Comparative analysis of in vitro conditions for rat adult neural progenitor cells

Various protocols have been published for in vitro expansion and maintenance of adult neural progenitor cells (ANPC). However, there are only few data comparing these protocols regarding their influence on proliferation, migration and differentiation. Freshly isolated ANPC from olfactory bulb (BO) and dentate gyrus (DG) of adult rat brains forming neurospheres and expressing the neural stem cell markers nestin and Sox-2 were used in a comparative analysis of five different medium combinations. Medium containing N2 and fetal calf serum (FCS), but no additional cytokines was unsuitable for an effective long-term expansion of ANPC due to a significantly reduced proliferation rate. Media containing BIT, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor AB (PDGF-AB) and leukemia inhibitory factor (LIF) or B27, bFGF and EGF are recommendable for the cultivation of DG-derived ANPC as neurospheres only. Unlike, culture media containing BIT, bFGF, EGF and PDGF-AB or N2, bFGF and EGF were suitable for all applications tested as they responded similarly regarding proliferation, migration and expression of differentiation markers. The results of the present study might help to improve the effective in vitro expansion of ANPC derived from rare human tissue samples.     

The gene for heparin-binding epidermal growth factor-like growth factor is stress-inducible: its role in cerebral ischemia.

The functions of the epidermal growth factor (EGF) family members in the adult brain are not known. This study investigated the changes in the expression of members of the EGF family following global ischemia employing in situ hybridization and immunohistochemical techniques to elucidate their roles in pathological conditions. EGF mRNA was not detected in either the control or the postischemic rat brain. Although transforming growth factor-alpha (TGF-alpha) mRNA was widely expressed in the normal brain, its expression did not change appreciably following ischemia. By contrast, heparin-binding EGF-like growth factor (HB-EGF) mRNA expression was rapidly increased in the CA3 sector and the dentate gyrus of the hippocampus, cortex, thalamus, and cerebellar granule and Purkinje cell layers. EGF receptor mRNA, which was widely expressed, also showed an increase in the CA3 sector and dentate gyrus. Conversely, HB-EGF mRNA did not show any increase prior to ischemic neuronal injury in the CA1 sector, the region most vulnerable to ischemia. Immunohistochemical detection of HB-EGF in the postischemic brain suggested a slight increase of immunostaining in the dentate gyrus of the hippocampus and the cortex. These findings showed that the gene encoding HB-EGF is stress-inducible, indicating the likelihood that HB-EGF is a neuroprotective factor in cerebral ischemia.     

Leukemia inhibitory factor is a key regulator of astrocytic, microglial and neuronal responses in a low-dose pilocarpine injury model

Insult to the central nervous system (CNS) induces many changes, including altered neurotransmitter expression, activation of astrocytes and microglia, neurogenesis and cell death. Cytokines and growth factors are candidates to be involved in astrocyte and microglial activation, and the up-regulation of glial fibrillary acidic protein (GFAP) is associated with brain damage. One of these candidates is leukemia inhibitory factor (LIF), a pro-inflammatory cytokine that is induced in astrocytes by brain damage or seizure. LIF also regulates expression of both neuropeptide Y (NPY) and galanin following peripheral nerve injury. To test the hypothesis that LIF regulates astrocyte, microglial and neuropeptide responses to a mild insult, we used a low-dose pilocarpine model to induce a brief seizure in LIF knock-out (KO) mice. Compared to wild type mice, the LIF KO mouse displays reduced astrocyte and microglial activation in the hippocampus. In addition, LIF KO mice display dramatically altered NPY, but not galanin, expression in response to injury. Thus, LIF is required for normal glial responses to brain damage, and, as in the periphery, LIF regulates NPY expression in the CNS.     

Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus

Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of Oenanthe javanica on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67(an endogenous marker for cell proliferation) and doublecortin(a marker for neuroblast). Our results showed that Oenanthe javanica extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the Oenanthe javanica extract-treated group compared with the control group. These results indicate that Oenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.     

