The role of adenosine in the maturation of sleep homeostasis in rats

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1 J Neurophysiol 117: , 217. First published October 26, 216; doi:1.1152/jn RESEARCH ARTICLE Control of Homeostasis The role of adenosine in the maturation of sleep homeostasis in rats X Irma Gvilia, 1,2,4 Natalia Suntsova, 1,2 Andrey Kostin, 1 Anna Kalinchuk, 5 Dennis McGinty, 1,3 Radhika Basheer, 5 and Ronald Szymusiak 1,2 1 Research Service (151A3), Veterans Affairs Greater Los Angeles Healthcare System, North Hills, California; 2 Department of Medicine, University of California, Los Angeles, California; 3 Department of Psychology, University of California, Los Angeles, California; 4 Ilia State University, Tbilisi, Georgia; and 5 Department of Psychiatry, Harvard Medical School, Boston, Massachusetts Submitted 23 August 216; accepted in final form 24 October 216 Gvilia I, Suntsova N, Kostin A, Kalinchuk A, McGinty D, Basheer DM, Szymusiak R. The role of adenosine in the maturation of sleep homeostasis in rats. J Neurophysiol 117: , 217. First published October 26, 216; doi:1.1152/jn Sleep homeostasis in rats undergoes significant maturational changes during postweaning development, but the underlying mechanisms of this process are unknown. In the present study we tested the hypothesis that the maturation of sleep is related to the functional emergence of adenosine (AD) signaling in the brain. We assessed postweaning changes in 1) wake-related elevation of extracellular AD in the basal forebrain (BF) and adjacent lateral preoptic area (LPO), and 2) the responsiveness of median preoptic nucleus (MnPO) sleep-active cells to increasing homeostatic sleep drive. We tested the ability of exogenous AD to augment homeostatic responses to sleep deprivation (SD) in newly weaned rats. In groups of postnatal day (P)22 and P3 rats, we collected dialysate from the BF/LPO during baseline (BSL) wake-sleep, SD, and recovery sleep (RS). HPLC analysis of microdialysis samples revealed that SD in P3 rats results in significant increases in AD levels compared with BSL. P22 rats do not exhibit changes in AD levels in response to SD. We recorded neuronal activity in the MnPO during BSL, SD, and RS at P22/P3. MnPO neurons exhibited adult-like increases in waking neuronal discharge across SD on both P22 and P3, but discharge rates during enforced wake were higher on P3 vs. P22. Central administration of AD (1 nmol) during SD on P22 resulted in increased sleep time and EEG slow-wave activity during RS compared with saline control. Collectively, these findings support the hypothesis that functional reorganization of an adenosinergic mechanism of sleep regulation contributes to the maturation of sleep homeostasis. NEW & NOTEWORTHY Brain mechanisms that regulate the maturation of sleep are understudied. The present study generated first evidence about a potential mechanistic role for adenosine in the maturation of sleep homeostasis. Specifically, we demonstrate that early postweaning development in rats, when homeostatic response to sleep loss become adult like, is characterized by maturational changes in wake-related production/release of adenosine in the brain. Pharmacologically increased adenosine signaling in developing brain facilitates homeostatic responses to sleep deprivation. sleep homeostasis; postweaning development; rats; adenosine; preoptic hypothalamus HOMEOSTATIC REGULATION OF sleep in rats undergoes significant maturational changes during early postweaning, but the brain Address for reprint requests and other correspondence: I. Gvilia, Research Service (151A3), V.A. Greater Los Angeles Healthcare System, Plummer St., North Hills, CA ( igvilia@ucla.edu). mechanisms that drive this process are largely unknown. Previous studies in the rat revealed that different indexes of sleep homeostatic regulation emerge at different time points of postnatal development (Blumberg et al. 24; Frank and Heller 1997; Frank et al. 1998; Gramsbergen 1976; Gvilia et al. 211; Mohns et al. 26; Seelke and Blumberg 28; Todd et al. 21). Compensatory increases in total sleep amount during recovery sleep (RS) following sleep deprivation (SD) were observed as early as postnatal day (P)2 (Todd et al. 21). Post-SD increases in non-rapid-eye-movement (NREM) sleep delta ( )-spectral power (.3 4. Hz), a measure of the intensity of sleep in adults (Borbely and Tobler 1985), were found in P24 but not P2 rats (Frank et al. 1998). We recently examined the homeostatic sleep response to SD on P22 and P3 by evaluating the increases in NREM sleep total time, NREM sleep -power, and mean duration of NREM sleep episodes (Gvilia et al. 211). We reported increased NREM sleep amount and NREM sleep -power during RS vs. baseline at both ages. However, increases in NREM sleep episode duration during RS were observed only at P3 (Gvilia et al. 211). Collectively, these findings suggested that P2-P3 is characterized by the emergence of brain mechanisms that regulate sleep intensity and continuity. Sleep homeostasis in adults is controlled by interaction of the endogenous sleep-regulatory substance adenosine (AD) and neuronal groups implicated in wake-sleep control (Basheer et al. 2, 24; Benington et al. 1995; Radulovacki 1985; Schwierin et al. 1996). AD is a product of brain metabolism, and its levels are elevated as a consequence of sustained wakefulness (Basheer