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Synucleinopathies such as Parkinson’s disease (PD) are characterized by pathologic production, aggregation, and cell-to-cell transmission of α-synuclein (α-syn) protein that results in impaired cellular function. While neurons of the substantia nigra pars compacta express high levels of α-synuclein and are highly vulnerable to its aberrant expression or conformation, brain-resident macrophages (microglia) are also sensitive to abnormal α-synuclein, with recent reports indicating that elevated levels impair phagocytic abilityin vivoandin vitro. To explore the impact of elevated α-syn on microglial function we employed a co-culture model containing iPSC-derived neurons and microglia-like cells. iPSCs from healthy control donors and a Parkinson’s donor with an allelic triplication of theSNCAgene locus were differentiated into neurons and microglia-like cells. In monoculture, neurons and microglia generated from theSNCAtriplication donor expressed higher levels ofSNCAtranscript and protein. Neurons were found to have significantly greater expression ofSNCAcompared to microglia, regardless of donor genotype. Co-cultures of neurons and microglia revealed that microglia cultured withSNCAtriplication neurons displayed reduction in phagocytosis of fluorescentE. coli, irrespective of microglia donor genotype.SNCAmRNA and protein expression could be reduced with treatment with an antisense oligonucleotide (ASO) targetingSNCA. ASO treatment partially rescued microglia phagocytosis inSNCAtriplication co-cultures and in co-cultures containingSNCAtriplication neurons and healthy control microglia. Our results complement and extend previous findings of impaired microglial function in the presence of elevated α-synuclein in a novel patient-derived co-culture model that utilizes more disease-relevant conditions rather than the relaying on the addition of exogenous α-synuclein.

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Nav1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund’s adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia and models of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The observed efficacy could be correlated with the mouse dorsal root ganglion exposure and NaV1.7 potency associated with 29.

(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169) and the phosphate prodrug 4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl dihydrogen phosphate (BMS-986163) were identified from a drug discovery effort focused on the development of novel, intravenous glutamate N-methyl-d-aspartate 2B receptor (GluN2B) negative allosteric modulators (NAMs) for treatment-resistant depression (TRD). BMS-986169 showed high binding affinity for the GluN2B subunit allosteric modulatory site (Ki = 4.03-6.3 nM) and selectively inhibited GluN2B receptor function in Xenopus oocytes expressing human N-methyl-d-aspartate receptor subtypes (IC50 = 24.1 nM). BMS-986169 weakly inhibited human ether-a-go-go-related gene channel activity (IC50 = 28.4 μM) and had negligible activity in an assay panel containing 40 additional pharmacological targets. Intravenous administration of BMS-986169 or BMS-986163 dose-dependently increased GluN2B receptor occupancy and inhibited in vivo [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding, confirming target engagement and effective cleavage of the prodrug. BMS-986169 reduced immobility in the mouse forced swim test, an effect similar to intravenous ketamine treatment. Decreased novelty suppressed feeding latency, and increased ex vivo hippocampal long-term potentiation was also seen 24 hours after acute BMS-986163 or BMS-986169 administration. BMS-986169 did not produce ketamine-like hyperlocomotion or abnormal behaviors in mice or cynomolgus monkeys but did produce a transient working memory impairment in monkeys that was closely related to plasma exposure. Finally, BMS-986163 produced robust changes in the quantitative electroencephalogram power band distribution, a translational measure that can be used to assess pharmacodynamic activity in healthy humans. Due to the poor aqueous solubility of BMS-986169, BMS-986163 was selected as the lead GluN2B NAM candidate for further evaluation as a novel intravenous agent for TRD.

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Fragile X syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5′‐UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP‐dependent mutant phenotypes. Here, we utilize FXS patient‐derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP‐positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP‐expressing neurons in a mosaic pattern is sufficient to normalize a FMRP‐dependent, hyperactive phenotype in FXS iPSC‐derived neurons., Previous studies have demonstrated that near full restoration of FMRP levels in Fragile X syndrome iPSC‐derived neuronal models can be achieved using gene editing tools; however, the minimum level of FMRP expression needed to normalize functional differences has not been established. In this study, we have confirmed a spontaneous, electrically hyperactive FXS phenotype in multiple iPSC‐derived neuronal lines. We then used CRISPR gene editing, cell mixing models, ASO‐mediated knockdown of FMR1 and targeted demethylation of CGG repeats to demonstrate that partial expression of FMRP can correct the hyperactive phenotype in iPSC‐derived FXS neurons.

