Developmental synaptic changes at the transient olivocochlear-inner hair cell synapse

In the mature mammalian cochlea, inner hair cells (IHCs) are mainly innervated by afferent fibers that convey sound information to the CNS. During postnatal development, however, medial olivocochlear (MOC) efferent fibers transiently innervate the IHCs. The MOC-IHC synapse, functional from postnatal day 0 (P0) to hearing onset (P12), undergoes dramatic changes in the sensitivity to acetylcholine (ACh) and in the expression of key postsynaptic proteins. To evaluate whether there are associated changes in the properties of ACh release during this period, we used a cochlear preparation from mice of either sex at P4, P6-P7, and P9-P11 and monitored transmitter release from MOC terminals in voltage-clamped IHCs in the whole-cell configuration. The quantum content increased 5.6× from P4 to P9-P11 due to increases in the size and replenishment rate of the readily releasable pool of synaptic vesicles without changes in their probability of release or quantum size. This strengthening in transmission was accompanied by changes in short-term plasticity properties, which switched from facilitation at P4 to depression at P9-P11. We have previously shown that at P9-P11, ACh release is supported by P/Q- and N-type voltage-gated calcium channels (VGCCs) and negatively regulated by BK potassium channels activated by Ca2+ influx through L-type VGCCs. We now show that at P4 and P6-P7, release is mediated by P/Q-, R- and L-type VGCCs. Interestingly, L-type VGCCs have a dual role: they both support release and fuel BK channels, suggesting that at immature stages presynaptic proteins involved in release are less compartmentalized.Fil: Kearney, Graciela Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Zorrilla de San Martín, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vattino, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Katz, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Functional postnatal maturation of the medial olivocochlear efferent-outer hair cell synapse

The organ of Corti, the auditory mammalian sensory epithelium, contains two types of mechanotransducer cells, inner and outer hair cells (IHCs and OHCs, respectively). IHCs are involved in conveying acoustic stimuli to the central nervous system while OHCs are implicated in the fine tuning and amplification of sounds. OHCs are innervated by medial olivocochlear (MOC) cholinergic efferent fibers. The functional characteristics of the MOC-OHC synapse during maturation were assessed by electrophysiological and pharmacological methods in mouse organs of Corti at postnatal days (P)11-13, hearing onset in altricial rodents, and at P20-22 when the OHCs are morphologically and functionally mature. Synaptic currents were recorded in whole-cell voltage-clamped OHCs while electrically stimulating the MOC fibers. A progressive increase in the number of functional MOC-OHC synapses, as well as in their strength and efficacy was observed between P11-13 and P20-22. At hearing onset, the MOC-OHC synapse presented facilitation upon MOC fibers high-frequency stimulation which disappeared at mature stages. In addition, important changes were found in the VGCC that are coupled to transmitter release. Ca2+ flowing in through L-type VGCCs contribute to trigger ACh release together with P/Q- and R-type VGCCs at P11-13 but not at P20-22. Interestingly, N-type VGCCs were found to be involved in this process at P20-22 but not at hearing onset. Moreover, the degree of compartmentalization of calcium channels with respect to BK channels and presynaptic release components significantly increased from P11-13 to P20-22. These results suggest that the MOC-OHC synapse is immature at the onset of hearing.Fil: Vattino, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Katz, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Positive modulation of the a9a10 nicotinic cholinergic receptor by ascorbic acid

Background and Purpose: The activation of α9α10 nicotinic cholinergic receptors (nAChRs) present at the synapse between efferent olivocochlear fibres and cochlear hair cells can prevent acoustic trauma. Hence, pharmacological potentiators of these receptors could be useful therapeutically. In this work, we characterize ascorbic acid as a positive modulator of recombinant α9α10 nAChRs. Experimental Approach: ACh-evoked responses were analysed under two-electrode voltage-clamp recordings in Xenopus laevis oocytes injected with α9 and α10 cRNAs. Key Results: Ascorbic acid potentiated ACh responses in X. laevis oocytes expressing α9α10 (but not α4β2 or α7) nAChRs, in a concentration-dependent manner, with an effective concentration range of 1–30 mM. The compound did not affect the receptor's current–voltage profile nor its apparent affinity for ACh, but it significantly enhanced the maximal evoked currents (percentage of ACh maximal response, 240 ± 20%). This effect was specific for the L form of reduced ascorbic acid. Substitution of the extracellular cysteine residues present in loop C of the ACh binding site did not affect the potentiation. Ascorbic acid turned into a partial agonist of α9α10 nAChRs bearing a point mutation at the pore domain of the channel (TM2 V13′T mutant). A positive allosteric mechanism of action rather than an antioxidant effect of ascorbic acid is proposed. Conclusions and Implications: The present work describes one of the few agents that activates or potentiates α9α10 nAChRs and leads to new avenues for designing drugs with potential therapeutic use in inner ear disorders.Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; Argentin

