Kimberly A. Leite-Morris

Reduction of alcohol’s reinforcing and motivational properties by the positive allosteric modulator of the GABAB receptor, BHF177, in alcohol-preferring rats.

BACKGROUND The positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, have been found to reduce alcohol self-administration in alcohol-preferring rats. The present study was designed to assess the effect of the newly synthesized positive allosteric modulator of the GABA(B) receptor, BHF177, on alcohols reinforcing and motivational properties in selectively bred Sardinian alcohol-preferring (sP) rats. METHODS sP rats were initially trained to respond on a lever [on a fixed ratio 4 (FR4) schedule of reinforcement] to orally self-administer alcohol (15%, v/v) or sucrose (1 to 3%, w/v) in daily 30-minute sessions. Once responding reached stable levels, rats were allocated to 2 different experiments: in the first experiment, rats were exposed to sessions with the FR4 schedule of reinforcement; in the second experiment, rats were exposed to sessions with a conventional progressive ratio (PR) schedule of reinforcement. In both experiments, the effect of BHF177 (0, 12.5, 25, and 50 mg/kg; i.g.) on responding for alcohol and sucrose (FR experiment: 1%, w/v; PR experiment: 3%, w/v) was determined. RESULTS In the FR experiment, pretreatment with 25 and 50 mg/kg BHF177 produced a 30 and 45% reduction, respectively, in responding for alcohol. In the PR experiment, pretreatment with 50 mg/kg BHF177 resulted in a 35% reduction in breakpoint for alcohol (defined as the lowest response requirement not achieved by each rat and used as index of the motivational strength of alcohol). In both experiments, the effect of BHF177 on alcohol self-administration was specific, since responding for sucrose was unaltered by BHF177 pretreatment. CONCLUSIONS The present results extend to BHF177 the capacity of the 2 previously tested positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, to specifically suppress alcohols reinforcing and motivational properties in alcohol-preferring rats.

Baclofen enhances extinction of opiate conditioned place preference

Conditioned opiate reward (COR) is rapidly acquired and slowly extinguished. The slow rate of extinction of the salience of drug-related cues contributes to drug craving and relapse. The gamma-aminobutyric acid receptor type B (GABA(B)) agonist, baclofen, attenuates the unconditioned rewarding actions of several drugs of abuse and was investigated for effects on the extinction of COR. C57BL/6 mice were utilized in an unbiased conditioned place preference (CPP) protocol using morphine (10mg/kg, s.c.) and saline. CPP was measured by increases in time spent in the morphine-associated (CS+) vs. the saline-associated (CS-) chamber in a 15-min test after four morphine and four saline alternated conditioning sessions. CPP and locomotor sensitization were produced to the CS+ chamber. Subsequently, sixteen daily extinction sessions were conducted with either vehicle or baclofen (1 or 2.5mg/kg, s.c.) treatment given either before or after the sessions. This design was used to create the baclofen drug state before or after the activation of the CPP memory trace in the extinction protocol. After morphine CPP development, its extinction was significantly facilitated in a dose-dependent manner by post-session, but not by pre-session, baclofen treatments. No significant sedative effects of baclofen were detected during any extinction training or testing phase. Baclofen treatment facilitated the extinction of COR and reduced conditioned sensitization during extinction when given after, but not before, the activation of the CPP memory trace. Baclofen appears to have disrupted reconsolidation of conditioned reward memory during extinction training and might similarly facilitate extinction learning in human opiate addiction.

Dendritic structural plasticity in the basolateral amygdala after fear conditioning and its extinction in mice.

Previous research suggests that morphology and arborization of dendritic spines change as a result of fear conditioning in cortical and subcortical brain regions. This study uniquely aims to delineate these structural changes in the basolateral amygdala (BLA) after both fear conditioning and fear extinction. C57BL/6 mice acquired robust conditioned fear responses (70-80% cued freezing behavior) after six pairings with a tone cue associated with footshock in comparison to unshocked controls. During fear acquisition, freezing behavior was significantly affected by both shock exposure and trial number. For fear extinction, mice were exposed to the conditioned stimulus tone in the absence of shock administration and behavioral responses significantly varied by shock treatment. In the retention tests over 3 weeks, the percentage time spent freezing varied with the factor of extinction training. In all treatment groups, alterations in dendritic plasticity were analyzed using Golgi-Cox staining of dendrites in the BLA. Spine density differed between the fear conditioned group and both the fear extinction and control groups on third order dendrites. Spine density was significantly increased in the fear conditioned group compared to the fear extinction group and controls. Similarly in Sholl analyses, fear conditioning significantly increased BLA spine numbers and dendritic intersections while subsequent extinction training reversed these effects. In summary, fear extinction produced enduring behavioral plasticity that is associated with a reversal of alterations in BLA dendritic plasticity produced by fear conditioning. These neuroplasticity findings can inform our understanding of structural mechanisms underlying stress-related pathology can inform treatment research into these disorders.

