Effects on sleep of microdialysis of adenosine A 1 and A 2a receptor analogs into the lateral preoptic area of rats

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1 Am J Physiol Regul Integr Comp Physiol 289: R1715 R1723, First published August 18, 2005; doi: /ajpregu Effects on sleep of microdialysis of adenosine A 1 and A 2a receptor analogs into the lateral preoptic area of rats Melvi M. Methippara, 1,2 Sunil Kumar, 1,3,4 Md. Noor Alam, 1,2 Ronald Szymusiak, 1,3 and Dennis McGinty 1,2 1 Research Service (151A3), Veteran Affairs Greater Los Angeles Healthcare System, Sepulveda, California; Departments of 2 Psychology and 3 Medicine, University of California, Los Angeles, California; and 4 Department of Zoology, Patna University, Patna, India Submitted 8 April 2005; accepted in final form 9 August 2005 Methippara, Melvi M., Sunil Kumar, Md. Noor Alam, Ronald Szymusiak, and Dennis McGinty. Effects on sleep of microdialysis of adenosine A 1 and A 2a receptor analogs into the lateral preoptic area of rats. Am J Physiol Regul Integr Comp Physiol 289: R1715 R1723, First published August 18, 2005; doi: /ajpregu Evidence suggests that adenosine (AD) is an endogenous sleep factor. The hypnogenic action of AD is mediated through its inhibitory A 1 and excitatory A 2A receptors. Although AD is thought to be predominantly active in the wake-active region of the basal forebrain (BF), a hypnogenic action of AD has been demonstrated in several other brain areas, including the preoptic area. We hypothesized that in lateral preoptic area (LPOA), a region with an abundance of sleep-active neurons, AD acting via A 1 receptors would induce waking by inhibition of sleep-active neurons and that AD acting via A 2A receptors would promote sleep by stimulating the sleep-active neurons. To this end, we studied the effects on sleep of an AD transport inhibitor, nitrobenzyl-thio-inosine (NBTI) and A 1 and A 2A receptor agonists/antagonists by microdialyzing them into the LPOA. The results showed that, in the sleep-promoting area of LPOA: 1)A 1 receptor stimulation or inhibition of AD transport by NBTI induced waking and 2) A 2A receptor stimulation induced sleep. We also confirmed that NBTI administration in the wake promoting area of the BF increased sleep. The effects of AD could be mediated either directly or indirectly via interaction with other neurotransmitter systems. These observations support a hypothesis that AD mediated effects on sleepwake cycles are site and receptor dependent. sleep-wake cycle; A 1 and A 2a receptor activation; nitrobenzyl-thioinosine; basal forebrain; site specificity SEVERAL LINES OF EVIDENCE support a hypothesis that adenosine (AD) is an endogenous sleep factor. AD levels increase during waking and decrease during sleep in all brain regions examined (13, 23, 39). Systemic AD agonist administration induces sleep and EEG synchronization (3, 36); this effect is reversed by AD antagonists such as caffeine (15, 36). Sleep is also induced by the local administration of AD or its agonists into basal forebrain (BF) (20, 25). In BF, increasing extracellular AD levels by administration of nitrobenzyl-thioinosine (NBTI), an AD transport inhibitor, also enhances sleep; sleep is unaffected after NBTI perfusion in thalamus even if it causes an increase in AD level (24). During sleep deprivation, AD levels increase in the BF, a region containing many wake-active neurons (40), but not in other sleep-wake-related regions such as the preoptic area (POA), pedunculopontine tegmental nucleus, or dorsal raphe nucleus (23). Therefore, AD is thought to have a predominant action in the wake-active region of the BF. However, additional targets for the hypnogenic action of AD have been suggested. Sleep is also induced by the administration of AD or its agonists into the preoptic