Strengthening of the Efferent Olivocochlear System Leads to Synaptic Dysfunction and Tonotopy Disruption of a Central Auditory Nucleus

The auditory system in many mammals is immature at birth but precisely organized in adults. Spontaneous activity in the inner ear plays a critical role in guiding this maturation process. This is shaped by an efferent pathway that descends from the brainstem and makes transient direct synaptic contacts with inner hair cells. In this work, we used an α9 cholinergic nicotinic receptor knock-in mouse model (of either sex) with enhanced medial efferent activity (Chrna9L9'T, L9'T) to further understand the role of the olivocochlear system in the correct establishment of auditory circuits. Wave III of auditory brainstem responses (which represents synchronized activity of synapses within the superior olivary complex) was smaller in L9'T mice, suggesting a central dysfunction. The mechanism underlying this functional alteration was analyzed in brain slices containing the medial nucleus of the trapezoid body (MNTB), where neurons are topographically organized along a mediolateral (ML) axis. The topographic organization of MNTB physiological properties observed in wildtype (WT) was abolished in L9'T mice. Additionally, electrophysiological recordings in slices indicated MNTB synaptic alterations. In vivo multielectrode recordings showed that the overall level of MNTB activity was reduced in the L9'T The present results indicate that the transient cochlear efferent innervation to inner hair cells during the critical period before the onset of hearing is involved in the refinement of topographic maps as well as in setting the properties of synaptic transmission at a central auditory nucleus.SIGNIFICANCE STATEMENT Cochlear inner hair cells of altricial mammals display spontaneous electrical activity before hearing onset. The pattern and firing rate of these cells are crucial for the correct maturation of the central auditory pathway. A descending efferent innervation from the CNS contacts the hair cells during this developmental window. The present work shows that genetic enhancement of efferent function disrupts the orderly topographic distribution of biophysical and synaptic properties in the auditory brainstem and causes severe synaptic dysfunction. This work adds to the notion that the transient efferent innervation to the cochlea is necessary for the correct establishment of the central auditory circuitry.Fil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Boero, Luis Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Castagna, Valeria C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Rodríguez Contreras, Adrián. City University Of New York. The City College Of New York.; Estados UnidosFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentin

Unravelling the molecular players at the cholinergic efferent synapse of the zebrafish lateral line

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons which aid to distinguish 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 (HC) share structural, functional and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Due to these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic cholinergic one (nAChR). However, the identities of the molecular players underlying acetylcholine (ACh)-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not α10 subunits, are enriched in zebrafish HC. 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, in vivo Ca2+ imaging on mechanically-stimulated zebrafish LL HC show that ACh elicits a decrease in evoked Ca2+ signals, irrespective of HC polarity. This effect is blocked by both α-Bungarotoxin and apamin, indicating coupling of ACh-mediated effects to SK potassium 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 the activation of Ca2+-dependent SK potassium channels.Fil: Carpaneto Freixas, Agustín Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Moglie, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Castagnola, Tais. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Salatino, Lucía. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Domene, Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Marcovich, Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gallino, Sofia Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Goutman, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Plazas, Paola Viviana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Activation of presynaptic GABAB receptors enables sustained synaptic transmission at high rate of stimulation at cholinergic olivocochlear-hair cell synapses

Outer hair cells (OHCs) receive an efferent input from the medial olivocochlear system (MOC) whose activation turns down the gain of the auditory system. MOC efferent fibers also transiently innervate the inner hair cells (IHCs) since birth to the onset of hearing (around P12 in altricial rodents). Although acetylcholine (ACh) is the main neurotransmitter at these synapses, an abundant GABA innervation is also present. Electrical stimulation of efferent fibers triggers the release of ACh, but also produces the activation of presynaptic GABAB receptors, that in turn downregulates the amount of ACh release. GABA-mediated mechanism is through the inhibition of P/Q type Ca2+ channels. In this work we discuss the consequences of GABAB-mediated inhibition in the short-term plasticity of these synapses. Inhibitory synaptic currents (IPSC) were recorded in IHCs and OHCs of acutely isolated organs of Corti at P9-P14, while MOC fibers were electrically stimulated. Preliminary results suggest that by reducing the amount of ACh release, activation of GABAB receptors reduces synaptic depression and enables sustained transmission during high-frequency stimulation at these synapses.Fil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Katz, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaSecond International Hearing Research Symposium: Closing the auditory efferent loop IIICiudad Autónoma de Buenos AiresArgentinaInternational Hearing ResearchAsociación Médica Argentin