Kainic acid induces leukemia inhibitory factor mRNA expression in the rat brain: differences in the time course of mRNA expression between the dentate gyrus and hippocampal CA1/CA3 subfields

Leukemia inhibitory factor (LIF) is a pluripotent cytokine which affects the survival and differentiation of various types of cells both in the hematopoietic and nervous systems. In this study, the time course and localization of LIF mRNA expression following kainic acid-induced seizures were examined by northern blot analyses and in situ hybridization. Northern blot analyses demonstrated that intraperitoneal injection of kainic acid at a convulsive dose induced LIF mRNA expression intensely in the hippocampus and moderately to weakly in the cerebral cortex, thalamus and hypothalamus. The expression peaked at 8-24 h after the injection in the hippocampus and cerebral cortex and at 8 h in the thalamus and hypothalamus. In situ hybridization revealed different time courses of LIF mRNA expression depending on the area of the hippocampus; that is, the expression peaked at 10 h in the granule cell layer of the dentate gyrus, then at 12 h in the polymorph and molecular layers of the dentate gyrus, and finally at 12-24 h in the strata oriens and radiatum of the CA1 and CA3 subfields. It is worth noting that the expression of LIF mRNA was intense in the dentate gyrus, the region where neurogenesis and aberrant network reorganization have been shown to be induced by seizures. The upregulation of LIF mRNA expression in the dentate granule cell layer followed by that in the dentate polymorph and molecular layers may be involved in activity-dependent neurogenesis in the granule cell layer and ectopic migration of granule cells to the polymorph and molecular layers in the dentate gyrus.     

Developmental lead exposure impairs contextual fear conditioning and reduces adult hippocampal neurogenesis in the rat brain

The effects of developmental lead exposure on the emotional reactivity, contextual fear conditioning and neurogenesis in the dentate gyrus of 60-80 days-old rats were studied. Wistar rat pups were exposed to 0.2% lead acetate via their dams' drinking water from postnatal day (PND) 1 to PND 21 and directly via drinking water from weaning until PND 30. At PND 60 and 80 the level of anxiety and contextual fear conditioning were studied, respectively. At PND 80 all animals received injections of BrdU to determine the effects of Pb on the generation of new cells in the dentate gyrus of hippocampus and on their survival and differentiation patterns. The results of the present study demonstrate that developmental lead exposure induces persistent increase in the level of anxiety and inhibition of contextual fear conditioning. Developmental lead exposure reduced generation of new cells in the dentate gyrus and altered the pattern of differentiation of BrdU-positive cells into mature neurons. A lower proportion of BrdU-positive cells co-expressed with the marker for mature neurons, calbindin. In contrast, the proportions of young not fully differentiated neurons and proportions of astroglial cells, generated from newly born cells, were increased in lead-exposed animals. Our results demonstrate that developmental lead exposure induces persistent inhibition of neurogenesis and alters the pattern of differentiation of newly born cells in the dentate gyrus of rat hippocampus, which could, at least partly, contribute to behavioral and cognitive impairments observed in adulthood.     

Proliferation of microglia and astrocytes in the dentate gyrus following entorhinal cortex lesion: a quantitative bromodeoxyuridine-labelling study

Entorhinal cortex lesion of adult rats induces glial activation and proliferation in the deafferented dentate molecular layer. Double-labelling immunocytochemistry for the astrocyte-specific antigen glial fibrillary acidic protein or the microglial cell marker Griffonia simplicifolia isolectin B4 with bromodeoxyuridine detection revealed that microglia counts and the proliferation rate in the ipsilateral dentate gyrus reached a maximum in the molecular layer at 3 days post-lesion (dpl) and returned to control levels by 30 dpl. Astrocyte counts in the ipsilateral dentate gyrus peaked at 30 dpl, with maximum proliferation at 7 dpl. At 100 dpl the astrocyte count had reverted to control levels. Glial proliferation was not restricted to the ipsilateral molecular layer but also occurred to some degree in the granule cell layer and the contralateral dentate gyrus. Thus entorhinal cortex lesion induces a rapid microglia reaction and long-lasting astrocyte activation in the deafferented termination zone of the perforant path. We conclude that glial proliferation after entorhinal cortex lesion follows a complex temporal and spatial pattern that coincides with processes of neuronal and axonal reorganization.     

