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Rationale: Neurosteroids and likely other lipid modulators access transmembrane sites on the GABA receptor (GABAR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABARs. Objectives: In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine. Methods: We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion. Results: We found no evidence that PIP2 depletion affected modulation of GABARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity. Conclusions: We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABARs by neurosteroids or related compounds. [ABSTRACT FROM AUTHOR]

Alcoholism is associated with acute and long-term cognitive dysfunction including memory impairment, resulting in substantial disability and cost to society. Thus, understanding how ethanol impairs cognition is essential for developing treatment strategies to dampen its adverse impact. Memory processing is thought to involve persistent, use-dependent changes in synaptic transmission, and ethanol alters the activity of multiple signaling molecules involved in synaptic processing, including modulation of the glutamate and gamma-aminobutyric acid (GABA) transmitter systems that mediate most fast excitatory and inhibitory transmission in the brain. Effects on glutamate and GABA receptors contribute to ethanolinduced changes in long-term potentiation (LTP) and long-term depression (LTD), forms of synaptic plasticity thought to underlie memory acquisition. In this paper, we review the effects of ethanol on learning-related forms of synaptic plasticity with emphasis on changes observed in the hippocampus, a brain region that is critical for encoding contextual and episodic memories. We also include studies in other brain regions as they pertain to altered cognitive and mental function. Comparison of effects in the hippocampus to other brain regions is instructive for understanding the complexities of ethanol's acute and long-term pharmacological consequences. [ABSTRACT FROM AUTHOR]

Synapses represent the main junctures of communication between neurons in the nervous system. In many neurotransmitter systems, a fraction of presynaptic terminals fails to release vesicles in response to action potential stimulation and strong calcium influx. These silent presynaptic terminals exhibit a reversible functional dormancy beyond low vesicle release probability, and dormancy status may have important implications in neural function. Recent advances have implicated presynaptic proteins interacting with vesicles downstream of cAMP and protein kinase A signaling cascades in modulating the number of these mute presynaptic terminals, and dormancy induction may represent a homeostatic neuroprotective mechanism active during pathological insults involving excitotoxicity. Interestingly, dormancy reversal may also be induced during Hebbian plasticity. Here, details of synaptic dormancy, recent insights into the molecular signaling cascades involved, and potential clinical and mechanistic implications of this form of synaptic plasticity are described. [ABSTRACT FROM PUBLISHER]

Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABAA receptor with maximum efficacy and potency similar to clinically used GABAA receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABAA receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABAA receptor function. Even dyes with no activity on GABAA receptors at baseline induced photodynamic enhancement of GABAA receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltagesensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity. [ABSTRACT FROM AUTHOR]

Older studies suggest that astrocytes act as potassium electrodes and depolarize with the potassium efflux accompanying neuronal activity. Newer studies suggest that astrocytes depolarize in response to neuronal glutamate release and the activity of electrogenic glial glutamate transporters, thus casting doubt on the fidelity with which astrocytes might sense extracellular potassium rises. Any K+-induced astrocyte depolarization might reflect a spatial buffering effect of astrocytes during neuronal activity. For these reasons, we studied stimulus-evoked currents in hippocampal CA1 astrocytes. Hippocampal astrocytes exhibited stimulus-evoked transient glutamate transporter currents and slower Ba2+-sensitive inward rectifier potassium (Kir) currents. In whole-cell astrocyte recordings, Ba2+ blocked a very weakly rectifying component of the astrocyte membrane conductance. The slow stimulus-elicited current, like measurements from K+-sensitive electrodes under the same conditions, predicted small bulk [K+]o increases (<0.5 mM) following the termination of short-stimulus trains. These currents indicate the potential for astrocyte spatial K+ buffering. However, Ba2+ did not significantly affect resting [K+]o or the [K+]o rises detected by the K+-sensitive electrode. To test whether local K+ rises may be significantly higher than those detected by glial recordings or by K+ electrodes, we assayed EPSCs and fiber volleys, two measures very sensitive to K+ increases. We found that Ba2+ had little effect on neuronal axonal or synaptic function during short-stimulus trains, indicating that Kirs do not influence local [K+]o rises enough, under these conditions to affect synaptic transmission. In conclusion, our results indicate that hippocampal astrocytes are faithful sensors of [K+]o rises, but we find little evidence for physiologically relevant spatial K+ buffering during brief bursts of presynaptic activity. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Vesicle availability partly determines the efficacy of synaptic communication in the CNS. The authors recently found that some hippocampal glutamate vesicles exhibit reluctance to exocytose during short, high-frequency action potential trains. These same vesicles can be "coaxed" into exocytosis by increased Ca2+ entry, by direct depolarization of synaptic terminals, or by challenge with hypertonic sucrose, a tool used to cause fusion of the population of release-ready synaptic vesicles. Interestingly, the authors did not find evidence of reluctance at hippocampal GABA synapses, suggesting that vesicle reluctance might be a negative feedback mechanism to prevent runaway excitation. It is also possible that synapses exhibit reluctance to retain a dormant population of readily accessible vesicles, ready to respond to triggers such as enhanced Ca2+ influx or neuromodulators. Recent work from the calyx of Held synapse suggests that reluctance might arise from inactivation of Ca2+ channels. The authors review this work, along with several other potential mechanisms of reluctance. [ABSTRACT FROM AUTHOR]

The size of the readily releasable pool (RRP) of vesicles is critically important for determining the size of postsynaptic currents generated in response to action potentials. However, discrepancies in RRP estimates exist among methods designed to measure RRP size. In glutamatergic hippocampal neurons, we found that hypertonic sucrose application yielded RRP size estimates approximately fivefold larger than values obtained with high-frequency action potential trains commonly assumed to deplete the RRP. This discrepancy was specific for glutamatergic neurons, because no difference was found between sucrose and train estimates of RRP size in GABAergic neurons. A small component of the difference in excitatory neurons was accounted for by postsynaptic receptor saturation. Train estimates of vesicle pool size obtained using more stimuli revealed that action potential-elicited EPSCs did not truly reach a steady state during shorter trains, and RRP estimates were closer to sucrose estimates made in the same neurons. This suggested that reluctant vesicles may contribute to the total available pool. Two additional lines of evidence supported this hypothesis. First, RRP estimates from strongly depolarizing hyperkalemic solutions closely matched those obtained with sucrose. Second, when Ca2+ influx was enhanced during trains, train estimates of pool size matched those obtained with sucrose. These data suggest that glutamatergic hippocampal neurons maintain a heterogeneous population of vesicles that can be differentially released with varying Ca 2+ influx, thereby increasing the range of potential synaptic responses. [ABSTRACT FROM AUTHOR]

