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1.  Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor 
Molecular Biology and Evolution  2014;31(12):3250-3265.
Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.
doi:10.1093/molbev/msu258
PMCID: PMC4245820  PMID: 25193338
nicotinic receptors; calcium permeability; molecular evolution; hearing
2.  Excitability of Type II Cochlear Afferents 
The Journal of Neuroscience  2014;34(6):2365-2373.
Two types of sensory hair cells in the mammalian cochlea signal through anatomically distinct populations of spiral ganglion afferent neurons. The solitary inner hair cell ribbon synapse uses multivesicular release to trigger action potentials that encode acoustic timing, intensity, and frequency in each type I afferent. In contrast, cochlear outer hair cells (OHCs) have a far weaker effect on their postsynaptic targets, the type II spiral ganglion afferents. OHCs typically release single vesicles with low probability so that extensive summation is required to reach the relatively high action potential initiation threshold. These stark differences in synaptic transfer call into question whether type II neurons contribute to the cognitive perception of sound. Given the sparse and weak synaptic inputs from OHCs, the electrical properties of type II afferents are crucial in determining whether synaptic responses can sum to evoke an action potential to convey information to the cochlear nucleus. In the present work, dual-electrode recordings determined that type II afferents of rats have length constants that exceed the length of the distal, spiral process, enabling spatial summation from widespread OHCs. Focal application of tetrodotoxin localized the spike initiation zone to the type II proximal, radial process, near the spiral ganglion, in agreement with the high voltage threshold measured in the spiral process. These measured membrane properties were incorporated into a compartmental model of the type II neuron to demonstrate that neurotransmitter release from at least six OHCs is required to trigger an action potential in a type II neuron.
doi:10.1523/JNEUROSCI.3428-13.2014
PMCID: PMC3913877  PMID: 24501375
auditory; cochlea; dendrite; excitability; outer hair cell; spiral ganglion
3.  Purposeful Variable Selection and Stratification to Impute Missing FAST Data in Trauma Research 
Background
The Focused Assessment with Sonography for Trauma (FAST) exam is an important variable in many retrospective trauma studies. The purpose of this study was to devise an imputation method to overcome missing data for the FAST exam. Due to variability in patients’ injuries and trauma care, these data are unlikely to be missing completely at random (MCAR), raising concern for validity when analyses exclude patients with missing values.
Methods
Imputation was conducted under a less restrictive, more plausible missing at random (MAR) assumption. Patients with missing FAST exams had available data on alternate, clinically relevant elements that were strongly associated with FAST results in complete cases, especially when considered jointly. Subjects with missing data (32.7%) were divided into eight mutually exclusive groups based on selected variables that both described the injury and were associated with missing FAST values. Additional variables were selected within each group to classify missing FAST values as positive or negative, and correct FAST exam classification based on these variables was determined for patients with non-missing FAST values.
Results
Severe head/neck injury (odds ratio, OR=2.04), severe extremity injury (OR=4.03), severe abdominal injury (OR=1.94), no injury (OR=1.94), other abdominal injury (OR=0.47), other head/neck injury (OR=0.57) and other extremity injury (OR=0.45) groups had significant ORs for missing data; the other group odds ratio was not significant (OR=0.84). All 407 missing FAST values were imputed, with 109 classified as positive. Correct classification of non-missing FAST results using the alternate variables was 87.2%.
Conclusions
Purposeful imputation for missing FAST exams based on interactions among selected variables assessed by simple stratification may be a useful adjunct to sensitivity analysis in the evaluation of imputation strategies under different missing data mechanisms. This approach has the potential for widespread application in clinical and translational research and validation is warranted.
Level of Evidence
Level II Prognostic or Epidemiological
doi:10.1097/TA.0b013e31828fa51c
PMCID: PMC3744188  PMID: 23778515
FAST exam; imputation; MCAR; MAR
4.  Activation of Presynaptic GABAB(1a,2) Receptors Inhibits Synaptic Transmission at Mammalian Inhibitory Cholinergic Olivocochlear–Hair Cell Synapses 
The Journal of Neuroscience  2013;33(39):15477-15487.
