Cholinergic Neurons in the Basal Forebrain Promote Wakefulness by Actions on Neighboring Non-Cholinergic Neurons: An Opto-Dialysis Study.

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.

Cholinergic neurons excite cortically projecting basal forebrain GABAergic neurons.

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP(+)) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 µm diameter) BF GFP(+) and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons.

Periodic properties of the histaminergic system of the mouse brain.

Brain histamine is involved in the regulation of the sleep-wake cycle and alertness. Despite the widespread use of the mouse as an experimental model, the periodic properties of major markers of the mouse histaminergic system have not been comprehensively characterized. We analysed the daily levels of histamine and its first metabolite, 1-methylhistamine, in different brain structures of C57BL/6J and CBA/J mouse strains, and the mRNA level and activity of histidine decarboxylase and histamine-N-methyltransferase in C57BL/6J mice. In the C57BL/6J strain, histamine release, assessed by in vivo microdialysis, underwent prominent periodic changes. The main period was 24 h peaking during the activity period. Additional 8 h periods were also observed. The release was highly positively correlated with active wakefulness, as shown by electroencephalography. In both mouse strains, tissue histamine levels remained steady for 24 h in all structures except for the hypothalamus of CBA/J mice, where 24-h periodicity was observed. Brain tissue 1-methylhistamine levels in both strains reached their maxima in the periods of activity. The mRNA level of histidine decarboxylase in the tuberomamillary nucleus and the activities of histidine decarboxylase and histamine-N-methyltransferase in the striatum and cortex did not show a 24-h rhythm, whereas in the hypothalamus the activities of both enzymes had a 12-h periodicity. These results show that the activities of histamine-metabolizing enzymes are not under simple direct circadian regulation. The complex and non-uniform temporal patterns of the histaminergic system of the mouse brain suggest that histamine is strongly involved in the maintenance of active wakefulness.

Histamine release in the basal forebrain mediates cortical activation through cholinergic neurons.

The basal forebrain (BF) is a key structure in regulating both cortical activity and sleep homeostasis. It receives input from all ascending arousal systems and is particularly highly innervated by histaminergic neurons. Previous studies clearly point to a role for histamine as a wake-promoting substance in the BF. We used in vivo microdialysis and pharmacological treatments in rats to study which electroencephalogram (EEG) spectral properties are associated with histamine-induced wakefulness and whether this wakefulness is followed by increased sleep and increased EEG delta power during sleep. We also investigated which BF neurons mediate histamine-induced cortical activation. Extracellular BF histamine levels rose immediately and remained constant throughout a 6 h period of sleep deprivation, returning to baseline levels immediately afterward. During the spontaneous sleep-wake cycle, we observed a strong correlation between wakefulness and extracellular histamine concentrations in the BF, which was unaffected by the time of day. The perfusion of histamine into the BF increased wakefulness and cortical activity without inducing recovery sleep. The perfusion of a histamine receptor 1 antagonist into the BF decreased both wakefulness and cortical activity. Lesioning the BF cholinergic neurons abolished these effects. Together, these results show that activation of the cholinergic BF by histamine is important in sustaining a high level of cortical activation, and that a lack of activation of the cholinergic BF by histamine may be important in initiating and maintaining nonrapid eye movement sleep. The level of histamine release is tightly connected to behavioral state, but conveys no information about sleep pressure.

Basal Forebrain Parvalbumin Neurons Mediate Arousals from Sleep Induced by Hypercarbia or Auditory Stimuli.

