Publications by authors named "Juan D Goutman"

20 Publications

  • Page 1 of 1

Encoding sound in the cochlea: from receptor potential to afferent discharge.

J Physiol 2021 Feb 28. Epub 2021 Feb 28.

INGEBI (CONICET), (1428) C. A. Buenos Aires, Argentina.

Ribbon-class synapses in the ear achieve analog to digital transformation of a continuously graded membrane potential to all-or-none spikes. In mammals, several auditory nerve fibres (ANFs) carry information from each inner hair cell (IHC) to the brain in parallel. Heterogeneity of transmission among synapses contributes to the diversity of ANF sound-response properties. In addition to the place code for sound frequency and the rate code for sound level, there is also a temporal code. In series with cochlear amplification and frequency tuning, neural representation of temporal cues over a broad range of sound levels enables auditory comprehension in noisy multi-speaker settings. The IHC membrane time constant introduces a low-pass filter that attenuates fluctuations of the receptor potential above 1-2 kHz. The ANF spike generator adds a high-pass filter via its depolarization-rate threshold that rejects slow changes in the postsynaptic potential and its phasic response property that ensures one spike per depolarization. Synaptic transmission involves several stochastic subcellular processes between IHC depolarization and ANF spike generation, introducing delay and jitter that limits the speed and precision of spike timing. ANFs spike at a preferred phase of periodic sounds in a process called phase-locking that is limited to frequencies below a few kilohertz by both the IHC receptor potential and the jitter in synaptic transmission. During phase-locking to periodic sounds of increasing intensity, faster and facilitated activation of synaptic transmission and spike generation may be offset by presynaptic depletion of synaptic vesicles, resulting in relatively small changes in response phase. Here we review encoding of spike-timing at cochlear ribbon synapses.
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http://dx.doi.org/10.1113/JP279189DOI Listing
February 2021

Divide and conquer acoustic diversity.

EMBO J 2021 Mar 8;40(5):e107531. Epub 2021 Feb 8.

Instituto de Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" (CONICET), CA Buenos Aires, Argentina.

Humans can recognize differences in sound intensity of up to 6 orders of magnitude. However, it is not clear how this is achieved and what enables our auditory systems to encode such a gradient. Özçete & Moser (2021) report in this issue that the key to this lies in the synaptic heterogeneity within individual sensory cells in the inner ear.
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http://dx.doi.org/10.15252/embj.2020107531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917547PMC
March 2021

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

J Neurosci 2021 01 17;41(1):47-60. Epub 2020 Nov 17.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas, 1428 Buenos Aires, Argentina

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, Ca imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels. The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.
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http://dx.doi.org/10.1523/JNEUROSCI.1772-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786207PMC
January 2021

Enhancement of the Medial Olivocochlear System Prevents Hidden Hearing Loss.

J Neurosci 2018 08 20;38(34):7440-7451. Epub 2018 Jul 20.

Instituto de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos Aires, Argentina, and

Cochlear synaptopathy produced by exposure to noise levels that cause only transient auditory threshold elevations is a condition that affects many people and is believed to contribute to poor speech discrimination in noisy environments. These functional deficits in hearing, without changes in sensitivity, have been called hidden hearing loss (HHL). It has been proposed that activity of the medial olivocochlear (MOC) system can ameliorate acoustic trauma effects. Here we explore the role of the MOC system in HHL by comparing the performance of two different mouse models: an α9 nicotinic receptor subunit knock-out (KO; KO), which lacks cholinergic transmission between efferent neurons and hair cells; and a gain-of-function knock-in (KI; ' KI) carrying an α9 point mutation that leads to enhanced cholinergic activity. Animals of either sex were exposed to sound pressure levels that in wild-type produced transient cochlear threshold shifts and a decrease in neural response amplitudes, together with the loss of ribbon synapses, which is indicative of cochlear synaptopathy. Moreover, a reduction in the number of efferent contacts to outer hair cells was observed. In KO ears, noise exposure produced permanent auditory threshold elevations together with cochlear synaptopathy. In contrast, the ' KI was completely resistant to the same acoustic exposure protocol. These results show a positive correlation between the degree of HHL prevention and the level of cholinergic activity. Notably, enhancement of the MOC feedback promoted new afferent synapse formation, suggesting that it can trigger cellular and molecular mechanisms to protect and/or repair the inner ear sensory epithelium. Noise overexposure is a major cause of a variety of perceptual disabilities, including speech-in-noise difficulties, tinnitus, and hyperacusis. Here we show that exposure to noise levels that do not cause permanent threshold elevations or hair cell death can produce a loss of cochlear nerve synapses to inner hair cells as well as degeneration of medial olivocochlear (MOC) terminals contacting the outer hair cells. Enhancement of the MOC reflex can prevent both types of neuropathy, highlighting the potential use of drugs that increase α9α10 nicotinic cholinergic receptor activity as a pharmacotherapeutic strategy to avoid hidden hearing loss.
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http://dx.doi.org/10.1523/JNEUROSCI.0363-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104299PMC
August 2018

