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Choline and acetylcholine: Regional distribution and effect of degeneration of cholinergic nerve terminals in the rat hippocampus
Nrurophormocology, 1973, 12, 819-823 Pergamon Press. Printed in Ct. Britain.
CHOLINE AND ACETYLCHOLINE: REGIONAL DISTRIBUTION AND EFFECT OF DEGENERATION OF CHOLINERGIC NERVE TERMINALS IN THE RAT HIPPOCAMPUS V. H. SETHY, R. H. ROTH, M. J. KUHAR and M. H. VAN WOERT Departments of Pharmacology, Psychiatry and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510 (Accepted
12 February
1973)
Summary-The regional distribution of choline and acetylcholine was examined in rat brain. Choline was found to have a distribution similar to that of acetylcholine. With the exception of the cerebellum the ratio of choline to acetylcholine in different brain regions ranged between 2.5 and 3.6. When the majority of the cholinergic nerve endings in the hippocampus were destroyed by placement of a lesion in the medial septal area the concentrations of acetylcholine and choline in the hippocampus were reduced by 70-74 and 20-32 %, respectively. These results suggest that a significant portion of the free choline in brain is associated with cholinergic neurones.
Recent studies in dur laboratory and elsewhere have indicated that brain synaptosomes possess a specific uptake mechanism for choline (Ch) (YAMAMURA and SNYDER, 1972) which is abolished when cholinergic nerve endings have degenerated (KUHAR, SETHY, ROTH and AGHAJANIAN, 1973). These observations suggest tliat a selective uptake mechanism for choline is present in cholinergic neurones. It is possible that this uptake process may provide the cholinergic neurones with a constant source of choline for use in acetylcholine (ACh) biosynthesis. Presently, it is not known whether a significant portion of the free choline found in brain is utilized for ACh biosynthesis. In fact, it is usually presumed that only a small portion of the total choline pool is actually utilized for ACh biosynthesis and the remainder is involved in other metabolic reactions. In order to gain some insight as to whether the cholinergic neurones contain a significant portion of the brain choline pool, we have (1) compared the regional distribution of ACh and choline in rat brain, and (2) examined the effect of chronic destruction of cholinergic input to the hippocampus on the concentration of both ACh and choline in the hippocampus. METHODS Male Sprague-Dawley rats weighing 225-275 g were used in this study. Animals were kept under constant diurnal lighting and temperature conditions for at least 1 week prior to their use for experimental purposes. All rats were killed at approximately the same time of the day.
Comparison of d$erent methods of sacrifice on ACh and choline levels of whole rat brain Rats were sacrificed microwave irradiation
either by decapitation with a guillotine or by exposing the rat to (Amana Radarange, model RR-4). After decapitation, the brains 819
