Role of GABA on circadian and homeostatic regulation of sleep

 描述(由申请人提供)：携带Scn1a基因杂合性功能丧失突变的动物(Scn1a+/-小鼠)，编码电压门控Na+通道Nav1.1的一个亚单位，表现出睡眠的动态平衡调节和静息-活动周期的昼夜调节的缺陷。在成年GABA能中间神经元中，NAV1.1通道是主要的电压门控性Na+通道，其活性降低导致GABA能张力降低，提示Scn1a+/-小鼠睡眠调节缺陷源于GABA能活性降低。然而，Scn1a+/-小鼠是一种被称为Dravet综合征(DS)的严重癫痫的模型，作为DS患者，不仅表现出睡眠调节失调，而且还表现出全身性癫痫发作。癫痫活动使对DS患者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与癫痫相关的神经损伤造成的睡眠中断的结果。我们的假设是，DS的睡眠障碍是睡眠调节中心内GABA能张力降低的结果，这与癫痫的存在无关。为了解决这一假设，我们建议有条件地将Scn1a+/-突变靶向特定的神经元和大脑区域。具体目标1将确定全球Scn1a+/-小鼠的睡眠异常是否源于突变对GABA能神经元的影响。我们将使用Scn1alox/-小鼠和Cre驱动鼠系将Scn1a+/-突变靶向这些细胞，该突变靶向整个大脑的GABA能神经元，并将评估这些突变体对睡眠的昼夜节律和稳态调节的完整性。这一方法将明确确定Scn1a+/-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能细胞是否也参与了这一表型。特定目标2将Scn1a+/-突变定位于视交叉上核(SCN)中的细胞，视交叉上核是调节睡眠的中央昼夜起搏器的位置。表达Cre重组酶的病毒，靶向所有SCN细胞或特定含有血管活性肠多肽(VIP)的细胞，将被注射到Scn1alox/+小鼠的SCN中。我们还将通过将Scn1alox/+小鼠与VIP启动子驱动Cre表达的小鼠系杂交，将突变靶向SCN VIPerable细胞。由于VIP神经元对于SCN振荡网络的完整性是必不可少的，我们预计这些VIP特异的Scn1a+/-突变体将显示出与所有SCN细胞都被靶向的突变体类似的效果。特定目标3将通过病毒将Scn1a+/-突变靶向丘脑网状核(RNT)，这是产生慢波睡眠和非REM睡眠中的纺锤波所必需的，在Scn1a+/-小鼠中两者都受到影响。AIMS 1和AIMS 2中的条件突变方法都不会诱发癫痫发作，这为在没有癫痫发作的情况下评估睡眠调节区Nav1.1通道活动减少的影响提供了一个独特的机会。我们预测，Scn1a+/-突变的条件性靶向SCN和RNT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。它们还将直接支持我们的假设，即DS患者的昼夜节律和稳态睡眠缺陷都源于特定脑区GABA能活动的减少，为DS患者睡眠障碍的治疗提供了新的途径。

 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.