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Effect of Ammonia on Motor Function in Adult Rats
Brain Research Bulletin, Vol. 43, No. 3, pp. 275–278, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00432-7
Effect of Ammonia on Motor Function in Adult Rats A. R. JAYAKUMAR, R. SUJATHA AND VANAJA PAUL 1 Department of Pharmacology & Environmental Toxicology, Dr. A. L. M. Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani, Madras 600 113, India [Received 21 August 1996; Accepted 21 November 1996] ABSTRACT: Changes in motor function were assessed in male rats after injecting graded doses (100, 200, 400, and 800 mg/kg, IP) of ammonium chloride and ammonium acetate. The effects were correlated with the concentrations of ammonia and glucose in the brain and blood. Spontaneous motor activity and motor coordination were inhibited after injecting 100 and 200 mg/kg, whereas with 400 and 800 mg/kg the animals exhibited convulsive movements. A dose-dependent increase was found in the concentrations of ammonia and glucose in both blood and brain. These were restored, 25 min after treatment, to control levels in the blood and not in the brain. A correlation was found between the time courses of inhibitory motor events and a rise in brain ammonia levels. Convulsant action of ammonium salts was accompanied by a marked elevation of ammonia and glucose concentration in the brain. The findings suggest that detoxication of diffused ammonia is a rate-limiting process in the brain and that ammonia, at toxic concentrations, decreases glucose utilization in the brain, resulting in an inhibition of motor function. A very high concentration of ammonia in the brain, although inhibiting glucose utilization, produces clonic convul sions probably by activating directly the motor neurons. Q 1997 Elsevier Science Inc.
and brain ammonia concentrations are correlated. In previous studies brain ammonia level was raised by acute dosing of ammonium salts [25]. Hyperammonemia resulting from portacaval shunting [2,7] or carbon tetrachloride-induced liver damage [10] was also used to raise brain ammonia. The latter techniques may produce several metabolic derangements also, as a result of liver impairment. Therefore, a defect in the motor function of these animals cannot be attributed solely to an increased level of ammonia in the brain. In view of these facts, in the present study changes in motor function were determined in groups of rats after injecting graded doses of ammonium chloride. The results were correlated with blood and brain ammonia data. The changes that occurred in brain and blood glucose concentrations were also measured in these animals to test whether ammonia loading has resulted in impairment of glucose metabolism also. To confirm the findings, tests were carried out after injecting ammonium acetate at the same dose level.
KEY WORDS: Brain ammonia, Brain glucose, Spontaneous motor activity, Motor coordination, Convulsions.
Wistar strain 5–6-month-old (150–160 g) male rats were used. Test (n Å 8) and control ( n Å 8) animals were selected randomly and were caged in groups of four at room temprature (22–307C). Animals were supplied ad lib with a balanced rat feed (Gold mohur pellets, Bangalore, India) and drinking water. Motor function tests and sacrifice for biochemical determinations were performed between 1000 and 1300 h.
MATERIALS AND METHODS Animals
INTRODUCTION Ammonia is generated as a normal product of many metabolic reactions in the body. Brain ammonia is derived principally from endogenous biochemical pathways and from diffusion from blood [6]. There appears to be a close relationship between nervous activity and ammonia formation. Brain ammonia content has been reported to decrease during sleep, hibernation, and anesthesia. It is increased by electrical stimulation of the brain and in convulsive seizures [28]. Although the functional activity of the nervous system is associated with its formation and utilization, in nonphysiological concentrations ammonia seems to disrupt neuronal function, because an elevation of brain ammonia concentration has been accompanied by derangement of cerebral function [3,24]. An elevation of brain ammonia following systemic administration of ammonium salts produced inhibition and excitation of motor function in humans [4,9], primates [5], and in rats [14,25]. Thus, controversy still exists in the action of ammonia on motor system. The characteristics of ammonia on motor function may be defined more appropriately if the time courses of motor impairment resulting from ammonia loading 1
Treatment Solutions of ammonium chloride and ammonium acetate were made in normal saline so as to inject intraperitoneally 100, 200, 400, and 800 mg/kg in a volume of 0.2 ml/100 g body weight. The pH of the solutions were not adjusted to 7.4, because both were acidic and because the aim of the study was to test the effects at their natural pH. Control animals received the same volume of the vehicle. Test animals were observed visually for convulsive movements. No convulsions occurred in rats treated with 100 and 200 mg/kg of ammonium salts. Spontaneous motor activity and motor coordination were measured in these animals. Spontaneous Motor Activity An activity chamber [22] was used to measure motor activity. The animal was placed in the chamber soon after injection and
To whom requests for reprints should be addressed.
