Search

Targeting the endocannabinoid (eCB) signaling system for pain relief is an important treatment option that is only now beginning to be mechanistically explored. In this review, we focus on two recently appreciated cannabinoid-based targeting strategies, treatments with cannabidiol (CBD) and α / β -hydrolase domain containing 6 (ABHD6) inhibitors, which have the exciting potential to produce pain relief through distinct mechanisms of action and without intoxication. We review evidence on plant-derived cannabinoids for pain, with an emphasis on CBD and its multiple molecular targets expressed in pain pathways. We also discuss the function of eCB signaling in regulating pain responses and the therapeutic promises of inhibitors targeting ABHD6, a 2-arachidonoylglycerol (2-AG)-hydrolyzing enzyme. Finally, we discuss how the novel cannabinoid biosensor GRAB eCB2.0 may be leveraged to enable the discovery of targets modulated by cannabinoids at a circuit-specific level. SIGNIFICANCE STATEMENT: Cannabis has been used by humans as an effective medicine for millennia, including for pain management. Recent evidence emphasizes the therapeutic potential of compounds that modulate endocannabinoid signaling. Specifically, cannabidiol and inhibitors of the enzyme ABHD6 represent promising strategies to achieve pain relief by modulating endocannabinoid signaling in pain pathways via distinct, nonintoxicating mechanisms of action., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: The endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid (CB) receptors with distinct pharmacological profiles, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRAB eCB2.0 , detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-CBs remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-CB action. Materials and Methods: GRAB eCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results: 2-AG increased GRAB eCB2.0 fluorescent signal (EC 50 =85 nM), and the cannabinoid 1 receptor (CB 1 R) antagonist, SR141716 (SR1), decreased GRAB eCB2.0 signal (IC 50 =3.3 nM), responses that mirror their known potencies at the CB 1 R. GRAB eCB2.0 fluorescent signal also increased in response to AEA (EC 50 =815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (EC 50 =632 and 868 nM, respectively), Δ 9 -tetrahydrocannabinol (Δ 9 -THC), and Δ 8 -THC (EC 50 =1.6 and 2.0 μM, respectively), and the artificial CB 1 R agonist, CP55,940 (CP; EC 50 =82 nM); however their potencies were less than what has been described at CB 1 R. Cannabidiol (CBD) did not affect basal GRAB eCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRAB eCB2.0 responses (IC 50 =9.7 nM). Conclusions: 2-AG and SR1 modulate the GRAB eCB2.0 fluorescent signal with EC 50 values that mirror their potencies at CB 1 R, whereas AEA, eCB analogues, THC, and CP increase GRAB eCB2.0 fluorescent signal with EC 50 values significantly lower than their potencies at CB 1 R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRAB eCB2.0 retains the negative allosteric modulator (NAM) property of CBD at CB 1 R. This study describes the pharmacological profile of GRAB eCB2.0 to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB 1 R ligands.

Background and Purpose: Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRAB eCB2.0 , can address this shortfall., Experimental Approach: 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRAB eCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader., Key Results: 2-AG and AEA increased GRAB eCB2.0 fluorescence in N2a cells with EC 50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB 1 R) antagonist SR141617 and absent in cells expressing the mutant-GRAB eCB2.0 . ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRAB eCB2.0 signal within minutes primarily via activation of P2X 7 receptors (P2X 7 R). This response was dependent on diacylglycerol lipase β activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/β-hydrolase domain containing 6 (ABHD6)., Conclusions and Implications: Considering that P2X 7 R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation., (© 2024 British Pharmacological Society.)

Stress has been shown to promote the development and persistence of binge eating behaviors. However, the neural circuit mechanisms for stress-induced binge-eating behaviors are largely unreported. The endogenous dynorphin (dyn)/kappa opioid receptor (KOR) opioid neuropeptide system has been well established to be a crucial mediator of the anhedonic component of stress. Here, we aimed to dissect the basis of dynorphinergic control of stress-induced binge-like eating behavior. We first established a mouse behavioral model for stress-induced binge-like eating behaviors. We found that mice exposed to stress increased their food intake of familiar palatable food (high fat, high sugar, HPD) compared to non-stressed mice. Following a brain-wide analysis, we isolated robust cFos-positive cells in the Claustrum (CLA), a subcortical structure with highly abundant KOR expression, following stress-induced binge-eating behavior. We report that KOR signaling in CLA is necessary for this elevated stress-induced binge eating behavior using local pharmacology and local deletion of KOR. In vivo calcium recordings using fiber photometry revealed a disinhibition circuit structure in the CLA during the initiation of HPD feeding bouts. We further established the dynamics of endogenous dynorphinergic control of this behavior using a genetically encoded dynorphin biosensor, Klight. Combined with 1-photon single-cell calcium imaging, we report significant heterogeneity with the CLA population during stress-induced binge eating and such behavior attenuates local dynorphin tone. Furthermore, we isolate the anterior Insular cortex (aIC) as the potential source of endogenous dynorphin afferents in the CLA. By characterizing neural circuits and peptidergic mechanisms within the CLA, we uncover a pathway that implicates endogenous opioid regulation stress-induced binge eating., Competing Interests: DECLARATION OF INTERESTS The authors have declared no competing interest.

