Research ReportSleep deprivation increases A1 adenosine receptor density in the rat brain
Introduction
Endogenous adenosine is considered a candidate molecule for the homeostatic sleep drive, inducing sleep after prolonged wakefulness (for review see Basheer et al., 2004).
In freely moving cats, microdialysis showed accumulating adenosine levels in the cortex during prolonged wakefulness (6 h) (Porkka-Heiskanen et al., 1997). Subsequent recovery-sleep restored the adenosine concentration to baseline levels. Similar experiments have been repeated in rat with comparable results in the basal forebrain (Basheer et al., 1999, Murillo-Rodriguez et al., 2004). An artificial increase of the adenosine concentration in the basal forebrain by infusion of adenosine in cats (Portas et al., 1997) or rats (Basheer et al., 1999) increased sleep. Inhibition of equilibrative nucleoside transporters (Porkka-Heiskanen et al., 2000) resulted in electroencephalographic and behavioral effects comparable to those under sleep deprivation.
It is a matter of debate which adenosine receptor subtype mediates the sleep inducing action of adenosine. The A1 receptor subtype (A1AR) has the widest distribution in the central nervous system with particularly high concentrations in the cortex, cerebellum, hippocampus, striatum and thalamus (Fredholm, 1995). Selective A1AR agonists increased the sleep drive in rats after either systemic or cerebroventricular administration (Benington et al., 1995, Schwierin et al., 1996). Inhibition of equilibrium nucleoside transporters (which in turn increased adenosine levels) had the same effect in cats, and correspondingly a selective A1AR antagonist infused into the basal forebrain decreased sleep propensity in cats (Strecker et al., 2000). Microdialysis perfusion of A1AR antisense oligonucleotides inhibiting the translation of A1AR mRNA, significantly decreased nonREM sleep and increased wakefulness in rats (Thakkar et al., 2003). However, A1AR knockout mice reacted similarly to sleep deprivation as their wild type littermates (Stenberg et al., 2003) and it has been reported that subarachnoidal infusion of the selective A2A adenosine receptor (A2AAR) agonist CGS21680 (Satoh et al., 1999) promoted deep sleep.
Intraperitonial injections of the A1AR agonist CPA over 5 days significantly decreased A1AR density in the rat cortex, suggesting desensitization (Roman et al., 2008). Since sleep deprivation increased cerebral adenosine levels it was initially hypothesized that A1AR density might similarly decrease with deprivation. However, 3 and 6 h of total sleep deprivation increased A1AR mRNA in the basal forebrain but not Bmax or KD values determined with [3H]DPCPX autoradiography (with and without GTP in the incubation medium) (Basheer et al., 2001). Moreover, A1AR density in the basal forebrain increased after sleep deprivations of 12 h (10%) and 24 h (14%) (Basheer et al., 2007). In addition, rapid eye movement sleep deprivation for 48 and 96 h was reported to increase A1AR density in cortical (15 and 20%) and subcortical (23 and 48%) areas of rat brains, as determined by [3H]L-PIA saturation binding experiments in membrane preparations. The KD value did not change significantly (Yanik and Radulovacki, 1987).
In humans, in vivo imaging of the A1AR with positron emission tomography and CPFPX showed an increase of A1AR availability in cortical and subcortical regions after one night of sleep deprivation (Elmenhorst et al., 2007). These findings also did not support the hypothesis that the increase in adenosine concentration during sleep deprivation will induce receptor desensitization due to a high agonist load. Instead, they were in accord with the results of Basheer et al. (2007) who found an increase in both A1AR mRNA and density after 12 and 24 h in the basal forebrain of the rat. The rationale of the present study was therefore to evaluate whether the widespread, particularly neocortical increase of A1ARs observed in humans could be replicated in rats. Such a finding would extend the role of adenosine from a focal sleep modulator in the basal forebrain to, with longer durations of sleep deprivation, a ubiquitous player in sleep regulation in many parts of the brain.
Section snippets
Results
The results of the effect of sleep deprivation on A1AR binding of [3H]CPFPX are given in Table 1, and relative regional changes are depicted in Fig. 1. A representative autoradiogram of [3H]CPFPX binding of a rat of the 12 h control group is shown in Fig. 2. The analysis of the data by two-way ANOVA showed that the means of the factors ‘group’ and ‘region’ are significantly different (F(3,276) = 267.12; p < 0.0001 and F(9,276) = 12.95; p < 0.0001, respectively) which is specified by the results from
Discussion
The results of the present study suggest that sleep deprivation for 24 h produces an increase of A1AR density in cortical and subcortical regions of the rat brain.
This finding is consistent with an increase of A1ARs in the basal forebrain (12 h, 10%; 24 h, 14%) established in a subset analysis in some of the animals reported here (Basheer et al., 2007). The results of the present study enlarge and extend these previous findings significantly, because 12 and 24 h of total sleep deprivation
Animals
Thirty-two male Sprague Dawley rats (250–350 g) were used. Rats were housed in a 12 h light/dark cycle (light on 7:00 AM to 7:00 PM), at 23 ± 1 °C with access to food and water ad libitum; treatment was according to the Association for Accreditation of Laboratory Animal Care and Use Committee as Boston VA Healthcare system, Harvard University and US National Institute of Health. In a subset of these animals (n = 24) an analysis of A1AR density changes in the basal forebrain has been performed and
Acknowledgments
Sabine Wilms, Markus Cremer and Ramkumar Karthikeyan are gratefully acknowledged for excellent technical assistance and Marcus Holschbach for providing Tritium labeled CPFPX.
This work was supported by the Department of Veterans Affairs Medical Research Service Award to RB, the National Institute of Mental Health (NIMH39683) to RWM, the Heinrich Hertz Foundation of the Ministry of Science and Technology, North-Rhine Westfalia, Germany to DE and the German Federal Ministry of Education and
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