Acetylcholine In Brain Slices Research Articles

Acetylcholinesterase loosens the brain's cholinergic anti-inflammatory response and promotes epileptogenesis

Recent studies show a key role of brain inflammation in epilepsy. However, the mechanisms controlling brain immune response are only partly understood. In the periphery, acetylcholine (ACh) release by the vagus nerve restrains inflammation by inhibiting the activation of leukocytes. Recent reports suggested a similar anti-inflammatory effect for ACh in the brain. Since brain cholinergic dysfunctions are documented in epileptic animals, we explored changes in brain cholinergic gene expression and associated immune response during pilocarpine-induced epileptogenesis. Levels of acetylcholinesterase (AChE) and inflammatory markers were measured using real-time RT-PCR, in-situ hybridization and immunostaining in wild type (WT) and transgenic mice over-expressing the “synaptic” splice variant AChE-S (TgS). One month following pilocarpine, mice were video-monitored for spontaneous seizures. To test directly the effect of ACh on the brain's innate immune response, cytokines expression levels were measured in acute brain slices treated with cholinergic agents. We report a robust up-regulation of AChE as early as 48 h following pilocarpine-induced status epilepticus (SE). AChE was expressed in hippocampal neurons, microglia, and endothelial cells but rarely in astrocytes. TgS mice overexpressing AChE showed constitutive increased microglial activation, elevated levels of pro-inflammatory cytokines 48 h after SE and accelerated epileptogenesis compared to their WT counterparts. Finally we show a direct, muscarine-receptor dependant, nicotine-receptor independent anti-inflammatory effect of ACh in brain slices maintained ex vivo. Our work demonstrates for the first time, that ACh directly suppresses brain innate immune response and that AChE up-regulation after SE is associated with enhanced immune response, facilitating the epileptogenic process. Our results highlight the cholinergic system as a potential new target for the prevention of seizures and epilepsy.

Effect of Anoxia on Choline Uptake and Release of Acetylcholine in Brain Slices Estimated With a Bioradiographic Technique Using [11C] Choline

The uptake of choline for the synthesis and release of acetylcholine and the metabolism of glucose under anoxic conditions was investigated in brain slices by bioradiography using [N-methyl-11C] choline ( [11C] choline) and [18F] 2-fluoro-2-deoxy-D-glucose ( [18F] FDG ) . [11C] Choline uptake and the release of accumulated11Cradioactivity in brain slices decreased with anoxic treatment, whereas [18F] FDG uptake increased. The decrease of [11C] choline uptake and the11Cradioactivity accumulated in striatal slices were recovered by acetyl-L-carnitine, an acetyl-donor. However, this effect was not seen in cerebral cortex. These results indicate that choline uptake for the synthesis and release of acetylcholine in brain are energy sensitive. The cholinergic dysfunction in ischemic brain might be improved by compensating for energy loss.

Assessment of choline uptake for the synthesis and release of acetylcholine in brain slices by a dynamic autoradiographic technique using [ 11C]choline

The uptake of choline for the synthesis and release of acetylcholine was investigated in brain slices by dynamic positron autoradiography using [ 11C]choline. Brain slices (330 μm) were incubated with [ 11C]choline in oxygenated Krebs–Ringer medium at 34 °C and serial two-dimensional time-resolved images of the uptake and release of radioactivity were recorded on Storage Phosphor screens. [ 11C]Choline uptake increased with the period of incubation and was 1.9 times higher in the striatum than cerebral cortex. The uptake in the striatum was significantly diminished by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline uptake. Pretreatment of brain slices with 50 mM K + for 20 min enhanced the uptake in striatum. The uptake of [ 11C]choline in brain slices was saturable using nonlabeled choline. Two uptake systems, a high-affinity and a low-affinity system, were confirmed to exist by kinetic analysis using Lineweaver–Burk plots. The 11C radioactivity that had accumulated in the striatum disappeared on treatment with veratridine, a depolarization agent, in the presence of HC-3. This pattern of disappearance was consistent with that of the appearance of unlabeled and labeled acetylcholine in the medium. These results indicate that this method is useful for obtaining information regarding the uptake of choline for the synthesis and release of acetylcholine in live brain tissues.

Negative effects of tacrine (tetrahydroaminoacridine) and methoxytacrine on the metabolism of acetylcholine in brain slices incubated under conditions stimulating neurotransmitter release.

The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of brain acetylcholine were investigated in experiments in which acetylcholine turnover was stimulated by tissue depolarization or by 4-aminopyridine. Rat cerebrocortical prisms were preincubated under "resting" conditions (Krebs-Ringer buffer with 3 mmol/L K+ and with paraoxon to inhibit cholinesterases) and then incubated in the presence of tacrine or methoxytacrine and of 50 mmol/L K+. Both drugs diminished the amount of acetylcholine released by depolarization and the amount of acetylcholine synthesized during incubation; in experiments in which [14C]choline was present in the incubation medium simultaneously with tacrine or methoxytacrine, the drugs diminished the uptake of [14C]choline by the tissue and the amount of [14C]-acetylcholine synthesized and released into the medium. In these experiments, it was not possible to distinguish whether the inhibitory effects of tacrine and methoxytacrine were primarily on the process of acetylcholine synthesis (particularly on the uptake of choline), or whether the drugs also acted directly on the process of neurotransmitter release. In subsequent experiments the prisms were preincubated with [14C]choline and only then subjected to a short depolarization in the presence of hemicholinium-3 and tacrine or methoxytacrine. Both drugs severely inhibited the release of preformed [14C]acetylcholine and prevented the diminution of tissue [14C]acetylcholine stores. Methoxytacrine was also found to diminish the release of acetylcholine induced by 4-aminopyridine while increasing the content of acetylcholine in the tissue. Tacrine and methoxytacrine had no effect on the activity of choline acetyltransferase (EC 2.3.1.6).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypoxia on uptake and acetylation of [ 3H]choline in brain slices from adult and newborn guinea pigs

The synthesis of [ 3H]acetylcholine in brain slices from adult and newborn guinea pigs were suppressed by anaerobiosis. The uptake and acetylation of [ 3H]choline in the newborn brain were very low, compared to findings in slices of the adult brain. The most sensitive tissue to hypoxia was the striatum followed by the hippocampus. There were significant differences in the case of the frontal cortex.

Phosphatidylethanolamine methyltransferase activity in developing, demyelinating, and diabetic mouse brain.

The catalysis by phosphatidylethanolamine methyltransferase (PEMT) of phosphatidylcholine (PC) synthesis by the successive methylation of phosphatidylethanolamine in the presence of S-adenosylmethionine (AdoMet) as methyl donor, was detected in actively myelinating mouse brains. PEMT activity in the microsome fraction of fetal mouse brain at 17 days of gestation was 253 mu u/mg protein and that of adult brain after 7 days of remyelination following 6 weeks cuprizone administration was 148 mu u/mg. These figures are much higher than found in normal adult brains (1.7 mu u/mg). An increase in PEMT activity was observed in the brains of genetically transmitted diabetic mice, C57BL/KsJ-db/db, and streptozotocin-induced diabetic mice; 16.3 and 9.2 mu u/mg, respectively. The methyl group of mecobalamin was transferred to homocysteine producing AdoMet and was further metabolized into choline and acetylcholine in brain slices. These results suggest that in the diabetic state, an increase in PC synthesis is probably required in order to replace damaged myelin or to supply choline or acetylcholine essential to for nerve functions. Mecobalamin might serve as the source of the methyl group utilized for PC synthesis.

Effects of tityustoxin (TsTX) from scorpion venom on the release and synthesis of acetylcholine in brain slices

Tityustoxin (TsTX) increased the release and synthesis of acetylcholine (ACh) in slices of rat brain hippocampus, hypothalamus, thalamus, cortex and striatum. The effect was highest in slices of hippocampus and cortex and smallest in hypothalamus. These effects of TsTX were dependent on the presence of Na + and Ca 2+ in the incubation medium. EGTA, 0.1 mM, blocked the effect of TsTX in all areas, except for the hippocampus and frontal striatum, where a concentration of 10mM was required. Tetrodotoxin blocked the increase in the release and synthesis of ACh induced by tityustoxin.

Relation between the content of acetyl-coenzyme A and acetylcholine in brain slices

Slices of rat caudate nuclei were incubated in vitro in media containing, among other constituents, three different concentrations of glucose (0.5, 2 and 10 mM), 0.2 mM-choline, paraoxon as an inhibitor of cholinesterase, and 5 mM- or 30 mM-K+. After 30 and 60 min of incubation, the concentrations of acetyl-CoA, acetylcholine and choline in the tissue and of acetylcholine in the incubation medium were measured. The content of acetyl-CoA in the sliced varied in direct relation to the concentration of glucose in the incubation medium. The content of acetylcholine in the slices and, in experiments with high K+, also the amount of acetylcholine released into the incubation medium varied in direct relation to the concentration of glucose in the incubation medium and to the concentration of acetyl-CoA in the slices; the relation between the concentrations of acetyl-CoA and of acetylcholine in the slices was linear. It was concluded that the availability of acetyl-CoA had a decisive influence on both the rate of synthesis of acetylcholine and its steady-state concentration. The observations accord with the view that, at the ultimate level, the synthesis of acetylcholine is controlled by the Law of Mass Action.

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).

The formation of choline and of acetylcholine by brain in vitro.

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four‐fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [3H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na+‐free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal‐Na+ medium (choline‐free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na+‐free medium.

A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool.

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.