et al. 24; Porkka-Heiskanen et al. 22, 211). Deprivation of sleep is accompanied by elevated AD levels in the basal forebrain (BF) and cortex, followed by a decline in AD levels during subsequent RS (Kallinchuk et al. 211; Porkka-Heiskanen et al. 2). Exogenous AD and its analogs promote sleep after systemic and central administration, and AD-induced sleep is accompanied by increased EEG slow-wave activity (SWA) (Benington et al. 1995; McCarley 27; Radulovacki 1985; Schwierin et al. 1996). The sleeppromoting effect of AD has been shown to involve an A 1 receptor (A 1- R)-mediated inhibition of wake-promoting systems (Alam et al. 1999; Bjorness et al. 29, 216; Rai et al. 21; Rainnie et al. 1994; Thakkar et al. 23; Arrigoni et al. 26; Hawryluk et al. 212; Yang et al. 213) and A 2A receptormediated activation of sleep-regulatory neurons in the preoptic 327

2 328 ADENOSINE AND MATURATION OF SLEEP area (POA) of the hypothalamus (Gallopin et al. 25; Kumar et al. 213; Methippara et al. 25; Scammell et al. 21). Sleep-regulatory neurons in the median preoptic nucleus (MnPO) and ventrolateral preoptic area (VLPO) are dynamically responsive to changes in homeostatic sleep pressure (Alam et al. 214; Gvilia et al. 26a,b; Suntsova et al, 22). Across the conditions of spontaneous sleep, acute SD, and post-sd RS, expression of c-fos in GABAergic neurons in the MnPO is maximal following SD suggesting that neuronal discharge of these neurons is elevated in response to increased homeostatic sleep drive (Gong et al. 24; Gvilia et al. 26a,b). Discharge of sleep-active neurons in the MnPO and VLPO is dynamically responsive to changes in homeostatic sleep pressure during SD and RS (Alam et al. 214). Local perfusion of the A 2A -R antagonist suppresses discharge of VLPO neurons during both SD and RS (Alam et al. 214). Developmental changes in AD signaling and the role of AD in the maturation of sleep homeostasis are unknown. To our knowledge, there are no published data on the course of developmental changes in single unit activity within POA sleep-regulatory sites. We hypothesized that the early postweaning period in rats is characterized by marked developmental changes in 1) wake-related elevation of extracellular AD levels in the BF and adjacent lateral preoptic area (LPO), and 2) the functional activity of POA sleepregulatory neurons. To test these hypotheses, we 1) measured SD-related changes in extracellular AD levels in the BF/LPO of P22 and P3 rats, 2) examined the ability of exogenous AD to augment the homeostatic responses to SD in P22 rats, and 3) characterized sleep-related neuronal discharge within the MnPO on P22 and P3. MATERIALS AND METHODS Animals and Experimental Environment Sprague-Dawley rat pups (n 29) were cross-fostered among eight different litters, within 3 days of birth. Day of birth was designated as P. The litter sizes were culled to 8 1 rat pups. Starting from P1, the rats were adapted to gentle-handling-induced SD procedures as described previously (Gvilia et al. 211). On P18, the rats were separated from dams and housed in pairs in cages containing bedding from their home cage. The cages were placed in environmental chambers maintaining an age-appropriate temperature (P18: 25 C; P19: 24 C; P2 P3: 22 C) and a 12-h light-dark cycle; lights-on at 6: AM designated as zeitgeiber time (ZT). After the weaning, the pups were fed crushed chow for rats. To verify that the pups could eat and reach water, they were monitored by video for the first 9 12 min after the separation from dams. Equal numbers of male and female rat pups were randomly assigned to three different sets of experiments. All experiments were approved by the Animal Care and Use Committee at the Veterans Affairs Greater Los Angeles Health Care System and were conducted in accordance with the National Research Council (US) Guide for the Care and Use of Laboratory Animals. Surgical and Postsurgical Procedures: Recordings All 29 experimental animals were anesthetized with methoxyflurane gas inhalation and implanted with cortical electroencephalogram (EEG) and dorsal neck electromyogram (EMG) electrodes for the assessment of wake-sleep states, using previously described techniques (Gvilia et al. 211). To measure extracellular AD levels in the rat brain on P22 and P3, two groups of rats (P19, n 8 and P27, n 7) were implanted with unilateral guide cannula for microdialysis probes targeting the BF/LPO. Stereotaxic coordinates for the guide cannula [ anterior-posterior (AP), length (L) 1.6, height (H) 7] were adjusted from adult rat atlas (Swanson 1998). To examine the effect of exogenous AD on homeostatic responses to SD on P22, a group of P19 rats (n 8) was implanted with a microinjection guide cannula (22-G stainless-steel tube) in the lateral ventricle at following coordinates: AP.92, L 1.4, H 3. The patency of the intracerebroventricular cannulas were assessed by administering angiotensin II (ANG II; 2 ng, human ANG II octapeptide; Peninsula Laboratories) before the experiments; angiotensin elicits a drinking response mediated by structures in the preoptic area (Epstein et al. 197). Another group of P19 rats (n 6) was prepared for a longitudinal recording of neuronal discharge within the MnPO; the rats were implanted with a preassembled bundle of 1 Formvarinsulated 22- m stainless steel microwires placed into a 23-G guide cannula and a miniature dual row electric