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Effects of antisera to S-100 protein and to synaptic membrane fraction on maze performance and EEG
Brain Research, 102 (1976) 313-321 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
313
EFFECTS OF ANT1SERA TO S-100 P R O T E I N A N D TO SYNAPTIC MEMB R A N E F R A C T I O N ON MAZE P E R F O R M A N C E A N D EEG
STEPHEN E. K A R P I A K , M A R K SEROKOSZ AND M A U R I C E M. RAPPORT
Division of Neuroscience, New York State Psychiatric Institute and the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, 722 West 168th Street, New York, N.Y. 10032 (U.S.A.) (Accepted July 25th, 1975)
SUMMARY
Topical cortical injection into rats of antiserum to the synaptic membrane fraction caused recurrent spiking activity as observed in the E E G ; 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.
INTRODUCTION
Our previous studies have shown that antiserum to the synaptic membrane fraction (anti-SMF) produces both behavioral and electrophysiological changes when injected into the brain, lntraventricular injection of anti-SMF into both rats and rabbits resulted in recurrent epileptiform activity in the EEG 6. Rats injected with anti-SMF also developed behavioral deficits on conditioned and spontaneous alternation tasks 7. Additionally, the 2-rnonth-old offspring of rats injected intravenously with anti-SMF during pregnancy showed behavioral changes on a classic DRL learning paradigm. The offspring of rats injected with antiserum to galactocerebroside or with isotonic saline did not develop any behavioral deficits 8. The following study was undertaken to (1) determine the electroencephalographic and behavioral effects of antisera directed against other brain constituents (e.g. S-100 protein and myelin), (2) utilize a topical (cortical) injection route for delivering the antibody to the brain, which minimizes the trauma to tissue and the blood-brain barrier associated with intraventricular injections, (3) observe the effects of these antibodies on a behavioral task (maze performance) known to be affected
314 by cortical lesions and (4) determine if local (topical) rather than diffuse (intra~entricular) 2-5,10-13 administration of antibodies alters behavior and EEG. Antiserum to myelin was selected for comparison with antiserum to SMF tor the following reasons. Since the antibodies in anti-SMF are, from the nature of the immunogen, presumably directed against a membrane structure, the study of an antiserum against a quite different membrane (CNS myelin) would provide an additional measure of specificity. Antiserum to the nerve tissue specific acidic protein, S-100 (ref. 14), was selected tbr 3 reasons: first, this is one of the relatively few antisera against specific brain constituents that is available, second, S-100 protein is, from recent evidencO 8, associated with membrane structures under certain conditions, and, in fact, the immunogen for anti-SMF contains small amounts of S-100 protein (Mahadik, unpublished data), and third, despite the fact that the S-100 protein was discovered in 1965 little evidence has yet been obtained concerning its function, and the proposed study could provide a clue. Since the antisera were to be injected topically onto the cortex we chose a behavioral task which has been shown to be affected by cortical lesions, namely, acquisition performance on the Lashley IlI maze 9. We found that both antiserum to SMF and antiserum to S-100 protein interfered with the behavioral performance of the rats on the maze task. However, only the antiserum to SMF produced abnormal EEG discharges; the antiserum to S-100 protein did not. Neither effect was demonstrable with the antiserum to myelin. MATERIALS AND METHODOLOGY
Anti-SMF. Rat brains (excluding stem and cerebellum) were homogenized and fractionated by differential centrifugation to obtain the synaptosome membrane fraction1, ~7. This procedure involved the preparation of the P2 fraction (containing mainly nerve endings and mitochondria), its disruption by repeated freezing and thawing, followed by the separation of myelin, synaptic membranes and mitochondria on a sucrose gradient. This synaptic membrane fraction was injected with complete Freund's adjuvant into all 4 footpads of several rabbits. After 4 weeks the animals were bled by cardiac puncture and the antisera were stored at --20 °C without preservative. Anti-myelin. Rabbits were immunized with rat brain myelin, prepared according to Norton and Podusolo 1~. One milligram of myelin was sonicated in 2 ml of water and then combined with an equal volume of Freund's complete adjuvant (FCA). Each rabbit received a total of l ml injected into all 4 footpads. After 3 weeks, ~t received a second injection of the same mixture intramuscularly. The animals were exsanguinated one week after the second injection by cardiac puncture, and the sera were stored without preservative at --20 °C. The injected animals showed signs of illness and paralysis. The antiserum used in this study (no. 1926) showed a high degree of reaction with pure rat myelin, 0.75 #g being detected at the 50yo endpoint in a test with 6 units of guinea pig complement x6. Anti-S-lO0 protein. Pure S-100 protein was prepared from bovine brain by a
315 3 step procedure involving precipitation with ammonium sulfate (60-100 ~o saturated, pH 4), column chromotography on DEAE cellulose (elution with pH gradient), and preparative polyacrylamide gel electrophoresis (Mahadik, G r a f and Rapport: to be published). The immunizing antigen was prepared by dissolving 1 mg of S-100 protein in 1 ml of saline and then combining it with an equal volume of FCA. Each rabbit received 1 ml of this mixture (500 #g of S-100) into the hind foot pads followed 3 weeks later by the same volume into the front foot pads. A third injection of 1 mg of S-100 was given intramuscularly 30 days later, and this was repeated after another 28 days. Animals were exsanguinated by cardiac puncture 12 days after the last injection, and the sera harvested and stored without preservative at --20 °C. The antiserum used here (no. 2045) gave a single line precipitate on an Ouchterlony plate when tested against 10 #g of pure S-100.
