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.

Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) in Drosophila. Null mutants of ent2 are lethal during late larval/early pupal stages, indicating that ent2 is essential for normal development. Hypomorphic mutant alleles of ent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock down ent2 expression in specific regions of the CNS and show that ent2 is required in the alpha/beta lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated in ent2 mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR(1)) into the ent2 mutant background. The results of the present study provide the first evidence of a role for ent2 function in Drosophila and suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

Cholesterol-dependent kinase activity regulates transmitter release from cerebellar synapses.

Changes in membrane cholesterol content can alter protein kinase activity, however, it is not known whether kinases regulating transmitter release are sensitive to membrane cholesterol content. Here we have used the cholesterol extracting agent methyl-beta-cyclodextrin to measure the effects of acute cholesterol reduction on transmitter release from cultured cerebellar neurons. Cholesterol depletion increased the frequency of spontaneous transmitter release without altering the amplitude and time course of mEPSCs. Evoked transmitter release was decreased by cholesterol extraction and the paired pulse ratio was also decreased. Alterations in synaptic transmission were not associated with failure of action potential generation or changes in presynaptic Ca(2+) signaling. Both the increase in mEPSC frequency and the change in paired pulse ratio were blocked by the broad spectrum protein kinase inhibitor staurosporine. The increase in mEPSC frequency was also sensitive to selective inhibitors of PKC and PKA. Our results therefore demonstrate that the activity of presynaptic protein kinases that regulate spontaneous and evoked neurotransmitter release is sensitive to changes of membrane cholesterol content.

Peptide-mediated transdermal delivery of botulinum neurotoxin type A reduces neurogenic inflammation in the skin.

Release of inflammatory pain mediators from peripheral sensory afferent endings contributes to the development of a positive feedback cycle resulting in chronic inflammation and pain. Botulinum neurotoxin type A (BoNT-A) blocks exocytosis of neurotransmitters and may therefore block the release of pain modulators in the periphery. Subcutaneous administration of BoNT-A (2.5, 5 and 10U) reduced plasma extravasation (PE) caused by electrical stimulation of the saphenous nerve or capsaicin in the rat hindpaw skin (ANOVA, Post hoc Tukey, p<0.05, n=6). Subcutaneous BoNT-A also reduced blood flow changes evoked by saphenous nerve stimulation (ANOVA, Post hoc Tukey, p<0.05, n=6). Subcutaneous BoNT-A had no effect on PE induced by local injection of substance P (SP) or vasodilation induced by local CGRP injection. Although BoNT-A is an effective treatment for a wide range of painful conditions, the toxin's large size necessitates that it be injected at numerous sites. We found that a short synthetic peptide (TD-1) can facilitate effective transdermal delivery of BoNT-A through intact skin. Coadministration of TD-1 and BoNT-A to the hindpaw skin resulted in a significant reduction in PE evoked by electrical stimulation. The findings show that BoNT-A can be administered subcutaneously or topically with a novel transdermal delivery peptide to reduce inflammation produced by activating nociceptors in the skin. Peptide-mediated delivery of BoNT-A is an easy and non-invasive way of administering the toxin that may prove to be useful in clinical practice.

Calcineurin and cytoskeleton in low-frequency depression.

Transmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the post-synaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in pre-synaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances post-synaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.

AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis.

AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.