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Lithium chloride has been widely used as a therapeutic mood stabilizer. Although cumulative evidence suggests that lithium plays modulatory effects on postsynaptic receptors, the underlying mechanism by which lithium regulates synaptic transmission has not been fully elucidated. In this work, by using the advantageous neuromuscular synapse, we evaluated the effect of lithium on the stability of postsynaptic nicotinic acetylcholine receptors (nAChRs) in vivo. We found that in normally innervated neuromuscular synapses, lithium chloride significantly decreased the turnover of nAChRs by reducing their internalization. A similar response was observed in CHO-K1/A5 cells expressing the adult muscle-type nAChRs. Strikingly, in denervated neuromuscular synapses, lithium led to enhanced nAChR turnover and density by increasing the incorporation of new nAChRs. Lithium also potentiated the formation of unstable nAChR clusters in non-synaptic regions of denervated muscle fibres. We found that denervation-dependent re-expression of the foetal nAChR γ-subunit was not altered by lithium. However, while denervation inhibits the distribution of β-catenin within endplates, lithium-treated fibres retain β-catenin staining in specific foci of the synaptic region. Collectively, our data reveal that lithium treatment differentially affects the stability of postsynaptic receptors in normal and denervated neuromuscular synapses in vivo, thus providing novel insights into the regulatory effects of lithium on synaptic organization and extending its potential therapeutic use in conditions affecting the peripheral nervous system., (© 2021. The Author(s).)

Atypical antipsychotics such as risperidone cause drug-induced metabolic syndrome. However, the underlying mechanisms remain largely unknown. Here, we report a new mouse model that reliably reproduces risperidone-induced weight gain, adiposity, and glucose intolerance. We found that risperidone treatment acutely altered energy balance in C57BL/6 mice and that hyperphagia accounted for most of the weight gain. Transcriptomic analyses in the hypothalamus of risperidone-fed mice revealed that risperidone treatment reduced the expression of Mc4r. Furthermore, Mc4r in Sim1 neurons was necessary for risperidone-induced hyperphagia and weight gain. Moreover, we found that the same pathway underlies the obesogenic effect of olanzapine-another commonly prescribed antipsychotic drug. Remarkably, whole-cell patch-clamp recording demonstrated that risperidone acutely inhibited the activity of hypothalamic Mc4r neurons via the opening of a postsynaptic potassium conductance. Finally, we showed that treatment with setmelanotide, an MC4R-specific agonist, mitigated hyperphagia and obesity in both risperidone- and olanzapine-fed mice., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Li et al.)

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

Autism spectrum disorder (ASD) is an immensely challenging developmental disorder characterized primarily by two core behavioral symptoms of social communication deficits and restricted/repetitive behaviors. Investigating the etiological process and identifying an appropriate therapeutic target remain as formidable challenges to overcome ASD due to numerous risk factors and complex symptoms associated with the disorder. Among the various mechanisms that contribute to ASD, the maintenance of excitation and inhibition balance emerged as a key factor to regulate proper functioning of neuronal circuitry. In this study, we employed prenatally exposed to valproic acid (VPA) to establish a validated ASD mouse model and found impaired inhibitory gamma-aminobutyric acid (GABAergic) neurotransmission through a presynaptic mechanism in these model mice, which was accompanied with decreased GABA release and GABA-A and GABA-B receptor subunits expression. And acute administration of individual GABA-A or GABA-B receptor agonists partially reversed autistic-like behaviors in the model mice. Furthermore, acute administration of the combined GABA-A and GABA-B receptor agonists palliated sociability deficits, anxiety and repetitive behaviors in the animal model of autistic-like behaviors, demonstrating the therapeutic potential of above cocktail in the treatment of ASD., (Copyright © 2020. Published by Elsevier B.V.)

GABA A receptors (GABA A Rs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABA A Rs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABA A Rs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABA A Rs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse event associated with treatment with paclitaxel and other chemotherapeutic agents. The prevention and treatment of CIPN are limited by a lack of understanding of the molecular mechanisms underlying this toxicity. In the current study, a human induced pluripotent stem cell-derived sensory neuron (iPSC-SN) model was developed for the study of chemotherapy-induced neurotoxicity. The iPSC-SNs express proteins characteristic of nociceptor, mechanoreceptor, and proprioceptor sensory neurons and show Ca 2+ influx in response to capsaicin, α,β-meATP, and glutamate. The iPSC-SNs are relatively resistant to the cytotoxic effects of paclitaxel, with half-maximal inhibitory concentration (IC 50 ) values of 38.1 µM (95% confidence interval (CI) 22.9-70.9 µM) for 48-hour exposure and 9.3 µM (95% CI 5.7-16.5 µM) for 72-hour treatment. Paclitaxel causes dose-dependent and time-dependent changes in neurite network complexity detected by βIII-tubulin staining and high content imaging. The IC 50 for paclitaxel reduction of neurite area was 1.4 µM (95% CI 0.3-16.9 µM) for 48-hour exposure and 0.6 µM (95% CI 0.09-9.9 µM) for 72-hour exposure. Decreased mitochondrial membrane potential, slower movement of mitochondria down the neurites, and changes in glutamate-induced neuronal excitability were also observed with paclitaxel exposure. The iPSC-SNs were also sensitive to docetaxel, vincristine, and bortezomib. Collectively, these data support the use of iPSC-SNs for detailed mechanistic investigations of genes and pathways implicated in chemotherapy-induced neurotoxicity and the identification of novel therapeutic approaches for its prevention and treatment., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

The brain functions can be reversibly modulated by the action of general anesthetics. Despite a wide number of pharmacological studies, an extensive analysis of the cellular determinants of anesthesia at the microcircuits level is still missing. Here, by combining patch-clamp recordings and mathematical modeling, we examined the impact of sevoflurane, a general anesthetic widely employed in the clinical practice, on neuronal communication. The cerebellar microcircuit was used as a benchmark to analyze the action mechanisms of sevoflurane while a biologically realistic mathematical model was employed to explore at fine grain the molecular targets of anesthetic analyzing its impact on neuronal activity. The sevoflurane altered neurotransmission by strongly increasing GABAergic inhibition while decreasing glutamatergic NMDA activity. These changes caused a notable reduction of spike discharge in cerebellar granule cells (GrCs) following repetitive activation by excitatory mossy fibers (mfs). Unexpectedly, sevoflurane altered GrCs intrinsic excitability promoting action potential generation. Computational modelling revealed that this effect was triggered by an acceleration of persistent sodium current kinetics and by an increase in voltage dependent potassium current conductance. The overall effect was a reduced variability of GrCs responses elicited by mfs supporting the idea that sevoflurane shapes neuronal communication without silencing neural circuits.