820
V. H.
SETHY,
R. H. ROTH,M. J. KUHARand M. H.
VAN WOERT
were quickly removed from the skull and then homogenized in 0.4 N perchloric acid (4°C). After microwave irradiation (SCHMIDT, SPETH, WELSCH and SCHMIDT, 1972) for 40 set, the heads were separated from the rest of the body and put in a dry-ice acetone bath for 15 set in order to cool the brain rapidly. Then the brains were removed from the skull, homogenized in 0.4 N perchloric acid and ACh and Ch were estimated as described below. Dissection
of discrete areas of rat brain
Our initial experiments indicated that brains of rats killed by microwave irradiation and dissected into discrete areas had slightly lower and less reproducible levels of ACh than those found in brains from rats killed by decapitation, and dissected in pentane at -5°C (CAMPBELL and JENDEN, 1970). The variability is probably in part explanable by the imprecise positioning of the rat in the oven during the irradiation procedure. Therefore the technique of decapitation was employed in all subsequent experiments. The method of GLOWINSKI and IVERSEN(1966) was used for dissection of cerebellum, brainstem, striatum, hippocampus, cortex, hypothalamus and midbrain. Brain regions were pooled (Table 2) in order to obtain detectable levels of ACh and choline in the sample. Estimation
of ACh and choline
Brain ACh and Ch were measured by the method of JENDEN, HANIN and LAMB (1968) and HANIN, MASSARELLI and COSTA (1972) with the following modifications. Propionylcholine was used as an internal standard and butyrylchloride was used for esterification of Ch. Analysis was done on a Packard (Model 7631) dual column gas-chromatograph equipped with dual flame ionization detectors and a Honeywell recorder. Coiled glass columns (6 ft, 6 mm o.d.) were packed with Pennwalt 223 amine packing (Applied Science). The flame detectors and injection ports were 210” and 250°C respectively, and the column temperature was 165°C. Nitrogen was used as a carrier gas (65 ml/min). Hydrogen flow was maintained at 30 ml/min and oxygen flow at 300 ml/min. Levels of ACh and Ch are expressed as nmol/g of brain. Placement
of lesion
High frequency (100 kHz) lesions were made by a Grass model LM4 Lesion maker (10 mA, 60 set) at the following coordinates, according to KONIG and KLIPPEL (1967) : anterior, 7890 pm; horizontal, 0 pm; vertical 0 pm. Clay-Adams insect pins (size 1) insulated to within 1 pm of the tip, were used as electrodes. Animals were sarificed by decapitation 5 days or more than 20 days after the placement of lesion (times at which biochemical changes such as reduction in ACh and choline acetylase have been shown to be complete (KUHAR et al., 1973). The brains were quickly removed from the skulls, put in cold (- 5°C) pentane and divided by a coronal section between the septum and the hippocampus (about anterior 6280 pm according to the coordinates of KONIG and KLIPPEL, 1967). The anterior portion of the brain was retained for histological examination. The lesion, which was reproducible with regard to size and location, destroyed the medial septal nucleus and part of the nucleus of the diagonal band and lateral septal nucleus (KUHAR et al., 1973). The hippocampus (including hippocampus proper, subiculum and dentate gyrus) was obtained from the posterior portion of the brain after peeling back the cerebral cortex and exposing the subcortical regions.
Brain distribution
821
of choline and acetylcholine RESULTS
Table 1 shows that ACh and Ch concentrations in whole rat brain were not significantly different when rats were sacrificed either by decapitation or by microwave irradiation. Since microwave irradiation destroys enzymatic activity within a few seconds (SCHMIDTet al., 1972) the comparable results obtained by the two methods suggests that there is no significant alteration in the content of ACh or choline in whole rat brain from the time of decapitation, until the brains are placed in cold pentane (less than 30 set). It should be Table I. Effect of different methods of sacrifice on rat whole brain acetylcholine and choline Method of sacrifice
Acetylcholine*
Decapitation Microwave (40 set)
20.2 i 2.9 22.2 + 3.2
* Mean of 3 observations
f
Choline* 11.1 i_ 6.1 86.4 f 16.7
S.E. (nmol/g).
emphasized however that in our study the rats were killed by total body irradiation as opposed to focusing the megnetron beam directly on the head of the animal as done by STAVINOHA,MODAK and WEINTRAUB(1972). Thus sufficient time might have elapsed to allow for some changes in choline and acetylcholine concentrations within the brains during the irradiation procedure. The regional distribution of rat brain ACh observed in our experiments (Table 2) was similar to that reported by others (CAMPBELLand JENDEN, Table 2. Regional distribution
Brain region
No. of bilateral regions pooled
of acetylcholine and choline in rat brain*
Acetylcholinet
Cholinet
Ch/ACh
Striatum
2
37.0 + 3.1
127.0 f 9.7
3.4
Hypothalamus Midbrain Hippocampus Brainstem Cortex Cerebellum
33 3 3
34.0 ii 27.1 20.4 * 20.0 i 9.8 f 4.7 +
111.3 + 67.8 l 54.9 * 66.6 f 35.1 + 38.1 f
;:: 2.7 3.3 3.6 8.0
I 2
3.8 1.3 2.0 0.8 1.2 0.5
12.9 5.7 6.0 8.0
1.8 3.1
* Rats were sacrificed by decapitation with guillotine. t Mean of 6 observations & S.E. (nmol/g).
1970; SCHMIDTet al., 1972). The ACh content was highest in the striatum and lowest in the cerebellum. It was of interest, however, that the regional distribution of choline closely paralleled the distribution of ACh (Table 2). With the exception of the cerebellum the ratio of choline to ACh concentration in different areas of rat brain varied between 2.5 and 36. These observations suggested that a significant portion of choline in brain might be stored in cholinergic neurones. For this reason we decided to destroy the cholinergic input to a given area of the brain and determine if this in any way altered the choline concentration in that region. A lesion was made in the septal area as outlined in the methods section. The ACh and choline concentrations were then measured in the hippocampus 5 days and
V. H. SETHY,R. H. ROTH, M. J. KUHAR and M. H. VAN WOERT
822
20 or more days after placement of the septal campus was reduced at 5 days and at 20 or septum by 74 and 70 ‘A respectively (Table 3). in the hippocampus was reduced by 31.5 and
lesion. The ACh more days after At the same time 20 % respectively
concentration in the hippoplacement of lesions in the the concentration of choline (Table 3).
Table 3. Effect of septal lesion on acetylcholine and choline concentration Acetylcholine
Duration (days) of lesion 5
> 20
Sham lesion
nmol/g mean i SE. ‘A reduction
Lesion
23.2 & 0.8 (3) 6.1 i 0.9 (7) 20.1 f I.7 (6) 6.0f 0.9 (5)
* P < 0.02. t P < oao1. Figures in parentheses
73.6t 70.27
in hippocampus
Choline nmol/g mean * SE. Sham lesion
Lesion
0/Oreduction
64.1 * 1.5 (5) 44.1 It 1.6(6) 50.8 i 2.1 (6) 41.2 i 2.4 (5)
31.5t 20.0*
indicate number of experiments. DISCUSSION
In the rat brain, previously published levels of choline ranged from 1.0 pmol/g (LUECKE and PEARSON, 1944) to 0.06 pmol/g (SETHY and VAN WOERT, 1973). This wide variation in the levels of choline may be due to the use of different methods for estimating choline or different techniques for sacrificing the animals. The gas-chromatographic technique of JENDEN et al. (1968) and HANIN et al. (1972), used in the present study, is a specific assay technique for estimation of choline and its esters. DROSS and KEWITZ (1972) recently reported that in the rat brain the concentration of choline increases with a velocity of 20.5 nmol/g x min immediately after decapitation. Since microwave irradiation of rats stops enzyme activity within 15 set or less depending upon the technique (NELSON and MANTZ, 1971; STAVINOHA et al., 1972), this should prevent further synthesis or destruction of ACh and choline in brain after microwave irradiation. We observed that the values of ACh and choline in whole rat brain after decapitation and microwave irradiation were not significantly different (Table 1), therefore it appears that no significant amount of ACh and choline was synthesized or destroyed in the short period (< 30 set) between decapitation and cooling the brain in pentane. However, we could not rule out the possibility that significant changes in the levels of ACh and choline might occur during the initial portion of the irradiation period prior to the complete inactivation of enzymes (i.e. the first 15-20 set of irradiation) which could make the results obtained by the two independent techniques similar. The method of decapitation was used for killing the animals in the remainder of the experiments. The order of distribution of ACh in discrete areas of rat brain was as follows: striatum > (Table hypothalamus > midbrain > hippocampus > brainstem > cortex > cerebellum 2). These results are in agreement with the findings of CAMPBELL and JENDEN (1970) and SCHMIDT et al. (1972) with the exception of the hippocampus which we found to contain larger amounts of ACh than brainstem. We also observed that the concentration of free choline in rat brain had a regional localization similar to that of ACh, i.e. the order of distribution was as follows: striatum > (Table hypothalamus > midbrain > brainstem > hippocampus > cerebellum > cortex 2). These results suggest that cholinergic neurones may store appreciable quantities of choline for the biosynthesis of ACh. Our observations in animals with lesions in the septum tend