No preclinical experimental approach enables the study of voluntary oral consumption of high-concentration Δ 9 -tetrahydrocannabinol (THC) and its intoxicating effects, mainly owing to the aversive response of rodents to THC that limits intake. Here, we developed a palatable THC formulation and an optimized access paradigm in mice to drive voluntary consumption. THC was formulated in chocolate gelatin (THC-E-gel). Adult male and female mice were allowed ad libitum access for 1 and 2 hr. Cannabimimetic responses (hypolocomotion, analgesia, and hypothermia) were measured following access. Levels of THC and its metabolites were measured in blood and brain tissue. Acute acoustic startle responses were measured to investigate THC-induced psychotomimetic behavior. When allowed access for 2 hr to THC-E-gel on the second day of a 3-day exposure paradigm, adult mice consumed up to ≈30 mg/kg over 2 hr, which resulted in robust cannabimimetic behavioral responses (hypolocomotion, analgesia, and hypothermia). Consumption of the same gelatin decreased on the following third day of exposure. Pharmacokinetic analysis shows that THC-E-gel consumption led to parallel accumulation of THC and its psychoactive metabolite, 11-OH-THC, in the brain, a profile that contrasts with the known rapid decline in brain 11-OH-THC levels following THC intraperitoneal (i.p.) injections. THC-E-gel consumption increased the acoustic startle response in males but not in females, demonstrating a sex-dependent effect of consumption. Thus, while voluntary consumption of THC-E-gel triggered equivalent cannabimimetic responses in male and female mice, it potentiated acoustic startle responses preferentially in males. We built a dose-prediction model that included cannabimimetic behavioral responses elicited by i.p. versus THC-E-gel to test the accuracy and generalizability of this experimental approach and found that it closely predicted the measured acoustic startle results in males and females. In summary, THC-E-gel offers a robust preclinical experimental approach to study cannabimimetic responses triggered by voluntary consumption in mice, including sex-dependent psychotomimetic responses., Competing Interests: AE, FU, AT, DS, AS, DP, MB, NS, BL No competing interests declared, (© 2023, English et al.)

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT + neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3 + neurons blocked subsequent cocaine CPP. SERT + /VGluT3 - expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT + neurons produced place aversion, whereas optical stimulation of SERT + terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT 1B receptors potentiated subsequent cocaine CPP. 5-HT 1B is known to be expressed on 5-HT terminals in NAc, and 5-HT 1B transcript was also detected in Pdyn + , Adora2a + and ChAT + (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT 1B transcript was selectively elevated in Pdyn + cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT 1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Behavioral stress exposure increases the risk of drug-taking in individuals with substance use disorders by mechanisms involving the dynorphins, which are the endogenous neuropeptides for the kappa opioid receptor (KOR). KOR agonists have been shown to encode dysphoria, aversion, and changes in reward valuation, and kappa opioid antagonists are in clinical development for treating substance use disorders. In this study, we confirmed that KORs were expressed in dopaminergic neurons in the ventral tegmental area (VTA) of male C57BL6/J mice. Genetic ablation of KORs from dopamine neurons blocked the potentiating effects of repeated forced swim stress on cocaine conditioned place preference (CPP). KOR activation inhibited dopamine neuron GCaMP6m calcium activity in VTA during swim stress and caused a rebound enhancement during the period after stress exposure. Transient optogenetic inhibition of VTA dopamine neurons with AAV5-DIO-SwiChR was acutely aversive in a real time place preference assay and blunted cocaine CPP when inhibition was administered concurrently with cocaine conditioning. However, when inhibition preceded cocaine conditioning by 30 min, cocaine CPP was enhanced. Retrograde tracing with CAV2-DIO-ZsGreen identified a population of prodynorphin Cre neurons in the dorsal raphe nucleus (DRN) projecting to the VTA. Optogenetic stimulation of dynorphinergic neurons within the DRN by Channelrhodopsin2 activated KOR in VTA and ablation of prodynorphin blocked stress potentiation of cocaine CPP. Together, these studies demonstrate the presence of a dynorphin/KOR midbrain circuit that projects from the DRN to VTA and is involved in altering the dynamic response of dopamine neuron activity to enhance drug reward learning., Competing Interests: Declaration of Competing Interest None.

Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In C57BL/6 male mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant-delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre- and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdyn Cre neurons using Channelrhodopsin-2 disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

The prototypical member of the receptor-inactivating kappa opioid receptor (KOR) antagonists, norbinaltorphimine (norBNI), produces prolonged receptor inactivation by a cJun kinase mechanism. These antagonists have potential therapeutic utility in the treatment of stress disorders; however, additional preclinical characterization is necessary to understand important aspects of their action. In this study, we report that norBNI does not work as effectively in female mice as in males because of estrogen regulation of G protein receptor kinase (GRK); pretreatment of ovary-intact female mice with the selective GRK2/3 inhibitor, Compound 101, made females equally sensitive to norBNI as males. Prior observations suggested that in vivo treatment with norBNI does not produce long-lasting inhibition of KOR regulation of dopamine release in the nucleus accumbens. We assessed the persistence of norBNI receptor inactivation in subcellular compartments. Fast-scan cyclic voltammetry recordings confirmed that presynaptic inhibition of dopamine release by the KOR agonist U69,593 was not blocked by in vivo pretreatment with norBNI under conditions that prevented KOR-mediated aversion and analgesia. We employed a novel in vivo proxy sensor of KOR activation, adenovirus associated double floxed inverted-HyPerRed, and demonstrated that KOR activation stimulates cJun kinase-dependent reactive oxygen species (ROS) production in somatic regions of ventral tegmental area dopamine neurons, but did not activate ROS production in dopamine terminals. The compartment selective action helps explain how dopamine somatic, but not terminally expressed, KORs are inactivated by norBNI. These results further elucidate molecular signaling mechanisms mediating receptor-inactivating KOR antagonist action and advance medication development for this novel class of stress-resilience medications. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) antagonists are being developed as novel proresilience therapeutics for the treatment of mood and substance use disorders. This study showed that the long-acting KOR antagonists are affected by both the sex of the animal and the subcellular compartment in which the receptor is expressed., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Stress is highly pervasive in humans, impacting motivated behaviors with an enormous toll on life quality. Many of the effects of stress are orchestrated by neuropeptides such as corticotropin-releasing factor (CRF). It has previously been shown that in stress-naïve male mice, CRF acts in the core of the nucleus accumbens (NAc) to produce appetitive effects and to increase dopamine release; yet in stress-exposed male mice, CRF loses its capacity to modulate NAc dopamine release and is aversive. In the current research, we tested whether this effect is comparable in females to males and whether the neuroadaptation is susceptible to social transmission. We found that, like in males, CRF increased dopamine release in stress-naïve but not stress-exposed female mice. Importantly, this persistent physiological change was not accompanied by overt behavioral changes that would be indicative of depression- or anxiety-like phenotype. Nonetheless, when these mice were housed for 7 days with stress-naïve conspecifics, the cage mates also exhibited a loss of dopamine potentiation by CRF. These data demonstrate the asymptomatic, yet pervasive transmission of stress-related neuroadaptations in the population., (Copyright © 2020 Steger, Land, Lemos, Chavkin and Phillips.)

Chronic pain affects a significant percentage of the United States population, and available pain medications like opioids have drawbacks that make long-term use untenable. Cannabinoids show promise in the management of pain, but long-term treatment of pain with cannabinoids has been challenging to implement in preclinical models. We developed a voluntary, gelatin oral self-administration paradigm that allowed male and female mice to consume ∆ 9 -tetrahydrocannabinol, cannabidiol, or morphine ad libitum. Mice stably consumed these gelatins over 3 weeks, with detectable serum levels. Using a real-time gelatin measurement system, we observed that mice consumed gelatin throughout the light and dark cycles, with animals consuming less THC-gelatin than the other gelatin groups. Consumption of all three gelatins reduced measures of allodynia in a chronic, neuropathic sciatic nerve injury model, but tolerance to morphine developed after 1 week while THC or CBD reduced allodynia over three weeks. Hyperalgesia gradually developed after sciatic nerve injury, and by the last day of testing, THC significantly reduced hyperalgesia, with a trend effect of CBD, and no effect of morphine. Mouse vocalizations were recorded throughout the experiment, and mice showed a large increase in ultrasonic, broadband clicks after sciatic nerve injury, which was reversed by THC, CBD, and morphine. This study demonstrates that mice voluntarily consume both cannabinoids and opioids via gelatin, and that cannabinoids provide long-term relief of chronic pain states. In addition, ultrasonic clicks may objectively represent mouse pain status and could be integrated into future pain models.

Activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) by the G i/o protein-coupled κ opioid receptor (KOR), μ opioid, and D2 dopamine receptors stimulates peroxiredoxin 6 (PRDX6)-mediated production of reactive oxygen species (ROS). ROS production by KOR-inactivating antagonists norbinaltorphimine (norBNI) and JDTic blocks Gα i protein activation, but the signaling mechanisms and consequences of JNK activation by KOR agonists remain uncharacterized. Binding of arrestins to KOR causes desensitization of G protein signaling and acts as a scaffold to initiate MAPK activation. Here, we found that the KOR agonists U50,488 and dynorphin B stimulated biphasic JNK activation with an early arrestin-independent phase, requiring the small G protein RAC family small GTPase 1 (RAC1) and protein kinase C (PKC), and a later arrestin-scaffolded phase, requiring RAC1 and Ras homolog family member (RHO) kinase. JNK activation by U50,488 and dynorphin B also stimulated PRDX6-dependent ROS production but with an inverted U-shaped dose-response relationship. KOR agonist-induced ROS generation resulted from the early arrestin-independent phase of JNK activation, and this ROS response was suppressed by arrestin-dependent activation of the MAPK p38. The apparent balance between p38 MAPK and JNK/ROS signaling has important physiological implications for understanding of dynorphin activities during the stress response. To visualize these activities, we monitored KOR agonist-mediated activation of ROS in transfected live cells by two fluorescent sensors, CellROX Green and HyPerRed. These findings establish an important aspect of opioid receptor signaling and suggest that ROS induction may be part of the physiological response to KOR activation., (© 2019 Schattauer et al.)