Beta-amyloid (Aβ) has been recognized as an early trigger in the pathogenesis of Alzheimer's disease (AD) leading to synaptic and cognitive impairments. Aβ can alter neuronal signaling through interactions with nicotinic acetylcholine receptors (nAChRs), contributing to synaptic dysfunction in AD. The three major nAChR subtypes in the hippocampus are composed of α7-, α4β2-, and α3β4-nAChRs. Aβ selectively affects α7- and α4β2-nAChRs, but not α3β4-nAChRs in hippocampal neurons, resulting in neuronal hyperexcitation. However, how nAChR subtype selectivity for Aβ affects synaptic function in AD is not completely understood. Here, we showed that Aβ associated with α7- and α4β2-nAChRs but not α3β4-nAChRs. Computational modeling suggested that two amino acids in α7-nAChRs, arginine 208 and glutamate 211, were important for the interaction between Aβ and α7-containing nAChRs. These residues are conserved only in the α7 and α4 subunits. We therefore mutated these amino acids in α7-containing nAChRs to mimic the α3 subunit and found that mutant α7-containing receptors were unable to interact with Aβ. In addition, mutant α3-containing nAChRs mimicking the α7 subunit interact with Aβ. This provides direct molecular evidence for how Aβ selectively interacted with α7- and α4β2-nAChRs, but not α3β4-nAChRs. Selective coactivation of α7- and α4β2-nAChRs also sufficiently reversed Aβ-induced AMPA receptor dysfunction, including Aβ-induced reduction of AMPA receptor phosphorylation and surface expression in hippocampal neurons. Moreover, costimulation of α7- and α4β2-nAChRs reversed the Aβ-induced disruption of long-term potentiation. These findings support a novel mechanism for Aβ's impact on synaptic function in AD, namely, the differential regulation of nAChR subtypes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons orchestrate various physiological events that control the onset of puberty. Previous studies showed that insulin-like growth factor 1 (IGF-1) induces the secretion of GnRH and accelerates the onset of puberty, suggesting a regulatory role of this hormone upon GnRH neurons., Methods: To reveal responsiveness of GnRH neurons to IGF-1 and elucidate molecular pathways acting downstream to the IGF-1 receptor (IGF-1R), in vitro electrophysiological experiments were carried out on GnRH-GFP neurons in acute brain slices from prepubertal (23-29 days) and pubertal (50 days) male mice., Results: Administration of IGF-1 (13 nM) significantly increased the firing rate and frequency of spontaneous postsynaptic currents and that of excitatory GABAergic miniature postsynaptic currents (mPSCs). No GABAergic mPSCs were induced by IGF-1 in the presence of the GABAA-R blocker picrotoxin. The increase in the mPSC frequency was prevented by the use of the IGF-1R antagonist, JB1 (1 µM), or the intracellularly applied PI3K blocker (LY294002, 50 µM), showing involvement of IGF-1R and PI3K in the mechanism. Blockade of the transient receptor potential vanilloid 1, an element of the tonic retrograde endocannabinoid machinery, by AMG9810 (10 µM) or antagonizing the cannabinoid receptor type-1 by AM251 (1 µM) abolished the effect., Discussion/conclusion: These findings indicate that IGF-1 arrests the tonic retrograde endocannabinoid pathway in GnRH neurons, and this disinhibition increases the release of GABA from presynaptic terminals that, in turn, activates GnRH neurons leading to the fine-tuning of the hypothalamo-pituitary-gonadal axis., (© 2020 S. Karger AG, Basel.)

Synaptic GABA A receptors are alternately exposed to short pulses of a high, millimolar concentration of GABA and prolonged periods of low, micromolar concentration of the transmitter. Prior work has indicated that exposure to micromolar concentrations of GABA can both activate the postsynaptic receptors generating sustained low-amplitude current and desensitize the receptors, thereby reducing the peak amplitude of subsequent synaptic response. However, the precise relationship between tonic activation and reduction of peak response is not known. Here, we have measured the effect of prolonged exposure to GABA or the combination of GABA and the neurosteroid allopregnanolone, which was intended to desensitize a fraction of receptors, on a subsequent response to a high concentration of agonist in human α 1 β 3 γ 2L receptors expressed in Xenopus oocytes. We show that the reduction in the peak amplitude of the post-exposure test response correlates with the open probability of the preceding desensitizing response. Curve fitting of the inhibitory relationship yielded an IC 50 of 12.5 µM and a Hill coefficient of -1.61. The activation and desensitization data were mechanistically analyzed in the framework of a three-state Resting-Active-Desensitized model. Using the estimated affinity, efficacy, and desensitization parameters, we calculated the amount of desensitization that would accumulate during a long (2-minute) application of GABA or GABA plus allopregnanolone. The results indicate that accumulation of desensitization depends on the level of activity rather than agonist or potentiator concentration per se. We estimate that in the presence of 1 µM GABA, approximately 5% of α 1 β 3 γ 2L receptors are functionally eliminated because of desensitization. SIGNIFICANCE STATEMENT: We present an analytical approach to quantify and predict the loss of activatable GABA A receptors due to desensitization in the presence of transmitter and the steroid allopregnanolone. The findings indicate that the peak amplitude of the synaptic response is influenced by ambient GABA and that changes in ambient concentrations of the transmitter and other GABAergic agents can modify tonically and phasically activated synaptic receptors in opposite directions., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Analysis of the presynaptic action potential's (AP syn ) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca 2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for K v 1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic K v 1 channel inactivation was mediated by the K v β1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of K v β1 blocked all broadening of the AP syn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic K v channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Snake venom three-finger α-neurotoxins (α-3FNTx) act on postsynaptic nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction (NMJ) to produce skeletal muscle paralysis. The discovery of the archetypal α-bungarotoxin (α-BgTx), almost six decades ago, exponentially expanded our knowledge of membrane receptors and ion channels. This included the localisation, isolation and characterization of the first receptor (nAChR); and by extension, the pathophysiology and pharmacology of neuromuscular transmission and associated pathologies such as myasthenia gravis, as well as our understanding of the role of α-3FNTxs in snakebite envenomation leading to novel concepts of targeted treatment. Subsequent studies on a variety of animal venoms have yielded a plethora of novel toxins that have revolutionized molecular biomedicine and advanced drug discovery from bench to bedside. This review provides an overview of nAChRs and their subtypes, classification of α-3FNTxs and the challenges of typifying an increasing arsenal of structurally and functionally unique toxins, and the three-finger protein (3FP) fold in the context of the uPAR/Ly6/CD59/snake toxin superfamily. The pharmacology of snake α-3FNTxs including their mechanisms of neuromuscular blockade, variations in reversibility of nAChR interactions, specificity for nAChR subtypes or for distinct ligand-binding interfaces within a subtype and the role of α-3FNTxs in neurotoxic envenomation are also detailed. Lastly, a reconciliation of structure-function relationships between α-3FNTx and nAChRs, derived from historical mutational and biochemical studies and emerging atomic level structures of nAChR models in complex with α-3FNTxs is discussed., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. Although previous studies have shown that postsynaptic neurotoxins account for 11-23% of N. atra venom, envenomed patients do not display marked signs of neurotoxicity. We have previously shown that the lack of clinical neurotoxicity following snake envenoming by some species with 'neurotoxic' venoms may be related to the high prevalence of short-chain postsynaptic neurotoxins in these venoms. In this study, we describe the isolation and characterization of α-Elapitoxin-Na1a (α-EPTX-Na1a; 6949 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 9% of N. atra crude venom. α-EPTX-Na1a (30-300 nM) produced concentration-dependent inhibition of indirect-twitches, with a t 90 value of 17 ± 2 min at 300 nM, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior addition of either Chinese N. atra monovalent antivenom (0.3 U/ml) or Australian polyvalent snake antivenom (2.4 U/ml), prevented the in vitro neurotoxic effects of α-EPTX-Na1a (30 nM). Addition of each of these antivenoms at the t 90 time point partially reversed the in vitro neurotoxicity caused by α-EPTX-Na1a (30 nM). The inhibition of indirect twitches by α-EPTX-Na1a (30 nM) was not reversed by repeatedly washing the tissue. α-EPTX-Na1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA 2 value of 8.21. De novo protein sequencing of α-EPTX-Na1a revealed a typical short-chain postsynaptic neurotoxin profile of 62 amino acids which shared >98% amino acid sequence similarity with short-chain postsynaptic neurotoxins from other Naja species. When compared to short-chain neurotoxins isolated from cobras in China, α-EPTX-Na1a contained novel residues K47Q (i.e. lysine to glutamine), N48T (i.e. asparagine to threonine) and G49A (i.e. glycine to alanine). In conclusion, α-EPTX-Na1a is a potent, pseudo-irreversible, short-chain neurotoxin. The high prevalence of α-EPTX-Na1a in Chinese N. atra venom is likely to explain the mild neurotoxicity experienced by envenomed patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Olesoxime is a cholesterol-like neuroprotective compound that targets to mitochondrial voltage dependent anion channels (VDACs). VDACs were also found in the plasma membrane and highly expressed in the presynaptic compartment. Here, we studied the effects of olesoxime and VDAC inhibitors on neurotransmission in the mouse neuromuscular junction. Electrophysiological analysis revealed that olesoxime suppressed selectively evoked neurotransmitter release in response to a single stimulus and 20 Hz activity. Also olesoxime decreased the rate of FM1-43 dye loss (an indicator of synaptic vesicle exocytosis) at low frequency stimulation and 20 Hz. Furthermore, an increase in extracellular Cl - enhanced the action of olesoxime on the exocytosis and olesoxime increased intracellular Cl - levels. The effects of olesoxime on the evoked synaptic vesicle exocytosis and [Cl - ] i were blocked by membrane-permeable and impermeable VDAC inhibitors. Immunofluorescent labeling pointed on the presence of VDACs on the synaptic membranes. Rotenone-induced mitochondrial dysfunction perturbed the exocytotic release of FM1-43 and cell-permeable VDAC inhibitor (but not olesoxime or impermeable VDAC inhibitor) partially mitigated the rotenone-driven alterations in the FM1-43 unloading and mitochondrial superoxide production. Thus, olesoxime restrains neurotransmission by acting on plasmalemmal VDACs whose activation can limit synaptic vesicle exocytosis probably via increasing anion flux into the nerve terminals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The large-conductance calcium-activated potassium (BK) channel is a critical regulator of neuronal action potential firing and follows two distinct trends in early postnatal development: an increase in total expression and a shift from the faster activating STREX isoform to the slower ZERO isoform. We analyzed the functional consequences of developmental trends in BK channel expression in hippocampal neurons isolated from neonatal rats aged 1 to 7 days. Following overnight cultures, action potentials and currents were recorded using whole cell patch-clamp electrophysiology. These neurons undergo a steady increase in excitability during this time, and the effect of blockade of BK channel activity with 100 nM iberiotoxin changes as the neurons mature. BK currents contribute significantly more to total potassium current and single action potentials in neurons of 1-day old rats (with BK blockade extending action potential duration by 0.46 ± 0.12 ms) than in those of 7-day old rats (with BK blockade extending action potential duration by 0.17 ± 0.05 ms). BK currents contribute consistently to maintain firing rates in neurons of 1-day old rats throughout extended action potential firing; BK blockade evenly depresses firing frequency across action potential trains. In neurons from 7-day old rats, BK blockade initially increases firing frequency and then progressively decreases frequency as firing continues, ultimately depressing neuronal firing rates to a greater extent than in the neurons from 1-day-old animals. These results are consistent with a transition from low expression of a fast-activating BK isoform (STREX) to high expression of a slower activating isoform (ZERO). NEW & NOTEWORTHY This work describes the early developmental trends of large-conductance calcium-activated potassium (BK) channel activity. Early developmental trends in expression of BK channels, both total expression and relative isoform expression, have been previously reported, but little work describes the effect of these changes in expression patterns on excitability. Here, we show that early changes in BK channel expression patterns lead to changes in the role of BK channels in determining the action potential waveform and neuronal excitability.

