Extracellular Concentration Of Acetylcholine Research Articles

Blockade of NMDA receptors in the prefrontal cortex increases dopamine and acetylcholine release in the nucleus accumbens and motor activity

The present study investigates the effects of injections of a specific N-methyl-D-aspartic acid (NMDA) antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phophonic acid (CPP) into the prefrontal cortex (PFC) on the extracellular concentrations of dopamine and acetylcholine in the nucleus accumbens (NAc) and on motor activity in the freely moving rat. Sprague-Dawley male rats were implanted with guide cannulas into the medial PFC and NAc to perform bilateral microinjections and microdialysis experiments. Spontaneous motor activity was monitored in the open field. Injections of CPP (1 microg/0.5 microL) into the PFC produced a significant increase of the baseline extracellular concentrations of dopamine (up to 130%), dihydroxyphenylacetic acid (DOPAC; up to 120%), homovanillic acid (HVA; up to 130%), and acetylcholine (up to 190%) in the NAc as well as motor hyperactivity. In the NAc, perfusion of the NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate antagonists CPP (50 microM)+6,7-dinitroquinoxaline-2,3-dione (DNQX; 50 microM) through the microdialysis probe blocked acetylcholine release, but not DOPAC and HVA increases produced by CPP injections into the PFC. Also, increases in motor activity produced by prefrontal injections of CPP were significantly reduced by bilateral injections into the NAc of a mixed D1/D2 antagonist, flupenthixol (5 and 25 microg/0.5 microL). Injections into the NAc of the muscarinic antagonist scopolamine (1 and 10 microg/0.5 microL) further increased, and of the nicotinic antagonist mecamylamine (1 and 10 microg/0.5 microL) did not change, the increases in motor activity produced by prefrontal CPP injections. These results suggest that the dysfunction of NMDA receptors in the PFC could be a key factor in the neurochemical and motor effects associated with corticolimbic hyperactivity.

Cotinus coggygria heartwood: a new source of acetylcholinesterase inhibiting compounds

Acetylcholinesterase (AChE) inhibition is today the main strategy in the clinical management of Alzheimer's disease. Its benefits extend from early stages of various dementia types into Parkinson's disease, ataxia, and myasthenia gravis1. Natural products have already established themselves as an excellent source for AChE inhibiting compounds (e.g. galantamine, huperzine A), but new substances with better efficacy and less side effects are still demanded2. While performing a screening of extracts from our plant collection using an enzyme assay with Ellman's reagent3, preparations made of sumac (Cotinus coggygria Scop., Anacardiaceae) were shown to inhibit AChE; the most potent was the methanol extract of the heartwood (IC50 of 89.3µg/ml; CI95 72.4–108.7µg/ml). Subsequent tests pointed to the ether partition as most active fraction of the above extract (IC50 of 25.4µg/ml; CI95% 20.8–32.2µg/ml). Bioactivity-guided isolation highlighted sulfuretin, its main component, as active compound (IC50 of 29.9µM; CI95% 26.4–33.7µM). Following these positive preliminary results, we investigated in vivo the ability of sumac extracts to enhance cholinergic transmission in the brain, using the push-pull technique. Injection into the ipsilateral cerebral ventricle of 10mg/kg ether partition increased the extracellular acetylcholine concentration in rat brain to about 220% compared to basal release. These are the first in vivo results demonstrating the elevation of cerebral acetylcholine level by an aurone-enriched C. coggygria fraction.

Extracts and constituents of Leontopodium alpinum enhance cholinergic transmission: Brain ACh increasing and memory improving properties

Leontopodium alpinum (‘Edelweiss’) was phytochemically investigated for constituents that might enhance cholinergic neurotransmission. The potency to increase synaptic availability of acetylcholine (ACh) in rat brain served as key property for the bioguided isolation of cholinergically active compounds using different chromatographic techniques. The dichlormethane (DCM) extract of the root, fractions and isolated constituents were injected i.c.v. and the effect on brain ACh was detected via the push–pull technique. The DCM extract enhanced extracellular ACh concentration in rat brain and inhibited acetylcholinesterase (AChE) in vitro. The extracellular level of brain ACh was significantly increased by the isolated sesquiterpenes, isocomene and 14-acetoxyisocomene, while silphiperfolene acetate and silphinene caused a small increasing tendency. Only silphiperfolene acetate showed in vitro AChE inhibitory activity, thus suggesting the other sesquiterpenes to stimulate cholinergic transmission by an alternative mechanism of action. Isocomene was further investigated with behavioural tasks in mice. It restored object recognition in scopolamine-impaired mice and showed nootropic effects in the T-maze alternation task in normal and scopolamine-treated mice. Additionally, this sesquiterpene reduced locomotor activity of untreated mice in the open field task, while the activity induced by scopolamine was abolished. The enhancement of synaptic availability of ACh, the promotion of alternation, and the amelioration of scopolamine-induced deficit are in accordance with a substance that amplifies cholinergic transmission. Whether the mechanism of action is inhibition of AChE or another pro-cholinergic property remains to be elucidated. Taken together, isocomene and related constituents of L. alpinum deserve further interest as potential antidementia agents in brain diseases associated with cholinergic deficits.

Environmental enrichment reduces the response to stress of the cholinergic system in the prefrontal cortex during aging

The present study was designed to evaluate the release of acetylcholine in the prefrontal cortex (PFC) induced by handling stress during aging and also to investigate whether this response changed as a result of the animals living in an enriched environment. Male Wistar rats of 3 months of age were housed in control and enriched conditions during the entire period of their adult life and experiments were performed at 6, 15 and 24 months of age. Spontaneous motor activity was first monitored in an open field arena. Then, rats were stereotaxically implanted with guide cannula to perform microdialysis experiments in the PFC and to evaluate the effects of stress on extracellular concentrations of acetylcholine. Handling stress increased the extracellular concentrations of acetylcholine in the PFC of control and enriched rats. These increases were not modified by aging in control rats. However, environmental enrichment (EE) reduced the effects of stress on acetylcholine concentrations in all groups of age. Spontaneous motor activity in the open field was reduced by aging. EE also decreased motor activity in all groups of age. These results suggest that EE reduces the reactivity to stress of the cholinergic system in the prefrontal cortex during aging.

Chronic treatment with a dopamine uptake blocker changes dopamine and acetylcholine but not glutamate and GABA concentrations in prefrontal cortex, striatum and nucleus accumbens of the awake rat

The present study was aimed to investigate the effects of a chronic treatment with the dopamine uptake blocker nomifensine on the in vivo extracellular concentrations of dopamine, acetylcholine, glutamate and GABA in the prefrontal cortex, striatum and nucleus accumbens. Male Wistar rats received intraperitoneal (i.p.) daily injections of nomifensine (10 mg/kg) or saline for 22 days. Microdialysis experiments were performed on days 1, 8, 15 and 22 of treatment to evaluate the effects of the injection of nomifensine or saline. Motor activity of the animals was monitored during microdialysis experiments. Injections of nomifensine increased extracellular concentration of dopamine in striatum and nucleus accumbens, but not in prefrontal cortex. Acetylcholine concentrations in striatum but not in nucleus accumbens were increased by nomifensine on days 15 and 22 of treatment. In prefrontal cortex, nomifensine increased acetylcholine levels without differences among days. No changes were found on glutamate and GABA concentrations in the three areas studied. Injections of nomifensine also increased spontaneous motor activity and stereotyped behaviour without differences among days. These results show that systemic chronic treatment with a dopamine uptake blocker produces differential effects on extracellular concentrations of dopamine and acetylcholine, but not glutamate and GABA, in different areas of the brain.

Donepezil‐ or rivastigmine‐induced acetylcholinesterase inactivation is not modulated by neramexane in rat brain

AbstractIn Alzheimer's disease (AD), the involvement of cholinergic deficit and overstimulation of N‐methyl‐D‐aspartate (NMDA) receptors by excessively released glutamate has been proposed. Since no existing drug currently addresses both pathologies, a combination therapy may be used clinically to target both mechanisms. The objective of the current study was to determine whether the NMDA receptor antagonist, neramexane affects the acetylcholinesterase (AChE) inhibition produced by donepezil and rivastigmine, two drugs used in the treatment of AD, or a prototype organophosphate compound diisopropylphosphorofluoridate (DFP) in rat cortex, hippocampus, striatum, and brainstem. Neramexane, at therapeutically relevant or higher doses (3.1, 6.2, or 12.4 mg/kg, ip), did not produce any apparent behavioral toxicity. Donepezil (0.75 mg/kg, ip), rivastigmine (0.35 mg/kg, ip) or DFP (1.5 mg/kg, ip) were administered at doses that produced approximately 50% inhibition of AChE and were well tolerated. Also, neramexane in combination with either of the AChE inhibitors did not produce any behavioral toxicity. This magnitude of AChE inhibition has been shown to increase extracellular acetylcholine (ACh) concentrations to therapeutically relevant levels. Neramexane (12.4 mg/kg) did not inhibit AChE activity in any of the brain regions tested when evaluated over a 24‐h period. Rats receiving neramexane alone or in combination with an AChE inhibitor (donepezil, rivastigmine, or DFP) were sacrificed 60 min after neramexane or DFP, 15 min after donepezil, and 30 min after rivastigmine administration. The times at which drug (donepezil, rivastigmine, or DFP) effects were assessed were based on maximal AChE inhibition established from previously reported time course studies. The current findings indicate that neramexane alone did not affect AChE activity at any dose tested nor did it influence the AChE inhibition produced by donepezil or rivastigmine in any brain region examined. However, a low dose of neramexane (3.1 mg/kg) significantly attenuated the inhibition of AChE produced by DFP in the hippocampus, striatum and brainstem, while higher doses (6.2 or 12.4 mg/kg) attenuated DFP‐induced AChE inhibition in all four of the brain regions evaluated. These results suggest that AChE inhibition produced by donepezil or rivastigmine is not altered by the co‐administration of neramexane. Drug Dev Res 68:253–260, 2007. © 2007 Wiley‐Liss, Inc.

Neurochemical and neurobehavioral effects of ganstigmine (CHF2819), a novel acetylcholinesterase inhibitor, in rat prefrontal cortex: An in vivo study

Ganstigmine (CHF2819) is a novel, orally active acetylcholinesterase inhibitor that induces a stimulation of brain cholinergic transmission. In vivo studies show that, in rat prefrontal cortex, extracellular acetylcholine (ACh) concentrations are significantly increased either after local (1 and 10 μM) or oral (1.5 and 3 mg/kg) administration. Moreover, repeated oral treatment (six consecutive days; 3 mg/kg) with ganstigmine significantly increases basal extracellular concentrations of ACh in rat prefrontal cortex. Then, acute ganstigmine administration induces a significant increase in extracellular ACh concentrations (actual values) with respect to the last sample in ganstigmine-treated rats. Concentrations of serotonin (5-HT) and noradrenaline (NA) are not affected by any oral dose of ganstigmine (1.5 and 3 mg/kg) used. Moreover, levels of dopamine (DA) and metabolites are not modified either. Basal extracellular concentrations of 5-HT, NA, DA and metabolites are not affected by repeated (six consecutive days) ganstigmine treatment (3 mg/kg). Furthermore, there is no effect of the challenge dose of ganstigmine (3 mg/kg) on 5-HT, NA, DA and metabolites levels. Finally, ganstigmine reverses the scopolamine-induced deficits of habituation and non-spatial working memory in rats. Taken together, these findings suggest that ganstigmine appears to be a suitable candidate for the treatment of the cholinergic deficit in patients with Alzheimer's disease.

Opened by a twist: a gating mechanism for the nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) play a central role in intercellular communications in the brain and at the neuromuscular junction. They are involved in nicotine addiction as well as in cognitive processes such as attention, access to consciousness, learning and memory and their pathologies include autism, schizophrenia, Parkinson and Alzheimer’s disease (references in Changeux and Edelstein 2005). Understanding the functional organization of the nAChR at the atomic level should thus be a source of insights for the development of new drug therapies. nAChRs are members of the cys-loop superfamily of ligand-gated ion channels. They are pentameric integral membrane proteins with a Wvefold axis of pseudo-symmetry perpendicular to the membrane. Heteroor homo-pentamers are based on the association of 1-10, 1-4, , and subunits which allows a large pharmacological variability and a diversity of control over activation and desensitization kinetics. The large number of subunits and of their combinations could also account for the diverse subunit distribution patterns, at the cellular and subcellular levels (Le Novere et al. 2002a). Each subunit can be subdivided into two principal domains: extracellular and transmembrane. The extracellular domain (ECD) carries the acetylcholine (ACh) binding site at the boundary between subunits and the transmembrane ion pore domain (IPD) delineates an axial cation-speciWc channel (Corringer et al. 2000; Wilson and Karlin 2001). These topologically distinct domains are allosterically coupled to each other. Therefore, nAChRs possess the structural elements necessary to convert a chemical signal, typically a local increase of extracellular ACh concentration, into an electrical signal generated by the opening of the ion channel. Electrophysiological analysis of nAChRs at the synapse shows that rapid release of ACh promotes fast opening of the channel, and that a prolonged application of nicotinic agonists and antagonists leads to a slow decrease of the response amplitude or “desensitization”. Several kinetic models have been proposed for the physiological processes of activation and desensitization (Katz and ThesleV 1957) and for the structural transitions related to general mechanisms of allosteric transitions known to mediate signal transmission (see Changeux and Edelstein 2005; Edelstein and Changeux 1996; Perutz 1989). The concerted Monod–Wyman– Changeux (MWC) model (Monod et al. 1965) extended to the nAChR assumes that the receptor protein exists spontaneously in reversible equilibrium between a basal state (B), an active open channel state (A) stabilized by ACh and nicotinic agonists, and one or several high aYnity desensitized state(s) with a closed channel (D) (see Changeux and Edelstein 2005; Edelstein and Changeux 1996). This mini-revue presents insight on the structure and gating of nAChR, based on the structures obtained experimentally Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.

Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples

Monitoring concentrations of acetylcholine (ACh) in specific brain regions is important in understanding disease pathology, as well as in designing and evaluating novel disease-modifying treatments where cholinergic dysfunction is a hallmark feature. We have developed a sensitive and quantitative liquid chromatography/tandem mass spectrometry method to analyze the extracellular concentrations of ACh, choline (Ch) and (3-carboxylpropyl)-trimethylammonium (iso-ACh) in brain microdialysis samples of freely moving animals. One immediate advantage of this new method is the ability to monitor ACh in its free form without having to use a cholinesterase inhibitor in the perfusate. The separation of ACh, Ch, iso-ACh and related endogenous compounds was carried out based on cation exchange chromatography with a volatile elution buffer consisting of ammonium formate, ammonium acetate and acetonitrile. An unknown interference of ACh, which was observed in brain microdialysates from many studies, was well separated from ACh to ensure the accuracy of the measurement. Optimization of electrospray ionization conditions for these quaternary ammonium compounds achieved the limits of detection (S/N = 3) of 0.2 fmol for ACh, 2 fmol for Ch and 0.6 fmol for iso-ACh using a benchtop tandem quadrupole mass spectrometer with moderate sensitivity. The limit of quantitation (S/N = 10) was 1 fmol for ACh, 3 fmol for iso-ACh and 10 fmol for Ch. This method was selective, precise (<10% R.S.D.), and sensitive over a range of 0.05–10 nM for ACh, 0.25–50 nM for iso-ACh and 15–3000 nM for Ch. To demonstrate that the developed method can be applied to monitoring changes in ACh concentrations in vivo, reference agents that have previously been shown to influence ACh levels were studied in rat dorsal hippocampus. This includes the 5-HT 6 receptor antagonist, SB-271046, and the cholinesterase inhibitor, donepezil. Moreover, levels of ACh were demonstrated to be sensitive to infusion of tetrodotoxin (TTX) suggesting that the ACh being measured in vivo was of neuronal origin. Collectively, these biological data provided in vivo validation of this analytical method.

Stress, prefrontal cortex and environmental enrichment: Studies on dopamine and acetylcholine release and working memory performance in rats

The aim of the present study was to investigate whether environmental enrichment changes the effects of acute stress on both the release of dopamine and acetylcholine in the prefrontal cortex (PFC) and working memory performance. Male Wistar rats (3 months of age) were housed in enriched or control conditions during 12 months. Behavioural testing was carried out to assess working memory performance in a delayed alternation task (water escape T-maze). Horizontal and vertical motor activity were also monitored in the open field. After behavioural testing (open field and water T-maze), animals were implanted with guide cannula in the PFC to perform microdialysis experiments and to monitor dopamine and acetylcholine extracellular concentrations. Handling stress (40 min) produced similar increases of extracellular concentrations of dopamine in the PFC of both enriched and control animals. In contrast, handling stress increased significantly the extracellular concentrations of acetylcholine in the PFC of control, but not enriched, animals. Exposing animals to a lit open field during 10 min significantly reduced working memory performance assessed immediately in the water T-maze just in control animals, though these effects were not significantly different between both groups of animals. Spontaneous motor activity in the open field was lower in enriched compared to control animals. These results suggest that environmental enrichment changes acetylcholine, but not dopamine, reactivity to stress in the PFC.

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.

Effect of a serotonin depleting regimen of 3,4-methylenedioxymethamphetamine (MDMA) on the subsequent stimulation of acetylcholine release in the rat prefrontal cortex

The amphetamine analog 3,4-methylenedioxymethamphetamine (MDMA) is considered to be selectively neurotoxic to serotonergic nerve terminals. Although the long term effects of MDMA on serotonin (5-HT) terminals have been well studied, other potential neurochemical consequences associated with MDMA-induced 5-HT depletion have been less well investigated. In view of the cognitive impairments in human MDMA abusers and the role of acetylcholine (ACh) in learning and memory, it was of interest to determine the influence of a 5-HT depleting regimen of MDMA on subsequent stimulation of ACh release in the prefrontal cortex (PFC). Male rats received vehicle or MDMA (10 mg/kg, i.p. every 2 h for four injections) and underwent in vivo microdialysis 7 days later to assess the subsequent drug- (e.g., MDMA, 5-HT 1A agonist) or stress- (e.g., tail pinch, presence of an intruder rat) induced stimulation of ACh release. The increase in the extracellular concentration of ACh in the PFC produced by MDMA (10 mg/kg, i.p.) was significantly less in rats previously exposed to the neurotoxic regimen of MDMA than that in control animals. In contrast, there was no difference in the magnitude of the stimulation of cortical ACh release elicited by the 5-HT 1A agonist, 8-hydroxy-2-(di- n-propyl-amino)tetralin (8-OH-DPAT, 0.3 mg/kg, s.c.), tail pinch (30 min) or the presence of an intruder rat (40 min) between control animals and animals previously exposed to a neurotoxic regimen of MDMA. These results suggest that although MDMA-induced 5-HT depletion diminishes subsequent MDMA-induced ACh release, there is little impact on cortical ACh release elicited by the stress of pain or the novelty of an environmental intruder.

Effects of the vasopressin (V 1B) receptor antagonist, SSR149415, and the corticotropin-releasing factor 1 receptor antagonist, SSR125543, on FG 7142-induced increase in acetylcholine and norepinephrine release in the rat

Arginine vasopressin and corticotropin-releasing factor are two neuroactive peptides that regulate hypothalamic–pituitary-axis and associated stress response. While the potential antidepressant and anxiolytic profiles of corticotropin-releasing factor 1 antagonists have been well studied, the concept of blockade of vasopressin system as another approach for the treatment of emotional processes has only been made available recently by the synthesis of the first non-peptide antagonist at the V 1b receptor, SSR149415. In the present study SSR149415 has been compared with the corticotropin-releasing factor 1 antagonist SSR125543 and with anxiolytic and antidepressant drugs on the response of hippocampal cholinergic and cortical noradrenergic systems to the anxiogenic benzodiazepine receptor inverse agonist FG 7142. Acute (0.3–10 mg/kg, i.p.) and long-term administration (10 mg/kg, i.p., 21 days) of SSR149415 and SSR125543 reduced the FG 7142-induced increase in extracellular concentrations of acetylcholine in the hippocampus of anesthetized rats measured by microdialysis. By contrast acute and long-term administration of SSR149415 failed to reduce the FG 7142-induced increase in the release of norepinephrine in the cortex of freely moving rats. The present results demonstrate that the two compounds have similar profiles in a model of activation by an anxiogenic drug of the hippocampal cholinergic system and they suggest that SSR149415 and SSR125543 may have anti-stress anxiolytic and antidepressant effects via a mechanism of action different from classical benzodiazepine ligands and noradrenergic antidepressants.

3,4-Methylenedioxymethamphetamine enhances the release of acetylcholine in the prefrontal cortex and dorsal hippocampus of the rat

The neurochemical effects produced by acute administration of 3,4-methylenedioxymethamphetamine (MDMA) on the monoaminergic systems in the brain are well documented; however, there has been little consideration of the potential effects of MDMA on other neurotransmitter systems. The present study was designed to investigate the acute effect of MDMA on cholinergic neurons by measuring acetylcholine (ACh) release in the medial prefrontal cortex (PFC) and dorsal hippocampus, terminal regions of cholinergic projection neurons originating in the basal forebrain. In vivo microdialysis and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used to assess the effects of MDMA on the extracellular concentration of ACh in the PFC and dorsal hippocampus of the rat. The systemic administration of MDMA (3-20 mg/kg, i.p.) resulted in an increased extracellular concentration of ACh in the PFC and dorsal hippocampus. Reverse dialysis of MDMA (100 microM) into the PFC and hippocampus also increased ACh release in these brain regions. Treatment with parachlorophenylalanine and alpha-methyl-para-tyrosine, inhibitors of serotonin (5-HT) and dopamine (DA) synthesis, respectively, significantly attenuated the release of ACh stimulated by MDMA in the PFC, but not in the dorsal hippocampus. MDMA exerts a stimulatory effect on the release of ACh in the PFC and dorsal hippocampus in vivo, possibly by mechanisms localized within these brain regions. In addition, these results suggest that the MDMA-induced release of ACh in the PFC involves both serotonergic and dopaminergic mechanisms.

Anti-inflammatory properties of cholinergic up-regulation: A new role for acetylcholinesterase inhibitors

We investigated the anti-inflammatory effects of acetylcholinesterase inhibitors (AChEI) at the cellular and molecular levels. AChEI suppressed lymphocyte proliferation and pro-inflammatory cytokine production, as well as extracellular esterase activity. Anti-inflammatory activity was mediated by the α7 nicotinic acetylcholine receptor (neuronal); the muscarinic receptor had the opposite effect. Treatment of the central nervous system (CNS) inflammatory disease, experimental autoimmune encephalomyelitis (EAE), with EN101, an anti-sense oligodeoxynucleotide, targeted to AChE mRNA, reduced the clinical severity of the disease and CNS inflammation intensity. The results of our experiments suggest that AChEI increase the concentration of extracellular acetylcholine (ACh), rendering it available for interaction with a nicotinic receptor expressed on lymphocytes. Our findings point to a novel role for AChEI which may be relevant in CNS inflammatory diseases such as EAE and multiple sclerosis. They also emphasize the importance of cholinergic balance in neurological disorders, such as Alzheimer's disease and myasthenia gravis, in which these drugs are used.

Dynorphin A (2-13) improves mecamylamine-induced learning impairment accompanied by reversal of reductions in acetylcholine release in rats

Accumulating evidence indicates that the endogenous opioid peptides dynorphin A (1-17) and synthetic dynorphin A (1-13) interact not only with opioid receptors but also with as yet poorly characterized non-opioid binding sites. Dynorphin A (1-13) improved impairments of learning and memory via not only kappa-opioid receptor-mediated, but also 'non-opioid' mechanisms. In the present study, the effects of des-tyrosine(1) dynorphin A (2-13) as a non-opioid metabolite of dynorphin A, and dynorphin A (1-13) on mecamylamine-induced impairment of the acquisition of learning in rats were investigated using a step-through type passive avoidance task. Further, hippocampal acetylcholine release was examined using in vivo microdialysis. Mecamylamine significantly shortened the step-through latency when given 30 min before the acquisition trial. Not only dynorphin A (1-13) but also dynorphin A (2-13) attenuated the mecamylamine-induced impairment of the acquisition of learning. The effect of dynorphin A (2-13) was not blocked by pre-treatment with nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist. Dynorphin A (2-13) completely abolished the decrease in the extracellular acetylcholine concentration induced by mecamylamine and this effect was not blocked by nor-BNI. Taken together with our previous findings, the present results may indicate that dynorphin A (2-13) improves impairment of learning and/or memory in 'non-opioid' mechanisms and dynorphin A (1-13) ameliorates impairment of the acquisition of learning via not only kappa-opioid receptor-mediated mechanisms but also 'non-opioid' mechanisms, by regulating the release of extracellular acetylcholine.

3,4‐Methylenedioxymethamphetamine (MDMA) enhances the release of acetylcholine by 5‐HT4 and D1 receptor mechanisms in the rat prefrontal cortex

3,4-Methylenedioxymethamphetamine (MDMA), an amphetamine analog, has been shown recently to increase the release of acetylcholine (ACh) in the prefrontal cortex (PFC). The present study further characterizes the stimulatory effect of MDMA on cortical ACh release and examines the role of serotonin (5-HT) and dopamine (DA) receptors in this response. The extracellular concentration of ACh was increased dose-dependently and similarly by the (+) and (-) enantiomers of MDMA (5 and 20 mg/kg, i.p.). The systemic administration of the 5-HT(4) antagonist SDZ 205,557 (1 mg/kg, i.p.), but not the 5-HT(2A/2B/2C) antagonist LY-53,857 (3 mg/kg, i.p.), significantly decreased cortical ACh release induced by MDMA. The MDMA-induced increase in the extracellular concentration of ACh also was significantly blunted in rats treated with the D(1) receptor antagonist SCH 23390 (0.5 mg/kg, i.p.). The extent to which the coadministration of SDZ 205,557 and SCH 23390 suppressed the MDMA-induced release of ACh in the PFC was no greater than that produced by either antagonist alone. These results suggest that the 5-HT(4) and D(1) receptor subtypes contribute to the mechanism by which MDMA increases ACh release in the PFC.

Effects of the metabotropic glutamate receptor agonist, ACPD, on the extracellular concentrations of GABA and acetylcholine in the prefrontal cortex of the rat during the normal process of aging

The aim of the present study was to investigate the effects of activation of metabotropic glutamate receptors (mGluR) on the extracellular concentrations of GABA and acetylcholine in the prefrontal cortex of freely moving rats of different groups of age. Perfusion, through the microdialysis probe, of the agonist of mGluR, (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 100, 500 and 1000 μM), in the prefrontal cortex of young rats produced a dose-related increase of the dialysate concentrations of GABA. The effects of perfusion of ACPD on the concentrations of GABA were attenuated in middle-aged rats. In the prefrontal cortex of aged rats, perfusion of ACPD produced no changes in dialysate concentrations of GABA at any of the doses used. Conversely, perfusion of ACPD (100, 500 and 1000 μM) in the prefrontal cortex of young, middle-aged and aged rats did not modify the dialysate concentrations of acetylcholine. Basal concentrations of acetylcholine in the prefrontal cortex of middle-aged and aged rats were significantly lower than those in young rats. In contrast, basal dialysate concentrations of GABA were not significantly different in young, middle-aged and aged rats. These results suggest that the interaction GABA–glutamate in the prefrontal cortex, mediated by mGluRs, changes with age.

In alcohol-treated rats, naloxone decreases extracellular dopamine and increases acetylcholine in the nucleus accumbens: evidence of opioid withdrawal

Withdrawal from ethanol is aversive. The question is why. As with the withdrawal from morphine, nicotine, diazepam and sugar, the ethanol withdrawal state may involve an increase in nucleus accumbens (NAc) acetylcholine (ACh) causing an alteration of the dopamine (DA)–ACh balance in favor of ACh. Therefore the effects of acute and chronic alcohol (1 gm/kg/day i.p.) treatment on extracellular concentrations of NAc ACh and DA were determined before and after naloxone-precipitated withdrawal. Ethanol initially increased DA to 119% of baseline as measured by microdialysis. This was still the case on the 21st day of ethanol injection when DA increased to 126%. There was no effect of ethanol on ACh. However, naloxone (3 mg/kg s.c.) injected the next day decreased extracellular DA to 83% of baseline and caused a significant rise in ACh to 119%. This state of high ACh combined with low DA may contribute to the aversive aspects of alcohol withdrawal.

Acetylcholinesterase Inhibitory Activity of Scopolin and Scopoletin Discovered by Virtual Screening of Natural Products

For the targeting selection of acetylcholinesterase (AChE) inhibitors from natural sources we generated a structure-based pharmacophore model utilizing an in silico filtering experiment for the discovery of promising candidates out of a 3D multiconformational database consisting of more than 110,000 natural products. In our study, scopoletin (1) and its glucoside scopolin (2) emerged as potential AChE inhibitors by the virtual screening procedure. They were isolated by different chromatographic methods from the medicinal plant Scopolia carniolica Jaqc. and tested in an enzyme assay using Ellman's reagent. They showed moderate, but significant, dose-dependent and long-lasting inhibitory activities. In the in vivo experiments (icv application of 2 micromol) 1 and 2 increased the extracellular acetylcholine (ACh) concentration in rat brain to about 170% and 300% compared to basal release, respectively. At the same concentration, the positive control galanthamine increased the ACh concentration to about the same level as 1. These are the first in vivo results indicating an effect of coumarins on brain ACh.