The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of behavioural arousal. The PF-LHA contains several cell types including neurones expressing the peptides, hypocretin (HCRT; also called orexin) and melanin-concentrating hormone (MCH). Evidence suggests that most of the PF-LHA neurones, including HCRT neurones, are active during waking and quiescent during non-rapid eye movement (non-NREM) sleep. The PF-LHA contains local GABAergic interneurones and also receives GABAergic inputs from sleep-promoting regions in the preoptic area of the hypothalamus. We hypothesized that increased GABA-mediated inhibition within PF-LHA contributes to the suppression of neuronal activity during non-REM sleep. EEG and EMG activity of rats were monitored for 2 h during microdialytic delivery of artificial cerebrospinal fluid (aCSF) or bicuculline, a GABA A receptor antagonist, into the PF-LHA in spontaneously sleeping rats during the lights-on period. At the end of aCSF or bicuculline perfusion, rats were killed and c-Fos immunoreactivity (Fos-IR) in HCRT, MCH and other PF-LHA neurones was quantified. In response to bicuculline perfusion into the PF-LHA, rats exhibited a dose-dependent decrease in non-REM and REM sleep time and an increase in time awake. The number of HCRT, MCH and non-HCRT/non-MCH neurones exhibiting Fos-IR adjacent to the microdialysis probe also increased dose-dependently in response to bicuculline. However, significantly fewer MCH neurones exhibited Fos-IR in response to bicuculline as compared to HCRT and other PF-LHA neurones. These results support the hypothesis that PF-LHA neurones, including HCRT neurones, are subject to increased endogenous GABAergic inhibition during sleep. In contrast, MCH neurones appear to be subject to weaker GABAergic control during sleep.
Previous work shows that sleep deprivation impairs hippocampal-dependent learning and long-term potentiation (LTP). Brain-derived neurotrophic factor (BDNF), cAMP response-element-binding (CREB) and calcium-calmodulin-dependent protein kinase II (CAMKII) are critical modulators of hippocampal-dependent learning and LTP. In the present study we compared the effects of short-(8 h) and intermediate-term (48 h) sleep deprivation (SD) on the expression of BDNF and its downstream targets, Synapsin I, CREB and CAMKII in the neocortex and the hippocampus. Rats were sleep deprived using an intermittent treadmill system which equated total movement in the SD and control treadmill animals (CT), but permitted sustained periods of rest in CT animals. Animals were divided into SD (treadmill schedule: 3 s on/12 s off) and two treadmill control groups, CT1 (15 min on/60 min off) and CT2 (30 min on/120 min off -permitting more sustained sleep). Real-time Taqman RT-PCR was used to measure changes in mRNA; BDNF protein levels were determined using ELISA. In the hippocampus, 8 h treatments reduced BDNF, Synapsin I, CREB and CAMKII gene expression in both SD and control groups. Following 48 h of experimental procedures, the expression of all these four molecular markers of plasticity was reduced in SD and CT1 groups compared to the CT2 and cage control groups. In the hippocampus, BDNF protein levels after 8 h and 48 h treatments paralleled the changes in mRNA. In neocortex, neither 8 h nor 48 h SD or control treatments had significant effects on BDNF, Synapsin I and CAMKII mRNA levels. Stepwise regression analysis suggested that loss of REM sleep underlies the effects of SD on hippocampal BDNF, Synapsin I and CREB mRNA levels, whereas loss of NREM sleep underlies the effects on CAMKII mRNA.
The adult hippocampal dentate gyrus (DG) is a site of continuing neurogenesis. This process is influenced by a variety of physiological and experiential stimuli including total sleep deprivation (TSD). In humans, sleep fragmentation (SF) is a more common sleep condition than TSD. SF is associated with several prevalent diseases. We assessed a hypothesis that SF would suppress adult neurogenesis in the DG of the adult rat. An intermittent treadmill system was used; the treadmill was on for 3 s and off for 30 s (SF). For sleep fragmentation control (SFC), the treadmill was on for 15 min and off for 150 min. SF was conducted for three durations: 1, 4 and 7 days. To label proliferating cells, the thymidine analog, 5-bromo-2-deoxyuridine (BrdU), was injected 2 h prior to the end of each experiment. Expression of the intrinsic proliferative marker, Ki67, was also studied. SF rats exhibited an increased number of non-rapid eye movement (NREM) sleep bouts with no change in the percent of time spent in this stage. The numbers of both BrdU-positive cells and Ki67-positive cells were reduced by approximately 70% (P<0.05) in the SF groups after 4 and 7 days of experimental conditions whereas no differences were observed after 1 day. In a second experiment, we found that the percentage of new cells expressing a neuronal phenotype 3 weeks after BrdU administration was lower in the SF in comparison with the SFC group for all three durations of SF. We also examined the effects of SF on proliferation in adrenalectomized (ADX) animals, with basal corticosterone replacement. ADX SF animals exhibited a 55% reduction in the number of BrdU-positive cells when compared with ADX SFC. Thus, elevated glucocorticoids do not account for most of the reduction in cell proliferation induced by the SF procedure, although a small contribution of stress is not excluded. The results show that sustained SF induced marked reduction in hippocampal neurogenesis.
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