plug. Microwires, at their tips, were cut at a 45 angle and their impedance was maintained within a narrow range (6 7 k at 1 khz). To prepare the animals for unit activity recording within the MnPO, a 2 2 mm hole was trephined in the skull, centered at bregma. The dura mater was incised.5 mm lateral to a sagittal sinus. The rostral part of the guide cannula was positioned at the level of bregma. During assembly implantation, the guide cannula was used to first displace the sagittal sinus and then was stereotaxically lowered in the midline to a point corresponding to H3. Stereotaxic coordinates for the MnPO in young rats (AP, L, H ) were adjusted from adult rat atlas (Swanson 1998). After fixation of the assembly to the skull, the bundle of microwires were advanced through the guide cannula until the preset limiter touched the blunt top of the cannula indicating that the tips of microwires reached the target depth (H 4. to 6.5). Sterile petroleum jelly was used to seal the top of the cannula. The method described allowed sustained recordings of well-differentiated single units across P22 P3. After the completion of surgery, the rats were housed individually and connected to the recording system. The system allowed the animals unimpeded movement throughout the cage. The EEG/EMG wires were connected to Grass amplifiers (15A94 Quad Neuroamplifiers; Grass Technologies: Astro-Med Industrial Park, West Warwick, RI). EEG and EMG activities were band-pass filtered at.3 3 and 1 1 Hz, respectively. Neuronal activity was recorded extracellularly using bipolar derivations from microwires and amplified by a 16-channel extracellular differential amplifier with a head stage (model 17; A-M Systems, Carlsborg, WA) with low and high cutoff filters of 1 Hz and 1 khz, respectively. Bioelectrical signals were digitized and stored on hard drive for offline analysis using Micro 141 data acquisition interface and Spike2 software package (Ver 5.; Cambridge Electronic Design, London, UK). Polygraphic data were digitized at a sampling rate 256 Hz and unit activity data at 1 or 25 khz for waveform and wavemark data channels, respectively. Experimental Paradigm Experiment 1. Experiment 1 was designed to assess SD-related changes in AD extracellular levels in the BF/LPO of P22 and P3 rats. In both age groups, microdialysis sample collection was carried out following 3 days recovery from surgery. Sixteen hours before ZT on the experimental day, a microdialysis probe (semipermeable membrane tip length: 1. mm; outer diameter:.22 mm; molecular cut off size: 5 kda; Eicom) was inserted into the guide cannula, cemented in place and perfused continuously with artificial cerebrospinal fluid (acsf; composition in mm: 145 NaCl, 2.7 KCl, 1.3 MgSO 4, 1.2 CaCl 2,and2Na 2 HPO 4 at ph, 7.2). Flow rate of acsf through the dialysis probe was 1. l/min. On P22 and P3, the rats were subjected to the collection of dialysate over consecutive 1-h epochs of 1-h pre-sd BSL wake-sleep cycle beginning at ZT, 3-h SD (ZT1-4), and 2-h RS (ZT4-6). Immediately after collection, samples were

3 ADENOSINE AND MATURATION OF SLEEP 329 stored at 7 F until high performance liquid chromatography (HPLC) analysis. To monitor the wake-to-sleep transitions and prevent sleep onset during the SD period, microdialysis sample collection was accompanied by EEG/EMG recordings. To interrupt sleep episodes, the rats were subjected to gentle arousing stimuli within 3 5 s of the first appearance of EEG/EMG signs of sleep, as described in our published studies (Gvilia et al. 26, 211). After completing the experimental protocol, all rats were given a lethal dose of anesthetic followed by transcardial perfusion of cold saline followed by 4% paraformaldehyde. Frozen sections, 4 m in thickness, collected through the BF/LPO were stained with thionin to determine location of microdialysis probes. Experiment 2. Experiment 2 was designed to determine if pharmacological elevation of brain extracellular AD levels during SD protocol on P22 would change the magnitude of the homeostatic responses to sleep loss. On P21, an injection cannula was inserted into the guide cannula at ZT and cemented in place. Three hours of SD was performed using the same protocol as in experiment 1, starting at ZT1. At ZT3, rats received intracerebroventricular injection of 5 l of.9% saline. SD was continued for one more hour and followed by the recording of recovery wake-sleep cycles at ZT4-ZT5.5. On P22, the rats were subjected to the same experimental protocol as on P21, but received intracerebroventricular AD (1 nmol in 5 l) at ZT3. The intracerebroventricular AD dose was based on studies in adult rats (Radulovacki at al. 1985). We chose the lowest effective dose reported in that study and tested it in a pilot experiment. At the end of the experiment, rats were euthanized using the same protocol as in experiment 1. Experiment 3. In experiment 3, we examined developmental changes in SD-related activity of MnPO sleep-regulatory cells. Longitudinal experiments started on P22 and ended on P3. Unit activity was recorded during pre-sd BSL at ZT-ZT2, SD at ZT2-ZT4, and post-sd recovery period at ZT4-ZT6. At the end of the experiments, the DC current was passed through the microwires (2 A for 15 s) to aid in visualization of final microwire position. Then, rats were perfused as described for experiment 1. Sections through the studied brain