Anna V. Kalinchuk

Adenosine, energy metabolism and sleep homeostasis

Adenosine is directly linked to the energy metabolism of cells. In the central nervous system (CNS) an increase in neuronal activity enhances energy consumption as well as extracellular adenosine concentrations. In most brain areas high extracellular adenosine concentrations, through A1 adenosine receptors, decrease neuronal activity and thus the need for energy. Adenosine may be a final common pathway for various sleep factors. We have identified a relatively specific area, the basal forebrain (BF), which appears to be central in the regulation/execution of recovery sleep after sleep deprivation (SD), or prolonged wakefulness. Adenosine concentration increases in this area during SD, and this increase induces sleep while prevention of the increase during SD abolishes recovery sleep. The increase in adenosine is associated with local changes in energy metabolism as indicated by increases in levels of pyruvate and lactate and increased phosphorylation of AMP-activated protein kinase. The increases in adenosine and sleep are associated with intact cholinergic system since specific lesion of the BF cholinergic cells abolishes both. Whether adenosine during SD is produced by the cholinergic neurons or astrocytes associated with them remains to be explored. An interesting, but so far unexplored question regards the relationship between the local, cortical regulation of sleep homeostasis and the global regulation of the state of sleep as executed by lower brain mechanisms, including the BF. The increase in adenosine concentration during SD also in cortical areas suggests that adenosine may have a role in the local regulation of sleep homeostasis. The core of sleep need is probably related to primitive functions of life, like energy metabolism. It can be noted that this assumption in no way excludes the possibility that later in evolution additional functions may have developed, e.g., related to complex neuronal network functions like memory and learning.

Sleep and Brain Energy Levels: ATP changes during sleep

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.

Local energy depletion in the basal forebrain increases sleep

Sleep saves energy, but can brain energy depletion induce sleep? We used 2,4‐dinitrophenol (DNP), a molecule which prevents the synthesis of ATP, to induce local energy depletion in the basal forebrain of rats. Three‐hour DNP infusions induced elevations in extracellular concentrations of lactate, pyruvate and adenosine, as well as increases in non‐REM sleep during the following night. Sleep was not affected when DNP was administered to adjacent brain areas, although the metabolic changes were similar. The amount and the timing of the increase in non‐REM sleep, as well as in the concentrations of lactate, pyruvate and adenosine with 0.5–1.0 mm DNP infusion, were comparable to those induced by 3 h of sleep deprivation. Here we show that energy depletion in localized brain areas can generate sleep. The energy depletion model of sleep induction could be applied to in vitro research into the cellular mechanisms of prolonged wakefulness.

Nitric oxide production in the basal forebrain is required for recovery sleep

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. Here, we assessed the role of the intercellular gaseous signaling agent NO in sleep homeostasis. We measured the concentration of nitrite and nitrate, indicative of NO production, in the basal forebrain (BF) of rats during sleep deprivation (SD), and found the level increased by 100 ± 51%. To test whether an increase in NO production might play a causal role in recovery sleep, we administered compounds into the BF that increase or decrease concentrations of NO. Infusion of either a NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide, or a NO synthase inhibitor, Nω‐nitro‐l‐arginine methyl ester (L‐NAME), completely abolished non‐rapid eye movement (NREM) recovery sleep. Infusion of a NO donor, (Z)‐1‐[N‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2diolate (DETA/NO), produced an increase in NREM that closely resembled NREM recovery after prolonged wakefulness. The effects of inhibition of NO synthesis and the pharmacological induction of sleep were effective only in the BF area. Indicators of energy metabolism, adenosine, lactate and pyruvate increased during prolonged wakefulness and DETA/NO infusion, whereas L‐NAME infusion during SD prevented the increases. We conclude that an increase in NO production in the BF is a causal event in the induction of recovery sleep.

The Role of Cholinergic Basal Forebrain Neurons in Adenosine-Mediated Homeostatic Control of Sleep: Lessons from 192 IgG-Saporin Lesions

A topic of high current interest and controversy is the basis of the homeostatic sleep response, the increase in non-rapid-eye-movement (NREM) sleep and NREM-delta activity following sleep deprivation (SD). Adenosine, which accumulates in the cholinergic basal forebrain (BF) during SD, has been proposed as one of the important homeostatic sleep factors. It is suggested that sleep-inducing effects of adenosine are mediated by inhibiting the wake-active neurons of the BF, including cholinergic neurons. Here we examined the association between SD-induced adenosine release, the homeostatic sleep response and the survival of cholinergic neurons in the BF after injections of the immunotoxin 192 immunoglobulin G (IgG)-saporin (saporin) in rats. We correlated SD-induced adenosine level in the BF and the homeostatic sleep response with the cholinergic cell loss 2 weeks after local saporin injections into the BF, as well as 2 and 3 weeks after i.c.v. saporin injections. Two weeks after local saporin injection there was an 88% cholinergic cell loss, coupled with nearly complete abolition of the SD-induced adenosine increase in the BF, the homeostatic sleep response, and the sleep-inducing effects of BF adenosine infusion. Two weeks after i.c.v. saporin injection there was a 59% cholinergic cell loss, correlated with significant increase in SD-induced adenosine level in the BF and an intact sleep response. Three weeks after i.c.v. saporin injection there was an 87% cholinergic cell loss, nearly complete abolition of the SD-induced adenosine increase in the BF and the homeostatic response, implying that the time course of i.c.v. saporin lesions is a key variable in interpreting experimental results. Taken together, these results strongly suggest that cholinergic neurons in the BF are important for the SD-induced increase in adenosine as well as for its sleep-inducing effects and play a major, although not exclusive, role in sleep homeostasis.

The effect of age on prepro-orexin gene expression and contents of orexin A and B in the rat brain

Orexin A and B (hypocretin 1 and 2) are hypothalamic peptides, which are synthesized in the lateral hypothalamus. Orexins participate in the regulation energy balance, food intake, vigilance and several endocrine and autonomic functions. The widespread projections of the orexin neurons suggest that they may have a role in coordination of different brain activities. The effects of ageing on the orexin system have not been studied previously. Prepro-orexin gene expression in the lateral hypothalamus, and the contents of orexin A and B peptides in the lateral hypothalamus and hypothalamus were measured in young, middle-aged and old (3, 12 and 24 months) rats. In the course of ageing, the expression of the prepro-orexin gene and the levels of orexin A and B decreased; the main decrease occurred by 12 months. Sleep deprivation for 6h increased slightly the expression of prepro-orexin gene in young rats. Deterioration of the orexin system may play a role in the phenomenon associated with aging, e.g. decreased consolidation of vigilance states, endocrine changes and dysfunctions of autonomic nervous system.

Inducible and neuronal nitric oxide synthases (NOS) have complementary roles in recovery sleep induction

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. We have previously shown that nitric oxide (NO) generation increases in the basal forebrain (BF) during sleep deprivation (SD). Moreover, both NO synthase (NOS) inhibition and a NO scavenger prevented recovery sleep induction, while administration of a NO donor during the spontaneous sleep–wake cycle increased sleep, indicating that NO is necessary and sufficient for the induction of recovery sleep. Next we wanted to know which NOS isoform is involved in the production of recovery sleep. Using in vivo microdialysis we infused specific inhibitors of NOS into the BF of rats during SD, and found that an inhibitor of inducible NOS (iNOS), 1400W, prevented non‐rapid eye movement (NREM) recovery, while an inhibitor of neuronal NOS (nNOS), L‐N‐propyl‐arginine, decreased REM recovery but did not affect NREM recovery. Using immunoblot analysis we found that iNOS was not expressed during the spontaneous sleep–wake cycle, but was induced by prolonged wakefulness (increased by 278%). A known iNOS inducer, lipopolysaccharide, evoked an increase in sleep that closely resembled recovery sleep, and its effects were abolished by 1400W. These results suggest that the elevation of NO produced by induction of iNOS in the BF during prolonged wakefulness is a specific mechanism for producing NREM recovery sleep and that the two NOS isoforms have a complementary role in NREM and REM recovery induction.

The time course of adenosine, nitric oxide (NO) and inducible NO synthase changes in the brain with sleep loss and their role in the non-rapid eye movement sleep homeostatic cascade.

J. Neurochem. (2011) 116, 260–272.

Sleep Deprivation Triggers Inducible Nitric Oxide-Dependent Nitric Oxide Production in Wake–Active Basal Forebrain Neurons

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.

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.