Negative allosteric modulation of CB1 cannabinoid receptor signaling suppresses opioid-mediated reward.

Blockade of cannabinoid type 1 (CB1)-receptor signaling decreases the rewarding properties of many drugs of abuse and has been proposed as an anti-addiction strategy. However, psychiatric side-effects limit the clinical potential of orthosteric CB1 antagonists. Negative allosteric modulators (NAMs) represent a novel and indirect approach to attenuate CB1 signaling by decreasing affinity and/or efficacy of CB1 ligands. We hypothesized that a CB1-NAM would block opioid reward while avoiding the unwanted effects of orthosteric CB1 antagonists. GAT358, a CB1-NAM, failed to elicit cardinal signs of direct CB1 activation or inactivation when administered by itself. GAT358 decreased catalepsy and hypothermia but not antinociception produced by the orthosteric CB1 agonist CP55,940, suggesting that a CB1-NAM blocked cardinal signs of CB1 activation. Next, GAT358 was evaluated using in vivo assays of opioid-induced dopamine release and reward in male rodents. In the nucleus accumbens shell, a key component of the mesocorticolimbic reward pathway, morphine increased electrically-evoked dopamine efflux and this effect was blocked by a dose of GAT358 that lacked intrinsic effects on evoked dopamine efflux. Moreover, GAT358 blocked morphine-induced reward in a conditioned place preference (CPP) assay without producing reward or aversion alone. GAT358-induced blockade of morphine CPP was also occluded by GAT229, a CB1 positive allosteric modulator (CB1-PAM), and absent in CB1-knockout mice. Finally, GAT358 also reduced oral oxycodone (but not water) consumption in a two-bottle choice paradigm. Our results support the therapeutic potential of CB1-NAMs as novel drug candidates aimed at preventing opioid reward and treating opioid abuse while avoiding unwanted side-effects.

A limited access oral oxycodone paradigm produces physical dependence and mesocorticolimbic region-dependent increases in DeltaFosB expression without preference.

The abuse of oral formulations of prescription opioids has precipitated the current opioid epidemic. We developed an oral oxycodone consumption model consisting of a limited access (4 h) two-bottle choice drinking in the dark (TBC-DID) paradigm and quantified dependence with naloxone challenge using mice of both sexes. We also assessed neurobiological correlates of withdrawal and dependence elicited via oral oxycodone consumption using immunohistochemistry for DeltaFosB (ΔFosB), a transcription factor described as a molecular marker for drug addiction. Neither sex developed a preference for the oxycodone bottle, irrespective of oxycodone concentration, bottle position or prior water restriction. Mice that volitionally consumed oxycodone exhibited hyperlocomotion in an open field test and supraspinal but not spinally-mediated antinociception. Both sexes also developed robust, dose-dependent levels of opioid withdrawal that was precipitated by the opioid antagonist naloxone. Oral oxycodone consumption followed by naloxone challenge led to mesocorticolimbic region-dependent increases in the number of ΔFosB expressing cells. Naloxone-precipitated withdrawal jumps, but not the oxycodone bottle % preference, was positively correlated with the number of ΔFosB expressing cells specifically in the nucleus accumbens shell. Thus, limited access oral consumption of oxycodone produced physical dependence and increased ΔFosB expression despite the absence of opioid preference. Our TBC-DID paradigm allows for the study of oral opioid consumption in a simple, high-throughput manner and elucidates the underlying neurobiological substrates that accompany opioid-induced physical dependence.

Selective targeting of NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in rodents.

The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified >200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics­without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.

Fecal microbiota transplantation and antibiotic treatment attenuate naloxone-precipitated opioid withdrawal in morphine-dependent mice.

Opioid addiction can produce severe side effects including physical dependence and withdrawal. Perturbations of the gut microbiome have recently been shown to alter opioid-induced side-effects such as addiction, tolerance and dependence. In the present study, we investigated the influence of the gut microbiome on opioid withdrawal by evaluating the effects of fecal microbiota transplantation (FMT), antibiotic and probiotic treatments, and pharmacological inhibition of gut permeability in a mouse model of opioid dependence. Repeated intraperitoneal (i.p.) morphine treatment produced physical dependence that was quantified by measuring somatic signs of withdrawal (i.e. number of jumps) precipitated using the opioid antagonist naloxone. Morphine-dependent mice that received FMT from morphine-treated donor mice exhibited fewer naloxone-precipitated jumps compared to morphine-dependent counterparts receiving FMT from saline-treated donor mice. Microbial contents in the mouse cecum were altered by morphine treatment but were not differentially impacted by FMT. A broad-spectrum antibiotic cocktail (ABX) regimen reduced the bacterial load and attenuated naloxone-precipitated morphine withdrawal in morphine-dependent mice, whereas commercially available probiotic strains did not reliably alter somatic signs of opioid withdrawal. ML-7, a pharmacological inhibitor of gut permeability, reduced the morphine-induced increase in gut permeability in vivo but did not reliably alter somatic signs of naloxone-precipitated opioid withdrawal. Our results suggest that the gut microbiome impacts the development of physical dependence induced by chronic morphine administration, and that therapeutic manipulations of the gut microbiome may reduce opioid withdrawal.

