ON THE TURNOVER OF ACETYLCHOLINE IN NERVE ENDINGS OF MOUSE BRAIN IN VIVO

Abstract— Acetylcholine is synthesized and stored in the nerve endings from which the liberation of the nerve transmittor is regulated by the nerve activity. The aim of the present investigation was to measure the in vivo turnover of acetylcholine in this subcellular acetylcholine pool. This has been carried out by injecting labelled choline intravenously and then by measuring at different time intervals the ratio between labelled choline and acetylcholine in the fractions obtained after subcellular fractionation. It was found that the ratio radioactive choline to radioactive acetylcholine was the same (2:1) in whole brain and in the nerve ending fraction 2 to 20 min after injection. Since it was assumed that the same ratio is true also for the endogenous compounds the choline pool in the nerve terminals was considered to make up 13 nmoles/g brain. The results also indicate that plasma choline is rapidly equilibrated with the nerve terminals and transformed to acetylcholine at a rate of about 5 nmoles/g brain/min.     

A TECHNIQUE FOR THE STUDY OF ACETYLCHOLINE TURNOVER IN MOUSE BRAIN IN VIVO

Abstract— —A method to measure the rate of acetylcholine turnover in mouse brain in vivo has been developed. It is based on the formation of labelled acetylcholine from intravenously injected labelled choline. The isotopic dilution of choline in the brain has been measured by assaying endogenous choline in the brain by an enzymatic method using tritium-labelled acetyl-CoA and purified choline acetyltransferase.
The rate of acetylcholine turnover in the brain could be calculated at 50 n-moles acetylcholine/g/min in conscious mice. In anaesthetized mice and in mice treated with oxotremorine, a decrease of acetylcholine turnover to about 10 n-moles/g/min was found.     

THE EFFECT OF PHOSPHOLIPASES ON THE UPTAKE OF ATROPINE AND ACETYLCHOLINE BY SLICES OF MOUSE BRAIN CORTEX

The uptake of [³H]atropine, [³H]acetylcholine and [¹⁴C]inulin in mouse brain cortex slices was studied in slices treated with phospholipases A or C. In control experiments the slices took up atropine and acetylcholine against a concentration gradient, indicating active uptake. This uptake was decreased by ouabain, by anaerobic conditions and by an increase in the potassium ion concentration. The phospholipases were found to decrease the uptake of atropine and particularly that of acetylcholine in the slices. The uptake of labelled inulin in enzyme-treated slices, as compared to untreated slices, was not decreased, indicating no change in the inulin space. The effect of the phospholipases was time dependent and, up to a certainlimit, concentration dependent. The effect of ouabain in decreasing the uptake of atropine was not eliminated by the enzyme treatment. The effect of anaerobic conditions in decreasing the uptake was weak after treatment with phospholipases. The effect of higher concentrations of potassium ions was abolished by treatment with phospholipase A. The results emphasize the importance of phospholipids as substances controlling structural order in membranes and suggest their participation in active transport.     

APPARENT IN VIVO ACETYLCHOLINE TURNOVER RATE IN WHOLE MOUSE BRAIN: EVIDENCE FOR A TWO COMPARTMENT MODEL BY TWO INDEPENDENT KINETIC ANALYSES

Abstract— Acetylcholine turnover has been determined in whole mouse brain using a newly available high specific activity [³H]choline (70 Ci/mmol). Animals were killed at various time points (0.25–10 min) after pulse adminstration of [³H]choline (Ch) by microwave irradiation of the head. Steady-state levels of ACh were determined by radioenzymatic analysis as described by G oldberg & M c C aman (1973) as modified by M c C aman & S tetzer , 1977. Ch levels were determined by a modification of the method of M c C aman & S tetzer (1977). Radiolabelled metabolites of [³H]Ch were separated by selective extraction of [³H]Ch and [³H]ACh inio tetraphenylboron in 3-heptanone (C arroll et al. , 1977) coupled with an enzymatic separation of [³H]Ch from [³H]ACh. A precursor-product relationship was verified for Ch and ACh specific activities. Acetylcholine turnover rate was determined by the biosynthesis ratio method (S chuberth et al. , 1969, Method 1) and by the finite-differences method (N eff et al. , 1971, Method 2). Both methods of kinetic analysis revealed two distinct turnover rates for acetylcholine. In the first phase (0.25–1.5 min post-[³H]Ch), the ACh turnover rate averaged 22nmol/g/min (both methods). During the second phase, (2–10 min) acetylcholine turnover rates were significantly ( P < 0.05 and P < 0.01) lower; i.e. 7nmol/g/min (Method 1) and 5.9 nmol/g/min (Method 2). The data are consistent with a 2-compartment model for ACh turnover in whole mouse brain. Additionally, the method described for the separation of radiolabelled metabolites of [³H]Ch allows an accurate determination of ACh turnover in as little as 2 mg of tissue.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

