Sara Faccidomo

Aggressive behavioral phenotypes in mice.

Aggressive behavior in male and female mice occurs in conflicts with intruding rivals, most often for the purpose of suppressing the reproductive success of the opponent. The behavioral repertoire of fighting is composed of intricately sequenced bursts of species-typical elements, with the resident displaying offensive and the intruder defensive acts and postures. The probability of occurrence as well as the frequency, duration, temporal and sequential patterns of aggressive behavior can be quantified with ethological methods. Classic selection and strain comparisons show the heritability of aggressive behavior, and point to the influence of several genes, including some of them on the Y chromosome. However, genetic effects on aggressive behavior critically depend upon the background strain, maternal environment and the intruder. These factors are equally important in determining changes in aggressive behavior in mice with a specific gene deletion. While changes in aggression characterize mutant mice involving a variety of genes, no pattern has emerged that links particular gene products (i.e. enzyme, peptide, receptor) to either an increase or a decrease in aggressive behavior, but rather emphasizes polygenic influences. A potentially common mechanism may be some components of the serotonin system, since alterations in 5-HT neurotransmission have been found in several of the KO mice that display unusual aggressive behavior.

Aggression heightened by alcohol or social instigation in mice: reduction by the 5-HT1B receptor agonist CP-94,253

Abstract  Rationale: Models of heightened aggression may be particularly relevant in exploring pharmacological options for the clinical treatment of aggressive and impulsive disorders. Objectives: To investigate and compare the effects of a 5-HT1B selective agonist, CP-94,253, on aggression that was heightened as a result of 1) social instigation or 2) alcohol treatment. Methods: Male CFW mice were administered 1.0 g/kg EtOH and were subsequently confronted by an intruder in their home cage. In a separate experimental procedure, resident male mice were instigated to aggressive behavior by brief exposure to a provocative stimulus male. To test the hypothesis that activation of the 5-HT1B receptor subtype would preferentially attenuate heightened aggression, in comparison to the moderate levels of species-typical aggressive behaviors, the selective agonist, CP-94,253 (1.0–30 mg/kg, IP), and antagonists to the 5-HT1B (GR 127935; 10 mg/kg, IP) and the 5-HT1A receptor (WAY 100,635; 0.1 mg/kg IP) were used. Results: CP-94,253 suppressed non-heightened aggressive behavior (ED50=7.2 mg/kg ). GR 127935, but not WAY 100,635 shifted the ED50 for CP-94,253 to 14.5 mg/kg. Importantly, the anti-aggressive effects of CP-94,253 were not accompanied by locomotor sedation. Alcohol-heightened and instigation-heightened aggression were suppressed at lower doses than those necessary to suppress non-heightened aggression (ED50=3.8 and 2.7 mg/kg, respectively). Conclusions: The current results support the hypothesis that activation of 5-HT1B receptors modulates very high levels of aggressive behavior in a pharmacologically and behaviorally specific manner.

Metabotropic Glutamate Receptor 5 Activity in the Nucleus Accumbens Is Required for the Maintenance of Ethanol Self-Administration in a Rat Genetic Model of High Alcohol Intake

