Eric W. Fish

Alcohol, Cocaine, and Brain Stimulation-Reward in C57Bl6/J and DBA2/J Mice

BACKGROUND Pleasure and reward are critical features of alcohol drinking that are difficult to measure in animal studies. Intracranial self-stimulation (ICSS) is a behavioral method for studying the effects of drugs directly on the neural circuitry that underlies brain reward. These experiments had 2 objectives: first, to establish the effects of alcohol on ICSS responding in the C57Bl6/J (C57) and DBA2/J (DBA) mouse strains; and second, to compare these effects to those of the psychostimulant cocaine. METHODS Male C57 and DBA mice were implanted with unipolar stimulating electrodes in the lateral hypothalamus and conditioned to spin a wheel for reinforcement by the delivery of rewarding electrical stimulation (i.e., brain stimulation-reward or BSR). Using the curve-shift method, the BSR threshold (theta(0)) was determined immediately before and after oral gavage with alcohol (0.3, 0.6, 1.0, 1.7 g/kg) or water. Blood alcohol concentration (BAC) was measured to determine the influence of alcohol metabolism on BSR threshold. Separately, mice were administered cocaine (1.0, 3.0, 10.0, 30.0 mg/kg) or saline intraperitoneally. RESULTS In C57 mice, the 0.6 g/kg dose of alcohol lowered BSR thresholds by about 20%, during the rising (up to 40 mg/dl), but not falling, phase of BAC. When given to the DBA mice, alcohol lowered BSR thresholds over the entire dose range; the largest reduction was by about 50%. Cocaine lowered BSR thresholds in both strains. However, cocaine was more potent in DBA mice than in C57 mice as revealed by a leftward shift in the cocaine dose-response curve. For both alcohol and cocaine, effects on BSR threshold were dissociable from effects on operant response rates. CONCLUSIONS In C57 and DBA mice, reductions in BSR threshold reflect the ability of alcohol to potentiate the neural mechanisms of brain reward. The DBA mice are more sensitive to the reward-potentiating effects of both alcohol and cocaine, suggesting that there are mouse strain differences in the neural mechanisms of brain reward that can be measured with the ICSS technique.

Pathway-specific dopaminergic deficits in a mouse model of Angelman syndrome

Angelman syndrome (AS) is a neurodevelopmental disorder caused by maternal deletions or mutations of the ubiquitin ligase E3A (UBE3A) allele and characterized by minimal verbal communication, seizures, and disorders of voluntary movement. Previous studies have suggested that abnormal dopamine neurotransmission may underlie some of these deficits, but no effective treatment currently exists for the core features of AS. A clinical trial of levodopa (L-DOPA) in AS is ongoing, although the underlying rationale for this treatment strategy has not yet been thoroughly examined in preclinical models. We found that AS model mice lacking maternal Ube3a (Ube3a(m-/p+) mice) exhibit behavioral deficits that correlated with abnormal dopamine signaling. These deficits were not due to loss of dopaminergic neurons or impaired dopamine synthesis. Unexpectedly, Ube3a(m-/p+) mice exhibited increased dopamine release in the mesolimbic pathway while also exhibiting a decrease in dopamine release in the nigrostriatal pathway, as measured with fast-scan cyclic voltammetry. These findings demonstrate the complex effects of UBE3A loss on dopamine signaling in subcortical motor pathways that may inform ongoing clinical trials of L-DOPA therapy in patients with AS.

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.

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.

Orexin-1 receptor antagonism does not reduce the rewarding potency of cocaine in Swiss-Webster mice

The orexin family of hypothalamic neuropeptides has been implicated in reinforcement mechanisms relevant to both food and drug reward. Previous behavioral studies with antagonists at the orexin A-selective receptor, OX(1), have demonstrated its involvement in behavioral sensitization, conditioned place-preference, and self-administration of drugs of abuse. Adult male Swiss-Webster mice were implanted with stimulating electrodes to the lateral hypothalamus and trained to perform intracranial self-stimulation (ICSS). The effects of the OX(1)-selective antagonist SB 334867 on brain stimulation-reward (BSR) and cocaine potentiation of BSR were measured. SB 334867 (10-30mg/kg, i.p.) alone had no effect on ICSS performance or BSR threshold. Cocaine (1.0-30mg/kgi.p.) dose-dependently potentiated BSR, measured as lowering of BSR threshold. This effect was not blocked by 30mg/kg SB 334867 at any cocaine dose tested. In agreement with previous reports, SB 334867 resulted in a reduction of body weight 24h after acute administration. Based on these data, it is concluded that orexins acting at OX(1) do not contribute to BSR; and are not involved in the reward-potentiating actions of cocaine on BSR. The data are discussed in the context of prior findings of SB 334867 effects on drug-seeking and drug-consuming behaviors.

