Monoacylglycerol Lipase Protects the Presynaptic Cannabinoid 1 Receptor from Desensitization by Endocannabinoids after Persistent Inflammation.

Cannabinoid-targeted pain therapies are increasing with the expansion of cannabis legalization, however, their efficacy may be limited by pain-induced adaptations in the cannabinoid system. Cannabinoid receptor subtype 1 (CB1R) inhibition of spontaneous, GABAergic miniature IPSCs (mIPSCs) and evoked IPSCs (eIPSCs) in the ventrolateral periaqueductal gray (vlPAG) were compared in slices from naive and inflamed male and female Sprague Dawley rats. Complete Freund's Adjuvant (CFA) injections into the hindpaw induced persistent inflammation. In naive rats, exogenous cannabinoid agonists robustly reduce both eIPSCs and mIPSCs. After 5-7 d of inflammation, the effects of exogenous cannabinoids are significantly reduced because of CB1R desensitization via GRK2/3, as function is recovered in the presence of the GRK2/3 inhibitor, Compound 101 (Cmp101). Inhibition of GABA release by presynaptic µ-opioid receptors in the vlPAG does not desensitize with persistent inflammation. Unexpectedly, while CB1R desensitization significantly reduces the inhibition produced by exogenous agonists, depolarization-induced suppression of inhibition protocols that promote 2-arachidonoylglycerol (2-AG) synthesis exhibit prolonged CB1R activation after inflammation. 2-AG tone is detected in slices from CFA-treated rats when GRK2/3 is blocked, suggesting an increase in 2-AG synthesis after persistent inflammation. Inhibiting 2-AG degradation with the monoacylglycerol lipase (MAGL) inhibitor JZL184 during inflammation results in the desensitization of CB1Rs by endocannabinoids that is reversed with Cmp101. Collectively, these data indicate that persistent inflammation primes CB1Rs for desensitization, and MAGL degradation of 2-AG protects CB1Rs from desensitization in inflamed rats. These adaptations with inflammation have important implications for the development of cannabinoid-based pain therapeutics targeting MAGL and CB1Rs.SIGNIFICANCE STATEMENT Presynaptic G-protein-coupled receptors are resistant to desensitization. Here we find that persistent inflammation increases endocannabinoid levels, priming presynaptic cannabinoid 1 receptors for desensitization on subsequent addition of exogenous agonists. Despite the reduced efficacy of exogenous agonists, endocannabinoids have prolonged efficacy after persistent inflammation. Endocannabinoids readily induce cannabinoid 1 receptor desensitization if their degradation is blocked, indicating that endocannabinoid concentrations are maintained at subdesensitizing levels and that degradation is critical for maintaining endocannabinoid regulation of presynaptic GABA release in the ventrolateral periaqueductal gray during inflammatory states. These adaptations with inflammation have important implications for the development of cannabinoid-based pain therapies.

Persistent inflammation selectively activates opioid-sensitive phasic-firing neurons within the vlPAG.

The ventrolateral periaqueductal gray (vlPAG) is a key brain area within the descending pain modulatory pathway and an important target for opioid-induced analgesia. The vlPAG contains heterogeneous neurons with respect to neurotransmitter content, receptor and channel expression, and in vivo response to noxious stimuli. This study characterizes intrinsic membrane properties of vlPAG neurons to identify neuron types that respond to inflammation and determine whether the pain-responsive neurons are inhibited by opioids. Surveying 382 neurons identified four neuron types with distinct intrinsic firing patterns: Phasic (48%), Tonic (33%), Onset (10%), and Random (9%). Mu-opioid receptor (MOR) expression was determined by the ability of a selective MOR agonist (DAMGO) to activate G protein-coupled inwardly rectifying potassium channel (GIRK) currents. Opioid-sensitive neurons were observed within each neuron type. Opioid sensitivity did not correlate with other intrinsic firing features, including low-threshold spiking that has been previously proposed to identify opioid-sensitive GABAergic neurons in the vlPAG of mice. Complete Freund's adjuvant (CFA)-induced acute inflammation (2 h) had no effect on vlPAG neuron firing patterns. However, persistent inflammation (5-7 days) selectively activated Phasic neurons through a significant reduction in their firing threshold. Opioid-sensitive neurons were strongly activated compared with the opioid-insensitive Phasic neurons. Overall, this study provides a framework to further identify neurons activated by persistent inflammation so that they may be targeted for future pain therapies.NEW & NOTEWORTHY Intrinsic firing properties define four distinct vlPAG neuron populations, and a subset of each population expresses MORs coupled to GIRK channels. Persistent, but not acute, inflammation selectively activates opioid-sensitive Phasic vlPAG neurons. Although the vlPAG is known to contribute to the descending inhibition of pain, the activation of a single physiologically defined neuron type in the presence of persistent inflammation represents a mechanism by which the vlPAG participates in descending facilitation of pain.

