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RationaleDesigner receptors exclusively activated by designer drugs (DREADDs) are a tool for “remote control” of defined neuronal populations during behavior. These receptors are inert unless bound by an experimenter-administered designer drug, most commonly clozapine-n-oxide (CNO). However, questions have emerged about the suitability of CNO as a systemically administered DREADD agonist.ObjectivesSecond-generation agonists such as JHU37160 (J60) have been developed, which may have more favorable properties than CNO. Here we sought to directly compare effects of CNO (0, 1, 5, & 10 mg/kg, i.p.) and J60 (0, 0.03, 0.3, & 3 mg/kg, i.p.) on operant food pursuit.MethodsMale and female TH:Cre+ rats and their wildtype (WT) littermates received cre-dependent hM4Di-mCherry vector injections into ventral tegmental area (VTA), causing inhibitory DREADD expression in VTA dopamine neurons in TH:Cre+ rats. Rats were trained to stably lever press for palatable food on a fixed ratio 10 schedule, and doses of both agonists were tested on separate days in a counterbalanced order.ResultsAll three CNO doses reduced operant food seeking in rats with DREADDs, and no CNO dose had behavioral effects in WT controls. The highest tested J60 dose significantly reduced responding in DREADD rats, but this dose alsoincreasedresponding in WTs, indicating non-specific effects. The magnitude of CNO and J60 effects in TH:Cre+ rats were correlated and were present in both sexes.ConclusionsFindings demonstrate the usefulness of directly comparing DREADD agonists when optimizing behavioral chemogenetics, and highlight the importance of proper controls, regardless of the DREADD agonist employed.

The reinforcing efficacy of cocaine is largely determined by its capacity to inhibit the dopamine transporter (DAT), and emerging evidence suggests that differences in cocaine potency are linked to several symptoms of cocaine use disorder. Despite this evidence, the neural processes that govern cocaine potencyin vivoremain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Usingex vivofast scan cyclic voltammetry, pharmacological probes of the DAT, biochemical assessments of DAT membrane availability and phosphorylation, and cocaine self-administration, we tested the effects of chemogenetic manipulations on cocaine potency at distal DATs in the nucleus accumbens as well as the behavioral economics of cocaine self-administration. We discovered that chemogenetic manipulation of dopamine neurons produced rapid, bidirectional modulation of cocaine potency at DATs in the nucleus accumbens. We then provided evidence that changes in cocaine potency are associated with alterations in DAT affinity for cocaine and demonstrated that this change in affinity coincides with DAT conformation biases and changes in DAT phosphorylation state. Finally, we showed that chemogenetic manipulation of dopamine neurons alters cocaine consumption in a manner consistent with changes in cocaine potency at distal DATs. Based on the spatial and temporal constraints inherent to our experimental design, we posit that changes in cocaine potency are driven by alterations in dopamine neuron activity. When considered together, these observations provide a novel mechanism through which GPCRs regulate cocaine's pharmacological and behavioral effects.SIGNIFICANCE STATEMENTDifferences in the pharmacological effects of cocaine are believed to influence the development and progression of cocaine use disorder. However, the biological and physiological processes that determine sensitivity to cocaine remain unclear. In this work, we use a combination of chemogenetics, fast scan cyclic voltammetry, pharmacology, biochemistry, and cocaine self-administration with economic demand analysis to demonstrate a novel mechanism by which cocaine potency is determinedin vivo. These studies identify a novel process by which the pharmacodynamics of cocaine are derivedin vivo, and thus this work has widespread implications for understanding the mechanisms that regulate cocaine consumption across stages of addiction.