Brain distribution
of choline and acetylcholine
823
to support this hypothesis. The concentration of ACh and choline was significantly reduced in the hippocampus after placement of a lesion in medial septal area. There was a 70-74% reduction in ACh levels but only 20-31.5’A reduction in choline levels in rats with chronic septal lesion. However, in terms of absolute amounts the observed reduction in ACh and choline was very similar. Thus there was a loss of 14-17 nmol of ACh and 10-20 nmol of choline. It is of interest that FRIESEN, LING and NAGAI (1967) also obtained similar results after chronic preganglionic denervation of the superior cervical ganglion of the cat. They found a 92% reduction in ACh content and 32% reduction in choline content in the superior cervical ganglion 14 days after denervation. The reduction in hippocampal choline produced by a septal lesion is probably due to the disappearance of endogenous stores of free choline or choline precursors such as phosphorylcholine located in the hippocampal cholinergic nerve terminals. Our results do not exclude the effects of cholinergic denervation on high or low affinity uptake mechanisms for choline or on choline storage in the postsynaptic hippocampal neurones. Acknowfedgemenrs-This research was supported in part by National Institute of Health Grants: MH-14092 and NS-07542. V. H. SETHYis a special fellow supported by Grant No: I-FlO-NSO 2597-01. M. J. KUHAR is a postdoctoral fellow supported by Basic Science Training grant MH-7114. REFERENCES CAMPBELL,L. B. and JENDEN,D. J. (1970). Gas chromatographic evaluation of the influence of oxotremorine upon the regional distribution of acetylcholine in rat brain. J. Neurochem. 17: 1697-1699. DROSS,K. and KEWITZ, H. (1972). Concentration and origin of choline in the rat brain. Naunyn-Schmiea’ebergs Arch. Pharmac.
274: 91-106.
FRIESEN,A. J. D., LING, G. M. and NAGAI, M. (1967). Choline and phospholipidcholine in a sympathetic ganglion and their relationship to acetylcholine synthesis. Nature, Land. 214: 722-724. GLOWINSKI,J. and IVERSEN,L. L. (1966). Regional studies of catecholamines in the rat brain--I. J. Neurothem. 13: 655-669. HANIN, I.,MASSARELLI, R. and COSTA,E. (1972). An approach to the in uiuo study of acetylcholine turnover in rat salivary glands by radio gas chromatography. J. Pharmac. exp. Ther. 181: 10-l 8. JENDEN,D. J., HANIN, I. and LAMB,S. I. (1968). Gas chromatographic microestimation of acetylcholine and related compounds. Analyt. Chem. 40: 125-128. KONIG, J. F. R. and KLIPPEL, R. A. (1967). The Raf Brain. Williams & Wilkins, Baltimore. KUHAR, M. J., SETHY,V. H., ROTH, R. H. and AGHAJANIAN,G. K. (1973). Choline: Selective accumulation by central cholinergic neurons. J. Neurochem. 20: 581-593. LUECKE,R. W. and PEARSON, P. B. (1944). The microbiological determination of free choline in plasma and urine. J. biol. Chem. 153: 259-264. NELSON,S. R. and MANTZ, M. L. (1971). Metabolite levels in brain after heating (microwave radiation) the decapitated mouse head. Fedn Proc. Fedn Am. Sots exp. Biol. 30: 496 (Abstr.). SCHMIDT, D. E., SPETH, R. C., WELSCH,F. and SCHMIDT,M. J. (1972). The use of microwave radiation in the determination of acetylcholine in the rat brain. Brain Res. 38: 377-389. SETHY,V. H. and VAN WOERT, M. H. (1973). Effect of L-DOPA on brain acetylcholine and choline in rats. Neuropharmacology 12: 27-3 1. STAVINOHA,W. B., MODAK, A. T. and WEINTRAUB,S. T. (1972). Studies on 2450 MHz microwave heating on the central cholinergic system. Fifth Int. Gong. Pharmacol. 1324 (Abstr.). YAMAMURA,H. I. and SNYDER, S. H. (1972). Choline: High affinity uptake by rat brain synaptosomes. Science 178: 626-628.
