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.     

Transplantation of septal cholinergic neurons to the hippocampus improves memory impairments of spatial learning in rats treated with AF64A

Embryonic septal neurons were transplanted into damaged hippocampus in adult rats which had received lateral ventricular administration of AF64A, a cholinergic neurotoxin. About 3 months after transplantation, the rats with bilateral septal grafts showed significant improvement in the radial maze and T-maze tasks. Many ingrowths of acetylcholinesterase (AChE)-positive fibers originating from the grafts were observed in the hippocampus of the rats which showed good performance in these learning tasks. These results indicate that transplantation of septal cholinergic neurons into the AF64A-treated hippocampus may induce at least partial recovery in learning tasks believed to involve the hippocampus.     

Septohippocampal adaptive GABAergic responses by AF64A treatment

A cholinergically disrupted laboratory animal has been produced by administration of the cholinotoxin ethylcholine aziridinium mustard (AF64A), which produced a dysfunction in the cholinergic forebrain system. After AF64A treatment, a reduction of choline acetyl transferase (ChAT) activity was measured in the hippocampal regions. ChAT activity was preferentially reduced in tissue samples of the dorsal with respect to the ventral hippocampus, and concomitantly with this reduction, a compensatory increase in ChAT activity in the medial septum was found. Tissue gamma‐aminobutyric acid (GABA) content in the hippocampal and septal brain areas was not affected by AF64A, indicating a specific effect on the cholinergic septohippocampal projection. The rate of GABA accumulation induced by aminooxyacetic acid administration was higher in the dorsal hippocampus and medial septum of AF64A‐treated animals, but not in their ventral hippocampus and lateral septum, where significant changes occurred in ChAT activity. Concomitantly with the changes in GABA metabolism, a significant B_max increase and K_d reduction of ³H‐flunitrazepam binding in the hippocampus of AF64A‐treated animals were associated with changes in the ChAT activity. This finding suggests an increase of GABA input on the cholinergic somas of the medial septum and an uncompensated GABAergic interneuron activity in the hippocampus. In this study, we present an adaptive mechanism of homotypic compensatory metabolism by cholinergic somas, and a heterotypic response of the GABAergic septohippocampal projection system, which was elicited by AF64A administration. J. Neurosci. Res. 55:178–186, 1999. © 1999 Wiley‐Liss, Inc.     

Transplantation of fetal cholinergic neurons into the hippocampus attenuates the cognitive and neurochemical deficits induced by AF64A.

The present experiments examined whether transplanted fetal cholinergic neurons would attenuate the behavioral and neurochemical deficits induced by the cholinotoxin AF64A (ethylcholine aziridinium ion). Bilateral injections of AF64A (3 nmol) into the lateral ventricles produced significant learning and memory impairments together with decreases in hippocampal high-affinity choline uptake (HAChU). AF64A-treated rats were impaired on both a standard radial arm maze (RAM) task and a working memory version in which a one-hour delay was imposed between the fourth and fifth arm choices. Transplantation of embryonic day E-17 septal/diagonal band tissue into the hippocampus (HPC) promoted recovery of performance on the standard version of the RAM task. However, this recovery was not observed when the animals were tested on the more difficult delay version of the task. Neurochemical analysis indicated that AF64A produced a significant (31%) decrease in hippocampal HAChU that was attenuated (14%) by transplantation of fetal cholinergic neurons. Histological analysis revealed that the transplants survived and innervated the HPC. There was no apparent relationship between fiber ingrowth into the HPC and behavioral recovery. These data indicate that transplant-induced behavioral recovery may be related to and limited by the cognitive demands of the testing situation. Generalized increases in cholinergic activity, transplant-mediated release of trophic factors, or a combination of both may underlie the behavioral recovery observed in the present studies.     

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.     

Dose-dependent effect of cholinotoxin AF64A on the cholinergic elements of the cingulum bundle in rat

Previous studies revealed that cholinergic neurons possessing long axons are extremely sensitive to ethylcholine aziridinium ion (AF64A) administration [Neuropharmacology 31 (1992) 397]. In the present paper we examined the effect of AF64A on the cholinergic elements of the cingulum bundle. Seven days after AF64A administration choline acetyltransferase (ChAT)-immunoreactive fibers were extensively damaged on the dorsal part of cingulum bundle. These findings are the first reporting damage by AF64A to this brain region.     

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.     

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.     