Kappa receptor activation by dynorphins contributes to the anxiogenic, dysphoric, and cognitive disrupting effects of repeated stress, suggesting that kappa receptor antagonists might have therapeutic utility in the treatment of stress disorders. Three classes of kappa antagonists have been distinguished: non-selective, selective-competitive (readily reversible), and non-competitive (receptor-inactivating); however, which would be the most effective medication has not been established. To assess the utility of receptor inactivating antagonists, we tested the effects of a range of doses in both male and female mice. As previously established, the antinociceptive effects of the kappa agonist U50,488 were blocked by a single injection of the long-acting antagonist norbinatorphimine (norBNI) (10 mg/kg i.p.) in male mice. Ten to 20-fold lower doses of norBNI were ineffective after a single administration, but daily administration of 1.0 or 0.5 mg/kg for 5 days completely blocked U50,488 antinociceptive effects. Daily administration of 0.1 mg/kg norBNI produced slowly accumulating inhibition and completely blocked the antinociceptive effect of U50,488 after 20-30 days. Estrogen reduces female sensitivity to kappa opioid effects, but 30 days of 0.1 mg/kg norBNI completely blocked U50,488 analgesia in ovariectomized mice. Receptor inactivation in both male and female mice treated for 30 days with 0.1 mg/kg norBNI persisted for at least 1-week. These results suggest that receptor-inactivating kappa antagonists are effective in both males and females when given at 100-fold lower doses than typically administered in preclinical studies. The enhanced safety of this low-dosing protocol has important clinical implications if receptor inactivating kappa antagonists advance in medication development.

Activation of κ opioid receptors (KORs) produces analgesia and aversion via distinct intracellular signaling pathways, but whether G protein-biased KOR agonists can be designed to have clinical utility will depend on a better understanding of the signaling mechanisms involved. We found that KOR activation produced conditioned place aversion and potentiated CPP for cocaine in male and female C57BL/6N mice. Consistent with this, males and females both showed arrestin-mediated increases in phospho-p38 MAPK following KOR activation. Unlike in males, however, KOR activation had inconsistent analgesic effects in females and KOR increased Gβγ-mediated ERK phosphorylation in males, but not females. KOR desensitization was not responsible for the lack of response in females because neither Grk3 nor Pdyn gene knock-out enhanced analgesia. Instead, responsiveness was estrous cycle dependent because KOR analgesia was evident during low estrogen phases of the cycle and in ovariectomized (OVX) females. Estradiol treatment of OVX females suppressed KOR-mediated analgesia, demonstrating that estradiol was sufficient to blunt Gβγ-mediated KOR signals. G protein-coupled receptor kinase 2 (GRK2) is known to regulate ERK activation, and we found that the inhibitory, phosphorylated form of GRK2 was significantly higher in intact females. GRK2/3 inhibition by CMPD101 increased KOR stimulation of phospho-ERK in females, decreased sex differences in KOR-mediated inhibition of dopamine release, and enhanced mu opioid receptor and KOR-mediated analgesia in females. In OVX females, estradiol increased the association between GRK2 and Gβγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gβγ by GRK2, blunts G protein-mediated signals. SIGNIFICANCE STATEMENT Chronic pain disorders are more prevalent in females than males, but opioid receptor agonists show inconsistent analgesic efficacy in females. κ opioid receptor (KOR) agonists have been tested in clinical trials for treating pain disorders based on their analgesic properties and low addictive potential. However, the molecular mechanisms underlying sex differences in KOR actions were previously unknown. Our studies identify an intracellular mechanism involving estradiol regulation of G protein-coupled receptor kinase 2 that is responsible for sexually dimorphic analgesic responses following opioid receptor activation. Understanding this mechanism will be critical for developing effective nonaddictive opioid analgesics for use in women and characterizing sexually dimorphic effects in other inhibitory G protein-coupled receptor signaling responses., (Copyright © 2018 the authors 0270-6474/18/388031-13$15.00/0.)

