Activation of cannabinoid-2 receptor protects against Pseudomonas aeruginosa induced acute lung injury and inflammation.

BACKGROUND: Bacterial pneumonia is a major risk factor for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pseudomonas aeruginosa (PA), an opportunistic pathogen with an increasing resistance acquired against multiple drugs, is one of the main causative agents of ALI and ARDS in diverse clinical settings. Given the anti-inflammatory role of the cannabinoid-2 receptor (CB2R), the effect of CB2R activation in the regulation of PA-induced ALI and inflammation was tested in a mouse model as an alternative to conventional antibiotic therapy. METHODS: In order to activate CB2R, a selective synthetic agonist, JWH133, was administered intraperitoneally (i.p.) to C57BL/6J mice. Furthermore, SR144528 (a selective CB2R antagonist) was administered in combination with JWH133 to test the specificity of the CB2R-mediated effect. PA was administered intratracheally (i.t.) for induction of pneumonia in mice. At 24 h after PA exposure, lung mechanics were measured using the FlexiVent system. The total cell number, protein content, and neutrophil population in the bronchoalveolar lavage fluid (BALF) were determined. The bacterial load in the whole lung was also measured. Lung injury was evaluated by histological examination and PA-induced inflammation was assessed by measuring the levels of BALF cytokines and chemokines. Neutrophil activation (examined by immunofluorescence and immunoblot) and PA-induced inflammatory signaling (analyzed by immunoblot) were also studied. RESULTS: CB2R activation by JWH133 was found to significantly reduce PA-induced ALI and the bacterial burden. CB2R activation also suppressed the PA-induced increase in immune cell infiltration, neutrophil population, and inflammatory cytokines. These effects were abrogated by a CB2R antagonist, SR144528, further confirming the specificity of the CB2R-mediated effects. CB2R-knock out (CB2RKO) mice had a significantly higher level of PA-induced inflammation as compared to that in WT mice. CB2R activation diminished the excess activation of neutrophils, whereas mice lacking CB2R had elevated neutrophil activation. Pharmacological activation of CB2R significantly reduced the PA-induced NF-κB and NLRP3 inflammasome activation, whereas CB2KO mice had elevated NLRP3 inflammasome. CONCLUSION: Our findings indicate that CB2R activation ameliorates PA-induced lung injury and inflammation, thus paving the path for new therapeutic avenues against PA pneumonia.

Psilocybin sex-dependently reduces alcohol consumption in C57BL/6J mice.

The classical psychedelic psilocybin is of interest as a treatment for alcohol use disorder (AUD). This study investigated the effects of psilocybin on voluntary ethanol consumption in adult male and female C57BL/6J mice administered saline or psilocybin intraperitoneally as a single dose of 0.1, 0.5, 1.0 or 2.0 mg/kg and provided 20% ethanol utilizing a two-bottle choice alcohol drinking paradigm. Ethanol was provided continuously for 3 days immediately following the administration of psilocybin, then withheld for 2 days, and then provided continuously for two subsequent additional days. A multilevel model (MLM) for repeated measures was used to compare ethanol consumption and preference in psilocybin-treated groups versus controls. Ethanol consumption and preference were reduced in male mice during the 3-day interval that immediately followed psilocybin administration. The effect of psilocybin on ethanol consumption was dose-related and was consistent across the 3-day interval at dosages of 0.5 mg/kg or greater. Psilocybin had no effect on consumption or preference when ethanol was subsequently reintroduced after 2 days of withdrawal. In contrast to males, psilocybin had no significant effect on ethanol consumption or preference in female mice at any dosage or time point. The lack of an effect of psilocybin on quinine preference, and its limited interaction with locomotor activity indicated that the observed reduction in voluntary ethanol consumption was not attributable to altered taste perception or motor effects. Total fluid consumption was increased in males at some time points and psilocybin dosages and unchanged in females, and the absence of any decrease in either group at any time point indicated that the observed reduction in ethanol consumption was not mediated by nonspecific effects on consummatory behavior. The finding of a sex-dependent effect of psilocybin on ethanol consumption suggests that the C57BL/6J mouse may provide a useful experimental approach to modeling sex differences in vulnerability to AUD in addition to investigation of the neurobiological basis of the effect of classical psychedelics on alcohol drinking behavior.

