- Email: [email protected]
Neuropharmacological consequences of choline administration
234
Brain Research, 184 (1980) 234-238 © Elsevier/North-Holland Biomedical Press
Neuropharmacological consequences of choline administration
L Y N N W E C K E R and D E N N I S E. S C H M I D T
Department of Pharmacology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, La. 70112 and the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn. 37232 (U.S.A.) (Accepted October 25th, 1979)
Key words: brain acetylcholine --- choline administration - - striatum - - hippocampus
Recent studies have shown that acute choline administration prevents atropineinduced acetylcholine (ACh) depletion in rat cerebral cortex and hippocampus, but the mechanisms involved have not been elucidated la. The effects of atropine on brain levels of ACh are presumably due to increased neurotransmitter release relative to synthesis1,2,4,10 and it has been suggested that the prophylactic actions of exogenous choline may involve alterations in ACh synthesis 1~. In light of evidence indicating that the utilization of exogenous choline and the regulatory mechanisms controlling the synthesis of ACh in the striatum differ from other brain regions 9,x2, we investigated and compared the effects of choline pretreatment on dose-dependent atropine-induced ACh depletion in rat striatum and hippocampus. Male Sprague-Dawley rats (180-230 g) were maintained on a 12 h light/dark cycle with standard rat chow (Wayne Lab Blox M R H 22/5, Allied Mills, Chicago, I11.) and water available ad libitum. All drugs were dissolved in saline and administered intraperitoneally (0.1 ml/100 g body weight) between 09.00 and 11.00. Animals were killed by microwave irradiation (2.4 sec) focused on the head in a modified Litton model 70-50 oven (2750 MHz, 1250 W). Under these conditions, the brain temperature reached 90 °C and all enzymes were inactivated 6. Following sacrifice, brains were removed, chilled in ice-cold pentane and dissected into discrete brain areas using a brain slicing apparatus s. Slices from the caudate-putamen and hippocampus were isolated, homogenized (Teflon-glass) in acetonitrile containing propionylcholine iodide as an internal standard and prepared for the simultaneous determination of ACh and choline by pyrolysis-gas chromatography7,11. Choline time course effects were determined in brain regions from rats injected with 60-120 mg/kg (free base) choline iodide and sacrificed 15-90 min following injection. Results were calculated as nmol ACh or choline/g brain tissue and expressed as group mean values ~- S.E.M. The effects of choline on atropine-induced ACh depletion were studied in animals injected with saline or choline iodide (60 mg/kg, free
235 base) 60 min p r i o r to the a d m i n i s t r a t i o n o f a t r o p i n e sulfate (5-30 mg/kg) a n d sacrificed 30 m i n following the a t r o p i n e injection. These results were calculated as n m o l A C h / g b r a i n tissue a n d expressed as g r o u p m e a n values 4- S.E.M. o f the per cent A C h d e p l e t i o n following a t r o p i n e a d m i n i s t r a t i o n . D a t a were a n a l y z e d on a D E C - 1 0 b y analysis o f variance ( A N O V A ) a n d statistical significance was d e t e r m i n e d b y a 2-tailed S t u d e n t ' s t-test. D o s e - r e s p o n s e p a r a m e t e r s were calculated b y l o g - d o s e p r o b i t analysis a. A c u t e a d m i n i s t r a t i o n o f choline i o d i d e (60-120 mg/kg, free base) to rats caused a significant ( P < 0.05) increase in the c o n c e n t r a t i o n o f free choline in b r a i n 15 min after (A) CAUDATE"PUTAMEN L
n moles 4~
g tissue J
6
l's
3'o
,'s
go
7~
9'o
~
7~
9~
time (minutes) (8) HIPPOCAMPUS 40
/
$
S--lld~lUe ~ 2(
?s
3'o
,'5
time(minutes)
Fig. 1. Effects of choline administration on ACh ( • 0 ) and choline ( 63 - - - Q) levels in: (A) caudate°putamen and (B) hippocampus. Rats were injected with choline iodide (60 mg/kg, free base) and sacrificed 15-90 min following injection by head-focused microwave irradiation. Each point is the mean of determinations from 6 animals and bars are standard errors. Asterisks denote significant differences as compared to controls (P < 0.05).