THE EFFECT OF ELECTROCONVULSIVE SHOCK ON PROTEIN SYNTHESIS IN MOUSE BRAIN

The effect of a single electroconvulsive shock on protein synthesis in mouse brain cortex was studied by observing the incorporation into protein of intraperitoneally injected [³H]- or [¹⁴C]leucine. When the precursor was injected immediately after the electroshock there was a 50 per cent inhibition of the incorporation which was not seen with injections at times later than 10 min. To investigate a possible specificity, the cerebral cortices of experimental and sham control animals which had been injected with different isotopes were homogenized together and fractionated by differential centrifugation. Cell fractions were then separately extracted with phosphate buffer and with Triton X-100. The ratio of ³H to ¹⁴C in each fraction was compared with that of the total homogenate to reveal any specific effects due to the electroconvulsive shock. The treatment produced a slight inhibition of the incorporation of the isotope into the heavier particulate fractions (i.e. nuclei, mitochondria, synaptosomes) relative to that in the microsome and cell sap fractions. A possible explanation of these results is given with a discussion of the limitations of the technique.     

THE EFFECT OF THIAMINE DEFICIENCY ON THE ACETYLCOENZYMEA AND ACETYLCHOLINE LEVELS IN THE RAT BRAIN

Abstract— It is shown that transketolase activities in red blood cells and whole brain of normal and thiamine-deficient rats correlate well with heart frequencies.
The effect of thiamine depletion on the levels of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh), and on the activities of pyruvate dehydrogenase, choline acetyl-transferase and acetylcholine esterase was studied in whole brains of thiamine-deficient, thiamine-supplemented ad libitum and pair-fed rats. The concentrations of acetyl-CoA and ACh decreased in thiamine-deficient brains by 42 and 35 per cent, respectively.
Total pyruvate dehydrogenase activity did not change during vitamin B₁ deficiency. The 'resolved' enzyme, reconstituted with thiamine diphosphate, had an association constant of 5.4 × 10⁻⁶ m . Choline acetyltransferase and acetylcholine esterase activities remained unchanged in thiamine deficiency.
Possible mechanisms which could explain the reduced Ach levels in vitamin B₁ deficiency are discussed.     

THE EFFECT OF dl-ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN

Abstract— dl -Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l -ornithine decarboxylase activity and a simultaneous decrease in S -adenosyl- l -methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl -allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl -allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.     

THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

THE EFFECT OF SULFHYDRYL REAGENTS ON THE TITYUSTOXIN-INDUCED ACETYLCHOLINE RELEASE IN RAT BRAIN SLICES1

Abstract— Pre-treatment of rat brain slices with organic mercurials prevents the increased acetylcholine release induced by tityustoxin. This inhibition is reversed by dithiothreitol. N-Ethylmaleimide blocks the tityustoxin effect irreversibly. Simultaneous incubation with mercurials and toxin reveals a competition of both ligands for a membrane sulfhydryl group apparently required for tityustoxin activity.     

EFFECT OF OESTRADIOL ON TURNOVER OF TYPE A MONOAMINE OXIDASE IN BRAIN

Abstract— Administration of oestrogen (oestradiol-17β or oestradiol-17β-benzoate) to ovariectomized (OVX) rats for 1–4 weeks results in an approx 30% decrease in the activity of monoamine oxidase (MAO) in the basomedial-hypothalamus (BM-Hyp) and corticomedial-amygdala (CM-Amy) but not in cerebral cortex. Further investigation shows that (1) decreased MAO activity in the BM-Hyp and CM-Amy occurs only in Type A MAO (serotonin as substrate) and does not occur in Type B MAO (phenylethylamine as substrate); (2) decreased MAO activity does not occur when a single large dose of oestrogen is given i. v. or when homogenates from oestrogen treated rats are mixed with homogenates from OVX rats suggesting that direct enzyme inhibition is not responsible for the change in activity; (3) oestrogen administration to OVX rats increases the rate constant of degradation for MAO in BM-Hyp and CM-Amy but not in cerebral cortex as determined in turnover studies using pargyline, an irreversible inhibitor of MAO. The increased rate of degradation results in shorter half lives ( t 1/2) for MAO in the BM-Hyp and CM-Amy of oestrogen treated rats. In OVX rats the t 1/2 is 9.8 days in BM-Hyp and 12.7 days in CM-Amy. Oestrogen administration results in a t 1/2 of 7.6 days in BM-Hyp and 7.8 days in CM-Amy. The possible relationship between oestrogen dependent decreased MAO activity and estrogen dependent lordosis behavior is discussed.     

THE EFFECT OF LIMB ISCHAEMIA ON THE TURNOVER OF NORADRENALINE IN THE HYPOTHALAMUS AND BRAIN STEM OF THE RAT

The effect of ischaemic limb injury on the turnover of noradrenaline in the hypothalamus and brain stem has been studied in rats. There are theoretical reasons for thinking that these regions are activated in trauma and previous work showed that during limb-ischaemia the concentration of noradrenaline in the hypothalamus decreased by 27 per cent. The tourniquets were applied to both hind-limbs 1 h after the injection of [¹⁴C]-tyrosine when the labelling of the noradrenaline was maximal. During 4 h limb ischaemia the endogenous tyrosine concentration in the plasma decreased while that in the hypothalamus first rose and then fell. Changes in a similar direction in the brain stem were not statistically significant. Limb ischaemia did not affect the decline in the specific activity of the plasma or tissue tyrosine. It was concluded that the injury increased the utilization of tyrosine by the body. During the 4 h bilateral hind-limb ischaemia the rate of decline of [¹⁴C]noradrenaline was significantly increased in the brain stem but not in the hypothalamus. Conditions in the brain stem were sufficiently close to ‘steady-state’ to be able to conclude that the injury increased the metabolism of noradrenaline in the brain stem. Conditions in the hypothalamus were too complicated for definite conclusions to be drawn. The possible reasons for this and the limitations of this method for studying noradrenaline turnover are discussed.     

EFFECT OF TRANQUILIZERS ON REGIONAL DISTRIBUTION OF ASCORBIC ACID IN THE RAT BRAIN

—Studies were made of the effects of fluphenazine, chlorpromazine and triflupromazine on tissue concentration, liver synthesis of ascorbic acid and its distribution in different areas of the brain. All the three drugs were found to increase liver concentration and synthesis of the vitamin at 24 hr after administration of a single oral dose of the vitamin, but only fluphenazine was found to increase its concentration in the adrenals and brain; the increase in the latter case was found to vary in different regions of the brain, the olfactory lobes, hypothalamus and residual brain showing maximum increases, andthe basal ganglia, visual cortex and remaining dorsal cortex showing minimum increases. The effects were found to be reversed 72 hr after drug treatment.     

微波辐射对小鼠脑内乙酰胆碱含量及胆碱乙酸转移酶活性的影响

用频率为2450MHz功率密度为10mW/Cm~2(WBASAR约11.4W/kg)的微波(连续波)对置于微波暗室内的昆明种雄性小鼠急性全身照射1小时后,立即按常规方法断头,取脑,制成样品,然后用放射免疫测定法测量小鼠脑内乙酰胆碱(ACh)含量及胆碱乙酰转移酶(ChAT)活性。结果表明:照射组的ACh含量为11.6±1.4pmol/mg(脑鲜重),ChAT活性为45.4±8.7pmolACh/min.mg(脑鲜重);而对照组的分别为16.0±2.1pmol/mg和61.0±13.8pmolACh/min.mg。证明微波照射后可引起动物脑内ACh水平和ChAT活性下降,提示微波辐射对中枢胆碱能系统确有不利影响。     

微波辐射对小鼠脑内乙酰胆碱含量及胆碱乙酸转移酶活性的影响

用频率为2450MHz功率密度为10mW/Cm~2(WBASAR约11.4W/kg)的微波(连续波)对置于微波暗室内的昆明种雄性小鼠急性全身照射1小时后,立即按常规方法断头,取脑,制成样品,然后用放射免疫测定法测量小鼠脑内乙酰胆碱(ACh)含量及胆碱乙酰转移酶(ChAT)活性。结果表明:照射组的ACh含量为11.6±1.4pmol/mg(脑鲜重),ChAT活性为45.4±8.7pmolACh/min.mg(脑鲜重);而对照组的分别为16.0±2.1pmol/mg和61.0±13.8pmolACh/min.mg。证明微波照射后可引起动物脑内ACh水平和ChAT活性下降,提示微波辐射对中枢胆碱能系统确有不利影响。     

THE EFFECT OF FASTING ON THE METABOLISM OF 5-HYDROXYTRYPTAMINE AND DOPAMINE IN THE BRAIN OF THE MOUSE

Abstract— The turnover of 5-hydroxytryptamine in the forebrain and of dopamine in the striatum was studied in mice fasted for 20 h. Such mice showed an increased tissue concentration of 5-hydroxyindoleacetic acid in the forebrain and an increased accumulation of this acid after probenecid. Fasted mice also showed a higher concentration of homovanillic acid in the striatum than fed mice. However, the administration of probenecid produced a smaller increase in homovanillic acid concentration in fasted than in fed mice. The decay of dopamine following α-methyl- p -tyrosine was reduced in fasted mice at 2 h, but not at 1 h or 6 h after administration of the inhibitor. The possibility that fasting increases the activity of some dopaminergic neurones while decreasing the activity of others is considered. The existence of a pool of homovanillic acid at a site within the striatum where the probenecid-sensitive transport is not effective is postulated.     

THE EFFECT OF ELEVATED AMINO ACIDS ON AMINOTRANSFERASE LEVELS IN BRAIN AND LIVER OF THE MOUSE

Abstract— Adult mice were fed standard diets that were enriched with selected amino acids, i.e. 3% methionine, 6% valine, or 8% lysine. These diets caused the following changes in the amino acid pool of the brain measured at 7 and 21 days. The high methionine diet resulted in 50-fold higher levels of methionine and cysteine and somewhat lower levels of serine and glutamine. The valine and lysine-enriched diets also caused 2- to 4-fold increases in valine and lysine contents of brain, respectively. In spite of the large changes in amino acid levels, however, there were essentially no changes in aspartate: α-ketoglutarate, alanine: α-ketoglutarate, ornithine: α-ketoglutarate, methionine: α-ketoglutarate, and the branched chain aminotransferase activities of brain 3, 10, and 21 days after the onset of the dietary regimen. In contrast, these diets produced significant changes in some of these enzyme activities in liver. Changes in liver included a 2-fold increase in ornithine and alanine aminotransferase activities with the methionine-enriched diet. Liver ornithine aminotransferase activity also increased slightly in animals fed the valine-enriched or lysine-enriched diet.     

EFFECT OF OLIGEMIA ON REGIONAL METABOLITE LEVELS IN CAT BRAIN

Abstract— Incomplete cerebral ischemia (oligemia) was produced in cat by carotid occlusion combined with arterial hypotension. Lowering arterial pressure to 50–60 Torr for 20 min caused marked alterations of the ATP, phosphocreatine, and lactate content of subcortical white matter. In contrast, metabolite levels in cerebral cortex and caudate nucleus were only moderately perturbed from control values. More severe oligemia resulted when arterial pressure was lowered to 30 Torr for 20 min following carotid occlusion. Metabolite levels in cortex, caudate nucleus, and white matter were greatly altered from control. In the gray matter there was regional heterogeneity of metabolic alteration, as evidenced from the pattern of NADH tissue fluorescence. The cortex contained micro-patches (0.1mm) of increased NADH, which frequently exhibited a columnar orientation.
These findings demonstrate two distinct types of cerebral inhomogeneity of metabolic failure with reduced blood flow; white matter fails before gray matter, and there is micro-heterogeneity of metabolic failure in the gray matter.