BACKGROUND Systemic modulation of Group I and II metabotropic glutamate receptors (mGluRs) regulate ethanol self-administration in a variety of animal models. Although these receptors are expressed in reward-related brain regions, the anatomical specificity of their functional involvement in ethanol self-administration remains to be characterized. This study sought to evaluate the functional role of Group I (mGluR5) and Group II (mGluR2/3) in mesocorticolimbic brain regions in ethanol self-administration. METHODS Alcohol-preferring (P) rats, a genetic model of high alcohol drinking, were trained to self-administer ethanol (15% v/v) versus water in operant conditioning chambers. Effects of brain site-specific infusion of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the mGluR2/3 agonist were then assessed on the maintenance of self-administration. RESULTS Microinjection of the mGluR5 antagonist MPEP in the nucleus accumbens reduced ethanol self-administration at a dose that did not alter locomotor activity. By contrast, infusion of the mGluR2/3 agonist LY379268 in the nucleus accumbens reduced self-administration and produced nonspecific reductions in locomotor activity. The mGluR5 involvement showed anatomical specificity as evidenced by lack of effect of MPEP infusion in the dorsomedial caudate or medial prefrontal cortex on ethanol self-administration. To determine reinforcer specificity, P-rats were trained to self-administer sucrose (.4% w/v) versus water, and effects of intra-accumbens MPEP were tested. The MPEP did not alter sucrose self-administration or motor behavior. CONCLUSIONS These results suggest that mGluR5 activity specifically in the nucleus accumbens is required for the maintenance of ethanol self-administration in individuals with genetic risk for high alcohol consumption.

Alcohol-heightened aggression in mice: attenuation by 5-HT1A receptor agonists

Abstract One of the critical mechanisms by which alcohol heightens aggression involves forebrain serotonin (5-HT) systems, possibly via actions on 5-HT1A receptors. The present experiments tested the hypothesis that activating 5-HT1A receptors by selective agonists will block the aggression-heightening effects of ethanol. Initially, the selective antagonist WAY 100635 was used to assess whether or not the changes in aggressive behavior after treatment with 8-OH-DPAT and flesinoxan result from action at the 5-HT1A receptors. Resident male CFW mice engaged in aggressive behavior (i.e. attack bites, sideways threats, tail rattle) during 5-min confrontations with a group-housed intruder male. Quantitative analysis of the behavioral repertoire revealed systematic reductions in all salient elements of aggressive behavior after treatment with 8-OH-DPAT (0.1–0.3 mg/kg, IP) or flesinoxan (0.1–1.0 mg/kg, IP). The 5-HT1A agonists also reduced motor activities such as walking, rearing and grooming, although to a lesser degree. Pretreatment with the antagonist WAY 100635 (0.1 mg/kg, IP) shifted the agonist dose-effect curves for behavioral effects to the right. In a further experiment, oral ethanol (1.0 g/kg, PO) increased the frequency of attacks in excess of 2 SD from their mean vehicle level of attacks in 19 out of 76 resident mice. Low doses of 8-OH-DPAT (0.03–0.3 mg/kg) and flesinoxan (0.1, 0.3, 0.6 mg/kg), given before the ethanol treatment, attenuated the alcohol-heightened aggression in a dose-dependent fashion. By contrast, these low 5-HT1A agonist doses affected motor activity in ethanol-treated resident mice to a lesser degree, suggesting behavioral specificity of these anti-aggressive effects. The current results support the hypothesized significant role of 5-HT1A receptors in the aggression-heightening effects of alcohol. If these effects are in fact due to action at somatodendritic 5-HT1A autoreceptors, then the anti-aggressive effects would be associated with decreased 5-HT neurotransmission.

Escalated Aggressive Behavior: New Pharmacotherapeutic Approaches and Opportunities

Abstract: Psychopharmacologic studies of aggressive behavior in animals under controlled laboratory conditions have been instrumental in developing and evaluating specific and effective novel drug treatments that reduce aggressive behavior. An initial contribution of this research is to create experimental conditions that enable the display of aggressive and defensive acts and postures in species that engage in either dominance or territorial or maternal aggression. Quantitative ethological analyses allow the precise delineation of the sequential organization of aggressive bursts, providing a benchmark for assessing excessive or pathological forms of aggressive behavior. A second contribution of preclinical research is the development of experimental models of escalated forms of aggressive behavior, such as focusing on genetic predispositions or social provocations and frustrative experiences. A critical role of preclinical research is in the pharmacological and neurochemical analysis of aggressive behavior; for example, a host of undesirable side effects prompted a shift from classic dopaminergic neuroleptic compounds to the more recently developed atypical neuroleptics with effective and more specific anti‐aggressive effects. The long‐established role of brain serotonin in impulsive and escalated forms of aggressive behavior continues to be a focus of preclinical studies. New evidence differentiates dynamic state changes in corticolimbic serotonergic neurons during the termination of aggressive behavior from the deficient‐serotonin trait in violence‐prone individuals. It can be anticipated that currently developed tools for targeting the genes that code for specific subtypes of serotonin receptors will offer new therapeutic options for reducing aggressive behavior, and the 5‐HT1B receptor appears to be a promising target. The modulation of GABA and GABAA receptors by 5‐HT in corticolimbic neurons promises to be particularly relevant for specific forms of escalated aggressive behavior such as alcohol‐heightened aggression.

