No abstract

We screened for novel circuits in the mouse brain that promote wakefulness. Chemogenetic activation experiments and EEG recordings pointed to glutamatergic/nitrergic (NOS1) and GABAergic neurons in the VTA. Activating glutamatergic/NOS1 neurons, which were wake- and REM-sleep-active, produced wakefulness through projections to the nucleus accumbens and the lateral hypothalamus. Lesioning the glutamate cells impaired the consolidation of wakefulness. By contrast, activation of GABAergic VTA neurons elicited long-lasting NREM-like sleep resembling sedation. Lesioning these neurons produced an increase in wakefulness that persisted for at least 4 months. Surprisingly, these VTA GABAergic neurons were wake-and REM-sleep-active. We suggest that GABAergic VTA neurons may limit wakefulness by inhibiting the arousal-promoting VTA glutamatergic and/or dopaminergic neurons and through projections to the lateral hypothalamus. Thus, in addition to its contribution to goal- and reward-directed behaviours, the VTA has a role in regulating sleep and wakefulness.

Endocannabinoids may serve as retrograde messengers to inhibit neurotransmitter release during depolarization-induced suppression of inhibition (DSI) or excitation (DSE). We therefore tested whether endocannabinoids inhibit N-type voltage-dependent Ca 2ϩ channels by activating G i/o -protein-coupled CB1 cannabinoid receptors (CB1R)-a possible mechanism underlying DSI/ DSE. Three putative endocannabinoids [2-arachidonylglycerol (2-AG), 2-arachidonyl glycerol ether (2-AGE), and anandamide (AEA)] and the cannabimimetic aminoalkylindole WIN 55,212-2 (WIN) inhibited whole-cell Ca 2ϩ currents in rat sympathetic neurons previously injected with cDNA encoding a human CB1R. Agonistmediated Ca 2ϩ current inhibition was blocked by a selective CB1R antagonist [SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride] and pertussis toxin (PTX) pretreatment. The rank order of potency was WIN (IC 50 ϭ 2 nM) Ͼ 2-AGE (350 nM) ϳ 2-AG (480 nM) Ͼ AEA (ϳ3 M), with each agonist displaying similar efficacy (approximately 50% maximal inhibition). Increasing CB1R expression level significantly enhanced AEA potency. AEA (10 M) also inhibited Ca 2ϩ channels in a voltageindependent, CB1R-independent, and PTX-insensitive manner, whereas 2-AG and 2-AGE were devoid of this activity. All three endocannabinoids activated G-protein-coupled inwardly rectifying potassium (GIRK) channels, GIRK1/4, heterologously expressed in sympathetic neurons. These results suggest a mechanism by which endocannibinoids might influence presynaptic function.

GPR35 is a G protein-coupled receptor recently "de-orphanized " using high-throughput intracellular calcium measurements in clonal cell lines expressing a chimeric G-protein ␣-subunit. From these screens, kynurenic acid, an endogenous metabolite of tryptophan, and zaprinast, a synthetic inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase, emerged as potential agonists for GPR35. To investigate the coupling of GPR35 to natively expressed neuronal signaling pathways and effectors, we heterologously expressed GPR35 in rat sympathetic neurons and examined the modulation of N-type (Ca V 2.2) calcium channels. In neurons expressing GPR35, calcium channels were inhibited in the absence of overt agonists, indicating a tonic receptor activity. Application of kynurenic acid or zaprinast resulted in robust voltage-dependent calcium current inhibition characteristic of G␤␥-mediated modulation. Both agonist-independent and -dependent effects of GPR35 were blocked by Bordetella pertussis toxin pretreatment indicating the involvement of G i/o proteins. In neurons expressing GPR35a, a short splice variant of GPR35, zaprinast was more potent (EC 50 ϭ 1 M) than kynurenic acid (58 M) but had a similar efficacy (approximately 60% maximal calcium current inhibition). Expression of GPR35b, which has an additional 31 residues at the N terminus, produced similar results but with much greater variability. Both GPR35a and GPR35b appeared to have similar expression patterns when fused to fluorescent proteins. These results suggest a potential role for GPR35 in regulating neuronal excitability and synaptic release.G protein-coupled receptors (GPCRs) comprise one of the largest families of cell surface proteins and represent a major target for both current therapeutic agents and drugs under development. Many cDNA clones are predicted to code for GPCRs based on high sequence similarity, especially in the transmembrane domains, to established GPCRs. For such orphan GPCRs, the identification of agonists, antagonists, and signal transduction pathways represents a major effort by industry for the discovery of novel drug targets (Stadel et al., 1997). GPR35, an orphan GPCR first discovered during a human genomic DNA screen, shares limited sequence homology with purinergic P2Y receptors, nicotinic acid receptor HM74, lysophosphatidic acid receptor GPR23, and an orphan receptor, GPR55. The highest levels of GPR35 mRNA were found in immune and gastrointestinal tissues with only limited expression in lung and neuronal tissues (O'Dowd et al., 1998;Wang et al., 2006). Subsequent to the initial description of GPR35 (now denoted GPR35a), Okumura et al. (2004) isolated a splice variant (GPR35b) from a human gastric cancer cDNA library that coded for an additional 31 amino acids at the N terminus. GPR35a and GPR35b mRNA levels were up-regulated in gastric cancer tissue, suggesting a role for both splice variants in malignant transformation. However, in the absence of an identified agonist, the functional role GPR35 plays in eithe...