236 injection. Maximal effects were noted following the administration of 60 mg/kg and choline levels increased to 132 ~ and 151 ~ of controls in the caudate-putamen and hippocampus, respectively (Fig. 1). Levels returned to control by 30 min and remained constant thereafter. As previously reported 13, no change in the steady-state concentration of ACh was detected in either brain region at any time following acute choline administration. The effects of choline pretreatment on atropine-induced ACh depletion in brain
(A) CAUDATE-PUTAMEN
4(
%
I
Depletion3( III 2O !
10
/
0
Atropine Sulfate Ong/kg)
(B) HIPPOCAMPUS 5C
4C
%
Depletion 3c 2C
iI
Atreplne Sulfate (mg/kg) Fig. 2. Effects of choline pretreatment on atropine-induced depletion of ACh in: (A) caudate-putamen and (B) hippocampus. Rats were injected with saline ( • • ) o r choline iodide ( (3- - - (3,60 mg/kg, free base) 60 min prior to the administration of atropine sulfate (5-30 mg/kg) and sacrificed 30 min following the second injection by head-focused microwave irradiation. Each point is the mean of determinations from 7 animals and bars are standard errors. Asterisks denote significant differences between saline and choline-injected rats (P < 0.05).
237 is shown in Fig 2. In the caudate-putamen, choline significantly (P < 0.05) prevented ACh depletion following the administration of 5-14 mg/kg atropine sulfate, but was ineffective at higher concentrations (Fig. 2A). The EDs0 of atropine sulfate was increased following choline pretreatment from control values of 9.1 to 12.7 mg/kg, but the per cent maximal depletion and slope of the log-dose probit curve were unaltered. The prophylactic effects of choline in preventing atropine-induced ACh depletion in the hippocampus were significant (P < 0.05) only at the lowest doses of atropine studied, i.e. 5-7 mg/kg (Fig. 2B). The ED~0 of atropine in the hippocampus, similar to the caudate-putamen, increased from 7.7 to 10.5 mg/kg following choline administration and no change was noted in the per cent maximal depletion. In contrast to the caudate-putamen, however, the slope of the atropine log-dose probit curve in the hippocampus was decreased from 2.41 to 1.45. These results, in confirmation of previous studiesl2,13, indicate that: (a) exogenous choline availability is a significant factor affecting the pharmacological responsiveness of central cholinergic neurons to the ACh depleting actions of atropine, and (b) the mechanism(s) involved in the choline-atropine interaction may differ in the striatum and hippocampus. Since atropine increases neuronal activity in cholinergic neurons, it is reasonable that this, in turn, increases the demand for choline necessary to maintain ACh synthesis. While the nature of the choline pool involved with the synthesis of ACh has not been characterized, it is possible that the supply of choline to this pool may not be sufficient to support synthesis under conditions of drug-induced increases in demand. Hence, reduced ACh levels result. If, however, exogenous choline could augment the supply of choline to this pool, then the steadystate concentration of ACh would be adequately maintained during periods of increased demand, viz. with moderate doses of atropine. However, as demand is increased further, i.e. with the administration of larger doses of atropine, the availability of choline may again be inadequate to maintain synthesis, and neurotransmitter depletion is evident. The exact mechanism for such a hypothetical augmentation of choline availability is open to speculation. Since this effect is evident for a considerable period of time following choline administration (60-90 min, unpublished observations) and after total free choline levels have returned to normal, it is likely that the additional choline supply may be present in a form other than free choline and may be located outside of the cholinergic neuron. Although the relationship between high affinity choline uptake and ACh synthesis has not been absolutely resolved, persuasive data indicate an obligatory role for sodit/m-dependent high affinity choline uptake in ACh synthesis5. Therefore, it is possible that during periods of increased demand, the augmented storage form of choline could be converted to free choline and transported by high affinity uptake for utilization in ACh synthesis. Alternatively, in addition to the proposed mechanism, one must also consider the possibility that choline administration dramatically increases the size of the free choline ACh precursor pool, but because this pool constitutes only a small percentage of total free choline in brain, the increase is undetected by conventional measurement. Experiments designed to investigate these possibilities are currently under way.