High-frequency synaptic transmission is depressed by moderate rises in the extracellular potassium concentration ([K[sup+]][subo]). Previous reports have indicated that depression of action potential signaling may underlie the synaptic depression. Here, we investigated the specific contribution of K[sup+]-induced action potential changes to synaptic depression. We found that glutamatergic transmission in the hippocampal area CA1 was significantly depressed by 8-10 mM [K[sup+]][subo], but that GABAergic transmission remained intact. Riluzole, a drug that slows recovery from inactivation of voltage-gated sodium channels (NaChs), interacts with subthreshold [K[sup+]][subo] to depress afferent volleys and EPSCs strongly. Thus, elevated [K[sup+]][subo] likely depresses synapses by slowing NaCh recovery from inactivation. It is unclear from previous studies whether [K[sup+]][subo]-induced action potential depression is caused by changes in initiation, reliability, or waveform. We investigated these possibilities explicitly. [K[sup+]][subo]-induced afferent volley depression was independent of stimulus strength, suggesting that changes in action potential initiation do not explain [K[sup+]][subo]-induced depression. Measurements of action potentials from single axons revealed that 8 mM [K[sup+]][subo] increased conduction failures in a subpopulation of fibers and depressed action potential amplitude in all fibers. Together, these changes quantitatively account for the afferent volley depression. We estimate that conduction failure explains more than half of the synaptic depression observed at 8 mM [K[sup+]][subo], with the remaining depression likely explained by waveform changes. These mechanisms of selective sensitivity of glutamate release to [K[sup+]][subo] accumulation represent a unique neuromodulatory mechanism and a brake on runaway excitation. [ABSTRACT FROM AUTHOR]

Pro-inflammatory changes contribute to multiple neuropsychiatric illnesses. Understanding how these changes are involved in illnesses and identifying strategies to alter inflammatory responses offer paths to potentially novel treatments. We previously found that acute pro-inflammatory stimulation with high (μg/ml) lipopolysaccharide (LPS) for 10–15 min dampens long-term potentiation (LTP) in the hippocampus and impairs learning. Effects of LPS involved non-canonical inflammasome signaling but were independent of toll-like receptor 4 (TLR4), a known LPS receptor. Low (ng/ml) LPS also inhibits LTP when administered for 2–4 h, and here we report that this LPS exposure requires TLR4. We also found that effects of low LPS on LTP involve the oxysterol, 25-hydroxycholesterol, akin to high LPS. Effects of high LPS on LTP are blocked by inhibiting synthesis of 5α-reduced neurosteroids, indicating that neurosteroids mediate LTP inhibition. 5α-Neurosteroids also have anti-inflammatory effects, and we found that exogenous allopregnanolone (AlloP), a key 5α-reduced steroid, prevented effects of low but not high LPS on LTP. We also found that activation of TLR2, TLR3 and TLR7 inhibited LTP and that AlloP prevented the effects of TLR2 and TLR7, but not TLR3. The enantiomer of AlloP, a steroid that has anti-inflammatory actions but low activity at GABAA receptors, prevented LTP inhibition by TLR2, TLR3 and TLR7. In vivo, both AlloP enantiomers prevented LPS-induced learning defects. These studies indicate that neurosteroids play complex roles in network effects of acute neuroinflammation and have potential importance for development of AlloP analogues as therapeutic agents. [ABSTRACT FROM AUTHOR]

Describes the glial contributions to excitatory neurotransmission in cultured hippocampal cells. Glial cells' Possession of neurotransmitter receptors and transporters; Recording of neuronal autaptic and glial responses from cultured hippocampal single-neuron micro-islands; Activation of rapid electrogenic glial glutamate transporter currents.

Synapses respond to brief, repetitive stimulation with synaptic depression when initial transmitter release probability is high. Vesicle depletion has been a long-standing hypothesis for depression, but results unexplained by the depletion hypothesis have been nagging. In this issue of Neuron, Xu and Wu show that, under some conditions, calcium current inactivation explains stimulus-dependent depression at the calyx of Held. [Copyright &y& Elsevier]

Among the proposed roles for astrocytes in the CNS is nutritive support for neurons. In this issue of Neuron, Voutsinos-Porche et al. provide evidence that astrocyte uptake of synaptic glutamate triggers astrocytic glycolysis and release of lactate, which in turn nourishes neurons and sustains neuronal activity. [Copyright &y& Elsevier]

Brain cholesterol metabolic products include neurosteroids and oxysterols, which play important roles in cellular physiology. In neurons, the cholesterol oxidation product, 24S-hydroxycholesterol (24S-HC), is a regulator of signaling and transcription. Here, we examined the behavioral effects of 24S-HC loss, using global and cell-selective genetic deletion of the synthetic enzyme CYP46A1. Mice that are globally deficient in CYP46A1 exhibited hypoactivity at young ages and unexpected increases in conditioned fear memory. Despite strong reductions in hippocampal 24S-HC in mice with selective loss of CYP46A1 in VGLUT1-positive cells, behavioral effects were not recapitulated in these conditional knockout mice. Global knockout produced strong, developmentally dependent transcriptional effects on select cholesterol metabolism genes. These included paradoxical changes in Liver X Receptor targets. Again, conditional knockout was insufficient to recapitulate most changes. Overall, our results highlight the complex effects of 24S-HC in an in vivo setting that are not fully predicted by known mechanisms. The results also demonstrate that the complete inhibition of enzymatic activity may be needed for a detectable, therapeutically relevant impact on gene expression and behavior. [ABSTRACT FROM AUTHOR]

Cortical electroencephalograms (EEGs) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/f) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than excitation/inhibition ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of excitation/inhibition ratio, we analyzed video-EEG during active exploration in mice of both sexes during various pharmacological manipulations with the fitting oscillations and one over f (FOOOF) algorithm. We found that GABAA receptor (GABAAR)-positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABAAR antagonists at subconvulsive doses) did not follow the prediction. To tilt excitation/inhibition ratio more selectively toward excitation, we suppressed the activity of parvalbumin-positive interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of cortical excitation/inhibition ratio. NEW & NOTEWORTHY Neuropsychiatric illness may be associated with altered excitation/inhibition balance. A single electroencephalogram (EEG) parameter, the aperiodic exponent of power spectra, may predict the ratio between excitation and inhibition. Here, we use cortical EEGs in mice to evaluate this hypothesis, using pharmacological manipulations of known mechanism. We show that the aperiodic exponent of EEG power spectra is not a reliable marker of excitation/inhibition ratio. Thus, alternative markers of this ratio must be sought. [ABSTRACT FROM AUTHOR]

Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Key points: Current views suggest γ2 subunit‐containing GABAA receptors mediate phasic IPSCs while extrasynaptic δ subunits mediate diffusional IPSCs and tonic current.We have re‐examined the roles of the two receptor populations using mice with picrotoxin resistance engineered into receptors containing the δ subunit.Using pharmacological separation, we find that in general δ and γ IPSCs are modulated in parallel by manipulations of transmitter output and diffusion, with evidence favouring modestly more diffusional contribution to δ IPSCs.Our findings also reveal that spontaneous δ IPSCs are mainly driven by channel deactivation, rather than by diffusion of GABA.Understanding the functional contributions of the two receptor classes may help us understand the actions of drug therapies with selective effects on one population over the other. GABAA receptors mediate transmission throughout the central nervous system and typically contain a δ subunit (δ receptors) or a γ2 subunit (γ2 receptors). δ IPSCs decay slower than γ2 IPSCs, but the reasons are unclear. Transmitter diffusion, rebinding, or slow deactivation kinetics of channels are candidates. We used gene editing to confer picrotoxin resistance on δ receptors in mice, then pharmacologically isolated δ receptors in mouse dentate granule cells to explore IPSCs. γ2 and δ components of IPSCs were modulated similarly by presynaptic manipulations and manipulations of transmitter lifetime, suggesting that GABA release recruits δ receptors proportionally to γ2 receptors. δ IPSCs showed more sensitivity to altered transmitter release and to a rapidly dissociating antagonist, suggesting an additional spillover contribution. Reducing GABA diffusion with 5% dextran increased the peak amplitude and decreased the decay of evoked δ IPSCs but had no effect on δ or dual‐component (mainly γ2‐driven) spontaneous IPSCs, suggesting that GABA actions can be local for both receptor types. Rapid application of varied [GABA] onto nucleated patches from dentate granule cells demonstrated a deactivation rate of δ receptors similar to that of δ spontaneous IPSCs, consistent with the idea that deactivation and local GABA actions drive δ spontaneous IPSCs. Overall, our results indicate that δ IPSCs are activated by both synaptic and diffusional GABA. Our results are consistent with a functional relationship between δ and γ2 GABAA receptors akin to that of slow NMDA and fast AMPA EPSCs at glutamate synapses. Key points: Current views suggest γ2 subunit‐containing GABAA receptors mediate phasic IPSCs while extrasynaptic δ subunits mediate diffusional IPSCs and tonic current.We have re‐examined the roles of the two receptor populations using mice with picrotoxin resistance engineered into receptors containing the δ subunit.Using pharmacological separation, we find that in general δ and γ IPSCs are modulated in parallel by manipulations of transmitter output and diffusion, with evidence favouring modestly more diffusional contribution to δ IPSCs.Our findings also reveal that spontaneous δ IPSCs are mainly driven by channel deactivation, rather than by diffusion of GABA.Understanding the functional contributions of the two receptor classes may help us understand the actions of drug therapies with selective effects on one population over the other. [ABSTRACT FROM AUTHOR]

The relative contributions of glycolysis and oxidative phosphorylation to neuronal presynaptic energy demands are unclear. In rat hippocampal neurons, ATP production by either glycolysis or oxidative phosphorylation alone sustained basal evoked synaptic transmission for up to 20 min. However, combined inhibition of both ATP sources abolished evoked transmission. Neither action potential propagation failure nor depressed Ca2+ influx explained loss of evoked synaptic transmission. Rather, inhibition of ATP synthesis caused massive spontaneous vesicle exocytosis, followed by arrested endocytosis, accounting for the disappearance of evoked postsynaptic currents (PSCs). In contrast to its weak effects on basal transmission, inhibition of oxidative phosphorylation alone depressed recovery from vesicle depletion. Local astrocytic lactate shuttling was not required. Instead, either ambient monocarboxylates or neuronal glycolysis was sufficient to supply requisite substrate. In summary basal transmission can be sustained by glycolysis, but strong presynaptic demands are met preferentially by oxidative phosphorylation, which can be maintained by bulk but not local monocarboxylates or by neuronal glycolysis. [ABSTRACT FROM AUTHOR]

Human studies examining the effects of the dissociative anesthetic ketamine as a model for psychosis and as a rapidly acting antidepressant have spurred great interest in understanding ketamine's actions at molecular, cellular, and network levels. Although ketamine has unequivocal uncompetitive inhibitory effects on N-methyl-D-aspartate receptors (NMDARs) and may preferentially alter the function of NMDARs on interneurons, recent work has questioned whether block of NMDARs is critical for its mood enhancing actions. In this viewpoint, we examine the evolving literature on ketamine supporting NMDARs as important triggers for certain psychiatric effects and the possibility that the antidepressant trigger is unrelated to NMDARs. The rapidly evolving story of ketamine offers great hope for untangling and treating the biology of both depressive and psychotic illnesses. [ABSTRACT FROM AUTHOR]

Nitrous oxide (N 2 O) is a noncompetitive inhibitor of NMDA receptors that appears to have ketamine-like rapid antidepressant effects in patients with treatment-resistant major depression. In preclinical studies, ketamine enhances glutamate-mediated synaptic transmission in the hippocampus and prefrontal cortex. In this study, we examined the effects of N 2 O on glutamate transmission in the hippocampus and compared its effects to those of ketamine. Glutamate-mediated synaptic transmission was studied in the CA1 region of hippocampal slices from adult albino rats using standard extracellular recording methods. Effects of N 2 O and ketamine at subanesthetic concentrations were evaluated by acute administration. Akin to 1 μM ketamine, 30% N 2 O administered for 15–20 minutes resulted in persistent enhancement of synaptic responses mediated by both AMPA receptors and NMDA receptors. Synaptic enhancement by both N 2 O and ketamine was blocked by co-administration of a competitive NMDA receptor antagonist at saturating concentration, but only ketamine was blocked by an AMPA receptor antagonist. Synaptic enhancement by both agents involved TrkB (tropomyosin receptor kinase B), mTOR (mechanistic target of rapamycin), and NOS (nitric oxide synthase) with some differences between N 2 O and ketamine. N 2 O potentiation occluded enhancement by ketamine, and in vivo N 2 O exposure occluded further potentiation by both N 2 O and ketamine. These results indicate that N 2 O has ketamine-like effects on hippocampal synaptic function at a subanesthetic, but therapeutically relevant concentration. These 2 rapid antidepressants have similar, but not identical mechanisms that result in persisting synaptic enhancement, possibly contributing to psychotropic actions. [ABSTRACT FROM AUTHOR]

The article discusses the findings of fluorescence studies of axon excitability. According to the author, axons have been intensely studied since the earliest days of neurophysiology because electrical transmission along axons to axon terminal is significant in neuronal signalling. The author mentions a study by Popovic and associates that exploit a voltage-sensitive dye that is loaded into single cells with sufficiently rapid and large modifications in fluorescence.