The dynorphin/κ-opioid receptor (KOR) system has been previously implicated in the regulation of cognition, but the neural circuitry and molecular mechanisms underlying KOR-mediated cognitive disruption are unknown. Here, we used an operational test of cognition involving timing and behavioral inhibition and found that systemic KOR activation impairs performance of male and female C57BL/6 mice in the differential reinforcement of low response rate (DRL) task. Systemic KOR antagonism also blocked stress-induced disruptions of DRL performance. KOR activation increased 'bursts' of incorrect responses in the DRL task and increased marble burying, suggesting that the observed disruptions in DRL performance may be attributed to KOR-induced increases in compulsive behavior. Local inactivation of KOR by injection of the long-acting antagonist nor-BNI in the ventral tegmental area (VTA), but not the infralimbic prefrontal cortex (PFC) or dorsal raphe nucleus (DRN), prevented disruption of DRL performance caused by systemic KOR activation. Cre-dependent genetic excision of KOR from dopaminergic, but not serotonergic neurons, also blocked KOR-mediated disruption of DRL performance. At the molecular level, we found that these disruptive effects did not require arrestin-dependent signaling, because neither global deletion of G-protein receptor kinase 3 (GRK3) nor cell-specific deletion of GRK3/arrestin-dependent p38α MAPK from dopamine neurons blocked KOR-mediated DRL disruptions. We then showed that nalfurafine, a clinically available G-biased KOR agonist, could also produce DRL disruptions. Together, these studies demonstrate that KOR activation in VTA dopamine neurons disrupts behavioral inhibition in a GRK3/arrestin-independent manner and suggests that KOR antagonists could be beneficial for decreasing stress-induced compulsive behaviors.

Inactivation of opioid receptors limits the therapeutic efficacy of morphine-like analgesics and mediates the long duration of kappa opioid antidepressants by an uncharacterized, arrestin-independent mechanism. Here we use an iterative, discovery-based proteomic approach to show that following opioid administration, peroxiredoxin 6 (PRDX6) is recruited to the opioid receptor complex by c-Jun N-terminal kinase (JNK) phosphorylation. PRDX6 activation generates reactive oxygen species via NADPH oxidase, reducing the palmitoylation of receptor-associated Gαi in a JNK-dependent manner. Selective inhibition of PRDX6 blocks Gαi depalmitoylation, prevents the enhanced receptor G-protein association and blocks acute analgesic tolerance to morphine and kappa opioid receptor inactivation in vivo. Opioid stimulation of JNK also inactivates dopamine D2 receptors in a PRDX6-dependent manner. We show that the loss of this lipid modification distorts the receptor G-protein association, thereby preventing agonist-induced guanine nucleotide exchange. These findings establish JNK-dependent PRDX6 recruitment and oxidation-induced Gαi depalmitoylation as an additional mechanism of Gαi-G-protein-coupled receptor inactivation.Opioid receptors are important modulators of nociceptive pain. Here the authors show that opioid receptor activation recruits peroxiredoxin 6 (PRDX6) to the receptor-Gαi complex by c-Jun N-terminal kinase, resulting in Gαi depalmitoylation and enhanced receptor-Gαi association.

The influence of micronutrients on dopamine systems is not well defined. Using mice, we show a potential role for reduced dietary vitamin D3 (cholecalciferol) in promoting diet-induced obesity (DIO), food intake, and drug consumption while on a high fat diet. To complement these deficiency studies, treatments with exogenous fully active vitamin D3 (calcitriol, 10 µg/kg, i.p.) were performed. Nondeficient mice that were made leptin resistant with a high fat diet displayed reduced food intake and body weight after an acute treatment with exogenous calcitriol. Dopamine neurons in the midbrain and their target neurons in the striatum were found to express vitamin D3 receptor protein. Acute calcitriol treatment led to transcriptional changes of dopamine-related genes in these regions in naive mice, enhanced amphetamine-induced dopamine release in both naive mice and rats, and increased locomotor activity after acute amphetamine treatment (2.5 mg/kg, i.p.). Alternatively, mice that were chronically fed either the reduced D3 high fat or chow diets displayed less activity after acute amphetamine treatment compared with their respective controls. Finally, high fat deficient mice that were trained to orally consume liquid amphetamine (90 mg/L) displayed increased consumption, while nondeficient mice treated with calcitriol showed reduced consumption. Our findings suggest that reduced dietary D3 may be a contributing environmental factor enhancing DIO as well as drug intake while eating a high fat diet. Moreover, these data demonstrate that dopamine circuits are modulated by D3 signaling, and may serve as direct or indirect targets for exogenous calcitriol.

The nucleus of the solitary tract (NTS) is a key gateway for meal-related signals entering the brain from the periphery. However, the chemical mediators crucial to this process have not been fully elucidated. We reveal that a subset of NTS neurons containing cholecystokinin (CCK(NTS)) is responsive to nutritional state and that their activation reduces appetite and body weight in mice. Cell-specific anterograde tracing revealed that CCK(NTS) neurons provide a distinctive innervation of the paraventricular nucleus of the hypothalamus (PVH), with fibers and varicosities in close apposition to a subset of melanocortin-4 receptor (MC4R(PVH)) cells, which are also responsive to CCK. Optogenetic activation of CCK(NTS) axon terminals within the PVH reveal the satiating function of CCK(NTS) neurons to be mediated by a CCK(NTS)→PVH pathway that also encodes positive valence. These data identify the functional significance of CCK(NTS) neurons and reveal a sufficient and discrete NTS to hypothalamus circuit controlling appetite.