238
Results indicating that the striatum has a greater capacity to utilize exogenous choline relative to the hippocampus are in agreement with previous studies ~2. Log-dose probit analysis indicates that the mechanism(s) involved in the choline-atropine interaction in the striatum is competitive in nature, while the hippocampal interaction is not. These findings support the premise that the regulation of and mechanisms involved in ACh synthesis in the striatum may differ from those in other brain regions 9,12. In conclusion, although the specific mechanism and site of action of exogenous choline has yet to be elucidated, evidence supports the hypothesis that choline availability is a significant factor determining the responsiveness of central cholinergic neurons to pharmacological manipulation.
1 Dudar, J. D. and Szerb, J. C., The effect of topically applied atropine on resting and evoked cortical acetylcholine release, J. Physiok (Lond.), 203 (1969) 741-762. 2 Karlen, B., Lundgren, G., Miyata, T., Lundin, J. and Holmstedt, B., Effect of atropine on acetylcholine metabolism in the mouse brain. In D. J. Jenden (Ed.), Cholinergic Mechanisms and Psychopharmacology, Advances in Behavioural Biology, Vol. 24, Plenum Press, New York, 1978, pp. 643-655. 3 Litchfield, J. T., Jr. and Wilcoxon, F. A., A simplified method of evaluating dose-effect experiments, J. Pharmacol. exp. Therap., 96 (1949) 99-113. 4 Lundholm, B. and Sparf, B., The effect of atropine on the turnover of acetylcholine in the mouse brain, Europ. J. Pharmacol., 32 (1975) 287-292. 5 Kuhar, M. J. and Murrin, L. C., Sodium-dependent, high affinity choline uptake, J. Neurochem., 30 (1978) 15-21. 6 Schmidt, D. E., Regional levels of choline and acetylcholine in rat brain following head focused microwave sacrifice: effect of amphetamine and parachloroamphetamine, Neuropharmacology, 15 (1976) 77-84. 7 Schmidt, D. E. and Speth, R. C., Simultaneous analysis of choline and acetylcholine levels in rat brain by pyrolysis gas chromatography, Analyt. Biochem., 67 (1975) 353-357. 8 Segal, D. S. and Kuczenski, R., Tyrosine hydroxylase activity: regional and subcellular distribution in brain, Brain Research, 68 (1974) 261-266. 9 Sherman, K. A., Hanin, I. and Zigmond, M. J., The effect of neuroleptics on acetylcholine concentration and choline uptake in striatum; implications for regulation of acetylcholine metabolism, J. Pharmacol. exp. Therap., 206 (1978) 677-686. 10 Szerb, J. C., Malik, H. and Hunter, E. G., Relationship between acetylcholine content and release in the cat's cerebral cortex, Canad. J. physiol. Pharmacol., 48 (1970) 780-790. l l Szilagyi, P. I. A., Schmidt, D. E. and Green, J. P., Microanalytical determination of acetylcholine, other choline esters, and choline by pyrolysis gas chromatography, Analyt. Chem., 40 (1968) 2009-2013. 12 Wecker, L. and Dettbarn, W.-D., Relationship between choline availability and acetylcholine synthesis in discrete regions of rat brain, J. Neurochem., 32 (1979) 961-967. 13 Wecker, L., Dettbarn, W.-D. and Schmidt, D. E., Choline administration: modification of the central actions of atropine, Science, 199 (1978) 86 87.
