Unraveling micro-endophenotypes of psychiatric disorders at the molecular, cellular and circuit levels

金沢大学附属病院前年度までに、薬理学的操作によるマイルドな慢性酸化ストレス負荷（2-cyclohexene-1-one；以下、CHX）が成熟ラットの様々な意思決定に影響を与えるが、単独では必ずしもうつ病などの特定の精神疾患を模倣しないこと、主にドパミン関連機能に影響を与えることを確認し、論文で発表した。さらに、慢性酸化ストレス負荷がコカイン慢性投与によって形成される逆耐性現象において、特に常同行動を遅発性に増強すること、線条体内で膜表面に発現するドパミン輸送体（糖鎖修飾で区別される）を増加させることを行動薬理実験及びウエスタン法で確認し、現在投稿準備中である。一方、in situ glutathionylationを可視化して検出する系の開発を目指し、ラット脳を用いて様々な条件検討を行ったところ、MPTPによる障害ラットモデルにて、血管内皮細胞に一致するシグナルを検出した。同様のシグナルはグルタチオン化タンパク質が多く存在すると予想される気管内皮細胞でも強く検出された。そこでタンパク質レベルでの追加確認のため、血管内皮細胞に存在しグルタチオン化が推定されているタンパク質（iNOS, eNOSなど）を幾つか選択し、抗グルタチオン抗体を用いた免疫沈降法を試みたが、残念ながらシグナルは検出されなかった。また、アポトーシスモデルでのin situ glutathionylation検出の試みも行ったが、期待したシグナルは得られなかった。これまでの最大の問題は成熟ラット脳内におけるポジティブコントロールとしてのグルタチオン化タンパク質が見つからないことであったが、最近ハンチントン舞踏病モデル動物及び患者脳の線条体にてカルシウムチャネルの一つであるTRPC5のグルタチオン化がin vivoで更新していることが報告された。そのため、高濃度CHX連続投与で強めの慢性酸化ストレスを負荷した成熟ラット脳を準備し、再度免疫沈降法と、in situ glutathionylation可視化法でグルタチオン化TRPC5の検出を再検討している。研究課題/領域番号:25116510, 研究期間(年度):2013-04-01 – 2016-03-3

Elucidation of neural computation for prediction and decision making: toward better human understanding and applications

金沢大学附属病院初年度に引き続き、cfos-LacZトランスジェニックラットを用いて道具学習時に脳内で誘導されるc-fosの発現部位とそのパターン変化について、訓練獲得状況との相関を見ながら検討を行った。まず、線条体でのc-fos発現は、訓練開始2,7,21,28日目では側坐核のmedial shellの他は介在神経とグリア細胞に発現が限局し、中型有棘細胞での発現は極めて乏しいことを確認した。ところが訓練１４日目では、線条体の全域で一致して中型有棘細胞に強い発現誘導を認めた。この発現はドパミンD1受容体陽性細胞と同D2受容体陽性細胞の両方で認められたが全体にD1優位で、特に目標指向性行動に関わる背内側線条体でD1/D2比が高く、反対に習慣に関わる背外側線条体でその比が最も低かった。この訓練１４日目前後で行動の質的変化が起きていないかを検討する目的にて、訓練反復がラットのレバー押しの速度にどのように影響を与えるかを解析した。動物は３０分のセッション間に最低数百から最高数千回のレバー押しを行うが、各レバー押しのタイミングデータから２回のレバー押しの間隔の逆数をレバー押し速度とし、其の速度変化について訓練初期から訓練後期に至るまでの推移について検討したところ（N=39-40）、動物は訓練当初は遅いレバー押し行動が行動全体の６０％近くを占めるが、訓練の反復に従い次第に早いレバー押し行動が増え、訓練１４日目前後で遅い行動から早い行動へのシフトが起きていることが確認された。この道具訓練はランダム間隔スケジュールを採用しているため、ラットは訓練初期の遅いレバー押しでも有効に餌を獲得することができており、すなわち遅いレバー押し行動はノイズ成分ではない。そこで現在「遅いレバー押し行動」が目標指向性行動に、「早いレバー押し行動」が習慣に相当すると仮説を立て、これを行動薬理学的に証明を試みている。研究課題/領域番号:26120712, 研究期間(年度):2014-04-01 – 2016-03-3

Identification of an unconventional process of instrumental learning characteristically initiated with outcome devaluation-insensitivity and generalized action selection.

