Ncuroscience Letters, 11 (1979) 317--321
317
© Elsevier/North-HollandScientific PuLlishers Ltd.
MYELIN BASIC PROTEIN DEPOLARIZES NEURONAL MEMBRA]~ES B.H.
G,~HWILERand C.G. HONEGGEI~
Biological and Medical Research l:h'vision Sandoz Ltd. and Department of Neurology. University o f Basle, Basle (Switzerland)
(Received November 3rd, 1978) (Revised version received November 30th, 1978) (Accepted November 30th, 1978)
SUMMARY Bath application of 10-SM myelin basic protein (MBP) to various types of cultured nerve cells resulted in a membrane depolarization amounting to a change of 41 + 15 inV. Excitability could be restored by repolarizing the membrane by means of current injection through the recording electrode. The action of MBP persisted in the presence of tetrodotoxin (TTX), Co 2÷ or D-600 as well as in low Na÷ and low Cl- solutions, whereas it was abolished by increasing extracellular Ca2÷ concentrations The action MBP was mimicked by ouabain. We propose that the effect of the protein might be generated by blockade of an ion pump. It is speculated that MBP may exert a direct effect on neuronal membranes in demyelinating diseases.
Myelin basic protein (MBP) is the major protein constituent of the nlyelin sheath [1,5]. Interest in this protein usually focusse3 on its ability to induce experimental allergic enceph~{omyelitis (EAE) [11]. The first indication for a possible bioeiec~ric effect of MBP was suggested by the work of Jankovid et al. [10] who found that intraventricular injection of MBP into rabbits caused behavioral changes. Earlier studi~s had demonstrated that MBP reduced the ventral root response to dorsal robt stimulation in rat spinal ford by depolarizing dorsal and ventral roots, and that MBP inhibited the firing of culturedrcerebellar Purkinje cells, an effect which was not sensitive to antagonists of various transmitters [8,9]. We now provide direct evidence that the inhibition is generated by exce~ive depolarization of neuronal membranes. Cultureswere prepared from various areas of the CNS of 1--10~iay-old ,rats as described for cerebellar cultures [ 6]. Stable intracellular records were taken from large nerve cells with 40- to 80-M~ microelectrodes filled with 3 M-KCI o r 1,6M potassium citrate solution. Standard electrophysiological techniques were used, current being injected through the recording electrode by means of an active bridge circuit. Electrophysiological experiments were carried out o n 3-'8-week-old cultures. MBP (histone content less than 5%)
318
was p r e p a x e d f r o m fresh b o v i n e spinal c o r d using a ~i$:ht m o d i f i c a t i o n o f t h e
standard method [3,4]. '~ Recordings were conducted at 36°C and the cul~m~s were continously perfused with Hanl~' baiancedsalt~solutionat2 ml/min. The Hanks' Solut.~on contained the following ion concentrations expressed in mmol/h Na ÷, 141.7; K ÷, 5.8; Cl-, 145.0; Ca 2., 1.3; Mg2+,:0.9; H : O ~ , 4.2; SO~-, 0.4; HPO~-, 0.2; H2PO7~, 0.4. For low Na* solutions, " NaCl' was replaced by cholinechloride, whereas for low CI" solutions,NaCl and K CI were replaced by Lh~.~ sodium and potassium saltsof isethionicacid. M B P (isoelectricpoint = 10.5-11.2), cyctochrome c from horse heart (Serva, IP ~ 10), protamine sulfate (Merck, IP ~ 12), histone from calfthymus (Sigma, Type III,IP = 10--11), spermine (EGA-Cher~ie, IP = 11.9) and spezmidine (EGA-Chemie, IP = 11.9) were directly dissolvedin Hanks' ~olution.The effect of the various substances a~td solutionswere tested on a minimum of 4 cells. In allexperiments reported in ~his study, M B P was used in a concentration of 10 -s M which was shown to produce maximal inhibition [8]. Addition of M B P to the bathing solution coy ~istentlyproduced membrane depolarization (Fig. 1) (mean 40.8 -+14.9 m V , -+S.D., n = 43). The action of the protein was slow in both onset and recovery. Initially,the cellsbecame excited and discharged at progressively faster rates (Fig. 1A). The amplitude of the action potentials diminished in proportion to the degree of depolarization until complete depolarization block occurred. Normal action potentials were observed, however, in the presence of the protein when the membrane potenti~l was repolarized by current injection through the recording electrode (Fig. 2). N o regularchange in membrane resistancecould be detected during the MBP-induced depolarization. The action of M B P persisted after applying 1 m M Co 2+, 10-s--10-6 g/m] tetrodotoxin (TTX) and 10-6--10-4 M D-600 (an organic calcium transport antagonist) snd was a]so observed in low:Na+ and low Ci- solutions [13]. Addition of 5 m M Ca2* bo thebathing solution (tota.lCa 2÷ concentration 6.3 m M ) not only reve=sed the depolarization (Fig. 1C), but also restored membrane excitability.In the presence of high concentration of M g 2÷ (up to 12 raM), M B P stillci~poiarizedthe cells,butthe effectwas reduced in amplitude. Prolonged application of high concentrations of tetraethylammonium (TEA) not only produced a membrane depolarization,but also prolonged the duration of action potentials presumably b y blocking the delayed increase n K -conductance dunng actlo~,potentials [7,12], an effect whlch was not observed w i t h M B P . . . . . ~ ~ . 'In view of the k n o w n depolarizing action of subst~mces Which inhibit (Na#, K" ~,ATPase [7], we compared the effectof ouabain with that of MfiP. Like M B P , ouabain in a concentration of 10" s M depolarized cultured nerve i
+
•
,
•
"
Ca 2+ concentrations, The e~ pe~-nents described were c~ied out on cult1~resderived from hippo-
319
A U MI
~'40. a-
0 ~
OUA
B ~.-ls 1 -65-~ .,,..,,..,/v
6
'
lb
'
2'0
'
''
3o MIN I
|
C
C o 2.
MBP
6
....
~'
'
lb
"
i~
MIN'
Fig. 1. Effect or MBP (10 -s M) and ouabain (OUA, 10 -s M~ on the average spontaneous firing rate (A) and on the membrane potential (B) of a hypothalamic neuron from the supraoptic nucleus area after 36 days in vitro. Compared with MB~ (B), the action of ouabain was much more slowly reversible and the cell only resume~ firing 40 rain after removal of ouabain. C: Addition of 5 mM Ca ~+ to the bathing solution first pre~ented and thei~ reversed the depolarization action of 10 -s M MBP. The recording was taken from a hypothalamie neuron afte~ 23 days in vitro. In B and C, action potentials are filtered out electronically t o illustrate more clearly the effect of MBP on the membrane potenti~.! c a m p u s (n -- 1 6 ) , h y p o t k l a l a m u s (n = 1 1 ) , i n f e r i o r olive (n = 6), l o c u s c o e r u l e u s (n := 6), a n d c e r e b e l l u m (n = 4). T h e a c t i o n o f M B P w a s n o t a r e a s p e c i f i c s i n c e t h e p r o i ~ i n w a s f o u n d t o d e p o l a r i z e all 4 3 cells t e s t e d ; n e i t h e r w a s it t h e o n l y b a s i c p r o t e i n e x h i b i t i n g t h i s p r o p e r t y . P r o t a m i n e (~.0 -s M) a n d h i s t o n e ( 1 0 -4 ) m i m i c k e d t h e M B P a c t i o n . O n t h e o t h e r h a n d c y t o , : h r o m e c ( 1 0 -4 M) a n d t h e
320 i
A CONTROL
B Ml~m
C MBP.REFOLARIZED
Db RECOVERY
Fig. 2. Excerpts of spike trains recorded from a call in a culture derived from locus coeruleus. Cultured 3 l days. Resting membrane potential!' '-74 mV. Application o f 10-SM MBP depolarized the cell (B)~and the ~ t i o n p o ~ n ~ gradually disappeared. Excitability was restored by repol~tzing the membrane ~C)by means 0f current injection through the recording e~.ectrode (current = "0.6 nA), When the protein was washed out, the cell slowly recovered, and after 24 min (D) the action potentialf~ closely resembled those observet~ during the control period (A).