Brain Research, 184 (1980) 234-238 © Elsevier/North-Holland Biomedical Press
Neuropharmacological consequences of choline administration
L Y N N W E C K E R and D E N N I S E. S C H M I D T
Department of Pharmacology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, La. 70112 and the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn. 37232 (U.S.A.) (Accepted October 25th, 1979)
Key words: brain acetylcholine --- choline administration - - striatum - - hippocampus
Recent studies have shown that acute choline administration prevents atropineinduced acetylcholine (ACh) depletion in rat cerebral cortex and hippocampus, but the mechanisms involved have not been elucidated la. The effects of atropine on brain levels of ACh are presumably due to increased neurotransmitter release relative to synthesis1,2,4,10 and it has been suggested that the prophylactic actions of exogenous choline may involve alterations in ACh synthesis 1~. In light of evidence indicating that the utilization of exogenous choline and the regulatory mechanisms controlling the synthesis of ACh in the striatum differ from other brain regions 9,x2, we investigated and compared the effects of choline pretreatment on dose-dependent atropine-induced ACh depletion in rat striatum and hippocampus. Male Sprague-Dawley rats (180-230 g) were maintained on a 12 h light/dark cycle with standard rat chow (Wayne Lab Blox M R H 22/5, Allied Mills, Chicago, I11.) and water available ad libitum. All drugs were dissolved in saline and administered intraperitoneally (0.1 ml/100 g body weight) between 09.00 and 11.00. Animals were killed by microwave irradiation (2.4 sec) focused on the head in a modified Litton model 70-50 oven (2750 MHz, 1250 W). Under these conditions, the brain temperature reached 90 °C and all enzymes were inactivated 6. Following sacrifice, brains were removed, chilled in ice-cold pentane and dissected into discrete brain areas using a brain slicing apparatus s. Slices from the caudate-putamen and hippocampus were isolated, homogenized (Teflon-glass) in acetonitrile containing propionylcholine iodide as an internal standard and prepared for the simultaneous determination of ACh and choline by pyrolysis-gas chromatography7,11. Choline time course effects were determined in brain regions from rats injected with 60-120 mg/kg (free base) choline iodide and sacrificed 15-90 min following injection. Results were calculated as nmol ACh or choline/g brain tissue and expressed as group mean values ~- S.E.M. The effects of choline on atropine-induced ACh depletion were studied in animals injected with saline or choline iodide (60 mg/kg, free
235 base) 60 min p r i o r to the a d m i n i s t r a t i o n o f a t r o p i n e sulfate (5-30 mg/kg) a n d sacrificed 30 m i n following the a t r o p i n e injection. These results were calculated as n m o l A C h / g b r a i n tissue a n d expressed as g r o u p m e a n values 4- S.E.M. o f the per cent A C h d e p l e t i o n following a t r o p i n e a d m i n i s t r a t i o n . D a t a were a n a l y z e d on a D E C - 1 0 b y analysis o f variance ( A N O V A ) a n d statistical significance was d e t e r m i n e d b y a 2-tailed S t u d e n t ' s t-test. D o s e - r e s p o n s e p a r a m e t e r s were calculated b y l o g - d o s e p r o b i t analysis a. A c u t e a d m i n i s t r a t i o n o f choline i o d i d e (60-120 mg/kg, free base) to rats caused a significant ( P < 0.05) increase in the c o n c e n t r a t i o n o f free choline in b r a i n 15 min after (A) CAUDATE"PUTAMEN L
n moles 4~
g tissue J
6
l's
3'o
,'s
go
7~
9'o
~
7~
9~
time (minutes) (8) HIPPOCAMPUS 40
/
$
S--lld~lUe ~ 2(
?s
3'o
,'5
time(minutes)
Fig. 1. Effects of choline administration on ACh ( • 0 ) and choline ( 63 - - - Q) levels in: (A) caudate°putamen and (B) hippocampus. Rats were injected with choline iodide (60 mg/kg, free base) and sacrificed 15-90 min following injection by head-focused microwave irradiation. Each point is the mean of determinations from 6 animals and bars are standard errors. Asterisks denote significant differences as compared to controls (P < 0.05).
