Tsuneyuki Yamamoto

Endocannabinoids selectively enhance sweet taste

Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known orexigenic mediators that act via CB1 receptors in hypothalamus and limbic forebrain to induce appetite and stimulate food intake. Circulating endocannabinoid levels inversely correlate with plasma levels of leptin, an anorexigenic mediator that reduces food intake by acting on hypothalamic receptors. Recently, taste has been found to be a peripheral target of leptin. Leptin selectively suppresses sweet taste responses in wild-type mice but not in leptin receptor-deficient db/db mice. Here, we show that endocannabinoids oppose the action of leptin to act as enhancers of sweet taste. We found that administration of AEA or 2-AG increases gustatory nerve responses to sweeteners in a concentration-dependent manner without affecting responses to salty, sour, bitter, and umami compounds. The cannabinoids increase behavioral responses to sweet-bitter mixtures and electrophysiological responses of taste receptor cells to sweet compounds. Mice genetically lacking CB1 receptors show no enhancement by endocannnabinoids of sweet taste responses at cellular, nerve, or behavioral levels. In addition, the effects of endocannabinoids on sweet taste responses of taste cells are diminished by AM251, a CB1 receptor antagonist, but not by AM630, a CB2 receptor antagonist. Immunohistochemistry shows that CB1 receptors are expressed in type II taste cells that also express the T1r3 sweet taste receptor component. Taken together, these observations suggest that the taste organ is a peripheral target of endocannabinoids. Reciprocal regulation of peripheral sweet taste reception by endocannabinoids and leptin may contribute to their opposing actions on food intake and play an important role in regulating energy homeostasis.

Phosholipase C-Related Inactive Protein Is Involved in Trafficking of γ2 Subunit-Containing GABAA Receptors to the Cell Surface

The subunit composition of GABAA receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABAA receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABAA receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABAA receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABAA receptors and GABAA receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABAA receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABAA receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABAA receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

Mice lacking the schizophrenia-associated protein FEZ1 manifest hyperactivity and enhanced responsiveness to psychostimulants

FEZ1 (fasciculation and elongation protein zeta 1), a mammalian ortholog of Caenorhabditis elegans UNC-76, interacts with DISC1 (disrupted in schizophrenia 1), a schizophrenia susceptibility gene product, and polymorphisms of human FEZ1 have been associated with schizophrenia. We have now investigated the role of FEZ1 in brain development and the pathogenesis of schizophrenia by generating mice that lack Fez1. Immunofluorescence staining revealed FEZ1 to be located predominantly in gamma-aminobutyric acid-containing interneurons. The Fez1(-/-) mice showed marked hyperactivity in a variety of behavioral tests as well as enhanced behavioral responses to the psychostimulants MK-801 and methamphetamine. In vivo microdialysis revealed that the methamphetamine-induced release of dopamine in the nucleus accumbens was exaggerated in the mutant mice, suggesting that enhanced mesolimbic dopaminergic transmission contributes to their hyperactivity phenotype. These observations implicate impairment of FEZ1 function in the pathogenesis of schizophrenia.

Increases of CRF in the amygdala are responsible for reinstatement of methamphetamine-seeking behavior induced by footshock

Recent evidence suggests the involvement of corticotropin-releasing factor (CRF) in drug abuse. Here, we evaluated whether CRF modulates the reinstatement of methamphetamine (METH)-seeking behavior induced by stress using a drug-self administration paradigm in rats. Rats were trained to lever-press for intravenous METH (0.02 mg/infusion) accompanied by light and tone (drug-associated cues) and then underwent extinction training (saline substituted for METH without cues). Under the extinction condition, the inhibitory effects of a CRF receptor antagonist on the stress-induced reinstatement of METH-seeking behavior were assessed. Anxiety-like behaviors during METH withdrawal in METH self-administered rats were also evaluated. The non-selective CRF receptor antagonist α-helical CRF(9-41) attenuated METH-seeking behavior induced by footshock stress. CRF levels both in the amygdala and in plasma were significantly increased on day 10 of withdrawal after METH self-administration. However, plasma corticosterone concentrations were unchanged during the withdrawal. In addition, METH-seeking behavior was not affected by an inhibitor of corticosterone synthesis, metyrapone. In the elevated plus maze test, METH self-administered rats showed a decrease in the duration of time spent in the open arms on day 10 of withdrawal. The increased CRF levels in the amygdala may, at least in part, contribute to the footshock-induced reinstatement of METH-seeking behavior and the increase in anxiety-like behavior. The present findings indicate that CRF receptor antagonists would be useful as a therapeutic agent for METH-dependence.

Methamphetamine-seeking behavior is due to inhibition of nicotinic cholinergic transmission by activation of cannabinoid CB1 receptors

We previously reported the involvement of cannabinoid CB1 receptors (CB1Rs) and nicotinic acetylcholine receptors (nAChRs) in the reinstatement of methamphetamine (MAP)-seeking behavior (lever-pressing response for MAP reinforcement under saline infusion). The present study examined whether the reinstatement involves interactions between these receptors. Rats were trained to self-administer MAP with a light and tone (MAP-associated cues). Then, extinction sessions under saline infusion without cues were conducted. After that, a reinstatement tests were conducted by either presenting the cues or a MAP-priming injection. Systemic and intracranial administration of HU210, a cannabinoid CB1R agonist, into the nucleus accumbens core (NAC) and prelimbic cortex (PrC) reinstated MAP-seeking behavior. The reinstatement caused by the systemic HU210 treatment was attenuated by intracranial administration of AM251, a cannabinoid CB1R antagonist, into each region mentioned above. Meanwhile, reinstatement induced by the MAP-associated cues and MAP-priming injection was also attenuated by intracranial administration of AM251 in each region. In these regions, the attenuating effects of AM251 on the reinstatement induced by each stimulus were blocked by the intracranial administration of mecamylamine, a non-selective nAChR antagonist, but not by scopolamine, a muscarinic ACh receptor (mAChR) antagonist. Furthermore, the intracranial administration of DHbetaE, an alpha4beta2 nAChR antagonist, but not MLA, an alpha7 nAChR antagonist, into each region blocked the AM251-induced attenuation of the reinstatement. These findings suggest that relapses to MAP-seeking behavior may be due to two steps, first inhibition of ACh transmission by the activation of cannabinoid CB1Rs and then the inactivation of alpha4beta2 nAChRs.

Association of cathepsin E deficiency with the increased territorial aggressive response of mice

Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E‐deficient (CatE−/−) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild‐type littermates when housed individually in a single cage. The increased aggressive response of CatE−/− mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK‐1‐specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE−/− mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE−/− mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK‐1 receptor signaling system. Double immunofluorescence microscopy also revealed the co‐localization of SP with synaptophysin but not with microtubule‐associated protein‐2. Our data thus indicate that cathepsin E is associated with the SP/NK‐1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.

A tryptamine-derived catecholaminergic enhancer, (-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP], attenuates reinstatement of methamphetamine-seeking behavior in rats.

Relapse to drug craving is problematic in treatment for drug abuse. Evidence suggests inactivation of dopaminergic neurotransmission during drug withdrawal. Meanwhile, a tryptamine analogue, (-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP], has been reported to enhance electrical stimulation of monoamine release. This study examined the effect of (-)-BPAP on reinstatement of methamphetamine-seeking behavior in an animal model of relapse to drug abuse. Rats were trained to i.v. self-administer methamphetamine paired with a light and tone (methamphetamine-associated cues) under a fixed-ratio 1 schedule of reinforcement for 10 days. After extinction session under saline infusions without cues, a reinstatement test under saline infusions was begun. Reinstatement induced by methamphetamine-associated cues or methamphetamine-priming injections was attenuated by repeated administration of (-)-BPAP during the extinction phase. Acute administration of (-)-BPAP on test day dose-dependently attenuated both reinstatements. Acute administration of (-)-BPAP neither reinstated methamphetamine-seeking behavior alone nor affected methamphetamine self-administration. Pretreatment with either R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-23390), a dopamine D(1)-like receptor antagonist, or amisulpride, a dopamine D(2)-like receptor antagonist, did not appreciably affected the acute effect of (-)-BPAP on both reinstatements. Co-pretreatment with the dopamine receptor antagonists failed to alter the effects of (-)-BPAP. Meanwhile, pretreatment with a dopamine D(1)-like receptor agonist, (+/-)-6-chloro-7,8-dihydroxy-l-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297), dose-dependently attenuated reinstatement induced by the cues or methamphetamine-priming injections. In contrast to (-)-BPAP, pretreatment with SCH-23390 reversed the effects of SKF-81297. Our findings suggest activation of dopamine D(1)-like receptors results in attenuation of the reinstatement of methamphetamine-seeking behavior. Additionally, our findings provide evidence to develop (-)-BPAP and dopamine D(1)-like receptor agonists as an anti-relapse medication for methamphetamine abusers.

Genetic or pharmacological depletion of cannabinoid CB1 receptor protects against dopaminergic neurotoxicity induced by methamphetamine in mice.

Abstract Accumulating evidence suggests that cannabinoid ligands play delicate roles in cell survival and apoptosis decisions, and that cannabinoid CB1 receptors (CB1R) modulate dopaminergic function. However, the role of CB1R in methamphetamine (MA)‐induced dopaminergic neurotoxicity in vivo remains elusive. Multiple high doses of MA increased phospho‐ERK and CB1R mRNA expressions in the striatum of CB1R (+/+) mice. These increases were attenuated by CB1R antagonists (i.e., AM251 and rimonabant), an ERK inhibitor (U0126), or dopamine D2R antagonist (sulpiride). In addition, treatment with MA resulted in dopaminergic impairments, which were attenuated by CB1R knockout or CB1R antagonists (i.e., AM251 and rimonabant). Consistently, MA‐induced oxidative stresses (i.e., protein oxidation, lipid peroxidation and reactive oxygen species) and pro‐apoptotic changes (i.e., increases in Bax, cleaved PKC&dgr;‐ and cleaved caspase 3‐expression and decrease in Bcl‐2 expression) were observed in the striatum of CB1R (+/+) mice. These toxic effects were attenuated by CB1R knockout or CB1R antagonists. Consistently, treatment with four high doses of CB1R agonists (i.e., WIN 55,212‐2 36 mg/kg and ACEA 16 mg/kg) also resulted in significant oxidative stresses, pro‐apoptotic changes, and dopaminergic impairments. Since CB1R co‐immunoprecipitates PKC&dgr; in the presence of MA or CB1R agonists, we applied PKC&dgr; knockout mice to clarify the role of PKC&dgr; in the neurotoxicity elicited by CB1Rs. CB1R agonist‐induced toxic effects were significantly attenuated by CB1R knockout, CB1R antagonists or PKC&dgr; knockout. Therefore, our results suggest that interaction between D2R, ERK and CB1R is critical for MA‐induced dopaminergic neurotoxicity and that PKC&dgr; mediates dopaminergic damage induced by high‐doses of CB1R agonist. HighlightsMA or CB1R agonists increased CB1R mRNA level in the striatum of wild type mice.Inhibition of CB1R attenuated dopaminergic degeneration induced by MA.CB1R co‐immunoprecipitated PKC&dgr; in the presence of MA or CB1R agonists.Oxidative burdens contributed to PKC&dgr; cleavage and apoptotic activity.CB1R agonists‐induced dopaminergic degeneration was protected by PKC&dgr; inhibition. Graphical abstract Figure. No Caption available.

Selective apoptosis induction in the hippocampal mossy fiber pathway by exposure to CT105, the C-terminal fragment of Alzheimer's amyloid precursor protein

Beta-amyloid protein (Abeta), a proteolytic byproduct of Alzheimers amyloid precursor protein (APP), has been shown to play a central role in the development of Alzheimers disease (AD). In addition, recent studies strongly suggest that other byproducts of proteolysis, such as C-terminal fragments of APP (APP-CTF), are also critically involved in the AD pathology. To explore this possibility, we investigated the histopathological changes induced by repeated low-dose intrahippocampal injection of a recombinant 105 amino acid C-terminal fragment of APP (CT105). First, we carried out a behavioral analysis by using the three-panel runway task, and found that the working memory was significantly impaired by CT105 exposure. Then, via propidium iodide staining, we encountered a number of cells exhibiting fragmented or shrank nuclei in the mossy fiber pathway (stratum lucidum and dentate hilus) in CT105-treated rats. These cells were positive for single-stranded DNA (ssDNA), an apoptosis-specific marker, and thus were considered to be apoptotic. Some of the ssDNA-positive cells were also positive for somatostatin. But neither ionized calcium-binding adapter molecule 1 (Iba1) nor S100beta occurred in ssDNA-positive cells. These findings suggest that CT105 induces apoptotic changes in cells of neuronal origin. Quantitative analysis showed that the densities of ssDNA-positive cells in the mossy fiber pathway were significantly higher in CT105-treated rats than in control animals. The present results suggest that CT105 causes dysfunction in the hippocampal mossy fiber system, and also provide some key to understand the relationship between APP-CTF and glutamatergic synaptic dysregulation in AD.

Prevention of drug priming- and cue-induced reinstatement of MDMA-seeking behaviors by the CB1 cannabinoid receptor antagonist AM251

BACKGROUND 3,4-Methylenedioxymethamphetamine (MDMA), a methamphetamine (METH) derivative, exhibits METH-like actions at monoamine transporters and positive reinforcing effects in rodents and primates. The purposes of the present study were to determine whether cross-reinstatement would be observed between MDMA and METH and if the cannabinoid receptor, a receptor known to play critical roles in the brain reward system, could modulate MDMA craving. METHODS Rats were trained to press a lever for intravenous MDMA (0.3mg/infusion) or METH (0.02mg/infusion) infusions under a fixed ratio 1 schedule paired with drug-associated cues (light and tone). Following drug self-administration acquisition training, rats underwent extinction training (an infusion of saline). Reinstatement tests were performed once the extinction criteria were achieved. RESULTS In MDMA-trained rats, the MDMA-priming injection (3.2mg/kg, i.p.) or re-exposure to MDMA-associated cues reinstated MDMA-seeking behavior. Additionally, a priming injection of METH (1.0mg/kg, i.p.) also reinstated MDMA-seeking behavior. In contrast, none of the MDMA doses reinstated METH-seeking behavior in the METH-trained rats. The CB1 cannabinoid receptor antagonist AM251 markedly attenuated the MDMA-seeking behaviors induced by MDMA-priming injection or re-exposure to MDMA-associated cues in a dose-dependent manner. CONCLUSIONS These findings show that MDMA has obvious addictive potential for reinstating drug-seeking behavior and that METH can be an effective stimulus for reinstating MDMA-seeking behaviors. Furthermore, based on the attenuating effect of AM251 in the reinstatement of MDMA-seeking behaviors, drugs that suppress CB1 receptors may be used in treatment of MDMA dependence.