Decrease In mEPSC Frequency Research Articles

SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions.

SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.

SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions

SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.

Dysfunction of the medial prefrontal cortex contributes to BPA-induced depression- and anxiety-like behavior in mice

Bisphenol A (BPA), a well-known environmental endocrine disruptor, has been implicated in anxiety-like behavior. But the neural mechanism remains elusive. Herein, we found that mice exposed to 0.5 mg/kg/day BPA chronically from postnatal days (PND) 21 to PND 80 exhibited depression- and anxiety-like behavior. Further study showed that medial prefrontal cortex (mPFC), was associated with BPA-induced depression- and anxiety-like behavior, as evidenced by decreased c-fos expression in mPFC of BPA-exposed mice. Both the morphology and function of glutamatergic neurons (also called pyramidal neurons) in mPFC of mice were impaired following BPA exposure, characterized by reduced primary branches, weakened calcium signal, and decreased mEPSC frequency. Importantly, optogenetic activation of the pyramidal neurons in mPFC greatly reversed BPA-induced depression- and anxiety-like behavior in mice. Furthermore, we reported that microglial activation in mPFC of mice may also have a role in BPA-induced depression- and anxiety-like behavior. Taken together, the results indicated that mPFC is the brain region that is greatly damaged by BPA exposure and is associated with BPA-induced depression- and anxiety-like behavior. The study thus provides new insights into BPA-induced neurotoxicity and behavioral changes.

ERα Stimulation Rapidly Modulates Excitatory Synapse Properties in Female Rat Nucleus Accumbens Core

Introduction: The nucleus accumbens core (NAcc) is a sexually differentiated brain region that is modulated by steroid hormones such as 17β-estradiol (estradiol), with consequential impacts on relevant motivated behaviors and disorders such as addiction, anxiety, and depression. NAcc estradiol levels naturally fluctuate, including during the estrous cycle in adult female rats, which is analogous to the menstrual cycle in adult humans. Across the estrous cycle, excitatory synapse properties of medium spiny neurons rapidly change, as indicated by analysis of miniature excitatory postsynaptic currents (mEPSCs). mEPSC frequency decreases during estrous cycle phases associated with high estradiol levels. This decrease in mEPSC frequency is mimicked by acute topical exposure to estradiol. The identity of the estrogen receptor (ER) underlying this estradiol action is unknown. Adult rat NAcc expresses three ERs, all extranuclear: membrane ERα, membrane ERβ, and GPER1. Methods: In this brief report, we take a first step toward addressing this challenge by testing whether activation of ERs via acute topical agonist application is sufficient for inducing changes in mEPSC properties recorded via whole-cell patch clamp. Results: An agonist of ERα induced large decreases in mEPSC frequency, while agonists of ERβ and GPER1 did not robustly modulate mEPSC properties. Conclusions: These data provide evidence that activation of ERα is sufficient for inducing changes in mEPSC frequency and is a likely candidate underlying the estradiol-induced changes observed during the estrous cycle. Overall, these findings extend our understanding of the neuroendocrinology of the NAcc and implicate ERα as a primary target for future studies.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Astroglial Regulation of Caloric Intake Following Acute High‐Fat Diet Exposure