The rise of ketamine and brexanolone as rapid antidepressant treatments raises the question of common mechanisms. Both drugs act without the long onset time of traditional antidepressants such as selective serotonin reuptake inhibitors. The drugs also share the interesting feature of benefit that persists beyond the initial drug lifetime. Here, we briefly review literature on functional changes that may mark the triggering mechanism of rapid antidepressant actions. Because ketamine has a longer history of study as a rapid antidepressant, we use this literature as a template to guide hypotheses about common action. Brexanolone has the complication of being a formulation of a naturally occurring neurosteroid; thus, endogenous levels need to be considered when studying the impact of exogenous administration. We conclude that network disinhibition and increased high‐frequency oscillations are candidates to mediate acute triggering effects of rapid antidepressants. [ABSTRACT FROM AUTHOR]

Detection of submillisecond interaural timing differences is the basis for sound localization in reptiles, birds, and mammals. Although comparative studies reveal that different neural circuits underlie this ability, they also highlight common solutions to an inherent challenge: processing information on timescales shorter than an action potential. Discrimination of small timing differences is also important for species recognition during communication among mormyrid electric fishes. These fishes generate a species-specific electric organ discharge (EOD) that is encoded into submillisecond-to-millisecond timing differences between receptors. Small, adendritic neurons (small cells) in the midbrain are thought to analyze EOD waveform by comparing these differences in spike timing, but direct recordings from small cells have been technically challenging. In the present study we use a fluorescent labeling technique to obtain visually guided extracellular recordings from individual small cell axons. We demonstrate that small cells receive 1-2 excitatory inputs from 1 or more receptive fields with latencies that vary by over 10 ms. This wide range of excitatory latencies is likely due to axonal delay lines, as suggested by a previous anatomic study. We also show that inhibition of small cells from a calyx synapse shapes stimulus responses in two ways: through tonic inhibition that reduces spontaneous activity and through precisely timed, stimulus-driven, feed-forward inhibition. Our results reveal a novel delay-line anticoincidence detection mechanism for processing submillisecond timing differences, in which excitatory delay lines and precisely timed inhibition convert a temporal code into a population code. [ABSTRACT FROM AUTHOR]

Inflammatory cells, including macrophages and microglia, synthesize and release the oxysterol 25-hydroxycholesterol (25HC), which has antiviral and immunomodulatory properties. Here, we examined the effects of lipopolysaccharide (LPS), an activator of innate immunity, on 25HC production in microglia, and the effects of LPS and 25HC on CA1 hippocampal synaptic plasticity and learning. In primary microglia, LPS markedly increases the expression of cholesterol 25-hydroxylase (Ch25h), the key enzyme involved in 25HC synthesis, and increases the levels of secreted 25HC. Wild-type microglia produced higher levels of 25HC than Ch25h knock-out (KO) microglia with or without LPS. LPS treatment also disrupts long-term potentiation (LTP) in hippocampal slices via induction of a form of NMDA receptor-dependent metaplasticity. The inhibitory effects of LPS on LTP were mimicked by exogenous 25HC, and were not observed in slices from Ch25h KO mice. In vivo, LPS treatment also disrupts LTP and inhibits one-trial learning in wild-type mice, but not Ch25h KO mice. These results demonstrate that the oxysterol 25HC is a key modulator of synaptic plasticity and memory under proinflammatory stimuli. [ABSTRACT FROM AUTHOR]

CNIH3 is an AMPA receptor (AMPAR) auxiliary protein prominently expressed in the dorsal hippocampus (dHPC), a region that plays a critical role in spatial memory and synaptic plasticity. However, the effects of CNIH3 on AMPAR-dependent synaptic function and behavior have not been investigated. We assessed a gain-of-function model of Cnih3 overexpression in the dHPC and generated and characterized a line of Cnih3 −/− C57BL/6 mice. We assessed spatial memory through behavioral assays, protein levels of AMPAR subunits and synaptic proteins by immunoblotting, and long-term potentiation in electrophysiological recordings. We also utilized a super-resolution imaging workflow, SEQUIN (Synaptic Evaluation and Quantification by Imaging of Nanostructure), for analysis of nanoscale synaptic connectivity in the dHPC. Overexpression of Cnih3 in the dHPC improved short-term spatial memory in female mice but not in male mice. Cnih3 −/− female mice exhibited weakened short-term spatial memory, reduced dHPC synapse density, enhanced expression of calcium-impermeable AMPAR (GluA2-containing) subunits in synaptosomes, and attenuated long-term potentiation maintenance compared with Cnih3 +/+ control mice; Cnih3 −/− males were unaffected. Further investigation revealed that deficiencies in spatial memory and changes in AMPAR composition and synaptic plasticity were most pronounced during the metestrus phase of the estrous cycle in female Cnih3 −/− mice. This study identified a novel effect of sex and estrous on CNIH3's role in spatial memory and synaptic plasticity. Manipulation of CNIH3 unmasked sexually dimorphic effects on spatial memory, synaptic function, AMPAR composition, and hippocampal plasticity. These findings reinforce the importance of considering sex as a biological variable in studies of memory and hippocampal synaptic function. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium (KATP) gain-of-function (GOF) mutations cause neonatal diabetes, with some individuals exhibiting developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. Mice expressing KATP-GOF mutations pan-neuronally (nKATP-GOF) demonstrated sensorimotor and cognitive deficits, whereas hippocampus-specific hKATP-GOF mice exhibited mostly learning and memory deficiencies. Both nKATP-GOF and hKATP-GOF mice showed altered neuronal excitability and reduced hippocampal long-term potentiation (LTP). Sulfonylurea therapy, which inhibits KATP, mildly improved sensorimotor but not cognitive deficits in KATP-GOF mice. Mice expressing KATP-GOF mutations in pancreatic β-cells developed severe diabetes but did not show learning and memory deficits, suggesting neuronal KATP-GOF as promoting these features. These findings suggest a possible origin of cognitive dysfunction in DEND and the need for novel drugs to treat neurological features induced by neuronal KATP-GOF. [ABSTRACT FROM AUTHOR]

Although action potential initiation and propagation are fundamental to nervous system function, there are few direct electrophysiological observations of propagating action potentials in small unmyelinated fibres, such as the axons within mammalian hippocampus. To circumvent limitations of previous studies that relied on extracellular stimulation, we performed dual recordings: whole-cell recordings from hippocampal CA3 pyramidal cell somas and extracellular recordings from their axons, up to 800 μm away. During brief spike trains under normal conditions, axonal spikes were more resistant to amplitude reduction than somatic spikes. Axonal amplitude depression was greatest at the axon initial segment < 150 μm from the soma, and initiation occurred ∼75 μm from the soma. Although prior studies, which failed to verify spike initiation, suggested substantial axonal depression during seizure-associated extracellular K+ ([K+]o) rises, we found that 8 m m[K+]o caused relatively small decreases in axonal spike amplitude during brief spike trains. However, during sustained, epileptiform spiking induced in 8 m m[K+]o, axonal waveforms decreased significantly in peak amplitude. During epileptiform spiking, bursts of two or more action potentials > 20 Hz failed to propagate in most cases. In normal [K+]o at 25 and 32°C, spiking superimposed on sustained somatic depolarization, but not spiking alone, produced similar axonal changes as the epileptiform activity. These results highlight the likely importance of steady-state inactivation of axonal channels in maintaining action potential fidelity. Such changes in axonal propagation properties could encode information and/or serve as an endogenous brake on seizure propagation. [ABSTRACT FROM AUTHOR]

Abstract Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABA A receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors. Highlights • KK-169 is a potent and efficacious positive allosteric modulator of NMDA receptors. • KK-169 is a clickable analogue of pregnenolone sulfate. • KK-169 reveals intracellular accumulation of sulfated steroids. [ABSTRACT FROM AUTHOR]

