Presynaptic Effects Research Articles

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.

Gβγ SNARE Interactions and Their Behavioral Effects.

Presynaptic terminals possess interlocking molecular mechanisms that control exocytosis. An example of such complexity is the modulation of release by presynaptic G Protein Coupled Receptors (GPCRs). GPCR ubiquity at synapses-GPCRs are present at every studied presynaptic terminal-underlies their critical importance in synaptic function. GPCRs mediate presynaptic modulation by mechanisms including via classical Gα effectors, but membrane-delimited actions of Gβγ can also alter probability of release by altering presynaptic ionic conductances. This directly or indirectly modifies action potential-evoked presynaptic Ca2+ entry. In addition, Gβγ can interact directly with SNARE complexes responsible for synaptic vesicle fusion to reduce peak cleft neurotransmitter concentrations during evoked release. The interaction of Gβγ with SNARE is displaced via competitive interaction with C2AB-domain containing calcium sensors such as synaptotagmin I in a Ca2+-sensitive manner, restoring exocytosis. Synaptic modulation of this form allows selective inhibition of postsynaptic receptor-mediated responses, and this, in combination with Ca2+ sensitivity of Gβγ effects on SNARE complexes allows for specific behavioral outcomes. One such outcome mediated by 5-HT receptors in the spinal cord seen in all vertebrates shows remarkable synergy between presynaptic effects of Gβγ and postsynaptic 5-HT-mediated changes in activation of Ca2+-dependent K+ channels. While acting through entirely separate cellular compartments and signal transduction pathways, these effects converge on the same effect on locomotion and other critical functions of the central nervous system.

Effects of isoflurane on dopaminergic neurone synaptic vesicle exocytosis

Despite widespread clinical use of anaesthetics, their pharmacological mechanisms of action are poorly understood. Volatile anaesthetics such as isoflurane induce a reversible state of amnesia, unconsciousness, and immobility in response to painful stimuli.1 At the cellular level, volatile anaesthetics exert prominent effects on synaptic transmission. While these synaptic effects involve predominately postsynaptic actions, data from our laboratory support presynaptic mechanisms as well.2 At presynaptic sites, anaesthetics differentially inhibit synaptic vesicle (SV) exocytosis depending on neurone phenotype, with greater inhibition of excitatory glutamatergic SV release than of inhibitory GABAergic SV release from hippocampal neurones.3,4 However, anaesthetic effects on dopamine release from dopaminergic (DA) neurones have not been studied, even though dopaminergic mechanisms have been implicated in emergence from anaesthesia.5 We investigated the effects of isoflurane on SV release in DA neurones (identified by post hoc tyrosine hydroxylase immunoreactivity) from rat ventral tegmental area (VTA) neurones using live-cell imaging to measure SV exocytosis and Ca2+ influx. We employed a pH-sensitive variant of eGFP (pHluorin) fused to the luminal domain of the vesicular monoamine transporter (vMAT) to measure exocytosis and the cell permeant fluorescent Ca2+ indicator Fluo-5F to measure intracellular Ca2+ concentration. Isoflurane differentially inhibited SV exocytosis and Ca2+ influx induced by electrically stimulated action potentials from both DA (by 29 ± 4%, n = 13, p < 0.0001 and 42 ± 3%, n = 6, p < 0.0001, respectively) and non-DA neurones (by 16 ± 5%, n = 10, p < 0.0055 and 58 ± 5%, n = 8, p < 0.0001, respectively). In contrast to other neurotransmitter phenotypes, isoflurane also inhibited SV exocytosis evoked by elevated K+ in DA neurones, suggesting a voltage-gated Na+ channel (Nav) independent mechanism of anaesthetic action in this neuronal population. This contrasts with SV exocytosis from glutamate or GABA releasing neurones, which requires Nav. SV release is known to be tightly coupled to Ca2+ entry6. The degree of inhibition of SV exocytosis by isoflurane in DA neurons was proportional to the reduction in Ca2+ influx, and could be mimicked by reducing extracellular Ca2+ concentration. Use of subtype-specific voltage-gated Ca2+ channel toxins revealed that SV exocytosis in DA neurones was solely mediated by P/Q-type and N-type Ca2+ channels. Isoflurane inhibited SV exocytosis mediated by both P/Q-type and N-type Ca2+ channels by a Nav-independent mechanism, supporting a role for inhibition of these Ca2+ channel subtypes in the presynaptic effects of isoflurane in DA neurones. These findings shed light on the presynaptic targets of isoflurane in DA neurones, and provide a molecular target for isoflurane induced unconsciousness and emergence from isoflurane anaesthesia. Funding: US NIH grant GM58055. 1.Hemmings HC Jr, Akabas MH, Goldstein PA, et al. Trends Pharmacol Sci. 2005; 26: 503–10.2.Hemmings HC Jr, Yan W, Westphalen RI, et al. Mol Pharmacol. 2005; 67: 1591–9.3.Westphalen RI, Hemmings HC Jr. J Pharmacol Exp Ther. 2006; 316: 216–23.4.Baumgart JP, Zhou ZY, Hara M, et al. Proc Natl Acad Sci USA. 2015; 112: 11959–64.5.Solt K, Van Dort CJ, Chemali JJ, et al. Anesthesiology. 2014; 121: 311–9.6.Dodge FA Jr, Rahamimoff R. J Physiol. 1967; 193: 419–32.

