Heljo Laev

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.

GM1 ganglioside reduces glutamate toxicity to cortical cells. Lowered LDH release and preserved membrane integrity.

As an in vitro model of CNS excitatory amino acid (EAA) injury, rat cortical neuronal cultures were challenged with glutamate (0.5 or 10 mM) and the levels of released lactate dehydrogenase (LDH) were monitored at 1 h, 1, 2, and 7 d. LDH release is correlated with levels of plasma membrane damage. GM1 has been shown to be continuously distributed on the outer surface of CNS cellular membranes. By staining for the distribution of endogenous GM1 ganglioside using cholera toxin/antitoxin immunohistochemistry, we were able to assess morphologically cellular plasma membrane integrity after damage. We used these two measures (LDH and GM1 localization) to study the neuroprotective effects of exogenous GM1 ganglioside to further elucidate its mechanism. Cortical cultures derived from 15-d rat fetuses were subjected to the glutamate challenge for 30 min. Parallel cultures were either pre- or post-treated with 80 microM of GM1. Exposure to 10 mM glutamate caused a highly significant increase in LDH release at 1-48 h. Pretreatment with GM1 reduced the release, whereas posttreatment reduced the LDH release even more. Plasma membrane changes observed by the GM1 immunohistochemistry reflected the LDH release data. All cultures treated with GM1 evidenced substantial structural integrity (continuous staining of GM1 along perikarya and processes) as compared to untreated cultures. These data support our hypothesis that GM1 treatment (pre- and post-) reduces plasma membrane damage.

Preparation and specificity of 11 monoclonal antibodies to GM1 ganglioside.

Abstract Eleven monoclonal antibodies to GM1 ganglioside were prepared from hybridoma clones obtained by fusion of spleen cells from mice immunized with GM1 with mouse myeloma cells. When the reactivities of these 11 monoclonal antibodies were determined by enzyme‐linked immunosorbent assay with six glycosphingolipids (GM1, GD1a, GD1b, GT1b, GM2, and asialo‐GM1), they showed different degrees of specificity. From their reactivity patterns, they could be divided into three groups: Group 1, those that react only with GM1 (C3 and D3); Group 2, those that react predominantly with GM1 (C6, B6, D1, e1, g1, g9, and e12); and Group 3, those that show poor discrimination (h2 and A4). The clones differed in their biological activities.

Topography of monosialoganglioside (GM1) in rat brain using monoclonal antibodies

Immunohistochemical localization of ganglioside GM1 using 3 monoclonal antibodies (C3 and D3 reacting exclusively with GM1 and C4h2 reacting also with other gangliosides) showed different staining patterns in rat brain regions (cerebellum, cerebral cortex and hippocampus). Staining in all brain areas was punctate and appeared to be restricted to surfaces of cells and their processes. In spite of similar reactivity to GM1, C3 and D3 showed qualitatively and quantitatively different and highly selective localization in all regions with no staining in white matter. In cerebellum, staining with C3 was predominantly associated with granular layer; staining with D3 was limited to Purkinje cell surfaces and surrounding structures. In cortex, staining with C3 was seen outlining large pyramidal neurons and fibers in cross-section, whereas sites stained with D3 appeared to be fewer, smaller and differed in location. In hippocampus, staining patterns were similar with both C3 and D3, outlining large pyramidal neurons of Ammons horn and granular cells of dentate gyrus as well as glomerular structures. Staining with C4h2 was topographically similar, but over 10-fold more extensive and was present in white matter.

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 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.

Cortical cell plasma membrane alterations after in vitro alcohol exposure: Prevention by GM1 ganglioside

Using choleratoxin/antitoxin immunohistochemistry, this study examined the effects of in vitro alcohol exposure on the morphology of cell plasma membranes in mixed fetal rat cortical cultures, and assessed the neuroprotective effects of exogenous monosialoganglioside (GM1). Gangliosides are involved in critical biological functions, including maintenance of membrane integrity. Plasma membranes are directly affected by alcohol exposure through multiple mechanisms. Results indicate that exposure to alcohol altered plasma membrane morphology as assessed by staining for the surface distribution of membrane GM1. Pretreatment with endogenous GM1 ameliorated the alcohol-induced alterations.

Traumatic injury of spinal cord cells in vitro reduced by GM1 ganglioside.

GM1 ganglioside (monosialoganglioside) is a significant endogenous component of central nervous system (CNS) cellular membranes, thereby contributing to the membranes integrity and function. Exogenous gangliosides have been shown to be incorporated into plasma membranes and can exert neuroprotective effects on damaged neuronal tissue(s). An in vitro method of physical injury (trauma) previously described which used cultures derived from fetal mouse spinal cord [38] was adapted for these studies in order for us to assess GMls neuroprotective efficacy. Injury was induced by uniformly crosshatching the spinal cell cultures with a 1 mm plastic pipette tip. The extent of injury and the effects of GM1 ganglioside posttreatment (80 μM) was assessed after 48 h by measuring lactate dehydrogenase (LDH) released and by observing changes in the plasma membrane surface distribution of endogenous GM1 using cholera toxin/antitoxin/fluorescent antibody immunohistochemistry. A gradient of injury, from the zone of maximum injury to partially traumatized or non-injured areas, was seen using immunohistochemistry. The primary injury zone in this gradient was characterized by areas of swollen or dead cells and abnormal or degenerating cell processes. At further distances, cells were observed to be nearly normal, with intact fibers. This gradient of injury may reflect proximate (at the locus of trauma) and distant effects of the release of neurotoxic levels of endogenous glutamate (Glu) and other excitatory amino acids. Ganglioside GM1 treatment resulted in a significantly reduced (>75%) release of LDH as well as enhanced cell and process integrity indicative of reduced tissue injury. These initial results indicate that GMls previously documented neuroprotective effects using neuronal culture systems can be generalized to injured spinal cells in vitro wherein there is evidence for preservation (rescue) of cellular plasma membranes after injury as reflected by reduced cell loss, swelling, and process degeneration, as well as decreased LDH release.