area (POA) (17, 29, 30, 45), the subarachnoid space underlying the POA (33 35, 45), or the shell of nucleus accumbens (34). AD acts via inhibitory A 1 and excitatory A 2A receptors (7, 11, 12). A 1 receptors are widespread in brain, whereas the distribution of A 2A receptors is limited (32). Both receptor types have been implicated in AD-mediated sleep promotion. Sleep induction by A 1 receptors is hypothesized to be mediated either by inhibition of BF wake-promoting neurons (1, 42) or by disinhibition of sleep-active neurons in the ventrolateral preoptic area, (VLPOA; Refs. 5 and 19). Application of an A 2A receptor agonist in the nucleus accumbens or subarachnoid space below the rostral forebrain or VLPOA induces sleep (34, 35). In the subarachnoid space, A 2A receptor agonist application induces c-fos in VLPOA (35), a region known to contain neurons that are predominantly active during sleep (37, 41). Although application of AD or NBTI to the BF consistently inhibits wake-active neurons, some sleep-active neurons were stimulated by AD and NBTI (1). This suggests that the action of AD could be determined by the AD receptor types in a given site. We hypothesized that AD acting via A 1 receptors would induce sleep by inhibiting the wake-active neurons in BF and would induce waking by inhibition of sleep-active neurons in lateral preoptic area (LPOA). We also reasoned that AD acting via A 2A receptors would promote sleep by stimulating the sleep-active neurons of LPOA. Therefore, we studied the effects on sleep of NBTI, and A 1 and A 2A receptor agonists/antagonists by microdialyzing them into the LPOA. Experiments were initially conducted to compare the effects on sleep of AD accumulation in a sleep-promoting (LPOA) vs. a wake-promoting area (BF). These experiments allowed us to confirm the earlier observations in the BF, which served as a benchmark. MATERIALS AND METHODS Subjects were male Sprague-Dawley rats ( g at the time of surgery) maintained on a 12:12-h light-dark cycle (lights on at 0600 AM and lights off at 0600 PM) with food and water freely available. All experiments were conducted in accordance with the National Research Council s Guide for the Care and Use of Laboratory Animals, and procedures were reviewed and approved by the Internal Animal Care and Use Committee of the Veterans Affairs Greater Los Angeles Healthcare System. Address for reprint requests and other correspondence: D. McGinty, Research Service (151A3), VAGLAHS, Plummer St., Sepulveda, CA ( dmcginty@ucla.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. R1715

2 R1716 Surgery Under ketamine/xylazine anesthesia (80/10 mg/kg ip) and aseptic conditions, rats were surgically prepared for polygraphic recording of sleep-waking states as described previously (18). Briefly, four stainless-steel screw electrodes were implanted in frontal and parietal bones of the skull for EEG recording, and three stainless steel wires were inserted into nuchal muscles for electromyogram (EMG) recording. One screw electrode fixed on the nasal bone served as ground. All electrodes were connected to a plug and fixed on the skull with dental acrylic. A microdialysis guide assembly consisting of a 23 G guide cannula with a stylet was implanted unilaterally 1 2 mm above the LPOA (AP 0.4 mm; DV 8.8 mm; L 1.3 mm from bregma) or BF (AP 0.2 mm; DV 8.5 mm; L 2.0 mm from bregma) (22). A copper-constantan thermocouple was implanted into hippocampus at AP 6.0 mm; DV 5.0 mm; L 5.0 mm (22) to record brain temperature. Recording During recovery from surgery (1 wk) and subsequent recordings, animals were housed individually in Plexiglas cages placed in an electrically shielded, sound-attenuated, temperature-controlled recording chamber (temperature: 25 2 C) and acclimatized to the recording environment. EEG signals were amplified