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.

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.

Compensatory eating behaviors in male and female rats in response to exercise training.

Exercise is often used as a strategy for weight loss maintenance. In preclinical models, we have shown that exercise may be beneficial because it counters the biological drive to regain weight. However, our studies have demonstrated sex differences in the response to exercise in this context. In the present study, we sought to better understand why females and males exhibit different compensatory food eating behaviors in response to regular exercise. Using a forced treadmill exercise paradigm, we measured weight gain, energy expenditure, food intake in real time, and the anorectic effects of leptin. The 4-wk exercise training resulted in reduced weight gain in males and sustained weight gain in females. In male rats, exercise decreased intake, whereas it increased food intake in females. Our results suggest that the anorectic effects of leptin were not responsible for these sex differences in appetite in response to exercise. If these results translate to the human condition, they may reveal important information for the use and application of regular exercise programs.

Cannabinoids in the descending pain modulatory circuit: Role in inflammation.

The legalization of cannabis in some states has intensified interest in the potential for cannabis and its constituents to lead to novel therapeutics for pain. Our understanding of the cellular mechanisms underlying cannabinoid actions in the brain have lagged behind opioids; however, the current opioid epidemic has also increased attention on the use of cannabinoids as alternatives to opioids for pain, especially chronic pain that requires long-term use. Endogenous cannabinoids are lipid signaling molecules that have complex roles in modulating neuronal function throughout the brain. In this review, we discuss cannabinoid functions in the descending pain modulatory pathway, a brain circuit that integrates cognitive and emotional processing of pain to modulate incoming sensory inputs. In addition, we highlight areas where further studies are necessary to understand cannabinoid regulation of descending pain modulation.

Running from fear: Exercise modulation of fear extinction.

Extinction-based exposure therapy is the most common behavioral therapy for anxiety and trauma-related disorders, but fear tends to resurface even after successful extinction. Identification of novel strategies to enhance fear extinction and reduce fear relapse is of paramount importance to mental health. Exercise can enhance cognitive function, but it is not yet well understood whether exercise can be an effective augmentation strategy for fear extinction. In the current review, we present the current state of knowledge on the effects of exercise on fear extinction. Effects of exercise duration, explanations for conflicting results, and potential mechanisms, focusing on a hypothesized role for dopamine, are all discussed. We also provide new data suggesting that the timing in which acute exercise occurs relative to fear extinction, is a crucial variable in determining whether exercise can enhance fear extinction. Clinical implications and ideas to guide future research endeavors in this area are provided.

Feeding the developing brain: Juvenile rats fed diet rich in prebiotics and bioactive milk fractions exhibit reduced anxiety-related behavior and modified gene expression in emotion circuits.

Early life nutrition is critical for brain development. Dietary prebiotics and bioactive milk fractions support brain development by increasing plasticity and altering activity in brain regions important for cognition and emotion regulation, perhaps through the gut-microbiome-brain axis. Here we examined the impact of a diet containing prebiotics, lactoferrin, and milk fat globule membrane (test diet) on beneficial gut bacteria, basal gene expression for activity and plasticity markers within brain circuits important for cognition and anxiety, and anxiety-related behavior in the open field. Juvenile male F344 rats were fed the test diet or a calorically matched control diet beginning postnatal day 24. After 4 weeks on diets, rats were sacrificed and brains were removed. Test diet significantly increased mRNA expression for cfos, brain derived neurotropic factor, and the GluN1 subunit of the NMDA receptor in the prefrontal cortex and reduced cfos mRNA within the amygdala. Diet-induced increases in fecal Lactobacillus spp., measured using selective bacterial culture, positively correlated with altered gene expression for cfos and serotonin receptors within multiple brain regions. In a separate cohort of juvenile rats, 4 weeks of the test diet increased time spent in the center of the open field, a behavior indicative of reduced anxiety. These data demonstrate that early life diets containing prebiotics and bioactive milk fractions can adaptively alter genes in neural circuits underlying emotion regulation and impact anxiety-related behavior.

Activation of Nigrostriatal Dopamine Neurons during Fear Extinction Prevents the Renewal of Fear.