Bidirectional control of infant rat social behavior via dopaminergic innervation of the basolateral amygdala.

Social interaction deficits seen in psychiatric disorders emerge in early-life and are most closely linked to aberrant neural circuit function. Due to technical limitations, we have limited understanding of how typical versus pathological social behavior circuits develop. Using a suite of invasive procedures in awake, behaving infant rats, including optogenetics, microdialysis, and microinfusions, we dissected the circuits controlling the gradual increase in social behavior deficits following two complementary procedures-naturalistic harsh maternal care and repeated shock alone or with an anesthetized mother. Whether the mother was the source of the adversity (naturalistic Scarcity-Adversity) or merely present during the adversity (repeated shock with mom), both conditions elevated basolateral amygdala (BLA) dopamine, which was necessary and sufficient in initiating social behavior pathology. This did not occur when pups experienced adversity alone. These data highlight the unique impact of social adversity as causal in producing mesolimbic dopamine circuit dysfunction and aberrant social behavior.

Exo- and Endo-cannabinoids in Depressive and Suicidal Behaviors.

Cannabis (marijuana) has been known to humans for thousands of years but its neurophysiological effects were sparsely understood until recently. Preclinical and clinical studies in the past two decades have indisputably supported the clinical proposition that the endocannabinoid system plays an important role in the etiopathogeneses of many neuropsychiatric disorders, including mood and addictive disorders. In this review, we discuss the existing knowledge of exo- and endo-cannabinoids, and role of the endocannabinoid system in depressive and suicidal behavior. A dysfunction in this system, located in brain regions such as prefrontal cortex and limbic structures is implicated in mood regulation, impulsivity and decision-making, may increase the risk of negative mood and cognition as well as suicidality. The literature discussed here also suggests that the endocannabinoid system may be a viable target for treatments of these neuropsychiatric conditions.

CB1 receptor neutral antagonist treatment epigenetically increases neuropeptide Y expression and decreases alcohol drinking.

Alcohol consumption is mediated by several important neuromodulatory systems, including the endocannabinoid and neuropeptide Y (NPY) systems in the limbic brain circuitry. However, molecular mechanisms through which cannabinoid-1 (CB1) receptors regulate alcohol consumption are still unclear. Here, we investigated the role of the CB1 receptor-mediated downstream regulation of NPY via epigenetic mechanisms in the amygdala. Alcohol drinking behavior was measured in adult male C57BL/6J mice treated with a CB1 receptor neutral antagonist AM4113 using a two-bottle choice paradigm while anxiety-like behavior was assessed in the light-dark box (LDB) test. The CB1 receptor-mediated changes in the protein levels of phosphorylated cAMP-responsive element binding protein (pCREB), CREB binding protein (CBP), H3K9ac, H3K14ac and NPY, and the mRNA levels of Creb1, Cbp, and Npy were measured in amygdaloid brain structures. Npy-specific changes in the levels of acetylated histone (H3K9/14ac) and CBP in the amygdala were also measured. We found that the pharmacological blockade of CB1 receptors with AM4113 reduced alcohol consumption and, in an ethanol-naïve cohort, reduced anxiety-like behavior in the LDB test. Treatment with AM4113 also increased the mRNA levels of Creb1 and Cbp in the amygdala as well as the protein levels of pCREB, CBP, H3K9ac and H3K14ac in the central and medial nucleus of amygdala, but not in the basolateral amygdala. Additionally, AM4113 treatment increased occupancy of CBP and H3K9/14ac at the Npy gene promoter, leading to an increase in both mRNA and protein levels of NPY in the amygdala. These novel findings suggest that CB1 receptor-mediated CREB signaling plays an important role in the modulation of NPY function through an epigenetic mechanism and further support the potential use of CB1 receptor neutral antagonists for the treatment of alcohol use disorder.