Pharmacological Antagonism of T-Type Calcium Channels Constrains Rebound Burst Firing in Two Distinct Subpopulations of GABA Neurons in the Rat Ventral Tegmental Area: Implications for α-Lipoic Acid.

The ventral tegmental area (VTA) is a midbrain region highly involved in motivation and reward. A large body of work has investigated synaptic plasticity and ion channel excitability in this area, which has strong implication in drug abuse. We recently provided electrophysiological and pharmacological evidence that the CaV3.1 isoform of T-type voltage-gated calcium channels contributes to the excitability of VTA dopamine (DA) neurons. However, the role of T-channels in excitability of VTA gamma-amino-butyric acid (GABA) neurons remained unaddressed. Here, with a population study of rat VTA GABA neurons, we provide evidence that T-channels contribute to rebound spiking activity in two phenotypically distinct subpopulations of GABAergic neurons, each with differing electrophysiological characteristics. Additionally, we provide the first study to investigate the effect of α-lipoic acid (ALA) on ion channels in mesolimbic reward circuitry. Taken together, our population study and pharmacology experiments implicate T-channels as a target for therapies aimed at tempering VTA and mesolimbic circuit excitability.

Neonatal general anesthesia causes lasting alterations in excitatory and inhibitory synaptic transmission in the ventrobasal thalamus of adolescent female rats.

Ample evidence has surfaced documenting the neurotoxic effects of various general anesthetic (GA) agents in the mammalian brain when administered at critical periods of synaptogenesis. However, little is known about how this neurotoxic insult affects persisting neuronal excitability after the initial exposure. Here we investigated synaptic activity and intrinsic excitability of the ventrobasal nucleus (VB) of the thalamus caused by neonatal GA administration. We used patch-clamp recordings from acute thalamic slices in young rats up to two weeks after neurotoxic GA exposure of isoflurane and nitrous oxide for 6 h at postnatal age of 7 (P7) days. We found that GA exposure at P7 increases evoked excitatory postsynaptic currents (eEPSCs) two fold by means through AMPA mediated mechanisms, while NMDA component was spared. In addition, miniature EPSCs showed a faster decay rate in neurons from GA treated animals when compared to sham controls. Likewise, we discovered that the amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased in VB neurons from GA animals about two-fold. Interestingly, these results were observed in female but not male rats. In contrast, intrinsic excitability and properties of T-type voltage gated calcium currents were minimally affected by GA exposure. Together, these data further the idea that GAs cause lasting alterations in synaptic transmission and neuronal excitability depending upon the placing and connectivity of neurons in the thalamus. Given that function of thalamocortical circuits critically depends on the delicate balance between excitation and inhibition, future development of therapies aimed at addressing consequences of altered excitability in the developing brain by GAs may be an attractive possibility.

Ventral Tegmental Area GABA Neurons Are Resistant to GABA(A) Receptor-Mediated Inhibition During Ethanol Withdrawal.

The neural mechanisms underlying alcohol dependence are not well-understood. GABAergic neurons in the ventral tegmental area (VTA) are a relevant target for ethanol. They are inhibited by ethanol at physiologically-relevant levels in vivo and display marked hyperexcitability during withdrawal. In the present study, we examined the effects of the GABA(A) receptor agonist muscimol on VTA neurons ex vivo following withdrawal from acute and chronic ethanol exposure. We used standard cell-attached mode electrophysiology in the slice preparation to evaluate the effects of muscimol on VTA GABA neuron firing rate following exposure to acute and chronic ethanol in male CD-1 GAD-67 GFP mice. In the acute condition, the effect of muscimol on VTA neurons was evaluated 24 h and 7 days after a single in vivo dose of saline or ethanol. In the chronic condition, the effect of muscimol on VTA neurons was evaluated 24 h and 7 days after either 2 weeks of twice-daily IP ethanol or saline or following exposure to chronic intermittent ethanol (CIE) vapor or air for 3 weeks. VTA GABA neuron firing rate was more sensitive to muscimol than DA neuron firing rate. VTA GABA neurons, but not DA neurons, were resistant to the inhibitory effects of muscimol recorded 24 h after a single ethanol injection or chronic ethanol exposure. Administration of the NMDA receptor antagonist MK-801 before ethanol injection restored the sensitivity of VTA GABA neurons to muscimol inhibition. Seven days after ethanol exposure, VTA GABA neuron firing rate was again susceptible to muscimol's inhibitory effects in the acute condition, but the resistance persisted in the chronic condition. These findings suggest that VTA GABA neurons exclusively undergo a shift in GABA(A) receptor function following acute and chronic exposure. There appears to be transient GABA(A) receptor-mediated plasticity after a single exposure to ethanol that is mediated by NMDA glutamate receptors. In addition, the resistance to muscimol inhibition in VTA GABA neurons persists in the dependent condition, which may contribute to the the hyperexcitability of VTA GABA neurons and inhibition of VTA DA neurons during withdrawal as well as the motivation to seek alcohol.

α6 subunit-containing nicotinic receptors mediate low-dose ethanol effects on ventral tegmental area neurons and ethanol reward.

Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10 mM) enhanced GABAA receptor (GABAA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100 mM) had no effect on optically evoked GABAA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse.