Releasable Pool Of Synaptic Vesicles Research Articles

Increased presynaptic excitability in a migraine with aura mutation.

Migraine is a common and disabling neurological disorder. The headache and sensory amplifications of migraine are attributed to hyperexcitable sensory circuits, but a detailed understanding remains elusive. A mutation in casein kinase 1 delta (CK1δ) was identified in non-hemiplegic familial migraine with aura and advanced sleep phase syndrome. Mice carrying the CK1δT44A mutation were more susceptible to spreading depolarization (the phenomenon that underlies migraine aura), but mechanisms underlying this migraine-relevant phenotype were not known. We used a combination of whole-cell electrophysiology and multiphoton imaging, in vivo and in brain slices, to compare CK1δT44A mice (adult males) to their wild-type littermates. We found that despite comparable synaptic activity at rest, CK1δT44A neurons were more excitable upon repetitive stimulation than wild-type, with a reduction in presynaptic adaptation at excitatory but not inhibitory synapses. The mechanism of this adaptation deficit was a calcium-dependent enhancement of the size of the readily releasable pool of synaptic vesicles, and a resultant increase in glutamate release, in CK1δT44A compared to wild-type synapses. Consistent with this mechanism, CK1δT44A neurons showed an increase in the cumulative amplitude of excitatory post-synaptic currents, and a higher excitation-to-inhibition ratio during sustained activity compared to wild-type. At a local circuit level, action potential bursts elicited in CK1δT44A neurons triggered an increase in recurrent excitation compared to wild-type, and at a network level, CK1δT44A mice showed a longer duration of 'up state' activity, which is dependent on recurrent excitation. Finally, we demonstrated that the spreading depolarization susceptibility of CK1δT44A mice could be returned to wild-type levels with the same intervention (reduced extracellular calcium) that normalized presynaptic adaptation. Taken together, these findings show a stimulus-dependent presynaptic gain of function at glutamatergic synapses in a genetic model of migraine, that accounts for the increased spreading depolarization susceptibility and may also explain the sensory amplifications that are associated with the disease.

Evidence That Ultrafast Nonquantal Transmission Underlies Synchronized Vestibular Action Potential Generation.

Amniotes evolved a unique postsynaptic terminal in the inner ear vestibular organs called the calyx that receives both quantal and nonquantal (NQ) synaptic inputs from Type I sensory hair cells. The nonquantal synaptic current includes an ultrafast component that has been hypothesized to underlie the exceptionally high synchronization index (vector strength) of vestibular afferent neurons in response to sound and vibration. Here, we present three lines of evidence supporting the hypothesis that nonquantal transmission is responsible for synchronized vestibular action potentials of short latency in the guinea pig utricle of either sex. First, synchronized vestibular nerve responses are unchanged after administration of the AMPA receptor antagonist CNQX, while auditory nerve responses are completely abolished. Second, stimulus evoked vestibular nerve compound action potentials (vCAP) are shown to occur without measurable synaptic delay and three times shorter than the latency of auditory nerve compound action potentials (cCAP), relative to the generation of extracellular receptor potentials. Third, paired-pulse stimuli designed to deplete the readily releasable pool (RRP) of synaptic vesicles in hair cells reveal forward masking in guinea pig auditory cCAPs, but a complete lack of forward masking in vestibular vCAPs. Results support the conclusion that the fast component of nonquantal transmission at calyceal synapses is indefatigable and responsible for ultrafast responses of vestibular organs evoked by transient stimuli.SIGNIFICANCE STATEMENT The mammalian vestibular system drives some of the fastest reflex pathways in the nervous system, ensuring stable gaze and postural control for locomotion on land. To achieve this, terrestrial amniotes evolved a large, unique calyx afferent terminal which completely envelopes one or more presynaptic vestibular hair cells, which transmits mechanosensory signals mediated by quantal and nonquantal (NQ) synaptic transmission. We present several lines of evidence in the guinea pig which reveals the most sensitive vestibular afferents are remarkably fast, much faster than their auditory nerve counterparts. Here, we present neurophysiological and pharmacological evidence that demonstrates this vestibular speed advantage arises from ultrafast NQ electrical synaptic transmission from Type I hair cells to their calyx partners.

