Sex differences in α-adrenergic receptor function contribute to impaired hypothalamic metaplasticity following chronic intermittent ethanol exposure.

BACKGROUND: Individuals with alcohol use disorder (AUD) exhibit maladaptive responses of the hypothalamic-pituitary-adrenal (HPA) axis to stress, which has been linked to high rates of relapse to drinking among abstinent individuals. Corticotropin-releasing factor (CRF) parvocellular neuroendocrine cells (PNCs) within the paraventricular nucleus of the hypothalamus (PVN) are critical to stress-induced HPA axis activation. Here, we investigate sex differences in synaptic transmission and plasticity in PNCs following the application of the stress-associated neurotransmitter norepinephrine (NE) in a rat model of AUD. METHODS: Adult Sprague-Dawley rats were exposed to 40 days of chronic intermittent ethanol (CIE) vapor and 30 to 108 days of protracted withdrawal. We measured changes in holding current, evoked synaptic currents, and short-term glutamatergic plasticity (STP) in putative PNCs following the application of NE (10 µM) with and without the selective α1 adrenergic receptor (AR) antagonist prazosin (10 µM) or the α2AR antagonist atipamezole (10 µM). The experiments were performed using whole-cell patch clamp recordings in slices from CIE rats and air-exposed controls. RESULTS: NE application caused two distinct effects: a depolarizing, inward, postsynaptic current and a reduction in amplitude of an evoked glutamatergic excitatory postsynaptic current (eEPSC). Both effects were sex- and CIE-specific. Prazosin blocked the postsynaptic inward current, while atipamezole blocked the NE-mediated suppression of eEPSCs. Additionally, STP formation was facilitated following NE application only in stress-naïve males and this response was lost in stressed animals exposed to a 30-min restraint stress following CIE exposure. Furthermore, NE + prazosin restored STP formation in stressed CIE males. CONCLUSIONS: NE exerts excitatory and inhibitory effects on CRF PVN PNCs, and both effects are influenced by sex and CIE. Behavioral and hormonal responses to stress are influenced by STP formation within the PVN, which is lost following CIE and restored with the preapplication of prazosin. The selective blockade of α1AR may, therefore, ameliorate CIE-induced deficits in HPA responses to stress in a sex-specific manner.

Circuit-Specific Early Impairment of Proprioceptive Sensory Neurons in the SOD1G93A Mouse Model for ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.

α2 Subunit-Containing GABAA Receptor Subtypes Are Upregulated and Contribute to Alcohol-Induced Functional Plasticity in the Rat Hippocampus.

Alcohol (EtOH) intoxication causes changes in the rodent brain γ-aminobutyric acid receptor (GABAAR) subunit composition and function, playing a crucial role in EtOH withdrawal symptoms and dependence. Building evidence indicates that withdrawal from acute EtOH and chronic intermittent EtOH (CIE) results in decreased EtOH-enhanced GABAAR δ subunit-containing extrasynaptic and EtOH-insensitive α1ßγ2 subtype synaptic GABAARs but increased synaptic α4ßγ2 subtype, and increased EtOH sensitivity of GABAAR miniature postsynaptic currents (mIPSCs) correlated with EtOH dependence. Here we demonstrate that after acute EtOH intoxication and CIE, upregulation of hippocampal α4ßγ2 subtypes, as well as increased cell-surface levels of GABAAR α2 and γ1 subunits, along with increased α2ß1γ1 GABAAR pentamers in hippocampal slices using cell-surface cross-linking, followed by Western blot and coimmunoprecipitation. One-dose and two-dose acute EtOH treatments produced temporal plastic changes in EtOH-induced anxiolysis or withdrawal anxiety, and the presence or absence of EtOH-sensitive synaptic currents correlated with cell surface peptide levels of both α4 and γ1(new α2) subunits. CIE increased the abundance of novel mIPSC patterns differing in activation/deactivation kinetics, charge transfer, and sensitivity to EtOH. The different mIPSC patterns in CIE could be correlated with upregulated highly EtOH-sensitive α2ßγ subtypes and EtOH-sensitive α4ßγ2 subtypes. Naïve α4 subunit knockout mice express EtOH-sensitive mIPSCs in hippocampal slices, correlating with upregulated GABAAR α2 (and not α4) subunits. Consistent with α2, ß1, and γ1 subunits genetically linked to alcoholism in humans, our findings indicate that these new α2-containing synaptic GABAARs could mediate the maintained anxiolytic response to EtOH in dependent individuals, rat or human, contributing to elevated EtOH consumption.

