Forebrain GABAergic cell-selective genetic manipulation in mice

小鼠前脑 GABA 能细胞选择性基因操作

• 批准号: 8556970
• 负责人: Kazutoshi Nakazawa
• 金额: $ 131.09万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8556970
• 关键词: Ablation; Acoustics; Adolescence; Adult; Affect; Amphetamines; Anhedonia; Animal Housing; Animal Model; Animals; Antioxidants; Anxiety; Attention; Auditory; Auditory area; Autistic Disorder; Autopsy; Behavior; Behavioral; Brain; Cell physiology; Cells; Chronic; Collaborations; Corpus striatum structure; Development; Disease; Dopamine; Dose; Down-Regulation; Drug Delivery Systems; Energy Metabolism; Enzymes; Etiology; Evoked Potentials; Exhibits; Frequencies; Functional disorder; Gene Targeting; Genes; Genetic; Glutamate Decarboxylase; Glutamate Receptor; Glutamates; Head; Hippocampus (Brain); Human; Impairment; Injection of therapeutic agent; Interneurons; Investigation; Knockout Mice; Lead; Link; Major Depressive Disorder; Measures; Mediating; Memory impairment; Mental disorders; Messenger RNA; Microdialysis; Mitochondria; Modafinil; Modeling; Mood Disorders; Motor Activity; Mus; Mutant Strains Mice; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NR1 gene; National Institute of Neurological Disorders and Stroke; Neurodevelopmental Disorder; Neurons; Nucleus Accumbens; Oxidation-Reduction; Oxidative Stress; PPAR gamma; Parvalbumins; Pathogenesis; Patients; Phenotype; Physiology; Play; Prosencephalon; Proteins; Psychotic Disorders; Rattus; Regulation; Reporting; Research; Rodent; Role; Schizophrenia; Sensory Process; Short-Term Memory; Signal Transduction; Site; Social Behavior; Social isolation; Sum; Symptoms; System; Testing; Training; Transgenic Mice; Transgenic Organisms; Weaning; Withdrawal; acetovanillone; awake; behavior test; cell type; excitatory neuron; frontal lobe; gamma-Aminobutyric Acid; genetic manipulation; in vivo; inhibitor/antagonist; interest; motivated behavior; mutant; neurodevelopment; neuropsychiatry; novel; oxidation; postnatal; prepulse inhibition; psychostimulant; receptor; recombinase; response; social; social stress; stem; synthetic enzyme

Since last year we have focused on the following four research questions. (1) Are there any other phenotypes which are reminiscent of schizophrenia in Ppp1r2-Cre/NR1 mutants? In human schizophrenia, impaired gamma oscillations are consistently reported and are thought to play a crucial role in a number of the symptoms. We already know that synchronized firing of cortical principal neurons is disturbed in the mutant NR1 mice, which may result in the abnormal oscillatory activity in the cortex. To investigate the integrity of gamma oscillations in mutant mice, last year we conducted local field potential (LFP) recoding in vivo before and after injection of a psychostimulant, Modafinil, which is known to induce robust and persistent gamma band oscillatory activity in mouse cortex. We found that, while the magnitude at the gamma frequency in floxed-NR1 control mice was enhanced 30-60 min after Modafinil injection, there was no such change in the mutant mice. To measure more physiologically-relevant gamma oscillatory activity, using recorded auditory-evoked gamma oscillations, we recorded spontaneous LFP activity and tone evoked potential from the auditory cortex of awake head-fixed mice placed in a stereotaxic apparatus. Forty Hz-click trains (80-dB)-evoked gamma oscillation, normalized to the tone-evoked response, was diminished in the mutant mice. On the other hand, the magnitude of spontaneous LFP fluctuations from mutant auditory cortex was enhanced. This suggests that GABAergic interneuron specific NMDAR hypofunction results in noisy spontaneous LFP fluctuations, which may impair synchronous gamma activity during sensory processing. Another major hallmark of human schizophrenia is an elevated subcortical dopamine release during psychosis. We explored the possibility of a similar dysregulation of dopaminergic tones in the Ppp1r2-Cre/NR1 KO mutants. Since last year, we assessed the psychostimulant-induced hyperlocomotion, which is supposed to be caused by increase in dopamine release in the striatum. We found that amphetamine (Amph)-evoked locomotor activity of the mutants is much higher than control littermates, suggesting an enhanced dopaminergic tone in the striatum in response to psychostimulant, which is seen in schizophrenia patients. Indeed, we confirmed an elevated dopamine release upon Amph injection by microdialysis, in collaboration with Daniel Paredes at NINDS. These two results, impaired gamma oscillation and dopamine dysregulation, provide additional schizophrenia-related pathophysiology to Ppp1r2-Cre/NR1 KO mutants. (2) How does social stress exacerbate the behavioral phenotypes of Ppp1r2-Cre/NR1 KO mutants? A growing body of evidence suggests that redox dysfunction and oxidative stress in brains during neurodevelopment may play a significant role in the etiology of schizophrenia. Previously we also demonstrated that social stress precipitates a number of the pathological phenotypes in the Ppp1r2-Cre/NR1 mutants. This environmental interaction has been hypothesized to involve alteration in oxidative stress mechanisms. To test this hypothesis, we investigated whether oxidative stress is involved in the emergence of schizophrenia-like behaviors in this animal model. Consistent with this idea, post-weaning social isolation (PWSI) augmented oxidative stress levels particularly in cortical PV interneurons and precipitated anxiety-like behavior in 8 week old mutants, a point at which group-housed animals displayed no such behavior. Interestingly, in mPFC of adult mutants we found decreased expression (both mRNA and protein) of a master regulator of mitochondria energy metabolism and anti-oxidation, PGC1 (peroxisome proliferator activated receptor gamma coactivator 1), which is normally highly expressed in fast-spiking PV interneurons. PWSI exacerbated the down-regulation of PGC1 in the mutants. Therefore, elevation of oxidative stress level in mutants appears to be due to impaired anti-oxidant defenses system, which occurs as a downstream consequence of the depletion of NMDARs in early postnatal corticolimbic interneurons. Chronic treatment with the antioxidant/NOX inhibitor apocynin, beginning from postnatal 2 weeks, largely diminished the signs of oxidative stress and alleviated anxiety and the spatial working memory deficit. Our results suggest that oxidative stress plays a critical role in PV interneuron dysfunction presumably through PGC1 downregulation and in the pathogenesis of schizophrenia-like phenotypes. (3) Is NMDAR hypofunction in interneurons really crucial for schizophrenia-related phenotypes? It is unclear whether NR1 ablation at other cell types, such as excitatory neurons, could also induce similar effects. To advance our investigation of whether NMDAR hypofunction-induced schizophrenia-like phenotypes may stem from interneuronal dysfunction, we utilized the Cre/loxP system to generate transgenic conditional knockout mice (G35-3-Cre/fNR1 KO), in which NR1 deletion is largely confined to cortical glutamatergic neurons. In contrast with interneuron-specific NR1 KO mice, we found that these mice do not exhibit comparable significant schizophrenia-like phenotypes in tests for working memory, social behavior, psychostimulant sensitivity, MK-801 response, and anhedonia or amotivation. Only prepulse inhibition (PPI) of acoustic startle was impaired in the mutants, suggesting attention deficits. This supports the refined hypothesis that NMDAR hypofunction must occur in cortical interneurons in order to model the hallmark symptoms of schizophrenia in rodents. (4) What is the impact of GABA decrease or Gad67 reduction on physiology and behavior? We previously observed Cre-targeted neuron-specific decrease in Gad67 levels in the Ppp1r2-Cre/NR1 mutants. Naturally, the next logical question was which, if any, of the phenotypes seen in the NR1 mutants can be explained by the decrease in Gad67. Alternatively, Gad67 reduction and further GABA level decrease have also been consistently reported in major depressive disorders (MDD) patients. Therefore, to directly assess the impact of Gad67 reduction in psychiatric disorder phenotypes, using the same Ppp1r2-Cre line we have been characterizing a novel transgenic mouse line in which Gad67, was ablated following postnatal day 7 selectively in 50% of cortical and hippocampal interneurons. Gad67 expression was reduced by 50% with a concomitant increase in Gad65 and the GABA levels in the mutant cortex and hippocampus were also significantly reduced after adolescence. This manipulation recapitulated several specific features common to major depression including anhedonia, a lack of motivated behavior, and pronounced social withdrawal. However, there were no impairments in tests of behavioral despair or schizophrenia-related behaviors (prepulse inhibition and spatial working memory). Perhaps consistent with this specific behavioral phenotype, in the nucleus accumbens amphetamine-evoked dopamine release. In all, these results suggest a relationship between the anhedonia aspect of major depressive disorders and impairments in subcortical dopamine regulation through alterations in cortical interneuron networks. We are investigating the underlying mechanisms of linking the cortical GABA reduction to striatal dopamine dysregulation. In sum, we suggest that the schizophrenia-like symptoms caused by disruptions in NMDA signaling may be uniquely due to cortical and hippocampal interneurons. Furthermore, it is becoming increasingly clear that alterations in oxidative stress may be crucial to the development of these phenotypes and that disruptions in gamma oscillations may play a fundamental role in the expression of the pathological condition.

