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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.

Topical cortical injection into rats of antiserum to the synaptic membrane fraction caused recurrent spiking activity as observed in the EEG; neither antiserum to S-100 protein nor antiserum to myelin caused any such abnormality. These same rats, when observed on a maze learning task (Lashley III) responded differently to the different antisera. Rats receiving injections of antiserum to the synaptic membrane fraction or antiserum to S-100 protein were inhibited in their performance, while rats injected with antiserum to myelin behaved like uninjected animals.

Two-month-old male offspring of rats injected intravenously on day 19 of gestation with antiserum to synaptic membrane fraction showed marked behavioral deficits on a DRL (differential reinforcement at low rates) training paradigm. These behavioral deficits included perseveration, slow acquisition rates, and poor retention. Offspring of pregnant rats injected with antiserum to galactocerebroside or isotonic saline did not show these behavioral effects.

Five-day-old rats received a single injection (50 microliter) of antiserum to ganglioside into the cisterna magna and were compared to control animals injected with the antiserum which had been absorbed with pure GM1 ganglioside to remove the specific antibodies. Both groups showed normal rates of body growth. However, animals receiving antiganglioside serum had, at 60 days of age, impaired performance when tested on a complex learning task (DRL) as well as chemical and morphological alterations in the somatosensory cerebral cortex. Gross morphology and wet weight of whole brain were normal in both groups. Microchemical analysis of somatosensory isocortex revealed a normal content of total solids, protein, and DNA. However, ganglioside sialic acid, galactocerebroside, and RNA were decreased by 31%, 32% and 25% of control values, respectively (P less than 0.01). Quantitative measurements of oblique dendrites of Golgi-stained cortical pyramidal neurons revealed a 31% decrease in the number of spines. Additionally, the majority of spines were of the stubby configuration, whereas dendrites from controls were populated predominately by thin spines. These observations suggest that antibodies to GM1 ganglioside interfere with optimal neonatal development on both dendrites and myelin in cerebral cortex. The results provide an animal model in which an immunologically-mediated disturbance of cortical development is associated with chronic behavioral impairment.

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.

The monosialoganglioside GM1 can potentiate the neuritogenic activity of media conditioned by several cell types: neonatal glia, C6 glioma, embryonic chick heart or skeletal muscle and the rat myogenic line L6. To probe further the neuritogenic activity of conditioned media (CM), 5 mouse monoclonal antibodies (mAbs) against GM1, designated B6, C3, C4h2, D1 and D3 were incorporated individually into nutrient medium (NM) supplemented with CM prior to incubation with sensory ganglia. Nine-day embryonic chick dorsal root ganglia were explanted onto collagen-coated coverslips and incubated at 35 degrees C for 5 h in NM supplemented with 150 micrograms/ml GM1. After washing with NM, the explants were re-fed with NM + CM containing 20% mAb and cultured for an additional 43 h. The resultant neuritogenesis was evaluated microscopically by determining mean neurite number and length of randomly mixed cultures. The 5 antibodies differed in their capacities to inhibit CM-mediated neuritogenesis of these primed target cells. D1 and D3 were most effective in reducing neurite length and number produced by all sources of the CM, while C3 and C4h2 were intermediate in their inhibition of neurite initiation (number). The effect of B6 on neurite initiation and elongation was the least. The ability of these mAbs to inhibit neuritogenic activity of CM derived from both glial and myogenic tissue suggests that gangliosides play a basic role in neuronal development. The differing responses elicited by the individual mAbs may reflect a relationship between the structural complexity of the GM1 molecule and the neuritogenic mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)