Reduced Motivation in Perinatal Fluoxetine-Treated Mice: A Hypodopaminergic Phenotype.

Early life is a sensitive period, in which enhanced neural plasticity allows the developing brain to adapt to its environment. This plasticity can also be a risk factor in which maladaptive development can lead to long-lasting behavioral deficits. Here, we test how early-life exposure to the selective-serotonin-reuptake-inhibitor (SSRI), fluoxetine, affects motivation, and dopaminergic signaling in adulthood. We show for the first time that mice exposed to fluoxetine in the early postnatal period exhibit a reduction in effort-related motivation. These mice also show blunted responses to amphetamine and reduced dopaminergic activation in a sucrose reward task. Interestingly, we find that the reduction in motivation can be rescued in the adult by administering bupropion, a dopamine-norepinephrine reuptake inhibitor used as an antidepressant and a smoke cessation aid but not by fluoxetine. Taken together, our studies highlight the effects of early postnatal exposure of fluoxetine on motivation and demonstrate the involvement of the dopaminergic system in this process.SIGNIFICANCE STATEMENT The developmental period is characterized by enhanced plasticity. During this period, environmental factors have the potential to lead to enduring behavioral changes. Here, we show that exposure to the SSRI fluoxetine during a restricted period in early life leads to a reduction in adult motivation. We further show that this reduction is associated with decreased dopaminergic responsivity. Finally, we show that motivational deficits induced by early-life fluoxetine exposure can be rescued by adult administration of bupropion but not by fluoxetine.

Dorsal and ventral mossy cells differ in their axonal projections throughout the dentate gyrus of the mouse hippocampus.

Glutamatergic hilar mossy cells (MCs) have axons that terminate both near and far from their cell body but stay within the DG, making synapses primarily in the molecular layer. The long-range axons are considered the primary projection, and extend throughout the DG ipsilateral to the soma, and project to the contralateral DG. The specificity of MC axons for the inner molecular layer (IML) has been considered to be a key characteristic of the DG. In the present study, we made the surprising finding that dorsal MC axons are an exception to this rule. We used two mouse lines that allow for Cre-dependent viral labeling of MCs and their axons: dopamine receptor D2 (Drd2-Cre) and calcitonin receptor-like receptor (Crlr-Cre). A single viral injection into the dorsal DG to label dorsal MCs resulted in labeling of MC axons in both the IML and middle molecular layer (MML). Interestingly, this broad termination of dorsal MC axons occurred throughout the septotemporal DG. In contrast, long-range axons of ventral MCs terminated in the IML, consistent with the literature. Taken together, these results suggest that dorsal and ventral MCs differ significantly in their axonal projections. Since MC projections in the ML are thought to terminate primarily on GCs, the results suggest a dorsal-ventral difference in MC activation of GCs. The surprising difference in dorsal and ventral MC projections should therefore be considered when evaluating dorsal-ventral differences in DG function.

Bidirectional Regulation of Cognitive and Anxiety-like Behaviors by Dentate Gyrus Mossy Cells in Male and Female Mice.

