurons after injury, but the mechanisms involved are not fully delineated. Macrophages in dorsal root ganglia (DRGs) are involved in the enhanced regeneration that occurs after a conditioning lesion (CL), but how macrophages stimulate this response is not known. Oncomodulin (Ocm) has been proposed as a proregenerative molecule secreted by macrophages and neutrophils, is expressed in the DRG after axotomy, and stimulates neurite outgrowth by DRG neurons in culture. Wild-type (WT) and Ocm knockout (KO) mice were used to investigate whether Ocm plays a role in the CL response in DRG neurons after sciatic nerve transection. Neurite outgrowth was measured after 24 and 48 h in explant culture 7 d after a CL. Sciatic nerve regeneration was also measured in vivo 7 d after a CL and 2 d after a subsequent sciatic nerve crush. The magnitude of the increased neurite outgrowth following a CL was significantly smaller in explants from Ocm KO mice than in explants from WT mice. In vivo after a CL, increased regeneration was found in WT animals but not in KO animals. Macrophage accumulation and levels of interleukin-6 (IL-6) mRNA were measured in axotomized DRG from WT and Ocm KO animals, and both were significantly higher than in sham-operated ganglia. At 6 h after axotomy, Il-6 mRNA was higher in WT than in Ocm KO mice. Our data support the hypothesis that Ocm plays a necessary role in producing a normal CL response and that its effects possibly result in part from stimulation of the expression of proregenerative macrophage cytokines such as IL-6.
Pre-symptomatic studies in mouse models of the neurodegenerative motor neuron (MN) disease, Amyot... more Pre-symptomatic studies in mouse models of the neurodegenerative motor neuron (MN) disease, Amyotrophic Lateral Sclerosis (ALS) highlight early alterations in intrinsic and synaptic excitability and have supported an excitotoxic theory of MN death. However, a role for synaptic inhibition in disease development is not sufficiently explored among other mechanisms. Since inhibition plays a role in both regulating motor output and in neuroprotection, we examined the age-dependent anatomical changes in inhibitory presynaptic terminals on MN cell bodies using fluorescent immunohistochemistry for GAD67 (GABA) and GlyT2 (glycine) presynaptic proteins comparing ALS-vulnerable trigeminal jaw closer (JC) motor pools with the ALS-resistant extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Our results indicate differential patterns of temporal changes of these terminals in vulnerable versus resilient MNs and relative differences between SOD1G93A and wild-type (WT) MNs. Notably, we found ...
ABSTRACTCa2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncom... more ABSTRACTCa2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncomodulin (OCM) is a Ca2+ binding protein preferentially expressed in outer hair cells of the cochlea and few other specialized cell types. Here, we expand on our previous reports and show that OCM prevents early progressive hearing loss in mice of two different genetic backgrounds: CBA/CaJ and C57BI/6J. In both backgrounds, genetic disruption of Ocm leads to early progressive hearing loss as measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). In both strains, loss of Ocm reduced hearing across lifetime (hearing span) by more than 50% relative to wild type (WT). Even though the two WT strains have very different hearing spans, OCM plays a considerable and similar role within their genetic environment to regulate hearing function. The accelerated ARHL of the Ocm KO illustrates the importance of Ca2+ signaling in maintaining hearing health.
Ca2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncomodulin (... more Ca2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncomodulin (OCM) is a Ca2+ binding protein (CaBP) preferentially expressed in outer hair cells (OHCs) of the cochlea and few other specialized cell types. Here, we expand on our previous reports and show that OCM delays hearing loss in mice of two different genetic backgrounds: CBA/CaJ and C57Bl/6J. In both backgrounds, genetic disruption of Ocm leads to early progressive hearing loss as measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). In both strains, loss of Ocm reduced hearing across lifetime (hearing span) by more than 50% relative to wild type (WT). Even though the two WT strains have very different hearing spans, OCM plays a considerable and similar role within their genetic environment to regulate hearing function. The accelerated age-related hearing loss (ARHL) of the Ocm KO illustrates the importance of Ca2+ signaling in maintaining hearing ...
Prenatal Exposure to Tobacco and Alcohol Alters Development of the Neonatal Auditory System
Developmental Neuroscience, 2021
Prenatal exposures to alcohol (PAE) and tobacco (PTE) are known to produce adverse neonatal and c... more Prenatal exposures to alcohol (PAE) and tobacco (PTE) are known to produce adverse neonatal and childhood outcomes including damage to the developing auditory system. Knowledge of the timing, extent, and combinations of these exposures on effects on the developing system is limited. As part of the physiological measurements from the Safe Passage Study, Auditory Brainstem Responses (ABRs) and Transient Otoacoustic Emissions (TEOAEs) were acquired on infants at birth and one-month of age. Research sites were in South Africa and the Northern Plains of the U.S. Prenatal information on alcohol and tobacco exposure was gathered prospectively on mother/infant dyads. Cluster analysis was used to characterize three levels of PAE and three levels of PTE. Repeated-measures ANOVAs were conducted for newborn and one-month-old infants for ABR peak latencies and amplitudes and TEOAE levels and signal-to-noise ratios. Analyses controlled for hours of life at test, gestational age at birth, sex, sit...
The deletion of both the α9 and α10 nAChR subunits results in a different phenotype than the single deletion of the α9 or α10 subunit
Biochemical Pharmacology, 2013
Early deficits in GABA inhibition parallels an increase in L-type Ca2+ currents in the jaw motor neurons of SOD1G93A mouse model for ALS
Outer hair cells (OHCs) enhance the sensitivity and the frequency tuning of the mammalian cochlea... more Outer hair cells (OHCs) enhance the sensitivity and the frequency tuning of the mammalian cochlea. r Similar to the primary sensory receptor, the inner hair cells (IHCs), the mature functional characteristics of OHCs are acquired before hearing onset. r We found that OHCs, like IHCs, fire spontaneous Ca 2+-induced action potentials (APs) during immature stages of development, which are driven by Ca V 1.3 Ca 2+ channels. r We also showed that the development of low-and high-frequency hair cells is differentially regulated during pre-hearing stages, with the former cells being more strongly dependent on experience-independent Ca 2+ action potential activity.
EF-hand Ca 2+-binding protein family members, αand β-parvalbumins have been studied for decades. ... more EF-hand Ca 2+-binding protein family members, αand β-parvalbumins have been studied for decades. Yet, considerable information is lacking distinguishing functional differences between mammalian α-parvalbumin (PVALB) and oncomodulin (OCM), a branded β-parvalbumin. Herein, we provide an overview detailing the current body of work centered around OCM as an EF-Hand Ca 2+-binding protein and describe potential mechanisms of OCM function within the inner ear and immune cells. Additionally, we posit that OCM is evolutionarily distinct from PVALB and most other β-parvalbumins. This review summarizes recent studies pertaining to the function of OCM and emphasizes OCM as a parvalbumin possessing a unique cell and tissue distribution, Ca 2+ buffering capacity and phylogenetic origin.
Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine tuning and high ... more Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca2+ spikes during a narrow period of immature development. Simultaneously, waves of Ca2+ activity in the non-sensory greater epithelial ridge act, via ATP-induced activation of P2X receptors, to synchronize OHC firing, resulting in the refinement of their afferent innervation. In the absence of connexin channels Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity in OHCs requires experience-independent Ca2+ signals ...
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Papers by Dwayne Simmons