Astroglial development in microencephalic rat brain after fetal methylazoxymethanol treatment

itfr.J.

Dd. Ncrtrosricwr. Printed in Great Britain.

073~S74XIRh $Ki.(nl+l~.Ol) PerBamon Journals Ltd. @ 19X6 ISDN

Vol. 4. NO. 4. PP. 1.5.3-362. IYXh.

ASTROGLIAL DEVELOPMENT IN MICROENCEPHALIC RAT BRAIN AFTER FETAL METHYLAZOXYMETHANOL TREATMENT M.

ERIKSDOTTER-NILSSON,

G.

JONSSON,

D.

DAHL*

and

H.

BJORKLUND

Department of Histology. Karolinska lnstitutet. Stockholm. Sweden. and *‘Department of Neur~~path~~l[~gy. Harvard Medical School and Spinal Cord Injury Research Lahoratory. West Roxhury Veterans Administratj~n Medical Center. Boston. MA 02132. U.S.A.

Abstract-Treatment of pregnant rats on gestation day 15 with methyluz~~xymethanol (MAM) leads to a marked micr~encephaiy in the offspring with a considerable atrophy in cerebral cortex. hipp~ampus and striatum. The development of the astrocytic populations in these atrophic regions was studied hy means of immunohistochemistry using an antiserum against glial fihrillary acidic protein (GFA). The distrihution and density of CFA-positive structures were not notahly altered in the parietal cortex. hippocampal formation and striatum after prenatal MAM-treatment as compared to control. Also the individual astrocytes were morphologically similar in experimental and control animals in all regions analyzed. We suggest that an adjustment of the astrocytic development has occurred in response to the changed neuronal environment. Alternatively, MAM-treatment may affect neuronal and glial precursor cells leading to a seemingly normal astrocytic cell density. Key w,or&: Methylazoxymethanol (MAM). Cerebral cortex. Hippocampal Astrocytes, Glial fihrillary acidic protein (GFA). Immunofluorescence.

formation,

Striatum.

The antimitotic agent mcthylazoxym~thanol (MAM) is selectively toxic for dividing cells by methylating guanine nucleotides. ” Administration of MAM to pregnant rats causes a severe and permanent atrophy of those regions in the central nervous system of the offspring where cells proliferate during the time of drug administration.“’ Injection on gestational day 15 will produce a marked atrophy in several forebrain structures including the cerebral cortex, the hippocampal formation and striatum while the brainstem is largely unaffected. 12~‘.f~2x*4’ Moreover, while the intrinsic neuronal population in cortex cerebri is considerably reduced, a relative hyperinnervation of monoamine nerve terminals is seen. if+-” Glial fibrillary acidic protein (GFA) is the major component of astrocyte intermediate filaments and GFA immunohistochemistry has been extensively used as a marker for astrocytes in studies of both normal and reactive astrocytes.‘.” Brain injury leads to an astrocytic hypertrophy and accumulation of GFA within individual cells.2~4*h~2’~27 Also during aging, the astrocytes increase in size, ‘.*” although the total number of cells does not seem to be signi~cantly altered.2”~-1”~J” In the adult animal neuronal cell reduction caused by trauma, old age and certain neurotoxins (e.g. the endogenous excitotoxin quinolinic acid)’ seems to play a crucial role for the development of a gliotic reaction (hypertrophy and/or increase in number of astrocytes). The present study was therefore undertaken to analyze the development of astroglial population visualized by GFA immunohistochemistry in MAM-induced microencephalic rat brain. It was hypothesized that this experimental model would provide information on neuron-astroglia interaction during development. The study would also elucidate whether or not a reduction in neuronal number initiated before the major astrocytic development might cause gliosis. MATERIALS

AND METHODS

MAM (Schwa~~Mann) was administered i.v. in a dose of 25 mglkg to pregnant rats (SpragueDawley, Alab, Stockholm, Sweden) on gestational day I5 under deep ether anesthesia. MAM was. dissolved in 0.9% NaCl and injected in a concentration of 10 mg/ml. Controls were injected with an equal volume of the solvent alone. The litters were culled to 8-10 pups and weaned at approximately 3 weeks of age. Address correspondence 60400. S-104 01 Stockholm,

to: Dr Maria Sweden.

Eriksdotter-Nilsson.

353

Department

of Histology.