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of pathological accumulations of misfolded, phosphorylated α-synuclein (αSyn) protein. Multiple lines of evidence indicate that synucleinopathy disease progression is driven by a prion-like process of transmission of a pathologic form of αSyn. One potential therapeutic approach to prevent cell-to-cell propagation is to target this transmissible species with selective antibodies. In this study, a rodent primary neuronal culture reporter system was developed to monitor induction of detergent-insoluble, phosphorylated (pS129) aggregates of αSyn. Induction of pS129 αSyn pathology was observed with both synthetic αSyn fibrils (PFFs) and brain lysates from multiple system atrophy (MSA) patients but not αSyn monomers or human brain lysate controls. The induction-competent species in MSA lysates could be enriched by high-speed centrifugation suggesting that it is present as a high molecular weight aggregate. Furthermore, samples derived from brain lysates from Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) patients also induced pS129 αSyn pathology, but required longer incubation times. Lastly, the potential of αSyn selective antibodies to immunodeplete induction-competent forms of αSyn from both PFF and synucleinopathy brain samples is described. The results demonstrate that antibodies targeting the C-terminal of αSyn are most effective for immunodepletion of pathology-inducing forms of αSyn from samples derived from human synucleinopathy brains. Furthermore, the data support the hypothesis that antibodies that recognize a C-terminal epitope and exhibit selectivity for oligomeric forms over monomeric forms of αSyn represent a desirable target for immunotherapy for synucleinopathy patients.

In vitro phenotypic assays of sensory neuron activity are important tools for identifying potential analgesic compounds. These assays are typically characterized by hyperexcitable and/or abnormally, spontaneously active cells. Whereas manual electrophysiology experiments provide high-resolution biophysical data to characterize both in vitro models and potential therapeutic modalities (e.g., action potential characteristics, the role of specific ion channels, and receptors), these techniques are hampered by their low throughput. We have established a spontaneously active dorsal root ganglia (DRG) platform using multiwell multielectrode arrays (MEAs) that greatly increase the ability to evaluate the effects of multiple compounds and conditions on DRG excitability within the context of a cellular network. We show that spontaneous DRG firing can be attenuated with selective Na+ and Ca2+ channel blockers, as well as enhanced with K+ channel blockers. In addition, spontaneous activity can be augmented with both the transient receptor potential cation channel subfamily V member 1 agonist capsaicin and the peptide bradykinin and completely blocked with neurokinin receptor antagonists. Finally, we validated the use of this assay by demonstrating that commonly used neuropathic pain therapeutics suppress DRG spontaneous activity. Overall, we have optimized primary rat DRG cells on a multiwell MEA platform to generate and characterize spontaneously active cultures that have the potential to be used as an in vitro phenotypic assay to evaluate potential therapeutics in rodent models of pain.

Duchenne Muscular Dystrophy (DMD) is a fatal disease of skeletal and cardiac muscle characterized by progressive muscle weakness and atrophy. Current average life expectancy is in the mid-20s and mortality is often linked to end stage cardiomyopathy. DMD is a monogenic disease caused by loss-of-function mutations in the dystrophin gene. While dystrophin is critical for muscle function by connecting the cytoskeleton to the sarcolemma and extracellular matrix, its absence can be largely compensated for by up-regulation of utrophin (UTRN), the fetal orthologue of dystrophin. Because of the importance of UTRN expression in the heart in both DMD patients and preclinical models, we utilized DMD patient iPSC-derived cardiomyocytes as a relevant cellular model to perform in vitro pharmacology as well as functional assessment. Human iPSC-derived cardiomyocytes recapitulate key properties of bona fide cardiomyocytes, such as expression of cardiac genes, spontaneous contractility, and response to chronotropic drugs (eg. isoproterenol and hERG channel blockers). We have created an isogenic pair of healthy control and DMD iPSC lines with exon 51 deletion using CRISPR/Cas9 technology. Functional measurements of cardiomyocyte electrophysiology were performed by multielectrode array (MEA) and fluorometric imaging plate reader (FLIPR) platforms. High-throughput assays were developed to measure UTRN expression in cardiomyocyte culture in 384-well plates. Importantly, we have identified small molecules that increased UTRN expression in both patient and isogenic DMD iPSC-derived cardiomyocytes. Our study represents a valuable strategy for small molecule drug discovery for the treatment of DMD cardiomyopathy based on iPSC technology.

