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.

Significance statement: 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.

Keywords: NREM to wake transitions; basal forebrain; cholinergic neurons; opto-dialysis.

Figure 1.

Design of the novel opto-dialysis probe and successful targeting of the BF sleep–wake control center. Diagram (a) and image (b) of the opto-dialysis probe. c, Sagittal view of the mouse brain illustrates placement of the opto-dialysis probe in the BF and recording of cortical EEG activity. d, Image of the opto-dialysis system combined with EEG/EMG in a freely moving mouse. e, Representative example of correct targeting of the BF in one mouse. The tip of the microdialysis probe is depicted by a black square in the coronal schematic drawing (left) and by a white asterisk in the image (right). Cholinergic neurons and fibers exhibit green (EYFP) fluorescence. f, Center of the probe tip (black squares) within BF for all mice (n = 6). All probe tips were located between −0.10 and 0.14 mm from bregma (Franklin and Paxinos, 2008). 3V, Third ventricle; aca, anterior commissure; CPu, caudate-putamen; CTX, cortex; HDB, horizontal limb of the diagonal band; LPO, lateral preoptic area; MCPO, magnocellular preoptic nucleus; SI, substantia innominata; Tu, olfactory tubercle; VP, ventral pallidum.

Figure 2.

Optogenetic stimulation of BF cholinergic neurons activates cholinergic neurons, increases wakefulness, and increases local ACh levels surrounding the opto-dialysis probe. a, Optogenetic stimulation activates BF cholinergic neurons (green, stained with ChAT). Staining of the immediate early gene product, c-Fos (red) illustrates neuronal activation in the nuclei of BF cholinergic neurons (overlay). b, The percentage of wakefulness significantly increased and NREM sleep decreased during the 2 h optogenetic stimulation period, compared with the same time period on the baseline (No Stim) day. c, Representative traces of LC-MS/MS measurements showing the increase in local BF ACh levels (mass to charge ratio, m/z) produced by optogenetic stimulation.^x unknown constituent of the microdialysates that generates an isobaric ion with ACh but separated from ACh chromatographically. d, Extracellular BF ACh levels were significantly increased during the first and the second hour of optical stimulation of BF cholinergic neurons, compared with the baseline (No Stim) day and compared with the prestimulation baseline (BL, set to 100%, indicated by the horizontal line). ACh levels returned to BL levels immediately after stimulation ended. e, The increased ACh levels caused by optogenetic stimulation are comparable with levels seen during a 3 h sleep deprivation (SD) period. Optogenetic stimulation paradigm: 10 Hz, 10 s repeated every minute for 2 h; n = 6 (BL), 5 (Stim/SD). *p < 0.05 (paired t test/one-way repeated-measures ANOVA post hoc Holm–Sidak). Data are mean ± SEM). Stim, Stimulation.

Figure 3.

Increased wakefulness induced by optogenetic stimulation of BF cholinergic neurons has a similar spectral profile to spontaneous wakefulness. a, The total amount of wakefulness increased during the stimulation period when BF cholinergic neurons were stimulated at 5, 8, or 10 Hz; but not at 2 Hz. The largest effect on wake was seen at 8 Hz 10 s. This frequency and duration were used for all further experiments (n = 5). *p < 0.05 (paired t test). Data are mean ± SEM. b, During the 2 h stimulation period on the baseline (No Stim) day and Stim day, power spectra of wakefulness periods were not significantly different, indicating that induced wakefulness was not different from spontaneous wakefulness (n = 6; 0.25 Hz bins). EEG power expressed as percent of total power (0.5–20 Hz) during the same experiment day for each animal. p > 0.05 (two-way repeated-measures ANOVA).

Figure 4.

Optogenetic stimulation of BF cholinergic neurons increases wakefulness, increases the probability of NREM to wake transitions, and reduces the latency to wakefulness. a, Optogenetic stimulation (8 Hz, 10 s/min, 2 h) increased wakefulness and decreased NREM sleep compared with the baseline (No stim) day. b, The cumulative probability distribution of NREM sleep to wakefulness transition latencies shows an increased probability to wake on the Stim day compared with the baseline day (513 and 402 trials starting in NREM for No Stim and Stim conditions, respectively). c, NREM sleep to wake latency decreased during the stimulation period compared with the same period on the baseline day. d, Transitions from NREM sleep to wakefulness within 10 s of the start of stimulation increases compared with the baseline day (n = 6). *p < 0.05 (paired t test/Wilcoxon signed rank test). Data are mean ± SEM.

Figure 5.

The wake-promoting effect of cholinergic stimulation is strongly attenuated by simultaneous reverse microdialysis of cholinergic receptor antagonists into the BF. Reverse microdialysis of atropine (atr, 50 μm), or a mixture of atropine (50 μm) and mecamylamine (mec, 1 mm) into the BF during optogenetic stimulation of cholinergic neurons abolishes the effect on the following: a, wakefulness; b, probability of NREM sleep to wakefulness transitions; c, NREM sleep to wakefulness latency; and d, transitions from NREM sleep to wakefulness within 10 s of the start of stimulation (Stim+ atr, n = 6; Stim day + atr and mec, n = 4). *p < 0.05 (one-way repeated-measures ANOVA post hoc Holm–Sidak). Data are mean ± SEM.