Psychopharmacologia (Berl.) 44, 4 3 - 4 5 (1975) - 9 by Springer-Verlag 1975
Cocaine and Amphetamine Modification of Cerebral Energy Metabolism in vivo LUCY J. KING, JUANITA L. CARL, and LAURO LAO Department of Psychiatry, School of Medicine, Washington University, St. Louis, Missouri Received May 1, 1975 Abstract. At the time of maximal behavioral stimulation after injection of amphetamine (5 mg/kg) in mice, elevation of cerebral cortical levels of malate in the citric acid cycle and of the amino acid, alanine, was observed, suggesting that this drug has widespread effects on energy metabolism. Cocaine
(20 mg/kg), in contrast, produced elevation of brain glucose but not of citric acid cycle substrates or amino acids at the time of maximal hyperactivity. These observations are discussed in terms of the mechanisms of action of these two central nervous system stimulants.
Key words: Amphetamine - Cocaine - Brain Energy Metabolism.
The central nervous system stimulants, cocaine and amphetamine, p r o d u c e obvious behavioral excitation, but few studies have attempted to correlate these changes with alterations in the basic brain processes o f energy metabolism. Those studies which have been carried out have focused on glycogen and anaerobic glycolysis (Nahorski and Rogers, 1973, 1974; Rogers and Nahorski, 1973). In the experiments described below citric acid cycle intermediates and related amino acids as well as glycolytic substrates were assayed at a time when behavioral stimulation appeared maximal after administration o f cocaine or amphetamine. In vitro studies have indicated that the effects o f cocaine on brain metabolism are enhanced by electrical stimulation (Bollard and McIlwain, 1959). Experiments in the present study measured alterations p r o d u c e d by cocaine and a m p h e t a m i n e on cerebral cortical energy metabolism during electrical stimulation in vivo. R a p i d freezing o f mice, intact, at intervals o f a few seconds during electrically induced convulsions, provides a preparation which allows for c h r o n o logic assessment o f changes in brain metabolism after electrical stimulation in vivo.
Methods
Adult white male mice ( 2 0 - 25 g body wt.) of the same strain (ICR) were used throughout. Mice were housed in the same cages of 5 mice, each, for several days prior to administration of drugs. Cocaine (20 mg/kg body wt.) or d-amphetamine (5 mg/kg body wt.), in approximately 0.25 ml aqueous solution, was injected, intraperitoneally. Control animals were injected at the same time with a similar volume of physiological saline. Twenty-five minutes after injection of saline, cocaine, or d-amphetamine, mice were plunged into Freon-12 (CClzF2)
that had been brought to its freezing point (-150~ with liquid N> The outer 3 - 4 mm of both cerebral hemispheres (approximately 100 rag) was removed in a cold chamber (Harris Lo Temp Freezer) at - 2 0 ~ with great care to prevent warming. Frozen animals o r dissected brains were kept at -80~ except for the few minutes necessary for dissection. For electrical stimulation, current was applied through metal electrodes, coated with Redox paste, and held firmly, bilaterally, just anterior to the ears. Unidirectional rectangular pulses of 0.1 msec duration and a frequency of 150/s were applied for 1 s by a Grass S 8 apparatus. For submaximal stimulation, a pulse amplitude of 50V was used. Only animals which showed some seizure activity without tonic hind limb extension (e.g. clonus, only) were used. For maximal (tonicclonic) convulsive stimulation a pulse amplitude of 100V was used. Only those mice which exhibited tonic hind limb extension were used. Mice were frozen as described above at 3, 10, 20, or 50 s after electrical stimulation. Preparation of HC104 extracts and enzymatic, fluorimetric assays were carried out by the methods of Lowry and Passonneau (1972). Substrates of anaerobic glycolosis, ATP, and P-creatine were assayed as described by Lowry et al. (1964); citric acid cycle intermediates, by the methods of Goldberg et al. (1966); and related amino acids, by the methods used in previous studies (King et al., 1974). Blood samples for serum glucose assay were obtained from severed bodies of decapitated mice which had received the same drugs at the same time as mice frozen for brain assays. Four mice, each, were sacrificed for saline, cocaine, or d-amphetamine injected groups. Statistical assessments used student's t test.