and band passfiltered at Hz, and EMG activity was recorded from electrode pairs on the nuchal muscles after band pass filtering at Hz (Model 78 D, Grass Instruments, Quincy, MA). For recording the brain temperature, signals from thermocouples were transmitted to Thermalert monitoring thermometers (Physitemp Instruments, Clifton, NJ) and through their analog output to a DC amplifier of the polygraph. All bioelectrical signals were analog-to-digital converted using a 1401 Plus data acquisition interface and Spike2 software (Cambridge Electronic Design, Cambridge, UK) and stored on a PC for off-line analysis. Polygraphic data were digitized at a sampling rate of 128 Hz for EEG and 256 Hz for EMG and brain temperature. Drugs All drugs were purchased from Sigma (St. Louis, MO). The drugs used were the adenosine transport inhibitor, NBTI (20 Mor50 M), the AD A1 receptor agonist, N(6)-cyclopentyladenosine (CPA; 25 M), the AD A 2A receptor agonist, 2-[p-(2-carbonylethyl) phenylethylamino]-5 -N-ethylcarboxamido adenosine (CGS21680, 0.5 M, 5.0 M, and 50 M), the AD A 1 antagonist, 8-cyclopentyl-1, 3-dimethylxanthine (CPDX, 1 M, 5 M, and 10 M) and the A 2A antagonist, 8-(3-Chlorostyryl) caffeine (CSC, 1 M, 5 M, or 10 M). Stock solutions of drugs were made as follows: NBTI was dissolved in DMSO to make 10 mm stock solution, CPA and CGS was dissolved in double-distilled water (1 mm stocks), CPDX was dissolved in 0.1 N NaOH (1 mm stock), and CSC was dissolved in DMSO (75.6 mm stock). All stock solutions were diluted in artificial cerebrospinal fluid (ACSF; in mm): 145 Na, 2.7 K, 1.0 Mg 2, 1.2 Ca 2,1.5Cl and 2Na 2 HPO 4,pH7 2 to their final working concentrations and kept at 20 C until further use. Experiments A total of 42 rats were used in three experiments (Table 1). In most experiments, rats were perfused with ACSF for 2 h, followed by 90 min of perfusion with one dose of drug or ACSF. For part of experiment 2, rats were microdialyzed for 90 min with a drug (A 1 or A 2A agonist), followed by 20 h of ACSF perfusion. The order of administration of drug doses and ACSF was randomized. ACSF perfusion was considered as the control treatment in all experiments. The amounts of DMSO in the final dilutions were 200 nl in 1 ml of 10 M CSC (0.02% DMSO) and 2 l in1mlof20 M of NBTI (0.2% DMSO). Because local application of DMSO into the POA at the higher doses of 5% (unpublished observations) and 25% (31) did not affect sleep, microdialysis with DMSO alone was not undertaken in this study. One week after surgery, the stylet, which was 2 mm longer than the guide cannula, was replaced by a microdialysis probe with 1-mm-long membrane (EiCom, Kyoto, Japan), so that the probe projected into the site of drug delivery (BF or LPOA). At least h after probe insertion, low dead volume Teflon tubing (100 cm, EiCom) was connected from inlet and outlet of the microdialysis probe to a remote pump, and the rats were connected to a polygraph cable for behavioral state recording. Rats were perfused with ACSF for at least an hour before the recordings started. The flow rate of the perfusate was set at Table 1. Summary of experiments Experiment Drug Duration Site Drug Delivery Time Dose, M n for Each Dose Total n 1: Adenosine NBTI 90 min BF Day and night transport 50 inhibitor LPOA Day and night : AD A 1 and A 2A agonists 3: AD A 1 and A 2A antagonists CPA (A 1 agonist) CGS (A 2A agonist) CPDX (A 1 antagonist) 90 min followed by 20 h of ACSF LPOA Night min followed by LPOA Night h of ACSF 90 min LPOA Night min Night min Night min LPOA Night CSC (A 2A 90 min LPOA Day antagonist) GRAND TOTAL 42 NBTI, nitrobenzyl-thio-inosine; CPA, N(6)-cyclopentyladenosine; CGS, 2-[p-(2-carbonylethyl) phenylethylamino]-5 -N-ethylcarboxamido adenosine; CPDX, 8-cyclopentyl-1, 3-dimethylxanthine; CSC, 8-(3-chlorostyryl caffeine.