Karolinska

institutet.

Box

354

M. Eriksdottcr-Nilsson

cf nl.

Seventeen animals (9 experimental and 8 controls) ranging in age from 6 to IX weeks (however. in each experiment, experimental and control animals were identical in age) were killed by exsanguination under deep ether anesthesia and the brains removed. Using razor blades. a frontal slice extending from the optic chiasm to a level 5 mm caudally was prepared. The tissue was frozen on dry ice and sectioned at 14 km on a cryostat. After fixation in acetone for 3 min, sections were processed according to the indirect immuno~uorescence technique of Coons.” GFA antiserum raised in rabbits”’ was used and diluted 1: 100 in phosphate-buffered saline (PBS). Preimmune serum and antiserum absorbed with its proper antigen were used as controls. After a quick rinsing in PBS, sections were incubated with the antiserum at 4°C in a humid atmosphere overnight. After rinsing in PBS 3 x IO min. sections were incubated in rhodamine-conjugated sheep antirabbit antibodies (Dako Patts, Denmark), diluted I:50 in PBS for 1 hr in darkness at room temperature. After a second rinse in PBS, sections were mounted in 90% glycerine in PBS. To reduce fading, 0.1% phenylene diamine was added to the mounting medium.” Both antisera contained 0.3% (v/v) Triton X-100. The sections were examined in fluorescence microscopes equipped with dark field and epi-illumination. Tri-X and Panatomic-X films (Kodak) were used for photography. Representative sections were stained with Cresyl Violet and examined by light microscopy. Although proper controls for testing specificity of the immunofluorescence were included, the present technique does not permit absolute identification of GFA. Thus, ‘positive’ findings should be interpreted as ‘GFA-like’ immunoreactivity. Stained coronal sections with parietal cortex and the hippocampal formation of four experimental and four control animals were subjected to computer-assisted image analysis using the IBAS interactive image-analysis system (Kontron). A Zeiss fluorescence microscope equipped with epi-illumination and a TV-camera was attached to it. The fluorescence microscopical image (primary magnification x 25) was picked up by the TV-camera and digitized into 512 x 728 pixels, each with a gray level between 0 and 255. The IBAS image analyzer was programmed to adjust for uneven background illumination and to enhance contrast. Thereafter a gray level threshold was chosen to select for GFA-positive fluorescent structures excluding virtually all background intensity. After proper gray level definition and generation of the binary picture, the computer calculated the area covered by GFA-immunofluorescence in percent of the field of view (rectangular measuring field; size: 255 x 364 pm at x 25). Assuming that the size and the fluorescence morphology of the astrocytes demonstrated by the procedure used are relatively uniform, the “% values calculated by the computer can be considered to be an index or a measure of the relative number of GFA-positive astrocytes per unit area in the region analyzed. From each animal three representative sections were selected and analyzed and in each section, three areas (areas l-3) of the cerebral cortex were measured: one area close to the midline (area I), and two areas (areas 2 and 3) at defined distances from the midline (Fig. 5). Because of the larger size of the control cortex. it was necessary to perform three measurements to cover the entire depth of the cortex. The three values obtained were averaged to represent one observation. In the hippocampal formation the CA4 area was measured (Fig. 5).

RESULTS As expected, the brains of the MAM-treated animals were macroscopically abnormal with a considerable reduction of the volume of the cerebral cortex. This abnormal development of the cerebral cortex was also clearly seen in Cresyl Violet-stained sections. showing that the thickness of the cerebral cortex was approximately 4%50% of that seen in control animals (Fig. 1). The immunocytochemical analysis showed that the astrocytes appeared to have adapted to the reduced cortical volume. It was thus consistently observed that the density of GFA-positive structures was largely unchanged as compared to control (Fig. 2). Only in the part of the parietal cortex immediately adjacent to the midline a relatively weak gliosis was occasionally observed. The fluorescence morphology (cell size, number, length and thickness of processes) of the individual astrocytes did not exhibit any discernable difference in experimental and control animals (Fig. 2c and d). Although both the hippocampal formation and the striatum were reduced in size and atrophic after MAM treatment, similar to the situation in cerebral cortex, no difference in

Astroglial

development

in MAM-treated

rats

Fig. 1. Cresyl Violet-stained coronal sections illustrating the decreased depth of the parietal cortex from a MAM-treated W-week-old rat (a). as compared to an IS-week-old control animal (b). Arrows indicate the border between cerebral cortex and corpus callosum. X 50.