Objective: The amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is increasingly recognized as a therapeutic target in drug-refractory pediatric epilepsy. Perampanel (PER) is a non-competitive AMPAR antagonist, and pre-clinical studies have shown the AMPAR-mediated anticonvulsant effects of decanoic acid (DEC), a major medium-chain fatty acid provided in the medium-chain triglyceride ketogenic diet., Methods: Using brain tissue resected from children with intractable epilepsy, we recorded the effects of PER and DEC in vitro., Results: We found resected pediatric epilepsy tissue exhibits spontaneous epileptic activity in vitro, and showed that DEC and PER inhibit this epileptiform activity in local field potential recordings as well as excitatory synaptic transmission., Interpretation: This study confirms AMPAR antagonists inhibit epileptiform discharges in brain tissue resected in a wide range of pediatric epilepsies., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

The flexibility of neuronal networks is believed to rely mainly on the plasticity of excitatory synapses. However, like their excitatory counterparts, inhibitory synapses also undergo several forms of synaptic plasticity. This review examines recent advances in the understanding of the molecular mechanisms leading to postsynaptic GABAergic plasticity. Specifically, modulation of GABAA receptor (GABAAR) number at postsynaptic sites plays a key role, with the interaction of GABAARs with the scaffold protein gephyrin and other postsynaptic scaffold/regulatory proteins having particular importance. Our understanding of these molecular interactions are progressing, based on recent insights into the processes of GABAAR lateral diffusion, gephyrin dynamics, and gephyrin nanoscale organization. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: The antiepileptic drug candidate, padsevonil, is the first in a novel class of drugs designed to interact with both presynaptic and postsynaptic therapeutic targets: synaptic vesicle 2 proteins and γ-aminobutyric acid type A receptors (GABA A Rs), respectively. Functional aspects of padsevonil at the postsynaptic target, GABA A Rs, were characterized in experiments reported here., Methods: The effect of padsevonil on GABA-mediated Cl - currents was determined by patch clamp on recombinant human GABA A Rs (α1β2γ2) stably expressed in a CHO-K1 cell line and on native GABA A Rs in cultured rat primary cortical neurons. Padsevonil selectivity for GABA A R subtypes was evaluated using a two-electrode voltage clamp on recombinant human GABA A Rs (α1-5/β2/γ2) in Xenopus oocytes., Results: In recombinant GABA A Rs, padsevonil did not evoke Cl - currents in the absence of the agonist GABA. However, when co-administered with GABA at effective concentration (EC) 20 , padsevonil potentiated GABA responses by 167% (EC 50 138 nmol/L) and demonstrated a relative efficacy of 41% compared with zolpidem, a reference benzodiazepine site agonist. Similarly, padsevonil demonstrated GABA-potentiating activity at native GABA A Rs (EC 50 208 nmol/L) in cultured rat cortical neurons. Padsevonil also potentiated GABA (EC 20 ) responses in GABA A Rs expressed in oocytes, with higher potency at α1- and α5-containing receptors (EC 50 295 and 281 nmol/L) than at α2- and α3-containing receptors (EC 50 1737 and 2089 nmol/L). Compared with chlordiazepoxide-a nonselective, full GABA A R agonist-the relative efficacy of padsevonil was 60% for α1β2γ2, 26% for α2β2γ2, 56% for α3β2γ2, and 41% for α5β2γ2; no activity was observed at benzodiazepine-insensitive α4β2γ2 receptors., Significance: Results of functional investigations on recombinant and native neuronal GABA A Rs show that padsevonil acts as a positive allosteric modulator of these receptors, with a partial agonist profile at the benzodiazepine site. These properties may confer better tolerability and lower potential for tolerance development compared with classic benzodiazepines currently used in the clinic., (© 2020 UCB Biopharma SRL. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying K+ channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs) - not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances - not antagonizes - the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.

The amygdala contributes toward emotional processes such as fear, anxiety, and social cognition. Furthermore, evidence suggests that increased excitability of basolateral amygdala (BLA) principal neurons underlie certain neuropsychiatric disorders. Gain-of-function mutations in neuronal L-type calcium channels (LTCCs) are linked to neurodevelopmental diseases, including autism spectrum disorders (ASDs). While LTCCs are expressed throughout the BLA, direct evidence for increased LTCC activity affecting BLA excitability and potentially contributing to disease pathophysiology is lacking. In this study, we utilized a pharmacological approach to examine the contributions of LTCCs to BLA principal cell excitability and synaptic activity at immature (postnatal day 7, P7) and juvenile (P21) developmental stages. Acute upregulation of LTCC activity in brain slices by application of the agonist ( S )-Bay K 8644 resulted in increased intrinsic excitability properties including firing frequency response, plateau potential, and spike-frequency adaptation selectively in P7 neurons. Contrastingly, for P21 neurons, the main effect of ( S )-Bay K 8644 was to enhance burst firing. ( S )-Bay K 8644 increased spontaneous inhibitory synaptic currents at both P7 and P21 but did not affect evoked synaptic currents at either stage. ( S )-Bay K 8644 did not alter P7 spontaneous excitatory synaptic currents, although it increased current amplitude in P21 neurons. Overall, the results provide support for the notion that alteration of LTCC activity at specific periods of early brain development may lead to functional alterations to neuronal network activity and subsequently contribute to underlying mechanisms of amygdala-related neurological disorders. NEW & NOTEWORTHY The role of L-type calcium channels (LTCCs) in regulating neuronal electrophysiological properties during development remains unclear. We show that in basolateral amygdala principal neurons, an increase of LTCC activity alters both neuronal excitability and synaptic activity. The results also provide evidence for the distinct contributions of LTCCs at different stages of neurodevelopment and shed insight into our understanding of LTCC dysfunction in amygdala-related neurological disorders.

PSD-95 is a major scaffolding protein of the post-synaptic density (PSD) of a glutamatergic synapse. PSD-95, via interactions with stargazin, anchors AMPA receptors at the synapse and regulates AMPAR currents. The expression of PSD-95 is regulated during synaptic plasticity. It is, however, unknown whether this regulation is required for induction of functional plasticity of glutamatergic synapses. Here, we show that NMDA-induced long-term depression of synaptic transmission (NMDA-LTD) is accompanied by downregulation of PSD-95 protein levels. Using pharmacologic and molecular manipulations, we further demonstrate that the NMDA-induced downregulation of PSD-95 depends on the activation of CaMKII and CaMKII-driven phosphorylation of PSD-95 serine 73. Surprisingly, neither CaMKII activity nor CaMKII-dependent phosphorylation of PSD-95 serine 73 are required for the expression of NMDA-LTD. These results support the hypothesis that synaptic plasticity of AMPARs may occur without dynamic regulation of PSD-95 protein levels.

Over the last two decades, the discovery of ketamine's antidepressant properties has galvanized research into the neurobiology of treatment-resistant depression. Nevertheless, the mechanism of action underlying antidepressant response to ketamine remains unclear. This study reviews electrophysiological studies of ketamine's effects in individuals with depression as well as healthy controls, with a focus on two putative markers of synaptic potentiation: gamma oscillations and long-term potentiation. The review focuses on: 1) measures of gamma oscillations and power and their relationship to both acute, psychotomimetic drug effects as well as delayed antidepressant response in mood disorders; 2) changes in long-term potentiation as a promising measure of synaptic potentiation following ketamine administration; and 3) recent efforts to model antidepressant response to ketamine using novel computational modeling techniques, in particular the application of dynamic causal modeling to electrophysiological data. The latter promises to better characterize the mechanisms underlying ketamine's antidepressant effects., Competing Interests: Declaration of competing interest Dr. Zarate is listed as a coinventor on a patent for the use of ketamine and its metabolites in major depression and suicidal ideation. Dr. Zarate is listed as a co-inventor on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine, and other stereoisomeric dehydro and hydroxylated metabolites of (R,S)-ketamine metabolites in the treatment of depression and neuropathic pain. Dr. Zarate is listed as co-inventor on a patent application for the use of (2R,6R)-hydroxynorketamine and (2S,6S)-hydroxynorketamine in the treatment of depression, anxiety, anhedonia, suicidal ideation, and post-traumatic stress disorders; he has assigned his patent rights to the U.S. government but will share a percentage of any royalties that may be received by the government. Dr. Gilbert has no conflict of interest to disclose, financial or otherwise., (Published by Elsevier Inc.)

Although migraine is a major global public health problem, its impact on cognitive abilities remains controversial. Thus, the present study investigated the effects of repeated administration of inflammatory soup to the dura of rats, over three weeks, on spatial cognition, hippocampal synaptic plasticity, and the expression of N-methyl-D-aspartate receptor subunits. Additionally, low doses of amitriptyline (5 mg/kg) were applied to assess its therapeutic effects. The inflammatory soup group exhibited significant reductions in the cutaneous stimulation threshold, presence of mild cognitive impairment, and decreased long-term potentiation in right hippocampus. However, amitriptyline improved pain behaviors, enhanced cognitive function, and increased synaptic plasticity in the inflammatory soup rats. On the other hand, the administration of amitriptyline to normal rats negatively influenced synaptic plasticity and reduced the expression of N-methyl-D-aspartate receptor subunits. The present results indicate that inflammatory soup-induced dural nociception led to impairments in spatial cognition that could be attributed to reductions in hippocampal long-term potentiation and the decreased expression of N-methyl-D-aspartate receptor subunits.

The consolidation of long-term memory is influenced by various neuromodulators. One of these is estradiol, a steroid hormone that is synthesized both in peripheral endocrine tissue and in the brain, including the hippocampus. Here, we examine the evidence regarding the role of estradiol in the hippocampus, specifically, in memory formation and its effects on the molecular mechanisms underlying synaptic plasticity. We conclude that estradiol improves memory consolidation and, thereby, long-term memory. Previous studies have shown that it does this in three, interconnected ways: (1) via functional changes in excitatory activity, (2) signaling changes in calcium dynamics, protein phosphorylation and protein expression, and (3) structural changes to synaptic morphology. Through a functional network analysis of proteins affected by estradiol, we identify potential protein-protein interactions that further support a role for estradiol in modulating synaptic plasticity as well as highlight signaling pathways that may be involved in these changes within the hippocampus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier Inc.)

Stress responses are associated with elevations in corticosterone levels and, as a consequence, increases in glutamate in the central nervous system which can lead to neurological impairment. Ceftriaxone promotes glutamate transport and has been used to reduce glutamate toxicity, but so far it is not known whether ceftriaxone is able to reverse the effects of corticosterone administration. Here we describe the separate and combined effects of acute ceftriaxone and acute corticosterone administration in local field potentials (LFPs) recorded from the somatosensory cortex (S1) of anesthetized mice. For this, LFPs were recorded from groups of anesthetized mice injected with saline, corticosterone, ceftriaxone, or both. Comparison of global state maps, and their displacements, as measured by ratios of different frequency bands (Ratio 1: 0.5-20 Hz/0.5-45 Hz; and Ratio 2: 0.5-4.5 Hz/0.5-9 Hz) revealed distinct and opposite effects for corticosterone and for ceftriaxone. Corticosterone specifically increased the displacement in Ratio 2, while ceftriaxone decreased it; in addition, when both corticosterone and ceftriaxone were injected, Ratio 2 displacement values were again similar to those of the control group. The present results suggest that ceftriaxone and corticosterone modulate specific frequency bands in opposite directions and reveal a potential role for ceftriaxone in counteracting the effects of corticosterone.

As the inhibitory γ-aminobutyric acid-ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In fact, the ultimate need for specificity is critical for the proper synaptic exploration. No caged allosteric modulators of the GABA A receptor have been reported so far; to introduce such an investigational approach, we exploited the structural motifs of the benzodiazepinic scaffold to develop a photocaged version of diazepam (CD) that was tested on basolateral amygdala (BLa) pyramidal cells in mouse brain slices. CD is devoid of any intrinsic activity toward the GABA A receptor before irradiation. Importantly, CD is a photoreleasable GABA A receptor-positive allosteric modulator that offers a different probing mechanism compared to caged GABA and photoswitchable ligands. CD potentiates the inhibitory signaling by prolonging the decay time of postsynaptic GABAergic currents upon photoactivation. Additionally, no effect on presynaptic GABA release was recorded. We developed a photochemical technology to individually study the GABA A receptor, which specifically expands the toolbox available to study GABAergic synapses., Competing Interests: The authors declare no competing interest.

