- Email: [email protected]
Activity of adenosine deaminase in the sleep regulatory areas of the rat CNS
Molecular Brain Research 80 (2000) 252–255 www.elsevier.com / locate / bres
Short communication
Activity of adenosine deaminase in the sleep regulatory areas of the rat CNS a,b , a,b b b Miroslaw Mackiewicz *, Elena V. Nikonova , Chris C. Bell , Raymond J. Galante , b d b,c Lin Zhang , Jonathan D. Geiger , Allan I. Pack a Division of Sleep and Chronobiology, Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, USA Center for Sleep and Respiratory Neurobiology, Hospital of the University of Pennsylvania, 991 Maloney Building, 3600 Spruce Street, Philadelphia, PA 19104 -4283, USA c Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA d Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada b
Accepted 13 June 2000
Abstract There are data to support the notion that adenosine (ADO), a neuromodulator in the CNS, is an important regulator of sleep homeostasis. It has been demonstrated that ADO agonists and antagonists strongly impact upon sleep. In addition, the level of adenosine varies across the sleep / wake cycle and increases following sleep deprivation. Adenosine deaminase (ADA) is a key enzyme involved in the metabolism of ADO. We questioned, therefore, whether there are differences in adenosine deaminase activity in brain regions relevant to sleep regulation. We found that ADA exhibits a characteristic spatial pattern of activity in the rat CNS with the lowest activity in the parietal cortex and highest in the region of the tuberomammillary nucleus (15.064.8 and 63.4628.0 nmoles / mg protein / 15 min, mean6S.D., respectively). There were significant differences among the brain regions by one-way ANOVA (F531.33, df56,123, P50.0001). The regional differences in ADA activity correlate with variations in the level of its mRNA. This suggests that spatial differences in ADA activity are the result of changes in the expression of the ADA gene. We postulate that adenosine deaminase plays an important role in the mechanism that controls regional concentration of adenosine in the brain and thus, it is a part of the sleep-wake regulatory mechanism. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Adenosine; Adenosine deaminase; Gene
An increased pressure to sleep is associated with accumulation in the CNS of molecules that promote sleep [1,2]. Adenosine (ADO) has been one of the most extensively studied sleep promoting molecules [10]. Perfusion of adenosine into the brain [10], inhibition of adenosine deaminase (ADA) [11] or of ADO transport [9], augments sleep. ADO receptor antagonists enhance wakefulness [10], whereas the receptor agonists promote sleep [10,14].
*Corresponding author. Tel.: 11-215- 615-3152 fax: 11-215- 6627749. E-mail address: [email protected] (M. Mackiewicz).
In addition, adenosine concentration in the CNS changes in relationship to the sleep-wake cycle [4,9]. Adenosine may impact upon sleep through: (1) inhibition of neurons in the major cholinergic and / or monoaminergic arousal systems; (2) disinhibition of neurons projecting to these systems; (3) local inhibition of cortical neurons. Adenosine deaminase, a key ADO metabolic enzyme, plays an important role in controlling ADO level [6]. Variations in its activity might be a part of sleep homeostasis. However, there is limited information on ADA activity in the regions relevant to sleep control. Thus, the goals of our study were to evaluate ADA
0169-328X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00142-X
M. Mackiewicz et al. / Molecular Brain Research 80 (2000) 252 – 255
activity in the key regions that are involved in sleep-wake control. We questioned whether differences in enzyme activity were paralleled by differences in its mRNA. Experiments were performed on male Sprague–Dawley rats, age 2 months, weighting 180–200 grams. Animals were housed in light / dark cycle of 12 h, a temperaturecontrolled room (228C), with food and water available ad libitum. Brain sectioning was performed according to the atlas of Paxinos and Watson [8]. The following regions were sampled for analyses: (1) vertical limb of the diagonal band (VDB), bregma 2.20 mm to 0.20 mm; (2) parietal cortex (CTX), horizontal limb of the diagonal band / magnocellular preoptic nucleus (HDB / MCPO), ventrolateral preoptic area (VLPO), bregma 0.20 mm to 21.80 mm; (3) tuberomammillary nucleus (TMN), bregma 22.80 mm to 24.8 mm; (4) dorsal raphe (DRN), bregma 26.8 to 28.8 mm; (5) locus coeruleus (LC), bregma 28.8 mm to 210.8 mm. The measurements of ADA enzymatic activity were performed as described previously by Geiger and Nagy [3]. A Waters high-pressure liquid chromatography system (Alliance), 2487 Absorbance Detector and Millenium 32 chromatography software was used in all analyses. Protein concentration was determined by the bicinchoninic acid method using bovine serum albumin as a standard. All assays were performed in duplicates. The overall differences in ADA activity among brain regions were estimated by one-way ANOVA followed by Tukey–Kramer post-hoc analyses. Detection of mRNA for ADA was achieved by reverse transcription-polymerase chain reaction (RT-PCR). The following primers were used: ADA (sense) 59-
253
