Sahebarao P. Mahadik

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.

Acute effects of GM1 ganglioside: reduction in both behavioral asymmetry and loss of Na+, K+-ATPase after nigrostriatal transection.

GM1 ganglioside injections (i.p.) reduce amphetamine-induced asymmetric rotation in rats 48 h after a partial unilateral transection of the nigrostriatal pathway. We found that this reduction was maximal when rats received their first GM1 injection within 2 h after surgery. Rats injected 4-12 h after surgery, or rats only pretreated with GM1, showed no significant effect on rotation. Striatal membrane Na+,K+-ATPase in rats injected with GM1 0-2 h after hemitransection showed only a 10% loss in activity (versus the untransected hemisphere) as compared to control losses of 38%. The maintenance of membrane Na+,K+-ATPase activity in GM1-treated rats may be one mechanism by which a balance between hemispheres in striatal dopaminergic transmission is preserved, resulting in reduced asymmetric rotation. The observation that there is a critical postsurgical period when GM1 administration results in optimal functional recovery supports our hypothesis that gangliosides are exerting an acute effect on damaged CNS tissue. This acute effect is further evidenced by the reduced loss of membrane Na+,K+-ATPase following injury.

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.

An Immunological Model of Epilepsy: Seizures Induced by Antibodies to GM1 Ganglioside

Summary: Following observations that the intracerebral injection into rats of antiserum to brain gangliosides resulted in recurrent epileptiform activity and that seizure activity was not seen if antibodies were removed by absorption of the antiserum with pure GM1 ganglioside, a study was undertaken to establish characteristics of the immunological agents used to produce this model of epilepsy. It was determined that the potencies (antibody titers with GM1ganglioside) of antiganglioside sera can be correlated with the intensities of epileptiform activity they induce; that immunoglobulin fractions from antiganglioside sera are even more effective biologically than the antisera; and that antibodies to GM1 ganglioside purified by affinity chromatography can also induce recurrent epileptiform discharges but are not as effective as either native antiserum or immunoglobulin fractions.

Abnormal growth of skin fibroblasts from schizophrenic patients

Fibroblast cultures were established from skin biopsies of 18 schizophrenic patients (14 on drug and 4 off drug) and 13 normal subjects, and growth parameters (initial growth and rate of growth) and morphology were studied. Fibroblasts from patients took significantly longer to grow than did normal fibroblasts. Cell lines were established within 2 weeks for all normal controls, but for only 6 (33%) of 18 schizophrenic patients. The rate of growth (doubling time) was also significantly longer for fibroblasts from patients than from normals. Neither time to establishment of initial growth nor doubling time was related to age, sex, age at onset, duration of illness, or medication status in the patients. Fibroblasts from normals showed uniform, long (slender), characteristic spindle-like, bipolar appearance, with unidirectional orientation, both while growing from explant as well as after subculturing. By contrast, fibroblasts from patients generally showed random size (shorter and flatter), mostly spiny, multipolar cells with short stubby projections, and an irregular orientation resulting in a criss-cross pattern, and often exhibited poor attachment. Fibroblasts from skin biopsies of patients who were drug free at the time of biopsy showed similar initial growth, doubling time, and morphology to those from patients who were receiving neuroleptic treatment. In vitro challenge of skin biopsies of normal subjects with haloperidol in culture resulted in slight delay in initial growth and marginal increase in doubling time. However, the morphology remained normal. Possible molecular mechanisms that may be associated with abnormal growth of fibroblasts in schizophrenia are discussed.

