Cannabinoid Receptor Signaling in Brain Functions

我的NSERC研究计划的长期目标是揭示大麻素受体介导的大脑功能的分子和细胞机制。在目前NSERC的资助下(2009-2014)，我们提供了第一个证据，证明大麻素1型受体(CB1R)也存在于星形胶质细胞中，并且合成大麻素类化合物(SCBS)在活体动物中的激活会诱导体内海马CA3-CA1突触的长期抑制，导致工作记忆障碍[han等，Cell(2012)148：1039-1050，它被选为2012 Cell 12篇最佳研究文章之一]。我们最近未发表的数据显示，在细胞外空间内内源性大麻素或内源性大麻素(ECB)急剧积累后，也发生了类似的级联信号事件。 人类一生中约有三分之一的时间是在睡眠中度过的，但其潜在的机制还远不清楚。自然选择在进化上分离了5亿年的脊椎动物和无脊椎动物中保存了CB1R，这表明CB1R对生命的重要性。CB1R是大脑中最丰富的G蛋白偶联受体。因此，发现CB1R在睡眠调节中发挥作用也就不足为奇了。CB1R是SCB和ECB的共同靶标。这项研究是新颖而重要的，因为它将回答SCB和ECB如何通过作用于下丘脑腹外侧视前核(VLPO)中的CB1R来调节睡眠-觉醒周期的完全未知的问题。 啮齿动物睡眠分为两大类：快速眼动睡眠(REM)和非快速眼动睡眠(NREM)，它们分别受VLPO的扩展部分和核心部分的调节。大脑中有两种主要的神经元ECB，即去雄激素(AEA)和2-花生四烯基甘油(2-AG)，它们根据需要在突触后胞浆中合成，释放到突触间隙，逆行激活突触前CB1R，在GABA能输入上去抑制(即兴奋)或在谷氨酸能输入上去激活(即抑制)突触后神经元。 我们最近的初步研究，结合各种证据，得出了这个项目的核心假说：SCB和ECB都激活VLPO CB1R，通过以下机制显著调节睡眠-觉醒周期：促进睡眠的VLPO GABA能神经元同时被抑制和兴奋，其中抑制是通过神经元AEA激活谷氨酸能突触CB1R产生失活效应，而兴奋是通过神经元2-AG激活GABA能突触CB1R产生去抑制效应；由于2-AG对VLPO的兴奋超过了AEA对VLPO的抑制，在正常的睡眠-觉醒周期或外源性ECB或SCB的作用下，这两条通路的同时激活可促进睡眠并抑制促进觉醒的食欲素能神经元以缩短觉醒。 利用电生理和行为学测试策略，我们将在我们最近用最先进的分子技术建立的16个突变小鼠系上对这一假说进行批判性检验：分别对CB1R或CB2R基因、2-AG合成酶基因、2-AG降解酶基因和AEA降解酶基因进行标记，建立4个突变系小鼠，然后与4个品系的小鼠杂交，这些小鼠接受改良的CreERT2(ICreERT2)精确插入(敲入)到大脑谷氨酸或GABA神经元或星形胶质细胞或小胶质细胞的4个基因中的每一个；给成年突变小鼠全身注射他莫昔芬，可以选择性地从大脑谷氨酸或GABA神经元或星形胶质细胞或小胶质细胞中删除其中一个“标记”基因。

The long-term goal of my NSERC research program is to reveal the molecular and cellular mechanisms underlying cannabinoid receptor-mediated brain functions. With current NSERC funding (2009-2014), we have provided the first evidence that the cannabinoid type 1 receptor (CB1R) also exists in astroglial cells and its activation in living animals by synthetic cannabinoids (sCBs) induces in vivo long-term depression at hippocampal CA3-CA1 synapses, leading to working memory impairment [Han et al, Cell (2012) 148:1039-1050, which was selected as 1 of the best 12 research articles in 2012 Cell]. Our recent unpublished data show a similar cascade signaling events following an acute accumulation of endogenous cannabinoids or endocannabinoids (eCBs) in extracellular space. Human spends about one-third of lives asleep, but its underlying mechanisms are far from clear. Natural selection has conserved CB1R, the most abundant G protein-coupled receptor in the brain, in vertebrates and invertebrates that have been evolutionarily separate for 500 million years, indicating the importance of CB1R to life. It is thus not surprising to find that CB1R, the common target of both sCB and eCB, plays a role in sleep modulation. The proposed research is novel and important, as it will answer the entirely unknown questions of how sCB and eCB regulate sleep-wake cycle via their action on CB1R in the ventrolateral preoptic nucleus (VLPO) of the hypothalamus. Rodent sleep is divided into two broad types: rapid eye movement (REM) and non-rapid eye movement (NREM) sleep, which are primarily regulated by the extended and core parts of the VLPO, respectively. The brain has two major neuronal eCBs, i.e., anandamide (AEA) and 2-arachidonylglycerol (2-AG), which are synthesized in postsynaptic cytoplasm on demand, released into synaptic cleft, and travel retrogradely to activate presynaptic CB1R either on GABAergic input to disinhibit (i.e., excite) or on glutamatergic input to deactivate (i.e., inhibit) postsynaptic neurons. Our recent pilot studies, together with various lines of evidence, lead to the core hypothesis of this project that both sCB and eCB activate VLPO CB1R to prominently modulate sleep-wake cycle through the following mechanism: sleep-promoting VLPO GABAergic neurons are simultaneously inhibited and excited via two pathways, with the inhibition being achieved by neuronal AEA activation of glutamatergic synaptic CB1R to produce deactivation effects, and the excitation being achieved by neuronal 2-AG activation of GABAergic synaptic CB1R to produce disinhibition effects; because VLPO excitation by 2-AG overpasses VLPO inhibition by AEA, a simultaneous activation of these two pathways in normal sleep-wake cycle or when exogenous eCB or sCB is applied enhances sleep and suppresses wake-promoting orexinergic neurons to shorten waking. Employing electrophysiological and behavioral testing strategies, we will critically test the hypothesis on 16 lines of mutant mice that we have recently established with the most advanced molecular technology: each of CB1R or CB2R gene, 2-AG synthesis enzyme gene, 2-AG degradative enzyme gene and AEA degradative enzyme gene was “labeled” to establish 4 lines of “floxed” mice, which were then crossed with 4 lines of mice receiving a precise insertion (knock-in) of the improved CreERT2 (iCreERT2) into each of 4 genes for brain glutamate or GABA neurons or astroglial or microglial cells; systemic tamoxifen injections to the adult mutant mice induce a deletion of one of the “labeled” gene selectively from brain glutamate or GABA neurons or astroglial or microglial cells.