Ethylcholine Mustard Aziridinium Ion Research Articles

Ferulic acid reverses the cognitive dysfunction caused by amyloid β peptide 1-40 through anti-oxidant activity and cholinergic activation in rats.

Cholinergic dysfunction and oxidation stress are the dominant mechanisms of memory deficit in Alzheimer's disease (AD). This study describes how ferulic acid (FA) ameliorates cognitive deficits induced by mecamylamine (MECA), scopolamine (SCOP), central acetylcholinergic neurotoxin ethylcholine mustard aziridinium ion (AF64A) and amyloid β peptide (Aβ1-40). This study also elucidates the role of anti-oxidant enzymes and cholinergic marker acetylcholinesterase (AChE) in the reversal of FA from Aβ1-40-induced cognitive deficits in rats. At 100 mg/kg, FA attenuated impairment induced by MECA and SCOP plus MECA; however, this improvement was not blocked by the peripheral muscarinic receptor antagonist scopolamine methylbromide (M-SCOP). At 100 and 300 mg/kg, FA also attenuated the impairment of inhibitory passive avoidance induced by AF64A. Further, FA attenuated the performance impairment and memory deficit induced by Aβ1-40 in rats, as did vitamin E/C. FA reversed the deterioration of superoxide dismutase (SOD) and AChE activities, and the glutathione disulfide (GSSG) and glutathione (GSH) levels in the cortex and hippocampus. Vitamin E/C only selectively reversed deterioration in the hippocampus. We suggest that FA reduced the progression of cognitive deficits by activating central muscarinic and nicotinic receptors and anti-oxidant enzymes.

Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Nicotine improves AF64A-induced spatial memory deficits in Morris water maze in rats

Ethylcholine mustard aziridinium ion (AF64A) is a neurotoxic derivative of choline that produces not only long-term presynaptic cholinergic deficits, but also various memory deficits in rats similar to some characteristics observed in Alzheimer's disease patients. This study investigated whether nicotine (NCT) administration attenuated spatial learning deficits induced by intracerebroventricular AF64A treatment. AF64A (6nmol/6μl)-or saline (SAL)-treated rats were trained in Morris water maze task. NCT (0.025–0.25mg/kg) was subcutaneously injected 5min before the training every day. The results showed that moderate dose (0.10mg/kg) of NCT attenuated AF64A-induced prolongation of escape latency. Furthermore, NCT dose-dependently recovered the AF64A-induced decrease of time spent in the target quadrant in the probe test. These results suggest that NCT improves AF64A-induced spatial memory deficits, and thus it is a potential therapeutic agent for the treatment of memory deficits in dementia.

Effects of the destruction of starburst-cholinergic amacrine cells by the toxin AF64A on rabbit retinal directional selectivity.

The effects of intraocular injections of ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of choline uptake, on the rabbit retina were assessed electrophysiologically, pharmacologically, anatomically, and behaviorally. Survival times from 1 day to 30 days were investigated. After 24 h, the shortest time tested, the directional selectivity of On-Off responding ganglion cells having the characteristic morphology of On-Off directionally selective directionally selective (DS) ganglion cells, as revealed by intracellular dye injection, was significantly reduced, both by an apparent decrease of preferred direction responses and an increase in responses to null-direction movement. No toxin-mediated changes in the dendritic trees of these cells were noted. Cells in AF64A-affected retinas having the DS morphology did not respond significantly to GABAergic or cholinergic agents such as picrotoxin and eserine, but did respond to nicotine. Recordings from a small random sample of other ganglion cell classes in the same retinas yielded no obvious changes in response properties. The direct effects on starburst (cholinergic) amacrine cells, which were identified by intraocular injection of the fluorescent dye DAPI with the AF64A, were investigated by intracellular injections of Lucifer yellow, and by immunohistochemical staining with antibodies to choline acetyltransferase (ChAT). Although starburst amacrine cell somas survived the AF64A treatment for at least several days, the dendrites could not be visualized by fluorescent dye injection in affected retinas due to dye leakage of the injected fluorescent dye from either the soma or proximal dendritic region. ChAT staining revealed a sequence in which ChAT-positive cells were undetectable first in the inner nuclear layer, and then in the ganglion cell layer. Cholinergic amacrine cells in the central retina were also affected before those in the periphery. The electrophysiological changes observed typically preceded the loss of ChAT activity. Behavioral tests for optokinetic nystagmus responses also revealed a lack of such responses in the affected eyes.