3 R1717 Fig. 1. Location of tips of microdialysis probes in the rat brain. Sagittal sections of rat brain showing the tracks of microdialysis probes through (A) lateral preoptic area (LPOA) and (C) basal forebrain (BF). Arrows indicate the location of tips of probes. Distribution of probe tips in stereotaxic planes of rat brain through (B) LPOA and (D) BF. 3V, third ventricle; ac, anterior commissure; HDB, horizontal diagonal band of Broca; LPO, lateral preoptic area; MPA, medial preoptic area; MnPO, median preoptic area; ox, optic chaism; SO, supraoptic nucleus; VDB, vertical diagonal band of Broca; VLPO, ventrolateral preoptic nucleus. 2 l/min, and the time taken by the perfusate to reach the rat from the reservoir vial was 600 s. Experiment 1. Effect of the adenosine transport inhibitor NBTI into LPOA vs. BF. It was previously shown that administration of NBTI increased extracellular AD levels (24). The objective of this experiment was to compare the effects on the sleep-wake state of increasing extracellular AD levels in the sleep-promoting LPOA vs. the wakepromoting BF. Because NBTI doses in the micromolar range are required to inhibit both NBTI-sensitive and -insensitive equilibrative AD transporters (44), we used 20- and 50- M doses. Two groups of rats were used: one group (n 8) received two doses of NBTI in LPOA, and the other group (n 6) was dialyzed with the two doses in the BF. Rats were microdialyzed with ACSF for 2 h followed by 90 min of perfusion with one dose of NBTI. The recordings were performed both during day (9:00 AM to 2:00 PM) and night (6:00 PM to 10:00 PM). NBTI was administered twice in 24 h; once during day and the other at night. Brain temperature was not recorded in this experiment. Experiment 2. Effects of adenosine agonists into LPOA.The aim of these experiments was to study the short-term (90 min) and long-term (20 h) effects on sleep-wake behavior of agonists of AD A 1 and A 2A receptors after administering them in LPOA. The rats were adapted for 10 days to a 4-h phase-advanced light-dark cycle with lights on at 2:00 AM and lights off at 2:00 PM. A 1 and A 2A agonists were administered immediately after lights off and continued for 90 min. For studying the short-term effects, three doses of the A2 A agonist were used: 0.5 M, 5.0 M, and 50 M; for comparison, data from the 90-min microdialysis period with 25 M of the A 1 agonist (CPA) was used. For studying the long-term effects, after the 90-min microdialysis with the drug, ACSF was perfused for 20 h. For long-term studies, only one dose of either A 1 (CPA; 25 M) or A2 A (CGS; 50 M) agonist was used. Experiments 3. Effects of adenosine antagonists into LPOA. As in the experiment 2, rats were adapted for 10 days to a shifted light-dark cycle (2:00 AM lights on and 2:00 PM lights off). All antagonist

4 R1718 Fig. 2. Effects of nitrobenzyl-thio-inosine (NBTI) on hypnograms. Effects on sleep-wake of 20 M of NBTI into BF at night (A) and LPOA during day (B). Arrows indicate the beginning of NBTI microdialysis. Thin lines represent artificial cerebrospinal fluid (ACSF) and thick lines represent NBTI. Abscissa is the duration of experiment in minutes. studies involved short-term drug perfusions lasting for 90 min followed by 2hofACSF perfusion. Microdialysis administration of A 1 antagonist (CPDX) was performed in the dark phase (beginning at 3:00 PM) and of the A 2A antagonist (CSC) in the light phase (8:00 AM). One of the three doses (1.0 M, 5.0 M, and 10.0 M ) of both drugs was given on any one day preceded or followed by ACSF. Histology After the completion of all recordings, rats were injected with heparin (500 U ip), followed by pentobarbital sodium anesthesia (100 Fig. 3. Effects of NBTI on sleep-wake cycle of rats. A comparison of effects on sleep-wake of microdialysis of 20 M of NBTI into BF (A) and LPOA (B) during both day and night. Asterisks indicate statistically significant differences (P 0.05) from the corresponding ACSF values (one-way ANOVA followed by Holm-Sidak s post hoc test). TSleep, total sleep Fig. 4. Short-term effects on sleep-wake during the microdialysis into LPOA of 20 M of N(6)-cyclopentyladenosine (CPA) and 50 M 2-[p-(2-carbonylethyl) phenylethylamino]-5 -N-ethylcarboxamido adenosine (CGS). The sleep-wake effects were averaged for the entire duration of recording (90 min). Microdialysis was performed in the dark phase. #Statistically significant difference for the CPA is P *Statistically significant difference for the CGS (One-way ANOVA followed by Holm-Sidak or Dunnet s post hoc test).