M. Eriksdotter-Nilsson

et al.

Fig. 2. Comparison of GFA-positive structures in parietal cortex of a 6-week-old rat treated with MAM (a and c), or NaCl (b and d). Although the depth of the cerebral cortex is clearly reduced in (a), no changes in density of GFA-positive structures can be observed as compared to (b). In (c) and (d) individual GFA-positive astrocytes are shown. Arrows indicate the border between cerebral cortex and corpus callosum. (a) and (b) x 135; (c) and (d) X 330.

Astroglial

development

in MAM-treated

rats

Fig. 3. GFA-positive structures in the hippocampal formation of a 6-week-old MAM-treated (a and c) and a control animal (b and d). Stratum moleculare (SM) is greatly reduced in size following MAM treatment (a). No difference in density and distribution of GFA-positive cells and fibers is observed as compared to the control brain (b). g= granular layer. (a) and (b) x 100; (c) and (d) X 135.

357

358

M. Eriks~ott~r-Noises

er al.

Fig. 4. GFA-like immunoreactivity in striatum of a Itweek-old MAM-treated rat (a) as compared to a control rat (b). No differences in the density and distribution of GFA-positive cells and fibers are observed. x 135.

Astroglial

development

in MAM-treated

rats

359

terms

of GFA-like immunoreactivity could be detected between MAM-treated and control in any of the two regions (Figs 3 and 4). Similar results were obtained in all animals studied, although the age of the rats analyzed varied between 6 and I8 weeks. The postnatal age thus did not seem to influence the relation between the density and morphology of GFA-positive structures in experimental as compared to age-matched control animals. The results from the image analysis of GFA-immunoreactivity in the parietal cortex and the hippocampal formation were in agreement with our microscopical findings. The percentage of GFA immunofluorescence (index of astrocyte density) after MAM was similar to the corresponding regions in controls. No significant difference between the percentage values obtained from the different areas of the cerebral cortex was recorded, neither in MAM-treated nor in controls (Fig. 5, Table 1). CONTROL

MAM

Fig. 5. Schematic illustration of coronal sections of control and MAM-treated rat brain at approximately mid-hypothalamic level. The dotted areas indicate the regions of the cerebral cortex (Cx. areas l-3) and hippocampal formation (Hf. CA4) where the quantitative measurements of GFA-immunoreactivity were carried out by image analysis.

Table

I. Effect of MAM

treatment on GFA immunofluorescence in rat cerebral hippocampus as analyzed by image analysis Cerebral

Area MAM Control

1

9.9 + 0.7 g.9r I.3

cortex and

cortex

Area 2

Area 3

10.2 2 0.8 9.4 k 0.x

10.3 + 0.8 8.92 0.8

Hippocampal formation (CA4) 12.8+-0.8 12.0-t 0.8

Mean k S.E.M. (n = 12). expressed as the area covered by GFA-immunofluorescence in percent of the field of view (measuring field). For definition of the various areas where the measurements were made. see Fig. 5 and Materials and Methods.

DISCUSSION As reported earlier,I2.I~.I7.‘X.~~.4I treatment with the antimitotic neurotoxin MAM on gestational day I5 causes a profound and permanent forebrain atrophy, especially severe in the cerebral cortex and the hippocampal formation, where a weight reduction of approximately 50% is observed; however, striatum is also moderately affected.lh In the present paper, we demonstrate that these gross morphological changes are accompanied by a corresponding reduction in the total amount of GFA-positive astrocytes. Thus, no changes in relative density or distribution of GFA-positive astrocytes in either parietal cortex cerebri, the hippocampal formation or striatum were seen in the MAM-treated animals. Furthermore, individual astrocytes had apparently unaltered morphology. Consequently, the total number of astrocytes in the affected areas is in all probability reduced since a relative gliosis would otherwise have occurred. It is generally believed that glial precursor cells are localized in the subependymal layer in the external wall of the lateral ventricles.‘.3’ Data available indicate that astrocytic proliferation occurs relatively late in ontogeny, one exception being radial glial cells, which in the primate have been shown to exist during the first third of gestation prior to the last neuronal cell division.2’ Radial glial cells have been suggested to later develop into morphologically normal astrocytes.‘h In the rat, GFA-positive astrocytes are first observed on gestational day 18 in the medial wall of the lateral ventricle.‘.“J Since only cells in active mitosis are affected by the MAM treatment, it