Spinal endogenous acetylcholine contributes to the analgesic effect of systemic morphine in rats.

Systemic morphine is known to cause increased release of acetyicholine in the spinal cord. Intrathecal injection of the cholinergic receptor agonists or acetyicholinesterase inhibitors produces antinociception in both animals and humans. In the present study, we explored the functional importance of spinal endogenous acetylcholine in the analgesic action produced by intravenous morphine. Rats were implanted with intravenous and intrathecal catheters. The antinociceptive effect of morphine was determined by the paw-withdrawal latency in response to a radiant heat stimulus after intrathecal treatment with atropine (a muscarinic receptor antagonist), mecamylamine (a nicotinic receptor antagonist), or cholinergic neurotoxins (ethylcholine mustard aziridinium ion [AF64A] and hemicholinium-3). Intravenous injection of 2.5 mg/kg morphine increased significantly the paw-withdrawal latency. Intrathecal pretreatment with 30 microg atropine (n = 7) or 50 microg mecamylamine (n = 6) both attenuated significantly the antinociceptive effect of morphine. The inhibitory effect of atropine on the effect of morphine was greater than that of mecamylanilne. Furthermore, the antinociceptive effect of morphine was significantly reduced in rats pretreated with intrathecal AF64A (n = 7) or hemicholinium-3 (n = 6) to inhibit the high-affinity choline transporter and acetylcholine synthesis. We found that intrathecal AF64A reduced significantly the [3H]hemicholinium-3 binding sites but did not affect its affinity in the dorsal spinal cord. The data in the current study indicate that spinal endogenous acetylcholine plays an important role in mediating the analgesic effect of systemic morphine through both muscarinic and nicotinic receptors.

An obligatory role for spinal cholinergic neurons in the antiallodynic effects of clonidine after peripheral nerve injury.

Indirect evidence supports a role of spinal cholinergic neurons in tonically reducing response to noxious mechanical stimulation and in effecting analgesia from alpha2-adrenergic agonists. This study directly assessed the role of cholinergic neurons in regulating the level of mechanical allodynia and in participating in the antiallodynic effect of the clinically used alpha2-adrenergic agonist, clonidine, in an animal model of neuropathic pain. Allodynia was produced in rats by ligation of the left L5 and L6 spinal nerves. Rats received a single intrathecal injection of saline or one of three different doses of the cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64-A; 2, 5, and 15 nmol). Seven days later, allodynia was assessed before and after intrathecal injection of 15 microg clonidine. The spinal cord was removed, and spinal cord acetylcholine content, cholinergic neuron number and distribution, and alpha2-adrenergic receptor expression were determined. AF64-A administration reduced both the number of cholinergic cells and the acetylcholine content of the lumbar dorsal spinal cord by 20-50% but did not affect level of mechanical allodynia. AF64-A did, however, completely block the anti-allodynic effect of clonidine. AF64-A did not reduce alpha2-adrenergic ligand binding in dorsal lumbar cord. These data suggest that spinal cholinergic tone does not affect the level of mechanical allodynia after peripheral nerve injury. There is a quantitative reliance on spinal cholinergic neurons in the allodynia relieving properties of intrathecal clonidine, and this reliance does not depend on alpha2-adrenergic receptors colocalized on spinal cholinergic interneurons.

Effect of AF64A, a cholinergic neurotoxin, on footshock stimulation-induced locus coeruleus excitation in rats

We studied the effect of ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin, on the footshock stimulation (FS)-induced excitation of the locus coeruleus (LC) neurons in rats. The FS-evoked LC excitation was significantly reduced in AF64A-treated rats, in comparison with normal rats. In particular, the early component of LC excitation was less pronounced. The number of choline acetyltransferase immunoreactive neurons in the septal complex was significantly lower than those in normal rats, except for in the ventral subgroup. These findings suggest that the cholinergic neuron system is involved in the early component of LC excitation in rats.

Role of Cholinergic Mossy Fibers in Vestibular Nuclei in the Development of Vestibular Compensation

Several lines of evidence have suggested that acetylcholine is a possible neurotransmitter/neuromodulator involved in vestibular compensation. However, details of cholinergic effective sites during vestibular compensation remain unclear. In this study, we selectively damaged the rat vestibulo-floccular cholinergic mossy fibers using ethylcholine mustard aziridinium ion. In these animals, unilateral labyrinthectomy caused more severe vestibulo-ocular deficits, especially in the initial stage. These findings suggest that the vestibulo-floccular cholinergic mossy fibers serve to restore the balance between intervestibular nuclear activities in order to induce vestibular compensation in the initial stage.