Subjects Male rats (Sprague-Dawley, 250 g) were implanted under ether anesthesia bilaterally with 8 stainless steel screw electrodes into the calvarium. A permanent cannula constructed from a 22 gauge spinal needle was places onto the right cortex of the rat through a burr hole (bregma taken as 0, AP --4 mm, R --4 ram). The electrodes were connected to a microamphenol plug and the entire assembly was affixed to the rat's skull with dental acrylic cement. All rats recovered uneventfully and were tested two weeks after surgery.
Testing and injection protocol On day 1 all rats were initially trained on the behavioral task (Lashley IlI maze). Immediately following the training they were given either a 25 #1 subdural injection of anti-SMF, anti-myelin or anti-S-100 (injection was done in a free-moving animal using a Harvard perfusion pump to maintain a rate of 3/A/rain). On day 2 the rats were again tested on the behavioral task and then given a second 25 #1 injection of the appropriate antiserum. Testing continued on days 3-5, no injections being given on these days. EEG was monitored on all days, particularly before, during and after each injection.
Apparatus All stereotactic work was done on a K o p f no. 900 small animal stereotactic apparatus. E E G was taken by a Offner Type T 8-channel recorder. A standard Lashley II! Maze (36 in. × 44 in. × 12 in.) painted flat black was employed 9. The maze had 8 cul-de-sacs, and an equal number of left and right turns, a start and finish box, and was covered by a screen. The experimenter had control of the door leading from the start box into the maze, which was evenly illuminated from above.
Behavioral testing Animals were food deprived (water ad lib.) for 48 h prior to the day of testing. On day 1 they were allowed 15 rain of habituation in the maze and were subsequently tested for 10 trials (running the maze) with a food reward present in the goal box.
316 RAT No. 2 2 3 - - ANTI-$YNAPTIC MEMBRANE FRACTION Doy I
Day 2
~-2 ~ ~ ' - " ~ J
f
3-4 ~ ~ / ' - . . , " ~ ' ~
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Day 5
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4-8 ~ . , . , . . - - ' ~ . - " , . / - ~ ' % , . , ~ % . . , ~ ~
I
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Fig. l. Cortical EEG tracings from rat no. 223 showing normal EEG patterns (day 1), followed by focal spiking activity (days 2 and 5) at site of cannula implantation (electrodes 2-3) subsequent to injection of antiserum to the synaptic membrane fraction.
Once the goal box was reached all rats were allowed to eat for 30 sec before being returned to the start box for the next trial. Animals were not allowed to eat more than 3.0 g of food per day in the maze. Latency (time spent running the maze) and errors (incorrect choices and retracings) were measured for each rat. A group of implanted but uninjected rats were also run. There were 12 animals in each group (total N for all groups equalled 48).
Histological examination All rat brains were examined for morphologic changes using cresyl violet and Rasmussen stains; 10 # m paraffin embedded sections were taken 5 m m on either side of the site of cannula implantation.
317 RAT No. 2 5 0 - ANTI-SYNAPTIC MEMBRANE Day 2
s-S ~/'v~. ~ , ¢ . ~ , ~
Day
I-"~-%~"'-~'~-~,'~-/~,~'-
I
6-7 7-8 I-5 4-8
Day 4. I-2 2-3 q
2~-4 5-6 6-7
7-8 I-5 4-8 IIis¢c I
Fig. 2. Cortical EEG tracings from rat no. 250 showing initial focal spiking activity at the site of injection (electrodes 2-3, day 2), followed by ipsilateral and contralateral spread of abnormal discharge on subsequent days (days 3 and 4). RESULTS
EEG changes Rats injected with anti-SMF developed spiking activity no sooner than 24 h after the initial injection. This epileptiform activity was characterized (day 2) by sporadic spiking localized to the area of injection (Fig. 1, electrodes 2-3) which on subsequent days became more sustained and of higher amplitude (100/~V, 6-8 spikes sec). In a few animals (4) this focal activity (Fig. 2, electrodes 2-3) became more diffuse and of still higher amplitude, spreading ipsilaterally (Fig. 2, day 3, electrodes 1-2) and contralaterally (Fig. 2, day 4) 2-4 days after injection (Table I). In all rats, the abnormal activity receded by the seventh day after initial injection. This epileptiform activity could be reactivated 4 weeks after the EEG had returned to normal by
318 TABLE I TIME COURSE OF EPILtiPI'IFORM ACTIVITY IN RATS INJECTED W I T H ANTISERUM TO THE SYNAPTIC MFMBRANE FRACTION
Rat no.
Onset of ~pikin,¢
Spread ipsilaterally
Spread contralaterally Terminathm
811 812 813 814 815 816 817 818 819 820 821 822
Day 2 2 2 2 2 2 2 2 2 2 2 2
Day 3 3 3 4 3 3 2 4 4 4 3 3
Day 3 5 --4 5 -
Day 7 7 6 8 7 7 7 8 8 9 6 7
i.rn. injection of pentylenetetrazole (Metrazol, 20 mg/kg) but was then restricted to the site of injection. Rats injected with antiserum to S-100 or myelin did not show any EEG changes.
Behavioral effects On day 1 all rats were performing the task at the same rate (latency 5.1 ~ 0:45 min; errors 5.2 :~: 0.6). On subsequent days of testing for latency there was a signi.
L A S H L E Y TIT MAZE
5-
4-
u)
:E o
z2. ..J
O ANTI- S-~O0
• ANTI--MYELIN
~r
NO I N J [ C T I O N
DAY
Fig. 3. G r a p h o f m e a n latency r e s p o n s e s (time s p e n t r u n n i n g the maze) for the 4 experimental grouPs, (vertical line, S.E.M.). Significant inhibition o f behavioral p e r f o r m a n c e is seen in the rats injected with a n t i s e r u m to the synaptic m e m b r a n e fraction a n d a n t i s e r u m to S-100 protein. Rats injected with a n t i s e r u m to myelin b e h a v e d like the uninjected group.
319 LASHLEYITr MAZE 5
4
3"
O
I" V O ANTI -S-POO 11~ A N T i . MYIELIN 0 No IN,~ECTION
1
i
~
4
g
DAY Fig. 4, Graph of mean errors made by each of the experimental groups (vertical line, S.E.M.). Both the rats injected with antiserum to the synaptic membrane fraction and rats injected with antiserum to the S-100 protein are making a significantly greater number of errors as compared to rats injected with
antiserum to myelin whose performance does not differ from the uninjected animals. ficant difference across groups (ANOVA, P < 0.001) (Fig. 3). Tukey tests showed that there were no differences between the anti-myelin injected and uninjected rats, nor were there any differences between rats injected with anti-SMF or anti-S-100, across days. However, both the anti-SMF and the anti-S-100 groups were significantly different from the anti-myelin and uninjected groups. There was a significant interaction between latency and days (P < 0.01), indicating that even though all groups were learning the maze, they were learning at different rates. This was evidenced by the fact that the controls, anti-myelin injected and uninjected rats, had learning curves with slopes (Fig. 3) of --0.5 and --0.49 respectively, whereas the anti-SMF and anti-S-100 injected rats showed slopes of --0.65 and --0.41 respectively. Analysis of error data (ANOVA) showed a significant difference (P < 0.01) between groups (Fig. 4). The anti-SMF and anti-S-100 rats were making a significantly greater number (P < 0.01) of errors as compared to the anti-myelin and uninjected rats (no difference being found between these groups). The motor activity in all animals was judged to be normal. There were no clinical signs associated with any of the epileptiform discharges. No morphological changes were seen in the brain following histological examination. DISCUSSION
One of the goals of this study was to determine if antiserum against brain constituents other than synaptic membrane fraction causes effects similar to those previously found with anti-SMF. The electroencephalographic observations showed that