236 injection. Maximal effects were noted following the administration of 60 mg/kg and choline levels increased to 132 ~ and 151 ~ of controls in the caudate-putamen and hippocampus, respectively (Fig. 1). Levels returned to control by 30 min and remained constant thereafter. As previously reported 13, no change in the steady-state concentration of ACh was detected in either brain region at any time following acute choline administration. The effects of choline pretreatment on atropine-induced ACh depletion in brain
(A) CAUDATE-PUTAMEN
4(
%
I
Depletion3( III 2O !
10
/
0
Atropine Sulfate Ong/kg)
(B) HIPPOCAMPUS 5C
4C
%
Depletion 3c 2C
iI
Atreplne Sulfate (mg/kg) Fig. 2. Effects of choline pretreatment on atropine-induced depletion of ACh in: (A) caudate-putamen and (B) hippocampus. Rats were injected with saline ( • • ) o r choline iodide ( (3- - - (3,60 mg/kg, free base) 60 min prior to the administration of atropine sulfate (5-30 mg/kg) and sacrificed 30 min following the second injection by head-focused microwave irradiation. Each point is the mean of determinations from 7 animals and bars are standard errors. Asterisks denote significant differences between saline and choline-injected rats (P < 0.05).
237 is shown in Fig 2. In the caudate-putamen, choline significantly (P < 0.05) prevented ACh depletion following the administration of 5-14 mg/kg atropine sulfate, but was ineffective at higher concentrations (Fig. 2A). The EDs0 of atropine sulfate was increased following choline pretreatment from control values of 9.1 to 12.7 mg/kg, but the per cent maximal depletion and slope of the log-dose probit curve were unaltered. The prophylactic effects of choline in preventing atropine-induced ACh depletion in the hippocampus were significant (P < 0.05) only at the lowest doses of atropine studied, i.e. 5-7 mg/kg (Fig. 2B). The ED~0 of atropine in the hippocampus, similar to the caudate-putamen, increased from 7.7 to 10.5 mg/kg following choline administration and no change was noted in the per cent maximal depletion. In contrast to the caudate-putamen, however, the slope of the atropine log-dose probit curve in the hippocampus was decreased from 2.41 to 1.45. These results, in confirmation of previous studiesl2,13, indicate that: (a) exogenous choline availability is a significant factor affecting the pharmacological responsiveness of central cholinergic neurons to the ACh depleting actions of atropine, and (b) the mechanism(s) involved in the choline-atropine interaction may differ in the striatum and hippocampus. Since atropine increases neuronal activity in cholinergic neurons, it is reasonable that this, in turn, increases the demand for choline necessary to maintain ACh synthesis. While the nature of the choline pool involved with the synthesis of ACh has not been characterized, it is possible that the supply of choline to this pool may not be sufficient to support synthesis under conditions of drug-induced increases in demand. Hence, reduced ACh levels result. If, however, exogenous choline could augment the supply of choline to this pool, then the steadystate concentration of ACh would be adequately maintained during periods of increased demand, viz. with moderate doses of atropine. However, as demand is increased further, i.e. with the administration of larger doses of atropine, the availability of choline may again be inadequate to maintain synthesis, and neurotransmitter depletion is evident. The exact mechanism for such a hypothetical augmentation of choline availability is open to speculation. Since this effect is evident for a considerable period of time following choline administration (60-90 min, unpublished observations) and after total free choline levels have returned to normal, it is likely that the additional choline supply may be present in a form other than free choline and may be located outside of the cholinergic neuron. Although the relationship between high affinity choline uptake and ACh synthesis has not been absolutely resolved, persuasive data indicate an obligatory role for sodit/m-dependent high affinity choline uptake in ACh synthesis5. Therefore, it is possible that during periods of increased demand, the augmented storage form of choline could be converted to free choline and transported by high affinity uptake for utilization in ACh synthesis. Alternatively, in addition to the proposed mechanism, one must also consider the possibility that choline administration dramatically increases the size of the free choline ACh precursor pool, but because this pool constitutes only a small percentage of total free choline in brain, the increase is undetected by conventional measurement. Experiments designed to investigate these possibilities are currently under way.