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its activity was measured for a duration of 30 min. Activity countings recorded during 0–10, 10–20, and 20–30 min were correlated with the biochemical data obtained 5, 15, and 25 min after treatment, respectively. Motor Coordination A conventional rota-rod apparatus [8] was used to test motor coordination. The rational of this test is that the animals whose motor function is impaired drop off from a moving rod (3 cm diameter, moving in its axis 10 rpm). Animals were placed on a rotating rod 5, 15, and 25 min after injecting 100 and 200 mg/ kg of ammonium salts and the time of their fall during an allotted time of 60 s was recorded. Endurance time of each animal was measured as the time between it was placed on the moving rod and the moment it fell down. Ammonia and Glucose Determination A modified microdiffusion method [13] was used to determine ammonia concentrations in the blood and brain. Animals were sacrificed by decapitation 5, 15, and 25 min after injecting 100 and 200 mg/kg of ammonium salts. The higher dose (400 and 800 mg/kg) groups were sacrificed 5–10 s after clonic convulsions appeared, the time of which ranged from 4–6 and 12– 14 min in ammonium chloride and ammonium acetate-treated animals, respectively. Soon after decapitation, blood oozing from the neck wound was collected in a heparinized syringe. Whole brain was dissected out and processed immediately for ammonia determination. Blood and brain glucose levels were measured as described previously [26] in similarly treated animals at the appropriate time of ammonia determination. Statistical Analysis Data were analyzed using ANOVA and were further analyzed using Tukey’s multiple comparison test. RESULTS Animals that received higher doses ( 400 and 800 mg / kg ) of ammonium salts entered into convulsions. Convulsive movements appeared 4 – 6 and 12 – 14 min after injecting ammonium chloride and ammonium acetate, respectively. Induction of convulsion was accompanied by a marked increase in the concentrations of ammonia and glucose in the blood and in brain ( Fig. 1 ) . Spontaneous Motor Activity It was measured 3 times after treatment, each one for a duration of 10 min. Ammonium salts, at both 100 and 200 mg/kg dose levels, did not alter motor activity during 0–10 min of treatment. However, after 10–20 and 20—30 min the activity of these animals was decreased in a dose-dependent manner. The effect was more powerful 10–20 min after treatment (Fig. 1). Motor Coordination Rota-rod endurance time was shortened not 5 min but 15 min after treatment, in a dose-dependent manner. The potency decreased significantly but did not disappear 25 min after treatment (Fig. 1). The data presented in Fig. 1 showed a correlation between the time courses of motor impairment and an increase in brain and not blood ammonia concentrations.