Selective modulation of GABAergic tonic current by dopamine in the nucleus accumbens of alcohol-dependent rats.

The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system and plays an important role in mediating alcohol-seeking behaviors. Alterations in glutamatergic and GABAergic signaling were recently demonstrated in the NAcc of rats after chronic intermittent ethanol (CIE) treatment, a model of alcohol dependence. Here we studied dopamine (DA) modulation of GABAergic signaling and how this modulation might be altered by CIE treatment. We show that the tonic current (I(tonic)) mediated by extrasynaptic γ-aminobutyric acid type A receptors (GABA(A)Rs) of medium spiny neurons (MSNs) in the NAcc core is differentially modulated by DA at concentrations in the range of those measured in vivo (0.01-1 µM), without affecting the postsynaptic kinetics of miniature inhibitory postsynaptic currents (mIPSCs). Use of selective D1 receptor (D1R) and D2 receptor (D2R) ligands revealed that I(tonic) potentiation by DA (10 nM) is mediated by D1Rs while I(tonic) depression by DA (0.03-1 µM) is mediated by D2Rs in the same MSNs. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDPßS) to the recording pipettes eliminated I(tonic) decrease by the selective D2R agonist quinpirole (5 nM), leaving intact the quinpirole effect on mIPSC frequency. Recordings from CIE and vehicle control (CIV) MSNs during application of D1R agonist (SKF 38393, 100 nM) or D2R agonist (quinpirole, 2 nM) revealed that SKF 38393 potentiated I(tonic) to the same extent, while quinpirole reduced I(tonic) to a similar extent, in both groups of rats. Our data suggest that the selective modulatory effects of DA on I(tonic) are unaltered by CIE treatment and withdrawal.

Plasticity of GABA(A) receptor-mediated neurotransmission in the nucleus accumbens of alcohol-dependent rats.

Chronic alcohol exposure-induced changes in reinforcement mechanisms and motivational state are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system and plays an important role in mediating alcohol-seeking behaviors. Here we describe the long-lasting alterations of γ-aminobutyric acid type A receptors (GABA(A)Rs) of medium spiny neurons (MSNs) in the NAcc after chronic intermittent ethanol (CIE) treatment, a rat model of alcohol dependence. CIE treatment and withdrawal (>40 days) produced decreases in the ethanol and Ro15-4513 potentiation of extrasynaptic GABA(A)Rs, which mediate the picrotoxin-sensitive tonic current (I(tonic)), while potentiation of synaptic receptors, which give rise to miniature inhibitory postsynaptic currents (mIPSCs), was increased. Diazepam sensitivity of both I(tonic) and mIPSCs was decreased by CIE treatment. The average magnitude of I(tonic) was unchanged, but mIPSC amplitude and frequency decreased and mIPSC rise time increased after CIE treatment. Rise-time histograms revealed decreased frequency of fast-rising mIPSCs after CIE treatment, consistent with possible decreases in somatic GABAergic synapses in MSNs from CIE rats. However, unbiased stereological analysis of NeuN-stained NAcc neurons did not detect any decreases in NAcc volume, neuronal numbers, or neuronal cell body volume. Western blot analysis of surface subunit levels revealed selective decreases in α1 and δ and increases in α4, α5, and γ2 GABA(A)R subunits after CIE treatment and withdrawal. Similar, but reversible, alterations occurred after a single ethanol dose (5 g/kg). These data reveal CIE-induced long-lasting neuroadaptations in the NAcc GABAergic neurotransmission.

Ethanol-induced plasticity of GABAA receptors in the basolateral amygdala.