CHOLINE AND ACETYLCHOLINE: REGIONAL DISTRIBUTION AND EFFECT OF DEGENERATION OF CHOLINERGIC NERVE TERMINALS IN THE RAT HIPPOCAMPUS V. H. SETHY, R. H. ROTH, M. J. KUHAR and M. H. VAN WOERT Departments of Pharmacology, Psychiatry and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510 (Accepted
12 February
1973)
Summary-The regional distribution of choline and acetylcholine was examined in rat brain. Choline was found to have a distribution similar to that of acetylcholine. With the exception of the cerebellum the ratio of choline to acetylcholine in different brain regions ranged between 2.5 and 3.6. When the majority of the cholinergic nerve endings in the hippocampus were destroyed by placement of a lesion in the medial septal area the concentrations of acetylcholine and choline in the hippocampus were reduced by 70-74 and 20-32 %, respectively. These results suggest that a significant portion of the free choline in brain is associated with cholinergic neurones.
Recent studies in dur laboratory and elsewhere have indicated that brain synaptosomes possess a specific uptake mechanism for choline (Ch) (YAMAMURA and SNYDER, 1972) which is abolished when cholinergic nerve endings have degenerated (KUHAR, SETHY, ROTH and AGHAJANIAN, 1973). These observations suggest tliat a selective uptake mechanism for choline is present in cholinergic neurones. It is possible that this uptake process may provide the cholinergic neurones with a constant source of choline for use in acetylcholine (ACh) biosynthesis. Presently, it is not known whether a significant portion of the free choline found in brain is utilized for ACh biosynthesis. In fact, it is usually presumed that only a small portion of the total choline pool is actually utilized for ACh biosynthesis and the remainder is involved in other metabolic reactions. In order to gain some insight as to whether the cholinergic neurones contain a significant portion of the brain choline pool, we have (1) compared the regional distribution of ACh and choline in rat brain, and (2) examined the effect of chronic destruction of cholinergic input to the hippocampus on the concentration of both ACh and choline in the hippocampus. METHODS Male Sprague-Dawley rats weighing 225-275 g were used in this study. Animals were kept under constant diurnal lighting and temperature conditions for at least 1 week prior to their use for experimental purposes. All rats were killed at approximately the same time of the day.
Comparison of d$erent methods of sacrifice on ACh and choline levels of whole rat brain Rats were sacrificed microwave irradiation
either by decapitation with a guillotine or by exposing the rat to (Amana Radarange, model RR-4). After decapitation, the brains 819
820
V. H.
SETHY,
R. H. ROTH,M. J. KUHARand M. H.
VAN WOERT
were quickly removed from the skull and then homogenized in 0.4 N perchloric acid (4°C). After microwave irradiation (SCHMIDT, SPETH, WELSCH and SCHMIDT, 1972) for 40 set, the heads were separated from the rest of the body and put in a dry-ice acetone bath for 15 set in order to cool the brain rapidly. Then the brains were removed from the skull, homogenized in 0.4 N perchloric acid and ACh and Ch were estimated as described below. Dissection
of discrete areas of rat brain
Our initial experiments indicated that brains of rats killed by microwave irradiation and dissected into discrete areas had slightly lower and less reproducible levels of ACh than those found in brains from rats killed by decapitation, and dissected in pentane at -5°C (CAMPBELL and JENDEN, 1970). The variability is probably in part explanable by the imprecise positioning of the rat in the oven during the irradiation procedure. Therefore the technique of decapitation was employed in all subsequent experiments. The method of GLOWINSKI and IVERSEN(1966) was used for dissection of cerebellum, brainstem, striatum, hippocampus, cortex, hypothalamus and midbrain. Brain regions were pooled (Table 2) in order to obtain detectable levels of ACh and choline in the sample. Estimation
