Role of GABA on circadian and homeostatic regulation of sleep

 描述（由申请方提供）：Scn 1a基因（编码电压门控Na+通道NaV1.1的一个亚基）中携带杂合功能丧失突变的动物（Scn 1a +/-小鼠）在睡眠的稳态调节和静息-活动周期的昼夜节律调节方面均表现出缺陷。NaV1.1通道是成年GABA能中间神经元中的主要电压门控Na+通道，其活性降低导致GABA能张力降低，表明Scn 1a +/-小鼠的睡眠调节缺陷来自GABA能活性降低。然而，Scn 1a +/-小鼠是一种称为Dravet综合征（DS）的严重癫痫模型，作为DS患者，不仅表现出睡眠失调，而且还表现出全身性癫痫发作。癫痫活动使DS中睡眠障碍的解释复杂化，因为睡眠调节缺陷可能是癫痫发作或癫痫相关神经损伤导致的睡眠中断的结果。我们的假设是，DS的睡眠障碍是睡眠调节中心内GABA能紧张度降低的结果，这与癫痫发作的存在无关。为了解决这一假设，我们建议有条件地将Scn 1a +/-突变靶向特定的神经元和大脑区域。具体目标1将确定全局Scn 1a +/-小鼠中的睡眠异常是否源于突变对GABA能神经元的特异性影响。我们将使用Scn 1alox/-小鼠和Cre驱动小鼠系（靶向整个大脑的GABA能神经元）将Scn 1a +/-突变靶向这些细胞，并将评估这些突变体中昼夜节律和稳态睡眠调节的完整性。该方法将明确确定Scn 1a +/-小鼠的睡眠调节缺陷是否是GABA能细胞内NaV1.1通道活性降低的结果，或者表达该通道的非GABA能细胞是否也有助于该表型。具体目标2将靶向Scn 1a +/-突变到视交叉上核（SCN）中的细胞，SCN是调节睡眠的中央昼夜节律起搏器的部位。将表达Cre重组酶的病毒（靶向所有SCN细胞或特异性含血管活性肠多肽（VIP）的细胞）注射到Scn 1alox/+小鼠的SCN中。我们还将通过将Scn 1alox/+小鼠与VIP启动子驱动Cre表达的小鼠系杂交，将突变靶向SCN VIPergic细胞。由于VIP神经元对SCN振荡网络的完整性至关重要，我们预计这些VIP特异性Scn 1a +/-突变体将显示出与所有SCN细胞靶向的突变体相似的效应。特异性目标3将病毒靶向Scn 1a +/-突变至丘脑网状核（RNT），这对于在非REM睡眠期间产生慢波睡眠和纺锤波至关重要，两者在Scn 1a +/-小鼠中均受损。预期目标1和2中的条件突变方法均不会诱导癫痫发作，这为评估睡眠调节区中NaV1.1通道活性降低（不存在癫痫发作）的影响提供了独特的机会。我们预测Scn 1a +/-突变对SCN和RNT的条件性靶向作用将分别导致睡眠的昼夜节律和稳态调节的缺陷。这些结果将为这些大脑区域内的NaV1.1通道在睡眠调节中的作用提供直接支持。他们也将直接支持我们的假设，即昼夜节律和稳态睡眠缺陷在DS出现从大脑非依赖性减少GABA能活性在特定的大脑区域，提供了新的途径，治疗睡眠障碍的DS。

 DESCRIPTION (provided by applicant): Animals carrying a heterozygous loss-of-function mutation in the Scn1a gene (Scn1a+/- mice), which encodes a subunit of the voltage-gated Na+ channel NaV1.1, show deficits in both homeostatic regulation of sleep and circadian regulation of rest-activity cycles. The NaV1.1 channel is the primary voltage-gated Na+ channel in adult GABAergic interneurons and its reduced activity results in a decrease of GABAergic tone, suggesting that sleep regulatory deficits in Scn1a+/- mice emerge from reduced GABAergic activity. However, Scn1a+/- mice are a model of a severe form of epilepsy known as Dravet syndrome (DS) and, as DS patients, show not only dysregulation of sleep but also generalized seizures. Epileptic activity complicates the interpretation of sleep disorders in DS, as sleep regulatory deficits could be the result of sleep disruption by seizures or of seizure-associated neural damage. Our hypothesis is that sleep disorders in DS are the consequence of reduced GABAergic tone within sleep regulatory centers that is independent of the presence of seizures. To address this hypothesis, we propose to conditionally target the Scn1a+/- mutation to specific neurons and brain regions. Specific Aim 1 will determine whether sleep abnormalities in global Scn1a+/- mice emerge from the effect of the mutation specifically on GABAergic neurons. We will target the Scn1a+/- mutation to these cells using an Scn1alox/- mouse and a Cre driver mouse line that targets GABAergic neurons throughout the brain, and will assess the integrity of the circadian and homeostatic regulation of sleep in these mutants. This approach will unequivocally determine whether sleep regulatory deficits in Scn1a+/- mice are the result of reduced NaV1.1 channel activity within GABAergic cells or whether non- GABAergic cells that express the channel also contribute to this phenotype. Specific Aim 2 will target the Scn1a+/- mutation to cells in the suprachiasmatic nucleus (SCN), the site of the central circadian pacemaker that regulates sleep. Viruses expressing Cre recombinase, targeting either all SCN cells or specifically vasoactive intestinal polypeptide (VIP)-containing cells, will be injected wihin the SCN of Scn1alox/+ mice. We will also target the mutation to the SCN VIPergic cells by crossing Scn1alox/+ mice with a mouse line in which the VIP promoter drives the expression of Cre. Because VIP neurons are essential for the integrity of the SCN oscillatory network, we expect that these VIP-specific Scn1a+/- mutants will show similar effects to mutants in which all SCN cells are targeted. Specific Aim 3 will virally target the Scn1a+/- mutation to the reticular nucleus of the thalamus (RNT), which is essential for the generation of slow-wave sleep and spindles during non- REM sleep, both compromised in Scn1a+/- mice. None of the conditional mutant approaches in Aims 1 and 2 is expected to induce seizures, offering a unique opportunity to assess the effect of reduced NaV1.1 channel activity in sleep regulatory regions, in the absence of seizures. We predict that the conditional targeting of the Scn1a+/- mutation to the SCN and RNT will lead to deficits in circadian and homeostatic regulation of sleep, respectively. These results would provide direct support for the role of the NaV1.1 channel within these brain regions in the regulation of sleep. They would also directly support our hypothesis that both circadian and homeostatic sleep deficits in DS emerge from seizure-independent reduced GABAergic activity in specific brain regions, providing new avenues for the treatment of sleep disorders in DS.