Breaches of the pial basement membrane and disappearance of the glia limitans during development underlie the cortical lamination defect in the mouse model of muscle-eye-brain disease

Neuronal overmigration is the underlying cellular mechanism of cerebral cortical malformations in syndromes of congenital muscular dystrophies caused by defects in O-mannosyl glycosylation. Overmigration involves multiple developmental abnormalities in the brain surface basement membrane, Cajal-Retzius cells, and radial glia. We tested the hypothesis that breaches in basement membrane and the underlying glia limitans are the key initial events of the cellular pathomechanisms by carrying out a detailed developmental study with a mouse model of muscle-eye-brain disease, mice deficient in O-mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1). The pial basement membrane was normal in the knockout mouse at E11.5. It was breached during rapid cerebral cortical expansion at E13.5. Radial glial endfeet, which comprise glia limitans, grew out of the neural boundary. Neurons moved out of the neural boundary through these breaches. The overgrown radial glia and emigrated neurons disrupted the overlying pia mater. The overmigrated neurons did not participate in cortical plate (CP) development; rather they formed a diffuse cell zone (DCZ) outside the original cortical boundary. Together, the DCZ and the CP formed the knockout cerebral cortex, with disappearance of the basement membrane and the glia limitans. These results suggest that disappearance of the basement membrane and the glia limitans at the cerebral cortical surface during development underlies cortical lamination defects in congenital muscular dystrophies and a cellular mechanism of cortical malformation distinct from that of the reeler mouse, double cortex syndrome, and periventricular heterotopia.     

Breaches of the pial basement membrane and disappearance of the glia limitans during development underlie the cortical lamination defect in the mouse model of muscle-eye-brain disease

Neuronal overmigration is the underlying cellular mechanism of cerebral cortical malformations in syndromes of congenital muscular dystrophies caused by defects in O-mannosyl glycosylation. Overmigration involves multiple developmental abnormalities in the brain surface basement membrane, Cajal-Retzius cells, and radial glia. We tested the hypothesis that breaches in basement membrane and the underlying glia limitans are the key initial events of the cellular pathomechanisms by carrying out a detailed developmental study with a mouse model of muscle-eye-brain disease, mice deficient in O-mannose beta31,2-N-acetylglucosaminyltransferase 1 (POMGnT1). The pial basement membrane was normal in the knockout mouse at E11.5. It was breached during rapid cerebral cortical expansion at E13.5. Radial glial endfeet, which comprise glia limitans, grew out of the neural boundary. Neurons moved out of the neural boundary through these breaches. The overgrown radial glia and emigrated neurons disrupted the overlying pia mater. The overmigrated neurons did not participate in cortical plate (CP) development; rather they formed a diffuse cell zone (DCZ) outside the original cortical boundary. Together, the DCZ and the CP formed the knockout cerebral cortex, with disappearance of the basement membrane and the glia limitans. These results suggest that disappearance of the basement membrane and the glia limitans at the cerebral cortical surface during development underlies cortical lamination defects in congenital muscular dystrophies and a cellular mechanism of cortical malformation distinct from that of the reeler mouse, double cortex syndrome, and periventricular heterotopia.     

Fukutin expression in glial cells and neurons: implication in the brain lesions of Fukuyama congenital muscular dystrophy

Expression and localization of fukutin, a gene responsible for Fukuyama congenital muscular dystrophy (FCMD), was studied in the central nervous system by in situ hybridization and immunohistochemistry. In control cases, glial cells expressed fukutin and the expression continued from fetuses to adults. Double immunostaining revealed that some of these cells were astrocytes. The glia limitans was stained by immunohistochemistry. In contrast, neuronal expression was decreased with neuronal maturation. The glia limitans formed by endfeet of astrocytes is abnormal in the brain of fetal to adult FCMD cases. These findings suggest an important role of astrocytes for the genesis of FCMD brain, although immature neurons expressed fukutin. In FCMD cases, expression of fukutin looked decreased. In the brain of fetal FCMD cases, decreased expression of fukutin is considered to provoke the disruption of glia limitans. In post-natal FCMD cases, prominent superficial gliosis is observed in the cerebral surface, where fukutin was weakly positive. Reactive increase of astrocytes may be required to maintain the glia limitans for compensating the decrease of fukutin expression in individual astrocytes. In the cerebellum, Bergmann glia, which did not express fukutin in control cases, elongated their cytoplasmic processes to the surface to form glia limitans even in the polymicrogyric area.     

Distribution patterns of estrogen receptor alpha and beta in the human cortex and hippocampus during development and adulthood

The expression of estrogen receptors (ERs) in the developing and adult human brain has not been clearly established, although estrogens are crucial for neuronal differentiation, synapse formation, and cognitive functions. By using immunohistochemistry, we have studied the distribution of ER alpha and ER beta in human cerebral cortex and hippocampus from early prenatal stages to adult life. ER alpha was detected in the cortex at 9 gestational weeks (GW), with a high expression in proliferating zones and the cortical plate. The staining intensity decreased gradually during prenatal development but increased again from birth to adulthood. In contrast, ER beta was first detected at 15 GW in proliferating zones, and at 16/17 GW, numerous ER beta immunopositive cells were also observed in the cortical plate. ER beta expression persisted in the adult cortex, being widely distributed throughout cortical layers II-VI. In addition, from around 15 GW to adulthood, ER alpha and ER beta were expressed in human hippocampus mainly in pyramidal cells of Ammon's horn and in the dentate gyrus. Western blotting and immunohistochemistry in the adult cerebral cortex and hippocampus revealed lower protein expression of ER alpha compared with ER beta. Double immunostaining showed that during fetal life both ERs are expressed in neurons as well as in radial glia, although only ER alpha is expressed in the Cajal-Retzius neurons of the marginal zone. These observations demonstrate that the expression of ER alpha and ER beta displays different spatial-temporal patterns during human cortical and hippocampal development and suggest that both ERs may play distinct roles in several processes related to prenatal brain development.     

Expression of glial cell line-derived neurotrophic factor in the brain and cerebrospinal fluid of the developing rat

Expression of GDNF in developing rat brain from PND 1 to 14 and on PND 21 was examined immunocytochemically. At PND 1, intense diffuse immunoreactivity was noted within the cytoplasm of a diverse group of neuronal and nonneuronal cells, including choroid plexus epithelial cells, ependymal cells, tanycytes of the third ventricle, and cellular elements in the subarachnoid compartment. GDNF expression became more localized among these cells from PND 7-14 and was almost undetectable by PND 21. Although GDNF-positive small glial cells were scattered within the cerebral cortical plate and the striatum already at PND 1, GDNF expression among astroglial cells within the corpus callosum and in the white matter adjacent to the lateral ventricles was more prominent between PND 5 and 8. GDNF expression among the pyramidal neurons of the cerebral cortex was evident relatively early in the postnatal period, but the neurons of the hippocampus and thalamus showed more intense immunoreactivity at later periods between PND 8-14. ELISA of the CSF revealed a rapid rise in GDNF levels from 71.4 +/- 10.9 pg/ml (mean +/- S.E.M.) at PND 1 to peak levels of 138.4 +/- 18.5, 135.1 +/- 5.4 and 132.9 +/- 8.0 pg/ml, at PND 5, 7 and 9, respectively. Peak CSF levels of GDNF occurred when GDNF expression was intense within astroglial cells in the corpus callosum and cerebral white matter. Thereafter, the levels gradually decreased to 76.5 +/- 9.7 pg/ml at PND 21. The widespread expression of GDNF among different cellular elements in the developing brain suggest that GDNF probably plays diverse functional roles in many different neuronal systems in addition to its known effects on the dopaminergic system. Developmental shifts in GDNF expression further suggest that GDNF may be of critical importance at different stages of brain growth and differentiation.     

Developmental events during the early stages of cerebral cortical neurogenesis in man

Summary The telencephalic vesicles of early human embryos and fetuses ranging from 6–18 weeks of ovulation age were subjected to correlative light and electron microscopic, immunocytochemical and Golgi analysis. The cellular events that take place during the early stages of cortical neurogenesis are characterized by the early maturation of layer I. The deployment of radial glia, the formation of the glia limitans, the establishment of the pial-glial barrier, and the migration and maturation of Cajal-Retzius cells within the marginal zone (MZ) all take place by 6–7 weeks. Neurons destined to populate the cortical plate (CP) arrive within the MZ by 7–8 weeks of age and appear to divide the MZ into two layers. Progressive compaction of CP neurons and widening of the intermediate zone characterize the next stage. It is suggested that the early establishment of the pial-glial barrier and the early maturation of the layer I are critical to the subsequent growth and development of the CP.Abbreviations CP Cortical plate - CR Cajal-Retzius - EM electron microscopy - GFAP glial fibrillary acidic protein - GL glial limitans - IZ intermediate zone - ML molecular layer - MZ marginal zone - NSE neuron-specific enolase - SVZ subventricular zone - VZ ventricular zone Supported in part by USPHS grant no. ES 02928. Presented at the 62nd annual meeting of the American Association of Neuropathologists, Minneapolis, 1986     

Disabled-1 mRNA and protein expression in developing human cortex

Disabled-1 (Dab1) forms part of the Reelin-Dab1 signalling pathway that controls neuronal positioning during brain development; Dab1 deficiency gives rise to a reeler-like inversion of cortical layers. To establish a timetable of Dab1 expression in developing human brain, Dab1 mRNA and protein expression were studied in prenatal human cortex. The earliest Dab1 signal was detected at 7 gestational weeks (GW), the stage of transition from preplate to cortical plate, suggesting a role of the Reelin-Dab1 signalling pathway in preplate partition. From 12 to 20 GW, the period of maximum cortical migration, Dab1 expression was prominent in the upper tiers of the cortical plate, to decline after midgestation. Radially orientated apical dendrites of Dab1-expressing neurons indicated a predominant pyramidal phenotype. Pyramidal cells in hippocampus and entorhinal cortex displayed a more protracted time of Dab1 expression compared to neocortex. In addition, at later stages (18-25 GW), Dab1 was also expressed in large neurons scattered throughout intermediate zone and subplate. From 14 to 22 GW, particularly high levels of Dab1 mRNA and protein were observed in cells of the ventricular/subventricular zone displaying the morphology of radial glia. The partial colocalization of vimentin and Dab1 in cells of the ventricular zone supported a radial glia phenotype. The concentration of Dab1 protein in ventricular endfeet and initial portions of radial processes of ventricular-zone cells points to a possible involvement of Dab1 in neurogenesis. Furthermore, a subset of Cajal-Retzius cells in the marginal zone colocalized Dab1 and Reelin, and may thus represent a novel target of the Reelin-Dab1 signalling pathway.     

Development of glial cells in the cerebral wall of ferrets: direct tracing of their transformation from radial glia into astrocytes

Coronal sections of the cerebral wall from developing ferrets (newborn to adult) were double-stained with antibodies to vimentin and glial fibrillary acidic protein (GFAP). At birth, the dominant glial population was radial glia and these cells labeled only for vimentin. A small population of immature astrocytes in the cortical plate was double labeled for GFAP and vimentin. In successive days, the number of vimentin-positive radial glia gradually decreased and they disappeared entirely at about 21 days. During this same period, the double-stained astrocytes increased in number and were distributed throughout the cortical plate and intermediate zone. After 6 weeks of age the astrocytes were mostly confined to the developing white matter. Around this time they gradually lost their vimentin staining, and in the adult no vimentin-positive elements were seen except at the ependymal surface. In newborn ferrets single radial glial cells were also visualized by applying the carbocyanine dye DiI onto the pial surface of fixed brains. While most radial glia extended from the ventricular zone to the pial surface, a substantial fraction of them had lost their contact to the ventricular zone. Their somata were displaced into the subventricular zone and lower portion of the intermediate zone. The possibility that radial glia transform into astrocytes was directly tested by injecting fluorescent dyes under the pial surface of newborn ferrets at a time when virtually no GFAP-positive astrocytes are present. The tracer, which was taken up in the upper portion of the cortical plate, stained the radial glial cell somata in the ventricular zone in a similar way as the dye DiI did in the fixed brains. As the radial glial cells disappeared at successively longer survival times, the tracer was ultimately found within newly formed GFAP-positive astrocytes. These results provide strong support for the hypothesis that radial glia cells are the immature form of astrocytes (Choi and Lapham: Brain Res. 148:295-311, '78; Schmechel and Rakic: Anat. Embryol. (Berl.) 156:115-152, '79), and also show that, at least in the ferret cortex, the transformation is accompanied by a change in the expression of intermediate filament protein.     

