Midkine in the mouse ventral tegmental area limits ethanol intake and Ccl2 gene expression.

Midkine (MDK) is a cytokine and neurotrophic factor that is more highly expressed in the brains of alcoholics and in mice predisposed to drink large amounts of ethanol, suggesting that MDK may regulate ethanol consumption. Here we measured ethanol consumption in male and female Mdk knockout (-/-) mice using the two-bottle choice and the drinking in the dark (DID) tests. We found that Mdk -/- mice consumed significantly more ethanol than wild-type controls in both tests. To determine if MDK acts in the ventral tegmental area (VTA) to regulate ethanol consumption, we delivered lentivirus expressing a Mdk shRNA into the VTA of male C57BL/6J mice to locally knockdown Mdk and performed the DID test. Mice expressing a Mdk shRNA in the VTA consumed more ethanol than mice expressing a control non-targeting shRNA, demonstrating that the VTA is one site in the brain through which MDK acts to regulate ethanol consumption. Since MDK also controls the expression of inflammatory cytokines in other organs, we examined gene expression of interleukin-1 beta (Il1b), tumor necrosis factor alpha (Tnfα) and the chemokine (C-C motif) ligand 2 (Ccl2) in the VTA of Mdk -/- mice and in mice expressing Mdk shRNA in the VTA. Expression of Ccl2 was elevated in the VTA of Mdk -/- mice and in mice expressing Mdk shRNA in the VTA. These results demonstrate that MDK functions in the VTA to limit ethanol consumption and levels of CCL2, a chemokine known to increase ethanol consumption.

Defining the role of corticotropin releasing factor binding protein in alcohol consumption.

The corticotropin releasing factor (CRF) exerts its effects by acting on its receptors and on the binding protein (CRFBP), and has been implicated in alcohol use disorder (AUD). Therefore, identification of the exact contribution of each protein that mediates CRF effects is necessary to design effective therapeutic strategies for AUD. A series of in vitro/in vivo experiments across different species were performed to define the biological discrete role of CRFBP in AUD. First, to establish the CRFBP role in receptor signaling, we developed a novel chimeric cell-based assay and showed that CFRBP full length can stably be expressed on the plasma membrane. We discovered that only CRFBP(10 kD) fragment is able to potentiate CRF-intracellular Ca2+ release. We provide evidence that CRHBP gene loss increased ethanol consumption in mice. Then, we demonstrate that selective reduction of CRHBP expression in the center nucleus of the amygdala (CeA) decreases ethanol consumption in ethanol-dependent rats. CRFBP amygdalar downregulation, however, does not attenuate yohimbine-induced ethanol self-administration. This effect was associated with decreased hemodynamic brain activity in the CRFBP-downregulated CeA and increased hemodynamic activity in the caudate putamen during yohimbine administration. Finally, in alcohol-dependent patients, genetic variants related to the CRFBP(10 kD) fragment were associated with greater risk for alcoholism and anxiety, while other genetic variants were associated with reduced risk for anxiety. Taken together, our data provide evidence that CRFBP may possess both inhibitory and excitatory roles and may represent a novel pharmacological target for the treatment of AUD.

Increased behavioral responses to ethanol in Lmo3 knockout mice.

LIM-domain-only 3 (LMO3) is a transcriptional regulator involved in central nervous system development and neuroblastoma. Our previous studies implicated a potential role for LMO3 in regulating ethanol sensitivity and consumption. Here, we examined behavioral responses to ethanol in a line of Lmo3 null (Lmo3(Z) ) mice, utilizing the ethanol-induced loss-of-righting-reflex (LORR) test, two-bottle choice ethanol consumption and the drinking in the dark (DID) test, which models binge-like ethanol consumption. We found that Lmo3(Z) mice exhibited increased sedation time in response to ethanol in the LORR test and drank significantly more ethanol in the DID test compared with their wild-type counterparts, but showed no differences in two-bottle choice ethanol consumption. To explore where LMO3 may be acting in the brain to produce an ethanol phenotype, we also examined reporter gene (ß-galactosidase) expression in heterozygous Lmo3(Z) mice and found strong expression in subcortical areas, particularly in those areas implicated in drug abuse, including the nucleus accumbens (Acb), cortex, hippocampus and amygdala. We also examined Lmo3 expression in the brains of wild-type mice who had undergone the DID test and found a negative correlation between Lmo3 expression in the Acb and the amount of ethanol consumed, consistent with the increased binge-like drinking observed in Lmo3(Z) mice. These results support a role for LMO3 in regulating behavioral responses to ethanol, potentially through its actions in the Acb.

Lmo4 in the nucleus accumbens regulates cocaine sensitivity.

An estimated 2 million Americans use cocaine, resulting in large personal and societal costs. Discovery of the genetic factors that contribute to cocaine abuse is important for understanding this complex disease. Previously, mutations in the Drosophila LIM-only (dLmo) gene were identified because of their increased behavioral sensitivity to cocaine. Here we show that the mammalian homolog Lmo4, which is highly expressed in brain regions implicated in drug addiction, plays a similar role in cocaine-induced behaviors. Mice with a global reduction in Lmo4 levels show increased sensitivity to the locomotor stimulatory effects of cocaine upon chronic cocaine administration. This effect is reproduced with downregulation of Lmo4 in the nucleus accumbens by RNA interference. Thus, Lmo genes play conserved roles in regulating the behavioral effects of cocaine in invertebrate and mammalian models of drug addiction.

Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior.

Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCepsilon) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCepsilon activator, psiepsilonRACK, increased levels of pro-CRF, whereas reducing PKCepsilon levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCepsilon by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCepsilon(-/-) mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCepsilon control over anxiety-like behavior through regulation of CRF in the amygdala.

Downregulation of mu opioid receptor by RNA interference in the ventral tegmental area reduces ethanol consumption in mice.

Pharmacological and genetic studies have implicated the mu opioid receptor (MOR) in the regulation of ethanol intake in animal models and humans. Non-specific antagonists of opioid receptors have been shown to affect ethanol consumption when infused directly into the ventral tegmental area (VTA) of rats. However, administration of MOR-selective antagonists into the VTA has yielded mixed results. We used RNA interference (RNAi) to specifically decrease levels of MOR messenger RNA in the VTA of mice and examined the effect on ethanol consumption in a two-bottle choice paradigm. Mice were injected in the VTA with lentivirus expressing either a small hairpin RNA (shRNA) targeting MOR or a control shRNA. One week after virus injection, mice were examined for ethanol consumption in a two-bottle choice experiment with increasing concentrations of ethanol over the course of 1 month. Expression of an shRNA targeting MOR in the VTA led to a significant reduction in ethanol consumption. These results strengthen the hypothesis that MOR in the VTA is one of the key brain substrates mediating alcohol consumption. The RNAi combined with lentiviral delivery can be used successfully in brain to effect a sustained reduction in expression of specific genes for behavioral analysis.