5 mg/kg ip). The animals were perfused transcardially with 50 ml of PBS (ph 7.4) followed by 300 ml of 4% paraformaldehyde and 100 ml each of 10% and 30% sucrose in PBS. The brains were removed and equilibrated in 30% sucrose. 40- m-thick serial sections were cut on a freezing microtome through the coronal plane and stained for Nissl (cresyl violet). Sleep and temperature data used for analysis were selected from only those rats whose brain sections showed the dialysis probe tracks terminating in the LPOA or BF. Data Analysis Sleep and temperature. On the basis of EEG and EMG patterns (46), polysomnographic data were scored manually at every 10-s interval into five sleep-wake stages: active waking (AW) and quiet waking (QW) comprising awake, slow-wave sleep 1 (SWS 1), and slow-wave sleep 2 (SWS 2) comprising non-rapid eye movement (NREM), and rapid eye movement (REM) sleep. Brain temperature data were averaged every 10 s to correspond to the sleep-scoring interval. The episode durations of each of five sleep-wake stages were assigned to five groups: durations of 30 s, 30sto 1 min, 1 to 2 min, 2 to 4 min, and 4 to 8 min. The duration of a sleep/wake stage in each episode group was calculated for the period of 90 min of microdialysis. Sleep and temperature data were averaged in bin sizes of 90 or 120 min. EEG spectral analysis. Because an important indicator of the homeostatic regulation of sleep is the increase in delta power during recovery sleep (10), the EEG was subjected to fast Fourier transformation analysis using a Spike 2 script. Spectral power of the NREM was calculated for three EEG bands: delta ( Hz), theta ( Hz), and sigma ( Hz) (4, 47). EEG power spectrum was calculated as follows: NREM episodes of at least 90-s duration after awake episodes of s were identified. From such a NREM episode, spectral power from a 30-s segment of NREM episode preceded and followed by at least 30 s of NREM was calculated. Spectral power from several such episodes was averaged. The spectral data during the drug perfusion were calculated as the percentage of the corresponding data during the ACSF perfusion. A one-way ANOVA followed by a post hoc test of either Holm- Sidak s or Dunnet s method was used to determine significant differences between control (ACSF) and experimental (drug) treatments. RESULTS The tips of the microdialysis probes were located within the borders in BF in 6 rats (experiment 1) and in LPOA in 36 rats (all groups) (Table1, Fig. 1). In the LPOA, the rostrocaudal distribution of the location of the probe tips extended between the stereotaxic planes of 0.26 mm and 0.8 mm posterior to bregma: 5 probe tips were located at 0.26 mm plane, and 11 probe tips were located each at 0.30 mm, 0.40 mm, and 0.80 mm planes. All locations were within the boundaries of LPOA; in 12 rats, the probe tips touched or penetrated VLPOA (Fig.1B). The location of tips of the probes in LPOA generally followed the cluster of sleep-active neurons reported (41). In the BF, probe tips were located in horizontal limb of diagonal band at 0.20 mm, 0.30 mm, and 0.80 mm from bregma (Fig. 1D). Experiment 1. Effect of the adenosine transport inhibitor NBTI into LPOA vs. BF. Microdialysis of 20 M NBTI into BF in the daytime did not affect sleep-wake cycle, whereas nighttime perfusion significantly (P 0.05) reduced total awake and increased NREM, REM, and total sleep (Figs. 2 and 3). During the day, microdialysis of 20 M NBTI into LPOA induced a significant (P 0.05) increase in total awake and decreased NREM and total sleep, but it did not affect REM sleep. Nighttime perfusion of NBTI in LPOA did not affect sleep-wake behavior. Microdialysis of 50 M NBTI into LPOA or BF did not affect sleep-wake stages significantly. During the day, 50 M NBTI in LPOA induced the following effects on sleep-wake states (means SE) : Total wake (CSF) vs (NBTI); NREM (CSF) vs (NBTI); total sleep (CSF) vs (NBTI). Experiment 2. Effects of adenosine agonists into LPOA. A 1 AGONIST (CPA). During