Neuropeptide S and Wakefulness

1. Neuropeptide S and Wakefulness P. Zhao, Y.F. Shao, M. Zhang, K. Fan, X.P. Kong, R. Wang, and Y.P. Hou

2. Histamine and arousal • Plays a major role in Motivation and Arousal • Appetitive behavior, sleep/wake cycles, and food anticipatory behavior • Of interest are histaminergicneurons of the tuberomammillarynucleus (TMN) of the hypothalamus

3. Histamine (HA) cont. • Histamminergic axons innervate many brain regions associated with arousal. • Innervate many areas (PFC to spinal cord) • Excitatory tone through H1r and H2r • Modulatory tone through H3r • Main afferent connections from hypothalamic regions, infralimbic regions, hypothalamic preoptic region, and the lateral septum • Human Histamine 

4. Histamine cont. • Histamine is essential for appetitive and aversive phases of motivated behaviors.

5. Histamine

6. Red Box = inhibition • Green Star = stimulation

7. Orexin/Hypocretin cont. • Hypothalamic peptides. • Only a few thousand orexinergic neurons, mainly located in lateral hypothalamus • Of note, orexinergic neurons project to histaminergic neurons in the TMN • Interaction between the two suggests a strong role in cortical arousal

8. Orexin/Hypocretin cont. • Orexins role in arousal. • Orx1 causes central activation of HPA axis • Hyperarousal • Believed that Ox stimulates: • Histaminergic (TMN) • Noradrenergic (LC) • Serotonergic (RN) • Cholinergic (Basal Forebrain)

9. Orexin cont. • Some of the primary reciprocal projection bundles innervate nuclei of the ascending arousal system. • Cholinergic pedunculopontine • Laterodorsaltegmental nuclei • Serotonergic dorsal and median raphe nuclei • Noradrenergic locus coeruleus • Histaminergictuberomammillary nucleus • All the above have been implicated in promoting/inducing arousal

10. Main Paper / Experiment Proper • NPS implicated in arousal – group interested in looking at NPS role in sleep/wake cycles. • So clearly the best way to tackle this is to jam a cannula into a rat brain and pump it full of NPS while asleep • Previous NPS studies noted increased NPSR mRNA expression in the posterior hypothalamus. • Histaminergic (HA) tuberomamillary nucleus (TMN) • Orexinergic (Ox) perifornical nucleus (PeF), dorsomedial hypothalamic nucleus (DMH), and lateral hyothalamic area (LH)

11. Experimental Units/Setup • Sprague-Dawley rats, 250-300g, 8-10wks • EEG, EMG, Immunohistochemistry • c-Fos, HDC, Ox-A • Cortical electrodes inserted into dura through 2 holes. (frontal and parietal cortices). • Guide cannula into right lateral ventricle

12. Experimental Setup • 1 wk recovery • Data shown is for mouse NPS, rat NPS mirrored results. • .1-1nmol mix injected at 2.5microliters at 10a (2hrs after lights on) so to be in maximal sleep mode!

13. Experimental Setup • 1 1/2 hrs post ICV admin of NPS. Animals killed, prepped and brain regions of interest sliced. • First c-Fos staining, then HDC or Ox-A

14. Data Analysis • Poplygraphic records scored in 30sec epochs for wakefulness, slow wave sleep, and paradoxical sleep • Cell counting Fos-ir, HDC-ir, Ox-A-ir, Fos-ir+HDC-ir and Fos-ir+Ox-A-it • HA neurons in the VTMN • Ox neurons in the PeF, LH, and DMH

15. DATA TIME! Control. 14.5-60 = (cortical beta and gamma) 9-14 (cortical alpha) 4.5-8.5 (theta) .5-4 (delta)

16. Data Time 2 .1nmol NPS 14.5-60 = (cortical beta and gamma) 9-14 (cortical alpha) 4.5-8.5 (theta) .5-4 (delta)

17. Data Time 3 1nmol NPS. 14.5-60 = (cortical beta and gamma) 9-14 (cortical alpha) 4.5-8.5 (theta) .5-4 (delta)

18. Analysis (per 2 h) of sleep-wake amounts before and after NPS i.c.v. injection during the lights-on phase. Values represent means SEM (n10 rats in each group). • * P0.05, ** P0.01, *** P0.001 compared with saline; # P0.05, • dose of .1nmol NPS compared with dose of 1nmol NPS.

19. Total amount and episode duration and number of sleep-wake states for 3 hr post injection of NPS and saline. Open, gray, and black bars respectively show the profiles for saline-, 0.1, and 1 nmol of NPS-treated rats. Values are means SEM (n10 rats in each group). * P0.05, ** P0.01, *** P0.001 compared with saline; # P0.05, ### P0.001, dose of 1 nmol NPS compared with dose of 0.1 nmol NPS.

20. c-Fos expression • c-Fos – detectable product of neuronal activity. • Look for c-Fos find active neurons. • Following 1nmol NPS injection… • TMN, Arc, PeF, DMH, LH • Followed up with combo c-Fos/HDC and c-Fos/Ox-a

22. c-Fos and Dual Staining

23. Results • .1 and 1nmol administration of NPS had immediate fast and low voltage EEG and ECG activity. • 1hr duration (.1nmol) 3hr duration (1nmol) • Wakefulness increased significantly while SWS and PS were suppressed or decreased • 1 ½ hr Post Injectioaw an increase in c-Fos expression in TMN, Arc, PeF, DMH, LH • HA neurons in the V and DTMN • Ox neurons in the DMH, PeF, and LH

24. Conclusions • NPS injections significantly enhanced wakefulness, and reduced SWS and PS. • Marked increase in c-Fos expression in posterior Hypothalamus. • Histaminergic neurons in the tuberomamillary nucleus • Orexinergic neurons in the perifornical nucleus, lateral hypothalamic area, and dorsomedial hypothalamus

25. Long story short • NPS promotes the release of histamine and orexin which in turn leads to arousal/wakefulness through further downstream effects.