polyamines spermine and spermidine (10-~--10-4 M) did not influence the bioelectric activity. The data confirm earlier observations obtained with extracellular recording techniques in which MBP was shown to inhibit neuronal firing [8]. Intracellular recordings now establish that application of MBP results in a s~rong depolarization below a critical value necessary for excitability. Since excitability could be restored by repolarizing the membrane electrically, it is concluded that MBP leaves t h e spike generating mechanism intact and that the observed pause in firing was entirely caused by excessive depolarization, The fact that the action of MBP persisted after applying TTX or Co2+ indicates that it acts independently of ongoing spiking or synaptic activity. Experiments carried out with specific blockers of ion permeabilities as well as by alterations in the ionic environment of the cells suggest that Na÷, Cl- and Ca2÷ permeabilities do not play a major role in the action of MBP. The protein might depolarize the neurons by reducing the resting K* -conductance. However. unlike TEA, MBP did nbt prolong action po~ntials, The finding that an increase in extracellula~ Ca ~* reversed the effect of M]~P is difficult to interpret. It is conceivablethat~BP might bind Ca2. at sites ~ p o ~ t for the mainte nance of the rest~g m e m b ~ e po~ntial: ~ view of the close similarity ~ between the d e p 0 ~ m g effect 0fMBP ~nd that of o u a b ~ , one possibility is t h a t MBP inhibi~ the Na-Z pump. Th~s is current!y under investigation ir more appropriate model s y ~ m s using ion tracer ~chniques. .... The o b s e ~ e d d e p o ~ i n g ac~on d o ~ i~0t a p p ~ r o b e a general property of all basic proteins as evidenced by the lsck of effeCt of cytochrome c. in co n~ast to otl.er basic pro~ins, for e x h a l e histones, relativdy high concen-
neuronal m e m b ~ e s ~
321 ACKNOWLEDGEMENT
We would like to thank Dr. E. van Deosen and Prof. J.J. Dreifuss for their help in the preparation of this manuscript, Drs. R. Maurer, B. Richardson and P. Hiestand for valuable discussions and Ms. E. Hoffman and L. Rietschin for excellent technical assistance. MBP was gen,~rously provided by Mr. W. Bucher (Neurological Clinic, Basle) and Dr. E.L. Grinnan (Lilly Research Laboratories, Indianapolis). REFERENCES 1 Carnegie, P.R. and Sims, N.R., Proteins and enzymes of myelin. In E.J. Field (Ed.), Multiple Sclerosis, MTP Press, Lancaster, 1977, pp. 165--207. 2 Cohen, S.R., Herndon, R.M. and McKhann, G.M., Radioimmunoassay of myelin basic protein in spinal fluid: An index of active demyelination, New Engl. J. Med., 295 (1976) 1455--1457. 3 Deibler, G.E., Martenson, R.E. and Kies, M.W~ Large sc~de preparation of myelin basic protein from central nervous tissue of several mammalian species, Prep. Biochem., 2 (1972) 1"39--165. 