Balapal S. Basavarajappa

Cannabinoid CB1 receptor knockout mice exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus accumbens

The mechanisms underlying predisposition to alcohol abuse and alcoholism are poorly understood. In this study, we evaluated the role of cannabinoid (CB1) receptors in (i) voluntary alcohol consumption, and (ii) acute alcohol‐induced dopamine (DA) release in the nucleus accumbens, using mice that lack the CB1 receptor gene (CB1–/–). CB1–/– mice exhibited dramatically reduced voluntary alcohol consumption, and completely lacked alcohol‐induced DA release in the nucleus accumbens, as compared to wild‐type mice. The gender difference, with female mice consuming significantly more alcohol than wild‐type male mice, was observed in wild‐type mice, whereas this gender difference was nonexistent in CB1 mutant male and female mice. There was also a significant gender difference, with the wild‐type, heterozygous, and mutant females consuming significantly more liquid and food than wild‐type, heterozygous and mutant males. However, the total volume of fluid consumption and food intake did not differ between wild‐type, heterozygous, and mutant mice. These results strongly suggest that the CB1 receptor system plays an important role in regulating the positive reinforcing properties of alcohol.

Chronic ethanol increases the cannabinoid receptor agonist anandamide and its precursor N-arachidonoylphosphatidylethanolamine in SK-N-SH cells.

Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down‐regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N‐arachidonoylphosphatidylethanolamine (N‐ArPE) in SK‐N‐SH cells that were prelabeled with [3H]arachidonic acid. The results indicate that exposure of SK‐N‐SH cells to EtOH (100 mM) for 72 h significantly increased levels of [3H]AnNH and [3H]N‐ArPE (p < 0.05) (1.43‐fold for [3H]AnNH and 1.65‐fold for [3H]N‐ArPE). Exposure of SK‐N‐SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [3H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [3H]N‐ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [3H]AnNH. The formation of [3H]AnNH and [3H]N‐ArPE by EtOH was enhanced by the Ca2+ ionophore A23187 or by the depolarizing agent veratridine and the K+ channel blocker 4‐aminopyridine. Further, the EtOH‐induced formation of [3H]AnNH and [3H]N‐ArPE was inhibited by exogenous AnNH, whereas only [3H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and Gi/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK‐N‐SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.

Chronic ethanol administration down-regulates cannabinoid receptors in mouse brain synaptic plasma membrane

The effects of chronic ethanol (EtOH) consumption on the central nervous system may be related in part to its action on biological membranes by altering various receptor functions. In the current study, we examined the effects of chronic EtOH (4 day inhalation) on cannabinoid receptors (CB1) labeled with [3H]CP55,940 in synaptic plasma membranes (SPM) isolated from mouse brain. Our results indicate the presence of a high level of CB1 receptors in controls (Bmax=12.0+/-0.3 pmol mg-1 protein) which decreased significantly (-58%) in SPM from mouse brain chronically exposed to EtOH. This effect occurs without any changes in the receptor affinity (Kd=2. 3+/-0.3 nM for control and 2.9+/-0.3 nM for EtOH group, P>0.05). Dissociation kinetic results showed a dissociation rate constant (K-1) of 0.09+/-0.01 min-1 for control and this dissociation rate constant decreased significantly in the chronic EtOH treated mice brain (0.05+/-0.01 min-1, P<0.05). The competition studies with anandamide resulted in a substantial decrease in [3H]CP55,940 binding in both the control and EtOH group, with a decrease (P<0.05) in the Ki values in the SPM of chronic EtOH exposed mice. Hill transformation analysis showed an nH close to one in control (0. 92+/-0.01). This did not change significantly after chronic EtOH (0. 95+/-0.01) administration, which indicates the existence of a single class of receptor for [3H]CP55,940 binding in SPM from control and EtOH treated mice. The observed down-regulation of CB1 receptors by chronic EtOH may indicate the involvement of cannabinoid receptors in EtOH tolerance and dependence.

Calpain mediates calcium-induced activation of the Erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons relevance to Alzheimer's disease

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimers disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.

Chronic ethanol inhibits the anandamide transport and increases extracellular anandamide levels in cerebellar granule neurons

Ethanol increases extracellular anandamide levels in neuronal cells. However, the molecular mechanisms by which this occurs are unknown. Chronic exposure of cerebellar granule neurons to ethanol increased the levels of anandamide accumulated in the cellular medium. Anandamide uptake was saturable and was inhibited (30% at 3 min) in response to chronic exposure to ethanol. Chronic ethanol treatment did not alter the K(m), but significantly decreased V(max) of anandamide transport (33%) (P<0.0001). Fatty acid amide hydrolase activity was not affected by chronic ethanol treatment. Anandamide transport processes are independent of cannabinoid CB1 receptor, as cannabinoid CB1 receptor knockout mice exhibited time-dependent anandamide transport and cannabinoid CB1 receptor antagonists did not alter the effects of chronic ethanol on anandamide transport. Furthermore, anandamide transport was inhibited by acute ethanol in a time- and dose-dependent manner. Interestingly, acute ethanol-induced inhibition of anandamide transport was abolished in neurons exposed to chronic ethanol, suggesting that the anandamide transport processes may play a role in the development of long-term cellular tolerance to ethanol.

