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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.

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.

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.

BackgroundForced 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.MethodsMale 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.ResultsWe 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.ConclusionsFemales 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.

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.

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.

Many malignant cancers, including breast cancer, have a propensity to invade bones, leading to excruciating bone pain. Opioids are the primary analgesics used to alleviate this cancer-induced bone pain (CIBP) but are associated with numerous severe side effects, including enhanced bone degradation, which significantly impairs patients' quality of life. By contrast, agonists activating only peripheral CB1 receptors (CB1Rs) have been shown to effectively alleviate multiple chronic pain conditions with limited side effects, yet no studies have evaluated their role(s) in CIBP. Here, we demonstrate for the first time that a peripherally selective CB1R agonist can effectively suppress CIBP. Our studies using a syngeneic murine model of CIBP show that both acute and sustained administration of a peripherally restricted CB1R agonist, 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI), significantly alleviated spontaneous pain behaviors in the animals. This analgesic effect by PrNMI can be reversed by a systemic administration but not spinal injection of SR141716, a selective CB1R antagonist. In addition, the cancer-induced bone loss in the animals was not exacerbated by a repeated administration of PrNMI. Furthermore, catalepsy and hypothermia, the common side effects induced by cannabinoids, were measured at the supratherapeutic doses of PrNMI tested. PrNMI induced mild sedation, yet no anxiety or a decrease in limb movements was detected. Overall, our studies demonstrate that CIBP can be effectively managed by using a peripherally restricted CB1R agonist, PrNMI, without inducing dose-limiting central side effects. Thus, targeting peripheral CB1Rs could be an alternative therapeutic strategy for the treatment of CIBP.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and dose-limiting side effect of cancer treatment that affects millions of cancer survivors throughout the world and current treatment options are extremely limited by their side effects. Cannabinoids are highly effective in suppressing pain symptoms of chemotherapy-induced and other peripheral neuropathies but their widespread use is limited by central nervous system (CNS)-mediated side effects. Here, we tested one compound from a series of recently developed synthetic peripherally restricted cannabinoids (PRCBs) in a rat model of cisplatin-induced peripheral neuropathy. Results show that local or systemic administration of 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI) dose-dependently suppressed CIPN mechanical and cold allodynia. Orally administered PrNMI also dose-dependently suppressed CIPN allodynia symptoms in both male and female rats without any CNS side effects. Co-administration with selective cannabinoid receptor subtype blockers revealed that PrNMI's anti-allodynic effects are mediated by CB1 receptor (CB1R) activation. Expression of CB2Rs was reduced in dorsal root ganglia from CIPN rats, whereas expression of CB1Rs and various endocannabinoid synthesizing and metabolizing enzymes was unaffected. Daily PrNMI treatment of CIPN rats for two weeks showed a lack of appreciable tolerance to PrNMI's anti-allodynic effects. In an operant task which reflects cerebral processing of pain, PrNMI also dose-dependently suppressed CIPN pain behaviors. Our results demonstrate that PRCBs exemplified by PrNMI may represent a viable option for the treatment of CIPN pain symptoms.

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 α1βγ2 subtype synaptic GABAARs but increased synaptic α4βγ2 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 α4βγ2 subtypes, as well as increased cell-surface levels of GABAAR α2 and γ1 subunits, along with increased α2β1γ1 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 α2βγ subtypes and EtOH-sensitive α4βγ2 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, β1, 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.

Neuronal cells express considerable plasticity responding to environmental cues, in part, through subcellular mRNA regulation. Here we report on the extensive changes in distribution of mRNAs in the cell body and axon compartments of peripheral sensory neurons and the 3' untranslated region (3'UTR) landscapes after unilateral sciatic nerve entrapment (SNE) injury in rats. Neuronal cells dissociated from SNE-injured and contralateral L4 and L5 dorsal root ganglia were cultured in a compartmentalized system. Axonal and cell body RNA samples were separately subjected to high throughput RNA sequencing (RNA-Seq). The injured axons exhibited enrichment of mRNAs related to protein synthesis and nerve regeneration. Lengthening of 3'UTRs was more prevalent in the injured axons, including the newly discovered alternative cleavage and polyadenylation of NaV1.8 mRNA. Alternative polyadenylation was largely independent from the relative abundance of axonal mRNAs; but they were highly clustered in functional pathways related to RNA granule formation in the injured axons. These RNA-Seq data analyses indicate that peripheral nerve injury may result in highly selective mRNA enrichment in the affected axons with 3'UTR alterations potentially contributing to the mechanism of neuropathic pain.

Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system (CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity, peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay identified candidates with

Fear promotes adaptive responses to threats. However, when the level of fear is not proportional to the level of threat, maladaptive fear-related behaviors characteristic of anxiety disorders result. Post-traumatic stress disorder develops in response to a traumatic event, and patients often show sensitized reactions to mild stressors associated with the trauma. Stress-enhanced fear learning (SEFL) is a rodent model of this sensitized responding, in which exposure to a 15-shock stressor nonassociatively enhances subsequent fear conditioning training with only a single trial. We examined the role of corticosterone (CORT) in SEFL. Administration of the CORT synthesis blocker metyrapone prior to the stressor, but not at time points after, attenuated SEFL. Moreover, CORT co-administered with metyrapone rescued SEFL. However, CORT alone without the stressor was not sufficient to produce SEFL. In these same animals, we then looked for correlates of SEFL in terms of changes in excitatory receptor expression. Western blot analysis of the basolateral amygdala (BLA) revealed an increase in the GluA1 AMPA receptor subunit that correlated with SEFL. Thus, CORT is permissive to trauma-induced changes in BLA function.

Peripheral nerve injuries can result in lifelong disability. Primary coaptation is the treatment of choice when the gap between transected nerve ends is short. Long nerve gaps seen in more complex injuries often require autologous nerve grafts or nerve conduits implemented into the repair. Nerve grafts, however, cause morbidity and functional loss at donor sites, which are limited in number. Nerve conduits, in turn, lack an internal scaffold to support and guide axonal regeneration, resulting in decreased efficacy over longer nerve gap lengths. By comparison, peptide amphiphiles (PAs) are molecules that can self-assemble into nanofibers, which can be aligned to mimic the native architecture of peripheral nerve. As such, they represent a potential substrate for use in a bioengineered nerve graft substitute. To examine this, we cultured Schwann cells with bioactive PAs (RGDS-PA, IKVAV-PA) to determine their ability to attach to and proliferate within the biomaterial. Next, we devised a PA construct for use in a peripheral nerve critical sized defect model. Rat sciatic nerve defects were created and reconstructed with autologous nerve, PLGA conduits filled with various forms of aligned PAs, or left unrepaired. Motor and sensory recovery were determined and compared among groups. Our results demonstrate that Schwann cells are able to adhere to and proliferate in aligned PA gels, with greater efficacy in bioactive PAs compared to the backbone-PA alone. In vivo testing revealed recovery of motor and sensory function in animals treated with conduit/PA constructs comparable to animals treated with autologous nerve grafts. Functional recovery in conduit/PA and autologous graft groups was significantly faster than in animals treated with empty PLGA conduits. Histological examinations also demonstrated increased axonal and Schwann cell regeneration within the reconstructed nerve gap in animals treated with conduit/PA constructs. These results indicate that PA nanofibers may represent a promising biomaterial for use in bioengineered peripheral nerve repair.

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.

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) (GDPβS) 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.

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.

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.

Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs, particularly those associated with molecular pathways for metabolic processes, oxidative stress, and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts, with methylation on the promoter regions of chromosomes 2, 16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes, which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis.

