The subjects were four normal Doberman pinschers and four narcoleptic Doberman pinschers. The normals were 1.5–3.1 year old males, and the narcoleptics were one male and three females 0.9–8.2 years old. No age or sex differences were seen in the results. The dogs were adapted to standing in a sling for 2 h/day for a period of 2 weeks. On the experimental day, the dog rested in the sling and blood pressure (BP) and heart rate (HR) were measured every 2.5 min for the entire 2 hour period using an oscillometric non-invasive BP monitor (SurgiVet Model V60046, SurgiVet Inc. Waukesha, WI, USA) with a cuff on the tail or a limb. Systolic, diastolic and mean arterial pressure (MAP) measurements were recorded. HR was recorded polygraphically with electrodes placed on the chest and limbs. Respiration was measured in the normal dogs with a strain gauge placed around the chest. HR and respiration signals were filtered and amplified with Grass Model 7P4G EKG Tachograph pre-amplifier and Model 7P1F DC pre-amplifier (Grass/Astro-Med, RI, USA) and digitized with Spike2 software (Cambridge Electronic Design, UK). A 30 min baseline was taken. All studies were completed between 10 AM and 2 PM.
After the 30 min baseline period, dogs were given either saline (0.5 ml) or one of the following drugs intravenously: prazosin hydrochloride (α1 adrenergic antagonist; 40 μg/kg; Sigma, St. Louis, MO, USA), physostigmine salicylate (cholinesterase inhibitor; 50 μg/kg; Taylor Pharmaceuticals, Decatur, IL, USA), labetalol hydrochloride (β and α1 adrenergic antagonist; 250 μg/kg; Bedford Laboratories, Bedford, OH, USA), phenylephrine (α1 adrenergic agonist; 150 μg/kg; Sigma) or methamphetamine hydrochloride (monoamine release enhancer; 125 μg/kg; Sigma). A second dose was given 30 min later (labetalol) or 45 min later (phenylephrine, methamphetamine and physostigmine), except for prazosin, which was only given once because of its long half life. Doses were those found optimal for triggering or preventing cataplexy in narcoleptic dogs (our data and (Nishino and Mignot, 1997
)). Resting in the sling for 2 hr after saline injection served as a control. Three replications of each condition were performed. At least 1 week elapsed between trials in each dog. No food was given during trials. Administration of physostigmine or prazosin in the doses employed always produces cataplexy in the narcoleptic dogs without the need to present eliciting stimuli or food.
The dogs were continuously monitored and defining symptoms of cataplexy, loss of neck, jaw or limb support, were noted every 2.5 minutes at the time of blood pressure measurement. Cataplexy occurrence under drug conditions was compared to that after saline injections. Cataplexy never occurred in the normal dogs.
CSF collection after drug administration
CSF was taken from the cisterna magna 1.5 hr after the initial dose (45 or 60 min after the second dose) under thiopental anesthesia (12.5 mg/kg, IV). This timing was determined based on a preliminary experiment that determined the time course (0, 30, 60 and 90 min) of Hcrt level with two drugs: labetalol and methamphetamine. It established that the peak level of Hcrt-1 in cisterna magna CSF occurred 60–90 min after drug administration at a time when the behavioral effects of all the drugs were pronounced. All assays were done blind to experimental conditions.
CSF samples (0.5 ml) were acidified with 1% trifluoroacetic acid (TFA) and loaded onto a C18 SEP-Column (Waters, Milford, MA). The peptide was eluted with 1% TFA/40% acetonitrile. The eluant was then dried and resuspended in RIA buffer. The solid-phase radioimmunoassay (Maidment & Evans, 1991) provided an IC50 of 2–3 fmol and a limit of detection of ~0.1 fmol. The Hcrt-1, iodinated Hcrt-1, and Hcrt-1 antiserum were obtained from Phoenix Pharmaceuticals (Cat. #RK-003-30; Belmont, CA).
All data are presented as mean ± SEM. CSF Hcrt-1 levels after drug conditions were compared with their corresponding values taken under control conditions. The dependent t-test was used. Continuous recordings of BP and HR were averaged across repeated trials and post-drug values were compared to those during the pre-drug baseline. Because all three measures of BP are highly correlated, only the analysis of MAP is presented.