Adenosine A2A receptors modulate the dopamine D2 receptor-mediated inhibition of synaptic transmission in the mouse prefrontal cortex.

Prefrontal cortex (PFC) circuits are modulated by dopamine acting on D1 - and D2 -like receptors, which are pharmacologically exploited to manage neuropsychiatric conditions. Adenosine A2A receptors (A2A R) also control PFC-related responses and A2A R antagonists are potential anti-psychotic drugs. As tight antagonistic A2A R-D2 R and synergistic A2A R-D1 R interactions occur in other brain regions, we now investigated the crosstalk between A2A R and D1 /D2 R controlling synaptic transmission between layers II/III and V in mouse PFC coronal slices. Dopamine decreased synaptic transmission, a presynaptic effect based on the parallel increase in paired-pulse responses. Dopamine inhibition was prevented by the D2 R-like antagonist sulpiride but not by the D1 R antagonist SCH23390 and was mimicked by the D2 R agonist sumanirole, but not by the agonists of either D4 R (A-412997) or D3 R (PD128907). Dopamine inhibition was prevented by the A2A R antagonist, SCH58261, and attenuated in A2A R knockout mice. Accordingly, triple-labelling immunocytochemistry experiments revealed the co-localization of A2A R and D2 R immunoreactivity in glutamatergic (vGluT1-positive) nerve terminals of the PFC. This reported positive A2A R-D2 R interaction controlling PFC synaptic transmission provides a mechanistic justification for the anti-psychotic potential of A2A R antagonists.

Presynaptic Effects of N-Methyl-D-Aspartate Receptors Enhance Parvalbumin Cell–Mediated Inhibition of Pyramidal Cells in Mouse Prefrontal Cortex

BackgroundTesting hypotheses regarding the role of N-methyl-D-aspartate receptor (NMDAR) hypofunction in schizophrenia requires understanding the mechanisms of NMDAR regulation of prefrontal cortex (PFC) circuit function. NMDAR antagonists are thought to produce pyramidal cell (PC) disinhibition. However, inhibitory parvalbumin-positive basket cells (PVBCs) have modest NMDAR-mediated excitatory drive and thus are unlikely to participate in NMDAR antagonist–mediated disinhibition. Interestingly, recent studies demonstrated that presynaptic NMDARs enhance transmitter release at central synapses. Thus, if presynaptic NMDARs enhance gamma-aminobutyric acid release at PVBC-to-PC synapses, they could participate in NMDAR-dependent PC disinhibition. Here, we examined whether presynaptic NMDAR effects could modulate gamma-aminobutyric acid release at PVBC-to-PC synapses in mouse PFC. MethodsUsing whole-cell recordings from synaptically connected pairs in mouse PFC, we determined whether NMDA or NMDAR antagonist application affects PVBC-to-PC inhibition in a manner consistent with a presynaptic mechanism. ResultsNMDAR activation enhanced by ∼40% the synaptic current at PVBC-to-PC pairs. This effect was consistent with a presynaptic mechanism given that it was 1) observed with postsynaptic NMDARs blocked by intracellular MK801, 2) associated with a lower rate of transmission failures and a higher transmitter release probability, and 3) blocked by intracellular MK801 in the PVBC. NMDAR antagonist application did not affect the synaptic currents in PVBC-to-PC pairs, but it reduced the inhibitory currents elicited in PCs with simultaneous glutamate release by extracellular stimulation. ConclusionsWe demonstrate that NMDAR activation enhances PVBC-to-PC inhibition in a manner consistent with presynaptic mechanisms, and we suggest that the functional impact of this presynaptic effect depends on the activity state of the PFC network.