Activation of presynaptic GABAB receptors enables sustained transmission at high rate of stimulation in cholinergic olivocochlear-hair cell synapses

During development, medial olivocochlear (MOC) neurons transiently innervate cochlear inner hair cells (IHCs). Although acetylcholine is the main neurotransmitter at this synapse, an abundant GABA innervation is also present. Electrical stimulation of efferent fibers triggers the release of acetylcholine, but also activates presynaptic GABAB receptors, that in turn reduce the amount of ACh release. The mechanism of action of GABA is through the inhibition of P/Q type Ca2+ channels. We are now studying the consequences of GABAB-mediated inhibition in the short-term plasticity properties of this synapse. Inhibitory synaptic currents (IPSC) were recorded in IHCs of acutely isolated organs of Corti at P9-P11 while MOC fibers were electrically stimulated. In control conditions, 15 pulses applied at high frequency (50 or 100 Hz) resulted in a progressive decrease in IPSC amplitudes. At 50 Hz, the depression rate was 32% whereas it increased to 53% at 100 Hz. At low-frequency (10 Hz), the specific GABAB agonist, baclofen, reduced the amplitude of IPSCs throughout the train. However, at higher stimulus rates, IPSCs were always larger than controls with a maximal enhancement of 43% for the last IPSC of the train at 100 Hz. These results suggest that by reducing synaptic depression, activation of GABAB receptors would enable sustained transmission during high-frequency stimulation at the MOC-inner hair cell synapse.Fil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXIX Annual Meeting and SAN-ISN Small Conference and CourseHuerta GrandeArgentinaSociedad Argentina de Investigación en Neurosciencia

Activation of presynaptic GABAB receptors minimize depression and enables sustained transmission at high rate of stimulation in cholinergic olivocochlear-hair cell synapses

During development, medial olivocochlear (MOC) neurons transiently innervate cochlear inner hair cells (IHCs). Although acetylcholine (ACh) is the main neurotransmitter at this synapse, an abundant GABA innervation is also present. Electrical stimulation of MOC efferent fibers triggers the release of ACh, but also activates presynaptic GABAB receptors, that in turn reduce the amount of ACh release. GABA-mediated mechanism is through the inhibition of P/Q type Ca2+ channels. We are now studying the consequences of GABAB-mediated inhibition in the short-term plasticity of this synapse. Inhibitory synaptic currents (IPSC) were recorded in IHCs of acutely isolated organs of Corti at P9-P11 while MOC fibers were electrically stimulated. In control condition, 10 pulses applied at high frequency (50 Hz) resulted in a progressive decrease on IPSC amplitudes throughout the train (P10/P1= 0.54). Contrary, specific GABAB agonist, baclofen, increased facilitation rate and eliminated depression at the same frequency (P10/P1= 1). Moreover, application of CGP35348, a GABAB antagonist, produced a bigger depression even at low stimulation frequencies (10Hz). These results suggest that the activation of presynaptic GABAB receptors, minimizes depression and would enable sustained transmission during high-frequency stimulation at the MOC-inner hair cell synapse.Fil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaXXXII Congreso Anual de la Sociedad Argentina de Investigación en NeurocienciaMar del PlataArgentinaSociedad Argentina de Investigación en Neurociencia

Efferent innervation to the cochlea

The auditory system consists of ascending and descending neuronal pathways. The beststudied is the ascending pathway, whereby sounds that are transduced in the cochlea intoelectrical signals are sent to the brain via the auditory nerve. Before reaching the auditorycortex, auditory ascending information has several central relays: the cochlear nucleus andsuperior olivary complex in the brainstem, the lateral lemniscal nuclei and inferior colliculusin the midbrain, and the medial geniculate body in the thalamus. The function(s) of the descendingcorticofugal pathway is less well understood. It plays important roles in shaping oreven creating the response properties of central auditory neurons and in the plasticity of theauditory system, such as reorganizing cochleotopic and computational maps. Corticofugalprojections are present at different relays of the auditory system. This review focuses on thephysiology and plasticity of the medial efferent olivocochlear system.Fil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin