Evoked Transmitter Release Research Articles

Vesicular release probability sets the strength of individual Schaffer collateral synapses

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.

Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging

Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (Pr). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets Pr remain to be defined. To address this, we developed QuaSOR, a super-resolution method for determining Pr from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. We mapped the Pr onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). We find that Pr varies greatly between synapses made by a single axon, quantify the contribution of key AZ proteins to Pr diversity and find that one of these, Complexin, suppresses spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission.

Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function.

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10-25mM) reduced endplate potential (EPP) amplitudes by 10-40% and enhanced depression during repetitive (2-20Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole-cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74mM. Cyclohexanol (10-20mM) also caused prolonged episodes of reduced-current, multi-channel bursting in outside-out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (-6kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α-subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (-3.8kcal/mol) to voltage-sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. KEY POINTS: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+ -channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.

CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice

Attention-deficit hyperactivity disorder (ADHD) is a common childhood-onset psychiatric disorder characterized by inattention, impulsivity and hyperactivity. ADHD exhibits substantial heritability, with rare monogenic variants contributing to its pathogenesis. Here we demonstrate familial ADHD caused by a missense mutation in CDH2, which encodes the adhesion protein N-cadherin, known to play a significant role in synaptogenesis; the mutation affects maturation of the protein. In line with the human phenotype, CRISPR/Cas9-mutated knock-in mice harboring the human mutation in the mouse ortholog recapitulated core behavioral features of hyperactivity. Symptoms were modified by methylphenidate, the most commonly prescribed therapeutic for ADHD. The mutated mice exhibited impaired presynaptic vesicle clustering, attenuated evoked transmitter release and decreased spontaneous release. Specific downstream molecular pathways were affected in both the ventral midbrain and prefrontal cortex, with reduced tyrosine hydroxylase expression and dopamine levels. We thus delineate roles for CDH2-related pathways in the pathophysiology of ADHD.

An engineered channelrhodopsin optimized for axon terminal activation and circuit mapping

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. However, ChR2 variants selectively transported down a neuron’s long-range axonal projections for precise presynaptic activation remain lacking. As a result, ChR2 activation is often contaminated by the spurious activation of en passant fibers that compromise the accurate interpretation of functional effects. Here, we explored the engineering of a ChR2 variant specifically localized to presynaptic axon terminals. The metabotropic glutamate receptor 2 (mGluR2) C-terminal domain fused with a proteolytic motif and axon-targeting signal (mGluR2-PA tag) localized ChR2-YFP at axon terminals without disturbing normal transmission. mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas enabling lower levels of photostimulation. Circuit connectivity mapping in vivo with the Spike Collision Test revealed that mGluR2-PA-tagged ChR2 is useful for identifying axonal projection with significant reduction in the polysynaptic excess noise. These results suggest that the mGluR2-PA tag helps actuate trafficking to the axon terminal, thereby providing abundant possibilities for optogenetic experiments.

Serotonergic Modulation of Spontaneous and Evoked Transmitter Release in Layer II Pyramidal Cells of Rat Somatosensory Cortex.

As axons from the raphe nuclei densely innervate the somatosensory cortex, we investigated how serotonin (5-HT) modulates transmitter release in layer II pyramidal cells of rat barrel cortex. In the presence of tetrodotoxin and gabazine, 10μM 5-HT caused a waxing and waning in the frequency of miniature excitatory postsynaptic currents (mEPSC) with no effect on amplitude. Specifically, within 15 min of recording the mEPSC frequency initially increased by 28 ± 7%, then dropped to below control (-15 ± 3%), before resurging back to 27 ± 7% larger than control. These changes were seen in 47% of pyramidal cells (responders) and were mediated by 5-HT2C receptors (5-HT2CR). Waxing resulted from phospholipase C activation, IP3 production, and Ca2+ release from presynaptic stores. Waning was prevented if PKC was blocked. In contrast, in paired recordings, the unitary EPSC amplitude was reduced by 50 ± 3% after 5-HT exposure in almost all cases with no significant effect on paired-pulse ratio and synaptic dynamics. This sustained EPSC reduction was also caused by 5-HT2R, but was mediated by presynaptic Gβγ subunits likely limiting influx through CaV2 channels. EPSC reduction, together with enhanced spontaneous noise in a restricted subset of inputs, could temporarily diminish the signal-to-noise ratio and affect the computation in the neocortical microcircuit.