Acute and chronic ethanol (EtOH) administration is known to affect function, surface expression, and subunit composition of γ-aminobutyric acid (A) receptors (GABAARs) in different parts of the brain, which is believed to play a major role in alcohol dependence and withdrawal symptoms. The basolateral amygdala (BLA) participates in anxiety-like behaviors including those induced by alcohol withdrawal. In the present study we assessed the changes in cell surface levels of select GABAAR subunits in the BLA of a rat model of alcohol dependence induced by chronic intermittent EtOH (CIE) treatment and long-term (>40 days) withdrawal and investigated the time-course of such changes after a single dose of EtOH (5 g/kg, gavage). We found an early decrease in surface expression of α4 and δ subunits at 1 h following single dose EtOH treatment. At 48 h post-EtOH and after CIE treatment there was an increase in α4 and γ2, while α1, α2, and δ surface expression were decreased. To relate functional changes in GABAARs to changes in their subunit composition we analyzed miniature inhibitory postsynaptic currents (mIPSCs) and the picrotoxin-sensitive tonic current (Itonic) 48 h after EtOH intoxication. The Itonic magnitude and most of the mIPSC kinetic parameters (except faster mIPSC decay) were unchanged at 48 h post-EtOH. At the same time, Itonic potentiation by acute EtOH was greatly reduced, whereas mIPSCs became significantly more sensitive to potentiation by acute EtOH. These results suggest that EtOH intoxication-induced GABAAR plasticity in the BLA might contribute to the diminished sedative/hypnotic and maintained anxiolytic effectiveness of EtOH.

Dihydromyricetin prevents fetal alcohol exposure-induced behavioral and physiological deficits: the roles of GABAA receptors in adolescence.

Fetal alcohol exposure (FAE) can lead to a variety of behavioral and physiological disturbances later in life. Understanding how alcohol (ethanol, EtOH) affects fetal brain development is essential to guide the development of better therapeutics for FAE. One of EtOH's many pharmacological targets is the γ-aminobutyric acid type A receptor (GABAAR), which plays a prominent role in early brain development. Acute EtOH potentiates inhibitory currents carried by certain GABAAR subtypes, whereas chronic EtOH leads to persistent alterations in GABAAR subunit composition, localization and function. We recently introduced a flavonoid compound, dihydromyricetin (DHM), which selectively antagonizes EtOH's intoxicating effects in vivo and in vitro at enhancing GABAAR function as a candidate for alcohol abuse pharmacotherapy. Here, we studied the effect of FAE on physiology, behavior and GABAAR function of early adolescent rats and tested the utility of DHM as a preventative treatment for FAE-induced disturbances. Gavage administration of EtOH (1.5, 2.5, or 5.0 g/kg) to rat dams on day 5, 8, 10, 12, and 15 of pregnancy dose-dependently reduced female/male offspring ratios (largely through decreased numbers of female offspring) and offspring body weights. FAE (2.5 g/kg) rats tested on postnatal days (P) 25-32 also exhibited increased anxiety and reduced pentylenetetrazol (PTZ)-induced seizure threshold. Patch-clamp recordings from dentate gyrus granule cells (DGCs) in hippocampal slices from FAE (2.5 g/kg) rats at P25-35 revealed reduced sensitivity of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and tonic current (Itonic) to potentiation by zolpidem (0.3 µM). Interestingly, potentiation of mIPSCs by gaboxadol increased, while potentiation of Itonic decreased in DGCs from FAE rats. Co-administration of EtOH (1.5 or 2.5 g/kg) with DHM (1.0 mg/kg) in pregnant dams prevented all of the behavioral, physiological, and pharmacological alterations observed in FAE offspring. DHM administration alone in pregnant rats had no adverse effect on litter size, progeny weight, anxiety level, PTZ seizure threshold, or DGC GABAAR function. Our results indicate that FAE induces long-lasting alterations in physiology, behavior, and hippocampal GABAAR function and that these deficits are prevented by DHM co-treatment of EtOH-exposed dams. The absence of adverse side effects and the ability of DHM to prevent FAE consequences suggest that DHM is an attractive candidate for development as a treatment for prevention of fetal alcohol spectrum disorders.

Dihydromyricetin as a novel anti-alcohol intoxication medication.