Chronic high fat diet (HFD) exposure results in hyperphagia and the development of obesity. Caloric intake during acute (3-5 days) HFD exposure, however, is regulated. We have previously shown that increased activation of NMDA receptors on the motoneurons of the dorsal motor nucleus of the vagus (DMV) contributes to the regulation of caloric intake during acute HFD exposure (Clyburn, et. al. 2018). The mechanism that is responsible for altered excitatory drive to DMV neurons is unknown. HFD exposure is associated with inflammation. Neuroinflammation and astroglial activation have been shown to modulate neural activity and glutamatergic signaling. The purpose of this study is to test the hypothesis that during acute HFD exposure, astrocyte activation increases the glutamatergic drive to DMV neurons and regulates caloric intake. Male and female Sprague-Dawley rats were transfected with the astrocyte inhibitory DREADD, pAAV-GFAP-hM4D(Gi)-mCherry, (“hM4Di”; Addgene #50479-AAV5; titer 1x1013 diluted 1:10) or AAV-GFAP104-mCherry, (“empty vector”; UNC vector core, titer 2.7x1012 diluted 1:10). Microinjections were made into the dorsal vagal complex. After recovery, rats were fed a control or HFD (14% and 60% kcal from fat, respectively) for 3-5 days. Whole-cell patch clamp recordings were made of gastric-projecting DMV neurons in thin brainstem slices of transfected rats. The effects of the NMDA receptor antagonist, AP5 (25μM), in the presence of clozapine (10μM) or the DREADD agonist, clozapine-N-oxide (CNO; 10μM) on miniature excitatory postsynaptic current (mEPSC) kinetics and action potential (AP) firing rate were examined. The effects of CNO and clozapine on caloric intake and gastric emptying rates using the 13C octanoic acid breath test were also assessed. As shown previously, following acute HFD, AP5 decreases mEPSC frequency and AP firing rate. This AP5-mediated decrease was unaffected by clozapine or CNO application to empty vector control groups. CNO in the hM4Di group attenuated the AP5-mediated decrease in mEPSC frequency and AP firing rate (mEPSC frequency: 54±5.0% vs. 53±9.5% vs. 100±1.5% of baseline, respectively; AP firing rate: 8±5.3% vs. 41±14.9% vs. 111±3.5%; P<0.05 with two-way ANOVA and post-hoc Bonferroni; N=7-9 cells from 3-5 rats). In addition, the delay in gastric emptying rates observed 4 days following HFD exposure was attenuated by CNO administration to hM4Di rats, while empty vector or clozapine controls were uneffected (119±7.9% vs. 166±7.4% vs. 144±6.7% of baseline, respectively; P<0.05 with two-way ANOVA and post-hoc Bonferroni; N=6-7 rats). CNO administration to hM4Di rats also prevented the regulation of caloric intake normally observed 3-5 days of HFD exposure, compared to clozapine and empty vector controls (AUC: 964±34.4a.u. vs. 843±14.3a.u. vs. 815±17.9a.u., respectively; P<0.05 from Student's unpaired t-test; N=6). These data suggest that astrocyte activation is involved in the alteration of glutamatergic currents and the homeostatic regulation of food intake following acute HFD. Understanding the mechanism responsible for the regulation of caloric intake is critical in identifying potential therapeutic targets for the treatment of obesity when such neuroplasticity may be absent.

Acute High Fat Diet‐Dependent Activation of Synaptic NMDA Receptors Requires Activation of Purinergic and Metabotropic Glutamate Receptors on Rat Dorsal Motor Nucleus of the Vagus Neurons