Manipulations that increase dopamine (DA) signaling can enhance fear extinction, but the circuits involved remain unknown. DA neurons originating in the substantia nigra (SN) projecting to the dorsal striatum (DS) are traditionally viewed in the context of motor behavior, but growing data implicate this nigrostriatal circuit in emotion. Here we investigated the role of nigrostriatal DA in fear extinction. Activation of SN DA neurons with designer Gq-coupled receptors exclusively activated by designer drugs (Gq-DREADD) during fear extinction had no effect on fear extinction acquisition, but enhanced fear extinction memory and blocked the renewal of fear in a novel context; a pattern of data paralleled by cFos expression in the central amygdala. D1 receptors in the DS are a likely target mediating the effects of SN DA activation. D1-expressing neurons in the medial DS (DMS) were recruited during fear extinction, and Gq-DREADD-induced DA potentiated activity of D1-expressing neurons in both the DMS and the lateral DS (DLS). Pharmacological activation of D1 receptors in the DS did not impact fear extinction acquisition or memory, but blocked fear renewal in a novel context. These data suggest that activation of SN DA neurons and DS D1 receptors during fear extinction render fear extinction memory resistant to the disrupting effects of changes in contextual contingencies, perhaps by recruiting habitual learning strategies involving the DLS. Nigrostriatal DA thus represents a novel target to enhance long-term efficacy of extinction-based therapies for anxiety and trauma-related disorders.

Acute exercise enhances the consolidation of fear extinction memory and reduces conditioned fear relapse in a sex-dependent manner.

Fear extinction-based exposure therapy is the most common behavioral therapy for anxiety and trauma-related disorders, but fear extinction memories are labile and fear tends to return even after successful extinction. The relapse of fear contributes to the poor long-term efficacy of exposure therapy. A single session of voluntary exercise can enhance the acquisition and consolidation of fear extinction in male rats, but the effects of exercise on relapse of fear after extinction are not well understood. Here, we characterized the effects of 2 h of voluntary exercise during the consolidation phase of contextual or auditory fear extinction learning on long-term fear extinction memory and renewal in adult, male and female, Long-Evans rats. Results indicate that exercise enhances consolidation of fear extinction memory and reduces fear relapse after extinction in a sex-dependent manner. These data suggest that brief bouts of exercise could be used as an augmentation strategy for exposure therapy, even in previously sedentary subjects. Fear memories of discrete cues, rather than of contextual ones, may be most susceptible to exercise-augmented extinction, especially in males. Additionally, exercise seems to have the biggest impact on fear relapse phenomena, even if fear extinction memories themselves are only minimally enhanced.

Voluntary exercise during extinction of auditory fear conditioning reduces the relapse of fear associated with potentiated activity of striatal direct pathway neurons.

Relapse of previously extinguished fear presents a significant, pervasive obstacle to the successful long-term treatment of anxiety and trauma-related disorders. Thus, identification of a novel means to enhance fear extinction to stand the passage of time and generalize across contexts is of the utmost importance. Acute bouts of exercise can be used as inexpensive, noninvasive treatment strategies to reduce anxiety, and have been shown to enhance memory for extinction when performed in close temporal proximity to the extinction session. However, it is unclear whether acute exercise can be used to prevent relapse of fear, and the neural mechanisms underlying this potential effect are unknown. The current study therefore examined whether acute exercise during extinction of auditory fear can protect against the later relapse of fear. Male F344 rats lacking an extended history of wheel running were conditioned to fear a tone CS and subsequently extinguished within either a freely mobile running wheel, a locked wheel, or a control context lacking a wheel. Rats exposed to fear extinction within a freely mobile wheel ran during fear extinction, and demonstrated reduced fear as well as attenuated corticosterone levels during re-exposure to the extinguished CS during the relapse test in a novel context 1week later. Examination of cfos mRNA patterns elicited by re-exposure to the extinguished CS during the relapse test revealed that acute exercise during extinction decreased activation of brain circuits classically involved in driving fear expression and interestingly, increased activity within neurons of the direct striatal pathway involved in reward signaling. These data suggest that exercise during extinction reduces relapse through a mechanism involving the direct pathway of the striatum. It is suggested that a positive affective state could become associated with the CS during exercise during extinction, thus resulting in a relapse-resistant extinction memory.