Ammonia The data presented here show that blood and not brain ammonia concentrations were raised 5 min after injection of 100 and 200 mg/kg of ammonium salts. A much more increase that occurred in the blood 15 min after treatment was accompanied by a significant increase in the brain. Blood and not brain ammonia was restored to control level 25 min after treatment. At this time brain ammonia content was significantly lesser than that measured 15 min after treatment. Blood and brain ammonia levels were elevated markedly at the time of induction of convulsions in animals treated with 400 and 800 mg/kg of ammonium salts (Fig. 1). Glucose Ammonium salts increased brain and blood glucose concentrations in a dose-dependent manner 5 min after treatment. A linear increase occurred in the brain, whereas in the blood it was restored to control level 25 min after treatment (Fig. 1). DISCUSSION Penetration of blood-borne ammonia into the brain by diffusion [6] accounted for an elevation of ammonia concentrations in the brain following ammonium salts treatment. However, the time courses of ammonia rise in brain and blood did not seem to correlate, because in the present study a rise was found in the blood and not in the brain 5 min after treatment. A significant increase was found in both blood and brain 15 min after treatment. These findings suggests that a significant quantity of ammonia diffuses into the brain only after the blood level is markedly increased. The same has been reported by previous investigators also [6]. A ready detoxication of diffused ammonia may also account for the failure of brain to have a significant increase 5 min after ammonium salts treatment. In support of this suggestion, diffused ammonia has been shown to be disposed off immediately to glutamine by the enzyme glutamine synthetase [15], which seems to be activated by an increase in ammonia concentration in the brain [27]. A restorage of blood and not brain ammonia to control level, 25 min after treatment, indicates that the ability of brain to detoxicate ammonia has been weakened at this time. Detoxication of ammonia to glutamine is likely to be arrested if, as shown previously [6], there is a temporary block in glutamine synthetic activity as a result of saturation of glutamine synthetase. In this condition, further diffusion of ammonia from blood may result in a disproportionally high levels of ammonia in the brain. This suggestion accounted for a prolonged increase in the brain. A correlation was found in the present study between impairment of motor function and a rise in brain ammonia level. Exercise-induced fatigue, which is manifested by motor incoordination, ataxia, and stupor, has been found to be accompanied by a rise in ammonia level in the blood [1,21]. These investigators have suggested that there is excess formation of ammonia peripherally during exercise and that diffusion of ammonia into the brain results in fatigue. These findings support a notion that ammonia that diffuses into the brain is responsible for defective motor function. In the present study, a correlation was also found between ammonium salts-induced motor impairment and an increase in brain glucose concentration. A transfer of glucose from blood did not seem to account for a rise in the brain, because brain levels increased linearly in a time-dependent manner, whereas blood glucose was restored to control level 25 min after treatment. If a rise in brain glucose is indicative of a decreased glucose utilization by the brain, then an inhibition of energy pro-
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BRAIN AMMONIA AND MOTOR FUNCTION
FIG. 1. Motor effects and brain and blood ammonia and glucose concentrations in ammonium chloride and ammonium acetate-treated rats. Each point represents mean { SEM of eight observations. *p õ 0.05, **p õ 0.01 compard to control. / p õ 0.05 compared to that recorded at 15 min (ANOVA and Tukey’s multiple comparison test).
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duction may account for ammonium salts-induced inhibition of motor function. Supportingly, ammonium acetate and portacaval shunting-induced stupor has been accompanied by decrease in glucose utilization [7,11,17,19] and a lowering of high-energy phosphates in the brain [20]. Interestingly, in the present study, a further increase in brain ammonia and glucose concentrations following administration of a much higher doses of ammonium salts resulted not in a marked suppression of motor function but in clonic convulsive movements. This finding apparently indicates that ammonia at a very high concentration produces excitatory responces, even if glucose utilization is inhibited. Excitatory response seems to occur in the brain with very high concentrations of ammonia, because investigators who examined the actions of a series of concentrations of ammonia in the central nervous system of the cat demonstrated that plasma ammonia levels upto 100 mol/l had no detectable effect on postsynaptic inhibition, whereas values in excess of 2000 mol/l were associated with complete loss of inhibitory hyperpolarization [12]. Electrophysiological studies have also shown evidently that ammonia produces postsynaptic disinhibition by inactivating chloride extrusion in the motor neurons, bringing the transmembrane potential closer to the threshold for firing [16,18,23]. In conclution, ammonia that diffuses into the brain in hyperammonemia condition is toxic to motor neurons. A moderate elevation of brain ammonia seems to result in an inhibition of motor function. An inhibition of glucose utilization accounted for this. Excitation of motor system seems to occur after there is a marked elevation of ammonia. This may be responsible for the convulsant action of ammonia. ACKNOWLEDGEMENTS
The authors wish to thank Indian Council of Medical Research, India, for a grant supporting this research.
