Zant Janneke C, Kim Tae, Prokai Laszlo, Szarka Szabolcs, McNally James, McKenna James T, Shukla Charu, Yang Chun, Kalinchuk Anna V, McCarley Robert W, Brown Ritchie E, Basheer Radhika
Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, Texas 76107.
J Neurosci. 2016 Feb 10;36(6):2057-67. doi: 10.1523/JNEUROSCI.3318-15.2016.
Understanding the control of sleep-wake states by the basal forebrain (BF) poses a challenge due to the intermingled presence of cholinergic, GABAergic, and glutamatergic neurons. All three BF neuronal subtypes project to the cortex and are implicated in cortical arousal and sleep-wake control. Thus, nonspecific stimulation or inhibition studies do not reveal the roles of these different neuronal types. Recent studies using optogenetics have shown that "selective" stimulation of BF cholinergic neurons increases transitions between NREM sleep and wakefulness, implicating cholinergic projections to cortex in wake promotion. However, the interpretation of these optogenetic experiments is complicated by interactions that may occur within the BF. For instance, a recent in vitro study from our group found that cholinergic neurons strongly excite neighboring GABAergic neurons, including the subset of cortically projecting neurons, which contain the calcium-binding protein, parvalbumin (PV) (Yang et al., 2014). Thus, the wake-promoting effect of "selective" optogenetic stimulation of BF cholinergic neurons could be mediated by local excitation of GABA/PV or other non-cholinergic BF neurons. In this study, using a newly designed opto-dialysis probe to couple selective optical stimulation with simultaneous in vivo microdialysis, we demonstrated that optical stimulation of cholinergic neurons locally increased acetylcholine levels and increased wakefulness in mice. Surprisingly, the enhanced wakefulness caused by cholinergic stimulation was abolished by simultaneous reverse microdialysis of cholinergic receptor antagonists into BF. Thus, our data suggest that the wake-promoting effect of cholinergic stimulation requires local release of acetylcholine in the basal forebrain and activation of cortically projecting, non-cholinergic neurons, including the GABAergic/PV neurons.
Optogenetics is a revolutionary tool to assess the roles of particular groups of neurons in behavioral functions, such as control of sleep and wakefulness. However, the interpretation of optogenetic experiments requires knowledge of the effects of stimulation on local neurotransmitter levels and effects on neighboring neurons. Here, using a novel "opto-dialysis" probe to couple optogenetics and in vivo microdialysis, we report that optical stimulation of basal forebrain (BF) cholinergic neurons in mice increases local acetylcholine levels and wakefulness. Reverse microdialysis of cholinergic antagonists within BF prevents the wake-promoting effect. This important result challenges the prevailing dictum that BF cholinergic projections to cortex directly control wakefulness and illustrates the utility of "opto-dialysis" for dissecting the complex brain circuitry underlying behavior.
由于胆碱能、GABA能和谷氨酸能神经元相互交织存在，了解基底前脑（BF）对睡眠-觉醒状态的控制构成了一项挑战。这三种BF神经元亚型均投射至皮层，并与皮层觉醒及睡眠-觉醒控制有关。因此，非特异性刺激或抑制研究无法揭示这些不同神经元类型的作用。最近使用光遗传学的研究表明，对BF胆碱能神经元的“选择性”刺激会增加非快速眼动睡眠和觉醒之间的转换，这表明胆碱能向皮层的投射在促进觉醒方面发挥作用。然而，这些光遗传学实验的解释因BF内可能发生的相互作用而变得复杂。例如，我们小组最近的一项体外研究发现，胆碱能神经元强烈兴奋邻近的GABA能神经元，包括投射至皮层的神经元亚群，这些神经元含有钙结合蛋白小白蛋白（PV）（Yang等人，2014年）。因此，对BF胆碱能神经元的“选择性”光遗传学刺激的促觉醒作用可能是由GABA/PV或其他非胆碱能BF神经元的局部兴奋介导的。在本研究中，我们使用新设计的光透析探针将选择性光刺激与同步体内微透析相结合，证明对胆碱能神经元的光刺激会局部增加小鼠体内乙酰胆碱水平并增加觉醒。令人惊讶的是，通过将胆碱能受体拮抗剂反向微透析至BF中，胆碱能刺激引起的觉醒增强被消除。因此，我们的数据表明，胆碱能刺激的促觉醒作用需要基底前脑局部释放乙酰胆碱，并激活投射至皮层的非胆碱能神经元，包括GABA能/PV神经元。
光遗传学是一种革命性工具，用于评估特定神经元群在行为功能中的作用，如睡眠和觉醒控制。然而，对光遗传学实验的解释需要了解刺激对局部神经递质水平的影响以及对邻近神经元的影响。在此，我们使用新型“光透析”探针将光遗传学与体内微透析相结合，报告对小鼠基底前脑（BF）胆碱能神经元的光刺激会增加局部乙酰胆碱水平和觉醒。在BF内反向微透析胆碱能拮抗剂可阻止促觉醒作用。这一重要结果挑战了普遍观点，即BF胆碱能向皮层的投射直接控制觉醒，并说明了“光透析”在剖析行为背后复杂脑回路方面的效用。