Two major GABAA receptor classes mediate ionotropic GABA signaling, those containing a δ subunit and those with a γ2 subunit. The classical viewpoint equates γ2-containing receptors with IPSCs and δ-containing receptors with tonic inhibition because of differences in receptor localization, but significant questions remain because the populations cannot be pharmacologically separated. We removed this barrier using gene editing to confer a point mutation on the δ subunit in mice, rendering receptors containing the subunit picrotoxin resistant. By pharmacologically isolating δ-containing receptors, our results demonstrate their contribution to IPSCs in dentate granule neurons and weaker contributions to thalamocortical IPSCs. Despite documented extrasynaptic localization, we found that receptor localization does not preclude participation in isolated IPSCs, including mIPSCs. Further, phasic inhibition from δ subunit-containing receptors strongly inhibited summation of EPSPs, whereas tonic activity had little impact. In addition to any role that S-containing receptors may play in canonical tonic inhibition, our results highlight a previously underestimated contribution of S-containing receptors to phasic inhibition. [ABSTRACT FROM AUTHOR]

Endogenous neurosteroids and synthetic neuroactive steroids (NAS) are important targets for therapeutic development in neuropsychiatric disorders. These steroids modulate major signaling systems in the brain and intracellular processes including inflammation, cellular stress and autophagy. In this review, we describe studies performed using unnatural enantiomers of key neurosteroids, which are physiochemically identical to their natural counterparts except for rotation of polarized light. These studies led to insights in how NAS interact with receptors, ion channels and intracellular sites of action. Certain effects of NAS show high enantioselectivity, consistent with actions in chiral environments and likely direct interactions with signaling proteins. Other effects show no enantioselectivity and even reverse enantioselectivity. The spectrum of effects of NAS enantiomers raises the possibility that these agents, once considered only as tools for preclinical studies, have therapeutic potential that complements and in some cases may exceed their natural counterparts. Here we review studies of NAS enantiomers from the perspective of their potential development as novel neurotherapeutics. • Neuroactive steroids (NAS) are an important class of potential neurotherapeutics. • Certain effects of NAS show high, no, or reverse enantioselectivity. • Enantiomers of progesterone, allopregnanolone and estradiol are neuroprotective. • The enantiomer of pregenolone sulfate has cognitive enhancing effects. • NAS enantiomers could represent novel treatments for neuropsychiatric illnesses. [ABSTRACT FROM AUTHOR]

Neurons require a nearly constant supply of ATP. Glucose is the predominant source of brain ATP, but the direct effects of prolonged glucose deprivation on neuronal viability and function remain unclear. In sparse rat hippocampal microcultures, neurons were surprisingly resilient to 16 h glucose removal in the absence of secondary excitotoxicity. Neuronal survival and synaptic transmission were unaffected by prolonged removal of exogenous glucose. Inhibition of lactate transport decreased microculture neuronal survival during concurrent glucose deprivation, suggesting that endogenously released lactate is important for tolerance to glucose deprivation. Tandem depolarization and glucose deprivation also reduced neuronal survival, and trace glucose concentrations afforded neuroprotection. Mass cultures, in contrast to microcultures, were insensitive to depolarizing glucose deprivation, a difference attributable to increased extracellular lactate levels. Removal of local astrocyte support did not reduce survival in response to glucose deprivation or alter evoked excitatory transmission, suggesting that on-demand, local lactate shuttling is not necessary for neuronal tolerance to prolonged glucose removal. Taken together, these data suggest that endogenously produced lactate available globally in the extracellular milieu sustains neurons in the absence of glucose. A better understanding of resilience mechanisms in reduced preparations could lead to therapeutic strategies aimed to bolster these mechanisms in vulnerable neuronal populations. [ABSTRACT FROM AUTHOR]

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors important for synaptic plasticity, memory, and neuropsychiatric health. NMDAR hypofunction contributes to multiple disorders, including anti-NMDAR encephalitis (NMDARE), an autoimmune disease of the CNS associated with GluN1 antibody-mediated NMDAR internalization. Here we characterize the functional/pharmacological consequences of exposure to CSF from female human NMDARE patients on NMDAR function, and we characterize the effects of intervention with recently described positive allosteric modulators (PAMs) of NMDARs. Incubation (48 h) of rat hippocampal neurons of both sexes in confirmed NMDARE patient CSF, but not control CSF, attenuated NMDA-induced current. Residual NMDAR function was characterized by lack of change in channel open probability, indiscriminate loss of synaptic and extrasynaptic NMDARs, and indiscriminate loss of GluN2B-containing and GluN2B-lacking NMDARs. NMDARs tagged with N-terminal pHluorin fluorescence demonstrated loss of surface receptors. Thus, function of residual NMDARs following CSF exposure was indistinguishable from baseline, and deficits appear wholly accounted for by receptor loss. Coapplication of CSF and PAMs of NMDARs (SGE-301 or SGE-550, oxysterol-mimetic) for 24 h restored NMDAR function following 24 h incubation in patient CSF. Curiously, restoration of NMDAR function was observed despite washout of PAMs before electrophysiological recordings. Subsequent experiments suggested that residual allosteric potentiation of NMDAR function explained the persistent rescue. Further studies of the pathogenesis of NMDARE and intervention with PAMs may inform new treatments for NMDARE and other disorders associated with NMDAR hypofunction. [ABSTRACT FROM AUTHOR]

N-methyl-d-aspartate receptors (NMDARs) govern synaptic plasticity, development, and neuronal response to insult. Prolonged activation of NMDARs such as during an insult may activate secondary currents or modulate Mg2+ sensitivity, but the conditions under which these occur are not fully defined. We reexamined the effect of prolonged NMDAR activation in juvenile mouse hippocampal slices. NMDA (10 μM) elicited current with the expected negative-slope conductance in the presence of 1.2 mM Mg2+ However, several minutes of continued NMDA exposure elicited additional inward current at -70 mV. A higher concentration of NMDA (100 µM) elicited the current more rapidly. The additional current was not dependent on Ca2+, network activity, or metabotropic NMDAR function and did not persist on agonist removal. Voltage ramps revealed no alteration of either reversal potential or NMDA-elicited conductance between -30 mV and +50 mV. The result was a more linear NMDA current-voltage relationship. The current linearization was also induced in interneurons and in mature dentate granule neurons but not immature dentate granule cells, dissociated cultured hippocampal neurons, or nucleated patches excised from CA1 pyramidal neurons. Comparative simulations of NMDA application to a CA1 pyramidal neuron and to a cultured neuron revealed that linearization can be explained by space-clamp errors arising from gradual recruitment of distal dendritic NMDARs. We conclude that persistent secondary currents do not strongly contribute to NMDAR responses in juvenile mouse hippocampus and careful discernment is needed to exclude contributions of clamp artifacts to apparent secondary currents.NEW & NOTEWORTHY We report that upon sustained activation of NMDARs in juvenile mouse hippocampal neurons there is apparent loss of Mg2+ block at negative membrane potentials. However, the phenomenon is explained by loss of dendritic voltage clamp, leading to a linear current-voltage relationship. Our results give a specific example of how spatial voltage errors in voltage-clamp recordings can readily be misinterpreted as biological modulation. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs), a major subtype of glutamate receptor mediating excitatory transmission throughout the CNS, participate in ischemia-induced neuronal death. Unfortunately, undesired side effects have limited the strategy of inhibiting/blocking NMDARs as therapy. Targeting endogenous positive allosteric modulators of NMDAR function may offer a strategy with fewer downsides. Here, we explored whether 24S-hydroxycholesterol (24S-HC), an endogenous positive NMDAR modulator characterized recently by our group, participates in NMDAR-mediated excitotoxicity following oxygen-glucose deprivation (OGD) in primary neuron cultures. 24S-HC is the major brain cholesterol metabolite produced exclusively in neurons near sites of glutamate transmission. By selectively potentiating NMDAR current, 24S-HC may participate in NMDAR-mediated excitotoxicity following energy failure, thus impacting recovery after stroke. In support of this hypothesis, our findings indicate that exogenous application of 24S-HC exacerbates NMDAR-dependent excitotoxicity in primary neuron culture following OGD, an ischemic-like challenge. Similarly, enhancement of endogenous 24S-HC synthesis reduced survival rate. On the other hand, reducing endogenous 24S-HC synthesis alleviated OGD-induced cell death. We found that 25-HC, another oxysterol that antagonizes 24S-HC potentiation, partially rescued OGD-mediated cell death in the presence or absence of exogenous 24S-HC application, and 25-HC exhibited NMDAR-dependent/24S-HC-dependent neuroprotection, as well as NMDAR-independent neuroprotection in rat tissue but not mouse tissue. Our findings suggest that both endogenous and exogenous 24S-HC exacerbate OGD-induced damage via NMDAR activation, while 25-HC exhibits species dependent neuroprotection through both NMDAR-dependent and independent mechanisms. [ABSTRACT FROM AUTHOR]

Anesthetic, GABA-active neurosteroids potently augment GABA A receptor function, leading to important behavioral consequences. Neurosteroids and their synthetic analogues are also models for a wide variety of cell-permeant neuroactive compounds. Cell permeation and compartmentalization raise the possibility that these compounds' actions are influenced by their cellular partitioning, but these contributions are not typically considered experimentally or therapeutically. To examine the interplay between cellular accumulation and pharmacodynamics of neurosteroids, we synthesized a novel chemical biology analogue (bio-active, clickable photolabel) of GABA-active neurosteroids. We discovered that the analogue selectively photo-labels neuronal Golgi in rat hippocampal neurons. The active analogue's selective distribution was distinct from endogenous cholesterol and not completely shared by some non-GABA active, neurosteroid-like analogues. On the other hand, the distribution was not enantioselective and did not require energy, in contrast to other recent precedents from the literature. We demonstrate that the soma-selective accumulation can act as a sink or source for steroid actions at plasma-membrane GABA receptors, altering steady-state and time course of effects at somatic GABA A receptors relative to dendritic receptors. Our results suggest a novel mechanism for compartment-selective drug actions at plasma-membrane receptors. [ABSTRACT FROM AUTHOR]

Significance: Optical imaging of responses in fluorescently labeled neurons has progressed significantly in recent years. However, there is still a need to monitor neural activities at divergent spatial scales and at depths beyond the optical diffusion limit. Aim: To meet these needs, we aim to develop multiscale photoacoustic tomography (PAT) to image neural activities across spatial scales with a genetically encoded calcium indicator GCaMP. Approach: First, using photoacoustic microscopy, we show that depth-resolved GCaMP signals can be monitored in vivo from a fly brain in response to odor stimulation without depth scanning and even with the cuticle intact. In vivo monitoring of GCaMP signals was also demonstrated in mouse brains. Next, using photoacoustic computed tomography, we imaged neural responses of a mouse brain slice at depths beyond the optical diffusion limit. Results: We provide the first unambiguous demonstration that multiscale PAT can be used to record neural activities in transgenic flies and mice with select neurons expressing GCaMP. Conclusions: Our results indicate that the combination of multiscale PAT and fluorescent neural activity indicators provides a methodology for imaging targeted neurons at various scales. [ABSTRACT FROM AUTHOR]

N-methyl-Daspartate receptors (NMDARs), a major subtype of glutamate receptors mediating excitatory transmission throughout the central nervous system (CNS), play critical roles in governing brain function and cognition. Because NMDAR dysfunction contributes to the etiology of neurological and psychiatric disorders including stroke and schizophrenia, NMDAR modulators are potential drug candidates. Our group recently demonstrated that the major brain cholesterol metabolite, 24S-hydroxycholesterol (24S-HC), positively modulates NMDARs when exogenously administered. Here, we studied whether endogenous 24S-HC regulates NMDAR activity in hippocampal slices. In CYP46A1-/- (knockout; KO) slices where endogenous 24S-HC is greatly reduced, NMDAR tone, measured as NMDAR-to-α-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor (AMPAR) excitatory postsynaptic current (EPSC) ratio, was reduced. This difference translated into more NMDAR-driven spiking in wild-type (WT) slices compared with KO slices. Application of SGE-301, a 24S-HC analog, had comparable potentiating effects on NMDAR EPSCs in both WT and KO slices, suggesting that endogenous 24S-HC does not saturate its NMDAR modulatory site in ex vivo slices. KO slices did not differ from WT slices in either spontaneous neurotransmission or in neuronal intrinsic excitability, and exhibited LTP indistinguishable from WT slices. However, KO slices exhibited higher resistance to persistent NMDAR-dependent depression of synaptic transmission induced by oxygen-glucose deprivation (OGD), an effect restored by SGE- 301. Together, our results suggest that loss of positive NMDAR tone does not elicit compensatory changes in excitability or transmission, but it protects transmission against NMDAR-mediated dysfunction. We expect that manipulating this endogenous NMDAR modulator may offer new treatment strategies for neuropsychiatric dysfunction. [ABSTRACT FROM AUTHOR]

Neuron-astrocyte interactions are critical for proper CNS development and function. Astrocytes secrete factors that are pivotal for synaptic development and function, neuronal metabolism, and neuronal survival. Our understanding of this relationship, however, remains incomplete due to technical hurdles that have prevented the removal of astrocytes from neuronal circuits without changing other important conditions. Here we overcame this obstacle by growing solitary rat hippocampal neurons on microcultures that were comprised of either an astrocyte bed (+astrocyte) or a collagen bed (-astrocyte) within the same culture dish. -Astrocyte autaptic evoked EPSCs, but not IPSCs, displayed an altered temporal profile, which included increased synaptic delay, increased time to peak, and severe glutamate release asynchrony, distinct from previously described quantal asynchrony. Although we observed minimal alteration of the somatically recorded action potential waveform, action potential propagation was altered. We observed a longer latency between somatic initiation and arrival at distal locations, which likely explains asynchronous EPSC peaks, and we observed broadening of the axonal spike, which likely underlies changes to evoked EPSC onset. No apparent changes in axon structure were observed, suggesting altered axonal excitability. In conclusion, we propose that local astrocyte support has an unappreciated role in maintaining glutamate release synchrony by disturbing axonal signal propagation. [ABSTRACT FROM AUTHOR]

