Selective cholinergic neurotoxin: AF64A''s effects in rat striatum

The selective neurotoxic effects of the aziridinium ion of ethylcholine (AF64A) have been examined after stereotaxic injection into the rat striatum. In a dose-response study (2-26 nmol), 8 nmol caused a 46% decrease in striatal choline acetyltransferase (CAT) activity with minimal effects on the activities of glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH) at 7 days. Maximal CAT reductions of 78-82% occurred with doses of 16-26 nmol which also caused dose-related decreases in GAD and TH activities that paralleled the progressive decrements in CAT. A time course study with 8 nmol indicated a rapid 20% reduction of CAT activity by 12 h and an additional gradual fall of 20% over the next week; TH and GAD activities were not significantly reduced. The selective inhibition of CAT activity persisted for at least 3 months. Histological examination of Nissl stained sections revealed an area of nonspecific damage at the injection site with an abrupt border surrounded by apparently normal striatal neuropil; however; neuronal perikarya staining intensely for acetylcholinesterase were not reduced. These preliminary findings strongly suggest that AF64A has selective neurotoxic effects against striatal cholinergic neurons while relatively sparing striatal GABAergic intrinsic neurons or dopaminergic afferents.     

Delayed expression of NADPH-diaphorase in rat brain after administration of the cholinotoxin AF64A

Administration of cholinotoxin etylcholine aziridinium (AF64A) into the brain selectively induces nonrever-sible cholinergic deficit. Wistar rats were injected intracerebroventricularly bilaterally with AF64A at doses of 1–3 nmol/ventricle. 28 days later the number of neurons survived was counted in dorsolateral, intermediate and medial groups of cells of the medial septum. AF64A induced a decrease in neuronal density and expression of cholineacetyl transferase at all doses used as well as in all regions studied. Brain sections were also stained for NADPH-diaphorase representing neuronal NO-synthase. Effects of AF64A on NADPH-diaphorase expression depended on the region studied. The number of NADPH-diaphorase-positive cells increased in the medial cellular group where more cholineacetly transferase-positive cells survived. In contrast, decrease in NADPH-diaphorase expression in the dorsolateral group of cells coincided with low level of cholineacetyltransferase-po-sitive neurons. The data presented suggest that in the AF64A-dependent model of neurodegeneration NO may play a neuroprotective function.     

Effects of intrastriatal injections of the cholinergic neurotoxin AF64A on spontaneous nocturnal locomotor behavior in the rat

Rats with bilateral striatal cholinergic lesions induced by the neurotoxin AF64A were found to have increased spontaneous nocturnal locomotor activity as measured in terms of the amount of time spent moving, the total distance traveled and the number of episodes and time of stereotypic behaviors. No significant differences between control and AF64A injected rats were found in the actual velocities between the two groups indicating that the rats were simply spending more time moving rather than moving with greater speed. Striatal activity of choline acetyltransferase (CAT) was significantly decreased in AF64A treated rats compared to controls, whereas the activities of glutamate decarboxylase (GAD) were not. Furthermore, there were no significant differences between groups in enzymatic activities of CAT and GAD in either the cortex or the hippocampus, indicating that the lesion was restricted to the striatum. The hyperactivity found in these rats after intrastriatal injection of AF64A supports a role for the striatal cholinergic system in locomotor behavior.     

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.     

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.     

Neuronal and microvascular alterations induced by the cholinergic toxin AF64A in the rat retina

The choline analogue ethylcholine mustard aziridinium ion (AF64A) produces both neuronal and non-neuronal alterations in the rat retina⁹. The possible involvement of the retinal capillaries in the origin of the apparently non-specific lesions has been investigated. Two hours after a single intraocular injection of 5 nmol AF64A, ultrastructural alterations were observed in neurons of the inner nuclear layer and the ganglion cell layer, where cholinergic cells are located. One week later, the number of cholinergic neurons, identified by choline acetyltransferase immunohistochemistry, was decreased to 65% of control, the neurons located in the inner nuclear layer being more sensitive than those in the ganglion cell layer. The same dose of AF64A also induced ultrastructural changes in retinal capillaries, which showed a significant increase in the number of pinocytotic vesicles and microvilli in the endothelial cells, 2–5 h after the toxin administration. One day later, arterioles and capillaries presented contracted profiles and the lumen was occasionally lost. The sensitivity of endothelial cells to the toxic effects of AF64A may be explained by the presence in the cerebral endothelium of a choline transport mechanism with an affinity close to that of cerebral synaptosomes. In vitro, both neuronal and endothelial choline uptake systems were equally sensitive to the toxin inhibitory effect. The early and severe vascular alterations induced in the retinal microvessels by AF64A may produced changes in blood perfusion and capillary permeability that could account for the apparently non-specific histological damage.     

