Cannabinoid Receptor Signaling in Brain Functions

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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 Our recent unpublished data show a similar cascade signaling events following an acute accumulation of endogenous cannabinoids or endocannabinoids (eCBs) in extracellular space.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.