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.
In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic ␣9␣10 receptor coupled to the activation of SK2 Ca 2ϩ -activated K ϩ channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q-and N-type voltage-gated calcium channels (VGCCs), -agatoxin IVA and -conotoxin GVIA, respectively, we show that Ca 2ϩ entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca 2ϩ entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca 2ϩ -activated K ϩ channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons.
In neurons of the adult brain, somatodendritic GABA receptors (GABA Rs) mediate fast synaptic inhibition and play a crucial role in synaptic integration. GABA Rs are not only present in the somatodendritic compartment, but also in the axonal compartment where they modulate action potential (AP) propagation and transmitter release. Although presynaptic GABA Rs have been reported in various brain regions, their mechanisms of action and physiological roles remain obscure, particularly at GABAergic boutons. Here, using a combination of direct whole-bouton or perforated patch-clamp recordings and local GABA photolysis in single axonal varicosities of cerebellar Purkinje cells, we investigate the subcellular localization and functional role of axonal GABA Rs both in primary cultures and acute slices. Our results indicate that presynaptic terminals of PCs carry GABA Rs that behave as auto-receptors; their activation leads to a depolarization of the terminal membrane after an AP due to the relatively high cytoplasmic Cl concentration in the axon, but they do not modulate the AP itself. Paired recordings from different terminals of the same axon show that the GABA R-mediated local depolarizations propagate substantially to neighbouring varicosities. Finally, the depolarization mediated by presynaptic GABA R activation augmented Ca influx and transmitter release, resulting in a marked effect on short-term plasticity. Altogether, our results reveal a mechanism by which presynaptic GABA Rs influence neuronal computation.
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 Ca 2ϩ -permeable postsynaptic ␣9␣10 nicotinic receptors coupled to the opening of hyperpolarizing Ca 2ϩ -activated small-conductance type 2 (SK2)K ϩ 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 GABA B(1a,2) receptors [GABA B(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 GABA B Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA B1 -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 GABA B1a isoform selectively inhibits release at efferent cholinergic synapses.
Down syndrome (DS) results in various degrees of cognitive deficits. In DS mouse models, recovery of behavioral and neurophysiological deficits using GABAAR antagonists led to hypothesize an excessive activity of inhibitory circuits in this condition. Nonetheless, whether over-inhibition is present in DS and whether this is due to specific alterations of distinct GABAergic circuits is unknown. In the prefrontal cortex of Ts65Dn mice (a well-established DS model), we found that the dendritic synaptic inhibitory loop formed by somatostatin-positive Martinotti cells (MCs) and pyramidal neurons (PNs) was strongly enhanced, with no alteration in their excitability. Conversely, perisomatic inhibition from parvalbumin-positive (PV) interneurons was unaltered, but PV cells of DS mice lost their classical fast-spiking phenotype and exhibited increased excitability. These microcircuit alterations resulted in reduced pyramidal-neuron firing and increased phase locking to cognitive-relevant network oscillations in vivo. These results define important synaptic and circuit mechanisms underlying cognitive dysfunctions in DS.
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 5.6ϫ 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 Ca 2ϩ 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. Significance StatementDuring postnatal development before the onset of hearing, cochlear inner hair cells (IHCs) present spontaneous Ca 2ϩ action potentials that release glutamate at the first auditory synapse in the absence of sound stimulation. The IHC Ca 2ϩ action potential frequency pattern, which is crucial for the correct establishment and function of the auditory system, is regulated by the efferent medial olivocochlear (MOC) system that transiently innervates IHCs during this period. We show here that developmental changes in synaptic strength and synaptic plasticity properties at the MOC-IHC synapse upon MOC fiber activation at different frequencies might be crucial for tightly shaping the pattern of afferent activity during this critical period.
Background and Purpose Excessive GABAergic inhibition contributes to cognitive dysfunctions in Down syndrome (DS). Selective negative allosteric modulators (NAMs) of α5‐containing GABAA receptors such as the α5 inverse agonist (α5IA) restore learning and memory deficits in Ts65Dn mice, a model of DS. In this study we have assessed the long‐lasting effects of α5IA on in vivo LTP and behaviour in Ts65Dn mice. Experimental Approach We made in vivo LTP recordings for six consecutive days in freely moving Ts65Dn mice and their wild‐type littermates, treated with vehicle or α5IA. In parallel, Ts65Dn mice were assessed by various learning and memory tests (Y maze, Morris water maze, or the novel object recognition) for up to 7 days, following one single injection of α5IA or vehicle. Key Results LTP was not evoked in vivo in Ts65Dn mice at hippocampal CA3‐CA1 synapses. However, this deficit was sustainably reversed for at least six consecutive days following a single injection of α5IA. This long‐lasting effect of α5IA was also observed when assessing working and long‐term memory deficits in Ts65Dn mice. Conclusion and Implications We show for the first time in vivo LTP deficits in Ts65Dn mice. These deficits were restored for at least 6 days following acute treatment with α5IA and might be the substrate for the long‐lasting pharmacological effects of α5IA on spatial working and long‐term recognition and spatial memory tasks. Our results demonstrate the relevance of negative allosteric modulators of α5‐containing GABAA receptors to the treatment of cognitive deficits associated with DS.
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