The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses.
doi:10.1523/JNEUROSCI.2554-13.2013
PMCID: PMC3782624  PMID: 24068816
5.  Retrograde Facilitation of Efferent Synapses on Cochlear Hair Cells 
Cochlear inner hair cells (IHCs) are temporarily innervated by efferent cholinergic fibers prior to the onset of hearing. During low-frequency firing, these efferent synapses have a relatively low probability of transmitter release but facilitate strongly with repetitive stimulation. A retrograde signal from the hair cell to the efferent terminal contributes to this facilitation. When IHCs were treated with the ryanodine receptor agonist, cyclic adenosine phosphoribose (cADPR), release probability of the efferent terminal rose. This effect was quantified by computing the quantum content from a train of 100 suprathreshold stimuli to the efferent fibers. Quantum content was sevenfold higher when IHCs were treated with 100 μM cADPR (applied in the recording pipette). Since cADPR is membrane impermeant, this result implies that an extracellular messenger travels from the hair cell to the efferent terminal. cADPR is presumed to generate this messenger by increasing cytoplasmic calcium. Consistent with this presumption, voltage-gated calcium flux into the IHC also caused retrograde facilitation of efferent transmission. Retrograde facilitation was observed in IHCs of a vesicular glutamate transporter (VGlut3) null mouse and for wild-type rat hair cells subject to wide-spectrum glutamate receptor blockade, demonstrating that glutamate was unlikely to be the extracellular messenger. Rather, bath application of nitric oxide (NO) donors caused an increase in potassium-evoked efferent transmitter release while the NO scavenger carboxy-PTIO was able to prevent retrograde facilitation produced by cADPR or IHC depolarization. Thus, hair cell activity can drive retrograde facilitation of efferent input via calcium-dependent production of NO.
doi:10.1007/s10162-012-0361-0
PMCID: PMC3540278  PMID: 23183877
nitric oxide; hair cell; cochlea; efferent inhibition
6.  Efferent synapses return to inner hair cells in the aging cochlea 
Neurobiology of aging  2012;33(12):2892-2902.
Efferent innervation of the cochlea undergoes extensive modification early in development, but it is unclear if efferent synapses are modified by age, hearing loss, or both. Structural alterations in the cochlea affecting information transfer from the auditory periphery to the brain may contribute to age-related hearing deficits. We investigated changes to efferent innervation in the vicinity of inner hair cells (IHC) in young and old C57BL/6 mice using transmission electron microscopy to reveal increased efferent innervation of IHCs in older animals. Efferent contacts on IHCs contained focal pre-synaptic accumulations of small vesicles. Synaptic vesicle size and shape were heterogeneous. Postsynaptic cisterns were occasionally observed. Increased IHC efferent innervation was associated with a smaller number of afferent synapses per IHC, increased outer hair cell loss, and elevated auditory brainstem response thresholds. Efferent axons also formed synapses on afferent dendrites but with a reduced prevalence in older animals. Age-related reduction of afferent activity may engage signaling pathways that support the return to an immature state of efferent innervation of the cochlea.
doi:10.1016/j.neurobiolaging.2012.02.007
PMCID: PMC3398161  PMID: 22405044
Cochlear innervation; age-related hearing loss; olivocochlear; efferent; neural plasticity
7.  Synaptic Transfer from Outer Hair Cells to Type II Afferent Fibers in the Rat Cochlea 
Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the Type II fiber. The present work addresses these questions using whole-cell recordings from the spiral process of type II afferents that run below OHCs in the apical turn of young (5–9 days postnatal) rat cochlea. A ‘high potassium puffer’ was used to elicit calcium action potentials from individual OHCs and thereby show that the average probability of transmitter release was 0.26 (range 0.02 to 0.73). Electron microscopy showed relatively few vesicles tethered to ribbons in equivalent OHCs. A ‘receptive field’ map for individual type II fibers was constructed by successively puffing onto OHCs along the cochlear spiral, up to 180 µm from the recording pipette. These revealed a conservative estimate of 7 presynaptic OHCs per type II fiber (range 1–11). EPSCs evoked from presynaptic OHCs separated by more than 100 µm did not differ in amplitude or waveform, implying that the type II fiber’s length constant exceeded the length of the synaptic input zone. Taken together these data suggest that type II fibers could communicate centrally by maximal activation of their entire pool of presynaptic OHCs.
doi:10.1523/JNEUROSCI.6194-11.2012
PMCID: PMC3433252  PMID: 22787038
8.  Abdominal aorta as a recipient artery: Using a free latissimus dorsi myocutaneous flap to close hip and pelvic defects 
INTRODUCTION
Free tissue transfer (FTT) is now a common procedure in many surgical centres around the world and it has shown well established results especially in the field of reconstructive surgery. The choice of FTT depends on the size of defect, nature of tissue, length of pedicle and donor site morbidity. Notwithstanding, FTT is complex and always depending on a sufficient recipient vessel.
