Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle

Abstract

Although maintained by multiple arousal systems, wakefulness falters if orexin (hypocretin), orexin receptors, or orexin neurons are deficient; narcolepsy results with hypersomnolence or sudden onset of rapid eye movement sleep [or paradoxical sleep (PS)] and loss of muscle tonus. To learn how orexin neurons maintain wakefulness, we recorded neurons in head-fixed rats across the sleep-waking cycle and then labeled them with Neurobiotin to identify them by immunohistochemistry. We show that identified orexin neurons discharge during active waking, when postural muscle tone is high in association with movement, decrease discharge during quiet waking in absence of movement, and virtually cease firing during sleep, when postural muscle tone is low or absent. During PS, they remain relatively silent in association with postural muscle atonia and most often despite phasic muscular twitches. They increase firing before the end of PS and thereby herald by several seconds the return of waking and muscle tone. The orexin neurons would thus stimulate arousal, while antagonizing sleep and muscle atonia.

Figure 1.

Orx neurons recorded and labeled in the hypothalamus. A, Image showing red, Cy3-immunostained Orx neurons in the perifornical, lateral hypothalamic area. B, Magnified image of the box in A showing an Nb-labeled cell stained with green, Cy2, and red, Cy3, thus appearing yellow as a Nb+/Orx+ cell (#c38u24). C, Plots of six Nb+/Orx+ recorded neurons on a coronal section (approximately -2.8 mm from bregma) through the lateral hypothalamic area. DMH, Dorsomedial hypothalamic nucleus; f, fornix; ic, internal capsule; LH, lateral hypothalamus; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta. Scale bars: A, C, 1 mm; B, 20 μm.

Figure 2.

Discharge of Nb+/Orx+ neurons across sleep-wake states. A, Bar graph showing mean spike rate (in blue with SEM) that varied significantly as a function of sleep-wake stage across cells (n = 6; F = 16.05; df = 5, 25; p = 0.007). The rate during aW was significantly higher than that during qW and all sleep stages, including PS (p < 0.05). The line graph (in red) shows the discharge rate of the Nb+/Orx+ neuron (#c32u10) shown in B and C. B, Hypnogram, spike rate histogram, and EMG amplitude and EEG frequency spectra (split for different scaling from blue to red over the lower frequency range, 0-30 Hz from 1 to 100 uV, and gamma range, 30-60 Hz from 0 to 25 uV) over the recording session (of 400 s corresponding to 40 10 s epochs). C, Four 1 min segments (as indicated by time below) of unit, EEG, and EMG activity during state transitions: from qW through aW, tSWS to SWS (supplemental video 1, available at www.jneurosci.org as supplemental material) (1), from tPS to PS (supplemental video 2, available at www.jneurosci.org as supplemental material) (2), from PS through aW back to PS (supplemental video 3, available at www.jneurosci.org as supplemental material) (3), and from PS to aW (supplemental video 4, available at www.jneurosci.org as supplemental material) (4). Stages were scored per 10 s epoch as indicated by the stage names between dots marking the epochs. Note that the unit fires during aW (in 1) becomes quiet during SWS (in 1) and PS (in 2 and 3), except for occasional spikes sometimes associated with twitches (in 2) or a brief arousal (in 3). The increase in firing before arousal from PS (in 3 and 4) anticipates the transition from PS to aW judged by EEG (open arrowhead) and also EMG (filled arrowhead). Calibration: horizontal, 10 s; vertical, 1 mV for EEG, 0.5 mV for EMG, and 2 mV for unit. OB, Olfactory bulb; PF, prefrontal cortex; RS, retrosplenial cortex.