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1.  Activity of nAChRs Containing α9 Subunits Modulates Synapse Stabilization via Bidirectional Signaling Programs 
Developmental neurobiology  2009;69(14):931.
Although the synaptogenic program for cholinergic synapses of the neuromuscular junction is well known, little is known of the identity or dynamic expression patterns of proteins involved in non-neuromuscular nicotinic synapse development. We have previously demonstrated abnormal presynaptic terminal morphology following loss of nicotinic acetylcholine receptor (nAChR) α9 subunit expression in adult cochleae. However, the molecular mechanisms underlying these changes have remained obscure. To better understand synapse formation and the role of cholinergic activity in the synaptogenesis of the inner ear, we exploit the nAChR α9 subunit null mouse. In this mouse, functional acetylcholine (ACh) neurotransmission to the hair cells is completely silenced. Results demonstrate a premature, effusive innervation to the synaptic pole of the outer hair cells in α9 null mice coinciding with delayed expression of cell adhesion proteins during the period of effusive contact. Collapse of the ectopic innervation coincides with an age-related hyperexpression pattern in the null mice. In addition, we document changes in expression of presynaptic vesicle recycling/trafficking machinery in the α9 null mice that suggests a bidirectional information flow between the target of the neural innervation (the hair cells) and the presynaptic terminal that is modified by hair cell nAChR activity. Loss of nAChR activity may alter transcriptional activity, as CREB binding protein expression is decreased coincident with the increased expression of N-Cadherin in the adult α9 null mice. Finally, by using mice expressing the nondesensitizing α9 L9′T point mutant nAChR subunit, we show that increased nAChR activity drives synaptic hyperinnervation.
doi:10.1002/dneu.20753
PMCID: PMC2819290  PMID: 19790106
cochlea; acetylcholine receptors; synapse development; olivocochlear system; alpha9 nAChR
2.  Constitutive Expression of the α10 Nicotinic Acetylcholine Receptor Subunit Fails to Maintain Cholinergic Responses in Inner Hair Cells After the Onset of Hearing 
Efferent inhibition of cochlear hair cells is mediated by α9α10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the α10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 α10 transgenic mice backcrossed to a Chrna10−/− background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the α10 transgene restored nAChR function. However, in the α10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.
doi:10.1007/s10162-009-0173-z
PMCID: PMC3084387  PMID: 19452222
nicotinic cholinergic receptors; efferent medial olivocochlear; SK2 channel; acetylcholine; transgenic mice
3.  SK2 channels are required for function and long-term survival of efferent synapses on mammalian outer hair cells 
Cochlear hair cells use SK2 currents to shape responses to cholinergic efferent feedback from the brain. Using SK2-/- mice, we demonstrate that, in addition to their previously defined role in modulating hair cell membrane potentials, SK2 channels are necessary for long-term survival of olivocochlear fibers and synapses. Loss of the SK2 gene also results in loss of electrically driven olivocochlear effects in vivo, and down regulation of ryanodine receptors involved in calcium-induced calcium release, the main inducer of nAChR evoked SK2 activity. Generation of double-null mice lacking both the α10 nAChR gene, loss of which results in hypertrophied olivocochlear terminals, and the SK2 gene, recapitulates the SK2-/- synaptic phenotype and gene expression, and also leads to down regulation of α9 nAChR gene expression. The data suggest a hierarchy of activity necessary to maintain early olivocochlear synapses at their targets, with SK2 serving an epistatic, upstream, role to the nAChRs.
doi:10.1016/j.mcn.2008.08.011
PMCID: PMC2661972  PMID: 18848895
cochlea; small conductance potassium channels; nicotinic receptors; synaptic degeneration; synaptogenesis
4.  A Point Mutation in the Hair Cell Nicotinic Cholinergic Receptor Prolongs Cochlear Inhibition and Enhances Noise Protection 
PLoS Biology  2009;7(1):e1000018.
The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9′T line of knockin mice with a threonine for leucine change (L9′T) at position 9′ of the second transmembrane domain of the α9 nicotinic cholinergic subunit, rendering α9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9′T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9′T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the α9α10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9L9′T/L9′T mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter α9α10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.
Author Summary
Nicotinic cholinergic receptors are essential to higher order brain function. Structurally, these operate through a myriad of ligand-gated pentameric arrangements of different homologous subunits. Here, we report progress in understanding the structural properties of a neuronal nicotinic receptor at the synapse. Receptors assembled from two nicotinic cholinergic subunits (α9 and α10) serve exclusively at the synapse between central nervous system descending fibers and cochlear hair cells. This enabled us to show direct causality between a point mutation of the α9 subunit, and predicted alterations in the synaptic strength in sensory hair cells of the cochlea of α9 point mutant mice. Furthermore, this single mutation results in profound enhancement of central nervous system feedback to the cochlea. And finally, as a consequence, mutant mice possessing this altered receptor have substantially improved resistance to traumatic sound. Thus, central neuronal feedback on cochlear hair cells provides an opportunity to define one specific role that nicotinic receptors can play in the nervous system, enabling study from biophysical to behavioral levels and promoting a target for the prevention of noise-induced hearing loss.
A point mutation in the cochlear hair cell nicotinic cholinergic receptor leads to strengthened central nervous system feedback to the cochlea and enhances protection from noise-induced hearing loss.
doi:10.1371/journal.pbio.1000018
PMCID: PMC2628405  PMID: 19166271
5.  Ryanodine is a Positive Modulator of Acetylcholine Receptor Gating in Cochlear Hair Cells 
The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include ‘ryanodine receptors’, whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the α9α10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the α9α10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 μM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken ‘short’ hair cells likewise was enhanced in the presence of 100 μM ryanodine. This facilitatory effect on current through the AChR could enhance brief (∼1 s) activation of associated calcium-dependent K+ (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate α9α10-mediated responses and for potential inner ear therapeutics based on this interaction.
doi:10.1007/s10162-007-0090-y
PMCID: PMC2538347  PMID: 17647061
ryanodine; nicotinic receptors; α9α10 receptors; ion channels; acetylcholine; efferent system
6.  Linopirdine Blocks α9α10-Containing Nicotinic Cholinergic Receptors of Cochlear Hair Cells 
Studies of the electrophysiological response to acetylcholine (ACh) in mammalian outer hair cells (OHCs) are hindered by the presence of a large potassium current, IK,n, most likely mediated by channels containing the KCNQ4 subunit. Since IK,n can be blocked by linopirdine, cholinergic effects might be better revealed in the presence of this compound. The aim of the present work was to study the effects of linopirdine on the ACh-evoked responses through α9α10-containing native and recombinant nicotinic cholinergic receptors. Responses to ACh were blocked by linopirdine in both OHCs and inner hair cells (IHCs) of rats at postnatal days 21–27 (OHCs) and 9–11 (IHCs). In addition, linopirdine blocked responses of recombinant α9α10 nicotinic cholinergic receptors (nAChRs) in a concentration-dependent manner with an IC50 of 5.2 μM. Block by linopirdine was readily reversible, voltage independent, and surmountable at high concentrations of ACh, thus suggestive of a competitive type of interaction with the receptor. The present results contribute to the pharmacological characterization of α9α10-containing nicotinic receptors and indicate that linopirdine should be used with caution when analyzing the cholinergic sensitivity of cochlear hair cells.
doi:10.1007/s10162-004-4025-6
PMCID: PMC2504548  PMID: 15492885
nicotinic receptors; linopirdine; ion channels; acetylcholine; K+ channels

Results 1-6 (6)