Alcohol use disorders (AUDs) constitute the most common form of substance abuse. The development of AUDs involves repeated alcohol use leading to tolerance, alcohol withdrawal syndrome, and physical and psychological dependence, with loss of ability to control excessive drinking. Currently there is no effective therapeutic agent for AUDs without major side effects. Dihydromyricetin (DHM; 1 mg/kg, i.p. injection), a flavonoid component of herbal medicines, counteracted acute alcohol (EtOH) intoxication, and also withdrawal signs in rats including tolerance, increased anxiety, and seizure susceptibility; DHM greatly reduced EtOH consumption in an intermittent voluntary EtOH intake paradigm in rats. GABA(A) receptors (GABA(A)Rs) are major targets of acute and chronic EtOH actions on the brain. At the cellular levels, DHM (1 µM) antagonized both acute EtOH-induced potentiation of GABA(A)Rs and EtOH exposure/withdrawal-induced GABA(A)R plasticity, including alterations in responsiveness of extrasynaptic and postsynaptic GABA(A)Rs to acute EtOH and, most importantly, increases in GABA(A)R α4 subunit expression in hippocampus and cultured neurons. DHM anti-alcohol effects on both behavior and CNS neurons were antagonized by flumazenil (10 mg/kg in vivo; 10 µM in vitro), the benzodiazepine (BZ) antagonist. DHM competitively inhibited BZ-site [(3)H]flunitrazepam binding (IC(50), 4.36 µM), suggesting DHM interaction with EtOH involves the BZ sites on GABA(A)Rs. In summary, we determined DHM anti-alcoholic effects on animal models and determined a major molecular target and cellular mechanism of DHM for counteracting alcohol intoxication and dependence. We demonstrated pharmacological properties of DHM consistent with those expected to underlie successful medical treatment of AUDs; therefore DHM is a therapeutic candidate.

Stress increases voluntary alcohol intake, but does not alter established drinking habits in a rat model of posttraumatic stress disorder.

BACKGROUND: Life-altering anxiety disorders, such as posttraumatic stress disorder (PTSD), can co-occur at high rates with substance use disorders. Alcoholism, compared with other substance use disorders, is particularly common. Rodent studies of acute stress effects on alcohol consumption show that stress can alter ethanol (EtOH) consumption. This study examined voluntary EtOH consumption in male Long-Evans rats that had undergone a stress-enhanced fear learning (SEFL) procedure. METHODS: Adult Long-Evans rats were exposed to a stress that consisted of 15 inescapable foot-shocks (1 mA, 1 second) known to cause a long-lasting nonassociative enhancement of subsequent fear learning. Control animals received no shock. One day later, animals were placed in a novel and very different context and received a single foot-shock. On day 3, animals were returned to the single shock context and freezing was used as a measure of learned fear. The intermittent access 2-bottle choice (2BC) regimen consisted of 1 bottle of water and 1 bottle of experimental solution, either 19% EtOH or 28.4% sucrose-0.08% quinine, for a 24-hour period, 3 days a week, and all other times 2 water bottles. This regimen lasted until stable levels of experimental solution drinking were reached, at which point the experimental solution was removed for 40 days and then returned to measure the resumption of consumption. RESULTS: Rats that received stress prior to EtOH consumed significantly more EtOH than control rats before and after reinstatement. Rats that received stress after drinking was established did not consume significantly more EtOH when the drug was returned. Stress had no significant effect on sucrose-quinine drinking, our calorie and taste control for EtOH. CONCLUSIONS: A single traumatic event sufficient to produce long-lasting enhancement of fear learning increases voluntary EtOH consumption, but does not alter previously acquired EtOH drinking habits or alter the consumption of a calorically equivalent sweet-bitter-tasting solution.

Dissociable contributions of basolateral amygdala and ventrolateral orbitofrontal cortex to flexible learning under uncertainty.

