Traumatic Stress, Chronic Ethanol Exposure, or the Combination, Alter Cannabinoid System Components in Reward and Limbic Regions of the Mouse Brain.

The cannabinoid system is independently affected by stress and chronic ethanol exposure. However, the extent to which co-occurrence of traumatic stress and chronic ethanol exposure modulates the cannabinoid system remains unclear. We examined levels of cannabinoid system components, anandamide, 2-arachidonoylglycerol, fatty acid amide hydrolase, and monoacylglycerol lipase after mouse single-prolonged stress (mSPS) or non-mSPS (Control) exposure, with chronic intermittent ethanol (CIE) vapor or without CIE vapor (Air) across several brain regions using ultra-high-performance liquid chromatography tandem mass spectrometry or immunoblotting. Compared to mSPS-Air mice, anandamide and 2-arachidonoylglycerol levels in the anterior striatum were increased in mSPS-CIE mice. In the dorsal hippocampus, anandamide content was increased in Control-CIE mice compared to Control-Air, mSPS-Air, or mSPS-CIE mice. Finally, amygdalar anandamide content was increased in Control-CIE mice compared to Control-Air, or mSPS-CIE mice, but the anandamide content was decreased in mSPS-CIE compared to mSPS-Air mice. Based on these data we conclude that the effects of combined traumatic stress and chronic ethanol exposure on the cannabinoid system in reward pathway regions are driven by CIE exposure and that traumatic stress affects the cannabinoid components in limbic regions, warranting future investigation of neurotherapeutic treatment to attenuate these effects.

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.

Postnatal ethanol exposure simplifies the dendritic morphology of medium spiny neurons independently of adenylyl cyclase 1 and 8 activity in mice.

BACKGROUND: Fetal exposure to alcohol can have multiple deleterious effects, including learning disorders and behavioral and executive functioning abnormalities, collectively termed fetal alcohol spectrum disorders. Neonatal mice lacking both calcium-/calmodulin-stimulated adenylyl cyclases (ACs) 1 and 8 demonstrate increased vulnerability to ethanol (EtOH)-induced neurotoxicity in the striatum compared with wild-type (WT) controls. However, the developmental impact on surviving neurons is still unclear. METHODS: WT and AC1/8 double knockout (DKO) mice were administered 1 dose of EtOH (2.5 g/kg) between postnatal days 5 to 7 (P5-7). At P30, brains were removed and processed for Golgi-Cox staining. Medium spiny neurons (MSNs) from the caudate putamen were analyzed for changes in dendritic complexity; number of branches, branch points and terminals, total and average dendritic length; spine density and soma size. RESULTS: EtOH significantly reduced the dendritic complexity and soma size in surviving MSNs regardless of genotype without affecting spine density. In the absence of EtOH, genetic deletion of AC1/8 reduced the dendritic complexity, number of branch points, spine density, and soma size of MSNs compared with WT controls. CONCLUSIONS: These data indicate that neonatal exposure to a single dose of EtOH is sufficient to cause long-term alterations in the dendritic complexity of MSNs and that this outcome is not altered by the functional status of AC1 and AC8. Therefore, although deletion of AC1/8 demonstrates a role for the ACs in normal morphologic development and EtOH-induced neurodegeneration, loss of AC1/8 activity does not exacerbate the effects of EtOH on dendritic morphology or spine density.

Spiral laser scanning photoacoustic microscopy for functional brain imaging in rats.

Significance: There are many neuroscience questions that can be answered by a high-resolution functional brain imaging system. Such a system would require the capability to visualize vasculature and measure neural activity by imaging the entire brain continually and in rapid succession in order to capture hemodynamic changes. Utilizing optical excitation and acoustic detection, photoacoustic technology enables label-free quantification of changes in endogenous chromophores, such as oxyhemoglobin, deoxyhemoglobin, and total hemoglobin. Aim: Our aim was to develop a sufficiently high-resolution, fast frame-rate, and wide field-of-view (FOV) photoacoustic microscopy (PAM) system for the purpose of imaging vasculature and hemodynamics in a rat brain. Approach: Although the most PA microscopy systems use raster scanning (or less commonly Lissajous scanning), we have developed a simple-to-implement laser scanning optical resolution PAM system with spiral scanning (which we have named "spiral laser scanning photoacoustic microscopy" or sLS-PAM) to acquire an 18 mm diameter image at fast frame rate (more than 1 fps). Such a system is designed to permit continuous rat brain imaging without the introduction of photobleaching artifacts. Conclusion: We demonstrated the functional imaging capability of the sLS-PAM system by imaging cerebral hemodynamics in response to whisker and electrical stimulation and used it for vascular imaging of a modeled brain injury. We believe that we have demonstrated the development of a simple-to-implement PAM system, which could become an affordable functional neuroimaging tool for researchers.

Quantification of prefrontal cortical neuronal ensembles following conditioned fear learning in a Fos-LacZ transgenic rat with photoacoustic imaging in Vivo.

Understanding the neurobiology of complex behaviors requires measurement of activity in the discrete population of active neurons, neuronal ensembles, which control the behavior. Conventional neuroimaging techniques ineffectively measure neuronal ensemble activity in the brain in vivo because they assess the average regional neuronal activity instead of the specific activity of the neuronal ensemble that mediates the behavior. Our functional molecular photoacoustic tomography (FM-PAT) system allows direct imaging of Fos-dependent neuronal ensemble activation in Fos-LacZ transgenic rats in vivo. We tested four experimental conditions and found increased FM-PAT signal in prefrontal cortical areas in rats undergoing conditioned fear or novel context exposure. A parallel immunofluorescence ex vivo study of Fos expression found similar findings. These findings demonstrate the ability of FM-PAT to measure Fos-expressing neuronal ensembles directly in vivo and support a mechanistic role for the prefrontal cortex in higher-order processing of response to specific stimuli or environmental cues.

Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains.

Sinoatrial node (SAN) cells are the heart's primary pacemaker. Their activity is tightly regulated by ß-adrenergic receptor (ß-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the ß-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca2+-activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during ß-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with ACI as the predominant Ca2+-activated isoform. Although ACI-KO (ACI-/-) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of ACI results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with ACI, while ryanodine receptor and L-type Ca2+ channels likely compartmentalize with ACI and other AC isoforms. In contrast, there were no significant differences in T-type Ca2+ and Na+ currents at baseline or after ß-AR stimulation between WT and ACI-/- SAN cells. Due to its central characteristic feature as a Ca2+-activated isoform, ACI plays a unique role in sustaining the rise of local cAMP and heart rates during ß-AR stimulation. The findings provide insights into the critical roles of the Ca2+-activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes.

Direct measurement of neuronal ensemble activity using photoacoustic imaging in the stimulated Fos-LacZ transgenic rat brain: A proof-of-principle study.

Measuring neuroactivity underlying complex behaviors facilitates understanding the microcircuitry that supports these behaviors. We have developed a functional and molecular photoacoustic tomography (F/M-PAT) system which allows direct imaging of Fos-expressing neuronal ensembles in Fos-LacZ transgenic rats with a large field-of-view and high spatial resolution. F/M-PAT measures the beta-galactosidase catalyzed enzymatic product of exogenous chromophore X-gal within ensemble neurons. We used an ex vivo imaging method in the Wistar Fos-LacZ transgenic rat, to detect neuronal ensembles in medial prefrontal cortex (mPFC) following cocaine administration or a shock-tone paired stimulus. Robust and selective F/M-PAT signal was detected in mPFC neurons after both conditions (compare to naive controls) demonstrating successful and direct detection of Fos-expressing neuronal ensembles using this approach. The results of this study indicate that F/M-PAT can be used in conjunction with Fos-LacZ rats to monitor neuronal ensembles that underlie a range of behavioral processes, such as fear learning or addiction.

Chronic Intermittent Ethanol Exposure Increases Ethanol Consumption Following Traumatic Stress Exposure in Mice.

Individuals with post-traumatic stress disorder (PTSD) often use alcohol to cope with their distress. This aberrant use of alcohol often develops into alcohol use disorder (AUD) leading to high rates of PTSD-AUD co-occurrence. Individuals with comorbid PTSD-AUD have more intense alcohol cravings and increased relapse rates during withdrawal than those with AUD alone. Also, individuals with PTSD or AUD alone often show similar psychological behaviors, such as impulsivity and anhedonia. Extensive clinical studies on the behavioral effects of PTSD-AUD comorbidity, namely alcohol use, have been performed. However, these effects have not been well studied or mechanistically explored in animal models. Therefore, the present study evaluated the effects of traumatic stress comorbid with alcohol exposures on ethanol intake, impulsivity, and anhedonia in mice. Adult male C57Bl/6 mice were first exposed to either mouse single-prolonged stress (mSPS), an animal model that has been validated for characteristics akin to PTSD symptoms, or control conditions. Baseline two-bottle choice ethanol consumption and preference tests were conducted after a 7-day isolation period, as part of the mSPS exposure. Next, mice were exposed to air or chronic intermittent ethanol (CIE), a vapor-induced ethanol dependence and withdrawal model, for 4 weeks. Two-bottle choice ethanol drinking was used to measure dependence-induced ethanol consumption and preference during periods intervening CIE cycles. The novelty suppressed feeding (NSF) test was used to evaluate impulsivity and anhedonia behaviors 48 h after mSPS and/or repeated CIE exposure. Results showed that, compared to control conditions, mSPS did not affect baseline ethanol consumption and preference. However, mSPS-CIE mice increased Post-CIE ethanol consumption compared to Control-Air mice. Mice exposed to mSPS had a shorter latency to feed during the NSF, whereas CIE-exposed mice consumed less palatable food reward in their home cage after the NSF. These results demonstrate that mice exposed to both mSPS and CIE are more vulnerable to ethanol withdrawal effects, and those exposed to mSPS have increased impulsivity, while CIE exposure increases anhedonia. Future studies to examine the relationship between behavioral outcomes and the molecular mechanisms in the brain after PTSD-AUD are warranted.

Repeated stress exposure in mid-adolescence attenuates behavioral, noradrenergic, and epigenetic effects of trauma-like stress in early adult male rats.

Stress in adolescence can regulate vulnerability to traumatic stress in adulthood through region-specific epigenetic activity and catecholamine levels. We hypothesized that stress in adolescence would increase adult trauma vulnerability by impairing extinction-retention, a deficit in PTSD, by (1) altering class IIa histone deacetylases (HDACs), which integrate effects of stress on gene expression, and (2) enhancing norepinephrine in brain regions regulating cognitive effects of trauma. We investigated the effects of adolescent-stress on adult vulnerability to severe stress using the single-prolonged stress (SPS) model in male rats. Rats were exposed to either (1) adolescent-stress (33-35 postnatal days) then SPS (58-60 postnatal days; n = 14), or (2) no adolescent-stress and SPS (58-60 postnatal days; n = 14), or (3) unstressed conditions (n = 8). We then measured extinction-retention, norepinephrine, HDAC4, and HDAC5. As expected, SPS exposure induced an extinction-retention deficit. Adolescent-stress prior to SPS eliminated this deficit, suggesting adolescent-stress conferred resiliency to adult severe stress. Adolescent-stress also conferred region-specific resilience to norepinephrine changes. HDAC4 and HDAC5 were down-regulated following SPS, and these changes were also modulated by adolescent-stress. Regulation of HDAC levels was consistent with the pattern of cognitive effects of SPS; only animals exposed to SPS without adolescent-stress exhibited reduced HDAC4 and HDAC5 in the prelimbic cortex, hippocampus, and striatum. Thus, HDAC regulation caused by severe stress in adulthood interacts with stress history such that seemingly conflicting reports describing effects of adolescent stress on adult PTSD vulnerability may stem in part from dynamic HDAC changes following trauma that are shaped by adolescent stress history.

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.

A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing.

Glutamate generates fast postsynaptic depolarization throughout the CNS. The positive-feedback nature of glutamate signaling likely necessitates flexible adaptive mechanisms that help prevent runaway excitation. We have previously explored presynaptic adaptive silencing, a form of synaptic plasticity produced by ongoing neuronal activity and by strong depolarization. Unsilencing mechanisms that maintain active synapses and restore normal function after adaptation are also important, but mechanisms underlying such presynaptic reactivation remain unexplored. Here we investigate the involvement of the cAMP pathway in the basal balance between silenced and active synapses, as well as the recovery of baseline function after depolarization-induced presynaptic silencing. Activation of the cAMP pathway activates synapses that are silent at rest, and pharmacological inhibition of cAMP signaling silences basally active synapses. Adenylyl cyclase (AC) 1 and AC8, the major Ca2+-sensitive AC isoforms, are not crucial for the baseline balance between silent and active synapses. In cells from mice doubly deficient in AC1 and AC8, the baseline percentage of active synapses was only modestly reduced compared with wild-type synapses, and forskolin unsilencing was similar in the two genotypes. Nevertheless, after strong presynaptic silencing, recovery of normal function was strongly inhibited in AC1/AC8-deficient synapses. The entire recovery phenotype of the double null was reproduced in AC8-deficient but not AC1-deficient cells. We conclude that, under normal conditions, redundant cyclase activity maintains the balance between presynaptically silent and active synapses, but AC8 plays a particularly important role in rapidly resetting the balance of active to silent synapses after adaptation to strong activity.

Ca-stimulated type 8 adenylyl cyclase is required for rapid acquisition of novel spatial information and for working/episodic-like memory.

Ca-stimulated adenylyl cyclases (ACs) transduce neuronal stimulation-evoked increase in calcium to the production of cAMP, which impinges on the regulation of many aspects of neuronal function. Type 1 and type 8 AC (AC1 and AC8) are the only ACs that are directly stimulated by Ca. Although AC1 function was implicated in regulating reference spatial memory, the function of AC8 in memory formation is not known. Because of the different biochemical properties of AC1 and AC8, these two enzymes may have distinct functions. For example, AC1 activity is regulated by both Ca and G-proteins. In contrast, AC8 is a pure Ca sensor. It is neither stimulated by G(s) nor inhibited by G(i). Recent studies also suggested that AC1 and AC8 were differentially concentrated at different subcellular domains, implicating that Ca-stimulated signaling might be compartmentalized. In this study, we used AC8 knock-out (KO) mice and found behavioral deficits in memory retention for temporal dissociative passive avoidance and object recognition memory. When examined by Morris water maze, AC8 KO mice showed normal reference memory. However, the acquisition of newer spatial information was defective in AC8 KO mice. Furthermore, AC8 KO mice were severely impaired in hippocampus-dependent episodic-like memory when examined by the delayed matching-to-place task. Because AC8 is preferentially localized at the presynaptic active zone, our results suggest a novel role of presynaptic cAMP signaling in memory acquisition and retention, as well as distinct mechanisms underlying reference and working/episodic-like memory.

Adenylyl cyclases types 1 and 8 promote pro-survival pathways after ethanol exposure in the neonatal brain.

Although a wide range of developmental disabilities following fetal alcohol exposure are observed clinically, the molecular factors that determine the severity of these sequelae remain undefined. In mice exposed to ethanol, deletion of adenylyl cyclases (ACs) 1 and 8 exacerbates the neuroapoptosis that occurs in a prolonged post-treatment period; however, it remains unclear whether AC1 and AC8 are critical to the primary or secondary mechanisms underlying ethanol-induced neurodegeneration. Here we demonstrate that mice lacking AC1 and AC8 (DKO) display significantly increased apoptosis in the striatum, a region sensitive to neuroapoptosis in the acute post-treatment period, compared to WT controls. The enhanced neuroapoptotic response observed in the striatum of DKO mice is accompanied by significant reductions in phosphorylation of known pro-survival proteins, insulin receptor substrate-1 (IRS-1), Akt and extracellular signal-regulated kinases (ERKs). These data suggest that AC1/AC8 are crucial activators of cell survival signaling pathways acutely following ethanol exposure and represent molecular factors that may directly modulate the severity of symptoms associated with Fetal Alcohol Syndrome.

Divergent effects of repeated cocaine and novel environment exposure on locus coeruleus c-fos expression and brain catecholamine concentrations in rats.

