Systemic and Nasal Delivery of Orexin-A (Hypocretin-1) Reduces the Effects of Sleep Deprivation on Cognitive Performance in Nonhuman Primates

Sam A. Deadwyler,¹ Linda Porrino,¹ Jerome M. Siegel,² and Robert E. Hampson^{1 1}Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, and ²Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, California Veterans Administration Greater Los Angeles Healthcare System/Sepulveda, North Hills, California 91343

The Journal of Neuroscience, December 26, 2007, 27(52):14239-14247

Defense Advanced Research Projects Agency (DARPA) Grants DAAD19-02-1-0060 (Army Research Office) (S.A.D., L.P.) and BAA 04-12-F9034, National Institutes of Health Grants DA00119 and DA06634 (S.A.D., L.P.), DA09085 (L.P.), and NS14610 and MH64109 (J.M.S.)

Subjects.
Eight adult male rhesus monkeys (Macaca mulatta) weighing (8.0–11.0 kg) were used in this study. They were individually housed in stainless steel cages in temperature and humidity controlled colony rooms with lighting maintained on a 6:00 A.M/6:00 P.M. on/off schedule and fed a diet of monkey chow supplemented by fresh fruit to maintain daily monitored body weight. Fluid intake was restricted in time and amount such that a prescribed volume of an animal's normal daily fluid intake (80 ml/kg) was received either during the behavioral testing session, or within 2 h of being returned to the home cage.

Behavioral Testing.
Each monkey was exposed to six different testing conditions [Normal Vehicle, Alert orexin-A (i.v. and nasal), Sleep Deprivation, Sleep Deprivation + orexin-A (i.v. or nasal)], which required 11–14 test sessions for each of the eight monkeys. Animals were placed in a primate chair 1.5 m in front of an LCD-front-projection screen for daily testing on a multi-image visual delayed match-to-sample (DMS) task and performed 150–300 trials per session (Hampson et al., 2004; Porrino et al., 2005). Animals were trained to move a cursor tracked by a fluorescent marker attached to the back of the monkey's hand into the images by positioning the hand within a two dimensional coordinate system on the chair counter. Stimuli consisted of clip art images projected as 25 cm squares within a 3 x 3 position matrix onto a 1.0 x 1.0 m display. All images were unique to a particular trial during a session and no image was exposed on more than on one trial per session. Responses to appropriate stimuli were rewarded with diluted fruit juice delivered via a sipper tube placed in front of the mouth. All animals were trained to a stable baseline on the DMS task in which delay varied randomly from 1 to 90 s on a given trial, and the number of non-match stimuli (images) varied randomly from 1 to 7 in the Match phase of the task. As shown previously performance accuracy varied directly with duration of delay and number of non-match images (#images) presented in the Match phase (Hampson et al., 2004; Porrino et al., 2005). Sets of stimulus images were routinely changed every 2 weeks to maintain the trial-unique feature of each session. Animals performed the task on consecutive days each week.

Sleep Deprivation Procedure.
Sleep deprivation consisted of 30–36 h of continuous sleep prevention and wakefulness supervised continuously by laboratory personnel as previously verified using EEG recordings of sleep architecture. On sleep deprivation nights animals were maintained in a cage separate from their home cage in a continuously lighted room and kept awake with videos, music, occasional treats, gentle cage shaking, and interaction with technicians (working in 3 h shifts) through the night, until the usual daily testing time the next day. After testing animals were returned to their home cage and allowed to sleep. In a previous investigation (Porrino et al., 2005) evidence was provided that changes in brain imaging correlates of local glucose utilization during the DMS task (see below) were consistent with simultaneously recorded EEG changes produced by 30–36 h of sleep deprivation. Hence, replication of the same brain imaging correlates of sleep deprivation in this study verified that each animal was tested in the same sleep loss condition as reported preciously. Intervals of 10 d, mandated by IACUC regulations, were interspersed between sleep deprivation episodes for each animal. There were no residual effects of the 30–36 h sleep deprivation regimen on testing 24 h afterward at which time animals had returned to their normal sleep patterns.