Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3 252 J. K. SAELENS, J. P. SIMKE, 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-C, Maximal Ch-C™ levels were found in the whole mousc brain within 5 seconds. The total dpms of Ch-C™ 30,000 20,000 c!4 - CHOLINE 10,000 5,000 4,000 c!4- ACETYLCHOLINE TOTAL DPM OF TRACER/ WHOLE MOUSE BRAIN \ 10 20 30 40 50 60 SECONDS AFTER INTRAVENOUS INJECTION OF CHOLINE - METHYL — c!4 Fic. 1 Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3
eae nee, Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3 ACETYLCIIOLINE METABOLISM 253 remained relatively constant for 45-60 seconds and during that time the ACh- C™ 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-C" intravenously and sacrificed at different time intervals up to 60 minutes. The ACh-Ch"4/Ch-C" 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-4 to Ch-Cl4 rose rapidly, peaked between 5 and 15 minutes and fell slowly over the remainder of the time course. The absolute amounts of ACh-“ and Ch-C¥, 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 0.8 ee, 0.7 fo, oN \ O 10 20 30 40 50 60 ay Seconds after IV, O64 FS QO Ch- C!4 injection 0.5 0.4 0.3 RATIO OF Ach-c!4/ch-¢!4 0.2 4h a 1000 @——® WHOLE BRAIN H Fe @ CEREBELLUM 0.1 @-------@ MIDBRAIN 500 ‘ e-.-. ~@ CORTEX @----@ BRAINSTEM @-.--—@ ORGANIC TRACER ) 10 20 30 40 50 60 MINUTES AFTER 1.V. Ch-c!4 INJECTION a ° fo} dpm ORGANIC SOLUBLE TRACER (WHOLE BRAIN) Fie, 2 Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3
Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3 254 J. K. SAELENS, J. P. SIMKE, J. SCHUMAN AND M. P. ALLEN few minutes after administration of the tracer. ACh-C™“/Ch-C" 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-C™ 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-C™/Ch-C"™ ratio curves should be reasonable, albcit conservative, cstimates 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: — 14 AF Ch = [ew 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 ai. (11), suggest that post-morten increascs in Ch levels duc 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 IT 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-C™ 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-C* levels. All the data with scopolamine is consistent with a feedback activation of cholinergic neurons resulting from cholinergic receptor blockade. The conversion of Ch-C™ to ACh-C™ was clearly accclerated as illustrated by the large increase in ACh specific activity but the reduction in the Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3
Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3 ACETYLCHOLINE METABOLISM 255 endogenous Ch pool counterbalanced the significantly higher ACh-C#/Ch-C™ ratio resulting in no change in the apparent synthesis of ACh. ‘The reduction in ACh levels caused by scopolamine is most likely a mattcr 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. 'TaBce I Estimates of the AF Choline Pools and Rates of Synthesis of Acetylcholine in Various Brain . Areas of Control Mice ET a ae Estimates of Rate of ACh — cl#]” Estimate] [ACh — C14 Synthesis Mean ———_——- AF Ché || of ACh ACh® mp Ch — c™ mu Ch — Cc mp 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 Cercbellum 6.9 0.201 33.6 0.040 1.34 @ ACh = Acetylcholine endogenous. ® TACh — cu = the peak ratios from Figure 2. Ch — Ct peak e¢ AF Ch—Available Free Cholinc-—the hypothetical choline pool suggested to be available for acctylcholine synthesis. Naphthylvinylpyridine. (NVP). The choline-0-acctyltransferase inhibitor, NVP, caused a dose related increase in endogenous ACh levels. The @ 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-C#! 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-C™ to ACh-C" but was incapable of reducing storage levels of ACh. The Ch transport Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3