- Email: [email protected]
The nervous system-specific S-100 antigen in cerebrospinal fluid of multiple sclerosis patients
Neuroscience Letters, 11 (1979) 171--175 © Ekevier/North-Holland Scientific Publishers Ltd. .
•
,
.
.
.
•
.
.
.
. ~ .
•
171
..
•
,
.,
_
.
.
'
THE NERVOUS SYSTEM,SPECIFIC S-100 ANTIGEN IN CEREBROSPINAL FLUID O]F MULTIPLE SCLEROSIS PATIENTS F. MICHETTI, A. MASSARO and M. MURAZIO
Department o f Anatomy and (A.M.) Department of Neurology, Universita Cattolica, 00168 Rome (It¢;lyj (Received October 2rid, 1978) (Revised verl:ion received November 24th, 1978) (Acceptecl November 27th, 1978)
SUMMARY
The nervous system-specific S-100 antigen has been found in cerebrospinal fluid (CSF) of 13 out of 18 patients with multiple sclerosis{MS), whereas it was undetectable in either of the 11 control patients with minor psychic distur. bances or with neurological disorders not usually associated with apparent parenchymal lesion. The levelsof the antigen appeared to be higher in CSF of patients in the acute phase of the disease. Though the small number of cases hampers final statements, the S-100 in CSF might serve as a possible index of active cell injury in the central nervous system underlying the pathogenesis of MS. The nervous system-specific S-100 protein [12] is piesent in significant amounts in the cell types involved in the degenerative processes characteristic of the multiple sclerosis (MS), in astrocytes and oligodendrocytes at higher concentrations than in neurons [for reviews see 1,Y.,17]. it is ~herefore reasonable to foresee a release of this intracelluiar marker into the cerebrospinal fluid (CSF) of MS patients. Notably, its presence is expected in CSF during the phases of exacerbation as an index of active cel~ injury. In the present study, data are given indicating the actual presence of the antigen in CSF of MS patients as compared to adequate controls. Furthermore., in MS patients, a relationship of the S-100 levels to the phases of exacerbation might be suggested. CSF was taken from eighteen 21--48-year-old MS patients of both sexes. MS diagnosis: Was based both on clinical findings and laboratory tests [ for reyiews see i0~15] .Twelve Out of 18 M S patients were in the acute phase of the disease atth e time of the lumbar puncture, whereas the remaining 6 were in a stationary phase. None of the patients had been treated with immunosuppressive drugs, or~had had steroid therapy or A C T H in the last three
172 months. CSF from eleven 7--49-year-old control patients was also examined. Among the controls, 5 individuals were affected by minor psychic disturbances (dizziness, tension headache or psychasthenic symptoms), but neurological disease., had been ruled out; the other 6 were affected b y neurological disorders which are not usually associated with apparent parenchymal injury of the nervous tirsue (2 patients with malformations of the central nervous system and 4 patients with 'essential' epilepsy). CSF specimens were stored a t - 2 0 ° C . S-100 protein was measured in CSF by microcomplement fixation assay as described by Moore and Perez [ 13 ]. Ox S-100 protein was prepared according to the procedure of Moore [12] and used as a standard. Cross reactivity between ox and human S-100 protein has been observed [ 18]. A monospecific anti-S-100 antiserum was obtained from rabbit and characterized according to Zukerman et al. [ 18]. Samples with complement alone and with complement and antiserum were assayed to test the anticomplementary activity of CSF samples and antiserum. All S-100 values were corrected for anticomplemen~'cy activity, when present. Total protein and IgG concentrations were determine:d
B 18
S-100 • ang/mt
16
S-I00 not detected ~ J
fJ~ e
,,, 0,1
12 ¸
m U
i
O O
o
i
Z=
m 5
8
•
MS
patients
~rd
patients
MS patients in the acut~ phase
!
