| 研究概要 |
Adeno-associated virus production: in order to modulate the activity of neurons located in the dorsal striatum of Adora2a-Cre mice (expressing Cre under the control of Adora2a gene regulatory elements), we successfully generated 800uL of AAV-DIO-eNpHR3.0 (2.5e12 copies/ml) AAV-DIO-ChR2-mcherry (7.2e12 copies/ml), AAV10-DIO-hM3Dq (3.6e11 copies/ml) and AAV10-DIO-hM4Di (2.2e13 copies/ml).
To efficiently annihilate dopaminergic neurons projecting into the dorsal striatum, we stereotaxically micro-injected 6-OHDA (4ug diluted into 1uL of artificial CSF) bilaterally into the Nucleus Accumbens (NAc) or directly into the Substancia Nigra pars compacta (SNc) of C57BL/6 male mice. As a control we also injected mice with vehicle into the SNc.
After implantation of sleep recording apparatus, we recorded sleep in these animals and observed a dramatic reduction of the total sleep amount in mice injected with 6-OHDA compared to control mice. This effect is particularly severe during dark phase (NREM: -11% in SNc injected mice, -24% in NAc injected mice; REM: -40% in SNc injected mice, -58% in NAc injected mice).
Stages transition analysis reveals that the difference between control animals and 6-OHDA treated animals comes from a reduction of sleep episode numbers (REM: -72% in SNc injected mice, -76% in NAc injected mice) but not duration. All in all, 6-OHDA treated animals show a decrease in sleep and wakefulness episodes fragmentation compared to control animals, resulting in longer episode stages for wakefulness and NREM sleep.
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| 今後の研究の推進方策 |
Pharmacogenetic control of GPCRs and optogenetic activation and silencing in the indirect pathway: Using Adora2a-Cre line, we will microinject Adeno-Associated Virus (AAV) coding for Cre-dependent conditional “DREADD” system into the striatum. We will also use optogenetic activation and silencing of the indirect pathway by illuminating the subregion of striatum after targeted expression of the Arch/ChR2 expression in the indirect pathway by using the Adora2a-Cre x Ai35/Ai32 mice or by injecting AAV10-DIO-ChR2 into the Adora2a-cre mice. Light-activation (depolarization) and silencing (hyperpolarization) of the striatopallidal neurons during behavioral testing will be achieved by illuminating the striatum with a 100 mW DPSS blue Laser.
Using Adora2a-Cre mice (expressing Cre under the control of Adora2a gene regulatory elements), mutant muscarinic receptors and optogenetic modified receptors are expressed solely on the indirect pathway neurons by using stereotaxic microinjection of viral vectors into the striatum. We show that pharmacogenetic exciting A2AR-expressing neurons in NAc via hM3Dq by CNO produce a robust increase in NREM sleep in mice. To critically optogenetically activate and silence the indirect pathway in freely behaving animals, the Adora2a-Cre mice are microinjected with AAV-DIO-ChR2-mcherry and stereotaxically implanted with EEG recording electrodes and the guide cannula. The EEG recording and laser stimulation is performed 2 weeks after the surgery.
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