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

Acute Complexin Knockout Abates Spontaneous and Evoked Transmitter Release

SNARE-mediated synaptic vesicle (SV) fusion is controlled by multiple regulatory proteins that determine neurotransmitter release efficiency. Complexins are essential SNARE regulators whose mode of action is unclear, as available evidence indicates positive SV fusion facilitation and negative "fusion clamp"-like activities, with the latter occurring only in certain contexts. Because these contradictory findings likely originate in part from different experimental perturbation strategies, we attempted to resolve them by examining a conditional complexin-knockout mouse line as the most stringent genetic perturbation model available. We found that acute complexin loss after synaptogenesis in autaptic and mass-cultured hippocampal neurons reduces SV fusion probability and thus abates the rates of spontaneous, synchronous, asynchronous, and delayed transmitter release but does not affect SV priming or cause "unclamping" of spontaneous SV fusion. Thus, complexins act as facilitators of SV fusion but are dispensable for "fusion clamping" in mammalian forebrain neurons.

Neuronal overexpression of Alzheimer's disease and Down's syndrome associated DYRK1A/minibrain gene alters motor decline, neurodegeneration and synaptic plasticity in Drosophila

Down syndrome (DS) is characterised by abnormal cognitive and motor development, and later in life by progressive Alzheimer's disease (AD)-like dementia, neuropathology, declining motor function and shorter life expectancy. It is caused by trisomy of chromosome 21 (Hsa21), but how individual Hsa21 genes contribute to various aspects of the disorder is incompletely understood. Previous work has demonstrated a role for triplication of the Hsa21 gene DYRK1A in cognitive and motor deficits, as well as in altered neurogenesis and neurofibrillary degeneration in the DS brain, but its contribution to other DS phenotypes is unclear. Here we demonstrate that overexpression of minibrain (mnb), the Drosophila ortholog of DYRK1A, in the Drosophila nervous system accelerated age-dependent decline in motor performance and shortened lifespan. Overexpression of mnb in the eye was neurotoxic and overexpression in ellipsoid body neurons in the brain caused age-dependent neurodegeneration. At the larval neuromuscular junction, an established model for mammalian central glutamatergic synapses, neuronal mnb overexpression enhanced spontaneous vesicular transmitter release. It also slowed recovery from short-term depression of evoked transmitter release induced by high-frequency nerve stimulation and increased the number of boutons in one of the two glutamatergic motor neurons innervating the muscle. These results provide further insight into the roles of DYRK1A triplication in abnormal aging and synaptic dysfunction in DS.

Pre-clinical symptoms of SBMA may not be androgen-dependent: implications from two SBMA mouse models.

A distinguishing aspect of spinal and bulbar muscular atrophy (SBMA) is its androgen-dependence, possibly explaining why only males are clinically affected. This disease, which impairs neuromuscular function, is linked to a polyglutamine expansion mutation in the androgen receptor (AR). In mouse models of SBMA, motor dysfunction is associated with pronounced defects in neuromuscular transmission, including defects in evoked transmitter release (quantal content, QC) and fiber membrane excitability (based on the resting membrane potential, RMP). However, whether such defects are androgen-dependent is unknown. Thus, we recorded synaptic potentials intracellularly from adult muscle fibers of transgenic (Tg) AR97Q male mice castrated pre-symptomatically. Although castration largely protects both QC and the RMP of fibers, correlating with the protective effect of castration on motor function, significant deficits in QC and RMP remained. Surprisingly, comparable defects in QC and RMP were also observed in pre-symptomatic AR97Q males, indicating that such defects emerge early and are pre-clinical. Exposing asymptomatic Tg females to androgens also induces both motor dysfunction and comparable defects in QC and RMP. Notably, asymptomatic Tg females also showed significant deficits in QC and RMP, albeit less severe, supporting their pre-clinical nature, but also raising questions about the androgen-dependence of pre-clinical symptoms. In summary, current evidence indicates that disease progression depends on androgens, but early pathogenic events may be triggered by the mutant AR allele independent of androgens. Such early, androgen-independent disease mechanisms may also be relevant to females carrying the SBMA allele.

