Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer's disease: An autoradiographic study using3H-hemicholinium-3

Summary ³H-hemicholinium-3 (³H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer's disease (AD) patients and of 11 matched controls. A significant decrease in the density of³H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I–III of the frontal cortex. By contrast, in the caudate-putamen the number of³H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (–40%), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD.     

Effects of cholinergic enhancing drugs on cholinergic transporters in the brain and peripheral blood lymphocytes of spontaneously hypertensive rats

Cholinergic hypofunction is a trait of Alzheimer's disease and vascular dementia and countering it is one of the main therapeutic strategies available for these disorders. Cholinergic transporters control cellular mechanisms of acetylcholine (ACh) synthesis and release at presynaptic terminals. This study has assessed the influence of 4 week treatment with two different cholinergic enhancing drugs, the cholinergic precursor choline alphoscerate (alpha-glyceryl-phosphorylcholine) or the acetylcholinesterase (AChE) inhibitor galantamine on high affinity choline uptake transporter (CHT) and vesicular ACh transporter (VAChT) expression in the brain of spontaneously hypertensive rats (SHR). SHR represent an animal model of cerebrovascular injury characterized by cholinergic hypofunction. Analysis was performed by immunochemistry, ELISA and immunohistochemistry on frontal cortex, striatum and hippocampus. Immunochemical and ELISA analysis was extended to peripheral blood lymphocytes (PBL), used as a peripheral reference of changes of brain cholinergic markers. An increased expression of VAChT and CHT was observed in brain areas investigated and in PBL of SHR. The similar trend for cholinergic transporters observed in brain and PBL suggests these cells may represent a marker of brain cholinergic transporters. Treatment with choline alphoscerate increased CHT and to a greater extent VAChT expression. Treatment with galantamine countered the increase of CHT and VAChT. The different activity of the cholinergic precursor and of the AChE inhibitor on parameters investigated is likely related to their mechanism of action. Choline alphoscerate increases ACh synthesis and release. This requires an augmentation of systems regulating neurotransmitter uptake and storage. The effect of choline alphoscerate on CHT and VAChT observed in this study suggests an improved synaptic efficiency elicited by the compound. The AChE inhibitor slows-down ACh degradation in the synaptic cleft. A greater availability of neurotransmitter elicited by galantamine counters the enhanced activity of cholinergic transporters compensating cholinergic deficits. Differences in the activity of the cholinergic precursor and AChE inhibitor investigated on CHT and VAChT suggests that association between choline alphoscerate and AChE/cholinesterase inhibitors may represent a strategy for potentiating deficient cholinergic neurotransmission worthwhile of being investigated in clinical trials.     

Impairment of cholinergic neurotransmission in adult and aged transgenic Tg2576 mouse brain expressing the Swedish mutation of human beta-amyloid precursor protein

To address the question of whether beta-amyloid peptides also affect cholinergic neurotransmission in vivo, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages ranging from 7 to 24 months were examined by immuno- and histochemical staining for choline acetyltransferase (ChAT) and acetycholinesterase (AChE), by assaying cholinergic enzyme activities and high-affinity choline uptake as well muscarinic and nicotinic cholinergic receptor binding levels by quantitative autoradiography. Cortical and hippocampal activities of AChE and ChAT were not different between transgenic mice and non-transgenic littermates regardless of the postnatal ages examined. However, high-affinity choline uptake was reduced in the hippocampus of 21-month-old transgenic mice. In brains of 8-month-old transgenic mice which do not yet demonstrate cortical beta-amyloids, reduced binding levels of cortical and hippocampal M1-muscarinic cholinergic receptors were observed, which were still reduced in 17-month-old transgenic mouse brains with high plaque load as compared to non-transgenic littermates. M2-muscarinic cholinergic receptor binding was hardly affected in brains from 8-month-old transgenic mice, but in 17-month-old transgenic mice reduced cortical and hippocampal binding levels were observed as compared to non-transgenic controls. Decreased cortical nicotinic cholinergic receptor binding was detected in 17-month-old transgenic mice. The development of changes in cholinergic synaptic markers in transgenic Tg2576 mouse brain before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble beta-amyloid on cholinergic neurotransmission and may be referred to the deficits in learning and memory also observed in these mice before significant plaque load.     

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.     

Substrate-induced internalization of the high-affinity choline transporter

Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.     

Neurochemical and behavioral recovery after colchicine lesions of the nucleus basalis magnocellularis in rats

Experimentally-induced lesions of the basal forebrain have been used to test the hypothesis that the cholinergic system plays a critical role in learning and memory. In the present study, a basal forebrain infusion of colchicine, a microtubule assembly inhibitor, was used to characterize the relationship between a cholinergic marker and behavioral function. Bilateral infusions were made in the nucleus basalis magnocellularis (NBM) of male Long-Evans rats. At 4 weeks post-lesion, behavioral assessments were made on half of the rats in each group. These rats were sacrificed 1 week later and regional choline acetyltransferase (ChAT) activity was measured. The remaining rats were behaviorally tested 11 weeks post-lesion and sacrificed 12 weeks post-lesion. The brains of additional rats were studied for Nissl-staining, ChAT-, GAD- and metEnk immunoreactivity (IR) and AChE histochemistry. At 5 weeks after colchicine infusion, there was a significant decrease in parietal and frontal cortical ChAT activity, impaired acquisition of a water maze spatial navigation task and decreased passive avoidance cross-over latency. At 12 weeks after colchicine infusion, ChAT activity was decreased in frontal but not parietal cortex; acquisition of the water maze task was not significantly different from vehicle-infused rats, and a significant deficit was observed in passive avoidance latency. ChAT-IR in the NBM showed a significant decrease at both time points, while changes in AChE-stained cortical fibers paralleled the ChAT activity. GAD- and metEnk-IR were decreased but were not different between the two time points. These data show task-specific behavioral recovery associated in time with recovery of regional cholinergic markers.     

Degeneration of the basalocortical pathway from the cortex induces a functional increase in galaninergic markers in the nucleus basalis magnocellularis of the rat.

The present work aimed 1) to evaluate whether an increase in galanin or galanin receptors could be induced in the nucleus basalis magnocellularis (nbm) by degeneration of the basalocortical neurons from the cortex and 2) to analyze the consequences of such an increase on cortical activity. First, a mild ischemic insult to the frontoparietal cortex was performed to induce the degeneration of the basalocortical system; galanin immunoreactivity, galanin binding sites, and cholinergic muscarinic receptors were quantified through immunocytochemistry and autoradiography. Second, galanin infusions in the nbm were undertaken to mimic a local increase of the galaninergic innervation; cortical acetylcholine release, cerebral glucose use, and cerebral blood flow were then measured as indices of cortical activity. As a result of the cortical ischemic lesion, the postsynaptic M1 and presynaptic M2 muscarinic receptors were found to be reduced in the altered cortex. In contrast, galaninergic binding capacity and fiber density were found to be increased in the ipsilateral nbm in parallel with a local decrease in the cholinergic markers such as the muscarinic M1 receptor density. Galanin infusion into the nbm inhibited the cortical acetylcholine release and cerebral blood flow increases elicited by the activation of the cholinergic basalocortical system but failed to affect acetylcholine release, cerebral blood flow, and cerebral glucose use when injected alone in the nbm. These results demonstrate that degeneration of the basalocortical system from the cortex induces an increase in galaninergic markers in the nbm, a result that might suggest that the galaninergic overexpression described in the basal forebrain of patients with Alzheimer's disease can result from a degeneration of the cholinergic basalocortical system from the cortex. Because galanin was found to reduce the activity of the basalocortical cholinergic system only when this one is activated, galanin might exert its role rather during activation deficits than under resting conditions such as the resting cortical hypometabolism, which is characteristic of Alzheimer's disease.     