Results
Mice given cocaine exhibited increased m o t o r activity at the time o f sacrifice. Brain glucose was significantly elevated in these animals (Table 1). Serum glucose in cocaine treated mice (9.8 + 1.2 ram/l) was elevated
44
Psychopharmacologia (Berl.), Vol. 44, Fasc. 1 (1975) Table 1. Substrates of brain energy metabolism after cocaine or d-amphetamine
Substrate (mM/kg brain) Glycogen Glucose Glucose-6-P Fructose-di-P Pyruvate Lactate ATP P-Creatine Citrate c~-Ketoglutarate Succinate Malate Gaba Glutamate Alanine Ammonia
Saline 1.72 + 0.17 1.82 • 0.08* 0.12 _+ 0.01 0.10 + 0.01 0.08 ___0.01 1.51 + 0.12 2.86 _+ 0.09 2.74 _+ 0.15 0.30 ,+ 0.01 0.17 ,+ 0.01 0.83 _+ 0.05 0.57 _+ 0.03** 1.62 ,+ 0.07 12.63 _+ 0.44 0.43 ,+ 0.03*** 0.12 ,+ 0.04
Cocaine 1.47 • 0.11 2.11 + 0.11" 0.11 -+ 0.01 0.11 _+ 0.01 0.08 _+ 0.01 1.51 _ 0.17 2.86 _+ 0.06 2.62 _+ 0.21 0.33 ,+ 0.01 0.17 + 0.01 0.88 ,+ 0.06 0.62 ,+ 0.03 1.64 ,+ 0.07 13.56 ,+ 0.50 0.49 ,+ 0.04 0.15 ,+ 0.04
d-Amphetamine 1.57 _+ 0.12 1.95 + 0.14 0.14 -+ 0.01 0.09 __ 0.01 0.09 • 0.01 1.80 + 0.16 2.89 _+ 0.05 2.71 ,+ 0.15 0.33 ,+ 0.03 0.17 ,+ 0.01 0.94 ,+ 0.07 0.67 ,+ 0.02** 1.57 ,+ 0.08 14.75 ,+ 1.09 0.62 ,+ 0.05*** 0.14 _4- 0.02
*P0.05; **P0.05; ***P0.01. Figures represent means of 9 or t0 mice _+ S.E.M. Mice were given 0.25 ml physiologic saline, 20 mg/kg cocaine, or 5 mg/kg d-amphetamine, intraperitoneally, 25 rain prior to being frozen intact. but was not statistically significantly different from that in saline controls (7.3 + 0.6 mm/1). Rogers and Nahorski (1973) likewise observed elevated brain and blood glucose but no change in glycogen, ATP, P-creatine, or lactate at 15 and 30 rain after injection of the same dose of cocaine. Mice which had been given amphetamine appeared extremely agitated at 25 min after injection. Malate, in the citric acid cycle, and the amino acid, alanine, were significantly elevated in cerebral cortex (Table 1). Serum glucose in these mice (9.5 _+ 0.8 mM/1) was elevated but not significantly different from saline controls (7.3 _+ 0.6 mM/1). The results differ from those of Nahorski and Rogers (1973) who observed elevated lactate, and decreased glycogen, glucose, and P-creatine at 15 and 30 min and decreased glucose-6-P at 30 min after injection of the same dose of amphetamine. They did not assay citric acid cycle metabolites or amino acids. In mice given maximal convulsive electrical stimulation (100 V), patterns of changes of brain substrates were similar to those reported previously (King et al., 1973, 1974), and changes in brain substrate levels in cocaine or amphetamine injected mice were similar to those in saline controls. After submaximal convulsive stimulation (50 V), there were no significant changes in metabolites in saline controls or in cocaine or amphetamine injected mice. In vitro findings (Bollard and McIlwain, 1959) of changes in metabolic rate in stimulated tissue brought about by cocaine were not demonstrated in this particular preparation of in vivo stimulation.