Compartmentalization of antagonistic Ca signals in developing cochlear hair cells.

Proc Natl Acad Sci U S A 2018 02 8;115(9):E2095-E2104. Epub 2018 Feb 8.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres," Consejo Nacional de Investigaciones Científicas y Técnicas (INGEBI-CONICET), 1428 Buenos Aires, Argentina;

During a critical developmental period, cochlear inner hair cells (IHCs) exhibit sensory-independent activity, featuring action potentials in which Ca ions play a fundamental role in driving both spiking and glutamate release onto synapses with afferent auditory neurons. This spontaneous activity is controlled by a cholinergic input to the IHC, activating a specialized nicotinic receptor with high Ca permeability, and coupled to the activation of hyperpolarizing SK channels. The mechanisms underlying distinct excitatory and inhibitory Ca roles within a small, compact IHC are unknown. Making use of Ca imaging, afferent auditory bouton recordings, and electron microscopy, the present work shows that unusually high intracellular Ca buffering and "subsynaptic" cisterns provide efficient compartmentalization and tight control of cholinergic Ca signals. Thus, synaptic efferent Ca spillover and cross-talk are prevented, and the cholinergic input preserves its inhibitory signature to ensure normal development of the auditory system.
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http://dx.doi.org/10.1073/pnas.1719077115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834711PMC
February 2018

Otoferlin acts as a Ca sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses.

Elife 2017 11 7;6. Epub 2017 Nov 7.

Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C-domain, Ca-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice () with lower Ca-binding affinity of the CC domain. The IHC ribbon synapse structure, synaptic Ca currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca concentration, by varying Ca influx through voltage-gated Ca-channels or Ca uncaging. Otoferlin thus functions as a Ca sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.
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http://dx.doi.org/10.7554/eLife.31013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700815PMC
November 2017

Mechanisms of synaptic depression at the hair cell ribbon synapse that support auditory nerve function.

Authors:
Juan D Goutman

Proc Natl Acad Sci U S A 2017 09 21;114(36):9719-9724. Epub 2017 Aug 21.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Héctor N. Torres" (INGEBI) (Consejo Nacional de Investigaciones Científicas y Técnicas), C1428ADN C. A. Buenos Aires, Argentina

Inner hair cells (IHCs) in the cochlea are the mammalian phono-receptors, transducing sound energy into graded changes in membrane potentials, the so called "receptor potentials." Ribbon synapses between IHCs and auditory nerve neurons are responsible for converting receptor potentials into spike rates. The characteristics of auditory nerve responses to sound have been described extensively. For instance, persistent acoustic stimulation produces sensory adaptation, which is revealed as a reduction in neuronal spike rate with time constants in the range of milliseconds to seconds. Since the amplitude of IHC receptor potentials is invariant during this period, the classic hypothesis pointed to vesicle depletion at the IHC as responsible for auditory adaptation. In this study, we observed that fast synaptic depression occurred in responses to stimuli of varying intensities. Nevertheless, release continued after this initial depression, via synaptic vesicles with slower exocytotic kinetics. Heterogeneity in kinetic elements, therefore, favored synaptic responses with an early peak and a sustained phase. The application of cyclothiazide (CTZ) revealed that desensitization of postsynaptic receptors contributed to synaptic depression, which was more pronounced during stronger stimulation. Thus, desensitization had a twofold effect: It abbreviated signaling between IHC and the auditory nerve and also balanced differences in decay kinetics between responses to different stimulation strengths. We therefore propose that both pre- and postsynaptic mechanisms at the IHC ribbon synapse contribute to synaptic depression at the IHC ribbon synapse and spike rate adaptation in the auditory nerve.
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http://dx.doi.org/10.1073/pnas.1706160114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594669PMC
September 2017

mGluR1 enhances efferent inhibition of inner hair cells in the developing rat cochlea.