4 Dunkley, P.R. and Carnegie, P.R., Isolation of myelin basic protein. In N. Marks and R. Rodnight (Eds.), Research Methods in Neurochemistry, Plenum Press, New York, 1974, pp. 219--245. 5 Eylar, E.H., Kniskern, P.S. and Zackson, J.J., Myelin basic proteins, Methods Enzymol., 32, (!974) 323--341. 6 Gtihwiler, B.H., Marnoon, A.M. and Tobias, C.A., Spontaneous 5ioe~.~t~ical activity of cultured cerebellar Purkinje cells during exposure to agents which prevent synaptic transmission, Brain Re~., 53 (1973) 71--79. 7 Godfraind, J.M., Kawamura, H., Krnjevid, K. and Purmain, R., Actions of dinitrophenol and some other metabolic inhibitors on cortical neurons, J. Physiol. (Lond.), 215 (1971) 199--222. 8 Honegger, C.G., Gihwiler, B.H. and Isler, II., The effect of myelin basic protein (MBP) ov the bioelectric activity of spinal cord and cerebellar neurones, Neurosci. Lett., 4 (1977) 303--307. 9 Islet, H. and Honegger, C.G., The effect of myelin basic protein (MBP) on the bioelectrical activity of the frog spinal cord in vitro, Experientia, 34 (1978) 900. 10 Jankovi~, B.D., Rabid, Lj., Janjic, M., Mitrovi4, K. and Ivonu~, J., Immuno-neurophysiological studies of the experimental ~dlergic encephalomyelitis following the injection of antibrain antibodies and myelin b~dc proteins into the cerebral cavity, Path. Europ., 2 (1966) 87--107. 11 Kies, M.W., Experimental allergic encephalomyelitis. In G.E. Gaul (Ed.), Biology of Brain Dysfunction~ Plenum Preu, New York, 1973, Vol. 2, pp. 185--224. 12 Krnjevi4; K., Pumain, R. and Renaud, L., Effects of Ba a÷ and tetraethylammonium on cortical neurones, J. Physiol. (Lond.), 215 (1971) 223--~:45. 13 Narahashi, T., Chemicals as tools in the study of excitable m~mbranes, Physiol. Rev., 54 (1974) 813--889. 14 Westall, F.C., Released myelin basic protein: the imm:mogenic factor? Immunochemistry, 11 (1974) 513--515.
317
© Elsevier/North-HollandScientific PuLlishers Ltd.
MYELIN BASIC PROTEIN DEPOLARIZES NEURONAL MEMBRA]~ES B.H.
G,~HWILERand C.G. HONEGGEI~
Biological and Medical Research l:h'vision Sandoz Ltd. and Department of Neurology. University o f Basle, Basle (Switzerland)
(Received November 3rd, 1978) (Revised version received November 30th, 1978) (Accepted November 30th, 1978)
SUMMARY Bath application of 10-SM myelin basic protein (MBP) to various types of cultured nerve cells resulted in a membrane depolarization amounting to a change of 41 + 15 inV. Excitability could be restored by repolarizing the membrane by means of current injection through the recording electrode. The action of MBP persisted in the presence of tetrodotoxin (TTX), Co 2÷ or D-600 as well as in low Na÷ and low Cl- solutions, whereas it was abolished by increasing extracellular Ca2÷ concentrations The action MBP was mimicked by ouabain. We propose that the effect of the protein might be generated by blockade of an ion pump. It is speculated that MBP may exert a direct effect on neuronal membranes in demyelinating diseases.