Previous work from this lab has demonstrated that, in rats, restoration of caloric intake following acute high fat diet (aHFD) is dependent upon upregulated glutamatergic signaling in the dorsal motor nucleus of the vagus (DMV), which recruits previously silent synaptic NMDA receptors (NMDARs). We have further shown that NMDARs activation is driven by the activation of extrasynaptic NMDA receptors (NMDARex) in an astrocyte-dependent process, although the exact mechanism involved is still unknown. The current study was designed to test the hypothesis that activation of purinergic and/or metabotropic glutamate receptors (mGluR) by the gliotransmitters ATP and glutamate is responsible for activation of NMDARex and NMDARs. Whole cell patch clamp recordings were made from DMV neurons in thin brainstem slices prepared from male and female Sprague-Dawley rats fed either a control diet (14% kcal from fat) or following acute, 3-5 days, high fat diet (aHFD; 60% kcal from fat) exposure. The presence of activated NMDARs was assessed by the ability of the antagonist AP5 (25µM) to decrease the frequency of miniature excitatory synaptic currents (mEPSC) as well as action potential (AP) firing. The involvement of P2X receptors was investigated using the antagonist PPADS (10µM) and agonist αβMeATP (10µM) while the involvement of group I mGluR was investigated using the antagonist, AIDA (300µM). In aHFD DMV neurons, perfusion with AP5 decreased action potential firing in 8/9 neurons (1.2±0.1 vs 0.2±0.13 AP/sec; P<0.05) and decreased mEPSC frequency in 8/9 neurons (3.18±1.71 vs 2.11±1.12; P<0.05). Application of PPADS blocked the ability of AP5 to decrease AP firing in 6/10 neurons (1.8±0.18 vs 1.5±0.20 AP/sec; P>0.05; P<0.05 vs AP5 alone) and mEPSC frequency (2.57±2.10 vs 2.49±1.63; P>0.05; P<0.05 vs AP5 alone). In contrast, in control DMV neurons, application of the agonist, αβMeATP, uncovered the ability of AP5 to decrease AP firing (1.09±1.31 vs 1.56±1.12 AP/sec; P<0.05) although it did not uncover actions to decrease mEPSC frequency (1.94±0.82 vs 2.17±0.91 P>0.05). In contrast, application of the group 1 mGluR antagonist, AIDA, blocked the ability of AP5 to decrease mEPSC frequency in 7/10 aHFD DMV neurons (1.88±1.45 vs 2.18±1.42; P>0.05), although it failed to block the actions of AP5 to inhibit AP firing in 7/9 neurons (0.74±0.71 vs 0.39±0.53; P<0.05). Taken together, these results show that purinergic and metabotropic glutamatergic signaling are involved in activation of NMDARs on DMV neurons following aHFD exposure. The current study provides a potential mechanism by which caloric balance is restored following aHFD exposure. Specifically, aHFD-dependent release of the brainstem gliotransmitters ATP and glutamate are required for the activation of DMV extrasynaptic and synaptic NMDA receptors. This upregulates NTS-DMV glutamatergic signaling and alters the excitability of central vagal neurocircuits involved in the modulation of gastrointestinal functions including feeding behavior.

Differential and synergistic roles of 17β-estradiol and progesterone in modulating adult female rat nucleus accumbens core medium spiny neuron electrophysiology.

Naturally occurring cyclical changes in sex steroid hormones such as 17β-estradiol and progesterone can modulate neuron function and behavior in female mammals. One example is the estrous cycle in rats, which is composed of multiple phases. We previously reported evidence of differences between estrous cycle phases in excitatory synapse and intrinsic electrophysiological properties of rat nucleus accumbens core (AcbC) medium spiny neurons (MSNs). The AcbC is a nexus between the limbic and premotor systems and is integral for controlling motivated and reward-associated behaviors and disorders, which are sensitive to the estrous cycle and hormones. The present study expands our prior findings by testing whether circulating levels of estradiol and progesterone correlate with changes in MSN electrophysiology across estrous cycle phases. As part of this project, the excitatory synapse and intrinsic excitability properties of MSNs in late proestrus of adult female rats were assessed. Circulating levels of estradiol correlate with resting membrane potential, the time constant of the membrane, and rheobase. Circulating levels of progesterone correlate with miniature excitatory postsynaptic current (mEPSC) frequency and amplitude. Circulating levels of estradiol and progesterone together correlate with mEPSC amplitude, resting membrane potential, and input resistance. The late proestrus phase features a prominent and unique decrease in mEPSC frequency. These data indicate that circulating levels of estradiol and progesterone alone or in combination interact with specific MSN electrophysiological properties, indicating differential and synergistic roles of these hormones. Broadly, these findings illustrate the underlying endocrine actions regarding how the estrous cycle modulates MSN electrophysiology.NEW & NOTEWORTHY This research indicates that estradiol and progesterone act both differentially and synergistically to modulate neuron physiology in the nucleus accumbens core. These actions by specific hormones provide key data indicating the endocrine mechanisms underlying how the estrous cycle modulates neuron physiology in this region. Overall, these data reinforce that hormones are an important influence on neural physiology.

α-Synuclein (αSyn) Preformed Fibrils Induce Endogenous αSyn Aggregation, Compromise Synaptic Activity and Enhance Synapse Loss in Cultured Excitatory Hippocampal Neurons.

