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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 <1% blood-brain barrier penetration for testing in a rat neuropathy induced by unilateral sciatic nerve entrapment (SNE). The SNE-induced mechanical allodynia was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked, while the brain-permeant CB1R agonist HU-210 (1) exhibited strong CNS side effects, in catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ∼0.001 cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene's antiallodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for neuropathic pain.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 the American Physiological Society.)

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., (Copyright © 2014 the American Physiological Society.)

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.

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.

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., (Copyright © 2014. Published by Elsevier B.V.)

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.

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.

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.

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.

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.

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.

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2-/- mice is similar to that of their Cox-2+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2-/- versus Cox-2+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.

Background: The alpha 4 subunit-containing gamma-aminobutyric acid (A) receptors (GABA(A)Rs) are highly expressed primarily at extrasynaptic sites in the dentate gyrus (DG) and thalamus and are suspected to contribute to tonic inhibition that is sensitive to potentiation by gaboxadol and ethanol (EtOH). Global alpha 4 subunit knockout (KO) mice exhibit greatly reduced tonic currents and insensitivity to ataxic, sedative and analgesic effects of gaboxadol compared to wild type (WT) controls. The alpha 4 KO mice were also significantly more sensitive to pentylenetetrazol-induced seizures. However, no differences were observed between alpha 4 KO and WT mice in other baseline behaviors or in the effects of EtOH on these behaviors. To examine possible functional and pharmacological GABA(A)R alterations, and search for causes for the lack of differences in EtOH behaviors we studied the effects of acute EtOH application on GABA(A)R-currents of DG cells from alpha 4 KO and WT control mice complemented by Western blot measurements., Methods: We studied the consequences of alpha 4 subunit deletion using Western immunoblotting and whole cell patch recordings from DG cells in brain slices from alpha 4 KO and WT mice., Results: The magnitude of tonic current and its potentiation by EtOH (10 to 100 mM), alphaxalone (3 microM), and Ro15-4513 (0.3 microM) was greatly attenuated in alpha 4 KO mice. The kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in alpha 4 KO mice were significantly slower compared to WT mice. Potentiation of mIPSCs by alphaxalone was greatly reduced in alpha 4 KO mice. Ro15-4513 had no effect on mIPSCs from WT or KO mice. However, mIPSCs of alpha 4 KO mice were significantly more sensitive to EtOH than those from WT mice. The gamma 2 subunit protein levels were selectively increased in hippocampus and thalamus, but not cortex of alpha 4 KO mice., Conclusions: These data suggest that the global loss of alpha 4 subunits leads to region- and cell location-specific compensatory increases in gamma 2 subunits, which in turn alter the pharmacological sensitivity of synaptic and extrasynaptic GABA(A)R-currents. Our data also suggests that while enhancement of tonic inhibitory currents by gaboxadol, alphaxalone, and EtOH are reduced, and behavioral sensitivity to gaboxadol and alphaxalone may be reduced, compensatory changes in synaptic GABA(A)R subunits may prevent similar reductions in behavioral sensitivity to EtOH.

Background: gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) have been implicated in mediating some of the behavioral effects of ethanol (EtOH), but the contribution of specific GABA(A)-R subunits is not yet fully understood. The GABA(A)-R alpha 4 subunit often partners with beta2/3 and delta subunits to form extrasynaptic GABA(A)-Rs that mediate tonic inhibition. Several in vitro studies have suggested that these extrasynaptic GABA(A)-Rs may be particularly relevant to the intoxicating effects of low doses of EtOH. In alpha 4 subunit knockout mice, tonic inhibition was greatly reduced, as were the potentiating effects of EtOH. We therefore hypothesized that those behavioral responses to EtOH that are mediated by alpha 4-containing GABA(A)-Rs would be diminished in alpha 4 knockout mice., Methods: We investigated behavioral responses to acute administration of moderate/high dose EtOH or pentylenetetrazol in alpha 4 subunit knockout mice. We compared behavioral responses to EtOH in alpha 4 knockout and wild-type littermates in the elevated plus maze (0.0, 1.0 g/kg EtOH), screen test (1.5, 2.0 g/kg), hypothermia (1.5, 2.0 g/kg), fixed speed rotarod (1.5, 2.0, 2.5 g/kg), open field (0.0, 1.0, 2.0 g/kg), radiant tail flick (2.0 g/kg), loss of righting reflex (3.5 g/kg), and EtOH metabolism and clearance assays. Sensitivity to pentylenetetrazol-induced seizures was also analyzed., Results: No differences were observed between alpha 4 knockout mice and wild-type controls in terms of the baseline behavior in the absence of EtOH treatment or in the behavioral effects of EtOH in the assays tested. In contrast, alpha 4 knockout mice were significantly more sensitive to pentylenetetrazol-induced seizures., Conclusions: We conclude that GABA(A)-Rs containing the alpha 4 subunit are not absolutely required for the acute behavioral responses to moderate/high dose EtOH that were assessed with the elevated plus maze, screen test, hypothermia, fixed speed rotarod, open field, radiant tail flick, and loss of right reflex assays. We further suggest that these findings are complicated by the demonstrated compensatory alterations in synaptic GABA(A)-R EtOH sensitivity and function in alpha 4 knockout mice.

Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.

The time-dependent effects of ethanol (EtOH) intoxication on GABA(A) receptor (GABA(A)R) composition and function were studied in rats. A cross-linking assay and Western blot analysis of microdissected CA1 area of hippocampal slices obtained 1 h after EtOH intoxication (5 g/kg, gavage), revealed decreases in the cell-surface fraction of alpha4 and delta, but not alpha1, alpha5, or gamma2 GABA(A)R subunits, without changes in their total content. This was accompanied (in CA1 neuron recordings) by decreased magnitude of the picrotoxin-sensitive tonic current (I(tonic)), but not miniature IPSCs (mIPSCs), and by reduced enhancement of I(tonic) by EtOH, but not by diazepam. By 48 h after EtOH dosing, cell-surface alpha4 (80%) and gamma2 (82%) subunit content increased, and cell-surface alpha1 (-50%) and delta (-79%) and overall content were decreased. This was paralleled by faster decay of mIPSCs, decreased diazepam enhancement of both mIPSCs and I(tonic), and paradoxically increased mIPSC responsiveness to EtOH (10-100 mm). Sensitivity to isoflurane- or diazepam-induced loss of righting reflex was decreased at 12 and 24 h after EtOH intoxication, respectively, suggesting functional GABA(A)R tolerance. The plastic GABA(A)R changes were gradually and fully reversible by 2 weeks after single EtOH dosing, but unexplainably persisted long after withdrawal from chronic intermittent ethanol treatment, which leads to signs of alcohol dependence. Our data suggest that early tolerance to EtOH may result from excessive activation and subsequent internalization of alpha4betadelta extrasynaptic GABA(A)Rs. This leads to transcriptionally regulated increases in alpha4 and gamma2 and decreases in alpha1 subunits, with preferential insertion of the newly formed alpha4betagamma2 GABA(A)Rs at synapses.

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.