PRESENTATION OF CASE
Herein, we report a case in which the abdominal aorta was used as arterial recipient vessel for microvascular transfer of a free latissimus dorsi myocutaneous flap. It was utilized to reconstruct an extensive pelvic and hip defect following a massive gas gangrene with a prior debridement of other potential recipient vessels.
DISCUSSION
In this case, the patient had a large defect that demanded a choice of a large flap such as the free latissimus dorsi myocutaneous flap. The iliac system has been sacrificed during the debridement procedure together with other potential recipient vessels. In the presented case, arterial anastomosis of the free latissimus dorsi myocutaneous flap was performed to the distal part of the aorta without complications.
CONCLUSION
Using the abdominal aorta as a recipient arterial vessel seems to be a reliable alternative that should be considered in difficult reconstructive scenarios such as the “vessel-depleted” pelvis.
doi:10.1016/j.ijscr.2012.09.008
PMCID: PMC3537943  PMID: 23103629
Abdominal aorta; Free latissimus dorsi flap; Recipient vessel
9.  Modulation of hair cell efferents 
Hearing research  2010;279(1-2):1-12.
Outer hair cells (OHCs) amplify the sound-evoked motion of the basilar membrane to enhance acoustic sensitivity and frequency selectivity. Medial olivocochlear (MOC) efferents inhibit OHCs to reduce the sound-evoked response of cochlear afferent neurons. OHC inhibition occurs through the activation of postsynaptic α9α10 nicotinic receptors tightly coupled to calcium-dependent SK2 channels that hyperpolarize the hair cell. MOC neurons are cholinergic but a number of other neurotransmitters and neuromodulators have been proposed to participate in efferent transmission, with emerging evidence for both pre- and postsynaptic effects. Cochlear inhibition in vivo is maximized by repetitive activation of the efferents, reflecting facilitation and summation of transmitter release onto outer hair cells. This review summarizes recent studies on cellular and molecular mechanisms of cholinergic inhibition and the regulation of those molecular components, in particular the involvement of intracellular calcium. Facilitation at the efferent synapse is compared in a variety of animals, as well as other possible mechanisms of modulation of ACh release. These results suggest that short-term plasticity contributes to effective cholinergic inhibition of hair cells.
doi:10.1016/j.heares.2010.12.018
PMCID: PMC3125486  PMID: 21187136
10.  Onset of cholinergic efferent synaptic function in sensory hair cells of the rat cochlea 
In the developing mammalian cochlea, the sensory hair cells receive efferent innervation originating in the superior olivary complex. This input is mediated by α9/α10 nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent SK2 potassium channels. We examined the acquisition of this cholinergic efferent input using whole-cell voltage-clamp recordings from inner hair cells (IHCs) in acutely excised apical turns of the rat cochlea from embryonic day 21 to postnatal day 8 (P8). Responses to 1 mM acetylcholine (ACh) were detected from P0 on in almost every IHC. The ACh-activated current amplitude increased with age and demonstrated the same pharmacology as α9-containing nAChRs. Interestingly, at P0, the ACh response was not coupled to SK2 channels, so that the initial cholinergic response was excitatory and could trigger action potentials in IHCs. Coupling to SK current was detected earliest at P1 in a subset of IHCs and by P3 in every IHC studied. Clustered nAChRs and SK2 channels were found on IHCs from P1 on using Alexa Fluor 488 conjugated α-bungarotoxin and SK2 immunohistochemistry. The number of nAChRs clusters increased with age to 16 per IHC at P8. Cholinergic efferent synaptic currents first appeared in a subset of IHCs at P1 and by P3 in every IHC studied, contemporaneously with ACh-evoked SK currents, suggesting that SK2 channels may be necessary at onset of synaptic function. An analogous pattern of development was observed for the efferent synapses that form later (P6–P8) on outer hair cells in the basal cochlea.
