RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals.

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

Mechanistic insights into cAMP-mediated presynaptic potentiation at hippocampal mossy fiber synapses.

Presynaptic plasticity is an activity-dependent change in the neurotransmitter release and plays a key role in dynamic modulation of synaptic strength. Particularly, presynaptic potentiation mediated by cyclic adenosine monophosphate (cAMP) is widely seen across the animals and thought to contribute to learning and memory. Hippocampal mossy fiber-CA3 pyramidal cell synapses have been used as a model because of robust presynaptic potentiation in short- and long-term forms. Moreover, direct presynaptic recordings from large mossy fiber terminals allow one to dissect the potentiation mechanisms. Recently, super-resolution microscopy and flash-and-freeze electron microscopy have revealed the localizations of release site molecules and synaptic vesicles during the potentiation at a nanoscale, identifying the molecular mechanisms of the potentiation. Incorporating these growing knowledges, we try to present plausible mechanisms underlying the cAMP-mediated presynaptic potentiation.

Increased vesicle fusion competence underlies long-term potentiation at hippocampal mossy fiber synapses.

Presynaptic long-term potentiation (LTP) is thought to play an important role in learning and memory. However, the underlying mechanism remains elusive because of the difficulty of direct recording during LTP. Hippocampal mossy fiber synapses exhibit pronounced LTP of transmitter release after tetanic stimulation and have been used as a model of presynaptic LTP. Here, we induced LTP by optogenetic tools and applied direct presynaptic patch-clamp recordings. The action potential waveform and evoked presynaptic Ca2+ currents remained unchanged after LTP induction. Membrane capacitance measurements suggested higher release probability of synaptic vesicles without changing the number of release-ready vesicles after LTP induction. Synaptic vesicle replenishment was also enhanced. Furthermore, stimulated emission depletion microscopy suggested an increase in the numbers of Munc13-1 and RIM1 molecules within active zones. We propose that dynamic changes in the active zone components may be relevant for the increased fusion competence and synaptic vesicle replenishment during LTP.

Subcortical glutamatergic inputs exhibit a Hebbian form of long-term potentiation in the dentate gyrus.

The hippocampus receives glutamatergic and GABAergic inputs from subcortical regions. Despite the important roles of these subcortical inputs in the regulation of hippocampal circuit, it has not been explored whether associative activation of the subcorticohippocampal pathway induces Hebbian plasticity of subcortical inputs. Here, we demonstrate that the hypothalamic supramammillary nucleus (SuM) to the dentate granule cell (GC) synapses, which co-release glutamate and GABA, undergo associative long-term potentiation (LTP) of glutamatergic, but not GABAergic, co-transmission. This LTP is induced by pairing of SuM inputs with GC spikes. We found that this Hebbian LTP is input-specific, requires NMDA receptors and CaMKII activation, and is expressed postsynaptically. By the net increase in excitatory drive of SuM inputs following LTP induction, associative inputs of SuM and the perforant path effectively discharge GCs. Our results highlight the important role of associative plasticity at SuM-GC synapses in the regulation of dentate gyrus activity and for the encoding of SuM-related information.

Excitatory selective LTP of supramammillary glutamatergic/GABAergic cotransmission potentiates dentate granule cell firing.

SignificanceIt is now established that many neurons can release multiple transmitters. Recent studies revealed that fast-acting neurotransmitters, glutamate and GABA, are coreleased from the same presynaptic terminals in some adult brain regions. The dentate gyrus (DG) granule cells (GCs) are innervated by the hypothalamic supramammillary nucleus (SuM) afferents that corelease glutamate and GABA. However, how these functionally opposing neurotransmitters contribute to DG information processing remains unclear. We show that glutamatergic, but not GABAergic, cotransmission exhibits long-term potentiation (LTP) at SuM-GC synapses. By the excitatory selective LTP, the excitation/inhibition balance of SuM inputs increases, and GC firing is enhanced. This study provides evidence that glutamatergic/GABAergic cotransmission balance is rapidly changed in an activity-dependent manner, and such plasticity may modulate DG activity.

Quantal analysis estimates docking site occupancy determining short-term depression at hippocampal glutamatergic synapses.

