Modulation of Dopamine Release from Neuron-Enriched Tissue Cultures by Cholinergic Agents

Modulation of [3H]dopamine release by cholinergic agents (acetylcholine, atropine, d-tubocurarine, oxotremorine, and nicotine) was studied in primary cell cultures derived from whole brains of foetal rats (17 days of gestation). Monolayer and aggregated neuron-enriched cultures were maintained for 17 days in vitro [3H]Dopamine basal outflow was enhanced by acetylcholine, nicotine, and atropine and was unaffected by oxotremorine, hexamethonium, and d-tubocurarine. The action of nicotine was antagonized by d-tubocurarine, and that of atropine was partially blocked by oxotremorine. A similar picture was seen when the influence of cholinergic agents was studied under depolarizing conditions. The action of oxotremorine was dependent on nerve activity. The presence of both muscarinic and nicotinic antagonists was necessary for abolishing the effect of acetylcholine on the dopamine outflow. These results show that dopamine release in both types of neuron-enriched cultures can be influenced by cholinergic agents and that both muscarinic and nicotinic receptors are involved in regulation of the amine's outflow.     

Agonist-mediated regulation of presynaptic receptor function during development of rat septal neurons in culture

Presynaptic receptors modulating the release of acetylcholine (ACh) were studied in fetal septal neurons cultured in a growth medium to which various drugs were added from day 3 in vitro (DIV 3) to DIV 14. The influence of these drugs on the function of the presynaptic muscarinic (M-) autoreceptor was determined at DIV 14 by measuring the inhibitory effect of the M-agonist oxotremorine on the electrically-evoked release of [(3)H]ACh from cultures pre-incubated with [(3)H]choline. The presence of the M-agonists oxotremorine (100 micromol/L) or carbachol (100 micromol/L) from DIV 3 to DIV 14, or from DIV 13 to DIV 14, abolished M-autoreceptor function at DIV 14, whereas the presence of the M-antagonist atropine (10 micromol/L from DIV 3 to DIV 14) during growth left M-autoreceptor function unaltered. Inhibition of ACh esterase by donepezil (1 micromol/L from DIV 3 to DIV 14) weakly decreased M-autoreceptor function at DIV 14; inhibition of neuronal firing by 0.1 tetrodotoxin (0.1 micromol/L from DIV 3 to DIV 14) did not tend to affect M-autoreceptor function at DIV 14. Co-cultivation of fetal septal and raphe neurons for 2 weeks yielded cell cultures containing both vesicular ACh transporter- and tryptophan hydroxylase-immunopositive cells. From these cultures, the release of both [(3)H]ACh and [(3)H]5-HT could be induced by electrical field stimulation. In co-cultured neurons versus septal-only ones the inhibitory effect of oxotremorine on the evoked release of [(3)H]ACh appeared almost normal, whereas that of the selective 5-HT(1B) agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one (CP-93,129) was completely abolished. The effects of CP-93,129 were also absent on DIV 14 in septal mono-cultures grown in the presence of CP-93,129 (10 micromol/L) from DIV 3 to DIV 14. It is therefore concluded that the regulation of presynaptic receptor function strongly depends on the concentrations of endogenous transmitters in the neuronal environment.     

Effects of Nucleus Basalis Lesions on the Muscarinic and Nicotinic Modulation of [3H] Acetylcholine Release in the Rat Cerebral Cortex

Presynaptic muscarinic and nicotinic receptors in the cerebral cortex reportedly inhibit and increase acetylcholine (ACh) release, respectively. In this study, we investigated whether these receptors reside on cholinergic nerve terminals projecting to the cerebral cortex from the nucleus basalis magnocellularis (nbm). Adult male rats received unilateral infusions of ibotenic acid (5 micrograms/1 microliter) in the nbm. Two weeks later, cerebral cortical cholinergic markers (choline acetyltransferase activity, high-affinity choline uptake, and coupled ACh synthesis) were significantly reduced in synaptosomes prepared from the lesioned hemispheres compared to contralateral controls. The depolarization-induced release of [3H]ACh from these synaptosomes was also reduced in the lesioned hemispheres, reflecting the reduced synthesis of transmitter. However, the nbm lesions had no effect on the inhibition of release induced by 100 microM oxotremorine. Synaptosomal [3H]ACh release was not altered by nicotine or the nicotinic agonists anabaseine and 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine. Nicotine (10-100 microM) did increase [3H]ACh release in control and lesioned hemispheres in cortical minces, but to a similar extent. These results suggest that neither muscarinic nor nicotinic receptors modulating ACh release reside on nbm-cholinergic terminals.     

Effect of Adenosine, Adenosine Derivatives, and Caffeine on Acetylcholine Release from Brain Synaptosomes: Interaction with Muscarinic Autoregulatory Mechanisms

Synaptosomes, prepared from rat cerebral cortex and hippocampus, were preincubated with [methyl-3H]choline. The effect of adenosine, cyclohexyladenosine, N-ethylcarboxamide adenosine, 2'-deoxyadenosine, and oxotremorine on K+-evoked 3H efflux was investigated. High-voltage electrophoretic separation showed that in the presence of physostigmine, the K+-evoked 3H efflux from hippocampal synaptosomes was 90% [3H]acetylcholine and 10% [3H]choline. Adenosine (30 microM) and oxotremorine (100 microM) both decreased [3H]acetylcholine release from hippocampal synaptosomes. The effect was inversely proportional to the KCl concentration and disappeared at a KCl concentration of 50 mM. Cyclohexyladenosine was approximately 3,000 times more active than adenosine, whereas N-ethylcarboxamide adenosine and 2'-deoxyadenosine were inactive. This indicates that A1 adenosine receptors were involved in the inhibitory effect. Caffeine antagonized the adenosine effect, and at a concentration of 100 microM, it stimulated [3H]acetylcholine efflux. The inhibitory effect of oxotremorine was as great in cortical as in hippocampal synaptosomes. In contrast, adenosine was much less active in cortical than in hippocampal synaptosomes. When inhibitory concentrations of adenosine and oxotremorine were added together into the incubation medium, the effect of adenosine on [3H]acetylcholine release was consistently reduced. An interaction between muscarinic and A1 adenosine presynaptic receptors at a common site modulating acetylcholine release can be assumed.     

Calcitonin gene-related peptide (CGRP) modulates cholinergic neurotransmission in the small intestine of man, pig and guinea-pig via presynaptic CGRP receptors.

The effect of calcitonin gene-related peptide (CGRP) on the cholinergically mediated twitch contraction in longitudinal muscle strips of the small intestine (duodenum, jejunum, ileum) of guinea-pig, pig and man was investigated. Independently of the anatomical region, CGRP inhibited the twitch response in the different specimens of all three species by about 40% with similar IC50 values (1.5-2.4 nmol/l). Only in the guinea-pig small intestine CGRP induced a contraction of the smooth muscle which was sensitive to scopolamine and tetrodotoxin. The electrically evoked [3H]acetylcholine release from jejunal longitudinal muscle strips with myenteric plexus attached of the guinea-pig, which were incubated with [3H]choline, was concentration-dependently inhibited by CGRP. A direct relaxant effect of CGRP on smooth muscle tone of carbachol precontracted preparations was only observed in specimens of the guinea-pig. In conclusion, presynaptic inhibitory CGRP receptors on cholinergic neurones modulate the release of acetylcholine in different parts of the small intestine.     

Acetylcholine Synthesis by Adult Bovine Adrenal Chromaffin Cell Cultures

Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures. Hemicholinium-3 (IC50 less than 10 muM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.     

Eosinophil-induced release of acetylcholine from differentiated cholinergic nerve cells

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 +/- 2% of control at 10(-7) M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 +/- 23% of control at 10(-5) M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 +/- 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 +/- 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.     

A Search for Receptors Modulating the Release of γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Various putative striatal transmitters and related compounds were studied for their effects on the release of gamma-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal outflow whereas oxotremorine caused no consistent change; the effect of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, alpha 2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.     

SCH 57790: a novel M2 receptor selective antagonist

As a decrease in cholinergic neurons has been observed in Alzheimer's Disease (AD), therapeutic approaches to AD include inhibition of acetylcholinesterase to increase acetylcholine levels. Evidence suggests that acetylcholine release in the CNS is modulated by negative feedback via presynaptic M2 receptors, blockade of which should provide another means of increasing acetylcholine release. Structure-activity studies of [4-(phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl]sulfinyl]-phenyl]-1-piperazin eacetonitrile. This compound, SCH 57790, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M1 receptors is 40-fold lower. SCH 57790 is an antagonist at M2 receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M2 receptor blockade for enhancement of cognition.     

Both A1 and A2a Purine Receptors Regulate Striatal Acetylcholine Release

The receptors responsible for the adenosine-mediated control of acetylcholine release from immunoaffinity-purified rat striatal cholinergic nerve terminals have been characterized. The relative affinities of three analogues for the inhibitory receptor were (R)-phenylisopropyladenosine greater than cyclohexyladenosine greater than N-ethylcarboxamidoadenosine (NECA), with binding being dependent of the presence of Mg2+ and inhibited by 5'-guanylylimidodiphosphate [Gpp(NH)p] and adenosine receptor antagonists. Adenosine A1 receptor agonists inhibited forskolin-stimulated cholinergic adenylate cyclase activity, with an IC50 of 0.5 nM for (R)-phenylisopropyladenosine and 500 nM for (S)-phenylisopropyladenosine. A1 agonists inhibited acetylcholine release at concentrations approximately 10% of those required to inhibit the cholinergic adenylate cyclase. High concentrations (1 microM) of adenosine A1 agonists were less effective in inhibiting both adenylate cyclase and acetylcholine release, due to the presence of a lower affinity stimulatory A2 receptor. Blockade of the A1 receptor with 8-cyclopentyl-1,3-dipropylxanthine revealed a half-maximal stimulation by NECA of the adenylate cyclase at 10 nM, and of acetylcholine release at approximately 100 nM. NECA-stimulated adenylate cyclase activity copurified with choline acetyltransferase in the preparation of the cholinergic nerve terminals, suggesting that the striatal A2 receptor is localized to cholinergic neurones. The possible role of feedback inhibitory and stimulatory receptors on cholinergic nerve terminals is discussed.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

Modulation of electrically evoked serotonin release in cultured rat raphe neurons

Electrically evoked release of serotonin (5-HT) and its modulation via 5-HT autoreceptors and alpha(2)-heteroreceptors was studied in primary cell cultures prepared from the embryonic (ED 15) rat mesencephalic brain region comprising the raphe nuclei. Cultures were grown for up to 3 weeks on circular glass coverslips. They developed a dense network of non-neuronal and neuronal cells, some of which were positive for tryptophan hydroxylase. To measure 5-HT release, the cultures were pre-incubated with [(3)H]5-HT (in the presence of the selective noradrenaline reuptake inhibitor oxaprotiline [1 micromol/L]), superfused with modified Krebs-Henseleit medium containing 6-nitroqipazine [1 micromol/L] and electrically stimulated using two conditions. Condition A: 360 pulses, 3 Hz, 0.5 ms, 90 mA, or condition B: 4 pulses 100 Hz, 0.5 ms, 90 mA (a condition which diminishes interactions with endogenously released transmitters during ongoing stimulation). After only 1 week in culture, the electrically evoked overflow of [(3)H] was Ca(2+) dependent and tetrodotoxin sensitive, suggesting an action-potential-induced exocytotic release of 5-HT. Using stimulation condition A in cultures grown for 2 weeks, both basal and evoked 5-HT release were strongly enhanced by methiotepine (1 micromol/L) but unaffected by the 5-HT(1B) autoreceptor agonist CP-93, 129 (1 micromol/L) and the alpha(2)-adrenoceptor agonist UK-14, 304 (1 micromol/L). Conversely, using stimulation condition B, not only CP-93, 129 (IC(50) 8.1 +/- 1.4 nmol/L) and UK-14, 304 (IC(50) 14.9 +/- 1.6 nmol/L) had inhibitory effects on cells grown for 2 weeks, but also the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin. In conclusion, we describe for the first time electrically evoked release of 5-HT from primary cultures of fetal raphe cells and its modulation via 5-HT(1B) and 5-HT(1A) auto- and alpha(2)-heteroreceptors. Such cultured raphe cells may represent a suitable model to study expression and development of presynaptic receptors on serotonergic neurons in-vitro.     

Bisquaternary Pyridinium Oximes as Presynaptic Agonists and Postsynaptic Antagonists of Muscarinic Receptors

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.     

Effects of oxotremorine and RMI 12330 A on [3H]acetylcholine release and adenylate cyclase activity in guinea pig superior cervical ganglion

There is considerable evidence that adenosine 3, 5-cyclic monophosphate (cAMP) is involved in the modulation of synaptic transmission in the guinea pig superior cervical ganglion (SCG). Presynaptic muscarinic receptors are known to attenuate, when activated, acetylcholine (ACh) release in the periphery as well as in the brain. Thus, the possible relationship between ganglionic adenylate cyclase activity and the output of ACh from electrically stimulated ganglia, preloaded with [³H]choline, was investigated. The muscarinic agonist oxotremorine significantly reduced in a dose-dependent manner the electrically evoked neurotransmitter release. The adenylate cyclase inhibitor N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (RMI 12330 A) also decreased ACh output. The inhibitory effects of these two drugs were additive. In crude ganglion membrane fractions oxotremorine significantly inhibited adenylate cyclase activity. The results indicate that drugs capable of inhibiting adenylate cyclase, significantly decrease ACh output from preganglionic nerve terminals in guinea pig SCG.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

HT22 hippocampal neuronal cell line possesses functional cholinergic properties

AimsHippocampal cholinergic hypofunction is known to be involved in the cognitive deficits of Alzheimer's disease, but the detailed mechanisms remain to be elucidated. In order to establish an in vitro hippocampal cholinergic neuronal model for the relevant mechanistic studies, we have characterized a widely used hippocampal neuronal cell line, HT22, a sub-line derived from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture.Main methodsWestern blot and immunocytochemistry were used to examine expression of cholinergic markers in HT22 cells. High potassium-evoked [³H]ACh release was used to evaluate the cholinergic functional properties of the cells.Key findingsWe found that HT22 cells express essential cholinergic markers, such as the high affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter, and muscarinic acetylcholine receptors. Exposure of HT22 cells to high potassium evoked [³H]ACh release in a dose-dependent manner. In addition, the [³H]ACh release was significantly potentiated when presynaptic autoreceptors were blocked.SignificanceOur results suggest that HT22 cells possess functional cholinergic properties, and can be used for an in vitro model for defining the mechanisms in cognitive deficits of Alzheimer's disease.     

Effect of Presynaptic P2 Receptor Stimulation on Transmitter Release

Because ATP is degraded to adenosine, its effect could be mediated by both P1 and P2 receptors. Hence, the actions of an ATP analogue, resistant to enzymatic breakdown (alpha, beta-methylene ATP), were studied on the resting and electrically evoked release of radioactivity from longitudinal muscle strips of guinea pig ileum, preloaded either with [3H]choline or with [3H]noradrenaline. Their effects were compared with the actions of adenosine and ATP. Although adenosine and ATP markedly decreased the [3H]acetylcholine release evoked by field stimulation, alpha,beta-methylene-ATP, a potent and selective agonist of P2x receptors, enhanced this release. However, 2-methyl-2-thio-ATP, an agonist of the P2y receptors, neither enhanced nor inhibited the [3H]-acetylcholine release. 8-Phenyltheophylline, an antagonist of P1 receptors, increased the stimulation-evoked release of acetylcholine, indicating that the release of acetylcholine is tonically controlled by endogenous adenosine via P1 receptors. When alpha,beta-methylene-ATP and 8-phenyltheophylline were added together, their potentiating effect on the acetylcholine release proved to be additive. Because alpha,beta-methylene-ATP failed to antagonize the presynaptic effect of adenosine on P1 purinoceptors, it seems very likely that its effect to enhance transmitter release is mediated via separate receptors, i.e., via P2x receptors, located on the axon terminals. Similarly, the stimulation-evoked release of [3H]noradrenaline was enhanced slightly by alpha,beta-methylene-ATP. Our results suggest that both cholinergic and noradrenergic axon terminals are equipped with P2 receptors through which the stimulation-evoked release of transmitter can be modulated by ATP in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of hemicholinium-3 on presynaptic nicotinic acetylcholine receptors located on the terminal region of myenteric motoneurons

Previously we have demonstrated the presence of presynaptic nicotinic acetylcholine receptors on the terminals of myenteric neurons in Auerbach's plexus of guinea-pig ileum. During these studies we observed, that the presence of hemicholinium-3, an inhibitor of the high affinity choline uptake significantly influences the contraction of the longitudinal muscle strip preparation. Our aim was to investigate the neurochemical background of this effect and quantitatively characterize the action of HC-3. We studied the effect of HC-3 on epibatidine- and electrical stimulation-evoked contraction and release of [3H]acetylcholine from the guinea-pig longitudinal muscle strip preparation. We found that in the presence of tetrodotoxin, when the contribution of somatodendritic nicotinic acetylcholine receptors to the response was prevented due to the inhibition of axonal conduction, HC-3 inhibited the epibatidine-evoked contraction and [3H]acetylcholine release in the submicromolar range (IC50 = 897 nM and IC50 = 693 nM, respectively), whereas the electrical stimulation-evoked contraction was not affected by HC-3, and the release of [3H]acetylcholine was apparently enhanced. Our data indicate that HC-3 inhibits the presynaptic nicotinic acetylcholine receptors of myenteric neurons. Since these receptors play an important role in the regulation of cholinergic neurotransmission in the enteric nervous system, the use of HC-3 in [3H]acetylcholine release experiments might bias the interpretation of data.     

Depression of potassium-evoked striatal acetylcholine release by delta-receptor activation: inhibition by cholinoactive agents

To examine the role of delta-opioid receptors in the modulation of striatal acetylcholine (ACh) release, the action of D-Pen2,L-Pen5-enkephalin, a selective delta-opioid receptor agonist, was tested on [3H]ACh release from slices of the rat caudate-putamen. Slices, incubated with [3H]choline, were superfused with a physiological buffer and stimulated twice by exposure to a high potassium (K+) concentration. In the absence of a cholinesterase inhibitor, 1 microM D-Pen2,L-Pen5-enkephalin produced a 46 and 35% decrease in the release of [3H]ACh evoked by 15 and 25 mM K+, respectively. The depressant action of the enkephalin analogue was concentration dependent, with a maximal effect on K+-evoked [3H]ACh release occurring at 1.0 microM, and was completely blocked in the presence of the delta-opioid receptor selective antagonist, ICI 174864 (1 microM). In the presence of the cholinesterase inhibitors physostigmine (10 microM) and neostigmine (10 microM), or the muscarinic receptor agonist oxotremorine (10 microM), D-Pen2,L-Pen5-enkephalin did not depress the K+-evoked release of [3H]ACh. Atropine (1 microM) blocked the inhibitory effect of physostigmine on the depressant action of D-Pen2,L-Pen5-enkephalin. The results of this study indicate that delta-opioid receptor activation is associated with an inhibition of striatal ACh release, but this opioid-cholinergic interaction is not apparent under conditions of presynaptic muscarinic receptor activation.     

Changes in Cholinergic but Not in GABAergic Markers in Amygdala, Piriform Cortex, and Nucleus Basalis of the Rat Brain Following Systemic Administration of Kainic Acid

Three days after systemic administration of kainic acid (15 mg/kg, s.c.), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high-affinity choline uptake) and GABAergic parameters [benzodiazepine and gamma-aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle-injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium-dependent high-affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid-induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and cholinoceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.