Neurexins control the strength and precise timing of glycinergic inhibition in the auditory brainstem.

Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca2+ sensitivity of release and a loosened coupling between voltage-gated Ca2+ channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses.

Diverse organization of voltage-gated calcium channels at presynaptic active zones.

Synapses are highly organized but are also highly diverse in their organization and properties to allow for optimizing the computing power of brain circuits. Along these lines, voltage-gated calcium (CaV) channels at the presynaptic active zone are heterogeneously organized, which creates a variety of calcium dynamics profiles that can shape neurotransmitter release properties of individual synapses. Extensive studies have revealed striking diversity in the subtype, number, and distribution of CaV channels, as well as the nanoscale topographic relationships to docked synaptic vesicles. Further, multi-protein complexes including RIMs, RIM-binding proteins, CAST/ELKS, and neurexins are required for coordinating the diverse organization of CaV channels at the presynaptic active zone. In this review, we highlight major advances in the studies of the functional organization of presynaptic CaV channels and discuss their physiological implications for synaptic transmission and short-term plasticity.

Neurexins regulate presynaptic GABAB-receptors at central synapses.

Diverse signaling complexes are precisely assembled at the presynaptic active zone for dynamic modulation of synaptic transmission and synaptic plasticity. Presynaptic GABAB-receptors nucleate critical signaling complexes regulating neurotransmitter release at most synapses. However, the molecular mechanisms underlying assembly of GABAB-receptor signaling complexes remain unclear. Here we show that neurexins are required for the localization and function of presynaptic GABAB-receptor signaling complexes. At four model synapses, excitatory calyx of Held synapses in the brainstem, excitatory and inhibitory synapses on hippocampal CA1-region pyramidal neurons, and inhibitory basket cell synapses in the cerebellum, deletion of neurexins rendered neurotransmitter release significantly less sensitive to GABAB-receptor activation. Moreover, deletion of neurexins caused a loss of GABAB-receptors from the presynaptic active zone of the calyx synapse. These findings extend the role of neurexins at the presynaptic active zone to enabling GABAB-receptor signaling, supporting the notion that neurexins function as central organizers of active zone signaling complexes.

Neurexins cluster Ca2+ channels within the presynaptic active zone.

To achieve ultrafast neurotransmission, neurons assemble synapses with highly organized presynaptic and postsynaptic nanomachines that are aligned by synaptic adhesion molecules. How functional assembly of presynaptic active zones is controlled via trans-synaptic interactions remains unknown. Here, we conditionally deleted all three neurexin adhesion molecules from presynaptic neurons of the calyx of Held in the mouse auditory system, a model synapse that allows precise biophysical analyses of synaptic properties. The pan-neurexin deletion had no effect on synapse development or the basic release machinery, but dramatically impaired fast neurotransmitter release. The overall properties of presynaptic calcium ion channels appeared normal, as reflected by the similar characteristics of calcium currents recorded at the nerve terminals. However, the pan-neurexin deletion significantly impaired the tight coupling of calcium influx to exocytosis, thereby suppressing neurotransmitter release. Furthermore, the pan-neurexin deletion reduced the function of calcium-activated BK potassium channels, whose activation depends on their tight association with presynaptic calcium channels. Together, these results suggest that neurexins perform a major function at the calyx synapse in coupling presynaptic calcium channels to release sites.

The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors.

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

RIM-binding proteins recruit BK-channels to presynaptic release sites adjacent to voltage-gated Ca2+-channels.

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca2+-triggered synaptic vesicle exocytosis. BK-channels are Ca2+-activated large-conductance K+-channels that require close proximity to Ca2+-channels for activation and control Ca2+-triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK-channels are recruited to presynaptic Ca2+-channels, however, is unknown. Here, we show that RBPs (for RIM-binding proteins), which are evolutionarily conserved active zone proteins containing SH3- and FN3-domains, directly bind to BK-channels. We find that RBPs interact with RIMs and Ca2+-channels via their SH3-domains, but to BK-channels via their FN3-domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK-currents and depleted BK-channels from active zones. Our data suggest that RBPs recruit BK-channels into a RIM-based macromolecular active zone complex that includes Ca2+-channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio-temporal coupling of Ca2+-influx to Ca2+-triggered neurotransmitter release in a presynaptic terminal.

Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+ channels.

Fast neurotransmitter release from ribbon synapses via Ca2+-triggered exocytosis requires tight coupling of L-type Ca2+ channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca2+ channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca2+ buffer EGTA. These findings suggest that RBPs tether L-type Ca2+ channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

Synaptotagmin-7-Mediated Asynchronous Release Boosts High-Fidelity Synchronous Transmission at a Central Synapse.

Synchronous release triggered by Ca2+ binding to synaptotagmin-1, -2, or -9 is thought to drive fast synaptic transmission, whereas asynchronous release induced by Ca2+ binding to synaptotagmin-7 is thought to produce delayed synaptic signaling, enabling prolonged synaptic computations. However, it is unknown whether synaptotagmin-7-dependent asynchronous release performs a physiological function at fast synapses lacking a prolonged signaling mode, such as the calyx of Held synapse. Here, we show at the calyx synapse that synaptotagmin-7-dependent asynchronous release indeed does not produce a prolonged synaptic signal after a stimulus train and does not contribute to short-term plasticity, but induces a steady-state, asynchronous postsynaptic current during stimulus trains. This steady-state postsynaptic current does not increase overall synaptic transmission but instead sustains reliable generation of postsynaptic spikes that are precisely time locked to presynaptic spikes. Thus, asynchronous release surprisingly functions, at least at some synapses, to sustain high-fidelity neurotransmission driven by synchronous release during high-frequency stimulus trains.

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release.

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon-specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca(2+)-buffer EGTA, suggesting that synaptic ribbons mediate nano-domain coupling of Ca(2+) channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano-domains that position release-ready synaptic vesicles adjacent to Ca(2+) channels.

Synaptotagmin-7 Is Essential for Ca2+-Triggered Delayed Asynchronous Release But Not for Ca2+-Dependent Vesicle Priming in Retinal Ribbon Synapses.

Most synapses release neurotransmitters in two phases: (1) a fast synchronous phase lasting a few milliseconds; and (2) a delayed "asynchronous" phase lasting hundreds of milliseconds. Ca(2+) triggers fast synchronous neurotransmitter release by binding to synaptotagmin-1, synaptotagmin-2, or synaptotagmin-9, but how Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Recent results suggested that consistent with the Ca(2+)-sensor function of synaptotagmin-7 in neuroendocrine exocytosis, synaptotagmin-7 also functions as a Ca(2+) sensor for synaptic vesicle exocytosis but operates during delayed asynchronous release. Puzzlingly, a subsequent study postulated that synaptotagmin-7 is not a Ca(2+) sensor for release but mediates Ca(2+)-dependent vesicle repriming after intense stimulation. To address these issues, we here analyzed synaptic transmission at rod bipolar neuron-AII amacrine cell synapses in acute mouse retina slices as a model system. Using paired recordings, we show that knock-out of synaptotagmin-7 selectively impairs delayed asynchronous release but not fast synchronous release. Delayed asynchronous release was blocked in wild-type synapses by intracellular addition of high concentrations of the slow Ca(2+)-chelator EGTA, but EGTA had no effect in synaptotagmin-7 knock-out neurons because delayed asynchronous release was already impaired. Moreover, direct measurements of vesicle repriming failed to uncover an effect of the synaptotagmin-7 knock-out on vesicle repriming. Our data demonstrate that synaptotagmin-7 is selectively essential for Ca(2+)-dependent delayed asynchronous release in retinal rod bipolar cell synapses, that its function can be blocked by simply introducing a slow Ca(2+) buffer into the cells, and that synaptotagmin-7 is not required for normal vesicle repriming. SIGNIFICANCE STATEMENT: How Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Synaptotagmin-7 has been implicated recently as Ca(2+) sensor in mediating delayed asynchronous release, or vesicle repriming, in cultured neurons. To test the precise function of synaptotagmin-7 in a physiologically important synapse in situ, we have used pair recordings to study the synaptic transmission between retinal rod bipolar cells and AII amacrine cells. Our data demonstrate that the knock-out of synaptotagmin-7 selectively impaired delayed asynchronous release but not synchronous release. In contrast, the readily releasable vesicles after depletion recover normally in knock-out mice. Therefore, our findings extend our knowledge of synaptotagmins as Ca(2+) sensors in vesicle fusion and support the idea that synapses are governed universally by different synaptotagmin Ca(2+) sensors mediating distinct release.

