Recent studies of the actions of cholinomimetic drugs on adrenergic nerves and their implications for the cholinergic link hypothesis

This review analyzes the results of recent studies of the actions of cholinomimetic drugs on adrenergic nerve terminals and their implications for the cholinergic link hypothesis. Thus far, evidence suggests that the only possible action of endogenous acetylcholine (ACh) present near noradrenaline (NA) stores is an inhibition of the release of NA from the adrenergic nerve terminals and that NA is released only when the action of acetylcholinesterase is inhibited. Nicotinic agents have been shown to act on adrenergic nerve terminal membranes, a finding that casts doubt on the proposed intraneuronal cholinergic sites for the action of endogenous ACh. Evidence also indicates that the mode of adrenergic neurone blocking action of bretylium and guanethidine is independent of the proposed cholinergic process in NA release. Current findings do not support the proposal that nicotinic agents in higher concentrations interfere with adrenergic neurotransmission. It is therefore concluded that nicotinic agents, in causing the release of NA from adrenergic nerve terminals, are merely exhibiting a pharmacological action and not mimicking the physiological function of ACh, as proposed by the cholinergic link hypothesis.     

Pharmacological correction of ketamine-impaired spatial memory of rats

Ketamine in dose 10 mg/kg increased the number of erroneous choices of rats with spatial strategy and didn't effect searching accuracy of rats with non-spatial strategy in 8-arm radial maze. Ketamine in doses 1 and 5 mg/kg disrupted rat short-term memory in delayed response reaction. Physostigmine and aspartic acid, but not haloperidol, diminished ketamine amnesia, therefore ketamine impaired the interaction of cholinergic and glutamate/aspartatergic neurons of hippocampal areas.     

The effects of muscarinic receptor blockers on the turnover rate of acetylcholine in various regions of the rat brain.

The actions of antimuscarinic agents (benztropine, trihexyphenidyl, and scopolamine) on the dynamics of acetylcholine (ACh) in central cholinergic neurons were examined in various rat brain areas. It was found that the pattern of changes in ACh turnover (TRACh) elicited by these drugs exhibited marked regional variations. After administration of the anticholinergic drugs, the TRACh in hippocampus and thalamus was increased, in cortex it was decreased, and in striatum it was unchanged. ACh concentration in the cortex and striatum was decreased while in hippocampus and thalamus ACh levels were unaltered. Further analysis of the cholinergic septo-hippocampal pathway using lesions of the fimbria-fornix and local drug injections into the septum argue against an in vivo action of these drugs on presynaptic or cell body muscarinic autoreceptors. Moreover, the data suggest that muscarinic receptor blockers cause an increased TRACh only in those areas where a feedback loop is operative, possibly by inhibiting a neuronal feedback loop involving at least one noncholinergic interneuron.     

Dopaminergic Regulation of Striatal Acetylcholine Release: The Critical Role of Acetylcholinesterase Inhibition

Abstract: This study examined the effects of different levels of acetylcholinesterase (AChE) inhibition on dopaminergic regulation of striatal acetylcholine (ACh) release as estimated by in vivo brain microdialysis. Systemic administration of d-amphetamine (2 or 10 mg/kg) increased the striatal output of ACh when the AChE inhibitor neostigmine (0.1 µM) was present in the perfusion fluid. In contrast, when the same experiments were conducted at 0.01 µM neostigmine, d-amphetamine failed to affect (2 mg/kg) or significantly decreased (10 mg/kg) striatal ACh output. The inhibitory action of the D₂ receptor agonist quinpirole (0.2 mg/kg) was significantly greater at 0.01 µM than at 0.1 µM neostigmine. Similarly, there was a nonsignificant trend for the D₂ antagonist raclopride (1 mg/kg) to stimulate ACh release to a greater extent at the low neostigmine concentration. In contrast, the stimulant effects of systemic administration of the D₁ agonist A-77636 (1.46 mg/kg) on striatal ACh release were the same at the two neostigmine concentrations. These results demonstrate that the concentration of an AChE inhibitor in the perfusion solution can quantitatively and even qualitatively influence the manner in which dopaminergic agents regulate ACh overflow in the striatum. On comparing the present results with earlier reports concerning the effects of d-amphetamine on tissue concentrations of ACh, it is tentatively concluded that a low neostigmine concentration is the more physiologically relevant condition. Under such conditions, at moderate doses d-amphetamine does not appear to alter striatal ACh release, with this likely being due to the opposing actions of D₁ and D₂ receptors. Nevertheless, until the endogenous interstitial concentrations of striatal ACh can be measured by other methods, the physiological relevance of ACh microdialysis studies in the striatum will remain uncertain.     

Alteration of Acetylcholine Penetration into, and Effects on, Venom-Treated Squid Axons by Physostigmine and Related Compounds

Choline and neostigmine markedly antagonize the effect of acetylcholine (ACh) on the action potential of the venom-treated squid axon, although they themselves have no effect on conduction. Physostigmine also antagonizes the blocking action of ACh at a concentration well below that which has any effect on conduction. In contrast, d-tubocurarine (curare) increases the effect of ACh on the action potential. Choline, neostigmine, and physostigmine markedly decrease the penetration of C¹⁴-labeled ACh into the axoplasm of the squid axon. Curare, in contrast, increases the penetration of ACh, whereas dimethylcurare gives variable results. The results provide an explanation why physostigmine and neostigmine do not influence the action of ACh on axonal conduction in a way similar to that observed at the junction. The additive effect of curare and ACh on the action potential may be due either to the greater rate of penetration of ACh or to an additive effect of the two compounds on the receptor, or to a combination of both factors.     

The associations acetylcholine-eserine and acetylcholine-morphine studied on the frog rectus abdominis muscle

The interaction of morphine and eserine with acetylcholine (Ach) on frog rectus abdominis muscle was studied. Both eserine and morphine potentiate the effects of exogenous Ach and this effect is certainly not due to the anticholinesterase action of the two drugs. Morphine is less effective than eserine in potentiating the response of the muscle to the Ach introduced in the organ-bath. The synergisms Ach-eserine and Ach-morphine are synergisms with potentiation: it is concluded that eserine and morphine in amphibian muscle interact with specific receptors. These binding sites are different from both the esterase and the ACh binding sites of the cholinergic receptor but appear to influence its function.     

Measurement of fasciculations as motor nerve ending discharges in the rat: a dose related effect of neostigmine

The muscle fasciculations caused by neostigmine and similar agents are the result of a primary drug action on motor nerve endings. Asynchronous, repetitive firing of action potentials are evoked at motor nerve endings which are then transmitted to muscle. A dose-response relationship between neostigmine dose and the rate of/or total neural activity has been established in the rat. This fasciculatory response to neostigmine can serve as an index of motor nerve ending excitability and may be useful in assessing the effects of certain pathological states or drug actions at the neuromuscular junction.     

Pharmacological sensitivity of the articular capsule of the primary spines of Eucidaris tribuloides

1. This paper describes the effects of several cholinergic agonists and antagonists, and of β-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides.2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely.3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M₃ muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine.4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules.5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of aeetyl-cholinesterase in the ligament.6. β-Phenylethy lamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament.7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.     

Lack of antagonism by naloxone of the analgesic and locomotor stimulant actions of ketamine

Ketamine hydrochloride caused dose-related analgesia and ataxia in rats. In mice, ketamine caused dose-related analgesia and stimulation of locomotor activity. None of these actions of ketamine were appreciably altered by the narcotic antagonist naloxone. Thus, these actions of ketamine do not appear to be mediated by opiate receptors.     

Phencyclidine-induced depressions of cerebellar purkinje neurons

Local administration of phencyclidine (PCP) by pressure ejection elicited a dose-dependent slowing of the spontaneous discharge of cerebellar Purkinje neurons. Ketamine also depressed firing and was much less potent than PCP. Effects of both PCP and ketamine were antagonized by local or parenteral administration of antipsychotic drugs. The similarities between the electrophysiological and behavioral actions of phencyclidine suggest that alterations in neuronal discharge may underlie its psychotomimetic properties.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Striatal Slices Is Independent of Dopaminergic Tone

The effects of neurotensin (NT) alone or in combination with the dopamine antagonist sulpiride were tested on the release of endogenous acetylcholine (ACh) from striatal slices. NT enhanced potassium (25 mM)-evoked ACh release from striatal slices in a dose-dependent manner. This effect was tetrodotoxin-insensitive, suggesting an action directly on cholinergic elements. The dopamine antagonist sulpiride (5 x 10(-5) M) significantly increased (63%) potassium-evoked ACh release from striatal slices; potassium-evoked ACh release was further increased (90%) in the presence of NT (10(-5) M) and sulpiride (5 x 10(-5) M). The second set of experiments tested the effects of 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra on NT-induced increases of potassium-evoked ACh release. These lesions did not alter the NT regulation of potassium-evoked ACh release from striatal slices, but did significantly increase spontaneous (33%) and potassium-evoked (40%) ACh release from striatal slices. Striatal choline acetyltransferase activity was not affected by 6-OHDA lesions. In addition, following 6-OHDA lesions, sulpiride was ineffective in altering ACh release from striatal slices. Furthermore, evoked ACh release in the presence of the combination of NT and sulpiride was not different from that in the presence of NT alone. These results suggest that in the rat striatum, NT regulates cholinergic interneuron activity by interacting with NT receptors associated with cholinergic elements. Moreover, the NT modulation of cholinergic activity is independent of either an interaction of NT with D2 dopamine receptors or the sustained release of dopamine.     

Microdialysis measures of functional increases in ACh release in the hippocampus with and without inclusion of acetylcholinesterase inhibitors in the perfusate

Because brain extracellular acetylcholine (ACh) levels are near detection limits in microdialysis samples, an acetylcholinesterase (AChE) inhibitor such as neostigmine is often added to microdialysis perfusates to increase ACh levels in the dialysate, a practice that raises concerns that the inhibitor might alter the results. Two experiments compared functional differences in ACh release with and without neostigmine. In the first experiment, 30-60% increases in extracellular ACh concentrations in the hippocampus were evident during food-rewarded T-maze training with 20-500 nm neostigmine in the perfusate but no increases were seen without neostigmine. In the second experiment, 78% increases in ACh release in the hippocampus were seen after injections of the GABA(A) receptor antagonist, bicuculline, into medial septum only if neostigmine (50 nm) was included in the perfusate. These findings suggest that, in the hippocampus, endogenous brain AChEs are very efficient at removing extracellular ACh, obscuring differences in ACh release in these experiments. Therefore, inclusion of AChE inhibitors in the microdialysis perfusate may be necessary under some conditions for observations of functional changes in release of ACh in the hippocampus.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Cerebral Cortex: Effect of Quinolinic Acid Lesions of the Basal Forebrain

The effects of neurotensin (NT) on endogenous acetylcholine (ACh) release from basal forebrain, frontal cortex, and parietal cortex slices were tested. The results show that NT differentially regulates evoked ACh release from frontal and parietal cortex slices without altering either spontaneous or evoked ACh release from basal forebrain slices. In the frontal cortex, NT significantly inhibited evoked ACh release by a tetrodotoxin (TTX)-insensitive mechanism, suggesting an action directly on cholinergic terminals. In the parietal cortex, NT enhanced evoked ACh release by a TTX-sensitive mechanism, suggesting an action of NT on the cholinergic neuron or in close proximity to the cholinergic neuron. The effects of NT on ACh release were confined to evoked ACh release; that is, spontaneous ACh release was not affected. NT did not affect spontaneous or potassium-evoked ACh release from occipital cortex slices. The second set of experiments tested the effects of quinolinic acid (QUIN) lesions of the basal forebrain cell bodies on the NT-induced regulation of evoked ACh release in the cerebral cortex. QUIN lesions of basal forebrain cell bodies caused decreases in choline acetyltransferase activity (27 and 28%), spontaneous ACh release (14 and 21%), and evoked ACh release (38 and 44%) in frontal and parietal cortex, respectively. In addition, 11 days following QUIN lesions of basal forebrain cell bodies, the action of NT to regulate evoked ACh release in frontal cortex or parietal cortex was no longer observed. The results suggest that in the rat frontal and parietal cortex, NT differentially regulates the activity of cholinergic neurons by decreasing and increasing evoked ACh release, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of pretreatments with carbamates,atropine and mecamylamine on survival and on Soman-induced alterations in rat and rabbit brain acetylcholine

A three component pretreatment regimen composed of a carbamate, atropine and mecamylamine offered complete protection against a multiple lethal doses of Soman in rats. In animals, given chemical pretreatment containing physostigmine in the drug regimen, Soman-induced cerebral acetylcholine (ACh) levels were initially elevated but were back down to normal by 30 min post Soman, but in rats given neostigmine in the pretreatment regimen, ACh concentrations were found to be the highest at 30 min after Soman exposure. The data suggest that peripheral acetylcholinesterase (AChE) and nicotinic and muscarinic ACh receptors are critical sites in organophosphorus (OP) anticholinesterase exposure in rats and should be protected to maximize efficacy against OP intoxication. The data also suggest that carbamates which penetrate the blood-brain barrier may be superior to quaternary carbamates in antagonizing OP exposure in that they could be expected to dampen and rapidly abolish OP-induced rises in total brain ACh which in turn should restore normal neural activity in the brain.     

Muscarinic-mediated analgesia

Systemic administration of cholinesterase inhibitors which cross the blood brain barrier have long been known to produce analgesia and enhance analgesia from opiates. A major site of analgesic action of cholinergic agents is the spinal cord. Muscarinic receptors are concentrated in the superficial layers of the dorsal horn of the spinal cord, an area of noxious sensory processing, and these reflect innervation primarily from cholinergic neurons with cell bodies deep in the neck of the dorsal horn. Spinal injection of cholinergic agonists results in analgesia which primarily reflects muscarinic receptor activation. Analgesia occurs in animal models of acute noxious stimulation and of chronic hypersensitivity pain. Although no cholinergic agonists have been tested for safety in humans, the cholinesterase inhibitor, neostigmine, has undergone such testing, and produces analgesia to experimental, acute postoperative, and chronic pain. Thus, muscarinic cholinergic agonists and cholinesterase inhibitors hold promise as non-opiate agents for the treatment of moderate to severe acute and chronic pain.     

Studies on rat sympathetic neurons developing in cell culture. III. Cholinergic transmission.

Principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in culture with several types of non-neuronal cells. As described in the second paper of this series, the neurons in such mixed cultures formed two types of excitatory synapses with each other, electrical and chemical. Evidence is presented here that transmission at the chemical synapses was cholinergic. Four nicotinic ganglionic blocking agents (curare, hexamethonium, tetraethylammonium, and mecamylamine) strongly attenuated or eliminated the excitatory postsynaptic potentials (e.p.s.p.'s) at moderate concentrations; atropine at relatively high concentrations also blocked transmission. Iontophoretic application of acetylcholine (ACh) to the surface of the neurons gave rise to depolarizations that could be made to resemble the e.p.s.p.'s in size and time course; the ACh potentials and the e.p.s.p.'s were then similarly affected by nicotinic blocking agents. The sensitivity to ACh was often distributed nonuniformly on the neuronal surface; it was common to find small, sharply localized regions of high sensitivity. Catecholamines (norepinephrine, epinephrine, and dopamine) had only inhibitory actions; in a few experiments adrenergic blocking agents (phenoxybenzamine, propranolol) were found to have no effect on the e.p.s.p.'s. These observations leave no doubt that the neurons released ACh and had ganglionic, nicotinic ACh receptors on their surfaces. The significance of the fact that a high proportion of the sympathetic neurons in mixed cultures formed cholinergic synapses is discussed.     

Stimulus-Secretion Coupling in Isolated Adrenal Chromaffin Cells: Calcium Channel Activation and Possible Role of Cytoskeletal Elements

Abstract: The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus-secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel-blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh- and K-evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage-dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K-evoked secretion to levels comparable to that of ACh but did not induce a vinblastine block of K-evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.     

Muscarinic Cholinoceptors That Mediate Pigment Aggregation Are Present in the Melanophores of Cyprinids (Zacco spp.)

Like melanophores of many teleosts, those of the dark chub, Zacco temmincki, and the common minnow, Z. platypus (Cyprinidae, Cypriniformes) responded to norepinephrine (NE) by the aggregation of pigment. It was further found that some melanophores were responsive to acetylcholine (ACh) in the same way. The response to NE was blocked by an alpha-adrenergic blocker, phentolamine, whereas the response to ACh was not. By contrast, two muscarinic cholinoceptor antagonists, namely, atropine and scopolamine, effectively blocked the action of ACh. The pigment aggregation due to the liberated sympathetic neurotransmitter was blocked by phentolamine but not by cholinergic blockers. These results suggest that, although the melanophores of these species are controlled in an orthodox manner by the sympathetic nervous system, some of them possess extra muscarinic cholinoceptors that also mediate the aggregation of pigment. The present report is the first to describe the presence of cholinoceptors on the chromatophores in species of fish other than those that belong to the order Siluriformes. The evolutionary implications of these findings are discussed.     

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.     

Acetylcholine/dopamine interaction in planaria

Planaria represents the most primitive example of centralization and cephalization of nervous system. Previous reports indicate that planaria shows specific behavioral patterns, analogous to mammalian stereotypes, in response to drugs acting on acetylcholine or dopamine transmission. Here we further characterized these responses, and investigated the interactions between cholinergic and dopaminergic systems by means of behavioral methods. Exposure to cholinergic agonists physostigmine or nicotine produced hypokinesia with ‘bridge-like’ and ‘walnut’ positions, respectively. Blockade of muscarinic receptors by atropine produced ‘screw-like’ hyperkinesia. Exposure to dopamine agonists (nomifensine, apomorphine) produced marked hyperkinesia with ‘screw-like’ movements. Finally, exposure to dopamine antagonists produced immobility or ‘bridge-like’ position. Pre-exposure to physostigmine blocked the behavioral effects of nomifensine and reduced and markedly delayed the behavioral effects of apomorphine. Pre-exposure to apomorphine slightly reduced and delayed the behavioral changes by physostigmine. Finally, planaria exposed to atropine after either SCH23388 or sulpiride showed ‘C-like’ or ‘screw-like’ hyperkinesia, respectively. Thus, reduction of cholinergic transmission seems to play a pivotal role in determining hyperkinesia in planaria. Under these conditions, different patterns of hyperkinetic activities occur, according to the subpopulation of dopamine receptors stimulated by drugs. These findings suggest that interactions between cholinergic and dopaminergic systems occur very early in animal phylogeny.