The microtubule network represents a key scaffolding structure that forms part of the neuronal cytoskeleton and contributes to biomolecule exchange within neurons. However, researchers have not determined whether an intact microtubule network is required for late associative plasticity. Therefore, the late associative plasticity of field excitatory postsynaptic potentials from two synaptic inputs was analyzed. Synaptic potentiation was induced through alternating tetanization of hippocampal Schaffer-collateral CA1 synaptic populations in acute slices prepared from young-adult C57BL/6 mice. Vincristine was applied to depolymerize microtubules. Vincristine did not alter the phosphorylation levels of plasticity-related pre- or postsynaptic proteins but reduced the level of a protein marker of the ER-Golgi intermediate compartment (ERGIC-53/p58). Vincristine did not alter the magnitude or maintenance of the synaptic potentiation evoked by repeated tetanization (3 × 100 stimuli at 100 Hz) of one synaptic population. However, this synaptic potentiation was sensitive to the coapplication of a protein synthesis inhibitor, such as rapamycin, anisomycin or cycloheximide, indicating that protein synthesis has become essential in depolymerized microtubules during the first hour of the synaptic potentiation. The application of vincristine up to a 70 stimuli, 100 Hz tetanization of a second synaptic input prevented the transformation of short-term potentiation into long-term potentiation (LTP), further indicating that intact microtubules are required for the late associative properties of synaptic plasticity. Therefore, activity-dependent synaptic plasticity does not rely on microtubules within the first two hours after tetanization; however, the associative interaction of independent synaptic inputs relies on their proper function. In addition, either new protein synthesis or microtubule-based processes are sufficient to stabilize LTP within the first 3 h after tetanization, and a deficit in synaptic plasticity is only observable when both processes are blocked., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, exerts rapid antidepressant effects in human patients and ameliorates depressive-like behavioral effects of chronic stress in animal models. Chronic stress and elevated corticosterone levels have been shown to modify serotonin (5-HT) neurotransmission, and ketamine's antidepressant-like activity involves a 5-HT-dependent mechanism. However, it is not known if and how ketamine affects the electrophysiological characteristics of neurons and synaptic transmission within the dorsal raphe nucleus (DRN), the main source of 5-HT forebrain projections. Our study was aimed at investigating the effects of a single ketamine administration on excitatory and inhibitory transmission in the DRN of rats which had previously been administered corticosterone twice daily for 7 days. Spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) were then recorded from DRN projection cells in ex vivo slice preparations obtained 24 h after ketamine injection. Repeated corticosterone administration increased sEPSC frequency and decreased sIPSC frequency in DRN projection cells. There were no changes either in the amplitude of postsynaptic currents or in the excitability of these cells. In slices prepared from rats with ketamine administered after the end of corticosterone treatment, the frequencies of sEPSCs and sIPSCs were similar to those in control preparations. These data indicate that a single administration of ketamine reversed the effects of corticosterone on excitatory and inhibitory transmission in the DRN., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Adenosine A 1 receptors (A 1 AR) are well characterized for their role in cytoprotection. Previous studies have demonstrated the presence of these receptors in the cochlea where their activation were shown to suppress cisplatin-induced inflammatory response and the resulting ototoxicity. Inhibition of A 1 AR by caffeine, a widely consumed psychoactive substance, could antagonize the endogenous protective role of these receptors in cochlea and potentiate cisplatin-induced hearing loss. This hypothesis was tested in a rat model of cisplatin ototoxicity following oral administration of caffeine. We report here that single-dose administration of caffeine exacerbates cisplatin-induced hearing loss without increasing the damage to outer hair cells (OHCs), but increased synaptopathy and inflammation in the cochlea. These effects of caffeine were mediated by its blockade of A 1 AR, as co-administration of R-PIA, an A 1 AR agonist, reversed the detrimental actions of caffeine and cisplatin on hearing loss. Multiple doses of caffeine exacerbated cisplatin ototoxicity which was associated with damage to OHCs and cochlear synaptopathy. These findings highlight a possible drug-drug interaction between caffeine and cisplatin for ototoxicity and suggest that caffeine consumption should be cautioned in cancer patients treated with a chemotherapeutic regimen containing cisplatin.

Tonic inhibitory currents, mediated by extrasynaptic GABA A receptors, are elevated at a delay following stroke. Flavonoids minimise the extent of cellular damage following stroke, but little is known about their mode of action. We demonstrate that the flavonoid, 2'-methoxy-6-methylflavone (0.1-10 µM; 2'MeO6MF), increases GABA A receptor tonic currents presumably via δ-containing GABA A receptors. Treatment with 2'MeO6MF 1-6 h post focal ischaemia dose dependently decreases infarct volume and improves functional recovery. The effect of 2'MeO6MF was attenuated in δ -/- mice, indicating that the effects of the flavonoid were mediated via δ-containing GABA A receptors. Further, as flavonoids have been shown to have multiple modes of action, we investigated the anti-inflammatory effects of 2'MeO6MF. Using a macrophage cell line, we show that 2'MeO6MF can dampen an LPS-induced elevation in NFkB activity. Assessment of vehicle-treated stroke animals revealed a significant increase in circulating IL1β, TNFα and IFγ levels. Treatment with 2'MeO6MF dampened the stroke-induced increase in circulating cytokines, which was blocked in the presence of the pan-AKT inhibitor, GSK690693. These studies support the hypothesis that compounds that potentiate tonic inhibition via δ-containing GABA A receptors soon after stroke can afford neuroprotection.

Inflammation inhibits the expression of some, but not all forms of respiratory motor plasticity. For example, systemic application of lipopolysaccharide (LPS) inhibits the phrenic long-term facilitation induced by moderate-intermittent hypoxia in vivo. There are multiple pro-inflammatory processes triggered by the systemic application of LPS, including neuroinflammation in the CNS. Considering that microglia can be activated by the systemic application of LPS, it is likely that this cell type influences the response of the respiratory circuits to intermittent hypoxia (IH). Thus, we aimed to test whether modulators of microglial function would affect the response to IH of the preBötzinger complex (preBötC) isolated in a brainstem slice preparation. This experimental approach avoids the systemic influences of these microglial modulators and limits their effects on cells, mostly microglia, included in the slice. First, we found that IH (3 × 5-min episodes of bubbling with 95% N 2 and 5% CO 2 , mixed with 5-min normoxic intervals by bubbling with 95% O 2 and 5% CO 2 ) induces a long-lasting increase in the respiratory rhythm frequency recorded directly from the preBötC, called in vitro long-term facilitation (LTF), which occurs simultaneously with a long-lasting decrease in burst amplitude. Moreover, we found that bath applications of "microglial activators" (LPS and fractalkine), "microglial inhibitors" (minocycline and fucoidan) and a microgliotoxin (liposomal clodronate) partially reduce in vitro LTF. These findings reveal a complex scenario in which both the activation and the inhibition of microglia halts IH-induced preBötC plasticity and suggest that experimental or pathological conditions that affect this cell type, almost in any way, could affect breathing and its plastic responses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Understanding the neurobiological processes that incite drug craving and drive relapse has the potential to help target efforts to treat addiction. The NAc serves as a critical substrate for reward and motivated behavior, in part due to alterations in excitatory synaptic strength within cortical-accumbens pathways. The present studies investigated a causal link between cocaine-induced reinstatement of conditioned place preference and rapid reductions of cocaine-dependent increases in NAc shell synaptic strength in male mice. Cocaine-conditioned place preference behavior and ex vivo whole-cell electrophysiology showed that cocaine-primed reinstatement and synaptic depotentiation were disrupted by inhibiting AMPAR internalization via intra-NAc shell infusion of a Tat-GluA2 3Y peptide. Furthermore, reinstatement was driven by an mGluR5-dependent reduction in AMPAR signaling. Intra-NAc shell infusion of the mGluR5 antagonist MTEP blocked cocaine-primed reinstatement and corresponding depotentiation, whereas infusion of the mGluR5 agonist CHPG itself promoted reinstatement and depotentiated synaptic strength in the NAc shell. Optogenetic examination of circuit-specific plasticity showed that inhibition of infralimbic cortical input to the NAc shell blocked cocaine-primed reinstatement, whereas low-frequency stimulation (10 Hz) of this pathway in the absence of cocaine triggered a reduction in synaptic strength akin to that observed with cocaine, and was sufficient to promote reinstatement in the absence of a cocaine challenge. These data support a model in which mGluR5-mediated reduction in GluA2-containing AMPARs at NAc shell synapses receiving input from the infralimbic cortex is a critical factor in triggering reinstatement of cocaine-primed conditioned approach behavior. SIGNIFICANCE STATEMENT These studies identified a sequence of neural events whereby reexposure to cocaine activates a signaling cascade that alters synaptic strength in the NAc shell and triggers a behavioral response driven by a drug-associated memory., (Copyright © 2019 the authors.)