It is widely appreciated that neuronal networks exhibit patterns of bursting and synchrony that are not captured by simple measures such as average spike rate. These patterns can encode information or represent pathological behavior such as seizures. However, methods for quantifying bursting and synchrony are not agreed upon and can be confounded with spike rate measures. Previous validation has largely relied on in silico networks and single experimental conditions. How published measures of bursting and synchrony perform when applied to biological networks of varied average spike rate and subjected to varied experimental challenges is unclear. In multielectrode array recordings of network activity, we found that two mechanistically distinct drugs, cyclothiazide and bicuculline, produced equivalent increases in average spike rate but differed in bursting and synchrony. We applied several measures of bursting to the recordings (2 threshold interval methods and a surprise-based method) and found that a measure based on an average critical interval, adjusted for the array-wide spike rate, performed best in quantifying differential drug effects. To quantify synchrony, we compared a coefficient of variation-based measure, the recently proposed spike time tiling coefficient, the SPIKE-distance measure, and a global synchrony index. The spike time tiling coefficient, the SPIKE-distance measure, and the global synchrony index all captured a difference between drugs with the best performance exhibited by the global synchrony index. In summary, our exploration should aid other investigators by highlighting strengths and limitations of current methods. [ABSTRACT FROM AUTHOR]

Background and Purpose Memantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators ( PAMs) might affect the pharmacodynamics of these NMDA blockers. Experimental Approach We used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24 S-hydroxycholesterol, along with pregnenolone sulphate ( PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques. Key Results SGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks - measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection. Conclusions and Implications Positive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour. [ABSTRACT FROM AUTHOR]

Oxysterols have emerged as important biomarkers in disease and as signaling molecules. We recently showed that the oxysterol 24(S)-hydroxycholesterol, the major brain cholesterol metabolite, potently and selectively enhances NMDA receptor function at a site distinct from other modulators. Here we further characterize the pharmacological mechanisms of 24(S)-hydroxycholesterol and its synthetic analog SGE201. We describe an oxysterol antagonist of this positive allosteric modulation, 25-hydroxycholesterol. We found that 24(S)-hydroxycholesterol and SGE201 primarily increased the efficacy of NMDAR agonists but did not directly gate the channel or increase functional receptor number. Rather than binding to a direct aqueous-accessible site, oxysterols may partition into the plasma membrane to access the NMDAR, likely explaining slow onset and offset kinetics of modulation. Interestingly, oxysterols were ineffective when applied to the cytosolic face of inside-out membrane patches or through a whole-cell pipette solution, suggesting a non-intracellular site. We also found that another natural oxysterol, 25-hydroxycholesterol, although exhibiting slight potentiation on its own, non-competitively and enantioselectively antagonized the effects of 24(S)-hydroxycholesterol analogs. In summary, we suggest two novel allosteric sites on NMDARs that separately modulate channel gating, but together oppose each other. [ABSTRACT FROM AUTHOR]

Rationale: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABA) receptors, the binding sites that mediate these actions have not been definitively identified. Objectives: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABA receptors. Results: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with αβγ subunits, modulated [S] t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [H]CW12 and [H]CW14 showed that these reagents covalently label GABA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α and β subunits or β subunits of the GABA receptor. Photolabeling of rat brain GABA receptors was shown to be both concentration-dependent and stereospecific. Conclusions: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABA receptors. [ABSTRACT FROM AUTHOR]

Few other neurotransmitters are of as intense interest to neuropsychiatry and neurology as dopamine, yet existing techniques to monitor dopamine release leave an important spatiotemporal gap in our understanding. Electrochemistry and fluorescence imaging tools have been developed to fill the gap, but these methods have important limitations. We circumvent these limitations by introducing a dopamine-gated chloride channel into rat dorsal striatal medium spiny neurons, targets of strong dopamine innervation, thereby transforming dopamine from a slow transmitter into a fast transmitter and revealing new opportunities for studying moment-tomoment regulation of dopamine release. We demonstrate pharmacological and biophysical properties of the channel that make it suitable for fast, local dopamine measurements, and we demonstrate for the first time spontaneous and evoked responses to vesicular dopamine release in the dorsal striatum. Evoked dopamine currents were separated into a fast, monosynaptic component and a slowerrising and decaying disynaptic component mediated by nicotinic receptor activation. In summary, LGC-53 represents a dopamine biosensor with properties suitable for temporal separation of distinct dopamine signals in targets of dopamine innervation. [ABSTRACT FROM AUTHOR]

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K+ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders. [ABSTRACT FROM AUTHOR]

A model of the alignment of neurosteroidsand ent-neurosteroids at the same binding site onγ-aminobutyric acidtype A (GABAA) receptors was evaluated for its abilityto identify the structural features in ent-neurosteroidsthat enhance their activity as positive allosteric modulators of thisreceptor. Structural features that were identified included: (1) aketone group at position C-16, (2) an axial 4α-OMe group, and(3) a C-18 methyl group. Two ent-steroids were identifiedthat were more potent than the anesthetic steroid alphaxalone in theirthreshold for and duration of loss of the righting reflex in mice.In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50values similar to those foundfor allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anestheticagents and as drugs to treat brain disorders that are amelioratedby positive allosteric modulators of GABAAreceptor function. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are critical to the regulation of excitatory synaptic function in the CNS. NMDARs govern experience-dependent synaptic plasticity and have been implicated in the pathophysiology of various neuropsychiatric disorders including the cognitive deficits of schizophrenia and certain forms of autism. Certain neurosteroids modulateNMDARsexperimentally but their low potency, poor selectivity, and very low brain concentrations make them poor candidates as endogenous ligands or therapeutic agents. Hereweshow that the major brain-derived cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC) is a very potent, direct, and selective positive allosteric modulator ofNMDARswith a mechanism that does not overlap that of other allosteric modulators. At submicromolar concentrations 24(S)-HC potentiates NMDAR-mediated EPSCs in rat hippocampal neurons but fails to affect AMPAR or GABAA receptors (GABAARs)-mediated responses. Cholesterol itself and other naturally occurring oxysterols present in brain do not modulateNMDARsat concentrations≤10μM. In hippocampal slices, 24(S)-HC enhances the ability of subthreshold stimuli to induce long-term potentiation (LTP). 24(S)-HC also reverses hippocampal LTP deficits induced by the NMDAR channel blocker ketamine. Finally, we show that synthetic drug-like derivatives of 24(S)-HC, which potently enhance NMDAR-mediated EPSCs and LTP, restore behavioral and cognitive deficits in rodents treated withNMDARchannel blockers. Thus, 24(S)-HC may function as an endogenous modulator of NMDARs acting at a novel oxysterol modulatory site that also represents a target for therapeutic drug development. [ABSTRACT FROM AUTHOR]