J Physiol 2017 06 21;595(11):3483-3495. Epub 2017 Apr 21.

Department of Otolaryngology, Head and Neck Surgery, Department of Neuroscience, The Center for Hearing and Balance and the Center for Sensory Biology, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

Key Points: Spontaneous activity of the sensory inner hair cells shapes maturation of the developing ascending (afferent) auditory system before hearing begins. Just before the onset of hearing, descending (efferent) input from cholinergic neurons originating in the brainstem inhibit inner hair cell spontaneous activity and may further refine maturation. We show that agonist activation of the group I metabotropic glutamate receptor mGluR1 increases the strength of this efferent inhibition by enhancing the presynaptic release of acetylcholine. We further show that the endogenous release of glutamate from the inner hair cells may increase the strength of efferent inhibition via the activation of group I metabotropic glutamate receptors. Thus, before the onset of hearing, metabotropic glutamate signalling establishes a local negative feedback loop that is positioned to regulate inner hair cell excitability and refine maturation of the auditory system.

Abstract: Just before the onset of hearing, the inner hair cells (IHCs) receive inhibitory efferent input from cholinergic medial olivocochlear (MOC) neurons originating in the brainstem. This input may serve a role in the maturation of the ascending (afferent) auditory system by inhibiting spontaneous activity of the IHCs. To investigate the molecular mechanisms regulating these IHC efferent synapses, we combined electrical stimulation of the efferent fibres with patch clamp recordings from the IHCs to measure efferent synaptic strength. By examining evoked responses, we show that activation of metabotropic glutamate receptors (mGluRs) by general and group I-specific mGluR agonists enhances IHC efferent inhibition. This enhancement is blocked by application of a group I mGluR1-specific antagonist, indicating that enhancement of IHC efferent inhibition is mediated by group I mGluRs and specifically by mGluR1s. By comparing spontaneous and evoked responses, we show that group I mGluR agonists act presynaptically to increase neurotransmitter release without affecting postsynaptic responsiveness. Moreover, endogenous glutamate released from the IHCs also enhances IHC efferent inhibition via the activation of group I mGluRs. Finally, immunofluorescence analysis indicates that the efferent terminals are sufficiently close to IHC glutamate release sites to allow activation of mGluRs on the efferent terminals by glutamate spillover. Together, these results suggest that glutamate released from the IHCs activates group I mGluRs (mGluR1s), probably present on the efferent terminals, which, in turn, enhances release of acetylcholine and inhibition of the IHCs. Thus, mGluRs establish a local negative feedback loop positioned to regulate IHC activity and maturation of the ascending auditory system in the developing cochlea.
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http://dx.doi.org/10.1113/JP272604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451706PMC
June 2017

Whole-Cell Patch-Clamp Recording of Mouse and Rat Inner Hair Cells in the Intact Organ of Corti.

Methods Mol Biol 2016 ;1427:471-85

Department of Otorhinolaryngology, University Medical Center Groningen, Hanzeplein 1, Groningen, GZ, 9713, The Netherlands.

Whole-cell patch clamping is a widely applied method to record currents across the entire membrane of a cell. This protocol describes application of this method to record currents from the sensory inner hair cells in the intact auditory sensory epithelium, the organ of Corti, isolated from rats or mice. This protocol particularly outlines the basic equipment required, provides instructions for the preparation of solutions and small equipment items, and methodology for recording voltage-activated and evoked synaptic currents from the inner hair cells.
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http://dx.doi.org/10.1007/978-1-4939-3615-1_26DOI Listing
December 2017

Cochlear hair cells: The sound-sensing machines.