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5′ UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS. Rescue of fragile X syndrome neurons by CRISPR-mediated DNA methylation editing of the FMR1 gene., National Institutes of Health (Grant HD045022), National Institutes of Health (Grant MH104610), National Institutes of Health (Grant R01NS088538), National Institutes of Health (Grant R01GM123511)

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Here we validate the design and use of a novel, customized electrophysiology system (Slice XVIvo™) that is capable of recording from 16 independent brain slices. The system consists of 16 independent recording chambers in which individual electrodes can be manually manipulated and fixed in order to stimulate and record extracellular responses from 16 brain slices simultaneously. Responses from each brain slice are elicited with individual stimulus isolator units and recorded through separate channels, thus allowing for independent control and analysis of the evoked extracellular activity from each slice. The system was designed to fit on a standard anti-vibration table, thus the Slice XVIvo™ system occupies considerably less space than other currently available multi-slice recording systems. We have demonstrated the utility of the system to obtain stable, extracellular responses from the CA1 region of the hippocampus, as well as induce long-term potentiation. Additionally, we show the utility of the Slice XVIvo™ system to significantly improved throughput for testing compounds in an oxygen and glucose deprivation assay. Overall, we have designed, created and validated a considerably cost- and space-efficient electrophysiology system that greatly improves throughput while minimizing the number of animals used in experiments.

The alpha7 (α7) nicotinic acetylcholine receptor is a therapeutic target for cognitive disorders. Here we describe 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide (B-973), a novel piperazine-containing molecule that acts as a positive allosteric modulator of the α7 receptor. We characterize the action of B-973 on the α7 receptor using electrophysiology and radioligand binding. At 0.1 mM acetylcholine, 1 μM B-973 potentiated peak acetylcholine-induced currents 6-fold relative to maximal acetylcholine (3 mM) and slowed channel desensitization, resulting in a 6900-fold increase in charge transfer. The EC 50 of B-973 was approximately 0.3 μM at acetylcholine concentrations ranging from 0.03 to 3 mM. At a concentration of 1 μM, B-973 shifted the acetylcholine EC 50 of peak currents from 0.30 mM in control to 0.007 mM. B-973 slowed channel deactivation upon acetylcholine removal (τ=50 s) and increased the affinity of the α7 agonist [ 3 H]A-585539. In the absence of exogenously added acetylcholine, application of B-973 at concentrations >1 μM induced large methyllycaconitine-sensitive currents, suggesting B-973 can function as an Ago-PAM at high concentrations. B-973 will be a useful probe for investigating the biological consequences of increasing α7 receptor activity through allosteric modulation.

Background Chronic ethanol (EtOH) leads to disruptions in resting electroencephalogram (EEG) activity and in sleep patterns that can persist into the withdrawal period. These disruptions have been suggested to be predictors of relapse. The thalamus is a key structure involved in both normal brain oscillations, such as sleep-related oscillations, and abnormal rhythms found in disorders such as epilepsy and Parkinson's disease. Previously, we have shown progressive changes in mouse thalamic T-type Ca2+ channels during chronic intermittent EtOH exposures that occurred in parallel with alterations in theta (4 to 8 Hz) EEG patterns. Methods Two groups of 8-week-old male C57BL/6 mice were implanted with wireless EEG/electromyogram (EMG) telemetry and subjected to 4 weeks of chronic, intermittent EtOH vapor exposure and withdrawal. During the week after the final withdrawal, mice were administered ethosuximide (ETX; 200 mg/kg) or saline. EEG data were analyzed via discrete Fourier transform, and sleep-scored for further analysis. Results Chronic intermittent EtOH exposure produced changes in the diurnal rhythms of the delta (0.5 to 4 Hz) and theta bands that persisted into a subsequent week of sustained withdrawal. These disruptions were restored with the T-channel blocker ETX. Repeated EtOH exposures preferentially increased the relative proportion of lower frequency power (delta and theta), whereas higher frequencies (8 to 24 Hz) were decreased. The EtOH-induced decreases in relative power for the higher frequencies continued into the sustained withdrawal week for both groups. Increases in absolute delta and theta power were observed in averaged nonrapid eye movement and rapid eye movement sleep spectral data during withdrawal in ETX-treated animals, suggesting increased sleep intensity. Conclusions These results suggest that persistent alterations in delta and theta EEG rhythms during withdrawal from chronic intermittent EtOH exposure can be ameliorated with ETX and that this treatment might also increase sleep intensity during withdrawal.