of ACh and choline
Brain ACh and Ch were measured by the method of JENDEN, HANIN and LAMB (1968) and HANIN, MASSARELLI and COSTA (1972) with the following modifications. Propionylcholine was used as an internal standard and butyrylchloride was used for esterification of Ch. Analysis was done on a Packard (Model 7631) dual column gas-chromatograph equipped with dual flame ionization detectors and a Honeywell recorder. Coiled glass columns (6 ft, 6 mm o.d.) were packed with Pennwalt 223 amine packing (Applied Science). The flame detectors and injection ports were 210” and 250°C respectively, and the column temperature was 165°C. Nitrogen was used as a carrier gas (65 ml/min). Hydrogen flow was maintained at 30 ml/min and oxygen flow at 300 ml/min. Levels of ACh and Ch are expressed as nmol/g of brain. Placement
of lesion
High frequency (100 kHz) lesions were made by a Grass model LM4 Lesion maker (10 mA, 60 set) at the following coordinates, according to KONIG and KLIPPEL (1967) : anterior, 7890 pm; horizontal, 0 pm; vertical 0 pm. Clay-Adams insect pins (size 1) insulated to within 1 pm of the tip, were used as electrodes. Animals were sarificed by decapitation 5 days or more than 20 days after the placement of lesion (times at which biochemical changes such as reduction in ACh and choline acetylase have been shown to be complete (KUHAR et al., 1973). The brains were quickly removed from the skulls, put in cold (- 5°C) pentane and divided by a coronal section between the septum and the hippocampus (about anterior 6280 pm according to the coordinates of KONIG and KLIPPEL, 1967). The anterior portion of the brain was retained for histological examination. The lesion, which was reproducible with regard to size and location, destroyed the medial septal nucleus and part of the nucleus of the diagonal band and lateral septal nucleus (KUHAR et al., 1973). The hippocampus (including hippocampus proper, subiculum and dentate gyrus) was obtained from the posterior portion of the brain after peeling back the cerebral cortex and exposing the subcortical regions.
Brain distribution
821
of choline and acetylcholine RESULTS
Table 1 shows that ACh and Ch concentrations in whole rat brain were not significantly different when rats were sacrificed either by decapitation or by microwave irradiation. Since microwave irradiation destroys enzymatic activity within a few seconds (SCHMIDTet al., 1972) the comparable results obtained by the two methods suggests that there is no significant alteration in the content of ACh or choline in whole rat brain from the time of decapitation, until the brains are placed in cold pentane (less than 30 set). It should be Table I. Effect of different methods of sacrifice on rat whole brain acetylcholine and choline Method of sacrifice
Acetylcholine*
Decapitation Microwave (40 set)
20.2 i 2.9 22.2 + 3.2
* Mean of 3 observations
f
Choline* 11.1 i_ 6.1 86.4 f 16.7
S.E. (nmol/g).
emphasized however that in our study the rats were killed by total body irradiation as opposed to focusing the megnetron beam directly on the head of the animal as done by STAVINOHA,MODAK and WEINTRAUB(1972). Thus sufficient time might have elapsed to allow for some changes in choline and acetylcholine concentrations within the brains during the irradiation procedure. The regional distribution of rat brain ACh observed in our experiments (Table 2) was similar to that reported by others (CAMPBELLand JENDEN, Table 2. Regional distribution
Brain region
No. of bilateral regions pooled
of acetylcholine and choline in rat brain*
Acetylcholinet
Cholinet
Ch/ACh
Striatum
2
37.0 + 3.1
127.0 f 9.7
3.4
Hypothalamus Midbrain Hippocampus Brainstem Cortex Cerebellum
33 3 3
34.0 ii 27.1 20.4 * 20.0 i 9.8 f 4.7 +
111.3 + 67.8 l 54.9 * 66.6 f 35.1 + 38.1 f
;:: 2.7 3.3 3.6 8.0
I 2
3.8 1.3 2.0 0.8 1.2 0.5
12.9 5.7 6.0 8.0
1.8 3.1
* Rats were sacrificed by decapitation with guillotine. t Mean of 6 observations & S.E. (nmol/g).
