Regulation of Acetylcholine Quantal Release by Coupled Thrombin/BDNF Signaling in Mouse Motor Synapses.

The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25-30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25-30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30-40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.

Estrogen-Dependent Functional Spine Dynamics in Neocortical Pyramidal Neurons of the Mouse.

Surgical ovariectomy has been shown to reduce spine density in hippocampal CA1 pyramidal cells of rodents, and this reduction is reversed by 17ß-estradiol (E2) treatment in a model of human estrogen replacement therapy. Here, we report reduction of spine density in apical dendrites of layer 5 pyramidal neurons of several neocortical regions that is reversed by subsequent E2 treatment in ovariectomized (OVX) female Thy1M-EGFP mice. We also found that OVX-associated reduction of spine density in somatosensory cortex was accompanied by a reduction in miniature EPSC (mEPSC) frequency (but not mIPSC frequency), indicating a change in functional synapses. OVX-associated spine loss in somatosensory cortex was also rescued by an agonist of the G-protein-linked estrogen receptor (GPER) but not by agonists of the classic estrogen receptors ERα/ERß, whereas the opposite selectivity was found in area CA1. Acute treatment of neocortical slices with E2 also rescued the OVX-associated reduction in mEPSC frequency, which could be mimicked by a GPER agonist and abolished by a GPER antagonist. Time-lapse in vivo two-photon imaging showed that OVX-associated reduction in spine density is achieved by both an increase in spine loss rate and a decrease in spine gain rate and that subsequent rescue by E2 reversed both of these processes. Crucially, the spines added after E2 rescue were no more likely to reappear at or nearby the sites of pre-OVX spines than those in control mice treated with vehicle. Thus, a model of estrogen replacement therapy, although restoring spine density and dynamics, does not entirely restore functional connectivity.SIGNIFICANCE STATEMENT Estrogen replacement therapy following menopause or surgical removal of the ovaries is a widespread medical practice, yet little is known about the consequences of such treatment for cells in the brain. Here, we show that estrogen replacement reverses some of the effects of surgical removal of the ovaries on the structure and function of brain cells in the mouse. Yet, importantly, the fine wiring of the brain is not returned to the presurgery state by estrogen treatment, suggesting lasting functional consequences.

The Lactate Receptor HCAR1 Modulates Neuronal Network Activity through the Activation of Gα and Gßγ Subunits.

The discovery of a G-protein-coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) in neurons has pointed to additional nonmetabolic effects of lactate for regulating neuronal network activity. In this study, we characterized the intracellular pathways engaged by HCAR1 activation, using mouse primary cortical neurons from wild-type (WT) and HCAR1 knock-out (KO) mice from both sexes. Using whole-cell patch clamp, we found that the activation of HCAR1 with 3-chloro-5-hydroxybenzoic acid (3Cl-HBA) decreased miniature EPSC frequency, increased paired-pulse ratio, decreased firing frequency, and modulated membrane intrinsic properties. Using fast calcium imaging, we show that HCAR1 agonists 3,5-dihydroxybenzoic acid, 3Cl-HBA, and lactate decreased by 40% spontaneous calcium spiking activity of primary cortical neurons from WT but not from HCAR1 KO mice. Notably, in neurons lacking HCAR1, the basal activity was increased compared with WT. HCAR1 mediates its effect in neurons through a Giα-protein. We observed that the adenylyl cyclase-cAMP-protein kinase A axis is involved in HCAR1 downmodulation of neuronal activity. We found that HCAR1 interacts with adenosine A1, GABAB, and α2A-adrenergic receptors, through a mechanism involving both its Giα and Gißγ subunits, resulting in a complex modulation of neuronal network activity. We conclude that HCAR1 activation in neurons causes a downmodulation of neuronal activity through presynaptic mechanisms and by reducing neuronal excitability. HCAR1 activation engages both Giα and Gißγ intracellular pathways to functionally interact with other Gi-coupled receptors for the fine tuning of neuronal activity.SIGNIFICANCE STATEMENT Expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) was recently described in neurons. Here, we describe the physiological role of this G-protein-coupled receptor (GPCR) and its activation in neurons, providing information on its expression and mechanism of action. We dissected out the intracellular pathway through which HCAR1 activation tunes down neuronal network activity. For the first time, we provide evidence for the functional cross talk of HCAR1 with other GPCRs, such as GABAB, adenosine A1- and α2A-adrenergic receptors. These results set HCAR1 as a new player for the regulation of neuronal network activity acting in concert with other established receptors. Thus, HCAR1 represents a novel therapeutic target for pathologies characterized by network hyperexcitability dysfunction, such as epilepsy.

