Kazuto Sakoori

Endogenous Nociceptin (Orphanin FQ) Suppresses Basal Hedonic State and Acute Reward Responses to Methamphetamine and Ethanol, but Facilitates Chronic Responses

The opioid peptide nociceptin (orphanin FQ) suppresses drug reward, drug self-administration, and impedes some of the processes believed to underlie the transition to addiction. As virtually all previous studies have used administration of nociceptin receptor agonists to evaluate the role of nociceptin on addiction-like behavior, the current study used a pharmacological (nociceptin receptor antagonist) and genetic (nociceptin receptor knockout mice) approach to elucidate the role of endogenous nociceptin. The nociceptin receptor antagonist UFP-101 induced a modest place preference, and enhanced the conditioned place preference induced by methamphetamine. In agreement with this, nociceptin receptor knockout mice had slightly enhanced methamphetamine and ethanol conditioned place preferences compared to wild-type mice. This effect did not appear to depend on differences in learning ability, as nociceptin receptor knockout mice had slightly weaker-conditioned place aversions to lithium chloride, the κ-opioid receptor agonist, U50488H, and the general opiate antagonist, naloxone. The development of behavioral sensitization to methamphetamine was lower in nociceptin receptor knockout mice, and attenuated by UFP-101 administration to wild-type mice. Additionally, ethanol consumption and preference in a two-bottle choice test was lower in nociceptin receptor knockout mice, though ethanol-stimulated locomotion was stronger. Whereas the rewarding effect of methamphetamine and ethanol following chronic treatment, as measured by place conditioning, strengthened in wild-type mice, this effect was absent in nociceptin receptor knockout mice. These results suggest that endogenous N/OFQ suppresses basal and drug-stimulated increases in hedonic state, and plays either a permissive or facilitatory role in the development of addiction.

Maintenance of conditioned place preferences and aversion in C57BL6 mice : effects of repeated and drug state testing

In order to determine the factors affecting the expression of place conditioning (i.e. conditioned place preference or aversion), groups of C57BL6 mice were conditioned to morphine, cocaine or naloxone (all drugs 4 mg/kg s.c.) and tested intermittently (up to 4 weeks) or repeatedly (daily). When tested once only in a drug-free state, the expression of place conditioning to all drugs generally strengthened with the passing of time and was only marginally different when tested once more later. The expression of place conditioning was remarkably resistant to extinction during repeated daily testing, though decreases were observed over 2 weeks of testing. Subsequently, a small and non-significant degree of spontaneous recovery of the expression of place conditioning was observed when animals were undisturbed for an extended period of time. Administering animals the same drug to which they were conditioned 20 min prior to testing (i.e. drug state testing), either prior to or following repeated testing, enhanced expression of morphine-induced conditioned place preference, whereas expression of cocaine-induced conditioned place preference was unaffected. When vehicle was administered prior to testing, there was no evidence of naloxone-induced conditioned place aversion, whereas naloxone administration maintained conditioned place aversion. These results show that the expression of place conditioning in C57BL6 mice does not readily diminish over time, but on the contrary, tends to strengthen if tested only once or intermittently. Thus, the hedonic properties of drugs may be more clearly revealed during long-term, rather than short-tem testing, and in some instances, when tested in the drugged state.

SLITRK6 mutations cause myopia and deafness in humans and mice

Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness.

The NOP (ORL1) receptor antagonist Compound B stimulates mesolimbic dopamine release and is rewarding in mice by a non-NOP-receptor-mediated mechanism.

Compound B (1‐[(3R, 4R)‐1‐cyclooctylmethyl‐3‐hydroxymethyl‐4‐piperidyl]‐3‐ethyl‐1,3‐dihydro‐2H‐benzimidazol‐2‐one, CompB) is a nociceptin/orphanin FQ (N/OFQ) antagonist showing high selectivity for the NOP (ORL1) receptor over classical opioid receptors. We studied the effect of subcutaneous CompB administration on the release of mesolimbic dopamine (DA) and the expression of hedonia in mice. CompB (0.3–30 mg kg−1) dose dependently stimulated mesolimbic DA release as measured by in vivo freely moving microdialysis, without any change in locomotor activity. However, intracerebroventricular administered N/OFQ (endogenous agonist of the NOP receptor, 6 nmol) did not influence CompB‐ (10 mg kg−1) induced DA release, despite clearly suppressing release when administered alone. Studies using NOP receptor knockout mice and no‐net‐flux microdialysis revealed mildly, but not statistically significantly higher endogenous DA levels in mice lacking the NOP receptor compared to wild‐type mice. Administration of CompB (10 mg kg−1) induced identical increases in mesolimbic DA release in wild‐type and NOP receptor knockout mice. CompB was rewarding in approximately the same dose range in which CompB induced major increases in mesolimbic DA release when assayed using a conditioned place preference paradigm. The rewarding effect of CompB (30 mg kg−1) was maintained in NOP receptor knockout mice. These results show that CompB stimulates mesolimbic DA release and is rewarding by an action independent of the NOP receptor, the precise site of which is unclear. Consequently, caution should be exercised when interpreting the results of studies using this drug, particularly when administered by a peripheral route.

Impaired cognitive function and altered hippocampal synapse morphology in mice lacking Lrrtm1, a gene associated with schizophrenia.

Recent genetic linkage analysis has shown that LRRTM1 (Leucine rich repeat transmembrane neuronal 1) is associated with schizophrenia. Here, we characterized Lrrtm1 knockout mice behaviorally and morphologically. Systematic behavioral analysis revealed reduced locomotor activity in the early dark phase, altered behavioral responses to novel environments (open-field box, light-dark box, elevated plus maze, and hole board), avoidance of approach to large inanimate objects, social discrimination deficit, and spatial memory deficit. Upon administration of the NMDA receptor antagonist MK-801, Lrrtm1 knockout mice showed both locomotive activities in the open-field box and responses to the inanimate object that were distinct from those of wild-type mice, suggesting that altered glutamatergic transmission underlay the behavioral abnormalities. Furthermore, administration of a selective serotonin reuptake inhibitor (fluoxetine) rescued the abnormality in the elevated plus maze. Morphologically, the brains of Lrrtm1 knockout mice showed reduction in total hippocampus size and reduced synaptic density. The hippocampal synapses were characterized by elongated spines and diffusely distributed synaptic vesicles, indicating the role of Lrrtm1 in maintaining synaptic integrity. Although the pharmacobehavioral phenotype was not entirely characteristic of those of schizophrenia model animals, the impaired cognitive function may warrant the further study of LRRTM1 in relevance to schizophrenia.

Enhanced nicotine sensitivity in nociceptin/orphanin FQ receptor knockout mice

The opioid peptide nociceptin (orphanin FQ) has been implicated in reward, reinforcement and addiction. The current study sought evidence of a role of endogenous nociceptin in nicotine responses by studying nociceptin receptor (NOP) knockout mice. The results were: (1) NOP receptor knockout mice showed enhanced anxiety-like behavior on an elevated plus maze. Whereas nicotine (0.05-0.5 mg/kg) tended to be anxiogenic in wild-type mice, NOP receptor KO mice were resistant to this effect, though interpretation was confounded by their stronger anxiety-like behavior. (2) When presented increasing nicotine concentrations (3-50 microg/ml) in a bottle choice drinking paradigm, there were no genotype-dependent differences in nicotine preference. However, NOP receptor knockout mice consumed more 3 microg/ml nicotine solution when considered in absolute terms. (3) NOP receptor knockout mice showed stronger hypothermic responses to nicotine (1 or 2 mg/kg) administration. (4) There was modest evidence that NOP receptor KO mice showed attenuated behavioral sensitization to a low dose of nicotine (0.05 mg/kg) during repeated daily treatment. (5) NOP receptor knockout mice more rapidly tolerated the sedative effect of nicotine (1 mg/kg), due partially to slightly lower locomotion on first treatment. (6) NOP receptor knockout mice, unlike wild-type mice, showed a significant mecamylamine (2.5 mg/kg) induced conditioned place aversion to nicotine (24 mg/kg/day) withdrawal. These results show that mice lacking the influence of endogenous N/OFQ mice are hypersensitive to nicotine in most measures, showing a role of endogenous nociceptin in modulating or mediating the acute effects of nicotine, and possibly nicotine addiction.

Lack of evidence of a role for the neurosteroid allopregnanolone in ethanol-induced reward and c-fos expression in DBA/2 mice

Previous studies using the 5alpha-reductase inhibitor finasteride suggest that progesterone metabolites, particularly the endogenous neurosteroid allopregnanolone, mediate some of the effects of ethanol. Consequently, we studied the effect of finasteride (2 x 25 mg/kg s.c., 12 h apart) pretreatment on the acquisition and expression of ethanol (2 g/kg i.p.) induced conditioned place preference and c-fos expression in DBA/2 mice; a strain known to be particularly sensitive to ethanol. Ethanol administration induced a clear conditioned place preference and widespread c-fos expression, with elements of the extended amygdala, Edinger-Westphal nucleus and paraventricular nucleus being especially sensitive. However, despite an approximately 99% decrease in whole brain allopregnanolone content, finasteride pretreatment had remarkably little effect on either ethanol-induced conditioned place preference or ethanol-induced c-fos expression. Thus, aside from a general stimulatory effect on c-fos expression in the ventral tegmental area, and generally mild depression of locomotor activity, no other effects of finasteride or interaction with ethanol effects were identifiable. Together, these studies suggest that endogenous allopregnanolone plays little part in mediating acute ethanol-induced reward or neural activation in DBA/2 mice.

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

Expression of morphine-conditioned place preference is more vulnerable than naloxone-conditioned place aversion to disruption by nociceptin in mice

The opioid peptide nociceptin (orphanin FQ) suppresses the incentive and rewarding properties of drugs. Thus, targeting the nociceptin system may be beneficial in treating drug addiction. The effects of nociceptin (0-1.5nmol intracerebroventricular) on the expression of morphine- (6mg/kg subcutaneous) and naloxone-(6mg/kg subcutaneous) induced place conditioning were examined in mice. Whereas doses of 0.5nmol nociceptin and above disrupted expression of morphine-conditioned place preference (CPP), naloxone-conditioned place aversion (CPA) remained intact at all doses of nociceptin tested. Doses of 0.5nmol nociceptin and above suppressed locomotion, though this appeared unrelated to the expression of place conditioning. These results suggest that nociceptin more potently blocks the ability of reward-associated cues than aversion-associated cues to influence behavioral biases.

Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients

ZIC2 is a causal gene for holoprosencephaly and encodes a zinc-finger-type transcriptional regulator. We characterized Zic2kd/+ mice with a moderate (40%) reduction in Zic2 expression. Zic2kd/+ mice showed increased locomotor activity in novel environments, cognitive and sensorimotor gating dysfunctions, and social behavioral abnormalities. Zic2kd/+ brain involved enlargement of the lateral ventricle, thinning of the cerebral cortex and corpus callosum, and decreased number of cholinergic neurons in the basal forebrain. Because these features are reminiscent of schizophrenia, we examined ZIC2 variant-carrying allele frequencies in schizophrenia patients and in controls in the Japanese population. Among three novel missense mutations in ZIC2, R409P was only found in schizophrenia patients, and was located in a strongly conserved position of the zinc finger domain. Mouse Zic2 with the corresponding mutation showed lowered transcription-activating capacity and had impaired target DNA-binding and co-factor-binding capacities. These results warrant further study of ZIC2 in the pathogenesis of schizophrenia.