Electroencephalogram monitoring during propofol (PRO) anesthesia typically features low-frequency oscillations, which may be involved with thalamic reticular nucleus (TRN) modulation. TRN receives noradrenergic inputs from the locus coeruleus (LC). We hypothesized that specific noradrenergic connections in the TRN may contribute to the emergence from PRO anesthesia. Intranuclei norepinephrine (NE) injections ( n = 10) and designer receptors exclusively activated by designer drugs (DREADDs) ( n = 10) were used to investigate the role of noradrenergic inputs from the LC to the TRN during PRO anesthesia. Whole-cell recording in acute brain slice preparations was used to identify the type of adrenoceptor that regulates noradrenergic innervation in the TRN. An intracerebral injection of NE into the TRN delays arousal in mice recovering from PRO anesthesia (means ± sd; 486.6 ± 57.32 s for the NE injection group vs. 422.4 ± 48.19 s for the control group; P = 0.0143) and increases the cortical-δ (0.1-4 Hz, 25.4 ± 2.9 for the NE injection group vs. 21.0 ± 1.7 for the control group; P = 0.0094) oscillation. An intra-TRN injection of NE also decreased the EC 50 of PRO-induced unconsciousness (57.05 ± 1.78 mg/kg for the NE injection group vs. 72.44 ± 3.23 mg/kg for the control group; P = 0.0096). Moreover, the activation of LC-noradrenergic nerve terminals in the TRN using DREADDs increased the recovery time [466.1 ± 44.57 s for the clozapine N-oxide (CNO) injection group vs. 426.1 ± 38.75 s for the control group; P = 0.0033], decreased the EC 50 of PRO-induced unconsciousness (64.77 ± 3.40 mg/kg for the CNO injection group vs. 74.00 ± 2.08 mg/kg for the control group; P = 0.0081), and increased the cortical-δ oscillation during PRO anesthesia (23.29 ± 2.58 for the CNO injection group vs. 19.56 ± 1.9 for the control group; P = 0.0213). In addition, whole-cell recording revealed that NE augmented the inhibitory postsynaptic currents in the TRN neurons via the α1-adrenoceptor. Our data indicated that enhanced NE signaling at the noradrenergic terminals of the LC-TRN projection delays arousal from general anesthesia, which is likely mediated by the α1-adrenoceptor activation. Our findings open a door for further understanding of the functions of various LC targets in both anesthesia and arousal.-Zhang, Y., Fu, B., Liu, C., Yu, S., Luo, T., Zhang, L., Zhou, W., Yu, T. Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

The transient receptor potential vanilloid 4 (TRPV4) channel may be opened by mechanical stimuli to mediate Ca 2+ and Na + influxes, and it has been suggested to mediate glaucoma retinopathy. However, it has been mostly unclear how TRPV4 activities affect the function of primate retinal ganglion cells (RGCs). We studied RGCs and bipolar cells (BCs) in the peripheral retina of the old-world primate using whole-cell current-clamp and voltage-clamp recordings, immunomarkers and confocal microscopy. RGCs were distinguished from displaced amacrine cells (ACs) by the absence of GABA and glycine immunoreactivity and possession of an axon and a large soma in the RGC layer. Strong TRPV4 signal was concentrated in medium to large somas of RGCs, and some TRPV4 signal was found in BCs (including PKCα-positive rod BCs), as well as the end feet, soma and outer processes of Mȕller cells. TRPV4 immunoreactivity quantified by the pixel intensity histogram revealed a high-intensity component for the plexiform layers, a low-intensity component for the soma layers of ACs and Mȕller cells, and both components in the soma layers of RGCs and BCs. In large RGCs, TRPV4 agonists 4α-phorbol 12,13 didecanoate (4αPDD) and GSK1016790A reversibly enhanced the spontaneous firing and shortened the delay of voltage-gated Na + (Nav) currents under current-clamp conditions, and under voltage-clamp conditions, 4αPDD largely reversibly increased the amplitude and frequency of spontaneous excitatory postsynaptic currents. In BCs, changes in the membrane tension induced by either applying pressure or releasing the pressure both activated a transient cation current, which reversed at ~ -10 mV and was enhanced by heating from 24 °C to 30 °C. The pressure for the half-maximal effect was ~18 mmHg. These data indicate that functional TRPV4 channels are variably expressed in primate RGCs and BCs, possibly contributing to pressure-related changes in RGCs in glaucoma.

A growing body of literature has demonstrated the potential for ketamine in the treatment of major depression. Sub-anesthetic doses produce rapid and sustained changes in depressive behavior, both in patients and rodent models, associated with reorganization of glutamatergic synapses in the prefrontal cortex (PFC). While ketamine is known to regulate N-methyl-D-aspartate (NMDA) -type glutamate receptors (NMDARs), the full complement of downstream cellular consequences for ketamine administration are not well understood. Here, we combine electrophysiology with 2-photon imaging and glutamate uncaging in acute slices of mouse PFC to further examine how ketamine alters glutamatergic synaptic transmission. We find that four hours after ketamine treatment, glutamatergic synapses themselves are not significantly affected. However, levels of the neuromodulatory Regulator of G-protein Signaling (RGS4) are dramatically reduced. This loss of RGS4 activity is associated with disruption of the normal compartmentalization of synaptic neuromodulation. Thus, under control conditions, α2 adrenergic receptors and type B γ-aminobutyric acid (GABAB) receptors selectively inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type glutamate receptors (AMPARs) and NMDARs, respectively. After ketamine administration and reduction in RGS4 activity, this selectivity is lost, with both modulatory systems broadly inhibiting glutamatergic transmission. These results suggest a novel mechanism by which ketamine may influence synaptic signaling and provide new avenues for the exploration of therapeutics directed at treating neuropsychiatric disorders, such as depression., Competing Interests: The authors have declared that no competing interests exist.

Acute opioid withdrawal syndrome is a series of neurological symptoms caused by the abrupt cessation of the chronic administration of opioids such as morphine. The locus coeruleus (LC) in the brain stem receives a dense projection of orexinergic fibers from the hypothalamus and is a candidate site for the expression of the somatic aspects of morphine withdrawal. Previous studies have shown that orexin-A contributes to the behavioral symptoms of naloxone-induced morphine withdrawal, partly by reducing the activity of GABAergic neurons, suggesting that orexin-A may negatively modulate fast GABAergic neurotransmission during morphine withdrawal. We used whole-cell patch-clamp recordings of LC neurons in brainstem slices to investigate the effect of orexin-A on bicuculline-sensitive GABAergic inhibitory postsynaptic currents (IPSCs) during naloxone-induced morphine withdrawal. Male Wistar rats (P14-P21) were given morphine (20 mg/kg, i.p.) daily for seven consecutive days to create dependency on the drug. The application of naloxone (1 µM) to brain slices of morphine-treated rats reduced the amplitude of evoked IPSCs (eIPSCs) as well as spontaneous IPSCs (sIPSCs) frequency but did not change sIPSCs amplitude. Orexin-A (100 nM) significantly enhanced the suppressive effect of naloxone on eIPSCs amplitude and sIPSCs frequency but had no effect on the presence of the orexin type 1 receptor (OX1R) antagonist, SB-334867. Orexin-A alone had no significant effect on eIPSCs and sIPSCs in the absence of naloxone. In summary, our results show that orexin-A, via OX1R, potentiates the suppressive effect of naloxone on GABAergic IPSCs of LC neurons in morphine-treated rats. We conclude that orexins may have a critical role in regulating GABAergic neurotransmission to LC neurons during naloxone-induced morphine withdrawal.

Preclinical and clinical evidence has demonstrated that ketamine has rapid antidepressant effects. Studies using pre-treatment with an AMPA inhibitor suggest that enhancing AMPA throughput is crucial to ketamine's effects, including increases in both basal and evoked gamma power. This study sought to replicate previous findings of increased gamma response to a somatosensory stimulus at 230 min and Day 1 in ketamine responders versus non-responders in 31 depressed subjects and 25 healthy controls. A significant difference in peak gamma power was seen in the depressed ketamine responders versus non-responders. These results implicate AMPA throughput in ketamine's mechanism of antidepressant action., (Copyright © 2018. Published by Elsevier B.V.)

