Caroline E. Bass

CaMKII: a biochemical bridge linking accumbens dopamine and glutamate systems in cocaine seeking

Increases in dopamine and glutamate transmission in the nucleus accumbens independently promote the reinstatement of cocaine seeking, an animal model of relapse. Here we have tested whether cocaine reinstatement in rats depends on interactions between accumbal dopamine and glutamate systems that are mediated by Ca2+/calmodulin-mediated kinase II (CaMKII). We show that stimulation of D1-like dopamine receptors in the nucleus accumbens shell reinstates cocaine seeking by activating L-type Ca2+ channels and CaMKII. Cocaine reinstatement is associated with D1-like dopamine receptor–dependent increases in accumbens shell CaMKII phosphorylated on Thr286 and glutamate receptor 1 (GluR1) phosphorylated on Ser831 (a known CaMKII phosphorylation site), in addition to increases in cell-surface expression of GluR1-containing AMPA receptors in the shell. Consistent with these findings, cocaine reinstatement is attenuated by intra-shell administration of AAV10-GluR1-C99, a vector that impairs the transport of GluR1-containing AMPA receptors. Thus, CaMKII may be an essential link between accumbens shell dopamine and glutamate systems involved in the neuronal plasticity underlying cocaine craving and relapse.

EP3 prostaglandin receptors in the median preoptic nucleus are critical for fever responses

Fever is a result of the action of prostaglandin E2 (PGE2) on the brain and appears to require EP3 prostaglandin receptors (EP3Rs), but the specific neurons on which PGE2 acts to produce fever have not been definitively established. Here we report that selective genetic deletion of the EP3Rs in the median preoptic nucleus of mice resulted in abrogation of the fever response. These observations demonstrate that the EP3R-bearing neurons in the median preoptic nucleus are required for fever responses.

Arousal Effect of Caffeine Depends on Adenosine A2A Receptors in the Shell of the Nucleus Accumbens

Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A2A receptors (A2ARs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A2ARs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A2ARs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A2ARs were focally removed from the NAc core or other A2AR-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

Targeting cells with single vectors using multiple-feature Boolean logic

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

Cocaine-Induced Chromatin Remodeling Increases Brain-Derived Neurotrophic Factor Transcription in the Rat Medial Prefrontal Cortex, Which Alters the Reinforcing Efficacy of Cocaine

Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.

Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer

Cannabinoids have been reported to possess antitumorogenic activity. Not much is known, however, about the effects and mechanism of action of synthetic nonpsychotic cannabinoids on breast cancer growth and metastasis. We have shown that the cannabinoid receptors CB1 and CB2 are overexpressed in primary human breast tumors compared with normal breast tissue. We have also observed that the breast cancer cell lines MDA-MB231, MDA-MB231-luc, and MDA-MB468 express CB1 and CB2 receptors. Furthermore, we have shown that the CB2 synthetic agonist JWH-133 and the CB1 and CB2 agonist WIN-55,212-2 inhibit cell proliferation and migration under in vitro conditions. These results were confirmed in vivo in various mouse model systems. Mice treated with JWH-133 or WIN-55,212-2 showed a 40% to 50% reduction in tumor growth and a 65% to 80% reduction in lung metastasis. These effects were reversed by CB1 and CB2 antagonists AM 251 and SR144528, respectively, suggesting involvement of CB1 and CB2 receptors. In addition, the CB2 agonist JWH-133 was shown to delay and reduce mammary gland tumors in the polyoma middle T oncoprotein (PyMT) transgenic mouse model system. Upon further elucidation, we observed that JWH-133 and WIN-55,212-2 mediate the breast tumor-suppressive effects via a coordinated regulation of cyclooxygenase-2/prostaglandin E2 signaling pathways and induction of apoptosis. These results indicate that CB1 and CB2 receptors could be used to develop novel therapeutic strategies against breast cancer growth and metastasis. [Mol Cancer Ther 2009;8(11):3117–29]

The GABAergic parafacial zone is a medullary slow wave sleep–promoting center

Work in animals and humans has suggested the existence of a slow wave sleep (SWS)-promoting/electroencephalogram (EEG)-synchronizing center in the mammalian lower brainstem. Although sleep-active GABAergic neurons in the medullary parafacial zone (PZ) are needed for normal SWS, it remains unclear whether these neurons can initiate and maintain SWS or EEG slow-wave activity (SWA) in behaving mice. We used genetically targeted activation and optogenetically based mapping to examine the downstream circuitry engaged by SWS-promoting PZ neurons, and we found that this circuit uniquely and potently initiated SWS and EEG SWA, regardless of the time of day. PZ neurons monosynaptically innervated and released synaptic GABA onto parabrachial neurons, which in turn projected to and released synaptic glutamate onto cortically projecting neurons of the magnocellular basal forebrain; thus, there is a circuit substrate through which GABAergic PZ neurons can potently trigger SWS and modulate the cortical EEG.

