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At least 253 records · Page 14Linked to original sources
PubMed1986

Cholinergic neurotoxicity induced by ethylcholine aziridinium (AF64A) in neuron-enriched cultures.

The sequence of events in neuronal changes induced by the cholinotoxin ethylcholine aziridinium (AF64A) was studied. Neuron-enriched cultures derived from 8-day-embryonic chick cerebra were treated with AF64A at concentrations of 10(-5), 10(-4) and 10(-3) M. Choline acetyltransferase (ChAT) was used as an index of cholinergic neurons. Changes in cell morphology, the immunocytochemical and biochemical presence of ChAT, and DNA and protein content were assessed. Neuron-enriched cultures exposed to AF64A showed a dose-dependent response; after 24 h of exposure to 10(-3) M toxin all cells were dead, whereas a concentration of 10(-5) M did not alter culture morphology or DNA and protein contents. Despite the lack of cytological changes and the presence of ChAT immunoreactivity, biochemically assessed ChAT activity was reduced 36% in 10(-5) M treated cultures. Thus, the implicated decrease in acetylcholine synthesis in these cells cannot entirely account for the neuronal degeneration. Simultaneous exposure of cultures to both AF64A and 10 times higher concentrations of choline chloride delayed or diminished the neurotoxic changes. The protective effect of high choline concentrations was interpreted as evidence of competition between choline and AF64A for the high affinity choline transport system and as constituents in the cell membrane. Examination of the temporal sequence of cytotoxic changes in 10(-4) M exposed cultures revealed that disruption of neuronal aggregates and fragmentation of neurites occurred between 4 and 8 hours of exposure. After 24 h, some neurons survived but with attenuated arbors; in contrast, astrocytes appeared intact, suggesting that glial cells are more resistant than neurons to the toxic effects of AF64A. These findings suggest this culture model may be useful to further elucidate the mechanisms of AF64A drug action and study differentiation of cultured neuronal populations in the absence of cholinergic cells.

Animals↗
PubMed1986

Immunohistochemical evidence for degeneration of cholinergic neurons in the forebrain of the rat following injection of AF64A-picrylsulfonate into the dorsal hippocampus.

The cholinergic neurotoxin, AF64A-picrylsulfonate, was unilaterally infused into the dorsal hippocampus of Wistar rats (2 nmol/2 microliters/4 min; A 6.2, Ls 1.5, H 6.5, Paxinos and Watson). After 19 days the for immunohistochemical staining of choline acetyltransferase (ChAT). Morphometry and counting of ChAT-immunoreactive profiles revealed shrinkage and disappearance of cholinergic neurons in the medial septum and diagonal band of Broca at the lesioned brain side. These data indicate a retrograde degeneration of cholinergic neurons following injection of AF64A-picrylsulfonate into the dorsal hippocampus of the rat.

Alzheimer Disease↗
PubMed1986

Specific toxic effects of ethylcholine nitrogen mustard on cholinergic neurons of the nucleus basalis of Meynert.

The putative cholinergic neurotoxin, ethylcholine aziridinium ion (AF64A), was injected unilaterally into the nucleus basalis of Meynert (nbM) in order to determine whether it would produce specific damage to the cholinergic cell bodies of this nucleus. Injections of small amounts of AF64A (0.01 nmol in 1 microliter) or of its vehicle had little effect on the appearance of the nbM or on the levels of choline acetyltransferase (ChAT) in the cortex. Injections of larger amounts of AF64A (0.02 and 0.05 nmol in 1 microliter and 0.02 nmol in 10 microliters) produced a loss of diffuse acetylcholinesterase staining in the nbM and a loss of large positively staining neurons. Furthermore, these injections produced a significant reduction of ChAT activity in the central portion of the cortex. However, non-cholinergic neurons in the area of the nbM were not affected by these AF64A injections. In addition, cortical uptake of monoamines was not affected by these lesions. Further increases in the amount of AF64A injected (0.1 nmol in 1 microliter and 0.035 nmol in 10 microliters) caused damage at the site of the injection which was not limited to the cholinergic elements of the nbM. These results suggest that AF64A can be used to produce specific lesions of cholinergic neurons, and therefore may be useful in developing animal models of human disorders involving cholinergic hypofunction, such as senile dementia of the Alzheimer type. However, there is a narrow dose range for producing these specific effects.

Animals↗
PubMed1987

Effect of cholinergic deficit induced by ethylcholine aziridinium (AF64A) on noradrenergic and dopaminergic parameters in rat brain.

