Sleep is one of the most pervasive biological phenomena, but one whose function remains elusive. Although many theories of function, indirect evidence, and even common sense suggest sleep is needed for an increase in brain energy, brain energy levels have not been directly measured with modern technology. We here report that ATP levels, the energy currency of brain cells, show a surge in the initial hours of spontaneous sleep in wake-active but not in sleep-active brain regions of rat. The surge is dependent on sleep but not time of day, since preventing sleep by gentle handling of rats for 3 or 6 h also prevents the surge in ATP. A significant positive correlation was observed between the surge in ATP and EEG non-rapid eye movement delta activity (0.5–4.5 Hz) during spontaneous sleep. Inducing sleep and delta activity by adenosine infusion into basal forebrain during the normally active dark period also increases ATP. Together, these observations suggest that the surge in ATP occurs when the neuronal activity is reduced, as occurs during sleep. The levels of phosphorylated AMP-activated protein kinase (P-AMPK), well known for its role in cellular energy sensing and regulation, and ATP show reciprocal changes. P-AMPK levels are lower during the sleep-induced ATP surge than during wake or sleep deprivation. Together, these results suggest that sleep-induced surge in ATP and the decrease in P-AMPK levels set the stage for increased anabolic processes during sleep and provide insight into the molecular events leading to the restorative biosynthetic processes occurring during sleep.

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 recentin vitrostudy 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 simultaneousin vivomicrodialysis, 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 STATEMENTOptogenetics 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 andin vivomicrodialysis, 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.

The thalamic reticular nucleus (TRN) is implicated in schizophrenia pathology. However, it remains unclear whether alterations of TRN activity can account for abnormal electroencephalographic activity observed in patients, namely reduced spindles (10–15 Hz) during sleep and increased delta (0.5–4 Hz) and gamma-band activity (30–80 Hz) during wakefulness. Here, we utilized optogenetic and reverse-microdialysis approaches to modulate activity of the major subpopulation of TRN GABAergic neurons, which express the calcium-binding protein parvalbumin (PV), and are implicated in schizophrenia dysfunction. An automated algorithm with enhanced efficiency and reproducibility compared to manual detection was used for sleep spindle assessment. A novel, low power, waxing-and-waning optogenetic stimulation paradigm preferentially induced spindles that were indistinguishable from spontaneously occurring sleep spindles without altering the behavioral state, when compared to a single pulse laser stimulation used by us and others. Direct optogenetic inhibition of TRN-PV neurons was ineffective in blocking spindles but increased both wakefulness and cortical delta/gamma activity, as well as impaired the 40 Hz auditory steady-state response. For the first time we demonstrate that spindle density is markedly reduced by (i) optogenetic stimulation of a major GABA/PV inhibitory input to TRN arising from basal forebrain parvalbumin neurons (BF-PV) and; (ii) localized pharmacological inhibition of low-threshold calcium channels, implicated as a genetic risk factor for schizophrenia. Together with clinical findings, our results support impaired TRN-PV neuron activity as a potential cause of schizophrenia-linked abnormalities in cortical delta, gamma, and spindle activity. Modulation of the BF-PV input to TRN may improve these neural abnormalities.

Sleep deprivation increases the levels of extracellular adenosine and A1 receptor (A1R)mRNA in the cholinergic zone of the basal forebrain, a region involved in sleep homeostasis. To evaluate homeostatic control mechanisms, we examined the sleep deprivation-induced changes in the A1R density in rodent brain using [3H]CPFPX receptor autoradiography. We also examined the role of nuclear factor-κB (NF-κB) in transcriptional upregulation of A1R mRNA by use of the inhibitor peptide SN50 to inhibit nuclear translocation of NF-κB. We found a significant increase in cholinergic basal forebrain A1R density following 24 h of sleep deprivation and evidence that the upregulation of A1R is mediated by NF-κB. The A1R increase may be important in sleep homeostasis, since the increase in A1R density would increase the inhibitory effect of given level of adenosine, thus increasing the gain of the homeostat.

J. Neurochem. (2011) 116, 260–272. Abstract Both adenosine and nitric oxide (NO) are known for their role in sleep homeostasis, with the basal forebrain (BF) wakefulness center as an important site of action. Previously, we reported a cascade of homeostatic events, wherein sleep deprivation (SD) induces the production of inducible nitric oxide synthase (iNOS)‐dependent NO in BF, leading to enhanced release of extracellular adenosine. In turn, increased BF adenosine leads to enhanced sleep intensity, as measured by increased non‐rapid eye movement sleep EEG delta activity. However, the presence and time course of similar events in cortex has not been studied, although a frontal cortical role for the increase in non‐rapid eye movement recovery sleep EEG delta power is known. Accordingly, we performed simultaneous hourly microdialysis sample collection from BF and frontal cortex (FC) during 11 h SD. We observed that both areas showed sequential increases in iNOS and NO, followed by increases in adenosine. BF increases began at 1 h SD, whereas FC increases began at 5 h SD. iNOS and Fos‐double labeling indicated that iNOS induction occurred in BF and FC wake‐active neurons. These data support the role of BF adenosine and NO in sleep homeostasis and indicate the temporal and spatial sequence of sleep homeostatic cascade for NO and adenosine.

Sleep loss negatively impacts performance, mood, memory, and immune function, but the homeostatic factors that impel sleep after sleep loss are imperfectly understood. Pharmacological studies had implicated the basal forebrain (BF) inducible nitric oxide (NO) synthase (iNOS)-dependent NO as a key homeostatic factor, but its cellular source was obscure. To obtain direct evidence about the cellular source of iNOS-generated NO during sleep deprivation (SD), we used intracerebroventricular perfusion in rats of the cell membrane-permeable dye diaminofluorescein-2/diacetate (DAF-2/DA) that, once intracellular, bound NO and fluoresced. To circumvent the effects of neuronal NOS (nNOS), DAF-2/DA was perfused in the presence of an nNOS inhibitor. SD led to DAF-positive fluorescence only in the BF neurons, not glia. SD increased expression of iNOS, which colocalized with NO in neurons and, more specifically, in prolonged wakefulness-active neurons labeled by Fos. SD-induced iNOS expression in wakefulness-active neurons positively correlated with sleep pressure, as measured by the number of attempts to enter sleep. Importantly, SD did not induce Fos or iNOS in stress-responsive central amygdala and paraventricular hypothalamic neurons, nor did SD elevate corticosterone, suggesting that the SD protocol did not provoke iNOS expression through stress. We conclude that iNOS-produced neuronal NO is an important homeostatic factor promoting recovery sleep after SD.