Cellular and circuit diversity determines the impact of endogenous opioids in the descending pain modulatory pathway.

The descending pain modulatory pathway exerts important bidirectional control of nociceptive inputs to dampen and/or facilitate the perception of pain. The ventrolateral periaqueductal gray (vlPAG) integrates inputs from many regions associated with the processing of nociceptive, cognitive, and affective components of pain perception, and is a key brain area for opioid action. Opioid receptors are expressed on a subset of vlPAG neurons, as well as on both GABAergic and glutamatergic presynaptic terminals that impinge on vlPAG neurons. Microinjection of opioids into the vlPAG produces analgesia and microinjection of the opioid receptor antagonist naloxone blocks stimulation-mediated analgesia, highlighting the role of endogenous opioid release within this region in the modulation of nociception. Endogenous opioid effects within the vlPAG are complex and likely dependent on specific neuronal circuits activated by acute and chronic pain stimuli. This review is focused on the cellular heterogeneity within vlPAG circuits and highlights gaps in our understanding of endogenous opioid regulation of the descending pain modulatory circuits.

Mice Expressing Regulators of G protein Signaling-insensitive Gαo Define Roles of µ Opioid Receptor Gαo and Gαi Subunit Coupling in Inhibition of Presynaptic GABA Release.

Regulators of G protein signaling (RGS) proteins modulate signaling by G protein-coupled receptors. Using a knock-in transgenic mouse model with a mutation in Gαo that does not bind RGS proteins (RGS-insensitive), we determined the effect of RGS proteins on presynaptic µ opioid receptor (MOR)-mediated inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). The MOR agonists [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and met-enkephalin (ME) inhibited evoked inhibitory postsynaptic currents (eIPSCs) in the RGS-insensitive mice compared with wild-type (WT) littermates, respectively. Fentanyl inhibited eIPSCs similarly in both WT and RGS-insensitive mice. There were no differences in opioid agonist inhibition of spontaneous GABA release between the genotypes. To further probe the mechanism underlying these differences between opioid inhibition of evoked and spontaneous GABA release, specific myristoylated Gα peptide inhibitors for Gαo1 and Gαi1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-Gα complexes. The Gαo1 inhibitor reduced DAMGO inhibition of eIPSCs, but Gαi1-3 inhibitors had no effect. Both Gαo1 and Gαi1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. Gαi1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both Gαo1 and Gαi1-3 inhibitors were needed to block the effects of DAMGO. Finally, baclofen-mediated inhibition of GABA release is unaffected in the RGS-insensitive mice and in the presence of Gαo1 and Gαi1-3 inhibitor peptides, suggesting that GABAB receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT: Presynaptic µ opioid receptors (MORs) in the ventrolateral periaqueductal gray are critical for opioid analgesia and are negatively regulated by RGS proteins. These data in RGS-insensitive mice provide evidence that MOR agonists differ in preference for Gαo versus Gαi and regulation by RGS proteins in presynaptic terminals, providing a mechanism for functional selectivity between agonists. The results further define important differences in MOR and GABAB receptor coupling to G proteins that could be exploited for new pain therapies.

Regulators of G-Protein Signaling (RGS) Proteins Promote Receptor Coupling to G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channels.