Preclinical and clinical research has shown that females are more vulnerable to the rewarding effects of stimulants, and it has been proposed that estrogens may play a role in this enhanced sensitivity; however sex differences in methamphetamine (METH)-induced neuroplasticity have not been explored. To address this gap in knowledge, we recorded from the prelimbic area of the prefrontal cortex (PL-PFC) of male and female rats following long access METH self-administration (SA) and investigated the resulting long-term synaptic neuroadaptations. Males and females took similar amounts of METH during SA; however, female rats exhibit significant synaptic baseline differences when compared to males. Furthermore, females exhibited a significant increase in evoked excitatory currents. This increase in evoked glutamate was correlated with increases in NMDA currents and was not affected by application of a GluN2B selective blocker. We propose that METH SA selectively upregulates GluN2B-lacking NMDA receptors (NMDAR) in the PFC of female rats. Our results may provide a mechanistic explanation for the sex differences reported for METH addiction in females., Competing Interests: The authors do not have any conflict of interest to report.

The nucleus accumbens (NAc) is critical for motivated behavior and is rewired following exposure to drugs of abuse. Medium spiny neurons (MSNs) in the NAc express either D1 or D2 receptors and project to distinct downstream targets. Differential activation of these MSNs depends on both excitation from long-range inputs and inhibition via the local circuit. Assessing how long-range excitatory inputs engage inhibitory circuitry is therefore important for understanding NAc function. Here, we use slice electrophysiology and optogenetics to study ventral hippocampal (vHPC)-evoked feedforward inhibition in the NAc of male and female mice. We find that vHPC-evoked excitation is stronger at D1+ than D1- MSNs, whereas inhibition is unbiased at the two cell types. vHPC inputs contact both parvalbumin-positive (PV+) and somatostatin-positive (SOM+) interneurons, but PV+ cells are preferentially activated. Moreover, suppressing PV+ interneurons indicates they are primarily responsible for vHPC-evoked inhibition. Finally, repeated cocaine exposure alters the excitation of D1+ and D1- MSNs, without concomitant changes to inhibition, shifting the excitation/inhibition balance. Together, our results highlight the contributions of multiple interneuron populations to feedforward inhibition in the NAc. Moreover, they demonstrate that inhibition provides a stable backdrop on which drug-evoked changes to excitation occur within this circuit. SIGNIFICANCE STATEMENT Given the importance of the nucleus accumbens (NAc) in reward learning and drug-seeking behaviors, it is critical to understand what controls the activity of cells in this region. While excitatory inputs to projection neurons in the NAc have been identified, it is unclear how the local inhibitory network becomes engaged. Here, we identify a sparse population of interneurons responsible for feedforward inhibition evoked by ventral hippocampal input and characterize their connections within the NAc. We also demonstrate that the balance of excitation and inhibition that projection neurons experience is altered by exposure to cocaine. Together, this work provides insight into the fundamental circuitry of this region as well as the effects of drugs of abuse., (Copyright © 2018 the authors 0270-6474/18/389091-14$15.00/0.)

Continuous (tonic) charge transfer through ionotropic receptors of γ-aminobutyric acid (GABA A Rs) is an important mechanism of inhibitory signalling in the brain. The conventional view has been that tonic GABA-ergic inhibitory currents are mediated by low concentrations of ambient GABA. Recently, however, it was shown that the GABA-independent, spontaneously opening GABA A Rs (s-GABA A Rs), may contribute significantly to the tonic GABA A R current. One of the common approaches to temporal lobe epilepsy (TLE) therapy is an increase of GABA concentration in the cerebrospinal fluid to augment tonic current through GABA A Rs. Such an increase, however, generates multiple side effects, which impose significant limitations on the use of correspondent drugs. In contrast, activation/deactivation of s-GABA A Rs in a GABA-independent manner may provide a mechanism of regulation of tonic conductance without modification of extracellular GABA concentration, thus avoiding connected side effects. Although s-GABA A Rs have been detected in our earlier work, it is unclear whether they modulate neural signalling, or, due to their independence from the neurotransmitter, they provide just a stable background effect without much impact on neural crosstalk dynamics. Here, we focused on the causal relationship between s-GABA A R activity and signal integration in the rat's dentate gyrus granule cells to find that s-GABA A Rs play an important role in neural signal transduction. s-GABA A Rs shape the dynamics of phasic inhibitory responses, regulate the action potential generation machinery and control the coincidence detection window pertinent to excitatory input summation. Our results demonstrate that tonic inhibition delivered by s-GABA A Rs contributes to the key mechanisms that ensure implementation of neural signal filtering and integration, in a GABA-independent manner. This makes s-GABA A R a new and important actor in the regulation of long-term neural plasticity and a perspective target for TLE therapy.

Encoding of spatial information in the superficial layers of the medial entorhinal cortex (sMEC) involves theta-modulated spiking and gamma oscillations, as well as spatially tuned grid cells and border cells. Little is known about the role of the arousal-promoting histaminergic system in the modification of information encoded in the sMEC in vivo, and how such histamine-regulated information correlates with behavioral functions. Here, we show that histamine upregulates the neural excitability of a significant proportion of neurons (16.32%, 39.18%, and 52.94% at 30 μM, 300 μM, and 3 mM, respectively) and increases local theta (4-12 Hz) and gamma power (low: 25-48 Hz; high: 60-120 Hz) in the sMEC, through activation of histamine receptor types 1 and 3. During spatial exploration, the strength of theta-modulated firing of putative principal neurons and high gamma oscillations is enhanced about 2-fold by histamine. The histamine-mediated increase of theta phase-locking of spikes and high gamma power is consistent with successful spatial recognition. These results, for the first time, reveal possible mechanisms involving the arousal-promoting histaminergic system in the modulation of spatial cognition.

Mature granule cells are poorly excitable neurons that were recently shown to fire action potentials, preferentially in bursts. It is believed that the particularly pronounced short-term facilitation of mossy fiber synapses makes granule cell bursting a very effective means of properly transferring information to CA3. However, the mechanism underlying the unique bursting behavior of mature granule cells is currently unknown. Here, we show that Cav3.2 T-type channels at the axon initial segment are responsible for burst firing of mature granule cells in rats and mice. Accordingly, Cav3.2 knockout mice fire tonic spikes and exhibit impaired bursting, synaptic plasticity and dentate-to-CA3 communication. The data show that Cav3.2 channels are strong modulators of bursting and can be considered a critical molecular switch that enables effective information transfer from mature granule cells to the CA3 pyramids.

Astrocytes respond to neuronal activity and were shown to be necessary for plasticity and memory. To test whether astrocytic activity is also sufficient to generate synaptic potentiation and enhance memory, we expressed the Gq-coupled receptor hM3Dq in CA1 astrocytes, allowing their activation by a designer drug. We discovered that astrocytic activation is not only necessary for synaptic plasticity, but also sufficient to induce NMDA-dependent de novo long-term potentiation in the hippocampus that persisted after astrocytic activation ceased. In vivo, astrocytic activation enhanced memory allocation; i.e., it increased neuronal activity in a task-specific way only when coupled with learning, but not in home-caged mice. Furthermore, astrocytic activation using either a chemogenetic or an optogenetic tool during acquisition resulted in memory recall enhancement on the following day. Conversely, directly increasing neuronal activity resulted in dramatic memory impairment. Our findings that astrocytes induce plasticity and enhance memory may have important clinical implications for cognitive augmentation treatments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Leptin is an adipose-derived hormone that controls appetite and energy expenditure. Leptin receptors are expressed on extra-hypothalamic ventrobasal (VB) and reticular thalamic (RTN) nuclei from embryonic stages. Here, we studied the effects of pressure-puff, local application of leptin on both synaptic transmission and action potential properties of thalamic neurons in thalamocortical slices. We used whole-cell patch-clamp recordings of thalamocortical VB neurons from wild-type (WT) and leptin-deficient obese (ob/ob) mice. We observed differences in VB neurons action potentials and synaptic currents kinetics when comparing WT vs. ob/ob. Leptin reduced GABA release onto VB neurons throughout the activation of a JAK2-dependent pathway, without affecting excitatory glutamate transmission. We observed a rapid and reversible reduction by leptin of the number of action potentials of VB neurons via the activation of large conductance Ca 2+ -dependent potassium channels. These leptin effects were observed in thalamocortical slices from up to 5-week-old WT but not in leptin-deficient obese mice. Results described here suggest the existence of a leptin-mediated trophic modulation of thalamocortical excitability during postnatal development. These findings could contribute to a better understanding of leptin within the thalamocortical system and sleep deficits in obesity.

