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Forebrain GABAergic cell-selective genetic manipulation in mice

小鼠前脑 GABA 能细胞选择性基因操作

基本信息

批准号：
8745739
负责人：
Kazutoshi Nakazawa
金额：
$ 113.91万
依托单位：
NATIONAL INSTITUTE OF MENTAL HEALTH
依托单位国家：
美国
项目类别：
财政年份：
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/8745739
关键词：
Ablation Abnormal coordination Acoustic Stimulation Acoustics Action Potentials 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 Defect Depressed mood Development Disease Dopamine Dose Down-Regulation Drug Targeting Energy Metabolism Environmental Risk Factor Enzymes Event Exhibits Frequencies Functional disorder Gene Targeting Genes Genetic Glutamate Decarboxylase Glutamate Receptor Glutamates Hippocampus (Brain)Human Impairment Interneurons Knockout Mice Lead Major Depressive Disorder Manuscripts Measures Mediating Memory impairment Mental disorders Messenger RNA Mitochondria Modeling Mood Disorders Mus Mutant Strains Mice N-Methyl-D-Aspartate Receptors N-Methylaspartate NR1 gene Neurodevelopmental Disorder Neurons Noise Nucleus Accumbens Oxidation-Reduction Oxidative Stress PPAR gamma Parvalbumins Pathogenesis Pattern Phase Phenotype Physiologic pulse Physiology Play Preparation Presynaptic Terminals Prosencephalon Proteins Psychiatry Pyramidal Cells Rattus Regulation Research Rodent Rodent Model Role Schizophrenia Short-Term Memory Signal Transduction Site Slice Social Behavior Social isolation Symptoms Synapses System Testing Tissues Training Transgenic Mice Transgenic Organisms Translating Weaning Withdrawal acetovanillone behavior test cell type excitatory neuron frontal lobe frontier gamma-Aminobutyric Acid genetic manipulation hippocampal pyramidal neuron induced pluripotent stem cell inhibitor/antagonist interest motivated behavior mutant neural circuit neuropsychiatry novel oxidation postnatal postsynaptic prepulse inhibition presynaptic psychostimulant receptor recombinase response social stem synthetic enzyme theories transmission process

项目摘要

Since last year we have focused on the following four research questions. (1) How does oxidative stress exacerbate the behavioral phenotypes of Ppp1r2-Cre/fGluN1 KO mutants? 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 (Jiang et al, Biol Psychiatry, 2013). We have also submitted a review manuscript entitled Convergence of genetic and environmental factors on parvalbumin-positive interneurons in schizophrenia to Frontier in Behav Neurosci. (2) Periodic auditory stimulation entrains the electro-encephalogram (EEG) to a specific phase and frequency, often referred to as the auditory steady-state response (ASSR). In both human and animal models, the ASSR has been used to assess the functional integrity of neural circuits that support synchronization. In order to investigate this phenomenon in a rodent model, we recorded LFPs directly from the primary auditory cortex of GABAergic interneuron-specific NMDAR hypofunction mice (Ppp1r2-Cre/fGluN1 KO mice), using the ASSR paradigm to assess both tone-evoked ASSRs and baseline LFP fluctuations. We then compared the relation of these two measures subjects. In mice with NMDAR hypofunction in cortical GABA neurons, we found that click train-evoked oscillatory LFP responses from auditory cortex are diminished. Specifically, these mutants show diminished power and phase locking at 40-Hz. Although the broadband baseline LFP power is also elevated, the signal-to-noise ratio reduction in the mutants appears to be largely due to external stimulis inability to evoke normal oscillatory power (Nakao et al., submitted). Because Ppp1r2-Cre-mediated genetic manipulation is confined to interneurons, it is unlikely that the defect is intrinsic to excitatory pyramidal neurons. Why, then, is the synchronized power of postsynaptic excitatory neurons diminished in Ppp1r2-cre/fGluN1 KO mutants? Postulating that the defect is one of synchrony between GABAergic inputs and excitatory neurons, we used double-patch recordings of sIPSCs from nearby pyramidal cells in tissue slices to test the theory. If synchronous GABAergic inputs are disturbed, IPSC events between nearby pyramidal neurons will be desynchronized. As expected, we found this to be true in mPFC slices from mutants at P28-P42. These results suggest that in our mutants, spike transmission, which must be precise to translate impulses into the correct postsynaptic responses, is disturbed. In order to identify the cellular mechanisms and impairments that best explain these mutant phenotypes, we assessed several mechanisms implicated in the desynchronization of IPSC events in postsynaptic pyramidal neurons. We applied brief pulses of current to presynaptic fast-spiking neurons to evoke unitary IPSCs in postsynaptic pyramidal neurons. This allowed us to see whether or not action potential impulses arriving at presynaptic terminals fail to evoke proper GABA release. In the mutants P28-P42, we found, GABA release is initially depressed, but subsequently IPSC amplitudes gradually increase. This pattern stands in sharp contrast to the unitary IPSC patterns observed in floxed controls, in which synapse transmission in response to the first impulse is robust and highly reliable. These results suggest that in these mutants, precise high-fidelity spike transmission at fast-spiking neuron/pyramidal neuron synapses is impaired (Zsiros et al, in preparation). (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. Since last year we utilized the Cre/loxP system to generate transgenic conditional knockout mice (G35-3-Cre/fGluN1 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 (Rompala et al, PLOS One, 2013). (4) What is the impact of GABA decrease or Gad67 reduction on physiology and behavior? 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 (Kolata et al, submitted).

