Adenylyl Cyclase 1 Is Required for Ethanol-Induced Locomotor Sensitization and Associated Increases in NMDA Receptor Phosphorylation and Function in the Dorsal Medial Striatum.

Neuroadaptive responses to chronic ethanol, such as behavioral sensitization, are associated with N-methyl-D-aspartate receptor (NMDAR) recruitment. Ethanol enhances GluN2B-containing NMDAR function and phosphorylation (Tyr-1472) of the GluN2B-NMDAR subunit in the dorsal medial striatum (DMS) through a protein kinase A (PKA)-dependent pathway. Ethanol-induced phosphorylation of PKA substrates is partially mediated by calcium-stimulated adenylyl cyclase 1 (AC1), which is enriched in the dorsal striatum. As such, AC1 is poised as an upstream modulator of ethanol-induced DMS neuroadaptations that promote drug responding, and thus represents a therapeutic target. Our hypothesis is that loss of AC1 activity will prevent ethanol-induced locomotor sensitization and associated DMS GluN2B-NMDAR adaptations. We evaluated AC1's contribution to ethanol-evoked locomotor responses and DMS GluN2B-NMDAR phosphorylation and function using AC1 knockout (AC1KO) mice. Results were mechanistically validated with the AC1 inhibitor, NB001. Acute ethanol (2.0 g/kg) locomotor responses in AC1KO and wild-type (WT) mice pretreated with NB001 (10 mg/kg) were comparable to WT ethanol controls. However, repeated ethanol treatment (10 days, 2.5 g/kg) failed to produce sensitization in AC1KO or NB001 pretreated mice, as observed in WT ethanol controls, following challenge exposure (2.0 g/kg). Repeated exposure to ethanol in the sensitization procedure significantly increased pTyr-1472 GluN2B levels and GluN2B-containing NMDAR transmission in the DMS of WT mice. Loss of AC1 signaling impaired ethanol-induced increases in DMS pGluN2B levels and NMDAR-mediated transmission. Together, these data support a critical and specific role for AC1 in striatal signaling that mediates ethanol-induced behavioral sensitization, and identify GluN2B-containing NMDARs as an important AC1 target.

Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8-deficient mice.

The cAMP/protein kinase A pathway regulates methamphetamine (METH)-induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild-type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH-induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)-evoked accumbal dialysate dopamine levels were similar between genotypes, saline-treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH-sensitized DKO mice compared to saline-treated controls, an effect not observed in WT mice. Notably, saline-treated DKO mice had significantly increased phosphorylated Dopamine- and cAMP-regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug-induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin-stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine-induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP-32 activation. These findings indicate AC 1/8 signaling regulates the sensitivity of the nigrostriatal pathway subserving stimulant and neuroadaptive sensitizing effects of methamphetamine. 3-MT, 3-methoxytyramine; Ca(2+), calcium; CaM, calmodulin; cdk5; cyclin-dependent kinase 5; DA, dopamine; DARPP-32, dopamine- and cAMP-regulated phosphoprotein; D1R, dopamine D1 receptor; HVA, homovanillic acid; PKA, protein kinase A.

Cocaine-induced locomotor sensitization in rats correlates with nucleus accumbens activity on manganese-enhanced MRI.

