STUDIES WITH AGENTS WHICH INFLUENCE ACETYLCHOLINE METABOLISM IN MOUSE BRAIN. ARTICLE FROM CIBA-GEIGY CORP.

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• Publication Date: June 1, 1974
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Reprinted from Archives internationales de Pharmacodynamie et de Thdrapie
Vol. 209 - No. 2 - June 1974
Studies with Agents which Influence Acetylcholine Metabolism
in Mouse Brain
J. K. SAELENS, J. P. SIMILE, J. SCIIUMAN AND M. P. ALLEN
Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit,
New Jersey, 07901-U.S.A.
Abstract-By using an intravenous single pulse injection of choline-C'4
into the tail vein of mice and measuring the endogenous levels of
choline and acetylcholine, the effects of agents which influence acetyl-
choline metabolism in mouse brain were studied. The results were
consistent with a feedback activation of cholinergic neurons with scopola-
mine, choline-0-acetyltransferase inhibition with naphthylvinylpyridine,
inhibition of acetylcholine release by morphine and cholinesterase
inhibition with physostigmine. The interactions of these agents with the
cholinergic system further suggests certain characteristics of cholinergic
neurons, e.g. that choline-0-acetyltransferase activity is accelerated by
a decrease in intracellular acetylcholine levels and retarded by an
increase in intracellular acetylcholine levels, that the choline transport
mechanism is functioning at maximum capacity under normal physiol-
ogical conditions and that the synaptic levels of endogenous choline play
a role in tracer choline capture.
Introduction
In 1969 Schuberth, et al. (1) introduced a technique for measuring the rate
of synthesis of acetylcholine (ACh) in whole mouse brain. In their procedure the
rate of radiactive ACh formation in the brain was measured following an intra-
venous pulse injection of tritium-labelled choline (Ch). After determination of
the isotope dilution of the Ch in the brain, the ACh rate of synthesis, in vivo,
was calculated. We have now applied this technique involving four agents
purported to affect central cholinergic neurons in different ways. The agents are
the muscarinic cholinergic receptor blocking agent, scopolamine; the choline-0-
acetyltransferase inhibitor, naphthylvinylpyridine (2, 3) ; the cholinesterase
inhibitor, physosti.gmine; and an agent reported. to block the release of ACh,
morphine (4, 5, 6). The results presented below are consistent with the proposed
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mechanisms of the various agents and help to further define certain character-
istics of central cholinergic neurons.
Administration of Choline-Methyl-C'4 (Ch-C14). Male CF1S (Carworth Farms)
mice, 18-20 g were used. Each mouse received 5?c (0.66 ? moles) of Ch-C14
chloride (New England. Nuclear, specific activity = 7.6 ?c/? mole) intravenously
in a tail vein. The tracer was dissolved in distilled water and administered in a
volume of 0.1. ml with a gas-tight Hamilton syringe through a 30 gauge needle.
Preparation of Brain Samples. The mice were sacrificed at different times after
injection by rapid immersion of the whole animal into a Dewar flask containing
liquid N2 for 3.5 seconds. Individual mouse brains were removed as rapidly as
possible, dissected on a thermoelectric cold plate maintained at 1-2? C, and
frozen in liquid N2 . Where whole brain analysis was performed, the brain was
placed directly in liquid N2. Timing was critical with this method. If the
immersion lasted for more than 4 seconds, the surface of the cortex began to
freeze and stick to the skull making removal of the tissue difficult. The frozen
brains or brain parts were weighed on a Mettler electronic balance and then
pulverized in a Thermovac (R) tissue crusher cooled with dry ice and liquid
nitrogen. The remainder of the procedure was as described for rats by Saelens
et al. (7).
Determination of ACh and Ch. ACh and Ch were determined by the enzymatic
method of Feigcnson and Saclens (8) using the modifications recommended by
Saelens et al. (9).
Statistical Analysis. The disposition of Ch-C14, and the levels of Ch and ACh
were determined in different groups of mice. In order to obtain the appropriate
parameters, the product of two independent variables were analyzed by the
method of Goodman (10). At least 5-10 mice were used in all measurements.
Agents. Test agents were dissolved in distilled water, and administered
subcutaneously, or intraperitoneally, in a volume of 0.1 ml/10 g of body weight
or intravenously in a volume of 0.05 ml/10 g of body weight. Mice received food
and water ad libitum up until the time of sacrifice.
The agents studied were scopolamine hydrobromide (Merck), naphthylvinyl-
pyridine (Aldrich), physostigminc sulfate (Merck) and morphine sulfate (Merck).
Control Studies: The Schuberth model for estimating the rate of synthesis of
ACh is essentially a single closed compartment model. To be valid, the level of
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J. K. SAELENS, J. P. SIMI{E, J. SCHUMAN AND M. P. ALLEN
precursor tracer should be reasonably constant during the time course of product
tracer formation. Further, product tracer formation should be linear over the
same time course and should be as far below the equilibrium point as possible.
Figure 1 shows that the first 2 criteria were satisfied for the first 45-60 seconds
after intravenous administration of the Ch-C14. Maximal Ch-C14 levels were
found in the whole mouse brain within 5 seconds. The total dpms of Ch-C14
30,000
20,000
C14 - CHOLINE
10,000
0 5,000
J
2,000
0
1,000
40
10 20 30 40 50 60
SECONDS AFTER INTRAVENOUS INJECTION OF CHOLINE -
METHYL- C14
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ACETYLCIIOLINE METABOLISM 253
remained relatively constant for 45-60 seconds and during that time the ACh-
C14 levels increased in a linear fashion.
The question of equilibrium necessitated looking at the tracer product-
precursor relationship over a longer time course. Mice were injected with
Ch-C14 intravenously and sacrificed at different time intervals up to 60 minutes.
The ACh-Ch'4/Ch-C14 ratio vs. time is shown in Figure 2 for various parts of
mouse brain and whole mouse brain. In all cases, the ratio of ACh-14 to Ch-C14
rose rapidly, peaked between 5 and 15 minutes and fell slowly over the remainder
of the time course. The absolute amounts of ACh-14 and Ch-C14, meanwhile,
fell continously throught the time course (data not shown). These data suggested
that the model behaved as a single closed compartment system only for the first
0 10 20 30 40 50 60
Second after I.V. Ch- C~4 injection
2500 0
S-f WHOLE BRAIN
?-----i CEREBELLUM
?--?----? MIDBRAIN
??---?-? CORTEX
10 20 30 40 50 60
MINUTES AFTER I.V. Ch-C14 INJECTION
U
500 a
0
E
CL
v
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few minutes after administration of the tracer. ACh-C14/Ch-C14 ratios obtained
at one minute or less were clearly below the peak ratios and therefore data
generated using this time interval satisfied the criteria of being below equilibrium
for product tracer formation. When the pharmacological agents were used, the
mice were always sacrificed 45 seconds after administration of the Ch-C14 and
30 minutes after administration of the agent.
As a working hypothesis, it is suggested that the tracer was equilibrating with
a part of the free Ch pool which was readily available for ACh synthesis during
the rise of the ratio curve, and the peak and. fall were the result of the tracer
beginning to equilibrate with other parts of the Ch pool. This interpretation is
supported by the delayed appearance of organic soluble tracer (in the acetone-
chloroform extract usually discarded when measuring endogenous ACh and Ch).
If this hypothesis is correct, then the peaks in the ACh-C14/Ch-C14 ratio curves
should be reasonable, albeit conservative, estimates of the ratio between en-
dogenous ACh and the portion of the Ch pool suggested to be available for
ACh synthesis. The peak ratios, therefore, allowed an indirect estimate of the
size of the available free Ch pool (AF Ch) using the following equation:
AF Ch - C11
= LACK - C14] X ACh (endogenous)
at peak
Using this indirect technique, the sizes of the AF Ch pools and the synthesis
rates of ACh in the whole brain, cortex, midbrain, brainstem and cerebellum
were estimated and are shown in Table I. Under these conditions, it was esti-
mated that the stores of ACh in mouse brain would be replaced every 2-3 minutes.
Using the direct technique, it should be noted that the endogenous brain Ch
levels of control animals shown in Table 2 were approximately twice the AF Ch
estimated above. Recent investigators, particularly Ewetz et at. (11), suggest that
post-morten increases in Ch levels due to catabolic destruction of Ch containing
compounds in the brain, are very difficult to avoid. It is possible that, despite
the precautions taken, the Ch levels in Table II include some Ch originating
from such sources. For this reason, when the single closed compartment calcula-
tions for rate of synthesis of ACh were applied. to the data presented here the
results were expressed as "apparent rate of synthesis."
Scopolamine: At 10 mg/kg i.p., scopolamine caused a 50 % decrease in endog-
enous ACh levels. However, the amount of ACh-C14 found in the brain at
45 seconds was actually slightly increased. Thus, the specific activity of the
reduced endogenous ACh pool was 2.4 times the specific activity of the corre-
sponding control ACh pool. This dose of scopolamine also caused a statistically
significant decrease in the endogenous Ch levels and a slight decrease in the
45 second Ch-C14 levels. All the data with scopolamine is consistent with a
feedback activation of cholinergic neurons resulting from cholinergic receptor
blockade. The conversion of Ch-C74 to ACh-C14 was clearly accelerated as
illustrated by the large increase in ACh specific activity but the reduction in the
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endogenous Ch pool counterbalanced the significantly higher ACh-CY4/Ch-C14
ratio resulting in no change in the apparent synthesis of ACh. The reduction in
ACh levels caused by scopolamine is most likely a matter of synthesis being
unable to keep up with the drug-induced facilitation of release. These results
suggest 2 characteristics of cholinergic neurons. First, the choline-0-acetyl-
transferase activity is accelerated by a decrease in ACh levels and/or Ch levels,
the former, alone, being the most logical. Second, Ch capture is not accelerated
by the decrease in ACh or Ch levels suggesting that the Ch transport mechanism
is functioning at maximum capacity under normal physiological conditions.
Estimates of the AFCholine Pools and Rates of Synthesis of Acetylcholine in Various Brain
Areas of Control Mice
Estimates
of Rate of
rACh - Cla b
Estimate
rACh C4
Synthesis
Mean
I
AF Che
-
of ACh
ACha m?
Ch. C''
J
m?
Ch C'4 J
L
m? Moles/
Area
Moles/g
peak
Moles/g
1 minute
g/minute
Whole Brain
13.6
0.550
24.7
0.263
6.50
Cortex
12.5
0.635
19.6
0.339
6.64
Midbrain
18.5
0.720
25.6
0.244
6.25
Brainstem
19.7
0.661
29.8
0.189
5.63
Cerebellum
6.9
0.201
33.6
0.040
1.34
u rACh-C"
IL Ch - Cln
peak
AF Ch-Available Free Choline-the hypothetical choline pool suggested to be
available for acetylcholine synthesis.
Nap hthylvinylpyridine (NVP). The choline-0-acetyltransferase inhibitor,
NVP, caused a dose related increase in endogenous ACh levels. The a priori
expectation would be just the opposite, i.e. a decrease in endogenous ACh levels.
However, others have also failed to demonstrate ACh depletion with NVP (2, 3).
The increase in endogenous ACh was associated with a dose related decrease in
the 45 second ACh-CIA levels which was statistically significant at 40 mg/kg i.v.
The specific activity of ACh in NVP treated animals also showed a dose related
decrease which dropped to 40 % of control at 40 mg/kg i.v. Thus, NVP, at
reasonable doses, was quite capable of depressing the conversion of Ch-C14 to
ACh-C14 but was incapable of reducing storage levels of ACh. The Ch transport
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256 J. K. SAELENS, J. P. SIMKE, J. SCHUMAN AND M. P. ALLEN
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ACETYLCIIOLINE METABOLISM 257
mechanism is also apparently independent of choline-0-acetyltransferase activity
as NVP clearly depressed the conversion of Ch-C14 to ACh-C14 but had no effect
on the capture of Ch-C14 from the bloodstream. Therefore, there is no evidence
here, that choline-0-acetyltransferase is a Ch acceptor inside the nerve ending
necessary for the Ch transport mechanism to function properly. NVP did cause
a significant reduction in the apparent rate of synthesis at 40 mg/kg i.v. Why the
ACh levels went up instead of down is still unclear.