Nonselective suppression of operant ethanol and sucrose self-administration by the mGluR7 positive allosteric modulator AMN082.

Emerging evidence indicates that specific metabotropic glutamate receptors (mGluRs) modulate ethanol self-administration. In general, inhibition of glutamate transmission through blockade of postsynaptic mGluRs, or activation of presynaptic mGluRs, inhibits ethanol self-administration. The goal of this preclinical study was to further characterize mGluR regulation of ethanol self-administration by examining effects of AMN082, an allosteric positive modulator of presynaptic mGluR7 activity. Separate groups of C57BL/6J male mice were trained to self-administer ethanol or sucrose on a fixed-ratio 4 schedule of reinforcement during 1 h sessions. On test days, mice were pretreated with AMN082 (0, 1.0, 3.0, 5.6, or 10 mg/kg) 30 min prior to self-administration sessions. Functional specificity and activity was examined by testing the effects of AMN082 (0-10 mg/kg) on open-field locomotor activity and HPA axis function as measured by plasma corticosterone levels. AMN082 (10 mg/kg) produced a significant reduction in ethanol and sucrose reinforced responding, and inhibited locomotor activity. Plasma corticosterone levels were significantly increased following AMN082 (5.6 and 10 mg/kg) suggesting a dose-dependent dissociation between the behavioral and hormonal effects of the compound. These data suggest that activation of mGluR7 by AMNO82 produces nonspecific reductions in motivated behavior that are associated with negative effects on motor activity.

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.

Escalated Aggression after Alcohol Drinking in Male Mice: Dorsal Raphé and Prefrontal Cortex Serotonin and 5-HT1B Receptors

A significant minority of individuals engages in escalated levels of aggression after consuming moderate doses of alcohol (Alc). Neural modulation of escalated aggression involves altered levels of serotonin (5-HT) and the activity of 5-HT1B receptors. The aim of these studies was to determine whether 5-HT1B receptors in the dorsal raphé (DRN), orbitofrontal (OFC), and medial prefrontal (mPFC) cortex attenuate heightened aggression and regulate extracellular levels of 5-HT. Male mice were trained to self-administer Alc by performing an operant response that was reinforced with a delivery of 6% Alc. To identify Alc-heightened aggressors, each mouse was repeatedly tested for aggression after consuming either 1.0 g/kg Alc or H2O. Next, a cannula was implanted into either the DRN, OFC, or mPFC, and subsets of mice were tested for aggression after drinking either Alc or H2O prior to a microinjection of the 5-HT1B agonist, CP-94,253. Additional mice were implanted with a microdialysis probe into the mPFC, through which CP-94,253 was perfused and samples were collected for 5-HT measurement. Approximately 60% of the mice were more aggressive after drinking Alc, confirming the aggression-heightening effects of 1.0 g/kg Alc. Infusion of 1 μg CP-94,253 into the DRN reduced both aggressive and motor behaviors. However, infusion of 1 μg CP-94,253 into the mPFC, but not the OFC, after Alc drinking, increased aggressive behavior. In the mPFC, reverse microdialysis of CP-94,253 increased extracellular levels of 5-HT; levels decreased immediately after the perfusion. This 5-HT increase was attenuated in self-administering mice. These results suggest that 5-HT1B receptors in the mPFC may serve to selectively disinhibit aggressive behavior in mice with a history of Alc self-administration.