Reversal learning measures the ability to form flexible associations between choice outcomes with stimuli and actions that precede them. This type of learning is thought to rely on several cortical and subcortical areas, including highly interconnected orbitofrontal cortex (OFC) and basolateral amygdala (BLA), and is often impaired in various neuropsychiatric and substance use disorders. However, unique contributions of these regions to stimulus- and action-based reversal learning have not been systematically compared using a chemogenetic approach and particularly before and after the first reversal that introduces new uncertainty. Here, we examined the roles of ventrolateral OFC (vlOFC) and BLA during reversal learning. Male and female rats were prepared with inhibitory DREADDs targeting projection neurons in these regions and tested on a series of deterministic and probabilistic reversals during which they learned about stimulus identity or side (left or right) associated with different reward probabilities. Using a counterbalanced within-subject design, we inhibited these regions prior to reversal sessions. We assessed initial and pre-post reversal changes in performance to measure learning and adjustments to reversals, respectively. We found that inhibition of vlOFC, but not BLA, eliminated adjustments to stimulus-based reversals. Inhibition of BLA, but not vlOFC, selectively impaired action-based probabilistic reversal learning, leaving deterministic reversal learning intact. vlOFC exhibited a sex-dependent role in early adjustment to action-based reversals, but not in overall learning. These results reveal dissociable roles for BLA and vlOFC in flexible learning and highlight a more crucial role for BLA in learning meaningful changes in the reward environment.

Relationship of axonal voltage-gated sodium channel 1.8 (NaV1.8) mRNA accumulation to sciatic nerve injury-induced painful neuropathy in rats.

Painful peripheral neuropathy is a significant clinical problem; however, its pathological mechanism and effective treatments remain elusive. Increased peripheral expression of tetrodotoxin-resistant voltage-gated sodium channel 1.8 (NaV1.8) has been shown to associate with chronic pain symptoms in humans and experimental animals. Sciatic nerve entrapment (SNE) injury was used to develop neuropathic pain symptoms in rats, resulting in increased NaV1.8 mRNA in the injured nerve but not in dorsal root ganglia (DRG). To study the role of NaV1.8 mRNA in the pathogenesis of SNE-induced painful neuropathy, NaV1.8 shRNA vector was delivered by subcutaneous injection of cationized gelatin/plasmid DNA polyplex into the rat hindpaw and its subsequent retrograde transport via sciatic nerve to DRG. This in vivo NaV1.8 shRNA treatment reversibly and repeatedly attenuated the SNE-induced pain symptoms, an effect that became apparent following a distinct lag period of 3-4 days and lasted for 4-6 days before returning to pretreatment levels. Surprisingly, apparent knockdown of NaV1.8 mRNA occurred only in the injured nerve, not in the DRG, during the pain alleviation period. Levels of heteronuclear NaV1.8 RNA were unaffected by SNE or shRNA treatments, suggesting that transcription of the Scn10a gene encoding NaV1.8 was unchanged. Based on these data, we postulate that increased axonal mRNA transport results in accumulation of functional NaV1.8 protein in the injured nerve and the development of painful neuropathy symptoms. Thus, targeted delivery of agents that interfere with axonal NaV1.8 mRNA may represent effective neuropathic pain treatments.

Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment.

Chronic pain arising from peripheral nerve injuries represents a significant clinical challenge because even the most efficacious anticonvulsant drug treatments are limited by their side effects profile. We investigated pain behavior, changes in axonal signal conduction and excitability of trigeminal neurons, and expression of voltage-gated sodium channels (NaVs) in the infraorbital nerve and trigeminal ganglion (TG) after infraorbital nerve entrapment (IoNE). Compared to Sham, IoNE rats had increased A- and C-fiber compound action potentials (CAPs) and Aδ component of A-CAP area from fibers innervating the vibrissal pad. After IoNE, A- and C-fiber CAPs were more sensitive to blockade by tetrodotoxin (TTX), and those fibers that were TTX-resistant were more sensitive to blockade by the NaV1.8 selective blocker, A-803467. Although NaV1.7 blocker, ICA-121431 alone, did not affect Aδ-fiber signal propagation, cumulative application with A-803467 and 4,9-anhydro-TTX significantly reduced the Aδ-fiber CAP in IoNE rats. In patch clamp recordings from small- and medium-sized TG neurons, IoNE resulted in reduced action potential (AP) depolarizing current threshold, hyperpolarized AP voltage threshold, increased AP duration, and a more depolarized membrane potential. While the transcripts of most NaVs were reduced in the ipsilateral TG after IoNE, NaV1.3, NaV1.7, and NaV1.8 mRNAs, and NaV1.8 protein, were significantly increased in the nerve. Altogether, our data suggest that axonal redistribution of NaV1.8, and to a lesser extent NaV1.3, and NaV1.7 contributes to enhanced nociceptive signal propagation in peripheral nerve after IoNE.