Synucleinopathies are characterized by the accumulation of insoluble α-synuclein (αSyn). To test whether αSyn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing αSyn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human αSyn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of αSyn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of αSyn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of αSyn pathology within 1 d. Moreover, by direct addition of αSyn PFFs into culture medium, the formation of misfolded αSyn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with αSyn PFFs. Our data indicate that intraneuronal αSyn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.SIGNIFICANCE STATEMENT Under pathological conditions, the presynaptic protein α-synuclein (αSyn) aggregates to form intraneuronal inclusions. To understand how and to what extent αSyn aggregates modulate synaptic activity before neuron loss, we demonstrate that αSyn preformed fibrils (PFFs) reduced synaptic activity in a dose- and time-dependent manner. The magnitude of these deficits correlated with the deposition of αSyn pathology, which dramatically compromised the colocalization of synaptic markers and altered the dendritic spine dynamics. The present work further highlights the impact of αSyn PFFs on synaptogenesis and physiological function, which may be applicable to other types of synucleinopathies.

Specific downregulation of hippocampal ATF4 reveals a necessary role in synaptic plasticity and memory.

Prior studies suggested that the transcription factor ATF4 negatively regulates synaptic plastic and memory. By contrast, we provide evidence from direct in vitro and in vivo knockdown of ATF4 in rodent hippocampal neurons and from ATF4-null mice that implicate ATF4 as essential for normal synaptic plasticity and memory. In particular, hippocampal ATF4 downregulation produces deficits in long-term spatial memory and behavioral flexibility without affecting associative memory or anxiety-like behavior. ATF4 knockdown or loss also causes profound impairment of both long-term potentiation (LTP) and long-term depression (LTD) as well as decreased glutamatergic function. We conclude that ATF4 is a key regulator of the physiological state necessary for neuronal plasticity and memory.

Gonadal steroids differentially modulate the actions of orphanin FQ/nociceptin at a physiologically relevant circuit controlling female sexual receptivity.

Orphanin FQ/nociceptin (OFQ/N) inhibits the activity of pro-opiomelanocortin (POMC) neurones located in the hypothalamic arcuate nucleus (ARH) that regulate female sexual behaviour and energy balance. We tested the hypothesis that gonadal steroids differentially modulate the ability of OFQ/N to inhibit these cells via presynaptic inhibition of transmitter release and postsynaptic activation of G protein-gated, inwardly-rectifying K(+) (GIRK)-1 channels. Whole-cell patch clamp recordings were performed in hypothalamic slices prepared from ovariectomised rats. OFQ/N (1 μm) decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs), and also caused a robust outward current in the presence of tetrodotoxin, in ARH neurones from vehicle-treated animals. A priming dose of oestradiol benzoate (EB; 2 μg) increased basal mEPSC frequency, markedly diminished both the OFQ/N-induced decrease in mEPSC frequency and the activation of GIRK-1 currents, and potentiated the OFQ/N-induced decrease in mIPSC frequency. Steroid treatment regimens that facilitate sexual receptivity reinstate the basal mEPSC frequency, the OFQ/N-induced decrease in mEPSC frequency and the activation of GIRK-1 currents to levels observed in vehicle-treated controls, and largely abolish the ability of OFQ/N to decrease mIPSC frequency. These effects were observed in an appreciable population of identified POMC neurones, almost one-half of which projected to the medial preoptic nucleus. Taken together, these data reveal that gonadal steroids influence the pleiotropic actions of OFQ/N on ARH neurones, including POMC neurones, in a disparate manner. These temporal changes in OFQ/N responsiveness further implicate this neuropeptide system as a critical mediator of the gonadal steroid regulation of reproductive behaviour.