the first 90 min of microdialysis, 25 M CPA induced a significant (P 0.05) increase in waking and reduced SWS1 and SWS2 stages, but it did not affect QW or REM (Fig. 4). The observed effects were due to a significant (P 0.05) increase in the number of AW episodes of 1 4 min duration and a suppression of short-duration episodes of SWS1 ( 30 s duration) and SWS2 (40 s 2 min) (Table 2). CPA did not have any delayed effect on sleep-wake stages (Fig. 5), nor did it affect the EEG power spectra in the sigma, delta, and theta frequency bands (Table 3). In addition, 40 min after the Table 2. Effects of AD A 1 agonist (25 M) and A 2A antagonist (10 M) into IPOA on sleep-wake episodes AWAKE QWAKE SWS 1 SWS 2 REM R1719 Episode Duration AD A1 agonist (CPA), night time microdialysis CSF CPA CSF CPA CSF CPA CSF CPA CSF CPA 30 s s 1 min min min min AWAKE QWAKE SWS 1 SWS 2 REM Episode Duration AD A2A antagonist (CSC), daytime microdialysis CSF CSC CSF CSC CSF CSC CSF CSC CSF CSC 30 s s 1 min min min min Bold values indicate significant difference (P 0.05) from CSF control (one-way ANOVA, followed by Holm-Sidak post hoc test). CSF, cerebrospinal fluid.

6 R1720 onset of microdialysis, CPA induced a significant, but shortlasting (30 min) increase in brain temperature (Fig. 6). A 2A AGONIST (CGS). In the first 90 min of microdialysis, CGS perfusion significantly (P 0.05) reduced AW and increased SWS1; it did not affect QW, SWS2, or REM (Fig. 4). CGS perfusion did not affect the durations of sleep-wake episodes (data not shown). CGS microdialysis induced long-term effects on sleep-wake stages; 2 h after the termination of CGS perfusion, AW was significantly (P 0.05) decreased, while there were significant increases in SWS1 and SWS2 (Fig. 5). CGS did not affect the brain temperature (Fig. 6). Of the three doses of CGS used for microdialysis, only 50 M induced significant changes in sleep-wake stages, while concentrations of 0.5 M and 5.0 M were ineffective (data not shown). CGS significantly reduced power in the theta band of the EEG spectrum of NREM episodes during the drug perfusion, while power in the sigma and delta bands was unaffected compared with the ACSF control (Table 3). Experiment 3. Effects of adenosine antagonists into LPOA. Microdialysis of 1- and 5- M concentrations of both A 1 antagonist, CPDX, and the A 2A antagonist CSC did not affect Fig. 6. Effects of A 1 and A 2A agonists on brain temperature. Comparison of effects of microdialysis into LPOA of 20 M of CPA and 50 M CGS. #Statistically significant difference for the CPA is indicated by P 0.05 (One-way ANOVA followed by Holm-Sidak post hoc test). sleep-wake structure compared with ACSF (Fig.7, A D); 10 M of CPDX also did not affect the sleep-wake stages (Fig. 7E). On the other hand, the A 2A antagonist, CSC, markedly (P 0.05) elevated AW and suppressed SWS 2, but it did not affect QW, SWS1, or REM (Fig. 7F). Microdialysis of 10- M CSC significantly (P 0.05) increased long-duration AW episodes (2 8 min) and reduced short-duration episodes of Fig. 5. Long-term (20 h) effects on sleep-wake cycle of microdialysis of 20 M CPA and 50 M CGS into LPOA. Statistically significant differences (P 0.05; one-way ANOVA followed by Holm-Sidak or Dunnett s post hoc test) from the ACSF are represented by # for the CPA and by * for the CGS. Hatched bar on the abscissa indicates the duration of drug perfusion. Solid and open bars represent dark and light portions of L/D cycle. AW, active waking; QW, quiet waking; SWS 1, slow-wave sleep 1; SWS 2, slow-wave sleep 2; REM, rapid eye movement. Fig. 7. Effects on sleep of three doses of A 1 and A 2A antagonists. Comparison of dose responses on sleep-wake during microdialysis into LPOA of three doses (1.0 M, 5.0 M, and 10.0 M) of 8-cyclopentyl-1, 3-dimethylxanthine (CPDX) (A, C, and E) and 8-(3-Chlorostyryl) caffeine (CSC) (B, D, and F). *Statistically significant difference (P 0.05; one-way ANOVA followed by Holm-Sidak post hoc test) from the ACSF control.