Bidirectional modulation of glutamatergic synaptic transmission by metabotropic glutamate type 7 receptors at Schaffer collateral-CA1 hippocampal synapses.

Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu7 ) receptors that reduce Ca2+ influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu7 receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission. Neurotransmitter release is driven by Ca2+ influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu7 ) receptors provoke a reduction in Ca2+ influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu7 receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca2+ from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu7 receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu7 receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122.

Presynaptic GABAA Receptors Modulate Thalamocortical Inputs in Layer 4 of Rat V1.

Fast inhibitory GABAergic transmission plays a fundamental role in neural circuits. Current theories of cortical function assume that fast GABAergic inhibition acts via GABAA receptors on postsynaptic neurons, while presynaptic effects of GABA depend on GABAB receptor activation. Manipulations of GABAA receptor activity in vivo produced different effects on cortical function, which were generally ascribed to the mode of action of a drug, more than its site of action. Here we show that in rodent primary visual cortex, α4-containing GABAA receptors can be located on subsets of glutamatergic and GABAergic presynaptic terminals and decrease synaptic transmission. Our data provide a novel mechanistic insight into the effects of changes in cortical inhibition; the ability to modulate inputs onto cortical circuits locally, via presynaptic regulation of release by GABAA receptors.

Presynaptic serotonin 5-HT1B/D receptor-mediated inhibition of glycinergic transmission to the frog spinal motoneurons.

Endogenous monoamine 5-hydroxytryptamine (5-HT, serotonin) is a phylogenetically ancient neurotransmitter present in vertebrates. The functions of 5-HT in central nervous system are intensively studied; however, the presynaptic effects of 5-HT in frog spinal motoneurons are practically unexplored. We have previously shown that 5-HT decreases the frequency of glycinergic miniature inhibitory postsynaptic potentials (mIPSPs), but does not affect the frequency of GABAergic mIPSPs and increases the frequency of glutamatergic postsynaptic potentials. In the present study, using pharmacological methods and intracellular recordings in motoneurons from an adult frog's isolated spinal cord, we aimed to identify the 5-HT receptor subtype responsible for inhibiting the release of glycine. Аn agonist of 5-HT1A and 5-HT7 receptors, 8-OH-DPAT, and a selective agonist of 5-HT2 receptors, α-Ме-5-НТ, did not show any significant effect on inhibitory transmission, indicating that 5-HT1A, 5-HT2, and 5-HT7 receptors are not involved in the modulation of glycine release in the adult frog spinal cord. An agonist of 5-HT1B/D receptors sumatriptan decreased the frequency (but not the amplitude) of glycinergic mIPSPs similar to 5-HT. An antagonist of 5-HT1,2 receptors, methysergide, abolished the effect of sumatriptan. Together our results suggest that 5-HT inhibits the release of glycine by activation of 5-HT1B/D receptors.

Differential sensitivity of presynaptic and postsynaptic GABAB receptors in rat ventrolateral periaqueductal gray.