Ketamine produces rapid and sustained antidepressant actions in depressed patients, but the precise cellular mechanisms underlying these effects have not been identified. Here we determined if modulation of neuronal activity in the infralimbic prefrontal cortex (IL-PFC) underlies the antidepressant and anxiolytic actions of ketamine. We found that neuronal inactivation of the IL-PFC completely blocked the antidepressant and anxiolytic effects of systemic ketamine in rodent models and that ketamine microinfusion into IL-PFC reproduced these behavioral actions of systemic ketamine. We also found that optogenetic stimulation of the IL-PFC produced rapid and long-lasting antidepressant and anxiolytic effects and that these effects are associated with increased number and function of spine synapses of layer V pyramidal neurons. The results demonstrate that ketamine infusions or optogenetic stimulation of IL-PFC are sufficient to produce long-lasting antidepressant behavioral and synaptic responses similar to the effects of systemic ketamine administration.

Background: Neuropeptide Y (NPY) is a hypothalamic neuropeptide that plays a prominent role in feeding and energy homeostasis. Expression of the NPY Y1 receptor (Y1R) is highly concentrated in the nucleus accumbens (Acb), a region important in the regulation of palatable feeding. In this study, we performed a number of experiments to investigate the actions of NPY in the Acb., Methods: First, we determined caloric intake and food choice after bilateral administration of NPY in the Acb in rats on a free-choice diet of saturated fat, 30% sucrose solution, and standard chow and whether this was mediated by the Y1R. Second, we measured the effect of intra-Acb NPY on neuronal activity using in vivo electrophysiology. Third, we examined co-localization of Y1R with enkephalin and dynorphin neurons and the effect of NPY on preproenkephalin messenger RNA levels in the striatum using fluorescent and radioactive in situ hybridization. Finally, using retrograde tracing, we examined whether NPY neurons in the arcuate nucleus projected to the Acb., Results: In rats on the free-choice, high-fat, high-sugar diet, intra-Acb NPY increased intake of fat, but not sugar or chow, and this was mediated by the Y1R. Intra-Acb NPY reduced neuronal firing, as well as preproenkephalin messenger RNA expression in the striatum. Moreover, Acb enkephalin neurons expressed Y1R and arcuate nucleus NPY neurons projected to the Acb., Conclusions: NPY reduces neuronal firing in the Acb resulting in increased palatable food intake. Together, our neuroanatomical, pharmacologic, and neuronal activity data support a role and mechanism for intra-Acb NPY-induced fat intake., (Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Optogenetics is an extremely powerful tool for selective neuronal activation/inhibition and dissection of neural circuits. However, a limitation of in vivo optogenetics is that an animal must be tethered to an optical fiber for delivery of light. Here, we describe a new method for in vivo, optogenetic inhibition of neural activity using an internal, animal-generated light source based on firefly luciferase. Two adeno-associated viruses encoding luciferase were tested and both produced concentration-dependent light after administration of the substrate, luciferin. Mice were co-infected with halorhodopsin- and luciferase-expressing viruses in the striatum, and luciferin administration significantly reduced Fos activity compared to control animals infected with halorhodopsin only. Recordings of neuronal activity in behaving animals confirmed that firing was greatly reduced after luciferin administration. Finally, amphetamine-induced locomotor activity was reduced in halorhodopsin/luciferase mice pre-injected with luciferin compared to controls. This demonstrates that virally encoded luciferase is able to generate sufficient light to activate halorhodopsin and suppress neural activity and change behavior. This approach could be used to generate inhibition in response to activation of specific molecular pathways.

Although the prefrontal cortex influences motivated behavior, its role in food intake remains unclear. Here, we demonstrate a role for D1-type dopamine receptor-expressing neurons in the medial prefrontal cortex (mPFC) in the regulation of feeding. Food intake increases activity in D1 neurons of the mPFC in mice, and optogenetic photostimulation of D1 neurons increases feeding. Conversely, inhibition of D1 neurons decreases intake. Stimulation-based mapping of prefrontal D1 neuron projections implicates the medial basolateral amygdala (mBLA) as a downstream target of these afferents. mBLA neurons activated by prefrontal D1 stimulation are CaMKII positive and closely juxtaposed to prefrontal D1 axon terminals. Finally, photostimulating these axons in the mBLA is sufficient to increase feeding, recapitulating the effects of mPFC D1 stimulation. These data describe a new circuit for top-down control of food intake.

