title: Cholinergic Neurons in the Basal Forebrain Promote Wakefulness by Actions on Neighboring Non-Cholinergic Neurons: An Opto-Dialysis Study.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Janneke C
authors: JC
authors: Zant
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Tae
authors: http://orcid.org/0000-0003-0201-5401
authors: T
authors: Kim
authors: Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, Texas 76107.
authors: Laszlo
authors: http://orcid.org/0000-0002-4559-3458
authors: L
authors: Prokai
authors: Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, Texas 76107.
authors: Szabolcs
authors: S
authors: Szarka
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: James
authors: J
authors: McNally
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: James T
authors: JT
authors: McKenna
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Charu
authors: C
authors: Shukla
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Chun
authors: C
authors: Yang
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Anna V
authors: AV
authors: Kalinchuk
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Robert W
authors: RW
authors: McCarley
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and.
authors: Ritchie E
authors: http://orcid.org/0000-0002-7164-4132
authors: RE
authors: Brown
authors: Veterans Administration Boston Healthcare System and Harvard Medical School Department of Psychiatry, West Roxbury, Massachusetts 02132, and Radhika_Basheer@hms.harvard.edu.
authors: Radhika
authors: R
authors: Basheer
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D018680:Cholinergic Antagonists / Q000008:administration & dosage / Q000494:pharmacology; D059329:Cholinergic Neurons / Q000187:drug effects / Q000502:physiology*; D004569:Electroencephalography; D004576:Electromyography; D005260:Female; D008297:Male; D051379:Mice; D017551:Microdialysis; D062308:Optogenetics; D010320:Parvalbumins / Q000378:metabolism; D010775:Photic Stimulation; D016548:Prosencephalon / Q000187:drug effects / Q000502:physiology*; D012894:Sleep Stages / Q000502:physiology; D014851:Wakefulness / Q000187:drug effects / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000502:physiology
chemical_list: D018680:Cholinergic Antagonists; D010320:Parvalbumins; D005680:gamma-Aminobutyric Acid; D000109:Acetylcholine
keywords: NREM to wake transitions; basal forebrain; cholinergic neurons; opto-dialysis