Reduced cell proliferation in the dentate gyrus is not correlated with the development of learned helplessness.

BACKGROUND: A plethora of indirect findings suggests that mood disorders may be caused by or result in structural changes in the brain, namely decreased hippocampal cell proliferation. METHODS: To test for these hypotheses, we used a rat model of depression, learned helplessness. Moderate unpredictable and inescapable foot shocks induced learned helplessness only in a portion of the rats. Rats that showed helpless behavior were compared to those behaving normally after inescapable shock. Proliferating cells in the dentate gyrus were labeled with BrdU (bromodeoxyuridine). RESULTS: Helpless behavior appeared before the decrease of dentate gyrus cell proliferation was maximal. Cell proliferation was decreased to the same extent in animals that developed helplessness as those that were not helpless. Furthermore, immobilization stress, which reduced the rate of cell proliferation, did not induce learned helplessness. CONCLUSION: These results are in line with reports that the rate of dentate gyrus cell proliferation is acutely down-regulated by stress, but the development of helpless behavior does not correlate with this process. Further studies will have to clarify if during learned helpless behavior neurogenesis is impaired by altered differentiation or survival of cells.     

Neurodegeneration and production of the new cells in the dentate gyrus of juvenile rat hippocampus after a single administration of ethanol

Administration of ethanol during brain development induces widespread neuronal loss in various structures of the brain. Here, we show that a single administration of ethanol given during the early postnatal period can induce not only neuronal death, but also an increase in proliferation of the progenitor cells in the dentate gyrus of hippocampal formation in rats. Ethanol (1.5 or 3 g/kg, i.p.) administered to 10-day-old rats induced massive neuronal degeneration as evidenced by TUNEL assay in the dentate gyrus. The neuronal death induced by a high dose of ethanol (3 g/kg) was accompanied by an enhanced proliferation of the progenitor cells labeled by bromodeoxyuridine (BrdU, 50 mg/kg, i.p.) in dentate gyrus. One and 3 weeks following ethanol or saline administration, ethanol-treated rats still had significantly more BrdU-labeled cells than control animals. In ethanol-treated rats, a higher proportion of newly born cells acquired the phenotype of immature postmitotic neurons whereas the final differentiation into calbindin-expressing granule cells remained unchanged. The proportion of astroglial cells was also increased in ethanol-treated rats. Thus, ethanol given in high doses not only induces neurodegeneration but also initiates the process of neuro- and gliogenesis, which might be responsible for the neuronal and glial reorganization of the dentate gyrus.     

Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells

Human fetal neural stem cells (hNSCs) can be expanded in vitro by mitogens or growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and/or leukemia inhibitory factor (LIF). Their effects on proliferation rate and differentiation pattern of hNSCs, however, have not been fully characterized. In this study, we cultured hNSCs in seven regimens, including bFGF, EGF, and LIF, either alone or in combinations. Cells were maintained as neurospheres in treatment media for various periods, up to six passages. A combination of bFGF, EGF, and LIF expanded hNSCs more efficiently than any other treatment as determined by counting total cell numbers using a trypan blue exclusion assay, a WST-1 cell viability assay, and a bromodeoxyuridine incorporation flow cytometric analysis. Differentiation patterns of hNSCs expanded under different conditions were also analyzed. We reported previously that hNSCs primed in vitro with a combination of bFGF, heparin, and laminin (FHL) induced neuronal differentiation toward a cholinergic phenotype. In this study, we show that the FHL priming increases neuronal differentiation while decreasing astroglial generation in all treatment groups as determined by immunostaining. However, cells proliferated under different growth factor conditions do vary in their phenotypic differentiation patterns. Particularly, significant generation of cholinergic cells was observed only in hNSCs expanded with EGF/bFGF or EGF/bFGF/LIF, but not with other treatment regimens, even when they are exposed to the same priming procedure. Our results indicate that hNSCs are highly plastic, with their proliferation and differentiation potential dependent on different growth factor treatments.     