320 though the topical injection of anti-SMF caused recurrent spiking activity. J:leithel anti-S-100 nor anti-myelin administered under identical conditions caused any st~ch abnormality. However, these same animals, when observed on a behavioral rusk (maze learning) responded differently to the different antisera. Rats receiving either anti-SMF or anti-S-100 were inhibited in their performance, while rats injected with anti-myelin behaved like uninjected animals. These observations further show. as compared to studies by other investigators, that these effects are produced even when the antibodies are administered by a route that is more confined, and perhaps better controlled than intraventricular injection. namely, topical application of the antibody to the cortex, Both behavioral and electrophysiological effects can be obtained this way. The production of EEG abnormalities by antiserum to SMF but not by either antiserum to myelin or antiserum to S-100 protein suggests that the antibodies in the anti-SMF serum responsible for this effect are directed against a specific antigenic component of the synaptic membrane and that this component is involved in the elicitation of epileptogenic activity. Two lectors prevent any estimate of whether qualitative differences observed between the effects of different antisera will eventually find some explanation in the differences between their specific antibody contents. One is that the degree of reaction of different antisera with specific brain constituents in vivo is not yet susceptible to evaluation. The other is that there are limitations to the amount of antiserum that can be injected into the brain, preventing any extensive dose-response study. Our results show that neurological and behavioral effects of antisera against different brain constituents can be dissociated, and therefore suggest that the specificity of the actions of such antisera on brain structures may be of a high order. ACKNOWLEDGEMENTS
We wish to thank Dr. S. Mahadik for the preparation of the S-100, and Dr. L. Graf for the preparation of the antisera. Financial support tbr this study was provided in part by a grant from the National Institute of Mental Health (MH 10315).
REFERENCES 1 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve endings from rat brain: an electron
2 3 4 5
microscopic study of cell-fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 0960) 79-85. HEATH,R. G., ANDKRUPP, I. M., Catatonia induced in monkeys by antibrain antibody, Amer. J.Psychiat., 123 (1967) 1499-1504. HYDEN,H., ANDLANGE,P., Correlation of the S-100 brain protein with behavior~ Exp. Cell Res., 62 (1970) 125-132. JANKOVIC,B. D., Biologicalactivity of antibrain antibody - - An introduction to immunoneurology, In L. GArro (Ed,), Macromolecu/es and Behavior, Appleton-Centry-Crofts, New York, 1972. pp. 99-130. JANKOVIC,B. D., RAKIC,L., VESKOV,R., ANDHORVAT,J., Effect of intraventricular injection ot" anti-brain antibody on defensive conditioned reflexes, Nature (Lond.), 218 (1968) 270-271.
321 6 KARPIAK, S. E., JR., BOWEN~F. P., AND RAPPORT, M. M., Epileptiform activity induced by antiserum to synaptic membrane, Brain Research, 59 (1973) 303-310. 7 KARPIAK,S. E., JR., Rapport, M. M., AND BOWEN, F. P., Irnmunologically induced behavioral and electrophysiological changes in the rat, Neuropsychologia, 12 (1974) 313-322. 8 KARPIAK, S. E., JR., AND RAPPORT, M. M., Behavioral changes in 2-month-old rats following prenatal exposure to antibodies against synaptic membranes, Brain Research, 92 (1975) 405~13. 9 LASHLEY,K. S., In F. A. BEACH, D. O. I-IEBB, C. T. MORGAN AND H. W. NISSEN (Eds.L The Neuropsychology of Lashley, McGraw-Hill, New York, 1960. 10 MACPHERSON,F. C., AND CHINERMAN~J., Effect of intraventricular injections of brain iso-antibodies on learning, Exp. Neurol., 31 (1970) 45-52. II MIHAILOVIC,L., AND CURPIC, D., Epileptiform activity evoked by intracerebral injection of antibrain antibodies, Brain Research: 32 (1971) 97-124. 12 MIHAILOVIC,L.,DIvAc, I., MITROVIC, E., MILOSEVIC,D., AND JANKOVIC,B. O., Effects of intraventricularly injected anti-brain antibodies on delayed alternation and visual discrimination tests performed in rhesus monkeys, Exp. NeuroL, 24 0969) 325 336. 13 MIHA1LOVIC,L., AND JANKOVIC,B. O., Effects of intraventricularly injected anti-n, caudatus antibody on the electrical activity of the cat brain, Nature (Lond.), 192 (1961) 665-666. 14 MOORE, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 0965) 739-744. 15 NORTON, W. T., AND PODUSOLO,•. E., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-757. 16 RAPPORT,M. M., GRAF, L., AUTILIO,L. A., AND NORTON, W. T., Immunological studies of organ and tumor lipids - - XIV. Galactocerebroside determinants in the myelin sheath of central nervous system, J. Neurochem., 11 (1964) 855-864. 17 RODRIGUEZ, G., ALBERICI, M., AND DEROBERTIS, E., Ultrastructural and enzymic studies of cholinergic and non-cholinergic synaptic membranes isolated from brain cortex, J. Neurochem., 4 (1967) 215-225. 18 RUSCA, G., CALISSANO,P., AND ALEMA, S., Identification of a membrane bound fraction of the S-100 protein, Brain Research, 49 (1972) 223-227.