MS patients in the stationaryphase
Fig. 1. Distribution of 8-100 protein in CSF of MS patients in various phases and control patients with neuropsychiatric disorders not usually associated •with apparent parenchymal injury. Control patienta are detailed in the text. S-100 protein was measured by microcomplement fixation atomy. B: S-100 protein levels in CSF of MS patient.q in the acute phase and in the stationary phase of at least three determinations, The dashed line indicates the limit of sensitivity of the assay. S-100 levels in individual cases, e. ~
:[ 7 3
in all samples respectively by the method of Lowry et al. [7 ] and by single immunodiffusion [9]. Fig. 1A indicates that the S-100 was detected in the C S F of 13 out of 18 M S patients in various phases of the disease, whereas it was undetectable in either of the 11 control patients. The variations of S-100 levelsin C S F of individual M S patients during the phases of the disease cannot be determined ~dthout long-term studies on a larger number of cases. However, in the group of subjects tested in the present study, different levels of S-100 were observed when patients in an active phase of demyelination were compared to clinicallystable M S patients (Fig. 1B). The antigen was undetectable in C S F of 3 out of 6 M S patients w h o wele i',,ot in the acute phase. The other 3 had borderline levelsfrom 6.4 to 7.4 ng/ml. A m o n g the M S patients in the acute phase, the S-100 was undetectable in 2 out of 12 patients,whereas in the remaining 10 it was detected in concentrations ranging from the limit of sensitivity(6 ng/ml) to 18.8 ng/ml. Of course, the possibilitythat proteolytic activitieslead to a general underestimation of S-100 concentrations in all samples cannot be ruled out. In the group of M S patients, there was no clearly significant statistical correlation of S-100 levels with C S F total protein or IgG concentrations. Nevertheless, also IgG percentages, like S-100 levels,were generally higher in acute phase patients,as was to be expected (not shown). With the limitations of the method in mind, the follcwing conclusions result from the above data: (1) The S-100 seems to be usually absent from h u m a n CSF. Likewise the protein is not usually released into CSF during functional diseases of the nervous system not associated with extensive tissue lesions. (2) Consequently, the S-100 found in CSF of M S patients might be regarded as a result of cell injury accompanying the pathogenesis of MS. The release of an intracellularconstituent into the exlxacellularenvironment during cellinjury is a trivialobservation. Nevertheless the present data appear to be worthy of consideration for two reasons: (1) As the S-100 proteb~ is unique to the nervous tissue,its release into CSF specificallyindicates a lesion occurring in the parenchyma. Moreover, since the S-100 is normally absent from plasma, the possibility that its presence in CSF is merely due to an alteration of the blood-CSF barrier can be ruled out. (2) The typical course of MS strongly calls for markers able to monitor cell injury. Recently, a relationship between the levels of myelin encephalitogenic protein fragments in CSF and MS phases has been reported [16]. In the group of patients tested in the present study S-100 levels in CSF appear also to be bigher in the acute phase of the disease. It is intriguing that CSF concentrations of both S-100 and encephalitogenic protein are not clearly related to IgC concentrations, although the small number of S-100 determinations hampers final statements. A possible explanation comes from the different factors leading to the release of brain protein and IgG in CSF. In fact the release of intracellular brain constituents might be directly attr;,buted to cell injury, whereas IgG synthesis and secretion is known to be conditioned by multiple factors.
174
T h e cellular and subcellularlocalizations: ofthe S-100 in laboratory animals have been extensivelyl studied in recent years. The protein has been Shown.to be a component both, ofglial icells andneurons,where~it!has been foun d in the cytosol asweU as in particulate matter[ 3,5,11]- ~Nevertheless, there is general agreement that a prominent aliquot of the S-100 is o f glial origin, (for ireviews see refs. 2 and 17). ~ g in,mind thelnature of typicaiMS lesions, it is therefore likely thatglial~ceUs a r e t h e main ~source o f S-100•in CSF. Whether the ;appearance o f S,100in CSF follows a change in concentration o f the protein in tissue affected by MS remains to be' determined. Nevertheless it seems likely at present that the release o f t h e protein may be attributed to mere diffusion fromdamaged tissuerather than to~ active cellular secretion. Moreover, cwing t o the presence of proteases in MS damaged tissue [ 4,6,14], the possibility exists that aportion or all of the S-100 in CSF is present as an antigenically active fragment, rather than as the native protein. The occurrence of S.100 and/or its fragt~lents in CSF of diseased subjects also opens the possibility that an aliquot of the antigen is filtered into theblood. Since the S-100 i~ a normally sequestered immunogen, its appearance in biological fluids during a disease in which •immunological mechanisms are involved migbtiead to s~eculations. In this respect, work is in progress to detect anti.S-100 antibodies in CSF and serum of patients with various neurological diseases. However, although a specific interaction of S-100 with myelin basic protein has been reported [8], data are lacking at present to suggest a specific role of the S-100 protein in the pathogenesis of MS. In conc'~usion, the 8-100 in CSF might be a candidate for an index of active cell injury during t,he exacerbations in MS patients. Nevertheless, owing to the various localizatic:is actually reported for the protein in normal tissue, it ought to be regarded as ~ general marker of parenchymal injury in the nervous tissue rather than a selective index of demyelination, More extensive studies on selected patients affe~ted by various diseases are yet required to establish the usefulnes~ of the S-1,)0 as amarker of active cell injury specific to nervous parenchyma. Progress in basic studies on S-100 and other nervous systemspecific proteins (for reviews see refs. 2 and 17)might provide useful tools for a deeper understanding of the molecular features of neurological lesions. i
~.
