- Email: [email protected]
Determination of acetylcholine in small samples of fresh brain tissue
ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS 66, 388-395 (1957)
Determination of Acetylcholine in Small Samples of Fresh Brain Tissue M. H. Aprison and P. NathanI From the Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Illinois Received June 8, 1956 INTRODUCTION
The purpose of this paper is to present the details of a bioassay method for the determination of the acetylcholine (ACh) concentration in small samples of fresh brain tissue. This method was developed to be used primarily in neurochemical studies in which the brain specimens available for analyses were not large enough to be processed by the usual techniques described in the literature, and secondarily, with central nervous system tissue extracts containing substances which interfere with the determination of ACh. At present, samples as small as 40 rag. of caudate nucleus from the brain of normal rabbits have been analyzed. Although the method can be easily adapted to determine total ACh, the procedure given below is for "free" or soluble ACh. The latter is biochemically separated from interfering substances prior to subjecting the samples to analysis by a bioassay procedure (the test object is a 3-4-cm. portion of guinea pig ileum). Many of the commonly used bioassay procedures employ tissue from animals that are difficult to obtain the year around or to find in all localities (frogs, leech, mollusk, etc.). Therefore, the authors felt that a method using smooth muscle tissue from an easily obtainable animal such as the guinea pig, applied in the above procedure, should be of interest. In addition, the method can be used to determine the ACh concentration in such small tissue samples taken from the brain of experimental animals previously poisoned by anticholinesterases such as diisopropyl fluorophosphate (isopropyl phosphorofluoridate) (1). ~ 1 Present address: May Institute for Medical Research of the Jewish Hospital Association, Cincinnati, Ohio. Also, manuscript in preparation. 388
ACtI IN SMALL BRAIN SAMPLES
389
EXPERIMENTAL Guinea pig ileum was employed in the bioassay. It was found that smooth muscle obtained from animals weighing between 200 and 450 g. were best suited for this purpose. These animals were sacrificed by decapitation, and the small intestine was tied off in segments about 20 in. long. The latter were removed within a few minutes with a minimum amount of manipulation. The outer surface of the ileum was washed with Tyrode solution several times. The long cylinders of muscle tissue were kept at room temperature with several washings with Tyrode solution per hour until used. A piece of intestine 3-4 cm. long was cut off from the longer length after tying it with cotton thread. The small segment was placed into the aerated tissue chamber and connected to the ink writing lever. It was then allowed to equilibrate for approximately 30 rain. with frequent changes of Tyrode. A constant response to ACh was established before the assay was begun. The Tyrode solution used in the bioassay was made up as follows: 8 g. NaC1, 1 g. glucose, 1 g. NaHCO3 , 0.2 g. KC1, 0.1 g. NaHfPO4, and 0.7 g. CaCI~ were dissolved in 1 1. of distilled water. This solution was kept at 30°C. which was the same temperature as that of the tissue bath. The brain tissue to be analyzed was obtained from rabbits. The animal was securely tied to a board. After pontocaine was administered subcutaneously in the middle region of the scalp, a craniotomy was performed. The upper portion of the skull was removed and the dura carefully cut, exposing the brain. Left and right cerebral cortical samples were taken immediately by cutting thin sections parallel to the upper surface of the brain by means of a scalpel. The sample contained mostly gray matter and was usually about 2 mm. thick. Each piece was immediately frozen between two pieces of Dry Ice. Then the brain was rapidly removed from the skull, and the left and right caudate nuclei were excised. The last two steps took approximately 30-40 sec. As each caudate nucleus was removed, it was also immediately frozen. Each tissue sample was quickly weighed, while still frozen, on a Roller-Smith torsion balance and then dropped into a glass hand homogenizer containing 1 ml. of ice cold eserine-water or eserine-Tyrode solution (2 X 10-4 M eserine). The eserine-water method was used predominantly since both yield comparable results. The homogenizer was precooled to 0°C. and kept in a container of cracked ice throughout the homogenization procedure. The homogenate was then transferred to a plastic tube which was kept in a mixture of crushed ice and salt until it was ready for centrifugation. The homogenizer was rinsed with 0.8 ml. of ice-cold solution. When several samples were obtained, they were spun down at 9500 r.p.m, for 5 min. on a Servall centrifuge kept in a 4°C. cold room. This step removed all of the water-insoluble material leaving the free ACh in solution. The supernatant of each tube was decanted into a 15-ml. calibrated conical centrifuge tube (preeooled to 4°C.). The volume was carefully adjusted to 1.9 ml. with cold Tyrode solution. Since the guinea pig ileum method is very sensitive to many interfering compounds, the reineckate precipitation procedure described by Bentley and Shaw was used to remove such substances. This latter step permits the separation of ACh from eserine, atropine, histamine, and epinephrine (2). To the 1.9 ml. of the sample in the conical centrifuge tube the following were added: 0.1 ml. choline chloride (1.6 mg.), 1.5 ml. freshly prepared saturated solu-
390
APRISON AND NATHAN
