Facilitation of transmitter release at squid synapses

Facilitation is shown to decay as a compound exponential with two time constants (T1, T2) at both giant and non-giant synapses in squid steilate ganglia bathed in solutions having low extracellular calcium concentrations ([Ca++]o). Maximum values of facilitation (F~) were significantly larger, and T1 was significantly smaller in giant than non-giant synapses. Decreases in [Ca++]o or increases in [Mn++]o had variable effects on T1 and F1, whereas decreases in temperature increased T~ but had insignificant effects on/'1. The growth of facilitation during short trains of equal interval stimuli was adequately predicted by the linear summation model developed by Mallart and Martin (1967.J. Physiol. (Lond.). 193: 676-694) for frog neuromuscular junctions. This result suggests that the underlying mechanisms of facilitation are similar in squid and other synapses which release many transmitter quanta.This work was supported by National Science Foundation research grant GB-36949, National Research Council (Canada) and Grass Fellowships to Dr. Charlton, and a National Institutes of Health career award (NS-00070) to Dr. Bittner.Neuroscienc

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

SNARE zippering and synaptic strength.

Synapses vary widely in the probability of neurotransmitter release. We tested the hypothesis that the zippered state of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex determines initial release probability. We tested this hypothesis at phasic and tonic synapses which differ by 100-1000-fold in neurotransmitter release probability. We injected, presynaptically, three Clostridial neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by the zippering of the SNARE complex or open to proteolytic attack. Under low stimulation conditions, the catalytic light-chain fragment of botulinum B (BoNT/B-LC) inhibited evoked release at both phasic and tonic synapses and cleaved VAMP; however, neither BoNT/D-LC nor tetanus neurotoxin (TeNT-LC) were effective in these conditions. The susceptibility of VAMP to only BoNT/B-LC indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end to approximately the zero-layer with the C-terminal end exposed under resting state. Therefore, the existence of the same partially zippered state of the trans-SNARE complex at both phasic and tonic synapses indicates that release probability is not determined solely by the zippered state of the trans-SNARE complex at least to the zero-layer

Presynaptic potentials and facilitation of transmitter release in the squid giant synapse

release in the squid giant synapse. Changes in action potentials were found to cause some, but not all, of the facilitation during twin-pulse stimulation. During trains of action potentials, there were no progressive changes in presynaptic action potentials which could account for the growth of facilitation. Facilitation could still be detected in terminals which had undergone conditioning depolarization or hyperpolarization. Facilitation could be produced by small action potentials in low [Ca++]o and by small depolarizations in the presence of tetrodotoxin. Although the production of facilitation varied somewhat with presynaptic depolarization, nevertheless, approximately equal amounts of facilitation could be produced by depolarizations which caused the release of very different amounts of transmitter

Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity

Many forms of neurodegeneration are ascribed to excessive cellular Ca2+ loading (Ca2+ hypothesis). We examined quantitatively whether factors other than Ca2+ loading were determinants of excitotoxic neurodegeneration. Cell survival, morphology, free intracellular Ca2+ concentration ([Ca2+](i)), and 45Ca2+ accumulation were measured in cultured cortical neurons loaded with known quantities of Ca2+ through distinct transmembrane pathways triggered by excitatory amino acids, cell membrane depolarization, or Ca2+ ionophores. Contrary to the Ca2+ hypothesis, the relationships between Ca2+ load and cell survival, free [Ca2+](i), and Ca2+-induced morphological alterations depended primarily on the route of Ca2+ influx, not the Ca2+ load. Notably, Ca2+ loading via NMDA receptor channels was toxic, whereas identical Ca2+ loads incurred through voltage-sensitive Ca2+ channels were completely innocuous. Furthermore, accounting quantitatively for Ca2+ loading via NMDA receptors uncovered a previously unreported component of L-glutamate neurotoxicity apparently not mediated by ionotropic or metabotropic glutamate receptors. It was synergistic with toxicity attributable to glutamate-evoked Ca2+ loading, and correlated with enhanced cellular ATP depletion. This previously unrecognized toxic action of glutamate constituted a chief excitotoxic mechanism under conditions producing submaximal Ca2+ loading. We conclude that (a) Ca2+ neurotoxicity is a function of the Ca2+ influx pathway, not Ca2+ load, and (b) glutamate toxicity may not be restricted to its actions on glutamate receptors

Determination of the time course and extent of neurotoxicity at defined temperatures in cultured neurons using a modified multiwell plate fluorescence scanner

The cellular and molecular mechanisms of hypoxic/ischemic neurodegeneration are sensitive to numerous factors that modulate the time course and degree of neuronal death. Among such factors is hypothermia, which can dramatically protect neurons from injury. To examine and control for temperature-dependent effects, we developed a technique that provides for a high-throughput, accurate, and reproducible determination of the time course and degree of neurotoxicity in cultured cortical neurons at precisely defined temperatures. We used a fluorescence multiwell plate scanner, modified by us to permit the control of temperature, to perform serial quantitative measurements of propidium iodide (PI) fluorescence in cortical neuronal cultures exposed to excitotoxic insults. In validating this approach, we show that these time course measurements correlate highly with manual counts of PI-stained cells in the same cultures (r = 0.958. p \u3c 0.0001) and with lactate dehydrogenase release (r = 0.964, p \u3c 0.0001). This method represents an efficient approach to mechanistic and quantitative studies of cell death as well as a high-throughput technique for screening new neuroprotective therapies in vitro

Susceptibility of VAMP to <i>Clostridial</i> neurotoxins.