Morphine: In a dose range of 1-10 mg/kg s.c. morphine was noteably ineffective
in altering all parameters involving ACh but dramatically increased the 45 second
Ch-C14 levels without altering endogenous Ch levels. The apparent rate of ACh
synthesis was significantly decreased by all doses of morphine tested but clearly
this was a consequence of increased Ch-C14 capture. There is considerable
evidence that morphine impairs the release of ACh in the brain (4, 5, 6). There
is also some evidence that ACh released into the synaptic cleft is hydrolyzed by
cholinesterase and the resulting Ch transported back into the cholinergic nerve
ending (e.g. 7). Morphine is almost doubled in the 45 second Ch-C14 capture at
10 mg/kg s.c. It is tempting to speculate that these 3 events are interrelated. The
tracer pulse most probably passes by the brain as a bolus where the peak specific
activity is exposed to the nervous tissue for a very short period of time. Once
into the extracellular space, including cholinergic neuron synapses, it is taken
up by the Ch transport mechanism into the neuron. At this juncture, the tracer
Ch-C14 is suggested to undergo some amount of isotope dilution from the non-
radioactive Ch hydrolyzed from the non-radioactive ACh being continuously
released during transmission. If morphine impairs the release of ACh, the tracer
should undergo a lesser amount of isotope dilution as the source of dilution, the
released ACh, is diminished. It is suggested that the cholinergic nerve endings
are not taking up a larger quantity of Ch but rather are taking up the same
quantity of Ch which has a higher proportion of Ch-C14. This hypothesis is
consistent with the fact that the endogenous Ch levels do not change under the
influence of morphine.
Physostigmine: The above hypothesis regarding morphine and Ch-C14 capture
was challenged by reducing the availability of synaptic Ch in a different way.
Physostigmine does not impair the release of ACh (2), but does prevent the
hydrolysis of ACh to Ch in the synapse. With 0.1 mg/kg s.c. of physostigmine,
again, the 45 second Ch-C14 levels were substantially higher than corresponding
controls. It was also of interest to note that physostigmine significantly decrease
the 45 second ACh-C14 levels and specific activity of ACh supporting the
contention of Kaita and Goldberg (12) that feedback inhibition by intracellular
ACh plays a role in ACh synthesis. The effects of physostigmine on ACh related
parameters also clearly distinguishes it from morphine.
Acknowledgment-The authors would like to acknowledge the valuable assistance of
Mr. C. Patel who performed the statistical analysis on the data presented here and the
invaluable skills and efficiency of Miss C. Laspina.
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258 J. K. SAELENS, J. P. SIMKE, J. SCHUMAN AND M. P. ALLEN
1. SCIIUBER'rII, J., SPARF, B. and SUNDWALL, A. Y. Neurochem. 16, 695 (1967).
2. KRELL, R. D. and GOLDBERG, A. M. Trans. Amer. Soc. Neurochem. 4, 129 (1973).
3. GLICIK, S. D., MITTAG, T. W. and GREEN, J. P. Neuropharmacol. 12, 291 (1973).
4. BELESIIIN, D. and POLAR, R. L. Y. Physiol., Lond., 177, 411 (1965).
5. BELESIIIN, D., POLAR, R. L. and SPROULL, D. H. Y. Physiol., Lond., 177, 420 (1965).
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7. SAELENS, J. K., SIl4IKE, J. P., ALLEN, M. P. and CONROY, C. A. Arch. int. Pharmacodyn.
203, 305 (1973).
8. FIIIGENSON, M. E. and SAELENS, J. K. Biochem. Pharmacol. 18, 1479 (1969).
9. SAELENS, J. K., ALLEN, M. P. and SIMI{E, J. P. Arch. int. Pharmacodyn. 186, 279 (1970).
10. GOODMAN, L. A. Y. Amer. Statist. Assoc. 55, 708 (1960).
11. Ewu'rz, L.; SPARr, B. and SoRBO, B. Symposium on in vitro Procedures With Radioiso-
topes in Medicine in Vienna, 1969. Ed. Int. Atomic Energy Agency SM-124/39, 175
(1970).
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