tion of ammonium reineekate (Fisher) in absolute alcohol, and 1.0 ml. absolute alcohol. The final alcohol concentration was 55.6%. The samples were stirred by hand and then placed in a deep freeze (-15°C.) for 1 hr. to permit complete precipitation to occur. The tubes were then spun at 4500 r.p.m, for about 3 rain. in the 4°C. cold room. The supernatant liquid was discarded, and the choline-AChreineckate precipitates were dissolved by adding a 3: 2 mixture of absolute ethanol and Tyrode solution until the total volume was 3 ml. The tubes were then placed in a shallow water bath kept at approximately 30°C. and stirred by hand periodically for about 1 hr. to aid in redissolving the reineckate complex. Since the samples still contained alcohol-precipitated proteins, the tubes were centrifuged again for a minute and the resulting supernatant decanted into calibrated centrifuge tubes. The precipitate was triturated for another 10-20 rain. with an equal volume of 3:2 mixture of absolute ethanol and Tyrode solution. After centrifugation the supernatants were combined, yielding 3.0 ml. of an unknown sample. The latter was kept at 30°C. until ready for use in the bioassay. The samples were usually run within a few hours, but could be kept overnight. The blank and standards for each run were prepared in the following manner. After the brain parts to be analyzed for ACh were taken and frozen, a piece of cortex from each hemisphere was removed and allowed to remain at room temperature for 5-10 min. By this procedure a considerable amount of the free ACh was presumably hydrolyzed by ChE. Both samples were then frozen with Dry Ice, weighed, and carried through the homogenization and first centrifugation procedures separately but in the same manner as the unknowns. At this point, the supernatants from both tubes were combined, brought to pH 9-10 with dilute NaOH, and boiled for approximately 4-5 rain. to insure destruction of the ACh. After cooling, the pit was lowered to 6.5-7.0 by adding dilute HC1 of approximately the same normality as the base. The absence of ACh was checked by bioassay. A 1.8-ml. aliquot of this adjusted supernatant was used to prepare the standard and 1.9 ml. for the blank. To prepare the standard, 0.1 ml. of a 6.0-/zg./ml. solution of ACh chloride (Merck) was added. This sample was used to check the ACh recovered during the reineckate precipitation procedure. These two samples were carried through exactly as the unknowns. In this manner six samples were obtained for analysis from each rabit: four unknowns, one standard, and one blank. The unknown samples or standards were added in small aliquots (0.i~0.3 In].) to 10-12 ml. of Tyrode in the mixing vessel. The Tyrode solution was then added until a total volume of 15 ml. was reached. The mixture was stirred and added to the chamber containing a portion of the ileum. The solution was allowed to remain in contact with the ileum for about 25-30 see. before it was removed. Two additional Tyrode rinses were employed and spaced about 20-30 see. apart before the next sample to be analyzed was added. Each unknown was followed by different aliquots of the standard. The procedure permitted the operator to arrive by trial and error at three different aliquots of the standard solution that produced contractions that were slightly smaller than the unknown sample, slightly larger, and one that was essentially identical. In most eases aliquots of all unknown samples were run at two different volumes and, when possible, these were run in duplicate. This procedure was necessary because the sensitivity of the ileum would
391
ACH IN SMALL BRAIN SAMPLES
change d u r i n g the course of t h e experiment. T h e m e t h o d p e r m i t t e d the p l o t t i n g of s t a n d a r d curves a t different times d u r i n g t h e bioassay. T h e m a x i m u m height of c o n t r a c t i o n was measured as well as t h a t a t 10- a n d 20-see. i n t e r v a l s following the a d d i t i o n of the samples. T h e m a x i m u m was found to occur between 20 a n d 40 sec. The u n k n o w n c o n c e n t r a t i o n of ACh was calculated b y comparing the height of c o n t r a c t i o n to t h e s t a n d a r d if identical in h e i g h t or b y i n t e r p o l a t i o n on t h e s t a n d a r d curves as explained above. T h r e e special t e s t s were used to i d e n t i f y t h e active substances as ACh. F i r s t t h e a c t i v i t y of t h e u n k n o w n solution was destroyed when i t was t r e a t e d w i t h NaOH. I t was also lost if the gut was p r e t r e a t e d with a solution c o n t a i n i n g atropine which is a specific antagonis t to ACh. Finally, if t h e b r a i n samples were n o t frozen i m m e d i a t e l y b u t left at room t e m p e r a t u r e for some time, the C h E in t h e tissue hydrolyzed the ACh, reducing its c o n c e n t r a t i o n markedly. T h e degree of d e s t r u c t i o n of the n e u r o h o r m o n e was m u c h less in the case of t h e animals receiving an antieholinesterase drug. RESULTS
The ACh recovery data for the reineckate precipitation procedure are presented in Table I. The per cent recovery of added ACh varied from 87 to 108 %. However, the majority of results obtained fell between 92 to 100 %. Since most of the unknowns contained approximately 0.6 pg. of ACh in the tissue sample taken for analysis, this amount of neurohormone was used per 3 m]. of final alcohol-Tyrode solution. The latter concentration of known ACh was determined ten times. The recoveries of ACh were found to be satisfactory. ,In Table II, the data from the experiments designed to test the reproducibility of the ACh determinations are given. Ten caudate nuclei TABLE I
Acetylcholine Recovery Data No. of t r ~ 15 1 1 1 10 1 2 1 1
ACh addeda
ACh recove~d
0 0.20 0.40 0.50 0.60 0.80 0.90 1.20 2.00
0 0.216 0.40 0.50 0.55-0.63 0.70 0.90, 0.94 1.14 1.92
#g.[3ml.
#go/3ml.
Per cent recovery -108 100 100 92-105 87 100, 105 95 96
T h e a l i q u o t a n a l y z e d was e i t h e r 0.1, 0.2, or 0.3 ml. T h e b i o a s s a y is sensitive t o 0.001 ~g./ml., a n d since t h e b a t h c o n t a i n s 15 ml., t h e a l i q u o t used in the analysis m u s t contain at least 0.015 ~g.
392
APRISON
AND NATHAN
TABLE II Reproducibility of Acetylcholine Determinations ° Sample No. b
10 11 14 15 18 19 23 24 Mean S.D.
Contraction Contraction Maximum Calculated ACh Calculated ACh Calculated ACh height at 10 he~ht at 20 contraction from data in from data in from data sec.C sec. e height e coLA col. B col. C ram. ram. ram. ~g./O.15 ml. ~g./O.I5 mL ~g./O.15 ~l. A B C
10 10 8.5 8.5 9 8 9 10 9.1 4-0.78
16.5 16 16 16.5 16 13.5 15 16 15.7 -4-0.99
23 25 27 27 25.5 25.5 24 23
0.031 0.031 0.029 0.029 0.038 0.035 0.030 0.033
0.030 0.030 0.032 0.033 0.039 0,035 0.031 0.035
0.029 0.032 0.035 0.035 0.035 0.035 ---
25 4-1.67
0.032 4-0.0031
0.033 4-0.0030
0.034 4-0.0026
The calculated values in the last three columns were obtained by comparison to the standard aliquots run after each set of unknown samples. b Each sample added was a 0.15-ml. aliquot of eaudate nucleus (28.6 mg./ml.). c Heights measured to nearest 0.5 mm. from five normal rabbits were obtained and carried through the procedure used in preparing a sample for ACh bioassay as previously described. The contents of these tubes were combined, and an aliquot containing 28.6 mg./ml, of tissue was taken for analysis. A C h was measured eight separate times; this was done by determining the unknown aliquot twice, preceded and followed by two standards (see Table II). The height of the contractions was measured as described above, and comparison with the standards were made. The results obtained are given in the last three columns of Table I I . The means agree very well and the three standard deviations are of the same order of magnitude. Consequently, the point in time of the comparison is not critical and the experimental conditions limit the length of time the ileum is allowed to contract due to the presence of ACh and other substances such as alcohol, reineekate, and choline that have been added or carried through the procedure. I t should be mentioned that too long a contact time of sample with the ileum will invariably cause it to go into a condition of spontaneous contractions and interfere with the bioassay. In addition, it was sometimes noted that in the case of some cerebral cortex samples, the rate of contraction of the ileum was not the same as that produced by the standard or pure ACh solution. In such cases maximum contraction
393
ACH I N SMALL BRAIN SAMPLES TABLE
III
Effect of Different Guinea Pig Ilia on the Bioassay of Acetylcholine Acetylchollne concentration found, ~g./ml. Gut number
0.1 ral. CI'~a
0.2 ml. CNa
Mean
1
--
0.183
2 3
0.206 0.212
0.167 0. 200 0.215 0.188
0.210 0.200
C N = c a u d a t e n u c l e u s s a m p l e (27.4 m g . / m l . ) ,
heights were used. The concentration of ACh found in the sample of combined caudate nuclei (five normal rabbits) was 7.70 ~g./g. The data from a typical experiment which indicate the effect of the use of different guinea pig ilia is shown in Table III. The unknown caudate nuclei sample was prepared as described above. The ilia from five guinea pigs were used in this experiment. Two different aliquots of the unknown were run along with the proper standards for each gut. The results were calculated in each ease as acetylcholine concentration (ug./ml.) found and are presented in Table III. The concentration of ACh (caudate nuclei from three normal rabbits) was 7.22 ~g./g. Spontaneous contractions occurred with one gut, while the sensitivity was too low in the case of another. These facts are not given in Table III and the reasons for their occurrence are unknown. Sometimes keeping a particularly active ileum in a large amount of Tyrode solution overnight at 7°C. will result in a preparation that has no spontaneous movement the next morning. Furthermore, samples and standards may be kept overnight (4°C.) without a significant change in activity. The ACh concentration and size of tissue sample from the right and left cortex as well as right and left caudate nucleus of normal albino rabbits are presented in Table IV. (Caudate samples were not determined in the first three animals.) The two results for the caudate nuclei given above may also be considered here. The recovery of ACh was found to vary between 92 and 105 %. Asymmetry in ACh content between right and left cortices was not noted in these normal rabbits. However, the ACh results varied considerably from animal to animal. The mean for t h e right cortex was 0.89 ~g./g, and for the left cortex was 0.87 ~g./g. The weight of the tissue samples from the cortex in these control rabbits varied in size from approximately 156 to 560 mg. The agreement in the means is better than one would expect since the bioassay procedures can have an inherent error of 10 %.
394
APRISON AND NATHAN TABLE IV
A C h Concentrations i n the B r a i n of N o r m a l A l b i n o Rabbits Left Caudate Right Caudate Left cortex Right Cortex Nucleus Nucleus Tissue Tissue Rabbit No. Tissue wt. ACh cone, Tissue wt. ACh conc. wt. ACh conc. wt. ACh conc. rag. l~g./g, rag. i~g./g, rag. ~g./g. rag. ~g./g. N-1 377.2 0.59 362.8 0.62 . . . . N-2 260.8 0.97 309.0 0.97 . . . . N-3 163.2 0.84 155.8 0.92 . . . . N-4 238.5 1.72 297.2 1.72 54.8 9.15 41.6 8.80 N-5 415.4 0.40 560.4 0.34 92.6 8.10 73.8 10.57 N-6 365.4 0.56 399.2 ' 0.46 72.0 4.68 65.6 6.35 N -7 241.4 0.55 312.2 0.54 43.4 3.04 53.4 2.78 1W-8 368.6 1.14 406.0 1.18 75.8 3.53 61.8 3.54 N-9 318.2 0.86 293.4 0.94 57.6 6.76 63.6 5.80 N-10 222.0 1.15 332.0 1.25 58.9 9.15 68.2 8.82
In the case of the caudate nuclei, the ACh results showed greater variability between animals. Furthermore, the difference in neurohormone concentration between corresponding right and left caudate nuclei was larger than in the case of the right and left cortices. However, this difference was not considered significant. The mean obtained in the case of the right caudate nuclei was 6.67 ~g./g. while for the left caudate nuclei it was 6.34 ~g./g. The concentration of ACh determined in the experiments described in Tables I and I i falls within the range of results shown in Table IV. Tissue samples of the eaudate nuclei varied in weight between 41.6 to 92.6 mg. DISCUSSION
The free ACh concentration found in the cortex of normal albino rabbits agrees with the results reported by Michaelis et al. (3). This value is about 25 % of the total ACh found in rabbit cortices by Tower and Elliott (4). The caudate nucleus which is functionally related to the cortex contains about seven times the free ACh concentration of the latter under normal conditions. When this comparison between the two brain areas is also made for the cholinesterase activity, it was found that the ratio is of the same order of magnitude (5). The method for measuring ACh as described in this paper has a number of advantages. It is sensitive, reproducible, accurate, rapid, and employs tissue from an easily obtainable experimental animal. Furthermore, the method permits an approach to the "ideal extraction." First, by rapidly freezing the excised area between pieces of Dry Ice (matter
ACH IN SMALL BRAIN SAMPLES
395
of seconds) and then homogenizing the tissue finely at 0°C. in the presence of eserine, the a m o u n t of ACh present in the extract should correspond closely to t h a t in the brain p a r t at the m o m e n t of excision. Stone (6) reported t h a t the difference in the average value for cortical tissue frozen i n situ and t h a t from tissue frozen a few seconds after excision is not statistically significant. Secondly, centrifugation to remove the particulate m a t t e r also removes most of the cholinesterase activity (7). 8 Finally, b y performing the reineckate-choline-acetylcholine precipitation procedure, material which can interfere with the biological assay of ACh is removed or reduced to noninterfering concentrations. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Dr. H. E. Himwich for his sustained interest and encouragement throughout the course of this work. The authors also gratefully acknowledge the technical assistance of Mrs. Madeline Griffin.
SUMMARY A method for the determination of "free" or soluble ACh in small pieces of fresh brain tissue has been developed and patterned in part after the one reported by Bentley and Shaw. Brain samples as small as 40 mg. of caudate nucleus and 155 mg. of cortex have been analyzed. Values for "free" ACh in the right and left cerebral cortex and caudate nucleus are given for normal albino rabbits. REFERENCES 1. API~ISON,M. I-I., AND NATHAN, P., Federation Proc. 15, 5 (1956). 2. BENTLEY, G. A., ANDSHAW,F. I-I., J. Pharmacol. Exptl. Therap. 106, 193 (1952). 3. MICI-IAELIS,M., FINESINGER,J. E., VERSTER, F. DE B., AND ERICKSON,R. W., J. Pharmacol. Exptl. Therap. 111, 169 (1954). 4. TowEn, D. B., AND ELLIOTT, K. A. C., Am. J. Physiol. 168, 747 (1952). 5. A•nISON, M. H., AND HIMWICH,H. E., Am. J. Physiol. 179, 502 (1954). 6. STONE, W. E., Arch. Biochem. and Biophys. 59, 181 (1955). 7. NATHAN, P., AND APRISON, M. H., Federation Proc. 14, 106 (1955). a Also, manuscript in preparation.