Involvement of the cAMP-Dependent Pathway in Dextromethorphan-Induced Inhibition of Spontaneous Glutamate Transmission in the Nucleus Tractus Solitarius Neurons of Guinea Pigs.

Dextromethorphan (DEX) presynaptically decreases glutamatergic transmission in second-order neurons of the nucleus tractus solitarius (TS). To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and TS-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin counteracted the inhibitory effect of DEX on mEPSCs. Eight-Bromo-cAMP and N-ethylmaleimide also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. This suggests that DEX decreases spontaneous glutamate release by inhibiting the cAMP-dependent pathway and synchronous release by another unknown mechanism.

ErbB4 Receptors in the Medial Habenula Regulate Contextual Fear Memory.

The Medial Habenular (MHb) and the Lateral Habenular nuclei are 2 main parts of the habenular complex (Hb). Recent studies showed that MHb plays an important role in memory, and in the expression of ErbB4. However, the expression of MHb ErbB4 receptor and its role in fear memory is not well understood. In this study, western blotting and quantitative real-time polymerase chain reaction were used to assess the protein and mRNA levels of ErbB4 in the process of contextual fear conditioning. A pharmacological approach was used to block and stimulate the ErbB4 receptor. Contextual fear conditioning tests induced a significant increase on the expression of ErbB4 at various times in the Hb and the MHb. Moreover, the blockade and stimulation of MHb ErbB4 receptors did not affect the fear formation but impaired and improved the contextual-dependent fear expression. Furthermore, in vitro electrophysiological recordings showed that the blockade of the MHb ErbB4 receptor reduced the presynaptic gamma-amino butyric acid release. ErbB4 is a susceptible gene for schizophrenia and the above findings may provide new insights into the mechanisms of fear-related responses.

Modulation of synaptic inputs in magnocellular neurones in a rat model of cancer cachexia.

In cancer cachexia, abnormal metabolism and neuroendocrine dysfunction cause anorexia, tissue damage and atrophy, which can in turn alter body fluid balance. Arginine vasopressin, which regulates fluid homeostasis, is secreted by magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus. Arginine vasopressin secretion by MNCs is regulated by both excitatory and inhibitory synaptic activity, alterations in plasma osmolarity and various peptides, including angiotensin II. In the present study, we used whole-cell patch-clamp recordings of brain slices to determine whether hyperosmotic stimulation and/or angiotensin II potentiate excitatory synaptic input in a rat model of cancer cachexia, similar to their effects in normal (control) rats. Hyperosmotic (15 and 60 mmol L-1   mannitol) stimulation and angiotensin II (0.1 µmol L-1 ) increased the frequency, but not the amplitude, of miniature excitatory postsynaptic currents in normal rats; in model rats, both effects were significantly attenuated. These results suggest that cancer cachexia alters supraoptic MNC sensitivity to osmotic and angiotensin II stimulation.

Cannabinoid-induced increase of quantal size and enhanced neuromuscular transmission.

Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal amplitudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction.

Endogenous purines modulate K+ -evoked ACh secretion at the mouse neuromuscular junction.

