Changes in Sensitivity of Reward and Motor Behavior to Dopaminergic, Glutamatergic, and Cholinergic Drugs in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is a leading cause of intellectual disability. FXS is caused by loss of function of the FMR1 gene, and mice in which Fmr1 has been inactivated have been used extensively as a preclinical model for FXS. We investigated the behavioral pharmacology of drugs acting through dopaminergic, glutamatergic, and cholinergic systems in fragile X (Fmr1-/Y) mice with intracranial self-stimulation (ICSS) and locomotor activity measurements. We also measured brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. Fmr1-/Y mice were more sensitive than wild type mice to the rewarding effects of cocaine, but less sensitive to its locomotor stimulating effects. Anhedonic but not motor depressant effects of the atypical neuroleptic, aripiprazole, were reduced in Fmr1-/Y mice. The mGluR5-selective antagonist, 6-methyl-2-(phenylethynyl)pyridine (MPEP), was more rewarding and the preferential M1 antagonist, trihexyphenidyl, was less rewarding in Fmr1-/Y than wild type mice. Motor stimulation by MPEP was unchanged, but stimulation by trihexyphenidyl was markedly increased, in Fmr1-/Y mice. Numbers of midbrain TH+ neurons in the ventral tegmental area were unchanged, but were lower in the substantia nigra of Fmr1-/Y mice, although no changes in TH levels were found in their forebrain targets. The data are discussed in the context of known changes in the synaptic physiology and pharmacology of limbic motor systems in the Fmr1-/Y mouse model. Preclinical findings suggest that drugs acting through multiple neurotransmitter systems may be necessary to fully address abnormal behaviors in individuals with FXS

Nociceptin receptor antagonist SB 612111 decreases high fat diet binge eating

Binge eating is a dysregulated form of feeding behavior that occurs in multiple eating disorders including binge-eating disorder, the most common eating disorder. Feeding is a complex behavioral program supported through the function of multiple brain regions and influenced by a diverse array of receptor signaling pathways. Previous studies have shown the overexpression of the opioid neuropeptide nociceptin (orphanin FQ, N/OFQ) can induce hyperphagia, but the role of endogenous nociceptin receptor (NOP) in naturally occurring palatability-induced hyperphagia is unknown. In this study we adapted a simple, replicable form of binge eating of high fat food (HFD). We found that male and female C57BL/6J mice provided with daily one-hour access sessions to HFD eat significantly more during this period than those provided with continuous 24 hour access. This form of feeding is rapid and entrained. Chronic intermittent HFD binge eating produced hyperactivity and increased light zone exploration in the open field and light-dark assays respectively. Treatment with the potent and selective NOP antagonist SB 612111 resulted in a significant dose-dependent reduction in binge intake in both male and female mice, and, unlike treatment with the serotonin selective reuptake inhibitor fluoxetine, produced no change in total 24-hour food intake. SB 612111 treatment also significantly decreased non-binge-like acute HFD consumption in male mice. These data are consistent with the hypothesis that high fat binge eating is modulated by NOP signaling and that the NOP system may represent a promising novel receptor to explore for the treatment of binge eating

Molecular mechanism of biased signaling at the kappa opioid receptor

The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR

DREADD agonist 21 is an effective agonist for muscarinic-based DREADDs in vitro and in vivo

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern

Receptor Reserve Moderates Mesolimbic Responses to Opioids in a Humanized Mouse Model of the OPRM1 A118G Polymorphism

The OPRM1 A118G polymorphism is the most widely studied μ-opioid receptor (MOR) variant. Although its involvement in acute alcohol effects is well characterized, less is known about the extent to which it alters responses to opioids. Prior work has shown that both electrophysiological and analgesic responses to morphine but not to fentanyl are moderated by OPRM1 A118G variation, but the mechanism behind this dissociation is not known. Here we found that humanized mice carrying the 118GG allele (h/mOPRM1-118GG) were less sensitive than h/mOPRM1-118AA littermates to the rewarding effects of morphine and hydrocodone but not those of other opioids measured with intracranial self-stimulation. Reduced morphine reward in 118GG mice was associated with decreased dopamine release in the nucleus accumbens and reduced effects on GABA release in the ventral tegmental area that were not due to changes in drug potency or efficacy in vitro or receptor-binding affinity. Fewer MOR-binding sites were observed in h/mOPRM1-118GG mice, and pharmacological reduction of MOR availability unmasked genotypic differences in fentanyl sensitivity. These findings suggest that the OPRM1 A118G polymorphism decreases sensitivity to low-potency agonists by decreasing receptor reserve without significantly altering receptor function

A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior

DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity and behavior. Here we used a structure-based approach to develop a new Gi coupled DREADD using the kappa-opioid receptor as template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally-expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors. Additionally, co-expression of the KORD and the Gq coupled M3-DREADD within the same neuronal population facilitated the sequential and bi-directional remote control of behavior. The availability of DREADDs activated by different ligands provides enhanced opportunities for investigating diverse physiological systems using multiplexed chemogenetic actuators

Different Contributions of Dopamine D1 and D2 Receptor Activity to Alcohol Potentiation of Brain Stimulation Reward in C57BL/6J and DBA/2J Mice