Determination of Acetylcholine in Small Samples of Fresh Brain Tissue M. H. Aprison and P. NathanI From the Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Illinois Received June 8, 1956 INTRODUCTION
The purpose of this paper is to present the details of a bioassay method for the determination of the acetylcholine (ACh) concentration in small samples of fresh brain tissue. This method was developed to be used primarily in neurochemical studies in which the brain specimens available for analyses were not large enough to be processed by the usual techniques described in the literature, and secondarily, with central nervous system tissue extracts containing substances which interfere with the determination of ACh. At present, samples as small as 40 rag. of caudate nucleus from the brain of normal rabbits have been analyzed. Although the method can be easily adapted to determine total ACh, the procedure given below is for "free" or soluble ACh. The latter is biochemically separated from interfering substances prior to subjecting the samples to analysis by a bioassay procedure (the test object is a 3-4-cm. portion of guinea pig ileum). Many of the commonly used bioassay procedures employ tissue from animals that are difficult to obtain the year around or to find in all localities (frogs, leech, mollusk, etc.). Therefore, the authors felt that a method using smooth muscle tissue from an easily obtainable animal such as the guinea pig, applied in the above procedure, should be of interest. In addition, the method can be used to determine the ACh concentration in such small tissue samples taken from the brain of experimental animals previously poisoned by anticholinesterases such as diisopropyl fluorophosphate (isopropyl phosphorofluoridate) (1). ~ 1 Present address: May Institute for Medical Research of the Jewish Hospital Association, Cincinnati, Ohio. Also, manuscript in preparation. 388
ACtI IN SMALL BRAIN SAMPLES
389
EXPERIMENTAL Guinea pig ileum was employed in the bioassay. It was found that smooth muscle obtained from animals weighing between 200 and 450 g. were best suited for this purpose. These animals were sacrificed by decapitation, and the small intestine was tied off in segments about 20 in. long. The latter were removed within a few minutes with a minimum amount of manipulation. The outer surface of the ileum was washed with Tyrode solution several times. The long cylinders of muscle tissue were kept at room temperature with several washings with Tyrode solution per hour until used. A piece of intestine 3-4 cm. long was cut off from the longer length after tying it with cotton thread. The small segment was placed into the aerated tissue chamber and connected to the ink writing lever. It was then allowed to equilibrate for approximately 30 rain. with frequent changes of Tyrode. A constant response to ACh was established before the assay was begun. The Tyrode solution used in the bioassay was made up as follows: 8 g. NaC1, 1 g. glucose, 1 g. NaHCO3 , 0.2 g. KC1, 0.1 g. NaHfPO4, and 0.7 g. CaCI~ were dissolved in 1 1. of distilled water. This solution was kept at 30°C. which was the same temperature as that of the tissue bath. The brain tissue to be analyzed was obtained from rabbits. The animal was securely tied to a board. After pontocaine was administered subcutaneously in the middle region of the scalp, a craniotomy was performed. The upper portion of the skull was removed and the dura carefully cut, exposing the brain. Left and right cerebral cortical samples were taken immediately by cutting thin sections parallel to the upper surface of the brain by means of a scalpel. The sample contained mostly gray matter and was usually about 2 mm. thick. Each piece was immediately frozen between two pieces of Dry Ice. Then the brain was rapidly removed from the skull, and the left and right caudate nuclei were excised. The last two steps took approximately 30-40 sec. As each caudate nucleus was removed, it was also immediately frozen. Each tissue sample was quickly weighed, while still frozen, on a Roller-Smith torsion balance and then dropped into a glass hand homogenizer containing 1 ml. of ice cold eserine-water or eserine-Tyrode solution (2 X 10-4 M eserine). The eserine-water method was used predominantly since both yield comparable results. The homogenizer was precooled to 0°C. and kept in a container of cracked ice throughout the homogenization procedure. The homogenate was then transferred to a plastic tube which was kept in a mixture of crushed ice and salt until it was ready for centrifugation. The homogenizer was rinsed with 0.8 ml. of ice-cold solution. When several samples were obtained, they were spun down at 9500 r.p.m, for 5 min. on a Servall centrifuge kept in a 4°C. cold room. This step removed all of the water-insoluble material leaving the free ACh in solution. The supernatant of each tube was decanted into a 15-ml. calibrated conical centrifuge tube (preeooled to 4°C.). The volume was carefully adjusted to 1.9 ml. with cold Tyrode solution. Since the guinea pig ileum method is very sensitive to many interfering compounds, the reineckate precipitation procedure described by Bentley and Shaw was used to remove such substances. This latter step permits the separation of ACh from eserine, atropine, histamine, and epinephrine (2). To the 1.9 ml. of the sample in the conical centrifuge tube the following were added: 0.1 ml. choline chloride (1.6 mg.), 1.5 ml. freshly prepared saturated solu-
390
APRISON AND NATHAN