FEBS Lett 2015 Nov 31;589(22):3354-61. Epub 2015 Aug 31.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, "Dr. Héctor N Torres" (CONICET-UBA), Vuelta de Obligado 2490, Buenos Aires, Argentina; Tercera Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, Buenos Aires, Argentina. Electronic address:

The sensory epithelium of the mammalian inner ear contains two types of mechanosensory cells: inner (IHC) and outer hair cells (OHC). They both transduce mechanical force generated by sound waves into electrical signals. In their apical end, these cells possess a set of stereocilia representing the mechanosensing organelles. IHC are responsible for detecting sounds and transmitting the acoustic information to the brain by converting graded depolarization into trains of action potentials in auditory nerve fibers. OHC are responsible for the active mechanical amplification process that leads to the fine tuning and high sensitivity of the mammalian inner ear. This active amplification is the consequence of the ability of OHC to alter their cell length in response to changes in membrane potential, and is controlled by an efferent inhibitory innervation. Medial olivocochlear efferent fibers, originating in the brainstem, synapse directly at the base of OHC and release acetylcholine. A very special type of nicotinic receptor, assembled by α9α10 subunits, participates in this synapse. Here we review recent knowledge and the role of both afferent and efferent synapse in the inner ear.
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http://dx.doi.org/10.1016/j.febslet.2015.08.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641020PMC
November 2015

Benzodiazepine modulation of homomeric GABAAρ1 receptors: differential effects of diazepam and 4'-chlorodiazepam.

Eur J Pharmacol 2014 Nov 22;743:24-30. Epub 2014 Sep 22.

Laboratorio de Neurobiología Celular y Molecular, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" (INGEBI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, Ciudad Autónoma de Buenos Aires CP 1428, Argentina. Electronic address:

GABA(A) receptors (GABA(A)Rs) are ligand-gated ion channels that mediate inhibitory neurotransmission in the central nervous system (CNS). They are members of the Cys-loop receptor family and display marked structural and functional heterogeneity. Many GABA(A)Rs receptor subtypes are allosterically modulated by benzodiazepines (BDZs), which are drugs extensively used as anxiolytics, sedative-hypnotics and anticonvulsants. One high-affinity site and at least three additional low-affinity sites for BDZ recognition have been identified in several heteromeric and homomeric variants of the GABA(A)Rs (e.g.: α1β2γ2, α1β2/3, β3, etc.). However, the modulation of homomeric GABA(A)ρRs by BDZs was not previously revealed, and these receptors, for a long a time, were assumed to be fully insensitive to the actions of these drugs. In the present study, human homomeric GABA(A)ρ1 receptors were expressed in Xenopus oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of BDZs. GABA(A)ρ1 receptor-mediated responses were modulated by diazepam and 4'-chlorodiazepam in the micromolar range, in a concentration-dependent, voltage-independent and reversible manner. Diazepam produced potentiating effects on GABA-evoked Cl(-) currents and 4'-Cl diazepam induced biphasic effects depending on the GABA concentration, whereas Ro15-4513 and alprazolam were negative modulators. BDZ actions were insensitive to flumazenil. Other BDZs showed negligible activity at equivalent experimental conditions. Our results suggest that GABA(A)ρ1 receptor function can be selectively and differentially modulated by BDZs.
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http://dx.doi.org/10.1016/j.ejphar.2014.09.017DOI Listing
November 2014

Transmitter release from cochlear hair cells is phase locked to cyclic stimuli of different intensities and frequencies.

Authors:
Juan D Goutman

J Neurosci 2012 Nov;32(47):17025-35a

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad de Buenos Aires, C1428ADN Buenos Aires, Argentina.

The auditory system processes time and intensity through separate brainstem pathways to derive spatial location as well as other salient features of sound. The independent coding of time and intensity begins in the cochlea, where afferent neurons can fire action potentials at constant phase throughout a wide range of stimulus intensities. We have investigated time and intensity coding by simultaneous presynaptic and postsynaptic recording at the hair cell-afferent synapse from rats. Trains of depolarizing steps to the hair cell were used to elicit postsynaptic currents that occurred at constant phase for a range of membrane potentials over which release probability varied significantly. To probe the underlying mechanisms, release was examined using single steps to various command voltages. As expected for vesicular release, first synaptic events occurred earlier as presynaptic calcium influx grew larger. However, synaptic depression produced smaller responses with longer first latencies. Thus, during repetitive hair cell stimulation, as the hair cell is more strongly depolarized, increased calcium channel gating hurries transmitter release, but the resulting vesicular depletion produces a compensatory slowing. Quantitative simulation of ribbon function shows that these two factors varied reciprocally with hair cell depolarization (stimulus intensity) to produce constant synaptic phase. Finally, we propose that the observed rapid vesicle replenishment would help maintain the vesicle pool, which in turn would equilibrate with the stimulus intensity (and therefore the number of open Ca(2+) channels), so that for trains of different levels the average phase will be conserved.
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http://dx.doi.org/10.1523/JNEUROSCI.0457-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705563PMC
November 2012

Short-term facilitation modulates size and timing of the synaptic response at the inner hair cell ribbon synapse.

