This study investigated the effects of 6h of TSD on metabolic activity, antioxidant responses and working memory. We chose to study 6h of TSD because this time period of sleep deprivation can be easily achieved by gentle handling, while longer periods of sleep deprivation would require more stressful procedures. Also, 6h of TSD has been shown to induce a significant increase in NREM sleep and slow wave activity, while 3h of TSD causes only a minor increase in total sleep time [30
In this study we showed that 6h of TSD did not affect spontaneous alternation behavior (SAB) in the Y maze. Pierard et al [19
] similarly reported that 3h of TSD in mice had no effect on SAB, in contrast 24h of TSD caused major disruptions in the ability of mice to run the alternation task. The mice chose to sleep rather than to explore the maze. Ten hours of TSD, on the other hand, decreased the SAB in deprived mice compared to non deprived mice. This suggests that the amount of sleep loss is an important factor affecting performance in the Y-maze. Guan et al [11
] reported that 6h of TSD, by gentle handling, impaired spatial memory, but not spatial learning, and did not influence nonspatial learning or memory in rats, as assessed by performance in the Morris water maze. Smith and Rose [25
] similarly reported that REM sleep is involved in spatial but not in non spatial learning in rats in the Morris water maze. The Y maze is a useful index of responsiveness to novelty, reflected by increased locomotor and exploratory behavior, as well as SAB, which is a measure of working memory [13
]. SAB is the innate tendency of rodents to remember the position of the arm selected in the preceding choice and therefore serves as a measure of cognitive impairment. The Morris water maze, on the other hand, is used to assess spatial and non spatial learning and memory (acquisition, retention).
Our study also showed that 6h of TSD increased exploratory behavior in a new environment. This is consistent with a previous study by Albert et al [1
] who reported that REM sleep deprived rats showed increased locomotor and exploratory activity and greater sensitivity to environmental stimuli compared to non deprived rats. Similarly, Tartar et al [28
] recently reported that 24h of treadmill-induced TSD or sleep fragmentation in rats, increased exploratory behavior in an open field test. Sleep deprived rats showed increased number of entries into and time spent in the open field.
This is the first study showing that acute (6h) TSD, by gentle handling, increases antioxidant responses (SOD, GPx and GSHt) in multiple rat brain regions. Free radicals have been shown to regulate the activities of antioxidative enzymes, (SOD and GPx) and endogenous antioxidant (GSHt) [12
]. Superoxide anions are free radicals produced in the mitochondria and endoplasmic reticulum as a by product of ATP synthesis. SOD converts superoxide anions into hydrogen peroxide and oxygen, while GPx, using glutathione as a cofactor, converts hydrogen peroxide and lipid hydroperoxides into oxygen and water or ROH respectively. Hydrogen peroxide and lipid hydroperoxides if not removed can produce the more reactive hydroxyl free radical, which can lead to oxidative stress. Superoxide anions can also react with nitric oxide to form peroxynitrite which can produce both hydroxyl radicals as well as nitrotyrosine, resulting in both oxidative and nitrosative stress.
The increase in total glutathione levels was the most pronounced antioxidant response after 6h of TSD, and may account for the ability of rats to compensate for any deficit in working memory and/or increased exploratory behavior. Cruz-Aguado et al [3
] reported that diethylmaleate (DEM)-treated rats, who had reduced total glutathione levels, exhibited a motivational or sensorimotor deficit, leading to a reduction in their exploratory or locomotor behavior. These authors [3
] further showed that glutathione depletion did not influence performance of animals in the passive avoidance test, although it resulted in impaired spatial acquisition but not retention in the Morris water maze. Dean et al [5
] similarly reported that 2-cyclohexene-1-one (CHX)-treated rats and mice, who had reduced total glutathione levels, showed disruption of short term spatial memory.. These findings support our hypothesis that increased total glutathione levels may prevent deficits in working memory in rats subjected to 6h of TSD.
