Brain fixation for acetylcholine measurements.

Brain fixation using a commercially available microwave oven (power output: 750 W) has been investigated as a means for enzyme inactivation preventing post-mortem changes in brain acetylcholine (ACh) and choline (Ch) levels. Rats and mice were decapitated, and the severed heads immediately irradiated for 5.5 and 3 s, respectively, resulting in a complete inactivation of brain acetylcholine esterase (AChE) and choline acetyltransferase (ChAT). The ACh and Ch contents measured in various brain regions of rat and mouse were: (in rat) striatum 60.5 and 32.4 nmol/g, hippocampus 20.4 and 30.9 nmol/g, cortex 24.2 and 19.6 nmol/g; (in mouse) striatum 70 and 47.2 nmol/g, hippocampus 22. 1 and 30.2 nmol/g, cortex 22.9 and 27.9 nmol/g. These values were found in accordance with those reported in the literature by irradiating whole animals in instruments of higher power capabilities. Thus, the procedure described in the present work may be a simple and valuable means of brain fixation for neurochemical studies of brain ACh in small animal species.

In vivo studies of the effect of magnetic field exposure on ontogeny of choline acetyltransferase in the rat brain.

Developmental increases of the activity of choline acetyltransferase (ChAT) were examined in the brains of fetuses and offspring from parent rats continuously exposed to a 500 mG, 60 Hz circularly polarized (CP) magnetic field (MF) prior to pregnancy, and further, during pregnancy and lactation. In developing rats between 12 days and 20 days of embryogenesis that were housed in a control unit, i.e., nonexposed to MF, the specific activity of ChAT in whole brain specimens increased from 2.4% to 6.9% of adult activity, while specific activity of ChAT in rat brain specimens between 12 days of embryogenesis and 10 days of postpartum increased from 2.4% to 21.6% of adult activity. On the other hand, the specific activity of ChAT in whole brain specimens from rats under housed MF exposure conditions was found to increase from 2.6% to 6.7% of adult activity between 12 days and 20 days of embryogenesis and from 2.6% to 21.6% of adult activity between 12 days of embryogenesis and 10 days postpartum. Furthermore, the effect of the same test magnetic environment on the specific activity of ChAT in the brains of parental rats was examined in order to determine whether magnetic field exposure of parental rats might reflect onto the development of fetal brain. It was observed that continuous exposure of parental rats to a 500 mG, 60 Hz CP MF did not show any significant changes in the specific activity of ChAT in the septodiagonal band complex, dorsal and ventral hippocampus, striatum, and frontoparietal cerebral cortex, as compared with the same brain regions of control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Choline acetyltransferase.

Acetylcholine is essential to neural function. It synthesis is catalyzed by choline acetyltransferase, the enzyme responsible for the acetylation of choline by acetyl coenzye A, a reaction favored slightly thermodymodynamically and not at all kinetically. An analytically pure enzyme still has not been obtained; however, method of purification have been greatly improved recently. Numerous inhibitors of the enzyme have been synthesized and their structure-action relationships examained. Evidence has been accumulated showing the essential involvement of an imidazole group in the active site of choline acetyltransferase. The literature regarding the controversial role to thiol groups in choline acetyltransferase is reviewed. Recently, derivatives of coenzyme A have been introduced as inhibitors of this enzyme and the specificity of coenzyme A binding has been examined. Possible mechanisms responsible for the control fo acetylcholine synthesis are discussed.

Microwave fixation of brain tissue as a neurochemical technique- a review.

Microwave devices have been developed for rapidly inactivating brain enzymes by focusing the power output into the heads of small laboratory animals. The rapid inactivation achieved prevents postmortem changes and permits the measurement of neurochemicals such as acetylcholine at concentrations close to those obtained in vivo. The technique promises the assay of neurochemical parameters not possible before.

Use of 300-msec microwave irradiation for enzyme inactivation: a study of effects of sodium pentobarbital on acetylcholine concentration in mouse brain regions.

Microwave irradiation of 6 kw at 2450 MHz for 300 msec was sufficient to completely inactivate mouse brain cholinesterase and choline acetyltransferase. After this method of sacrifice, the acetylcholine contents of mouse brain regions, given in nanomoles per gram, were found to be: striatum, 81; medulla-pons, 44; diencephalon-midbrain, 34; hippocampus, 31; cerebral cortex, 26; and cerebellum, 17. Sodium pentobarbital caused a dose-dependent increase in whole brain acetylcholine. A maximal increase of 81% in whole brain was seen at 15 minutes with 80 mg/kg of sodium pentobarbital. The increase in acetylcholine after sodium pentobarbital treatment was not caused by anoxia from respiratory depression or by hypothermia. All brain regions except the cerebellum exhibited an increase in acetylcholine after pentobarbital treatment. Fifteen minutes after treatment, cerebellar acetylcholine was significantly decreased. However, at the time when half of the animals had regained the righting reflex, the unconscious mice showed an increase in cerebellar acetylcholine which was statistically significant as compared to control. The relative accumulation rate of acetylcholine calculated for cerebral cortex and hippocampus was higher than that for striatum although the absolute rate of accumulation of ACh was higher in the striatum. Thus, after sodium pentobarbital treatment, the cerebral cortex and hippocampus exhibit a greater cholinergic response than the striatum.