doi:10.1523/JNEUROSCI.2743-11.2011
PMCID: PMC3213862  PMID: 22016543
onset of synaptic function; sensory hair cell; efferent synapse; synaptogenesis; α9/α10 nicotinic acetylcholine receptor
11.  Short term synaptic plasticity regulates the level of olivocochlear inhibition to auditory hair cells 
In the mammalian inner ear, the gain control of auditory inputs is exerted by medial olivocochlear (MOC) neurons that innervate cochlear outer hair cells (OHCs). OHCs mechanically amplify the incoming sound waves by virtue of their electromotile properties while the MOC system reduces the gain of auditory inputs by inhibiting OHCs function. How this process is orchestrated at the synaptic level remains unknown. In the present study, MOC firing was evoked by electrical stimulation in an isolated mouse cochlear preparation, while OHCs postsynaptic responses were monitored by whole-cell recordings. These recordings confirmed that electrically evoked inhibitory postsynaptic currents (eIPSCs) are mediated solely by α9α10 nicotinic acetylcholine receptors (nAChRs) functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1Hz. However, as the stimulation frequency was raised, the reliability of release increased due to presynaptic facilitation. In addition, the relatively slow decay of eIPSCs gave rise to temporal summation at stimulation frequencies above 10 Hz. The combined effect of facilitation and summation resulted in a frequency-dependent increase in the average amplitude of inhibitory currents in OHCs. Thus, we have demonstrated that short-term plasticity is responsible for shaping MOC inhibition and, therefore, encodes the transfer function from efferent firing frequency to the gain of the cochlear amplifier.
doi:10.1523/JNEUROSCI.6788-10.2011
PMCID: PMC3224807  PMID: 21994392
short-term synaptic plasticity; gain control; cochlea; outer hair cells; efferent innervation
12.  The diverse roles of ribbon synapses in sensory neurotransmission 
Nature reviews. Neuroscience  2010;11(12):812-822.
Summary
Sensory synapses of the visual and auditory systems must faithfully encode a wide dynamic range of graded signals, and must be capable of sustained transmitter release over long periods of time. Functionally and morphologically, these sensory synapses are unique: their active zones are specialized in several ways for sustained, rapid vesicle exocytosis, but their most striking feature is an organelle called the synaptic ribbon, which is a proteinaceous structure that extends into the cytoplasm at the active zone and tethers a large pool of releasable vesicles. But precisely how does the ribbon function to support tonic release at these synapses? Recent genetic and biophysical advances have begun to open the ‘black box’ of the synaptic ribbon with some surprising findings, and promise to resolve its function in vision and hearing.
doi:10.1038/nrn2924
PMCID: PMC3065184  PMID: 21045860
13.  A Rare Case of Toxic Epidermal Necrolysis with Unexpected Fever Resulting from Dengue Virus 
Case Reports in Dermatology  2010;2(3):189-194.
Toxic epidermal necrolysis (TEN), also known as Lyell's syndrome, is a life-threatening disease with common development of large wounds. Thus, affected patients are usually treated in specialized centers. Herein, we present a case of TEN in a patient infected with human immunodeficiency virus with the additional, unexpected diagnosis of dengue fever. In this context, we discuss cause, diagnosis, pathology, and treatment of TEN and highlight the role of rare and unexpected findings, as in this case an additional tropical virus infection. We underpin the importance of an interdisciplinary approach involving dermatologists, ophthalmologists, intensive care physicians, burn specialists and various other departments and emphasize the challenge of TEN treatment, especially if rare pathological findings occur.
doi:10.1159/000322963
PMCID: PMC3079534  PMID: 21509201
Toxic epidermal necrolysis; Dengue fever; Human immunodeficiency virus
14.  BK Channels Mediate Cholinergic Inhibition of High Frequency Cochlear Hair Cells 
PLoS ONE  2010;5(11):e13836.
Background
Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K+ channels that control the membrane potential and also ligand-gated K+ channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.
Methodology/Principal
Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K+ currents.
Conclusions/Significance
Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.
doi:10.1371/journal.pone.0013836
PMCID: PMC2973960  PMID: 21079807
15.  The Nicotinic Receptor of Cochlear Hair Cells: A Possible Pharmacotherapeutic Target? 
Biochemical pharmacology  2009;78(7):712-719.
Mechanosensory hair cells of the organ of Corti transmit information regarding sound to the central nervous system by way of peripheral afferent neurons. In return, the central nervous system provides feedback and modulates the afferent stream of information through efferent neurons. The medial olivocochlear efferent system makes direct synaptic contacts with outer hair cells and inhibits amplification brought about by the active mechanical process inherent to these cells. This feedback system offers the potential to improve the detection of signals in background noise, to selectively attend to particular signals, and to protect the periphery from damage caused by overly loud sounds. Acetylcholine released at the synapse between efferent terminals and outer hair cells activates a peculiar nicotinic cholinergic receptor subtype, the α9α10 receptor. At present no pharmacotherapeutic approaches have been designed that target this cholinergic receptor to treat pathologies of the auditory system. The potential use of α9α10 selective drugs in conditions such as noise-induced hearing loss, tinnitus and auditory processing disorders is discussed.
doi:10.1016/j.bcp.2009.05.023
PMCID: PMC2737545  PMID: 19481062
nicotinic cholinergic receptors; noise trauma; cochlea; tinnitus; efferent feedback
16.  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
17.  Hair Cell Afferent Synapses 
Current opinion in neurobiology  2008;18(4):389-395.
