Immunoglobulin class-switch recombination (CSR) requires activation-induced cytidine deaminase (AID). Deamination of DNA by AID in transcribed switch (S) regions leads to double-stranded breaks in DNA that serve as obligatory CSR intermediates. Here we demonstrate that the catalytic and regulatory subunits of protein kinase A (PKA) were specifically recruited to S regions to promote the localized phosphorylation of AID, which led to binding of replication protein A and subsequent propagation of the CSR cascade. Accordingly, inactivation of PKA resulted in considerable disruption of CSR because of decreased AID phosphorylation and recruitment of replication protein A to S regions. We propose that PKA nucleates the formation of active AID complexes specifically on S regions to generate the high density of DNA lesions required for CSR.
Background Corticotropin-releasing factor (CRF) mediates anxiogenic responses by activating CRF1 receptors in limbic brain regions. Anxiety is further modulated by the endogenous cannabinoid (eCB) system that attenuates the synaptic effects of stress. In the amygdala, acute stress activates the enzymatic clearance of the eCB N-arachidonoylethanolamine (anandamide; AEA) via fatty acid amide hydrolase (FAAH), although it is unclear whether chronic stress induces maladaptive changes in amygdalar eCB signaling to promote anxiety. Here, we used genetically-selected Marchigian Sardinian P (msP) rats carrying an innate overexpression of CRF1 receptors to study the role of constitutive upregulation in CRF systems on amygdalar eCB function and persistent anxiety-like effects. Methods We applied behavioral, pharmacological, and biochemical methods to broadly characterize anxiety-like behaviors and amygdalar eCB clearance enzymes in msP versus non-selected Wistar rats. Subsequent studies examined the influence of dysregulated CRF and FAAH systems in altering excitatory transmission in the central amygdala (CeA). Results MsPs display an anxious phenotype accompanied by elevations in amygdalar FAAH activity and reduced dialysate AEA levels in the CeA. Elevations in CRF-CRF1 signaling dysregulate FAAH activity, and this genotypic difference is normalized with pharmacological blockade of CRF1 receptors. MsPs also exhibit elevated baseline glutamatergic transmission in the CeA, and dysregulated CRF-FAAH facilitates stress-induced increases in glutamatergic activity. Treatment with a FAAH inhibitor relieves sensitized glutamatergic responses in msPs and attenuates the anxiety-like phenotype. Conclusions Pathological anxiety and stress hyper-sensitivity are driven by constitutive increases in CRF1 signaling that dysregulate AEA signaling mechanisms and disable neuronal constraint of CeA glutamatergic synapses.
The present experiments employed in vivo microdialysis to characterize the effects of commonly used endocannabinoid clearance inhibitors on basal and depolarization-induced alterations in interstitial endocannabinoid levels in the nucleus accumbens of rat brain. Compounds targeting the putative endocannabinoid transporter and hydrolytic enzymes (FAAH and MAGL) were compared. The transporter inhibitor AM404 modestly enhanced depolarization-induced increases in 2-arachidonoyl glycerol (2-AG) levels but did not alter levels of N-arachidonoyl-ethanolamide (anandamide, AEA). The transport inhibitor UCM707 did not alter dialysate levels of either endocannabinoid. The FAAH inhibitors URB597 and PF-3845 robustly increased AEA levels during depolarization without altering 2-AG levels. The MAGL inhibitor URB602 significantly enhanced depolarization-induced increases in 2-AG, but did not alter AEA levels. In contrast, the MAGL inhibitor JZL184 did not alter 2-AG or AEA levels under any condition tested. Finally, the dual FAAH/MAGL inhibitor JZL195 significantly enhanced depolarization-induced increases in both AEA and 2-AG levels. In contrast to the present observations in rats, prior work in mice has demonstrated a robust JZL184-induced enhancement of depolarization-induced increases in dialysate 2-AG. Thus, to further investigate species differences, additional tests with JZL184, PF-3845, and JZL195 were performed in mice. Consistent with prior reports, JZL184 significantly enhanced depolarization-induced increases in 2-AG without altering AEA levels. PF-3845 and JZL195 produced profiles in mouse dialysates comparable to those observed in rats. These findings confirm that interstitial endocannabinoid levels in the brain can be selectively manipulated by endocannabinoid clearance inhibitors. While PF-3845 and JZL195 produce similar effects in both rats and mice, substantial species differences in JZL184 efficacy are evident, which is consistent with previous studies.