去年以来，我们重点研究了以下四个问题。 (1)氧化应激如何加剧Ppp1r2-Cre/fGluN1 KO突变体的行为表型？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，特别是在皮层PV中间神经元中，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，该调节因子通常在快速峰值的 PV 中间神经元中高度表达。 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，氧化应激在PV中间神经元功能障碍中发挥着关键作用，可能是通过PGC1下调以及在精神分裂症样表型的发病机制中发挥着关键作用（Jiang等人，Biol Psychiatry，2013）。我们还向《行为神经学前沿》提交了一篇题为《精神分裂症中小清蛋白阳性中间神经元的遗传和环境因素的融合》的综述手稿。 (2) 周期性听觉刺激将脑电图 (EEG) 引入特定相位和频率，通常称为听觉稳态响应 (ASSR)。在人类和动物模型中，ASSR 已被用于评估支持同步的神经回路的功能完整性。为了在啮齿动物模型中研究这一现象，我们直接从 GABA 能中间神经元特异性 NMDAR 功能障碍小鼠（Ppp1r2-Cre/fGluN1 KO 小鼠）的初级听觉皮层记录了 LFP，使用 ASSR 范例来评估音调诱发的 ASSR 和基线 LFP 波动。然后我们比较了这两个测量主题的关系。在皮质 GABA 神经元 NMDAR 功能低下的小鼠中，我们发现听觉皮层的点击列车诱发的振荡 LFP 反应减弱。具体来说，这些突变体在 40 Hz 时表现出功率和锁相减弱。尽管宽带基线 LFP 功率也升高，但突变体的信噪比降低似乎主要是由于外部刺激无法激发正常振荡功率（Nakao 等人提交）。由于 Ppp1r2-Cre 介导的基因操作仅限于中间神经元，因此该缺陷不太可能是兴奋性锥体神经元固有的。那么，为什么 Ppp1r2-cre/fGluN1 KO 突变体中突触后兴奋性神经元的同步能力减弱呢？假设该缺陷是 GABA 能输入与兴奋性神经元之间的同步性之一，我们使用组织切片中附近锥体细胞的 sIPSC 双片记录来测试该理论。如果同步 GABA 能输入受到干扰，附近锥体神经元之间的 IPSC 事件将不同步。正如预期的那样，我们发现 P28-P42 突变体的 mPFC 切片中确实存在这种情况。这些结果表明，在我们的突变体中，必须精确地将脉冲转化为正确的突触后反应的尖峰传输受到干扰。为了确定最能解释这些突变表型的细胞机制和损伤，我们评估了与突触后锥体神经元 IPSC 事件去同步有关的几种机制。我们对突触前快速尖峰神经元施加短暂的电流脉冲，以唤起突触后锥体神经元中的单一 IPSC。这使我们能够了解到达突触前末梢的动作电位冲动是否无法引起适当的 GABA 释放。在突变体 P28-P42 中，我们发现 GABA 释放最初受到抑制，但随后 IPSC 幅度逐渐增加。这种模式与 floxed 对照中观察到的单一 IPSC 模式形成鲜明对比，其中响应第一脉冲的突触传输是稳健且高度可靠的。这些结果表明，在这些突变体中，快速尖峰神经元/锥体神经元突触的精确高保真尖峰传输受到损害（Zsiros 等人，准备中）。 (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。自去年以来，我们利用Cre/loxP系统生成转基因条件敲除小鼠（G35-3-Cre/fGluN1 KO），其中NR1缺失主要局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，以便模拟啮齿类动物精神分裂症的标志性症状（Rompala 等人，PLOS One，2013）。 (4) GABA减少或Gad67减少对生理和行为有什么影响？为了直接评估 Gad67 减少精神障碍表型的影响，我们使用相同的 Ppp1r2-Cre 系来表征一种新型转基因小鼠系，其中在出生后第 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（Kolata 等人提交）。