Synaptotagmin2 (Syt2) Drives Fast Release Redundantly with Syt1 at the Output Synapses of Parvalbumin-Expressing Inhibitory Neurons.

Parvalbumin-expressing inhibitory neurons in the mammalian CNS are specialized for fast transmitter release at their output synapses. However, the Ca2+ sensor(s) used by identified inhibitory synapses, including the output synapses of parvalbumin-expressing inhibitory neurons, have only recently started to be addressed. Here, we investigated the roles of Syt1 and Syt2 at two types of fast-releasing inhibitory connections in the mammalian CNS: the medial nucleus of the trapezoid body to lateral superior olive glycinergic synapse, and the basket/stellate cell-Purkinje GABAergic synapse in the cerebellum. We used conditional and conventional knock-out (KO) mouse lines, with viral expression of Cre-recombinase and a light-activated ion channel for optical stimulation of the transduced fibers, to produce Syt1-Syt2 double KO synapses in vivo Surprisingly, we found that KO of Syt2 alone had only minor effects on evoked transmitter release, despite the clear presence of the protein in inhibitory nerve terminals revealed by immunohistochemistry. We show that Syt1 is weakly coexpressed at these inhibitory synapses and must be genetically inactivated together with Syt2 to achieve a significant reduction and desynchronization of fast release. Thus, our work identifies the functionally relevant Ca2+ sensor(s) at fast-releasing inhibitory synapses and shows that two major Syt isoforms can cooperate to mediate release at a given synaptic connection.SIGNIFICANCE STATEMENT During synaptic transmission, the influx of Ca2+ into the presynaptic nerve terminal activates a Ca2+ sensor for vesicle fusion, a crucial step in the activity-dependent release of neurotransmitter. Synaptotagmin (Syt) proteins, and especially Syt1 and Syt2, have been identified as the Ca2+ sensor at excitatory synapses, but the Ca2+ sensor(s) at inhibitory synapses in native brain tissue are not well known. We found that both Syt1 and Syt2 need to be genetically inactivated to cause a significant reduction of activity-evoked release at two types of fast inhibitory synapses in mouse brain. Thus, we identify Syt2 as a functionally important Ca2+ sensor at fast-releasing inhibitory synapses, and show that Syt1 and Syt2 can redundantly control transmitter release at specific brain synapses.

Tau pathology-mediated presynaptic dysfunction

Brain tauopathies are characterized by abnormal processing of tau protein. While somatodendritic tau mislocalization has attracted considerable attention in tauopathies, the role of tau pathology in axonal transport, connectivity and related dysfunctions remains obscure. We have previously shown using the squid giant synapse that presynaptic microinjection of recombinant human tau protein (htau42) results in failure of synaptic transmission. Here, we evaluated molecular mechanisms mediating this effect. Thus, the initial event, observed after htau42 presynaptic injection, was an increase in transmitter release. This event was mediated by calcium release from intracellular stores and was followed by a reduction in evoked transmitter release. The effect of htau42 on synaptic transmission was recapitulated by a peptide comprising the phosphatase-activating domain of tau, suggesting activation of phosphotransferases. Accordingly, findings indicated that htau42-mediated toxicity involves the activities of both GSK3 and Cdk5 kinases.

Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission.