[Curative effect of anti-HBV treatment in advanced schistosomiasis patients with ascites and HIBV infection].

OBJECTIVE: To improve the curative effect of advanced schistosomiasis patients with ascites and HBV infection. METHODS: A total of 27 advanced schistosomiasis patients with ascites and HBV infection were selected as a trial group and given with anti-HBV treatment, and 31 corresponding patients were as the controls and did not received anti-HBV treatment from February 2003 to December 2012. RESULTS: The incidence of ascites recurrence, spontaneous peritonitis, hepatic encephalopathy, hepatorenal syndrome, upper gastrointestinal bleeding, and primary liver cancer in the trial group were significantly lower than those in the control group. The detection indexes of liver function, renal function and prothrombin time in the trial group were superior to those in the control group. The mortality in the treatment group was also significantly lower than that in the control group. CONCLUSION: The anti-HBV treatment in the advanced schistosomiasis patients with ascites and HBV infection can obviously improve their physical conditions, the survival rate, and their life qualities.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

[Curative effect of Ruangan pills in treatment of schistosomiasis liver fibrosis].

OBJECTIVE: To explore the efficacy, mechanism and safety of silibinin combined with Ruangan pills (a Chinese herbal preparation) in the treatment of schistosomiasis liver fibrosis. METHODS: A total of 200 patients with schistosomiasis liver fibrosis were randomly divided into a control group and a treatment group, and 100 patients in each group were respectively administered with oral silibinin alone and oral silibinin combined with Ruangan pills, respectively. The curative effects in the two groups were evaluated in 3 months, 6 months, 9 months and 12 months respectively. RESULTS: The common five clinical symptoms of schistosomiasis liver fibrosis patients significantly relieved in the treatment group 12 months after the therapy, and the total efficiency reached more than 75%, which were significantly higher than that in the control group. In the treatment group and the control group, there was no improvement in the liver B ultrasonic classification 3 months and 6 months after the therapy (P > 0.05); however, in 9 months and 12 months, the liver B ultrasonic classification in the treatment group was better than that in the control group (P < 0.05, P < 0.01, respectively). For the four serum indexes of liver fibrosis, there was no significant differences between the two groups in 3 months, however, in 6 months, 9 months, and 12 months, there was a significant improvement in the treatment group compared with the control group. There were no obviously adverse effects in two groups. CONCLUSION: Silibinin combined with Ruangan pills has a better curative effect in the treatment of schistosomiasis liver fibrosis.

[Long-term therapeutic effect of Ruangan pills on advanced schistosomiasis patients with ascites].

OBJECTIVE: To evaluate the long-term therapeutic effect of Ruangan pills on advanced schistosomiasis combined with ascites. METHODS: The data of 54 advanced schistosomiasis patients with ascites were collected, and the patients were divided into two groups namely a treatment group and a control group according to whether taking Ruangan pills. The effective rates, improvement status of symptoms and levels of serum albumin (ALB) and hyaluronic acid (HA) of the two groups were compared. RESULTS: The effective rates of the treatment group and control group were 92.59% and 44.44%, respectively, and the difference between them was statistically significant (P < 0.05). After the treatment for 9 and 12 months, the percentages of patients without ascites and patients with symptom improvement in the treatment group were 77.78% and 92.59%, 92.59% and 96.30%, respectively, while those in the control group were 29.63% and 37.00%, 48.15% and 51.85%, respectively, and the differences between the two groups by different time were all statistical significant (all P < 0.05). After the treatment for 9 months, the percentages of patients with the normal levels of ALB and HA were 88.89% and 59.26%, while those in the control group were 40.74% and 14.81%, respectively, and the differences between the two groups were statistically significant (both P < 0.05). CONCLUSION: The long-term treatment of Ruangan pills can not only improve clinical symptoms but also control the ascites recurrence, however, the therapeutic effect and the recurrence rate of ascites in longer-term still need further observation.

Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.

Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

Most vesicles in a central nerve terminal participate in recycling.