去年以来，我们重点研究了以下四个问题。 (1) Ppp1r2-Cre/NR1 突变体中是否还有其他让人联想到精神分裂症的表型？ 在人类精神分裂症中，伽马振荡受损的情况不断被报道，并且被认为在许多症状中发挥着至关重要的作用。我们已经知道，NR1突变小鼠的皮质主要神经元的同步放电受到干扰，这可能导致皮质振荡活动异常。 为了研究突变小鼠伽马振荡的完整性，去年我们在注射精神兴奋剂莫达非尼之前和之后进行了体内局部场电位（LFP）重新编码，莫达非尼已知会在小鼠皮层中诱导强烈且持久的伽马带振荡活动。 我们发现，虽然 floxed-NR1 对照小鼠的伽马频率幅度在莫达非尼注射后 30-60 分钟有所增强，但突变小鼠中没有这种变化。 为了测量更多生理相关的伽马振荡活动，使用记录的听觉诱发伽马振荡，我们记录了放置在立体定位仪中的清醒头部固定小鼠的听觉皮层的自发 LFP 活动和音调诱发电位。四十赫兹的点击序列 (80-dB) 诱发的伽马振荡，标准化为音调诱发的反应，在突变小鼠中减弱。另一方面，突变听觉皮层自发 LFP 波动的幅度增强。这表明 GABA 能中间神经元特异性 NMDAR 功能减退导致嘈杂的自发 LFP 波动，这可能会损害感觉处理过程中的同步伽马活动。 人类精神分裂症的另一个主要特征是精神病期间皮质下多巴胺释放升高。我们探索了 Ppp1r2-Cre/NR1 KO 突变体中多巴胺能音调类似失调的可能性。自去年以来，我们评估了精神兴奋剂引起的过度运动，这被认为是由纹状体中多巴胺释放增加引起的。我们发现突变体的安非他明 (Amph) 诱发的运动活性远高于对照同窝小鼠，表明纹状体中对精神兴奋剂的反应增强的多巴胺能张力，这在精神分裂症患者中可见。 事实上，我们与 NINDS 的 Daniel Paredes 合作，证实通过微透析注射 Amph 后多巴胺释放增加。 伽玛振荡受损和多巴胺失调这两个结果为 Ppp1r2-Cre/NR1 KO 突变体提供了额外的精神分裂症相关病理生理学。 (2) 社会压力如何加剧Ppp1r2-Cre/NR1 KO突变体的行为表型？ 越来越多的证据表明，神经发育过程中大脑的氧化还原功能障碍和氧化应激可能在精神分裂症的病因学中发挥重要作用。此前我们还证明，社会压力会导致 Ppp1r2-Cre/NR1 突变体出现多种病理表型。假设这种环境相互作用涉及氧化应激机制的改变。为了检验这一假设，我们研究了氧化应激是否与该动物模型中精神分裂症样行为的出现有关。 与这一观点一致的是，断奶后社会隔离（PWSI）会增加氧化应激水平，尤其是皮质PV中间神经元的氧化应激水平，并在8周大的突变体中引发焦虑样行为，而群养动物此时不会表现出此类行为。有趣的是，在成年突变体的 mPFC 中，我们发现线粒体能量代谢和抗氧化的主要调节因子 PGC1（过氧化物酶体增殖物激活受体 γ 辅激活因子 1）的表达（mRNA 和蛋白质）降低，该蛋白通常在快速尖峰放电中高度表达。 PV中间神经元。 