~
~
•
ACKNOWLEDGEMENTS The authors are deeply indebted with Prof. G. Macchi and Prof. N. Miani for their encouragement and critical review of the manuscript. This investigation w u partially supported by C.N.R. contract no. 75.00637.04. REFERENCES
1 Adams,C,E.M., PatholOgy ofmultiple sclerosis: progress of the lesion. Brit. M~d. Bull., 33 (1977) 15--20. 2 Book,E., Nervous system specific proteins. J. Neurochem., 30 (1978) 7--14. 3 Cicero,T.J., Cowan, W., Moore, B.W. and Suntzeff; V., The cellular localization of the
175 two l~rain-specific proteins S-100 and 14.3.2,'Brain Res., 18 (1970) 25--34. 4 Cuzner, M.L. and Davison, A.N., Changes in ceJ'ebral lysosomal enzyme activity and lipids in multiple sclerosis, J. neurol. Sci., 19 (1973) 29--36. 5 Donato, R., Michetti, F. and Miani, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Res., 98 (1975) 661--573. 6 Einstein, E.R., Dalai, K.B. and Csejtey, J., Increased protease activity and changes in basic proteins and lipids in multiple sclerosis.plaques, J. neurol. Sci., 11 (1970) 109--121 7 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265--275. 8 Mahdik, S.P., Graf, L. :ind Rapport, M.M., Immunochemical studies of the interaction between myelin basic protein and S-100 protein, J. Neurochem., 27 (1976) 405--40P 9 Mancini, G.A., Carbon~ra, A.O. and Heremans, J.F., Immunochemical quantitation o antigens by single radi~ immunodiffusion, Immuno~hemistry, 2 (1965) 235--254. 10 McDonald, W.I. and H~,lliday, A.M., Diagnosis and classification of multiple sclerosis, Brit. IVied. Bull. 33 (1977) 4--8. 11 Michetti, F., Miani, N., DeRenzis, G., Caniglia, A. and Correr, S., Nuclear localization of S-100 protein, J. Neurochem. 22 (1974) 239--244. 12 Moore, B.W., A soluble protein characteristic of the nervous system: Biochem. Biophys. Res. Commun., 19 (1965) 739--744. 13 Moore, B.W. and Perez, V.J., Complement fixation for antigens at a picogram level, J. Immunol., 96 (1966) 1000--1005. 14 Riekkinen, P.J., Clausen, J., Fog, T. and Rinne, U.K., Acid proteinase activity of white matter and plaques in multiple sclerosis, Acta neurol, seand., 46 (1970) 349--353. 15 Thompson, E.J., Laboratory diagnosis of multiple sclerosis: immunological and biochemical aspects, Brit. Med. Bull., 33 (1977) 28--33. 16 Whitaker, J.N., Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis, Neurology, 27 (1977) 911--920. 17 Zomzely-Neurath, C. and Keller, A., Nervous system-specific proteins of vertebrate~: A search for functions and physiological roles, Neurochem. Res. 2 (1977) 353--377. 18 Zuckerman, J.E., Hersehman, H.R. and Levine, L., Appearance of a brain-specific antigen (the S-100 protein) during human foetal development, J. Neurochem. 17 (1970) 247--251.