The ability to rapidly arouse from sleep is important for survival. However, increased arousals in patients with sleep apnea and other disorders prevent restful sleep and contribute to cognitive, metabolic, and physiologic dysfunction [1, 2]. Little is currently known about which neural systems mediate these brief arousals, hindering the development of treatments that restore normal sleep. The basal forebrain (BF) receives inputs from many nuclei of the ascending arousal system, including the brainstem parabrachial neurons, which promote arousal in response to elevated blood carbon dioxide levels, as seen in sleep apnea [3]. Optical inhibition of the terminals of parabrachial neurons in the BF impairs cortical arousals to hypercarbia [4], but which BF cell types mediate cortical arousals in response to hypercarbia or other sensory stimuli is unknown. Here, we tested the role of BF parvalbumin (PV) neurons in arousal using optogenetic techniques in mice. Optical stimulation of BF-PV neurons produced rapid transitions to wakefulness from non-rapid eye movement (NREM) sleep but did not affect REM-wakefulness transitions. Unlike previous studies of BF glutamatergic and cholinergic neurons, arousals induced by stimulation of BF-PV neurons were brief and only slightly increased total wake time, reminiscent of clinical findings in sleep apnea [5, 6]. Bilateral optical inhibition of BF-PV neurons increased the latency to arousal produced by exposure to hypercarbia or auditory stimuli. Thus, BF-PV neurons are an important component of the brain circuitry that generates brief arousals from sleep in response to stimuli, which may indicate physiological dysfunction or danger to the organism.

Altered Electroencephalographic Activity Associated with Changes in the Sleep-Wakefulness Cycle of C57BL/6J Mice in Response to a Photoperiod Shortening.

AIM: Under natural conditions diurnal rhythms of biological processes of the organism are synchronized with each other and to the environmental changes by means of the circadian system. Disturbances of the latter affect hormonal levels, sleep-wakefulness cycle and cognitive performance. To study mechanisms of such perturbations animal models subjected to artificial photoperiods are often used. The goal of current study was to understand the effects of circadian rhythm disruption, caused by a short light-dark cycle regime, on activity of the cerebral cortex in rodents. METHODS: We used electroencephalogram to assess the distribution of vigilance states, perform spectral analysis, and estimate the homeostatic sleep drive. In addition, we analyzed spontaneous locomotion of C57BL/6J mice under symmetric, 22-, 21-, and 20-h-long light-dark cycles using video recording and tracking methods. RESULTS AND CONCLUSIONS: We found that shortening of photoperiod caused a significant increase of slow wave activity during non-rapid eye movement sleep suggesting an elevation of sleep pressure under such conditions. While the rhythm of spontaneous locomotion was completely entrained by all light-dark cycles tested, periodic changes in the power of the θ- and γ-frequency ranges during wakefulness gradually disappeared under 22- and 21-h-long light-dark cycles. This was associated with a significant increase in the θ-γ phase-amplitude coupling during wakefulness. Our results thus provide deeper understanding of the mechanisms underlying the impairment of learning and memory retention, which is associated with disturbed circadian regulation.

The Leeds food preference questionnaire after mild sleep restriction - A small feasibility study.

Besides the increased sedentary lifestyle and increased caloric intake, changes in dietary composition may play an important role in the increased prevalence of obesity. Because inadequate sleep could be a risk factor in the aetiology of obesity, reliable methods for assessing food intake and food choice after sleep restriction are needed. We translated the Leeds food preference questionnaire (LFPQ), addressing preferences for sweet/savoury tastes and low-fat/high-fat foods, into Dutch, and tested it in 15 mildly sleep-restricted psychology students. The participants completed the LFPQ in our laboratory on two separate occasions, with approximately one week in between. Sleep on the preceding night was not controlled, but mild sleep-restriction was confirmed by a short sleep latency test (sSLT) or a short maintenance of wakefulness test (sMWT). Each participant completed the sSLT and sMWT once, just before the LFPQ, in a cross-over design randomised for the first test. Differences were present in preferences for food items from different categories (sweet/savoury and low-fat/high-fat; p<0.001). The choice frequencies for various food categories were comparable on both occasions (p=0.27). The choice frequencies for individual items were also comparable on both occasions (p=0.27). The LFPQ is easily implemented under mild sleep-restricted conditions, and translation is straightforward. Future studies using the LFPQ after sleep restriction could elucidate if restricting sleep or longer periods affects food choice, which could underlie increases in obesity risk.