Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer’s disease (AD). A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14–15 month) endothelial nitric oxide synthase deficient (eNOS−/−) mice. APP, β-site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS−/− mice as compared to LMA wild type controls. APP and Aβ1-40 were increased in hippocampal tissue of eNOS−/− mice as compared to wild type mice. LMA eNOS−/− mice displayed an increased inflammatory phenotype as compared to LMA wild type mice. Importantly, LMA eNOS−/− mice performed worse in a radial arm maze test of spatial learning and memory as compared to LMA wild type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline.

Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. Here, we identified that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1−/−). Utilizing mice expressing β-galactosidase (lacZ) under the control of seven-repeated CRE sites in both genotypes (CRE-lacZ/ENT1+/+ mice and CRE-lacZ/ENT1−/− mice) as well as dnCREB (dominant negative form of CREB), we found that reduced CREB activity in the DMS is causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we demonstrated that A2AR antagonist (ZM241385) dampened PKA-activity mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1+/+ mice, but not in ENT1−/− mice. Taken together, our studies indicate that A2AR-mediated CREB signaling in the DMS is a key determinant to enhance the development of goal-directed ethanol drinking in mice.

Alcohol-sensitive type 1 equilibrative nucleoside transporter (ENT1) regulates adenosine-mediated glutamate neurotransmission in the brain. Our behavioral studies suggest that the diminished aversive effects of ethanol and the increased resistance to acute ethanol intoxication in mice lacking ENT1, could be related to increased voluntary ethanol self-seeking behavior. In addition, we found that ENT1 null mice were resistant to the ataxic effects of glutamate antagonists when tested on a rotarod. Using microdialysis experiments, we examined glutamate levels in the dorsal and ventral striatum in response to ethanol. In the dorsal striatum of ENT1 null mice, a low intoxicating dose of ethanol (1.5 g/kg) induced a greater increase of glutamate levels, while a higher hypnotic dose of ethanol (3.0 g/kg) decreased to a lesser degree the glutamate levels, compared with that of wild-type mice. In the ventral striatum, however, the low (1.5 g/kg) and the high (3.0 g/kg) ethanol doses altered glutamate levels similarly in both genotypes. Our results suggest that adenosine-regulated glutamatergic signaling contributes to a reduced level of alcohol response, which might be associated with a higher susceptibility for alcoholism in humans.

Background Mice lacking type 1 equilibrative nucleoside transporter (ENT1−/−) exhibit increased ethanol-preferring behavior compared to wild-type littermates. This phenotype of ENT1−/− mice appears to be correlated with increased glutamate levels in the nucleus accumbens (NAc). However, little is known about the downstream consequences of increased glutamate signaling in the NAc. Methods To investigate the significance of the deletion of ENT1 and its effect on glutamate signaling in the NAc, we employed microdialysis and iTRAQ proteomics. We validated altered proteins using Western blot analysis. We then examined the pharmacological effects of the inhibition of the N-Methyl-D-Aspartate (NMDA) glutamate receptor and protein kinase Cγ (PKCγ) on alcohol drinking in wild-type mice. In addition, we investigated in vivo cAMP response element binding (CREB) activity using CRE-lacZ mice in an ENT1−/− background. Results We identified that NMDA glutamate receptor-mediated down-regulation of intracellular PKCγ-neurogranin (Ng)-Ca2+-calmodulin dependent protein kinase type II (CaMKII) signaling is correlated with reduced CREB activity in ENT1−/− mice. Inhibition of PKCγ promotes ethanol drinking in wild-type mice to levels similar to those of ENT1−/− mice. In contrast, an NMDA glutamate receptor antagonist reduces ethanol drinking of ENT1−/− mice. Conclusion These findings demonstrate that the genetic deletion or pharmacological inhibition of ENT1 regulates NMDA glutamate receptor-mediated signaling in the NAc which provides a molecular basis that underlies the ethanol-preferring behavior of ENT1−/− mice.