William B. Stavinoha

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.

Behavioural Pharmacological Characteristics of Honokiol, an Anxiolytic Agent Present in Extracts of Magnolia Bark, Evaluated by an Elevated Plus-maze Test in Mice

Honokiol, a neolignane derivative of Magnolia bark, has central depressant action and, at much lower doses, anxiolytic activity. We have investigated the characteristics of the behavioural effects of honokiol by means of an elevated plus‐maze test.

THE LEVELS OF LABILE INTERMEDIARY METABOLITES IN MOUSE BRAIN FOLLOWING RAPID TISSUE FIXATION WITH MICROWAVE IRRADIATION

The levels of several labile glycolytic and organic phosphate metabolites in mouse brain were determined following rapid inactivation with 2450 MHz microwave irradiation. The levels of ATP in mouse brain following a 0·25 s exposure in a 6 kW microwave oven was found to be 2·416 ± 0·061. Whole brain levels of 8 labile intermediary metabolites in 0·4 s irradiated samples were comparable to those reported using the previously‐described methods of freeze‐blowing or whole‐animal immersion. Analysis of these same metabolites in 4 gross areas of brain did not reveal any anoxic changes betwen superficial and deeper brain areas. The advantages of the mcrowave irradiation inactivation technique for regional brain studies of labile intermediary metabolites is discussed.

Choline Content of Rat Brain

The free choline concentration in the rat brain was found to be 26.3 nanomoles per gram of brain tissue. This value was obtained through use of 6-second microwave irradiation for killing animals and inactivating enzymes, followed by a pyrolysis-gas chromatographic assay procedure. The identities of compounds measured from brain samples were verified by mass spectrometry.

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

Abstract 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.

Honokiol, a putative anxiolytic agent extracted from Magnolia bark, has no diazepam-like side-effects in mice

Use of the elevated plus-maze experiment and activity and traction tests in mice have revealed that seven daily treatments with 0.2 mg kg(-1) and higher doses of honokiol, a neolignane derivative extracted from Magnolia bark, had an anxiolytic effect without change in motor activity or muscle tone. Diazepam, 1 mg kg(-1), had the same anxiolytic potential as 0.2 mg kg(-1) honokiol but induced muscle relaxation. The aim of this study was to determine whether honokiol had diazepam-like side-effects. Mice treated with 1-10 mg kg(-1) diazepam, but not those treated with 0.1-2 mg kg(-1) honokiol, for 12 days showed withdrawal symptoms characterized by hyperactivity and running-fit when they were challenge-administered intraperitoneal flumazenil (10 mg kg(-1)) 24 h after the last treatment with diazepam. Oral diazepam (0.5-2 mg kg(-1), 10 min before) dose-dependently prolonged hexobarbital (100 mg kg(-1), i.p.)-induced sleeping, disrupted learning and memory, and inhibited (+)-bicuculline (40 mg kg(-1), i.p.)-induced death. Honokiol (0.2-20 mg kg(-1), p.o., 3 h before) had no such effects. The prolongation by diazepam (1 mg kg(-1)) of hexobarbital-induced sleeping was not modified by honokiol (0.2-20 mg kg(-1)). These results suggest that honokiol is less likely than diazepam to induce physical dependence, central depression and amnesia at doses eliciting the anxiolytic effect. It is also considered that honokiol might have no therapeutic effect in the treatment of convulsion.

RATE OF ACCUMULATION OF ACETYLCHOLINE IN DISCRETE REGIONS OF THE RAT BRAIN AFTER DICHLORVOS TREATMENT

Abstract– The time course for accumulation of acetylcholine was measured in rat brain regions after treatment with 15 mg/kg, i.v., dichlorvos. With this dose of dichlorvos 84‐96% of the brain cholinester‐ase is inhibited within 1 min. After killing and concomitant enzyme inactivation through microwave irradiation, the acetylcholine levels were measured by pyrolysis‐gas chromatography. In the brain regions studied, the striatum had the highest rate of accumulation of acetylcholine and the cerebellum had the lowest. The calculated turnover time in minutes for the regions of the brain were cerebral cortex 0.9; hippocampus 1; striatum 1.4; cerebellum 1.7; medulla‐pons 2.2; midbrain 4.5; thalamus 5.6.

Effect of chronic ingestion of lead on the central cholinergic system in rat brain regions

Abstract The effect of chronic lead ingestion on the central cholinergic system of immature rats was investigated. Rats were given 1% lead acetate solution in the drinking water before and after weaning. Tap water and sodium acetate controls were used. The growth of rats treated with lead was significantly inhibited as compared to controls. After chronic lead ingestion, a significant decrease in cholinesterase activity was seen in the medulla-pons, midbrain, and diencephalon, and a significant increase in choline acetyltransferase activity was found in the medulla-pons, hippocampus, and cerebral cortex. The only brain region exhibiting a change in acetylcholine content after chronic lead administration was the diencephalon, where a significant increase was observed. Thus, significant changes in the central cholinergic system have been found after chronic lead ingestion.

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

Abstract 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.

Endorphins in brains of decapitated and microwave-killed mice.

Abstract The size distribution of opioid peptides (endorphins) in mouse brain was determined by gel filtration, using the inhibition of stereospecific 3H-etorphine binding to guinea pig brain membranes as a measure of endorphin activity. The largest endorphin was about the same size as β-LPH-(61–91), the next largest was about half this size and the smallest was the size of the pentapeptide enkephalins. There was no important difference in the size distribution between brains of mice killed by rapid microwave irradiation and those killed by decapitation. Thus, opioid peptides of all three size classes appear to be present in the brain in vivo .