C57BL/6J (C57) and DBA/2J (DBA) mice respond differently to drugs that affect dopamine systems, including alcohol. The current study compared effects of D1 and D2 receptor agonists and antagonists, and the interaction between D1/D2 antagonists and alcohol, on intracranial self-stimulation in male C57 and DBA mice to determine the role of dopamine receptors in the effects of alcohol on brain stimulation reward (BSR). In the initial strain comparison, dose effects on BSR thresholds and maximum operant response rates were determined for the D1 receptor agonist SKF-82958 (±-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 0.1–0.56 mg/kg) and antagonist SCH 23390 (+-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrochloride; 0.003–0.056 mg/kg), and the D2 receptor agonist quinpirole (0.1–3.0 mg/kg) and antagonist raclopride (0.01–0.56 mg/kg). For the alcohol interaction, SCH 23390 (0.003 mg/kg) or raclopride (0.03 mg/kg) was given before alcohol (0.6–2.4 g/kg p.o.). D1 antagonism dose-dependently elevated and SKF-82958 dose-dependently lowered BSR threshold in both strains; DBA mice were more sensitive to SKF-82958 effects. D2 antagonism dose-dependently elevated BSR threshold only in C57 mice. Low doses of quinpirole elevated BSR threshold equally in both strains, whereas higher doses of quinpirole lowered BSR threshold only in C57 mice. SCH 23390, but not raclopride, prevented lowering of BSR threshold by alcohol in DBA mice. Conversely, raclopride, but not SCH 23390, prevented alcohol potentiation of BSR in C57 mice. These results extend C57 and DBA strain differences to D1/D2 sensitivity of BSR, and suggest differential involvement of D1 and D2 receptors in the acute rewarding effects of alcohol in these two mouse strains

Effects of the atypical neuroleptic aripiprazole on ICSS and locomotor behavior in wild type (WT, <i>white</i><i>circles</i>) and <i>Fmr1</i>^-/Y mice (red circles).

<div><p>Changes in BSR threshold (<b>A</b>) and maximum operant response rate (MAX, <b>B</b>) 46-60 minutes after i.p. aripiprazole injections are shown. Values are expressed as mean percentages of pre-injection baselines ± SEM. </p> <p>Asterisks (*) indicate <i>p</i> < 0.05 vs. vehicle (V). <b>C</b>. Mean distance traveled (± SEM) before and after injection of vehicle (VEH) or aripiprazole (ARI, 0.1 mg/kg i.p.) in 15-minute intervals. Shading indicates post-injection time points. <b>D</b>. Mean change in locomotion (± SEM) as a percentage of pre-injection baseline activity for 60 minutes after injection of vehicle (VEH) or aripiprazole (ARI, 0.1 mg/kg i.p.).</p></div

Changes in Sensitivity of Reward and Motor Behavior to Dopaminergic, Glutamatergic, and Cholinergic Drugs in a Mouse Model of Fragile X Syndrome

<div><p>Fragile X syndrome (FXS) is a leading cause of intellectual disability. FXS is caused by loss of function of the FMR1 gene, and mice in which <i>Fmr1</i> has been inactivated have been used extensively as a preclinical model for FXS. We investigated the behavioral pharmacology of drugs acting through dopaminergic, glutamatergic, and cholinergic systems in fragile X (<i>Fmr1</i>^-/Y) mice with intracranial self-stimulation (ICSS) and locomotor activity measurements. We also measured brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. <i>Fmr1</i>^-/Y mice were more sensitive than wild type mice to the rewarding effects of cocaine, but less sensitive to its locomotor stimulating effects. Anhedonic but not motor depressant effects of the atypical neuroleptic, aripiprazole, were reduced in <i>Fmr1</i>^-/Y mice. The mGluR5-selective antagonist, 6-methyl-2-(phenylethynyl)pyridine (MPEP), was more rewarding and the preferential M1 antagonist, trihexyphenidyl, was less rewarding in <i>Fmr1</i>^-/Y than wild type mice. Motor stimulation by MPEP was unchanged, but stimulation by trihexyphenidyl was markedly increased, in <i>Fmr1</i>^-/Y mice. Numbers of midbrain TH+ neurons in the ventral tegmental area were unchanged, but were lower in the substantia nigra of <i>Fmr1</i>^-/Y mice, although no changes in TH levels were found in their forebrain targets. The data are discussed in the context of known changes in the synaptic physiology and pharmacology of limbic motor systems in the <i>Fmr1</i>^-/Y mouse model. Preclinical findings suggest that drugs acting through multiple neurotransmitter systems may be necessary to fully address abnormal behaviors in individuals with FXS.</p> </div

Effects of the partially M1-selective antagonist trihexyphenidyl on ICSS and locomotor behavior in wild type (WT, <i>white</i><i>circles</i>) and <i>Fmr1</i>^-/Y mice (red circles).

<p>Changes in BSR threshold (<b>A</b>) and maximum operant response rate (MAX, <b>B</b>) 31-45 minutes after i.p. trihexyphenidyl injections are shown. Values are expressed as mean percentages of pre-injection baselines ± SEM. Asterisks (*) indicate <i>p</i> < 0.05 vs. vehicle (V); daggers (†) indicate <i>p</i> < 0.05 vs. WT (dose x genotype interaction <i>post </i><i>hoc</i>). <b>C</b>. Mean distance traveled (± SEM) before and after injection of dH₂O (VEH) or trihexyphenidyl (THX, 10.0 mg/kg i.p.) in 15-minute intervals. Shading indicates post-injection time points. Daggers (†) indicate <i>p</i> < 0.05 vs. WT (time x genotype interaction <i>post </i><i>hoc</i>). <b>D</b>. Mean change in locomotion (± SEM) as a percentage of pre-injection baseline for 60 minutes after injection of dH₂O (VEH) or trihexyphenidyl (THX, 10.0 mg/kg i.p.).</p