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Background: Attentional modulation in the visual cortex of primates is characterized by multiplicative changes of sensory responses with changes in the attentional state of the animal. The cholinergic system has been linked to such gain changes in V1. Here, we aim to determine if a similar link exists in macaque area MT. While rhesus monkeys performed a top-down spatial attention task, we locally injected a cholinergic agonist or antagonist and recorded single-cell activity., Results: Although we confirmed cholinergic influences on sensory responses, there was no additional cholinergic effect on the attentional gain changes. Neither a muscarinic blockage nor a local increase in acetylcholine led to a significant change in the magnitude of spatial attention effects on firing rates., Conclusions: This suggests that the cellular mechanisms of attentional modulation in the extrastriate cortex cannot be directly inferred from those in the primary visual cortex.

Acetylcholine (ACh) is an abundant neurotransmitter and neuromodulator in many species. In Drosophila melanogaster ACh is the neurotransmitter used in peripheral sensory neurons and is a primary excitatory neurotransmitter and neuromodulator within the central nervous system (CNS). The receptors that facilitate cholinergic transmission are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs). This receptor classification is shared in both mammals and insects; however, both the pharmacological and functional characterization of these receptors within the Drosophila nervous system has lagged behind its mammalian model counterparts. In order to identify the impact of ACh receptor subtypes in regulating the performance of neural circuits within the larval CNS, we used a behavioral and electrophysiological approach to assess cholinergic modulation of locomotion and sensory-CNS-motor circuit excitability. We exposed intact and semi-intact 3 rd instar larvae to ACh receptor agonists and antagonists to observe their roles in behavior and regulation of neural circuit excitability and to investigate AChR pharmacological properties in vivo. We combined this with targeted AChR RNAi-mediated knockdown to identify specific receptor subtypes facilitating ACh modulation of circuit efficacy. We identify a contribution by both mAChRs and nAChRs in regulation of locomotor behavior and reveal they play a role in modulation of the excitability of a sensory-CNS-motor circuit. We further reveal a conspicuous role for mAChR-A and mAChR-C in motor neurons in modulation of their input-output efficacy., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Key Points: This study investigates the effects of cholinergic transmission on the expiratory oscillator, the parafacial respiratory group (pFRG) in urethane anaesthetized adult rats. Local inhibition of the acetyl cholinesterase enzyme induced activation of expiratory abdominal muscles and active expiration. Local application of the cholinomimetic carbachol elicited recruitment of late expiratory neurons, expiratory abdominal muscle activity and active expiration. This effect was antagonized by local application of the muscarinic antagonists scopolamine, J104129 and 4DAMP. We observed distinct physiological responses between the more medial chemosensitive region of the retrotrapezoid nucleus and the more lateral region of pFRG. These results support the hypothesis that pFRG is under cholinergic neuromodulation and the region surrounding the facial nucleus contains a group of neurons with distinct physiological roles., Abstract: Active inspiration and expiration are opposing respiratory phases generated by two separate oscillators in the brainstem: inspiration driven by a neuronal network located in the preBötzinger complex (preBötC) and expiration driven by a neuronal network located in the parafacial respiratory group (pFRG). While continuous activity of the preBötC is necessary for maintaining ventilation, the pFRG behaves as a conditional expiratory oscillator, being silent in resting conditions and becoming rhythmically active in the presence of increased respiratory drive (e.g. hypoxia, hypercapnia, exercise and through release of inhibition). Recent evidence from our laboratory suggests that expiratory activity in the principal expiratory pump muscles, the abdominals, is modulated in a state-dependent fashion, frequently occurring during periods of REM sleep. We hypothesized that acetylcholine, a neurotransmitter released in wakefulness and REM sleep by mesopontine structures, contributes to the activation of pFRG neurons and thus acts to promote the recruitment of expiratory abdominal muscle activity. We investigated the stimulatory effect of cholinergic neurotransmission on pFRG activity and recruitment of active expiration in vivo under anaesthesia. We demonstrate that local application of the acetylcholinesterase inhibitor physostigmine into the pFRG potentiated expiratory activity. Furthermore, local application of the cholinomimetic carbachol into the pFRG activated late expiratory neurons and induced long lasting rhythmic active expiration. This effect was completely abolished by pre-application of the muscarinic antagonist scopolamine, and more selective M3 antagonists 4DAMP and J104129. We conclude that cholinergic muscarinic transmission contributes to excitation of pFRG neurons and promotes both active recruitment of abdominal muscles and active expiratory flow., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Acetylcholine, a major neurotransmitter of the parasympathetic and sympathetic nervous systems, was discovered in the early 1900s. Over the years, researchers have revealed much about its regulation, properties of its receptors and features of the downstream signaling that influence its terminal effects. The acetylcholine system, traditionally associated with neuromuscular communication, is now known to play a crucial part in modulation of the immune system and other 'rest and digest' effects. Recent research seeks to elucidate the system's role in brain functions including cognition, sleep, arousal, motivation, reward and pain. We highlight clinically approved and experimental drugs that modulate the acetylcholine receptors. The complexities in targeting the acetylcholine receptors are vast and finding future indications for drug development associated with specific acetylcholine receptors remains a challenge., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.