•
,
.
.
.
•
.
.
.
. ~ .
•
171
..
•
,
.,
_
.
.
'
THE NERVOUS SYSTEM,SPECIFIC S-100 ANTIGEN IN CEREBROSPINAL FLUID O]F MULTIPLE SCLEROSIS PATIENTS F. MICHETTI, A. MASSARO and M. MURAZIO
Department o f Anatomy and (A.M.) Department of Neurology, Universita Cattolica, 00168 Rome (It¢;lyj (Received October 2rid, 1978) (Revised verl:ion received November 24th, 1978) (Acceptecl November 27th, 1978)
SUMMARY
The nervous system-specific S-100 antigen has been found in cerebrospinal fluid (CSF) of 13 out of 18 patients with multiple sclerosis{MS), whereas it was undetectable in either of the 11 control patients with minor psychic distur. bances or with neurological disorders not usually associated with apparent parenchymal lesion. The levelsof the antigen appeared to be higher in CSF of patients in the acute phase of the disease. Though the small number of cases hampers final statements, the S-100 in CSF might serve as a possible index of active cell injury in the central nervous system underlying the pathogenesis of MS. The nervous system-specific S-100 protein [12] is piesent in significant amounts in the cell types involved in the degenerative processes characteristic of the multiple sclerosis (MS), in astrocytes and oligodendrocytes at higher concentrations than in neurons [for reviews see 1,Y.,17]. it is ~herefore reasonable to foresee a release of this intracelluiar marker into the cerebrospinal fluid (CSF) of MS patients. Notably, its presence is expected in CSF during the phases of exacerbation as an index of active cel~ injury. In the present study, data are given indicating the actual presence of the antigen in CSF of MS patients as compared to adequate controls. Furthermore., in MS patients, a relationship of the S-100 levels to the phases of exacerbation might be suggested. CSF was taken from eighteen 21--48-year-old MS patients of both sexes. MS diagnosis: Was based both on clinical findings and laboratory tests [ for reyiews see i0~15] .Twelve Out of 18 M S patients were in the acute phase of the disease atth e time of the lumbar puncture, whereas the remaining 6 were in a stationary phase. None of the patients had been treated with immunosuppressive drugs, or~had had steroid therapy or A C T H in the last three
172 months. CSF from eleven 7--49-year-old control patients was also examined. Among the controls, 5 individuals were affected by minor psychic disturbances (dizziness, tension headache or psychasthenic symptoms), but neurological disease., had been ruled out; the other 6 were affected b y neurological disorders which are not usually associated with apparent parenchymal injury of the nervous tirsue (2 patients with malformations of the central nervous system and 4 patients with 'essential' epilepsy). CSF specimens were stored a t - 2 0 ° C . S-100 protein was measured in CSF by microcomplement fixation assay as described by Moore and Perez [ 13 ]. Ox S-100 protein was prepared according to the procedure of Moore [12] and used as a standard. Cross reactivity between ox and human S-100 protein has been observed [ 18]. A monospecific anti-S-100 antiserum was obtained from rabbit and characterized according to Zukerman et al. [ 18]. Samples with complement alone and with complement and antiserum were assayed to test the anticomplementary activity of CSF samples and antiserum. All S-100 values were corrected for anticomplemen~'cy activity, when present. Total protein and IgG concentrations were determine:d
B 18
S-100 • ang/mt
16
S-I00 not detected ~ J
fJ~ e
,,, 0,1
12 ¸
m U
i
O O
o
i
Z=
m 5
8
•
MS
patients
~rd
patients
MS patients in the acut~ phase
!