313
EFFECTS OF ANT1SERA TO S-100 P R O T E I N A N D TO SYNAPTIC MEMB R A N E F R A C T I O N ON MAZE P E R F O R M A N C E A N D EEG
STEPHEN E. K A R P I A K , M A R K SEROKOSZ AND M A U R I C E M. RAPPORT
Division of Neuroscience, New York State Psychiatric Institute and the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, 722 West 168th Street, New York, N.Y. 10032 (U.S.A.) (Accepted July 25th, 1975)
SUMMARY
Topical cortical injection into rats of antiserum to the synaptic membrane fraction caused recurrent spiking activity as observed in the E E G ; 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.
INTRODUCTION
Our previous studies have shown that antiserum to the synaptic membrane fraction (anti-SMF) produces both behavioral and electrophysiological changes when injected into the brain, lntraventricular injection of anti-SMF into both rats and rabbits resulted in recurrent epileptiform activity in the EEG 6. Rats injected with anti-SMF also developed behavioral deficits on conditioned and spontaneous alternation tasks 7. Additionally, the 2-rnonth-old offspring of rats injected intravenously with anti-SMF during pregnancy showed behavioral changes on a classic DRL learning paradigm. The offspring of rats injected with antiserum to galactocerebroside or with isotonic saline did not develop any behavioral deficits 8. The following study was undertaken to (1) determine the electroencephalographic and behavioral effects of antisera directed against other brain constituents (e.g. S-100 protein and myelin), (2) utilize a topical (cortical) injection route for delivering the antibody to the brain, which minimizes the trauma to tissue and the blood-brain barrier associated with intraventricular injections, (3) observe the effects of these antibodies on a behavioral task (maze performance) known to be affected
314 by cortical lesions and (4) determine if local (topical) rather than diffuse (intra~entricular) 2-5,10-13 administration of antibodies alters behavior and EEG. Antiserum to myelin was selected for comparison with antiserum to SMF tor the following reasons. Since the antibodies in anti-SMF are, from the nature of the immunogen, presumably directed against a membrane structure, the study of an antiserum against a quite different membrane (CNS myelin) would provide an additional measure of specificity. Antiserum to the nerve tissue specific acidic protein, S-100 (ref. 14), was selected tbr 3 reasons: first, this is one of the relatively few antisera against specific brain constituents that is available, second, S-100 protein is, from recent evidencO 8, associated with membrane structures under certain conditions, and, in fact, the immunogen for anti-SMF contains small amounts of S-100 protein (Mahadik, unpublished data), and third, despite the fact that the S-100 protein was discovered in 1965 little evidence has yet been obtained concerning its function, and the proposed study could provide a clue. Since the antisera were to be injected topically onto the cortex we chose a behavioral task which has been shown to be affected by cortical lesions, namely, acquisition performance on the Lashley IlI maze 9. We found that both antiserum to SMF and antiserum to S-100 protein interfered with the behavioral performance of the rats on the maze task. However, only the antiserum to SMF produced abnormal EEG discharges; the antiserum to S-100 protein did not. Neither effect was demonstrable with the antiserum to myelin. MATERIALS AND METHODOLOGY
Anti-SMF. Rat brains (excluding stem and cerebellum) were homogenized and fractionated by differential centrifugation to obtain the synaptosome membrane fraction1, ~7. This procedure involved the preparation of the P2 fraction (containing mainly nerve endings and mitochondria), its disruption by repeated freezing and thawing, followed by the separation of myelin, synaptic membranes and mitochondria on a sucrose gradient. This synaptic membrane fraction was injected with complete Freund's adjuvant into all 4 footpads of several rabbits. After 4 weeks the animals were bled by cardiac puncture and the antisera were stored at --20 °C without