Poster Presentations

Loss of hypocretin (HCRT) neurons has been linked to narcolepsy. These neurons project widely throughout brain, but it is not known which projection to which target site produces what symptom. We showed that HCRT receptors are present in brainstem areas implicated in REM sleep Begin superscript 1 End superscript. Since abnormal REM sleep triggering characterizes narcolepsy, we used HCRT2-saporin (HCRT2SAP), a toxin that selectively lesions HCRT receptor bearing cells, to assess the effects of such lesions on sleep. We also used α-DBH-sap to specifically destroy NA-LC neurons, which are the major brainstem targets of HCRT neurons. Methods: Twenty-three male Sprague-Dawley rats (350-620 g) instrumented for sleep recordings were given a single bilateral microinjection of either saline (n=11), or α-DBH-sap (n=5; 100 ng), or HCRT2-SAP (n=6; 46 ng). All injections were stereotaxically aimed to dorsolateral pons (A=-0.7; L=±1.4-1.6;V=+3.0). Then 24 h sleep recordings were done on 3rd, 6th, 9th, 12th and 18th days post-injections (12:12h lights on/off). Scoring was made visually on a computer (Icelus software) in 12s epochs for waking, slow wave sleep (SWS) and REM sleep (REMS) by one technician blind to treatment. ANOVA and t-test were used to compare changes in sleep parameters. Then brain were fixed, removed and sectioned for immunohistochemistry against DBH (1:50K;Chemicon) TH (1:12K; Chemicon) and NeuN (1:1K;Chemicon) proteins. Histochemistry for NADPH was made as well. A technician blind to treatment counted DBH-ir and NADPH+ cells in a 1:5 sections across the mesopontine tegmentum. Results: α-DBH-sap lesioned DBH-ir cells in the locus coeruleus (LC) region but did not affect the number of NADPH+ cells (cholinergic) in the LDTg (Fig 1). In addition α-DBH-sap produced a major loss in DBH fibers-ir among several LC-projection sites. TH and NeuN-ir neurons were not evident in the LC after α-DBH-sap either. Despite all these major degenerative signs observed in the LC after α-DBHsap, sleep parameters over the long-term were not different from saline-injected rats. In contrast rats lesioned with HCRT2-SAP showed a significant increase in nighttime sleep time across three weeks after injections (Table 1). Nighttime SWS+REMS time percent increased by 44% in HCRT2-SAP lesioned rats (P<0.01). The nighttime increase in sleep was associated with a significant increase in SWS and REMS bouts (P<0.05) but not with any change in bout duration. Daytime sleep was not affected by HCRT2-SAP. This toxin lesioned NADPH+ neurons in LDTg as well as NeuN-ir neurons in the parabrachial nucleus although DBH-ir cells were spared in the LC. Figure 1