Microtubule depolymerization contributes to spontaneous neurotransmitter release in vitro

Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.

Emerging targets in drug discovery against neurodegenerative diseases: Control of synapsis disfunction by the RhoA/ROCK pathway.

Synaptic spine morphology is controlled by the activity of Rac1, Cdc42 and RhoA, which need to be finely balanced, and in particular RhoA/ROCK prevents the formation of new protrusions by stabilizing actin formation. These processes are crucial to the maturation process, slowing the de novo generation of new spines. The RhoA/ROCK also influences plasticity processes, and selective modulation by ROCK1 of MLC-dependent actin dynamics leads to neurite retraction, but not to spine retraction. ROCK1 is also responsible for the reduction of the readily releasable pool of synaptic vesicles. These and other evidences suggest that ROCK1 is the main isoform acting on the presynaptic neuron. On the other hand, ROCK2 seems to have broad effects on LIMK/cofilin-dependent plasticity processes such as cofilin-dependent PSD changes. The RhoA/ROCK pathway is an important factor in several different brain-related pathologies via both downstream and upstream pathways. In the aggregate, these evidences show that the RhoA/ROCK pathway has a central role in the etiopathogenesis of a large group of CNS diseases, which underscores the importance of the pharmacological modulation of RhoA/ROCK as an important pathway to drug discovery in the neurodegenerative disease area. This article aims at providing the first review of the role of compounds acting on the RhoA/ROCK pathway in the control of synaptic disfunction.

RIM-Binding Proteins Are Required for Normal Sound-Encoding at Afferent Inner Hair Cell Synapses

The afferent synapses between inner hair cells (IHC) and spiral ganglion neurons are specialized to faithfully encode sound with sub-millisecond precision over prolonged periods of time. Here, we studied the role of Rab3 interacting molecule-binding proteins (RIM-BP) 1 and 2 – multidomain proteins of the active zone known to directly interact with RIMs, Bassoon and CaV1.3 – in IHC presynaptic function and hearing. Recordings of auditory brainstem responses and otoacoustic emissions revealed that genetic disruption of RIM-BPs 1 and 2 in mice (RIM-BP1/2–/–) causes a synaptopathic hearing impairment exceeding that found in mice lacking RIM-BP2 (RIM-BP2–/–). Patch-clamp recordings from RIM-BP1/2–/– IHCs indicated a subtle impairment of exocytosis from the readily releasable pool of synaptic vesicles that had not been observed in RIM-BP2–/– IHCs. In contrast, the reduction of Ca2+-influx and sustained exocytosis was similar to that in RIMBP2–/– IHCs. We conclude that both RIM-BPs are required for normal sound encoding at the IHC synapse, whereby RIM-BP2 seems to take the leading role.

β-Adrenergic Receptors/Epac Signaling Increases the Size of the Readily Releasable Pool of Synaptic Vesicles Required for Parallel Fiber LTP

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca2+-induced increase in presynaptic cAMP that is mediated by Ca2+-sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by β-adrenergic receptors (βARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that βARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these βARs occluded PF-PC LTP, while the β1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in Epac2 -/- mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the βAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP.SIGNIFICANCE STATEMENT G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to β-adrenergic receptor (βAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by βARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this βAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of βARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon.

Presynaptically silent synapses are modulated by the density of surrounding astrocytes

Astrocytes, comprising the primary glial-cell type, are involved in the formation and maturation of synapses, and thus contribute to sustainable synaptic transmission between neurons. Given that the animals in higher phylogenetic tree have brains with a higher density of glial cells with respect to neurons, there is a possibility that the relative astrocytic density directly influences synaptic transmission. However, the notion has not been tested thoroughly. Here we addressed it, by using a primary culture preparation where single hippocampal neurons are surrounded by a variable but a countable number of cortical astrocytes in dot-patterned microislands, and recording synaptic transmission by patch-clamp electrophysiology. Neurons with a higher astrocytic density showed a higher amplitude of the evoked excitatory postsynaptic current than that of neurons with a lower astrocytic density. The size of the readily releasable pool of synaptic vesicles per neuron was significantly larger. The frequency of spontaneous synaptic transmission was higher, but the amplitude was unchanged. The number of morphologically identified glutamatergic synapses was comparable, but the percentage of functional ones was increased, indicating a lower ratio of presynaptically silent synapses. Taken together, the higher astrocytic density enhanced excitatory synaptic transmission by increasing the fraction of functional synapses through presynaptic un-silencing.