MS patients in the stationaryphase
Fig. 1. Distribution of 8-100 protein in CSF of MS patients in various phases and control patients with neuropsychiatric disorders not usually associated •with apparent parenchymal injury. Control patienta are detailed in the text. S-100 protein was measured by microcomplement fixation atomy. B: S-100 protein levels in CSF of MS patient.q in the acute phase and in the stationary phase of at least three determinations, The dashed line indicates the limit of sensitivity of the assay. S-100 levels in individual cases, e. ~
:[ 7 3
in all samples respectively by the method of Lowry et al. [7 ] and by single immunodiffusion [9]. Fig. 1A indicates that the S-100 was detected in the C S F of 13 out of 18 M S patients in various phases of the disease, whereas it was undetectable in either of the 11 control patients. The variations of S-100 levelsin C S F of individual M S patients during the phases of the disease cannot be determined ~dthout long-term studies on a larger number of cases. However, in the group of subjects tested in the present study, different levels of S-100 were observed when patients in an active phase of demyelination were compared to clinicallystable M S patients (Fig. 1B). The antigen was undetectable in C S F of 3 out of 6 M S patients w h o wele i',,ot in the acute phase. The other 3 had borderline levelsfrom 6.4 to 7.4 ng/ml. A m o n g the M S patients in the acute phase, the S-100 was undetectable in 2 out of 12 patients,whereas in the remaining 10 it was detected in concentrations ranging from the limit of sensitivity(6 ng/ml) to 18.8 ng/ml. Of course, the possibilitythat proteolytic activitieslead to a general underestimation of S-100 concentrations in all samples cannot be ruled out. In the group of M S patients, there was no clearly significant statistical correlation of S-100 levels with C S F total protein or IgG concentrations. Nevertheless, also IgG percentages, like S-100 levels,were generally higher in acute phase patients,as was to be expected (not shown). With the limitations of the method in mind, the follcwing conclusions result from the above data: (1) The S-100 seems to be usually absent from h u m a n CSF. Likewise the protein is not usually released into CSF during functional diseases of the nervous system not associated with extensive tissue lesions. (2) Consequently, the S-100 found in CSF of M S patients might be regarded as a result of cell injury accompanying the pathogenesis of MS. The release of an intracellularconstituent into the exlxacellularenvironment during cellinjury is a trivialobservation. Nevertheless the present data appear to be worthy of consideration for two reasons: (1) As the S-100 proteb~ is unique to the nervous tissue,its release into CSF specificallyindicates a lesion occurring in the parenchyma. Moreover, since the S-100 is normally absent from plasma, the possibility that its presence in CSF is merely due to an alteration of the blood-CSF barrier can be ruled out. (2) The typical course of MS strongly calls for markers able to monitor cell injury. Recently, a relationship between the levels of myelin encephalitogenic protein fragments in CSF and MS phases has been reported [16]. In the group of patients tested in the present study S-100 levels in CSF appear also to be bigher in the acute phase of the disease. It is intriguing that CSF concentrations of both S-100 and encephalitogenic protein are not clearly related to IgC concentrations, although the small number of S-100 determinations hampers final statements. A possible explanation comes from the different factors leading to the release of brain protein and IgG in CSF. In fact the release of intracellular brain constituents might be directly attr;,buted to cell injury, whereas IgG synthesis and secretion is known to be conditioned by multiple factors.
174
T h e cellular and subcellularlocalizations: ofthe S-100 in laboratory animals have been extensivelyl studied in recent years. The protein has been Shown.to be a component both, ofglial icells andneurons,where~it!has been foun d in the cytosol asweU as in particulate matter[ 3,5,11]- ~Nevertheless, there is general agreement that a prominent aliquot of the S-100 is o f glial origin, (for ireviews see refs. 2 and 17). ~ g in,mind thelnature of typicaiMS lesions, it is therefore likely thatglial~ceUs a r e t h e main ~source o f S-100•in CSF. Whether the ;appearance o f S,100in CSF follows a change in concentration o f the protein in tissue affected by MS remains to be' determined. Nevertheless it seems likely at present that the release o f t h e protein may be attributed to mere diffusion fromdamaged tissuerather than to~ active cellular secretion. Moreover, cwing t o the presence of proteases in MS damaged tissue [ 4,6,14], the possibility exists that aportion or all of the S-100 in CSF is present as an antigenically active fragment, rather than as the native protein. The occurrence of S.100 and/or its fragt~lents in CSF of diseased subjects also opens the possibility that an aliquot of the antigen is filtered into theblood. Since the S-100 i~ a normally sequestered immunogen, its appearance in biological fluids during a disease in which •immunological mechanisms are involved migbtiead to s~eculations. In this respect, work is in progress to detect anti.S-100 antibodies in CSF and serum of patients with various neurological diseases. However, although a specific interaction of S-100 with myelin basic protein has been reported [8], data are lacking at present to suggest a specific role of the S-100 protein in the pathogenesis of MS. In conc'~usion, the 8-100 in CSF might be a candidate for an index of active cell injury during t,he exacerbations in MS patients. Nevertheless, owing to the various localizatic:is actually reported for the protein in normal tissue, it ought to be regarded as ~ general marker of parenchymal injury in the nervous tissue rather than a selective index of demyelination, More extensive studies on selected patients affe~ted by various diseases are yet required to establish the usefulnes~ of the S-1,)0 as amarker of active cell injury specific to nervous parenchyma. Progress in basic studies on S-100 and other nervous systemspecific proteins (for reviews see refs. 2 and 17)might provide useful tools for a deeper understanding of the molecular features of neurological lesions. i
~.