ctgtgctgcatgcgccaccagccc-39, (antisense) 59-gtgaggtagctggaccagggccag-39]; a-tubulin (sense) 59-gtgtcttccatcactgcttccctca-39, (antisense) 59-gaactctccctcctccatgccctct39. RNA from CTX and the region of TMN was isolated by the guanidinium thiocyanate method and cDNA was synthesized with random hexanucleotides [13]. Amplification (35–45 cycles; 948C, 1 sec; 558C, 15 sec; 728C, 20 sec) was performed in the LightCycler (Roche). PCR kinetics for ADA and a-tubulin were determined by monitoring the binding of SYBR Green I dye to PCR products [16]. The relative differences in the amount of ADA mRNA between the CTX and TMN was performed by assessing the difference in the number of PCR cycles that were needed to reach a log-linear phase of amplification reaction. The ADA level in different brain regions is presented in Fig. 1. Differences in ADA activity among brain regions were examined by one-way ANOVA with a significant effect (F531.33, df56,123, P50.0001) indicating overall differences. The level of ADA ranged from 15.064.8 in CTX to 63.4628.0 and 38.067.4 (mean6S.D.) nmoles / mg protein / 15 min in the TMN and VLPO, respectively. ADA activity in the TMN was significantly higher than in all other brain regions tested (TMN vs. VLPO post-hoc P50.001). In contrast, the level of ADA activity in the CTX was significantly lower than in all other brain regions (CTX vs. TMN / VLPO post-hoc P50.00001). The relative differences in the level of ADA mRNA in CTX and the region containing TMN were estimated by analysis of the kinetics of ADA amplification using quantitative PCR [16]. Fig. 2 shows ADA amplification
Fig. 1. Adenosine deaminase activity (nmoles / mg protein / 15 min; mean6S.D.) in the rat CNS (n520–28). Abbreviations: TMN-tuberomammillary nucleus; VLPO-ventrolateral preoptic area; DRN-dorsal raphe nucleus; VDB-vertical limb of diagonal band; HDB-horizontal limb of diagonal band; LC-locus coeruleus; CTX-cerebral cortex.
254
M. Mackiewicz et al. / Molecular Brain Research 80 (2000) 252 – 255
Fig. 2. Amplification curves of adenosine deaminase. The log-linear phase of accumulation of ADA products using cDNA from CTX and TMN is between 34 and 36 and 28–32 PCR cycles, respectively. The shift of the log-linear part of the amplification curves by six cycles indicates a large difference in the amount of starting material, i.e., ADA mRNA.
curves using cDNA samples from CTX and TMN, as well as the formation of non-specific PCR products in the absence of cDNA. The log-linear phase of accumulation of ADA products using cDNA prepared from CTX and TMN were estimated between 34–36 and 28–32 cycles, respectively. The shift of the log-linear part of the amplification curves indicates approximately 32-fold difference in the amount of ADA mRNA. There are data to support that adenosine, a neuromodulator in the CNS, is involved in sleep homeostasis [4,9–11,14]. Here, we report studies on the spatial differences in ADA activity, in brain regions involved in sleepwake regulation. In addition, we report a mechanism underlying such spatial differences. Adenosine deaminase activities presented here are in good agreement with previous results on ADA levels in the rat brain for those regions that were studied [3]. Our results confirm earlier observations that ADA enzymatic activity in the rat CNS exhibits robust spatial differences, with the highest level in the TMN region. TMN is the sole source of the histaminergic innervation in the CNS and it is a crucial component of the sleep regulatory mechanism [12]. We propose that high ADA activity in these neurons have functional significance such as a ‘compensatory’ role for metabolizing ADO as its
production increases during wakefulness. This can exert an inhibitory effect on the histaminergic arousal system. The second highest level of ADA was detected in the VLPO area. It is postulated that VLPO neurons affect sleep by inhibiting ascending arousal systems in particular, the histaminergic system [15]. Disinhibition of VLPO by adenosine during the onset of sleep may play a critical role in a rapid shifting of the balance from wakefulness to sleep. However, currently the role of ADO in control of TMN and VLPO neurons is unknown. It has been demonstrated that cortical arousal is strongly influenced by neuronal groups present in other regions of the CNS [5]. Adenosine may also contribute to sleep by acting on monoaminergic and cholinergic systems projecting directly to the thalamus and cortical areas, or may act locally on cortical neurons. These regions also have higher levels of ADA activity than cortex but significantly less than TMN. Thus, with respect to ADA, the histaminergic neurons are relatively unique among the arousal systems in the very high level of activity they have. To elucidate the mechanism underlying regional differences in ADA activity, we estimated the level of ADA gene transcript in various brain regions. Quantitative RTPCR was used in these experiments [16]. We have been able to show 32-fold differences in the level of mRNA for
M. Mackiewicz et al. / Molecular Brain Research 80 (2000) 252 – 255
ADA in CTX and TMN. It has been previously demonstrated that ADA activity varies markedly between various peripheral tissues and that the differences in enzymatic level are due to alterations of ADA gene transcript [7]; our results strongly support this notion. In conclusion, our studies have shown a high level of ADA activity in the key regions for control of sleep and wakefulness. Since adenosine is an important molecule for sleep regulation, these regional differences are likely to have important physiological consequences.