Myelin basic protein (MBP) is the major protein constituent of the nlyelin sheath [1,5]. Interest in this protein usually focusse3 on its ability to induce experimental allergic enceph~{omyelitis (EAE) [11]. The first indication for a possible bioeiec~ric effect of MBP was suggested by the work of Jankovid et al. [10] who found that intraventricular injection of MBP into rabbits caused behavioral changes. Earlier studi~s had demonstrated that MBP reduced the ventral root response to dorsal robt stimulation in rat spinal ford by depolarizing dorsal and ventral roots, and that MBP inhibited the firing of culturedrcerebellar Purkinje cells, an effect which was not sensitive to antagonists of various transmitters [8,9]. We now provide direct evidence that the inhibition is generated by exce~ive depolarization of neuronal membranes. Cultureswere prepared from various areas of the CNS of 1--10~iay-old ,rats as described for cerebellar cultures [ 6]. Stable intracellular records were taken from large nerve cells with 40- to 80-M~ microelectrodes filled with 3 M-KCI o r 1,6M potassium citrate solution. Standard electrophysiological techniques were used, current being injected through the recording electrode by means of an active bridge circuit. Electrophysiological experiments were carried out o n 3-'8-week-old cultures. MBP (histone content less than 5%)
318
was p r e p a x e d f r o m fresh b o v i n e spinal c o r d using a ~i$:ht m o d i f i c a t i o n o f t h e
standard method [3,4]. '~ Recordings were conducted at 36°C and the cul~m~s were continously perfused with Hanl~' baiancedsalt~solutionat2 ml/min. The Hanks' Solut.~on contained the following ion concentrations expressed in mmol/h Na ÷, 141.7; K ÷, 5.8; Cl-, 145.0; Ca 2., 1.3; Mg2+,:0.9; H : O ~ , 4.2; SO~-, 0.4; HPO~-, 0.2; H2PO7~, 0.4. For low Na* solutions, " NaCl' was replaced by cholinechloride, whereas for low CI" solutions,NaCl and K CI were replaced by Lh~.~ sodium and potassium saltsof isethionicacid. M B P (isoelectricpoint = 10.5-11.2), cyctochrome c from horse heart (Serva, IP ~ 10), protamine sulfate (Merck, IP ~ 12), histone from calfthymus (Sigma, Type III,IP = 10--11), spermine (EGA-Cher~ie, IP = 11.9) and spezmidine (EGA-Chemie, IP = 11.9) were directly dissolvedin Hanks' ~olution.The effect of the various substances a~td solutionswere tested on a minimum of 4 cells. In allexperiments reported in ~his study, M B P was used in a concentration of 10 -s M which was shown to produce maximal inhibition [8]. Addition of M B P to the bathing solution coy ~istentlyproduced membrane depolarization (Fig. 1) (mean 40.8 -+14.9 m V , -+S.D., n = 43). The action of the protein was slow in both onset and recovery. Initially,the cellsbecame excited and discharged at progressively faster rates (Fig. 1A). The amplitude of the action potentials diminished in proportion to the degree of depolarization until complete depolarization block occurred. Normal action potentials were observed, however, in the presence of the protein when the membrane potenti~l was repolarized by current injection through the recording electrode (Fig. 2). N o regularchange in membrane resistancecould be detected during the MBP-induced depolarization. The action of M B P persisted after applying 1 m M Co 2+, 10-s--10-6 g/m] tetrodotoxin (TTX) and 10-6--10-4 M D-600 (an organic calcium transport antagonist) snd was a]so observed in low:Na+ and low Ci- solutions [13]. Addition of 5 m M Ca2* bo thebathing solution (tota.lCa 2÷ concentration 6.3 m M ) not only reve=sed the depolarization (Fig. 1C), but also restored membrane excitability.In the presence of high concentration of M g 2÷ (up to 12 raM), M B P stillci~poiarizedthe cells,butthe effectwas reduced in amplitude. Prolonged application of high concentrations of tetraethylammonium (TEA) not only produced a membrane depolarization,but also prolonged the duration of action potentials presumably b y blocking the delayed increase n K -conductance dunng actlo~,potentials [7,12], an effect whlch was not observed w i t h M B P . . . . . ~ ~ . 'In view of the k n o w n depolarizing action of subst~mces Which inhibit (Na#, K" ~,ATPase [7], we compared the effectof ouabain with that of MfiP. Like M B P , ouabain in a concentration of 10" s M depolarized cultured nerve i
+
•
,
•
"
Ca 2+ concentrations, The e~ pe~-nents described were c~ied out on cult1~resderived from hippo-
319
A U MI
~'40. a-
0 ~
OUA
B ~.-ls 1 -65-~ .,,..,,..,/v
6
'
lb
'
2'0
'
''
3o MIN I
|
C
C o 2.