Regulators of G-protein signaling (RGS) proteins negatively modulate presynaptic µ-opioid receptor inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). Paradoxically, we find that G-protein-coupled receptor (GPCR) activation of G-protein-gated inwardly rectifying K+ channels (GIRKs) in the vlPAG is reduced in an agonist- and receptor-dependent manner in transgenic knock-in mice of either sex expressing mutant RGS-insensitive Gαo proteins. µ-Opioid receptor agonist activation of GIRK currents was reduced for DAMGO and fentanyl but not for [Met5]-enkephalin acetate salt hydrate (ME) in the RGS-insensitive heterozygous (Het) mice compared with wild-type mice. The GABAB agonist baclofen-induced GIRK currents were also reduced in the Het mice. We confirmed the role of Gαo proteins in µ-opioid receptor and GABAB receptor signaling pathways in wild-type mice using myristoylated peptide inhibitors of Gαo1 and Gαi1-3 The results using these inhibitors indicate that receptor activation of GIRK channels is dependent on the preference of the agonist-stimulated receptor for Gαo versus that for Gαi. DAMGO and fentanyl-mediated GIRK currents were reduced in the presence of the Gαo1 inhibitor, but not the Gαi1-3 inhibitors. In contrast, the Gαo1 peptide inhibitor did not affect ME activation of GIRK currents, which is consistent with results in the Het mice, but the Gαi1-3 inhibitors significantly reduced ME-mediated GIRK currents. Finally, the reduction in GIRK activation in the Het mice plays a role in opioid- and baclofen-mediated spinal antinociception, but not supraspinal antinociception. Thus, our studies indicate that RGS proteins have multiple mechanisms of modulating GPCR signaling that produce negative and positive regulation of signaling depending on the effector.SIGNIFICANCE STATEMENT Regulators of G-protein signaling (RGS) proteins positively modulate GPCR coupling to GIRKs, and this coupling is critical for opioid- and baclofen-mediated spinal antinociception, whereas µ-opioid receptor-mediated supraspinal antinociception depends on presynaptic inhibition that is negatively regulated by RGS proteins. The identification of these opposite roles for RGS proteins has implications for signaling via other GPCRs.

Bidirectional Modulation of Intrinsic Excitability in Rat Prelimbic Cortex Neuronal Ensembles and Non-Ensembles after Operant Learning.

Learned associations between environmental stimuli and rewards drive goal-directed learning and motivated behavior. These memories are thought to be encoded by alterations within specific patterns of sparsely distributed neurons called neuronal ensembles that are activated selectively by reward-predictive stimuli. Here, we use the Fos promoter to identify strongly activated neuronal ensembles in rat prelimbic cortex (PLC) and assess altered intrinsic excitability after 10 d of operant food self-administration training (1 h/d). First, we used the Daun02 inactivation procedure in male FosLacZ-transgenic rats to ablate selectively Fos-expressing PLC neurons that were active during operant food self-administration. Selective ablation of these neurons decreased food seeking. We then used male FosGFP-transgenic rats to assess selective alterations of intrinsic excitability in Fos-expressing neuronal ensembles (FosGFP+) that were activated during food self-administration and compared these with alterations in less activated non-ensemble neurons (FosGFP-). Using whole-cell recordings of layer V pyramidal neurons in an ex vivo brain slice preparation, we found that operant self-administration increased excitability of FosGFP+ neurons and decreased excitability of FosGFP- neurons. Increased excitability of FosGFP+ neurons was driven by increased steady-state input resistance. Decreased excitability of FosGFP- neurons was driven by increased contribution of small-conductance calcium-activated potassium (SK) channels. Injections of the specific SK channel antagonist apamin into PLC increased Fos expression but had no effect on food seeking. Overall, operant learning increased intrinsic excitability of PLC Fos-expressing neuronal ensembles that play a role in food seeking but decreased intrinsic excitability of Fos- non-ensembles.SIGNIFICANCE STATEMENT Prefrontal cortex activity plays a critical role in operant learning, but the underlying cellular mechanisms are unknown. Using the chemogenetic Daun02 inactivation procedure, we found that a small number of strongly activated Fos-expressing neuronal ensembles in rat PLC play an important role in learned operant food seeking. Using GFP expression to identify Fos-expressing layer V pyramidal neurons in prelimbic cortex (PLC) of FosGFP-transgenic rats, we found that operant food self-administration led to increased intrinsic excitability in the behaviorally relevant Fos-expressing neuronal ensembles, but decreased intrinsic excitability in Fos- neurons using distinct cellular mechanisms.

Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories.

