Selective activation of the peripheral cannabinoid receptor 1 (CB1R) has been shown to suppress neuropathic pain symptoms in rodents. However, relatively little is known about changes in CB1R and its endogenous ligands during development or maintenance of neuropathic pain. Using immunohistochemistry, Western blot, real-time reverse transcription polymerase chain reaction, as well as liquid chromatography/mass spectrometry, we studied the changes in CB1Rs and endocannabinoids N-arachidonoylethanolamine/anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat lumbar (L4 and L5) dorsal root ganglia (DRG) after neuropathic pain induction (L5 spinal nerve ligation: SNL). Immunohistochemistry revealed that in control rats, CB1R is expressed in the majority (76-83%) of nociceptive neurons as indicated by co-labeling with isolectin B4 (IB4) or antibodies recognizing transient receptor potential vanilloid (TRPV1), calcitonin gene related peptide (CGRP), and the NR2C/2D subunits of the N-methyl-D-aspartate receptor. After L5 SNL, CB1R mRNA and protein increases in the ipsilateral uninjured L4 DRG whereas the percentages of CB1R immunoreactive (CB1R-ir) neurons remain unchanged in L4 and L5 DRG. However, for these CB1R-ir neurons, we observe significant increases in percentage of TRPV1-ir cells in ipsilateral L4 DRG, and decreases in percentage of IB4- and CGRP-co-labeled cells in ipsilateral L5 DRG. Levels of both AEA and 2-AG increase significantly only in the ipsilateral L5 DRG. These results are consistent with the preserved analgesic effects of cannabinoids in neuropathic pain and provide a rational framework for the development of peripherally acting endocannabinoid-based therapeutic interventions for neuropathic pain.

Alcohol withdrawal syndrome (AWS) symptoms include hyperexcitability, anxiety, and sleep disorders. Chronic intermittent ethanol (CIE) treatment of rats with subsequent withdrawal of ethanol (EtOH) reproduced AWS symptoms in behavioral assays, which included tolerance to the sleep-inducing effect of acute EtOH and its maintained anxiolytic effect. Electrophysiological assays demonstrated a CIE-induced long-term loss of extrasynaptic GABA_Areceptor (GABA_AR) responsiveness and a gain of synaptic GABA_AR responsiveness of CA1 pyramidal and dentate granule neurons to EtOH that we were able to relate to behavioral effects. After CIE treatment, the α4 subunit-preferring GABA_AR ligands 4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridin-3-ol, La³⁺, and Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5α][1,4]benzodiazepine-3-carboxylate) exerted decreased effects on extrasynaptic currents but had increased effects on synaptic currents. Electron microscopy revealed an increase in central synaptic localization of α4 but not δ subunits within GABAergic synapses on the dentate granule cells of CIE rats. Recordings in dentate granule cells from δ subunit-deficient mice revealed that this subunit is not required for synaptic GABA_AR sensitivity to low [EtOH]. The profound alterations in EtOH sensitivity and α4 subunit localization at hippocampal GABA_ARs of CIE rats suggest that such changes in these and other relevant brain circuits may contribute to the development of tolerance to the sleep-inducing effects and long-term dependence on alcohol.

The time-dependent effects of ethanol (EtOH) intoxication on GABA_Areceptor (GABA_AR) 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 α4 and δ, but not α1, α5, or γ2 GABA_AR 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 ofI_tonicby EtOH, but not by diazepam. By 48 h after EtOH dosing, cell-surface α4 (80%) and γ2 (82%) subunit content increased, and cell-surface α1 (−50%) and δ (−79%) and overall content were decreased. This was paralleled by faster decay of mIPSCs, decreased diazepam enhancement of both mIPSCs andI_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_AR tolerance. The plastic GABA_AR 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 α4βδ extrasynaptic GABA_ARs. This leads to transcriptionally regulated increases in α4 and γ2 and decreases in α1 subunits, with preferential insertion of the newly formed α4βγ2 GABA_ARs at synapses.

Background: Neuropathic 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. Results: After 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. Conclusion: Cuff 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.

The δ subunit of the γ-aminobutyric acid (A) receptor (GABA_AR) is expressed postnatally mostly in the cerebellum, thalamus, and dentate gyrus. Previous studies in mice with a targeted disruption of the δ subunit revealed a considerable attenuation of behavioral responses to neuroactive steroids but not to other neuromodulatory drugs. Here we show that δ subunit loss leads to a concomitant reduction in hippocampal α4 subunit levels. These changes were accompanied by faster decay of evoked inhibitory postsynaptic potentials (IPSPs) in dentate granule neurons of –/– mutants (decay τ = 25 ms) compared with +/+ controls (τ = 50 ms). Furthermore, the GABA_AR-mediated miniature inhibitory postsynaptic currents (mIPSCs) also decayed faster in δ-mutants (τ = 6.3 ms) than controls (τ = 7.2 ms) and had decreased frequency (controls, 10.5 Hz; mutants, 6.6 Hz). Prolongation of mIPSCs by the neuroactive steroid anesthetic, alphaxalone (1–10 μM), was smaller in δ-mutants (at 10 μM, 65% increase) compared with +/+ littermates (308% increase). In competition binding experiments, alphaxalone (0.03–1 μM) modulation of [³⁵S]t-butylbicyclophosphorothionate binding was reduced in δ-mutant brain homogenates, indicating that the decreased alphaxalone effects on mIPSCs were due to changes in the GABA_AR protein. Faster decay of evoked IPSPs and mIPSCs in δ-mutants suggests presence of the δ subunit at both synaptic and extrasynaptic GABA_ARs. Decreased synaptic and extrasynaptic inhibition likely contributes to the pro-epileptic phenotype of δ-mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. While the simplest explanation is that δ subunit-containing GABA_ARs represent the actual target of neurosteroids, it is possible that the behavioral and physiological sensitivity to neuroactive steroids is indirectly altered in the δ –/– mice.

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.