5-Hydroxytryptamine–Mediated Neurotransmission Modulates Spontaneous and Vagal-Evoked Glutamate Release in the Nucleus of the Solitary Tract Effect of Uptake Blockade

The effect of blockade of either 5-hydroxytryptamine (5-HT)/serotonin transporter (SERT) with citalopram or the organic cation transporter 3 (OCT3)/plasma membrane monoamine transporter (PMAT) with decynium-22 (D-22) on spontaneous and evoked release of 5-HT in the nucleus tractus solitarius (NTS) was investigated in rat brainstem slices treated with gabazine. 5-HT release was measured indirectly by changes in the frequency and amplitude of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) [in the presence of tetrodotoxin (TTX)] and evoked EPSCs. Blockade of 5-HT3 receptors with granisetron reduced, whereas the 5-HT3 agonist phenylbiguanide increased, the frequency of mEPSCs. 5-HT decreased mEPSC frequency at low concentrations and increased frequency at high concentrations. This inhibition was blocked by the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635), which was ineffective on its own, whereas the excitation was reversed by granisetron. The addition of citalopram or D-22 caused inhibition, which was prevented by 5-HT1A blockade. Thus, in the NTS, the spontaneous release of 5-HT is able to activate 5-HT3 receptors, but not 5-HT1A receptors, as the release in their vicinity is removed by uptake. The ineffectiveness of corticosterone suggests that the low-affinity, high-capacity transporter is PMAT, not OCT3. For evoked 5-HT release, only D-22 caused an increase in the amplitude of EPSCs, with a decrease in the paired pulse ratio, and increased the number of spontaneous EPSCs after 20-Hz stimulation. Thus, for the evoked release of 5-HT, the low-affinity, high-capacity transporter PMAT, but not 5-HT transporter (5-HTT)/SERT, is important in the regulation of changes in 5-HT extracellular concentration.

Evidence that 5‐HT neurotransmission within the nucleus tractus solitarii (NTS) differentially modulates glutamate release via 5‐HT3 and 5‐HT1A receptors

The effects of spontaneously released 5‐HT were investigated in NTS containing slices (300 μm). Recordings were made of glutamatergic mEPSCs (abolished by CNQX) from voltage clamped (−60 mv) NTS neurones in the presence of gabazine (25 μmol) and TTX (1 μmol). Low doses (300μM; n=5) of 5‐HT inhibited (frequency decreased by 53±12%), while high doses 10μM increased mEPSC frequency (1285±276%) and amplitude (21±5 %). In presence of the 5‐HT3 antagonist granisetron (3μM) 5‐HT now only caused a decrease (31±11%) in frequency. The 5‐HT3 receptor agonist PBG (1μM) increased mEPSC frequency by 497±112% and amplitude by 18±4%, which was also blocked by granisetron. Granisetron (1μM; n=6) alone decreased mEPSC frequency by 35±4% as did the SSRI citalopram (0.5μM, n=5; 54±5%) and the OCT3 inhibitor decynium 22 (1μM; n=4; 27±2). The effects of the reuptake inhibitors were blocked by WAY‐100635 (1μM). The data indicates that the spontaneous release of 5‐HT in the NTS only activates presynaptically located 5‐HT3 and 5‐HT1A receptors. However, the ability of 5‐HT uptake blockade to only increase 5‐HT1A receptor activation suggests that the ability of spontaneously released 5‐HT to activate presynaptic 5‐HT3 receptors is not subject to modulation by reuptake. This would be consistent with these 5‐HT3 receptors being heteroreceptors. These data shows that endogenous 5‐HT has two distinct modulatory actions on glutamate release.

Rapid glucocorticoid-induced activation of TRP and CB1 receptors causes biphasic modulation of glutamate release in gastric-related hypothalamic preautonomic neurons

Glucocorticoids rapidly regulate synaptic input to neuroendocrine cells in the hypothalamic paraventricular nucleus (PVN) by inducing the retrograde release of endogenous messengers. Here we investigated the rapid effects of dexamethasone (DEX) on excitatory synaptic input to feeding-related, preautonomic PVN neurons using whole-cell patch-clamp recordings. In ∼50% of identified gastric-related preautonomic PVN neurons, DEX elicited a biphasic synaptic response characterized by an initial rapid and transient increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs), followed by a decrease in mEPSC frequency within 9 min; remaining cells displayed only a decrease in mEPSC frequency. The late-phase decrease in mEPSC frequency was mimicked by the cannabinoid receptor agonists anandamide (AEA) and WIN 55,212-2, and it was blocked by the CB1 receptor antagonist AM251. The biphasic DEX effect was mimicked by AEA. The early increase in mEPSCs was mimicked by activation of transient receptor potential vanilloid type 1 (TRPV1) receptors with capsaicin and by activation of TRPV4 receptors with 4-α-PDD. The increase was reduced, but not blocked, by selective TRPV1 antagonists and in TRPV1 knockout mice; it was blocked completely by the broad-spectrum TRPV antagonist ruthenium red and by combined application of selective TRPV1 and TRPV4 antagonists. The DEX effects were prevented entirely by intracellular infusion of the G-protein inhibitor, GDPβS. Thus, DEX biphasically modulates synaptic glutamate onto a subset of gastric-related PVN neurons, which is likely mediated by induction of a retrograde messenger. The effect includes a TRPV1/4 receptor-mediated transient increase and subsequent CB1 receptor-mediated suppression of glutamate release. Multiphasic modulation of glutamate input to PVN neurons represents a previously unappreciated complexity of control of autonomic output by glucocorticoids and endogenous cannabinoids.