“Flash and freeze” synaptic plasticity

Neuroscience The eponymous mossy fibers in the hippocampus of the brain connect the dentate gyrus to the CA3 pyramidal region and are implicated in short-term memories spanning a few seconds to minutes. How such memories are held in the circuit is a puzzle. One candidate is post-tetanic potentiation, a form of synaptic plasticity that decays within tens to hundreds of seconds and regulates synaptic strength at hippocampal mossy fiber–CA3 pyramidal neuron synapses. Vandael et al. simultaneously recorded from pairs of mossy fiber terminals and postsynaptic CA3 pyramidal neurons and followed measurements with “flash and freeze” electron microscopy. The authors observed that post-tetanic potentiation was induced by an increase in the size of the readily releasable pool of synaptic vesicles in granule cells. Neuron 10.1016/j.neuron.2020.05.013 (2020).

Short-Term Plasticity at Hippocampal Mossy Fiber Synapses Is Induced by Natural Activity Patterns and Associated with Vesicle Pool Engram Formation

SummaryPost-tetanic potentiation (PTP) is an attractive candidate mechanism for hippocampus-dependent short-term memory. Although PTP has a uniquely large magnitude at hippocampal mossy fiber-CA3 pyramidal neuron synapses, it is unclear whether it can be induced by natural activity and whether its lifetime is sufficient to support short-term memory. We combined in vivo recordings from granule cells (GCs), in vitro paired recordings from mossy fiber terminals and postsynaptic CA3 neurons, and “flash and freeze” electron microscopy. PTP was induced at single synapses and showed a low induction threshold adapted to sparse GC activity in vivo. PTP was mainly generated by enlargement of the readily releasable pool of synaptic vesicles, allowing multiplicative interaction with other plasticity forms. PTP was associated with an increase in the docked vesicle pool, suggesting formation of structural “pool engrams.” Absence of presynaptic activity extended the lifetime of the potentiation, enabling prolonged information storage in the hippocampal network.

Role of GluA3 AMPA Receptor Subunits in the Presynaptic and Postsynaptic Maturation of Synaptic Transmission and Plasticity of Endbulb-Bushy Cell Synapses in the Cochlear Nucleus.

The AMPA receptor (AMPAR) subunit GluA3 has been suggested to shape synaptic transmission and activity-dependent plasticity in endbulb-bushy cell synapses (endbulb synapses) in the anteroventral cochlear nucleus, yet the specific roles of GluA3 in the synaptic transmission at endbulb synapses remains unexplored. Here, we compared WT and GluA3 KO mice of both sexes and identified several important roles of GluA3 in the maturation of synaptic transmission and short-term plasticity in endbulb synapses. We show that GluA3 largely determines the ultrafast kinetics of endbulb synapses glutamatergic currents by promoting the insertion of postsynaptic AMPARs that contain fast desensitizing flop subunits. In addition, GluA3 is also required for the normal function, structure, and development of the presynaptic terminal which leads to altered short term-depression in GluA3 KO mice. The presence of GluA3 reduces and slows synaptic depression, which is achieved by lowering the probability of vesicle release, promoting efficient vesicle replenishment, and increasing the readily releasable pool of synaptic vesicles. Surprisingly, GluA3 also makes the speed of synaptic depression rate-invariant. We propose that the slower and rate-invariant speed of depression allows an initial response window that still contains presynaptic firing rate information before the synapse is depressed. Because this response window is rate-invariant, GluA3 extends the range of presynaptic firing rates over which rate information in bushy cells can be preserved. This novel role of GluA3 may be important to allowing the postsynaptic targets of spherical bushy cells in mice use rate information for encoding sound intensity and sound localization.SIGNIFICANCE STATEMENT We report novel roles of the glutamate receptor subunit GluA3 in synaptic transmission in synapses between auditory nerve fibers and spherical bushy cells (BCs) in the cochlear nucleus. We show that GluA3 contributes to the generation of ultrafast glutamatergic currents at these synapses, which is important to preserve temporal information about the sound. Furthermore, we demonstrate that GluA3 contributes to the normal function and development of the presynaptic terminal, whose properties shape short-term plasticity. GluA3 slows and attenuates synaptic depression, and makes it less dependent on the presynaptic firing rates. This may help BCs to transfer information about the high rates of activity that occur at the synapse in vivo to postsynaptic targets that use rate information for sound localization.