Discussion
Elevation of malate in brain during increased flux through the citric acid cycle (in hyperthermia) was observed by Goldberg etal. (1966) who postulated that the level of malate might be an indicator of citric acid cycle flux in the oxidative state. If the elevation of malate observed after amphetamine injection represents an increase in citric acid cycle flux, then oxidative metabolism might well play an important role in the mechanism of action of amphetamine. In addition, elevation of alanine suggests changes in amino acids related to energy metabolites; perhaps, increased conversion of glucose metabolites to amino acids or exchange of amino acids and citric acid cycle metabolites in transamination reactions. There is evidence that amphetamine alters enzymes of glycogen metabolism (Nahorski and Rogers, 1974). It is possible that elevation of malate and alanine are related to changes in glycogen metabolism inasmuch as citric acid cycle metabolism is intimately related to anaerobic glycolysis and alanine can be metabolized to the glycolytic pathway substrate, pyruvate. However, the results of the present study suggest that there might well be widespread, not necessarily directly related, changes in brain energy metabolism brought about by amphetamine. In the present experiments, cocaine produced elevation of brain glucose at the beginning of the glycolytic pathway, but amphetamine produced elevation of malate, in the citric acid cycle, and of the amino acid, alanine. These differences might well
L. J. King et al. : Cocaine and Amphetamine Modification of Cerebral Energy Metabolism in vivo relate to mechanisms of action of the two drugs. Cocaine and amphetamine have m a n y effects in c o m m o n both behavioral (e.g. increased locomotor activity) and chemical (e.g. block of reuptake of the catecholamines, norepinephrine and dopamine, in nervous tissue). However, there is increasing evidence of different mechanisms of action in the two drugs. F o r example, recent in vitro studies indicate that cocaine affects indoleamines (as well as catecholamines) by inhibiting turn over of brain serotonin (Friedman et al., 1975). It seems reasonable that demands made upon, or alterations produced in, energy metabolism might affect reactions in different parts of the pathways of glucose metabolism. If elevation of malate level by amphetamine but not by cocaine reflects increased oxidative (citric acid cycle) metabolism associated with amphetamine administration, it is possible that the primary mechanisms of action of amphetamine require greater energy production than do the chief mechanisms of action of cocaine. Synthesis and release of neurotransmitter amines like norepinephrine, dopamine, and serotonin, are energy-requiring processes. A drug enhancing synthesis or release of amines would require more energy expenditure than one which did not. M e m b r a n e transport also requires energy expenditure. Thus, a drug which blocked transport across membranes (e.g. reuptake into neurons) might be associated with diminished energy expenditure. Differences in energy metabolism might well offer clues about which of the m a n y biochemical effects of amphetamine or of cocaine are of major importance.
45
Acknowledgement. This research was supported by a grant from S.A.O.D.A.P. (grant number DA 4 RG 008).
References Bollard, B. M., McIlwain, H. : Cocaine and procaine on the electrically stimulated metabolism of cerebral tissues. Biochem. Pharmacol. 2, 81-88 (1959) Friedman, E., Gershon, S., Rotrosen, J.: Effects of acute cocaine treatment on the turnover of 5-hydroxytryptamine in the rat brain. Brit. J. Pharmacol. 54, 61 - 6 4 (1975) Goldberg, N. D., Passonneau, J. V., Lowry, O.H.: Effects of changes in brain metabolism on the levels of citric acid cycle intermediates. J. biol. Chem. 241, 3997-4003 (1966) King, L. J., Carl, J. L., Lao, L.: Carbohydrate metabolism in brain during convulsions and its modification by phenobarbitone. J. Neurochem. 20, 477-485 (1973) King, L. J., Carl, J. L., Lao, L. : Brain amino acids during convulsions. J. Neurochem. 22, 307-309 (1974) Lowry, O. H., Passonneau, J. V. : A flexible system of enzymatic analysis. New York: Academic Press 1972 Lowry, O. H., Passonneau, J. V., Hasselberger, F. X., Schulz, D. W. : Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J. biol. Chem. 239, 18-30 (1964) Nahorski, S. R., Rogers, K. J. : In vivo effects of amphetamine on metabolites and metabolic rate in brain. J. Neurochem. 21, 679-686 (1973) Nahorski, S. R., Rogers, K. J. : The incorporation of glucose into brain glycogen and the activities of cerebral glycogen phosphorylase and synthetase: some effects of amphetamine. J. Neurochem. 23, 579-587 (1974) Rogers, K. J., Nahorski, S. R. : Depression of cerebral metabolism by stimulant doses of cocaine. Brain Res. 57, 255-258 (1973)
Dr. King, Departments of Psychiatry and Pharmacology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, U.S.A.