Transplantation of ventral forebrain cholinergic neurons to the hippocampus ameliorates impairment of radial-arm maze learning in rats with AF64A treatment

Two types of cholinergic neurons were transplanted into the hippoccampus of adult rats chemically damaged by lateral ventricular administration of AF64A, a cholinergic neurotoxin, and the effects were compared with respect to their ability to reinnervate the hippocampus and to repair behavioral deficit. Pieces of brain tissue containing the nucleus basalis magnocellularis (NBM) or the striatum were taken for grafting from 17-day rat fetuses. About 3 months after transplantation, the rats with bilateral NBM grafts showed significant amelioration in radial-arm maze performance and habituation to a novel environment in an open field box, although they had not recovered to the control level. In rats with NBM grafts that showed a good performance, there were surviving grafts and many ingrowths of AChE-positive fibers in the hippocampus. By contrast, rats with striatal grafts showed hardly any significant improvement in these behavioral measures. The AChE staining revealed poor outgrowth of the striatal grafts into the hippocampus. These results indicate that grafting of NBM cholinergic neurons, which are anatomically similar to septal neurons, into the hippocampus produces a partial restorative effect on the cognitive impairment associated with hypofunction of the septohippocampal system.     

Ultrastructural and neurochemical effects of the presumed cholinergic toxin AF64A in the rat interpeduncular nucleus

We have studied the effect of the presumptive cholinergic neurotoxin, ethylcholine mustard aziridinium ion (compound AF64A), on ultrastructure and neurochemical markers in the rat interpeduncular nucleus (IPN). Stereotaxic injections of 1 nmol of AF64A resulted in extensive degeneration of synaptic terminals within 40 h. Ultrastructural damage to neuronal cell bodies, dendrites and axons was also sometimes observed at this stage. Five days after the injection, more severe degenerative changes were observed in a larger number of neuronal cell bodies, axons and dendrites. High affinity uptake of [³H]choline, but not [³H]GABA, was significantly decreased 24 h after toxin injection. Five days after the injection, not only choline acetyltransferase but also glutamate decar☐ylase levels were significantly decreased. Our results suggest that, in addition to presynaptic cholinergic neurotoxicity, AF64A also leads to degenerative alterations of non-cholinergic neurons. Our electron microscopic observations constitute the first ultrastructural report on neuropathological damage caused by AF64A.     

Behavioral and neuroanatomical consequences of a unilateral intraventricular infusion of AF64A and limitations on the neuroprotective effects of nimodipine

The monoethylcholine aziridinium ion, AF64A, (3 nmol in 1 μl) or artificial CSF (1 μl) was infused unilaterally into the right dorsal lateral ventricle of male adult rats. Treatment with the L-type calcium channel antagonist, nimodipine (70 μg/kg b.wt.) or its vehicle was administered beginning before and for seven days following surgery. The infusion of AF64A reduced spontaneous alternation rates in the T-maze when compared to CSF and sham infused animals. F64A-treated animals also took longer to reach the goal area in a complex maze task on specific trials relative to CSF and sham-infused animals. Locomotion and habituation to the open field did not differ between surgery groups. Unilateral AF64A significantly depleted acetylcholinesterase (AChE) positive terminals in the ipsilateral hippocampus and cell bodies in the ipsilateral medial septal area (MSA). Receptors for nerve growth factor (NGF-R), often colocalized with cholinergic cell bodies and terminals, also were depleted in the ipsilateral MSA of AF64A infused animals. Treatment with nimodipine did not have a neuroprotective effect on AF64A animals in either behavioral or histological results. However, some degree of protection was found in the vehicle-treated rats. This effect was likely a consequence of the stress of the injection procedure rather than the content of the vehicle, largely polyethylene glycol 400. Nimodipine-treated animals, regardless of surgery group, exhibited fewer emotional responses and had lower spontaneous alternation rates than untreated animals. The behavioral alterations found in the nimodipine groups are most easily explained in terms of altered emotionality. Overall our findings indicate that AF64A is a potent cholinotoxin that can selectively eliminate the ipsilateral septohippocampal cholinergic system when unilaterally infused into the lateral ventricle. It is possible that the mechanism of action of AF64A, like other nitrogen mustard analogues, involves disruption of basic processes involved in protein synthesis and DNA activities. Because of this, the toxic effects of the aziridinium mustard are independent of extracellular calcium and thus may not be susceptible to protection by calcium channel antagonists.     

Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (Bmax) in non-M1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M1 subtype.     

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.     