Brain Penetrant, but not Peripherally Restricted, Synthetic Cannabinoid 1 Receptor Agonists Promote Morphine-Mediated Respiratory Depression.

Introduction: Cannabis acceptance and use continues to rise despite the gaps in knowledge regarding the mechanisms of cannabinoids and the endocannabinoid system in many physiological functions, including respiratory influence. Methods: With recent evidence of cannabinoid receptor 1 (CB1R) presence in the collection of respiratory neurons in the brainstem, as well as in the peripheral lung tissue, it is vital that the mechanisms involved in central and peripheral CB1R modulation of respiratory function be delineated. In this study we sought to define the roles of central versus peripheral CB1R activation on respiratory depression alone and in combination with morphine using whole body plethysmography. Results: We show that the peripherally restricted CB1 agonist (4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine [PrNMI] 0.3, 0.6, and 1 mg/kg) does not induce respiratory depression, while our previous studies showed that a central penetrating synthetic cannabinoid does induce respiratory depression. Significantly, the combination of morphine with the peripheral CB1 agonist, PrNMI, attenuated morphine-induced respiratory depression. Conclusions: These studies support that a peripherally restricted CB1R agonist may be a unique strategy to attenuate the respiratory depression associated with opioid therapy.

Plasticity of GABAA receptors after ethanol pre-exposure in cultured hippocampal neurons.

Alcohol use causes many physiological changes in brain with behavioral sequelae. We previously observed (J Neurosci 27:12367-12377, 2007) plastic changes in hippocampal slice recordings paralleling behavioral changes in rats treated with a single intoxicating dose of ethanol (EtOH). Here, we were able to reproduce in primary cultured hippocampal neurons many of the effects of in vivo EtOH exposure on GABA(A) receptors (GABA(A)Rs). Cells grown 11 to 15 days in vitro demonstrated GABA(A)R δ subunit expression and sensitivity to enhancement by short-term exposure to EtOH (60 mM) of GABA(A)R-mediated tonic current (I(tonic)) using whole-cell patch-clamp techniques. EtOH gave virtually no enhancement of mIPSCs. Cells pre-exposed to EtOH (60 mM) for 30 min showed, 1 h after EtOH withdrawal, a 50% decrease in basal I(tonic) magnitude and tolerance to short-term EtOH enhancement of I(tonic), followed by reduced basal mIPSC area at 4 h. At 24 h, we saw considerable recovery in mIPSC area and significant potentiation by short-term EtOH; in addition, GABA(A)R currents exhibited reduced enhancement by benzodiazepines. These changes paralleled significant decreases in cell-surface expression of normally extrasynaptic δ and α4 GABA(A)R subunits as early as 20 min after EtOH exposure and reduced α5-containing GABA(A)Rs at 1 h, followed by a larger reduction of normally synaptic α1 subunit at 4 h, and then by increases in α4γ2-containing cell-surface receptors by 24 h. Measuring internalization of biotinylated GABA(A)Rs, we showed for the first time that the EtOH-induced loss of I(tonic) and cell-surface δ/α4 20 min after withdrawal results from increased receptor endocytosis rather than decreased exocytosis.

Impact of stress resilience and susceptibility on fear learning, anxiety, and alcohol intake.

Post-traumatic stress disorder (PTSD) can develop after exposure to traumatic events and severely impacts the quality of life. PTSD is frequently comorbid with substance use disorders, with alcoholism being particularly common. However, not everyone who experiences trauma develops PTSD and the factors that render individuals susceptible or resilient to the effects of stress are unknown although gender appears to play an important role. Rodent models of stress exposure such as stress-enhanced fear learning (SEFL) recapitulate some aspects of PTSD symptomology, making them an invaluable tool for studying this disorder. This study examined whether exposure to a modified version of the SEFL procedure (4 footshocks instead of the standard 15 over 90 min) would reveal both susceptible and resilient subjects. Following stress exposure, distinct susceptible and resilient groups emerged that differed in fear learning and anxiety-related behavior as well as voluntary alcohol intake. Some aspects of stress susceptibility manifested differently in males compared to females, with susceptibility associated with increased alcohol intake in males and increased baseline anxiety in females.