REFERENCES 1. Banister, F. W.; Cameron, R. L. Exercise-induced hyperammonemia: Peripheral and central effects. Int. J. Sports Med. 2:S129–S142; 1990. 2. Blei, A. T.; Olafsson, S.; Therrien, G,; Butterworth, R. F. Ammonia induced brain edema and intracranial hypertension in rats after portacaval anastomosis. Hepatology 19:1437–1444; 1994. 3. Butterworth, R. F.; Giguere, J. F.; Michaud, J.; Lavoie, J.; Layrargues, P. G. Ammonia: Key factor in the pathogenesis of hepatic encephalopathy. Neurochem. Pathol. 6:1–12; 1987. 4. Cohn, R.; Castell, D. O. The effects of acute hyperammonemia on the electroencephalogram. J. Lab. Clin. Med. 68:195–205; 1966. 5. Cole, M.; Rutherford, R. B.; Owen Smith, F. Experimental ammonia encephalopathy in the primate. Arch. Neurol. Chicago 26:130–136; 1972. 6. Cooper, A. J. L.; Mora, S. N.; Cruz, N. F.; Gelbard, A. S. Cerebral ammonia metabolism in hyperammonemia rats. J. Neurochem. 44:1716–1723; 1985. 7. Cruz, N. F.; Dienel, G. A. Brain glucose levels in portacaval-shunted rats with chronic, moderate hyperammonemia: Implications for determination of local cerebral glucose utilization. J Cereb. Blood Flow Metab. 14:113–124; 1994.
8. Dunham, N. W.; Miya, T. S. A note on a simple apparatus for detecting neurological deficit in rats and mice. J. Am. Pharmaceut. Assoc. Sci. Ed. XIVI:208–209; 1957. 9. Eichler, M. Psychological changes associated with induced hyperammonemia. Science 144:886–888; 1964. 10. Gebhardt, R.; Reichen, J. Changes in distribution and activity of glutamine synthetase in carbon tetrachloride-induced cirrhosis in the rat: Potential role in hyperammonemia. Hepatology 20:684–691; 1994. 11. Hawkins, R. A.; Jessy, J.; Mans, A. M.; De Joseph, M. R. Effects of reducing brain glutamine synthesis on metabolic symptoms of hepatic encephalopthy. J. Neurochem. 60:1000–1006; 1993. 12. Iles, J. F.; Jack, J. J. B. Ammonia: Assessment of its action on postsynaptic inhibition as a cause of convulsions. Brain 103:555–578; 1980. 13. Jayakumar, A. R.: Sujatha, R.; Paul, V. Colorimetric determination of blood and brain ammonia. Ind. J. Biochem. Biophys. 33:231– 233; 1996. 14. Jessy, J.; Murthy, Ch. R. K. Elevation of transamination of branched chain amino acids in brain in acute ammonia toxicity. Neurochem. Int. 7:1027–1031; 1985. 15. Kelleher, J. A.; Gregory, G. A.; Chan, P. H. Effect of fructose-1, 6biphosphate on glutamate uptake and glutamine synthetase activity in hypoxic astrocyte cultures. Neurochem. Res. 19:209–215; 1994. 16. Llinas, R.; Baker, R.; Precht, W. Blockade of inhibition by ammonium acetate: Action on chloride pump in cat trochlear motoneurons. J. Neurophysiol. 37:522–532; 1974. 17. Lockwood, A. H.; Ginsberg, M. D.; Butler, C. M.; Gutierrez, M. T. Selective effects of ammonia on regional brain glucose metabolism (abstr). Ann. Neurol. 12:114; 1982. 18. Lux, H. D.; Loracher, C.; Neher, E. The action of ammonium in postsynaptic inhibition of cat spinal motoneurons. Exp. Brain Res. 11:431–447; 1970. 19. Mans, A. M.; DeJoseph, M. R.; Hawkins, R. A. Metabolic abnormalities and grade of encephalopthy in acute hepatic failure. J. Neurochem. 63:1829–1838; 1994. 20. McCandless, D. W.; Schenker, S. Effect of acute ammonia intoxication on energy stores in the cerebral reticular activating system. Exp. Brain Res. 44:325–330; 1981. 