1970; SCHMIDTet al., 1972). The ACh content was highest in the striatum and lowest in the cerebellum. It was of interest, however, that the regional distribution of choline closely paralleled the distribution of ACh (Table 2). With the exception of the cerebellum the ratio of choline to ACh concentration in different areas of rat brain varied between 2.5 and 36. These observations suggested that a significant portion of choline in brain might be stored in cholinergic neurones. For this reason we decided to destroy the cholinergic input to a given area of the brain and determine if this in any way altered the choline concentration in that region. A lesion was made in the septal area as outlined in the methods section. The ACh and choline concentrations were then measured in the hippocampus 5 days and
V. H. SETHY,R. H. ROTH, M. J. KUHAR and M. H. VAN WOERT
822
20 or more days after placement of the septal campus was reduced at 5 days and at 20 or septum by 74 and 70 ‘A respectively (Table 3). in the hippocampus was reduced by 31.5 and
lesion. The ACh more days after At the same time 20 % respectively
concentration in the hippoplacement of lesions in the the concentration of choline (Table 3).
Table 3. Effect of septal lesion on acetylcholine and choline concentration Acetylcholine
Duration (days) of lesion 5
> 20
Sham lesion
nmol/g mean i SE. ‘A reduction
Lesion
23.2 & 0.8 (3) 6.1 i 0.9 (7) 20.1 f I.7 (6) 6.0f 0.9 (5)
* P < 0.02. t P < oao1. Figures in parentheses
73.6t 70.27
in hippocampus
Choline nmol/g mean * SE. Sham lesion
Lesion
0/Oreduction
64.1 * 1.5 (5) 44.1 It 1.6(6) 50.8 i 2.1 (6) 41.2 i 2.4 (5)
31.5t 20.0*
indicate number of experiments. DISCUSSION
In the rat brain, previously published levels of choline ranged from 1.0 pmol/g (LUECKE and PEARSON, 1944) to 0.06 pmol/g (SETHY and VAN WOERT, 1973). This wide variation in the levels of choline may be due to the use of different methods for estimating choline or different techniques for sacrificing the animals. The gas-chromatographic technique of JENDEN et al. (1968) and HANIN et al. (1972), used in the present study, is a specific assay technique for estimation of choline and its esters. DROSS and KEWITZ (1972) recently reported that in the rat brain the concentration of choline increases with a velocity of 20.5 nmol/g x min immediately after decapitation. Since microwave irradiation of rats stops enzyme activity within 15 set or less depending upon the technique (NELSON and MANTZ, 1971; STAVINOHA et al., 1972), this should prevent further synthesis or destruction of ACh and choline in brain after microwave irradiation. We observed that the values of ACh and choline in whole rat brain after decapitation and microwave irradiation were not significantly different (Table 1), therefore it appears that no significant amount of ACh and choline was synthesized or destroyed in the short period (< 30 set) between decapitation and cooling the brain in pentane. However, we could not rule out the possibility that significant changes in the levels of ACh and choline might occur during the initial portion of the irradiation period prior to the complete inactivation of enzymes (i.e. the first 15-20 set of irradiation) which could make the results obtained by the two independent techniques similar. The method of decapitation was used for killing the animals in the remainder of the experiments. The order of distribution of ACh in discrete areas of rat brain was as follows: striatum > (Table hypothalamus > midbrain > hippocampus > brainstem > cortex > cerebellum 2). These results are in agreement with the findings of CAMPBELL and JENDEN (1970) and SCHMIDT et al. (1972) with the exception of the hippocampus which we found to contain larger amounts of ACh than brainstem. We also observed that the concentration of free choline in rat brain had a regional localization similar to that of ACh, i.e. the order of distribution was as follows: striatum > (Table hypothalamus > midbrain > brainstem > hippocampus > cerebellum > cortex 2). These results suggest that cholinergic neurones may store appreciable quantities of choline for the biosynthesis of ACh. Our observations in animals with lesions in the septum tend
