The Ditribution Of Activated Neurons Following Central Administration Of Neuropeptide S—evidenced By C-fos Immunohistochemistry

Backgroud and Objective:Neuropeptide S (NPS) is a newly identified neuromodulator and its precursor mRNA is expressed between locus coeruleus and Barrington’s nuclei, in specific, the substantia innominata, the principle sensory trigeminal nuclei and the lateral parabrachial nuclei. Neuropeptide S receptor (NPSR) mRNA is widely distributed in the brain, e.g., the cortex, thalamus, hypothalamus, amygdala, and subiculum. That NPS selectively binds with high affinity to NPSR could produce the mobilization of intracellular Ca2+and increase in cAMP levels. Current researches confirm that NPS involves in regulating sleep-wake cycle, locomotor activity, anxiolytic-like effects, study and memory, feeding and energy metabolism, and also is concerned with addiction, analgesia, neuroendocrine, immune, antioxidant and other physiological and pathological processes. Based on previous studies, we hypothesized that NPS may involve its regulation in those physiological and pathological processes through its receptor. The aim of our study was to identify the distribution of brain neurons activated by central administration of NPS in rats using c-fos immunohistochemistry and thus to provide the evidence of morphological localization of NPS involving in those physiological and pathological processes.Methods:Adult male Sprague-Dawley rats, weighing250-300g (n=12), were randomly selected into NPS group (n=6) and saline group (n=6). Respectively injected with lnmol NPS and saline at10:00through an implanted guide cannula (diameter=0.6mm, length=28mm, i.c.v. AP-0.9mm, ML+1.5mm, DV-3.3mm according to the atlases of Paxinos and Watson),1.5hours later, the animals were deeply anesthetized with chloral hydrate and perfused via the ascending aorta with200ml saline and then300ml ice-cold4%paraformaldehyde(PFA) in0.1M phosphate(ph7.4). Their brains were removed, immersed in35%sucrose solution in PBS48h at4℃, and coronally sectioned (35μm) at-20℃. The floating sections were rinsed in0.01M PB saline for Fos immunostaining, and the light microscope was used to observe the distribution of c-fos immunoreactivity neurons, then count the numbers and analyze statistically the variations of fos-ir neurons number among different nuclei.Results:1.The number of Fos-IR neurons induced by NPS increased in telencephalonThe number of Fos-IR neurons induced by NPS in the piriform cortex was increased by399%(P<0.0001) and in Motor cx1, Motor cx2, Somatosensory cx1, Somatosensory cx2were respectively increased by200%,198%,197%,198%compared with saline (P<0.0001); In amygdala and extended including cortical amygdala, basomedial amygdala, lateral amygdala, medial amygdala, bed stria terminalis increased by296%,214%,305%,413%(P<0.0001),110%(p<0.05);In hippocampal formation, including CA1, CA2,CA3,and subiculum increased by203%,203%,562%,268%(p<0.0001).2.The number of Fos-IR neurons induced by NPS increased in diencephalonCompared with saline, the number of Fos-IR neurons-induced by NPS in the hypothalamic including suprachiasmatic nuclei, paraventricular nuclei, dorsomedial hypothalamic nuclei, ventromedial hypothalamic nuclei, arcuate nuclei, perifornical nuclei, ventral and dorsal tuberomammillary nuclei and lateral hypothalamus area were respectively increased by322%,108%,274%,126%,267%,520%,641%,586%and378%(P<0.0001).3. The number of Fos-IR neurons-induced by NPS increased in brainstemThe number of Fos-IR neurons induced by NPS in the brainstem including superficial gray layer, insuperficial gray layer, periaqueductal gray, locus coeruleus, dorsal raphe nuclei, caudal linear raphe nuclei was increased by203%,479%,263%,202%,210%and157%compared with saline(p<0.0001).Conclusion:1,That NPS activated the histaminergic neurons, orexinergic neurons and suprachiasmatic nuclei of hypothalamus, locus coeruleus and dorsal raphe nuclei and caudal linear raphe nuclei indicates its regulation in the sleep-wake cycle.2,That NPS activated the amygdala and extended, paraventricular hypothalamic nuclei, locus coeruleus, dorsal raphe nuclei and caudal linear raphe nuclei suggests its role in the regulation of emotion.3,That NPS activated the piriform cortex, amygdala and extended neurons indicates it may involve in the regulation of olfactory sensation.4,That NPS activated the hippocampal formation, amygdala and extended neurons suggests it may involve in the study and memory process. 5,That NPS activated the arcuate nuclei, dorsomedial hypothalamic nuclei, ventromedial hypothalamic nuclei and paraventricular nuclei suggests its role in the regulation of food intake.6,That NPS activated the orexinergic neurons and paraventricular nuclei indicates it may involve in the regulation of drug addiction and dependence.7,That NPS activated the superior and inferior colliculus suggests it role in the regulation of auditory and visual sensation.8,That NPS activated the periaqueductal gray and arcuate nuclei shows it may involve in the regulation of algesia9,That NPS activated the arcuate nuclei indicates its role in the regulation of neuroendocrine and autonomic nervous system.