Immature astrocytes in Fukuyama congenital muscular dystrophy: an immunohistochemical study

Recent studies of Fukuyama congenital muscular dystrophy have focused on abnormalities of the basement membrane in muscle and brain. The cerebral cortex has a unique basement membrane at the glia limitans, which is intimately related to astrocytes in the developing brain, and the basement membrane may be partially produced by the astrocyte. In this study the cerebral astrocytes in six patients with Fukuyama congenital muscular dystrophy, including two fetal patients, were characterized by immunohistochemical study. In fetal Fukuyama congenital muscular dystrophy, astrocytes reacted less to antibodies of glial fibrillary acidic protein, S-100 protein, and alphaB-crystallin than control astrocytes, but in postnatal Fukuyama congenital muscular dystrophy, astrocytes reacted more to these antibodies and displayed beading of processes. Moreover, vimentin was positive in the astrocytes of two postnatal Fukuyama congenital muscular dystrophy patients. This astrocytic appearance may suggest immaturity of astrocytes in Fukuyama congenital muscular dystrophy. Astrocytes exhibiting beaded cytoplasmic processes were prominent at the subpia of the cortex and around vessels. The authors hypothesize that these immature astrocytes are unable to participate in the function of the cortical basement membrane, which is defective in Fukuyama congenital muscular dystrophy. Studies of neurons and meninges were similar to those of control subjects.     

Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol

In order to study estrogen effects on developing human neurons, we have established primary cultures of neurons and glia from 8-13-week human embryo cortex and spinal cord. The neuronal identity of the cultures was verified using the neuronal synaptic vesicle and neuronal endosomal membrane markers synaptotagmin, synapsin and synaptophysin, and the glial contribution to the mixed glial-neuronal cultures was verified using the glial marker glial fibrillary acidic protein (GFAP). We here report expression of estrogen receptor beta (ERbeta) in these cells using RT-PCR and sequencing, RNAse protection assay, immunohistochemistry and immunoblotting. We found that both neuronal and mixed glial-neuronal cultures expressed ERbeta. Treatment with 17beta-estradiol gave an increased expression of ERbeta in both types of cultures. These results suggest that ERbeta is expressed in fetal brain and thus may mediate effects of estrogen in the developing nervous system. Furthermore, the results suggest that expression of ERbeta in fetal brain may be regulated by estrogen.     

Are breaches in the glia limitans the primary cause of the micropolygyria in Fukuyama-type congenital muscular dystrophy (FCMD)? – Pathological study of the cerebral cortex of an FCDM fetus

A light and electron microscopic study of the brain of an 18-week fetus with a prenatal genetic diagnosis of Fukuyama-type congenital muscular dystrophy revealed a widespread mantle of abnormal neurogliomesenchymal tissue that covered a dysplastic cerebral cortex. In this area alone, the glia limitans that adjoined the abnormal mantle via one or two layers of basal lamina had frequent breaches, through which neuroglial elements extruded. In the most severely affected cortical region, which had only a rudimentary and fragmentary glia limitans, the majority of cortical neurons had migrated into the neurogliomesenchymal tissue. The massive overmigrated neurons still maintained a somewhat columnar arrangement, and the marked dysplasia abruptly shifted to a neurogliomesenchymal tissue-free normal cortical structure with an intact glia limitans, thus indicating essentially vertical overmigration of neurons without significant tangential migration of them. Together the above findings imply that breaches in the glia limitans may be the primary cause of the micropolygyria seen in this genetic disorder. Received: 2 June 1995 / Revised, accepted: 17 August 1995     

Induction of glial cell line-derived neurotrophic factor receptor proteins in cerebral cortex and striatum after permanent middle cerebral artery occlusion in rats

In an attempt to elucidate whether glial cell line-derived neurotrophic factor (GDNF) receptors are induced after ischemic brain injury, possible expression of immunoreactive GDNF receptor-alpha1 (GFRalpha-1) and c-ret (RET) was examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Immunohistochemical study showed that both GFRalpha-1 and RET staining cells which were not detected in sham control brain, were present in the ipsilateral cortex and caudate at 3 to 8 h after permanent MCAO, and then decreased but remained to some extent at 24 h. Positive cells for both GDNF receptors were predominantly in cortical neurons of ischemic penumbral area. Western blot analysis confirmed the induction of those receptors after permanent MCAO. This rapid induction of GFRalpha-1 and RET, which correlates with the similar induction of GDNF under these conditions, may play a role in the early response to ischemic brain injury.     