Leukemia inhibitory factor promotes oligodendrocyte survival after spinal cord injury

Injury to the mammalian spinal cord is accompanied by a delayed, secondary wave of oligodendrocyte apoptosis that arises several days after the initial injury. A strong candidate to support oligodendrocyte survival after spinal cord injury is the pleiotropic cytokine, leukemia inhibitory factor (LIF). In vitro, LIF potentiates the differentiation and survival of oligodendrocyte precursors. LIF can also prevent oligodendrocyte apoptosis in response to either growth factor removal or cytotoxic challenge. More recently, in vivo studies have demonstrated that LIF is effective in preventing oligodendrocyte death in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We therefore asked whether systemic delivery of LIF could ameliorate oligodendrocyte death in a mouse model of spinal cord injury. We have found that daily administration of LIF (25 microg/kg/day) promotes oligodendrocyte survival after spinal cord injury. Interestingly however, this effect does not appear to be mediated by a direct action of LIF on the oligodendrocyte but rather via an ancillary cell type, which results in augmented expression of another trophic factor capable of supporting oligodendrocyte survival, insulin-like growth factor 1 (IGF-1).     

Long-term administration of scopolamine interferes with nerve cell proliferation,differentiation and migration in adult mouse hippocampal dentate gyrus,but it does not induce cell death

Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperitoneal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen(Neu N; a neuronal marker) and Fluoro-Jade B(a marker for the localization of neuronal degeneration) was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67(a marker for proliferating cells)-immunoreactive cells were reduced in number and reac hed the lowest level at 4 weeks. Doublecortin(DCX; a marker for newly generated neurons)-immunoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2′-deoxyuridine(Brd U)-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature Brd U/Neu N double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death.     

Transforming growth factor-β1 inhibits the proliferation of rat astrocytes induced by serum and growth factors

A number of cytokines and growth factors may affect astrocyte proliferation and functions. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine which exerts multiple effects on growth and differentiation of different cell types. TGF-β1 is present in low amounts in the normal brain. TGF-β1 gene expression, however, is increased in the central nervous system (CNS) in several pathological conditions. In this study we examined the in vitro effects of TGF-β1 on the proliferative response of rat astrocytes to serum and growth factors. Astrocyte cultures were established from the cerebellum and cortex of newborn Lewis rats. The proliferative response of these cultures to serum and growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), IGF-2, interleukin 1 (IL-1)] was studied by [³H]-thymidine incorporation test in the presence or absence of TGF-β1. TGF-β1 significantly inhibited the proliferative response of astrocyte cultures to both autologous and heterologous serum. In addition, a strong inhibition of bFGF-, EGF-, and PDGF-induced proliferation was observed. The effect of TGF-β1 on the proliferative response to IL-1 was less evident but still significant. No effect was observed when TGF-β1 was added to IGF-1 and IGF-2 stimulated cultures. These data confirm previous reports showing a down-regulating activity of TGF-β on astrocyte proliferation and suggest that this cytokine may play physiological and pharmacological roles in the regulation of reactive astrocytosis in the CNS. © 1995 Wiley-Liss, Inc.     

