A1 receptor and adenosinergic homeostatic regulation of sleep-wakefulness: effects of antisense to the A1 receptor in the cholinergic basal forebrain

Abstract

We hypothesized that adenosine, acting via the A1 receptor, is a key factor in the homeostatic control of sleep. The increase in extracellular levels of adenosine during prolonged wakefulness is thought to facilitate the transition to sleep by reducing the discharge activity of wakefulness-promoting neurons in the basal forebrain. Adenosine A1 receptor control of the homeostatic regulation of sleep was tested by microdialysis perfusion of antisense oligonucleotides against the mRNA of the A1 receptor in the magnocellular cholinergic region of the basal forebrain of freely behaving rats. After microdialysis perfusion of A1 receptor antisense in the basal forebrain, spontaneous levels of sleep-wakefulness showed a significant reduction in non-rapid eye movement (REM) sleep with an increase in wakefulness. After 6 hr of sleep deprivation, the antisense-treated animals spent a significantly reduced amount of time in non-REM sleep, with postdeprivation recovery sleep hours 2-5 showing a reduction of approximately 50-60%. There was an even greater postdeprivation reduction in delta power (60-75%) and a concomitant increase in wakefulness. All behavioral state changes returned to control (baseline) values after the cessation of antisense administration. Control experiments with microdialysis perfusion of nonsense (randomized antisense) oligonucleotides and with artificial CSF showed no effect during postdeprivation recovery sleep or spontaneously occurring behavioral states. Antisense to the A1 receptor suppressed A1 receptor immunoreactivity but did not show any neurotoxicity as visualized by Fluoro-Jade staining. These data support our hypothesis that adenosine, acting via the A1 receptor, in the basal forebrain is a key component in the homeostatic regulation of sleep.

Figure 1.

A, Schematic representation of the anatomical location of the histologically identified probe tip sites in 28 animals. All sites were located between anteroposterior -0.2 and -0.8 mm and were mapped onto this one coronal brainstem section (-0.4 mm; adapted from Paxinos and Watson, 1997). Probes were placed bilaterally in each animal. The bilateral antisense perfusion sites (total on each side, n = 12; n = 6 each for experiments 1 and 2) are represented by solid red circles, the nonsense perfusion sites (total on each side, n = 10; n = 6 for experiment 1 and n = 4 for experiment 2) are represented by solid black circles, and the ACSF perfusion sites (n = 6 for experiment 2) are represented by solid blue circles. HDB, Nucleus of horizontal limb of the diagonal band; LPO, lateral preoptic nucleus; MCPO, magnocellular preoptic nucleus; SIB, substantia innominata basal; VP, ventral pallidum; VLPO, ventrolateral preoptic region. For identification of unlabeled structures, see Paxinos and Watson (1997). B, Representative photomicrograph of an antisense perfusion site in the cholinergic BF. The lesion (arrow) caused by the microdialysis probe is located in the midst of cholinergic neurons, the intact staining of which for ChAT indicates an absence of antisense-induced toxic effects on synthesis of this protein. Scale bar, 50 μm.

Figure 2.

Effect of microdialys is perfusion of antisense oligonucleotides against the mRNA of the A₁ receptor on spontaneous bouts of sleep-wakefulness in freely behaving animals during both the light period (top) and the dark period (bottom). During the control day (open bar), ACSF was perfused for 8 hr (from 10:00 A.M. to 6:00 P.M.). This was followed by microdialysis perfusion of antisense for 16 hr, delivered over 2d (8hr each day, from 10:00 A.M. to 6:00 P.M.). The top graph shows that, during the light period, there was a significant increase in wakefulness (p < 0.05; paired t test) and a significant decrease in non-REM sleep (p < 0.05; paired t test) on the first day after antisense perfusion (post-ODN day 1; solid bars) compared with the control (ACSF) day, whereas waking (W) and non-REM sleep were not different from control levels on post-ODN day 2 (gray bars). There was no effect on REM sleep. During the dark period (bottom), there was a significant (p < 0.05; paired t test) increase in wakefulness (W) but no change in non-REM sleep on post-ODN day 1 (solid bar) compared with control day (open bar), whereas both waking and non-REM sleep were not different from control levels on post-ODN day 2. There was a significant decrease in REM sleep (p < 0.05; paired t test) on both post-ODN day 1 and 2 (gray bar). For details, see Results. *p < 0.05.

Figure 3.

Absence of differences in the behavioral state effects of microdialysis perfusion of nonsense oligonucleotides against the mRNA of the adenosine A₁ receptor (NS; solid circles) compared with ACSF perfusion (open triangles) during the first 5 hr of recovery sleep after 6 hr of sleep deprivation. Symbols indicate the mean ± SEM percentage of total time spent in wakefulness (A), non-REM sleep (B), and REM sleep (C). An hour-by-hour t test comparison for the two groups in each state showed no significant differences.

Figure 4.

Increased wakefulness (A) and decreased non-REM sleep (B) effects of antisense oligonucleotides against the mRNA of the adenosine A₁ receptor during the first 5 hr of the recovery sleep period (left part of each graph) after 6 hr of sleep deprivation compared with control animals (pooled ACSF and nonsense treatment groups, ACSF + NS). There was a significant increase in wakefulness and a decrease in non-REM sleep during the second, third, fourth, and fifth hours. REM sleep (C) did not show significant differences. The right part of the graphs (within box) shows that, for the subsequent 7 hr, there was no compensation for the antisense-induced changes in wakefulness and non-REM. Ordinate is mean ± SEM percentage of time spent in each behavioral state, and abscissa is time of day, with lights off occurring at 7:00 P.M. and lights on occurring at 7:00 A.M. **p < 0.01.

Figure 5.

EEG delta power (1–4 Hz, mean ± SEM) after sleep deprivation, expressed as percentage of each animal's delta power during the same time period on a nondeprivation day (previous day). Open triangles indicate control animals (ACSF and nonsense), and solid squares indicate antisense-treated animals. There was a significant decrease in delta activity in antisense-treated animals during all 5 hr of recovery sleep compared with the pooled controls (**p < 0.01). For details, see Results.

Figure 6.

Effect of A₁ receptor antisense ODN perfusion (A, C) compared with nonsense ODN perfusion (B, D) on A₁ receptor immunoreactivity in the BF. Note the substantial suppression of A₁ receptor immunoreactivity in and around the probe lesion site (black arrow) on the antisense-perfused side compared with the nonsense-perfused side (B; arrow, probe lesion site). The rectangular area in A and B is magnified and displayed in C and D. The cell bodies with A₁ receptor immunoreactivity are clearly visible (D) in the side that received nonsense ODNs, whereas the side with antisense treatment (C) showed marked suppression of cell body A₁ immunoreactivity, leaving fuzzy-appearing background activity. acp, Anterior commissure posterior part (Paxinos and Watson, 1997). Scale bars: A, B, 100 μm; C, D, 50 μm

Figure 7.

Effects of A₁ receptor antisense perfusion on the integrity of neurons. A, Fluoro-Jade labeling is constrained to the immediate vicinity of the microdialysis probe track. This restricted location in and just around the track was present regardless of whether ACSF or antisense was perfused. B, Cresyl violet staining of a section 120 μm from the Fluoro-Jade section revealed the presence of intact neurons (arrows) outside the probe track. Scale bar, 20 μm.