region were stained with thionin. Data Analysis Adenosine measurements using HPLC: experiment 1. Microdialysis samples were analyzed with a HPLC system coupled to a fluorescence detector Waters 2475 (Waters, Milford, MA) as described elsewhere (Kalinchuk at al. 211; Savelyev et al. 212). Briefly, we performed the derivatization of AD in microdialysis samples using 1% chloracetaldehyde, 1 M HCl, and 1 M EDTA. After derivatization reagents were added, we incubated samples for 4 min at 8 C. AD levels were measured at an excitation/emission wavelength of 265/399 nm. The mobile phase consisted of 5 mm ammonium acetate,.2 mm tetrabutylammonium hydrogen sulfate, 1 M EDTA, and 15% methanol. The analysis of chromatograms was performed using the PowerChrom software (EDAQ, Denistone East, Australia). We analyzed only the samples from rats that showed a proper location of microdialysis probes in the BF/LPO (n 5/age group). Wake-sleep cycle analysis: experiment 2. behavioral states of the rat pups were determined in 4-s epochs of EEG/EMG recordings by an experienced scorer blind to the experimental condition and group identity of the animal. Wakefulness, NREM sleep, and REM sleep were defined according to the criteria described previously (Gvilia et al. 211). EEG records of consecutive 4-s epochs of NREM sleep, for the entire post-sd recovery sleep, were subjected to a fast-fourier transform routine to obtain EEG power spectra in the -frequency range. Epochs containing EEG artifacts were excluded from spectral analysis. The average SWA value for all NREM sleep epochs subjected to spectral analysis was determined for each animal, and individual group mean SWA values were calculated. To assess the magnitude of compensatory responses to sleep loss, post-sd recordings were analyzed for total amounts of the wake-sleep states (as percentage of total recording time) and the level of sleep consolidation. Analysis of single unit activity within the MnPO: experiment 3. Action potentials were discriminated from background activity with the spike-sorting algorithm of the Spike 2 software. Stability of the neuronal recording was assessed comparing averaged action potential waveforms from different time points. The major outcome measures for unit activity were discharge rates during different baseline sleep-wake states, during different time points within the SD protocol, and during different time points within the post-sd recovery period. For each recorded cell, mean discharge rates were calculated during baseline active wake (AW), quiet wake (QW), NREM sleep, and REM sleep. From six to ten 3-s epochs of each state were selected for analysis. The criterion for selecting sleep-active cells for further analysis was a statistically significant (one-way ANOVA) increase in discharge rate during NREM and/or REM sleep compared with both AW and QW. The 2-h SD period was divided into 3-min segments and mean waking (W), and AW and QW discharge rates were determined within 1-min sample in the middle portion of each segment. Similarly, post-sd period was portioned into 3-min segments, and mean discharge rates were calculated for all episodes of NREM sleep occurring within 1 min in the middle of the segments. Statistical Analysis Experiment 1. For experiment 1, statistical analysis was performed using Sigma Plot 13. Statistical software (Systat Software, San Jose, CA). To compare averaged AD levels during 1-h pre-sd BSL, 3-h SD period (average of 3 hourly values), and 2-h RS period (average of 2 hourly values) in P22 and P3 age groups, we used two-way repeated-measures ANOVA with condition as a within-subjects factor with three levels (BSL, SD, and RS) and age as a between-subjects factor followed by simple main effect analysis and Fisher least significant difference (LSD) post hoc test. Experiment 2. For Experiment 2, wake-sleep data were averaged for the 9-min recordings following the SD protocol. Student s paired t-test was used to assess differences between the treatment (intracerebroventricular AD) and control (intracerebroventricular saline) conditions in the measured wake-sleep parameters. P.5 was considered to be significant for all tests. Experiment 3. For Experiment 3, a two-way repeated-measures ANOVA with sleep-wake state as a within-subjects factor with 4 levels (AW, QW, NREM, and REM sleep) and age as a betweensubjects factor followed by simple main effect analysis and Tukey s post hoc test was used to compare the mean firing rates of MnPO sleep-related cells in the course of BSL sleep-waking cycle. To compare the mean firing rates of MnPO sleep-related cells across BSL wakefulness and four quarters of SD period, and across BSL NREM sleep and four quarters of RS, we used a two-way repeated-measures ANOVA with time as a within-subjects factor with five levels (BSL, SD/ RS quarters 1 4) and age as a betweensubjects factor followed by simple main effect analysis and Tukey s post hoc test. RESULTS SD-Related Changes in Extracellular AD Levels in the BF/LPO of P22 and P3 Rats: Experiment 1 The localization of tips of microdialysis probes within BF/ LPO was histologically confirmed in five out of eight animals in the P22 group and in five out of seven animals in the P3

