Data were downloaded to a PC through an inductively coupled Actiwatch reader and further analyzed by a program of our design. Actigraphs were secured to a neck collar that was placed on the dogs throughout the period of study. The effects of Hcrt-1 on sleep-wake periods were monitored continuously for 24 h/day with collar mounted actigraphs (Actiwatch, Mini Mitter Inc, Sundriver, OR) while the animals remained in their home dog runs. Hcrt-1 or control injections were administered between 15:30 and 16:30 h. On control days, saline was administered in the same manner. This treatment combined with the large volume of the diluent used minimizes problems caused by the “stickiness” of the peptide. The glass syringe was pre-soaked in 1% BSA, rinsed in Milli-Q water, then dried at 60☌ prior to use. One to 4 /ug/kg of Hcrt-1 (orexin-A, #003–30, Phoenix Pharmaceuticals, Mountain View, CA) dissolved in normal saline (100 /ug in 2 ml) was administered through the cephalic vein using a glass syringe. We determined the effect of Hcrt-1 administration on activity levels and the duration of sleep-waking states over the 24-hour period with actigraphy. We determined the effect of Hcrt-1 administration on sleep organization using polygraph recording. Hcrt-1 is pharmacologically stable whereas Hcrt-2 is known to degrade rapidly ( Kastin and Akerstrom, 1999). Hcrt-1 has a high affinity for both Hcrt-1 and Hcrt-2 receptors ( Sakurai et al. We analyzed the effect of systemically administered Hcrt-1 on cataplexy with a modified food-elicited cataplexy test (FECT) ( Baker and Dement, 1985). Dogs were between 1.5 years and 10 years of age, with an average of 3.8 years. Six genetically narcoleptic Doberman pinschers (5 males and 1 female) served as subjects.
In the current study, we tested the effect of intravenous administration of Hcrt-1 on narcolepsy/cataplexy in canine narcoleptics. However, a recent study suggested that non-saturable mechanisms for Hcrt-1 transport across the BBB exist, at least for iodinated Hcrt-1 ( Kastin and Akerstrom, 1999). Basic research on the behavioral effects of the hypocretins has generally used intracerebroventricular or intra-parenchymal microinjection of the peptide ( Hagan et al., 1999 Dube et al., 1999), and some studies have concluded that Herts administered systemically do not cross the blood-brain barrier (BBB) at sufficient levels to affect physiological function ( Chen et al., 1999 Takahashi et al., 1999), making development of an Hcrt receptor agonist with good BBB permeability a high priority. This work suggests that administration of Hcrt might reverse symptoms of narcolepsy by compensating for either inefficient receptor transduction or diminished levels of the agonist. Human narcoleptics have reduced levels of Hcrt-1 in their cerebrospinal fluid ( Nishino et al., 2000). A null mutation of the gene encoding the two known hypocretin (Hcrt) peptides produces aspects of the narcolepsy syndrome in mice ( Chemelli et al., 1999). Drug side effects, residual sleepiness and cataplexy episodes continue to be major problems for most treated narcoleptics ( Aldrich, 1998).Ī mutation in the gene responsible for the hypocretin-2 (orexin-2) receptor is the genetic cause of canine narcolepsy ( Lin et al., 1999). Current drug treatments can be dichotomized into those that are aimed at daytime sleepiness, typically using dopamine agonists or Modafinil (Provigil, by Cephalon, West Chester, PA), and those that are aimed at cataplexy, typically using tricyclic antidepressants ( Siegel, 1999). Narcoleptic patients experience cataplexy, which is a sudden loss of muscle tone most commonly in response to the sudden onset of strong emotions, excessive daytime sleepiness and fragmentation of sleep during the night.
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