Cocaine and Amphetamine Modification of Cerebral Energy Metabolism in vivo LUCY J. KING, JUANITA L. CARL, and LAURO LAO Department of Psychiatry, School of Medicine, Washington University, St. Louis, Missouri Received May 1, 1975 Abstract. At the time of maximal behavioral stimulation after injection of amphetamine (5 mg/kg) in mice, elevation of cerebral cortical levels of malate in the citric acid cycle and of the amino acid, alanine, was observed, suggesting that this drug has widespread effects on energy metabolism. Cocaine
(20 mg/kg), in contrast, produced elevation of brain glucose but not of citric acid cycle substrates or amino acids at the time of maximal hyperactivity. These observations are discussed in terms of the mechanisms of action of these two central nervous system stimulants.
Key words: Amphetamine - Cocaine - Brain Energy Metabolism.
The central nervous system stimulants, cocaine and amphetamine, p r o d u c e obvious behavioral excitation, but few studies have attempted to correlate these changes with alterations in the basic brain processes o f energy metabolism. Those studies which have been carried out have focused on glycogen and anaerobic glycolysis (Nahorski and Rogers, 1973, 1974; Rogers and Nahorski, 1973). In the experiments described below citric acid cycle intermediates and related amino acids as well as glycolytic substrates were assayed at a time when behavioral stimulation appeared maximal after administration o f cocaine or amphetamine. In vitro studies have indicated that the effects o f cocaine on brain metabolism are enhanced by electrical stimulation (Bollard and McIlwain, 1959). Experiments in the present study measured alterations p r o d u c e d by cocaine and a m p h e t a m i n e on cerebral cortical energy metabolism during electrical stimulation in vivo. R a p i d freezing o f mice, intact, at intervals o f a few seconds during electrically induced convulsions, provides a preparation which allows for c h r o n o logic assessment o f changes in brain metabolism after electrical stimulation in vivo.
Methods
Adult white male mice ( 2 0 - 25 g body wt.) of the same strain (ICR) were used throughout. Mice were housed in the same cages of 5 mice, each, for several days prior to administration of drugs. Cocaine (20 mg/kg body wt.) or d-amphetamine (5 mg/kg body wt.), in approximately 0.25 ml aqueous solution, was injected, intraperitoneally. Control animals were injected at the same time with a similar volume of physiological saline. Twenty-five minutes after injection of saline, cocaine, or d-amphetamine, mice were plunged into Freon-12 (CClzF2)
that had been brought to its freezing point (-150~ with liquid N> The outer 3 - 4 mm of both cerebral hemispheres (approximately 100 rag) was removed in a cold chamber (Harris Lo Temp Freezer) at - 2 0 ~ with great care to prevent warming. Frozen animals o r dissected brains were kept at -80~ except for the few minutes necessary for dissection. For electrical stimulation, current was applied through metal electrodes, coated with Redox paste, and held firmly, bilaterally, just anterior to the ears. Unidirectional rectangular pulses of 0.1 msec duration and a frequency of 150/s were applied for 1 s by a Grass S 8 apparatus. For submaximal stimulation, a pulse amplitude of 50V was used. Only animals which showed some seizure activity without tonic hind limb extension (e.g. clonus, only) were used. For maximal (tonicclonic) convulsive stimulation a pulse amplitude of 100V was used. Only those mice which exhibited tonic hind limb extension were used. Mice were frozen as described above at 3, 10, 20, or 50 s after electrical stimulation. Preparation of HC104 extracts and enzymatic, fluorimetric assays were carried out by the methods of Lowry and Passonneau (1972). Substrates of anaerobic glycolosis, ATP, and P-creatine were assayed as described by Lowry et al. (1964); citric acid cycle intermediates, by the methods of Goldberg et al. (1966); and related amino acids, by the methods used in previous studies (King et al., 1974). Blood samples for serum glucose assay were obtained from severed bodies of decapitated mice which had received the same drugs at the same time as mice frozen for brain assays. Four mice, each, were sacrificed for saline, cocaine, or d-amphetamine injected groups. Statistical assessments used student's t test.