Acknowledgements We are grateful to Dr. Jacqueline Cater for help in statistical analysis. We would like to thank Mr. Daniel C. Barrett for his editorial help. Supported by NHLBI grant HL / MH59609 and SCOR grant HL-60287.
References [1] A.A. Borbely, A two process model of sleep regulation, Hum. Neurobiol. 1 (1996) 195–204. [2] A.A. Borbely, I. Tobler, Endogenous sleep-promoting substances and sleep regulation, Physiol. Rev. 69 (1989) 605–670. [3] J.D. Geiger, J.I. Nagy, Distribution of adenosine deaminase activity in rat brain and spinal cord, J. Neurosci. 6 (1986) 2707–2714. [4] J.P. Huston, H.L. Haas, F. Boix, M. Pfister, U. Decking, J. Schrader, R.K.W. Schwarting, Extracellular adenosine levels in neostriatum and hippocampus during rest and activity periods of rats, Neuroscience 73 (1996) 99–107.
255
[5] B.E. Jones, Basic mechanisms of sleep-wake states, in: M.H. Kryger (Ed.), Principles practice of sleep medicine, W.B. Saunders Co, Philadelphia, 1994, pp. 145–162. [6] P. Meghji, Adenosine production and metabolism, in: T. Stone (Ed.), Adenosine in the Nervous System, Academic Press, San Diego, CA, 1991, pp. 25–39. [7] K.A. Mohamedali, O.M. Guicherit, R.E. Kellems, F.B. Rudolph, The highest levels of purine catabolic enzymes in mice are present in the proximal small intestine, J. Biol. Chem. 268 (1993) 23728– 23733. [8] G. Paxinos, C. Watson, The Rat Brain In Stereotaxic Coordinates, Academic Press, San Diego, CA, 1986. [9] T. Porkka-Heiskanen, R.E. Strecker, M. Thakkar, R.W. McCarley, Adenosine: a mediator of sleep inducing effect of prolonged wakefulness, Science 276 (1997) 1265–1268. [10] M. Radulovacki, Adenosine and sleep, in: J.W. Phillis (Ed.), Adenosine and adenine nucleotides as regulators of cellular function, CRC Press, Boca Raton, FL, 1991, pp. 381–390. [11] M. Radulovacki, R.M. Virus, M. Djuricic-Nedelson, R.D. Green, Hypnotic effects of deoxycorformycin in rats, Brain Res. 271 (1983) 392–395. [12] K. Sakkai, M. Mansari, J.S. Lin, J.G. Zhang, G. Vanni-Mercier, The posterior hypothalamus in the regulation of wakefulness and paradoxical sleep, in: M. Mancia, G. Marini (Eds.), The Diencephalon and Sleep, Raven Press, New York, 1990, pp. 171–198. [13] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [14] S. Satoh, H. Matsumura, O. Hayaishi, Involvement of adenosine A 2a receptor in sleep promotion, Eur.J. Pharmacol. 351 (1998) 155–162. [15] J.E. Sherin, J.K. Elmquist, F. Torrealba, C.B. Saper, Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat, J. Neurosci. 18 (1998) 4705–4721. [16] C.T. Witter, M.G. Herrmann, A.A. Moss, R.P. Rasunssen, Continuous fluorescence monitoring of rapid cycle of DNA amplification, Biotechniques 22 (1997) 130–137.
