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Activation of cortical interneurons during sleep: an anatomical link to homeostatic sleep regulation?

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Although slow wave activity in the EEG has been linked to homeostatic sleep regulation, the neurobiological substrate of sleep homeostasis is not well understood. Whereas cortical neurons typically exhibit reduced discharge rates during slow wave sleep (SWS), a subpopulation of GABAergic interneurons, which express the enzyme neuronal nitric oxide synthase (nNOS), has recently been found to be activated during SWS. The extent of activation of these nNOS neurons is proportional to homeostatic sleep ‘drive’. These cells are an exception among cortical interneurons in that they are projection neurons. We propose that cortical nNOS neurons are positioned to influence neuronal activity across widespread brain areas. They could thus provide a long-sought anatomical link for understanding homeostatic sleep regulation.

Introduction

Numerous studies of sleep deprivation (SD) in humans and animals have provided evidence that sleep is homeostatically regulated. Sleep loss produces proportional increases in the ‘drive’ to sleep, in the subsequent occurrence of sleep, and in slow wave activity (SWA) recorded in the electroencephalogram (EEG) during non-rapid eye movement (NREM) sleep (Glossary). This property of homeostatic regulation, along with a circadian input, was incorporated into the ‘two-process model’ of sleep regulation (Figure 1) in which the homeostatic sleep-related ‘process S’ was proposed to interact with input from the circadian system (‘process C’) to gate the occurrence of sleep and wakefulness [1]. Process S is suggested to reflect a biochemical process(es) that begins to increase at the onset of wakefulness. Once a threshold value is reached, sleep occurs only if process C is in the appropriate circadian phase (Figure 1b). Although seemingly simplistic, this model accounts remarkably well for the timing of sleep in humans and other species.

Cortical EEG is widely accepted to reflect thalamocortical activity 2, 3, 4. A number of studies have suggested that SWA is related to the underlying process S. SWA increases in proportion to prior wake duration and decreases over the course of night-time sleep period (Figure 1a), reflecting a diminution of process S during sleep. Thus, SWA has been interpreted to represent the cortical manifestation of recovery processes from prior waking activities that occur during sleep.

Despite elegant cellular electrophysiological studies indicating a role for a corticothalamocortical loop in the generation of cortical slow waves 2, 3, 4, the identification of the neurobiological basis of homeostatic sleep regulation has been elusive. Sleep homeostasis probably involves a distributed network of sleep-active brain areas. Lesion, electrical stimulation and Fos immunohistochemistry experiments have identified sleep-active areas in the preoptic anterior hypothalamus (POAH) and the medulla. Curiously, despite the relationship between SWA and sleep homeostasis, sleep-active neurons have only recently been described in the cerebral cortex (discussed below).

Section snippets

Evidence from electrophysiological studies

The concept of a ‘sleep-active’ neuron is straightforward: a cell whose firing rate is greater during either rapid eye movement (REM) or non-REM (NREM) sleep than during wakefulness. Because neurons discharge in a variety of modes (including tonic, phasic, and burst firing), such studies typically calculate an average discharge rate over a defined period of time that is used to establish a threshold ratio between arousal states to label a cell as wake-active versus NREM-active or REM-active.

Identification of sleep-active neurons in the cerebral cortex

Fos immunhistochemistry has been used to identify sleep-active neurons in the POAH 15, 17, and we used a similar approach to identify other sleep-active brain areas that might be related to homeostatic sleep regulation [42]. Using Fos expression in conjunction with immunolabeling for phenotypic markers to identify cortical interneurons [26] we found that a subset of GABAergic interneurons, which express neuronal nitric oxide synthase (nNOS), showed greatly elevated Fos expression during

Anatomy and physiology of cortical nNOS neurons

nNOS neurons have been identified in the cerebral cortex using two methods: nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and immunohistochemistry for NOS proteins [49]. NADPH-d-positive neurons in the cortex have been classified as type I or type II [50]. Type I neurons have large somata, intense NADPH-d activity as well as intense nNOS immunoreactivity, and are located in the deeper layers of the cortex and even in the white matter. By contrast, type II

GABAergic cortical projection neurons and nNOS

Communication between cortical areas depends largely on glutamatergic neurons because these neurons constitute the majority of long-distance connections with either other pyramidal neurons or inhibitory interneurons. Until recently the general view was that glutamatergic activation of local circuit GABAergic interneurons was required to produce inhibitory effects between distant cortical areas. However, several studies have now demonstrated that inhibitory effects can also be produced

nNOS in the cortex: more than a phenotypic marker of sleep-active neurons?

Because Fos expression is increased only in the cortical and none of the subcortical nNOS neuronal populations during sleep [48], we propose that the placement of the cortical nNOS neurons in a network (instead of the presence of nNOS per se) is more likely to account for activation of these cells during sleep. Nonetheless, strong evidence indicates that NO is an endogenous sleep-promoting substance [71]. Intracerebroventricular (icv) injection of the NO precursor, L-arginine, increases NREM

Behavioral state-dependent activation of nNOS neurons

As described above, the activation of nNOS neurons during sleep appears to be an exception among cortical neurons and is probably a consequence of inputs from its afferent network. Unlike cortical nNOS neurons, the wake-active monoaminergic neurons in the LC, DRN and TMN decrease their discharge during sleep. Serotonergic and cholinergic inputs to the nNOS cells have been established 57, 58, although it unknown whether these inputs contact the sleep-active population of nNOS cells specifically.

Concluding remarks

More than 25 years after the two-process model of sleep regulation was proposed [1], our understanding of the neurobiological substrates underlying sleep homeostasis remains incomplete. Recent studies have demonstrated activation of a rare population of GABAergic cortical interneurons during sleep. Furthermore, the number of nNOS interneurons activated was found to be directly proportional to NREM delta energy, an index of homeostatic sleep drive. At this point it is unclear whether Fos

Acknowledgements

Research supported by the National Institutes of Health (grants R01 HL059658 and R01 NS064193), the Human Frontiers Science Program (RGY0070/2007) and the CNRS ‘Nitrex’ project. We are grateful to Drs. Lars Dittrich for very helpful comments.

Glossary

Dorsal raphe nuclei (DRN)
midbrain nuclei that synthesize serotonin and have both ascending projections and descending projections. DRN neurons have their highest discharge rate during active wakefulness and are quiescent during REM sleep.
Electroencephalogram (EEG)
integrated measure of brain electrical activity, usually recorded from the scalp or brain surface.
Fos immunohistochemisty
Immunological detection of c-Fos, an immediate-early gene transcription factor, is commonly used as an indicator

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      In a recent study, chemogenetic activation of somatostatin-positive cells in the cerebral cortex increased SWA, slope of individual slow waves, and non-REM sleep duration, whereas chemogenetic inhibition of these cells decreased SWA and slow wave incidence without changing time spent in non-REM sleep (Funk et al., 2017). Since all type I nNOS-positive cells are also somatostatin-positive cells (Kilduff et al., 2011), it is likely that the changes in SWA observed in this study depended, at least partially, on the change in the activity of nNOS cells. Nevertheless, the relative contribution of nNOS-positive vs. nNOS-negative somatostatin cells in the regulation of SWA is not known.

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    Present address: Harvard Medical School/VA Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132, USA.

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