4 33 ADENOSINE AND MATURATION OF SLEEP nm AP=-.11 mm AP=-.26 mm 3 P22 P3 Concentration of adenosine CA CA 1 5 Och Och BL SD RS Fig. 1. Sleep-deprivation (SD)-related changes in extracellular adenosine (AD) levels in the basal forebrain (BF)/lateral preoptic area (LPO) of postnatal day (P)22 and P3 rats. A: coronal section showing the localization of the tip of microdialysis probe in the BF (arrow) of a P22 rat. B: reconstruction of the locations of the tips of microdialysis probes in the BF/LPO in groups of P22 (grey bars) and P3 (black bars) rats (n 5/age group). C: group mean molar concentrations of AD for the BF/LPO of P22/P3 rats in different experimental conditions. Scale bar 5 m. Error bars represent means SE. CA, anterior commissure; Och, optic chiasm; AP, anterior/posterior; BSL, baseline; RS, recovery sleep. Asterisks with brackets indicate significance level of differences in mean molar concentrations of AD between experimental conditions within an age group. Stars indicate significance level of differences in mean molar concentrations of AD between age groups. P.5. group (Fig. 1, A and B). The comparison of AD levels across BSL, SD (average of 3 hourly values) and RS (average of 2 hourly values) conditions (Fig. 1C) revealed statistically significant age condition interaction [F(2,16) 4.6, P.5, two-way repeated-measures ANOVA]. Simple main effect analysis showed that in the P22 group AD levels did not change significantly across the experimental conditions. In the P3 group, main effect of factor condition was significant [F(2,8) 4.7, P.5, one-way repeated-measures ANOVA]. Specifically, SD resulted in increased AD levels compared with BSL (P.5, Fisher LSD post hoc test). During RS, compared with SD, AD levels decreased (P.5, Fisher LSD post hoc test) and did not differ from BSL values. Age differences in AD levels were not statistically significant during pre-sd BSL and RS. During SD, AD levels were higher in the P3 vs. P22 age group [F(1,8) 5.8, P.5, one-way ANOVA]. A ICV saline on P21 The Effect of Exogenous AD on Sleep Homeostatic Responses to Acute SD: Experiment 2 Administration of AD (1 nmol icv) during SD protocol on P22 resulted in significant changes in post-sd wake-sleep characteristics, compared with the control (intracerebroventricular saline) condition. Examples of the RS response to intracerebroventricular saline vs. AD injection in one rat are shown in Fig. 2. Intracerebroventricular AD vs. intracerebroventricular saline caused significant a decrease in the percentage of time spent in wakefulness (t-test, P.1) and increase in the percentage of NREM sleep time (t-test, P.5; Table 1). AD injection also increased NREM sleep EEG SWA during post-sd recovery period (P.1), but the treatment did not cause compensatory increases in NREM sleep bout duration (see Table 1). The percentage of time spent in REM sleep tended to increase following intracerebroventricular AD, but B ICV adenosine on P22 Wake NREM REM V Delta power EEG EMG Time min Fig. 2. The effect of intracerebroventricular adenosine on sleep homeostatic responses to SD in newly weaned rats. A, 1st row: hypnogram of 9-min continuous recording of RS following central administration of saline, during the last quarter of SD, on P21. A, 2nd row: shows non-rapid-eye-movement (NREM) sleep EEG delta power values for RS on P21. A, 3rd and 4th rows: cortical EEG and neck muscle EMG recordings during RS on P21, respectively. B, 1st row: hypnogram of 9-min continuous recording of RS following central administration of adenosine, during the last quarter of SD, on P22. B, 2nd row: NREM sleep EEG delta power values for RS on P22. B, 3rd and 4th rows: cortical EEG and neck muscle EMG recordings during RS on P22, respectively. min

5 ADENOSINE AND MATURATION OF SLEEP 331 Table 1. Homeostatic responses to SD following intracerebroventricular AD on P22: experiment 2 Wake-Sleep Characteristics During Recovery Period, ZT4 5.5 Intracerebroventricular Saline at ZT3 on P21 Intracerebroventricular Adenosine at ZT3 on P22 Wakefulness, % NREM sleep, % REM sleep, % EEG SWA, V 2 2, , Mean duration of NREM sleep episodes, min Mean duration of REM sleep episodes, min Values are expressed as means SE; n 6. ZT, zeitgeber time; P, postnatal day; SD, sleep deprivation. NREM, non-rapid eye movement; REM, rapid eye movement; EEG SWA, slow-wave activity represented by power density in the.5- to 4.-Hz band. Intracerebroventricular adenosine resulted in significantly lower percentage of wakefulness total time [t(1)5.2, P.1] and higher percentage of NREM sleep total time]t(1)-2.6, P.5], compared with the control condition. NREM sleep EEG SWA also increased following intracerebroventricular adenosine (AD) compared with the control [t(1)4.8, P.1]. All calculations were done for the 9-min period that followed the termination of SD protocol. P.1; P.5. the difference between values obtained in the experimental and control conditions did not reach statistical significance. Mean duration of REM sleep episodes also did not differ between the two conditions (see Table 1). Developmental Changes in Sleep-Related Neuronal discharge in the MnPO: Experiment 3 Figure 3 shows examples of localization of microwires (Fig. 3A) and recording of neuronal activity during BSL wake-sleep cycle within the MnPO (Fig. 3B). Discharge rates of sleepactive cells recorded on P22 (n 7) and P3 (n 7) were analyzed for 2-h pre-sd BSL, 2-h SD, and 2-h post-sd recovery period. During pre-sd BSL, all sleep-active neurons discharged at low rates ( 1 spikes/s) in AW (Fig. 4A). Comparison of the mean