Cannabinoid CB1 receptor inhibition blunts adolescent-typical increased binge alcohol and sucrose consumption in male C57BL/6J mice

Increased binge alcohol consumption has been reported among adolescents as compared to adults in both humans and rodent models, and has been associated with serious long-term health consequences. However, the neurochemical mechanism for age differences in binge drinking between adolescents and adults has not been established. The present study was designed to evaluate the mechanistic role of the cannabinoid CB1 receptor in adolescent and adult binge drinking. Binge consumption was established in adolescent and adult male C57BL/6J mice by providing access to 20% alcohol or 1% sucrose for 4h every other day. Pretreatment with the CB1 antagonist/inverse agonist AM-251 (0, 1, 3, and 10mg/kg) in a Latin square design dose-dependently reduced adolescent alcohol consumption to adult levels without altering adult intake. AM-251 (3mg/kg) also reduced adolescent but not adult sucrose consumption. Adolescent reductions in alcohol and sucrose were not associated with alterations in open-field locomotor activity or thigmotaxis. These findings point to age differences in CB1 receptor activity as a functional mediator of adolescent-typical increased binge drinking as compared to adults. Developmental alterations in endocannabinoid signaling in the adolescent brain may therefore be responsible for the drinking phenotype seen in this age group

CaMKII inhibition in the prefrontal cortex specifically increases the positive reinforcing effects of sweetened alcohol in C57BL/6J mice

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is required for synaptic plasticity and has been proposed to be a primary molecular component of the etiology of alcohol addiction. Chronic alcohol intake upregulates CaMKIIα protein expression in reward-related brain regions including the amygdala and nucleus accumbens, and CaMKIIα activity in the amygdala is required for the positive reinforcing effects of alcohol, suggesting this system promotes consumption in the early stages of alcohol addiction. Alternatively, the medial prefrontal cortex (mPFC) is known to inhibit limbic activity via CaMKII-dependent excitatory projections and may, therefore, enable top-down regulation of motivation. Here we sought to remove that regulatory control by site-specifically inhibiting CaMKII activity in the mPFC, and measured effects on the positive reinforcing effects of sweetened alcohol in C57BL/6J mice. Infusion of the CAMKII inhibitor KN-93 (0–10.0 μg) in the mPFC primarily increased alcohol+sucrose reinforced response rate in a dose- and time-dependent manner. KN-93 infusion reduced response rate in behavior-matched sucrose-only controls. Importantly, potentiation of operant responding for sweetened alcohol occurred immediately after infusion, at a time during which effects on sucrose responding were not observed, and persisted through the session. These results suggest that endogenous CaMKII activity in the mPFC exerts inhibitory control over the positive reinforcing effects of alcohol. Downregulation of CaMKII signaling in the mPFC might contribute to escalated alcohol use

CaMKII α -GluA1 Activity Underlies Vulnerability to Adolescent Binge Alcohol Drinking

Binge drinking during adolescence is associated with increased risk for developing alcohol use disorders (AUDs); however, the neural mechanisms underlying this liability are unclear. In this study, we sought to determine if binge-drinking alters expression or phosphorylation of two molecular mechanisms of neuroplasticity, calcium/calmodulin dependent kinase II alpha (CaMKIIα) and the GluA1 subunit of AMPA receptors (AMPAR) in addiction-associated brain regions. We also asked if activation of CaMKIIα-dependent AMPAR activity escalates binge-like drinking

Mephedrone (4-methylmethcathinone) and intracranial self-stimulation in C57BL/6J mice: Comparison to cocaine

The recreational use of cathinone-derived synthetic stimulants, also known as "bath salts", has increased during the last five years. A commonly abused drug in this class is mephedrone (4-methylmethcathinone or "meow-meow"), which alters mood and produces euphoria in humans. Intracranial self-stimulation (ICSS) measures the behavioral effects of neuroactive compounds on brain reward circuitry. We used ICSS to investigate the ability of mephedrone and cocaine to alter responding for electrical stimulation of the medial forebrain bundle in C57BL/6J mice. Adult male C57BL/6J mice (n=6) implanted with unipolar stimulating electrodes at the level of the lateral hypothalamus responded for varying frequencies of brain stimulation reward (BSR). The frequency that supported half maximal responding (EF50), the BSR threshold (θ(0)), and the maximum response rate were determined before and after intraperitoneal administration of saline, mephedrone (1.0, 3.0, or 10.0 mg/kg), or cocaine (1.0, 3.0, or 10.0 mg/kg). Mephedrone dose-dependently decreased EF50 (max. effect=72.3% of baseline), θ(0) (max. effect=59.6% of baseline), and the maximum response rate (max. effect=67.0% of baseline) beginning 15 min after administration. Beginning immediately after administration, cocaine dose-dependently lowered EF50 (max. effect=66.4% of baseline) and θ(0) (max. effect=60.1% of baseline) but did not affect maximum response rate. These results suggest that mephedrone, like cocaine, potentiates BSR, which may indicate its potential for abuse. Given the public health concern of stimulant abuse, future studies will be necessary to determine the cellular and behavioral effects of acute and chronic mephedrone use