The dentate gyrus (DG) of the hippocampus is important for cognition and behavior. However, the circuits underlying these functions are unclear. DG mossy cells (MCs) are potentially important because of their excitatory synapses on the primary cell type, granule cells (GCs). However, MCs also activate GABAergic neurons, which inhibit GCs. We used viral delivery of designer receptors exclusively activated by designer drugs (DREADDs) in mice to implement a gain- and loss-of-function study of MCs in diverse behaviors. Using this approach, manipulations of MCs could bidirectionally regulate behavior. The results suggest that inhibiting MCs can reduce anxiety-like behavior and improve cognitive performance. However, not all cognitive or anxiety-related behaviors were influenced, suggesting specific roles of MCs in some, but not all, types of cognition and anxiety. Notably, several behaviors showed sex-specific effects, with females often showing more pronounced effects than the males. We also used the immediate early gene c-Fos to address whether DREADDs bidirectionally regulated MC or GC activity. We confirmed excitatory DREADDs increased MC c-Fos. However, there was no change in GC c-Fos, consistent with MC activation leading to GABAergic inhibition of GCs. In contrast, inhibitory DREADDs led to a large increase in GC c-Fos, consistent with a reduction in MC excitation of GABAergic neurons, and reduced inhibition of GCs. Together, these results suggest that MCs regulate anxiety and cognition in specific ways. We also raise the possibility that cognitive performance may be improved by reducing anxiety.SIGNIFICANCE STATEMENT The dentate gyrus (DG) has many important cognitive roles as well as being associated with affective behavior. This study addressed how a glutamatergic DG cell type called mossy cells (MCs) contributes to diverse behaviors, which is timely because it is known that MCs regulate the activity of the primary DG cell type, granule cells (GCs), but how MC activity influences behavior is unclear. We show, surprisingly, that activating MCs can lead to adverse behavioral outcomes, and inhibiting MCs have an opposite effect. Importantly, the results appeared to be task-dependent and showed that testing both sexes was important. Additional experiments indicated what MC and GC circuitry was involved. Together, the results suggest how MCs influence behaviors that involve the DG.

Dual pharmacological inhibitor of endocannabinoid degrading enzymes reduces depressive-like behavior in female rats.

Major depressive disorder (MDD) is common, often under-treated and a leading cause of disability and mortality worldwide. The causes of MDD remain unclear, including the role of the endocannabinoid system. Intriguingly, the prevalence of depression is significantly greater in women than men. In this study we examined the role of endocannabinoids in depressive behavior. The levels of endocannabinoids, N-arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) were measured along with brain derived neurotrophic factor (BDNF) in postmortem ventral striata of female patients with MDD and non-psychiatric controls, and in Wistar Kyoto (WKY) rat, a selectively inbred strain of rat widely used for testing the depressive behavior. The effect of pharmacological elevation of endocannabinoids through inhibition of their catabolizing enzymes (fatty acid amide hydrolase [FAAH] and monoacyl glycerol lipase [MAGL]) on depressive-like phenotype was also assessed in WKY rat. The findings showed lower levels of endocannabinoids and BDNF in the ventral striata of MDD patients and WKY rats. A dual inhibitor of FAAH and MAGL, JZL195, elevated the endocannabinoids and BDNF levels in ventral striatum, and reduced the depressive-like phenotype in female WKY rats. Collectively, our study suggests a blunted ventral striatal endocannabinoid and BDNF signaling in depressive behavior and concludes that endocannabinoid enhancing agents may have an antidepressant effect.

Cannabinoid exposure during pregnancy and its impact on immune function.

Cannabinoids are the most commonly abused illicit drugs worldwide. While cannabis can be beneficial for certain heath conditions, abuse of potent synthetic cannabinoids has been on the rise. Exposure to cannabinoids is also prevalent in women of child-bearing age and pregnant women. These compounds can cross the placental barrier and directly affect the fetus. They mediate their effects primarily through G-protein coupled cannabinoid receptors, CB1 and CB2. In addition to significant neurological effects, cannabinoids can trigger robust immunomodulation by altering cytokine levels, causing apoptosis of lymphoid cells and inducing suppressor cells of the immune system. Profound effects of cannabinoids on the immune system as discussed in this review, suggest that maternal exposure during pregnancy could lead to dysregulation of innate and adaptive immune system of developing fetus and offspring potentially leading to weakening of immune defenses against infections and cancer later in life. Emerging evidence also indicates the underlying role of epigenetic mechanisms causing long-lasting impact following cannabinoid exposure in utero.