Studies over the last decade using FM dyes to label vesicles at many terminals, including the calyx-type nerve terminal, led to a well accepted "principle" that only a small fraction of vesicles (∼5-20%) participate in recycling under physiological conditions. This principle imposes a large challenge in maintaining synaptic transmission during repetitive firing, because the small recycling pool may limit the number of available vesicles for release and nerve terminals would have to distinguish the recycling pool from the reserve pool and keep reserve pool vesicles from being used. By recording the presynaptic capacitance changes and the postsynaptic EPSC at rat calyx of Held synapses in the absence or presence of transmitter glutamate in nerve terminals, we developed a new method to count functional recycling vesicles. We found that essentially all vesicles in calyces participated in recycling, challenging the small-recycling-pool principle established by FM dye labeling. Nerve terminals may use all available vesicles to maximize their ability in maintaining synaptic transmission during repetitive firing.

SNARE proteins synaptobrevin, SNAP-25, and syntaxin are involved in rapid and slow endocytosis at synapses.

Rapid endocytosis, which takes only a few seconds, is widely observed in secretory cells. Although it is more efficient in recycling vesicles than in slow clathrin-mediated endocytosis, its underlying mechanism, thought to be clathrin independent, is largely unclear. Here, we report that cleavage of three SNARE proteins essential for exocytosis, including synaptobrevin, SNAP-25, and syntaxin, inhibited rapid endocytosis at the calyx of Held nerve terminal, suggesting the involvement of the three SNARE proteins in rapid endocytosis. These SNARE proteins were also involved in slow endocytosis. In addition, SNAP-25 and syntaxin facilitated vesicle mobilization to the readily releasable pool, most likely via their roles in endocytosis and/or exocytosis. We conclude that both rapid and slow endocytosis share the involvement of SNARE proteins. The dual role of three SNARE proteins in exo- and endocytosis suggests that SNARE proteins may be molecular substrates contributing to the exocytosis-endocytosis coupling, which maintains exocytosis in secretory cells.

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis.

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.

Calcium-channel number critically influences synaptic strength and plasticity at the active zone.

How synaptic-vesicle release is controlled at the basic release structure, the active zone, is poorly understood. By performing cell-attached current and capacitance recordings predominantly at single active zones in rat calyces, we found that single active zones contained 5-218 (mean, 42) calcium channels and 1-10 (mean, 5) readily releasable vesicles (RRVs) and released 0-5 vesicles during a 2-ms depolarization. Large variation in the number of calcium channels caused wide variation in release strength (measured during a 2-ms depolarization) by regulating the RRV release probability (P(RRV)) and the RRV number. Consequently, an action potential opened ∼1-35 (mean, ∼7) channels, resulting in different release probabilities at different active zones. As the number of calcium-channels determined P(RRV), it critically influenced whether subsequent release would be facilitated or depressed. Regulating calcium channel density at active zones may thus be a major mechanism to yield synapses with different release properties and plasticity. These findings may explain large differences reported at synapses regarding release strength (release of 0, 1 or multiple vesicles), P(RRV), short-term plasticity, calcium transients and the requisite calcium-channel number for triggering release.

A membrane pool retrieved via endocytosis overshoot at nerve terminals: a study of its retrieval mechanism and role.

Endocytosis overshoot, which retrieves more membrane than vesicles just being exocytosed, occurs at nerve terminals and non-neuronal secretory cells. The mechanism that retrieves the overshoot membrane pool and the role of this pool remain largely unknown. We addressed this issue at the rat calyx of Held nerve terminal with capacitance measurements. We found that every calyx contained an overshoot pool ∼1.8 times the readily releasable pool. Retrieval of this pool required large calcium influx, and was inhibited by blockers of calcium/calmodulin-activated calcineurin and dynamin, suggesting the involvement of calcineurin and dynamin in endocytosis overshoot. Depletion of the overshoot pool slowed down compensatory endocytosis, whereas recovery of the overshoot pool via exocytosis that deposited stranded vesicles to the plasma membrane led to recovery of compensatory endocytosis, suggesting that the overshoot pool enhances endocytosis efficiency. These results suggest that the overshoot pool exists at every nerve terminal, is of limited size arising from vesicles stranded at the plasma membrane, is retrieved via calcium/calmodulin/calcineurin and dynamin signaling pathway, and can enhance endocytosis efficiency. Potential mechanisms for how the endocytosis overshoot pool enhances endocytosis efficiency are discussed.