Fos expression in orexin neurons varies with behavioral state

Abstract

The neuropeptide orexin (also known as hypocretin) is hypothesized to play a critical role in the regulation of sleep-wake behavior. Lack of orexin produces narcolepsy, which is characterized by poor maintenance of wakefulness and intrusions of rapid eye movement (REM) sleep or REM sleep-like phenomena into wakefulness. Orexin neurons heavily innervate many aminergic nuclei that promote wakefulness and inhibit REM sleep. We hypothesized that orexin neurons should be relatively active during wakefulness and inactive during sleep. To determine the pattern of activity of orexin neurons, we recorded sleep-wake behavior, body temperature, and locomotor activity under various conditions and used double-label immunohistochemistry to measure the expression of Fos in orexin neurons of the perifornical region. In rats maintained on a 12 hr light/dark cycle, more orexin neurons had Fos immunoreactive nuclei during the night period; in animals housed in constant darkness, this activation still occurred during the subjective night. Sleep deprivation or treatment with methamphetamine also increased Fos expression in orexin neurons. In each of these experiments, Fos expression in orexin neurons correlated positively with the amount of wakefulness and correlated negatively with the amounts of non-REM and REM sleep during the preceding 2 hr. In combination with previous work, these results suggest that activation of orexin neurons may contribute to the promotion or maintenance of wakefulness. Conversely, relative inactivity of orexin neurons may allow the expression of sleep.

Fig. 1.

Camera lucida drawing illustrating the cell counting box in the perifornical region. In experiment 1, cells also were counted within boxes that sampled the lateral hypothalamic area and the area medial to the perifornical region. VMH, Ventromedial hypothalamic nucleus; Arc, arcuate hypothalamic nucleus; f, fornix; opt, optic tract; 3V, third ventricle. Eachdot represents an orexin-IR neuron.

Fig. 2.

A, Fos-IR nuclei are uncommon in perifornical orexin-IR neurons from a rat killed at ZT 3.B, A rat killed at ZT 15 has many Fos-IR nuclei in orexin-IR neurons. Arrow, Orexin-IR neuron;arrowhead, Fos-IR nucleus; double arrow, Fos-IR/orexin-IR neuron. Scale bar, 50 μm.

Fig. 3.

Fos expression in perifornical orexin neurons is higher during the night in rats maintained on a 12 hr LD cycle (ZT 3 through ZT 21 groups). In rats maintained in constant darkness (CT 3 and CT 15 groups), the percentage of double-labeling also is higher during the subjective night.

Fig. 4.

Orexin neurons in the perifornical region have the greatest diurnal variation in Fos expression. Orexin neurons medial to the perifornical region have a less striking diurnal variation, but Fos expression does not vary in orexin neurons of the lateral hypothalamic area. *p = 0.05; **p = 0.01.

Fig. 5.

Methamphetamine increases Fos expression in perifornical orexin neurons. *p = 0.003.

Fig. 6.

Sleep deprivation from ZT 5 to ZT 7 increases Fos expression in perifornical orexin-IR neurons. Sleep deprivation from ZT 17 to ZT 19 marginally increases Fos immunoreactivity in orexin-IR neurons. *p = 0.04; **p < 0.0001.

Fig. 7.

Across all three experiments, Fos expression in perifornical orexin-IR neurons correlates positively with the amount of wakefulness (r = 0.78; p < 0.0001) and negatively with the amounts of NREM (r= −0.78; p < 0.0001) and REM sleep (r = −0.63, p < 0.0001). ○, Experiment 1; ●, experiment 2; ■, experiment 3.