~
~
•
ACKNOWLEDGEMENTS The authors are deeply indebted with Prof. G. Macchi and Prof. N. Miani for their encouragement and critical review of the manuscript. This investigation w u partially supported by C.N.R. contract no. 75.00637.04. REFERENCES
1 Adams,C,E.M., PatholOgy ofmultiple sclerosis: progress of the lesion. Brit. M~d. Bull., 33 (1977) 15--20. 2 Book,E., Nervous system specific proteins. J. Neurochem., 30 (1978) 7--14. 3 Cicero,T.J., Cowan, W., Moore, B.W. and Suntzeff; V., The cellular localization of the
175 two l~rain-specific proteins S-100 and 14.3.2,'Brain Res., 18 (1970) 25--34. 4 Cuzner, M.L. and Davison, A.N., Changes in ceJ'ebral lysosomal enzyme activity and lipids in multiple sclerosis, J. neurol. Sci., 19 (1973) 29--36. 5 Donato, R., Michetti, F. and Miani, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Res., 98 (1975) 661--573. 6 Einstein, E.R., Dalai, K.B. and Csejtey, J., Increased protease activity and changes in basic proteins and lipids in multiple sclerosis.plaques, J. neurol. Sci., 11 (1970) 109--121 7 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265--275. 8 Mahdik, S.P., Graf, L. :ind Rapport, M.M., Immunochemical studies of the interaction between myelin basic protein and S-100 protein, J. Neurochem., 27 (1976) 405--40P 9 Mancini, G.A., Carbon~ra, A.O. and Heremans, J.F., Immunochemical quantitation o antigens by single radi~ immunodiffusion, Immuno~hemistry, 2 (1965) 235--254. 10 McDonald, W.I. and H~,lliday, A.M., Diagnosis and classification of multiple sclerosis, Brit. IVied. Bull. 33 (1977) 4--8. 11 Michetti, F., Miani, N., DeRenzis, G., Caniglia, A. and Correr, S., Nuclear localization of S-100 protein, J. Neurochem. 22 (1974) 239--244. 12 Moore, B.W., A soluble protein characteristic of the nervous system: Biochem. Biophys. Res. Commun., 19 (1965) 739--744. 13 Moore, B.W. and Perez, V.J., Complement fixation for antigens at a picogram level, J. Immunol., 96 (1966) 1000--1005. 14 Riekkinen, P.J., Clausen, J., Fog, T. and Rinne, U.K., Acid proteinase activity of white matter and plaques in multiple sclerosis, Acta neurol, seand., 46 (1970) 349--353. 15 Thompson, E.J., Laboratory diagnosis of multiple sclerosis: immunological and biochemical aspects, Brit. Med. Bull., 33 (1977) 28--33. 16 Whitaker, J.N., Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis, Neurology, 27 (1977) 911--920. 17 Zomzely-Neurath, C. and Keller, A., Nervous system-specific proteins of vertebrate~: A search for functions and physiological roles, Neurochem. Res. 2 (1977) 353--377. 18 Zuckerman, J.E., Hersehman, H.R. and Levine, L., Appearance of a brain-specific antigen (the S-100 protein) during human foetal development, J. Neurochem. 17 (1970) 247--251.