Anna Korenovsky

Haloperidol alters rat CNS cholinergic system: Enzymatic and morphological analyses

Chemical and morphological changes in cholinergic marker enzymes, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT) of striatum, hippocampus, and cerebral cortex were studied following haloperidol treatment of rats. After short-term (7-21 days) haloperidol treatment, the levels of both enzymes (AChE and ChAT) were increased in striatum and hippocampus (greater than 25%), but not in cortex. After long-term (+40 days) haloperidol treatment, the level of AChE activity returned to control levels in all brain areas, whereas the levels of striatal and hippocampal ChAT decreased by 26% and 29%, respectively. No change in levels of both enzymes was detected after acute treatment (single dose) of haloperidol or chronic treatment with either clozapine or imipramine. Morphological analysis of cholinergic neurons and their processes using monoclonal antibody to ChAT showed two types of changes following 40 days of haloperidol treatment. First, parallel to the observed decrease in the levels of ChAT activity there was a visual decrease in the immunoreactivity in neurons as well as in their processes in striatum and hippocampus. Second, there was an apparent reduction in the size and number of stained neurons and their processes. No changes were seen in immunoreactivity after an acute treatment with haloperidol. These results indicate that the chronic haloperidol treatment in rats causes changes in central cholinergic systems that may be relevant to the pathophysiology of schizophrenia and its treatment.

Slab gel analysis of the polypeptide components of rat brain subcellular organelles.

Abstract Electrophoresis on polyacrylamide slab gels containing SDS with a discontinuous buffer ( Laemmli (1970) Nature 227 , 680–685) has been modified for analysis of the polypeptides in subcellular fractions of rat brain cortex, with attention to sample preparation (delipidation, ratio of SDS to protein, presence of EDTA and mercaptoethanol, storage under N 2 , pH), electrophoresis conditions, and staining technique. With these modifications, subcellular fractions (nuclei, mitochondria, microsomes, synaptic membranes) gave reproducible electrophoretograms having sharp, well-resolved bands, On 10% acrylamide slab gels, over 50 polypeptide bands were resolved and, on 9% to 25% gradient slabs, over 70: ranging in molecular weight from 200.000. Banding patterns showed substantial differences among subcellular fractions, including microsomes and synaptic membranes. Gradlent slab-gel electrophoresis has sufficient sensitivity and resolving power to be the method of choice for comparing subcellular fractions prepared by different procedures and for establishing new criteria of purity.

Serum antibodies to nicotinic acetylcholine receptors in schizophrenic patients

Although elevated serum levels of antibodies to the nicotinic acetylcholine receptor (nAChR) have been reported in neuroleptic treated patients with tardive dyskinesia, such antibodies have not been determined in comparable nondyskinetic patients. Using a toxin-binding inhibition assay, we examined serum anti-nAChR antibody levels in 17 DSM-III-R chronic schizophrenic patients, seven of whom had persistent tardive dyskinesia, and 10 normal controls. On the average, anti-nAChR antibody levels were significantly higher in schizophrenic patients than in normal controls, but but not differ between patients with and without tardive dyskinesia and was not related to age, sex, or duration of illness in patients.

IMMUNOCHEMICAL PROPERTIES OF S-100 PROTEINS AND THEIR SUBUNITS

Abstract— The immunological activities of two populations of bovine S‐100 proteins with anti‐S‐100 serum were studied by complement fixation and rocket immunoelectrophoresis. The reactivities of subunits of these two populations were studied by crossed immunoelectrophoresis and rocket immunoelectrophoresis. Although the two populations conformed in all respects to the properties of S‐100 protein, the immunological reactivity of one, III‐IVa‐1, was significantly lower than that of the other, III‐IVb‐1. The difference was much larger when the S‐100 protein fractions were isolated in the absence of aids (mercaptoethanol, EDTA, EGTA, protease inhibitors). With bovine S‐100 fractions, the three subunits separated by differences in charge as well as the four subunits separated by differences in molecular weight all reacted with the same antibody molecules in the antiserum. The reactivities of the subunits showed large quantitative differences.

Quantitative analyses of plasma cholinesterase isozymes in haloperidol-treated rats

We describe a quantitative slab gel electrophoresis procedure that allows quantitative determination of plasma levels of discrete cholinesterase isozymes. Using this method, the effects of haloperidol treatment on plasma cholinesterase isozyme levels were examined in normal rats. Eight isozymes were detected by enzymatic reaction with either of two substrates (alpha-naphthyl acetate, NA; acetylthiocholine iodide, AcTCh), and then quantified using densitometric scanning. With AcTCh substrate, the activities of two major isozymes (1 and 2) were found to be linear with increasing quantities of applied plasma. With NA as substrate, Iso-OMPA (a pseudocholinesterase inhibitor) inhibited activities of all isozymes, except isozymes 2 and 8. With either substrate, BW284C51 (acetylcholinesterase inhibitor) inhibited 100% and 13% of activity of isozymes 2 and 8, respectively, and with AcTCh substrate, 37% of isozyme 1. Based on the differential patterns of substrate specificity and action of inhibitors, and the reproducibility of patterns, we propose that these isozymes represent distinct molecular species. Short-term (14 days) and long-term (45 days) haloperidol treatment both resulted in altered levels of specific cholinesterase (ChE) isozymes. On the average, with AcTCh substrate, haloperidol treatment increased levels of isozymes 1 and 2 by 30% and 8%, respectively, after 14 days, and by 50% and 30%, respectively, after 45 days. Isozymes 3 through 8 showed minor changes. Plasma levels of isozymes 1 and 2 returned to baseline pretreatment values after a 40-day drug-free period. No significant change was observed after either short- or long-term treatment with clozapine, imipramine, or saline, or after an acute (less than 5 days) haloperidol treatment. No change was noted in RBC-ChE levels as function of treatment. These findings indicate that, in the rat, chronic haloperidol treatment results in differential changes in the plasma levels of discrete ChE isozymes. We have suggested that these changes reflect an alteration of central dopaminergic-cholinergic balance.

Synaptic membrane antigens: detection and characterization.

Analysis of the antigenic components of synaptic membranes presents several problems caused by detergents required to solubilize the proteins. These problems involve detection of the reaction between antibodies and proteins in the detergent extract as well as characterization of the individual antigens after further purification. We found a satisfactory solution for analysis of rat synaptic membranes by using rocket immunoelectrophoresis with an intermediate gel containing 0.5% berol to sequester sodium dodecyl sulfate (SDS) and an antibody gel containing 0.1% berol to maintain the solubility of the proteins. With antiserum prepared against a highly purified synaptic membrane fraction, these conditions gave rocket heights proportional to protein concentration (for the major rocket) and showed no reactions with membrane extracts of liver, kidney and spleen. By separation on gradient slab gels containing SDS, 4 antigenic polypeptides were found in Berol extracts of synaptic membrane, having apparent molecular sizes of 56,000, 58,000, 62,000 and 66,000 daltons. The SDS extract of the berol insoluble residue contained 3 antigenic polypeptides (12,500; 15,000, 16,500 daltons). The combined use of gradient slab gel electrophoresis in SDS and rocket immunoelectrophoresis with an intermediate gel to remove SDS provides a sensitive and rapid method for definitive identification of individual antigenic components in detergent extracts of membrane and for further purification of these antigens.

Synaptic Membrane Antigens in Developing Rat Brain Cerebral Cortex and Cerebellum

Abstract: The contents of five synaptic membrane antigens (56K, 58K, 62K, 63K, and 64K) were determined in rat cerebral cortex and cerebellum at eight developmental time points: E9, E14, P < 1, P5, P14, P28, P60, and P180 (E, embryonic; P, postnatal). In cerebral cortex, the five antigens showed five different developmental patterns with respect both to specific content (i.e., quantity per unit of membrane) and total content (i.e., quantity per cortex). The 56K, 58K, and 62K polypeptides were first detected at E14, increased slightly to P5, then increased rapidly from P5 to P28 by 14‐, 11‐, and 18‐fold, respectively. From P28 to PI80, the patterns of these antigens showed very large differences. The 63K and 64K antigens were first detected at P14 and P28, respectively. The specific content of 63K antigen continued to increase steadily throughout adult life; in contrast, the specific content of the 64K antigen did not change appreciably. In cerebellum only three antigens (56K, 58K, and 62K) were detected. These three antigens showed different developmental patterns. The 56K polypeptide was first detected at E14; its specific content increased very rapidly to a maximum at P < 1; it then decreased, first slowly, and then more rapidly, disappearing at P60. The 58K polypeptide also was detectable at E14 and increased very rapidly to a maximum at P < 1. It then decreased markedly to P5, followed by an increase, returning almost to its maximum level at P14. It then slowly decreased disappearing at P180. The 62K antigen was first detected at P14 and then it slowly decreased with disappearance at P60. The patterns with respect to total contents per cerebellum were similar for the three antigens, with a maximum at P28. We conclude that the highest increase in the contents of these antigens roughly corresponds to the period of maximal synaptogenesis (P9 to P28) in both regions. Differences among developmental patterns probably reflect changing molecular machinery required for development and functional differentiation of synapses in different brain regions. The fine structure of these patterns suggests that the quantitative measurement of synaptic membrane antigens will be useful for delineating complex processes occurring during synaptogenesis.

Plasma cholinesterase isozymes and REM latency in schizophrenia

The relation between electroencephalographic sleep parameters and plasma cholinesterase isozymes was examined in a group of 19 unmedicated schizophrenic patients. Rapid eye movement (REM) latency was found to be significantly inversely correlated with isozyme 3 (mainly acetylcholinesterase). The results are discussed in relation to cholinergic involvement in the regulation of REM sleep and in the pathophysiology of schizophrenia.

Distribution of Six Synaptic Membrane Antigens in Subcellular Fractions of Rat Brain Cortex

Abstract: The subcellular distribution in rat brain cortex of six synaptic membrane antigens (56K, 58K, 62K, 63K, 64K, 66K) was studied by rocket immunoelectrophoresis, using antiserum to a highly purified synaptic plasma membrane fraction. Initial analysis of the insoluble portion of subcellular fractions showed that these antigens were also present in smooth microsomes, rough microsomes, and synaptic vesicles; that only traces were present in synaptic junctions; and that none was present in nuclei, mitochondria, and myelin. A trace amount of activity was also present in synaptic vesicle cytosol, but none in whole brain cytosol. Quantitative measurements of synaptic plasma membranes, smooth microsomes, and synaptic vesicles showed that all six antigens were present in synaptic plasma membranes and smooth microsomes, but that the 66K antigen was absent from synaptic vesicles. The 56K, 58K, 62K, 63K, and 64K antigens were present in highest concentration in synaptic plasma membranes, whereas the 66K antigen content was highest in smooth microsomes. Only the 58K, 62K, and 63K antigens were detectable in the membrane fraction of whole brain. Their enrichments in synaptic plasma membranes were 10.9, 5.4, and 5.9, respectively. We conclude that the 56K, 58K, 62K, 63K and 64K antigens are primary components of synaptic plasma membranes. The presence of synaptic plasma membrane antigens in smooth microsomes and synaptic vesicles probably represents material being actively transported, consistent with the hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are transported via smooth endoplasmic reticulum.