Opiate Sensitization Induces FosB/ΔFosB Expression in Prefrontal Cortical, Striatal and Amygdala Brain Regions

Sensitization to the effects of drugs of abuse and associated stimuli contributes to drug craving, compulsive drug use, and relapse in addiction. Repeated opiate exposure produces behavioral sensitization that is hypothesized to result from neural plasticity in specific limbic, striatal and cortical systems. ΔFosB and FosB are members of the Fos family of transcription factors that are implicated in neural plasticity in addiction. This study examined the effects of intermittent morphine treatment, associated with motor sensitization, on FosB/ΔFosB levels using quantitative immunohistochemistry. Motor sensitization was tested in C57BL/6 mice that received six intermittent pre-treatments (on days 1, 3, 5, 8, 10, 12) with either subcutaneous morphine (10 mg/kg) or saline followed by a challenge injection of morphine or saline on day 16. Mice receiving repeated morphine injections demonstrated significant increases in locomotor activity on days 8, 10, and 12 of treatment (vs. day 1), consistent with development of locomotor sensitization. A morphine challenge on day 16 significantly increased locomotor activity of saline pre-treated mice and produced even larger increases in motor activity in the morphine pre-treated mice, consistent with the expression of opiate sensitization. Intermittent morphine pre-treatment on these six pre-treatment days produced a significant induction of FosB/ΔFosB, measured on day 16, in multiple brain regions including prelimbic (PL) and infralimbic (IL) cortex, nucleus accumbens (NAc) core, dorsomedial caudate-putamen (CPU), basolateral amygdala (BLA) and central nucleus of the amygdala (CNA) but not in a motor cortex control region. Opiate induced sensitization may develop via Fos/ΔFosB plasticity in motivational pathways (NAc), motor outputs (CPU), and associative learning (PL, IL, BLA) and stress pathways (CNA).

Repeated valproate treatment facilitates fear extinction under specific stimulus conditions.

Single dose treatment with histone deacetylase inhibitor (HDACi) agents has been shown to enhance extinction learning in rodent models under certain conditions. The present novel studies were designed to examine the effects of repeated HDACi treatment, with valproate or sodium butyrate, on the extinction of conditioned fear. In Experiments 1 and 2, short duration CS exposure (30s) in combination with vehicle administration progressively attenuated conditioned fear responses over 40 or more sessions. This effective extinction training was not augmented by HDACi treatments. In Experiment 3, we used a long duration CS exposure (120 s) to weaken extinction training. With these extinction parameters, repeated valproate treatment substantially facilitated the acquisition and retention of fear extinction. Results of this study extend previous work suggesting that HDACis have utility in augmenting the efficiency of fear extinction, although their apparent benefits are critically dependent upon specific parameters of extinction training.

Changes in expression of c-Fos protein following cocaine-cue extinction learning.

Extinguishing abnormally strengthened learned responses to cues associated with drugs of abuse remains a key tactic for alleviating addiction. To assist in developing pharmacotherapies to augment exposure therapy for relapse prevention, investigation into neurobiological underpinnings of drug-cue extinction learning is needed. We used regional analyses of c-Fos and GluR2 protein expression to delineate neural activity and plasticity that may be associated with cocaine-cue extinction learning. Rats were trained to self-administer cocaine paired with a light cue, and later underwent a single 2h extinction session for which cocaine was withheld but response-contingent cues were presented (cocaine-cue extinction). Control groups consisted of rats yoked to animals self-administering cocaine and receiving saline non-contingently followed by an extinction session, or rats trained to self-administer cocaine followed by a no-extinction session for which levers were retracted, and cocaine and cues were withheld. Among 11 brain sites examined, extinction training increased c-Fos expression in basolateral amygdala and prelimbic prefrontal cortex of cocaine-cue extinguished rats relative to both control conditions. In dorsal subiculum and infralimbic prefrontal cortex, extinction training increased c-Fos expression in both cocaine-cue and saline-cue extinguished rats relative to the no-extinction control condition. GluR2 protein expression was not altered in any site examined after extinction or control training. Findings suggest that basolateral amygdala and prelimbic prefrontal cortex neurons are activated during acquisition of cocaine-cue extinction learning, a process that is independent of changes in GluR2 abundance. Other sites are implicated in processing the significance of cues that are present early in extinction training.

Effects of Ethanol and NAP on Cerebellar Expression of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10−12 M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression.

Alterations in expression and phosphorylation of GluA1 receptors following cocaine-cue extinction learning

Brain regional analyses of total GluA1 and GluA1-pSer(845) were used to delineate plasticity of the AMPA receptor in conjunction with cocaine-cue extinction learning. Rats were trained to self-administer cocaine paired with a 2-s light cue and later underwent a single 2 h extinction session for which cocaine was withheld but response-contingent cues were presented. Control groups received yoked-saline sessions or received cocaine self-administration training without undergoing extinction training. Extinction-related increases and decreases, respectively, in total GluA1 were observed in the ventromedial prefrontal cortex (vmPFC) and basolateral amygdala (BLA). Phosphorylation of GluA1 at Ser(845) was increased in the vmPFC and nucleus accumbens (NAc). Though total GluA1 did not change in NAc, there was a positive association between the number of responses during extinction training and the magnitude of total GluA1 in NAc. No significant changes were evident in the dorsal hippocampus. We conclude that the BLA and vmPFC, in particular, appear to be loci for the inhibition of learned behavior induced via extinction training, but each site may have different signaling functions for cocaine-cue extinction learning.

Extinction of opiate reward reduces dendritic arborization and c-Fos expression in the nucleus accumbens core

Recurrent opiate use combined with environmental cues, in which the drug was administered, provokes cue-induced drug craving and conditioned drug reward. Drug abuse craving is frequently linked with stimuli from a prior drug-taking environment via classical conditioning and associative learning. We modeled the conditioned morphine reward process by using acquisition and extinction of conditioned place preference (CPP) in C57BL/6 mice. Mice were trained to associate a morphine injection with a drug context using a classical conditioning paradigm. In morphine conditioning (0, 0.25, 0.5, 1, 5, or 10 mg/kg) experimental mice acquired a morphine CPP dose response with 10mg/kg as most effective. During morphine CPP extinction experiments, mice were divided into three test groups: morphine CPP followed by extinction training, morphine CPP followed by sham extinction, and saline controls. Extinction of morphine CPP developed within one extinction experiment (4 days) that lasted over two more trials (another 8 days). However, the morphine CPP/sham extinction group retained a place preference that endured through all three extinction trials. Brains were harvested following CPP extinction and processed using Golgi-Cox impregnation. Changes in dendritic morphology and spine quantity were examined in the nucleus accumbens (NAc) Core and Shell neurons. In the NAcCore only, morphine CPP/extinguished mice produced less dendritic arborization, and a decrease in neuronal activity marker c-Fos compared to the morphine CPP/sham extinction group. Extinction of morphine CPP is associated with decreased structural complexity of dendrites in the NAcCore and may represent a substrate for learning induced structural plasticity relevant to addiction.

Targeting the GABAB Receptor for the Treatment of Alcohol Use Disorder

Accumulating lines of experimental and clinical evidence suggest that the prototypic, orthosteric GABA type B (GABAB) receptor agonist, baclofen, may possess therapeutic potential for treatment of alcohol use disorder (AUD). At preclinical level, acute or repeated treatment with nonsedative doses of baclofen has repeatedly been reported to suppress several alcohol-motivated behaviors, including alcohol drinking, in rats and mice. At clinical level, administration of doses of baclofen ranging from low to extremely high doses has been found to suppress alcohol consumption, craving for alcohol, and alcohol withdrawal signs and symptoms in patients affected by AUD, confirming that baclofen may represent a promising option for treatment of AUD. A more recent avenue of research is represented by generalization of baclofen effects to the positive allosteric modulators of the GABAB receptor: preclinical data collected to date suggest that these compounds reproduce, with a much higher therapeutic index, several suppressing effects of baclofen on alcohol-motivated behaviors.