Abstract: Neurosteroids are potent and effective neuromodulators that are synthesized from cholesterol in the brain. These agents and their synthetic derivatives influence the function of multiple signaling pathways including receptors for γ-aminobutyric acid (GABA) and glutamate, the major inhibitory and excitatory neurotransmitters in the central nervous system (CNS). Increasing evidence indicates that dysregulation of neurosteroid production plays a role in the pathophysiology of stress and stress-related psychiatric disorders, including mood and anxiety disorders. In this paper, we review the mechanisms of neurosteroid action in brain with an emphasis on those neurosteroids that potently modulate the function of GABAA receptors. We then discuss evidence indicating a role for GABA and neurosteroids in stress and depression, and focus on potential strategies that can be used to manipulate CNS neurosteroid synthesis and function for therapeutic purposes. [Copyright &y& Elsevier]

G-protein-activated inward-rectifying K+ (GIRK) channels hyperpolarize neurons to inhibit synaptic transmission throughout the nervous system. By accelerating G-protein deactivation kinetics, the regulator of G-protein signaling (RGS) protein family modulates the timing of GIRK activity. Despite many investigations, whether RGS proteins modulate GIRK activity in neurons by mechanisms involving kinetic coupling, collision coupling, or macromolecular complex formation has remained unknown. Here we show that GIRK modulation occurs by channel assembly with R7-RGS/ Gβ5 complexes under allosteric control of R7 RES-binding protein (R7BP). Elimination of R7BP occludes the Gβ5 subunit that interacts with GIRK channels. R7BP-bound R7-RGS/Gβ5 complexes and Gβγ dimers interact noncompetitively with the intracellular domain of GIRK channels to facilitate rapid activation and deactivation of GIRK currents. By disrupting this allosterically regulated assembly mechanism, R7BP ablation augments GIRK activity. This enhanced GIRK activity increases the drug effects of agonists acting at Gprotein-coupled receptors that signal via GIRK channels, as indicated by greater antinociceptive effects of GABA(B) or μ-opioid receptor agonists. These findings show that GIRK current modulation in vivo requires channel assembly with allosterically regulated RGS protein complexes, which provide a target for modulating GIRK activity in neurological disorders in which these channels have crucial roles, including pain, epilepsy, Parkinson's disease and Down syndrome. [ABSTRACT FROM AUTHOR]

Synaptic function and plasticity are crucial for information processing within the nervous system. In glutamatergic hippocampal neurons, presynaptic function is silenced, or muted, after strong or prolonged depolarization. This muting is neuroprotective, but the underlying mechanisms responsible for muting and its reversal, unmuting, remain to be clarified. Using cultured rat hippocampal neurons, we found that muting induction did not require protein synthesis; however, slow forms of unmuting that depend on protein kinase A (PKA), including reversal of depolarization-induced muting and forskolin-induced unmuting of basally mute synapses, required protein synthesis. In contrast, fast unmuting of basally mute synapses by phorbol esters was protein synthesis-independent. Further studies of recovery from depolarization-induced muting revealed that protein levels of Rim1 and Munc13-1, which mediate vesicle priming, correlated with the functional status of presynaptic terminals. Additionally, this form of unmuting was prevented by both transcription and translation inhibitors, so proteins are likely synthesized de novo after removal of depolarization. Phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB), a nuclear transcription factor, was elevated after recovery from depolarization-induced muting, consistent with a model in which PKA-dependent mechanisms, possibly including pCREBactivated transcription, mediate slow unmuting. In summary, we found that protein synthesis was required for slower, PKAdependent unmuting of presynaptic terminals, but it was not required for muting or a fast form of unmuting. These results clarify some of the molecular mechanisms responsible for synaptic plasticity in hippocampal neurons and emphasize the multiple mechanisms by which presynaptic function is modulated. [ABSTRACT FROM AUTHOR]

Astrocytes contribute to many neuronal functions, including synaptogenesis, but their role in the development of synaptic plasticity remains unclear. Presynaptic muting of hippocampal glutamatergic terminals defends against excitotoxicity. Here we studied the role of astrocytes in the development of presynaptic muting at glutamatergic synapses in rat hippocampal neurons. We found that astrocytes were critical for the development of depolarization-dependent and Gvo-dependent presynaptic muting. The ability of cAMP analogues to modulate presynaptic function was also impaired by astrocyte deficiency. Although astrocyte deprivation resulted in postsynaptic glutamate receptor deficits, this effect appeared independent of astrocytes' role in presynaptic muting. Muting was restored with chronic, but not acute, treatment with astrocyte-conditioned medium, indicating that a soluble factor is permissive for muting. Astrocyte-derived thrombospondins (TSPs) are likely responsible because TSP1 mimicked the effect of conditioned medium, and gabapentin, a high-affinity antagonist of TSP binding to the α2δ-1 calcium channel subunit, mimicked astrocyte deprivation. We found evidence that protein kinase A activity is abnormal in astrocyte-deprived neurons but restored by TSP1, so protein kinase A dysfunction may provide a mechanism by which muting is disrupted during astrocyte deficiency. In summary our results suggest an important role for astrocyte-derived TSPs, acting through α2δ-1, in maturation of a potentially important form of presynaptic plasticity. [ABSTRACT FROM AUTHOR]

Excessive NMDA receptor activation and excitotoxicity underlies pathology in many neuropsychiatric and neurological disorders, including hypoxia/ischemia. Thus, the development of effective therapeutics for these disorders demands a complete understanding of NMDAreceptor (NMDAR) activation during excitotoxic insults. The extrasynaptic NMDA Rhypothesis posits that synaptic NMDARsare neurotrophic/neuroprotective and extrasynaptic NMDARs are neurotoxic. The extrasynaptic hypothesis is built in part on observed selectivity for extrasynaptic receptors of a neuroprotective use-dependent NMDAR channel blocker, memantine. In rat hippocampal neurons, we found that a neuroprotective concentration of memantine shows little selectivity for extrasynaptic NMDARs when all receptors are tonically activated by exogenous glutamate. This led us to test the extrasynaptic NMDAR hypothesis using metabolic challenge, where the source of excitotoxic glutamate buildup may be largely synaptic. Three independent approaches suggest strongly that synaptic receptors participate prominently in hypoxic excitotoxicity. First, block of glutamate transporters with a nonsubstrate antagonist exacerbated rather than prevented damage, consistent with a primarily synaptic source of glutamate. Second, selective, preblock of synaptic NMDARs with a slowly reversible, use-dependent antagonist protected nearly fully against prolonged hypoxic insult. Third, glutamate pyruvate transaminase, which degrades ambient but not synaptic glutamate, did not protect against hypoxia but protected against exogenous glutamate damage. Together, these results suggest that synaptic NMDARs can mediate excitotoxicity, particularly when the glutamate source is synaptic and when synaptic receptor contributions are rigorously defined. Moreover, the results suggest that in some situations therapeutically targeting extrasynaptic receptors may be inappropriate. [ABSTRACT FROM AUTHOR]