firing rates of these cells across wake-sleep states on P22 and P3, using two-way repeated measures ANOVA, revealed statistically significant age behavioral state interaction [F(3,36) 7.625, P.1]. Simple main effect analysis showed that the mean firing rate in the course of the wake-sleep cycle changed significantly in both age groups [on P22: F(3,18) 33,4 P.1; on P3, F(3,18) 24.4, P.1]. Specifically, neuronal discharge increased during QW compared with AW and was significantly higher in NREM and REM sleep compared with both AW and QW (Fig. 4B). Age differences in the firing rates of sleep-active neurons depended on behavioral state. In AW and QW, the between-group differences in firing rates were insignificant [F(1,12).1, P.5 and F(1,12).6, P.5, respectively]. During both NREM and REM sleep, discharge rates were higher on P3 vs. P22 [F(1,12) 1., P.1 and F(1,12) 5., P.5, respectively]. The comparison of the mean firing rates of sleep-active cells across pre-sd BSL waking and four quarters of SD period (Fig. 5, A, C, and D) revealed statistically significant age time interaction for W, AW, and QW discharge rates [F(4,48) 2.6, P.5, F(4 48) 3.7, P.5, and F(4,48) 4., P.1, respectively]. The simple main effect of factor time was statistically significant for W, AW, and QW discharge on both P22 and P3. During SD, compared with pre-sd BSL, mean W discharge (Fig. 5A) did not change significantly during the first quarter of SD both on P22 and P3. During the second, third, and fourth quarters of the SD period, compared with BSL, W discharge significantly increased by 39 57% on P22 and by 69 84% on P3 (P.1.5, Tukey post hoc test) and was higher on P3 vs. P22 although the difference was significant only during the second quarter of SD [F(1,12) 5.1, P.5, one-way ANOVA]. AW discharge (Fig. 5C) also significantly increased by 49 75% on P22 and by % on P3 during second, third, and fourth quarters of SD period (P.1.5, Tukey post hoc test) and was significantly higher on P3 vs. P22 during the last quarter [F(1,12) 4.9, P.5, one-way ANOVA]. QW discharge rate (Fig. 5D) significantly increased, compared with BSL, by 35% during the last quarter of SD on P22 and by 7% in the second quarter of SD on P3. The age difference was significant only during the second quarter of SD with higher discharge in P3 rats [F(1,12) 5.7, P.5]. It is important to note that despite a significant increase in neuronal discharge during SD, it remained about two times lower than during NREM sleep. The comparison of the mean firing rates of sleep-active cells in NREM sleep during BSL and four quarters of RS period (Fig. 5B) revealed statistically significant age time interaction [F(4,48) 4., P.1]. During post-sd RS, the mean firing rate of sleep-active cells did not exhibit significant changes vs. BSL in both ages. Sleep-active cells discharged at significantly higher rates on P3 vs. P22 across RS [F(1,12) , P.1.5]. DISCUSSION In the present study we tested the hypothesis that maturational changes in sleep homeostasis during postweaning development in rats are related to the functional emergence of AD signaling in the brain. We demonstrate that P3 but not P22 rats exhibited adult-like increases in brain extracellular AD in response to acute SD (Fig. 1) and that central administration of exogenous AD during SD in P22 rats resulted in enhanced sleep rebound and EEG SWA response during RS compared with saline control (Fig. 2). Thus, at P22, the brain is capable of responding to AD, even if AD release is not yet increased during SD. These findings identify a potential mechanistic role for AD in the maturation of sleep homeostasis across P22-P3. We recorded state-dependent neuronal activity in the MnPO on P22 and P3 during BSL wake-sleep, SD and RS. We identified neurons with sleep-related discharge under BSL conditions at both ages, but discharge rates during sleep were higher on P3 compared with P22 (Fig. 4). MnPO neurons exhibited adult-like increases in waking neuronal discharge across a 2-h period of SD on both P22 and P3, but discharge rates during enforced wake were higher on P3 vs. P22 (Fig. 5). Discharge rates during RS were also elevated at P3 vs. P22 (Fig. 5). These findings suggest that functional maturation of preoptic sleep regulatory neurons occurs during the fourth week of postnatal life in rats. We have previously shown that increased sleep consolidation during RS following acute SD was present at P3 but not at P22 (Gvilia et al. 211). In the present study, we

6 332 ADENOSINE AND MATURATION OF SLEEP A Rostral MnPO Caudal MnPO Fig. 3. median preoptic nucleus (MnPO) neuronal activity during baseline wake-sleep cycle in a P22 rat. A: coronal sections showing the localization of microwires in rostral and caudal parts of the MnPO. Arrows and arrowhead indicate microwire tracks and electrolytic lesion at the recording site, respectively. CA, anterior commissure; Fx, fornix. B, top to bottom: neck muscle EMG and cortical EEG recordings during baseline wake-sleep cycle on P22 and representative recordings of MnPO unit activity in a P22 rat. B EMG EEG AW NREM REM AW NREM REM Unit 1 Unit 2 Unit 3 5 s assessed postweaning changes in the level of AD signaling in the BF/LPO. In groups of P22 and P3 rats, we collected dialysate over consecutive 1-h epochs of 1-h BSL wakesleep cycle, 3-h SD, and 2-h RS. HPLC analysis of microdialysis samples revealed that SD in P3 rats