Ca2+- and Phospholipid-Dependent Mechanisms for the Coupling of Synaptic Vesicle Consumption and Re-Supply Rates

The efficacy and reliability of synaptic transmission depend on the ability of the transmitter release machinery to replenish the releasable pool of synaptic vesicles (SV) during and following activity. To sustain transmitter release during periods of high-frequency activity, a mechanism must exist that increases the recovery rate of the releasable SV pool. We sought to identify Ca2+-dependent SV replenishment mechanisms that operate in vivo, study how they dynamically interact during periods of synaptic activity, and illustrate how they affects transmitter release dynamics and short-term synaptic plasticity. Munc13s are a family of presynaptic proteins with three domains that are directly or indirectly regulated by Ca2+. Munc13s are functionally absolutely essential for the priming of SVs, and have a central role in the organization of SVs prior to and during transmitter release. Here, we present novel genetically modified mouse lines in which Ca2+- and phospholipid sensing by the regulatory domains of Munc13-1 is altered. Our analyses demonstrate that the SV replenishment rates can be controlled bi-directionally by Munc13-1, with consequent effects on short-term synaptic plasticity and information processing.

Outside-in regulation of the readily releasable pool of synaptic vesicles by α2δ-1.

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N-type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin-1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ-1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin-1α are mutually exclusive. The overexpression of α2δ-1 enhances the effect of pregabalin and the ectodomain of neurexin-1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ-1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ-1 as an outside-in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage-gated calcium channels to synaptic vesicles.

Target-wide Induction and Synapse Type-Specific Robustness of Presynaptic Homeostasis.

Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. In this work, we make several fundamental advances by interrogating the synapse specificity of PHP. We define how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. We describe a general PHP property that includes global induction and synapse-specific expression mechanisms. Finally, we detail a novel synapse-specific PHP expression mechanism that enables the conversion from short- to long-term PHP expression. If our data can be extended to other systems, including the mammalian central nervous system, they suggest that PHP can be broadly induced and expressed to sustain the function of complex neural circuitry.

Capacitance measurement of dendritic exocytosis in an electrically coupled inhibitory retinal interneuron: an experimental and computational study.

Exocytotic release of neurotransmitter can be quantified by electrophysiological recording from postsynaptic neurons. Alternatively, fusion of synaptic vesicles with the cell membrane can be measured as increased capacitance by recording directly from a presynaptic neuron. The “Sine + DC” technique is based on recording from an unbranched cell, represented by an electrically equivalent RC‐circuit. It is challenging to extend such measurements to branching neurons where exocytosis occurs at a distance from a somatic recording electrode. The AII amacrine is an important inhibitory interneuron of the mammalian retina and there is evidence that exocytosis at presynaptic lobular dendrites increases the capacitance. Here, we combined electrophysiological recording and computer simulations with realistic compartmental models to explore capacitance measurements of rat AII amacrine cells. First, we verified the ability of the “Sine + DC” technique to detect depolarization‐evoked exocytosis in physiological recordings. Next, we used compartmental modeling to demonstrate that capacitance measurements can detect increased membrane surface area at lobular dendrites. However, the accuracy declines for lobular dendrites located further from the soma due to frequency‐dependent signal attenuation. For sine wave frequencies ≥1 kHz, the magnitude of the total releasable pool of synaptic vesicles will be significantly underestimated. Reducing the sine wave frequency increases overall accuracy, but when the frequency is sufficiently low that exocytosis can be detected with high accuracy from all lobular dendrites (~100 Hz), strong electrical coupling between AII amacrines compromises the measurements. These results need to be taken into account in studies with capacitance measurements from these and other electrically coupled neurons.