Expression of the glial cell line-derived neurotrophic factor gene in rat brain after transient MCA occlusion

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.     

Abnormalities of cerebellar foliation in rats prenatally exposed to ethanol

Pregnant rats were given a liquid diet containing 5% (w/v) ethanol between gestational days 10 and 21. Cerebella of their offspring were examined at 7 weeks of age. Rats exposed prenatally to ethanol showed a fusion of folia V and VI in the cerebellar vermis. Around the fusion, the cortical laminar structure was disrupted: Purkinje cell dendrites derived from each adjacent folium were tangled, and solitary or clustered ectopic granule cells were in the molecular layer. Some ectopic granule cells surrounded basophilic rosette-like structures. Glial fibrillary acidic protein immunostaining revealed defects in the glia limitans, which is formed by Bergmann glial endfeet on the cerebellar surface. Absence of the glia limitans was observed corresponding to the fusion area. These findings suggested that prenatal exposure to ethanol might impair the formation of the glia limitans in the cerebellum, resulting in the fusion of folia and the disruption of the cortical structure. These malformations may be involved in the delayed motor development and ataxia seen in human fetal alcohol syndrome.     

GDNF对谷氨酸介导的皮层运动神经元损伤的保护作用

目的:利用肌萎缩侧索硬化症的脑片培养模型,观察胶质细胞源性神经营养因子(GDNF)对运动神经元的保护作用。方法取出生后1天乳鼠的大脑组织切片做脑片器官型培养,随机分为对照组、模型组、不同浓度GDNF组(1ng/ml、5ng/ml和50ng/ml),倒置显微镜观察脑片形态变化,SMI-32免疫组化观察皮层大锥体细胞数目的变化。结果:对照组体外生长良好,而模型组生长不良,SMI-32阳性细胞较对照组减少,GDNF5 ng/ml和50 ng/ml各组皮层大锥体细胞存活数目较模型组显著增多,且具有量效关系。结论:GDNF能保护皮层运动神经元免受谷氨酸兴奋毒性的损伤。     

The development of glial fibrillary acidic protein-positive cells and the appearance of laminin-like immunoreactivity in fetal olfactory bulb transplants

Embryonic rat olfactory bulbs were transplanted into the site vacated by aspiration of an olfactory bulb from a neonatal rat. This paper presents our findings related to the development of glial fibrillary acidic protein (GFAP) positive (+ve) glial cells and the appearance of laminin-like immunoreactivity in these transplants. The GFAP + ve glial cells formed perivascular end-feet on the invading vasculature and formed a glia limitans along the glia surface of the transplant. This reconstituted glia limitans was continuous with that of the host brain, there being no glia limitans at the donor-host interface. Thus, the donor tissue was well-integrated with that of the host brain.     

Cortical dysplasia associated with massive ectopia of neurons and glial cells within the subarachnoid space

Summary A detailed neuropathological study of the brain of a 31-day-old premature newborn infant revealed the presence of massive ectopia of neurons and glial cells within the subarachnoid space. The extrusion of neural tissue into the subarachnoid space appeared to have taken place through multiple pialglial bridges. The laminar cortical pattern was also severely disturbed at these sites. Narrow strips of normal and dysplastic cortex alternated in direct relationship to the presence or absence of the pial-glial gaps. Migration of postmitotic neurons and the final positioning of postmigratory neurons appear to take place within highly specified and restricted pathways entrained in a radial direction. Our findings suggest that the pial-glial barrier plays an important role in the control of neuronal migration, and that its disruption may lead to the development of neuronal and glial cell ectopias in the subarachnoid space. The crucial role played by radial glia, the glia limitans and the basal lamina during cortical neurogenesis is emphasized.Supported in part by USPHS grant ES02928Presented in part at the annual meeting of the American Association of Neuropathologists, Minneapolis, MN, June 1986     

Intracellular marking with lucifer yellow CH and horseradish peroxidase of cells electrophysiologically characterized as glia in the cerebral cortex of the cat.

Intracellular microelectrodes filled with either Lucifer Yellow CH, a highly florescent dye, or horseradish peroxidase (HRP) were used to electrophysiologically characterize and mark cells in the cerebral cortex of cat. Fifty-eight cells, characterized electrophysiologically as glia, were marked with Lucifer Yellow CH. All were identified as protoplasmic astrocytes, and included cells in the glia limitans of the molecular layer. An additional 54 cells, similarly characterized as glia, were labeled with HRP. The results were the same; only protoplasmic astrocytes were labeled. The "staining quality" of the glia labeled with HRP was superior to that of cells injected with Lucifer Yellow; greater lengths of individual processes were revealed, and they could often be followed to blood vessels where they ended on the walls of vessels with expanded perivascular end-feet. The observations indicate that the many previously reported studies on presumed glial cells in the cat cerebral cortex have characterized the behavior of protoplasmic astrocytes. Neurons were also marked during these experiments. The "staining" quality of the Lucifer Yellow filled neurons was excellent; dendritic spines, axons, and axon collaterals were clearly visible. These fine neuronal details were not as well revealed after HRP labeling. High resting membrane potentials (RMP's) were not a prerequisite for obtaining well-marked neurons (mean RMP of Lucifer Yellow filled neurons was -33.6 mV; mean RMP of HRP filled neurons was 42.3 mV). In contrast, the mean RMPs of Lucifer Yellow and HRP marked glia was -68 Mv and -75 mV respectively, and the quality of "staining" appeared to be more closely related to the RMP.     

Ontogeny of inter-alpha inhibitor protein (IAIP) expression in human brain

Inter-alpha inhibitor proteins (IAIPs) are naturally occurring immunomodulatory molecules found in most tissues. We have reported ontogenic changes in the expression of IAIPs in brain during development in sheep and abundant expression of IAIPs in fetal and neonatal rodent brain in a variety of cellular types and brain regions. Although a few studies identified bikunin, light chain of IAIPs, in adult human brain, the presence of the complete endogenous IAIP protein complex has not been reported in human brain. In this study, we examined the immunohistochemical expression of endogenous IAIPs in human cerebral cortex from early in development through the neonatal period and in adults using well-preserved postmortem brains. We examined total, nuclear, and cytoplasmic staining of endogenous IAIPs and their expression in neurofilament light polypeptide–positive neurons and glial fibrillary acidic protein (GFAP)–positive astrocytes. IAIPs were ubiquitously detected for the first time in cerebral cortical cells at 24–26, 27–28, 29–36, and 37–40 weeks of gestation and in adults. Quantitative analyses revealed that IAIPs were predominately localized in the nucleus in all age groups, but cytoplasmic IAIP expression was more abundant in adult than in the younger ages. Immunoreactivity of IAIPs was expressed in neurons and astrocytes in all age groups. In addition, IAIP co-localization with GFAP-positive astrocytes was more abundant in adults than in the developing brain. We conclude that IAIPs exhibit ubiquitous expression, and co-localize with neurons and astrocytes in the developing and adult human brain suggesting a potential role for IAIPs in development and endogenous neuroprotection.     

Appearance of neuronal S-100β during development of the rat brain

In addition to being an astroglial protein, S-100β is localised in distinct populations of neurons in the adult rat hindbrain. We report, here, the expression of S-100β in both neurons and glia of the rat brain during development. Prenatally, S-100β immunoreactivity was confined to glial cells close to the germinal zone. After birth, S-100β positive glial cells were seen mainly in the brainstem and cerebellum, while only a few were detected in cerebral cortex and hippocampus. The number of S-100β containing glial cells increased steadily during the first 2 postnatal weeks after which the adult pattern was attained. No S-100β containing neurons were present prenatally. The first S-100β containing neurons were seen in the mesencephalic trigeminal nucleus at postnatal day 1 (P1), and in the motor trigeminal nucleus at P3. Neuronal S-100β immunoreactivity in other nuclei was mostly attained from the 10th to the 21st postnatal day. The neuronal S-100β immunoreactivity was first detected in the cell nuclei during development, then increased in the cytoplasm with ages. A nuclear staining in many immunoreactive neurons persisted until the adult. It usually took 1 to 2 weeks for neuronal S-100β to attain the adult staining pattern, i.e., heavy staining of the cytoplasm and processes, after its appearance. The forebrain never contained S-100β positive neurons. The S-100β is first expressed in glial cells, suggesting it is primarily of the glial origin. Coupled with neurotrophic effects of the protein, the time course of neuronal S-100β expression during the critical period of neuronal development implies that it may be involved in neuronal differentiation and maturation.