360

M. Eriksdotter-Nilsson

et ai.

seems less likely that the reduced number of GFA-positive astrocytes is due to a direct effect of the neurotoxin. It can however not be completely ruled out that some actively dividing glial precursor cells”.“’ in the subependymal layer are eliminated by the MAM treatment. If so, a reduced number of GFA-positive astrocytes could eventually be the result. However, if this mechanism is the sole reason for the readjusted total number of astrocytes. it seems highly improbable that astrocytic precursors are affected in the correct proportion, resulting in a normal final cell density. Since, at least in the monkey, radial glial cells do not divide during the peak of neurogenesiz? they are probably not affected by the drug treatment. A predestined program at the precursor level determining the final number of astrocytes is less likely in view of our findings, since this would have led to a relative increase in astrocytic numbers in MAM-treated animals. Earlier studies have shown that animals treated with MAM on gestational day 15 show a monoaminergic hyperinnervation in those brain regions affected by the toxin.‘J.‘h-‘” The monamine cell-body groups in the rat brain have been shown to appear and mature between gestational day I2 and 14.“.3’ Therefore such neurons should be insensitive to the antimitotic effect of MAM administered on gestational day 15. Although noradrenergic axons at this time have reached the primordial neocortex and an extensive cortical innervation during the neonatal period will develop,3’ further studies have suggested that the development of the noradrenergic axonal arborization essentially is insensitive to the neuronal loss in its terminal field.” Similarly, a relative cholinergic hyperinnervation of the cortex is also seen, although to a lesser extent.” It is hypothesized that there is one intrinsic and one extrinsic component of cortical cholinergic innervation, the intrinsic population being reduced by MAM treatment. and the extrinsic innervation unaffected, since the latter invades the cortex at approximately day 12 postnatally.” Consequently, the monoaminergic and the extrinsic cholinergic systems are unlikely to influence the astrocytic development in MAM-treated animals. GABAergic neurons, which are intrinsic to the neocortex and distributed uniformly throughout all layers,‘5.3” are reduced in proportion to the reduction in cortical mass.” Of course, interneurons other than GABA-containing ones also are severely reduced in number. One might speculate that the number of local neurons influences the astrocytic development. The mechanism is unknown, but one possibility is that some trophic factors stimulating proliferation are released in proportion to the amount of local neurons. Alternatively, the reduced cortical tissue, and thus the available space, may be the limiting factor if the astrocytes actually are predetermined to a certain cell density. Thus, the reduced intrinsic neuronal population in the brain regions affected by MAM may play an important role in determining growth and development of the astrocytic population, whereas the extrinsic innervation may have little influence on this development. In conclusion, prenatal exposure to the neurotoxin MAM causes a considerable reduction of the astroglial populations demonstrable by GFA-immunohistochemistry in several brain regions including cortex cerebri, the hippocampal formation and striatum, regions exhibiting a marked atrophy after MAM. The fluorescence morphology of the individual astrocytes also appear to be unaltered in the atrophic regions. Astrocytic development thus appears to adapt to the severe neuronal losses in these brain areas. resulting in an apparently unaltered density and distribution of GFA-immunoreactive structures. A direct effect of MAM treatment on astrocytic precursors cannot be fully ruled out, although this alternative appears less likely. We therefore suggest that on gestational day I5 the astrocytic development is not predetermined, and that instead a readjustment to the changed neuronal development occurs. Ack,low(c~~~rmc,,tr,s-This work was supported by the Swedish Medical Research Council (14X-03185, 12P-7310. 04X2295). Loo and Hans Ostermans Foundation, the Expressen Prenatal Research Foundation and Karolinska lnstitutcts fonder. Dr Dahl was supported by the Veterans Administration. For valuable discussions, we are grateful to Dr Lars Olson. We wish to thank Lena Hultgren. Karin Lundstriimer. Barbro Standwerth and Eva Lindqvist for skillful technical assistance and Ida Engqvist for secretarial help.

REFERENCES I.

Aitman J. (1966) Proliferation and migration giiogenesis. E.t-p. Ne~m~/. 16, 26% 27X. 2. Amaducci L., Forno K. I. and Eng L. F. (1981) Nettrosci. Let!. 21. 27-32.

of undifferentiated Glial fibrillary

precursor

acidic protein

cells in the in crytlgenic

rat during

postnatal

lesions of the rat brain.

Astroglial

development

in MAM-treated

rats

361

3. Bignami A. and Dahl D. (1974)

Astrocyte specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary protein. J. camp. Neltrol. 153, 27-38. 4. Bignami A. and Dahl D. (1976) The astroglial response to stabbing. lmmunofluorescence studies with antibodies to astrocyte-specific protein (GFA) in mammalian and submammalian vertebrates. N<*~roparh. appl. Newohio/. 2, 99-

I I I. 5. Bignami A.. Dahl D. and Rueger C. (1980) Glial tibrillary acidic (GFA) protein in normal neural cells and in pathological conditions. In Advmccs in Celhdrrr Neurobiology. Vol. I (eds Fedoroff S. and Hertz L.). pp, 285-319. Academic Press. New York. 6. Bjiirklund H.. Dahl D. and Olson L. (1984) Morphometry of GFA and vimentin positive astrocytes in grafted and lesioned cortex cerebri. /tar. J. devl Nrwosci. 2, 1X1-192. 7. Bjiirklund H.. Eriksdotter-Nilsson M., Dahl D.. Rose G.. Hoffer B. and Olson L. (1985) Image analysis of GFApositive astrocytes from adolescence to senescence. Esp. Bruin Res. 58, 163-170. x. BjBrklund H.. Olson L.. Dahl D. and Schwartz R. (1985) Short- and long-term consequences of intracranial injections of the excitotoxin quinolinic acid as evidenced by GFA immunohistochemistry of astrocytes. Brcrin Res. (in press). 9. Coons A. H. (1958) Fluorescent antibody methods. In Gc*rlc,m/ Cyrochetnical Me/hod.y (ed. J. F. Danielli). pp. 399422. Academic Press, New York. 10. Dahl D. and Bignami A. (1976) Immunogenic properties of the glial fibrillary acidic protein. Brnin Rev. 116, 150-157. II. Eng L. F. and DeArmond S. J. (1982) lmmunocytochemical studies of astrocytes in normal development and disease. In Advcmces in Cell~rkrr Newohiology. Vol. 3 (eds Fedoroff S. and Hertz L.). pp. 14.5-170. Academic Press. New York. 12. Fischer M. H.. Welker C. and Waisman H. A. (1972) Generalized growth retardation in rats induced by prenatal exposurc to methylazoxymethanol acetate. Teratology 5, 223-232. IS. Haddad R. K.. Rabe A.. Laquer G. L., Spatz M. and Valsamis M. P. (1969) Intellectual deficit associated with transplacentally induced microcephaly in the rat. Science 163, 8X-90. 14. Hallman H. and Jonsson G. (1984) Monoamine neurotransmitter metabolism in microencephalic rat brain after prenatal methylazoxymethanol treatment. Braitl Res. Bull. 13, 3X3-389. IS. Johnson G. D. and de la Nogueira-Araujo G. M. (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J. /mmrno/. Merh. 43, 349-350. 16. Jonsson G. and Hallman H. (1982) Effects of prenatal methylazoxymethanol treatment on the development of central monoamine neurons. Devl Bruin Res. 2, 513-530. 17. Johnston M. V. and Coyle J. T. (1979) Histological and neurochemical effects of fetal treatment with methylazoxymethanol on rat neocortex in adulthood. Brain Res. 170, 13.5-155. 18. Johnston M. V.. Grzanna R. and Coyle J. T. (1979) Methylazoxymethanol treatment of fetal rats results in abnormally dense noradrenergic innervation of neocortex. Science 203, 369-371. 19. Johnston M. V. and Coyle J. T. (1980) Ontogeny of neurochemical markers for noradrenergic GABAergic and cholinergic neurons in neocortex lesioned with methylazoxymethanol acetate. J. Nectrochem. 34, 1429-1441. 20. Landfield P. W.. Rose G., Sandles L., Wohlstadter T. C. and Lynch G. (1977) Patterns of astroglial hypertrophy and neuronal degeneration in the hippocampus of aged, memory-deficient rats. J. Gcrontol. 32, .3-12. 21. Latov N.. Nilaver G.. Zimmerman E. A.. Johnson W. G., Silverman A.