| Sleep plays important roles during physiological and pathophysiological processes. It is well known that sleep plays a key role in learning and memory.Neuropeptide, a member of a class of protein-like molecules made in the brain, are tightly related with sleep and wakefulness. Neuropeptide S (NPS) and its receptor (NPSR) form a newly discovered neuropeptide system. The 20 residue peptide NPS, is the endogenous ligand of the NPSR, a former orphan G-protein-coupled receptor. NPS can be found in all vertebrate species (human, rat, rat, chick, chimpanzee and cattle), mainly distributing in brain, glandula thyreoidea, salivary gland and mammary gland. NPS/NPSR system modulates many physiological functions, such as wakefulness and sleep, motoring, anxiety, pain, food intake and oxidative damage. The most important one is modulating wakefulness and sleep. However, as a newly discovered neuropeptide, controversy exists about the expression and functions of neuropeptide S in the central nervous system (CNS).As the complete expression pattern and the distribution of NPS and NPS mRNA in the in the rat central nervous system has not been described in detail so far, we planed to investigate the expression of NPS in CNS to lay foundation for future functional researches. We describe in this paper a comprehensive anatomical mapping of NPS in rat CNS using immunohistochemistry and in situ hybridization together. We investigated the effects of different durations of rapid eye movement (REM) sleep deprivation on the expression of NPS and NPSmRNA in rat hypothalamus. We also discussed the connection between cognition disorder and hippocampal p-CREB changes caused by REM sleep deprivation and studied the role of NPS in modulating hippocampus-dependent spatial learning and memory by the MWM, determined whether NPS could affect spatial learning and memory impairments induced by REMSD and examined the change of p-CREB expression in the rat hippocampus in the NPS treated group after 72h REMSD.The present study investigates the NPS expression pattern in the rat CNS using immunohistochemistry and in situ hybridization histochemistry. We found that NPS were widely distributed in rat CNS, and the distribution patterns and levels of NPS immunolabeling of native protein had significant correlation with its mRNA counterpart. The main olfactory bulb showed NPS immunostaining. The immunostaining was observed mainly in the dendrites and cell bodies. Heavy NPS immunostaining was observed in the motor, somatosensory, piriform and entorhinal cortex. Dense immuno labeling in layers V, suggests a role for NPS in interconnection of local cortical areas. In the hippocampus, the cell body and distal dendrites of CA1-4 pyramidal cells had different levels of NPS immunostaining, suggesting that NPS may contribute to hippocampal excitatory neurotransmission or modulation in learning and memory. NPS was expressed extensively in most subnuclei of the thalamus, particularly heavy immunostaining was observed in the anteroventral, anteromedial, anterodorsal and ventral-lateral thalamic nuclei. In different regions of the hypothalamus, NPS immunostaining neurons and fibers were widely. NPS immunostaining was widely distributed in the rat brain stem, heavy NPS immunostaining was detected in the locus caeruleus, parabrachial nucle, mesencephalic trigeminal nucleus, motor trigeminal nucleus. The study showed heavy immunostaining for NPS in the Purkinje cell.NPS immunostaining was observed at the cervical, thoracic, lumbar and sacral levels. The most prominent staining was seen in the dorsal horn.Adult male Sprague-Dawley rats were randomly divided into cage control group (CC), tank contral group (TC), REM sleep deprivation group (SD).Each group were divided into three duration groups:1d,3d and 5d. The rats were deprived of REM sleep for different durations by the modified multiple platform method (MMPM). Immunohistochemistry, in situ hybridization histochemistry and RT-PCR were used to test the expression of neuropeptide S in rat hypothalamus. Resluts show that:1. There was no significant difference between CC and TC in the expression of neuropeptide S in rats hypothalamus; 2. The expression of neuropeptide S in rats hypothalamus of SD1 were not significantly different compared to that of CC and TC3; 3. The expression of neuropeptide S in rats hypothalamus of SD3 and SD5 were significantly higher than that of SD1,CC and TC.At last, we used the Morris water maze (MWM) to determine the effects of NPS on spatial learning and memory following intracerebroventricular (i.c.v.) injection in rats after 72 h REMSD. We also investigated the changes of phosphorylation of cAMP-response element binding protein (CREB) in the rat hippocampus in the NPS treated group after 72h REMSD. Adult male Sprague-Dawley rats were randomly divided into rats were randomly divided into six groups (CC-aCSF group, TC-aCSF group, SD-aCSF group, CC-NPS group, TC-NPS group and SD-NPS group). Rats were deprived of REM sleep by the modified multiple platform method (MMPM) for 72h while NPS or aCSF was injected every day. The Morris water maze task was performed to test the spatial learning and memory. After certain duration of REM sleep deprivation, hippocampal CREB phosphorylation level and quantity were analyzed by immunohistochemistry and Western-blot. Results show that 1.72h REMSD impaired spatial learning in rats and NPS mitigated the deficit.2.72h REMSD impaired hippocampus-dependent spatial memory, and NPS mitigated spatial memory impairment induced by REMSD in rats.3. By immunostaining, There were p-CREB-ir cells in CC-aCSF group, TC-aCSF group, CC-NPS group and TC-NPS group in CA1 and DG, fewer positive cells were observed in SD-aCSF group and positive cells increased in the SD-NPS group.4. After 72h REM sleep deprivation, the expression of p-CREB in the hippocampus significantly decreased, while the expression of p-CREB increased in the SD-NPS group compared to the SD-aCSF group.Firstly, this is the first extensive study undertaken to investigate the combined distribution of NPS protein and mRNA in the rat CNS using immunohistochemistry and in situ hybridization histochemistry together. The distribution of NPS shown here considerably extends previous studies which reported NPS exists in limited populations of neurons and may show the directions for explore the new function of NPS.Secondly, we first found that after 72h REM sleep deprivation, the expression of NPS and NPS mRNA increased in rat hypothalamus. This convinced the relationship between sleep and NPS. NPS may be participates in the body self-regulation after REM sleep deprivation.Lastly, in summary, our findings demonstrate that neuropetide S plays an important role in REM sleep deprivation-induced memory impairment, and imply that NPS/NPSR system may be a potential target for treatment of spatial memory impairment caused by REM sleep deprivation. The protective effect of NPS on spatial memory impairment in REMSD rats may be mediated by increasing the p-CREB expression in the hippocampus. |
PDF Full Text Request