7 Table 3. Effects of microdialysis of AD A 1 and A 2A agonists (CPA and CGS) and A 2A antagonist (CSC) on non-rapid eye movement EEG spectral power Spectral Bands of EEG CPA CGS CSC Sigma (11 16 Hz) Theta ( Hz) * Delta ( Hz) *Significant difference (P 0.05) from the baseline; one-way ANOVA followed by Holm-Sidak post hoc test. SWS1 (10 30 s) and SWS 2 (10 s 1 min) (Table 2). However, CSC did not affect the EEG power spectra (Table 3). Neither CPDX nor CSC significantly affected the brain temperature (data not shown). DISCUSSION % Difference From the Baseline (100 %) NBTI effects. In this study, we compared the effects of the adenosine transport inhibitor NBTI in a wake-related (BF) vs. a sleep-related (LPOA) area. The microdialysis of NBTI induced opposite effects in the two regions; sleep was enhanced in the BF and wakefulness was enhanced in the LPOA. The effect induced in the BF replicated and confirmed earlier reports (24), indicating that AD accumulation in the BF could facilitate sleep. Sleep facilitation could be mediated through inhibition of wake-related neurons of the region (1, 42), including cholinergic neurons. However, the observation of waking induced by NBTI in the LPOA is inconsistent with the hypothesis of AD as a general sleep factor (2, 24). This unexpected finding could be explained by a hypothesis that buildup of AD in the sleeppromoting area inhibits the sleep-active neurons via their resident A 1 receptors (see below). A higher dose (50 M) of NBTI application in BF and LPOA was ineffective. This may have resulted from coincident activation of opposing effects of AD at A 1 and A 2A receptors, nullifying the general inhibitory effect of AD buildup. At the lower dose, the A 1 effect would predominate; at the higher dose, the A 2A effect would counteract the A 1 effect. A 1 agonist/antagonist effects. Earlier studies have indicated that A 1 receptors could mediate the sleep-inducing action of AD by either postsynaptic inhibition of wake-active neurons in R1721 the BF (1, 42, 43) or presynaptic disinhibition of GABAergic inputs to sleep-active neurons of the ventrolateral POA (5, 19). Accordingly, stimulation of A 1 receptors in the sleep-promoting LPOA would be expected to induce sleep. However, in this study, administration of A 1 agonist or the AD transport inhibitor NBTI in the LPOA induced waking and suppressed sleep. This observation supports a hypothesis that AD in LPOA may inhibit sleep-active neurons, which are prominent in LPOA (41), via A 1 receptors. Although A 1 receptor stimulation of LPOA suppressed sleep, it did not affect sleep intensity during residual NREM, as indicated by the EEG markers of sleep depth in rat (Table 3). It has been shown that in hypothalamic neurons, glutamatergic neurotransmission is antagonized by A 1 agonist actions (21). Possibly in LPOA, AD antagonizes the excitatory input to sleep-active neurons. A 2A agonist/antagonist effects. We have also examined the involvement of AD A 2A receptors of LPOA in sleep-wake and body temperature regulation. Whereas stimulation of A 1 receptors induced waking and suppressed sleep, A 2A receptor activation produced sleep enhancement, particularly SWS1. The lack of effect of A 2A receptor stimulation on sleep intensity indicated by delta and sigma power in the EEG spectrum could be due to the absence of its effect on SWS2, the stage most rich in these frequency bands in the rat. The effects of the A 2A antagonist were opposite to those of the agonist, namely, an increase in waking with a concomitant decrease in sleep. These results complement the earlier reports of sleep induction by A 2A agonist in subarachnoid space near the VLPOA and BF (33 35). However, microinjection of an A 2A agonist, CV 1808 (2-phenylaminoadenosine) into POA of rats was reported not to induce significant changes in sleep, although the resulting amount of SWS 2 was strongly elevated (45). Differences between the two studies could be due to the fact CV 1808 