Δ9-tetrahydrocannabinol (THC) is the intoxicating constituent of cannabis and is responsible for the drug's reinforcing effects. Retrospective human studies suggest that cannabis use during adolescence is linked to long-term negative psychological outcomes, but in such studies it is difficult to distinguish the effects of THC from those of coexisting factors. Therefore, translationally relevant animal models are required to properly investigate THC effects in adolescents. However, though the relevance of these studies depends upon human-relevant dosing, surprisingly little is known about THC pharmacology and its effects on behavior and brain activity in adolescent rodents-especially in females. Here, we conducted a systematic investigation of THC pharmacokinetics, metabolism and distribution in blood and brain, and of THC effects upon behavior and neural activity in adolescent Long Evans rats of both sexes. We administered THC during an early-middle adolescent window (postnatal days 27-45) in which the brain may be particularly sensitive to developmental perturbation by THC. We determined the pharmacokinetic profile of THC and its main first-pass metabolites (11-hydroxy-THC and 11-nor-9-carboxy-THC) in blood and brain following acute injection (0.5 or 5 mg/kg, intraperitoneal). We also evaluated THC effects on behavioral assays of anxiety, locomotion, and place conditioning, as well as c-Fos expression in 14 brain regions. Confirming previous work, we find marked sex differences in THC metabolism, including a female-specific elevation in the bioactive metabolite 11-hydroxy-THC. Furthermore, we find dose-dependent and sex-dependent effects on behavior, neural activity, and functional connectivity across multiple nodes of brain stress and reward networks. Our findings are relevant for interpreting results of rat adolescent THC exposure studies, and may lend new insights into how THC impacts the brain in a sex-dependent manner.

RationaleAdolescent exposure to ∆9-tetrahydrocannabinol (THC), the psychotropic constituent of cannabis, might affect brain development, and in rodent models leads to long-term behavioral and physiological alterations. Yet, the basic pharmacology of this drug in adolescent rodents, especially when ingested via ecologically relevant routes like aerosol inhalation, commonly referred to as "vaping," is still poorly characterized. Moreover, sex differences exist in THC metabolism, kinetics, and behavioral effects, but these have not been rigorously examined after vapor dosing in adolescents.ObjectivesWe investigated the pharmacokinetics and pharmacodynamics of aerosolized THC (30min inhalation exposure, 25 or 100mg/ml) in adolescent Wistar rats of both sexes.MethodsLiquid chromatography/mass spectrometry analysis of THC and its major metabolites was conducted on blood plasma and brain tissue at 5, 30, 60, and 120min following a 30-min aerosol dosing session. Effects on activity in a novel environment for 120min after aerosol, and temperature, were measured in separate rats.ResultsWe found sex-dependent differences in the pharmacokinetics of THC and its active (11-OH-THC) and inactive (11-COOH-THC) metabolites in the blood and brain, along with dose- and sex-dependent effects on anxiety-like and exploratory behaviors; namely, greater 11-OH-THC levels accompanied by greater behavioral effects in females at the low dose but similar hypothermic effects in both sexes at the high dose.ConclusionsThese results provide a benchmark for dosing adolescent rats with aerosolized (or "vaped") THC, which could facilitate adoption by other labs of this potentially human-relevant THC exposure model to understand cannabis effects on the developing brain.

Adult rats emit ultrasonic vocalizations (USVs) related to their affective states, potentially providing information about their subjective experiences during behavioral neuroscience experiments. If so, USVs might provide an important link between invasive animal preclinical studies and human studies in which subjective states can be readily queried. Here, we induced USVs in male and female Long Evans rats using acute amphetamine (2 mg/kg), and asked how reversibly inhibiting nucleus accumbens neurons using designer receptors exclusively activated by designer drugs (DREADDs) impacts USV production. We analyzed USV characteristics using “Deepsqueak” software, and manually categorized detected calls into four previously defined subtypes. We found that systemic administration of the DREADD agonist clozapine-n-oxide, relative to vehicle in the same rats, suppressed the number of frequency-modulated and trill-containing USVs without impacting high frequency, unmodulated (flat) USVs, nor the small number of low-frequency USVs observed. Using chemogenetics, these results thus confirm that nucleus accumbens neurons are essential for production of amphetamine-induced frequency-modulated USVs. They also support the premise of further investigating the characteristics and subcategories of these calls as a window into the subjective effects of neural manipulations, with potential future clinical applications.