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y13 and A1 , A2A , and A3 receptors, respectively. To elucidate the action of endogenous purines on K+ -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K+ , the P2Y13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A1 , A3 , and A2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K+ -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K+ , endogenous ATP/ADP and adenosine bind to inhibitory P2Y13 and A1 and A3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K+ , suggesting that more adenosine is needed to activate excitatory A2A receptors. At high K+ concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K+ concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.

α2δ-1 Signaling Drives Cell Death, Synaptogenesis, Circuit Reorganization, and Gabapentin-Mediated Neuroprotection in a Model of Insult-Induced Cortical Malformation.

Developmental cortical malformations (DCMs) result from pre- and perinatal insults, as well as genetic mutations. Hypoxia, viral infection, and traumatic injury are the most common environmental causes of DCMs, and are associated with the subsyndromes focal polymicrogyria and focal cortical dysplasia (FCD) Type IIId, both of which have a high incidence of epilepsy. Understanding the molecular signals that lead to the formation of a hyperexcitable network in DCMs is critical to devising novel treatment strategies. In a previous study using the freeze-lesion (FL) murine model of DCM, we found that levels of thrombospondin (TSP) and the calcium channel auxiliary subunit α2δ-1 were elevated. TSP binds to α2δ-1 to drive the formation of excitatory synapses during development, suggesting that overactivation of this pathway may lead to exuberant excitatory synaptogenesis and network hyperexcitability seen in DCMs. In that study, antagonizing TSP/α2δ-1 signaling using the drug gabapentin (GBP) reduced many FL-induced pathologies. Here, we used mice with a genetic deletion of α2δ-1 to determine how α2δ-1 contributes to cell death, elevated excitatory synapse number, and in vitro network function after FL and to examine the molecular specificity of GBP's effects. We identified a critical role for α2δ-1 in FL-induced pathologies and in mediating the neuroprotective effects of GBP. Interestingly, genetic deletion of α2δ-1 did not eliminate GBP's effects on synaptogenesis, suggesting that GBP can have α2δ-1-independent effects. Taken together these studies suggests that inhibiting α2δ-1 signaling may have therapeutic promise to reduce cell death and network reorganization associated with insult-induced DCMs.

The Marine Guanidine Alkaloid Crambescidin 816 Induces Calcium Influx and Cytotoxicity in Primary Cultures of Cortical Neurons through Glutamate Receptors.

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

Sensitizing exposure to amphetamine increases AMPA receptor phosphorylation without increasing cell surface expression in the rat nucleus accumbens.

Exposure to psychostimulants like cocaine or amphetamine leads to long-lasting sensitization of their behavioral and neurochemical effects. Here we characterized changes in AMPA receptor distribution and phosphorylation state in the rat nucleus accumbens (NAcc) weeks after amphetamine exposure to assess their potential contribution to sensitization by this drug. Using protein cross-linking, biochemical, subcellular fractionation, and slice electrophysiological approaches in the NAcc, we found that, unlike cocaine, previous exposure to amphetamine did not increase cell surface levels of either GluA1 or GluA2 AMPA receptor subunits, redistribution of these subunits to the synaptic or perisynaptic cellular membrane domains, protein-protein associations required to support the accumulation and retention of AMPA receptors in the PSD, or the peak amplitude of AMPA receptor mediated mEPSCs recorded in NAcc slices. On the other hand, exposure to amphetamine significantly slowed mEPSC decay times and increased levels in the PSD of PKA and CaMKII as well as phosphorylation by these kinases of the GluA1 S845 and S831 residues selectively in this cellular compartment. As the latter effects are known to respectively regulate channel open probability and duration as well as conductance, they provide a novel mechanism that could contribute to the long-lasting AMPA receptor dependent expression of sensitization by amphetamine. Rather than increase the number of surface and synaptic AMPA receptors as with cocaine, this mechanism could increase NAcc medium spiny neuron reactivity to glutamate afferents by increasing the phosphorylation state of critical regulatory sites in the AMPA receptor GluA1 subunit in the PSD.

ß-Amyloid triggers aberrant over-scaling of homeostatic synaptic plasticity.