Results
Mice given cocaine exhibited increased m o t o r activity at the time o f sacrifice. Brain glucose was significantly elevated in these animals (Table 1). Serum glucose in cocaine treated mice (9.8 + 1.2 ram/l) was elevated
44
Psychopharmacologia (Berl.), Vol. 44, Fasc. 1 (1975) Table 1. Substrates of brain energy metabolism after cocaine or d-amphetamine
Substrate (mM/kg brain) Glycogen Glucose Glucose-6-P Fructose-di-P Pyruvate Lactate ATP P-Creatine Citrate c~-Ketoglutarate Succinate Malate Gaba Glutamate Alanine Ammonia
Saline 1.72 + 0.17 1.82 • 0.08* 0.12 _+ 0.01 0.10 + 0.01 0.08 ___0.01 1.51 + 0.12 2.86 _+ 0.09 2.74 _+ 0.15 0.30 ,+ 0.01 0.17 ,+ 0.01 0.83 _+ 0.05 0.57 _+ 0.03** 1.62 ,+ 0.07 12.63 _+ 0.44 0.43 ,+ 0.03*** 0.12 ,+ 0.04
Cocaine 1.47 • 0.11 2.11 + 0.11" 0.11 -+ 0.01 0.11 _+ 0.01 0.08 _+ 0.01 1.51 _ 0.17 2.86 _+ 0.06 2.62 _+ 0.21 0.33 ,+ 0.01 0.17 + 0.01 0.88 ,+ 0.06 0.62 ,+ 0.03 1.64 ,+ 0.07 13.56 ,+ 0.50 0.49 ,+ 0.04 0.15 ,+ 0.04
d-Amphetamine 1.57 _+ 0.12 1.95 + 0.14 0.14 -+ 0.01 0.09 __ 0.01 0.09 • 0.01 1.80 + 0.16 2.89 _+ 0.05 2.71 ,+ 0.15 0.33 ,+ 0.03 0.17 ,+ 0.01 0.94 ,+ 0.07 0.67 ,+ 0.02** 1.57 ,+ 0.08 14.75 ,+ 1.09 0.62 ,+ 0.05*** 0.14 _4- 0.02
*P0.05; **P0.05; ***P0.01. Figures represent means of 9 or t0 mice _+ S.E.M. Mice were given 0.25 ml physiologic saline, 20 mg/kg cocaine, or 5 mg/kg d-amphetamine, intraperitoneally, 25 rain prior to being frozen intact. but was not statistically significantly different from that in saline controls (7.3 + 0.6 mm/1). Rogers and Nahorski (1973) likewise observed elevated brain and blood glucose but no change in glycogen, ATP, P-creatine, or lactate at 15 and 30 rain after injection of the same dose of cocaine. Mice which had been given amphetamine appeared extremely agitated at 25 min after injection. Malate, in the citric acid cycle, and the amino acid, alanine, were significantly elevated in cerebral cortex (Table 1). Serum glucose in these mice (9.5 _+ 0.8 mM/1) was elevated but not significantly different from saline controls (7.3 _+ 0.6 mM/1). The results differ from those of Nahorski and Rogers (1973) who observed elevated lactate, and decreased glycogen, glucose, and P-creatine at 15 and 30 min and decreased glucose-6-P at 30 min after injection of the same dose of amphetamine. They did not assay citric acid cycle metabolites or amino acids. In mice given maximal convulsive electrical stimulation (100 V), patterns of changes of brain substrates were similar to those reported previously (King et al., 1973, 1974), and changes in brain substrate levels in cocaine or amphetamine injected mice were similar to those in saline controls. After submaximal convulsive stimulation (50 V), there were no significant changes in metabolites in saline controls or in cocaine or amphetamine injected mice. In vitro findings (Bollard and McIlwain, 1959) of changes in metabolic rate in stimulated tissue brought about by cocaine were not demonstrated in this particular preparation of in vivo stimulation.