results in significant increases in extracellular AD levels compared with BSL. During RS, compared with SD, AD levels decrease and do not differ from BSL values (see Fig. 1). In contrast, P22 rats do not exhibit significant changes in AD levels in response to SD suggesting that previously described immature responses to sleep loss in this age group are related to low production of AD. Age differences in AD Fig. 4. Discharge rates of MnPO sleep-active cells during baseline wake-sleep cycles recorded on P22 and P3. A: discharge rates (spikes/s) of individual MnPO neurons across baseline wake-sleep states on P22 and P3 (n 7/age group). B: group means SE discharge rates of MnPO sleep-active cells recorded on P22/P3. AW, active wake; QW, quiet wake; NREM, non-rapid-eye-movement sleep; REM, rapid-eye-movement sleep. Asterisks with brackets indicate the significance level of differences in mean discharge rates between behavioral states within an age group. Stars indicate the significance level of differences in mean discharge rates between age groups. P.5; P.1; P.1.

7 ADENOSINE AND MATURATION OF SLEEP 333 A B spikes/s 2.5 P22 P3 W 5 NREM C AW D QW Fig. 5. Discharge rates of MnPO sleep-active cells during sleep deprivation and following recovery sleep on P22 and P3. A: group means SE discharge rates of MnPO sleep-active neurons across baseline wake and four quarters of sleep deprivation period on P22 and P3 (n 7/age group). B: group means SE discharge rates of MnPO sleep-active neurons across baseline sleep and four quarters of recovery sleep. C and D: group mean discharge rates of MnPO sleep-active neurons across baseline active and quiet wake, respectively, and four quarters of sleep deprivation period on P22 and P3. W, wake; NREM, non-rapid-eye-movement sleep; AW, active wake; QW, quiet wake. Asterisks indicate the significance level of differences in mean discharge rates across a behavioral state in each age group. Stars indicate the significance level of differences in mean discharge rates between age groups. P.5; P.1; P.1. levels are not statistically significant during BSL and RS. During SD, the levels of AD are higher in P3 group. These findings indicate that endogenous AD production during sustained waking significantly increases during P22-P3, in association with maturation of homeostatic responses to SD. Levels of AD receptor expression at P22 do not appear to be the limiting factor in the strength of the homeostatic responses to SD, as exogenous AD administration during SD on P22 results in increased sleep rebound and increased EEG SWA during RS compared with saline control. This is supported by the finding that the distribution of A 1 -R at P2 widely resembles the heterogeneous pattern observed in the adult (Weaver 1996). Studies in adult rats have demonstrated that exogenous AD and its analogs promote homeostatic responses of increased sleep amount, sleep depth, and sleep continuity (Benington et al. 1995; McCarley et al. 27; Radulovacki 1985; Schwierin et al. 1996). Findings from the present study indicate that central administration of AD vs. saline during SD does not facilitate increases in mean duration of sleep episodes during RS in P22 rats (Table 1), suggesting that the brain mechanisms that regulate sleep homeostasis at this age are not sufficiently mature to produce all indexes of sleep rebound in response to acute SD. Present findings on the regulation of sleep homeostasis during early postweaning are in agreement with previous reports (Frank et al. 1998; Gvilia et al. 211). These studies demonstrated that on P2-24, rats exhibit some, but not all, components of the homeostatic sleep response to SD. Frank et al. (1998) examined the response to 3-h SD on P12, P16, P2, and P24 in Long Evan rats. They reported increases in NREM sleep -power in response to SD only in P24 rats although adult-like levels of spontaneous NREM sleep -power were found at P14 (Frank and Heller 1997). Hence, the brain is capable of increasing -power at P14 but not able to increase cortical synchrony further in response to SD until P24. A progressive decline in NREM sleep -activity during the 12-h rest-light phase of the 24-h light-dark cycle, which is typical for adult rats and reflective of adult sleep homeostasis, was also absent in rats younger than P24 (Frank and Heller 1997). Frank et al. (1998) did not find increased sleep consolidation during RS in P24 rats, and we did not observe it in P22 rats, even following intracerebroventricular administration of AD. Our findings and the findings by Frank et al. are in substantial agreement with each other and indicate that the mechanisms regulating sleep intensity in response to sleep loss emerge between P2 and P24, while mechanisms regulating sleep continuity emerge between P24 and P3. A limitation of our microdialysis study was that we only measured extracellular AD levels in the BF/LPO. We selected this region because robust changes in BF AD levels in response to SD have been documented previously (Porkka-Heiskanen et al. 1997, 2; Basheer et al. 1999; Kalinchuk et al. 28, 215) and the BF is adjacent to regions of the preoptic hypothalamus implicated in sleep regulation. Increased AD levels in response to SD have been detected in the neocortex as well (Kalinchuck et al. 211) and the developmental time course of extracellular AD signaling may differ in cortical and subcortical sites.

