Bim23042

Bag-1 and bantag-1, BRS3 agonist and antagonist, respectively, were a kind gift from Dr Marc Reitman (Merck, Rahway, NJ USA). Bombesin, GRP, BIM 23042 were purchased from Tocris (Ellisville, MO, USA). RC-3095, TTX, CNQX, AP-5 and gabazine, BAPTA-AM were purchased from Sigma (St Louis, MO, USA).

For systemic antagonism of hypocretin signaling in awake animals, mice were gavage fed 100 mg/kg hypocretin receptor antagonist almorexant (ALM, Selleckchem) dissolved in 0.25% methylcellulose in PBS. After 2 hours, breathing behavior and response to tube confinement were monitored and recorded by plethysmography.
For anesthetized conditions, urethane was administered by intraperitoneal injection and breathing monitored through the tracheostomy tube as described above. For LHA disinhibition, 200 nl of GABA receptor antagonist bicuculline (Tocris, 1mM) was unilaterally injected into LHA of wild-type adult mice. To antagonize hypocretin signaling, animals were gavage fed ALM 2 hours before the bicuculline injection as described above.
For local pharmacological experiments combined with optogenetics, 2–4 weeks before the pharmacological injection AAV-DIO-ChR2 virus was injected into the LHA of Hcrt-IRES-Cre mice and a cannula implanted. To locally antagonize hypocretin signaling, 200 nl of ALM (20mM) was stereotactically injected into the RTN. Baseline breathing behavior was recorded for 10 minutes before the response to photoactivation of hypocretin neurons was analyzed. To locally inhibit NMBR, 500 nl of NMBR antagonist BIM23042 (Tocris, 10uM) was stereotactically injected into the preBötC immediately before the response to photoactivation of hypocretin neurons was analyzed.