Selective targeting of peripheral cannabinoid receptors prevents behavioral symptoms and sensitization of trigeminal neurons in mouse models of migraine and medication overuse headache.

ABSTRACT: Migraine affects ∼15% of the world's population greatly diminishing their quality of life. Current preventative treatments are effective in only a subset of migraine patients, and although cannabinoids seem beneficial in alleviating migraine symptoms, central nervous system side effects limit their widespread use. We developed peripherally restricted cannabinoids (PRCBs) that relieve chronic pain symptoms of cancer and neuropathies, without appreciable central nervous system side effects or tolerance development. Here, we determined PRCB effectiveness in alleviating hypersensitivity symptoms in mouse models of migraine and medication overuse headache. Long-term glyceryl trinitrate (GTN, 10 mg/kg) administration led to increased sensitivity to mechanical stimuli and increased expression of phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor potential ankyrin 1 proteins in trigeminal ganglia. Peripherally restricted cannabinoid pretreatment, but not posttreatment, prevented behavioral and biochemical correlates of GTN-induced sensitization. Low pH-activated and allyl isothiocyanate-activated currents in acutely isolated trigeminal neurons were reversibly attenuated by PRCB application. Long-term GTN treatment significantly enhanced these currents. Long-term sumatriptan treatment also led to the development of allodynia to mechanical and cold stimuli that was slowly reversible after sumatriptan discontinuation. Subsequent challenge with a previously ineffective low-dose GTN (0.1-0.3 mg/kg) revealed latent behavioral sensitization and increased expression of phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor potential ankyrin 1 proteins in trigeminal ganglia. Peripherally restricted cannabinoid pretreatment prevented all behavioral and biochemical correlates of allodynia and latent sensitization. Importantly, long-term PRCB treatment alone did not produce any behavioral or biochemical signs of sensitization. These data validate peripheral cannabinoid receptors as potential therapeutic targets in migraine and medication overuse headache.

Chronic alcohol disrupts hypothalamic responses to stress by modifying CRF and NMDA receptor function.

The chronic inability of alcoholics to effectively cope with relapse-inducing stressors has been linked to dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and corticotropin-releasing factor (CRF) signaling. However, the cellular mechanisms responsible for this dysregulation are yet to be identified. After exposure of male Sprague Dawley rats to chronic intermittent ethanol (CIE; 5-6 g/kg orally for 35 doses over 50 days) or water, followed by 40-60 days of protracted withdrawal, we investigated CIE effects on glutamatergic synaptic transmission, stress-induced plasticity, CRF- and ethanol-induced NMDAR inhibition using electrophysiological recordings in parvocellular neurosecretory cells (PNCs) of the paraventricular nucleus. We also assessed CIE effects on hypothalamic mRNA expression of CRF-related genes using real-time polymerase chain reaction, and on HPA axis function by measuring stress-induced increases in plasma adrenocorticotropic hormone, corticosterone, and self-grooming. In control rats, ethanol-mediated inhibition of NMDARs was prevented by CRF1 receptor (CRFR1) blockade with antalarmin, while CRF/CRFR1-mediated NMDAR blockade was prevented by intracellularly-applied inhibitor of phosphatases PP1/PP2A, okadaic acid, but not the selective striatal-enriched tyrosine protein phosphatase inhibitor, TC-2153. CIE exposure increased GluN2B subunit-dependent NMDAR function of PNCs. This was associated with the loss of both ethanol- and CRF-mediated NMDAR inhibition, and loss of stress-induced short-term potentiation of glutamatergic synaptic inputs, which could be reversed by intracellular blockade of NMDARs with MK801. CIE exposure also blunted the hormonal and self-grooming behavioral responses to repeated restraint stress. These findings suggest a cellular mechanism whereby chronic alcohol dysregulates the hormonal and behavioral responses to repetitive stressors by increasing NMDAR function and decreasing CRFR1 function.