Alcohol Alters the Activation of ERK1/2, a Functional Regulator of Binge Alcohol Drinking in Adult C57BL/6J Mice

Binge alcohol drinking is a particularly risky pattern of alcohol consumption that often precedes alcohol dependence and addiction. The transition from binge alcohol drinking to alcohol addiction likely involves mechanisms of synaptic plasticity and learning in the brain. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to be involved in learning and memory, as well as the response to drugs of abuse, but their role in binge alcohol drinking remains unclear. The present experiments were designed to determine the effects of acute alcohol on extracellular signaling related kinases (ERK1/2) expression and activity, and to determine whether ERK1/2 activity functionally regulates binge-like alcohol drinking

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

Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood

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, or DID) and intermittent access (IA) drinking. In the first experiment (DID), mice had access to a single bottle containing alcohol or sucrose for four hours every-other day. In the second experiment (IA), mice had intermittent access to two bottles, one containing alcohol or sucrose and one containing water, for 24 h on Mon/Wed/Fri. In both experiments, mice were administered LEV (0.3 – 100 mg/kg i.p.) 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, while 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 g/kg to approximately 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

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the precise role of CRF1-expressing neuronal populations in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat, which expresses a bicistronic iCre-2A-tdTomato transgene directed by 200kb of promoter and enhancer sequence surrounding the Crhr1 cDNA present within a BAC clone, that has been transgenically inserted into the rat genome. We report that Crhr1 and Cre mRNA expression are highly colocalized in CRF1-Cre-tdTomato rats within both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be selectively targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like behavior and nocifensive responses. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the rostrocaudal axis of the CRF1-Cre-tdTomato rat brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons and circuits can now be performed in assays that require the use of rats as the model organism

Comparison of the adolescent and adult mouse prefrontal cortex proteome

<div><p>Adolescence is a developmental period characterized by unique behavioral phenotypes (increased novelty seeking, risk taking, sociability and impulsivity) and increased risk for destructive behaviors, impaired decision making and psychiatric illness. Adaptive and maladaptive adolescent traits have been associated with development of the medial prefrontal cortex (mPFC), a brain region that mediates regulatory control of behavior. However, the molecular changes that underlie brain development and behavioral vulnerability have not been fully characterized. Using high-throughput 2D DIGE spot profiling with identification by MALDI-TOF mass spectrometry, we identified 62 spots in the PFC that exhibited age-dependent differences in expression. Identified proteins were associated with diverse cellular functions, including intracellular signaling, synaptic plasticity, cellular organization and metabolism. Separate Western blot analyses confirmed age-related changes in DPYSL2, DNM1, STXBP1 and CFL1 in the mPFC and expanded these findings to the dorsal striatum, nucleus accumbens, motor cortex, amygdala and ventral tegmental area. Ingenuity Pathway Analysis (IPA) identified functional interaction networks enriched with proteins identified in the proteomics screen, linking age-related alterations in protein expression to cellular assembly and development, cell signaling and behavior, and psychiatric illness. These results provide insight into potential molecular components of adolescent cortical development, implicating structural processes that begin during embryonic development as well as plastic adaptations in signaling that may work in concert to bring the cortex, and other brain regions, into maturity.</p></div

Adolescent development impacts a functional protein network involved in behavior, cell-to-cell signaling and interaction, and nervous system development and function.

<p>Visualization of a protein interaction network identified by Ingenuity Pathway Analysis as being altered by adolescent brain development (<i>p</i> score = 31). Proteins shown in red were up-regulated in the adult mPFC, proteins in green were up-regulated in the adolescent mPFC, and proteins in yellow indicate statistically significant interaction proteins identified by IPA network analysis. Solid lines indicate a direct interaction, and dashed lines indicate an indirect interaction mediated by additional, non-significant proteins. Asterisks denote proteins that were identified multiple times in the proteomic analysis that have been consolidated into a single point in the functional network. Focus molecule syntaxin binding protein 1 (STXBP1) is indicated in bold.</p