Sun Choi

Altered glutamatergic neurotransmission in the striatum regulates ethanol sensitivity and intake in mice lacking ENT1

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.

Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking Through Accumbal N-Methyl-D-Aspartate Receptor Signaling

BACKGROUND Mice lacking type 1 equilibrative nucleoside transporter (ENT1(-/-)) exhibit increased ethanol-preferring behavior compared with 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 cyclic adenosine monophosphate response element binding activity using cyclic adenosine monophosphate response element-β-galactosidase mice in an ENT1(-/-) background. RESULTS We identified that NMDA glutamate receptor-mediated downregulation of intracellular PKCγ-neurogranin-calcium-calmodulin dependent protein kinase type II signaling is correlated with reduced cyclic adenosine monophosphate response element binding 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. CONCLUSIONS 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.

ENT1 Regulates Ethanol-Sensitive EAAT2 Expression and Function in Astrocytes

BACKGROUND Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum. METHODS We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real-time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol-induced EAAT2 expression. RESULTS An ENT1-specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up-regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol-induced EAAT2 up-regulation. CONCLUSIONS Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.

Striatal Adenosine Signaling Regulates EAAT2 and Astrocytic AQP4 Expression and Alcohol Drinking in Mice

Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.

Attenuation of Ethanol Withdrawal by Ceftriaxone-Induced Upregulation of Glutamate Transporter EAAT2

Alcohol withdrawal syndrome (AWS) is a potentially fatal outcome of severe alcohol dependence that presents a significant challenge to treatment. Although AWS is thought to be driven by a hyperglutamatergic brain state, benzodiazepines, which target the GABAergic system, comprise the first line of treatment for AWS. Using a rat model of ethanol withdrawal, we tested whether ceftriaxone, a β-lactam antibiotic known to increase the expression and activity of glutamate uptake transporter EAAT2, reduces the occurrence or severity of ethanol withdrawal manifestations. After a 2-week period of habituation to ethanol in two-bottle choice, alcohol-preferring (P) and Wistar rats received ethanol (4.0 g/kg) every 6 h for 3–5 consecutive days via gavage. Rats were then deprived of ethanol for 48 h during which time they received ceftriaxone (50 or 100 mg/kg, IP) or saline twice a day starting 12 h after the last ethanol administration. Withdrawal manifestations were captured by continuous video recording and coded. The evolution of ethanol withdrawal was markedly different for P rats vs Wistar rats, with withdrawal manifestations occurring >12 h later in P rats than in Wistar rats. Ceftriaxone 100 mg/kg per injection twice per day (200 mg/kg/day) reduced or abolished all manifestations of ethanol withdrawal in both rat variants and prevented withdrawal-induced escalation of alcohol intake. Finally, ceftriaxone treatment was associated with lasting upregulation of ethanol withdrawal-induced downregulation of EAAT2 in the striatum. Our data support the role of ceftriaxone in alleviating alcohol withdrawal and open a novel pharmacologic avenue that requires clinical evaluation in patients with AWS.

Adenosine A2A receptor and ERK-driven impulsivity potentiates hippocampal neuroblast proliferation

Dampened adenosine A2A receptor (A2AR) function has been implicated in addiction through enhancement of goal-directed behaviors. However, the contribution of the A2AR to the control of impulsive reward seeking remains unknown. Using mice that were exposed to differential reward of low rate (DRL) schedules during Pavlovian-conditioning, second-order schedule discrimination, and the 5-choice serial reaction time task (5-CSRTT), we demonstrate that deficits of A2AR function promote impulsive responses. Antagonism of the A2AR lowered ERK1 and ERK2 phosphorylation in the dorsal hippocampus (dHip) and potentiated impulsivity during Pavlovian-conditioning and the 5-CSRTT. Remarkably, inhibition of ERK1 and ERK2 phosphorylation by U0126 in the dHip prior to Pavlovian-conditioning exacerbated impulsive reward seeking. Moreover, we found decreased A2AR expression, and reduced ERK1 and ERK2 phosphorylation in the dHip of equilibrative nucleoside transporter type 1 (ENT1–/–) null mice, which displayed exacerbated impulsivity. To determine whether impulsive response behavior is associated with hippocampal neuroblast development, we investigated expression of BrdU+ and doublecortin (DCX+) following 5-CSRTT testing. These studies revealed that impulsive behavior driven by inhibition of the A2AR is accompanied by increased neuroblast proliferation in the hippocampus.