Short communication
Activity of adenosine deaminase in the sleep regulatory areas of the rat CNS a,b , a,b b b Miroslaw Mackiewicz *, Elena V. Nikonova , Chris C. Bell , Raymond J. Galante , b d b,c Lin Zhang , Jonathan D. Geiger , Allan I. Pack a Division of Sleep and Chronobiology, Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, USA Center for Sleep and Respiratory Neurobiology, Hospital of the University of Pennsylvania, 991 Maloney Building, 3600 Spruce Street, Philadelphia, PA 19104 -4283, USA c Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA d Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada b
Accepted 13 June 2000
Abstract There are data to support the notion that adenosine (ADO), a neuromodulator in the CNS, is an important regulator of sleep homeostasis. It has been demonstrated that ADO agonists and antagonists strongly impact upon sleep. In addition, the level of adenosine varies across the sleep / wake cycle and increases following sleep deprivation. Adenosine deaminase (ADA) is a key enzyme involved in the metabolism of ADO. We questioned, therefore, whether there are differences in adenosine deaminase activity in brain regions relevant to sleep regulation. We found that ADA exhibits a characteristic spatial pattern of activity in the rat CNS with the lowest activity in the parietal cortex and highest in the region of the tuberomammillary nucleus (15.064.8 and 63.4628.0 nmoles / mg protein / 15 min, mean6S.D., respectively). There were significant differences among the brain regions by one-way ANOVA (F531.33, df56,123, P50.0001). The regional differences in ADA activity correlate with variations in the level of its mRNA. This suggests that spatial differences in ADA activity are the result of changes in the expression of the ADA gene. We postulate that adenosine deaminase plays an important role in the mechanism that controls regional concentration of adenosine in the brain and thus, it is a part of the sleep-wake regulatory mechanism. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Adenosine; Adenosine deaminase; Gene
An increased pressure to sleep is associated with accumulation in the CNS of molecules that promote sleep [1,2]. Adenosine (ADO) has been one of the most extensively studied sleep promoting molecules [10]. Perfusion of adenosine into the brain [10], inhibition of adenosine deaminase (ADA) [11] or of ADO transport [9], augments sleep. ADO receptor antagonists enhance wakefulness [10], whereas the receptor agonists promote sleep [10,14].
*Corresponding author. Tel.: 11-215- 615-3152 fax: 11-215- 6627749. E-mail address: [email protected] (M. Mackiewicz).
In addition, adenosine concentration in the CNS changes in relationship to the sleep-wake cycle [4,9]. Adenosine may impact upon sleep through: (1) inhibition of neurons in the major cholinergic and / or monoaminergic arousal systems; (2) disinhibition of neurons projecting to these systems; (3) local inhibition of cortical neurons. Adenosine deaminase, a key ADO metabolic enzyme, plays an important role in controlling ADO level [6]. Variations in its activity might be a part of sleep homeostasis. However, there is limited information on ADA activity in the regions relevant to sleep control. Thus, the goals of our study were to evaluate ADA
0169-328X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00142-X
M. Mackiewicz et al. / Molecular Brain Research 80 (2000) 252 – 255
activity in the key regions that are involved in sleep-wake control. We questioned whether differences in enzyme activity were paralleled by differences in its mRNA. Experiments were performed on male Sprague–Dawley rats, age 2 months, weighting 180–200 grams. Animals were housed in light / dark cycle of 12 h, a temperaturecontrolled room (228C), with food and water available ad libitum. Brain sectioning was performed according to the atlas of Paxinos and Watson [8]. The following regions were sampled for analyses: (1) vertical limb of the diagonal band (VDB), bregma 2.20 mm to 0.20 mm; (2) parietal cortex (CTX), horizontal limb of the diagonal band / magnocellular preoptic nucleus (HDB / MCPO), ventrolateral preoptic area (VLPO), bregma 0.20 mm to 21.80 mm; (3) tuberomammillary nucleus (TMN), bregma 22.80 mm to 24.8 mm; (4) dorsal raphe (DRN), bregma 26.8 to 28.8 mm; (5) locus coeruleus (LC), bregma 28.8 mm to 210.8 mm. The measurements of ADA enzymatic activity were performed as described previously by Geiger and Nagy [3]. A Waters high-pressure liquid chromatography system (Alliance), 2487 Absorbance Detector and Millenium 32 chromatography software was used in all analyses. Protein concentration was determined by the bicinchoninic acid method using bovine serum albumin as a standard. All assays were performed in duplicates. The overall differences in ADA activity among brain regions were estimated by one-way ANOVA followed by Tukey–Kramer post-hoc analyses. Detection of mRNA for ADA was achieved by reverse transcription-polymerase chain reaction (RT-PCR). The following primers were used: ADA (sense) 59-
253
ctgtgctgcatgcgccaccagccc-39, (antisense) 59-gtgaggtagctggaccagggccag-39]; a-tubulin (sense) 59-gtgtcttccatcactgcttccctca-39, (antisense) 59-gaactctccctcctccatgccctct39. RNA from CTX and the region of TMN was isolated by the guanidinium thiocyanate method and cDNA was synthesized with random hexanucleotides [13]. Amplification (35–45 cycles; 948C, 1 sec; 558C, 15 sec; 728C, 20 sec) was performed in the LightCycler (Roche). PCR kinetics for ADA and a-tubulin were determined by monitoring the binding of SYBR Green I dye to PCR products [16]. The relative differences in the amount of ADA mRNA between the CTX and TMN was performed by assessing the difference in the number of PCR cycles that were needed to reach a log-linear phase of amplification reaction. The ADA level in different brain regions is presented in Fig. 1. Differences in ADA activity among brain regions were examined by one-way ANOVA with a significant effect (F531.33, df56,123, P50.0001) indicating overall differences. The level of ADA ranged from 15.064.8 in CTX to 63.4628.0 and 38.067.4 (mean6S.D.) nmoles / mg protein / 15 min in the TMN and VLPO, respectively. ADA activity in the TMN was significantly higher than in all other brain regions tested (TMN vs. VLPO post-hoc P50.001). In contrast, the level of ADA activity in the CTX was significantly lower than in all other brain regions (CTX vs. TMN / VLPO post-hoc P50.00001). The relative differences in the level of ADA mRNA in CTX and the region containing TMN were estimated by analysis of the kinetics of ADA amplification using quantitative PCR [16]. Fig. 2 shows ADA amplification
Fig. 1. Adenosine deaminase activity (nmoles / mg protein / 15 min; mean6S.D.) in the rat CNS (n520–28). Abbreviations: TMN-tuberomammillary nucleus; VLPO-ventrolateral preoptic area; DRN-dorsal raphe nucleus; VDB-vertical limb of diagonal band; HDB-horizontal limb of diagonal band; LC-locus coeruleus; CTX-cerebral cortex.
254
M. Mackiewicz et al. / Molecular Brain Research 80 (2000) 252 – 255
Fig. 2. Amplification curves of adenosine deaminase. The log-linear phase of accumulation of ADA products using cDNA from CTX and TMN is between 34 and 36 and 28–32 PCR cycles, respectively. The shift of the log-linear part of the amplification curves by six cycles indicates a large difference in the amount of starting material, i.e., ADA mRNA.
curves using cDNA samples from CTX and TMN, as well as the formation of non-specific PCR products in the absence of cDNA. The log-linear phase of accumulation of ADA products using cDNA prepared from CTX and TMN were estimated between 34–36 and 28–32 cycles, respectively. The shift of the log-linear part of the amplification curves indicates approximately 32-fold difference in the amount of ADA mRNA. There are data to support that adenosine, a neuromodulator in the CNS, is involved in sleep homeostasis [4,9–11,14]. Here, we report studies on the spatial differences in ADA activity, in brain regions involved in sleepwake regulation. In addition, we report a mechanism underlying such spatial differences. Adenosine deaminase activities presented here are in good agreement with previous results on ADA levels in the rat brain for those regions that were studied [3]. Our results confirm earlier observations that ADA enzymatic activity in the rat CNS exhibits robust spatial differences, with the highest level in the TMN region. TMN is the sole source of the histaminergic innervation in the CNS and it is a crucial component of the sleep regulatory mechanism [12]. We propose that high ADA activity in these neurons have functional significance such as a ‘compensatory’ role for metabolizing ADO as its
production increases during wakefulness. This can exert an inhibitory effect on the histaminergic arousal system. The second highest level of ADA was detected in the VLPO area. It is postulated that VLPO neurons affect sleep by inhibiting ascending arousal systems in particular, the histaminergic system [15]. Disinhibition of VLPO by adenosine during the onset of sleep may play a critical role in a rapid shifting of the balance from wakefulness to sleep. However, currently the role of ADO in control of TMN and VLPO neurons is unknown. It has been demonstrated that cortical arousal is strongly influenced by neuronal groups present in other regions of the CNS [5]. Adenosine may also contribute to sleep by acting on monoaminergic and cholinergic systems projecting directly to the thalamus and cortical areas, or may act locally on cortical neurons. These regions also have higher levels of ADA activity than cortex but significantly less than TMN. Thus, with respect to ADA, the histaminergic neurons are relatively unique among the arousal systems in the very high level of activity they have. To elucidate the mechanism underlying regional differences in ADA activity, we estimated the level of ADA gene transcript in various brain regions. Quantitative RTPCR was used in these experiments [16]. We have been able to show 32-fold differences in the level of mRNA for
M. Mackiewicz et al. / Molecular Brain Research 80 (2000) 252 – 255
ADA in CTX and TMN. It has been previously demonstrated that ADA activity varies markedly between various peripheral tissues and that the differences in enzymatic level are due to alterations of ADA gene transcript [7]; our results strongly support this notion. In conclusion, our studies have shown a high level of ADA activity in the key regions for control of sleep and wakefulness. Since adenosine is an important molecule for sleep regulation, these regional differences are likely to have important physiological consequences.
Acknowledgements We are grateful to Dr. Jacqueline Cater for help in statistical analysis. We would like to thank Mr. Daniel C. Barrett for his editorial help. Supported by NHLBI grant HL / MH59609 and SCOR grant HL-60287.
References [1] A.A. Borbely, A two process model of sleep regulation, Hum. Neurobiol. 1 (1996) 195–204. [2] A.A. Borbely, I. Tobler, Endogenous sleep-promoting substances and sleep regulation, Physiol. Rev. 69 (1989) 605–670. [3] J.D. Geiger, J.I. Nagy, Distribution of adenosine deaminase activity in rat brain and spinal cord, J. Neurosci. 6 (1986) 2707–2714. [4] J.P. Huston, H.L. Haas, F. Boix, M. Pfister, U. Decking, J. Schrader, R.K.W. Schwarting, Extracellular adenosine levels in neostriatum and hippocampus during rest and activity periods of rats, Neuroscience 73 (1996) 99–107.
255
[5] B.E. Jones, Basic mechanisms of sleep-wake states, in: M.H. Kryger (Ed.), Principles practice of sleep medicine, W.B. Saunders Co, Philadelphia, 1994, pp. 145–162. [6] P. Meghji, Adenosine production and metabolism, in: T. Stone (Ed.), Adenosine in the Nervous System, Academic Press, San Diego, CA, 1991, pp. 25–39. [7] K.A. Mohamedali, O.M. Guicherit, R.E. Kellems, F.B. Rudolph, The highest levels of purine catabolic enzymes in mice are present in the proximal small intestine, J. Biol. Chem. 268 (1993) 23728– 23733. [8] G. Paxinos, C. Watson, The Rat Brain In Stereotaxic Coordinates, Academic Press, San Diego, CA, 1986. [9] T. Porkka-Heiskanen, R.E. Strecker, M. Thakkar, R.W. McCarley, Adenosine: a mediator of sleep inducing effect of prolonged wakefulness, Science 276 (1997) 1265–1268. [10] M. Radulovacki, Adenosine and sleep, in: J.W. Phillis (Ed.), Adenosine and adenine nucleotides as regulators of cellular function, CRC Press, Boca Raton, FL, 1991, pp. 381–390. [11] M. Radulovacki, R.M. Virus, M. Djuricic-Nedelson, R.D. Green, Hypnotic effects of deoxycorformycin in rats, Brain Res. 271 (1983) 392–395. [12] K. Sakkai, M. Mansari, J.S. Lin, J.G. Zhang, G. Vanni-Mercier, The posterior hypothalamus in the regulation of wakefulness and paradoxical sleep, in: M. Mancia, G. Marini (Eds.), The Diencephalon and Sleep, Raven Press, New York, 1990, pp. 171–198. [13] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [14] S. Satoh, H. Matsumura, O. Hayaishi, Involvement of adenosine A 2a receptor in sleep promotion, Eur.J. Pharmacol. 351 (1998) 155–162. [15] J.E. Sherin, J.K. Elmquist, F. Torrealba, C.B. Saper, Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat, J. Neurosci. 18 (1998) 4705–4721. [16] C.T. Witter, M.G. Herrmann, A.A. Moss, R.P. Rasunssen, Continuous fluorescence monitoring of rapid cycle of DNA amplification, Biotechniques 22 (1997) 130–137.