Gangliosides Accelerate Rat Neonatal Learning and Levels of Cortical Acetylcholinesterases

Several studies have shown that exogenous gangliosides can stimulate neurite outgrowth (in vitro), accelerate peripheral nerve regeneration (in vivo) and facilitate CNS recovery after lesioning. Experiments were designed to assess the effects of ganglioside administration on neonatal development. Rat neonates received daily subcutaneous injections of gangliosides from PN day 5 through 15. Learning behavior (acquisition and retention) was facilitated in rats which had received injections of either total, GM1 or GD1b ganglioside. Rats injected with GD1a and GT1b ganglioside were not different from controls. Levels of total AChE activity as well as its 4s and 10s molecular forms in cortex, were assayed at PN days 9, 14, 21 and 28 in rats injected with GM1 ganglioside. These animals had consistently higher levels of enzyme activity as compared to saline controls. It is hypothesized that exogenous gangliosides accelerate CNS maturation.

Ganglioside treatment: Reduction of CNS injury and facilitation of functional recovery

Increased attention has focused on the use of brain gangliosides as a treatment for brain injury. This review traces the progression of in vitro and in vivo research which led to studies which have demonstrated that ganglioside treatment can facilitate recovery after CNS damage in animal models (for example, lesions, transections, hypoxia, ischemia). Hypotheses regarding the ability of gangliosides to stimulate neuronal plasticity, modulate trophic factors and reduce injury processes are presented.

Acute Effects of Gangliosides on CNS Injury

Recently investigators have focused their research efforts on exogenous ganglioside effects on processes associated with CNS injury. These CNS studies were prompted by reports that ganglioside-treatments enhance peripheral nerve regeneration after damage (Gorio et al., 1981; 1983), and that when gangliosides are added to some cell or tissue cultures, increased neurite outgrowth occurs (Ferrari et al., 1983; Leon et al., 1982; Roisen et al., 1981).

Topographic studies of glycoproteins of intact synaptosomes from rat brain cortex

Glycoproteins in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups by galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components were fractionated on diatrizoate to give four synaptosomal membrane fractions (A to D) and a mitochondrial pellet (E). Fractions A and B represent highly purified synaptosomal plasma membranes. After separation of their polypeptides by electrophoresis, 4/5 of the label was present in two bands: one about 72 000 and the other between 7800 and 3200 daltons. Seven other bands were labeled to various degrees: 160 000, 96 000, 53 000, 39 000, 34 000, 23 000 and 16 000 daltons. With isolated membranes (which incorporate 5--6 times more label) 4/5 of label was present in polypeptides in three ranges: 160 000--96 000, 70 000--40 000 and 7800--3200. The number of polypeptides that can be labeled by treatment of isolated membranes is very large. In comparison, glycoproteins whose topographical distribution permits interaction with large molecules at the synpatic surface are very limited. It is further suggested that the external synaptosome membrane involves a relatively tight network of interacting molecules that cannot be readily penetrated by large molecules.

HETEROGENEITY OF S‐100 PROTEINS OF BRAIN: SUBUNIT COMPOSITIONS

Abstract— Two populations of S‐100 proteins (III‐IVa‐1) and (III‐IVb‐1) were isolated from bovine brain by a simple three‐step procedure involving precipitation with ammonium sulfate, ion exchange chromatography and gel electrophoresis. These two fractions reacted with anti‐S‐100 serum and showed all the properties of S‐100 protein, but differed in the distribution of protein among the four subunits obtained in SDS‐gels. These subunits had apparent molecular weights of 7800, 6800, 6100, and 5200. Separations based on charge differences revealed only three subunits. Similar fractions isolated from rat brain contained only two subunits with apparent molecular weights of 7800 and 6500. The two S‐100 protein fractions also differed in the specific extinction at 280 nm and in the protein distribution among the separate bands which formed either in a 14% acrylamide‐agarose gel‐discontinuous buffer system or in 7.5% gels in the presence of calcium. The yield of S‐100 protein isolated in the presence of aids (EDTA, EGTA, mercaptoethanol, protease inhibitors) was considerably higher than in their absence (80 mg vs 60 mg/kg tissue), the predominant loss occurring in fraction III‐IVa‐1. It is concluded that S‐100 protein’contains a number of molecular species which have different subunit compositions and differences in stability.