INTRODUCTION: Chronic administration of cocaine causes a disinhibited, hyperexploratory response to novel environments. As the norepinephrine (NE) system regulates exploration and is dysregulated following cocaine exposure, we hypothesized that this cocaine-mediated hyperexploratory response is associated with increased locus coeruleus (LC) reactivity. METHODS: To test this hypothesis, we used dual fluorescent in situ hybridization immunofluorescence to analyze novelty-induced c-fos and tyrosine hydroxylase expression in the LC and high-pressure liquid chromatography to measure dopamine (DA) and NE concentrations in key catecholamine projection regions following exposure to cocaine. RESULTS: Repeated cocaine exposure followed by a 14-day drug-free period increased exploration of novel environments, replicating previous findings. Novelty exposure increased LC c-fos expression, increased anterior cingulate NE, and decreased ventral tegmental area DA. Cocaine exposure decreased amygdala (AMY) DA, but had no effect on LC c-fos expression or NE in any tested brain region. No interactions between cocaine and novelty were found. Open arm exploration was positively correlated with LC c-fos expression and NE concentrations in both the anterior cingulate and nucleus accumbens, and negatively correlated with AMY DA concentration. CONCLUSIONS: Our findings confirm that exposure to novel environments increases LC activity and NE in the anterior cingulate cortex, that long-term exposure to cocaine dysregulates AMY DA, and that disinhibited exploration in novel environments correlates with NE and DA in regions that modulate risk-taking and avoidance behavior. Further studies investigating the effects of cocaine on brain catecholamine systems are important in understanding the long-lasting effects of cocaine on brain function.

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.

cAMP response element-binding protein deficiency allows for increased neurogenesis and a rapid onset of antidepressant response.

cAMP response element-binding protein (CREB) has been implicated in the molecular and cellular mechanisms of chronic antidepressant (AD) treatment, although its role in the behavioral response is unclear. CREB-deficient (CREB(alpha delta) mutant) mice demonstrate an antidepressant phenotype in the tail suspension test (TST) and forced-swim test. Here, we show that, at baseline, CREB(alpha delta) mutant mice exhibited increased hippocampal cell proliferation and neurogenesis compared with wild-type (WT) controls, effects similar to those observed in WT mice after chronic desipramine (DMI) administration. Neurogenesis was not further augmented by chronic DMI treatment in CREB(alpha delta) mutant mice. Serotonin depletion decreased neurogenesis in CREB(alpha delta) mutant mice to WT levels, which correlated with a reversal of the antidepressant phenotype in the TST. This effect was specific for the reversal of the antidepressant phenotype in these mice, because serotonin depletion did not alter a baseline anxiety-like behavior in CREB(alpha delta) mutant mice. The response to chronic AD treatment in the novelty-induced hypophagia (NIH) test may rely on neurogenesis. Therefore, we used this paradigm to evaluate chronic AD treatment in CREB(alpha delta) mutant mice to determine whether the increased neurogenesis in these mice alters their response in the NIH paradigm. Whereas both WT and CREB(alpha delta) mutant mice responded to chronic AD treatment in the NIH paradigm, only CREB(alpha delta) mutant mice responded to acute AD treatment. However, in the elevated zero maze, DMI did not reverse anxiety behavior in mutant mice. Together, these data show that increased hippocampal neurogenesis allows for an antidepressant phenotype as well as a rapid onset of behavioral responses to AD treatment.

Requirement for the POZ/BTB protein NAC1 in acute but not chronic psychomotor stimulant response.

NAC1 is a novel member of the POZ/BTB (Pox virus and Zinc finger/Bric-a-bracTramtrack Broad complex) but varies from other proteins of this class in that it lacks the characteristic DNA-binding motif, suggesting a novel role. We have employed constitutive gene deletion to elucidate the role of NAC1 in vivo. Nac1 mutant mice are viable with no obvious developmental or physiological impairments. Previous studies suggest a role for NAC1 in cocaine-mediated behaviors. Therefore, we evaluated a variety of behaviors associated with psychomotor stimulant effects in Nac1 mutant mice. Acute locomotor activating effects of cocaine or amphetamine are absent in Nac1 mutant mice, however longer exposure to these psychomotor stimulants result in the development of behavioral sensitization. Acute rewarding properties of cocaine and amphetamine are also blunted in mutant mice, yet repeated exposure resulted in conditioned place preference similar to that observed in wild-type mice. Lastly, increases in extracellular dopamine in the nucleus accumbens, which accompany acute cocaine administration, are blunted in mutant mice, but following chronic cocaine extracellular dopamine levels are increased to the same extent as in wild-type mice. Together these data indicate involvement of NAC1 in the acute behavioral and neurochemical responses to psychomotor stimulants.