Israel Hanin

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 the 15 and the 30 nmol groups. Their step-through latencies were significantly 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 and/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 disorders such as senile dementia of the Alzheimers type.

Selective Presynaptic Cholinergic Neurotoxicity Following Intrahippocampal AF64A Injection in Rats

Abstract: Compound AF64A, ethylcholine mustard aziridinium ion (0.4–8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10–fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+‐dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r= 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histo‐chemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long‐term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.

Choline analogs as potential tools in developing selective animal models of central cholinergic hypofunction

Abstract A chronic deficiency in central cholinergic function has been implicated in a number of neuropsychiatric disease states. This deficiency most probably exists at the presynaptic nerve terminal in the brain, where acetylcholine metabolism is known to occur. To date there are no reports on animals that could simulate the neurochemical conditions which appear to cause these diseases in humans, as a result of a direct manipulation of the central cholinergic system. Several compounds related to choline have however been studied, which might be useful agents for developing such an animal model, through their specific action on the high-affinity choline transport system in the brain. This minireview presents an overview of results obtained with these potentially neurotoxic choline analogs, and provides a critical analysis of current knowledge in this area of investigation.

Progress in Alzheimer's and Parkinson's diseases

Pathology in Alzheimers Disease: General: 1. The Pathogenesis of Alzheimers Disease R.D. Terry. 2. Molecular and Cellular Abnormalities of Tau in Early Alzheimers Disease P. Davis, et al. Apoliprotein E: 3. ApoE and Memory in Alzheimers Disease H.S. Soininen, P.J. Riekkinen, Sr. 4. Divergent Metabolism of Apolipoproteins E3 and E4 by Cells R.E. Pitas, et al. 5. APP, ApoE, and Presenilin Transgenics: Toward a Genetic Model of Alzheimers Disease L. Pradier, et al. Apoptosis Oxidative Stress Amyloids, Inflammation and Other Defects: 8. Apoptosis in Alzheimers Disease: Inductive Agents and Antioxidant Protective Factors C.W. Cotman, C.J. Pike. 9. Membrane Constituencies and Receptor Subtype Contribute to Age-Related Increases in Vulnerability to Oxidative Stress: Implications for Neurodegenerative Disease J.A. Joseph, et al. 10. Mitochondrial Dysfunction and Alzheimers Disease S.S. Ghosh, et al. 11. Mitochondrial Dysfunction in Parkinsons Disease: Potential Applications for Cybrid Modeling of the Disease R.H. Swerdlow, et al. Amyloid Aggregation: 27. Amyloid-beta Hypothesis of Alzheimers Disease M.S. Shearman. 28. Discovery and Characterization of Peptidoorganic Inhibitors of Amyloid beta-Peptide Polymerization M.A. Findeis, S.M. Molineaux. 29. Anthracyclines and Amyloidosis C. Post, et al. 30. The Amino Terminus of the beta-Amyloid Peptide Contains an Essential Epitope for Maintaining Its Solubility B. Solomon, et al. 31. Animal Models of Amyloid Aggregation and Deposition R. Kisilevsky Tau in Alzheimers Disease: 32. The Conformations of Tau Protein and Its Aggregation into Alzheimers Paired Helical Filaments E. Mandelkow, et al. Alzheimers Disease/Parkinsonss Disease and Related Disorders: Clinical and Etiological Aspects: 37. Different Cognitive Profiles on Memory Tests in Parkinsons Disease and Alzheimers Disease R.F. Allegri, et al. Parkinsons Disease--Etiology, Genetics, Treatment: 56. Genetic Aspects of Parkinsons Disease Y. Mizuno, et al. Cholinergic Strategies in Alzheimers and Parkinsons Disease: 64. The Rationale for Development of Cholinergic Therapies in AD A. Enz, P.T. Francis. Alzheimers Disease--Muscarinic Treatment and Therapy--beta-Amyloids: 70. APP Localization and Trafficking in the Central Nervouse System J.D. Buxbaum, et al. Cholinesterase Inhibitors for Treatment of Alzheimers Disease: 75. Crystallographic Studies on Complexes of Acetylcholinesterase with the Natural Cholinesterase Inhibitors Fasciculin and Huperzine A I. Silman, et al. Neurotrophic Factor Therapy--Neuroprotection, Neurotransplantation and Other Treatment Strategies: 86. Functional Effects of GDNF on Dopamine Neurons in Animal Models of Parkinsons Disease A.F. Hoffman, et al. Animal Models in Alzheimers and Parkinsons Diseases: 93. Age Dependence of Muscarinic Plasticity in the Rat Hippocampus J.M. Auerbach, M. Segal. Diagnostic Too

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.

AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase

Abstract: Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuronspecific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma × glioma cell line, NG‐108‐15, with 100 μM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60–85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration‐ and time‐dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl‐coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64As capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.

Amitriptyline plasma levels and clinical response in primary depression.

Sixteen patients with primary depression were treated for 4 wk with amitriptyline. After clinical diagnoses were determined, patients entered a double‐blind protocol (amitriptyline or placebo) and their clinical status was determined with the Hamilton Depression Rating Scale by raters blind to the drug type, its dosage and plasma levels. Amitriptyline (AT) and nortriptyline (NT) plasma levels were assayed twice weekly by gas chromatography‐mass spectrometry. In the 16 patients, a negative correlation between the Hamilton Score and the mean total tricyclic level (p < 0.01), as well as with individual plasma levels, was found at the end of the treatment period. When the group was divided into clinical responders and nonresponders, the mean total tricyclic (AT + NT) levels discriminated the two groups by day 12 (p < 0.001) as well as at the end of the protocol (day 26, 88% of the patients were classified correctly if an arbitrary level of200 ng/ml total tricyclic plasma level was chosen). These results strongly suggest the presence of a positive correlation between plasma levels and clinical improvement in patients with primary depression.

Pre-treatment neurotransmitter metabolites and response to imipramine or amitriptyline treatment

Preliminary data are presented from the NIMH Collaborative Study on the psychobiology of depression, biological studies, dealing with relationships between the pre-treatment levels of the neurotransmitter metabolites 3-methoxy-4-hydrophenethyleneglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) and the subsequent therapeutic response of depressed patients to imipramine or amitriptyline. Eighty-seven depressed patients were studied during pre-treatment and treatment periods. It has been found that (1) both low pre-treatment urinary MHPG and low CSF 5-HIAA values are associated with a response to imipramine; these relationships were not artefacts due to sex or age; (2) there were no significant relationships between pre-treatment urinary MHPG, CSF MHPG, 5-HIAA, or HVA values and the subsequent response, or failure of response, to amitriptyline; (3) there was not a bimodal distribution for CSF 5-HIAA. For both males and females, there were positive and statistically significant correlations between CSF MHPG and urinary MHPG; for the females, there were positive and significant correlations between both urinary and CSF MHPG and CSF 5-HIAA. The theoretical and practical implications of these findings are discussed.

Cholinergic lesion of the striatum impairs acquisition and retention of a passive avoidance response.

Significant impairments in the acquisition and retention of a step-down passive avoidance task were found in rats with striatal lesions induced by the cholinergic neurotoxin AF64A. No significant differences between control and AF64A-injected rats were found in sensitivity to electric shock or in various measures of spontaneous locomotor activity. Striatal choline acetyltransferase (CAT) activity was significantly decreased in AF64A-treated rats compared with controls, whereas glutamic acid decarboxylase (GAD) activities were not. Furthermore, there were no significant differences between groups in activities of CAT and GAD in either the cortex or the hippocampus, results that support the specificity of the lesion to the striatum. The passive avoidance deficits found in these rats after intrastriatal injection of AF64A support a role for the striatal cholinergic system in complex behavioral processes.

Analysis of microquantities of choline and its esters utilizing gas chromatography-chemical ionization mass spectrometry

Gas chromatography-chemical ionization mass spectrometry has been applied successfully in the analysis of choline and its esters. This approach serves to extend further the potential of existing gas chromatographic procedures which are capable of the microestimation of choline esters following their N-demethylation by either chemical or physical means. Typical fragmentation patterns with ions at me = 72 and me = (M + 1) were obtained for each choline ester derivative. When methane was used as the reactant gas, the above fragments were approximately of equal abundance for each ester. Use of isobutane as reactant gas yielded almost 80% of the (M + 1) fragment, and only approximately 5% of the fragment ion at me = 72. Recovery of all fragments was linear for nondeuterated as well as deuterated analogs of choline ester derivatives. Recovery, as evident from the analysis of records of relative ratios of injected isotopic variants of these esters, indicated that this analysis of choline esters using chemical ionization mass spectrometry coupled with gas chromatography is quantitative and highly reproducible.