Discussion
Elevation of malate in brain during increased flux through the citric acid cycle (in hyperthermia) was observed by Goldberg etal. (1966) who postulated that the level of malate might be an indicator of citric acid cycle flux in the oxidative state. If the elevation of malate observed after amphetamine injection represents an increase in citric acid cycle flux, then oxidative metabolism might well play an important role in the mechanism of action of amphetamine. In addition, elevation of alanine suggests changes in amino acids related to energy metabolites; perhaps, increased conversion of glucose metabolites to amino acids or exchange of amino acids and citric acid cycle metabolites in transamination reactions. There is evidence that amphetamine alters enzymes of glycogen metabolism (Nahorski and Rogers, 1974). It is possible that elevation of malate and alanine are related to changes in glycogen metabolism inasmuch as citric acid cycle metabolism is intimately related to anaerobic glycolysis and alanine can be metabolized to the glycolytic pathway substrate, pyruvate. However, the results of the present study suggest that there might well be widespread, not necessarily directly related, changes in brain energy metabolism brought about by amphetamine. In the present experiments, cocaine produced elevation of brain glucose at the beginning of the glycolytic pathway, but amphetamine produced elevation of malate, in the citric acid cycle, and of the amino acid, alanine. These differences might well
L. J. King et al. : Cocaine and Amphetamine Modification of Cerebral Energy Metabolism in vivo relate to mechanisms of action of the two drugs. Cocaine and amphetamine have m a n y effects in c o m m o n both behavioral (e.g. increased locomotor activity) and chemical (e.g. block of reuptake of the catecholamines, norepinephrine and dopamine, in nervous tissue). However, there is increasing evidence of different mechanisms of action in the two drugs. F o r example, recent in vitro studies indicate that cocaine affects indoleamines (as well as catecholamines) by inhibiting turn over of brain serotonin (Friedman et al., 1975). It seems reasonable that demands made upon, or alterations produced in, energy metabolism might affect reactions in different parts of the pathways of glucose metabolism. If elevation of malate level by amphetamine but not by cocaine reflects increased oxidative (citric acid cycle) metabolism associated with amphetamine administration, it is possible that the primary mechanisms of action of amphetamine require greater energy production than do the chief mechanisms of action of cocaine. Synthesis and release of neurotransmitter amines like norepinephrine, dopamine, and serotonin, are energy-requiring processes. A drug enhancing synthesis or release of amines would require more energy expenditure than one which did not. M e m b r a n e transport also requires energy expenditure. Thus, a drug which blocked transport across membranes (e.g. reuptake into neurons) might be associated with diminished energy expenditure. Differences in energy metabolism might well offer clues about which of the m a n y biochemical effects of amphetamine or of cocaine are of major importance.
45
Acknowledgement. This research was supported by a grant from S.A.O.D.A.P. (grant number DA 4 RG 008).
References Bollard, B. M., McIlwain, H. : Cocaine and procaine on the electrically stimulated metabolism of cerebral tissues. Biochem. Pharmacol. 2, 81-88 (1959) Friedman, E., Gershon, S., Rotrosen, J.: Effects of acute cocaine treatment on the turnover of 5-hydroxytryptamine in the rat brain. Brit. J. Pharmacol. 54, 61 - 6 4 (1975) Goldberg, N. D., Passonneau, J. V., Lowry, O.H.: Effects of changes in brain metabolism on the levels of citric acid cycle intermediates. J. biol. Chem. 241, 3997-4003 (1966) King, L. J., Carl, J. L., Lao, L.: Carbohydrate metabolism in brain during convulsions and its modification by phenobarbitone. J. Neurochem. 20, 477-485 (1973) King, L. J., Carl, J. L., Lao, L. : Brain amino acids during convulsions. J. Neurochem. 22, 307-309 (1974) Lowry, O. H., Passonneau, J. V. : A flexible system of enzymatic analysis. New York: Academic Press 1972 Lowry, O. H., Passonneau, J. V., Hasselberger, F. X., Schulz, D. W. : Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J. biol. Chem. 239, 18-30 (1964) Nahorski, S. R., Rogers, K. J. : In vivo effects of amphetamine on metabolites and metabolic rate in brain. J. Neurochem. 21, 679-686 (1973) Nahorski, S. R., Rogers, K. J. : The incorporation of glucose into brain glycogen and the activities of cerebral glycogen phosphorylase and synthetase: some effects of amphetamine. J. Neurochem. 23, 579-587 (1974) Rogers, K. J., Nahorski, S. R. : Depression of cerebral metabolism by stimulant doses of cocaine. Brain Res. 57, 255-258 (1973)
Dr. King, Departments of Psychiatry and Pharmacology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, U.S.A.
