| A-19 |
Hypocretin neurons and CSF hypocretin levels in genetically (hypocretin receptor-2 mutated) narcoleptic Dobermans |
| Center for Narcolepsy, Sleep Research Center, Department of Psychiatry and Behavioral Sciences, Stanford University, Palo Alto, CA, USA | |
| Beth Ripley, Mutsumi Okura, Nobuhiro Fujiki, Mariko Ozu, Emmanuel Mignot,and Seiji Nishino | |
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Introduction: We recently discovered that mutations in the hypocretin receptor-2 gene (Hcrtr 2) cause
narcolepsy in Dobermans and Labradors (1). Shortly thereafter, Chemelli et al. reported that
preprohypocretin knockout mice display narcolepsy-like symptoms (2). Furthermore, we have
demonstrated that cerebrospinal (CSF) hypocretin levels are undetectably low in most human
narcoleptics (3). These findings suggest that a deficiency in hypocretin neurotransmission is involved
in the etiology/pathophysiology of narcolepsy.
Hypocretins -1 and -2 (orexins A and B) are neuropeptides that are derived from the same precursor peptide (prepro-hypocretin) by proteolytic processing(4, 5). Prepro-hypocretin mRNA and immunoreactive hypocretin-1 and -2 neurons are specifically localized within and around the lateral hypothalamic area (LHA) (6). Hypocretin -1 and -2 bind and activate two related G-protein-coupled receptors, termed Hcrtr 1 and Hcrtr 2. Hcrtr 1 is selective for hypocretin-1 whereas Hcrtr 2 has equal affinity for both hypocretin-1 and D2. Hcrtr-1 and -2 mRNA have been shown to be differentially localized, suggesting distinct physiological roles for hcrtr-1 and hcrtr-2 (5). In this study, we examined CSF levels of hypocretins -1 and -2 and hypocretin neurons in narcoleptic and control dogs to study whether hypocretin neurons and ligand production are altered in receptor-2 mutated narcoleptic dogs. Methods: For measuring hypocretins -1 and -2 in the CSF, cisternal CSF taps were carried out in 33 genetically narcoleptic (1.42 } 0.30 years) and 20 asymptomatic (11 heterozygous and 9 control; 1.63 } 0.25 years) dogs between 9:00a.m. and 2:00p.m., and samples were immediately frozen. Peptides were extracted from CSF using a reversed phase SEP-PAK C-18 column, and were then measured using radioimmunoassay (RIA) kits (Phoenix Pharmaceuticals, Mountain View, CA). For immunocytochemical staining of neurons, two narcoleptic Dobermans (32 months, two males) and two control Dobermans (38.5 months, one male and one female) were used. Immunohistochemical detection of hypocretin was done using either anti-hypocretin 1 antiserum (Phoenix Pharmaceuticals, CA) or mouse/rat hypocretin-2 antibodies (Alpha Diagnostic, TX). Results: Hypocretin -1, but not hypocretin-2, was detectable in 1ml samples of CSF in both narcoleptic and control dogs. CSF hypocretin-1 levels were similar in narcoleptic (273.5 } 5.75 pg/ml) and asymptomatic (260.0 } 5.51pg/ml) dogs. Additionally, there was no difference in hypocretin-1 levels between homozygous (257 } 9.5 pg/ml) and heterozygous (261.6 } 8.7 pg/ml) dogs born in the same backcross litters. Hypocretin-1 and hypocretin-2 immunoreactive neurons were found to be specifically localized in the perifornical nucleus and dorsal and lateral hypothalamic areas in adult control and narcoleptic dog brains. Gross microscopic inspection showed no clear difference in morphology, number, or distribution of neurons in narcoleptic and control dog brains. Our results suggest that no massive cell death or proliferation in hypocretin neurons occurs and that no altered ligand production is present in receptor-2 mutated adult narcoleptic animals. These results suggest that deficits in hypocretin receptor-2 mediated functions, versus receptor-1 mediated functions, are critical for developing the narcolepsy phenotype. 1. Lin L et al. Cell 1999;98:365-376. 2. Chemelli RM et al. Cell 1998; 98:437-451. 3.Nishino S et al. | |