Antipsychotic-like effects of a neurotensin receptor type 1 agonist.

Although neurotensin (NT) analogs are known to produce antipsychotic-like effects, the therapeutic possibility of a brain penetrant NTS1 agonist in treating psychiatric disorders has not been well studied. Here, we examined whether PD149163, a brain-penetrant NTS1-specific agonist, displays antipsychotic-like effects in C57BL/6J mice by investigating the effect of PD149163 on amphetamine-mediated hyperactivity and amphetamine-induced disruption of prepulse inhibition. In addition, we assessed the effect of PD149163 on glycogen synthase kinase-3 (GSK-3) activity, a downstream molecular target of antipsychotics and mood stabilizers, using phospho-specific antibodies. PD149163 (0.1 and 0.5mg/kg) inhibited amphetamine-induced hyperactivity in mice, indicating that NTS1 activation inhibits psychomotor agitation. PD149163 (0.5mg/kg) also increased prepulse inhibition, suggesting that NTS1 activation reduces prepulse inhibition deficits which often co-occur with psychosis in humans. Interestingly, PD149163 increased the inhibitory serine phosphorylation on both GSK-3α and GSK-3β in a dose- and time-dependent manner in the nucleus accumbens and medial prefrontal cortex of the mice. Moreover, PD149163 inhibited GSK-3 activity in the nucleus accumbens and medial prefrontal cortex in the presence of amphetamine. Thus, like most current antipsychotics and mood stabilizers, PD149163 inhibited GSK-3 activity in cortico-striatal circuitry. Together, our findings indicate that PD149163 may be a novel antipsychotic.

Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter

The neural circuit of the dorsal hippocampus (dHip) and nucleus accumbens (NAc) contributes to cue-induced learning and addictive behaviors, as demonstrated by the escalation of ethanol-seeking behaviors observed following deletion of the adenosine equilibrative nucleoside transporter 1 (ENT1-/-) in mice. Here we perform quantitative LC-MS/MS neuroproteomics in the dHip and NAc of ENT1-/- mice. Using Ingenuity Pathway Analysis, we identified proteins associated with increased long-term potentiation, ARP2/3-mediated actin cytoskeleton signaling and protein expression patterns suggesting deficits in glutamate degradation, GABAergic signaling, as well as significant changes in bioenergetics and energy homeostasis (oxidative phosphorylation, TCA cycle, and glycolysis). These pathways are consistent with previously reported behavioral and biochemical phenotypes that typify mice lacking ENT1. Moreover, we validated decreased expression of the SNARE complex protein VAMP1 (synaptobrevin-1) in the dHip as well as decreased expression of pro-dynorphin (PDYN), neuroendocrine convertase (PCSK1), and Leu-Enkephalin (dynorphin-A) in the NAc. Taken together, our proteomic approach provides novel pathways indicating that ENT1-regulated signaling is essential for neurotransmitter release and neuropeptide processing, both of which underlie learning and reward-seeking behaviors.

Regulation of hippocampal neural stem/progenitor cell proliferation by adenosine via equilibrative nucleoside transporter 1

O2-4-2-3 Nemo-like kinase promotes neurogenesis by interfering with formation of Notch transcription complex Tohru Ishitani 1 , Tomoko Hirao 2, Maho Suzuki 3, Miho Isoda 2, Shizuka Ishitani 1, Motoo Kitagawa 4, Kunihiro Matsumoto 3, Motoyuki Itoh 2 1 Div. of Cell Reg. Sys., M.I.B., Kyushu University, Fukuoka, Japan 2 Inst. for Advanced Research, Nagoya University, Nagoya, Japan 3 Group of Signal Transduction, Laboratory of Cell Regulation, Div. of Biol. Sci., Grad. Sch. of Sci., Nagoya University, Naguya, Japan 4 Dep. of Mol. and Tumor Path., Chiba University Grad. Sch. of Med., Chiba, Japan