J Neurosci 2011 Jun;31(22):7974-81

Center for Hearing and Balance and Center for Sensory Biology, The Johns Hopkins School of Medicine, Department of Otolaryngology-Head and Neck Surgery and Neuroscience, Baltimore, Maryland 21205, USA.

Inner hair cells (IHCs) in the mammalian cochlea are able to continuously release neurotransmitter in the presence of constant stimuli. Nonetheless, strong synaptic depression is observed over the first few milliseconds of stimulation. This process most likely underlies adaptation in the auditory nerve. In the present study we demonstrate that under certain conditions of stimulation, facilitation can occur at the IHC ribbon synapse. Using simultaneous whole-cell, voltage-clamp recordings from IHCs and afferent fiber endings in excised postnatal rat cochleae, we stimulated IHCs with 2 ms long test depolarizations from a holding potential of -89 mV. Synaptic currents in afferent fibers occurred with high failure rates of ∼ 50%. However, when a pre-depolarization to values of -55 to -49 mV was implemented before the test pulse, success rates of the synaptic response increased to 100%, the strength of the synaptic response increased ∼ 2.8-fold, and synaptic latency was reduced by ∼ 50%. When calcium influx was minimized during pre-depolarization, none of these effects were found, suggesting that calcium influx during pre-depolarizations is required for synaptic conditioning. Similarly, in response to paired-pulse protocols, short term facilitation occurred. The response to the second stimulus increased up to ∼ 5-fold, and its latency was reduced by up to 35% compared to the response to the first stimulus. We propose that at the IHC resting membrane potential, the ribbon synapse operates in a constantly facilitated mode caused by Ca(2+) influx, optimizing the size and timing of the postsynaptic response in auditory nerve fibers.
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http://dx.doi.org/10.1523/JNEUROSCI.0604-11.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125715PMC
June 2011

Postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse.

J Vis Exp 2011 Feb 10(48). Epub 2011 Feb 10.

Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins School of Medicine, USA.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood. The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 μm in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.
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http://dx.doi.org/10.3791/2442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110417PMC
February 2011

Interaction between facilitation and depression at a large CNS synapse reveals mechanisms of short-term plasticity.

J Neurosci 2010 Feb;30(6):2007-16

Laboratory of Synaptic Mechanisms, Brain-Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.

The two fundamental forms of short-term plasticity, short-term depression and facilitation, coexist at most synapses, but little is known about their interaction. Here, we studied the interplay between short-term depression and facilitation at calyx of Held synapses. Stimulation at a "low" frequency of 10 or 20 Hz, which is in the range of the spontaneous activity of these auditory neurons in vivo, induced synaptic depression. Surprisingly, an instantaneous increase of the stimulation frequency to 100 or 200 Hz following the low-frequency train uncovered a robust facilitation of EPSCs relative to the predepressed amplitude level. This facilitation decayed rapidly ( approximately 30 ms) and depended on presynaptic residual Ca(2+), but it was not caused by Ca(2+) current facilitation. To probe the release probability of the remaining readily releasable vesicles following the low-frequency train we made presynaptic Ca(2+) uncaging experiments in the predepressed state of the synapse. We found that low-frequency stimulation depletes the fast-releasable vesicle pool (FRP) down to approximately 40% of control and that the remaining FRP vesicles are released with approximately 2-fold slower release kinetics, indicating a hitherto unknown intrinsic heterogeneity among FRP vesicles. Thus, vesicles with an intrinsically lower release probability predominate after low frequency stimulation and undergo facilitation during the onset of subsequent high-frequency trains. Facilitation in the predepressed state of the synapse might help to stabilize the amount of transmitter release at the onset of high-frequency firing at these auditory synapses.
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http://dx.doi.org/10.1523/JNEUROSCI.4378-09.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634054PMC
February 2010

Time course and calcium dependence of transmitter release at a single ribbon synapse.