preservative. Anti-myelin. Rabbits were immunized with rat brain myelin, prepared according to Norton and Podusolo 1~. One milligram of myelin was sonicated in 2 ml of water and then combined with an equal volume of Freund's complete adjuvant (FCA). Each rabbit received a total of l ml injected into all 4 footpads. After 3 weeks, ~t received a second injection of the same mixture intramuscularly. The animals were exsanguinated one week after the second injection by cardiac puncture, and the sera were stored without preservative at --20 °C. The injected animals showed signs of illness and paralysis. The antiserum used in this study (no. 1926) showed a high degree of reaction with pure rat myelin, 0.75 #g being detected at the 50yo endpoint in a test with 6 units of guinea pig complement x6. Anti-S-lO0 protein. Pure S-100 protein was prepared from bovine brain by a
315 3 step procedure involving precipitation with ammonium sulfate (60-100 ~o saturated, pH 4), column chromotography on DEAE cellulose (elution with pH gradient), and preparative polyacrylamide gel electrophoresis (Mahadik, G r a f and Rapport: to be published). The immunizing antigen was prepared by dissolving 1 mg of S-100 protein in 1 ml of saline and then combining it with an equal volume of FCA. Each rabbit received 1 ml of this mixture (500 #g of S-100) into the hind foot pads followed 3 weeks later by the same volume into the front foot pads. A third injection of 1 mg of S-100 was given intramuscularly 30 days later, and this was repeated after another 28 days. Animals were exsanguinated by cardiac puncture 12 days after the last injection, and the sera harvested and stored without preservative at --20 °C. The antiserum used here (no. 2045) gave a single line precipitate on an Ouchterlony plate when tested against 10 #g of pure S-100.
Subjects Male rats (Sprague-Dawley, 250 g) were implanted under ether anesthesia bilaterally with 8 stainless steel screw electrodes into the calvarium. A permanent cannula constructed from a 22 gauge spinal needle was places onto the right cortex of the rat through a burr hole (bregma taken as 0, AP --4 mm, R --4 ram). The electrodes were connected to a microamphenol plug and the entire assembly was affixed to the rat's skull with dental acrylic cement. All rats recovered uneventfully and were tested two weeks after surgery.
Testing and injection protocol On day 1 all rats were initially trained on the behavioral task (Lashley IlI maze). Immediately following the training they were given either a 25 #1 subdural injection of anti-SMF, anti-myelin or anti-S-100 (injection was done in a free-moving animal using a Harvard perfusion pump to maintain a rate of 3/A/rain). On day 2 the rats were again tested on the behavioral task and then given a second 25 #1 injection of the appropriate antiserum. Testing continued on days 3-5, no injections being given on these days. EEG was monitored on all days, particularly before, during and after each injection.
Apparatus All stereotactic work was done on a K o p f no. 900 small animal stereotactic apparatus. E E G was taken by a Offner Type T 8-channel recorder. A standard Lashley II! Maze (36 in. × 44 in. × 12 in.) painted flat black was employed 9. The maze had 8 cul-de-sacs, and an equal number of left and right turns, a start and finish box, and was covered by a screen. The experimenter had control of the door leading from the start box into the maze, which was evenly illuminated from above.
Behavioral testing Animals were food deprived (water ad lib.) for 48 h prior to the day of testing. On day 1 they were allowed 15 rain of habituation in the maze and were subsequently tested for 10 trials (running the maze) with a food reward present in the goal box.
316 RAT No. 2 2 3 - - ANTI-$YNAPTIC MEMBRANE FRACTION Doy I
Day 2
~-2 ~ ~ ' - " ~ J
f
3-4 ~ ~ / ' - . . , " ~ ' ~
5-6 --t"~'~'~/'-'-~',~
I
j
,Pr"~%"'v'~/',-,~
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......~
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I
J
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! •
o
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--e
Day 5
!
I-2 2-3
3'-4 5-6 6-7
7-8 ' - " ' ~ " ' , ~ " ' ' " - ~ ' ~ ' - ~ ~ - ~ ' ~ " ~
4-8 ~ . , . , . . - - ' ~ . - " , . / - ~ ' % , . , ~ % . . , ~ ~
I
~
~
J BOD
Fig. l. Cortical EEG tracings from rat no. 223 showing normal EEG patterns (day 1), followed by focal spiking activity (days 2 and 5) at site of cannula implantation (electrodes 2-3) subsequent to injection of antiserum to the synaptic membrane fraction.
Once the goal box was reached all rats were allowed to eat for 30 sec before being returned to the start box for the next trial. Animals were not allowed to eat more than 3.0 g of food per day in the maze. Latency (time spent running the maze) and errors (incorrect choices and retracings) were measured for each rat. A group of implanted but uninjected rats were also run. There were 12 animals in each group (total N for all groups equalled 48).
Histological examination All rat brains were examined for morphologic changes using cresyl violet and Rasmussen stains; 10 # m paraffin embedded sections were taken 5 m m on either side of the site of cannula implantation.
317 RAT No. 2 5 0 - ANTI-SYNAPTIC MEMBRANE Day 2
s-S ~/'v~. ~ , ¢ . ~ , ~
Day
I-"~-%~"'-~'~-~,'~-/~,~'-
I
6-7 7-8 I-5 4-8
Day 4. I-2 2-3 q
2~-4 5-6 6-7
7-8 I-5 4-8 IIis¢c I
Fig. 2. Cortical EEG tracings from rat no. 250 showing initial focal spiking activity at the site of injection (electrodes 2-3, day 2), followed by ipsilateral and contralateral spread of abnormal discharge on subsequent days (days 3 and 4). RESULTS
EEG changes Rats injected with anti-SMF developed spiking activity no sooner than 24 h after the initial injection. This epileptiform activity was characterized (day 2) by sporadic spiking localized to the area of injection (Fig. 1, electrodes 2-3) which on subsequent days became more sustained and of higher amplitude (100/~V, 6-8 spikes sec). In a few animals (4) this focal activity (Fig. 2, electrodes 2-3) became more diffuse and of still higher amplitude, spreading ipsilaterally (Fig. 2, day 3, electrodes 1-2) and contralaterally (Fig. 2, day 4) 2-4 days after injection (Table I). In all rats, the abnormal activity receded by the seventh day after initial injection. This epileptiform activity could be reactivated 4 weeks after the EEG had returned to normal by
318 TABLE I TIME COURSE OF EPILtiPI'IFORM ACTIVITY IN RATS INJECTED W I T H ANTISERUM TO THE SYNAPTIC MFMBRANE FRACTION
Rat no.
Onset of ~pikin,¢
Spread ipsilaterally
Spread contralaterally Terminathm
811 812 813 814 815 816 817 818 819 820 821 822
Day 2 2 2 2 2 2 2 2 2 2 2 2
Day 3 3 3 4 3 3 2 4 4 4 3 3
Day 3 5 --4 5 -
Day 7 7 6 8 7 7 7 8 8 9 6 7
i.rn. injection of pentylenetetrazole (Metrazol, 20 mg/kg) but was then restricted to the site of injection. Rats injected with antiserum to S-100 or myelin did not show any EEG changes.
Behavioral effects On day 1 all rats were performing the task at the same rate (latency 5.1 ~ 0:45 min; errors 5.2 :~: 0.6). On subsequent days of testing for latency there was a signi.
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• ANTI--MYELIN
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NO I N J [ C T I O N
DAY
Fig. 3. G r a p h o f m e a n latency r e s p o n s e s (time s p e n t r u n n i n g the maze) for the 4 experimental grouPs, (vertical line, S.E.M.). Significant inhibition o f behavioral p e r f o r m a n c e is seen in the rats injected with a n t i s e r u m to the synaptic m e m b r a n e fraction a n d a n t i s e r u m to S-100 protein. Rats injected with a n t i s e r u m to myelin b e h a v e d like the uninjected group.
