Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.     

Nitric Oxide-Induced Release of Acetylcholine in the Nucleus Accumbens: Role of Cyclic GMP, Glutamate, and GABA

Abstract: We have previously shown that the basal acetylcholine release in the ventral striatum is under the enhancing influence of endogenous nitric oxide (NO) and that NO donors cause pronounced increases in the acetylcholine release rate. To investigate the role of cyclic GMP, glutamate, and GABA in the NO-induced acetylcholine release, we superfused the nucleus accumbens, (Nac) of the anesthetized rat with various compounds through a push-pull cannula and determined the neurotransmitter released in the perfusate. Superfusion of the Nac with the NO donors diethylamine/NO (DEANO; 100 µmol/L), S-nitroso-N-acetylpenicillamine (SNAP; 200 µmol/L), or 3-morpholinosydnonimine (SIN-1; 200 µmol/L) enhanced the acetylcholine release rate. The guanylyl cyclase inhibitor 1H-(1,2,4)-oxodiazolo(4,3-a)quinoxalin-1-one (ODQ; 10 µmol/L) abolished the effects of DEANO and SIN-1. 6-(Phenylamino)-5,8-quinolinedione (LY-83583; 100 µmol/L), which also inhibits cyclic GMP synthesis, inhibited the releasing effects of DEANO and of SNAP, whereas the effect of SIN-1 on acetylcholine release was not influenced. The DEANO-induced release of acetylcholine was also abolished in the presence of 20 µmol/L 6,6-dinitroquinoxaline-2,3-dione (DNQX) and 10 µmol/L (±)-2-amino-5-phosphonopentanoic acid (AP-5). Simultaneous superfusion with 50 µmol/L quinpirole and 10 µmol/L 7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF 83566) was ineffective. Superfusion with 500 µmol/L DEANO decreased the release of acetylcholine. The inhibitory effect of 500 µmol/L DEANO was reversed to an enhanced release on superfusion with 20 µmol/L bicuculline. Bicuculline also enhanced the basal release rate. These findings indicate that cyclic GMP mediates the NO-induced release of acetylcholine by enhancing the outflow of glutamate. Dopamine is not involved in this process. Only high concentrations of NO increase the output of GABA, which in turn decreases acetylcholine release. Our results suggest that cells that are able to release glutamate, such as glutamatergic neurons, are the main target of NO in the Nac.     

Evidence for acetylcholine as a neurotransmitter in the statocyst of Octopus vulgaris

Summary Evidence is presented for acetylcholine as neurotransmitter in the sensory epithelia (macula and crista) of the statocyst of Octopus vulgaris, based on the following techniques: (i) histochemical assay of acetylcholinesterase at light- and electron-microscopical levels, in combination with the detailed knowledge of the ultrastructural and neuronal organization of the receptor epithelia; (ii) lesion/degeneration experiments of the efferent fibre system; (iii) radiochemical assay of acetylcholine; and (iv) bioassay of acetylcholine. All data support the hypothesis that in the statocyst of O. vulgaris acetylcholine acts as a neurotransmitter in the efferent fibre system.This paper is dedicated to Professor Franz Huber, Seewiesen, on the occasion of his 60th birthday     

Excitatory and inhibitory receptive fields of tectal cells are differentially modified by magnocellular and parvocellular divisions of the pigeon nucleus isthmi

It has been known that magnocellular and parvocellular divisions of the pigeon nucleus isthmi exert excitatory and inhibitory actions on tectal cells, respectively. The present study shows that injection of N-methyl-D-aspartate into the parvocellular division results in an increase in responsive strength and extent of the inhibitory receptive fields, which expand into the excitatory receptive fields of tectal cells. This injection concurrently leads to a decrease in responsiveness and extent of the excitatory fields. On the other hand, injection of acetylcholine into the magnocellular division enhances visual responsiveness, although the excitatory field is not obviously changed in extent. Meanwhile, strength and extent of the inhibitory fields are decreased by acetylcholine. The excitatory and inhibitory fields are reduced in both strength and extent by magnocellular and parvocellular injection of lidocaine, respectively. It suggests that isthmic inputs from both parvocellular and magnocellular divisions converge onto the same tectal cells, and the magnocellular and parvocellular subnuclei can modulate excitatory and inhibitory receptive fields of tectal cells, respectively, with some interactions between both fields. Accepted: 1 March 2000     

