Synergistic control of neurotransmitter release by different members of the synaptotagmin family

Quantal neurotransmitter release at nerve terminals is tightly regulated by the presynaptic Ca2+ concentration. Here, we summarise current advances in understanding how the interplay between presynaptic Ca2+ dynamics and different Ca2+ release sensors shapes action potential-evoked release on a timescale from hundreds of microseconds to hundreds of milliseconds. In particular, we review recent studies that reveal the synergistic roles of the low Ca2+ affinity/fast release sensors synaptotagmins 1, 2 and 9 and the high affinity/slow release sensor synaptotagmin 7 in the regulation of synchronous and asynchronous release and of short-term synaptic plasticity. We also examine new biochemical and structural data and outline a working model that could potentially explain the cooperative roles of different synaptotagmins in molecular terms

Action potential broadening in a presynaptic channelopathy

Brain development and interictal function are unaffected in many paroxysmal neurological channelopathies, possibly explained by homoeostatic plasticity of synaptic transmission. Episodic ataxia type 1 is caused by missense mutations of the potassium channel Kv1.1, which is abundantly expressed in the terminals of cerebellar basket cells. Presynaptic action potentials of small inhibitory terminals have not been characterized, and it is not known whether developmental plasticity compensates for the effects of Kv1.1 dysfunction. Here we use visually targeted patch-clamp recordings from basket cell terminals of mice harbouring an ataxia-associated mutation and their wild-type littermates. Presynaptic spikes are followed by a pronounced afterdepolarization, and are broadened by pharmacological blockade of Kv1.1 or by a dominant ataxia-associated mutation. Somatic recordings fail to detect such changes. Spike broadening leads to increased Ca2+ influx and GABA release, and decreased spontaneous Purkinje cell firing. We find no evidence for developmental compensation for inherited Kv1.1 dysfunction

Preparation of dissociated mouse primary neuronal cultures from long-term cryopreserved brain tissue

BACKGROUND: Dissociated primary neuronal cultures are widely used as a model system to investigate the cellular and molecular properties of diverse neuronal populations and mechanisms of action potential generation and synaptic transmission. Typically, rodent primary neuronal cultures are obtained from freshly-dissociated embryonic or postnatal brain tissue, which often requires intense animal husbandry. This can strain resources when working with genetically modified mice. NEW METHOD: Here we describe an experimental protocol for frozen storage of mouse hippocampi, which allows fully functional dissociated primary neuronal cultures to be prepared from cryopreserved tissue. RESULTS: We show that thawed hippocampal neurons have functional properties similar to those of freshly dissociated neurons, including neuronal morphology, excitability, action potential waveform and synaptic neurotransmitter release, even after cryopreservation for several years. COMPARISON TO THE EXISTING METHODS: In contrast to the existing methods, the protocol described here allows for efficient long-term storage of samples, allowing researchers to perform functional experiments on neuronal cultures from brain tissue collected in other laboratories. CONCLUSIONS: We anticipate that this method will facilitate collaborations among laboratories based at distant locations and will thus optimise the use of genetically modified mouse models, in line with the 3Rs (Replacement, Reduction and Refinement) recommended for scientific use of animals in research

Alternative Splicing of P/Q-Type Ca2+ Channels Shapes Presynaptic Plasticity

Alternative splicing of pre-mRNAs is prominent in the mammalian brain, where it is thought to expand proteome diversity. For example, alternative splicing of voltage-gated Ca2+ channel (VGCC) α1 subunits can generate thousands of isoforms with differential properties and expression patterns. However, the impact of this molecular diversity on brain function, particularly on synaptic transmission, which crucially depends on VGCCs, is unclear. Here, we investigate how two major splice isoforms of P/Q-type VGCCs (Cav2.1[EFa/b]) regulate presynaptic plasticity in hippocampal neurons. We find that the efficacy of P/Q-type VGCC isoforms in supporting synaptic transmission is markedly different, with Cav2.1[EFa] promoting synaptic depression and Cav2.1[EFb] synaptic facilitation. Following a reduction in network activity, hippocampal neurons upregulate selectively Cav2.1[EFa], the isoform exhibiting the higher synaptic efficacy, thus effectively supporting presynaptic homeostatic plasticity. Therefore, the balance between VGCC splice variants at the synapse is a key factor in controlling neurotransmitter release and presynaptic plasticity

Independent regulation of Basal neurotransmitter release efficacy by variable Ca²+ influx and bouton size at small central synapses.