The over-production of ß-amyloid (Aß) has been strongly correlated to neuronal dysfunction and altered synaptic plasticity in Alzheimer's disease (AD). Accordingly, it has been proposed that disrupted synaptic transmission and neuronal network instability underlie memory failure that is evident in the early phases of AD. Homeostatic synaptic plasticity (HSP) serves to restrain neuronal activity within a physiological range. Therefore a disruption of this mechanism may lead to destabilization in synaptic and neural circuit function. Here, we report that during HSP by neuronal activity deprivation, application of Aß results in an aberrant over-response of the up-regulation of AMPA receptor (AMPAR)-mediated synaptic currents and cell-surface AMPAR expression. In the visual cortex, in vivo HSP induced by visual deprivation shows a similar over-response following an Aß local injection. Aß increases the expression of GluA2-lacking, calcium permeable AMPARs (CP-AMPARs), which are required for the initiation, but not maintenance of HSP. Both GluA2-lacking and GluA2-containing AMPARs contribute to the Aß-mediated over-scaling of HSP. We also find that Aß induces the dissociation of HDAC1 from the miR124 transcription factor EVI1, leading to an up-regulation of miR124 expression and increased amount of CP-AMPARs. Thus, via aberrant stimulation of miR124 expression and biogenesis of CP-AMPARs, Aß is able to induce an over response in HSP. This Aß-mediated dysregulation in homeostatic plasticity may play an important role in the pathogenesis of altered neural function and memory deficits in the early stages of AD.

Presynaptic G Protein-Coupled Receptors Differentially Modulate Spontaneous Glutamate Release in the Supraoptic Nucleus.

Spontaneous glutamate release in the supraoptic nucleus is modulated by a number of inhibitory G protein coupled receptors (GPCR), including GABAB , adenosine A1 and group III metabotropic glutamate receptors (mGluR). It remains unclear whether they have distinct roles or are redundant mechanisms that protect from hyperexcitation. To address this question, we facilitated spontaneous glutamate release using nifedipine or forskolin, which act in a protein kinase A (PKA)-independent and -dependent manner, respectively, and tested the effects of inhibitory GPCR agonists. We found that a GABAB receptor (GABAB R) agonist specifically inhibited forskolin-induced miniature excitatory postsynaptic currents (mEPSC), in contrast to an adenosine A1 receptor (A1R) agonist, which specifically inhibited nifedipine-induced mEPSCs. This suggests that GABAB Rs and A1 Rs modulate independent mechanisms activated by forskolin and nifedipine, respectively. However, the inhibitory effects of GABAB R and A1 R agonists on basal mEPSCs occluded each other, suggesting that these receptors also have an overlapping role. Group III mGluRs appear to have a greater control over glutamate release because agonists to these receptors inhibited both nifedipine- and forskolin-induced mEPSCs. mEPSCs induced by norepinephrine had the same characteristics as those induced by forskolin [i.e. PKA-dependence and sensitivity to GABAB R and group III mGluR agonists, but not an A1 R agonist]. In summary, the present study highlights the differential effects of GABAB R, A1 R and mGluR agonists on glutamate release stimulated by different secretagogues, including the endogenous neuromodulator norepinephrine. These results suggest that the roles of these inhibitory GPCRs are not completely redundant, and also indicate the physiological implications of having different excitatory and inhibitory GPCRs on the same synapse.

Adenosine A1 Receptor Suppresses Tonic GABAA Receptor Currents in Hippocampal Pyramidal Cells and in a Defined Subpopulation of Interneurons.