Addiction is a chronic relapsing disorder, and during recovery many people experience several relapse events as they attempt to voluntarily abstain from drug. New preclinical relapse models have emerged that capture this common human experience, and mounting evidence indicates that resumption of drug seeking after voluntary abstinence recruits neural circuits distinct from those recruited during reinstatement after experimenter-imposed abstinence, or abstinence due to extinction training. Ventral pallidum (VP), a key limbic node involved in drug seeking, has well-established roles in conventional reinstatement models tested following extinction training, but it is unclear whether this region also participates in more translationally relevant models of relapse. Here we show that chemogenetic inhibition of VP neurons decreased cocaine-, context-, and cue-induced relapse tested after voluntary, punishment-induced abstinence. This effect was strongest in the most compulsive, punishment-resistant rats, and reinstatement was associated with neural activity in anatomically defined VP subregions. VP inhibition also attenuated the propensity of rats to display "abortive lever pressing," a species-typical risk assessment behavior seen here during punished drug taking, likely resulting from concurrent approach and avoidance motivations. These results indicate that VP, unlike other connected limbic brain regions, is essential for resumption of drug seeking after voluntary abstinence. Since VP inhibition effects were strongest in the most compulsively cocaine-seeking individuals, this may also indicate that VP plays a particularly important role in the most pathological, addiction-like behavior, making it an attractive target for future therapeutic interventions.

Δ(9)-tetrahydrocannabinol (THC) is the intoxicating constituent of cannabis and is responsible for the drug’s reinforcing effects. Retrospective human studies suggest that cannabis use during adolescence is linked to long-term negative psychological outcomes, but in such studies it is difficult to distinguish the effects of THC from those of coexisting factors. Therefore, translationally relevant animal models are required to properly investigate THC effects in adolescents. However, though the relevance of these studies depends upon human-relevant dosing, surprisingly little is known about THC pharmacology and its effects on behavior and brain activity in adolescent rodents—especially in females. Here, we conducted a systematic investigation of THC pharmacokinetics, metabolism and distribution in blood and brain, and of THC effects upon behavior and neural activity in adolescent Long Evans rats of both sexes. We administered THC during an early-middle adolescent window (postnatal days 27–45) in which the brain may be particularly sensitive to developmental perturbation by THC. We determined the pharmacokinetic profile of THC and its main first-pass metabolites (11-hydroxy-THC and 11-nor-9-carboxy-THC) in blood and brain following acute injection (0.5 or 5 mg/kg, intraperitoneal). We also evaluated THC effects on behavioral assays of anxiety, locomotion, and place conditioning, as well as c-Fos expression in 14 brain regions. Confirming previous work, we find marked sex differences in THC metabolism, including a female-specific elevation in the bioactive metabolite 11-hydroxy-THC. Furthermore, we find dose-dependent and sex-dependent effects on behavior, neural activity, and functional connectivity across multiple nodes of brain stress and reward networks. Our findings are relevant for interpreting results of rat adolescent THC exposure studies, and may lend new insights into how THC impacts the brain in a sex-dependent manner.

Early-life adversity increases the risk for emotional disorders such as depression and schizophrenia. Anhedonia, thought to be a core feature of these disorders, is provoked by our naturalistic rodent model of childhood adversity (i.e., rearing pups for one week in cages with limited bedding and nesting, LBN). Drug use and addiction are highly comorbid with psychiatric disorders featuring anhedonia, yet effects of LBN on drug-seeking behavior and the reward and stress-related circuits that underlie it remain unknown. Here we examined the effects of LBN on cocaine intake and seeking, using a battery of behavioral tests measuring distinct aspects of cocaine reward, and for comparison, chocolate intake. We also examined activation of neurons within the pleasure/reward and stress circuits following cocaine in LBN and control rats. Early-life adversity reduced spontaneous intake of palatable chocolate, extending prior reports of sucrose and social-play anhedonia. In a within-session cocaine behavioral economic test, LBN rats self-administered lower dosages of cocaine under low-effort conditions, consistent with a reduced hedonic set-point for cocaine, and potentially anhedonia. In contrast, cocaine demand elasticity was not consistently affected, indicating no major changes in motivation to maintain preferred cocaine blood levels. These changes were selective, as LBN did not cause an overt anxiety-like phenotype, nor did it affect sensitivity to self-administered cocaine dose, responding for cocaine under extinction conditions, cocaine- or cue-induced reinstatement of cocaine seeking, or locomotor response to acute cocaine. However, high Fos expression was seen after cocaine in both reward- and stress-related brain regions of LBN rats, including nucleus accumbens core, central amygdala, and lateral habenula. In contrast, hypothalamic orexin neuron activation after cocaine was significantly attenuated in LBN rats. Together, these findings demonstrate enduring effects of early-life adversity on both reward- and fear/anxiety-related neural circuits, as well as anhedonia-like reductions in consumption of natural and drug rewards., Highlights • Adversity during a sensitive developmental period provokes persistent anhedonia. • This adversity reduces cocaine hedonic set point, but not motivation. • Cocaine-associated Fos is altered in reward- and anxiety/fear circuits. • Cocaine-dose sensitivity, reinstatement, and locomotion are unchanged. • Effects are selective, as anxiety-related behaviors were unaltered.

RationaleAdolescence is characterized by endocannabinoid (ECB)-dependent refinement of neural circuits underlying emotion, learning, and motivation. As a result, adolescent cannabinoid receptor stimulation (ACRS) with phytocannabinoids or synthetic agonists like "Spice" cause robust and persistent changes in both behavior and circuit architecture in rodents, including in reward-related regions like medial prefrontal cortex and nucleus accumbens (NAc).Objectives and methodsHere, we examine persistent effects of ACRS with the cannabinoid receptor 1/2 specific agonist WIN55-212,2 (WIN; 1.2 mg/kg/day, postnatal day (PD) 30-43), on natural reward-seeking behaviors and ECB system function in adult male Long Evans rats (PD 60+).ResultsWIN ACRS increased palatable food intake, and altered attribution of incentive salience to food cues in a sign-/goal-tracking paradigm. ACRS also blunted hunger-induced sucrose intake, and resulted in increased anandamide and oleoylethanolamide levels in NAc after acute food restriction not seen in controls. ACRS did not affect food neophobia or locomotor response to a novel environment, but did increase preference for exploring a novel environment.ConclusionsThese results demonstrate that ACRS causes long-term increases in natural reward-seeking behaviors and ECB system function that persist into adulthood, potentially increasing liability to excessive natural reward seeking later in life.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping., Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a powerful tool for neuroscience, but the standard DREADD ligand, CNO, has significant drawbacks. Here the authors report two novel high-potency DREADD ligands and a novel DREADD radiotracer for imaging purposes.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.

Ventral tegmental area (VTA) dopamine (DA) neurons perform diverse functions in motivation and cognition, but their precise roles in addiction-related behaviors are still debated. Here, we targeted VTA DA neurons for bidirectional chemogenetic modulation during specific tests of cocaine reinforcement, demand, and relapse-related behaviors in male rats, querying the roles of DA neuron inhibitory and excitatory G-protein signaling in these processes. Designer receptor stimulation of Gqsignaling, but not Gssignaling, in DA neurons enhanced cocaine seeking via functionally distinct projections to forebrain limbic regions. In contrast, engaging inhibitory Gi/osignaling in DA neurons blunted the reinforcing and priming effects of cocaine, reduced stress-potentiated reinstatement, and altered behavioral strategies for cocaine seeking and taking. Results demonstrate that DA neurons play several distinct roles in cocaine seeking, depending on behavioral context, G-protein-signaling cascades, and DA neuron efferent targets, highlighting their multifaceted roles in addiction.SIGNIFICANCE STATEMENTG-protein-coupled receptors are crucial modulators of ventral tegmental area (VTA) dopamine neuron activity, but how this metabotropic signaling impacts the complex roles of dopamine in reward and addiction is poorly understood. Here, we bidirectionally modulate dopamine neuron G-protein signaling with DREADDs (designer receptors exclusively activated by designer drugs) during a variety of cocaine-seeking behaviors, revealing nuanced, pathway-specific roles in cocaine reward, effortful seeking, and relapse-like behaviors. Gqand Gsstimulation activated dopamine neurons, but only Gqstimulation robustly enhanced cocaine seeking. Gi/oinhibitory signaling reduced some, but not all, types of cocaine seeking. Results show that VTA dopamine neurons modulate numerous distinct aspects of cocaine addiction- and relapse-related behaviors, and point to potential new approaches for intervening in these processes to treat addiction.