Brain distribution
of choline and acetylcholine
823
to support this hypothesis. The concentration of ACh and choline was significantly reduced in the hippocampus after placement of a lesion in medial septal area. There was a 70-74% reduction in ACh levels but only 20-31.5’A reduction in choline levels in rats with chronic septal lesion. However, in terms of absolute amounts the observed reduction in ACh and choline was very similar. Thus there was a loss of 14-17 nmol of ACh and 10-20 nmol of choline. It is of interest that FRIESEN, LING and NAGAI (1967) also obtained similar results after chronic preganglionic denervation of the superior cervical ganglion of the cat. They found a 92% reduction in ACh content and 32% reduction in choline content in the superior cervical ganglion 14 days after denervation. The reduction in hippocampal choline produced by a septal lesion is probably due to the disappearance of endogenous stores of free choline or choline precursors such as phosphorylcholine located in the hippocampal cholinergic nerve terminals. Our results do not exclude the effects of cholinergic denervation on high or low affinity uptake mechanisms for choline or on choline storage in the postsynaptic hippocampal neurones. Acknowfedgemenrs-This research was supported in part by National Institute of Health Grants: MH-14092 and NS-07542. V. H. SETHYis a special fellow supported by Grant No: I-FlO-NSO 2597-01. M. J. KUHAR is a postdoctoral fellow supported by Basic Science Training grant MH-7114. REFERENCES CAMPBELL,L. B. and JENDEN,D. J. (1970). Gas chromatographic evaluation of the influence of oxotremorine upon the regional distribution of acetylcholine in rat brain. J. Neurochem. 17: 1697-1699. DROSS,K. and KEWITZ, H. (1972). Concentration and origin of choline in the rat brain. Naunyn-Schmiea’ebergs Arch. Pharmac.
274: 91-106.
FRIESEN,A. J. D., LING, G. M. and NAGAI, M. (1967). Choline and phospholipidcholine in a sympathetic ganglion and their relationship to acetylcholine synthesis. Nature, Land. 214: 722-724. GLOWINSKI,J. and IVERSEN,L. L. (1966). Regional studies of catecholamines in the rat brain--I. J. Neurothem. 13: 655-669. HANIN, I.,MASSARELLI, R. and COSTA,E. (1972). An approach to the in uiuo study of acetylcholine turnover in rat salivary glands by radio gas chromatography. J. Pharmac. exp. Ther. 181: 10-l 8. JENDEN,D. J., HANIN, I. and LAMB,S. I. (1968). Gas chromatographic microestimation of acetylcholine and related compounds. Analyt. Chem. 40: 125-128. KONIG, J. F. R. and KLIPPEL, R. A. (1967). The Raf Brain. Williams & Wilkins, Baltimore. KUHAR, M. J., SETHY,V. H., ROTH, R. H. and AGHAJANIAN,G. K. (1973). Choline: Selective accumulation by central cholinergic neurons. J. Neurochem. 20: 581-593. LUECKE,R. W. and PEARSON, P. B. (1944). The microbiological determination of free choline in plasma and urine. J. biol. Chem. 153: 259-264. NELSON,S. R. and MANTZ, M. L. (1971). Metabolite levels in brain after heating (microwave radiation) the decapitated mouse head. Fedn Proc. Fedn Am. Sots exp. Biol. 30: 496 (Abstr.). SCHMIDT, D. E., SPETH, R. C., WELSCH,F. and SCHMIDT,M. J. (1972). The use of microwave radiation in the determination of acetylcholine in the rat brain. Brain Res. 38: 377-389. SETHY,V. H. and VAN WOERT, M. H. (1973). Effect of L-DOPA on brain acetylcholine and choline in rats. Neuropharmacology 12: 27-3 1. STAVINOHA,W. B., MODAK, A. T. and WEINTRAUB,S. T. (1972). Studies on 2450 MHz microwave heating on the central cholinergic system. Fifth Int. Gong. Pharmacol. 1324 (Abstr.). YAMAMURA,H. I. and SNYDER, S. H. (1972). Choline: High affinity uptake by rat brain synaptosomes. Science 178: 626-628.