Effects of mGlu1-receptor blockade on ethanol self-administration in inbred alcohol-preferring rats

The Group I family of metabotropic glutamate receptors includes subtype 1 (mGlu1) and subtype 5 (mGlu5) receptors. This family of receptors has generated interest as potential targets for different areas of therapeutic development, including intervention for alcohol and drug abuse. Most of this interest is driven by findings showing involvement of mGlu5 receptors in the regulation of drug self-administration; however, studies examining the role of mGlu1 receptors in drug self-administration are limited. The purpose of this work was to examine the role of mGlu1-receptor antagonism in the maintenance of ethanol self-administration and the self-administration of an alternate nondrug reward, sucrose. Male alcohol-preferring inbred rats were trained to self-administer ethanol (15% vol/vol) versus water on a concurrent schedule of reinforcement, and the effect of the mGlu1-receptor antagonist JNJ16259685 (0.1-1.0mg/kg intraperitoneal [IP]) was evaluated on self-administration. The rats were then trained to self-administer sucrose (0.4% wt/vol) versus water, and the same dose range of JNJ16259685 was tested. Locomotor activity was tested in a separate assessment to evaluate potential nonspecific motor effects of the antagonist. Ethanol self-administration was dose dependently reduced by JNJ16259685. This reduction was likely due to a motor impairment as the lowest effective dose (0.1mg/kg) significantly reduced locomotor behavior. Sucrose self-administration was reduced by the highest JNJ16259685 dose (1.0mg/kg), and this reduction was also likely due to a motor impairment. Interestingly, ethanol self-administration was more sensitive to mGlu1-receptor antagonism than sucrose self-administration as lower JNJ16259685 doses reduced ethanol-reinforced responding and motor behavior. Together, these results suggest that mGlu1 receptors do not play a specific role in modulating ethanol self-administration or the self-administration of an alternate nondrug reward (i.e., sucrose).

Moderate Alcohol Drinking and the Amygdala Proteome: Identification and Validation of Calcium/Calmodulin Dependent Kinase II and AMPA Receptor Activity as Novel Molecular Mechanisms of the Positive Reinforcing Effects of Alcohol

BACKGROUND Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood. METHODS Here, we conducted a high-throughput screen of the amygdala proteome in mice after moderate alcohol drinking (n = 12/group) followed by behavioral studies (n = 6-8/group) to uncover novel molecular mechanisms of the positive reinforcing properties of alcohol that strongly influence the development of addiction. RESULTS Two-dimensional difference in-gel electrophoresis with matrix assisted laser desorption ionization tandem time-of-flight identified 29 differentially expressed proteins in the amygdala of nondependent C57BL/6J mice following 24 days of alcohol drinking. Alcohol-sensitive proteins included calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and a network of functionally linked proteins that regulate neural plasticity and glutamate-mediated synaptic activity. Accordingly, alcohol drinking increased α-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) in central amygdala (CeA) and phosphorylation of AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in CeA and lateral amygdala. Further, CaMKIIα-Thr286 and GluA1-Ser831 phosphorylation was increased in CeA and lateral amygdala of mice that lever-pressed for alcohol versus the nondrug reinforcer sucrose. Mechanistic studies showed that targeted pharmacologic inhibition of amygdala CaMKII or AMPAR activity specifically inhibited the positive reinforcing properties of alcohol but not sucrose. CONCLUSIONS Moderate alcohol drinking increases the activity and function of plasticity-linked protein networks in the amygdala that regulate the positive reinforcing effects of the drug. Given the prominence of positive reinforcement in the etiology of addiction, we propose that alcohol-induced adaptations in CaMKIIα and AMPAR signaling in the amygdala may serve as a molecular gateway from use to abuse.