The ventrolateral periaqueductal gray (PAG) in the midbrain plays a key role in the descending pain modulatory system. γ-Aminobutyric acid type B (GABAB) receptors belong to a metabotropic receptor subfamily and mediate both presynaptic and postsynaptic effects in PAG. It has been well documented that activation of GABAB receptors yields analgesia in some PAG subdivisions. In the present study, employing whole-cell patch-clamp recordings on acute rat PAG slices, we simultaneously monitored the responses of presynaptic and postsynaptic GABAB receptors. We found that the GABAB agonist, baclofen, exhibits less efficacy and potency at GABAB postsynaptic versus presynaptic receptors. This sensitivity bias may contribute to synapse homeostasis and implicate a novel pharmacotherapy treatment.

17β-Estradiol Acutely Potentiates Glutamatergic Synaptic Transmission in the Hippocampus through Distinct Mechanisms in Males and Females

Estradiol (E2) acutely potentiates glutamatergic synaptic transmission in the hippocampus of both male and female rats. Here, we investigated whether E2-induced synaptic potentiation occurs via presynaptic and/or postsynaptic mechanisms and which estrogen receptors (ERs) mediate E2's effects in each sex. Whole-cell voltage-clamp recordings of mEPSCs in CA1 pyramidal neurons showed that E2 increases both mEPSC frequency and amplitude within minutes, but often in different cells. This indicated that both presynaptic and postsynaptic mechanisms are involved, but that they occur largely at different synapses. Two-photon (2p) glutamate uncaging at individual dendritic spines showed that E2 increases the amplitude of uncaging-evoked EPSCs (2pEPSCs) and calcium transients (2pCaTs) at a subset of spines on a dendrite, demonstrating synapse specificity of E2's postsynaptic effects. All of these results were essentially the same in males and females. However, additional experiments using ER-selective agonists indicated sex differences in the mechanisms underlying E2-induced potentiation. In males, an ERβ agonist mimicked the postsynaptic effects of E2 to increase mEPSC, 2pEPSC, and 2pCaT amplitude, whereas in females, these effects were mimicked by an agonist of G protein-coupled ER-1. The presynaptic effect of E2, increased mEPSC frequency, was mimicked by an ERα agonist in males, whereas in females, an ERβ agonist increased mEPSC frequency. Thus, E2 acutely potentiates glutamatergic synapses similarly in both sexes, but distinct ER subtypes mediate the presynaptic and postsynaptic aspects of potentiation in each sex. This indicates a latent sex difference in which different molecular mechanisms converge to the same functional endpoint in males versus females.SIGNIFICANCE STATEMENTSome sex differences in the brain may be latent differences, in which the same functional endpoint is achieved through distinct underlying mechanisms in males versus females. Here we report a latent sex difference in molecular regulation of excitatory synapses in the hippocampus. The steroid 17β-estradiol is known to acutely potentiate glutamatergic synaptic transmission in both sexes. We find that this occurs through a combination of increased presynaptic glutamate release probability and increased postsynaptic sensitivity to glutamate in both sexes, but that distinct estrogen receptor subtypes underlie each aspect of potentiation in each sex. These results indicate that therapeutics targeting a specific estrogen receptor subtype or its downstream signaling would likely affect synaptic transmission differently in the hippocampus of each sex.

Human Brain-Derived Aβ Oligomers Bind to Synapses and Disrupt Synaptic Activity in a Manner That Requires APP.

Compelling genetic evidence links the amyloid precursor protein (APP) to Alzheimer's disease (AD) and several theories have been advanced to explain the relationship. A leading hypothesis proposes that a small amphipathic fragment of APP, the amyloid β-protein (Aβ), self-associates to form soluble aggregates that impair synaptic and network activity. Here, we used the most disease-relevant form of Aβ, protein isolated from AD brain. Using this material, we show that the synaptotoxic effects of Aβ depend on expression of APP and that the Aβ-mediated impairment of synaptic plasticity is accompanied by presynaptic effects that disrupt the excitatory/inhibitory (E/I) balance. The net increase in the E/I ratio and inhibition of plasticity are associated with Aβ localizing to synapses and binding of soluble Aβ aggregates to synapses requires the expression of APP. Our findings indicate a role for APP in AD pathogenesis beyond the generation of Aβ and suggest modulation of APP expression as a therapy for AD.SIGNIFICANCE STATEMENT Here, we report on the plasticity-disrupting effects of amyloid β-protein (Aβ) isolated from Alzheimer's disease (AD) brain and the requirement of amyloid precursor protein (APP) for these effects. We show that Aβ-containing AD brain extracts block hippocampal LTP, augment glutamate release probability, and disrupt the excitatory/inhibitory balance. These effects are associated with Aβ localizing to synapses and genetic ablation of APP prevents both Aβ binding and Aβ-mediated synaptic dysfunctions. Our results emphasize the importance of APP in AD and should stimulate new studies to elucidate APP-related targets suitable for pharmacological manipulation.

Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.

Ethanol Mediated Inhibition of Synaptic Vesicle Recycling at Amygdala Glutamate Synapses Is Dependent upon Munc13-2.

Chronic exposure to alcohol produces adaptations within the basolateral amygdala (BLA) that are associated with the development of anxiety-like behaviors during withdrawal. In part, these adaptations are mediated by plasticity in glutamatergic synapses occurring through an AMPA receptor mediated form of post-synaptic facilitation in addition to a unique form of presynaptic facilitation. In comparison to the post-synaptic compartment, relatively less is understood about the mechanisms involved in the acute and chronic effects of ethanol in the presynaptic terminal. Previous research has demonstrated that glutamatergic terminals in the mouse BLA are sensitive to ethanol mediated inhibition of synaptic vesicle recycling in a strain-dependent fashion. Importantly, the strain-dependent differences in presynaptic ethanol sensitivity are in accordance with known strain-dependent differences in ethanol/anxiety interactions. In the present study, we have used a short-hairpin RNA to knockdown the expression of the presynaptic Munc13-2 protein in C57BL/6J mice, whose BLA glutamate terminals are normally ethanol-insensitive. We injected this shRNA, or a scrambled control virus, into the medial prefrontal cortex (mPFC) which sends dense projections to the BLA. Accordingly, this knockdown strategy reduces the expression of the Munc13-2 isoform in mPFC terminals within the BLA and alters presynaptic terminal function in C57BL/6J mice in a manner that phenocopies DBA/2J glutamate terminals which are normally ethanol-sensitive. Here, we provide evidence that manipulation of this single protein, Munc13-2, renders C57BL/6J terminals sensitive to ethanol mediated inhibition of synaptic vesicle recycling and post-tetanic potentiation. Furthermore, we found that this ethanol inhibition was dose dependent. Considering the important role of Munc13 proteins in synaptic plasticity, this study potentially identifies a molecular mechanism regulating the acute presynaptic effects of ethanol to the long lasting adaptations in the BLA that occur during chronic ethanol exposure.

Characterization of serotonin-induced inhibition of excitatory synaptic transmission in the anterior cingulate cortex

Excitatory synaptic transmission in central synapses is modulated by serotonin (5-HT). The anterior cingulate cortex (ACC) is an important cortical region for pain perception and emotion. ACC neurons receive innervation of projecting serotonergic nerve terminals from raphe nuclei, but the possible effect of 5-HT on excitatory transmission in the ACC has not been investigated. In the present study, we investigated the role of 5-HT on glutamate neurotransmission in the ACC slices of adult mice. Bath application of 5-HT produced dose-dependent inhibition of evoked excitatory postsynaptic currents (eEPSCs). Paired pulse ratio (PPR) was significantly increased, indicating possible presynaptic effects of 5-HT. Consistently, bath application of 5-HT significantly decreased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. After postsynaptic application of G protein inhibitor GDP-β-S, 5-HT produced inhibition of eEPSCs was significantly reduced. Finally, NAN-190, an antagonist of 5-HT1A receptor, significantly reduced postsynaptic inhibition of 5-HT and abolished presynaptic inhibition. Our results strongly suggest that presynaptic as well as postsynaptic 5-HT receptor including 5-HT1A subtype receptor may contribute to inhibitory modulation of glutamate release as well as postsynaptic responses in the ACC.

CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9±3.5%) than wild-type cells (1.1±0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.

Soluble Ectodomain of Neuroligin 1 Decreases Synaptic Activity by Activating Metabotropic Glutamate Receptor 2.