These observations demonstrate that long-term intermittent exposure to clinically meaningful Δ9-THC doses induces persistent impairments in impulse control and attentional function. If present in humans, these disruptions may impact academic and professional performance.
Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotineinduced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.T he mesocorticolimbic dopamine (DA) system provides a critical link between the brain regions that process cognitive information and those controlling motor behavior. Precise control of these ventral tegmental area (VTA) projections facilitates seeking rewarding stimuli, retreating from aversive stimuli, constraint of motivational state, and behavioral flexibility necessary for survival. GABAergic signaling provides robust inhibition that gates VTA DA cell excitability (1, 2), and loss of this inhibition leads to pathological dysregulation of mesocorticolimbic circuitry (3, 4).Endocannabinoids (eCBs) regulate DAergic activity through retrograde signaling from DA cell bodies onto presynaptic cannabinoid type 1 (CB 1 ) receptors expressed on both inhibitory and excitatory inputs. Although both 2-arachidonoylglycerol (2-AG) and anandamide (AEA) function as endogenous CB 1 agonists in the brain (5-7), these lipids exhibit distinct pharmacological profiles in vivo (8, 9) and mediate differential behavioral effects (10, 11). Endocannabinoids are produced and degraded ondemand, and the primary enzymes responsible for eCB degradation have been well-characterized using selective pharmacological tools that inactivate monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) (11-13). However, a complete evaluation of the influence of eCB signaling in the brain has been hampered by the lack of appropriate corresponding tools for selectively inactivating on-demand eCB biosynthesis.Substantial evidence implicates eCB signaling in the etiology of nicotine ...
Impulsivity is a risk factor for alcoholism and long-term alcohol exposure may further impair impulse control in a manner that propels problematic alcohol use. The present study employed the rat 5-Choice Serial Reaction Time Task (5-CSRTT) to measure behavioral inhibition and attentional capacity during abstinence from repeated 5d cycles of alcohol liquid diet consumption. Task performance was not disrupted following the first cycle of alcohol exposure, however, evidence of impaired behavioral inhibition emerged following the third cycle of alcohol exposure. In comparison with controls, alcohol rats exhibited deficits in inhibitory control during cognitively challenging 5-CSRTT tests employing variable inter-trial intervals (varITI). This behavioral disruption was not present during early abstinence (3d) but was evident by 7d abstinence and persisted for at least 34d. Interestingly, renewed alcohol consumption ameliorated these disruptions in impulse control, though deficient behavioral inhibition re-emerged during subsequent abstinence. Indices of increased impulsivity were no longer present in tests conducted after 49 days of abstinence. Alcohol-related impairments in impulse control were not evident in sessions employing highly familiar task parameters regardless of abstinence period and control experiments confirmed that performance deficits during the challenge sessions were unlikely to result from alcohol-related disruption in the adaptation to repeated varITI testing. Together, the current findings demonstrate that chronic intermittent alcohol consumption results in decreased behavioral inhibition in rats that is temporally similar to clinical observations of disrupted impulsive control in abstinent alcoholics performing tasks of behavioral inhibition.
Impaired cognitive processing is a hallmark of addiction. In particular, deficits in inhibitory control can propel continued drug use despite adverse consequences. Clinical evidence shows that detoxified alcoholics exhibit poor inhibitory control in the Continuous Performance Task (CPT) and related tests of motor impulsivity. Animal models may provide important insight into the neural mechanisms underlying this consequence of chronic alcohol exposure though pre-clinical investigations of behavioral inhibition during alcohol abstinence are sparse. The present study employed the rat 5 Choice-Continuous Performance Task (5C-CPT), a novel pre-clinical variant of the CPT, to evaluate attentional capacity and impulse control over the course of protracted abstinence from chronic intermittent alcohol consumption. In tests conducted with familiar 5C-CPT conditions EtOH-exposed rats exhibited impaired attentional capacity during the first hours of abstinence and impaired behavioral restraint (increased false alarms) during the first 5d of abstinence that dissipated thereafter. Subsequent tests employing visual distractors that increase the cognitive load of the task revealed significant increases in impulsive action (premature responses) at 3 and 5 weeks of abstinence, and the emergence of impaired behavioral restraint (increased false alarms) at 7 weeks of abstinence. Collectively, these findings demonstrate the emergence of increased impulsive action in alcohol-dependent rats during protracted alcohol abstinence and suggest the 5C-CPT with visual distractors may provide a viable behavioral platform for characterizing the neurobiological substrates underlying impaired behavioral inhibition resulting from chronic intermittent alcohol exposure.
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