Oxygen-glucose deprivation (OGD) leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca2+]i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs). Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca2+]i, (shown by calcium imaging), increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca2+ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM) partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX), also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca2+]i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca2+ buffering which also speeds recovery.

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster.

Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.

The Same Synaptic Vesicles Originate Synchronous and Asynchronous Transmitter Release

Transmitter release and synaptic vesicle exo- and endocytosis during high-frequency stimulation (20 pulses/s) in the extracellular presence of different bivalent cations (Ca(2+), Sr2+ or Ba2+) were studied in frog cutaneous pectoris nerve-muscle preparations. It was shown in electrophysiological experiments that almost only synchronous transmitter release was registered in a Ca(2+)-containing solution; a high intensity of both synchronous and asynchronous transmitter release was registered in a Sr2+-containing solution, and asynchronous transmitter release almost only was observed in a Ba2+-containing solution. It was shown in experiments with a FM 1-43 fluorescent dye that the synaptic vesicles that undergo exocytosis-endocytosis during synchronous transmitter release (Ca-solutions) are able to participate in asynchronous exocytosis in Ba-solutions. The vesicles that had participated in the asynchronous transmitter release (Ba-solutions) could subsequently participate in a synchronous release (Ca-solutions). It was shown in experiments with isolated staining of recycling and reserve synaptic vesicle pools that both types of evoked transmitter release originate from the same synaptic vesicle pool.

Distinct Regulation of Transmitter Release at the Drosophila NMJ by Different Isoforms of nemy.

Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. In the present study, we have examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, we generated two independent excisions of nemy that differentially affect the expression of nemy isoforms. We show that the nemy 45 excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy 26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. We further show that nemy 45 mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy 26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine β hydroxylase activity (TβH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators.

Mechanisms of hydrogen sulfide (H2S) action on synaptic transmission at the mouse neuromuscular junction.

Hydrogen sulfide (H2S) is a widespread gasotransmitter also known as a powerful neuroprotective agent in the central nervous system. However, the action of H2S in peripheral synapses is much less studied. In the current project we studied the modulatory effects of the H2S donor sodium hydrosulfide (NaHS) on synaptic transmission in the mouse neuromuscular junction using microelectrode technique. Using focal recordings of presynaptic response and evoked transmitter release we have shown that NaHS (300 μM) increased evoked end-plate currents (EPCs) without changes of presynaptic waveforms which indicated the absence of NaHS effects on sodium and potassium currents of motor nerve endings. Using intracellular recordings it was shown that NaHS increased the frequency of miniature end-plate potentials (MEPPs) without changing their amplitudes indicating a pure presynaptic effect. Furthermore, NaHS increased the amplitude of end-plate potentials (EPPs) without influencing the resting membrane potential of muscle fibers. L-cysteine, a substrate of H2S synthesis induced, similar to NaHS, an increase of EPC amplitudes whereas inhibitors of H2S synthesis (β-cyano-L-alanine and aminooxyacetic acid) had the opposite effect. Inhibition of adenylate cyclase using MDL 12,330A hydrochloride (MDL 12,330A) or elevation of cAMP level with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (pCPT-cAMP) completely prevented the facilitatory action of NaHS indicating involvement of the cAMP signaling cascade. The facilitatory effect of NaHS was significantly diminished when intracellular calcium (Ca(2+)) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Activation of ryanodine receptors by caffeine or ryanodine increased acetylcholine release and prevented further action of NaHS on transmitter release, likely due to an occlusion effect. Inhibition of ryanodine receptors by ryanodine or dantrolene also reduced the action of NaHS on EPC amplitudes. Our results indicate that in mammalian neuromuscular synapses endogenously produced H2S increases spontaneously and evoked quantal transmitter release from motor nerve endings without changing the response of nerve endings. The presynaptic effect of H2S appears mediated by intracellular Ca(2+) and cAMP signaling and involves presynaptic ryanodine receptors.