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.     

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 sacrified 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     

Lesion with the neurotoxin AF64A alters hippocampal cholinergic receptor function

The effect of selective lesion of cholinergic inputs to the hippocampus on the function of hippocampal cholinergic receptors was examined. Hippocampal cholinergic neurons were lesioned in the rat by administration of the selective cholinergic neurotoxin AF64A (ethylcholine mustard azirtdinium). Cholinergic receptor function was examined by assessing the ability of cholinergic agonists and antagonists to modulate the evoked release of radiolabelled acetylcholine (ACh) from hippocampal slices. Nicotine enhanced release, with a bell-shaped dose-response curve. The dose-response curve and EC₅₀ for nicotine was shifted 10-fold to the left in lesioned rats, suggesting an increased sensitivity to nicotine. However, there were no differences in either the number or affinity of nicotinic receptors as determined with binding studies. The muscarinic agonist oxotremorine inhibited the evoked release of ACh in control tissues, but had much less effect in AF64A-lesioned tissues. Binding to the M1 receptor subtype was not changed. However, the K_d for binding to the high affinity subtype of the M2 receptor was increased 10-fold, suggesting that the receptor has become less sensitive to stimulation. Loss of M2 function may allow an increase in the effect of stimulating nicotinic receptors that modulate ACh release.     

杏仁核和扣带回毁损对甲基苯丙胺大鼠边缘区多巴胺受体的影响

目的　探讨立体定向杏仁核和扣带回的联合毁损对甲基苯丙胺 (MAP)大鼠脑内边缘区多巴胺D2受体表达的影响。方法　 4 0只SD大鼠随机分为对照组、MAP组、MAP加毁损组和MAP加假毁损组 ,每组各 10只 ;采用经腹腔注射MAP制备精神分裂症MAP模型 ,立体定向 射频毁损杏仁核和扣带回 ,免疫组织化学ABC法观察边缘区D2受体的表达。结果　与对照组比较 ,MAP组及MAP加假毁损组大鼠边缘区D2受体表达有非常显著性差异(P <0 0 1) ;MAP加毁损组大鼠边缘区D2受体阳性细胞数目与对照组比较无显著性差异 (P <0 0 1)。结论　杏仁核和扣带回的联合毁损可以抑制使用MAP而诱发的边缘区D2表达的亢进。     

Modulation of hippocampal norepinephrine release by cholinergic agonists is altered by AF64A lesion

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

Differential long-term effect of AF64A on [H]ACh synthesis and release in rat hippocampal synaptosomes

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 μl/side) or vehicle (saline). Synaptosomes were preloaded with [³H]choline ([³H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [³H]Ch and [³H]acetylcholine ([³H]ACh) and for their ability to synthesize and release [³H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [³H]ACh was reduced to 60% of control; (ii) residing [³H]ACh levels were 43% of control while residing [³H]Ch levels were 72% of control; (iii) basal and K⁺-induced [³H]ACh release were 77% and 73% of control, respectively; (iv) high K⁺-induced synthesis of [³H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.     

Degeneration of beta-amyloid-associated cholinergic structures in transgenic APP SW mice

Cholinergic dysfunction is a consistent feature of Alzheimer's disease, and the interrelationship between beta-amyloid deposits, inflammation and early cholinergic cell loss is still not fully understood. To characterize the mechanisms by which beta-amyloid and pro-inflammatory cytokines may exert specific degenerating actions on cholinergic cells ultrastructural investigations by electron microscopy were performed in brain sections from transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein and progressively develop beta-amyloid plaques during aging. Both light and electron microscopical investigations of the cerebral cortex of 19-month-old transgenic mice revealed a number of pathological tissue responses in close proximity of beta-amyloid plaques, such as activated microglia, astroglial proliferation, increased number of fibrous astrocytes, brain edema, degeneration of nerve cells, dendrites and axon terminals. Ultrastructural detection of choline acetyl transferase (ChAT)-immunostaining in cerebral cortical sections of transgenic mice clearly demonstrated degeneration of ChAT-immunoreactive fibres in the environment of beta-amyloid plaques and activated glial cells suggesting a role of beta-amyloid and/or inflammation in specific degeneration of cholinergic synaptic structures.     