tion of ammonium reineekate (Fisher) in absolute alcohol, and 1.0 ml. absolute alcohol. The final alcohol concentration was 55.6%. The samples were stirred by hand and then placed in a deep freeze (-15°C.) for 1 hr. to permit complete precipitation to occur. The tubes were then spun at 4500 r.p.m, for about 3 rain. in the 4°C. cold room. The supernatant liquid was discarded, and the choline-AChreineckate precipitates were dissolved by adding a 3: 2 mixture of absolute ethanol and Tyrode solution until the total volume was 3 ml. The tubes were then placed in a shallow water bath kept at approximately 30°C. and stirred by hand periodically for about 1 hr. to aid in redissolving the reineckate complex. Since the samples still contained alcohol-precipitated proteins, the tubes were centrifuged again for a minute and the resulting supernatant decanted into calibrated centrifuge tubes. The precipitate was triturated for another 10-20 rain. with an equal volume of 3:2 mixture of absolute ethanol and Tyrode solution. After centrifugation the supernatants were combined, yielding 3.0 ml. of an unknown sample. The latter was kept at 30°C. until ready for use in the bioassay. The samples were usually run within a few hours, but could be kept overnight. The blank and standards for each run were prepared in the following manner. After the brain parts to be analyzed for ACh were taken and frozen, a piece of cortex from each hemisphere was removed and allowed to remain at room temperature for 5-10 min. By this procedure a considerable amount of the free ACh was presumably hydrolyzed by ChE. Both samples were then frozen with Dry Ice, weighed, and carried through the homogenization and first centrifugation procedures separately but in the same manner as the unknowns. At this point, the supernatants from both tubes were combined, brought to pH 9-10 with dilute NaOH, and boiled for approximately 4-5 rain. to insure destruction of the ACh. After cooling, the pit was lowered to 6.5-7.0 by adding dilute HC1 of approximately the same normality as the base. The absence of ACh was checked by bioassay. A 1.8-ml. aliquot of this adjusted supernatant was used to prepare the standard and 1.9 ml. for the blank. To prepare the standard, 0.1 ml. of a 6.0-/zg./ml. solution of ACh chloride (Merck) was added. This sample was used to check the ACh recovered during the reineckate precipitation procedure. These two samples were carried through exactly as the unknowns. In this manner six samples were obtained for analysis from each rabit: four unknowns, one standard, and one blank. The unknown samples or standards were added in small aliquots (0.i~0.3 In].) to 10-12 ml. of Tyrode in the mixing vessel. The Tyrode solution was then added until a total volume of 15 ml. was reached. The mixture was stirred and added to the chamber containing a portion of the ileum. The solution was allowed to remain in contact with the ileum for about 25-30 see. before it was removed. Two additional Tyrode rinses were employed and spaced about 20-30 see. apart before the next sample to be analyzed was added. Each unknown was followed by different aliquots of the standard. The procedure permitted the operator to arrive by trial and error at three different aliquots of the standard solution that produced contractions that were slightly smaller than the unknown sample, slightly larger, and one that was essentially identical. In most eases aliquots of all unknown samples were run at two different volumes and, when possible, these were run in duplicate. This procedure was necessary because the sensitivity of the ileum would
391
ACH IN SMALL BRAIN SAMPLES
change d u r i n g the course of t h e experiment. T h e m e t h o d p e r m i t t e d the p l o t t i n g of s t a n d a r d curves a t different times d u r i n g t h e bioassay. T h e m a x i m u m height of c o n t r a c t i o n was measured as well as t h a t a t 10- a n d 20-see. i n t e r v a l s following the a d d i t i o n of the samples. T h e m a x i m u m was found to occur between 20 a n d 40 sec. The u n k n o w n c o n c e n t r a t i o n of ACh was calculated b y comparing the height of c o n t r a c t i o n to t h e s t a n d a r d if identical in h e i g h t or b y i n t e r p o l a t i o n on t h e s t a n d a r d curves as explained above. T h r e e special t e s t s were used to i d e n t i f y t h e active substances as ACh. F i r s t t h e a c t i v i t y of t h e u n k n o w n solution was destroyed when i t was t r e a t e d w i t h NaOH. I t was also lost if the gut was p r e t r e a t e d with a solution c o n t a i n i n g atropine which is a specific antagonis t to ACh. Finally, if t h e b r a i n samples were n o t frozen i m m e d i a t e l y b u t left at room t e m p e r a t u r e for some time, the C h E in t h e tissue hydrolyzed the ACh, reducing its c o n c e n t r a t i o n markedly. T h e degree of d e s t r u c t i o n of the n e u r o h o r m o n e was m u c h less in the case of t h e animals receiving an antieholinesterase drug. RESULTS
The ACh recovery data for the reineckate precipitation procedure are presented in Table I. The per cent recovery of added ACh varied from 87 to 108 %. However, the majority of results obtained fell between 92 to 100 %. Since most of the unknowns contained approximately 0.6 pg. of ACh in the tissue sample taken for analysis, this amount of neurohormone was used per 3 m]. of final alcohol-Tyrode solution. The latter concentration of known ACh was determined ten times. The recoveries of ACh were found to be satisfactory. ,In Table II, the data from the experiments designed to test the reproducibility of the ACh determinations are given. Ten caudate nuclei TABLE I
Acetylcholine Recovery Data No. of t r ~ 15 1 1 1 10 1 2 1 1
ACh addeda
ACh recove~d
0 0.20 0.40 0.50 0.60 0.80 0.90 1.20 2.00
0 0.216 0.40 0.50 0.55-0.63 0.70 0.90, 0.94 1.14 1.92
#g.[3ml.