How to Build a Fast and Highly Sensitive Sound Detector That Remains Robust to Temperature Shifts.

Frogs must have sharp hearing abilities during the warm summer months to successfully find mating partners. This study aims to understand how frog hair cell ribbon-type synapses preserve both sensitivity and temporal precision during temperature changes. Under room (∼24°C) and high (∼32°C) temperature, we performed in vitro patch-clamp recordings of hair cells and their afferent fibers in amphibian papillae of either male or female bullfrogs. Afferent fibers exhibited a wide heterogeneity in membrane input resistance (Rin) from 100 mΩ to 1000 mΩ, which may contribute to variations in spike threshold and firing frequency. At higher temperatures, most fibers increased their frequency of spike firing due to an increase in spontaneous EPSC frequencies. Hair cell resting membrane potential (Vrest) remained surprisingly stable during temperature increases, because Ca2+ influx and K+ outflux increased simultaneously. This increase in Ca2+ current likely enhanced spontaneous EPSC frequencies. These larger "leak currents" at Vrest also lowered Rin and produced higher electrical resonant frequencies. Lowering Rin will reduce the hair cells receptor potential and presumably moderate the systems sensitivity. Using membrane capacitance measurements, we suggest that hair cells can partially compensate for this reduced sensitivity by increasing exocytosis efficiency and the size of the readily releasable pool of synaptic vesicles. Furthermore, paired recordings of hair cells and their afferent fibers showed that synaptic delays shortened and multivesicular release becomes more synchronous at higher temperatures, which should improve temporal precision. Together, our results explain many previous in vivo observations on the temperature dependence of spikes in auditory nerves.SIGNIFICANCE STATEMENT The vertebrate inner ear detects and transmits auditory information over a broad dynamic range of sound frequency and intensity. It achieves remarkable sensitivity to soft sounds and precise frequency selectivity. How does the ear of cold-blooded vertebrates maintain its performance level as temperature changes? More specifically, how does the hair cell to afferent fiber synapse in bullfrog amphibian papilla adjust to a wide range of physiological temperatures without losing its sensitivity and temporal fidelity to sound signals? This study uses in vitro experiments to reveal the biophysical mechanisms that explain many observations made from in vivo auditory nerve fiber recordings. We find that higher temperature facilitates vesicle exocytosis and electrical tuning to higher sound frequencies, which benefits sensitivity and selectivity.

Dystrobrevin is required postsynaptically for homeostatic potentiation at the Drosophila NMJ

Evolutionarily conserved homeostatic systems have been shown to modulate synaptic efficiency at the neuromuscular junctions of organisms. While advances have been made in identifying molecules that function presynaptically during homeostasis, limited information is currently available on how postsynaptic alterations affect presynaptic function. We previously identified a role for postsynaptic Dystrophin in the maintenance of evoked neurotransmitter release. We herein demonstrated that Dystrobrevin, a member of the Dystrophin Glycoprotein Complex, was delocalized from the postsynaptic region in the absence of Dystrophin. A newly-generated Dystrobrevin mutant showed elevated evoked neurotransmitter release, increased bouton numbers, and a readily releasable pool of synaptic vesicles without changes in the function or numbers of postsynaptic glutamate receptors. In addition, we provide evidence to show that the highly conserved Cdc42 Rho GTPase plays a key role in the postsynaptic Dystrophin/Dystrobrevin pathway for synaptic homeostasis. The present results give novel insights into the synaptic deficits underlying Duchenne Muscular Dystrophy affected by a dysfunctional Dystrophin Glycoprotein complex.