PWSI 加剧了突变体中 PGC1 的下调。因此，突变体中氧化应激水平的升高似乎是由于抗氧化防御系统受损，这是出生后早期皮质边缘中间神经元中 NMDAR 耗尽的下游结果。从出生后两周开始，使用抗氧化剂/氮氧化物抑制剂夹竹桃麻素进行长期治疗，可大大减少氧化应激的迹象，减轻焦虑和空间工作记忆缺陷。 我们的结果表明，氧化应激可能通过 PGC1 下调而在 PV 中间神经元功能障碍和精神分裂症样表型的发病机制中发挥关键作用。 (3) 中间神经元中的 NMDAR 功能减退真的对精神分裂症相关表型至关重要吗？ 目前尚不清楚其他细胞类型（例如兴奋性神经元）的 NR1 消融是否也会引起类似的效果。 为了进一步研究 NMDAR 功能低下诱导的精神分裂症样表型是否可能源于神经元间功能障碍，我们利用 Cre/loxP 系统生成转基因条件敲除小鼠 (G35-3-Cre/fNR1 KO)，其中 NR1 缺失大部分仅限于皮质谷氨酸能神经元。与中间神经元特异性 NR1 KO 小鼠相比，我们发现这些小鼠在工作记忆、社会行为、精神兴奋剂敏感性、MK-801 反应以及快感缺乏或动机缺失测试中没有表现出类似显着的精神分裂症表型。突变体中只有声惊吓的前脉冲抑制（PPI）受损，表明注意力缺陷。 这支持了一个完善的假设，即 NMDAR 功能减退必须发生在皮质中间神经元中，才能模拟啮齿类动物精神分裂症的标志性症状。 (4) GABA减少或Gad67减少对生理和行为有什么影响？ 我们之前观察到 Ppp1r2-Cre/NR1 突变体中 Gad67 水平的 Cre 靶向神经元特异性降低。 当然，下一个逻辑问题是 NR1 突变体中出现的哪些表型（如果有的话）可以用 Gad67 的减少来解释。另外，在重度抑郁症 (MDD) 患者中也一致报道了 Gad67 减少和 GABA 水平进一步下降。因此，为了直接评估 Gad67 对精神疾病表型减少的影响，我们使用相同的 Ppp1r2-Cre 系来表征一种新型转基因小鼠系，其中出生后第 7 天，50% 的皮质和海马中间神经元中的 Gad67 被选择性消除。青春期后，Gad67 表达减少 50%，同时 Gad65 增加，突变皮层和海马中的 GABA 水平也显着降低。这种操纵概括了重度抑郁症常见的几个具体特征，包括快感缺乏、缺乏动机行为和明显的社交退缩。然而，在行为绝望或精神分裂症相关行为（前脉冲抑制和空间工作记忆）的测试中没有出现任何损害。也许与这种特定的行为表型一致，在伏隔核中苯丙胺诱发的多巴胺释放。 总而言之，这些结果表明，重度抑郁症的快感缺乏方面与通过皮质中间神经元网络的改变而导致的皮质下多巴胺调节受损之间存在关系。 我们正在研究皮质 GABA 减少与纹状体多巴胺失调之间联系的潜在机制。 总之，我们认为由 NMDA 信号传导中断引起的精神分裂症样症状可能是由皮质和海马中间神经元独特引起的。此外，越来越清楚的是，氧化应激的改变可能对这些表型的发展至关重要，而伽马振荡的破坏可能在病理状况的表达中发挥重要作用。