#go/3ml.
Per cent recovery -108 100 100 92-105 87 100, 105 95 96
T h e a l i q u o t a n a l y z e d was e i t h e r 0.1, 0.2, or 0.3 ml. T h e b i o a s s a y is sensitive t o 0.001 ~g./ml., a n d since t h e b a t h c o n t a i n s 15 ml., t h e a l i q u o t used in the analysis m u s t contain at least 0.015 ~g.
392
APRISON
AND NATHAN
TABLE II Reproducibility of Acetylcholine Determinations ° Sample No. b
10 11 14 15 18 19 23 24 Mean S.D.
Contraction Contraction Maximum Calculated ACh Calculated ACh Calculated ACh height at 10 he~ht at 20 contraction from data in from data in from data sec.C sec. e height e coLA col. B col. C ram. ram. ram. ~g./O.15 ml. ~g./O.I5 mL ~g./O.15 ~l. A B C
10 10 8.5 8.5 9 8 9 10 9.1 4-0.78
16.5 16 16 16.5 16 13.5 15 16 15.7 -4-0.99
23 25 27 27 25.5 25.5 24 23
0.031 0.031 0.029 0.029 0.038 0.035 0.030 0.033
0.030 0.030 0.032 0.033 0.039 0,035 0.031 0.035
0.029 0.032 0.035 0.035 0.035 0.035 ---
25 4-1.67
0.032 4-0.0031
0.033 4-0.0030
0.034 4-0.0026
The calculated values in the last three columns were obtained by comparison to the standard aliquots run after each set of unknown samples. b Each sample added was a 0.15-ml. aliquot of eaudate nucleus (28.6 mg./ml.). c Heights measured to nearest 0.5 mm. from five normal rabbits were obtained and carried through the procedure used in preparing a sample for ACh bioassay as previously described. The contents of these tubes were combined, and an aliquot containing 28.6 mg./ml, of tissue was taken for analysis. A C h was measured eight separate times; this was done by determining the unknown aliquot twice, preceded and followed by two standards (see Table II). The height of the contractions was measured as described above, and comparison with the standards were made. The results obtained are given in the last three columns of Table I I . The means agree very well and the three standard deviations are of the same order of magnitude. Consequently, the point in time of the comparison is not critical and the experimental conditions limit the length of time the ileum is allowed to contract due to the presence of ACh and other substances such as alcohol, reineekate, and choline that have been added or carried through the procedure. I t should be mentioned that too long a contact time of sample with the ileum will invariably cause it to go into a condition of spontaneous contractions and interfere with the bioassay. In addition, it was sometimes noted that in the case of some cerebral cortex samples, the rate of contraction of the ileum was not the same as that produced by the standard or pure ACh solution. In such cases maximum contraction
393
ACH I N SMALL BRAIN SAMPLES TABLE
III
Effect of Different Guinea Pig Ilia on the Bioassay of Acetylcholine Acetylchollne concentration found, ~g./ml. Gut number
0.1 ral. CI'~a
0.2 ml. CNa
Mean
1
--
0.183
2 3
0.206 0.212
0.167 0. 200 0.215 0.188
0.210 0.200
C N = c a u d a t e n u c l e u s s a m p l e (27.4 m g . / m l . ) ,
heights were used. The concentration of ACh found in the sample of combined caudate nuclei (five normal rabbits) was 7.70 ~g./g. The data from a typical experiment which indicate the effect of the use of different guinea pig ilia is shown in Table III. The unknown caudate nuclei sample was prepared as described above. The ilia from five guinea pigs were used in this experiment. Two different aliquots of the unknown were run along with the proper standards for each gut. The results were calculated in each ease as acetylcholine concentration (ug./ml.) found and are presented in Table III. The concentration of ACh (caudate nuclei from three normal rabbits) was 7.22 ~g./g. Spontaneous contractions occurred with one gut, while the sensitivity was too low in the case of another. These facts are not given in Table III and the reasons for their occurrence are unknown. Sometimes keeping a particularly active ileum in a large amount of Tyrode solution overnight at 7°C. will result in a preparation that has no spontaneous movement the next morning. Furthermore, samples and standards may be kept overnight (4°C.) without a significant change in activity. The ACh concentration and size of tissue sample from the right and left cortex as well as right and left caudate nucleus of normal albino rabbits are presented in Table IV. (Caudate samples were not determined in the first three animals.) The two results for the caudate nuclei given above may also be considered here. The recovery of ACh was found to vary between 92 and 105 %. Asymmetry in ACh content between right and left cortices was not noted in these normal rabbits. However, the ACh results varied considerably from animal to animal. The mean for t h e right cortex was 0.89 ~g./g, and for the left cortex was 0.87 ~g./g. The weight of the tissue samples from the cortex in these control rabbits varied in size from approximately 156 to 560 mg. The agreement in the means is better than one would expect since the bioassay procedures can have an inherent error of 10 %.