Changes in Cholinergic Neurons and Failure in Learning Function After Microsphere Embolism-Induced Cerebral Ischemia

Central cholinergic neurons play an important role in learning and memory functions. The present study was undertaken to elucidate the pathological changes in learning function and acetylcholine metabolism of the cerebral cortex and hippocampus, following microsphere embolism in rats. Microspheres (48

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) were injected into the right internal carotid artery of the rats. Learning function was determined using a passive avoidance task on the seventh day after the embolism. In the biochemical study, acetylcholine and choline contents, and choline acetyltransferase activity were measured in the cerebral cortex and hippocampus. Cortical acetylcholinesterase-containing fibers were quantitatively estimated in the embolized rat. Passive avoidance was impaired in the microsphere-embolized rat. Microsphere embolism decreased the acetylcholine concentration and choline acetyltransferase activity in the cerebral cortex and hippocampus. In the histochemical study, the length of cortical acetylcholinesterase-containing fibers was decreased, but cell density was unchanged in the ipsilateral hemisphere of the microsphere-embolized rat. The results suggest that microsphere embolism induces severe damage to cholinergic neurons, which may be related to the impairment of learning function in the ischemic brain.     

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.     

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.     

AF64A(ethylcholine aziridinium ion)-induced basal forebrain lesion impairs maze performance

Rats were given bilateral injections of ethylcholine aziridinium ion, AF64A (1 nmol/side) into the basal forebrain (BF). One month later, choline acetyltransferase activity was reduced by 25% in the frontal cortex (FC). There was a marked decrease in cortical uptake of [3H]choline, but [3H]GABA and [3H]dopamine uptake was not affected by the injection. Histological analysis confirmed that this dose of AF64A caused acetylcholinesterase staining in the FC to disappear. Acquisition and retention of a T-maze task were impaired in the rats with BF lesions one month after the injection. Acquisition of the water-filled multiple T-maze task was also impaired by AF64A. These observations suggest that the cholinergic component in the BF is involved in spatial memory.     

Neurotransmitter chemistry of lissencephalic cortex induced in ferrets by fetal treatment with methylazoxymethanol acetate

Treatment of pregnant ferrets with 15 mg/kg of methylazoxymethanol acetate (MAM) at 33 days of fetal gestation results in offspring with cortical hypoplasia and lissencephally. Neurochemical analysis of 5 areas of cortex from 8-week-old offspring of MAM- or vehicle-treated jills indicated an overall enrichment in markers for catecholaminergic (tyrosine hydroxylase, norepinephrine) and cholinergic (choline acetyltransferase, acetylcholine) terminals but minimal change in the concentration of GABAergic markers (glutamate decarboxylase, gamma-aminobutyric acid); however, there did not appear to be a direct, inverse relationship between the concentration of catecholaminergic and cholinergic markers and the degree of hypoplasia in cortical subareas unlike what has been found previously in the rat.     

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.     

Development of the cholinergic fibres innervating the cerebral cortex of the rat

The ontogeny of innervation of the cholinergic fibres from the basal forebrain into the cingulate, frontal, parietal and piriform cortices of the rat has been examined using a modified histochemical method of acetylcholinesterase (AChE). The method produced crisp fibre staining with enhanced visibility and a clear back-ground, and a pattern of the distribution of these fibres was comparable to that achieved by choline acetyltransferase (ChAT) immunocytochemistry. In the rat, the AChE-stained fibres developed progressively from the deep cortical white matter towards the cortex itself. In general, a few AChE-positive fibres were seen in the subcortical white matter and the cingulum bundle, entering into the cerebral cortex by about 5 postnatal days. The number of these AChE-positive processes increased dramatically during the following two weeks. Thereafter, the general appearance of the overall pattern of distribution of the AChE fibres changed little, but the staining density became gradually more intense and by about 28 days after birth it was virtually indistinguishable from that in the adult. The onset and the development of the AChE-positive fibre network varied considerably between individual cortical regions, and indicated, in general, an anterior to posterior gradient. Within the dispersed AChE fibre network in the cerebral cortex, three bands of relatively enriched cholinergic processes, namely the deep cortical, mid-cortical and superficial layers, developed in an 'inside-out' fashion. The exact position of some of these AChE-rich bands varied from one cortical region to another and during development. A striking correlation during ontogeny was observed in the cerebral cortex between the changing patterns of AChE fibre network and the activity of ChAT, the enzyme synthesizing acetylcholine. The present findings can also provide an important anatomical baseline for future studies related to the factors controlling the expression of ChAT activity and the development of cholinergic neurotransmitter system in the rat.     

Increased activity of choline acetyltransferase and acetylcholinesterase in actively epileptic human cerebral cortex

We measured the activities of the cholinergic marker synthetic and catabolic enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in surgical specimens obtained from 38 patients immediately following anterior temporal lobectomy for intractable epilepsy. Samples from patients with actively spiking lateral temporal cortex were compared to non-spiking lateral temporal cortex obtained from patients in whom the epileptic discharges were confined to the hippocampus. Mean activities of ChAT and AChE were increased by 25% (P less than 0.01) and 30% (P less than 0.025) respectively in the spiking vs. non-spiking cortex. We suggest that the above-normal activity of these cholinergic marker enzymes may reflect sprouting of cholinergic nerve terminals in spontaneously spiking cortex of some patients and/or increased acetylcholine metabolism secondary to the stimulatory effect of the ongoing epileptic discharge.     

Selective degeneration of a putative cholinergic pathway in the chinchilla cochlea following infusion with ethylcholine aziridinium ion

Ethylcholine aziridinium ion (AF64A) diluted in artificial perilymph, or artificial perilymph alone was infused into the cochlea of chinchillas. After a survival time of 7 days, the cochleas were fixed with aldehydes, post-fixed in osmium and embedded in epoxy resin for light and electron microscopy. The ultrastructure of the cochleas infused with artificial perilymph was normal. Infusion of 1 microM AF64A resulted in massive degeneration of the axons of the lateral efferent system, a putative cholinergic pathway that originates in the brainstem and terminates on dendrites of the spiral ganglion innervating cochlear inner hair cells. The axons and terminals of a second putative cholinergic pathway, the medial efferent system which terminates on the outer hair cells, were normal. Infusion of AF64A in a concentration of 10 microM resulted in significant pathology of cochlear and supporting cells as well as the loss of efferent terminals at both inner and outer hair cell regions. The results suggest that AF64A is a selective neurotoxin when used under low-dosage conditions, and that certain pathways may be more susceptible to the effects of AF64A than others. One interpretation of these findings is that lateral efferent axons may have a higher rate of high-affinity choline uptake than terminals of the medial efferent axons.     

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.     

Selective cortical infarction reduces [3H]sulpiride binding in rat caudate-putamen: autoradiographic evidence for presynaptic D2 receptors on corticostriate terminals

Although the existence of presynaptic D2 dopamine receptors on corticostriate terminals has been supported by numerous receptor-binding studies, recent autoradiographic data has failed to demonstrate loss of striatal D2 receptors following cortical lesions. In the present study, Long-Evans rats were subjected to unilateral middle cerebral artery (MCA) infarction in order to produce reproducible lesions of the neocortex without damaging subcortical structures. Animals were sacrificed 2 and 4 wk following lesion and brains were prepared for receptor autoradiography. D2 receptors were studied using the selective ligand [3H]sulpiride, while D1 dopamine receptors were examined using [3H]SCH 23390. Sodium-dependent, high-affinity choline uptake sites were labeled with [3H]hemicholinium-3, thereby providing a quantitative measure of cholinergic neuronal integrity. Unilateral cortical infarction resulted in approximately a 20% reduction in [3H]sulpiride binding in several discrete regions of the ipsilateral caudate-putamen (CPu), but not in the nucleus accumbens. D2 receptor binding was also reduced significantly in some areas of the contralateral CPu when compared with [3H]sulpiride binding in sham-operated, control animals. In contrast, D1 receptors (as identified by [3H]SCH 23390 and high-affinity choline uptake sites (labeled with [3H]-HC-3) were not affected by the cortical lesion. The results provide autoradiographic confirmation of the existence of presynaptic D2 receptors on corticostriate terminals.     

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.