The distinction between goal-directed action and habitual response, particularly with respect to moderate or extended appetitive instrumental training, is well documented; however, the propensity toward instrumental behavior in the early training stage has not been elucidated. In this study, we trained Sprague Dawley rats to press a lever to obtain food as an outcome for various time periods and monitored the changes in their sensitivity to outcome devaluation and choice between the levers they had been trained with and unfamiliar levers. After the extensive training with a random interval schedule, the rats were insensitive to outcome devaluation, and exhibited a typical habit-like phenotype, as previously reported, and the untrained leverpresses were relatively rare and sporadic. During the initial stage of training (≤1 week), the rats exhibited a similar insensitivity to the devaluation; however, in contrast to the overtrained condition, they performed distinctive unbiased leverpresses on both the trained and untrained levers. Thus, we propose a possibility that, contrary to the authentic concept that instrumental learning is initiated with an outcome devaluation-sensitive goal-directed stage, under some conditions, this learning can unconventionally begin with the initial stage that is distinct from both goal-directed action and habitual response. © The Author(s) 2017

Repeated Exposure of Adult Rats to Transient Oxidative Stress Induces Various Long-Lasting Alterations in Cognitive and Behavioral Functions

Exposure of neonates to oxidative stress may increase the risk of psychiatric disorders such as schizophrenia in adulthood. However, the effects of moderate oxidative stress on the adult brain are not completely understood. To address this issue, we systemically administrated 2-cyclohexen-1-one (CHX) to adult rats to transiently reduce glutathione levels. Repeated administration of CHX did not affect the acquisition or motivation of an appetitive instrumental behavior (lever pressing) rewarded by a food outcome under a progressive ratio schedule. In addition, response discrimination and reversal learning were not affected. However, acute CHX administration blunted the sensitivity of the instrumental performance to outcome devaluation, and this effect was prolonged in rats with a history of repeated CHX exposure, representing pro-depression-like phenotypes. On the other hand, repeated CHX administration reduced immobility in forced swimming tests and blunted acute cocaine-induced behaviors, implicating antidepressant-like effects. Multivariate analyses segregated a characteristic group of behavioral variables influenced by repeated CHX administration. Taken together, these findings suggest that repeated administration of CHX to adult rats did not cause a specific mental disorder, but it induced long-term alterations in behavioral and cognitive functions, possibly related to specific neural correlates

Pre-stress performance in an instrumental training predicts post-stress behavioral alterations in chronically stressed rats

Stress is a major factor in the development of major depressive disorder (MDD), but few studies have assessed individual risk based on pre-stress behavioral and cognitive traits. To address this issue, we employed appetitive instrumental lever pressing with a progressive ratio (PR) schedule to assess these traits in experimentally naive Sprague-Dawley rats. Based on four distinct traits that were identified by hierarchical cluster analysis, the animals were classified into the corresponding four subgroups (Low Motivation, Quick Learner, Slow Learner, and Hypermotivation), and exposed to chronic unpredictable stress (CUS) before monitoring their post-stress responses for 4 weeks. The four subgroups represented the following distinct behavioral phenotypes after CUS: the Low Motivation subgroup demonstrated weight loss and a late-developing paradoxical enhancement in PR performance that may be related to inappropriate decision-making in human MDD. The Quick Learner subgroup exhibited a transient loss of motivation and the habituation of serum corticosterone (CORT) response to repeated stress. The Slow Learner subgroup displayed resistance to demotivation and a suppressed CORT response to acute stress. Finally, the Hypermotivation subgroup exhibited resistance to weight loss, habituated CORT response to an acute stress, and a long-lasting amotivation. Overall, we identified causal relationships between pre-stress traits in the performance of the instrumental training and post-stress phenotypes in each subgroup. In addition, many of the CUS-induced phenotypes in rats corresponded to or had putative relationships with representative symptoms in human MDD. We concluded that the consequences of stress may be predictable before stress exposure by determining the pre-stress behavioral or cognitive traits of each individual in rats. ©2015 Iguchi, Kosugi, Lin, Nishikawa, Minabe and Toda

Altered dendritic spine plasticity in cocaine-withdrawn rats

金沢大学附属病院神経科精神科Chronic cocaine treatment is associated with changes in dendritic spines in the nucleus accumbens, but it is unknown whether this neuroplasticity alters the effect of a subsequent cocaine injection on spine morphology and protein content. Three weeks after daily cocaine or saline administration, neurons in the accumbens were filled with the lipophilic dye, DiI. Although daily cocaine pretreatment did not alter spine density compared with daily saline, there was a shift from smaller to larger diameter spines. During the first 2 h after an acute cocaine challenge, a bidirectional change in spine head diameter and increase in spine density was measured in daily cocainepretreated animals. In contrast, no change in spine diameter or density was elicited by a cocaine challenge in daily saline animals during the first 2 h after injection. However, spine density was elevated at 6 h after a cocaine challenge in daily saline-pretreated animals. The time-dependent profile of proteins in the postsynaptic density subfraction elicited by a cocaine challenge in daily cocaine-pretreated subjects indicated that the changes in spine diameter and density were associated with a deteriorating actin cytoskeleton and a reduction in glutamate signaling-related proteins. Correspondingly, the amplitude of field potentials in accumbens evoked by stimulating prefrontal cortex was reduced for up to 6 h after acute cocaine in daily cocaine-withdrawn animals. These data indicate that daily cocaine pretreatment dysregulates dendritic spine plasticity elicited by a subsequent cocaine injection. Copyright © 2009 Society for Neuro science