Chronic alcohol exposure causes marked changes in reinforcement mechanisms and motivational state that 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. Although the NAcc plays an important role in mediating alcohol-seeking behaviors, little is known about the molecular mechanisms underlying alcohol-induced neuroadaptive changes in NAcc function. The aim of this study was to investigate the effects of chronic intermittent ethanol (CIE) treatment, a rat model of alcohol withdrawal and dependence, on intrinsic electrical membrane properties and glutamatergic synaptic transmission of medium spiny neurons (MSNs) in the NAcc core during protracted withdrawal. We show that CIE treatment followed by prolonged withdrawal increased the inward rectification of MSNs observed at hyperpolarized potentials. In addition, MSNs from CIE-treated animals displayed a lower input resistance, faster action potentials (APs), and larger fast afterhyperpolarizations (fAHPs) than MSNs from vehicle-treated animals, all suggestive of increases in K(+)-channel conductances. Significant increases in the Cs(+)-sensitive inwardly rectifying K(+)-current accounted for the increased input resistance, while increases in the A-type K(+)-current accounted for the faster APs and increased fAHPs in MSNs from CIE rats. We also show that the amplitude and the conductance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated mEPSCs were enhanced in CIE-treated animals due to an increase in a small fraction of functional postsynaptic GluA2-lacking AMPARs. These long-lasting modifications of excitability and excitatory synaptic receptor function of MSNs in the NAcc core could play a critical role in the neuroadaptive changes underlying alcohol withdrawal and dependence.

There is considerable evidence that ethanol (EtOH) potentiates γ-aminobutyric acid type A receptor (GABA(A)R) action, but only GABA(A)Rs containing δ subunits appear sensitive to low millimolar EtOH. The α4 and δ subunits co-assemble into GABA(A)Rs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABA(A)R function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABA(A)Rs. Based on this hypothesis, any highly EtOH-sensitive GABA(A)Rs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABA(A)Rs in mice with a global deletion of the α4 subunit (KO). The dentate granule cells of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT) controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABA(A)Rs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p.) revealed increased intracellular fraction of the α1, α4, and δ, but not α2, α5, or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 μM, requiring α1/α2, inactive at α4/5 GABA(A)Rs) than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice were significantly less sensitive to zolpidem, suggesting decreases in functional α1/2βγ GABA(A)Rs. These data further support our hypothesis that EtOH intoxication induces GABA(A)R plasticity via internalization of highly EtOH-sensitive GABA(A)Rs.

Seizure-induced hilar basal dendrites on dentate granule cells are observed in several rodent models of temporal lobe epilepsy. Ultrastructural evidence showed that basal dendrites receive predominantly excitatory synapses, including many from mossy fibers. Such highly interconnected granule cells with basal dendrites are suggested to enhance hyperexcitability within the dentate network. For an expanded treatment of this topic see Jasper's Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at). © 2010 International League Against Epilepsy.

Microglial cells are constantly monitoring the central nervous system for sick or dying cells and pathogens. Previous studies showed that the microglial cells in the dentate gyrus have a heterogeneous morphology with multipolar cells in the hilus and fusiform cells apposed to the granule cell layer both at the hilar and at the molecular layer borders. Although previous studies showed that the microglia in the dentate gyrus were not activated, the data in the present study show dying granule cells apposed by Iba1-immunolabeled microglial cell bodies and their processes both at hilar and at molecular layer borders of the granule cell layer. Initially, these Iba1-labeled microglial cells surround individual, intact granule cell bodies. When small openings in the plasma membrane of granule cells are observed, microglial cells are apposed to these openings. When larger openings in the plasma membrane occur at this site of apposition, the granule cells display watery perikaryal cytoplasm, watery nucleoplasm and damaged organelles. Such morphological features are characteristic of neuronal edema. The data also show that following this localized disintegration of the granule cell’s plasma membrane, the Iba1-labeled microglial cell body is found within the electron-lucent perikaryal cytoplasm of the granule cell, where it is adjacent to the granule cell’s nucleus which is deformed. We propose that granule cells are dying by a novel microglia-associated mechanism that involves lysis of their plasma membranes followed by neuronal edema and nuclear phagocytosis. Based on the morphological evidence, this type of cell death differs from either apoptosis or necrosis.

Hilar basal dendrites form on dentate granule cells following seizures. To determine whether other brain insults cause the formation of hilar basal dendrites, a model of global cerebral hypoxia/ischemia was used. Rats underwent a transient induction of ischemia by occlusion of both common carotid arteries followed by reperfusion. Hippocampal slices were prepared from these animals 1 month after the ischemic insult, and granule cells were labeled with a retrograde tracing technique after biocytin injections into stratum lucidum of CA3b. Ischemic rats had numerous biocytin-labeled granule cells with hilar basal dendrites located at the hilar border of the granule cell layer. Quantitative analysis of ischemic rats compared to controls showed a significant increase in the percentage of biocytin-labeled granule cells with hilar basal dendrites. These data demonstrate that other brain insults in addition to epilepsy may result in the formation of hilar basal dendrites on granule cells.

BackgroundNeuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.ResultsAfter unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.ConclusionCuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

BackgroundNeuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.ResultsAfter unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.ConclusionCuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

BackgroundAdenosine 5'-triphosphate (ATP) has a ubiquitous role in metabolism and a major role in pain responses after tissue injury. We investigated the changes in basal and KCl-evoked ATP release from rat dorsal root ganglia (DRG) after peripheral neuropathy induction by unilateral sciatic nerve entrapment (SNE).ResultsAfter SNE, rats develop long-lasting decreases in ipsilateral hindpaw withdrawal thresholds to mechanical and thermal stimulation. At 15-21 days after neuropathy induction, excised ipsilateral L4-L5 DRG display significantly elevated basal extracellular ATP levels compared to contralateral or control (naive) DRG. However, KCl-evoked ATP release is no longer observed in ipsilateral DRG. We hypothesized that the differential SNE effects on basal and evoked ATP release could result from the conversion of extracellular ATP to adenosine with subsequent activation of adenosine A1 receptors (A1Rs) on DRG neurons. Adding the selective A1R agonist, 2-chloro-N(6)-cyclopentyladenosine (100 nM) significantly decreased basal and evoked ATP release in DRG from naïve rats, indicating functional A1R activation. In DRG ipsilateral to SNE, adding a selective A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (30 nM), further increased basal ATP levels and relieved the blockade of KCl-evoked ATP release suggesting that increased A1R activation attenuates evoked ATP release in neurons ipsilateral to SNE. To determine if altered ATP release was a consequence of altered DRG metabolism we compared O(2) consumption between control and neuropathic DRG. DRG ipsilateral to SNE consumed O(2) at a higher rate than control or contralateral DRG.ConclusionThese data suggest that peripheral nerve entrapment increases DRG metabolism and ATP release, which in turn is modulated by increased A1R activation.

Granule cells with hilar basal dendrites (HBDs) are found after status epilepticus (SE) in three rat models of temporal lobe epilepsy. These granule cells are commonly located at the hilar border and could be newly born granule cells based on their location. The aim of this study was to determine how long it takes for HBDs to form on granule cells after SE. Pilocarpine was injected to induce SE and rats were killed at different times: 3 days, 1, 2, and 3 weeks after SE. Biocytin was injected into CA3 stratum lucidum of hippocampal slices to label granule cells with HBDs. The number, morphology, and length of HBDs were analyzed at the different time points. Basal processes of granule cells from rats killed 3 days after pilocarpine injection were judged not to be HBDs because they were short in length and did not ramify in the hilus. "True" HBDs were detected as early as 7 and 8 days after pilocarpine-induced SE. Similar frequencies of granule cells with HBDs were observed at the later time points. This study shows that HBDs can form on granule cells as early as 1 week following SE. These results are consistent with the hypothesis that HBDs on granule cells may be generated from seizure-induced, de novo granule cells, however, alternative explanations that some or all HBDs arise from pre-SE generated granule cells cannot be ruled out at this time and will require further examination.

The stereotyped morphology of dentate granule cells in rodents consists of apical dendrites arborizing in the molecular layer and an axon arising from the opposite pole of the soma. Recently, we showed that epilepsy induces the formation of basal dendrites on granule cells and that these dendrites extend into the hilus of the dentate gyrus. The present Golgi study of granule cells from adult rats shows two atypical features for granule cells in control rats. One is the occurrence of recurrent basal dendrites (RBDs) that are defined as basal dendrites arising at or near the hilar pole of the soma and then curving back to the molecular layer. The frequency of granule cells with RBDs was 3.8% in control rats. The second is apical axons of granule cells that were observed to originate from either the apical pole of the soma or an apical dendrite. The incidence of these "apical" axons was about 1%. These morphological findings in the present study suggest that rat granule cells are more heterogeneous than previously indicated. Furthermore, their frequency was not increased in epileptic rats.