238
Results indicating that the striatum has a greater capacity to utilize exogenous choline relative to the hippocampus are in agreement with previous studies ~2. Log-dose probit analysis indicates that the mechanism(s) involved in the choline-atropine interaction in the striatum is competitive in nature, while the hippocampal interaction is not. These findings support the premise that the regulation of and mechanisms involved in ACh synthesis in the striatum may differ from those in other brain regions 9,12. In conclusion, although the specific mechanism and site of action of exogenous choline has yet to be elucidated, evidence supports the hypothesis that choline availability is a significant factor determining the responsiveness of central cholinergic neurons to pharmacological manipulation.
1 Dudar, J. D. and Szerb, J. C., The effect of topically applied atropine on resting and evoked cortical acetylcholine release, J. Physiok (Lond.), 203 (1969) 741-762. 2 Karlen, B., Lundgren, G., Miyata, T., Lundin, J. and Holmstedt, B., Effect of atropine on acetylcholine metabolism in the mouse brain. In D. J. Jenden (Ed.), Cholinergic Mechanisms and Psychopharmacology, Advances in Behavioural Biology, Vol. 24, Plenum Press, New York, 1978, pp. 643-655. 3 Litchfield, J. T., Jr. and Wilcoxon, F. A., A simplified method of evaluating dose-effect experiments, J. Pharmacol. exp. Therap., 96 (1949) 99-113. 4 Lundholm, B. and Sparf, B., The effect of atropine on the turnover of acetylcholine in the mouse brain, Europ. J. Pharmacol., 32 (1975) 287-292. 5 Kuhar, M. J. and Murrin, L. C., Sodium-dependent, high affinity choline uptake, J. Neurochem., 30 (1978) 15-21. 6 Schmidt, D. E., Regional levels of choline and acetylcholine in rat brain following head focused microwave sacrifice: effect of amphetamine and parachloroamphetamine, Neuropharmacology, 15 (1976) 77-84. 7 Schmidt, D. E. and Speth, R. C., Simultaneous analysis of choline and acetylcholine levels in rat brain by pyrolysis gas chromatography, Analyt. Biochem., 67 (1975) 353-357. 8 Segal, D. S. and Kuczenski, R., Tyrosine hydroxylase activity: regional and subcellular distribution in brain, Brain Research, 68 (1974) 261-266. 9 Sherman, K. A., Hanin, I. and Zigmond, M. J., The effect of neuroleptics on acetylcholine concentration and choline uptake in striatum; implications for regulation of acetylcholine metabolism, J. Pharmacol. exp. Therap., 206 (1978) 677-686. 10 Szerb, J. C., Malik, H. and Hunter, E. G., Relationship between acetylcholine content and release in the cat's cerebral cortex, Canad. J. physiol. Pharmacol., 48 (1970) 780-790. l l Szilagyi, P. I. A., Schmidt, D. E. and Green, J. P., Microanalytical determination of acetylcholine, other choline esters, and choline by pyrolysis gas chromatography, Analyt. Chem., 40 (1968) 2009-2013. 12 Wecker, L. and Dettbarn, W.-D., Relationship between choline availability and acetylcholine synthesis in discrete regions of rat brain, J. Neurochem., 32 (1979) 961-967. 13 Wecker, L., Dettbarn, W.-D. and Schmidt, D. E., Choline administration: modification of the central actions of atropine, Science, 199 (1978) 86 87.