Synaptic cell adhesion molecules represent important targets for neuronal activity-dependent proteolysis. Postsynaptic neuroligins (NLs) form trans-synaptic complexes with presynaptic neurexins (NXs). Both NXs and NLs are cleaved from the cell surface by metalloproteases in an activity-dependent manner, releasing a soluble extracellular fragment and membrane-tethered C-terminal fragment. The cleavage of NL1 depresses synaptic transmission, but the mechanism by which this occurs is unknown. Metabotropic glutamate receptor 2 (mGluR2) are located primarily at the periphery of presynaptic terminals, where they inhibit the formation of cyclic adenosine monophosphate (cAMP) and consequently suppress the release of glutamate and decrease synaptic transmission. In the present study, we found that the soluble ectodomain of NL1 binds to and activates mGluR2 in both neurons and heterologous cells, resulting in a decrease in cAMP formation. In a slice preparation from the hippocampus of mice, NL1 inhibited the release of glutamate from mossy fibers that project to CA3 pyramidal neurons. The presynaptic effect of NL1 was abolished in the presence of a selective antagonist for mGluR2. Thus, our data suggest that the soluble extracellular domain of NL1 functionally interacts with mGluR2 and thereby decreases synaptic strength.

Analgesic Effects Mediated by Alpha 7 Nicotinic Acetylcholine Receptors: A Potential Non‐Opioid Treatment for Pain

Nicotinic acetylcholine receptors (nAChRs) are expressed abundantly throughout the nervous system. A wide variety of neurobehavioral processes involve nAChRs, including analgesic effects in chronic pain models. However, nAChR subunit composition and cellular mechanisms underlying nAChR mediated analgesia are not well understood. Ventrolateral periaqueductal gray (vlPAG) is an important target for analgesic drugs in the descending pain modulatory pathway. Thus, we investigated the nAChRs in vlPAG and tested their antinociceptive effects in an animal model of pain.Adult male Sprague Dawley rats (&gt; 8weeks) were used for all the experiments. Whole cell recordings were performed from backlabeled vlPAG neurons that project to the RVM in a brain slice preparation. nAChR expressing cells were identified by focal application of ACh. The contribution of the α7 nAChR subtype was tested with selective antagonists. In addition, μ‐opioid receptors (MOR) expression was assessed using selective MOR agonists. Further, we tested the behavioral relevance of these physiological effects using the formalin assay while modulating α7 nAChR activity using both systemic and focal (within the vlPAG) pharmacological interventions.We found that 63% of vlPAG‐RVM projection neurons expressed functional α7 nAChRs and were largely non‐overlapping with those expressing MORs. Nicotine enhanced spontaneous inhibitory postsynaptic current (sIPSC) frequency onto vlPAG neurons that lack α7 nAChRs (12/19), but less so in α7 expressing neurons (7/24 neurons, Fisher's test p&lt;0.05). However, nicotinic modulation of spontaneous excitatory postsynaptic current (EPSC) frequency was similar between α7+ and α7‐neurons. Pharmacological testing indicated that these presynaptic effects of nicotine were mediated by β2‐containing nAChRs. In the in vivo behavioral assay, both systemic and focal administration of the α7 nAChR‐selective agonist, PHA‐543613 reduced nocifensive responses to the formalin injection. Furthermore, focal α7 antagonist pretreatment blocked PHA‐543613‐induced antinociception via either systemic or focal administration methods. We then tested a combination of sub‐maximal doses of the α7 agonist and morphine, which produced additive antinociceptive effects. Interestingly, focal administration of the α7 antagonist blocked the antinociceptive effects of this drug combination. The α7 antagonist did not affect morphine induced antinociception when administered alone at a maximal effective concentration.Our study demonstrates two populations of vlPAG‐RVM projection neurons that differentially express α7 nAChRs and MORs. Our working model is that α7‐expressing neurons contribute to a pain inhibiting pathway, while MOR‐expressing neurons enhance pain signaling. The additive antinociceptive effects of combining sub‐maximal doses of α7 and MOR agonists supports the idea of independent mechanisms and these observations may lead to novel treatment strategies to limit opioid drug use for the relief of chronic pain.Support or Funding InformationThis study was funded by a Frank Family Fellowship to I.U., a Gates Foundation Fellowship to B.M., and the NIH grants DA07255 to C.A.D., DA019695, DA036978, and DA015918 to D.S.M.