Differential calcium dependence in basal and forskolin-potentiated spontaneous transmitter release in basolateral amygdala neurons

Action potential-independent transmitter release, or spontaneous release, is postulated to produce multiple postsynaptic effects (e.g., maintenance of dendritic spines and suppression of local dendritic protein synthesis). Potentiation of spontaneous release may contribute to the precise modulation of synaptic function. However, the expression mechanism underlying potentiated spontaneous release remains unclear. In this study, we investigated the involvement of extracellular and intracellular calcium in basal and potentiated spontaneous release. Miniature excitatory postsynaptic currents (mEPSCs) of the basolateral amygdala neurons in acute brain slices were recorded. Forskolin, an adenylate cyclase activator, increased mEPSC frequency, and the increase lasted at least 25min after washout. Removal of the extracellular calcium decreased mEPSC frequency in both naïve and forskolin-treated slices. On the other hand, chelation of intracellular calcium by BAPTA-AM decreased mEPSC frequency in naïve, but not in forskolin-treated slices. A blockade of the calcium-sensing receptor (CaSR) resulted in an increase in mEPSC frequency in forskolin-treated, but not in naïve slices. These findings indicate that forskolin-induced potentiation is accompanied by changes in the mechanisms underlying Ca2+-dependent spontaneous release.

Presynaptic GABAB receptors decrease neurotransmitter release in vestibular nuclei neurons during vestibular compensation

Unilateral damage to the peripheral vestibular receptors precipitates a debilitating syndrome of oculomotor and balance deficits at rest, which extensively normalize during the first week after the lesion due to vestibular compensation. In vivo studies suggest that GABAB receptor activation facilitates recovery. However, the presynaptic or postsynaptic sites of action of GABAB receptors in vestibular nuclei neurons after lesions have not been determined. Accordingly, here presynaptic and postsynaptic GABAB receptor activity in principal cells of the tangential nucleus, a major avian vestibular nucleus, was investigated using patch-clamp recordings correlated with immunolabeling and confocal imaging of the GABAB receptor subunit-2 (GABABR2) in controls and operated chickens shortly after unilateral vestibular ganglionectomy (UVG). Baclofen, a GABAB agonist, generated no postsynaptic currents in principal cells in controls, which correlated with weak GABABR2 immunolabeling on principal cell surfaces. However, baclofen decreased miniature excitatory postsynaptic current (mEPSC) and GABAergic miniature inhibitory postsynaptic current (mIPSC) events in principal cells in controls, compensating and uncompensated chickens three days after UVG, indicating the presence of functional GABAB receptors on presynaptic terminals. Baclofen decreased GABAergic mIPSC frequency to the greatest extent in principal cells on the intact side of compensating chickens, with concurrent increases in GABABR2 pixel brightness and percentage overlap in synaptotagmin 2-labeled terminals. In uncompensated chickens, baclofen decreased mEPSC frequency to the greatest extent in principal cells on the intact side, with concurrent increases in GABABR2 pixel brightness and percentage overlap in synaptotagmin 1-labeled terminals. Altogether, these results revealed changes in presynaptic GABAB receptor function and expression which differed in compensating and uncompensated chickens shortly after UVG. This work supports an important role for GABAB autoreceptor-mediated inhibition in vestibular nuclei neurons on the intact side during early stages of vestibular compensation, and a role for GABAB heteroreceptor-mediated inhibition of glutamatergic terminals on the intact side in the failure to recover function.