-J.. Defendini R. and Cotc L. (1979) Fibrillary astrocytes proliferate in response to brain injury: a study combining immunoperoxidase technique for glial fibrillary acidic protein and radioautography of tritiated thymidine. Devl Biol. 72, 381-384. 22. Lauder J. M. and Bloom F. E. (1974) Ontogeny of monoamine neurons in the locus coeruleus. raphe nuclei and suhstantia nigra of the rat. I. Cell differentiation. J. camp. Nerrrol. 155, 469-482. 23. Levitt P.. Cooper M. L. and Rakic P. (1981) Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey. An ultrasrructural immunoperoxidase analysis. J. Nectrosci. I, 27-39. 24. Levitt P., Cooper M. L. and Rakic P. (1983) Early divergence and changing proportions of neuronal and glial precursor cells in the primate cerebral ventricular zone. Devl Biol. %, 472484. 25. Levitt P. and Rakic P. (1980) lmmunopcroxidase localization of glial fibrillary acidic protein in radial glial cells and astrocytes of the developing rhesus monkey brain. J. camp. Netrrol. 193, 815-840. 26. Ling E. A.. Paterson J. A.. Privat A., Mori S. and Leblond C. P. (1973) Investigation of glial cells in semithin sections. I. Identification of glial cells in the brain of young rats. J. camp. Newel. 149, 43-71. 27. Luse S. (196X) Pathology of rhe Nervous Sysfem. Vol. I (ed. Winkler J.). pp. 538-553. McGraw-Hill. New York. 28. Matsumoto H.. Spatz M. and Laquer G. L. (1972) Quantitative changes with age in the DNA content of methylazoxymethanol-induced microencephalic rat brain. J. Neuroche,n. 19, 297-306. 29. Nagata Y. and Matsumoto H. (1969) Studies on methylazoxymethanol: methylation of nucleic acids in the fetal rat brain. Proc. Sot. cxp. hiol. Med. 132, X3-385. 30. O’Kusky J. and Colonnier M. (1982) Postnatal changes in the number of astrocytes. oligodendrocytes and microglia in the visual cortex (area 17) of the macaque monkey: a stereological analysis of normal and monocularly deprived animals. J. cornp. Neural. 210, 307-315. 31. Olson L. and Seiger A. (1972) Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations. Z. Anat. EnrwGe.sch. 137, 301-316. 32. Olson L. and Seiger A. (1973) Late prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations. Z. Am/. EnrwGesch. 140, 281-318. 33. Privat A. and Leblond C. P. (1972) The subependymal layer and neighboring region in the brain of the young rat. J. camp. Neltrol. 146, 277-302. 34. Raju T.. Bignami A. and Dahl D. (1981) In vivo and iu vitro differentiation of neurons and astrocytes in the rat embryo: immunofluorescence study with neurofilament and glial filament antisera. Devl Biol. 85, 344-357. 3.5. Ribak C. E. (1978) Aspinous and sparsely spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase. Neurocytology 7, 461478. 36. Schmechel D. E. and Rakic P. (1979) A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes. Anar. Embryo/. 156, 115-152. 37. Schultze B.. Nowak B. and Maurer W. (1974) Cycle times of the neural epithelial cells of various types of neurons in the rat. An autoradiographic study. J. camp. Nerrrol. 158, 207-218. DN 4:4-o

362

M. Eriksdotter-Nilsson

et al.

3X. Spatz M. and Luquer G. L. (iY68) Transplacental chemical induction of microencephaly in two strains of rats. Proc. Sot. exp. hid. Med. 129, 70.5-7 IO. 39. Storm-Muthisen J. (1976) Distrihution of the components of the GABA system in neuronal tissue: cerebellum and hippocampus effects of axotomy. In GARA in Akrvotrs Sysfwn Ftmctiorc (cds Roberts E.. Chase T. N. and Tower D. B.). pp. IJY-168. Raven Press. New York. 40. Vaughan D. W. and Peters A. (1974) Neuroglial cell in the cerebral cortex of rats from young adulthood to old age: an electron microscope study. J. Neuror~rol. 3. 405429. 41. Welker C.. Fischer M. H. and Waisman H. A. (lY71) Postnatal development of the brain in microencephalic rats. Amt. Rev. 169. 452.