Background: Mounting evidence suggests that overeating may be conceptualized within the same behavioral and neurobiological framework as drug addiction. One potentially important difference between overeating versus drug abuse refers to the sensory stimulation of oral receptors by palatable foods, a feature that may be required for reinforcement during intake. Likewise, postingestive effects and caloric content of food also contribute to reinforcing behavior and might influence the development of compulsive eating behavior. The purpose of the current study was to establish whether intragastric self-administration of fat emulsions, that is, bypassing the oral cavity, recapitulates some of the behavioral and neurobiological hallmarks of psychostimulant self-administration., Methods: We used behavioral assays in mice to assess acquisition, maintenance, extinction, and reinstatement of intragastric self-administration of lipid emulsions to determine the extent to which postoral fat self-administration recapitulates psychostimulant self-administration. Striatal dopamine efflux during behavioral tasks was determined by brain microdialysis coupled to chromatographic-electrochemical analyses., Results: We show that in direct analogy to drug self-administration, 1) decreases in fat dose concentration were met with compensatory increases in response rates aimed at maintaining constant hourly caloric intake; 2) rates of responding markedly increased during both extinction and progressive ratio schedules of reinforcement; and 3) elevations in striatal dopamine levels observed during maintenance were markedly attenuated during extinction sessions, only to be restored on reinstatement., Conclusions: Our data thus support the contention that stimulation of oral receptors by caloric foods may not be required for the expression of certain addiction-related neurobehavioral markers., (Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Temporal control, or how organisms guide movements in time to achieve behavioral goals, depends on dopamine signaling. The medial prefrontal cortex controls many goal-directed behaviors and receives dopaminergic input primarily from the midbrain ventral tegmental area. However, this system has never been linked with temporal control. Here, we test the hypothesis that dopaminergic projections from the ventral tegmental area to the prefrontal cortex influence temporal control. Rodents were trained to perform a fixed-interval timing task with an interval of 20 s. We report several results: first, that decreasing dopaminergic neurotransmission using virally mediated RNA interference of tyrosine hydroxylase impaired temporal control, and second that pharmacological disruption of prefrontal D1 dopamine receptors, but not D2 dopamine receptors, impaired temporal control. We then used optogenetics to specifically and selectively manipulate prefrontal neurons expressing D1 dopamine receptors during fixed-interval timing performance. Selective inhibition of D1-expressing prefrontal neurons impaired fixed-interval timing, whereas stimulation made animals more efficient during task performance. These data provide evidence that ventral tegmental dopaminergic projections to the prefrontal cortex influence temporal control via D1 receptors. The results identify a critical circuit for temporal control of behavior that could serve as a target for the treatment of dopaminergic diseases.

A recent paper in Nature (Lim et al., 2012) describes the effects of melanocortin receptors in the nucleus accumbens. The studies connect a hypothalamic peptide system with brain reward centers and show effects on specific neuronal populations and behavioral components of mood., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Maladaptive responses to stress adversely affect human behavior, yet the signaling mechanisms underlying stress-responsive behaviors remain poorly understood. Using a conditional gene knockout approach, the α isoform of p38 mitogen-activated protein kinase (MAPK) was selectively inactivated by AAV1-Cre-recombinase infection in specific brain regions or by promoter-driven excision of p38α MAPK in serotonergic neurons (by Slc6a4-Cre or ePet1-Cre) or astrocytes (by Gfap-CreERT2). Social defeat stress produced social avoidance (a model of depression-like behaviors) and reinstatement of cocaine preference (a measure of addiction risk) in wild-type mice, but not in mice having p38α MAPK selectively deleted in serotonin-producing neurons of the dorsal raphe nucleus. Stress-induced activation of p38α MAPK translocated the serotonin transporter to the plasma membrane and increased the rate of transmitter uptake at serotonergic nerve terminals. These findings suggest that stress initiates a cascade of molecular and cellular events in which p38α MAPK induces a hyposerotonergic state underlying depression-like and drug-seeking behaviors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Stress is a complex experience that carries both aversive and motivating properties. Chronic stress causes an increase in the risk of depression, is well known to increase relapse of drug seeking behavior, and can adversely impact health. Several brain systems have been demonstrated to be critical in mediating the negative affect associated with stress, and recent evidence directly links the actions of the endogenous opioid neuropeptide dynorphin in modulating mood and increasing the rewarding effects of abused drugs. These results suggest that activation of the dynorphin/kappa opioid receptor (KOR) system is likely to play a major role in the pro-addictive effects of stress. This review explores the relationship between dynorphin and corticotropin-releasing factor (CRF) in the induction of dysphoria, the potentiation of drug seeking, and stress-induced reinstatement. We also provide an overview of the signal transduction events responsible for CRF and dynorphin/KOR-dependent behaviors. Understanding the recent work linking activation of CRF and dynorphin/KOR systems and their specific roles in brain stress systems and behavioral models of addiction provides novel insight to neuropeptide systems that regulate affective state., (Copyright 2009 Elsevier B.V. All rights reserved.)

