Effects of the designer drug 4-methylamphetamine on core temperature and serotonin levels in the striatum and hippocampus of rats.

New psychoactive substances (NPS) are typically synthesized in clandestine laboratories in an attempt to chemically modify already federally regulated drugs in an effort to circumvent the law. Drugs derived from a phenethylamine pharmacophore, such as 4-chloroamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), reliably induce thermogenesis and serotonergic deficits in the striatum and hippocampus of rodents. 4-methylamphetamine (4-MA), a relative newcomer to the NPS scene, was originally investigated in the mid-1900 s as a potential anorexigenic agent. With its phenethylamine pharmacophore, 4-MA was hypothesized to produce similar toxicological alterations as its chemical analogs. In the present study, three doses (1.0, 2.5, and 5.0 mg/kg, ip.) of 4-MA were administered to rats twice daily for two days. Core temperature data were calculated and analyzed as temperature area under the curve (TAUC). On the second day of dosing, a hypothermic response to 4-MA (2.5 and 5.0 mg/kg) was noted between 0.5 and 2.0 h post-treatment. Only the highest dose of 4-MA decreased body weight on the second day of treatment and maintained this reduction in weight for seven days after treatment ceased. None of the doses of 4-MA evaluated significantly altered serotonin levels in the hippocampus or striatum seven days after final treatment. The present findings demonstrate that the 4-methyl substitution to amphetamine generates a pharmacological and toxicological profile that differs from other similar phenethylamine analogs.

Clozapine N-oxide, compound 21, and JHU37160 do not influence effortful reward-seeking behavior in mice.

RATIONALE: Clozapine N-oxide (CNO) has been developed as a ligand to selectively activate designer receptors exclusively activated by designer drugs (DREADDs). However, previous studies have revealed that peripherally injected CNO is reverse-metabolized into clozapine, which, in addition to activating DREADDs, acts as an antagonist at various neurotransmitter receptors, suggesting potential off-target effects of CNO on animal physiology and behaviors. Recently, second-generation DREADD agonists compound 21 (C21) and JHU37160 (J60) have been developed, but their off-target effects are not fully understood. OBJECTIVES: The present studies assessed the effect of novel DREADD ligands on reward-seeking behavior. METHODS: We first tested the possible effect of acute i.p. injection of low-to-moderate (0.1, 0.3, 1, 3 mg/kg) of CNO, C21, and J60 on motivated reward-seeking behavior in wild-type mice. We then examined whether a high dose (10 mg/kg) of these drugs might be able to alter responding. RESULTS: Low-to-moderate doses of all drugs and a high dose of CNO or C21 did not alter operant lick responding for a reward under a progressive ratio schedule of reinforcement, in which the number of operant lick responses to obtain a reward increases after each reward collection. However, high-dose J60 resulted in a total lack of responding that was later observed in an open field arena to be due to a sedative effect. CONCLUSIONS: This study provides definitive evidence that commonly used doses of CNO, C21, and J60 have negligible off-target effects on motivated reward-seeking but urges caution when using high doses of J60 due to sedative effects.

Designer Drug, 25D-NBOMe, Has Reinforcing and Rewarding Effects through Change of a Dopaminergic Neurochemical System.

2-(2,5-Dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)_ethanamine (25D-NBOMe), an analogue of the 2C family, is a newly synthesized psychoactive substance. It acts as an agonist at the 5-HT2A receptor and has a similar mechanism to that of NBOMe compounds. However, the pharmacological mechanism for its rewarding and reinforcing effects has not been revealed. In the present study, intravenous self-administration (IVSA) test and conditioned place preference (CPP) test were performed to investigate whether 25D-NBOMe has abuse potential. We also evaluated the effects of 25D-NBOMe on neurochemical changes using western blot analysis and microdialysis. The IVSA test revealed increased self-administration in 25D-NBOMe (0.03 mg/kg)-treated rats. In addition, the CPP test revealed rewarding effects in 25D-NBOMe (1 mg/kg)-treated mice. In the neurochemical studies, 25D-NBOMe treatment affected the expression of dopamine (DA) receptor D1 (DRD1), DA receptor D2 (DRD2), tyrosine hydroxylase, DA transporter (DAT), and phospho-DAT (p-DAT) in the nucleus accumbens (NAc). In addition, microdialysis revealed that treatment with progressively increasing doses (1, 3, and 10 mg/kg) of 25D-NBOMe increased the extracellular levels of DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the rat NAc. Taken together, our results show the abuse potential and neurochemical changes related to addictive behavior after administration of 25D-NBOMe.

Chemogenetic modulation of astrocytes and microglia: State-of-the-art and implications in neuroscience.

Insights into the role astrocytes and microglia play in normal and diseased brain functioning has expanded drastically over the last decade. Recently, chemogenetic tools have emerged as cutting-edge techniques, allowing targeted and spatiotemporal precise manipulation of a specific glial cell type. As a result, significant advances in astrocyte and microglial cell function have been made, showing how glial cells can intervene in central nervous system (CNS) functions such as cognition, reward and feeding behavior in addition to their established contribution in brain diseases, pain, and CNS inflammation. Here, we discuss the latest insights in glial functions in health and disease that have been made through the application of chemogenetics. We will focus on the manipulation of intracellular signaling pathways induced by activation of the designer receptors exclusively activated by designer drugs (DREADDs) in astrocytes and microglia. We will also elaborate on some of the potential pitfalls and the translational potential of the DREADD technology.

Validation of DREADD agonists and administration route in a murine model of sleep enhancement.

BACKGROUND: Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZGABA-hM3Dq) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration. NEW METHODS: The present study is designed to validate the use of alternative DREADDs ligands-deschloroclozapine (DCZ) and compound 21 (C21)-and a new administration route, the voluntary oral administration. RESULTS: We show that IP injections of DCZ and C21 dose-dependently enhance SWS in PZGABA-hM3Dq mice, similar to CNO. We also show that oral administration of CNO, DCZ and C21 induces the same sleep phenotype as compared with IP injection. COMPARISON WITH EXISTING METHODS AND CONCLUSION: Therefore, DCZ and C21 are powerful alternatives to the use of CNO. Moreover, the voluntary oral administration is suitable for repeated dosing of DREADDs ligands.

Evaluation of Cytotoxic, Necrotic, Apoptotic, and Autophagic Effects of Methamphetamine and 3,4-Methylenedioxymethamphetamine on U-87 MG (Glial) and B104-1-1 (Neuronal) Cell Lines.

Methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) are empathogen (entactogen) psychoactive designer drugs which are mainly used for recreational purposes. Both MA and MDMA are central nervous system stimulants which are classified as monoamine neurotransmitter reuptake inhibitors. They have strong cytotoxic effects on dopaminergic and serotonergic neurons. Neurotoxicities of MA and MDMA by glial activation have been shown. The present work has investigated and measured cytotoxic, necrotic and apoptotic, and autophagic effects of MA and MDMA on U-87 MG (glial) and B104-1-1 (neuronal) cell lines by janus green, ethidium bromide/acridine orange, and monodansylcadaverine/propidium iodide staining to evaluate and compare their effects on glial and neuronal cells, respectively. The results of the present work showed that: (1) MDMA induced more potent mitochondrial toxicity, stronger necrotic and autophagic effects than MA in both B104-1-1 (neuronal) and U-87 MG (glial) cell lines; (2) although MDMA induced stronger apoptotic effect than MA on U-87 MG cell line, it had equal apoptotic effect on B104-1-1 cell line with MA; and (3) MDMA induced more potent mitochondrial toxicity, stronger necrotic, apoptotic, and autophagic effects than MA in B104-1-1 cell line than U-87 MG cell line.

Resting-State fMRI-Based Screening of Deschloroclozapine in Rhesus Macaques Predicts Dosage-Dependent Behavioral Effects.

Chemogenetic techniques, such as designer receptors exclusively activated by designer drugs (DREADDs), enable transient, reversible, and minimally invasive manipulation of neural activity in vivo Their development in nonhuman primates is essential for uncovering neural circuits contributing to cognitive functions and their translation to humans. One key issue that has delayed the development of chemogenetic techniques in primates is the lack of an accessible drug-screening method. Here, we use resting-state fMRI, a noninvasive neuroimaging tool, to assess the impact of deschloroclozapine (DCZ) on brainwide resting-state functional connectivity in 7 rhesus macaques (6 males and 1 female) without DREADDs. We found that systemic administration of 0.1 mg/kg DCZ did not alter the resting-state functional connectivity. Conversely, 0.3 mg/kg of DCZ was associated with a prominent increase in functional connectivity that was mainly confined to the connections of frontal regions. Additional behavioral tests confirmed a negligible impact of 0.1 mg/kg DCZ on socio-emotional behaviors as well as on reaction time in a probabilistic learning task; 0.3 mg/kg DCZ did, however, slow responses in the probabilistic learning task, suggesting attentional or motivational deficits associated with hyperconnectivity in fronto-temporo-parietal networks. Our study highlights both the excellent selectivity of DCZ as a DREADD actuator, and the side effects of its excess dosage. The results demonstrate the translational value of resting-state fMRI as a drug-screening tool to accelerate the development of chemogenetics in primates.SIGNIFICANCE STATEMENT Chemogenetics, such as designer receptors exclusively activated by designer drugs (DREADDs), can afford control over neural activity with unprecedented spatiotemporal resolution. Accelerating the translation of chemogenetic neuromodulation from rodents to primates requires an approach to screen novel DREADD actuators in vivo Here, we assessed brainwide activity in response to a DREADD actuator deschloroclozapine (DCZ) using resting-state fMRI in macaque monkeys. We demonstrated that low-dose DCZ (0.1 mg/kg) did not change whole-brain functional connectivity or affective behaviors, while a higher dose (0.3 mg/kg) altered frontal functional connectivity and slowed response in a learning task. Our study highlights the excellent selectivity of DCZ at proper dosing, and demonstrates the utility of resting-state fMRI to screen novel chemogenetic actuators in primates.

Designer Benzodiazepines' Activity on Opioid Receptors: A Docking Study.

BACKGROUND: Previous studies have reported that benzodiazepines (BZDs) seem to enhance euphoric and reinforcing properties of opioids in opioid users so that a direct effect on opioid receptors has been postulated, together with a possible synergistic induction of severe side effects due to co use of BDZs and opioids. This is particularly worrisome given the appearance on the market of designer benzodiazepines (DBZDs), whose activity/toxicity profiles are scarcely known. OBJECTIVES: This study aimed to evaluate, through computational studies, the binding affinity (or lack thereof) of 101 DBZDs identified online on the kappa, mu, and delta opioid receptors (K, M, DOR); and to assess whether their mechanism of action could include activation of the latter. METHODS: MOE® was used for the computational studies. Pharmacophore mapping based on strong opioids agonist binders' 3D chemical features was used to filter the DBZDs. Resultant DBZDs were docked into the crystallised 3D active conformation of KOR (PDB6B73), DOR (PDB6PT3) and MOR (PDB5C1M). Co-crystallised ligands and four strong agonists were used as reference compounds. A score (S, Kcal/mol) representative of the predicted binding affinity, and a description of ligand interactions were obtained from MOE®. RESULTS: The docking results, filtered for S < -8.0 and the interaction with the Asp residue, identified five DBZDs as putative binders of the three ORs : ciclotizolam, fluloprazolam, JQ1, Ro 48-6791, and Ro 48-8684. CONCLUSION: It may be inferred that at least some DBZDs may have the potential to activate opioid receptors. This could mediate/increase their anxiolytic, analgesic, and addiction potentials, as well as worsen the side effects associated with opioid co-use.

Behavioral and slice electrophysiological assessment of DREADD ligand, deschloroclozapine (DCZ) in rats.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) have become a premier neuroscience research tool for enabling reversible manipulations of cellular activity following experimenter-controlled delivery of a DREADD-specific ligand. However, several DREADD ligands, e.g., clozapine-N-oxide (CNO), have metabolic and off-target effects that may confound experimental findings. New DREADD ligands aim to reduce metabolic and potential off-target effects while maintaining strong efficacy for the designer receptors. Recently a novel DREADD ligand, deschloroclozapine (DCZ), was shown to induce chemogenetic-mediated cellular and behavioral effects in mice and monkeys without detectable side effects. The goal of the present study was to examine the effectiveness of systemic DCZ for DREADD-based chemogenetic manipulations in behavioral and slice electrophysiological applications in rats. We demonstrate that a relatively low dose of DCZ (0.1 mg/kg) supports excitatory DREADD-mediated cFos induction, DREADD-mediated inhibition of a central amygdala-dependent behavior, and DREADD-mediated inhibition of neuronal activity in a slice electrophysiology preparation. In addition, we show that this dose of DCZ does not alter gross locomotor activity or induce a place preference/aversion in control rats without DREADD expression. Together, our findings support the use of systemic DCZ for DREADD-based manipulaations in rats, and provide evidence that DCZ is a superior alternative to CNO.

Activation of the Locus Coeruleus Mediated by Designer Receptor Exclusively Activated by Designer Drug Restores Descending Nociceptive Inhibition after Traumatic Brain Injury in Rats.

Disruption of endogenous pain control mechanisms including descending pain inhibition has been linked to several forms of pain including chronic pain after traumatic brain injury (TBI). The locus coeruleus (LC) is the principal noradrenergic (NA) nucleus participating in descending pain inhibition. We therefore hypothesized that selectively stimulating LC neurons would reduce nociception after TBI. All experiments used a well-characterized rat lateral fluid percussion model of TBI. NA neurons were stimulated by administering clozapine N-oxide (CNO) to rats selectively expressing a designer receptor exclusively activated by designer drug (DREADD) viral construct in their LC's. Mechanical nociceptive thresholds were measured using von Frey fibers. The efficacy of diffuse noxious inhibitory control (DNIC), a critical endogenous pain control mechanism, was assessed using the hindpaw administration of capsaicin. Immunohistochemical analyses demonstrated the selective expression of the DREADD construct in LC neurons after stereotactic injection. During the 1st week after TBI, when rats demonstrated hindlimb (HL) nociceptive sensitization, CNO administration provided transient anti-allodynia in DREADD-expressing rats but not in rats injected with control virus. Seven weeks after TBI we observed a complete loss of DNIC in response to capsaicin. However, CNO administration largely restored DNIC in TBI DREADD-expressing rats but not those injected with control virus. Unexpectedly, the effects of LC activation in the DREADD-expressing rats were blocked by the α-1 adrenergic receptor antagonist prazosin, but not the α-2 adrenergic receptor antagonist atipamezole. These results suggest that directly stimulating the LC after TBI can reduce both early and late manifestations of dysfunctional endogenous pain regulation. Clinical approaches to activating descending pain circuits may reduce suffering in those with pain after TBI.

Chronic Behavioral Manipulation via Orally Delivered Chemogenetic Actuator in Macaques.

The chemogenetic technology referred to as designer receptors exclusively activated by designer drugs (DREADDs) offers reversible means to control neuronal activity for investigating its functional correlation with behavioral action. Deschloroclozapine (DCZ), a recently developed highly potent and selective DREADD actuator, displays a capacity to expand the utility of DREADDs for chronic manipulation without side effects in nonhuman primates, which has not yet been validated. Here we investigated the pharmacokinetics and behavioral effects of orally administered DCZ in female and male macaque monkeys. Pharmacokinetic analysis and PET occupancy examination demonstrated that oral administration of DCZ yielded slower and prolonged kinetics, and that its bioavailability was 10%-20% of that in the case of systemic injection. Oral DCZ (300-1000 µg/kg) induced significant working memory impairments for at least 4 h in monkeys with hM4Di expressed in the dorsolateral prefrontal cortex (Brodmann's area 46). Repeated daily oral doses of DCZ consistently caused similar impairments over two weeks without discernible desensitization. Our results indicate that orally delivered DCZ affords a less invasive strategy for chronic but reversible chemogenetic manipulation of neuronal activity in nonhuman primates, and this has potential for clinical application.SIGNIFICANCE STATEMENT The use of designer receptors exclusively activated by designer drugs (DREADDs) for chronic manipulation of neuronal activity for days to weeks may be feasible for investigating brain functions and behavior on a long time-scale, and thereby for developing therapeutics for brain disorders, such as epilepsy. Here we performed pharmacokinetics and in vivo occupancy study of orally administered deschloroclozapine to determine a dose range suitable for DREADDs studies. In monkeys expressing hM4Di in the prefrontal cortex, single and repeated daily doses significantly induced working-memory impairments for hours and over two weeks, respectively, without discernible desensitization. These results indicate that orally delivered deschloroclozapine produces long-term stable chemogenetic effects, and holds great promise for the translational use of DREADDs technology.

Non-conserved residues dictate dopamine transporter selectivity for the potent synthetic cathinone and psychostimulant MDPV.

Clandestine chemists are currently exploiting the pyrrolidinophenone scaffold to develop new designer drugs that carry the risk of abuse and overdose. These drugs promote addiction through the rewarding effects of increased dopaminergic neurotransmission. 3,4-Methylenedioxypyrovalerone (MDPV) and its analogs are illicit psychostimulants of this class that are ∼50-fold more potent than cocaine at inhibiting the human dopamine transporter (hDAT). In contrast, MDPV is a weak inhibitor at both the human serotonin transporter (hSERT) and, as it is shown here, the Drosophila melanogaster DAT (dDAT). We studied three conserved residues between hSERT and dDAT that are unique in hDAT (A117, F318, and P323 in dDAT), and one residue that is different in all three transporters (D121 in dDAT). hDAT residues were replaced in the dDAT sequence at these positions using site-directed mutagenesis and stable cell lines were generated expressing these mutant transporters. The potencies of MDPV and two of its analogs were determined using a Ca2+-mobilization assay. In this assay, voltage-gated Ca2+ channels are expressed to sense the membrane electrical depolarization evoked when dopamine is transported through DAT. Each individual mutant slightly improved MDPV's potency, but the combination of all four increased its potency ∼100-fold (2 log units) in inhibiting dDAT activity. Molecular modeling and docking studies were conducted to explore the possible mode of interaction between MDPV and DAT in silico. Two of the studied residues (F318 and P323) are at the entrance of the S1 binding site, whereas the other two (A117 and D121) face the aryl moiety of MDPV when bound to this site. Therefore, these four non-conserved residues can influence MDPV selectivity not only by stabilizing binding, but also by controlling access to its binding site at DAT.

Chemogenetic stimulation of the Gi pathway in astrocytes suppresses neuroinflammation.

Engineered G protein-coupled receptors (GPCRs) are commonly used in chemogenetics as designer receptors exclusively activated by designer drugs (DREADDs). Although several GPCRs have been studied in astrocytes using a chemogenetic approach, the functional role of the astrocytic Gi pathway is not clear, as the literature is conflicting depending on the brain regions or behaviors investigated. In this study, we evaluated the role of the astrocytic Gi pathway in neuroinflammation using a Gi -coupled DREADD (hM4Di). Gi -DREADD was expressed in hippocampal astrocytes of a lipopolysaccharide (LPS)-induced neuroinflammation mouse model using adeno-associated viruses. We found that astrocyte Gi -DREADD stimulation using clozapine N-oxide (CNO) inhibits neuroinflammation, as characterized by decreased levels of proinflammatory cytokines, glial activation, and cognitive impairment in mice. Subsequent experiments using primary astrocyte cultures revealed that Gi -DREADD stimulation significantly downregulated LPS-induced expression of Nos2 mRNA and nitric oxide production. Similarly, in vitro calcium imaging showed that activation of the astrocytic Gi pathway attenuated intracellular calcium transients triggered by LPS treatment, suggesting a positive correlation between enhanced calcium transients and the inflammatory phenotype of astrocytes observed in the inflamed brain. Taken together, our results indicate that the astrocytic Gi pathway plays an inhibitory role in neuroinflammation, providing an opportunity to identify potential cellular and molecular targets to control neuroinflammation.

The Spicy Story of Cannabimimetic Indoles.

The Sterling Research Group identified pravadoline as an aminoalkylindole (AAI) non-steroidal anti-inflammatory pain reliever. As drug design progressed, the ability of AAI analogs to block prostaglandin synthesis diminished, and antinociceptive activity was found to result from action at the CB1 cannabinoid receptor, a G-protein-coupled receptor (GPCR) abundant in the brain. Several laboratories applied computational chemistry methods to ultimately conclude that AAI and cannabinoid ligands could overlap within a common binding pocket but that WIN55212-2 primarily utilized steric interactions via aromatic stacking, whereas cannabinoid ligands required some electrostatic interactions, particularly involving the CB1 helix-3 lysine. The Huffman laboratory identified strategies to establish CB2 receptor selectivity among cannabimimetic indoles to avoid their CB1-related adverse effects, thereby stimulating preclinical studies to explore their use as anti-hyperalgesic and anti-allodynic pharmacotherapies. Some AAI analogs activate novel GPCRs referred to as "Alkyl Indole" receptors, and some AAI analogs act at the colchicine-binding site on microtubules. The AAI compounds having the greatest potency to interact with the CB1 receptor have found their way into the market as "Spice" or "K2". The sale of these alleged "herbal products" evades FDA consumer protections for proper labeling and safety as a medicine, as well as DEA scheduling as compounds having no currently accepted medical use and a high potential for abuse. The distribution to the public of potent alkyl indole synthetic cannabimimetic chemicals without regard for consumer safety contrasts with the adherence to regulatory requirements for demonstration of safety that are routinely observed by ethical pharmaceutical companies that market medicines.

Side-by-side comparison of the effects of Gq- and Gi-DREADD-mediated astrocyte modulation on intracellular calcium dynamics and synaptic plasticity in the hippocampal CA1.

Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca2+. In addition, Ca2+-dependent and/or Ca2+-independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca2+ signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca2+ dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca2+ events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca2+-dependent and Ca2+-independent mechanisms, respectively.

Vesicular glutamate transporter 2-containing neurons of the centrally-projecting Edinger-Westphal nucleus regulate alcohol drinking and body temperature.

Previous studies in rodents have repeatedly demonstrated that the centrally-projecting Edinger-Westphal nucleus (EWcp) is highly sensitive to alcohol and is also involved in regulating alcohol intake and body temperature. Historically, the EWcp has been known as the main site of Urocortin 1 (Ucn1) expression, a corticotropin-releasing factor-related peptide, in the brain. However, the EWcp also contains other populations of neurons, including neurons that express the vesicular glutamate transporter 2 (Vglut2). Here we transduced the EWcp with adeno-associated viruses (AAVs) encoding Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the EWcp in alcohol drinking and in the regulation of body temperature. Activation of the EWcp with excitatory DREADDs inhibited alcohol intake in a 2-bottle choice procedure in male C57BL/6J mice, whereas inhibition of the EWcp with DREADDs had no effect. Surprisingly, analysis of DREADD expression indicated Ucn1-containing neurons of the EWcp did not express DREADDs. In contrast, AAVs transduced non-Ucn1-containing EWcp neurons. Subsequent experiments showed that the inhibitory effect of EWcp activation on alcohol intake was also present in male Ucn1 KO mice, suggesting that a Ucn1-devoid population of EWcp regulates alcohol intake. A final set of chemogenetic experiments showed that activation of Vglut2-expressing EWcp neurons inhibited alcohol intake and induced hypothermia in male and female mice. These studies expand on previous literature by indicating that a glutamatergic, Ucn1-devoid subpopulation of the EWcp regulates alcohol consumption and body temperature.

VTA MC3R neurons control feeding in an activity- and sex-dependent manner in mice.

Increasing evidence indicates that the melanocortin and mesolimbic dopamine (DA) systems interact to regulate feeding and body weight. Because melanocortin-3 receptors (MC3R) are highly expressed in the ventral tegmental area (VTA), we tested whether VTA neurons expressing these receptors (VTA MC3R neurons) control feeding and body weight in vivo. We also tested whether there were sex differences in the ability of VTA MC3R neurons to control feeding, as MC3R -/- mice show sex-dependent alterations in reward feeding and DA levels, and there are clear sex differences in multiple DA-dependent behaviors and disorders. Designer receptors exclusively activated by designer drugs (DREADD) were used to acutely activate and inhibit VTA MC3R neurons and changes in food intake and body weight were measured. Acutely altering the activity of VTA MC3R neurons decreased feeding in an activity- and sex-dependent manner, with acute activation decreasing feeding, but only in females, and acute inhibition decreasing feeding, but only in males. These differences did not appear to be due to sex differences in the number of VTA MC3R neurons, the ability of hM3Dq to activate VTA MC3R neurons, or the proportion of VTA MC3R neurons expressing tyrosine hydroxylase (TH). These studies demonstrate an important role for VTA MC3R neurons in the control of feeding and reveal important sex differences in behavior, whereby opposing changes in neuronal activity in male and female mice cause similar changes in behavior.

Effects of α-pyrrolidino-phenone cathinone stimulants on locomotor behavior in female rats.

The α-pyrrolidino-phenone cathinone stimulants first came to widespread attention because of bizarre behavior consequent to the use of α-pyrrolidinopentiophenone (α-PVP, "flakka") reported in popular press. As with other designer drugs, diversification of cathinones has been driven by desirable subjective effects, but also by attempts to stay ahead of legal controls of specific molecules. The α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinopropiophenone (α-PPP) compounds have been relatively under-investigated relative to α-PVP and provide a key opportunity to also investigate structure-activity relationships, i.e., how the extension of the alpha carbon chain may affect potency or efficacy. Female rats were used to contrast the effects of α-PHP and α-PPP with those of α-PVP in altering wheel activity and effects on spontaneous locomotion, temperature and intracranial self-stimulation reward. The α-PPP, α-PHP and α-PVP compounds (5, 10 mg/kg, i.p.) suppressed wheel activity. Inhalation of α-PHP or α-PVP also suppressed wheel activity, but for an abbreviated duration compared with the injection route. Spontaneous activity was increased, and brain reward thresholds decreased, in a dose-dependent manner by all three compounds; only small decrements in body temperature were observed. These data show that all three of the α-pyrrolidino-phenone cathinones exhibit significant stimulant-like activity in female rats. Differences were minor and abuse liability is therefore likely to be equivalent for all three α-pyrrolidino-phenones.

Disrupting the medial prefrontal cortex with designer receptors exclusively activated by designer drug alters hippocampal sharp-wave ripples and their associated cognitive processes.

The hippocampus and medial prefrontal cortex (mPFC) interact during a myriad of cognitive processes including decision-making and long-term memory consolidation. Exactly how the mPFC and hippocampus interact during goal-directed decision-making remains to be fully elucidated. During periods of rest, bursts of high-frequency oscillations, termed sharp-wave ripple (SWR), appear in the local field potential. Impairing SWRs on the maze or during post-learning rest can interfere with memory-guided decision-making and memory consolidation. We hypothesize that the hippocampus and mPFC bidirectionally interact during SWRs to support memory consolidation and decision-making. Rats were trained on the neuroeconomic spatial decision-making task, Restaurant Row, to make serial stay-skip decisions where the amount of effort (delay to reward) varied upon entry to each restaurant. Hippocampal cells and SWRs were recorded in rats with the mPFC transduced with inhibitory DREADDs. We found that disrupting the mPFC impaired consolidating SWRs in the hippocampus. Hippocampal SWR rates depended on the internalized value of the reward (derived from individual flavor preferences), a parameter important in decision-making, and disrupting the mPFC changed this relationship. Additionally, we found a dissociation between SWRs that occurred while rats were on the maze dependent upon whether those SWRs occurred while the rat was anticipating food reward or during post-reward consumption.

Abnormalities in the composition of the gut microbiota in mice after repeated administration of DREADD ligands.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are known as genetically modified G-protein-coupled receptors (GPCRs), which can be activated by synthetic ligands such as clozapine N-oxide (CNO) and DREADD agonist 21 (compound 21: C21). The brain-gut-microbiota axis has a crucial role in bidirectional interactions between the brain and the gastrointestinal microbiota. In this study, we investigated whether repeated administration of CNO or C21 could influence the gut microbiota and short-chain fatty acids (SCFAs) in feces of adult mice. Repeated administration of CNO or C21 as drinking water did not alter the α- and ß-diversity of gut microbiota in mice compared with control mice. However, we found significant changes in relative abundance for several bacteria in the CNO (or C21) group at the taxonomic level compared to the control group. The linear discriminant analysis effect size (LEfSe) algorithm distinguished the family Prevotellaceae, the genus Anaerocolumna, the genus Prevotella, and the genus Frisingicoccus, these four specific microbial markers for the CNO group relative to the control group. In addition, the LEfSe algorithm identified the family Clostridiaceae, the genus Faecalicatena and the genus Marinisporobacter, these three bacteria of different taxonomic as potential microbial markers for the C21 group relative to the control group. In contrast, repeated administration of CNO (or C21) did not alter SCFAs in feces samples of adult mice. The data suggest that repeated administration of CNO or C21 contributes to an unusual organization of the gut microbiota in adult mice. Therefore, abnormalities in the composition of gut microbiota by repeated dosing of DREADD ligands should be taken into consideration for behavioral and biological functions in rodents treated with DREADD ligands.