21. Okamura, K.; Matsubara, F.; Yoshioka, Y.; Kikuchi, N,; Kikuchi, Y.; Kohri, H. Exercise-induced changes in branced chain amino acid/aromatic amino acid ratio in the rat brain and plasma. Jpn. J. Pharmacol. 45:243–248; 1987. 22. Paul, V.; Balasubramaniam, E.; Kazi, M. The nuerobehavioral toxicity of endosulfan in rats: A serotonergic involvement in learning impairment. Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Sect. 270:1–7; 1994. 23. Raabe, W.; Gumnit, R. J. Disinhibition in cat motor cortex by ammonia, J. Neurophysiol. 38:347–355; 1975. 24. Rao, V. L. R.; Murthy, Ch. R. K.; Butterworth, R. F. Glutamatergic synaptic dysfunction in hyperammonemic syndromes. Metab. Brain Dis. 7:1–20; 1992. 25. Rukmini Devi, R. P.; Murthy, Ch. R. K. Ammonia-induced alterations in the activities of synaptosomal cholinesterases of rat brain under in vitro and in vivo conditions. Neurosci. Lett. 159:131–134; 1993. 26. Sasaki, T.; Matsuy, S.; Sanae, A. Effect of acetic acid concentration on the colour reaction in the o-toluidine-boric acid method for blood glucose determinations. Rinsho Kagaku 1:346–353; 1972. 27. Subbalakshmi, G. Y. C. V.; Murthy, Ch. R. K. Differential response of enzymes of glutamate metabolism in neuronal perikarya and synaptosomes in acute hyperammonemia in rat. Neurosci. Lett. 59:121– 126; 1985. 28. Tsukada, Y. Ammonia metabolism. In: Lajtha, A., ed. Handbook of neurochemistry, vol. 5A. New York: Plenum Press; 1971:215–233.
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PII S0361-9230(96)00432-7
Effect of Ammonia on Motor Function in Adult Rats A. R. JAYAKUMAR, R. SUJATHA AND VANAJA PAUL 1 Department of Pharmacology & Environmental Toxicology, Dr. A. L. M. Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani, Madras 600 113, India [Received 21 August 1996; Accepted 21 November 1996] ABSTRACT: Changes in motor function were assessed in male rats after injecting graded doses (100, 200, 400, and 800 mg/kg, IP) of ammonium chloride and ammonium acetate. The effects were correlated with the concentrations of ammonia and glucose in the brain and blood. Spontaneous motor activity and motor coordination were inhibited after injecting 100 and 200 mg/kg, whereas with 400 and 800 mg/kg the animals exhibited convulsive movements. A dose-dependent increase was found in the concentrations of ammonia and glucose in both blood and brain. These were restored, 25 min after treatment, to control levels in the blood and not in the brain. A correlation was found between the time courses of inhibitory motor events and a rise in brain ammonia levels. Convulsant action of ammonium salts was accompanied by a marked elevation of ammonia and glucose concentration in the brain. The findings suggest that detoxication of diffused ammonia is a rate-limiting process in the brain and that ammonia, at toxic concentrations, decreases glucose utilization in the brain, resulting in an inhibition of motor function. A very high concentration of ammonia in the brain, although inhibiting glucose utilization, produces clonic convul sions probably by activating directly the motor neurons. Q 1997 Elsevier Science Inc.
