Endogenous Acetylcholine Levels Research Articles

Development and aging of cholinergic synapses. I. Endogenous levels of acetylcholine and choline in developing autonomic ganglia and iris of the chick.

Acetylcholine (ACh) and choline (Ch) levels were measured by means of a sensitive micromethod in developing irises, ciliary and sympathetic ganglia of the chick, starting at 5--7 days of incubation (DI) up to 1 year of age. The neurotransmitter is present in all three organs at relatively low levels (1--10 pmol) from 5 DI (iris) or 7 DI (ciliary and sympathetic ganglion). This is followed by a rapid and sustained 9-fold increase in all three tissues up to 14 DI. Thus, it appears that low levels of ACh may be sufficient for neurotransmission to occur in the primitive ciliary ganglion and iris. After hatching, total ACh levels continue to increase up to 1 year of age in the iris and ciliary ganglion and up to 3 months in the sympathetic ganglion. The increase may depend on either an increased functional demand for the neurotransmitter or an increased number of preganglionic terminals. In general Ch levels parallel closely the levels of ACh in each organ throughout development. It is concluded that ACh and Ch are present since the earliest detectable stages of development in the ganglia and iris, and their first increase seems to be correlated to the phase of innervation of the organs. The subsequent increase probably correlates to synaptogenesis. The close relationship between Ch and ACh levels indicates a regulatory role of Ch for ACh synthesis during neuronal development.

Development and Aging of Cholinergic Synapses I. Endogenous Levels of Acetylcholine and Choline in Developing Autonomic Ganglia and Iris of the Chick (Part 2 of 2)

Acetylcholine (ACh) and choline (Ch) levels were measured by means of a sensitive micromethod in developing irises, ciliary and sympathetic ganglia of the chick, strung at 5–7 days of incubation (DI) up to 1 year of age. The neurotransmitter is present in all three organs at relatively low levels (1–10 pmol) from 5 DI (iris) or 7 DI (ciliary and sympathetic ganglion). This is followed by a rapid and sustained 9-fold increase in all three tissues up to 14 DI. Thus, it appears that low levels of ACh may be sufficient for neurotransmission to occur in the primitive ciliary ganglion and iris. After hatching, total ACh levels continue to increase up to 1 year of age in the iris and ciliary ganglion and up to 3 months in the sympathetic ganglion. The increase may depend on either an increased functional demand for the neurotransmitter or an increased number of preganglionic terminals. In general Ch levels parallel closely the levels of ACh in each organ throughout development. It is concluded that ACh and Ch are present since the earliest detectable stages of development in the ganglia and iris, and their first increase seems to be correlated to the phase of innervation of the organs. The subsequent increase probably correlates to synaptogenesis. The close relationship between Ch and ACh levels indicates a regulatory role of Ch for ACh synthesis during neuronal development.

Microinjection of kainic acid into the rat hippocampus

The effects of unilateral injection of kainic acid into the rate hippocampus have been examined in terms of morphologic, neurochemical and behavioral sequelae. Infusion of 10 nmoles if kainate causes a rapid and complete degeneration of neuronal perikarya in the entire hippocampal formation followed by gliosis and atrophy of the region. This unilateral neuronal loss is accompanied by a 50% decrease in the specific activity of the biochemical markers for GABAergic neurons including glutamic acid decarboxylase, endogenous GABA and synaptosomal uptake of [ 3H]GABA. The extrinsic hippocampal cholinergic and noradrenergic afferents also exhibit significant alteration. Although the specific activity of choline acetyltransferase is unaffected and the specific activity of tyrosine hydroxylase is significantly increased in the injected hippocampus, the synaptosomal high affinity uptake process for [ 3H]choline and [ 3H]norepinephrine are significantly reduced at 10 days after injection. Whereas the level of endogenous acetylcholine is elevated in the lesioned hippocampus at 2 days after injection, the level of endogenous norepinephrine is reduced. For several hours after intrahippocampal injections of 5 nmoles or more of kainate, rats exhibit epileptiform behavior. Intrahippocampal injection of kainate may be a useful rodent model for temporal lobe seizure disorders.

Modulation of neostriatal acetylcholine in the rat by dopamine and 5-hydroxytryptamine afferents

Endogenous and deuterium labelled acetylcholine (ACh) and choline (Ch) in the neostriatum were chemically assayed after radio-frequency lesions in the substantia nigra-ventral tegmental area and in the B7 or B8 5-hydroxytryptamine (5-HT) regions of the brainstem. Lesions in the substantia nigra-ventral tegmental region or in the B7 area, which provide dopamine (DA) and 5-HT afferents to the caudate-putamen nucleus, respectively, caused a decrease in 2H 9-ACh synthesis, while endogenous levels of ACh and Ch were unchanged. A unilateral lesion of the B8 5-HT region, which projects in part to the substantia nigra, produced an increase in endogenous ACh levels, as well as a decrease in 2H 9-ACh synthesis on the side ipsilateral to the lesion. On the basis of these data, we conclude that DA and 5-HT projections to the caudate-putamen nucleus have a net excitatory effect on cholinergic interneurons in that area. Furthermore, we suggest that the putative B8 5-HT projection is excitatory upon nigral neurons that, in turn, project to the neostriatum.

Quantitative Measurements of Endogenous Levels of Acetylcholine and Choline in Tetrathyridia of Mesocestoides corti (Cestoda) by Means of Combined Gas Chromatograph-Mass Spectrometry

Endogenous levels of choline and acetylcholine in tetrathyridia of Mesocestoides corti were determined under different environmental conditions. The highest values of choline and ACh were obtained when tetrathyridia were washed in media NCTC 135 and Eagle's Basal Medium. The lowest values were obtained when organisms were washed in 0.85% NaCl, or BME without choline chloride. Incubation of organisms in different concentrations of physostigmine showed an increase in the ACh level. The levels of choline and ACh decreased with decreasing drug concentration. DFP also showed a similar effect on choline and ACh. Oxotremorine increased choline and ACh levels but the increase was considerably lower than in physostigmine or DFP-treated organisms. While the histochemical association of acetylcholinesterase (AChE) with the nervous system has been the primary means of claiming the presence of acetylcholine (ACh) in helminthic tissue, a few workers have demonstrated ACh by biochemical isolation. Bueding (1952) showed that extracts of Schistosoma mansoni contain high levels of AChE and choline acetylase activities. Barker et al. (1966) reported the level of ACh in S. mansoni to be 5.3 jug/g wet weight. Chance and Mansour (1953) demonstrated AChE and choline acetylase activities in the liver fluke, Fasciola hepatica. Furthermore, they reported that this parasite contains an ACh-like substance. The presence of ACh was demonstrated in Ascaris by Mellanby (1955) who showed that the head region contains a much higher level than the body. Several biochemical methods are routinely used for determining ACh-like activity in extremely small amounts, but their reliability has been questioned (Hosein and Orzek, 1966; Stockley, 1969) in view of the fact that their response is not specific to one compound alone. Definitive identification of ACh and choline Received for publication 2 November 1973. * This investigation was aided in part by research grant USPHS AI-11107-01 from the NIH, Bethesda, Maryland. t Recipient of USPHS-NIAID Training Grant No. TOI-A1-00249. + Grand Island Biological Company, 519 Aldo Avenue, Santa Clara, California 95050. was made in tetrathyridia of Mesocestoides corti by means of a combined gas chromatograph-mass spectrometry (GC-MS) system. The functional aspects of ACh in this parasite will be reported elsewhere. MATERIALS AND METHODS Tetrathyridia of Mesocestoides, originally isolated from lizards (Specht and Voge, 1965), were maintained in Swiss albino mice. The animals were killed by cervical dislocation and tetrathyridia were transferred from the body cavity into petri dishes containing GIBCOt NCTC 135, "Eagle's Basal Medium" (BME) with or without choline chloride, or physiological saline solution (0.85% NaCl). Worms were preincubated in BME without choline chloride for a maximum of 1/2 hr before exposure to drug. The number of worms never exceeded 20 per ml of medium. The maximum period of incubation was 1 hr. For each drug concentration, the motility of worms was observed under the dissecting microscope at 15, 30, 45, and 60 min. The effect on choline and ACh levels by 3 different concentrations of physostigmine salicylate and 2 concentrations of oxotremorine and diisopropylfluorophosphate (DFP) was tested. The effect of each concentration was tested 3 times and organisms for each concentration were obtained from a different mouse. After incubation at 37 C, parasites were washed and dried by use of aspiration of fluids or by means of a Millipore filter. Organisms were then removed with a frozen spatula and immediately immersed in liquid nitrogen. The entire process of washing and drying lasted 6 to 8 min. Organisms were pulverized by the method of Campbell et al. (1970). The finely powdered tissue, 0.066 to 1.11 g, was transferred to a tared, chilled glass homogenizer to prevent thawing, and weighed. With few exceptions, the

Endogenous levels of acetylcholine and choline in individual neurons of Aplysia.

Endogenous levels of acetylcholine and choline in individual neurons of Aplysia.

Choline: selective accumulation by central cholinergic neurons.

Abstract— Most of the cholinergic input to the hippocampus was destroyed by placement of lesions in the medial septal area. In animals with such lesions we found that hippocampal ChAc activity was reduced by 85–90% and endogenous acetylcholine levels were reduced by more than 80 %. When hippocampal synaptosomes from animals with lesions were incubated with [3H]choline at concentrations of 7.5 nm, 1 μm and 10 μm there was approximately a 60 % reduction in the uptake of [3H]choline, suggesting that cholinergic nerve endings were mainly responsible for [3H]choline uptake. At 0.1 mm concentrations of [3H]choline, there was only a 25 % reduction of choline uptake, suggesting that at higher concentrations of choline there was more nonspecific uptake. The uptake of radiolabelled tryptophan, glutamate and GABA were only slightly or not at all affected by the lesions. There was a significant reduction of uptake of radiolabelled serotonin and norepinephrine, since known monoaminergic tracts were disrupted. Choline uptake was reduced only in brain regions in which cholinergic input was interrupted (i.e. the cerebral cortex and hippocampus) and remained unchanged in other regions (i.e. the cerebellum and striatum). The time course of the reduction in choline uptake was similar to that of the reductions in ChAc activity and endogenous ACh levels; there was no decrease at 1 day, a significant decrease at 2 days, and the maximal decrease at 4 days postlesion. There was a close correlation among choline uptake, ChAc activity and ACh levels in the four brain regions examined (i.e. the striatum, cerebral cortex, hippocampus and cerebellum). Our results suggest that when hippocampal synaptosomes (and perhaps synaptosomes from other brain areas as well) are incubated in the presence of choline, at concentrations of 10 μm m or lower, then cholinergic nerve endings are responsible for the bulk of the choline accumulated by the tissue.

On the role of acetylcholine in regulating the rate of the early chick embryo heart.

AbstractA study has been made of the effect of acetylcholine upon the heart rate of the early chick embryo, and it has been noted that under certain experimental conditions acetylcholine stimulates the heart rate during the period of vagal innervation, while depressing the heart rate after the nerve supply is established. Isolated chick embryo hearts freely suspended in a phosphate‐buffered (bicarbonate‐free) medium beat at only about one‐third their in ovo rates, and acetylcholine produces biphasic effects of stimulation and inhibition of the heart rate depending upon the concentration of the drug and the age of the hearts, the maximal stimulation occurring at five days. When suspended in Krebs bicarbonate medium the hearts beat at rates of about 72 to 74% their in ovo rates, and acetylcholine produces only slowing of the heart rate. This effect of acetylcholine can be diminished or reversed when an inhibitor of endogenous acetylcholine, hemicholinium‐3 is added. We have postulated that both inhibitory and stimulatory receptors for acetylcholine exist in the early chick embryo heart and that the response to added acetylcholine depends in part on levels of endogenous acetylcholine already present in the heart. The role of endogenous acetylcholine in regulating heart rate is discussed.