exhibits more than 10-fold less selectivity as A 2A ligand compared with CGS 21680, the A 2A agonist used in this study (14). In the rat hypothalamus, presence of mrna for A 2A receptors was indicated by RT PCR studies (6), and A 2A receptors were weakly radiolabeled by the selective A 2A antagonist [ 3 H]SCH in mouse hypothalamus (8). Weak immunocytochemical labeling of A 2A receptors was seen in the magnocellular preoptic nucleus (32), which is adjacent to our microdialysis probes. The strong effects observed after A 2A agonist/antagonist application in LPOA in the present study Fig. 8. Schematic showing the hypothetical involvement of adenosine (AD) A 1 and A 2A receptors of BF wake active neurons and preoptic area (POA) sleep promoting neurons in state transitions. During waking to sleep transition, AD accumulation around the BF wake active neurons inhibits them by their resident A 1 receptors and stimulates sleep active neurons of POA by A 2A receptors precipitating sleep. Oval with a line across indicates inhibition of adenylate cyclase, and cone with serrations indicates stimulation. Ad cyc, adenylate cyclase; Gi, inhibitory G protein; Gs, stimulating G protein.

8 R1722 also suggests the presence of A 2A receptors in the LPOA. A 2A agonist/antagonist-induced effects could also be mediated indirectly via metabotropic glutamate receptor 5 (mglur5) in LPOA (49), which is known to interact with A 2A receptors (9). Another possibility is that the effects observed could be via A 2A receptor-mediated alteration of the hypothalamic blood flow (16). Involvement of A 2A receptors of LPOA in sleep promotion is significant in view of the recent finding that caffeine-induced waking is greatly attenuated in A 2A receptor knockout mice, but not in A 1 receptor knockouts (26). Moreover, knockout mice lacking A 2A receptors were also shown to have altered sleep (48), in contrast to the A 1 receptor knockout mice whose homeostatic regulation of NREM sleep remained unaffected (38). Methodological considerations. In this report, microdialysis was used for drug delivery to the brain parenchyma of LPOA and BF. The distinct advantage offered by microdialysis is the continuous and steady rate of drug administration without restraining or otherwise disturbing the animals, as opposed to the bolus delivery of the microinjection method (45), which may induce release of prostaglandins affecting the body temperature (27, 28) that, in turn, can alter the sleep-wake stages. In this study, the effects of drugs were consistent and reversible; microdialysis of ACSF alone did not affect the sleep-wake stages; and the effects of the adenosine A 2A agonist were opposite to those of the antagonist. Schematic of the mechanism of sleep-promoting action of AD. During waking, activity of the cholinergic wake-active neurons increases the extracellular AD levels. AD acts upon A 1 receptors on these wake-active neurons inhibiting them through their negative coupling to adenylate cyclase. Simultaneously, extracellular AD excites nearby sleep-active neurons via their resident A 2A receptors by increasing adenylate cyclase activity, causing the transition to sleep (Fig. 8). In conclusion, the results of the present study show that, in the LPOA sleep-promoting area, A 1 receptor activation induces waking, and A 2A receptor activation induces sleep. These observations support the hypothesis that AD-mediated effects on sleep-wake states are site and receptor dependent. ACKNOWLEDGMENTS We thank Chris Angara for technical support. GRANTS This study was supported by the grants MH 47480, MH 63323, HL and Research Service of Veterans Administration. REFERENCES 1. Alam MN, Szymusiak R, Gong H, King J, and McGinty D. Adenosinergic modulation of rat basal forebrain neurons during sleep and waking: neuronal recording with microdialysis. J Physiol 521: , Basheer R, Porkka-Heiskanen T, Strecker RE, Thakkar MM, and McCarley RW. 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