Increased ethanol consumption and preference and decreased ethanol sensitivity in female FAAH knockout mice.

Previous studies have shown that mice lacking cannabinoid (CB1) receptor gene consume markedly reduced levels of ethanol. Mice lacking the enzyme fatty acid amidohydrolase (FAAH) are severely impaired in their ability to degrade anandamide (AEA) and therefore represent a unique animal model in which to examine the function of AEA in vivo on ethanol-drinking behavior. In the current study, FAAH(-/-) mice were tested for ethanol, saccharin or quinine consumption and preference. Ethanol-induced hypothermia, and sleep time were used to evaluate the sensitivity to acute effects of ethanol. Ethanol intake and preference were increased only in female FAAH(-/-) mice. No significant difference in saccharin or quinine consumption or preference was observed between genotypes. Female FAAH(-/-) mice were less sensitive to the hypothermic and sedative/hypnotic effects of acute ethanol. Supersensitivity to exogenous AEA was noted in both male and female FAAH(-/-) mice. Following voluntary ethanol consumption, CB1 receptor levels and function were down-regulated in male FAAH(+/+), FAAH(-/-), and female FAAH(+/+) mice but not in female FAAH(-/-) mice. Our results suggest that absence of an effect in male mice indicates a sex-linked mechanism that is secondary (or modulatory) to FAAH function. Thus, the data suggest that FAAH may be indirectly related to ethanol intake and sensitivity and central endocannabinoidergic-mediated pathways may regulate ethanol consumption.

G9a-Mediated Histone Methylation Regulates Ethanol-Induced Neurodegeneration in the Neonatal Mouse Brain

Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces neuronal cell loss. However, the molecular mechanisms underlying these neuronal losses are still poorly understood. Here, we tested the possibility of histone methylation mediated by G9a (lysine dimethyltransferase) in regulating neuronal apoptosis in P7 mice exposed to ethanol. G9a protein expression, which is higher during embryogenesis and synaptogenic period compared to adult brain, is entirely confined to the cell nuclei in the developing brain. We found that ethanol treatment at P7, which induces apoptotic neurodegeneration in neonatal mice, enhanced G9a activity followed by increased histone H3 lysine 9 (H3K9me2) and 27 (H3K27me2) dimethylation. In addition, it appears that increased dimethylation of H3K9 makes it susceptible to proteolytic degradation by caspase-3 in conditions in which ethanol induces neurodegeneration. Further, pharmacological inhibition of G9a activity prior to ethanol treatment at P7 normalized H3K9me2, H3K27me2 and total H3 proteins to basal levels and prevented neurodegeneration in neonatal mice. Together, these data demonstrate that G9a mediated histone H3K9 and K27 dimethylation critically regulates ethanol-induced neurodegeneration in the developing brain. Furthermore, these findings reveal a novel link between G9a and neurodegeneration in the developing brain exposed to postnatal ethanol and may have a role in fetal alcohol spectrum disorders.

Role of Endocannabinoids and Cannabinoid CB1 Receptors in Alcohol-Related Behaviors

Abstract: This review presents the remarkable research during the past several years indicating that some of the pharmacological and behavioral effects of alcohol, including alcohol drinking and alcohol‐preferring behavior, are mediated through one of the most abundant neurochemical systems in the central nervous system, the endocannabinoid signaling system. The advances, with the discovery of specific receptors and the existence of naturally occurring cannabis‐like substances in the mammalian system and brain, have helped in understanding the neurobiological basis for drugs of abuse, including alcoholism. The cDNA and genomic sequences encoding G‐protein‐coupled cannabinoid receptors (CB1 and CB2) from several species have now been cloned. This has facilitated discoveries of endogenous ligands (endocannabinoids). To date, two fatty acid derivatives characterized to be arachidonylethanolamide and 2‐arachidonylglycerol have been isolated from both nervous and peripheral tissues. Both these compounds have been shown to mimic the pharmacological and behavioral effects of Δ9‐tetrahydrocannabinol, the psychoactive component of marijuana. The involvement of the endocannabinoid signaling system in tolerance development to drugs of abuse, including alcohol, were unknown until recently. Studies from our laboratory demonstrated for the first time the downregulation of CB1 receptor function and its signal transduction by chronic alcohol. The observed downregulation of CB1 receptor binding and its signal transduction results from the persistent stimulation of receptors by the endogenous CB1 receptor agonists arachidonylethanolamide and 2‐arachidonylglycerol, the synthesis of which is increased by chronic alcohol treatment. The deletion of CB1 receptor has recently been shown to block voluntary alcohol intake in mice, which is consistent with our previous findings where the DBA/2 mice known to avoid alcohol intake had significantly reduced brain CB1 receptor function. These findings suggest a role for the CB1 receptor gene in excessive alcohol drinking behavior and development of alcoholism. Ongoing investigations may lead to the development of potential therapeutic agents to modulate the endocannabinoid signaling system, which will be helpful for the treatment of alcoholism.

Effect of chronic ethanol exposure on mouse brain arachidonic acid specific phospholipase A2

The enzyme phospholipase A2 (PLA2), which catalyzes the hydrolysis of an ester bond at the sn-2 position of 1,2-sn-diacylglycerols, has been suggested to play an important role in regulating cellular functions. Although ethanol (EtOH)-induced activation of PLA2 activity was reported previously by us in mouse brain (Hungund et al., Neurochem Int 25: 321-325, 1994), its subcellular localization and biochemical properties have not been investigated. Therefore, in the present study, we examined the subcellular localization and characterization of EtOH-activated PLA2 activity in mouse brain. The results indicated that EtOH treatment decreased the specific activity of PLA2 for the first 48 hr, and then the activity increased and reached a peak level in both cytosol (1.6-fold) and membrane (1.7-fold) fractions at 96 hr of exposure. Specific activity was found to be higher in the membrane fraction than in the cytosol. Using differential density gradient centrifugation, subcellular localization of the membrane-associated PLA2 revealed that most of the EtOH-activated PLA2 specific activity was associated with the synaptic membrane (44%) followed by the nuclear membrane (13%). No significant increase in the PLA2 specific activity of mitochondrial and microsomal membranes was observed. No activity was detected in the myelin membrane. PLA2 specific activity of membranes from control and EtOH-exposed mouse brain exhibited preference for arachidonic acid over linoleic acid at the sn-2 position of glycero-3-phosphocholine (PC). No detectable PLA2 specific activity was found when PC containing oleic acid at the sn-2 position was used as a substrate. The present results also indicated that the PLA2 specific activity of membrane from control and EtOH-exposed mouse brain was insensitive to dithiothreitol, strongly stimulated by Ca2+, enhanced by glycerol, and inhibited by the cytosolic PLA2 (cPLA2) inhibitor methyl arachidonyl fluorophosphonate with an IC50 value of 3.33 microM. In summary, results suggest that the properties of EtOH-activated PLA2 activity found in mouse brain membrane fraction are similar to those of cPLA2 found in variety of cells, including mammalian brain.

Cannabinoid receptor agonist-stimulated [35S]guanosine triphosphateγs binding in the brain of C57BL/6 and DBA/2 mice

The two inbred strains of mice C57BL/6 (alcohol‐preferring) and DBA/2 (alcohol‐avoiding) mice have been shown to differ significantly in their preference for alcohol (EtOH). We have previously demonstrated the differences in the density and the affinity of cannabinoid (CB1) receptors in the brains of the two inbred C57BL/6 and DBA/2 mouse strains. In the present study, we investigated the CB1 receptor agonist‐stimulated guanosine‐5′‐O‐(3‐[35S]thio)‐triphosphate ([35S]GTPγS) binding in plasma membranes (PM) from C57BL/6 and DBA/2 mice. The results indicate that the net CP55,940‐stimulated [35S]GTPγS binding was increased with increasing concentrations of CB1 receptor agonists and GDP. The net CB1 receptor agonist (WIN55,212‐2 or HU‐210 or CP55,940)‐stimulated [35S]GTPγS binding was reduced significantly (–10% to –12%, P < 0.05) in PM from DBA/2 mice; no significant differences were observed in basal [35S]GTPγS binding among these strains. Nonlinear regression analysis of net CP55,940‐stimulated [35S]GTPγS binding showed that the Bmax of cannabinoid agonist‐stimulated binding was significantly reduced (–24%) in DBA/2 mice (Bmax = 12.43 ± 0.64 for C57BL/6 and 9.46 ± 0.98 pmol/mg protein for DBA/2; P < 0.05) without any significant changes in the G protein affinity. The pharmacological specificity of CP55,940‐stimulated [35S]GTPγS binding was examined with CB1 receptor antagonist SR141716A, and these studies indicated that CP55,940‐stimulated [35S]GTPγS binding was blocked by SR141716A, with a decrease in the IC50 values in the PM from the DBA/2 mouse strain. These results suggest that a signal transduction pathway(s) downstream from the CB1 receptor system may play an important role in controlling the voluntary EtOH consumption by these strains of mice. J. Neurosci. Res. 64:429–436, 2001.