UNLABELLED: In operant learning, initial reward-associated memories are thought to be distinct from subsequent extinction-associated memories. Memories formed during operant learning are thought to be stored in "neuronal ensembles." Thus, we hypothesize that different neuronal ensembles encode reward- and extinction-associated memories. Here, we examined prefrontal cortex neuronal ensembles involved in the recall of reward and extinction memories of food self-administration. We first trained rats to lever press for palatable food pellets for 7 d (1 h/d) and then exposed them to 0, 2, or 7 daily extinction sessions in which lever presses were not reinforced. Twenty-four hours after the last training or extinction session, we exposed the rats to either a short 15 min extinction test session or left them in their homecage (a control condition). We found maximal Fos (a neuronal activity marker) immunoreactivity in the ventral medial prefrontal cortex of rats that previously received 2 extinction sessions, suggesting that neuronal ensembles in this area encode extinction memories. We then used the Daun02 inactivation procedure to selectively disrupt ventral medial prefrontal cortex neuronal ensembles that were activated during the 15 min extinction session following 0 (no extinction) or 2 prior extinction sessions to determine the effects of inactivating the putative food reward and extinction ensembles, respectively, on subsequent nonreinforced food seeking 2 d later. Inactivation of the food reward ensembles decreased food seeking, whereas inactivation of the extinction ensembles increased food seeking. Our results indicate that distinct neuronal ensembles encoding operant reward and extinction memories intermingle within the same cortical area. SIGNIFICANCE STATEMENT: A current popular hypothesis is that neuronal ensembles in different prefrontal cortex areas control reward-associated versus extinction-associated memories: the dorsal medial prefrontal cortex (mPFC) promotes reward seeking, whereas the ventral mPFC inhibits reward seeking. In this paper, we use the Daun02 chemogenetic inactivation procedure to demonstrate that Fos-expressing neuronal ensembles mediating both food reward and extinction memories intermingle within the same ventral mPFC area.

Associative Learning Drives the Formation of Silent Synapses in Neuronal Ensembles of the Nucleus Accumbens.

BACKGROUND: Learned associations between environmental stimuli and rewards play a critical role in addiction. Associative learning requires alterations in sparsely distributed populations of strongly activated neurons, or neuronal ensembles. Until recently, assessment of functional alterations underlying learned behavior was restricted to global neuroadaptations in a particular brain area or cell type, rendering it impossible to identify neuronal ensembles critically involved in learned behavior. METHODS: We used Fos-GFP transgenic mice that contained a transgene with a Fos promoter driving expression of green fluorescent protein (GFP) to detect neurons that were strongly activated during associative learning, in this case, context-independent and context-specific cocaine-induced locomotor sensitization. Whole-cell electrophysiological recordings were used to assess synaptic alterations in specifically activated GFP-positive (GFP+) neurons compared with surrounding nonactivated GFP-negative (GFP-) neurons 90 min after the sensitized locomotor response. RESULTS: After context-independent cocaine sensitization, cocaine-induced locomotion was equally sensitized by repeated cocaine injections in two different sensitization contexts. Correspondingly, silent synapses in these mice were induced in GFP+ neurons, but not GFP- neurons, after sensitization in both of these contexts. After context-specific cocaine sensitization, cocaine-induced locomotion was sensitized exclusively in mice trained and tested in the same context (paired group), but not in mice that were trained in one context and then tested in a different context (unpaired group). Silent synapses increased in GFP+ neurons, but not in GFP- neurons from mice in the paired group, but not from mice in the unpaired group. CONCLUSIONS: Our results indicate that silent synapses are formed only in neuronal ensembles of the nucleus accumbens shell that are related to associative learning.

Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.

Context-induced reinstatement of drug seeking is a well established animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in striatum has previously been shown to contribute to context-induced reinstatement of heroin, cocaine, and alcohol seeking, but not yet for methamphetamine seeking. In this study, we found that context-induced reinstatement of methamphetamine seeking increased expression of the neural activity marker Fos in dorsal but not ventral striatum. Reversible inactivation of neural activity in dorsolateral but not dorsomedial striatum using the GABA agonists muscimol and baclofen decreased context-induced reinstatement. Based on our previous findings that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in dorsal striatum and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the immediate early genes Fos and FosB and the NMDA receptor subunit gene Grin2a in only Fos-positive neurons. RNAscope in situ hybridization confirmed that Grin2a, as well as Grin2b, expression were increased in only Fos-positive neurons from dorsolateral, but not dorsomedial, striatum. Our results demonstrate an important role of dorsolateral striatum in context-induced reinstatement of methamphetamine seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons.