Acetylcholinesterase Receptor Research Articles

Neonicotinoid Poisoning and Management.

ABSTRACTNeonicotinoids are a newer class of insecticides, which act on postsynaptic nicotinic acetylcholine esterase receptors. Its use is gradually increasing over recent years due to its better safety profile compared to other commonly used pesticides like organophosphates, organochlorides, carbamates, and pyrethroids. The better toxicological profile is attributed to more selectivity for insects compared to mammals and decreased penetration through the blood–brain barrier. Common symptoms of self-poisoning described are dizziness, hypertension, tachycardia, nausea, vomiting, eye irritation, dermatitis, and oral mucosal lesions. Mortality due to poisoning is less than 3%. Till date, there is no specific antidote for neonicotinoid poisoning and management of poisoning is symptomatic and supportive.How to cite this articleSelvam V, Srinivasan S. Neonicotinoid Poisoning and Management. Indian J Crit Care Med 2019;23(Suppl 4):S260–S262.

Evaluation of cholinergic functions in patients with Japanese encephalitis and Herpes simplex encephalitis

Cognitive and memory impairment are related to cholinergic dysfunction and are important complications of viral encephalitis, In view of paucity of studies on cholinergic dysfunction in encephalitis, this study has been undertaken.We report acetyl choline esterase (AChE) and muscurinic 2 (M2) receptor levels in herpes simplex encephalitis (HSE) and Japanese encephalitis (JE) patients, and correlate these with cognitive functions and MRI findings.Patients with JE and HSE were evaluated for consciousness, neurological and MRI findings, plasma AChE and M2 receptor levels on admission and after one year. Twenty-nine patients with JE and 23 with HSE were included. Admission AChE levels in JE (48.32 ± 5.36 nmol/min/ml) and HSE (41.92 ± 5.12 nmol/min/ml) were significantly lower compared with controls (70.50 ± 8.30 nmol/min/ml). M2 receptor levels were also low in JE (4.52 ± 0.56 ng/ml) and HSE (4.35 ± 0.57 ng/ml) compared with controls (7.95 ± 0.41 ng/ml). In JE, AChE activity (r = 0.43, p = 0.02) and M2 receptor levels (r = 0.43, p = 0.02) correlated with caudate involvement, and AChE activity (r = 0.76, p = 0.03) with Mini Mental State Examination ( MMSE) score. In HSE, M2 receptor levels (r = 0.53, p = 0.03) correlated with MMSE. The levels of AChE and M2 receptors increased at one year compared to the baseline, which was greater in JE than in HSE.Both AChE and M2 receptors were reduced in JE and HSE and correlated with cognition at one year. Recovery of these biomarkers was more in JE than HSE.

Study on neurotoxicity of dinotefuran, thiamethoxam and imidacloprid against Chinese lizards (Eremias argus)

The neurotoxicity of dinotefuran, thiamethoxam and imidacloprid against Chinese lizards (Eremias argus) were evaluated in acute oral exposure and 28d subchronic exposure. Dinotefuran was not easily metabolized and showed strong persistence in the lizard brain. Thiamethoxam and imidacloprid were rapidly absorbed and excreted in lizards, and were not easily enriched in the lizard brain. Dinotefuran and thiamethoxam could directly increase the concentrations of acetylcholine in the brain and blood by up-regulating the expression of the ach gene, which in turn enhanced the binding of acetylcholine and acetylcholinesterase receptors, eventually causing the release of dopamine. The effect of dinotefuran was more pronounced than thiamethoxam. Clothianidin was a major metabolite of thiamethoxam in the brain and aggravated the neurotoxic effects of thiamethoxam. Imidacloprid desnitro olefin was the only metabolite of imidacloprid that enriched in the brain. The protonation effect of imidacloprid desnitro olefin was stronger than that of the parent imidacloprid, which increased its binding ability to lizard acetylcholinesterase receptors. Competitive inhibition of imidacloprid desnitro olefin and acetylcholine led to the down-regulation of ach gene expression. Although neonicotinoids caused the opening of ligand-gated ion channel through the activation of acetylcholinesterase receptors, the body would alleviate these effects by the inhibition of voltage-dependent channel activity for compensatory mechanisms. This study provided a new perspective on the neotoxic effects of neonicotinoids.

Molecular Docking Studies of <i>Alpinia galanga</i> Phytoconstituents for Psychostimulant Activity

Dopamine is a neurotransmitter responsible for sending signals from the central nervous system. It allows human beings to stay attentive and focused. Caffeine, the most widely consumed psychoactive substance in the world, is known to improve alertness by enhancing dopamine signaling in the brain. EnXtra®, an Alpinia galanga extract has been clinically proven to promote alertness however the mechanism for such action required elucidation. The current study was designed to explore the mechanism for the neurocognitive enhancing property of EnXtra® by the in-silico interaction of its potential compounds with various targets involved in such process namely Dopamine and Acetylcholinesterase (AchE). As evident by the outcomes of the study, active compounds of EnXtra® can block the dopamine reuptake thereby increasing the dopamine levels which further can enhance the visuospatial performance and mental clarity, leading to improved mental alertness. At the same time, its strong effect on Acetylcholinesterase receptors is indicative of its nootropic potential.

Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

The observation of acetylcholinesterase (AChE) type H (AChEH), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChEH The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChEH, accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChEH, and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

Larvicidal activity and possible mode of action of four flavonoids and two fatty acids identified in Millettia pinnata seed toward three mosquito species.

BackgroundAedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit dengue fever and West Nile virus diseases, respectively. This study was conducted to determine the toxicity and mechanism of action of four flavonoids and two fatty acids from Millettia pinnata (Fabaceae) seed as well as six pure fatty acids and four fatty acid esters toward third instar larvae from insecticide-susceptible C. pipiens pallens and A. aegypti as well as wild A. albopictus. Efficacy of 12 experimental liquid formulations containing M. pinnata seed methanol extract and hydrodistillate (0.5–10.0% liquids) was also assessed.MethodsThe contact toxicities of all compounds and 12 formulations were compared with those of two larvicides, temephos and fenthion and the commercial temephos 200 g/L emulsifiable concentrate (EC). The possible mode of larvicidal action of the constituents was elucidated using biochemical methods. Larval mortality and cAMP level were analyzed by the Bonferroni multiple-comparison method.ResultsPotent toxicity was produced by karanjin, oleic acid, karanjachromene, linoleic acid, linolenic acid, pongamol, pongarotene, and elaidic acid toward C. pipiens pallens larvae (24 h LC50, 14.61–28.22 mg/L) and A. aegypti larvae (16.13–37.61 mg/L). Against wild A. albopictus larvae, oleic acid (LC50, 18.79 mg/L) and karanjin (35.26 mg/L) exhibited potent toxicity. All constituents were less toxic than either temephos or fenthion. Structure–activity relationship indicates that the degree of saturation, the side chain length, and the geometric isomerism of fatty acids appear to play a role in determining the fatty acid toxicity. Acetylcholinesterase (AChE) is the main site of action of the flavonoids, oleic acid, and palmitic acid. The mechanism of larvicidal action of elaidic acid, arachidic acid, and behenic acid might be due to interference with the octopaminergic system. Linoleic acid and linolenic acid might act on both AChE and octopaminergic receptor. M. pinnata seed extract or hydrodistillate applied as 10% liquid provided 100% mortality toward the three mosquito species larvae and the efficacy of the liquids was comparable to that of temephos 200 g/L EC.ConclusionFurther studies will warrant possible applications of M. pinnata seed-derived products as potential larvicides for the control of mosquito populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0848-8) contains supplementary material, which is available to authorized users.

Effect of some Plants and Pesticides on Acetylcholinesterase

The principal role of acetylcholinesterase (AChE)is the termination of nerve impulse transmission at the cholinergic synapses by rapid hydrolysis of acetylcholine (ACh). Inhibition of AChE serves as a strategy for the treatment of Alzheimer's Disease (AD). Several acetylcholinesterase inhibitors (AChEIs) are used for the symptomatic treatment of AD A potential source of AChE inhibitors is certainly provided by the abundance of plants in nature. The aim of this article is study the effect of some plants and pesticides on acetylcholinesterase. Several plants considered as a source of acetylcholinesterase inhibitors and pesticides special organophosphorus. Cholinesterase inhibitors are the only approved drugs for treating patients with mild to moderately severe Alzheimer's disease, a disorder associated with progressive degeneration of memory and cognitive function. The memory impairment in patients with Alzheimer's disease results from a deficit of cholinergic function in the brain. The most important changes observed in the brain are a decrease in cortical levels of the neurotransmitter acetylcholine and associated enzyme choline transferees. Acetylcholinesterase inhibitors can restore the level of acetylcholine by inhibiting acetylcholinesterase. Principal role of acetyl cholinesterase (AChE) is the termination of nerve impulse transmission at the cholinergic synapses by rapid hydrolysis of acetylcholine (ACh). Inhibition of AChE serves as a strategy for the treatment of Alzheimer's Disease (AD), senile dementia, ataxia, myasthenia gravis and Parkinson's disease (Kumar et al., 2011). There are a few synthetic medicines, e.g., tacrine, donepezil and the natural product-based rivastigmine for treatment of cognitive dysfunction and memory loss associated with AD. There are two types of cholinesterase, AChE and butyrylcholinestrase (BuChE). The AChE is found primarily in the blood and neural synapses. The BuChE is found primarily in the liver. The biggest difference between the two is the substrates. The AChE hydrolyzes acetylcholine (ACh) more quickly and BuChE hydrolyzes butyrylcholine (BuCh) more quickly. The BuCh is a synthetic compound used to distinguish AChE receptors from BuChE receptors. Many of the drugs that are available for treatment of AD target both AChE and BuChE but some are more (Mehta et al., 2012). These compounds have been reported to have their adverse effects including gastrointestinal disturbances and problems associated with bioavailability, which necessitates the interest in finding better AChE inhibitors from natural resources. In traditional practices numerous plants have been used to treat cognitive disorders, including neurodegenerative diseases and different neuron pharmacological disorders.

Cholinergic properties of new 7-methoxytacrine-donepezil derivatives.

Organophosphorus nerve agents inhibit acetylcholinesterase (AChE) which causes the breakdown of the transmitter acetylcholine (ACh) in the synaptic cleft. Overstimulation of cholinergic receptors (muscarinic and nicotinic) by excessive amounts of ACh causes several health problems and may even cause death. Reversible AChE inhibitors play an important role in prophylaxis against nerve agents. The presented study investigated whether 7-methoxytacrine (7-MEOTA) and 7-MEOTA-donepezil derivatives can act as central and peripheral reversible AChE inhibitors and simultaneously antagonize muscarinic and nicotinic receptors. The possible mechanism of action was studied on cell cultures (patch clamp technique, calcium mobilization assay) and on isolated smooth muscle tissue (contraction study). Furthermore, the kinetics of the compounds were also examined. CNS availability was predicted by determining the passive blood-brain barrier penetration estimated via a modified PAMPA assay. In conclusion, this study provides promising evidence that the new synthesized 7-MEOTA-donepezil derivatives have the desired anticholinergic effect; they can inhibit AChE, and nicotinic and muscarinic receptors in the micromolar range. Furthermore, they seem to penetrate readily into the CNS. However, their real potency and benefit must be verified by in vivo experiments.

Radiosynthesis and in vivo evaluation of fluorinated huprine derivates as PET radiotracers of acetylcholinesterase

Developing positron emission tomography (PET) radiotracers for non-invasive study of the cholinergic system is crucial to the understanding of neurodegenerative diseases. Although several acetylcholinesterase (AChE) PET tracers radiolabeled with carbon-11 exist, no fluorinated radiotracer is currently used in clinical imaging studies. The purpose of the present study is to describe the first fluorinated PET radiotracer for this brain enzyme. Three structural analogs of huprine, a specific AChE inhibitor presenting high affinity towards AChE in vitro, were synthesized and labeled with fluorine-18 via a mesylate/fluoro-nucleophilic aliphatic substitution: ([(18)F]-FHUa, [(18)F]-FHUb and [(18)F]-FHUc). Initial biological evaluation included in vitro autoradiography in rat with competition with an AChE inhibitor at different concentrations, and microPET-scan on anesthetized rats. In vivo PET studies in anesthetized cat focused on [(18)F]-FHUa. Although radiosynthesis of these huprine analogs was straightforward, they showed poor brain penetration potential, partially reversed after pharmacological inhibition of P-glycoprotein. These results indicated that current huprine analogs are not suitable for PET mapping of brain AChE receptors, but require physicochemical modulation in order to increase brain penetration.

Effects of radiofrequency thermocoagulation on trigeminal ganglions in rabbits

To study the histological effects of different temperatures and durations of radiofrequency on the trigeminal ganglions in rabbits. Trigeminal ganglions of 80 rabbits were treated by R-2000B RF thermocoagulation at 60, 70, 80°C respectively and the thermal electrode of 50 mm long and 0.4 mm in diameter. The treaded trigeminal ganglions were removed for HE (hematoxylin & eosin) dying at Days 1, 7, 14 and 28. And the expressions of neurofilament protein (NF200), acetylcholinesterase (ACHE) and muscarinic receptors (M-receptor) were observed after an immunohistochemical detection of HE positive samples. The positive manifestations of HE were degeneration of trigeminal ganglionic cells, the distribution of nerve fibers and a loss of myelinic membrane. The positive features of immunohistochemistry were as follows: NF200: dark yellow-brown particles within cytoplasm of ganglionic cells and nerve fibers cells; ACHE: deep yellow-brown particles within cytoplasm of ganglion cells and nerve fibers cells; M-receptor: yellow-brown particles within cytoplasm of ganglionic cells and nerve fibers cells. The expressions of NF200, ACHE and M-receptor increased while the temperature and radio frequency time remained constant. There was no significant difference between the expression rates of NF200, ACHE, M receptor at the same temperature for 1 and 3 minutes (P > 0.05). When treated for the same time, the expressions of NF200, ACHE and M receptor decreased more significantly at 70°C versus 60°C (P < 0.05). However, there was no significant difference between 70°C and 80°C (P > 0.05). 70°C may be a suitable temperature at which radiofrequency causes the greatest harm to trigeminal ganglions so that nerve activity decreases significantly.

Thyrotoxic Dysphagia in an 82-Year-Old Male

Dysphagia is a common problem in elderly patients and a rare manifestation of Graves' disease. We report a case of an 82-year-old male who presented with a 4-week history of dysphagia and weight loss. Workup for his dysphagia with upper endoscopy, MRI brain, electromyography, acetyl-cholinesterase receptor antibodies, and voltage-gated calcium channel antibodies were negative. Modified Barium swallow test showed oropharyngeal dysphagia. Thyroid function tests that revealed hyperthyroidism and antibodies to TSH-receptor were positive. Based on the above findings, we considered Graves' disease as the most likely diagnosis. Patient was treated with methimazole and beta-blockers and subsequently his dysphagia resolved. This paper highlights the importance to clinicians of considering thyrotoxicosis as possible diagnosis in an elderly patient presenting with unexplained dysphagia.

Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and gamma-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [(3)H]MK-801 with a K(i) value of 0.27 mum, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.

Acetylcholinesterase inhibitor is a potentially useful therapeutic agent for nicotine-induced periodontal disease

Many cell types possess the essential components for a non-neuronal cholinergic system. These components include acetylcholine, choline acetyltransferase, acetylcholinesterase (AChE) and nicotinic acetylcholine receptors. Studies have indicated that AChE may be crucial for various types of apoptosis and that tobacco smoking augments the severity of periodontal disease. We hypothesize that there is a non-neuronal cholinergic system in periodontal tissue that is activated by excess nicotine, which increases expression of AChE, thus increasing apoptosis of periodontal cells. If so, the introduction of an AChE inhibitor should decrease periodontal cell apoptosis, lessening periodontal disease.

Lymph heart in chick - somitic origin, development and embryonic oedema

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.

Multiple cascade effects of oxidative stress on astroglia

Many neurodegenerative diseases share common underlying features, most prominent of which are dysregulation of calcium homeostasis and reactive astrogliosis, ultimately triggered by oxidative stress. Interestingly, an additional feature of the early response to stress conditions is the upregulation and release of acetylcholinesterase (AChE). This study therefore investigates the link between oxidative stress, calcium influx, gene expression, protein synthesis, and AChE release. We report that, in astroglia and in an immortalized cell line, GH4-halpha7, acute oxidative stress causes influx of extracellular calcium through L-type voltage-gated calcium channels (L-VGCC), followed by increased release of AChE into the extracellular medium. Moreover, rapid and sustained changes in mRNA expression of AChE, L-VGCC, and melastatin-like transient receptor potential 2 (TRPM2) accompany profound suppression of global protein synthesis. Application of L-VGCC blockers selectively reduces stress-induced calcium influx and AChE release, mitigates changes in gene expression, and facilitates recovery from protein synthesis suppression. Although glia exhibit greater sensitivity in their responses, the results are comparable in astroglia and GH4-halpha7 cells, and suggest a generalized and integrated cellular response to stress conditions that characterizes changes observed in neurodegeneration.

Identification of Acetylcholinesterase Receptors in Rotifera

Identification of Acetylcholinesterase Receptors in Rotifera

Acetylcholine and choline effects on erythrocyte nitrite and nitrate levels

Acetylcholine has been detected in human blood. Acetylcholine receptors and acetylcholinesterase are present in erythrocyte membranes. We tested the acetylcholine and choline effects on nitric oxide metabolites (NOx), namely nitrites and nitrates, and observed if they are dependent on interactions with muscarinic receptors and acetylcholinesterase. Human erythrocyte suspensions were incubated with acetylcholine and choline in the absence or presence of 10 microM atropine or 10 microM velnacrine maleate. The nitrite and nitrate concentrations were determined by the Griess method. Acetylcholine or choline increased NOx control concentrations (P <0.001). The nitrite concentrations decreased in the presence of atropine or velnacrine maleate (P <0.03). The nitrate concentrations only decreased when velnacrine maleate was incubated with acetylcholine or choline (10 microM, P <0.03). These results demonstrated that acetylcholine and choline modulate nitric oxide metabolites on erythrocytes and this effect is mediated by interactions with erythrocyte membrane muscarinic receptors and membrane enzyme acetylcholinesterase. A hypothesis for the signal transduction mechanism has been discussed for acetylcholinesterase and muscarinic receptor (M1) participation.

Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET, and permethrin, alone and in combination.

Since their return from Persian Gulf War (PGW), many veterans have complained of symptoms including muscle and joint pain, ataxia, chronic fatigue, headache, and difficulty with concentration. The causes of the symptoms remain unknown. Because these veterans were exposed to a combination of chemicals including pyridostigmine bromide (PB), DEET, and permethrin, we investigated the effects of these agents, alone and in combination, on the sensorimotor behavior and central cholinergic system of rats. Male Sprague-Dawley rats (200-250 gm) were treated with DEET (40 mg/kg, dermal) or permethrin (0.13 mg/kg, dermal), alone and in combination with PB (1.3 mg/kg, oral, last 15 days only), for 45 days. Sensorimotor ability was assessed by a battery of behavioral tests that included beam-walk score, beam-walk time, incline plane performance, and forepaw grip on days 30 and 45 following the treatment. On day 45 the animals were sacrificed, and plasma and CNS cholinesterase, and brain choline acetyl transferase, muscarinic and nicotinic acetylcholine receptors were evaluated. Animals treated with PB, alone or in combination with DEET and permethrin, showed a significant deficit in beam-walk score as well as beam-walk time as compared with controls. Treatment with either DEET or permethrin, alone or in combination with each other, did not have a significant effect on beam-walk score. All chemicals, alone or in combination, resulted in a significant impairment in incline plane testing on days 30 and 45 following treatment. Treatment with PB, DEET, or permethrin alone did not have any inhibitory effect on plasma or brain cholinesterase activities, except that PB alone caused moderate inhibition in midbrain acetylcholinesterase (AChE) activity. Treatment with permethrin alone caused significant increase in cortical and cerebellar AChE activity. A combination of DEET and permethrin or PB and DEET led to significant decrease in AChE activity in brainstem and midbrain and brainstem, respectively. A significant decrease in brainstem AChE activity was observed following combined exposure to PB and permethrin. Coexposure with PB, DEET, and permethrin resulted in significant inhibition in AChE in brainstem and midbrain. No effect was observed on choline acetyl transferase activity in brainstem or cortex, except combined exposure to PB, DEET, and permethrin caused a slight but significant increase in cortical choline acetyltransferase activity. Treatment with PB, DEET, and permethrin alone caused a significant increase in ligand binding for m2 muscarinic acetylcholine receptor (mAChR) in the cortex. Coexposure to PB, DEET, and permethrin did not have any effect over that of PB-induced increase in ligand binding. There was no significant change in ligand binding for nicotinic acetylcholine receptor (nAChR) associated with treatment with the chemical alone; a combination of PB and DEET or coexposure with PB, DEET, and permethrin caused a significant increase in nAChR ligand binding in the cortex. Thus, these results suggest that exposure to physiologically relevant doses of PB, DEET, and permethrin, alone or in combination, leads to neurobehavioral deficits and region-specific alterations in AChE and acetylcholine receptors.

Ontogenic distribution of muscarinic receptors and acetylcholinesterase in the rabbit hippocampus

Ontogenic development of muscarinic receptors was examined in the hippocampus of rabbits (from P2 to P60) using radioautographic method. Muscarinic sites were labelled with (3H)-quinuclinidyl-benzilate and pharmacologically defined M1 and M2 receptor subtypes with (3H)-pirenzepine and (3H)-oxotremorine, respectively. The distribution of binding sites was compared to acetylcholinesterase (AChE) staining in adjacent hippocampal sections. The two cholinergic components are progressively set up in the hippocampus during the first three postnatal weeks. The AChE staining was very low in all hippocampal fields in P2 rabbits. At P8 and after, the AChE staining was more pronounced in CA3 and CA4 than in CA1 and CA2. On the contrary, the M1 muscarinic binding sites were more abundant in CA1 and CA2 hippocampal fields than in CA3 and CA4 at all ages studied. M2 muscarinic binding sites were only distinguishable at P45 and have a relatively homogeneous distribution. This study shows a differential developmental evolution in the distribution of AChE and muscarinic M1 receptors, and no obvious correspondence between these two cholinergic markers was observed.

Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor

We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1–7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30–40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18–20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occured in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrated that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.