Before fusion, synaptic vesicles (SVs) pause at discrete release/docking sites. During repetitive stimulation, the probability of site occupancy changes following SV fusion and replenishment. The occupancy probability is considered to be one of the crucial determinants of synaptic strength, but it is difficult to estimate separately because it usually blends with other synaptic parameters. Thus, the contribution of site occupancy to synaptic function, particularly to synaptic depression, remains elusive. Here, we directly estimated the occupancy probability at the hippocampal mossy fibre-CA3 interneuron synapse showing synaptic depression, using statistics of counts of vesicular events detected by deconvolution. We found that this synapse had a particularly high occupancy (∼0.85) with a high release probability of a docked SV (∼0.8) under 3 mm external calcium conditions. Analyses of quantal amplitudes and SV counts indicated that quantal size reduction decreased the amplitudes of all responses in a train to a similar degree, whereas release/docking site number was unchanged during trains, suggesting that quantal size and release/docking site number had little influence on the extent of synaptic depression. Model simulations revealed that the initial occupancy with high release probability and slow replenishment determined the time course of synaptic depression. Consistently, decreasing external calcium concentration reduced both the occupancy and release probability, and the reductions in turn produced less depression. Based on these results, we suggest that the occupancy probability is a crucial determinant of short-term synaptic depression at glutamatergic synapses in the hippocampus. KEY POINTS: The occupancy probability of a release/docking site by a synaptic vesicle at presynaptic terminals is considered to be one of the crucial determinants of synaptic strength, but it is difficult to estimate separately from other synaptic parameters. Here, we directly estimate the occupancy probability at the hippocampal mossy fibre-interneuron synapse using statistics of vesicular events detected by deconvolution. We show that the synapses have particularly high occupancy (0.85) with high release probability (0.8) under high external calcium concentration ([Ca2+ ]o ) conditions, and that both parameter values change with [Ca2+ ]o , shaping synaptic depression. Analyses of the quantal amplitudes and synaptic vesicle counts suggest that quantal sizes and release/docking site number have little influence on the extent of synaptic depression. The results suggest that the occupancy probability is a crucial determinant of short-term synaptic depression at glutamatergic synapses in the hippocampus.

Rapid Ca2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses.

The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca2+ uncaging experiments did not reveal changes in the intracellular Ca2+ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca2+ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca2+ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca2+ is explained by the simple mechanism of rapid Ca2+ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca2+ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca2+ channels.

Direct imaging of rapid tethering of synaptic vesicles accompanying exocytosis at a fast central synapse.

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.

Intersectin-Mediated Clearance of SNARE Complexes Is Required for Fast Neurotransmission.

The rapid replenishment of release-ready synaptic vesicles (SVs) at a limiting number of presynaptic release sites is required to sustain high-frequency neurotransmission in CNS neurons. Failure to clear release sites from previously exocytosed material has been shown to impair vesicle replenishment and, therefore, fast neurotransmission. The identity of this material and the machinery that removes it from release sites have remained enigmatic. Here we show that the endocytic scaffold protein intersectin 1 clears release sites by direct SH3 domain-mediated association with a non-canonical proline-rich segment of synaptobrevin assembled into the SNARE complex for neuroexocytosis. Acute structure-based or sustained genetic interference with SNARE complex recognition by intersectin 1 causes a rapid stimulation frequency-dependent depression of neurotransmission due to impaired replenishment of release-ready SVs. These findings identify a key molecular mechanism that underlies exo-endocytic coupling during fast neurotransmitter release at central synapses.

Ca-dependence of synaptic vesicle exocytosis and endocytosis at the hippocampal mossy fibre terminal.

KEY POINTS: We used presynaptic capacitance measurements at the hippocampal mossy fibre terminal at room temperature to measure Ca-dependence of exo- and endocytotic kinetics. The readily releasable pool (RRP) of synaptic vesicles was released with a time constant of 30-40 ms and was sensitive to Ca buffers, BAPTA and EGTA. Our data suggest that recruitment of the vesicles to the RRP was Ca-insensitive and had a time constant of 1 s. In addition to the RRP, the reserve pool of vesicles, which had a similar size to RRP, was depleted during repetitive stimulation. Our data suggest that synaptic vesicle endocytosis was also Ca-insensitive. ABSTRACT: Hippocampal mossy fibre terminals comprise one of the cortical terminals, which are sufficiently large to be accessible by patch clamp recordings. To measure Ca-dependence of exo- and endocytotic kinetics quantitatively, we applied presynaptic capacitance measurements to the mossy fibre terminal at room temperature. The time course of synaptic vesicle fusion was slow, with a time constant of tens of milliseconds, and was sensitive to Ca buffers EGTA and BAPTA, suggesting a loose coupling between Ca channels and synaptic vesicles. The size of the readily-releasable pool (RRP) of synaptic vesicles was relatively insensitive to Ca buffers. Once the RRP was depleted, it was recovered by a single exponential with a time constant of ∼1 s independent of the presence of Ca buffers, suggesting Ca independent vesicle replenishment. In addition to the RRP, the reserve pool of vesicles was released slowly during repetitive stimulation. Endocytosis was also insensitive to Ca buffers and had a slow time course, excluding the involvement of rapid vesicle cycling in vesicle replenishment. Although mossy fibre terminals are known to have various forms of Ca-dependent plasticity, some features of vesicle dynamics are robust and Ca-insensitive.