Deficient Wnt signalling triggers striatal synaptic degeneration and impaired motor behaviour in adult mice.

Synapse degeneration is an early and invariant feature of neurodegenerative diseases. Indeed, synapse loss occurs prior to neuronal degeneration and correlates with the symptom severity of these diseases. However, the molecular mechanisms that trigger synaptic loss remain poorly understood. Here we demonstrate that deficient Wnt signalling elicits synaptic degeneration in the adult striatum. Inducible expression of the secreted Wnt antagonist Dickkopf1 (Dkk1) in adult mice (iDkk1) decreases the number of cortico-striatal glutamatergic synapses and of D1 and D2 dopamine receptor clusters. Synapse loss occurs in the absence of axon retraction or cell death. The remaining excitatory terminals contain fewer synaptic vesicles and have a reduced probability of evoked transmitter release. IDkk1 mice show impaired motor coordination and are irresponsive to amphetamine. These studies identify Wnts as key endogenous regulators of synaptic maintenance and suggest that dysfunction in Wnt signalling contributes to synaptic degeneration at early stages in neurodegenerative diseases.

Presynaptic T-type Ca2+ channels modulate dendrodendritic mitral-mitral and mitral-periglomerular connections in mouse olfactory bulb.

Mitral cells express low-voltage activated Cav3.3 channels on their distal apical tuft dendrites (McKay et al., 2006; Johnston and Delaney, 2010). They also discharge Na(+)-dependent dendritic action potentials and release glutamate from these dendrites. Around resting membrane potentials, between -65 and -50 mV, Cav3.x channels are a primary determinant of cytoplasmic [Ca(2+)]. In this study using C57 mice, we present evidence that subthreshold Cav3.x-mediated Ca(2+) influx modulates action potential evoked transmitter release and directly drives asynchronous release from distal tuft dendrites. Presynaptic hyperpolarization and selective block of Cav3.x channels with Z941 (Tringham et al., 2012) reduce mitral-to-mitral EPSP amplitude, increase the coefficient of variation of EPSPs, and increase paired-pulse ratios, consistent with a reduced probability of transmitter release. Both hyperpolarization and Cav3.x channel blockade reduce steady-state cytoplasmic [Ca(2+)] in the tuft dendrite without reducing action potential evoked Ca(2+) influx, suggesting that background [Ca(2+)] modulates evoked release. We demonstrate that Cav3.x-mediated Ca(2+) influx from even one mitral cell at membrane potentials between -65 and -50 mV is sufficient to produce feedback inhibition from periglomerular neurons. Deinactivation of Cav3.x channels by hyperpolarization increases T-type Ca(2+) influx upon repolarization and increases feedback inhibition to produce subthreshold modulation of the mitral-periglomerular reciprocal circuit.

Effects of Oxidation of Membrane Cholesterol on the Vesicle Cycle in Motor Nerve Terminals in the Frog Rana Ridibunda

Experiments on neuromuscular preparations from the frog Rana ridibunda assessed the effects of oxidation of membrane cholesterol on the presynaptic vesicle cycle. Application of cholesterol oxidase (1 activity unit) for 30 min oxidized about 0.007 mg of cholesterol per g of preparation and decreased the stability of lipid rafts in nerve terminals. Electrophysiological studies demonstrated that oxidation of cholesterol decreased evoked transmitter release. Experiments using fluorescent FM dyes demonstrated suppression of synaptic vesicle exo- and endocytosis processes, along with dispersal of clusters of synaptic vesicles. Comparative analysis of electrophysiological and optical data, along with experiments using a dye quencher, demonstrated that transmitter could be released from some synaptic vesicles through transient fusion pores (the kiss-and-run mechanism). It is suggested that oxidation of cholesterol suppresses evoked exocytosis and delivery of synaptic vesicles of the reserve pool to the exocytosis site, thus interfering with their clustering. Vesicles of the recycling pool release transmitter by the “kiss-and-run” mechanism.