LSD Administered as a Single Dose Reduces Alcohol Consumption in C57BL/6J Mice.

There is a substantive clinical literature on classical hallucinogens, most commonly lysergic acid diethylamide (LSD) for the treatment of alcohol use disorder. However, there has been no published research on the effect of LSD on alcohol consumption in animals. This study evaluated the effect of LSD in mice using a two-bottle choice alcohol drinking paradigm. Adult male C57BL/6J mice were exposed to ethanol to develop preference and divided into three groups of equal ethanol consumption, and then treated with single intraperitoneal injection of saline or 25 or 50 µg/kg LSD and offered water and 20% ethanol. The respective LSD-treated groups were compared to the control group utilizing a multilevel model for repeated measures. In mice treated with 50 µg/kg LSD ethanol consumption was reduced relative to controls (p = 0.0035), as was ethanol preference (p = 0.0024), with a group mean reduction of ethanol consumption of 17.9% sustained over an interval of 46 days following LSD administration. No significant effects on ethanol consumption or preference were observed in mice treated with 25 µg/kg LSD. Neither total fluid intake nor locomotor activity in the LSD-treated groups differed significantly from controls. These results suggest that classical hallucinogens in the animal model merit further study as a potential approach to the identification of targets for drug discovery and investigation of the neurobiology of addiction.

Cannabinoid-1 receptor neutral antagonist reduces binge-like alcohol consumption and alcohol-induced accumbal dopaminergic signaling.

Binge alcohol (ethanol) drinking is associated with profound adverse effects on our health and society. Rimonabant (SR141716A), a CB1 receptor inverse agonist, was previously shown to be effective for nicotine cessation and obesity. However, studies using rimonabant were discontinued as it was associated with an increased risk of depression and anxiety. In the present study, we examined the pharmacokinetics and effects of AM4113, a novel CB1 receptor neutral antagonist on binge-like ethanol drinking in C57BL/6J mice using a two-bottle choice drinking-in-dark (DID) paradigm. The results indicated a slower elimination of AM4113 in the brain than in plasma. AM4113 suppressed ethanol consumption and preference without having significant effects on body weight, ambulatory activity, preference for tastants (saccharin and quinine) and ethanol metabolism. AM4113 pretreatment reduced ethanol-induced increase in dopamine release in nucleus accumbens. Collectively, these data suggest an important role of CB1 receptor-mediated regulation of binge-like ethanol consumption and mesolimbic dopaminergic signaling, and further points to the potential utility of CB1 neutral antagonists for the treatment of binge ethanol drinking.

Marijuana, Spice 'herbal high', and early neural development: implications for rescheduling and legalization.

Marijuana is the most widely used illicit drug by pregnant women in the world. In utero exposure to Δ9-tetrahydrocannabinol (Δ9-THC), a major psychoactive component of marijuana, is associated with an increased risk for anencephaly and neurobehavioural deficiencies in the offspring, including attention deficit hyperactivity disorder (ADHD), learning disabilities, and memory impairment. Recent studies demonstrate that the developing central nervous system (CNS) is susceptible to the effects of Δ9-THC and other cannabimimetics, including the psychoactive ingredients of the branded product 'Spice' branded products. These exocannabinoids interfere with the function of an endocannabinoid (eCB) system, present in the developing CNS from E12.5 (week 5 of gestation in humans), and required for proliferation, migration, and differentiation of neurons. Until recently, it was not known whether the eCB system is also present in the developing CNS during the initial stages of its ontogeny, i.e. from E7.0 onwards (week 2 of gestation in humans), and if so, whether this system is also susceptible to the action of exocannabinoids. Here, we review current data, in which the presence of an eCB system during the initial stage of development of the CNS is demonstrated. Furthermore, we focus on recent advances on the effect of canabimimetics on early gestation. The relevance of these findings and potential adverse developmental consequences of in utero exposure to 'high potency' marijuana, Spice branded products and/or cannabinoid research chemicals during this period is discussed. Finally, we address the implication of these findings in terms of the potential dangers of synthetic cannabinoid use during pregnancy, and the ongoing debate over legalization of marijuana.