The consequences of reduced cholinergic function on noradrenergic and dopaminergic neurons has been studied in various rat brain areas for a period of up to 28 days following bilateral intracerebroventricular infusion of various doses of ethylcholine aziridinium ion (AF64A; 1-5 nmol/ventricle). This treatment resulted in a dose-dependent, persistent decrease in acetylcholine (ACh) content ranging from 50.3 +/- 6.0% to 76.9 +/- 3.8% when compared to vehicle-injected rats. Concomitantly, there was a transient, dose-dependent decrease (up to 46.7 +/- 6.4%) in norepinephrine (NE) levels in hippocampus, cortex and hypothalamus. Whereas the noradrenergic system recovered fully within 28 days after 1-3 nmol AF64A/ventricle, the decrease in NE levels persisted after 5 nmol/ventricle. In striatum, a small decrease in ACh levels 4 days after AF64A infusion was accompanied by a transient, dose-dependent decrease in the levels of dopamine (DA) and its metabolites dihydroxyphenylacetic acid and homovanillic acid, suggesting a decrease in DA synthesis and release. Dopaminergic function was fully restored within 14 days after all doses of AF64A used. These data suggest that reduction of cholinergic function might have a considerable impact on noradrenergic and dopaminergic neurons, causing an increase in NE release as well as depression of dopaminergic function.

Acetylcholine↗
PubMed1987

AF64A (ethylcholine aziridinium ion), a cholinergic neurotoxin, selectively impairs working memory in a multiple component T-maze task.

The present study examined the nature of the cognitive deficits associated with a selective decrease of cholinergic activity in the hippocampus. Male Fischer rats were trained to perform a multiple component T-maze task which simultaneously assessed their ability to perform on the basis of trial-specific information (working memory) and trial-independent information (reference memory). Following 125 acquisition trials rats were bilaterally injected with AF64A (3 nmol/side) or artificial CSF into the lateral ventricles and allowed 14 days to recover before behavioral testing resumed. The controls rapidly returned to their preoperative level of performance on both components of the maze task. AF64A-treated animals were transiently impaired on the reference memory task. Their performance rapidly improved and they were performing at preoperative levels within 4 days of testing. In contrast, these animals exhibited a marked and long-lasting impairment in their performance of the working memory component. After behavioral testing was completed, neurochemical analysis revealed that AF64A produced a significant decrease in choline acetyltransferase (ChAT) activity in the hippocampus (43%) 42 days following surgery. This dosing regimen produced no alterations of striatal or cortical ChAT activity. These data suggest that alterations of hippocampal cholinergic activity severely impair an animal's ability to perform working memory tasks.

Animals↗
PubMed1988

AF64A-induced working memory impairment: behavioral, neurochemical and histological correlates.

The present studies examined the behavioral, neurochemical and histological consequences of intraventricular administration of ethylcholine aziridinium ion (AF64A). Male Long-Evans rats were trained to perform a radial arm maze task in which a one hour delay was imposed between the fourth and fifth arm selections. Following acquisition, animals were bilaterally injected with AF64A (3 nmol/side) or CSF into the lateral ventricles and allowed 14 days to recover before behavioral testing resumed. AF64A-treated animals were markedly impaired in their ability to perform this working/episodic memory task at a variety of delay intervals. In contrast to a long-lasting impairment on the radial maze task, these animals showed no impairment in their ability to acquire a simple discrimination task (reference/skill memory). Neurochemical analysis revealed a significant (50%) decrease in choline acetyltransferase (ChAT) activity in the hippocampus (HPC) 90 days following surgery. ChAT activity was not affected in the striatum, frontal and parietal cortices, cingulate or amygdala. Regional concentrations of catecholamines and indoleamines were not affected in any of these brain regions. Histological analysis of animals receiving unilateral injections of AF64A (3 nmol) into the right lateral ventricle revealed decreases in ChAT-immunoreactive (ChAT-IR) cells within the medial septum/vertical limb diagonal band (MS/VLDB), but not in nucleus accumbens, striatum or basal nucleus regions. These data suggest that: (1) intraventricular administration of AF64A can markedly impair working/episodic, as opposed to reference/skill memory, processes; (2) AF64A can be used to selectively alter presynaptic cholinergic indices within the hippocampus; and (3) the behavioral deficits resulting from AF64A administration are most likely a consequence of altered septohippocampal cholinergic function.

Animals↗
PubMed1988

Cholinotoxicity of the ethylcholine aziridinium ion in primary cultures from rat central nervous system.

The cytotoxic effects of ethylcholine aziridinium ion (AF64A) were studied in primary cultures prepared from either whole brain, septum, or midbrain of fetal rats. AF64A, at concentrations up to 22.5 microM, significantly reduced the number of acetylcholinesterase-stained cells without affecting the number of dopaminergic neurons or their ability to take up and release [3H]dopamine. Many of the survived acetylcholinesterase-stained cells appeared with intact somata but damaged processes, indicating a retrograde degeneration starting at the nerve terminal. Higher concentrations of AF64A (greater than 22.5 microM), caused general toxicity which was expressed by degeneration of various neuronal and glial cells. Choline (500 microM), significantly protected the cells from AF64A induced cytotoxicity. The results are consistent with a previously described kinetic model, that predicted a dual action of AF64A: selective cholinotoxicity at low concentrations and non-selective cytotoxicity at higher concentrations.

Acetylcholinesterase↗
PubMed1988

Induction of cortical cholinergic hypofunction and memory retention deficits through intracortical AF64A infusions.

Ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of high-affinity choline uptake on cholinergic nerve terminals, appears to selectively decrease presynaptic cholinergic markers after intracerebral injection. To restrict AF64A's action to cholinergic terminals within the frontoparietal (FP) cortex, the present study utilized multiple-site cortical infusions of the agent. Following an extensive histological analysis, a dose of 1 nmol AF64A/1 microliter was selected for determining AF64A's effects on acetylcholinesterase (AChE) staining, cortical cholinergic/non-cholinergic markers, and passive avoidance behavior. Adult rats given two infusions of AF64A into the right FP cortex had reduced AChE staining throughout 75% of the ipsilateral FP cortex at 10 days following infusion, thus suggesting an extensive cortical diffusion of the agent; minimal non-specific damage was seen (totalling only 4% of the ipsilateral FP cortex for both infusion sites) and no effects on AChE staining were observed in the striatum or hippocampus. Three weeks after bilateral AF64A infusions into the FP cortex (two injections on each side), significant frontal cortex deficits were observed in high-affinity choline uptake, acetylcholine synthesis, acetylcholine release, and hemicholinium-3 binding compared to vehicle-infused controls. However, choline acetyltransferase activity within the anterior cortex did not appear to be consistently affected by AF64A infusion. Cortical glutamic acid decarboxylase activity, as well as cortical monoaminergic markers, and neuropeptide levels were also unaffected. Moreover, animals that received bilateral AF64A infusions and were tested two weeks afterwards showed marked memory retention deficits during both the 24-h and 48-h postshock trials of passive avoidance testing. These results indicate that cortical AF64A infusion induces a specific, long-term cholinergic hypofunction of presynaptic markers within the cortex, resulting in a significant long-term memory impairment. Since the primary cholinergic innervation to the FP cortex, originating in the nucleus basalis of Meynert, appears to become dysfunctional (but not totally degenerative) in Alzheimer's disease, cortical AF64A infusions may closely reflect this cholinergic dysfunction by 'functionally' eliminating cortical cholinergic terminals.

Acetylcholine↗
PubMed1988

Intraventricular administration of the cholinotoxin AF64A increases the accumulation of aluminum in the rat parietal cortex and hippocampus, but not in the frontal cortex.

Aluminum (Al) concentrations of the rat frontal cortex, parietal cortex, and hippocampus were measured by atomic absorption spectroscopy 16 days after a unilateral intracerebroventricular injection of Na gluconate, Al gluconate, or the cholinotoxin AF64A. A fourth group of rats were injected with AF64A 6 days before injection of Al gluconate and subsequently sacrificed 10 days later. The combined treatment of AF64A and Al gluconate resulted in enhanced intraneuronal accumulation of Al in the parietal cortex and hippocampus but not in the frontal cortex. Consequently, Al may not be considered to be a primary factor in the pathogenesis of Alzheimer's disease.

Aluminum↗
PubMed1988

Biochemical and histological modifications of the rat retina induced by the cholinergic neurotoxin AF64A.

Intraocular injections of ethylcholine mustard aziridinium ion (AF64A) in the rat depressed retinal choline acetyltransferase (ChAT) activity in a dose-dependent manner without any significant change in the content of amino acid neurotransmitters GABA, glycine, aspartate and glutamate. ChAT reduction was already detected 24 h after the injection and persisted for at least one month. In vitro AF64A also inhibited retinal ChAT activity. No changes in muscarinic receptor sites were detected. The histological study showed light cells, characterized by cytoplasmic swelling in the innermost part of the inner nuclear layer and in the ganglion cell layer. We suggest that these light cells are the cholinergic retinal neurons affected by the toxin. In addition, dark cells in the inner nuclear layer, large empty spaces in the outer nuclear layer, inflammatory infiltrate and vascular alterations were also observed in treated retinas. Choline uptake systems in photoreceptors and in endothelial cells or cholinergic perivascular nerve endings may explain the lesions observed in the outer nuclear layer and the vascular alterations.

Amino Acids↗
PubMed1989

Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A).

Ethylcholine aziridinium (AF64A) has been proposed as a specific cholinergic neurotoxin. In earlier studies, using AF64A, we reported that slow infusion of 1-2 nmol of this compound into each lateral ventricle of Sprague-Dawley rats resulted in small, and transient decreases in noradrenaline (NA) and serotonin (5-HT) levels in the hippocampus, while inducing a permanent and significant cholinergic hypofunction in the same brain region. The experiments described in this paper were designed to test the hypothesis that such noradrenergic and serotonergic changes after small doses of AF64A are secondary to the changes observed in cholinergic neurons. Levels of NA, and of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were measured concurrently with levels of acetylcholine (ACh), in various brain regions of rats in which the effect of AF64A was attenuated, and in respective control animals. The effect of AF64A was diminished by inhibiting the interaction of AF64A with the high affinity transport site for choline (HAChT). This was achieved using hemicholinium-3 (HC-3), which does not cross the blood-brain barrier, and A-4 (a bis 4-methylpiperidine analog of HC-3), which is centrally active following its peripheral administration. A-4 (20 or 40 mg/kg i.p.) or HC-3 (10 micrograms/ventricle) had no effect on ACh, NA, 5-HT or 5-HIAA levels in saline-treated rats. However, all treatments significantly attenuated the decrease in ACh content produced by AF64A pretreatment. Transient decreases in NA, 5-HT and 5-HIAA contents after AF64A treatment were prevented or reduced by prior treatment with A-4 or HC-3. These results indicate that changes in noradrenergic and serotonergic neurons following AF64A administration are not due to non-specific toxicity of AF64A, but may be the result of adaptation of these neurons to withdrawal of cholinergic input, which would normally inhibit the release of NA and 5-HT. These results also indicate that AF64A can be used to produce specific lesions of hippocampal cholinergic nerve terminals.

Animals↗
PubMed1989

The cholinergic neurotoxin ethylcholine mustard aziridinium (AF64A) induces an increase in MAO-B activity in the rat brain.

Recently it was reported that there is an increase in monoamine oxidase B (MAO-B) activity in post-mortem brains of patients with Alzheimer's disease. It was postulated that this increase in MAO-B activity was due to gliosis associated with neuronal degeneration. The aim of the present investigation was to evaluate the effect on MAO of neuronal degeneration primarily affecting the cholinergic system. The specific cholinergic toxin AF64A (3 and 4.5 nmol) was injected bilaterally into the cerebral ventricles of rats. We then estimated MAO-A, MAO-B, dopamine (DA) uptake rates and choline acetyltransferase (ChAT) activities in hippocampus, striatum and cortex, 1, 2.5 and 4.5 weeks after the injection. Marked long-lasting reduction in ChAT activities appeared only in hippocampus, consistent with previous reports. The MAO-A activity was unchanged as were DA uptake rates. Neither was there any change in MAO-B activity found 1 week after the injection. However, a significant increase in MAO-B activity appeared after 2.5 weeks and persisted after 4.5 weeks in all 3 brain regions investigated. This result is likely to reflect progressive gliosis after cholinergic neuronal degeneration. Previous results have shown an increased MAO-B activity with age and a further accelerated increase in Alzheimer's disease. Experimentally, hemitransection and injection of kainic acid have been shown to cause a similar increase. The present results show that changes in MAO-B activity also reflect degenerative processes in brain mainly affecting the cholinergic system.

Acetylcholinesterase↗
PubMed1977

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.

Alkylating Agents↗
PubMed1977

Alkylating esters. X. The reaction of some aziridine alkylating agents with methionine and S-methyl cysteine.

Two biologically active aziridine ring-containing compounds, N,N-ethylene urethane (I) and N,N-ethylene urea (II), have been shown to react with methionine in dilute phosphate buffer (pH 7.4) at 37 degree C. Degradative procedures indicate that the aziridine ring effectively alylates the thio ether group of methionine and other thio ether-containing amino acids to produce sulphonium salts (V). By using [35S]methionine, the sulphonium salts have been shown to be quite stable under physiological conditions (t1/2 7--9 days) hydrolysing to convert the methionine residue to homoserine. It is proposed that similar alkylations of methionyl residues in vivo by aziridine-alkylating agents may explain the complex, and al yet unknwn, metabolic fate of the aziridine ring and could also be a factor contributing to the diverse effects that these agents have on living cells.

Alkylating Agents↗