Adenosine is an endogenous neuromodulator that decreases excitability of hippocampal circuits activating membrane-bound metabotropic A1 receptor (A1R). The presynaptic inhibitory action of adenosine A1R in glutamatergic synapses is well documented, but its influence on inhibitory GABAergic transmission is poorly known. We report that GABAA receptor (GABAAR)-mediated tonic, but not phasic, transmission is suppressed by A1R in hippocampal neurons. Adenosine A1R activation strongly inhibits GABAAR agonist (muscimol)-evoked currents in Cornu Ammonis 1 (CA1) pyramidal neurons and in a specific subpopulation of interneurons expressing axonal cannabinoid receptor type 1. In addition, A1R suppresses tonic GABAAR currents measured in the presence of elevated ambient GABA as well as in naïve slices. The inhibition of GABAergic currents involves both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and decreases GABAAR δ-subunit expression. On the contrary, no A1R-mediated modulation was detected in phasic inhibitory postsynaptic currents evoked either by afferent electrical stimulation or by spontaneous quantal release. The results show that A1R modulates extrasynaptic rather than synaptic GABAAR-mediated signaling, and that this modulation selectively occurs in hippocampal pyramidal neurons and in a specific subpopulation of inhibitory interneurons. We conclude that modulation of tonic GABAAR signaling by adenosine A1R in specific neuron types may regulate neuronal gain and excitability in the hippocampus.

Postsynaptic Potentiation in Mouse Motor Synapses Induced by ATP Accumulation in Synaptic Cleft.

In mouse motor synapses, a non-selective purinoceptor antagonist suramin increased the quantum content of endplate potentials (EPP) without changing the time course of synaptic potentials. An ectonucleotidase inhibitor ARL 67156 had no effect on the amplitude and quantum content of EPP and miniature endplate potentials (mEPP) evoked by single stimuli, but significantly prolonged their duration. Long-term high-frequency stimulation of the nerve in the presence of ARL 67156 persistently increased the amplitude and duration of EPP during the train of impulses, but did not change their quantum content. ATP-γ-S, a non-hydrolyzed ATP analogue, significantly increased the amplitudes and prolonged the rising and falling phases of EPP and mEPP. The ATP-induced postsynaptic potentiation in neuromuscular transmission can result from the increase in ATP content and its longer presence in the synaptic cleft.

Dexmedetomidine prevents post-ischemic LTP via presynaptic and postsynaptic mechanisms.

Increasing evidence indicates that dexmedetomidine (DEX), a selective α2-adrenergic receptor agonist, has a neuroprotective effect against cerebral injury. However, it remains unknown whether and how DEX functionally prevents the pathological form of synaptic plasticity caused by ischemia in the hippocampal CA1 neurons. To address this issue, we analyzed the role of DEX using a model of brain ischemia (oxygen and glucose deprivation, OGD) referred to as post-ischemic LTP (i-LTP). We found that DEX could reduce i-LTP by selectively activating α2 receptors. To clarify its detailed mechanisms, the presynaptic and postsynaptic roles of DEX were investigated. The activation of the α2 receptors of DEX decreased the frequency spontaneous mEPSCs, which exerted its presynaptic mechanisms. In addition, DEX also decreased the amplitude of mEPSCs and prevented the depolarization of postsynaptic membranes during OGD treatment, which exerted its postsynaptic mechanisms. More importantly, our results indicate that postsynaptic ß receptors, not α1 receptors, participated in i-LTP. Therefore, these results demonstrated that decreasing ß receptors activation by DEX-medicated pre- and post-synaptic α2 receptors activation is responsible for i-LTP. Because of the NMDARs required for i-LTP, we further examined the critical roles of postsynaptic ß receptors downstream PKA regulation of NMDA receptor-mediated EPSCs (NMDA EPSC). We clarified that it is attributable to the direct effect of DEX on NMDA EPSC as mediated by PKA inactivation. These findings suggest that DEX can protect neurons from functional damage caused by a relatively mild degree of transient cerebral ischemia, and this effect is mediated by both presynaptic reduction of NE and glutamate release and postsynaptic suppression of NMDAR activation by ß receptors and downstream PKA regulation.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses.

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+ -permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+ -permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

Resveratrol up-regulates AMPA receptor expression via AMP-activated protein kinase-mediated protein translation.

Resveratrol is a phytoalexin that confers overall health benefits including positive regulation in brain function such as learning and cognition. However, whether and how resveratrol affects synaptic activity remains largely unknown. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are glutamatergic receptors that mediate the majority of fast excitatory transmission and synaptic plasticity, and thus play a critical role in higher brain functions, including learning and memory. We find that in rat primary neurons, resveratrol can rapidly increase AMPAR protein level, AMPAR synaptic accumulation and the strength of excitatory synaptic transmission. The resveratrol effect on AMPAR protein expression is independent of sirtuin 1 (SIRT1), the conventional downstream target of resveratrol, but rather is mediated by AMP-activated protein kinase (AMPK) and subsequent downstream phosphoinositide 3-kinase (PI3K)/Akt signaling. Application of the AMPK specific activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis via regulation of the eukaryotic initiation factor (eIF) 4E/4G complex. Disruption of the translation initiation complex completely blocks resveratrol-dependent AMPAR up-regulation. These findings indicate that resveratrol may regulate brain function through facilitation of AMPAR biogenesis and synaptic transmission.

Activation of mu-opioid receptors in the ventrolateral orbital cortex inhibits the GABAergic miniature inhibitory postsynaptic currents in rats.

Previous studies have indicated that mu-opioid receptors in the ventrolateral orbital cortex (VLO) are involved in antinociception in tail flick tests and GABAergic neurons or terminals express mu-opioid receptors in the VLO. The current study examined the effect of selective mu-opioid receptor agonist DAMGO on the GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the VLO in rats using the whole-cell patch clamp. The results demonstrated that 5 µM DAMGO application into the rat VLO slices significantly reduced the GABAergic mIPSCs frequency, without any effect on its amplitude, and this effect of DAMGO was reversed by pretreatment with selective mu-opioid receptor antagonist 1 µM CTOP. Importantly, application of CTOP alone into the VLO slices did not produce any effect on the frequency and amplitude of GABAergic mIPSCs. These results indicate a presynaptic effect of mu-opioid receptor activation on the GABAergic neurons in the VLO. The current data suggests that a presynaptic inhibition of the GABA release may contribute to the mu-opioid receptor mediated effects in the VLO and provides novel electrophysiological evidence for the underlying mechanisms of mu-opioid receptors in the VLO.

Nf1 regulates alcohol dependence-associated excessive drinking and gamma-aminobutyric acid release in the central amygdala in mice and is associated with alcohol dependence in humans.

BACKGROUND: The neurofibromatosis type 1 (Nf1) gene encodes a GTPase activating protein that negatively regulates small GTPases of the Ras family. METHODS: We assessed alcohol-related behaviors including alcohol sensitivity, dependent and nondependent drinking, and basal and alcohol-induced gamma-aminobutyric acid (GABA) release in the central nucleus of the amygdala (CeA) in Nf1 heterozygous null mice (Nf1(+/-)). We also investigated the associations of NF1 polymorphisms with alcohol dependence risk and severity in humans. RESULTS: Nf1(+/-) mice do not differ from wild-type mice in nondependent drinking, such as 24-hour, 2-bottle choice drinking in the dark binge drinking or limited access 2-bottle choice. However, Nf1(+/-) mice failed to escalate alcohol drinking following chronic intermittent ethanol vapor exposure (CIE) to induce dependence. Alcohol acutely increases GABA release in the CeA and alcohol dependence is characterized by increased baseline GABA release in CeA. Interestingly, GABA release in Nf1(+/-) mice is greater at baseline than wild-type mice, is not elevated by induction of dependence by CIE, and failed to show alcohol-induced facilitation both before and after CIE. Additionally, we observed that multiple variants in the human NF1 gene are associated with a quantitative measure of alcohol dependence in both African Americans and European Americans. CONCLUSIONS: In this translational investigation, we found that Nf1 activity regulates excessive drinking and basal and ethanol-stimulated GABA release in the mouse central amygdala. We also found that genetic variation in NF1 may confer an inherent susceptibility to the transition from nondependent to dependent drinking in humans.