and brain ammonia concentrations are correlated. In previous studies brain ammonia level was raised by acute dosing of ammonium salts [25]. Hyperammonemia resulting from portacaval shunting [2,7] or carbon tetrachloride-induced liver damage [10] was also used to raise brain ammonia. The latter techniques may produce several metabolic derangements also, as a result of liver impairment. Therefore, a defect in the motor function of these animals cannot be attributed solely to an increased level of ammonia in the brain. In view of these facts, in the present study changes in motor function were determined in groups of rats after injecting graded doses of ammonium chloride. The results were correlated with blood and brain ammonia data. The changes that occurred in brain and blood glucose concentrations were also measured in these animals to test whether ammonia loading has resulted in impairment of glucose metabolism also. To confirm the findings, tests were carried out after injecting ammonium acetate at the same dose level.
KEY WORDS: Brain ammonia, Brain glucose, Spontaneous motor activity, Motor coordination, Convulsions.
Wistar strain 5–6-month-old (150–160 g) male rats were used. Test (n Å 8) and control ( n Å 8) animals were selected randomly and were caged in groups of four at room temprature (22–307C). Animals were supplied ad lib with a balanced rat feed (Gold mohur pellets, Bangalore, India) and drinking water. Motor function tests and sacrifice for biochemical determinations were performed between 1000 and 1300 h.
MATERIALS AND METHODS Animals
INTRODUCTION Ammonia is generated as a normal product of many metabolic reactions in the body. Brain ammonia is derived principally from endogenous biochemical pathways and from diffusion from blood [6]. There appears to be a close relationship between nervous activity and ammonia formation. Brain ammonia content has been reported to decrease during sleep, hibernation, and anesthesia. It is increased by electrical stimulation of the brain and in convulsive seizures [28]. Although the functional activity of the nervous system is associated with its formation and utilization, in nonphysiological concentrations ammonia seems to disrupt neuronal function, because an elevation of brain ammonia concentration has been accompanied by derangement of cerebral function [3,24]. An elevation of brain ammonia following systemic administration of ammonium salts produced inhibition and excitation of motor function in humans [4,9], primates [5], and in rats [14,25]. Thus, controversy still exists in the action of ammonia on motor system. The characteristics of ammonia on motor function may be defined more appropriately if the time courses of motor impairment resulting from ammonia loading 1
Treatment Solutions of ammonium chloride and ammonium acetate were made in normal saline so as to inject intraperitoneally 100, 200, 400, and 800 mg/kg in a volume of 0.2 ml/100 g body weight. The pH of the solutions were not adjusted to 7.4, because both were acidic and because the aim of the study was to test the effects at their natural pH. Control animals received the same volume of the vehicle. Test animals were observed visually for convulsive movements. No convulsions occurred in rats treated with 100 and 200 mg/kg of ammonium salts. Spontaneous motor activity and motor coordination were measured in these animals. Spontaneous Motor Activity An activity chamber [22] was used to measure motor activity. The animal was placed in the chamber soon after injection and
To whom requests for reprints should be addressed.
