Identification of a population of sleep-active cerebral cortex neurons

Abstract

The presence of large-amplitude, slow waves in the EEG is a primary characteristic that distinguishes cerebral activity during sleep from that which occurs during wakefulness. Although sleepactive neurons have been identified in other brain areas, neurons that are specifically activated during slow-wave sleep have not previously been described in the cerebral cortex. We have identified a population of cells in the cortex that is activated during sleep in three mammalian species. These cortical neurons are a subset of GABAergic interneurons that express neuronal NOS (nNOS). Because Fos expression in these sleep-active, nNOS-immunoreactive (nNOS-ir) neurons parallels changes in the intensity of slow-wave activity in the EEG, and these neurons are innvervated by neurotransmitter systems previously implicated in sleep/wake control, cortical nNOS-ir neurons may be part of the neurobiological substrate that underlies homeostatic sleep regulation.Fos ͉ interneuron ͉ neuronal NOS ͉ sleep deprivation ͉ wakefulness S leep is well known to have recuperative properties (1) and to facilitate performance of learned behaviors (2, 3); conversely, sleep deprivation results in cognitive and performance deficits (4). The presence of large-amplitude, slow waves in the EEG is the hallmark that distinguishes cerebral activity during sleep from wakefulness. Because the intensity of slow waves measured in the delta range of the EEG (1.0-4.0 Hz) appears to be homeostatically regulated and proportional to prior wake duration (5), slow-wave activity (SWA) has been hypothesized to be associated with the restorative function of sleep and with synaptic plasticity (2, 3, 6).The neural circuitry underlying SWA involves a corticothalamocortical loop and interplay between a hyperpolarizationactivated cation current (I h ) and a low-threshold Ca 2ϩ current (I t ) in thalamocortical neurons (7,8). It is currently unknown whether the cerebral cortex is an active participant or simply a passive conveyor of sleep history-dependent SWA dynamics. Identification of a discrete sleep-active population of cortical neurons, such as those in the basal forebrain (BF) (9) and the preoptic-anterior hypothalamus (POAH) (10-13), would help distinguish between these alternatives.In experiments conducted in three mammalian species, we found that GABAergic interneurons expressing the enzyme neuronal NOS (nNOS) are activated both during spontaneous sleep and during recovery sleep (RS) after sleep deprivation (SD). The proportion of nNOS-immunoreactive (nNOS-ir) neurons that are activated during sleep is correlated with a measure of SWA intensity known as ''delta energy' ' (14, 15). Because sleep-active, nNOS-ir neurons are innervated by neurotransmitter systems previously implicated in sleep/wake control, cortical nNOS-ir neurons may be a previously unrecognized cell type involved in SWA and part of the neurobiological substrate that underlies homeostatic sleep regulation. Results Fos Expression Is Increased in Cortical nNOS Neurons During RS.Fos expression has previ...

Homeostatic Sleep Pressure is the Primary Factor for Activation of Cortical nNOS/NK1 Neurons

“…We conclude that cortical nNOS neurons are not activated simply by the occurrence of NREM sleep, rather, the %Fos/nNOS depends on the magnitude of sleep pressure that has accumulated during the time preceding sleep onset. Together with our previous studies (Gerashchenko et al, 2008;Morairty et al, 2013), these results demonstrate that cortical nNOS neurons are responsive to homeostatic sleep drive.…”

“…Sleep factors are thought to act by inhibiting wake-promoting neurons (Porkka-Heiskanen and Kalinchuk, 2011;Rainnie et al, 1994), but may also act directly on the cerebral cortex (Clinton et al, 2011;Szymusiak, 2010). We have recently described a population of cortical GABAergic interneurons that is specifically activated during sleep (Gerashchenko et al, 2008;Pasumarthi et al, 2010). These neurons are identified by colocalized immunoreactivity for neuronal nitric oxide synthase (nNOS) and the substance P (SP) receptor NK1 (Dittrich et al, 2012).…”

“…The percentage of nNOS neurons expressing Fos was calculated as described previously (Gerashchenko et al, 2008;Pasumarthi et al, 2010). Micrographs for publication were taken at  200 magnification on a Leica DM 5000B microscope (Leica Microsystems, IL, USA) with a Microfire S99808 camera (Optronics, CA, USA) in Stereoinvestigator (MBF Bioscience, Williston, VT, USA).…”

“…Coronal brain sections were cut at 40-mm thickness. Double immunohistochemistry for Fos and nNOS was performed on serial sections of rat brain as described previously (Gerashchenko et al, 2008;Pasumarthi et al, 2010). Sections were first incubated overnight with rabbit anti-Fos antibody (1:4000-5000, sc-52, Santa Cruz Biotechnology, Dallas, TX, USA), then with biotinylated donkey anti-rabbit antibody (1:500, Jackson ImmunoResearch, West Grove, PA, USA), followed by avidinbiotinylated horseradish peroxidase complex (1:200, PK-6100, Vector Laboratories, Burlingame, CA, USA), and nickel-enhanced 3,3 0 diaminobenzidine (SK4100, Vector Laboratories) for a black reaction product.…”

“…These neurons are identified by colocalized immunoreactivity for neuronal nitric oxide synthase (nNOS) and the substance P (SP) receptor NK1 (Dittrich et al, 2012). Activation of these neurons, assessed by immunoreactivity for the functional activity marker Fos, correlates with time spent in NREM sleep as well as with NREM delta power (Gerashchenko et al, 2008;Morairty et al, 2013). Prior wake duration 'dose-dependently' increased the proportion of Fos-labeled nNOS neurons when rats were subjected to 2, 4, or 6 h of sleep deprivation (SD) followed by a 2-h recovery sleep (RS) opportunity .…”