Dysfunction in fatty acid amide hydrolase is associated with depressive-like behavior in Wistar Kyoto rats.

BACKGROUND: While the etiology of depression is not clearly understood at the present time, this mental disorder is thought be a complex and multifactorial trait with important genetic and environmental contributing factors. METHODOLOGY/PRINCIPAL FINDINGS: The role of the endocannabinoid (eCB) system in depressive behavior was examined in Wistar Kyoto (WKY) rat strain, a genetic model of depression. Our findings revealed selective abnormalities in the eCB system in the brains of WKY rats compared to Wistar (WIS) rats. Immunoblot analysis indicated significantly higher levels of fatty acid amide hydrolase (FAAH) in frontal cortex and hippocampus of WKY rats with no alteration in the level of N-arachidonyl phosphatidyl ethanolamine specific phospholipase-D (NAPE-PLD). Significantly higher levels of CB1 receptor-mediated G-protein coupling and lower levels of anandamide (AEA) were found in frontal cortex and hippocampus of WKY rats. While the levels of brain derived neurotropic factor (BDNF) were significantly lower in frontal cortex and hippocampus of WKY rats compared to WIS rats, pharmacological inhibition of FAAH elevated BDNF levels in WKY rats. Inhibition of FAAH enzyme also significantly increased sucrose consumption and decreased immobility in the forced swim test in WKY rats. CONCLUSIONS/SIGNIFICANCE: These findings suggest a critical role for the eCB system and BDNF in the genetic predisposition to depressive-like behavior in WKY rats and point to the potential therapeutic utility of eCB enhancing agents in depressive disorder.

Cannabinoid receptor 1 signaling in embryo neurodevelopment.

In utero exposure to tetrahydrocannabinol, the psychoactive component of marijuana, is associated with an increased risk for neurodevelopmental defects in the offspring by interfering with the functioning of the endocannabinoid (eCB) system. At the present time, it is not clearly known whether the eCB system is present before neurogenesis. Using an array of biochemical techniques, we analyzed the levels of CB1 receptors, eCBs (AEA and 2-AG), and the enzymes (NAPE-PLD, DAGLα, DAGLß, MAGL, and FAAH) involved in the metabolism of the eCBs in chick and mouse models during development. The findings demonstrate the presence of eCB system in early embryo before neurogenesis. The eCB system might play a critical role in early embryogenesis and there might be adverse developmental consequences of in utero exposure to marijuana and other drugs of abuse during this period.

Innate difference in the endocannabinoid signaling and its modulation by alcohol consumption in alcohol-preferring sP rats.

The present study was undertaken to examine whether genetically predetermined differences in components of the endocannabinoid system were present in the brain of Sardinian alcohol-preferring (sP) and Sardinian alcohol-non-preferring (sNP) rats, a pair of rat lines selectively bred for opposite alcohol preference. The effects of acquisition and maintenance of alcohol drinking, alcohol withdrawal, and alcohol re-exposure on the endocannabinoid system was also assessed in the striatum of sP rats. The findings revealed significantly higher density of the CB1 receptors and levels of CB1 receptor mRNA, CB1 receptor-mediated G-protein coupling, and endocannabinoids in the cerebral cortex, hippocampus and striatum of alcohol-naive sP rats than sNP rats. A significantly lower expression of mFAAH enzyme was evident in the hippocampus of alcohol-naive sP rats. Alcohol drinking (during both acquisition and maintenance phases) in sP rats resulted in a significant reduction in striatal CB1 receptor-mediated G-protein coupling whereas alcohol withdrawal attenuated this effect. Alcohol consumption was also associated with markedly increased levels of endocannabinoids in the striatum. Co-administration of the CB1 receptor antagonist, rimonabant (SR141716A) reduced alcohol intake, and reversed alcohol-induced changes in CB1 receptor-mediated G-protein activation. These findings provided a new insight into a potential genetic basis of excessive alcohol consumption, suggesting innate differences in the endocannabinoid system might be associated with higher alcohol preference in sP rats. The data also indicate a modulation of CB1 receptor-mediated signaling following alcohol consumption, and further strengthen the potential of the endocannabinoid system as a target for the treatment of alcohol related behaviors.