A long-standing goal of substance abuse research has been to link drug-induced behavioral outcomes with the activity of specific brain regions to understand the neurobiology of addiction behaviors and to search for drug-able targets. Here, we tested the hypothesis that cocaine produces locomotor (behavioral) sensitization that correlates with increased calcium channel-mediated neuroactivity in brain regions linked with drug addiction, such as the nucleus accumbens (NAC), anterior striatum (AST) and hippocampus, as measured using manganese-enhanced MRI (MEMRI). Rats were treated with cocaine for 5 days, followed by a 2-day drug-free period. The following day, locomotor sensitization was quantified as a metric of cocaine-induced neuroplasticity in the presence of manganese. Immediately following behavioral testing, rats were examined for changes in calcium channel-mediated neuronal activity in the NAC, AST, hippocampus and temporalis muscle, which was associated with behavioral sensitization using MEMRI. Cocaine significantly increased locomotor activity and produced behavioral sensitization compared with saline treatment of control rats. A significant increase in MEMRI signal intensity was determined in the NAC, but not AST or hippocampus, of cocaine-treated rats compared with saline-treated control rats. Cocaine did not increase signal intensity in the temporalis muscle. Notably, in support of our hypothesis, behavior was significantly and positively correlated with MEMRI signal intensity in the NAC. As neuronal uptake of manganese is regulated by calcium channels, these results indicate that MEMRI is a powerful research tool to study neuronal activity in freely behaving animals and to guide new calcium channel-based therapies for the treatment of cocaine abuse and dependence.

Aberrant striatal dopamine transmitter dynamics in brain-derived neurotrophic factor-deficient mice.

Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems, including forebrain dopamine-containing neurons. Recent evidence shows a strong correlation between neuropsychiatric disorders and BDNF hypofunction. The aim of the present study was to characterize the effect of low endogenous levels of BDNF on dopamine system function in the caudate-putamen using heterozygous BDNF (BDNF(+/-) ) mice. Apparent extracellular dopamine levels in the caudate-putamen, determined by quantitative microdialysis, were significantly elevated in BDNF(+/-) mice compared with wildtype controls (12 vs. 5 nM, respectively). BDNF(+/-) mice also had a potentiated increase in dopamine levels following potassium (120 mM)-stimulation (10-fold) relative to wildtype controls (6-fold). Slice fast-scan cyclic voltammetry revealed that BDNF(+/-) mice had reductions in both electrically evoked dopamine release and dopamine uptake rates in the caudate-putamen. Superfusion of BDNF led to partial recovery of the electrically stimulated dopamine release response in BDNF(+/-) mice. Conversely, tissue accumulation of L-3,4-dihydroxyphenylalanine, extracellular levels of dopamine metabolites, and spontaneous locomotor activity were unaltered. Together, this study indicates that endogenous BDNF influences dopamine system homeostasis by regulating the release and uptake dynamics of pre-synaptic dopamine transmission.

Age-related murine hippocampal CA1 laminae oxidative stress measured in vivo by QUEnch-assiSTed (QUEST) MRI: impact of isoflurane anesthesia.

Age-related impairments in spatial learning and memory often precede non-familial neurodegenerative disease. Ex vivo studies suggest that physiologic age-related oxidative stress in hippocampus area CA1 may contribute to prodromal spatial disorientation and to morbidity. Yet, conventional blood or cerebrospinal fluid assays appear insufficient for early detection or management of oxidative stress within CA1 sub-regions in vivo. Here, we address this biomarker problem using a non-invasive MRI index of CA1 laminae oxidative stress based on reduction in R1 (= 1/T1) after anti-oxidant administration. An R1 reduction reflects quenching of continuous and excessive production of endogenous paramagnetic free radicals. Careful motion-correction image acquisition, and avoiding repeated exposure to isoflurane, facilitates detection of hippocampus CA1 laminae oxidative stress with QUEnch-assiSTed (QUEST) MRI. Intriguingly, age- and isoflurane-related oxidative stress is localized to the stratum lacunosum of the CA1 region. Our data raise the possibility of using QUEST MRI and FDA-approved anti-oxidants to remediate spatial disorientation and later neurodegeneration with age in animals and humans.

Calcium/calmodulin-stimulated adenylyl cyclases 1 and 8 regulate reward-related brain activity and ethanol consumption.

Evidence suggests a predictive link between elevated basal activity within reward-related networks (e.g., cortico-basal ganglia-thalamic networks) and vulnerability for alcoholism. Both calcium channel function and cyclic adenosine monophosphate (cAMP)/protein kinase A-mediated signaling are critical modulators of reward neurocircuitry and reward-related behaviors. Calcium/calmodulin-stimulated adenylyl cyclases (AC) 1 and 8 are sensitive to activity-dependent increases in intracellular calcium and catalyze cAMP production. Therefore, we hypothesized AC1 and 8 regulate brain activity in reward regions of the cortico-basal ganglia-thalamic circuit and that this regulatory influence predicts voluntary ethanol drinking responses. This hypothesis was evaluated by manganese-enhanced magnetic resonance imaging and chronic, intermittent ethanol access procedures. Ethanol-naïve mice with genetic deletion of both AC1 and 8 (DKO mice) exhibited bilateral reductions in baseline activity within cortico-basal ganglia-thalamic regions associated with reward processing compared to wild-type controls (WT, C57BL/6 mice). Significant activity changes were not evident in regions either outside of the cortico-basal ganglia-thalamic network or within the network that are not associated with reward processing. Parallel studies demonstrated that reward network hypoactivity in DKO mice predicted a significant attenuation in consumption and preference levels to escalating ethanol concentrations (12, 20 and 30%) compared to WT mice, an effect that was maintained over extended access (14 sessions) to 20% ethanol. Summarizing, these data support a contribution of AC1 and 8 in cortico-basal ganglia-thalamic activity and the predictive value of this regulatory influence on ethanol drinking behavior, which merits the future evaluation of calcium-stimulated ACs in the neural processes that engender vulnerability to maladaptive alcohol drinking.

Single-Prolonged Stress Impairs Prefrontal Cortex Control of Amygdala and Striatum in Rats.

Medial prefrontal cortex (mPFC), amygdala, and striatum neurocircuitry has been shown to play an important role in post-traumatic stress disorder (PTSD) pathology in humans. Clinical studies show hypoactivity in the mPFC and hyperactivity in the amygdala and striatum of PTSD patients, which has been associated with decreased mPFC glutamate levels. The ability to refine neurobiological characteristics of PTSD in an animal model is critical in furthering our mechanistic understanding of the disease. To this end, we exposed male rats to single-prolonged stress (SPS), a validated model of PTSD, and hypothesized that traumatic stress would differentially activate mPFC subregions [prelimbic (PL) and infralimbic (IL) cortices] and increase striatal and amygdalar activity, which would be associated with decreased mPFC glutamate levels. in vivo, neural activity in the subregions of the mPFC, amygdala, and striatum was measured using manganese-enhanced magnetic resonance imaging (MEMRI), and glutamate and N-acetylaspartate (NAA) levels in the mPFC and the dorsal striatum (dSTR) were measured using proton magnetic resonance spectroscopy (1H-MRS) longitudinally, in rats exposed to SPS or control conditions. As hypothesized, SPS decreased MEMRI-based neural activity in the IL, but not PL, cortex concomitantly increasing activity within the basolateral amygdala (BLA) and dorsomedial striatum (dmSTR). 1H-MRS studies in a separate cohort revealed SPS decreased glutamate levels in the mPFC and increased NAA levels in the dSTR. These results confirm previous findings that suggest SPS causes mPFC hypoactivation as well as identifies concurrent hyperactivation in dmSTR and BLA, effects which parallel the clinical neuropathology of PTSD.

The selective delta opioid agonist SNC80 enhances amphetamine-mediated efflux of dopamine from rat striatum.

The highly selective delta opioid agonist, SNC80, elicits dopamine-related behaviors including locomotor stimulation and conditioned place-preference. In contrast, it has been reported that SNC80 fails to promote dopamine efflux from the striatum of freely moving rats. However, SNC80 does enhance behavioral responses to the stimulants, amphetamine and cocaine, suggesting an interaction between delta opioids and psychostimulants. Since the increase in locomotor activity elicited by amphetamine and related stimulants acting at the dopamine transporter is associated with increases in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances this activity by potentiating the overflow of dopamine through the transporter. To test this hypothesis, striatal preparations from Sprague Dawley rats were assayed for dopamine efflux in response to amphetamine challenge. SNC80 was given either in vivo or in vitro directly to rat striatal tissue, prior to in vitro amphetamine challenge. Both in vivo and in vitro administration of SNC80 enhanced amphetamine-mediated dopamine efflux in a concentration- and time-dependent manner. However, SNC80 in either treatment paradigm produced no stimulation of dopamine efflux in the absence of amphetamine. The effect of SNC80 on amphetamine-mediated dopamine overflow, but not the effect of amphetamine alone, was blocked by the delta selective antagonist, naltrindole and was also observed with other delta agonists. The results of this study demonstrate that even though SNC80 does not stimulate dopamine efflux alone, it is able to augment amphetamine-mediated dopamine efflux through a delta opioid receptor mediated action locally in the striatum.

Synergistic activity between the delta-opioid agonist SNC80 and amphetamine occurs via a glutamatergic NMDA-receptor dependent mechanism.

Glutamate is known to cause the release of dopamine through a Ca(2+)-sensitive mechanism that involves activation of NMDA ionotropic glutamate receptors. In the current study, we tested the hypothesis that the delta opioid agonist SNC80 acts indirectly, via the glutamatergic system, to enhance both amphetamine-stimulated dopamine efflux from striatal preparations and amphetamine-stimulated locomotor activity. SNC80 increased extracellular glutamate content, which was accompanied by a concurrent decrease in GABA levels. Inhibition of NMDA signaling with the selective antagonist MK801 blocked the enhancement of both amphetamine-induced dopamine efflux and hyperlocomotion observed with SNC80 pretreatment. Addition of exogenous glutamate also potentiated amphetamine-stimulated dopamine efflux in a Mg(2+)- and MK801-sensitive manner. After removal of Mg(2+) to relieve the ion conductance inhibition of NMDA receptors, SNC80 both elicited dopamine release alone and produced a greater enhancement of amphetamine-evoked dopamine efflux. The action of SNC80 to enhance amphetamine-evoked dopamine efflux was mimicked by the GABA(B) antagonist 2-hydroxysaclofen. These cumulative findings suggest SNC80 modulates amphetamine-stimulated dopamine efflux through an intra-striatal mechanism involving inhibition of GABA transmission leading to the local release of glutamate followed by subsequent activation of NMDA receptors.

Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure.

Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the maturational parameters examined in the current study may not be sensitive enough to detect effects of a single ethanol exposure during the brain growth spurt period. Genetic deletion of AC1/8 reveals a role for these cylases in attenuating ethanol-induced behavioral effects in the neonatally-exposed adolescent.

Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice.

Drugs of abuse like ethanol have the ability to stimulate forebrain dopaminergic pathways. Although the positive reinforcing properties of abused substances are largely attributed to their effects on dopamine transmission, alcohol addiction involves complex interactions between numerous molecular mediators. Brain-derived neurotrophic factor (BDNF) is suggested to have a protective role in regulating the reinforcing effects of ethanol. In the present study, we evaluated the effects of an acute, systemic injection of ethanol (2 g/kg) on BDNF protein levels and extracellular dopamine concentrations, measured by in vivo microdialysis, in the caudate-putamen of wildtype and heterozygous BDNF mice. In both genotypes, the peak increase in extracellular dopamine following ethanol coincided temporally with a decrease in BDNF protein levels following a similar ethanol treatment. Moreover, the effect of ethanol to increase extracellular dopamine was blunted in heterozygous BDNF mice compared to wildtype mice. While the magnitude of decrease in BDNF protein induced by ethanol was similar between genotypes (two-fold), ethanol treatment induced significantly lower BDNF protein levels in heterozygous BDNF mice overall. These findings suggest the effects of ethanol are influenced by an interaction between BDNF and dopamine transmission, which may relate to the pathway through which BDNF regulates ethanol intake.