MBP
6
....
~'
'
lb
"
i~
MIN'
Fig. 1. Effect or MBP (10 -s M) and ouabain (OUA, 10 -s M~ on the average spontaneous firing rate (A) and on the membrane potential (B) of a hypothalamic neuron from the supraoptic nucleus area after 36 days in vitro. Compared with MB~ (B), the action of ouabain was much more slowly reversible and the cell only resume~ firing 40 rain after removal of ouabain. C: Addition of 5 mM Ca ~+ to the bathing solution first pre~ented and thei~ reversed the depolarization action of 10 -s M MBP. The recording was taken from a hypothalamie neuron afte~ 23 days in vitro. In B and C, action potentials are filtered out electronically t o illustrate more clearly the effect of MBP on the membrane potenti~.! c a m p u s (n -- 1 6 ) , h y p o t k l a l a m u s (n = 1 1 ) , i n f e r i o r olive (n = 6), l o c u s c o e r u l e u s (n := 6), a n d c e r e b e l l u m (n = 4). T h e a c t i o n o f M B P w a s n o t a r e a s p e c i f i c s i n c e t h e p r o i ~ i n w a s f o u n d t o d e p o l a r i z e all 4 3 cells t e s t e d ; n e i t h e r w a s it t h e o n l y b a s i c p r o t e i n e x h i b i t i n g t h i s p r o p e r t y . P r o t a m i n e (~.0 -s M) a n d h i s t o n e ( 1 0 -4 ) m i m i c k e d t h e M B P a c t i o n . O n t h e o t h e r h a n d c y t o , : h r o m e c ( 1 0 -4 M) a n d t h e
320 i
A CONTROL
B Ml~m
C MBP.REFOLARIZED
Db RECOVERY
Fig. 2. Excerpts of spike trains recorded from a call in a culture derived from locus coeruleus. Cultured 3 l days. Resting membrane potential!' '-74 mV. Application o f 10-SM MBP depolarized the cell (B)~and the ~ t i o n p o ~ n ~ gradually disappeared. Excitability was restored by repol~tzing the membrane ~C)by means 0f current injection through the recording e~.ectrode (current = "0.6 nA), When the protein was washed out, the cell slowly recovered, and after 24 min (D) the action potentialf~ closely resembled those observet~ during the control period (A).
polyamines spermine and spermidine (10-~--10-4 M) did not influence the bioelectric activity. The data confirm earlier observations obtained with extracellular recording techniques in which MBP was shown to inhibit neuronal firing [8]. Intracellular recordings now establish that application of MBP results in a s~rong depolarization below a critical value necessary for excitability. Since excitability could be restored by repolarizing the membrane electrically, it is concluded that MBP leaves t h e spike generating mechanism intact and that the observed pause in firing was entirely caused by excessive depolarization, The fact that the action of MBP persisted after applying TTX or Co2+ indicates that it acts independently of ongoing spiking or synaptic activity. Experiments carried out with specific blockers of ion permeabilities as well as by alterations in the ionic environment of the cells suggest that Na÷, Cl- and Ca2÷ permeabilities do not play a major role in the action of MBP. The protein might depolarize the neurons by reducing the resting K* -conductance. However. unlike TEA, MBP did nbt prolong action po~ntials, The finding that an increase in extracellula~ Ca ~* reversed the effect of M]~P is difficult to interpret. It is conceivablethat~BP might bind Ca2. at sites ~ p o ~ t for the mainte nance of the rest~g m e m b ~ e po~ntial: ~ view of the close similarity ~ between the d e p 0 ~ m g effect 0fMBP ~nd that of o u a b ~ , one possibility is t h a t MBP inhibi~ the Na-Z pump. Th~s is current!y under investigation ir more appropriate model s y ~ m s using ion tracer ~chniques. .... The o b s e ~ e d d e p o ~ i n g ac~on d o ~ i~0t a p p ~ r o b e a general property of all basic proteins as evidenced by the lsck of effeCt of cytochrome c. in co n~ast to otl.er basic pro~ins, for e x h a l e histones, relativdy high concen-