Developmental changes in the excitatory short-term plasticity at input synapses in the rat inferior colliculus.

The inferior colliculus (IC) is the primal center of convergence and integration in the auditory pathway. Although extensive functional changes are known to occur at the relay synapses in the auditory brainstem during development, the changes in the IC remain to be investigated. Here, we have measured excitatory postsynaptic currents (EPSCs) of the neurons in the central nucleus of the IC in response to stimulation of either the lateral lemniscus or the commissure of the inferior colliculus. Before hearing onset, the lemniscus inputs exhibited short-term depression, whereas commissural inputs showed facilitation. After hearing onset, the N-methyl-d-aspartate-EPSCs exhibited faster decay for both pathways, whereas the decay of the AMPA-EPSCs were unaltered. Furthermore, the EPSCs showed more constant responses during repetitive stimulation in both pathways. These developmental changes ensure faster and more reliable signal transmission to the inferior colliculus after onset of hearing.

Hapln4/Bral2 is a selective regulator for formation and transmission of GABAergic synapses between Purkinje and deep cerebellar nuclei neurons.

Purkinje cells (PCs) convey the sole output of the cerebellar cortex to the deep cerebellar nuclei (DCN). DCN neurons are enwrapped in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast-spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the DCN and medial nucleus of the trapezoid body, which are the post-synaptic neurons of large axo-somatic synapses adapted for fast signaling. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast-signaling synapses. To elucidate the role of the PNN at these synapses, we investigated the electrophysiological and morphological properties of DCN synapses in hyaluronan and proteoglycan binding link protein 4 (Hapln4/Bral2) knockout (KO) mice around postnatal day (P)14. Hapln4/Bral2 is important for PNN structure, as it stabilizes the interaction between hyaluronan and proteoglycan. Here, using immunohistochemistry we show that Hapln4/Bral2 localized closely with GABAergic terminals. In DCN neurons of Hapln4/Bral2 KO mice, inhibitory synaptic strengths were reduced as compared to those in wild-type mice, whereas the properties of excitatory synapses were unaffected. The reduced IPSC amplitudes were mainly because of reduced numbers of releasable vesicles. Moreover, Hapln4/Bral2 deficiency reduced the number of PC GABAergic terminals in the DCN. These results demonstrate that Hapln4/Bral2 is a PNN component that selectively contributes to formation and transmission of PC-DCN synapses in the cerebellum. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Kinetics of transmitter release at the calyx of Held synapse.

Synaptic contacts mediate information transfer between neurons. The calyx of Held, a large synapse in the mammalian auditory brainstem, has been used as a model system for the mechanism of transmitter release from the presynaptic terminal for the last 20 years. By applying simultaneous recordings from pre- and postsynaptic compartments, the calcium-dependence of the kinetics of transmitter release has been quantified. A single pool of readily releasable vesicles cannot explain the time course of release during repetitive activity. Rather, multiple pools of vesicles have to be postulated that are replenished with distinct kinetics after depletion. The physical identity of vesicle replenishment has been unknown. Recently, it has become possible to apply total internal reflection fluorescent microscopy to the calyx terminal. This technique allowed the visualization of the dynamics of individual synaptic vesicles. Rather than recruitment of vesicles to the transmitter release sites, priming of tethered vesicles in the total internal reflection fluorescent field limited the number of readily releasable vesicles during sustained activity.

Fast Ca2+ Buffer-Dependent Reliable but Plastic Transmission at Small CNS Synapses Revealed by Direct Bouton Recording.

The small size of presynaptic structures and their rapid function have obscured the mechanisms underlying neurotransmission and plasticity. To dissect the function of conventional small presynaptic boutons, we performed direct recording using axon varicosities of cerebellar granule cells (GCs), a parallel-fiber bouton, in dissociated culture, in which pre- and postsynaptic paired recordings are feasible. Identification and accessibility of EGFP-labeled GC boutons allowed us to patch-clamp record presynaptic voltage-gated Ca2+ currents and membrane capacitances, together with excitatory postsynaptic currents. We find that GC boutons have 20 readily releasable vesicles, which are loosely coupled to Ca2+ channels and rapidly replenished, and that synaptic strength and short-term plasticity are tightly regulated by intracellular Ca2+ buffering. Our functional dissection of small boutons thus reveals the sophisticated design of small synapses capable of reliable but plastic outputs with limited resources.