The role of the acetylcholine-mediated system in the organization of postictal antinociception was investigated. For this purpose, nicotinic and muscarinic cholinergic receptor antagonists were microinjected into the nucleus raphe magnus (NRM), a key structure of the endogenous pain inhibitory system. After the tail-flick test baseline recording, male Wistar rats (N=8 per group) were submitted to stereotaxic surgery for the introduction of a guide cannula aiming at the NRM. Five days after surgery, atropine or mecamylamine (1 µg/0.2 µL, 3 µg/0.2 µL, or 5 µg/0.2 µL) was microinjected into the NRM. The tail-flick withdrawal latency was recorded immediately after peripheral treatment with pentylenetetrazole (PTZ) (64 mg/kg), in two different interictal time windows, and for 130 minutes after the last seizure evoked by intraperitoneal injection of PTZ. The blockade of GABA-mediated Cl(-) influx caused tonic-clonic convulsions in all animals followed by sustained postictal antinociception lasting 110 minutes after seizures; the nociceptive threshold was also found to be high in interictal periods. Pretreatment of the NRM with either atropine or mecamylamine antagonized both interictal and postictal antinociception, suggesting the involvement of cholinergic mechanisms recruiting muscarinic and nicotinic cholinergic receptors of the NRM in the organization of tonic-clonic seizure-induced antinociception., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes. Acetylcholine (ACh) is the primary parasympathetic neurotransmitter and also a non-neural paracrine factor produced by different cells. Here, we analyzed the expression of the cholinergic system in DCs. We found that DCs express the muscarinic receptors M(3), M(4) and M(5), as well as the enzymes responsible for the synthesis and degradation of ACh, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively. Differentiation of DCs in the presence of the cholinergic agonist carbachol, the synthetic analog of ACh, resulted in an increased expression of HLA-DR and CD86 and the stimulation of TNF-α and IL-8 production. All these effects were prevented by atropine, a muscarinic ACh receptor (mAChR) antagonist. Carbachol, was also able to modulate the function of DCs when added after the differentiation is accomplished; it increased the expression of HLA-DR, improved the T cell priming ability of DCs, and stimulated the production of TNF-α but not IL-12 or IL-10. By contrast, carbachol significantly inhibited the stimulation of HLA-DR expression and the enhancement in the T cell priming ability of DCs triggered by LPS. Interestingly, the TNF-α antagonist etanercept completely prevented the increased expression of HLA-DR induced by carbachol, suggesting that it promotes the phenotypic maturation of DCs by stimulating the production of TNF-α. ACh induced similar effects than carbachol; it stimulated the expression of HLA-DR and the production of TNF-α, while inhibiting the stimulation of HLA-DR expression and IL-12 production triggered by LPS. Similarly, neostigmine, an inhibitor of AChE, also stimulated the expression of HLA-DR and the production of TNF-α by DCs while inhibiting the production of TNF-α and IL-12 triggered by LPS. These results support the existence of an autocrine/paracrine loop through which ACh modulates the function of DCs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Acetylcholine (ACh) is the major neurotransmitter released from vestibular efferent terminals onto hair cells and afferents. Previous studies indicate that the two classes of acetylcholine receptors, nicotinic (nAChRs) and muscarinic receptors (mAChRs), are expressed by vestibular hair cells (VHCs). To identify if both classes of receptors are present in VHCs, whole cell, voltage-clamp- and current-clamp-patch recordings were performed on isolated pigeon vestibular type I and type II HCs during the application of the cholinergic agonists, acetylcholine and carbachol, and the cholinergic antagonists, D-tubocurarine and atropine. By applying in different combinations, these compounds were used to selectively activate either nAChRs or mAChRs. The effects of nAChR and mAChR activation on HC currents and zero electrode current potential (V(z)) were monitored. It was found that presumed mAChR activation decreased both inward and outward currents in both type I and type II HCs, resulting in either a depolarization or hyperpolarization. Conversely, nAChR activation mainly increased both inward and outward currents in type II HCs, resulting in a hyperpolarization of their V(z). nAChR activation also increased outward currents in type I HCs resulting in either a depolarization or hyperpolarization of their V(z). The decrease of inward and outward currents and the depolarization of the V(z) in type I pigeon HCs by activation of mAChRs represents a new finding. Ion channel candidates in pigeon vestibular HCs that might underlie the modulation of the macroscopic ionic currents and V(z) by different AChR activation are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The involvement of astrocytes in the cholinergic modulation of the cochlear nucleus has been studied using primary astrocyte cultures prepared from this nucleus. The cells were loaded with the membrane permeable form of the fluorescent Ca(2+) indicator Fluo-4, and carbachol-induced Ca(2+) concentration increases were monitored using an imaging system. In the presence of cholinergic stimulation 36.3% of the cells produced Ca(2+) transients. The time course of the transients was variable; 45.0% of the responding cells showed only a rapid Ca(2+) concentration increase, while in 50.5% of the astrocytes the fast component was followed by a slow plateau phase. Using muscarine as well as general and more specific cholinergic antagonists (atropine, pirenzepine, 4-DAMP and hexamethonium), the role of the M3 and (to a smaller extent) M1 muscarinic acetylcholine receptors could be demonstrated in the genesis of the carbachol-induced Ca(2+) transients. The presence of these two subtypes of muscarinic receptors has been confirmed at both mRNA (Q-PCR) and protein (immunocytochemistry) levels. Our data demonstrate the responsiveness of the cochlear astrocytes towards cholinergic stimulation, suggesting that they may have roles in mediating the effects of cholinergic modulation in the rat cochlear nucleus.

Antimuscarinic effects of ipratropium bromide and atropine are associated with prevention of the development of Arthus reaction, while the cytostatic effects of cyclophosphamide and doxorubicin lead to involution of the thymus and spleen, suppression of antibody production, and aggravation of inflammation, which causes edema of the ankle joint (cyclophosphamide treatment). Apoptosis of tumor cell and inhibition of inflammation can be essential for chemotherapy of malignant and autoimmune diseases.

It is suggested to use Daphnia magna Straus as a biotest object for the evaluation of heart rate (HR) as a functional parameter. The influence of cholinergic ligands (atropine and carbamylcholine) on the Daphnia cardiac rhythm has been studied. It is found that the cholinergic agonist and antagonist produced opposite influence on the HR and are capable of prevent the action of each other. The Daphnia HR variation test can be used in evaluating the effect of xenobiotics and in selecting agents for the pharmacological correction of this functional parameter.

S 24795 evoked methyllycaconitine-sensitive inward currents in voltage-clamped hippocampal interneurons with maximum amplitude about 14% that of ACh-evoked responses. Experiments with rat alpha7 receptors expressed in Xenopus oocytes confirmed that S 24795 is a partial agonist of alpha7 nAChR with an EC(50) of 34+/-11 microM and I(max) of approximately 10% relative to ACh. When 60 microM ACh was co-applied to alpha7-expressing oocytes along with increasing concentrations of S 24795, there was a progressive decrease in response compared to the responses to 60 microM ACh alone (IC(50) 45+/-9 microM). The positive allosteric modulator 5-hydroxyindole potentiated ACh- and S 24795-evoked responses of alpha7 receptors in both oocytes and hippocampal interneurons. In hippocampal slice experiments, depending on the ACh concentrations in the application pipette and the ratio of ACh to S 24795, co-application of S 24795 with ACh variously increased, decreased, or had no effect on responses, compared to ACh alone. In order to estimate the effective dilution factor for the pressure application experiments, we tested alpha7 receptors in oocytes with ACh alone and in co-application with S 24795 at the same ratios as in the slice experiments, but at varying dilution factors. The pattern of interaction seen in the slice experiments was most closely matched under the conditions of a 3:100 dilution, suggesting that the pipette solution was diluted approximately 30-fold at the site of action. This dilution factor was consistent with the potency of ACh and S 24795 in the oocyte expression system (EC(50)s approximately 30 microM).

The aim of the study was to investigate an action of 2-aminopyridine and its new sulfonylcarbamide derivatives 2-AP21, 2-AP22, 2-AP26, and 2-AP27 (10(-5)-10(-3) M) on carbachol-induced shortening of action potential duration and reduction of contraction force in guinea pig atrial muscles. Experiments were carried out using a standard method of myocardium electromechanical activity registration. Under control conditions (perfusion of atrial strips with Tyrode solution), an average of action potential duration, measured at 90% (AP90) and 50% (AP50) of repolarization, were 112.32+/-6.07 ms and 50.21+/-3.25 ms, (n=19), respectively, and contraction force was of 1.42+/-0.28 mN (n=20). Carbachol (10(-6)M), an agonist of muscarinic acetylcholine receptor and activator of K(Ach) channels, markedly decreased AP90 to 35.31+/-4.21%, AP50--to 26.42+/-2.66% (n=19) (P<0.001), and contraction force--to 24.23+/-2.0% (n=20) (P<0.001) vs. control. Modification of 2-aminopyridine structure by replacing 2-amino group by 4-toluolsulfonylcarbamide fragment and quaternization of nitrogen in pyridine ring increased anticholinergic effect on action potential duration and contraction force. According to their maximal prolongation of AP at 90% of repolarization, all new drugs ranked as follows: 2-AP27>>2-AP26>2-AP22> or =2-AP>2-AP21. 2-aminopyridine derivative 2-AP27, containing 4-toluolsulfonylcarbamide fragment and 4-nitrobenzyl radical at quaternized nitrogen of the pyridine, had the most potent anticholinergic effect on AP90 (936.60+/-178.23%). 2-AP22 and 2-AP26 (containing methyl or allyl radicals at quaternized nitrogen of the pyridine, respectively) showed a much weaker anticholinergic effect (231.39+/-28.48% and 318.25+/-63.81%, respectively). The weakest anticholinergic effect (63.59+/-34.38%) was induced by 2-aminopyridine derivative 2-AP21, which had non-quaternized nitrogen of the pyridine.

The control of ovarian steroidogenesis during pregnancy is mainly of endocrine origin. At present, there is little information about the influence of neural factors on the gestation physiology. The purpose of this work was to study the action of cholinergic agents in celiac ganglion upon the liberation of progesterone and ovarian androstenedione in the second half of pregnancy in rats. We used the ex vivo celiac ganglion-superior ovarian nerve-ovary integrated system (celiac ganglion-SON-ovary) that was incubated in buffer solution for 180 min, with the celiac ganglion and the ovary located in different compartments and linked by the SON. The results obtained indicate that the control values of ovarian androstenedione vary according to the pregnancy day analyzed. The addition of acetylcholine in ganglion decreased the liberation of both steroids on Day 15 whereas at the end of pregnancy it decreased the liberation of androstenedione without modifying progesterone. Due to the effect observed with atropine and hexametonium, acetylcholine action might occur through unspecific ganglionic pathways (Days 15 and 21) or through muscarinic ganglionic receptors (Days 19 and 20). Thus, we conclude that the cholinergic sympathetic system from the celiac ganglion might be a fine modulator of the pregnancy physiology.

A basic question regarding the integrative properties of the central nervous system is how transient motor commands or brief sensory stimuli are able to evoke persistent neural changes, mainly in the form of a sustained tonic rate of action potentials. Examples of this persistent neural activity have been reported in prefrontal (Fuster, 1997) and entorhinal (Egorov et al., 2002) cortices, as part of the neural mechanisms involved in short-term working memory (Goldman-Rakic, 1995). Interestingly enough, the general organization of motor systems assumes the presence of bursts of short-lasting motor commands encoding movement characteristics such as velocity, duration, and amplitude, followed by a maintained tonic firing encoding the position at which the moving appendage should be maintained (Robinson, 1981; Moschovakis, 1997). Thus, persistent neural activity seems to be necessary for both behavioral (positions of fixation) and cognitive (working memory) processes.

In big brown bats, tone-specific plastic changes [best frequency (BF) shifts] of cortical and collicular neurons can be evoked by auditory fear conditioning, repetitive acoustic stimuli or cortical electric stimulation. It has been shown that acetylcholine (ACh) plays an important role in evoking large long-term cortical BF shifts. However, the role of N-methyl-d-aspartate (NMDA) receptors in evoking BF shifts has not yet been studied. We found 1) NMDA applied to the auditory cortex (AC) or inferior colliculus (IC) augmented the auditory responses, as ACh did, whereas 2-amino-5-phosphovalerate (APV), an antagonist of NMDA receptors, reduced the auditory responses, as atropine did; 2) although any of these four drugs did not evoke BF shifts, they influenced the development of the long-term cortical and short-term collicular BF shifts elicited by conditioning; 3) like ACh, NMDA augmented the cortical and collicular BF shifts regardless of whether it was applied to the AC or IC; 4) endogenous ACh of the AC and IC is necessary to produce the long-term cortical and short-term collicular BF shifts; 5) blockade of collicular NMDA receptors by APV abolished the development of the collicular BF shift and made the cortical BF shift small and short-term; 6) blockade of cortical NMDA receptors by APV reduced the cortical and collicular BF shifts and made the cortical BF shift short-term; and 7) conditioning with NMDA + atropine applied to the AC evoked the small, short-term cortical BF shift, whereas conditioning with APV + ACh applied to the AC evoked the small, but long-term cortical BF shift.

Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the alpha7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human alpha7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by alpha4beta2, alpha3beta4, and alpha9alpha10 nAChRs. PNU-120596 increased the channel mean open time of alpha7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of alpha7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances alpha7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

In contrast to antagonists, agonists tend to induce considerable conformational changes in their receptors, resulting in opening of ion channels, either directly or via secondary messengers. These conformational transformations require great energy expenses. However, the experimentally determined free energies of complexation between agonists and receptors are often relatively smaller than those for the corresponding antagonists. To rationalize this so-called 'agonist paradox', which has not been clarified in the literature, we have developed an alternative model. Our model may help to discriminate between agonists and antagonists of the acetylcholine (ACh) and mu-opioid receptors. For this purpose, a series of ligands (1-18) have been analyzed both in structural terms and with respect to complexation geometry within the anionic binding sites of these two receptor types.

Miniature stimulating postsynaptic currents of Lumbricus terrestris somatic muscle cells were recorded. Atropine, d-tubocurarin, alpha-bungarotoxin, carbacholine, and proserin did not modify the amplitude and temporal parameters of miniature stimulatory postsynaptic currents, while carbacholine and nicotine depolarized the muscle membrane. Presumably, Lumbricus terrestris muscle cells contain acetylcholine-sensitive channel-receptor complexes not belonging to classical nicotinic or muscarinic acetylcholine receptors.

The mechanism by which muscarinic or nicotinic agonists produce antinociception has been the subject of several studies. In the present investigation, we used intrathecal administration of drugs to rats to show that muscarinic or nicotinic agonists such as bethanechol (BCh) and dimethylphenylpiperazinium (DM), respectively, dose-dependently increased the tail flick latency and reduced the pain produced by a surgical incision performed on the plantar aspect of a hind paw. The effects of BCh in both tests were inhibited by the previous intrathecal administration of atropine, but not mecamylamine (muscarinic and nicotinic antagonists, respectively). Mecamylamine significantly reduced the effects of DM in both tests. Atropine significantly reduced the effect of DM in the tail flick test and inhibited the effect of DM against the incisional pain. Intrathecal hemicholinium-3 (HC-3), a reversible inhibitor of choline transporter, did not change the effect of BCh in the tail flick test but produced a non-significant reduction of the effect of BCh against incisional pain. In contrast, HC-3 produced a non-significant reduction of the effect of DM in the tail flick test but fully inhibited the effect of DM against incisional pain. Therefore, the BCh-induced antinociception depends on a direct activation of muscarinic receptors, whereas DM-induced antinociception results in drug interaction with nicotinic receptors to activate the further release of acetylcholine from intrinsic spinal cholinergic terminals. The acetylcholine released by DM in turn induces antinociception via activation of muscarinic receptors.

Objectives: We studied the effects of beta-adrenergic and cholinergic stimulation and blockade on spontaneous atrial fibrillation (AF) in the intact dog heart., Background: Paroxysmal AF is often preceded by changes in autonomic tone, but the relative roles of adrenergic and cholinergic influences on AF induction are not well known., Methods: Perfusion of catecholamines and acetylcholine (ACh), as well as their combination, through the sinus node artery was used to induce AF in 20 anesthetized open-chest dogs without electrical stimulation of atria., Results: Isoproterenol and adrenaline (10 to 100 micromol/l) induced AF in 21% (3 of 14) and 17% (1 of 6) of dogs, respectively. Atropine (1 to 2 mg) treatment prevented catecholamine-mediated AF, indicating a critical role of cholinergic tone in these AF episodes. Acetylcholine (2.8 +/- 0.3 micromol/l) induced AF in all dogs. Beta-blockade by propranolol (1 mg/kg) did not prevent ACh-induced AF, but increased the threshold ACh concentration for AF induction to 23.5 +/- 3.4 micromol/l (p < 0.05). Acetylcholine-mediated AF was facilitated by isoproterenol (1 to 2 and 10 micromol/l), which decreased the threshold ACh concentration for AF induction to 0.5 +/- 0.1 and 0.4 +/- 0.1 micromol/l, respectively (p < 0.05) and increased the AF duration (from 25 +/- 7 to 141 +/- 54 and 233 +/- 60 s, respectively; p < 0.05). Epicardial mapping of the right atrium (112 unipolar electrodes) demonstrated similar activation patterns during arrhythmias induced by ACh and catecholamines., Conclusions: These data indicate that although both autonomic systems play a role in AF, cholinergic stimulation is likely the main factor for spontaneous AF initiation in this animal model. Adrenergic tone modulates the initiation and maintenance of cholinergically mediated AF.

Frog vestibular organs are endowed with a prominent cholinergic efferent innervation whose stimulation results in several different effects, thereby suggesting diversity in the expression of postsynaptic acetylcholine (ACh) receptors. The application of ACh can mimic efferent stimulation in producing both an inhibition and a facilitation of afferent discharge which are thought to be mediated by at least two distinct ACh receptors present on vestibular hair cells, i.e., alpha9-containing nicotinic receptors (alpha9nAChR) and muscarinic receptors (mAChR), respectively. Using patch-clamp and multiunit vestibular afferent recordings, we demonstrate the presence of an additional excitatory hair cell nicotinic ACh receptor pharmacologically distinct from both alpha9nAChR and mAChR. In order of increasing potency, this distinct receptor was activated by ACh, carbachol, and particularly by the selective nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP). This DMPP-sensitive nicotinic receptor (RDMPP) was antagonized by the classic nicotinic antagonist d-tubocurarine, but refractory to strychnine, atropine, and propylbenzilylcholine mustard, at concentrations that completely block alpha9nAChR and/or mAChR. Activation of RDMPP on application of ACh or DMPP to a subpopulation of isolated posterior semicircular canal (SCC) hair cells resulted in a large depolarization (18.0 +/- 1.2 mV). The current underlying this depolarization was typically small (80.1 +/- 21.6 pA) and showed an inward rectification starting around -45 mV. Given their respective EC50s (47 nM vs. 20 microM), RDMPP was nearly 400 times more sensitive to ACh than alpha9nAChR and thus responded to concentrations of ACh considered too low to be effective at stimulating alpha9nAChR. Despite this remarkable sensitivity, exogenous ACh readily stimulated the mAChR in the intact posterior SCC preparation but failed to activate RDMPP unless the acetylcholinesterase inhibitor physostigmine was present, or high concentrations of ACh were used (>3 mM). In frog, RDMPP most likely underlies the rapid excitatory response seen during efferent stimulation.

In a previous study, our laboratory demonstrated that the intraventricular infusion of nerve growth factor (NGF) accelerated kindling rates and enhanced mossy fiber sprouting in the absence of noticeable kindling-associated neuronal loss. The purpose of the present study was to investigate whether these NGF effects were mediated via the cholinergic system. This study evaluated the effects of the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine on kindling rates and kindling-induced mossy fiber sprouting in adult rats. The results showed that pilocarpine accelerated kindling rates and enhanced kindling-induced mossy fiber sprouting in the CA3 region of the hippocampus, whereas scopolamine retarded kindling rates and blocked kindling-induced mossy fiber sprouting in the CA3 and IML regions. These findings suggest that the cholinergic system may contribute to the long-term structural and functional alterations that are characteristic of the kindled state. Moreover, these data provide support for the hypothesis that NGF infusions may mediate kindling and kindling-induced mossy fiber sprouting via regulation of the cholinergic system., (Copyright 2002 Wiley-Liss, Inc.)

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3'5'(c)AMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3'5' cyclic adenosine monophosphate (3'5'(c)AMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M(1) and M(3), which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.

We have exploited the complementary arrangement of afferents in a coronal slice (300-400 microm) of the rat olfactory tubercle (OT) maintained in vitro to investigate transmission in two separate synaptic pathways. We recorded extracellular responses within the OT dense cell layer in slices and stimulated either the outermost layer to activate primary olfactory fibres or deeper to activate secondary input. Superficial stimulation produced a synaptic potential with superimposed population spike. This interpretation was based on blockade by calcium removal from the bathing medium and the use of the glutamate antagonist DNQX (10 microM); the spike was found to be selectively suppressed by tetrodotoxin applied near the cells. The spike, but not the synaptic wave, was depressed by 12 mM Ca2+ and enhanced by 1 mM Ba2+ in the bathing medium. Deep stimulation to activate association and intrinsic fibres elicited a nerve volley followed by a later response, also blocked by Ca2+ removal or 10 microM DNQX. It was unaffected by high Ca2+ or Ba2+, hence resulting from synaptic and not action current flow. Removal of Mg2+ from the bathing medium revealed an NMDA component of synaptic transmission at both loci that was selectively blocked by D-AP-5. The deep synaptic response, only, was depressed by carbachol IC50 7 microM or muscarine IC50 13 microM. This depression was also induced by AChE inhibitors eserine or tacrine and was antagonized by 1 microM atropine or 5-10 microM clozapine. These results characterize transmission in the OT and demonstrate a role for muscarinic modulation of deeper synapses in the OT that is influenced by psychotherapeutic drugs.

In an attempt to know the relation of seizure and gastric mucosal damage, we challenged arecoline (ACL) centrally to induce seizure and investigated gastric hemorrhagic injury in acid-irrigated stomachs of rats. The protective effects of several drugs also were evaluated. After deprivation of food for 24 h, rats were received laparotomy under diethylether-anesthesia. Both pylorus sphincters and carotid esophagus were ligated. The forestomach was equipped with a cannula for gastric irrigation. After recovery from anesthesia (approximately 1 h), the stomach was irrigated for 2 h with an acid solution containing 100 mM HCl and 54 mM NaCl or the same volume of normal saline. Intracerebroventricular (i.c.v.) ACL (0, 1, 3 or 10 mg/kg dissolved in 10 microl of CSF) was challenged to rats immediately after gastric irrigation. The seizure in rats was produced by ACL in a dose-related manner. The ulcerogenic parameters such as decrease of gastric mucosal glutathione levels and increase of histamine concentrations and lipid peroxide generations as well as the raise of luminal hemoglobin contents and exacerbated mucosal lesions were obtained depending on the doses of ACL challenged. These ulcerogenic parameters produced in ACL (10 mg/kg, i.c.v.) seizure rats were markedly ameliorated by gastric vagotomy or central anticholinergics. Intraperitoneal ketotifen, zinc sulfate, diphenhydramine or cimetidine also produced significant (p<0.05) inhibitions of these ulcerogenic parameters in ACL seizure rats. In conclusion, central ACL seizure may produce gastric oxidative stress and hemorrhagic lesions via vagal nervous activation and histamine release in acid-irrigated stomachs of rats.

The results of study of the selectivity of the effects of cholinonegative and cholinopositive agents obtained on Daphnia magna hydrobionts were compared with the results of investigations on rats. This allowed Daphnia to be recommended as an alternative test object for studying the selectivity of the effect of cholinotropic agents in relation to some subtypes of muscarine receptors in an intact organism.

Background: Chronic ethanol treatment (CET) for 28 weeks significantly increases electrically-stimulated 3H-GABA release from hippocampal slices. This increase in GABA release may be one of the mechanisms by which CET decreases the magnitude of long-term potentiation (LTP) in the hippocampus. The present study examined whether CET increases GABA release via an alteration in heterologous presynaptic cholinergic regulation., Methods: Animals were treated with ethanol or sucrose diet for 28 weeks followed by either no withdrawal or a 48-hr withdrawal period. The electrically-stimulated 3H-GABA release from preloaded superfused hippocampal slices of naive and CET rats was measured., Results: Carbachol increased 3H-GABA release in a concentration-dependent manner, and atropine modulated 3H-GABA release in a biphasic concentration-dependent manner. Atropine (10 microM) significantly blocked the effects of carbachol. Oxotremorine, a selective muscarinic receptor agonist, also increased 3H-GABA release. Mecamylamine, a selective nicotinic antagonist, did not modulate 3H-GABA release and did not block the effects of carbachol. The effects of these agents were also tested in rats 0 or 48 hrs after withdrawal from CET. The biphasic effects of atropine were decreased, whereas the facilitating effects of carbachol were significantly increased. There were no changes in the effects of these agents on 3H-acetylcholine release from hippocampal slices of CET rats compared to sucrose-treated rats., Conclusion: These results suggest that presynaptic muscarinic receptors facilitate GABA release, whereas nicotinic receptors do not play a significant role in modulating GABA release in hippocampus. CET selectively alters presynaptic muscarinic regulation of GABA release in hippocampus and may help us to further understand the mechanism underlying the disruption of LTP by CET.

Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80-90 dpm/mg, which constituted approximately 1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of NG-nitro-L-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAA receptors, which mediate inhibition of NO synthesis.

Human nicotinic acetylcholine receptor (AChR) subtypes alpha3 beta2, alpha3 beta2 alpha5, alpha3 beta4, and alpha3 beta4 alpha5 were stably expressed in cells derived from the human embryonic kidney cell line 293. alpha3 beta4 AChRs were found in prominent 2-micrometer patches on the cell surface, whereas most alpha3 beta2 AChRs were more diffusely distributed. The functional properties of the alpha3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on alpha3 beta4 AChRs and nearly a full agonist on alpha3 beta2 alpha5 AChRs. Chronic exposure of cells expressing alpha3 beta2 AChRs or alpha3 beta2 alpha5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of alpha3beta4 or alpha3 beta4 alpha5 AChRs, i.e. the up-regulation of alpha3 AChRs depended on the presence of beta2 but not beta4 subunits in the AChRs. This was also found to be true of alpha3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, alpha3 beta2 AChRs were expressed at much lower levels than alpha3 beta4 AChRs, but in the presence of nicotine, the amount of alpha3 beta2 AChRs exceeded that of alpha3 beta4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated alpha3beta2 AChRs were functional. The nicotinic antagonists curare and dihydro-beta-erythroidine also up-regulated alpha3 beta2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of alpha3 beta2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of alpha3 beta2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

The aim of the study was an evaluation of the influence that agonists and antagonists of the cholinergic and adrenergic transmission have on the acquisition and retention of habituation in a pigeon. Habituation training was performed by a rotatory test, under specific drugs influencing cholinergic and adrenergic transmission. The results show that after injection of a cholinergic antagonist or adrenergic agonist the acquisition of habituation is quicker; on the contrary, cholinergic agonists or adrenergic antagonists make this process slower. These drugs do not have any influence on the retention of habituation.

Acutely dissociated adult rat striatal cholinergic neurons labeled with [3H]choline were used in a perifusion system to study muscarinic regulation of basal and forskolin-stimulated fractional [3H]acetylcholine ([3H]-ACh) efflux in the absence of synaptic modulation. Carbachol inhibited basal (40% maximal inhibition; IC50 approximately 0.7 microM) and forskolin-evoked release (75% inhibition; IC50 approximately 0.05 microM) in a concentration-dependent manner, and both carbachol actions were abolished with atropine. Thus, activation of striatal muscarinic cholinergic autoreceptors potently inhibits basal and adenylate cyclase-stimulated ACh release. Tonic inhibitory control of cholinergic activity by functional striatal circuitry apparently prevents detection of these important physiological interactions in slices or in situ.

In the present study, we examined the effects of the agonists and antagonists of cholinergic receptors on central dopaminergic function using the 6-hydroxydopamine model of dopamine receptor supersensitivity. Unilateral lesioning of the substantia nigra with 6-hydroxydopamine was carried out in Wistar rats. Two weeks after surgery, the rats were tested for the presence of dopaminergic supersensitivity by their response to the dopamine receptor agonist, apomorphine. Apomorphine-induced rotations were significantly reinforced by the muscarinic receptor antagonist, atropine. In contrast to atropine, the muscarinic receptor agonist oxotremorine attenuated apomorphine's effects. Acute treatment of nicotine significantly reduced apomorphine-induced rotations. However, when increasing doses of nicotine were given for nine days, the rotations of the nicotine-dependent rats were significantly enhanced. So the fact that both muscarinic and nicotinic cholinergic activity could modulate apomorphine-induced rotations was readily apparent in these experiments.

The effect of lesioning hippocampal cholinergic neurons with the neurotoxin AF64A on the ability of cholinergic agonists to modulate stimulation-induced release of 3H-norepinephrine (NE) from rat hippocampal slices was studied. Rats received intracerebroventricular injections of either AF64A (ethylcholine mustard aziridinium, 2 nmol) or vehicle (sham operated). Six weeks after treatment, release of 3H-NE evoked by electrical stimulation (2 Hz, 2 min) in the presence or absence of cholinergic agonists and/or antagonists was measured. Activation of M2 receptors with oxotremorine (in the presence of the M1 antagonist pirenzepine) caused a small inhibition of NE release, which was abolished in hippocampi from AF64A-treated rats. The Kd for high-affinity binding of the selective M2 ligand [3H] AF-DX 384 was increased 10-fold in lesioned tissues. The M1 selective agonist McN-A-343 produced a significant enhancement of NE release, which was unchanged by AF64A lesion. Binding studies with [3H] pirenzepine showed no change in the affinity or number of M1 receptors. Nicotine also caused a significant enhancement of evoked NE release, but this effect was markedly reduced in tissues from AF64A-treated rats. AF64A treatment caused a twofold decrease in the number of [3H] nicotine binding sites. This study suggests that long-term lesion of hippocampal cholinergic neurons with AF64A alters the function of postsynaptic muscarinic M2 and nicotinic cholinergic receptors that modulate the release of NE in the hippocampus.

The effects of cholinergic agonists and antagonists on electrical coupling between horizontal cells were studied in dark-adapted mudpuppy retinas. Carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) uncoupled horizontal cells, but the muscarinic agonist oxotremorine did not. The uncoupling effects of carbachol and DMPP were blocked by the nicotinic antagonist D-tubocurarine and by the dopamine antagonist fluphenazine, indicating that carbachol uncoupled horizontal cells by stimulating dopamine release via nicotinic receptors. Carbachol also caused an increase in release of [3H]dopamine from retinas. D-Tubocurarine increased horizontal cell coupling, indicating that tonic cholinergic input was present in dark-adapted retinas. D-Tubocurarine did not reduce light-evoked uncoupling of horizontal cells, suggesting that cholinergic neurons are not an essential part of the direct pathway by which light causes an immediate increase in dopamine release.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Cholinergic compounds inhibit FMRFamide-induced contractions in dispersed muscle fibres isolated from adult Schistosoma mansoni. Acetylcholine (ACh) was the most effective cholinergic agonist tested with an EC50 < 100 nM. Less effective were propionylcholine and arecoline with EC50 < 1 microM and butyrylcholine and carbachol with EC50 < 10 microM. Choline, muscarine, pilocarpine, nicotine, DMPP (1,1-dimethylphenylpiperazine) and levamisole were all ineffective. Amongst tested antagonists, d-tubocurarine (100 microM), mecamylamine (1 mM), scopolamine (1 mM) and quinuclidinyl benzilate (10 microM) were all ineffective. Bicuculline, picrotoxin and strychnine were also ineffective. However alpha-bungarotoxin, at 100 nM, was able to block the inhibitory ACh effect. From these data it appears that the cholinergic receptor on the schistosome muscle fibres may be of the nicotinic type, but that its pharmacology is different from that of nicotinic receptors of vertebrates as well as of nematodes or a variety of other invertebrates.

The possible involvement of central cholinergic neurotransmission in lipomobilization in avian species was investigated by injecting carbachol into the brain lateral ventricle (i.c.v.) of conscious pigeons (N = 9 per experimental group). I.c.v. injections of carbachol (27 nmol in 1 microliter) induced an intense increase (nearly 91% above baseline levels) in the concentration of plasma free fatty acids (FFA). Previous intraperitoneal administration of hexamethonium (10 mg/kg) completely blocked the lipomobilizing effect of i.c.v. injections of carbachol. These results suggest that cholinergic neurons may be involved in central mechanisms controlling FFA mobilization in pigeons, and that this response is mainly mediated by activation of autonomic nervous system.

Objective: To characterize the response of equine jejunal smooth muscle to adrenergic and cholinergic mediators., Design: Evaluation of myogenic responses, using an in vitro model., Sample Population: Intestinal tissues were obtained from horses without gastrointestinal tract disorders or systemic disease., Procedure: Baseline myogenic tone and amplitude and frequency of contraction were determined for suspended jejunal muscle strips. The level of adrenergic and cholinergic regulation was assessed, using atropine and adrenoceptor antagonists. The response of the muscles to norepinephrine was characterized, using adrenergic blockade and alpha- and beta-agonists., Results: Adrenergic and cholinergic blockade had minimal effect on baseline myogenic activity. However, alpha 1- and beta 2-agonists induced significant (P < 0.05) decreases in the amplitude and frequency of contraction. Surprisingly, alpha 2-agonists caused an increase in the contraction amplitude of longitudinal muscle fibers (neurogenic in origin). Change in circular muscle activity was not induced by alpha 2-agonists. Norepinephrine induced a similar selective response and was inhibited by yohimbine., Conclusions: Baseline jejunal activity appears to be myogenic in origin and can function independently of sympathetic and parasympathetic input. However, intestinal smooth muscle can be affected by adrenergic agonists and potentially by increased concentrations of circulating catecholamines. Norepinephrine may act by altering the activity of other neurotransmitters. Differing responses between circular and longitudinal muscle fibers indicates a need to evaluate both components., Clinical Relevance: Selective alpha 2-agonists may be potentially useful for motility modification of the equine jejunum. Therapeutic use of adrenergic blockade will be effective only in cases of increased adrenergic stimulation.

To identify cholinergically mediated components in the optic nerve response (ONR) we studied effects of cholinergic agonists and antagonists in the arterially perfused cat eye. Acetylcholine, carbachol, scopolamine, quinuclidinylbenzilate and mecamylamine were applied intra-arterially in micromolar concentrations. Recordings of rod- and cone-driven ERG accompanied those of the ONR and revealed: (i) cholinergic agonists enhanced the b-wave, particularly under photopic conditions, whereas scopolamine decreased the b-wave. Mecamylamine induced biphasic effects (decrease followed by increase) in the amplitudes of the rod- and cone-driven b-waves. The effects on the cone-driven ERG were more marked than those on the rod-driven ERG. (ii) The ON-component of the ONR was increased, then decreased by acetylcholine. The cholinergic antagonists exerted complex changes in the ONR-ON component depending on dosage and adaptation. Scopolamine increased, then decreased the rod-driven ON-component, but mainly increased the cone-driven ON-component. Mecamylamine tended to increase the cone-driven, but to decrease the rod-driven ON-component of the ONR. (iii) The configuration of the rod- as well as for the cone-driven ONR, in particular the early plateau and OFF-components, were consistently and reversibly changed by cholinergic agonists, as well as by both muscarinic and nicotinic antagonists. Agonists decreased, and antagonists increased the amplitude of the plateau-component. We conclude that the ERG b-wave was enhanced by acetylcholine, but decreased by cholinergic antagonists. Cholinergic agonists and antagonists affect the same specific components of the ONR in a dose-related and reversible fashion, indicating a major contribution of cholinergic mechanisms to information processing in the cat retina.

This study evaluates the functional activity of the mouse sweat glands in response to cholinergic agonists and antagonists using the silicone imprint technique. In intact mice the response to acetylcholine, methacholine and pilocarpine did not differ significantly from control saline injection, indicating that immobilization induces high levels of sweating, masking the effects of cholinergic stimulation. Plantar emotional sweating was completely abolished by local anesthesia at the ankle. Under these conditions, administration of acetylcholine only provoked detectable sweating when injected locally into the sole skin. Methacholine activated an increasing number of sweat glands in a dose-dependent manner between 0.5 and 10 mg/kg; the response was maximal after 5-10 min of administration and decreased subsequently. With pilocarpine the maximum number of reactive sweat glands was observed at a dose of 2.5 mg/kg. The response was stable for 45 min with doses 2.5 and 5 mg/kg, but decreased exponentially with higher doses. The subtype of sweat gland muscarinic receptor was characterized by determining the inhibitory effect of different cholinergic antagonists on pilocarpine response. Atropine and 4-DAMP were equally potent inhibitors, showing a dose-related effect from 0.05 mg/kg. Pirenzepine only showed inhibitory effects with doses 10-times higher, whereas gallamine and hexamethonium did not induce inhibition at any of the doses tested. These findings suggest that the mouse eccrine sweat gland muscarinic receptors are predominantly M3.

Calcium responses of isolated rat pineal cells to noradrenergic, cholinergic and vasopressinergic stimulations were recorded by use of the fura-2 technique and an image analysis system. Subsequently the recorded cells were identified as pinealocytes by immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker. S-antigen immunoreactive pinealocytes were shown to respond to norepinephrine stimulation with an elevation of the intracellular free calcium concentration ([Ca2+]i). This response was dose-dependent and consisted of a rapid increase in [Ca2+]i (primary phase) followed by a decrease to an elevated plateau well above the basal level (secondary phase). The plateau persisted for at least 1 h when cells were constantly exposed to norepinephrine and dropped to basal level upon removal of the stimulus. Analysis of the calcium responses of cells treated with caffeine or thapsigargin suggested that the primary phase reflects mobilization of calcium from inositol 1,4,5-trisphosphate-sensitive intracellular calcium stores. Depletion of these calcium stores was a decisive and sufficient prerequisite to evoke the secondary phase which was apparently elicited by calcium influx. These data suggest that a capacitative calcium entry is involved in pineal calcium signalling. Acetylcholine induced an increase in [Ca2+]i in rat pinealocytes. Experiments with different cholinergic agonists and antagonists provided evidence that the acetylcholine-induced calcium response was mediated via nicotinic acetylcholine receptors. Stimulation of isolated rat pineal cells with arginine-vasopressin caused a rise in [Ca2+]i in approx. 5% of the cells. However, these cells remained unidentified because they contained neither immunoreactive S-antigen nor immunoreactive glial fibrillary acidic protein, a marker for interstitial (glial) cells of the rat pineal organ. Taken together, the results underline the pivotal role of norepinephrine for the regulation of pineal signal transduction, but they also support the notion that other neurotransmitters and neuropeptides are involved in the modulation of pineal calcium signalling.

Extracellular recordings were made from brain stem slices to test the effects of bath application of cholinergic agonists and antagonists on the firing rates of spontaneously active dorsal cochlear nucleus neurons. About 90% of neurons responded to carbachol. A higher proportion responded to muscarine than to nicotine. Muscarine elicited larger responses at lower concentrations than nicotine. Responses to either carbachol or muscarine were always blocked by atropine or scopolamine. The nicotinic antagonists d-tubocurarine, hexamethonium, and mecamylamine blocked the responses to nicotine, but did not decrease the responses to carbachol. Regularly firing neurons showed only increases of firing rate during exposure to cholinergic agonists. About half of responsive bursting neurons showed increased firing; half showed increased followed by decreased firing to 10 microM carbachol or muscarine. All phases of the responses of most bursting neurons were greatly decreased or abolished in low calcium, high magnesium medium, while responses of regular neurons were not detectably affected. Thus, cholinergic agonists appear to act directly on regularly firing neurons, while their actions on bursting neurons may require synaptic activity. The data suggest that cholinergic transmission in the dorsal cochlear nucleus is predominantly muscarinic, and that most regularly firing spontaneously active neurons have muscarinic receptors.