neuronal m e m b ~ e s ~
321 ACKNOWLEDGEMENT
We would like to thank Dr. E. van Deosen and Prof. J.J. Dreifuss for their help in the preparation of this manuscript, Drs. R. Maurer, B. Richardson and P. Hiestand for valuable discussions and Ms. E. Hoffman and L. Rietschin for excellent technical assistance. MBP was gen,~rously provided by Mr. W. Bucher (Neurological Clinic, Basle) and Dr. E.L. Grinnan (Lilly Research Laboratories, Indianapolis). REFERENCES 1 Carnegie, P.R. and Sims, N.R., Proteins and enzymes of myelin. In E.J. Field (Ed.), Multiple Sclerosis, MTP Press, Lancaster, 1977, pp. 165--207. 2 Cohen, S.R., Herndon, R.M. and McKhann, G.M., Radioimmunoassay of myelin basic protein in spinal fluid: An index of active demyelination, New Engl. J. Med., 295 (1976) 1455--1457. 3 Deibler, G.E., Martenson, R.E. and Kies, M.W~ Large sc~de preparation of myelin basic protein from central nervous tissue of several mammalian species, Prep. Biochem., 2 (1972) 1"39--165. 4 Dunkley, P.R. and Carnegie, P.R., Isolation of myelin basic protein. In N. Marks and R. Rodnight (Eds.), Research Methods in Neurochemistry, Plenum Press, New York, 1974, pp. 219--245. 5 Eylar, E.H., Kniskern, P.S. and Zackson, J.J., Myelin basic proteins, Methods Enzymol., 32, (!974) 323--341. 6 Gtihwiler, B.H., Marnoon, A.M. and Tobias, C.A., Spontaneous 5ioe~.~t~ical activity of cultured cerebellar Purkinje cells during exposure to agents which prevent synaptic transmission, Brain Re~., 53 (1973) 71--79. 7 Godfraind, J.M., Kawamura, H., Krnjevid, K. and Purmain, R., Actions of dinitrophenol and some other metabolic inhibitors on cortical neurons, J. Physiol. (Lond.), 215 (1971) 199--222. 8 Honegger, C.G., Gihwiler, B.H. and Isler, II., The effect of myelin basic protein (MBP) ov the bioelectric activity of spinal cord and cerebellar neurones, Neurosci. Lett., 4 (1977) 303--307. 9 Islet, H. and Honegger, C.G., The effect of myelin basic protein (MBP) on the bioelectrical activity of the frog spinal cord in vitro, Experientia, 34 (1978) 900. 10 Jankovi~, B.D., Rabid, Lj., Janjic, M., Mitrovi4, K. and Ivonu~, J., Immuno-neurophysiological studies of the experimental ~dlergic encephalomyelitis following the injection of antibrain antibodies and myelin b~dc proteins into the cerebral cavity, Path. Europ., 2 (1966) 87--107. 11 Kies, M.W., Experimental allergic encephalomyelitis. In G.E. Gaul (Ed.), Biology of Brain Dysfunction~ Plenum Preu, New York, 1973, Vol. 2, pp. 185--224. 12 Krnjevi4; K., Pumain, R. and Renaud, L., Effects of Ba a÷ and tetraethylammonium on cortical neurones, J. Physiol. (Lond.), 215 (1971) 223--~:45. 13 Narahashi, T., Chemicals as tools in the study of excitable m~mbranes, Physiol. Rev., 54 (1974) 813--889. 14 Westall, F.C., Released myelin basic protein: the imm:mogenic factor? Immunochemistry, 11 (1974) 513--515.