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its activity was measured for a duration of 30 min. Activity countings recorded during 0–10, 10–20, and 20–30 min were correlated with the biochemical data obtained 5, 15, and 25 min after treatment, respectively. Motor Coordination A conventional rota-rod apparatus [8] was used to test motor coordination. The rational of this test is that the animals whose motor function is impaired drop off from a moving rod (3 cm diameter, moving in its axis 10 rpm). Animals were placed on a rotating rod 5, 15, and 25 min after injecting 100 and 200 mg/ kg of ammonium salts and the time of their fall during an allotted time of 60 s was recorded. Endurance time of each animal was measured as the time between it was placed on the moving rod and the moment it fell down. Ammonia and Glucose Determination A modified microdiffusion method [13] was used to determine ammonia concentrations in the blood and brain. Animals were sacrificed by decapitation 5, 15, and 25 min after injecting 100 and 200 mg/kg of ammonium salts. The higher dose (400 and 800 mg/kg) groups were sacrificed 5–10 s after clonic convulsions appeared, the time of which ranged from 4–6 and 12– 14 min in ammonium chloride and ammonium acetate-treated animals, respectively. Soon after decapitation, blood oozing from the neck wound was collected in a heparinized syringe. Whole brain was dissected out and processed immediately for ammonia determination. Blood and brain glucose levels were measured as described previously [26] in similarly treated animals at the appropriate time of ammonia determination. Statistical Analysis Data were analyzed using ANOVA and were further analyzed using Tukey’s multiple comparison test. RESULTS Animals that received higher doses ( 400 and 800 mg / kg ) of ammonium salts entered into convulsions. Convulsive movements appeared 4 – 6 and 12 – 14 min after injecting ammonium chloride and ammonium acetate, respectively. Induction of convulsion was accompanied by a marked increase in the concentrations of ammonia and glucose in the blood and in brain ( Fig. 1 ) . Spontaneous Motor Activity It was measured 3 times after treatment, each one for a duration of 10 min. Ammonium salts, at both 100 and 200 mg/kg dose levels, did not alter motor activity during 0–10 min of treatment. However, after 10–20 and 20—30 min the activity of these animals was decreased in a dose-dependent manner. The effect was more powerful 10–20 min after treatment (Fig. 1). Motor Coordination Rota-rod endurance time was shortened not 5 min but 15 min after treatment, in a dose-dependent manner. The potency decreased significantly but did not disappear 25 min after treatment (Fig. 1). The data presented in Fig. 1 showed a correlation between the time courses of motor impairment and an increase in brain and not blood ammonia concentrations.
Ammonia The data presented here show that blood and not brain ammonia concentrations were raised 5 min after injection of 100 and 200 mg/kg of ammonium salts. A much more increase that occurred in the blood 15 min after treatment was accompanied by a significant increase in the brain. Blood and not brain ammonia was restored to control level 25 min after treatment. At this time brain ammonia content was significantly lesser than that measured 15 min after treatment. Blood and brain ammonia levels were elevated markedly at the time of induction of convulsions in animals treated with 400 and 800 mg/kg of ammonium salts (Fig. 1). Glucose Ammonium salts increased brain and blood glucose concentrations in a dose-dependent manner 5 min after treatment. A linear increase occurred in the brain, whereas in the blood it was restored to control level 25 min after treatment (Fig. 1). DISCUSSION Penetration of blood-borne ammonia into the brain by diffusion [6] accounted for an elevation of ammonia concentrations in the brain following ammonium salts treatment. However, the time courses of ammonia rise in brain and blood did not seem to correlate, because in the present study a rise was found in the blood and not in the brain 5 min after treatment. A significant increase was found in both blood and brain 15 min after treatment. These findings suggests that a significant quantity of ammonia diffuses into the brain only after the blood level is markedly increased. The same has been reported by previous investigators also [6]. A ready detoxication of diffused ammonia may also account for the failure of brain to have a significant increase 5 min after ammonium salts treatment. In support of this suggestion, diffused ammonia has been shown to be disposed off immediately to glutamine by the enzyme glutamine synthetase [15], which seems to be activated by an increase in ammonia concentration in the brain [27]. A restorage of blood and not brain ammonia to control level, 25 min after treatment, indicates that the ability of brain to detoxicate ammonia has been weakened at this time. Detoxication of ammonia to glutamine is likely to be arrested if, as shown previously [6], there is a temporary block in glutamine synthetic activity as a result of saturation of glutamine synthetase. In this condition, further diffusion of ammonia from blood may result in a disproportionally high levels of ammonia in the brain. This suggestion accounted for a prolonged increase in the brain. A correlation was found in the present study between impairment of motor function and a rise in brain ammonia level. Exercise-induced fatigue, which is manifested by motor incoordination, ataxia, and stupor, has been found to be accompanied by a rise in ammonia level in the blood [1,21]. These investigators have suggested that there is excess formation of ammonia peripherally during exercise and that diffusion of ammonia into the brain results in fatigue. These findings support a notion that ammonia that diffuses into the brain is responsible for defective motor function. In the present study, a correlation was also found between ammonium salts-induced motor impairment and an increase in brain glucose concentration. A transfer of glucose from blood did not seem to account for a rise in the brain, because brain levels increased linearly in a time-dependent manner, whereas blood glucose was restored to control level 25 min after treatment. If a rise in brain glucose is indicative of a decreased glucose utilization by the brain, then an inhibition of energy pro-
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BRAIN AMMONIA AND MOTOR FUNCTION
FIG. 1. Motor effects and brain and blood ammonia and glucose concentrations in ammonium chloride and ammonium acetate-treated rats. Each point represents mean { SEM of eight observations. *p õ 0.05, **p õ 0.01 compard to control. / p õ 0.05 compared to that recorded at 15 min (ANOVA and Tukey’s multiple comparison test).