The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca²⁺ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca²⁺ concentration transient. We further show that synapses with a high vesicular release probability exhibit a lower Ca²⁺ cooperativity, arguing that this is a direct consequence of increased Ca²⁺ influx at the active zone. We conclude that variability of neurotransmitter release under basal conditions at small central synapses is accounted for not only by the number of release-ready vesicles, but also by their fusion probabilities, which are set independently of bouton size by variable spike-evoked presynaptic Ca²⁺ influx

Lambert-Eaton syndrome IgG inhibits transmitter release via P/Q Ca²⁺ channels

Objective: To determine whether immunoglobulin G (IgG) from patients with Lambert Eaton Syndrome (LEMS) decreases action-potential evoked synaptic vesicle exocytosis, and whether the effect is mediated by P/Q-type voltage-gated calcium channels (VGCCs). Methods: IgG was obtained from four patients with LEMS (3M, 1F), including two patients with lung malignancy. Antibodies against P/Q-type VGCCs were detected in all four patients, and Ntype in two. We incubated neuronal cultures with LEMS IgG and determined the size of the total recycling pool of synaptic vesicles and the rate of action-potential evoked exocytosis using fluorescence imaging of the amphiphilic dye SynaptoRed C1. Pooled IgG from healthy volunteers was used as a control. We repeated the experiments on synapses lacking P/Q-type calcium channels from a Cacna1a knockout mouse to determine whether these channels account for the pathogenic effect of LEMS IgG. Results: LEMS IgG had no effect on the total recycling pool size but significantly reduced the rate of action potential-evoked synaptic exocytosis in wild type neurons when compared to neurons treated with control IgG. In contrast, LEMS IgG had no effect on the rate of synaptic vesicle exocytosis in neurons lacking P/Q-type channels. Conclusions: These data provide direct evidence that LEMS IgG inhibits neurotransmitter release by acting on P/Q-type VGCCs

Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery

Latrophilin, neurexin, and their signaling-deficient mutants facilitate α-latrotoxin insertion into membranes but are not involved in pore formation

Pure alpha -latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha -latrotoxin pores are permeable to Ca2+ and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha -latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin tin the presence of Ca2+). Interestingly, on subsequent removal of Ca2+, alpha -latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha -latrotoxin antibodies. Our results indicate that (i) c alpha -latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha -latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha -latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha -latrotoxin interaction with other neuronal proteins

The alpha-latrotoxin mutant LTXN4C enhances spontaneous and evoked transmitter release in CA3 pyramidal neurons.

Alpha-latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (1) receptor binding-stimulation and (2) membrane pore formation. Here, we use the toxin mutant LTX(N4C) to selectively study the receptor-mediated actions of LTX. LTX(N4C) binds to both LTX receptors (latrophilin and neurexin) and greatly enhances the frequency of spontaneous and miniature EPSCs recorded from CA3 pyramidal neurons in hippocampal slice cultures. The effect of LTX(N4C) is reversible and is not attenuated by La3+ that is known to block LTX pores. On the other hand, LTX(N4C) action, which requires extracellular Ca2+, is inhibited by thapsigargin, a drug depleting intracellular Ca2+ stores, by 2-aminoethoxydiphenyl borate, a blocker of inositol(1,4,5)-trisphosphate-induced Ca2+ release, and by U73122, a phospholipase C inhibitor. Furthermore, measurements using a fluorescent Ca2+ indicator directly demonstrate that LTX(N4C) increases presynaptic, but not dendritic, free Ca2+ concentration; this Ca2+ rise is blocked by thapsigargin, suggesting, together with electrophysiological data, that the receptor-mediated action of LTX(N4C) involves mobilization of Ca2+ from intracellular stores. Finally, in contrast to wild-type LTX, which inhibits evoked synaptic transmission probably attributable to pore formation, LTX(N4C) actually potentiates synaptic currents elicited by electrical stimulation of afferent fibers. We suggest that the mutant LTX(N4C), lacking the ionophore-like activity of wild-type LTX, activates a presynaptic receptor and stimulates Ca2+ release from intracellular stores, leading to the enhancement of synaptic vesicle exocytosis