脊髓神经干细胞培养及体外诱导分化实验研究

目的探讨胚胎大鼠脊髓神经干细胞(SNSCs)的分离培养及体外诱导分化的特点和规律。方法利用无血清培养技术和细胞免疫化学方法。结果在表皮生长因子(EGF),碱性成纤维细胞生长因子(bFGF)的作用下,SNSCs快速增殖,形成多细胞组成的细胞球,将这些细胞球分离成单细胞重新培养,单个的细胞又很快变成细胞球。虽然白血病抑制因子(LIF)不是SNSCs培养所必需的,但是它可明显促进SNSCs增殖。诱导分化实验显示:SNSCs可分化为神经元、星型胶质细胞和少突胶质细胞,而LIF组则分化很少。结论从胚鼠脊髓分离神经干细胞是可行的,这些细胞在体外经多次传代后仍具有较强的增殖能力和多向分化潜能;LIF可以促进SNSCs增殖并抑制其分化。     

Transforming growth factor α differentially affects GABAergic and cholinergic neurons in rat medial septal cell cultures

The effects of transforming growth factor α (TGFα) on low and high density cultures of fetal (embryonic day 17) rat medial septal cells were investigated and in some instances, compared to those of epidermal growth factor (EGF). In high density cultures, TGFα induces a significant increase in the number of astroglia and microglia. While the effects of TGFα on the astroglia are more pronounced when compared to EGF, those on the microglia are less notable. In addition, TGFα produces a time- and dose-dependent decrease in the activity of choline acetyltransferase (EC 2.3.1.6) and a proportional decrease in the number of acetylcholinesterase-positive neurons in these high density cultures. However, although both EGF and TGFα decreased choline acetyltransferase activity maximally at the same concentration (10 ng/ml), the latter was consistently more potent. TGFα does not affect cholinergic cell survival but the expression of their chemical phenotype and does so indirectly via the glial cells. On the other hand, TGFα directly induces a dose and time-dependent increase in glutamic acid decarboxylase activity in these high density cultures without affecting the number of glutamic acid decarboxylase immunoreactive neurons. In low density cultures, TGFα acts as a general neuronal survival factor, affecting both cholinergic and GABAergic neurons. Here TGFα's neurotrophic activity is more evident than its effects on their chemical phenotype. These results suggest that TGFα exerts distinct and differential effects on the biochemical expression of two neuronal populations in the developing medial septum maintained in high density culture. Finally, as TGFα acts as a general neuronal survival factor in low density cultures, cell to cell interactions appear to be important in the ultimate response of these cells to this growth factor.     

Effects of IL-1beta, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells.

Inflammation may affect the local presence of sensory nerve fibers in situ and inflammatory mediators influence sensory neurons in vitro. In the present study we have investigated effects of the cytokines interleukin-1beta (IL-1beta, interleukin-6 (IL-6), and leukemia inhibitory factor (LIF) on survival of and neurite growth from neonatal rat sensory neurons co-cultured with fibroblast-like cells prepared from neonatal rat skin (sFLCs) or perichondrium (pFLCs). The results showed that both FLC types expressed receptors for all three cytokines. Five ng/ml of either cytokine, but not lower or higher concentrations, supported survival of DRG neurons co-cultured with sFLCs. Neuronal survival was also enhanced by addition of the soluble IL-6 receptor (rsIL-6R) with or without IL-6. In co-cultures with pFLCs neuronal survival was promoted by IL-6, increasing with cytokine concentration. Addition of rsIL-6R without IL-6 did also stimulate neuronal survival. The growth of neurites from DRG neurons co-cultured with sFLCs was stimulated by 0.5 ng/ml LIF, unaffected by 5 ng/ml LIF and inhibited by 50 ng/ml LIF. Considering DRG neurons co-cultured with pFLCs, 50 ng/ml of either of the three cytokines, as well as rsIL-6R conditioned medium, stimulated neurite outgrowth. Some of the cytokine effects observed were reduced by application of antibodies against nerve growth factor (NGF). We conclude that that the cytokines examined affect DRG neurons in terms of survival or neuritogenesis, that the effects are influenced by cytokine concentration and the origin of the FLCs and that some of the effects are indirect, probably being mediated by factors released from FLCs.     