Glucocorticoid hypersecretion following intracerebroventricular injection of ethylcholine mustard aziridinium ion in rats

To investigate whether cholinergic hypofunctions in the brain influence hypothalamic-pituitary-adrenal activity, we examined the effects of cholinergic neurotoxin ethylcholine mustard aziridinium ion on basal and stress-induced levels of corticosterone in rats. Blood sampling from rats following intracerebroventricular injection of saline (5 μl, as a control) or this neurotoxin (5 nmol/5 μl) was performed over a day in one series, and was taken before, during and after an immobilization stress exposure in another series. Plasma levels of corticosterone and adrenocorticotropin were determined by the radioimmunoassay. The basal levels of plasma corticosterone and adrenocorticotropin over a day were significantly higher in the neurotoxin-treated rats, compared with the control rats (corticosterone, P<0.001; adrenocorticotropin, P<0.05). Further, relative adrenal gland weight of the neurotoxin-treated rats was significantly greater than that of the control rats ( P<0.05). However, responses in plasma corticosterone level caused by the immobilization stress in the neurotoxin-treated rats were not different from those in the control rats. The present study demonstrated that damage to the cholinergic neurons in the brain increased hypothalamic-pituitary-adrenal activity over a day, probably due to freedom from inhibitory influences of the hippocampal cholinergic system, but that this damage did not influence stress-induced changes in plasma glucocorticoid level.

The pedunculopontine tegmental nucleus and the role of cholinergic neurons in nicotine self-administration in the rat: a correlative neuroanatomical and behavioral study

The objective of this study was to determine whether the pedunculopontine tegmental nucleus plays a role in the maintenance of nicotine self-administration, and whether the ascending cholinergic projection from this nucleus to midbrain dopamine neurons in the ventral tegmental area might be involved. Studies were done with rats trained to self-administer nicotine intravenously. Self-administration was examined before and after the pedunculopontine tegmental nucleus was lesioned with the ethylcholine mustard aziridinium ion, a selective cholinergic toxin. Lesions were assessed qualitatively and quantitatively in histological sections stained for either nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry to identify cholinergic neurons, or for Nissl. Self-administration was also tested after an acute manipulation in which microinfusions of the nicotinic cholinergic antagonist dihydro-β-erythroidine were made into the pedunculopontine tegmentum. Infusions of neurotoxin into the pedunculopontine tegmentum reduced nicotine self-administration behaviour when tested weeks later. Toxin treatment reduced the number of cholinergic neurons in the tegmentum, while largely sparing the non-cholinergic population in this area. Lesions were limited to the pedunculopontine area and did not extend to the neighboring laterodorsal tegmental nucleus or to the substantia nigra. Acute manipulation of the pedunculopontine tegmental nucleus with microinfusions of dihydro-β-erythroidine also produced an attenuation of nicotine self-administration. Collectively these data show that the pedunculopontine tegmental nucleus is part of the neuronal circuitry mediating nicotine self-administration, and that the population of cholinergic neurons is likely a critical element.

Ethylcholine mustard aziridinium ion (AF64A) induces glucocorticoid hypersecretion in rats

Ethylcholine mustard aziridinium ion (AF64A) induces glucocorticoid hypersecretion in rats

Cholinergic Influence on Vestibular Stimulation-Induced Locus Coeruleus Inhibition in Rats

In our previous study, caloric stimulation (CS) of the vestibular apparatus inhibited noradrenergic neuronal activity in the locus coeruleus (LC) in urethane-anaesthetized rats. Therefore, the inhibition of LC noradrenergic neurons is involved in vestibulo-autonomic responses. Since motion sickness can be cured by scopolamine, cholinergic neuron system may also be involved in vestibulo-autonomic responses. The present study examined the effects of intracerebroventricular injection of ethylcholine mustard aziridinium ion (AF64A), a presynaptic cholinergic neurotoxin, on CS-induced LC inhibition. In AF64A-treated rats, the CS-induced LC inhibition was less pronounced than in normal rats. In a subsequent series of experiments, the intravenous injection of scopolamine blocked the CS-induced LC inhibition. These findings suggest that central cholinergic neurons are associated with noradrenergic neuronal inhibition during the vestibulo-autonomic reflex.

Neurochemical determination of the location of NMDA and GABA receptors on rat striatal cholinergic neurons

This paper reports on the protocol for neurochemical determination of the location of various receptors on cholinergic neurons in various brain regions. We applied this protocol to investigate whether NMDA and GABA receptors are located on rat striatal cholinergic neurons. When striatal cholinergic neurons were selectively destroyed by intrastriatal injection of cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A), the number of NMDA and GABA A receptors decreased. However, no significant changes were observed on the number of GABA B receptors. These results suggest that NMDA and GABA A, but not GABA B receptors are located on cholinergic neurons in the striatum. These results also indicate the usefulness and scientific applicability of the present protocol. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Regional localization of receptors and transmitters

P–96 - Changes in GABAa and GABAb receptors in rat striatum microinjected with ethylcholine mustard aziridinium ion, AF64A.

P–96 - Changes in GABAa and GABAb receptors in rat striatum microinjected with ethylcholine mustard aziridinium ion, AF64A.

Effects of AF64A on gene expression of choline acetyltransferase (ChAT) in the septo-hippocampal pathway and striatum in vivo.

AF64A (ethylcholine mustard aziridinium ion) was stereotaxically administered bilaterally (1 nmol/side) into rat lateral cerebral ventricles. Choline acetyltransferase (ChAT) activity and ChAT mRNA levels were measured at predetermined time points in the septo-hippocampal pathway and striatum, both well identified as rich in cholinergic neurons. AF64A caused a rapid but transient increase in ChAT mRNA (167%, P < 0.05) and ChAT activity (164%, P < 0.01) in the septum. By day 7 post treatment, there was a significant decrease in ChAT mRNA (42.5% of control, P < 0.05) in the septum although the ChAT activity still stayed high. This decreased ChAT mRNA level in the septum lasted for at least four weeks, and was paralleled by a long-lasting decrease in ChAT activity in the hippocampus. In the striatum, on the other hand, there were no observed changes in either ChAT activity or ChAT mRNA. These data suggest that the long term effect of AF64A on the septo-hippocampal cholinergic pathway may, at least in part, be due to an action of AF64A on gene expression in the cholinergic neuron. The difference in the response to AF64A between the septo-hippocampal and striatal cholinergic systems might be due to their difference in neuron types.

Effects of AF64A on the mRNA levels of muscarinic receptor subtypes in the rat iris sphincter.

The reduction of parasympathetic nerve activity by the treatment with ethylcholine mustard aziridinium ion (AF64A) in vivo induced both specific and non-specific supersensitivities in the rat iris sphincter (Tanaka et al., 1999). Changes in the expression of muscarinic receptor subtypes, which could be a cause of specific supersensitivity induced by the treatment with AF64A, were examined using competitive PCR techniques. Muscarinic receptor population is composed of m2, m3, and m4 subtypes in the rat iris (Furuta et al., 1998). Interestingly, m4 mRNA was much more abundantly expressed than m2 and m3 in the rat iris sphincter. The treatment with AF64A significantly increased the mRNA levels of m2 and m3 subtypes to 370 and 330% of the control but not that of m4 (approximately 90% of the control). In addition, the total protein contents were increased to approximately 125% of the control. The up-regulation of the mRNA levels of m2 and m3 subtypes by the treatment with AF64A was significant when they were compensated for the increase in total protein contents. The down regulation of m4 mRNA expression was not significant even after being corrected for the protein content. These results suggest that the up-regulation of the mRNA levels of m2 and m3 subtypes may be, at least in part, responsible for the supersensitivity to muscarinic agonists after the treatment with AF64A in vivo.

Modulation of acetylcholine release via GABA A and GABA B receptors in rat striatum

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABA A and GABA B receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABA A receptor binding without significantly altering GABA B receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABA A, but not GABA B receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABA A receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABA B receptors located on non-cholinergic neuronal elements in the striatum.

Supersensitivity by AF64A, a novel inhibitor of neuronal choline-uptake, of the rat iris sphincter smooth muscle.

Effects of ethylcholine mustard aziridinium ion (AF64A) on contractile responses to agonists or transmural nerve stimulation (TNS) were examined in rat iris sphincter muscle. The responses to TNS of isolated sphincter muscle were abolished within 1 hr after the addition of 0.1 mM AF64A to the bathing solution, while responses to acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) were not changed significantly. In sphincter muscles which were isolated 3 days after the micro-injection of 100 nmol AF64A into the anterior chamber of eyes in vivo, the response to TNS were decreased to about 30% of the control. The injection of AF64A at higher concentrations often resulted in serious chronic damage of the eye. When 100 nmol AF64A was injected twice with an interval of 3 days, the response to TNS was decreased to about 20% of the control. Maximum responses and sensitivities to ACh and 5-HT were markedly enhanced in sphincters from eyes which had been treated with AF64A twice. The sensitivity of depolarized sphincters to external Ca2+ was also increased significantly. Seven weeks after the second injection, responses to TNS recovered to more than 50% of the control and the effects of AF64A mostly disappeared. In conclusion, the acute inhibition of parasympathetic transmission without postsynaptic changes can be achieved within 1 hr after bath-application of 0.1 mM AF64A. The reduction of parasympathetic nerve activity by treatment with AF64A in vivo induces nonspecific supersensitivity in iris sphincter, whereas effects of AF64A were mostly reversible under the present experimental conditions.

Comparative Studies of Huperzine A, E2020, and Tacrine on Behavior and Cholinesterase Activities

Comparative effects of cholinesterase inhibitors (ChEI) huperzine A with E2020 and tacrine on the radial maze performance in ethylcholine mustard aziridinium ion (AF64A)-treated rat and inhibition of cholinesterase activity were studied. The intracerebroventricular (ICV) injection of AF64A (3 nmol/side) caused significant impairment in the rat’s ability to fulfill the partially baited maze paradigm. Oral huperzine A (0.5–0.8 mg/kg), E2020 (1.0–2.0 mg/kg), and tacrine (8.0 mg/kg) effectively reversed AF64A-induced working memory deficit. The doses that improved AF64A-induced memory deficit were correlated to about 25–30% (huperzine A) and less than 10% (E2020, tacrine) inhibition of acetylcholinesterase (AChE) activity in the cortex and hippocampus. Huperzine A, E2020 and tacrine all produced dose-dependent inhibition of brain AChE following ICV and oral administration. Oral huperzine A exhibited higher efficacy on the inhibition of AChE in the cortex and hippocampus than those of E2020 and tacrine. Tacrine was more effective in inhibiting plasma butyrylcholinesterase (BuChE) than it was brain AChE. Conversely, the BuChE activity was less affected by huperzine A and E2020. The results showed that huperzine A had high bioavailability and more selective inhibition on AChE activity in cortex and hippocampus. Huperzine A fits more closely with the established criteria for an ideal AChE inhibitor to be used in clinical studies.

Cholinergic amacrine cells are not required for the progression and atropine-mediated suppression of form-deprivation myopia

Muscarinic cholinergic pathways have been implicated in the visual control of ocular growth. However, the source(s) of acetylcholine and the tissue(s) which regulate ocular growth via muscarinic acetylcholine receptors (mAChRs) remain unknown. We sought to determine whether retinal sources of acetylcholine and mAChRs contribute to visually guided ocular growth in the chick. Cholinergic amacrine cells were ablated by intraocular injections of either ethylcholine mustard aziridinium ion (ECMA; a selective cholinotoxin) or quisqualic acid (QA; an excitotoxin that destroys many amacrine cells, including those that release acetylcholine). Disruption of cholinergic pathways was assessed immunocytochemically with antibodies to the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and three different isoforms of mAChR, and by biochemical assay for ChAT activity. ECMA (25 nmol) destroyed two of the four subtypes of cholinergic amacrine cells and attenuated retinal ChAT activity, but left retinal mAChR-immunoreactivity intact. QA (200 nmol) destroyed the majority of all four subtypes of cholinergic amacrine cells, and ablated most mAChR-immunoreactivity and ChAT activity in the retina. ECMA and QA had no apparent effect on mAChRs or cholinergic fibres in the choroid, only marginally reduced choroidal ChAT activity, and had little effect on ChAT activity in the anterior segment. Toxin-treated eyes remained emmetropic and responded to form-deprivation by growing excessively and becoming myopic. Furthermore, daily intravitreal injection of 40 μg atropine for 6 days into form-deprived toxin-treated eyes completely prevented ocular elongation and myopia. We conclude that neither cholinergic amacrine cells nor mAChRs in the retina are required for visual regulation of ocular growth, and that atropine may exert its growth-suppressing influence by acting upon extraretinal mAChRs, possibly in the choroid, retinal pigmented epithelium, or sclera.