Stress is a complex human experience and having both rewarding and aversive motivational properties. The adverse effects of stress are well documented, yet many of underlying mechanisms remain unclear and controversial. Here we report that the anxiogenic properties of stress are encoded by the endogenous opioid peptide dynorphin acting in the basolateral amygdala. Using pharmacological and genetic approaches, we found that the anxiogenic-like effects of Corticotropin Releasing Factor (CRF) were triggered by CRF(1)-R activation of the dynorphin/kappa opioid receptor (KOR) system. Central CRF administration significantly reduced the percent open-arm time in the elevated plus maze (EPM). The reduction in open-arm time was blocked by pretreatment with the KOR antagonist norbinaltorphimine (norBNI), and was not evident in mice lacking the endogenous KOR ligand dynorphin. The CRF(1)-R agonist stressin 1 also significantly reduced open-arm time in the EPM, and this decrease was blocked by norBNI. In contrast, the selective CRF(2)-R agonist urocortin III did not affect open arm time, and mice lacking CRF(2)-R still showed an increase in anxiety-like behavior in response to CRF injection. However, CRF(2)-R knockout animals did not develop CRF conditioned place aversion, suggesting that CRF(1)-R activation may mediate anxiety and CRF(2)-R may encode aversion. Using a phosphoselective antibody (KORp) to identify sites of dynorphin action, we found that CRF increased KORp-immunoreactivity in the basolateral amygdala (BLA) of wildtype, but not in mice pretreated with the selective CRF(1)-R antagonist, antalarmin. Consistent with the concept that acute stress or CRF injection-induced anxiety was mediated by dynorphin release in the BLA, local injection of norBNI blocked the stress or CRF-induced increase in anxiety-like behavior; whereas norBNI injection in a nearby thalamic nucleus did not. The intersection of stress-induced CRF and the dynorphin/KOR system in the BLA was surprising, and these results suggest that CRF and dynorphin/KOR systems may coordinate stress-induced anxiety behaviors and aversive behaviors via different mechanisms.

Although stress has profound effects on motivated behavior, the underlying mechanisms responsible are incompletely understood. In this study we elucidate a functional pathway in mouse brain that encodes the aversive effects of stress and mediates stress-induced reinstatement of cocaine place preference (CPP). Activation of the dynorphin/kappa opioid receptor (KOR) system by either repeated stress or agonist produces conditioned place aversion (CPA). Because KOR inhibition of dopamine release in the mesolimbic pathway has been proposed to mediate the dysphoria underlying this response, we tested dopamine-deficient mice in this study and found that KOR agonist in these mice still produced CPA. However, inactivation of serotonergic KORs by injection of the KOR antagonist norBNI into the dorsal raphe nucleus (DRN), blocked aversive responses to the KOR agonist U50,488 and blocked stress-induced reinstatement of CPP. KOR knockout (KO) mice did not develop CPA to U50,488; however, lentiviral re-expression of KOR in the DRN of KOR KO mice restored place aversion. In contrast, lentiviral expression in DRN of a mutated form of KOR that fails to activate p38 MAPK required for KOR-dependent aversion, did not restore place aversion. DRN serotonergic neurons project broadly throughout the brain, but the inactivation of KOR in the nucleus accumbens (NAc) coupled with viral re-expression in the DRN of KOR KO mice demonstrated that aversion was encoded by a DRN to NAc projection. These results suggest that the adverse effects of stress may converge on the serotonergic system and offers an approach to controlling stress-induced dysphoria and relapse.

Stress is a complex human experience having both positive and negative motivational properties. When chronic and uncontrollable, the adverse effects of stress on human health are considerable and yet poorly understood. Here, we report that the dysphoric properties of chronic stress are encoded by the endogenous opioid peptide dynorphin acting on specific stress-related neuronal circuits. Using different forms of stress presumed to evoke dysphoria in mice, we found that repeated forced swim and inescapable footshock both produced aversive behaviors that were blocked by a kappa-opioid receptor (KOR) antagonist and absent in mice lacking dynorphin. Injection of corticotropin-releasing factor (CRF) or urocortin III, key mediators of the stress response, produced place aversion that was also blocked by dynorphin gene deletion or KOR antagonism. CRF-induced place aversion was blocked by the CRF2 receptor antagonist antisauvigine-30, but not by the CRF1 receptor antagonist antalarmin. In contrast, place aversion induced by the KOR agonist U50,488 was not blocked by antisauvigine-30. These results suggest that the aversive effects of stress were mediated by CRF2 receptor stimulation of dynorphin release and subsequent KOR activation. Using a phospho-selective antibody directed against the activated KOR to image sites of dynorphin action in the brain, we found that stress and CRF each caused dynorphin-dependent KOR activation in the basolateral amygdala, nucleus accumbens, dorsal raphe, and hippocampus. The convergence of stress-induced aversive inputs on the dynorphin system was unexpected, implicates dynorphin as a key mediator of dysphoria, and emphasizes kappa-receptor antagonists as promising therapeutics.

The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both kappa-opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated kappa receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(-/-) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the kappa agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and the KOR-dependent swim stress-induced immobility while not affecting kappa-opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that beta-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin-kappa-opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress.

Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.