Hypofunction of the serotonergic (5-HT) system has close relationship with the symptoms in major depressive disorders (MDD), however, the underlying neural circuitry mechanisms are not fully understood. Lateral habenula (LHb) plays a crucial role in aversive behaviors and is activated in conditions of depression. It has been reported that 5-HT inhibits the excitability of LHb neurons, leading to the hypothesis that decreased transmission of 5-HT would elevate the activity of LHb and therefore mediates depressive symptoms. Using retrograde tract tracing with cholera toxin subunit B, we find that dorsal raphe nucleus (DRN) sends primary 5-HT projection to the LHb. In vitro slice patch-clamp recording reveals that opto-stimulation of DRN inputs to the LHb suppresses the frequency of miniature excitatory postsynaptic current, while increases paired pulse ratio in LHb neurons, indicating 5-HT projection presynaptically suppresses the excitability of LHb neurons. In chronic unpredictable mild stress (CUMS) rat model of depression, optogenetic stimulation of DRN-LHb projection alleviates the depressive symptoms in CUMS models. Meanwhile, opto-inhibition of this circuit results in elevated c-fos expression in LHb and induces depression-like behaviors. This study demonstrates that the 5-HT projection from DRN to LHb suppresses the excitability of LHb neurons, and hypofunction of 5-HT transmission induces depressive behavior via the activation of LHb. Our results reveal the functional connectivity of DRN-LHb circuit and its antidepressant action, which may provide a novel target for the treatment of depression.

Neuronal inclusions composed of α-synuclein (α-syn) characterize Parkinson's Disease (PD) and Dementia with Lewy bodies (DLB). Cognitive dysfunction defines DLB, and up to 80% of PD patients develop dementia. α-Syn inclusions are abundant in the hippocampus, yet functional consequences are unclear. To determine if pathologic α-syn causes neuronal defects, we induced endogenous α-syn to form inclusions resembling those found in diseased brains by treating hippocampal neurons with α-syn fibrils. At seven days after adding fibrils, α-syn inclusions are abundant in axons, but there is no cell death at this time point, allowing us to assess for potential alterations in neuronal function that are not caused by neuron death. We found that exposure of neurons to fibrils caused a significant reduction in mushroom spine densities, adding to the growing body of literature showing that altered spine morphology is a major pathologic phenotype in synucleinopathies. The reduction in spine densities occurred only in wild type neurons and not in neurons from α-syn knockout mice, suggesting that the changes in spine morphology result from fibril-induced corruption of endogenously expressed α-syn. Paradoxically, reduced postsynaptic spine density was accompanied by increased frequency of miniature excitatory postsynaptic currents (EPSCs) and presynaptic docked vesicles, suggesting enhanced presynaptic function. Action-potential dependent activity was unchanged, suggesting compensatory mechanisms responding to synaptic defects. Although activity at the level of the synapse was unchanged, neurons exposed to α-syn fibrils, showed reduced frequency and amplitudes of spontaneous Ca 2+ transients. These findings open areas of research to determine the mechanisms that alter neuronal function in brain regions critical for cognition at time points before neuron death.

Excessive alcohol consumption in humans induces deficits in decision making and emotional processing, which indicates a dysfunction of the prefrontal cortex (PFC). The present study aimed to determine the impact of chronic intermittent ethanol (CIE) inhalation on mouse medial PFC pyramidal neurons. Data were collected 6-8 days into withdrawal from 7 weeks of CIE exposure, a time point when mice exhibit behavioral symptoms of withdrawal. We found that spine maturity in prelimbic (PL) layer 2/3 neurons was increased, while dendritic spines in PL layer 5 neurons or infralimbic (IL) neurons were not affected. Corroborating these morphological observations, CIE enhanced glutamatergic transmission in PL layer 2/3 pyramidal neurons, but not IL layer 2/3 neurons. Contrary to our predictions, these cellular alterations were associated with improved, rather than impaired, performance in reversal learning and strategy switching tasks in the Barnes maze at an earlier stage of chronic ethanol exposure (5-7 days withdrawal from 3 to 4 weeks of CIE), which could result from the anxiety-like behavior associated with ethanol withdrawal. Altogether, this study adds to a growing body of literature indicating that glutamatergic activity in the PFC is upregulated following chronic ethanol exposure, and identifies PL layer 2/3 pyramidal neurons as a sensitive target of synaptic remodeling. It also indicates that the Barnes maze is not suitable to detect deficits in cognitive flexibility in CIE-withdrawn mice., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Drug addiction is a brain disorder characterized by chronic, compulsive use of drugs. Previous studies have found a number of chronic morphine-induced changes in the brain at molecular levels. A study from our lab showed that chronic morphine-induced increase in the expression of presynaptic D1 receptors in basolateral amygdala (BLA) neurons played an important role in environmental cue-induced retrieval of morphine withdrawal memory. However, the downstream neurocircuitry of chronic morphine-induced increase presynaptic D1 receptors in the BLA remains to be elucidated. Using retrogradely labelling technique combined with whole-cell patch-clamp methods, our results showed that (1) chronic morphine sensitized the effect of D1 receptor agonist on presynaptic glutamate release in BLA neurons that projected to the prelimbic cortex (PrL), but had no influence on that in BLA neurons that projected to the nucleus accumbens (NAc) or the CA1 of the hippocampus; (2) chronic morphine sensitized the effect of D1 receptor agonist on action potential firing in BLA neurons that projected to the PrL, but without affecting the intrinsic excitability and the sensitivity of postsynaptic glutamate receptors to glutamate in BLA neurons that projected to the PrL. These results suggest that chronic morphine selectively sensitizes the effect of D1 receptor agonist on presynaptic glutamate release in BLA neurons that project to PrL and induces a sensitization of the effect of D1 receptor agonist on action potential firing in BLA neurons that project to the PrL., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in spinal functions like locomotion. In Xenopus laevis tadpoles at two days old, a model vertebrate for motor control research, we investigated the role of mAChRs in the skin mechanosensory pathway. We found that mAChR activation by carbachol did not affect the sensory Rohon-Beard neuron properties. However, carbachol could hyperpolarise sensory interneurons and decrease their voltage responses to outward currents. Carbachol could increase the threshold for the mechanosensory pathway to start swimming, preventing the initiation of swimming at higher concentrations altogether. Recording from the sensory interneurons in carbachol showed that their spiking after skin stimulation was depressed. However, the general muscarinic antagonist atropine did not have a clear effect on the swimming threshold or the modulation of sensory interneuron membrane conductance. Our results suggest the skin mechanosensory pathway may be subject to muscarinic modulation in this simple vertebrate system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The central nucleus of the amygdala (CeA) is critical in the regulation of sodium appetite. Angiotensin II (Ang II) is important in the generation of sodium appetite and may function as a neurotransmitter or modulator to affect the synaptic transmission and the excitability of neurons. However, the role of Ang II in the CeA remains unclear. In this study, we determined the effects of Ang II on the excitatory and inhibitory synaptic inputs to the CeA neurons in brain slices with whole-cell patch-clamp recordings. Ang II (0.5-5 μM) significantly potentiated the amplitude of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) in a concentration-dependent manner. Ang II (2 μM) significantly increased the amplitude of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) without affecting the frequency. This effect was blocked by Ang II type 1 (AT 1 ) receptor antagonist, losartan. One mM guanosine 5'-O-(-2-thiodiphosphate) (GDP-β-s) in the pipette internal solution eliminated the facilitatory effect of Ang II on GABAergic synaptic transmission. In contrast, Ang II had no effect on the spontaneous glutamatergic excitatory postsynaptic currents (EPSCs) and did not alter the frequency and amplitude of miniature EPSCs at concentrations that facilitated IPSCs. Furthermore, Ang II decreased the firing activity of CeA neurons, and this effect was abolished by losartan and GDP-β-s. In addition, Ang II failed to inhibit CeA neurons in the presence of bicuculline. These data provide substantial new evidence that Ang II inhibits the CeA neurons by facilitation of GABAergic synaptic input efficacy through activation of postsynaptic AT 1 receptors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)