Declining phosphatases underlie aging-related hyperphosphorylation of neurofilaments.

Cytoskeletal protein phosphorylation is frequently altered in neuropathologic states but little is known about changes during normal aging. Here we report that declining protein phosphatase activity, rather than activation of kinases, underlies aging-related neurofilament hyperphosphorylation. Purified PP2A or PP2B dephosphorylated the heavy neurofilament (NFH) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting either phosphatase induced NFH phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 phosphoepitope associated with late axon maturation, more than doubled during the 12-month period after NFH expression plateaued at p21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.

Selective alterations of the CB1 receptors and the fatty acid amide hydrolase in the ventral striatum of alcoholics and suicides.

Recent studies in rodents have suggested a role for the central endocannabinoid system in the regulation of mood and alcohol related behaviors. Alcohol use disorder is often associated with suicidal behavior. In the present study, we examined whether abnormalities in the endocannabinoid system in the ventral striatum are associated with alcohol dependence and suicide. The levels of CB1 receptors, receptor-mediated G-protein signaling, and activity and level of the fatty acid amide hydrolase (FAAH) were analyzed postmortem in the ventral striatum of alcohol-dependent nonsuicides (CA, n=9), alcohol-dependent suicides (AS, n=9) and nonpsychiatric controls (C, n=9). All subjects underwent a psychological autopsy, and toxicological and neuropathological examinations. The levels of the CB1 receptors and the CB1 receptor-mediated G-protein signaling were significantly lower in the ventral striatum of CA compared to the control group. However, these parameters were elevated in AS when compared to CA group. The activity of FAAH enzyme was lower in CA compared to the control group while it was found to be significantly higher in AS compared with CA group. These findings suggest that alcohol dependence is associated with the downregulation of the CB1 receptors, while suicide is linked to the upregulation of these receptors in the ventral striatum. Alteration in the activity of FAAH enzyme that regulates the anandamide (AEA) content might in turn explain differences in the CB1 receptor function in alcohol dependence and suicide. These findings may have etiological and therapeutic implications for the treatment of alcohol addiction and suicidal behavior.

Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases.

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.

Genetic and pharmacological manipulations of the CB(1) receptor alter ethanol preference and dependence in ethanol preferring and nonpreferring mice.

Recent studies have indicated a role for the endocannabinoid system in ethanol-related behaviors. This study examined the effect of pharmacological activation, blockade, and genetic deletion of the CB(1) receptors on ethanol-drinking behavior in ethanol preferring C57BL/6J (B6) and ethanol nonpreferring DBA/2J (D2) mice. The deletion of CB(1) receptor significantly reduced the ethanol preference. Although the stimulation of the CB(1) receptor by CP-55,940 markedly increased the ethanol preference, this effect was found to be greater in B6 than in D2 mice. The antagonism of CB(1) receptor function by SR141716A led to a significant reduction in voluntary ethanol preference in B6 than D2 mice. A significant lower hypothermic and greater sedative response to acute ethanol administration was observed in both the strains of CB(1) -/- mice than wild-type mice. Interestingly, genetic deletion and pharmacological blockade of the CB(1) receptor produced a marked reduction in severity of handling-induced convulsion in both the strains. The radioligand binding studies revealed significantly higher levels of CB(1) receptor-stimulated G-protein activation in the striatum of B6 compared to D2 mice. Innate differences in the CB(1) receptor function might be one of the contributing factors for higher ethanol drinking behavior. The antagonists of the CB(1) receptor may have therapeutic potential in the treatment of ethanol dependence.