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 histaminergic neurons of the tuberomammillary nucleus (TMN) of the hypothalamus
Histamine neurons located exclusively in tmn

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. Green Star = stimulation
Orexin neurons mainly located in LH
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
Auto radiograph of Ox mRNA expression

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)
RN = Raphe Nucles

9. Orexin cont.
Some of the primary reciprocal projection bundles innervate nuclei of the ascending arousal system.
Cholinergic pedunculopontine
Laterodorsal tegmental nuclei
Serotonergic dorsal and median raphe nuclei
Noradrenergic locus coeruleus
Histaminergic tuberomammillary 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, g, 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
HDC = Histamine decarboxylase (histamine synthesizing enzyme)
Ox-A

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 = (cortical beta and gamma) 9-14 (cortical alpha) (theta) .5-4 (delta)

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

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

18. Analysis (per 2 h) of sleep-wake amounts before and after NPS i. c. v
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

21. Left 2 c-Fos then c-Fos histamnine
Right 2 c-Fos then c-Fos ox

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.