Evaluation of the necessity for rapid inactivation of brain enzymes prior to analysis of norepinephrine dopamine and serotonin in the mouse.

Norepinephrine, dopamine and serotonin concentrations were measured in mouse whole brain. Animals were killed either by decapitation or by exposure to 250 msec microwave irradiation which produces irreversible inactivation of brain enzymes. The biogenic amines were determined by mass fragmentometry, fluorometry and by a combination of high performance liquid chromatography and an electrochemical detector. No differences were found in the levels of these neurochemicals between the two methods of animal sacrifice. Therefore, rapid inactivation of brain enzymes is not necessary prior to analysis for catecholamines and serotonin in mouse whole brain.     

Levels of cyclic nucleotides in mouse regional brain following 300 ms microwave inactivation.

Abstract— The uniformity and speed of inactivation of mouse brain adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterase were measured after 6 kW microwave irradiation (MWR). Inactivation of enzymes was uniform throughout the brain during heating and 100% loss of activity was evident after 300 ms. MWR. For comparison of effects of inactivation times on levels of cyclic nucleotides measured in regional brain areas, cyclic AMP and cyclic GMP were estimated after 1.5 kW MWR requiring 4 s of heating and 6 kW MWR requiring 300 ms. Except for corpus striatum, uniformly lower levels of cyclic AMP were measured following 300 ms vs. 4s MWR . There was no change in cyclic GMP levels in regional brain areas after 4s vs. 300 ms MWR . Cyclic AMP and cyclic GMP were measured from the same regional brain tissue samples after 300 ms and ratios calculated. The finding of much lower cyclic AMP:cyclic GMP ratios than had previously been reported suggests that slow inactivation times provide for the measurement of regional brain cyclic nucleotide values which are not consistent with the in-vivo state.     

THE USE OF MICROWAVE HEATING TO INACTIVATE CHOLINESTERASE IN THE RAT BRAIN PRIOR TO ANALYSIS FOR ACETYLCHOLINE

Abstract— Heating with 2450 MHz microwave radiation has been investigated as a means for animal sacrifice concurrent with enzyme inactivation. Uniform inactivation of cholinesterase (EC 3.1.1.8) in the entire brain can be effected in the rat within 4 s and in the mouse within 2 s without destruction of acetylcholine. The acetylcholine content in the whole brain of a rat was found to be 25.4 ± 1.5 nmol/g after irradiation, in comparison to 13.8 ± 1.7 nmol/g after standard methods of sacrifice. In the mouse whole brain, the comparable acetylcholine contents were 25.5 ± 2.6 and 13.7 ± 1.7 nmol/g, respectively. The value of this procedure for rapid inactivation of enzymes in the study of acetylcholine turnover is discussed.     

Identification of acetylcholine and propionylcholine in bull spermatozoa by integrate pyrolysis, gas chromatography and mass spectrometry

Acetylcholine was identified in fertile bull spermatozoa using combined pyrolysis gas-chromatography and mass spectrometry. Bull spermatozoa contain other minor choline esters in smaller quantities than acetylcholine. One of the minor choline esters is possibly propionycholine. The spermatozoa from infertible bulls exhibited low motility and very low levels of acetylcholine.     

LEVELS OF NOREPINEPHRINE AND DOPAMINE IN MOUSE BRAIN REGIONS FOLLOWING MICROWAVE INACTIVATION-RAPID POST-MORTEM DEGRADATION OF STRIATAL DOPAMINE IN DECAPITATED ANIMALS

—The effects of 2 methods of killing on norepinephrine and dopamine in mouse brain regions were examined. One method utilized decapitation, while the other method utilized heating with microwave irradiation concentrated on the head. The norepinephrine and dopamine contents of the cerebellum, medulla-pons, midbrain, diencephalon, hippocampus, corpus striatum, and cerebral cortex were determined by methods using liquid chromatography with electrochemical detection. Dopamine content in striatum was also quantitated by the method of gas chromatography with mass fragmentography. A significantly lower value for decapitated animals, as compared to the microwave heated group, was found only for dopamine exclusively in the striatum. Activities of the enzymes tyrosine hydroxylase, DO PA decarboxylase, monoamine oxidase, and catechol-o-methyltransferase in the striatum were also examined. These enzymes were totally inactivated by the microwave heating, except catechol-o-methyltransferase which was decreased approx 80%. These results support either (1) the existence of a substantial pool of dopamine in the striatum with a very rapid turnover rate or (2) a decapitation-related release and destruction of striatal dopamine. Measurements of 3-methoxytyramine in the striatum exhibit post-mortem increases corresponding to the decreases of dopamine. Use of the rapid tissue enzyme inactivation technique suggests that in vivo levels of this O-methylated dopamine metabolite are an order of magnitude lower than the results normally obtained after killing by decapitation.     

Rate of inactivation of adenyl cyclase and phosphodiesterase: determinants of brain cyclic AMP

In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.