Proc Natl Acad Sci U S A 2007 Oct 2;104(41):16341-6. Epub 2007 Oct 2.

Department of Otolaryngology, Head and Neck Surgery, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 824, Baltimore, MD 21205, USA.

At the first synapse in the auditory pathway, the receptor potential of mechanosensory hair cells is converted into a firing pattern in auditory nerve fibers. For the accurate coding of timing and intensity of sound signals, transmitter release at this synapse must occur with the highest precision. To measure directly the transfer characteristics of the hair cell afferent synapse, we implemented simultaneous whole-cell recordings from mammalian inner hair cells (IHCs) and auditory nerve fiber terminals that typically receive input from a single ribbon synapse. During a 1-s IHC depolarization, the synaptic response depressed >90%, representing the main source for adaptation in the auditory nerve. Synaptic depression was slightly affected by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization; however, it was mostly caused by reduced vesicular release. When the transfer function between transmitter release and Ca(2+) influx was tested at constant open probability for Ca(2+) channels (potentials >0 mV), a super linear relation was found. This relation is presumed to result from the cooperative binding of three to four Ca(2+) ions at the Ca(2+) sensor. However, in the physiological range for receptor potentials (-50 to -30 mV), the relation between Ca(2+) influx and afferent activity was linear, assuring minimal distortion in the coding of sound intensity. Changes in Ca(2+) influx caused an increase in release probability, but not in the average size of multivesicular synaptic events. By varying Ca(2+) buffering in the IHC, we further investigate how Ca(2+) channel and Ca(2+) sensor at this synapse might relate.
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http://dx.doi.org/10.1073/pnas.0705756104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2042208PMC
October 2007

Functional activation by central monoamines of human dopamine D(4) receptor polymorphic variants coupled to GIRK channels in Xenopus oocytes.

Eur J Pharmacol 2007 May 8;562(3):165-73. Epub 2007 Feb 8.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

We studied the functional activation of different polymorphic variants of the human dopamine D(4) receptors by the three major central monoamines, dopamine, noradrenaline and serotonin. Dopamine D(4) receptors carrying two (D4.2), four (D4.4) or seven (D4.7) repeats within the third intracellular domain were co-expressed with G protein-regulated inwardly rectifying potassium channels (GIRK1) in frog oocytes. All the dopamine D(4) receptor variants coupled to oocyte G(i/o) proteins and modulated co-expressed GIRK1 channels. Monoamine-induced responses were detected as increases in voltage-clamp recorded GIRK1 currents. Dopamine, noradrenaline as well as serotonin stimulated dopamine D(4) receptors. Dose-response analysis showed that dopamine and noradrenaline are full agonists whereas serotonin acted as partial agonist. Dopamine was 5-fold more potent on D4.2 and D4.7 (EC(50)=1 nM) than on D4.4 (EC(50)=5 nM) suggesting that the actions of dopamine and therapeutic drugs on dopamine D(4) receptors might vary among individuals depending on their repertoire of expressed alleles. In contrast, noradrenaline and serotonin did not discriminate among dopamine D(4) receptor variants (EC(50 NA)=50 nM, EC(50 5-HT)=1.5 microM). All monoamine effects were blocked by the specific dopaminergic D(4) antagonist (S)-(-)-4-[4-[2-(Isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide (PNU101387). Sequence analyses of dopamine D(4) receptors and related monoamine receptors revealed that dopamine D(4) receptors have most aminoacidic residues necessary for binding of dopamine, noradrenaline and serotonin. Our data indicate that dopamine D(4) receptors can be pharmacologically stimulated by any the three major central monoamines.
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http://dx.doi.org/10.1016/j.ejphar.2007.01.055DOI Listing
May 2007

Analysis of macroscopic ionic currents mediated by GABArho1 receptors during lanthanide modulation predicts novel states controlling channel gating.

Br J Pharmacol 2005 Dec;146(7):1000-9

Laboratorio de Neurobiología Celular y Molecular, INGEBI, CONICET, Departamento de Fisiología, Biología Molecular y Celular, FCEyN, Universidad de Buenos Aires, Vuelta de Obligado 2490, CP 1428 Ciudad Autónoma de Buenos Aires, Argentina.