Production of acetylcholine in rat brain following thiamine deprivation and treatment with thiamine antagonists

Abstract— The effects of thiamine deprivation and of treatment with the thiamine antagonists, oxythiamine and pyrithiamine, on the storage and synthesis of acetylcholine were studied in rats. Rats treated with pyrithiamine always developed ataxia and convulsions, and they died in an average of 36 ± 5.0 hr after onset of convulsions. Injections of sublethal doses of eserine after onset of convulsions had no effect or shortened survival time. If injections were started before the onset of convulsions, the survival time was increased to 56 ± 3.3 hr. The content of total acetylcholine-like compounds, measured by bioassay, in the brain was decreased in all three types of thiamine deficiency. On the other hand, the amount of parenterally administered [¹⁴C]pyruvate converted to [¹⁴C]acetylcholine in vivo was affected only by treatment with pyrithiamine. The increase found was probably due to an increased permeability of the blood-brain barrier to the pyruvate. Conversion of [¹⁴C]pyruvate to [¹⁴C]acetylcholine in vitro was decreased significantly in homogenates of brains from both oxythiamine and pyrithiamine-treated animals.     

Theory for the feedback inhibition of fast release of neurotransmitter

Autoinhibition of neurotransmitter release occurs via binding of transmitter to appropriate receptors. Experiments have provided evidence suggesting that the control of neurotransmitter release in fast systems is mediated by these inhibitory autoreceptors. Earlier, the authors formulated and analysed a mathematical model for a theory of release control in which these autoreceptors played a key role. The key experimental findings on which the release-control theory is based are: (i) the inhibitory autoreceptor has high affinity for transmitter under rest potential and shifts to low affinity upon depolarization; (ii) the bound (with transmitter) autoreceptor associates with exocytotic machinery Ex and thereby blocks it, preventing release of neurotransmitter. Release commences when depolarization shifts the autoreceptor to a low-affinity state and thereby frees Ex from its association with the autoreceptors. Here we extend the model that describes control of release so that it also accounts for release autoinhibition. We propose that inhibition is achieved because addition of transmitter, above its rest level, causes transition of the complex of autoreceptor and Ex to a state of stronger association. Relief of Ex from this state requires higher depolarization than from the weakly associated complex. In contrast to the weakly associated complex that only requires binding of transmitter to the autoreceptor to be formed, the transition to the strongly associated complex is induced by a second messenger, which is produced as a result of the receptor binding to transmitter. The theory explains the following experimental results (among others): for inhibition via transmitter or its agonists, the magnitude of inhibition decreases with depolarization; a plot of inhibition as a function of the concentration of muscarine (an acetylcholine agonist) yields an S-shaped curve that shifts to the right for higher depolarizations; the time course of release does not change when transmitter is added; the time course of release also does not change when transmitter antagonists are added, although quantal content increases; however, addition of acetylcholine esterase (an enzyme that hydrolyses acetylcholine) prolongs release.     

Correlation between Choline Signal Intensity and Acetylcholine Level in Different Brain Regions of Rat

Acetylcholine is an important excitatory neurotransmitter, which plays a crucial role in synaptic transmission. The level of acetylcholine is decreased in the early stages of Alzheimer disease (AD), the most common neurodegenerative disease. Therefore, measurement of acetylcholine in the brain may help the clinical diagnosis of AD. However, the methods used till now to detect the brain acetylcholine level are invasive, which are neither recommended nor acceptable in the clinic. Acetylcholine is synthesized from choline-containing compounds (Cho), the latter can be estimated by noninvasive proton magnetic resonance spectroscopy (¹H MRS). To explore whether the Cho signal intensity could be used to represent the acetylcholine level in the brain, we employed ¹H MRS to detect the Cho signal, and simultaneously, we also used microdialysis and high-performance liquid chromatography (HPLC) to measure the level of acetylcholine in hippocampus, striatum, frontal cortex, and somatosensory barrel field (S1BF cortex) of rats, respectively. The results showed that the correlations between Cho signal intensity and acetylcholine level in hippocampus, striatum, frontal cortex, and S1BF cortex were, respectively, 0.823 (p = 0.044), 0.851 (p = 0.032), 0.817 (p = 0.047), and 0.822 (p = 0.045). The F-values of the regression model were, respectively, 8.404 (p = 0.044), 10.47 (p = 0.032), 8.000 (p = 0.047), and 8.326 (p = 0.045). And the derived regression equations were y = 0.67x + 1.363 (hippocampus), y = 5.398x + 6.684 (striatum), y = 0.656x + 0.564 (frontal cortex), and y = 0.394x + 1.127 (S1BF cortex), respectively (y means acetylcholine, and x means Cho). These data suggest that the Cho signal intensity observed by ¹H MRS may be used as an indicator of acetylcholine level in different brain regions of the rats. X.-C. Wang, X.-X. Du, and Q. Tian equally contributed to the work.     

Muscarinic acetylcholine responses in the early embryonic chic retina

The action of acetylcholine on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). Increases in [Ca²⁺]_i were evoked by the puff application of acetylcholine at concentration than 0.1 μM. The Ca²⁺ response became larger in dose–dependant manner up to 10 μM of acetylcholine applied. The rise in [Ca²⁺]_i was not due to the influx of Ca⁺² through calcium channels, but to the release of Ca²⁺ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca²⁺ rise caused by depolarization with 100 mM K⁺, had no effects on the acetylcholine response and the Ca²⁺ response to acetylcholine occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolished the response to 10 μM acetylcholine, whereas d-tubocurarine of 100 μM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 μM each), evoked comparable responses with that to 10 μM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 μM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca²⁺ mobilization we found in the early embryonic chick retina may be regarded as a part of the “embryonic muscarinic system” proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis. 1994 John Wiley & Sons, Inc.     

Bacterial expression of the water-soluble domain of lynx1, an endogenous neuromodulator of human nicotinic receptors

The membrane-associated protein Lynx1 is expressed in the human central nervous system and plays an important role in the regulation of the activity of nicotinic acetylcholine receptors. In the present study, ws-Lynx1 encoding the water-soluble domain of human Lynx1 was cloned and studied by heterological expression in E. coli. In milligram quantities, the ws-Lynx1 protein could only be obtained in cytoplasmic inclusion bodies of bacterial cells. To optimize the yield of ws-Lynx1 recombinant protein, a system was developed that allowed retrieving functionally active ws-Lynx1 from the inclusion bodies. After renaturation, the protein was characterized by mass spectrometry and by circular dichroism spectroscopy. ws-Lynx1 was shown to inhibit the binding of [¹²⁵I]-α-bungarotoxin to membranes from the electric organ of the Torpedo californica ray containing muscle-type nicotinic acetylcholine receptors (α1₂βγδ) in a competitive manner.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Effect of Pentylenetetrazole-Induced Kindling on Acetylcholine Release in the Hippocampus of Freely Moving Rats

Abstract: The role of γ-aminobutyric acid (GABA) modulation of septohippocampal cholinergic neurons in kindling was investigated. Hippocampal acetylcholine release was evaluated with the microdialysis technique in freely moving rats either after acute administration of isoniazid (an inhibitor of GABA synthesis) or pentylenetetrazole (PTZ)(a blocker of the GABA_A receptor-associated Cl⁻ channel) or after chronic administration of PTZ. Short-term treatment with PTZ (5–50 mg/kg, i.p.) or isoniazid (150–250 mg/kg, s.c.) increased hippocampal acetylcholine release in a dose-dependent manner. In contrast, the basal concentration of acetylcholine in the dialysate from the hippocampus of rats chronically treated with PTZ (kindled animals) was significantly reduced relative to that of vehicle-treated rats (2.39 ± 0.21 vs. 4.2 ± 0.31 pmol per 20-min sample; p < 0.01). Moreover, the release of acetylcholine was markedly more sensitive to the effect of a challenge injection of PTZ (10 or 20 mg/kg, i.p.) in kindled rats than in naive rats or rats chronically treated with vehicle. Abecarnil, a selective benzodiazepine receptor agonist with marked anticonvulsant activity, was administered together with chronic PTZ to evaluate whether persistent activation of GABA_A receptors and suppression of seizures during kindling might affect the sensitivity of septohippocampal cholinergic neurons to a challenge dose of PTZ. Abecarnil (1 mg/kg, i.p.) administered 40 min before each PTZ injection neither antagonized the decrease in basal acetylcholine release (2.26 ± 0.19 pmol per 20-min sample) nor prevented the development of kindling. In contrast, abecarnil prevented the chronic PTZ-induced increase in the sensitivity of acetylcholine release to a challenge dose of PTZ. These results provide novel in vivo data concerning the role of hippocampal acetylcholine function in the development of kindling and potentially in the learning and memory deficits associated with this phenomenon.     

The effect of light on the level of acetylcholine in seedlings of the wild-type and phytochrome mutants of tomato (Lycopersicon esculentum Mill.)

Applying the method of pyrolysis coupled with gas chromatography (PYR-GC) the content of endogenous acetylcholine (ACh) was investigated in the extracts obtained from tomato (Lycopersicon esculentum Mill.). Seven-day-old seedlings of wild type (WT) and phytochrome mutants au (aurea), hp (high pigment), fri (far-red light insensitive) and tri (temporarily red light insensitive) were studied. In the analyzed material the presence of choline and acetylcholine was discovered. The highest content of ACh (381 mmole/g of fresh weight) was found in tomato cotyledons, whereas the lowest amount (162 nmole/g of fresh weight) in roots. The level of ACh in the plants grown under the continuous light was higher than in etiolated ones. However, no considerable differences in the concentrations of ACh in au and tri seedlings grown under the continuous light and in darkness were observed. The irradiation of etiolated seedlings of wild type with red light was accompanied by the increase of endogenous level of ACh. The pulse of far-red light applied directly after red light reversed this stimulating effect. A similar effect of both light wavelengths on the content of ACh was also found in the case of the tri mutant. On the other hand, in the case of fri mutant, pulse of red light caused the drop in the content of ACh, whereas far-red applied after red light caused visible increase in the level of the investigated substance. In tissues of au mutant no effect of red and far-red lights on the concentration of ACh was established.     

EFFECT OF PHOSPHATIDYLSERINE ON ACETYLCHOLINE OUTPUT FROM THE CEREBRAL CORTEX OF THE RAT

The effect of sonicated suspensions of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine injected intravenously on acetylcholine release from the cerebral cortex was investigated in urethane anaesthetized rats. The electroeorticogram was also recorded. Phosphatidylserine caused a dose dependent, calcium dependent increase in acetylcholine output with no electrocorticografic changes. The increase, 75% peak effect after 150 mg/kg, was abolished by septal lesions and pretreatment with pimozide. Phosphatidylserine had no effect on acetylcholine release from brain slices in vitro. Phosphatidylethanolamine was approximately half as active as phosphatidylserine and phosphatidylcholine had no effect on acetylcholine output in vivo. It is concluded that phosphatidylserine exerts an indirect stimulating action on a septio-cortical cholinergic pathway.     

Hybridization of Euglena RNA to the heavy and the light chloroplast DNA components separated in alkaline CsC1 gradients

It has been suggested that increases in cyclic GMP levels are responsible for the negative inotropic effects of acetylcholine in the heart. This hypothesis was tested by monitoring the effects of acetylcholine and sodium nitroprusside on tension and cyclic nucleotide levels in strips of cat atrial appendage. Sodium nitroprusside markedly increased atrial cyclic GMP levels but did not decrease the twitch tension developed by the atrial strips. Low concentrations of acetylcholine, on the other hand, decreased twitch tension without increasing myocardial cyclic GMP levels. No significant change in cyclic AMP levels was observed in any of these experiments. These results are not consistent with the proposed role for cyclic GMP as the mediator of the negative inotropic effects of acetylcholine.     

Cholinergic mechanisms of interneuronal transmission in the retina

Acetylcholine, applied to the isolated perfused frog and human retina, induces a corneopositive potential. This electrogenic action of acetylcholine, in conjunction with existing data, confirms the view that transmission in the retina is cholinergic. The magnitude and the temporal course of the potential evoked by acetylcholine depends both on its concentration and on the state of adaptation of the retina. Photic stimulation reduces the response to acetylcholine; under these circumstances flashes are more effective than a steady illumination. On the other hand the response of the retina to light decreases during perfusion with acetylcholine. The positive component of the ERG is particularly strongly inhibited, leaving only the negative P_III. The results indicate that acetylcholine acts on synapses between the first and second retinal neurons. They can be explained in terms of the hypthesis of the desensitization of cholinergic receptors in the retina.     

Carbamylcholine and acetylcholine-sensitive,cation-selective ionophore as part of the purified acetylcholine receptor

Summary Black lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric rayTorpedo californica or its tryptic digest. In both cases, conductance of cations increased and was dependent on the concentration of the receptor protein. Conductance of Ca⁺⁺ was dependent on its concentration, but addition of carbamylcholine gave no reproducible or consistent effects. Only in the case of the tryptic digest of the acetylcholine receptor did carbamylcholine and acetylcholine consistently induce monovalent cation selective conductance (P _{Na, K}P _Cl=4.4). The induced monovalent cationic conductance due to carbamylcholine (10 m) varied from 10- to over 100-fold. Curare (10 m) prevented the action of carbamylcholine.Na-dodecyl sulfate gel electrophoresis of the acetylcholine receptor, before and after tryptic digestion, indicated that this mild enzyme treatment hydrolyzed the receptor molecule subunits. Nevertheless, the receptor molecule retained its full binding of [acetyl-³H]acetylcholine; and analytical gel electrophoresis indicated that it remained intact possibly through hydrogen, hydrophobic and disulfide bonding.     

Effects of saturated fatty acids on amylase release from exocrine pancreatic segments of sheep,rats, hamsters,field voles and mice

 Stimulatory effects of saturated fatty acids consisting of 4 (butyrate), 8 (octanoate), 12 (laurate) and 16 (palmitate) carbon atoms, as well as acetylcholine on pancreatic amylase release were assessed in tissue segments isolated from sheep, rats, hamsters, field voles and mice. The amount of amylase release induced by the fatty acids (1 μmol ⋅ l^-1 to 10 mml ⋅ l^-1) and by acetylcholine (10 nmol ⋅ l^-1 to 100 μmol ⋅ l^-1) increased in a concentration-dependent manner, and the maximum response in response to the fatty acids was obtained at the maximal dose used. The maximum increase in amylase release in response to butyrate or octanoate was highly and significantly (r=0.974, P<0.001) dependent on the log value of the mean body mass in the following order: sheep>rats>hamsters>field voles>mice. On the other hand, the response to laurate and palmitate was variable among animal species. Addition of atropine (1.4 μmol ⋅ l^-1) to the medium did not reduce the responses to octanoate stimulation, but significantly reduced acetylcholineinduced responses, implying that the effects of the fatty acids were not mediated through activation of muscarinic acetylcholine receptors. Reduction of calcium ion concentration in the medium significantly inhibited the responses induced by the fatty acids and acetylcholine, suggesting that amylase release depends on extracellular calcium ions. Accepted: 14 May 1996     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

LES COMPARTIMENTS D''ACETYLCHOLINE DE L''ORGANE ELECTRIQUE DE LA TORPILLE ET LEURS MODIFICATIONS PAR LA STIMULATION

Abstract— Changes in ‘free’ and ‘bound’ acetylcholine before and after stimulation have been investigated in vivo and in slices of electric organ of Torpedo marmorata incubated or superfused with physiological saline solutions. Spontaneous miniature end-plate potentials could be recorded and on electrical stimulation discharges of up to 30 V could be elicited. The electrical response fell off rapidly on repetitive stimulation. ‘Bound’ acetylcholine is that which relhains after the tissue has been homogenized since any ‘free’ acetylcholine is hydrolysed by the esterases when the tissue is disrupted. ‘Free’ acetylcholine can therefore be determined as the difference between the total acetylcholine found when the tissue is extracted with trichloroacetic acid and that which remains when the tissue is homogenized. Most of the ‘bound’ acetylcholine is present in synaptic vesicles. Stimulation of the tissue until the electrical response had fallen was accompanied by a drop in the level of ‘free’ acetylcholine. Lowered calcium and increased magnesium concentrations in the medium caused a decrease in the electrical response to stimulation and a decrease in the fall of ‘free’ acetylcholine. In other experiments, a decrease of both compartments was noticed at the end of the stimulation period. However the drop in ‘bound’ acetylcholine could also be elicited after the ‘free’ had fallen, by continuing the stimulation. When anticholinesterases were put in the medium, acetylcholine released on stimulation could be collected. On pre-incubation of the slice with [¹⁴C]choline, the acetylcholine stores became labelled. The specific radioactivity of the ‘free’ acetylcholine fluctuated on serial stimulations, whereas the specific radioactivity of the ‘bound’ acetylcholine remained stable under these experimental conditions. It is concluded that the ‘free’ compartment of acetylcholine is the most immediately available for release on stimulation.