Mechanisms of plasticity are important to the astounding capacity of the brain to adapt and learn. Ion channels are significant contributors to neuronal plasticity, but their dysfunction has been implicated in several nervous system diseases from movement disorders to epilepsy. Although many inherited ion channel mutations have been associated with these disorders, it has been recently recognized that channelopathies can also include aberrant ion channel function that is acquired after an insult or injury to the brain. These acquired alterations are being investigated in animal models of temporal lobe epilepsy, where studies have shown functional changes in voltage-gated ion channels that lead to increases in excitability. Studies of these hyperexcitable neurons have included recordings in the hippocampus, entorhinal cortex, and thalamus and support the existence of an extended seizure network with several nodes of altered activity that are established during epileptogenesis. A better understanding of the key ion channels and brain regions that are responsible for the development of this hyperexcitability, along with the molecular mechanisms involved, may provide novel treatment strategies for epilepsy.

Some epilepsies are linked to inherited traits, but many appear to arise through acquired alterations in neuronal excitability. Status epilepticus (SE) is associated with numerous changes that promote spontaneous recurrent seizures (SRS), and studies have suggested that hippocampal T-type Ca2+channels underlie increased bursts of activity integral to the generation of these seizures. The thalamus also contributes to epileptogenesis, but no studies have directly assessed channel alterations in the thalamus during SE or subsequent periods of SRS. We therefore investigated longitudinal changes in thalamic T-type channels in a mouse pilocarpine model of epilepsy. T-type channel gene expression was not affected during SE; however CaV3.2 mRNA was significantly upregulated at both 10 d post-SE (seizure-free period) and 31 d post-SE (SRS-period). Overall T-type current density increased during the SRS period, and the steady-state inactivation shifted from a more hyperpolarized membrane potential during the latent stage, to a more depolarized membrane potential during the SRS period. CaV3.2 functional involvement was verified with CaV3.2 inhibitors that reduced the native T-type current in mice 31 d post-SE, but not in controls. Burst discharges of thalamic neurons reflected the changes in whole-cell currents, and we used a computational model to relate changes observed during epileptogenesis to a decreased tendency to burst in the seizure-free period, or an increased tendency to burst during the period of SRS. We conclude that SE produces an acquired channelopathy by inducing long-term alterations in thalamic T-type channels that contribute to characteristic changes in excitability observed during epileptogenesis and SRS.

(+/-)-Mecamylamine is a racemic mixture of a widely used brain-permeant noncompetitive inhibitor of muscle-type and neuronal nicotinic receptors (NNRs). The present studies evaluated whether the stereoisomers of this drug show different profiles for inhibition of the high-sensitivity (HS) and low-sensitivity (LS) isoforms of the human alpha4beta2 NNR subtype expressed in subclonal human epithelial 1 cells. We found that at low concentrations (micromolar range), TC-5214 [S-(+)-mecamylamine] was more effective than TC-5213 [R-(-)-mecamylamine] in inhibiting the LS alpha4beta2 NNRs. In addition, we demonstrated that TC-5214 potentiated and TC-5213 inhibited agonist-induced activation of HS alpha4beta2 NNRs. The stereoselectivity of mecamylamine enantiomers at HS and LS alpha4beta2 receptors demonstrates that TC-5214 is the preferred stereoisomer for selective activation of HS, whereas it is more effective in suppressing LS receptor function. This feature could be relevant to therapeutic applications where such a selective mechanism of action is required.

The long lasting antidepressant response seen following acute, i.v. ketamine administration in patients with treatment-resistant depression (TRD) is thought to result from enhanced synaptic plasticity in cortical and hippocampal circuits. Using extracellular field recordings in rat hippocampal slices, we show that a single dose of the non-selective NMDA receptor antagonist ketamine or CP-101,606, a selective antagonist of the NR2B subunit of the NMDA receptor, enhances hippocampal synaptic plasticity induced with high frequency stimulation (HFS) 24h after dosing - a time at which plasma concentrations of the drug are no longer detectable in the animal. These results indicate that acute inhibition of NMDA receptors containing the NR2B subunit can lead to long-lasting changes in hippocampal plasticity.