8 334 ADENOSINE AND MATURATION OF SLEEP The sleep-promoting effect of AD has been shown to involve A 2A -mediated activation of sleep-regulatory neurons in the POA (Alam et al. 214; Kumar et al. 213). We hypothesized that the difference in the magnitude of sleep homeostatic responses to sleep loss, between P22 and P3 rats, may reflect not only increases in AD production across postweaning development, but it may also reflect changes in the responsiveness of POA sleep-regulatory neuronal groups to the elevation of sleep drive. We have previously quantified c-fos immunoreactivity (IR) in POA GABAergic neurons in P22 and P3 rats during BSL sleep-wake, acute SD, and RS (Gvilia et al. 211). Among these three experimental conditions, in both age groups, Fos-IR in MnPO GABAergic neurons was highest during SD with no recovery period. But, the numbers of Fos /GAD cells were higher in P3 vs. P22 sleep-deprived rats. Given that c-fos method cannot detect dynamic changes in neuronal activity, we now assessed single unit activity in the MnPO across BSL wake-sleep cycle, SD, and RS on P22 and P3. We demonstrate that waking discharge of MnPO sleepactive neurons during SD vs. BSL increases during second, third, and fourth quarters of the 2-h SD protocol on both P22 and P3 (see Fig. 5A). SD-related discharge rate of MnPO sleep-active cells on P3 vs. P22 is significantly higher for the second quarter of the SD protocol. These findings indicate that the level of responsiveness of MnPO sleep-active neurons to the elevation of sleep drive is higher in P3 vs. P22 rats. Our findings on the temporal relationships between discharge of MnPO sleep-active neurons and the extent of homeostatic sleep pressure are consistent with previously published reports in adult rats (Alam et al. 214; Gvilia et al. 26a,b; Suntsova et al, 22). One limitation of our study of POA single unit activity in response to SD is that we did not record the extracellular activity of VLPO sleep-active neurons. Previous findings in adult rats suggest that activation of VLPO neurons is strongly dependent on the expression of sleep (Gvilia et al. 26b; Sherin et al. 1996; Szymusiak et al. 1998) and that a subpopulation of these neurons is also responsive to changing homeostatic sleep pressure during waking (Alam et al. 214). Our previous study of sleep-related c-fos expression in developing rats indicated that VLPO Fos /GAD cell counts in P22 rats did not differ among the BSL, SD, and RS conditions. In P3 rats, the number of Fos /GAD cells in VLPO were elevated during RS following acute SD (Gvilia et al. 211). A second limitation is that our extracellular neuronal recording technique does not permit identification of the transmitter phenotype of recorded units. However, in the regions of the MnPO examined with electrophysiology in this study, we have previously documented sleep related c-fos expression in neurons that colocalized glutamic acid decarboxylase (GAD) a marker of GABAergic neurons (Gvilia et al. 26b, 211). This study provides first evidence about parallel maturation of 1) the homeostatic sleep responses to SD, 2) SD-related increased production/release of adenosine in the brain, and 3) the responsiveness of hypothalamic sleep-regulatory neurons to increased sleep drive during SD. Collectively, findings of this study support the hypothesis that developmental changes in adenosinergic systems in the rat may contribute to the maturation of sleep homeostasis during postweaning development. However, the relationship between maturation of the homeostatic response to sleep loss and changes in brain AD levels described here are correlational. Further studies examining the effects of manipulating (increasing and/or suppressing) AD signaling in the brain at different developmental stages are needed to determine the causal role of AD in the maturation of sleep homeostasis. GRANTS This work was supported by the Department of Veterans Affairs Medical Research Service Award BX1556 (to R. Szymusiak), National Institute of Mental Health Grant MH (to R. Szymusiak), Shota Rustaveli National Science Foundation Grant 31/61 (to I. Gvilia), Department of Veterans Affairs Medical Research Service Award 2I1BX144 (to R. Basheer), and National Institute of Neurological Disorders and Stroke Grant NS (to R. Basheer). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS I.G., N.S., and A. Kostin performed experiments; I.G., N.S., and A. Kalinchuk analyzed data; I.G., N.S., D.M., and R.B. interpreted results of experiments; I.G. drafted manuscript; I.G., N.S., A. 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