394
APRISON AND NATHAN TABLE IV
A C h Concentrations i n the B r a i n of N o r m a l A l b i n o Rabbits Left Caudate Right Caudate Left cortex Right Cortex Nucleus Nucleus Tissue Tissue Rabbit No. Tissue wt. ACh cone, Tissue wt. ACh conc. wt. ACh conc. wt. ACh conc. rag. l~g./g, rag. i~g./g, rag. ~g./g. rag. ~g./g. N-1 377.2 0.59 362.8 0.62 . . . . N-2 260.8 0.97 309.0 0.97 . . . . N-3 163.2 0.84 155.8 0.92 . . . . N-4 238.5 1.72 297.2 1.72 54.8 9.15 41.6 8.80 N-5 415.4 0.40 560.4 0.34 92.6 8.10 73.8 10.57 N-6 365.4 0.56 399.2 ' 0.46 72.0 4.68 65.6 6.35 N -7 241.4 0.55 312.2 0.54 43.4 3.04 53.4 2.78 1W-8 368.6 1.14 406.0 1.18 75.8 3.53 61.8 3.54 N-9 318.2 0.86 293.4 0.94 57.6 6.76 63.6 5.80 N-10 222.0 1.15 332.0 1.25 58.9 9.15 68.2 8.82
In the case of the caudate nuclei, the ACh results showed greater variability between animals. Furthermore, the difference in neurohormone concentration between corresponding right and left caudate nuclei was larger than in the case of the right and left cortices. However, this difference was not considered significant. The mean obtained in the case of the right caudate nuclei was 6.67 ~g./g. while for the left caudate nuclei it was 6.34 ~g./g. The concentration of ACh determined in the experiments described in Tables I and I i falls within the range of results shown in Table IV. Tissue samples of the eaudate nuclei varied in weight between 41.6 to 92.6 mg. DISCUSSION
The free ACh concentration found in the cortex of normal albino rabbits agrees with the results reported by Michaelis et al. (3). This value is about 25 % of the total ACh found in rabbit cortices by Tower and Elliott (4). The caudate nucleus which is functionally related to the cortex contains about seven times the free ACh concentration of the latter under normal conditions. When this comparison between the two brain areas is also made for the cholinesterase activity, it was found that the ratio is of the same order of magnitude (5). The method for measuring ACh as described in this paper has a number of advantages. It is sensitive, reproducible, accurate, rapid, and employs tissue from an easily obtainable experimental animal. Furthermore, the method permits an approach to the "ideal extraction." First, by rapidly freezing the excised area between pieces of Dry Ice (matter
ACH IN SMALL BRAIN SAMPLES
395
of seconds) and then homogenizing the tissue finely at 0°C. in the presence of eserine, the a m o u n t of ACh present in the extract should correspond closely to t h a t in the brain p a r t at the m o m e n t of excision. Stone (6) reported t h a t the difference in the average value for cortical tissue frozen i n situ and t h a t from tissue frozen a few seconds after excision is not statistically significant. Secondly, centrifugation to remove the particulate m a t t e r also removes most of the cholinesterase activity (7). 8 Finally, b y performing the reineckate-choline-acetylcholine precipitation procedure, material which can interfere with the biological assay of ACh is removed or reduced to noninterfering concentrations. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Dr. H. E. Himwich for his sustained interest and encouragement throughout the course of this work. The authors also gratefully acknowledge the technical assistance of Mrs. Madeline Griffin.
SUMMARY A method for the determination of "free" or soluble ACh in small pieces of fresh brain tissue has been developed and patterned in part after the one reported by Bentley and Shaw. Brain samples as small as 40 mg. of caudate nucleus and 155 mg. of cortex have been analyzed. Values for "free" ACh in the right and left cerebral cortex and caudate nucleus are given for normal albino rabbits. REFERENCES 1. API~ISON,M. I-I., AND NATHAN, P., Federation Proc. 15, 5 (1956). 2. BENTLEY, G. A., ANDSHAW,F. I-I., J. Pharmacol. Exptl. Therap. 106, 193 (1952). 3. MICI-IAELIS,M., FINESINGER,J. E., VERSTER, F. DE B., AND ERICKSON,R. W., J. Pharmacol. Exptl. Therap. 111, 169 (1954). 4. TowEn, D. B., AND ELLIOTT, K. A. C., Am. J. Physiol. 168, 747 (1952). 5. A•nISON, M. H., AND HIMWICH,H. E., Am. J. Physiol. 179, 502 (1954). 6. STONE, W. E., Arch. Biochem. and Biophys. 59, 181 (1955). 7. NATHAN, P., AND APRISON, M. H., Federation Proc. 14, 106 (1955). a Also, manuscript in preparation.