High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3β4 and hα4β2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC(50) values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHβE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

Fast solution exchange techniques have revolutionized the study of synaptic transmission and promise to remain an important neuroscience research tool. Here we provide evidence for the hypothesis that using continuous, rapid transitions through an agonist solution can significantly increase the exchange rate around a cell by reducing the diffusion boundary at the membrane. This novel approach of rapid solution exchange during whole-cell recordings – described as a “liquid bullet” (LB) application – takes advantage of a bidirectional solution flow around the cell, allowing for a full solution exchange within a range of several milliseconds. An exchange rate (10–90% rise time) of about 2 ms could be achieved during both agonist application and washout. We recorded whole-cell currents from cells expressing the rapidly desensitizing α7 neuronal nicotinic receptor (NNR) subtype that exhibited very fast rise times of around 4–5 ms. We further demonstrated the advantages of a LB application over conventional methods by the ability of this method to elicit concentration-dependent responses for rapidly desensitizing compounds that were not measurable with conventional agonist applications. In addition, we illustrate the utility of this approach for frequency-based assays through fast, repeated agonist applications at frequencies of 1 Hz and 30 Hz. This approach could therefore be useful for the study of rapid agonist–receptor interactions that closely mimic the physiological conditions in the synaptic cleft during bursts of neuronal activity.

Early-life stress has been shown to increase sus- ceptibility to anxiety and substance abuse. Disrupted activity within the anterior insular cortex (AIC) has been shown to play a role in both of these disorders. Altered serotonergic processing is implicated in controlling the activity levels of the associated cognitive networks. We therefore investigated changes in both serotonin receptor expression and glutama- tergic synaptic activity in the AIC of alcohol-drinking rhesus monkeys. We studied tissues from male rhesus monkeys raised under two conditions: Male rhesus monkeys (1) "mother reared" (MR) by adult females (n9) or (2) "Nursery reared" (NR), that is, separated from their mothers and reared as a separate group under surrogate/peer-reared conditions (n9). The NR condition represents a long-standing and well- validated nonhuman primate model of early life stress. All monkeys were trained to self-administer ethanol (4% w/v) or an isocaloric maltose-dextrin control solution. Subsets from each rearing condition were then given daily access to etha- nol, water, or maltose-dextrin for 12 months. Tissues were collected at necropsy and were further analyzed. Using real time RT-PCR we found that ethanol-naive, NR monkeys had lower AIC levels of 5-HT1A and 5-HT2A receptor mRNA com- pared with ethanol-naive, MR animals. Although NR monkeys consumed more ethanol over the 12-month period compared with MR animals, both MR and NR animals expressed greater 5-HT1A and 5-HT2A receptor mRNA levels following chronic alcohol self-administration. The interaction between nursery- rearing conditions and alcohol consumption resulted in a significant enhancement of both 5-HT1A and 5-HT2A receptor mRNA levels such that lower expression levels observed in nursery-rearing conditions were not found in the alcohol self-administration group. Using voltage clamp recordings in the whole cell configuration we recorded excitatory postsyn- aptic currents in both ethanol-naive and chronic self-admin- istration groups of NR and MR monkeys. Both groups that self-administered ethanol showed greater glutamatergic ac- tivity within the AIC. This AIC hyperactivity in MR alcohol- consuming monkeys was accompanied by an increased sen- sitivity to regulation by presynaptic 5-HT1A receptors that was not apparent in the ethanol-naive, MR group. Our data indicate that chronic alcohol consumption leads to greater AIC activity and may indicate a compensatory upregulation of presynaptic 5-HT1A receptors. Our results also indicate that AIC activity may be less effectively regulated by 5-HT in ethanol-naive NR animals than in NR monkeys in response to chronic ethanol self-administration. These data suggest pos- sible mechanisms for increased alcohol seeking and possi- ble addiction potential among young adults who had previ- ously experienced early-life stress that include disruptions in both AIC activity and serotonin system dynamics. © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Chronic ethanol exposure produces profound disruptions in both brain rhythms and diurnal behaviors. The thalamus has been identified as a neural pacemaker of both normal and abnormal rhythms with low-threshold, transient (T-type) Ca2+channels participating in this activity. We therefore examined T-type channel gene expression and physiology in the thalamus of C57Bl/6 mice during a 4-wk schedule of chronic intermittent ethanol exposures in a vapor chamber. We found that chronic ethanol disrupts the normal daily variations of both thalamic T-type channel mRNA levels and alters thalamic T-type channel gating properties. The changes measured in channel expression and function were associated with an increase in low-threshold bursts of action potentials during acute withdrawal periods. Additionally, the observed molecular and physiological alterations in the channel properties in wild-type mice occurred in parallel with a progressive disruption in the normal daily variations in theta (4–9 Hz) power recorded in the cortical electroencephalogram. Theta rhythms remained disrupted during a subsequent week of withdrawal but were restored with the T-type channel blocker ethosuximide. Our results demonstrate that a key ion channel underlying the generation of thalamic rhythms is altered during chronic ethanol exposure and withdrawal and may be a novel target in the management of abnormal network activity due to chronic alcoholism.