Long-Acting Glucagon-Like Peptide-1 Receptor Agonists Suppress Voluntary Alcohol Intake in Male Wistar Rats.

Alcohol use disorder (AUD) is a chronic relapsing condition characterized by compulsive alcohol-seeking behaviors, with serious detrimental health consequences. Despite high prevalence and societal burden, available approved medications to treat AUD are limited in number and efficacy, highlighting a critical need for more and novel pharmacotherapies. Glucagon-like peptide-1 (GLP-1) is a gut hormone and neuropeptide involved in the regulation of food intake and glucose metabolism via GLP-1 receptors (GLP-1Rs). GLP-1 analogs are approved for clinical use for diabetes and obesity. Recently, the GLP-1 system has been shown to play a role in the neurobiology of addictive behaviors, including alcohol seeking and consumption. Here we investigated the effects of different pharmacological manipulations of the GLP-1 system on escalated alcohol intake and preference in male Wistar rats exposed to intermittent access 2-bottle choice of 10% ethanol or water. Administration of AR231453 and APD668, two different agonists of G-protein receptor 119, whose activation increases GLP-1 release from intestinal L-cells, did not affect voluntary ethanol intake. By contrast, injections of either liraglutide or semaglutide, two long-acting GLP-1 analogs, potently decreased ethanol intake. These effects, however, were transient, lasting no longer than 48 h. Semaglutide, but not liraglutide, also reduced ethanol preference on the day of injection. As expected, both analogs induced a reduction in body weight. Co-administration of exendin 9-39, a GLP-1R antagonist, did not prevent liraglutide- or semaglutide-induced effects in this study. Injection of exendin 9-39 alone, or blockade of dipeptidyl peptidase-4, an enzyme responsible for GLP-1 degradation, via injection of sitagliptin, did not affect ethanol intake or preference. Our findings suggest that among medications targeting the GLP-1 system, GLP-1 analogs may represent novel and promising pharmacological tools for AUD treatment.

Sex-dependent effects of chronic intermittent voluntary alcohol consumption on attentional, not motivational, measures during probabilistic learning and reversal.

BACKGROUND: Forced alcohol (ethanol, EtOH) exposure has been shown to cause significant impairments on reversal learning, a widely-used assay of cognitive flexibility, specifically on fully-predictive, deterministic versions of this task. However, previous studies have not adequately considered voluntary EtOH consumption and sex effects on probabilistic reversal learning. The present study aimed to fill this gap in the literature. METHODS: Male and female Long-Evans rats underwent either 10 weeks of voluntary intermittent 20% EtOH access or water only (H2O) access. Rats were then pretrained to initiate trials and learn stimulus-reward associations via touchscreen response, and subsequently required to select between two visual stimuli, rewarded with probability 0.70 or 0.30. In the final phase, reinforcement contingencies were reversed. RESULTS: We found significant sex differences on several EtOH-drinking variables, with females reaching a higher maximum EtOH consumption, exhibiting more high-drinking days, and escalating their EtOH at a quicker rate compared to males. During early abstinence, EtOH drinkers (and particularly EtOH-drinking females) made more initiation omissions and were slower to initiate trials than H2O drinking controls, especially during pretraining. A similar pattern in trial initiations was also observed in discrimination, but not in reversal learning. EtOH drinking rats were unaffected in their reward collection and stimulus response times, indicating intact motivation and motor responding. Although there were sex differences in discrimination and reversal phases, performance improved over time. We also observed sex-independent drinking group differences in win-stay and lose-shift strategies specific to the reversal phase. CONCLUSIONS: Females exhibit increased vulnerability to EtOH effects in early learning: there were sex-dependent EtOH effects on attentional measures during pretraining and discrimination phases. We also found sex-independent EtOH effects on exploration strategies during reversal. Future studies should aim to uncover the neural mechanisms for changes in attention and exploration in both acute and prolonged EtOH withdrawal.

Molecular consequences of fetal alcohol exposure on amniotic exosomal miRNAs with functional implications for stem cell potency and differentiation.

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH's effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.