Environmental Enrichment Produces a Behavioral Phenotype Mediated by Low Cyclic Adenosine Monophosphate Response Element Binding (CREB) Activity in the Nucleus Accumbens

BACKGROUND Previous research has shown that rats reared in an enriched condition (EC) are more sensitive to the acute effects of amphetamine than rats reared in an isolated condition (IC); yet, EC rats self-administer less amphetamine than IC rats. The present study used cocaine to further explore this environmental enrichment behavioral phenotype, as well as the underlying molecular mechanisms involved. METHODS Enriched condition and IC rats were studied in a broad battery of behavioral tests, including cocaine conditioned place preference (CPP) and self-administration and several measures of anxiety- and depression-related behavior. The involvement of the transcription factor, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), in mediating EC versus IC differences was investigated. RESULTS Enriched condition rats exhibited less cocaine self-administration, despite showing enhanced cocaine CPP. Enriched condition rats also displayed less depression-like behavior but higher levels of anxiety-like behavior. This behavioral phenotype is consistent with low CREB activity in the nucleus accumbens, a key brain reward region. Indeed, EC rats have less phospho-CREB (the transcriptionally active form of the protein) in the nucleus accumbens than IC rats, and a selective knockdown of CREB in this brain region of normally reared rats, by use of a novel viral vector expressing a short hairpin RNA (shRNA) directed against CREB, reproduced the EC behavioral phenotype. CONCLUSIONS These studies identify a potential molecular mechanism for how rearing environment-a nonpharmacological, nonsurgical manipulation-can modify a wide range of complex emotional behaviors.

Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging

Recent studies demonstrate that age-related dysfunction of NF-E2-related factor-2 (Nrf2)-driven pathways impairs cellular redox homeostasis, exacerbating age-related cellular oxidative stress and increasing sensitivity of aged vessels to oxidative stress-induced cellular damage. Circulating levels of insulin-like growth factor (IGF)-1 decline during aging, which significantly increases the risk for cardiovascular diseases in humans. To test the hypothesis that adult-onset IGF-1 deficiency impairs Nrf2-driven pathways in the vasculature, we utilized a novel mouse model with a liver-specific adeno-associated viral knockdown of the Igf1 gene using Cre-lox technology (Igf1(f/f) + MUP-iCre-AAV8), which exhibits a significant decrease in circulating IGF-1 levels (~50%). In the aortas of IGF-1-deficient mice, there was a trend for decreased expression of Nrf2 and the Nrf2 target genes GCLC, NQO1 and HMOX1. In cultured aorta segments of IGF-1-deficient mice treated with oxidative stressors (high glucose, oxidized low-density lipoprotein, and H(2)O(2)), induction of Nrf2-driven genes was significantly attenuated as compared with control vessels, which was associated with an exacerbation of endothelial dysfunction, increased oxidative stress, and apoptosis, mimicking the aging phenotype. In conclusion, endocrine IGF-1 deficiency is associated with dysregulation of Nrf2-dependent antioxidant responses in the vasculature, which likely promotes an adverse vascular phenotype under pathophysiological conditions associated with oxidative stress (eg, diabetes mellitus, hypertension) and results in accelerated vascular impairments in aging.

Time course for the induction and maintenance of tolerance to Δ9-tetrahydrocannabinol in mice

The time course for the development of tolerance to delta-9-tetrahydrocannabinol (Δ9-THC) was studied in an effort to determine the role that length of dosing may have in the onset and maintenance of tolerance. Mice were chronically treated with either vehicle or 10 mg/kg of Δ9-THC subcutaneously twice a day. The mice were tested 24 h after the last injection for tolerance as assessed by the production of antinociception and suppression of spontaneous activity. Tolerance was first observed after three injections of Δ9-THC (1.5 days) resulting in a 7-fold and 23-fold decrease in potency for the measures of antinociception and hypoactivity, respectively. Seven injections of Δ9-THC (3.5 days of dosing) resulted in a 12-fold and 36-fold decrease in potency, respectively, while 13 injections of Δ9-THC (6.5 days of dosing) produced a 6.2-fold and 9.8-fold degree of tolerance. The time course for the recovery from Δ9-THC-induced tolerance was also determined with a separate group of animals. Mice were dosed for 6.5 days with 10 mg/kg of Δ9-THC and were not tested until 4.5, 7.5, and 11.5 days after cessation of drug treatment. After 4.5 days without drug treatment the mice exhibited a 7.5-fold and 2.3-fold degree of tolerance as measured by antinociception and hypoactivity, respectively. After 7.5 days without drug treatment a 3.4-fold degree of tolerance remained for the measure of antinociception, while no tolerance was detected for the measure of hypoactivity. No tolerance was observed for the measure of antinociception after 11.5 days without drug treatment. This time course indicates that the mechanisms responsible for either the production or maintenance of tolerance differ between the measures of antinociception and suppression of spontaneous activity.