Substance P plays a critical role in synaptic transmission in striatal neurons

Substance P is one of the major neuropeptides released by striatal neurons; however, its function in the striatum remains unclear. In this study, we found substance P triggers spontaneous neurotransmitter release and rapid synaptic vesicle exocytosis in cultured striatal neurons, as substance P knockdown in these neurons impaired spontaneous neurotransmitter release and calcium-dependent rapid synaptic neurotransmission. Furthermore, treatment with exogenous substance P completely rescued the synaptic dysfunction phenotype in striatal neurons lacking this neuropeptide. On the other hand, substance P knockdown had no effect on the size of the readily releasable pool of synaptic vesicles, but decreased the probability of presynaptic release of synaptic vesicles in cultured striatal neurons. Treatment with CP96345, a NK1 receptor antagonist, also resulted in synaptic defects in cultured striatal neurons. In summary, we propose substance P is critical for synaptic transmission in striatal neurons.

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.SIGNIFICANCE STATEMENT During postnatal development before the onset of hearing, cochlear inner hair cells (IHCs) present spontaneous Ca2+ action potentials that release glutamate at the first auditory synapse in the absence of sound stimulation. The IHC Ca2+ 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.

Altered Intracellular Calcium Homeostasis Underlying Enhanced Glutamatergic Transmission in Striatal-Enriched Tyrosine Phosphatase (STEP) Knockout Mice.

The striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in synaptic transmission. The current hypothesis on STEP function holds that it opposes synaptic strengthening by dephosphorylating and inactivating key neuronal proteins involved in synaptic plasticity and intracellular signaling, such as the MAP kinases ERK1/2 and p38, as well as the tyrosine kinase Fyn. Although STEP has a predominant role at the post-synaptic level, it is also expressed in nerve terminals. To better investigate its physiological role at the presynaptic level, we functionally investigated brain synaptosomes and autaptic hippocampal neurons from STEP knockout (KO) mice. Synaptosomes purified from mutant mice were characterized by an increased basal and evoked glutamate release compared with wild-type animals. Under resting conditions, STEP KO synaptosomes displayed increased cytosolic Ca2+ levels accompanied by an enhanced basal activity of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and hyperphosphorylation of synapsin I at CaMKII sites. Moreover, STEP KO hippocampal neurons exhibit an increase of excitatory synaptic strength attributable to an increased size of the readily releasable pool of synaptic vesicles. These results provide new evidence that STEP plays an important role at nerve terminals in the regulation of Ca2+ homeostasis and neurotransmitter release.

The presynaptic scaffolding protein Piccolo organizes the readily releasable pool at the calyx of Held.

Bassoon and Piccolo do not mediate basal synaptic vesicle release at a high-frequency synapse. Knockdown of Bassoon increases short-term depression at the calyx of Held. Both Bassoon and Piccolo have shared functions in synaptic vesicle replenishment during high-frequency synaptic transmission. Piccolo organizes the readily releasable pool of synaptic vesicles. It safeguards a fraction of them to be not immediately available for action potential-induced release. This enables the synapse to sustain high-frequency synaptic transmission over long periods. Synaptic vesicles (SVs) are released at the active zone (AZ), a specialized region of the presynaptic plasma membrane organized by a highly interconnected network of multidomain proteins called the cytomatrix of the active zone (CAZ). Two core components of the CAZ are the large, highly homologous scaffolding proteins Bassoon and Piccolo, whose function is not well understood. To investigate their role in synaptic transmission, we established the small hairpin RNA (shRNA)-mediated in vivo knockdown (KD) of Bassoon and Piccolo at the rat calyx of Held synapse. KD of Bassoon and Piccolo, separately or simultaneously, did not affect basic SV release. However, short-term depression (STD) was prominently increased by the KD of Bassoon, whereas KD of Piccolo only had a minor effect. The observed alterations in STD were readily explained by reduced SV replenishment in synapses deficient in either of the proteins. Thus, the regulation of SV refilling during ongoing synaptic activity is a shared function of Bassoon and Piccolo, although Bassoon appears to be more efficient. Moreover, we observed the recruitment of slowly-releasing SVs of the readily-releasable pool (RRP), which are normally not available for action potential-induced release, during high-frequency stimulation in Piccolo-deficient calyces. Therefore, the results obtained in the present study suggest a novel and specific role for Piccolo in the organization of the subpools of the RRP.