Here we show increased antioxidant responses in rats subjected to short term (6h) TSD, however, we previously reported decreased antioxidant responses in rats exposed to long term (5–11 days) TSD [20
]. We propose that acute (short term) sleep loss increases the production of free radicals which then induces the antioxidant responses. Chronic (long term) sleep loss further increases the levels of free radicals, and the elevated antioxidant responses would be incapable of successfully scavenging these enhanced free radicals, resulting in damage to the antioxidative enzymes, thus leading to decreased antioxidant responses. This differential effect of acute and chronic sleep deprivation varies across brain regions. Increased antioxidant responses were observed in the rat cortex, hippocampus, basal forebrain, brainstem and cerebellum with 6h of TSD, while decreased responses were observed in the rat hippocampus and brainstem with 5–11 days of TSD [20
]. The brain is not uniformly vulnerable to the effects of sleep deprivation. We speculate that this could be due to the fact that certain neuronal populations may be more susceptible to free radical production as a result of increased activity of these neurons due to prolonged waking. Cirelli [2
] showed that transcriptional changes, in the rat cerebral cortex, associated with prolonged sleep loss differed significantly from short term sleep deprivation. She suggested that sleep loss may trigger an oxidative stress response in some brain regions, but the brain is capable of responding to this acute stress effectively and thus prevents oxidative damage, while chronic stress may result in irreversible changes [2
Changes in antioxidant responses have also been observed with paradoxical sleep deprivation (PSD). D’Almeida et al [4
] reported that 96h of PSD significantly decreased GSHt levels in the rat hypothalamus. Decreased GSHt levels and SOD activity as well as higher lipid peroxidation (thiobarbituric acid reactive substances) were also observed in the hippocampus, thalamus and hypothalamus of rats subjected to 96h of PSD [24
]. These changes were greater in old (24 months) compared to adult (8 months) rats. On the other hand, increased SOD activity and lower lipid peroxidation was noted in the cortex and brainstem of PSD deprived rats, but these changes were greater in adult compared to old rats [24
]. Silva et al [23
] showed that 72h of PSD increased the ratio of oxidized/reduced glutathione and increased lipid peroxidation in the mouse hippocampus.
We hypothesize that elevated glucose metabolism, arising from increased energy demands during wakefulness, a period of high neuronal activity, may be a potential source of elevated free radicals. Ikeda et al. [14
] proposed that free radicals are produced during wakefulness, a period of high neuronal activity, while Scharf et al., [22
] proposed that free radicals may be activated by the depletion of cellular energy occurring during extended wakefulness. We previously reported that chronic TSD (>45h) causes degenerative changes in the rat supraoptic nucleus (SON) of the hypothalamus, a region of high metabolic activity [6
]. Gip et al [9
] reported that 6h of TSD decreased glycogen levels in the rat cerebellum and hippocampus, while increasing glucose levels in the cortex. They proposed that the regional effects of TSD on brain glycogen and glucose levels may be correlated with differing energy demands.
Hexokinase (HK), the enzyme that catalyzes the initial step in glucose metabolism, is the major factor governing the rate of glucose metabolism. In this study we showed that 6h of TSD increased HK activity in several rat brain regions. Thakkar and Mallick [29
] similarly reported that 4 days of PSD, by the flower pot technique, increased HK activity in the rat brainstem, cerebellum and cerebrum. Knull et al [15
] reported that intraperitoneal injection of glucose to galactose fed chicks increased HK activity in the cerebellum, while Mayer et al [16
] reported that the HK activity of the small intestine of starved rats increased significantly within the first 15 min. of perfusion with 50mM glucose.
We show for the first time that acute (6h) TSD in the rat increases antioxidant responses in multiple brain regions. This may reflect an enhanced production of free radicals, arising in part from elevated glucose metabolism (in the cortex). The absence of antioxidant responses in the hypothalamus, despite an increase in HK activity would suggest that endogenous antioxidants in the rat hypothalamus were sufficient to scavenge the free radicals produced by increased glucose metabolism, resulting from 6h of TSD. Conversely antioxidants, other than SOD, GPx or GSHt, may be involved. Also increased antioxidant responses in the rat brainstem, basal forebrain, hippocampus and cerebellum could be due to free radicals produced from sources other than increased glucose metabolism. Furthermore 6h of TSD increased locomotor and exploratory behavior in a new environment without affecting SAB in the Y maze. Thus acute (6h) sleep loss may trigger mechanisms (like increased antioxidant responses) that prevent initial deterioration in working memory and also lead to increased exploratory behavior.