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 μl/4 min; A 6.2, L^s 1.5, H 6.5, Paxinos and Watson). After 19 days the animals' brains were processed 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 AF64-A-picrylsulfonate into the dorsal hippocampus of the rat.     

Neurochemical Effects Following AF64A Injection into the Basal Nuclei of Rats

Abstract: AF64A, a specific cholinergic neurotoxin, was injected into the basal nuclei of rats. The injected sites were the bilateral nucleus basalis of Meynert (NBM) and the medial septal nucleus (MSN), well known to be the nuclei of origin of the two major cholinergic pathways. The remote effects of injection were estimated by the regional choline acetyltransferase (CAT) activity in the frontal cortex, striatum and hippocampus. The injection of AF64A (1 nmol in 1 ul) produced a reduction in the CAT activity in each projected site: NBM lesions in the frontal cortex and MSN lesions in the hippocampus after one and three weeks. Twelve weeks after the injection, the reduced CAT activity had returned to normal levels. This neurochemical effect shows plasticity and recovery with time. The injections of small amounts of AF64A (0.2 and 0.1 nmol in 1 μl) produced no chemical changes after one week.     

Ganglioside AGF2 promotes task-specific recovery and attenuates the cholinergic hypofunction induced by AF64A

Ganglioside AGF2 attenuated both the cognitive impairments and the cholinergic hypofunction induced by ethylcholine aziridinium ion (AF64A). Adult male rats were initially trained to perform a standard radial arm maze (RAM) task. Following training, they were injected intraperitoneally with 10 mg/kg AGF2 (AF/AGF2, CSF/AGF2) or the saline vehicle (AF/SAL, CSF/SAL) for 3 days prior to and for 14 days following bilateral injection of AF64A (3 nmol/side) or artificial CSF into the lateral ventricles. AF64A (AF/SAL) impaired performance of the standard RAM task and a working memory version of the task in which various delays were imposed between the fourth and fifth arm choices. In contrast, animals that received AGF2 and AF64A (AF/AGF2) were initially impaired on the standard RAM task but rapidly recovered and were performing as well as the control groups (CSF/SAL, CSF/AGF2) by the end of training. The AF/AGF2 group, however, exhibited persistent deficits on the working memory version of the RAM task. These data demonstrate that AGF2 promotes behavioral recovery in a task-dependent manner in this model system. Neurochemical analysis revealed that AF64A produced a significant 37% decrease in hippocampal ChAT activity that was significantly attenuated, but not prevented, by prior treatment with AGF2. Thus the behavioral recovery afforded by AGF2 might be related to increased cholinergic activity in the hippocampus that is sufficient for the performance of tasks which either lack or have a minimal working memory component. An analysis of the temporal profile of AGF2-induced neurochemical recovery revealed that ChAT activity was enhanced at 20, but not 2 or 11, weeks following AF64A. Since AGF2 did not attenuate the cholinergic cell loss (35%) induced by AF64A in the medial septum these data indicate that AGF2 might have (1) enhanced sprouting of cholinergic terminals following the initial insult, (2) directly increased ChAT activity in surviving neurons, or (3) induced behavioral and neurochemical recovery through a combination of these or other mechanisms.     

Method for the quantitation and characterization of the cholinergic neurotoxin, monoethylcholine mustard aziridinium ion (AF64A)

Monoethylcholine aziridinium ion (AF64A), which is generated from the precursor, acetylcholine mustard hydrochloride, exerts selective neurotoxic effects against brain cholinergic neurons when injected intracerebrally. Conditions associated with optimal generation of AF64A were examined. The results indicate that hydrolysis and cyclization of the precursor are optimal at 25 degrees C with the former occurring at pH 9.0 and the latter at pH 7.3. The aziridinium ion is best stabilized at pH 7.3 at 4 degrees C.     

扣带回毁损对甲基苯丙胺大鼠颞叶皮质多巴胺D2受体的影响

目的探讨扣带回立体定向毁损对甲基苯丙胺(MAP)大鼠脑内颞叶皮质多巴胺D2受体表达的影响。方法80只SD大鼠随机分为对照组、MAP组、MAP 毁损组和MAP 假毁损组,每组各20只;采用经腹腔注射MAP制备精神分裂症MAP模型,立体定向-射频毁损扣带回,免疫组织化学ABC法观察颞叶皮质D2受体的表达。结果与对照组比较,MAP组及MAP 假毁损组大鼠颞叶皮质D2受体表达差异有显著性(P<0.01);MAP 毁损组大鼠颞叶皮质DA受体阳性细胞数目与对照组比较差异无显著性(P>0.05)。结论扣带回毁损可以抑制使用MAP而诱发的颞叶皮质D2表达的亢进。     

Oxidative stress in the brain following intraventricular administration of ethylcholine aziridinium (AF64A)

AF64A is a toxic analog of choline that disrupts high affinity choline transport and produces a persistent presynaptic cholinergic hypofunction. The observed neuroprotectant effects of Vitamin E in the AF64A model suggested that oxidative stress contributed to the cholinotoxicity of AF64A. The studies presented here examined whether intraventricular injection of AF64A produces oxidative stress in the brain of male Wistar rats. Indices of oxidative stress including thiobarbituric acid reactive species TBARS), free radical generation using hydrogen peroxide-induced, luminol-dependent chemiluminescence (CL) and superoxide scavenging/generating activity were measured in cerebral cortex, hippocampus and the rest of the brain, without cerebellum, 1, 3 or 5 days after bilateral intraventricular injection of 3 nmol of AF64A or artificial CSF (sham surgery). The sham operation itself induced oxidative stress throughout the brain (increased TBARS, CL and superoxide generation). In addition to the oxidative stress of the sham surgery AF64A increased basal TBARS on day 1 and Fe/ascorbate-induced TBARS on days 3 and 5 throughout the brain. AF64A produced compensatory ‘antioxidative’ changes as well with increased superoxide scavenging activity observed on day 3 and decreased basal TBARS on day 5. AF64A also induced specific changes in the hippocampus including a decrease of CL and an increase of superoxide scavenging activity on day 5. The increased superoxide scavenging activity persisted up to 126 days. The results of the present study provide the first direct evidence that AF64A induces oxidative stress following intraventricular injection.     

AF64A, a cholinergic neurotoxin, selectively depletes acetylcholine in hippocampus and cortex, and produces long-term passive avoidance and radial-arm maze deficits in the rat

The behavioral and biochemical effects of AF64A, a presynaptic cholinergic neurotoxin, were investigated. Bilateral administration of this compound into the lateral cerebral ventricles produced transient and dose-related effects on sensorimotor function and long-term impairments of cognitive behavior. Male Fischer-F344 rats dosed with either 15 or 30 nmol of AF64A reacted 29–62% faster than CSF-injected controls in a hot-plate test 14 (but not 1, 7, 21 or 28) days following dosing. The group administered 15 nmol of AF64A was also significantly more active (41%) than controls 28 days following dosing. The activity level of this group was comparable to that of controls at other times and hyperactivity was never observed in the 30 nmol group. Retention of a step-through passive avoidance task, assessed 35 days after dosing, was impaired in both 15 and the 30 nmol groups. Their step-through latencies were significatlly shorter than the control latencies, and they exhibited more partial entries during the 24-h retention test. Radial-arm maze performance, measured 60–80 days following treatment, was markedly impaired in the treated groups. Animals treated with AF64A made fewer correct responses in their first 8 choices, required more total selections to complete the task, and had an altered pattern of spatial responding in the maze. The neurochemical changes produced by AF64A, determined 120 days after dosing, were specific to the cholinergic system and consisted of decreases of ACh in both the hippocampus (15 and 30 nmol groups) and the frontal cortex (30 nmol group). The concentrations of catecholamines, indoleamines, their metabolites and choline in various brain regions were not affected by AF64A. Furthermore, histological analysis revealed that the doses of AF64A used in the present study did not damage the hippocampus, the fimbria-fornix, the septum or the caudate nucleus. These data support the contention that cholinergic processes in the hippocampus, nd/or frontal cortex play an important role in learning and memory processes. Furthermore, based upon the behavioral and biochemical data presented, it is suggested that AF64A could be a useful pharmacological tool for examining the neurobiological substrates of putative cholinergic disorder such as senile dementia of the Alzheimer's type.