This review will cover advances in the study of hair cell afferent synaptic function occurring between 2005 and 2008. During this time capacitance measurements of vesicular fusion have continued to be refined, optical methods have added insights regarding vesicle trafficking, and paired intracellular recordings have established the transfer function of the afferent synapse at high resolution. Further, genes have been identified with forms of deafness known as auditory neuropathy, and their role in afferent signaling explored in mouse models. With these advances, our view of the hair cell afferent synapse has continued to be refined, and surprising properties have been revealed that emphasize the unique role of this structure in neural function.
doi:10.1016/j.conb.2008.09.006
PMCID: PMC2860955  PMID: 18824101
18.  The Postsynaptic Function of Type II Cochlear Afferents 
Nature  2009;461(7267):1126-1129.
The mammalian cochlea is innervated by two classes of sensory neurons. Type I neurons make up 90-95% of the cochlear nerve and contact single inner hair cells (IHCs) to provide acoustic analysis as we know it. In contrast, the far less numerous Type II neurons arborize extensively among outer hair cells (OHCs) 1,2 and supporting cells3,4. Their scarcity, and smaller caliber axons, have made them the subject of much speculation, but little experimental progress for the past 50 years. Here we record from Type II fibers near their terminal arbors under OHCs to show that these receive excitatory glutamatergic synaptic input. The Type II peripheral arbor conducts action potentials, but the small and infrequent glutamatergic excitation implies a requirement for strong acoustic stimulation. Further, we show that Type II neurons are excited by adenosine tri-phosphate (ATP). Exogenous ATP depolarized Type II neurons both directly, and by evoking glutamatergic synaptic input 5. The present results prove that Type II neurons function as cochlear afferents, and can be modulated by ATP. The lesser magnitude of synaptic drive dictates a fundamentally different role in auditory signaling from that of Type I afferents.
doi:10.1038/nature08487
PMCID: PMC2785502  PMID: 19847265
19.  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
20.  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
21.  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
22.  Topological and Developmental Gradients of Calbindin Expression in the Chick's Inner Ear  
Mobile intracellular calcium buffers play an important role in regulating calcium flux into mechanosensory hair cells and calbindin D-28k is expressed at high levels in the chick's basilar papilla. We have used RT-PCR, in situ hybridization, and immunohistology to demonstrate that calbindin expression varies systematically according to hair cell position and developmental age. RT-PCR using microdissected quarters of the posthatch basilar papilla showed that mRNA levels were lowest in the (low frequency) apex and higher in basal quadrants. In situ hybridization revealed calbindin mRNA in posthatch hair cells and supporting cells, with more intense labeling of hair cells from basal (high frequency) positions. A similar topology was obtained with calbindin antibodies. Neither calbindin riboprobe nor calbindin antibody labeled cochlear neurons. In contrast, a subset of large vestibular neurons and their calyciform endings onto Type I vestibular hair cells were strongly labeled by the calbindin antibody, while vestibular hair cells were negative for calbindin immunoreactivity. Likewise, calbindin in situ hybridization was negative for vestibular hair cells but positive in a subset of larger vestibular neurons. Calbindin mRNA was detected in hair cells of the basal half of the papilla at embryonic day 10 (E10) and calbindin immunoreactivity was detected at E12. Hair cells in the apical half of the papilla had equivalent calbindin expression two days later. Immunoreactivity appeared in abneural supporting cells days later than in hair cells, and not until E20 in neurally located supporting cells. These results demonstrate that calbindin message and protein levels are greater in high-frequency hair cells. This "tonotopic" gradient may result from the stabilization of a basal-to-apical developmental gradient and could be related at least in part to calcium channel expression along this axis.
doi:10.1007/s101620010071
PMCID: PMC3202366  PMID: 12083720
cochlea; hair cells; vestibule; mechanotransduction; embryonic; calcium buffering

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