<p><b><i>A</i></b>, Zippered states of the SNARE complex. <b><i>Ai</i></b>, SNARE complex tightly zippered beyond the zero-layer indicated by gray arrow head. VAMP (blue) is protected from cleavage because the binding (yellow and orange bars) and cleavage (scissors and white lines) sites of VAMP-specific <i>Clostridial</i> neurotoxins (TeNT, BoNT/B and BoNT/D) are occluded. <b><i>Aii</i></b>, Partially zippered SNARE complex. The binding sites of TeNT and BoNT/D are occluded but the binding and cleavage sites for BoNT/B are exposed such that VAMP is susceptible to cleavage. Green – syntaxin, red – SNAP25 (represents both SNARE binding motifs). <b><i>B</i></b>, <i>Clostridial</i> neurotoxins cleave crayfish VAMP <i>in-vitro</i>. Crayfish CNS protein sample was incubated with inactive or active neurotoxins (BoNT/B-LC (0.5 µg/μL), BoNT/D-LC (0.3 µg/μL) and TeNT-LC (0.5 µg/μL)) and stained for neuronal VAMP. The protein bands of 18 kDa represent VAMP. Cleaved VAMP does not appear on the blot when active neurotoxins were used because the VAMP antibody binds only to the uncleaved VAMP protein. Actin staining of 38 kDa below the VAMP blot shows that equal amounts (10 µg) of protein were loaded in each lane. <b><i>C</i></b>, Comparison of full-length crayfish VAMP amino acid sequence with VAMP sequences from other species. Crayfish VAMP is similar to VAMP from other species, especially in the conserved SNARE motif region (black bar). The cleavage sites of VAMP-specific neurotoxins are indicated in the alignment. The primary binding sites of the neurotoxins used in this study are indicated as boxed regions (V1 motif (aa 38–47) – TeNT and BoNT/D; V2 motif (aa 62–71) – BoNT/B) based on the human VAMP sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095130#pone.0095130-Arndt1" target="_blank">[29]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095130#pone.0095130-Foran1" target="_blank">[31]</a>. The multiple protein sequence alignment was performed using the online ClustalW2 Multiple Sequence Alignment tool (European Molecular Biology Laboratory - European Bioinformatics Institute, <a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>).</p

Phasic EPSPs are inhibited using TeNT-LC (<i>B</i>), BoNT/D-LC (<i>C</i>) and BoNT/B-LC (<i>D</i>) under high frequency stimulation.

<p><b><i>A</i></b>, Example showing the phasic action potential (AP) remains unchanged before (<i>i</i>) and after (<i>ii</i>) the injection of each neurotoxin (BoNT/B-LC used as the example). <b><i>E</i></b>, Percent difference between active and inactive neurotoxin, in which the inactive neurotoxin is the reference point at 0%. In <i>B-E,</i> a solid black line represents the time course of neurotoxin injection (90 min), the dotted lines represent the time course of each round of high frequency stimulation (40 min) and vertical arrows (↓) represent when EPSPs were recorded. Active neurotoxins: TeNT-LC (▪), BoNT/D-LC (▴) and BoNT/B-LC (•). Inactive neurotoxins: TeNT-LC (□), BoNT/D-LC (△) and BoNT/B-LC (○). Error bars represent S.E.M. An asterisk (*) denotes a significant difference (p<0.05) and ‘n’ represents sample size (active/inactive neurotoxin). Scale bars: horizontal – 10 ms; vertical – 10 mV (AP), 5 mV (EPSP).</p

VAMP in phasic and tonic axonal terminals is susceptible only to BoNT/B-LC under low frequency stimulation.

<p>Immunostaining of VAMP and synapsin after the injection of inactive and active TeNT-LC (<i>A</i>), BoNT/D-LC (<i>B</i>) and BoNT/B-LC (<i>C</i>) into the phasic or tonic axon during the low frequency stimulation experiments. In <i>A-C</i>, arrows denote phasic terminals and arrow heads denote tonic terminals. The yellow areas in the merged image represent an overlap of VAMP and synapsin immunoreactivity. In <i>C</i>, the injected boutons contain only synapsin indicating that active BoNT/B-LC cleaved VAMP under low frequency stimulation. Note that only active BoNT/B-LC reduced VAMP immunoreactivity under low frequency stimulation conditions. In <i>B</i>,<i>C</i>, no tonic terminals were present in the phasic image with active neurotoxin. Scale bars – <i>A</i> and <i>B</i>: 19 µm; <i>C</i>: 10 µm.</p