Glucocorticoid regulation of glial responses during hippocampal neurodegeneration and regeneration

Glucocorticoids can prevent or accelerate neurodegeneration in the adult rat hippocampus. To investigate these actions of glucocorticoids, we previously cloned genes from the hippocampus. Adrenalectomy specifically increased glial fibrillary acidic protein and transforming growth factor (TGF)-β1 mRNAs in the dentate gyrus and these effects were dependent on induced apoptosis. Corticosterone treatment prevented apoptosis, and decreased glial activation and the influx of activated microglia. Since these effects are opposite to injury and neurodegeneration, we propose that they represent adaptive actions of glucocorticoids, preventing cellular defense mechanisms from overshooting. We used adrenalectomy as a model to investigate how adult granule neurons die in vivo and the effects of neurotrophic factors in protecting against apoptosis. Neurotrophin-4/5 and TGF-β1 protected granule neurons against adrenalectomy-induced apoptosis. Since neurogenesis is also greatly increased in the dentate gyrus following adrenalectomy, we compared the time course of birth and death with glial responses. TGF-β1 mRNA increased before the detection of dying cells in the dentate gyrus, which was coincident with increased proliferation in the neurogenic zone. Glucocorticoids also increased Ndrg2 mRNA in glia in the neurogenic zone; Ndrg2 is a member of a novel gene family involved in neural differentiation and synapse formation. Therefore, studying the effects of glucocorticoid manipulation on the dentate gyrus is increasing our understanding of how mature neurons die by apoptosis and the role of glia in induced apoptosis and neurogenesis. Discovering how endocrine and inflammatory responses regulate neuron birth and survival is important for developing successful neuron replacement strategies to treat neurodegenerative diseases.     

大鼠胎脑皮质神经干细胞体外培养及诱导分化的实验研究

目的 从孕龄15d SD胚胎鼠脑皮质中分离并培养神经干细胞（neural stem cells。NSCs），观察其生长、增殖及分化。方法 采用包含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的无血清培养及单细胞克隆技术，对胚胎鼠脑皮质神经干细胞进行原代、传代培养及诱导其分化。用Nestin染色鉴定神经干细胞特性，用免疫组化方法（β-Ⅲ-tubulin、GFAP染色）检测神经干细胞分化为神经元及神经胶质细胞状况。结果 从孕龄15dSD胚胎鼠脑皮质中分离的组织，经原代及传代培养均可形成细胞克隆．切具有增殖能力。原代及传代培养细胞呈Nestin（神经上皮干细胞蛋白）表达阳性．诱导分化后的细胞表达神经元细胞、星形胶质细胞的特异性抗原。结论 本实验分离、培养的孕龄15dSD胚胎鼠脑皮质细胞Nestin表达阳性．分化后表达神经元和星形胶质细胞的标记物，是大鼠的神经干细胞，并具有多向分化潜能。     

Epidermal growth factor and its receptor in the developing human nervous system

Recent data suggest that epidermal growth factor and epidermal growth factor receptors (EGF/EGF-R) are present and functional in neurons within the central nervous system. Previously, EGF was detected in developing and mature rat brain and cerebrospinal fluid. Also, EGF-R was documented in discrete locations in normal adult human brain, as well as in senile plaques associated with Alzheimer's disease. Using two polyclonal sera, anti-EGF and anti-EGF-R, in conjunction with immunohistochemical staining, we examined formalin-fixed, paraffin-embedded neural tissues from 10 autopsied, human brains. These specimens were collected from patients who died during various stages of development ranging from 27 weeks of estimated gestational age to 63 years of age. Immunostaining for EGF and EGF-R was detected in hippocampal pyramidal cells. Purkinje cells, large multipolar neurons of the dentate nucleus, anterior horn cells, dorsal root ganglion cells, cells of the dorsal nucleus of Clark, intermediolateral column cells and ependymal cells. Positive binding studies with 125I-EGF confirmed that numerous EGF receptors are unoccupied, assessable, and available for interactions with potential ligands such as EGF and TGF alpha in developing rat brains. It appears that EGF and/or EGF-R may play a role during maturation and differentiation of the human central nervous system.