Maternal Loss of Ube3a Produces an Excitatory/Inhibitory Imbalance through Neuron Type-Specific Synaptic Defects

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3A. AS model mice, which carry a maternal Ube3a null mutation (Ube3a(m-/p+)), recapitulate major features of AS in humans, including enhanced seizure susceptibility. Excitatory neurotransmission onto neocortical pyramidal neurons is diminished in Ube3a(m-/p+) mice, seemingly at odds with enhanced seizure susceptibility. We show here that inhibitory drive onto neocortical pyramidal neurons is more severely decreased in Ube3a(m-/p+) mice. This inhibitory deficit follows the loss of excitatory inputs and appears to arise from defective presynaptic vesicle cycling in multiple interneuron populations. In contrast, excitatory and inhibitory synaptic inputs onto inhibitory interneurons are largely normal. Our results indicate that there are neuron type-specific synaptic deficits in Ube3a(m-/p+) mice despite the presence of Ube3a in all neurons. These deficits result in excitatory/inhibitory imbalance at cellular and circuit levels and may contribute to seizure susceptibility in AS.

Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.

Characterization and reversal of synaptic defects in the amygdala in a mouse model of fragile X syndrome

Fragile X syndrome (FXS), a common inherited form of mental impairment and autism, is caused by transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. Earlier studies have identified a role for aberrant synaptic plasticity mediated by the metabotropic glutamate receptors (mGluRs) in FXS. However, many of these observations are derived primarily from studies in the hippocampus. The strong emotional symptoms of FXS, on the other hand, are likely to involve the amygdala. Unfortunately, little is known about how exactly FXS affects synaptic function in the amygdala. Here, using whole-cell recordings in brain slices from adult Fmr1 knockout mice, we find mGluR-dependent long-term potentiation to be impaired at thalamic inputs to principal neurons in the lateral amygdala. Consistent with this long-term potentiation deficit, surface expression of the AMPA receptor subunit, GluR1, is reduced in the lateral amygdala of knockout mice. In addition to these postsynaptic deficits, lower presynaptic release was manifested by a decrease in the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), increased paired-pulse ratio, and slower use-dependent block of NMDA receptor currents. Strikingly, pharmacological inactivation of mGluR5 with 2-methyl-6-phenylethynyl-pyridine (MPEP) fails to rescue either the deficit in long-term potentiation or surface GluR1. However, the same acute MPEP treatment reverses the decrease in mEPSC frequency, a finding of potential therapeutic relevance. Therefore, our results suggest that synaptic defects in the amygdala of knockout mice are still amenable to pharmacological interventions against mGluR5, albeit in a manner not envisioned in the original hippocampal framework.

Synapse-Specific Adaptations to Inactivity in Hippocampal Circuits Achieve Homeostatic Gain Control while Dampening Network Reverberation

Synaptic homeostasis, induced by chronic changes in neuronal activity, is well studied in cultured neurons, but not in more physiological networks where distinct synaptic circuits are preserved. We characterized inactivity-induced adaptations at three sets of excitatory synapses in tetrodotoxin-treated organotypic hippocampal cultures. The adaptation to inactivity was strikingly synapse specific. Hippocampal throughput synapses (dentate-to-CA3 and CA3-to-CA1) were upregulated, conforming to homeostatic gain control in order to avoid extreme limits of neuronal firing. However, chronic inactivity decreased mEPSC frequency at CA3-to-CA3 synapses, which were isolated pharmacologically or surgically. This downregulation of recurrent synapses was opposite to that expected for conventional homeostasis, in apparent conflict with typical gain control. However, such changes contributed to an inactivity-induced shortening of reverberatory bursts generated by feedback excitation among CA3 pyramids, safeguarding the network from possible runaway excitation. Thus, synapse-specific adaptations of synaptic weight not only contributed to homeostatic gain control, but also dampened epileptogenic network activity.