Noradrenaline depletion protects cholinergic neurons in rat hippocampus against AF64A-induced damage

The role of the noradrenergic system in the cholinotoxicity of ethylcholine aziridinium ion (AF64A) was studied in rats. Male Sprague-Dawley rats were treated with the noradrenergic neurotoxin DSP-4 (N-(2-chloroethyl)-n-ethyl-2-bromobenzylamine; 50 mg/kg i.p.) in the presence of the serotonin uptake inhibitor fluoxetine, 14 days prior to bilateral intracerebroventricular injection of AF64A (2 nmol/lateral ventricle). In rats in which noradrenaline (NA) was depleted by 94%, the loss of acetylcholine (ACh) in hippocampus induced by AF64A was significantly attenuated (p less than 0.02). However, when there was only a partial depletion of NA (50% reduction), the AF64A-induced loss of ACh was a pronounced as in rats with intact noradrenergic function. These findings indicate that the noradrenergic lesion has to be complete before a protective effect is apparent. Moreover, they imply that noradrenergic input is involved in AF64A-induced cholinergic damage in the hippocampus.     

Reduction in striatal D2 dopamine receptor mRNA and binding following AF64A lesions

Unilateral lesions by a cholinotoxin, receptor autoradiography, andin situ hybridization techniques were employed to determine if dopaminergic receptors are located on cholinergic interneurons in the caudate-putamen (CPu). Lesion of the CPu with small amounts of the cholinotoxin AF64A resulted in a significant decrease in D₂ receptor mRNA and D₂ receptor binding. The loss was more pronounced in lateral and central portions of the CPu. Results obtained using [³H] SCH23390 binding to D₁ receptors indicated that there was no change in this dopamine receptor subtype in the AF64A-lesioned CPu. A decrease in D₂ receptor mRNA and receptor binding in AF64A-lesioned animals indicates that a population of postsynaptic D₂ receptors is associated with the cholinergic interneurons. Lack of any change in [³H]SCH23390 binding in the AF64A-lesioned animals suggests that D₁ receptors are not located on cholinergic neurons. These results provide evidence to support the selectivity of the lesion when used as indicated.     

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.     

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.     

Effects of nefiracetam on deficits in active avoidance response and hippocampal cholinergic and monoaminergic dysfunctions induced by AF64A in mice

Summary The effects of nefiracetam [DM-9384; N-(2,6-dimethyl-phenyl)-2-(2-oxo-pyrrolidinyl)acetamide] and of phosphatidylcholine on a step-up active avoidance response, locomotor activities and regional brain cholinergic and monoaminergic neurotransmitters in AF64A-treated mice were investigated. Intracerebroventricular (i.c.v.) injection of AF64A (ethylcholine mustard aziridinium ion; 8 nmol/ventricle) impaired acquisition and retention of the avoidance task, and increased vertical and horizontal locomotor activities. Regional levels of acetylcholine, noradrenaline, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were significantly decreased and homovanillic acid (HVA) levels were increased in the hippocampus but not in the septum, cerebral cortex or striatum of AF64A-treated animals. Administration of nefiracetam (3 mg/kg, p.o.) twice daily for 9 days to AF64A-treated animals ameliorated the deficit in active avoidance response in addition to attenuating the increase in locomotor activities. In parallel with these behavioural effects, nefiracetam reversed AF64A-induced alterations in the hippocampal profiles of cholinergic and monoaminergic neurotransmitters and their metabolites. In contrast, administration of phosphatidylcholine (30 mg/kg, p.o.) twice daily for 9 days had no significant effect on the deficit in active avoidance response, despite significantly reversing the decrease in acetylcholine levels in the hippocampus. These results indicate that the effects of nefiracetam on AF64A-induced behavioural deficits are probably due to its ability to facilitate both cholinergic and monoaminergic neurotransmitter systems.     

Neuroplasticity, the aging brain, and Alzheimer's disease.

A variety of neurological disorders are associated with the loss of specific populations of neurons. Alzheimer's, Parkinson's, and Huntington's diseases present unique constellations of behavioral and neurological abnormalities which result from the degeneration of neurons in specific regions of the brain. Approaches to the treatment of these neurodegenerative disorders have met with either limited or no success. New treatment strategies based upon a better understanding of the inherent mechanisms of neuroplasticity might provide more rational approaches to prevent, limit, or treat these and other neurodegenerative disorders. The development and standardization of appropriate animal models of neurodegenerative disorders will be essential to realize this possibility. Using the cholinergic neurotoxin AF64A we have developed a rodent model of cholinergic hypofunction that exhibits behavioral, anatomical, and neurochemical deficits very analogous to those observed in Alzheimer's disease. Furthermore, we have found that administration of neurotrophic factors, such as ganglioside AGF2, and the transplantation of fetal cholinergic neurons into the hippocampus can attenuate both the behavioral and neurobiological alterations induced by AF64A. These efforts should lead to the development of innovative clinical strategies and they should also help to elucidate the neurobiology of brain injury and recovery of function.     

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.