Perturbation of Behavior and Other CNS Functions by Antibodies to

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Perturbation of Behavior and Other CNS Functions by Antibodies to Ganglioside MAURICE M. RAPPORT, STEPHEN E. KARPIAK, and SAHEBARAO P. MAHADIK Departments of Psychiatry and Biochemistry, Columbia University, College of Physicians and Surgeons, New York, NY 10032

From the viewpoint of the chemist, the brain presents an a l most limitless frontier.

The brain, as a center for communica-

tion control, has been shown by anatomists and physiologists to be composed of a network of neurons that make contact with one another mostly by release of chemicals at synaptic junctions (neurotransmission).

There are astronomical numbers of these

synaptic junctions, and there is also a complex array of chemical transmitters and chemical modulators involved in neurotransmission. Many of these transmitters and modulators have not yet been identified.

The physiological actions of these substances are diverse

(they both excite and depress activity) so we must also postulate that many different molecular structures are involved in receptor functions even for the very same transmitter or modulator. In this extensive array of synaptic connections lie the mechanisms for plastic adaptations of the brain to the external environment -- modifications that subserve the processes of sensory reception, memory and learning, emotional responses, and abstract thought.

A major task of neurochemists is to sort out molecules

participating in the myriad synaptic connections and to identify them. Antibodies As A Bridge Between Structure And Function Methods of separating and characterizing molecules have developed rapidly in the past twenty years and new ones are appear-

0-8412-0556-6/ 80/ 47-128-40755.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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ing with regularity. Among these, Immunological methods are unique in representing relatively mature methods that s t i l l retain a large measure of unexploited potential. The specificity of antibodies for both large and small chemical structures has been well established during the last 30 to 40 years, and has proven of Inestimable value, especially in the field of endocrinology. However, the application of immunological techniques to study synaptic differences is s t i l l in Its infancy. Our laboratory has been addressing Itself for more than a decade to developing an immunological bridge that will make a connection between specific chemical structures in the synapse and various CNS functions. The basis for this effort lies in earlier demonstrations that antibodies or antisera can serve as Interventive agents that will perturb CNS functions — for example by inducing alterations in the EE6 or inhibiting performance on various tasks (]_,£,3). We have studied the interventive action of antibodies against an array of different antigens using EEG as well as a number of behavioral paradigms (4,5,6) and have demonstrated to our own satisfaction that an encouraging degree of specificity is associated with the actions of these different types of antibodies. For example, we found that functional alterations were Induced with antisera to gangliosides, to S-100 protein (a brain specific protein found mainly in glial c e l l s ) , and to synaptic membranes. No such effects were seen with antisera to galactocerebroside, to 14-3-2 protein (a neuron-specif1c protein), or to erythrocyte membranes. Furthermore, the antibodies to G ganglioside following intracortical Injection induced EEG spiking (7) and inhibited learning (8,9) whereas antiserum to S-100 protein inhibited learning but did not alter the EEG (10). If we accept this evidence that passive administration of antibodies is capable of at least some degree of discrimination among different CNS functions, the specificity of antibodies for molecular structure provides a bridge between structure and function. M1

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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G M I Ganglioside As A Synaptic Target For Antibodies We will make one further assumption in order to reduce the area of investigation to reasonable proportions. We will assume that synaptic contacts are the site of action of these passively transferred antibodies that are able to disrupt CNS functions. Therefore molecules that are directly involved in such contacts and are accessible to the extracellular space (synaptic cleft) become priority targets of our efforts. G^ ganglioside f i t s the category well. It Is a small stable molecule whose chemistry is well-established. It can be prepared in workable quantities by reproducible procedures, and criteria of purity are available and readily met. Gangliosides are present in substantial amounts in synaptic membranes,and the ganglioside molecule is accessible to the synaptic cleft as we have shown by labeling intact synaptosomes using enzymic oxidation (galactose oxidase) followed by reduction with tritium-labelled borohydride (]i). However, available knowledge suggests that gangliosides are present in a l l synaptic connections, and i f the hypothesis is correct that disturbances of CNS functions by antibodies result from perturbation of synaptic contacts, we might then expect that all CNS functions would be susceptible to disturbance by these antiganglioside antibodies. If this were true, i t would limit the usefulness of these antibodies as an interventive agent. Ant i ganglioside serum would s t i l l offer two major advantages stemming from 1) the relative ease and reproducibility of the methods for preparing i t and characterizing its antibody content (titer) and specificity and 2) its provision of a rigorous control reagent 1n the form of the antiserum from which antibodies to G M I are absorbed (removed) with pure Gm ganglioside. Antibodies To Ganglioside Interfere With CNS Functions Selectively We have now subjected rats to a number of behavioral tests in which we could show inhibition of learning by small quantities of antiganglioside serum and no inhibition by the absorbed serum.



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Among these may be listed inhibition of passive avoidance learning (9), inhibition of morphine analgesia (12) and blockade of sedation induced by reserpine (unpublished data). Schupf and Williams (13) have added blockade of cholinergic stimulation of drinking. However, and quite unexpectedly, we found the antiganglioslde reagent was not effective in a number of other tests, such as pattern discrimination, fixed-ratio conditioning, selfstimulation, pain threshold and activity levels (all unpublished results), and in the experiments of Schupf and Williams, eating and drinking (personal communication). These results, summarized in Table I, Indicate that despite the widespread distribution of M1 ganglioside in the brain, one cannot yet predict whether a particular behavior will or will not be affected by administration of antibodies to ganglioside. One infers from these results that G M I ganglioside receptors may provide a chemical basis for discriminating among different behaviors. If this proves to be true for G^n ganglioside, i t may also be true for other ganglioside species as well as for other molecules in synaptic membranes that can serve as "receptors" for antibody ligands. G

Mechanisms By Which Antibodies May Perturb CNS Functions How might one account for this discriminatory capability? One explanation might be found in the differences in topography of "antigenic receptors" in different synaptic contacts, differences both in the number of receptors and in their distribution. Another explanation might be found in differences among synaptic contacts with respect to the type of membrane process that is a l tered by the binding of antibody molecules. The number of such processes is substantial and continues to grow. If we consider that the binding of an antibody ligand to an antigenic site in the membrane may alter membrane conformation and/or membrane fluidity, then as a consequence of such alterations a number of properties of the membrane may change including its permeability to ions, its enzyme activities, and the distribution


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TABLE I BIOLOGICAL EFFECTIVENESS OF ANTIGANGLIOSIDE SERUM EFFECTIVE

INEFFECTIVE

Injection Site Test Procedure Test Procedure Injection Site cortex: sensorihypothalamus l.EEG seizures l.EEG seizures motor, frontal, visual; hippocampus; amygdala visual cortex 2.Inhibition of 2.Pattern i.vc. lateral learning discrimigeniculate (passive nation avoidance) 3.Inhibition 3.Activity periacqueductal i .vc. of morphine grey levels analgesia 4. Blockade of 4. Fixed^ratio i.vc. i.vc. reserpine conditioning sedation 5. Developmental lateral 5. Selfi.cist. hypothalamus interference stimulation (DRL behavior; a) PAG dendrogenesis 6. Pain threshold of pyramidal b) i.vc. cells in cortex) lateral 7. Eating and hypothalamus drinking 6. Blockade of lateral cholinergic hypothalamus stimulation of drinking



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of membrane components, Including receptor sites. These in turn may affect release and uptake of neurotransmitters, cause i n creased metabolism of receptor sites or trigger endocytosis. For most of these mechanisms examples are available from studies of various types of cells. In addition the antibody binding can activate the complement system leading to membranolysis. The l i s t of possible mechanisms, indicated in Figure 1, is by no means complete. It does, however, suggest a number of experiments that should be helpful in elucidating the basis for discriminatory capability. Antibodies To GMI Ganglioside Inhibit Dendritic Development The effect of antiganglioside serum on development, indicated in Table I, provides some suggestion that gangliosides may be i n volved in the signaling mechanisms that regulate the sequential developmental processes of dendrogenesis and myelinogenesis in the CNS. It was recently observed (14,15) that intracisternal injection of antiganglioside antibodies into 5 day-old rats caused chemical, morphological, and behavioral changes in the adult animals. Chemical studies of somatosensory cortex revealed decreases of about 30% (p

Perturbation of Behavior and Other CNS Functions by Antibodies to

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