319 LASHLEYITr MAZE 5
4
3"
O
I" V O ANTI -S-POO 11~ A N T i . MYIELIN 0 No IN,~ECTION
1
i
~
4
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DAY Fig. 4, Graph of mean errors made by each of the experimental groups (vertical line, S.E.M.). Both the rats injected with antiserum to the synaptic membrane fraction and rats injected with antiserum to the S-100 protein are making a significantly greater number of errors as compared to rats injected with
antiserum to myelin whose performance does not differ from the uninjected animals. ficant difference across groups (ANOVA, P < 0.001) (Fig. 3). Tukey tests showed that there were no differences between the anti-myelin injected and uninjected rats, nor were there any differences between rats injected with anti-SMF or anti-S-100, across days. However, both the anti-SMF and the anti-S-100 groups were significantly different from the anti-myelin and uninjected groups. There was a significant interaction between latency and days (P < 0.01), indicating that even though all groups were learning the maze, they were learning at different rates. This was evidenced by the fact that the controls, anti-myelin injected and uninjected rats, had learning curves with slopes (Fig. 3) of --0.5 and --0.49 respectively, whereas the anti-SMF and anti-S-100 injected rats showed slopes of --0.65 and --0.41 respectively. Analysis of error data (ANOVA) showed a significant difference (P < 0.01) between groups (Fig. 4). The anti-SMF and anti-S-100 rats were making a significantly greater number (P < 0.01) of errors as compared to the anti-myelin and uninjected rats (no difference being found between these groups). The motor activity in all animals was judged to be normal. There were no clinical signs associated with any of the epileptiform discharges. No morphological changes were seen in the brain following histological examination. DISCUSSION
One of the goals of this study was to determine if antiserum against brain constituents other than synaptic membrane fraction causes effects similar to those previously found with anti-SMF. The electroencephalographic observations showed that
320 though the topical injection of anti-SMF caused recurrent spiking activity. J:leithel anti-S-100 nor anti-myelin administered under identical conditions caused any st~ch abnormality. However, these same animals, when observed on a behavioral rusk (maze learning) responded differently to the different antisera. Rats receiving either anti-SMF or anti-S-100 were inhibited in their performance, while rats injected with anti-myelin behaved like uninjected animals. These observations further show. as compared to studies by other investigators, that these effects are produced even when the antibodies are administered by a route that is more confined, and perhaps better controlled than intraventricular injection. namely, topical application of the antibody to the cortex, Both behavioral and electrophysiological effects can be obtained this way. The production of EEG abnormalities by antiserum to SMF but not by either antiserum to myelin or antiserum to S-100 protein suggests that the antibodies in the anti-SMF serum responsible for this effect are directed against a specific antigenic component of the synaptic membrane and that this component is involved in the elicitation of epileptogenic activity. Two lectors prevent any estimate of whether qualitative differences observed between the effects of different antisera will eventually find some explanation in the differences between their specific antibody contents. One is that the degree of reaction of different antisera with specific brain constituents in vivo is not yet susceptible to evaluation. The other is that there are limitations to the amount of antiserum that can be injected into the brain, preventing any extensive dose-response study. Our results show that neurological and behavioral effects of antisera against different brain constituents can be dissociated, and therefore suggest that the specificity of the actions of such antisera on brain structures may be of a high order. ACKNOWLEDGEMENTS
We wish to thank Dr. S. Mahadik for the preparation of the S-100, and Dr. L. Graf for the preparation of the antisera. Financial support tbr this study was provided in part by a grant from the National Institute of Mental Health (MH 10315).
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2 3 4 5
microscopic study of cell-fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 0960) 79-85. HEATH,R. G., ANDKRUPP, I. M., Catatonia induced in monkeys by antibrain antibody, Amer. J.Psychiat., 123 (1967) 1499-1504. HYDEN,H., ANDLANGE,P., Correlation of the S-100 brain protein with behavior~ Exp. Cell Res., 62 (1970) 125-132. JANKOVIC,B. D., Biologicalactivity of antibrain antibody - - An introduction to immunoneurology, In L. GArro (Ed,), Macromolecu/es and Behavior, Appleton-Centry-Crofts, New York, 1972. pp. 99-130. JANKOVIC,B. D., RAKIC,L., VESKOV,R., ANDHORVAT,J., Effect of intraventricular injection ot" anti-brain antibody on defensive conditioned reflexes, Nature (Lond.), 218 (1968) 270-271.
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