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duction may account for ammonium salts-induced inhibition of motor function. Supportingly, ammonium acetate and portacaval shunting-induced stupor has been accompanied by decrease in glucose utilization [7,11,17,19] and a lowering of high-energy phosphates in the brain [20]. Interestingly, in the present study, a further increase in brain ammonia and glucose concentrations following administration of a much higher doses of ammonium salts resulted not in a marked suppression of motor function but in clonic convulsive movements. This finding apparently indicates that ammonia at a very high concentration produces excitatory responces, even if glucose utilization is inhibited. Excitatory response seems to occur in the brain with very high concentrations of ammonia, because investigators who examined the actions of a series of concentrations of ammonia in the central nervous system of the cat demonstrated that plasma ammonia levels upto 100 mol/l had no detectable effect on postsynaptic inhibition, whereas values in excess of 2000 mol/l were associated with complete loss of inhibitory hyperpolarization [12]. Electrophysiological studies have also shown evidently that ammonia produces postsynaptic disinhibition by inactivating chloride extrusion in the motor neurons, bringing the transmembrane potential closer to the threshold for firing [16,18,23]. In conclution, ammonia that diffuses into the brain in hyperammonemia condition is toxic to motor neurons. A moderate elevation of brain ammonia seems to result in an inhibition of motor function. An inhibition of glucose utilization accounted for this. Excitation of motor system seems to occur after there is a marked elevation of ammonia. This may be responsible for the convulsant action of ammonia. ACKNOWLEDGEMENTS
The authors wish to thank Indian Council of Medical Research, India, for a grant supporting this research.
REFERENCES 1. Banister, F. W.; Cameron, R. L. Exercise-induced hyperammonemia: Peripheral and central effects. Int. J. Sports Med. 2:S129–S142; 1990. 2. Blei, A. T.; Olafsson, S.; Therrien, G,; Butterworth, R. F. Ammonia induced brain edema and intracranial hypertension in rats after portacaval anastomosis. Hepatology 19:1437–1444; 1994. 3. Butterworth, R. F.; Giguere, J. F.; Michaud, J.; Lavoie, J.; Layrargues, P. G. Ammonia: Key factor in the pathogenesis of hepatic encephalopathy. Neurochem. Pathol. 6:1–12; 1987. 4. Cohn, R.; Castell, D. O. The effects of acute hyperammonemia on the electroencephalogram. J. Lab. Clin. Med. 68:195–205; 1966. 5. Cole, M.; Rutherford, R. B.; Owen Smith, F. Experimental ammonia encephalopathy in the primate. Arch. Neurol. Chicago 26:130–136; 1972. 6. Cooper, A. J. L.; Mora, S. N.; Cruz, N. F.; Gelbard, A. S. Cerebral ammonia metabolism in hyperammonemia rats. J. Neurochem. 44:1716–1723; 1985. 7. Cruz, N. F.; Dienel, G. A. Brain glucose levels in portacaval-shunted rats with chronic, moderate hyperammonemia: Implications for determination of local cerebral glucose utilization. J Cereb. Blood Flow Metab. 14:113–124; 1994.
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