Kinetics of Releasable Synaptic Vesicles and Their Plastic Changes at Hippocampal Mossy Fiber Synapses.

Hippocampal mossy fiber boutons (hMFBs) are presynaptic terminals displaying various forms of synaptic plasticity. The presynaptic mechanisms underlying synaptic plasticity still remain poorly understood. Here, we have combined high temporal resolution measurements of presynaptic capacitance and excitatory postsynaptic currents (EPSCs) to measure the kinetics of exocytosis. In addition, total internal reflection fluorescence (TIRF) microscopy was employed to directly visualize dynamics of single synaptic vesicles adjacent to the plasma membrane at high spatial resolution. Readily releasable vesicles mostly consisted of already-tethered vesicles in the TIRF field. Vesicle replenishment had fast and slow phases, and TIRF imaging suggests that the fast phase depends on vesicle priming from already-tethered vesicles. Application of cyclic AMP (cAMP), a molecule crucial for LTP, mainly increases the vesicular release probability rather than the number of readily releasable vesicles or their replenishment rate, likely by changing the coupling between Ca2+ channels and synaptic vesicles. Thus, we revealed dynamic properties of synaptic vesicles at hMFBs.

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.

Distinct modes of endocytotic presynaptic membrane and protein uptake at the calyx of Held terminal of rats and mice.

Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, compensatory retrieval of membranes and vesicular proteins is essential. We combined capacitance measurements and pH-imaging via pH-sensitive vesicular protein marker (anti-synaptotagmin2-cypHer5E), and compared the retrieval kinetics of membranes and vesicular proteins at the calyx of Held synapse. Membrane and Syt2 were retrieved with a similar time course when slow endocytosis was elicited. When fast endocytosis was elicited, Syt2 was still retrieved together with the membrane, but endocytosed organelle re-acidification was slowed down, which provides strong evidence for two distinct endocytotic pathways. Strikingly, CaM inhibitors or the inhibition of the Ca(2+)-calmodulin-Munc13-1 signaling pathway only impaired the uptake of Syt2 while leaving membrane retrieval intact, indicating different recycling mechanisms for membranes and vesicle proteins. Our data identify a novel mechanism of stimulus- and Ca(2+)-dependent regulation of coordinated endocytosis of synaptic membranes and vesicle proteins.

Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses.

Ca(2+) current facilitation determines short-term facilitation at inhibitory synapses between cerebellar Purkinje cells.

KEY POINTS: Short-term facilitation takes place at GABAergic synapses between cerebellar Purkinje cells (PCs). By directly patch clamp recording from a PC axon terminal, we studied the mechanism of short-term facilitation. We show that the Ca(2+) currents elicited by high-frequency action potentials were augmented in a [Ca(2+) ]i -dependent manner. The facilitation of synaptic transmission showed 4-5th power dependence on the Ca(2+) current facilitation, and was abolished when the Ca(2+) current amplitude was adjusted to be identical. Short-term facilitation of Ca(2+) currents predominantly mediates short-term facilitation at synapses between PCs. ABSTRACT: Short-term synaptic facilitation is critical for information processing of neuronal circuits. Several Ca(2+) -dependent positive regulations of transmitter release have been suggested as candidate mechanisms underlying facilitation. However, the small sizes of presynaptic terminals have hindered the biophysical study of short-term facilitation. In the present study, by directly recording from the axon terminal of a rat cerebellar Purkinje cell (PC) in culture, we demonstrate a crucial role of [Ca(2+) ]i -dependent facilitation of Ca(2+) currents in short-term facilitation at inhibitory PC-PC synapses. Voltage clamp recording was performed from a PC axon terminal visualized by enhanced green fluorescent protein, and the Ca(2+) currents elicited by the voltage command consisting of action potential waveforms were recorded. The amplitude of presynaptic Ca(2+) current was augmented upon high-frequency paired-pulse stimulation in a [Ca(2+) ]i -dependent manner, leading to paired-pulse facilitation of Ca(2+) currents. Paired recordings from a presynaptic PC axon terminal and a postsynaptic PC soma demonstrated that the paired-pulse facilitation of inhibitory synaptic transmission between PCs showed 4-5th power dependence on that of Ca(2+) currents, and was completely abolished when the Ca(2+) current amplitude was adjusted to be identical. Thus, short-term facilitation of Ca(2+) currents predominantly mediates short-term synaptic facilitation at synapses between PCs.