Acute alcohol exposure during neurulation: Behavioral and brain structural consequences in adolescent C57BL/6J mice.

Prenatal alcohol exposure (PAE) can induce physical malformations and behavioral abnormalities that depend in part on thedevelopmental timing of alcohol exposure. The current studies employed a mouse FASD model to characterize the long-term behavioral and brain structural consequences of a binge-like alcohol exposure during neurulation; a first-trimester stage when women are typically unaware that they are pregnant. Time-mated C57BL/6J female mice were administered two alcohol doses (2.8g/kg, four hours apart) or vehicle starting at gestational day 8.0. Male and female adolescent offspring (postnatal day 28-45) were then examined for motor activity (open field and elevated plus maze), coordination (rotarod), spatial learning and memory (Morris water maze), sensory motor gating (acoustic startle and prepulse inhibition), sociability (three-chambered social test), and nociceptive responses (hot plate). Regional brain volumes and shapes were determined using magnetic resonance imaging. In males, PAE increased activity on the elevated plus maze and reduced social novelty preference, while in females PAE increased exploratory behavior in the open field and transiently impaired rotarod performance. In both males and females, PAE modestly impaired Morris water maze performance and decreased the latency to respond on the hot plate. There were no brain volume differences; however, significant shape differences were found in the cerebellum, hypothalamus, striatum, and corpus callosum. These results demonstrate that alcohol exposure during neurulation can have functional consequences into adolescence, even in the absence of significant brain regional volumetric changes. However, PAE-induced regional shape changes provide evidence for persistent brain alterations and suggest alternative clinical diagnostic markers.

Levetiracetam results in increased and decreased alcohol drinking with different access procedures in C57BL/6J mice.

The antiepileptic levetiracetam (LEV) has been investigated for the treatment of alcohol abuse. However, little is known about how LEV alters the behavioral effects of alcohol in laboratory animals. The acute effects of LEV on alcohol drinking by male C57BL/6J mice were investigated using two different drinking procedures, limited access [drinking-in-the-dark (DID)] and intermittent access (IA) drinking. In the first experiment (DID), mice had access to a single bottle containing alcohol or sucrose for 4 h every other day. In the second experiment (IA), mice had IA to two bottles, one containing alcohol or sucrose and one containing water, for 24 h on Monday, Wednesday, and Friday. In both experiments, mice were administered LEV (0.3–100 mg/kg intraperitoneally) or vehicle 30 min before access to the drinking solutions. In the DID mice, LEV increased alcohol intake from 4.3 to 5.4 g/kg, whereas in the IA mice LEV decreased alcohol intake from 4.8 to 3.0 g/kg in the first 4 h of access and decreased 24 h alcohol intake from 20 to ∼15 g/kg. These effects appear specific to alcohol, as LEV did not affect sucrose intake in either experiment. LEV appears to differentially affect drinking in animal models of moderate and heavier alcohol consumption.

Levetiracetam Has Opposite Effects on Alcohol- and Cocaine-Related Behaviors in C57BL/6J Mice

The antiepileptic drug levetiracetam (LEV) is a potential treatment for alcohol use disorders, yet few preclinical studies exist on its effects in animal models relevant to drug or alcohol abuse. We investigated the effects of LEV on locomotor stimulation following acute and repeated administration of alcohol or cocaine and on alcohol- and cocaine-mediated changes in responding for brain stimulation reward (BSR) in C57BL/6J mice. LEV alone (10.0–100.0 mg/kg intraperitoneally) had no significant effect on locomotor activity or intracranial self-stimulation. Pretreatment with LEV reduced acute locomotor stimulation by 2.0 g/kg alcohol, attenuated the development of locomotor sensitization to alcohol with repeated exposure, and produced a shift in the dose-response curve for alcohol on BSR threshold without affecting maximum operant response rate (MAX). Conversely, LEV pretreatment enhanced both acute locomotor stimulation by 15 mg/kg cocaine and development of locomotor sensitization following repeated exposure and produced a leftward shift in the dose-response curve for cocaine on BSR threshold without affecting MAX. Electrophysiological recordings in vitro showed that LEV reduced excitatory currents in both ventral tegmental area (VTA) dopamine neurons and nucleus accumbens (NAc) medium spiny neurons, consistent with a presynaptic effect. The opposite effects of LEV pretreatment on alcohol- and cocaine-related behaviors may predict its clinical utility in the treatment of patients with alcohol, but not psychostimulant abuse disorders.

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.

Dose-dependent teratogenicity of the synthetic cannabinoid CP-55,940 in mice

Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n>10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose-response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB.