The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. & Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee

Differential Regulation of the Period Genes in Striatal Regions following Cocaine Exposure

Several studies have suggested that disruptions in circadian rhythms contribute to the pathophysiology of multiple psychiatric diseases, including drug addiction. In fact, a number of the genes involved in the regulation of circadian rhythms are also involved in modulating the reward value for drugs of abuse, like cocaine. Thus, we wanted to determine the effects of chronic cocaine on the expression of several circadian genes in the Nucleus Accumbens (NAc) and Caudate Putamen (CP), regions of the brain known to be involved in the behavioral responses to drugs of abuse. Moreover, we wanted to explore the mechanism by which these genes are regulated following cocaine exposure. Here we find that after repeated cocaine exposure, expression of the Period (Per) genes and Neuronal PAS Domain Protein 2 (Npas2) are elevated, in a somewhat regionally selective fashion. Moreover, NPAS2 (but not CLOCK (Circadian Locomotor Output Cycles Kaput)) protein binding at Per gene promoters was enhanced following cocaine treatment. Mice lacking a functional Npas2 gene failed to exhibit any induction of Per gene expression after cocaine, suggesting that NPAS2 is necessary for this cocaine-induced regulation. Examination of Per gene and Npas2 expression over twenty-four hours identified changes in diurnal rhythmicity of these genes following chronic cocaine, which were regionally specific. Taken together, these studies point to selective disruptions in Per gene rhythmicity in striatial regions following chronic cocaine treatment, which are mediated primarily by NPAS2. © 2013 Falcon et al

Comparison of ethanol-related behaviors and FosB mapping in hybrid mice with distinct drinking patterns

textDistinct alcohol self-administration behaviors are observed when comparing two F1 hybrid strains of mice: C57BL/6J x NZB/B1NJ (B6xNZB) show reduced alcohol preference (RAP) after experience with high concentrations of alcohol and abstinence periods and C57BL/6J x FVB/NJ (B6xFVB) show sustained alcohol preference (SAP), providing models of stable, high alcohol consumption and moderate drinking. The purpose of this dissertation is to characterize ethanol-related behaviors and define neurocircuits engaged by SAP and RAP. We performed a battery of behavioral tests to define behaviors that predict SAP and RAP. B6xFVB exhibited less severe ethanol-induced conditioned taste aversion and were less sensitive to ethanol-induced loss of righting reflex (LORR) than B6xNZB. Both hybrids demonstrated ethanol-induced place preference and low ethanol withdrawal severity. Hybrids differ in sensitivity to the aversive and sedative, but not rewarding, effects of ethanol. Results of elevated plus maze, mirror chamber, and locomotor tests reveal B6xFVB mice are less anxious and more active than B6xNZB mice. The validity of the SAP behavioral phenotype in B6xFVB mice was determined by testing whether chronic self-administration of ethanol produced tolerance or dependence. We measured responses from ethanol-naïve and ethanol-experienced mice in tests of ethanol-induced hypothermia, withdrawal severity, and LORR. Chronic ethanol self-administration resulted in tolerance to sedative and hypothermic effects of ethanol; however, physical dependence was not evident as measured by ethanol withdrawal severity. We tested the hypothesis that SAP and RAP behavioral differences are represented by differential production of the inducible transcription factor, FosB. FosB immunoreactivity was quantified in 16 brain structures after chronic ethanol consumption or only water. Neuronal activity (as measured by FosB levels) depended on ethanol experience, brain region, and genotype, further supporting the notion that neuronal circuitry underlies motivational aspects of ethanol consumption. For B6xNZB mice, ethanol consumption resulted in increased neuronal activity in the EW, VTA, and amygdala, known ethanol- reward-, and stress-related brain regions. In B6xFVB, ethanol consumption resulted in a larger network of correlated regional activity, whereas in B6xNZB ethanol consumption resulted in a smaller network. These studies characterized genetic models of stable, high consumption (SAP) and moderate drinking (RAP) in two hybrid mouse strains.Institute for Neuroscienc

The Effect of Apremilast on Circadian Rhythm in the Ventral Tegmental Area

Chronic binge drinking negatively impacts society and is a leading cause of preventable deaths. The purpose of the experiment is to determine if regulation of circadian rhythms in the ventral tegmental area (VTA) will contribute to less ethanol consumption. The VTA plays a major role in addiction. A simple genetic animal model examines iHDID-1 mice and their binge-like drinking intake. The mice undergo Drinking in the Dark (DID) for 6 weeks prior to administration of apremilast or saline treatment at weeks 7 and 8. The mice are exposed to DID and sacrificed at two separate time points (ZT 3 and ZT 15) to account for the peaks and troughs of circadian rhythms. Apremilast treatment has shown evidence to support amelioration of circadian rhythms leads to a decrease in ethanol intake. If circadian rhythms are increased in the VTA, then the amount of ethanol consumed will decrease

NPAS2 Regulation of Anxiety-Like Behavior and GABAA Receptors

Abnormal circadian rhythms and circadian genes are strongly associated with several psychiatric disorders. Neuronal PAS Domain Protein 2 (NPAS2) is a core component of the molecular clock that acts as a transcription factor and is highly expressed in reward- and stress-related brain regions such as the striatum. However, the mechanism by which NPAS2 is involved in mood-related behaviors is still unclear. We measured anxiety-like behaviors in mice with a global null mutation in Npas2 (Npas2 null mutant mice) and found that Npas2 null mutant mice exhibit less anxiety-like behavior than their wild-type (WT) littermates (in elevated plus maze, light/dark box and open field assay). We assessed the effects of acute or chronic stress on striatal Npas2 expression, and found that both stressors increased levels of Npas2. Moreover, knockdown of Npas2 in the ventral striatum resulted in a similar reduction of anxiety-like behaviors as seen in the Npas2 null mutant mouse. Additionally, we identified Gabra genes as transcriptional targets of NPAS2, found that Npas2 null mutant mice exhibit reduced sensitivity to the GABAa positive allosteric modulator, diazepam and that knockdown of Npas2 reduced Gabra1 expression and response to diazepam in the ventral striatum. These results: (1) implicate Npas2 in the response to stress and the development of anxiety; and (2) provide functional evidence for the regulation of GABAergic neurotransmission by NPAS2 in the ventral striatum

Effects of Chronic Stimulation of Nucleus Accumbens on Binge Drinking and Transcriptome

We previously found that stimulating activity in the nucleus accumbens (NAc) reduced binge-like alcohol drinking in mice. We manipulated the NAc using clozapine-n-oxide (CNO) and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). In a subsequent study, we tested the hypothesis that chronic administration of CNO (to stimulate excitatory DREADDs expressed in the NAc) could produce lasting reductions in binge drinking (as compared with mice receiving vehicle). We observed that 4 weeks of CNO administration resulted in reductions in binge-like drinking that lasted at least 1 week. Based on these results, we hypothesized that transcriptional changes may underlie the observed behavioral plasticity. To test this hypothesis, we conducted a study to further explore the effects of NAc stimulation on behavior and gene expression in mice selectively bred to drink to intoxication (High Drinking in the Dark; HDID mice). We stereotaxically injected AAVCre and AAV2DIO-hM3Dq into the NAc and subsequently measured ethanol intake for 6 weeks using the Drinking in the Dark (DID) paradigm. We employed 2 experimental conditions [drinking fluid (ethanol or water) and treatment (CNO or vehicle; IP)] with 11-12 mice/group. Vehicle groups were injected with 1% DMSO in saline daily for 6 weeks. For CNO groups, mice were treated with vehicle during weeks 1 (baseline) and 6 (washout) and CNO during weeks 2-5. At the end of the study we isolated NAc RNA and performed RNA Seq. To explore quantitative changes across samples in the NAc transcriptome and determine target genes associated with binge-like drinking, we performed Differential Expression analysis (DE) and Weighted gene co-expression analysis (WGCNA). RNA-Seq sample reads were aligned to the Mus musculus genome (via STAR aligner), filtered and normalized to produce a count matrix. Using DESeq2, the count matrix was used to determine significantly up or down regulated genes based on p-value threshold. WGCNA was then used to describe correlation patterns among expressed genes. To identify modules (co-regulated genes) and hubs (genes correlating strongly with a significant number of related expressed modules) the count matrix was processed with the WGCNA package in R. By using these analyses, we are working to identify changes in gene expression related to harmful binge-like drinking and CNO/DREADD-induced reductions in binge-drinking. Our results suggest that chronically increasing NAc activity (via CNO/DREADDs) can induce molecular and behavioral plasticity

FUNCTIONAL IMPLICATIONS OF THE CLOCK 3111T/C SINGLE-NUCLEOTIDE POLYMORPHISM

Circadian rhythm disruptions are prominently associated with Bipolar Disorder (BD). Circadian rhythms are regulated by the molecular clock, a family of proteins that function together in a transcriptional-translational feedback loop. The CLOCK protein is a key transcription factor of this feedback loop, and previous studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice (Roybal et al., 2007). The Clock 3111T/C single-nucleotide polymorphism (SNP; rs1801260) is a genetic variation of the human Clock gene that is significantly associated with increased frequency of manic episodes in BD patients (Benedetti et al., 2003). The 3111T/C SNP is located in the 3’ untranslated region of the Clock gene. In this study, we sought to examine the functional implications of the human Clock 3111T/C SNP by transfecting a mammalian cell line (mouse embryonic fibroblasts isolated from Clock -/- knockout mice) with pcDNA plasmids containing the human Clock gene with either the T or C SNP at position 3111. We then measured circadian gene expression over a 24 hour time period. We found that the Clock3111C SNP resulted in higher mRNA levels than the Clock 3111T SNP. Further, we found that Per2, a transcriptional target of CLOCK, was also more highly expressed with Clock 3111C expression, indicating the 3’UTR SNP affects the expression, function and stability of Clock mRNA

Chronic self-administration of alcohol results in elevated ΔFosB: comparison of hybrid mice with distinct drinking patterns

All authors are with the Waggoner Center for Alcoholism and Addiction Research, Institute for Neuroscience, University of Texas at Austin, Austin, TX, 78712, USA -- Angela R. Ozburn is with the Department of Psychiatry, University of Pittsburgh Medical Center, 450 Technology Dr. Ste. 223, Pittsburgh, PA, 15219-3143, USABackground: The inability to reduce or regulate alcohol intake is a hallmark symptom for alcohol use disorders. Research on novel behavioral and genetic models of experience-induced changes in drinking will further our knowledge on alcohol use disorders. Distinct alcohol self-administration behaviors were previously observed when comparing two F1 hybrid strains of mice: C57BL/6J x NZB/B1NJ (BxN) show reduced alcohol preference after experience with high concentrations of alcohol and periods of abstinence while C57BL/6J x FVB/NJ (BxF) show sustained alcohol preference. These phenotypes are interesting because these hybrids demonstrate the occurrence of genetic additivity (BxN) and overdominance (BxF) in ethanol intake in an experience dependent manner. Specifically, BxF exhibit sustained alcohol preference and BxN exhibit reduced alcohol preference after experience with high ethanol concentrations; however, experience with low ethanol concentrations produce sustained alcohol preference for both hybrids. In the present study, we tested the hypothesis that these phenotypes are represented by differential production of the inducible transcription factor, ΔFosB, in reward, aversion, and stress related brain regions. Results: Changes in neuronal plasticity (as measured by ΔFosB levels) were experience dependent, as well as brain region and genotype specific, further supporting that neuronal circuitry underlies motivational aspects of ethanol consumption. BxN mice exhibiting reduced alcohol preference had lower ΔFosB levels in the Edinger-Westphal nucleus than mice exhibiting sustained alcohol preference, and increased ΔFosB levels in central medial amygdala as compared with control mice. BxN mice showing sustained alcohol preference exhibited higher ΔFosB levels in the ventral tegmental area, Edinger-Westphal nucleus, and amygdala (central and lateral divisions). Moreover, in BxN mice ΔFosB levels in the Edinger-Westphal nucleus and ventral tegmental regions significantly positively correlated with ethanol preference and intake. Additionally, hierarchical clustering analysis revealed that many ethanol-naïve mice with overall low ΔFosB levels are in a cluster, whereas many mice displaying sustained alcohol preference with overall high ΔFosB levels are in a cluster together. Conclusions: By comparing and contrasting two alcohol phenotypes, this study demonstrates that the reward- and stress-related circuits (including the Edinger-Westphal nucleus, ventral tegmental area, amygdala) undergo significant plasticity that manifests as reduced alcohol preference.Waggoner Center for Alcohol and Addiction [email protected]

Chronic Chemogenetic Stimulation of the Nucleus Accumbens Produces Lasting Reductions in Binge Drinking and Ameliorates Alcohol-Related Morphological and Transcriptional Changes

Binge drinking is a dangerous pattern of behavior. We tested whether chronically manipulating nucleus accumbens (NAc) activity (via clozapine-N-oxide (CNO) and Designer Receptors Exclusively Activated by Designer Drugs (DREADD)) could produce lasting eects on ethanol binge-like drinking in mice selectively bred to drink to intoxication. We found chronically increasing NAc activity (4 weeks, via CNO and the excitatory DREADD, hM3Dq) decreased binge-like drinking, but did not observe CNO-induced changes in drinking with the inhibitory DREADD, hM4Di. The CNO/hM3Dq-induced reduction in ethanol drinking persisted for at least one week, suggesting adaptive neuroplasticity via transcriptional and epigenetic mechanisms. Therefore, we defined this plasticity at the morphological and transcriptomic levels. We found that chronic binge drinking (6 weeks) altered neuronal morphology in the NAc, an effect that was ameliorated with CNO/hM3Dq. Moreover, we detected significant changes in expression of several plasticity-related genes with binge drinking that were ameliorated with CNO treatment (e.g., Hdac4). Lastly, we found that LMK235, an HDAC4/5 inhibitor, reduced binge-like drinking. Thus, we were able to target specific molecular pathways using pharmacology to mimic the behavioral effects of DREADDs

<i>Clock</i>, <i>Npas2</i> and <i>Bmal1</i> expression after cocaine treatment.

<p>Real-time PCR analysis of <i>Clock, Npas2,</i> and <i>Bmal1</i> expression in the CP and NAc following saline, acute (15 mg/kg, 1 day), or chronic cocaine treatment (15 mg/kg, 7 days) in wild type mice. *p<0.05 by t-test, n = 6.</p