Lanthanide-induced modulation of GABA(C) receptors expressed in Xenopus oocytes was studied. We obtained two-electrode voltage-clamp recordings of ionic currents mediated by recombinant homomeric GABArho(1) receptors and performed numerical simulations of kinetic models of the macroscopic ionic currents.GABA-evoked chloride currents were potentiated by La(3+), Lu(3+) and Gd(3+) in the micromolar range. Lanthanide effects were rapid, reversible and voltage independent. The degree of potentiation was reduced by increasing GABA concentration.Lu(3+) also induced receptor desensitization and decreased the deactivation rate of GABArho(1) currents. In the presence of 300 microM Lu(3+), dose-response curves for GABA-evoked currents showed a significant enhancement of the maximum amplitude and an increase of the apparent affinity. The rate of onset of TPMPA and picrotoxin antagonism of GABArho(1) receptors was modulated by Lu(3+). These results suggest that the potentiation of the anionic current was the result of a direct lanthanide-receptor interaction at a site capable of allosterically modulating channel properties. Based on kinetic schemes, which included a second open state and a nonconducting desensitized state that closely reproduced the experimental results, two nonexclusive probable models of GABArho(1) channels gating are proposed.
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http://dx.doi.org/10.1038/sj.bjp.0706411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1751227PMC
December 2005

Studies on the mechanisms of action of picrotoxin, quercetin and pregnanolone at the GABA rho 1 receptor.

Br J Pharmacol 2004 Feb 19;141(4):717-27. Epub 2004 Jan 19.

Departamento de Fisiología, Biología Molecular y Celular, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, República Argentina.

1. The mechanisms of action of antagonists of the gamma-aminobutyric acid C (GABA(C)) receptor picrotoxin, quercetin and pregnanolone were studied. 2. Ionic currents (chloride), mediated through human homomeric GABA rho(1) receptors expressed in Xenopus oocytes, were recorded by two-electrode voltage clamp. 3. Dose-response (D-R) curves and kinetic measurements of GABA rho(1) currents were carried out in the presence or absence of antagonists. Use-dependent actions were also evaluated. 4. Picrotoxin, quercetin and pregnanolone exerted noncompetitive actions. 5. IC(50) values measured at the EC(50) for GABA (1 microM) were as follows: picrotoxin 0.6+/-0.1 microM (Hill coefficient n=1.0+/-0.2); quercetin 4.4+/-0.4 microM (n=1.5+/-0.2); pregnanolone 2.1+/-0.5 microM (n=0.8+/-0.1). 6. These antagonists produced changes only in the slope of the linear current-voltage relationships, which was indicative of voltage-independent effects. 7. The effect of picrotoxin on GABA rho(1) currents was use-dependent, strongly relied on agonist concentration and showed a slow onset and offset. The mechanism was compatible with an allosteric inhibition and receptor activation was a prerequisite for antagonism. 8. The effect of quercetin was use-independent, showed relatively fast onset and offset, and resulted in a slowed time course of the GABA-evoked currents. 9. The effect of pregnanolone was use-independent, presented fast onset and a very slow washout, and did not affect current activation. 10. All the antagonists accelerated the time course of deactivation of the GABA rho(1) currents.
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http://dx.doi.org/10.1038/sj.bjp.0705657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1574239PMC
February 2004

Flavonoid modulation of ionic currents mediated by GABA(A) and GABA(C) receptors.

Eur J Pharmacol 2003 Feb;461(2-3):79-87

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires) (INGEBI (CONICET-UBA)), Capital Federal (1428), Buenos Aires, Argentina.

The modulation of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA-gated Cl(-) channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors expressed in Xenopus laevis oocytes in the micromolar range. alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by alpha(4)beta(2) neuronal nicotinic acetylcholine, serotonin 5-HT(3A) and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized alpha(1)beta(1)gamma(2s) GABA(A) receptors. Effects of apigenin and quercetin on alpha(1)beta(1)gamma(2s) GABA(A) receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation.
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http://dx.doi.org/10.1016/s0014-2999(03)01309-8DOI Listing
February 2003