BDNF-induced endocannabinoid release modulates neocortical glutamatergic neurotransmission.

Endocannabinoids (eCBs) and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), are potent neuromodulators found throughout the mammalian neocortex. Both eCBs and BDNF play critical roles in many behavioral and neurophysiological processes and are targets for the development of novel therapeutics. The effects of eCBs and BDNF are primarily mediated by the type 1 cannabinoid (CB1) receptor and the trkB tyrosine kinase receptor, respectively. Our laboratory and others have previously established that BDNF potentiates excitatory transmission by enhancing presynaptic glutamate release and modulating NMDA receptors. In contrast, we have shown that BDNF attenuates inhibitory transmission by inducing postsynaptic release of eCBs that act retrogradely to suppress GABA release in layer 2/3 of somatosensory cortex. Here, we hypothesized that BDNF also induces release of eCBs at excitatory synapses, which could have a mitigating or opposing effect on the direct presynaptic effects of BDNF. We found the highest levels of expression of CB1 and trkB and receptors in layers 2/3 and 5. Surprisingly, BDNF did not increase the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) onto layer 5 pyramidal neurons in somatosensory cortex, in contrast to its effects in the hippocampus and visual cortex. However, the effect of BDNF on mEPSC frequency in somatosensory cortex was unmasked by blocking CB1 receptors or disrupting eCB release. Thus, BDNF-trKB signaling regulates glutamate release in the somatosensory cortex via opposing effects, a direct presynaptic enhancement of release probability, and simultaneous postsynaptically-induced eCB release that decreases release probability via presynaptic CB1 receptors.

Presynaptic Ethanol Actions: Potential Roles in Ethanol Seeking.

Ethanol produces intoxication through actions on numerous molecular and cellular targets. Adaptations involving these and other targets contribute to chronic drug actions that underlie continued and problematic drinking. Among the mechanisms involved in these ethanol actions are alterations in presynaptic mechanisms of synaptic transmission, including presynaptic protein function and excitation-secretion coupling. At synapses in the central nervous system (CNS), excitation-secretion coupling involves ion channel activation followed by vesicle fusion and neurotransmitter release. These mechanisms are altered by presynaptic neurotransmitter receptors and prominently by G protein-coupled receptors (GPCRs). Studies over the last 20-25years have revealed that acute ethanol exposure alters neurotransmitter secretion, with especially robust effects on synapses that use the neurotransmitter gamma-aminobutyric acid (GABA). Intracellular signaling pathways involving second messengers such as cyclic AMP and calcium are implicated in these acute ethanol actions. Ethanol-induced release of neuropeptides and small molecule neurotransmitters that act on presynaptic GPCRs also contribute to presynaptic potentiation at synapses in the amygdala and hippocampus and inhibition of GABA release in the striatum. Prolonged exposure to ethanol alters neurotransmitter release at many CNS GABAergic and glutamatergic synapses, and changes in GPCR function are implicated in many of these neuroadaptations. These presynaptic neuroadaptations appear to involve compensation for acute drug effects at some synapses, but "allostatic" effects that result in long-term resetting of synaptic efficacy occur at others. Current investigations are determining how presynaptic neuroadaptations contribute to behavioral changes at different stages of alcohol drinking, with increasing focus on circuit adaptations underlying these behaviors. This chapter will discuss the acute and chronic presynaptic effects of ethanol in the CNS, as well as some of the consequences of these effects in amygdala and corticostriatal circuits that are related to excessive seeking/drinking and ethanol abuse.

Cross-Neutralisation of In Vitro Neurotoxicity of Asian and Australian Snake Neurotoxins and Venoms by Different Antivenoms.

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to venom in an in vitro preparation, cross-neutralization of neurotoxicity means that antivenoms from one region may be effective in other regions which do not have effective antivenoms. TCAV only neutralized post-synaptic neurotoxicity and is potentially useful in distinguishing pre-synaptic and post-synaptic effects in the chick biventer cervicis preparation.