Stages Of Brain Development Research Articles

SK-ER3 Neuroblastoma Cells as a Model for the Study of Estrogen Influence on Neural Cells

The neuroblastoma SK-ER3 cell line obtained by stable transfection of the human SK-N-BE cell line is proposed as a model for the study of estrogen receptor activity in cells of neural origin. In the SK-ER3 cell line the estrogen receptor, once activated, initiates a differentiation program leading to growth arrest, morphological changes, and acquisition of the dopaminergic phenotype. In the absence of estrogens, this program can be triggered by IGF-I, which can activate the unliganded estrogen receptor via the ras-pathway. It is proposed that this model system might recapitulate the events occurring in vivo during the differentiation of the nervous system and that IGF-I may play an important role in the activation of estrogen receptor at the very early stage of brain development affecting the differentiation of a number of hypothalamic and extrahypothalamic brain regions.

Isolation of cDNA clones of the rat mRNAs expressed preferentially in the prenatal stages of brain development

For better understanding of the molecular mechanisms underlying the developmental processes of the mammalian brain, we isolated rat fetal brain-enriched (FBE) cDNA clones, whose corresponding mRNAs were expressed at least 5-fold more in the fetal brain than in the adult brain. Our modified differential screening procedure, which utilized a two-vector (pT7T3D and pBluescript) system and showed low background levels of colony hybridization for screening, efficiently identified 64 candidate FBE clones from a small number (475) of colonies in the fetal brain cDNA library. After subsequent second screening of the candidate FBE clones by Northern blot analysis, we successfully isolated 22 distinct FBE clones. The nucleotide sequence analysis of the 22 FBE clones revealed that 13 of them had no significant matches to the sequences reported in the databases, whereas 9 of them matched previously reported sequences (α-tubulin Mα1, β-tubulin Mβ5, thymosin-β10, stathmin, β-tubulin Mβ2, α-internexin, ferritin Lg chain, neuronatin and amphoterin), most of which have been shown to be down-regulated during brain development. We also found that the Northern blot analysis in the second screening could be replaced by cDNA library DNA-Southern blot analysis, in most clones corresponding to relatively abundantly expressed mRNAs. Thus, once the cDNA library is constructed, clone selection will be possible in such clones without the use of additional RNA or Northern blot in screening, allowing the analysis of small brain regions of interest.

Restricted Expression of the Actin-Regulatory Protein, Tropomyosin, Defines Distinct Boundaries, Evaginating Neuroepithelium, and Choroid Plexus Forerunners during Early CNS Development

In the hindbrain, rhombomeres represent morphological units that develop characteristic, segment-specific structures. Similar segments, known as prosomeres, have been proposed to exist in the forebrain. The neuroepithelial cells of the sharp boundary regions that form the borders between many segments often exhibit distinct shapes, reflecting unique cytoskeletal organization. The present investigation examined the expression of one family of actin-binding, regulatory proteins, the tropomyosins (TM), in boundaries. We found that high molecular weight TMs selectively concentrate in boundary cells and other neuroepithelial zones that exhibit unique cell shapes and movements. Specific TM expression is found at hindbrain boundaries as early as embryonic day 10 in the rat, whereas rhombomeres themselves were TM-negative. Highly restricted TM localization also defined some prosomere boundaries in the early forebrain, particularly those exhibiting unique cell shapes. Furthermore, several regions of the neuroepithelium that evaginate are TM-immunoreactive, including tuberal and preoptic neuroepithelium. Most striking, a subpopulation of neuroepithelial cells in the medial telencephalic wall expresses TM, apparently marking the neuroepithelial region that gives rise to the choroid plexus at least 2 d before its formation. This suggests that the medial cerebral wall is not entirely dedicated to generating cells that comprise allocortex. TM expression in the choroid plexus is maintained through initial evagination and appearance in all ventricles. The spatially restricted expression of TMs implicates that this actin-binding protein is involved in the dynamic regulation of cell shape or motility associated with boundary formation and morphogenesis of the neuroepithelium during critical stages of brain development.

Modulation of muscarinic receptor-stimulated phosphoinositide breakdown by sulfhydryl group modification is a general response in different rat brain regions and depends on the stage of brain development.

In a previous study we reported that muscarinic receptor-stimulated phosphoinositide breakdown in rat cortical slices is significantly reduced by modification of critical thiols located at the post-receptor level. We have now extended this observation by investigating whether this effect is a general response in different brain areas and is differentially regulated, within the cortex, in the neonatal and adult brain. Experimental results have demonstrated that indeed the effect of sulfhydryl group modification on muscarinic receptor-stimulated phosphoinositide breakdown appears to be mediated by a general mechanism operative in different brain regions (cortex, hippocampus and striatum). In addition, this response is more pronounced in the early stages of brain development, when muscarinic receptor agonists lead to the highest inositol phosphate accumulation.

MRI brain abnormalities in chronic schizophrenia: One process or more?

It has been suggested that schizophrenia is primarily a prefrontal-temporal-limbic circuitry disorder. Further, it has been argued that primary neurologic vulnerability to the illness is established only during early stages of brain development and is not progressive. We tested the hypothesis of whether brain volume losses in prefrontal and temporal-limbic regions have occurred either before or after brain growth was hypothesized to be complete in schizophrenia. Nineteen chronic schizophrenic patients and 19 age- and sex-matched normal controls underwent magnetic resonance imaging (MRI). All scans were segmented into gray and white matter and cerebrospinal fluid (CSF) compartments for the frontal and temporal lobes and posterior cerebral hemispheres. Multivariate analysis of variance was used to analyze absolute intracranial cerebrum and subregion volumes, i.e., gray, white and CSF, absolute tissue (i.e., gray plus white) volumes, and tissue to intracranial volume (TCV) ratios. Patients showed significant intracranial volume reductions only in the frontal lobes but highly significantly lower TCV ratios (i.e., greater relative tissue loss) in all three major regions. It is suggested that the observed decreases in frontal intracranial volumes reflect a pathologic process in schizophrenia that impacted the frontal regions before brain growth was complete. We hypothesize that the generalized lower patient TCV ratios are attributable to a process that affected the whole cerebrum over a time period after brain volume had reached its maximum levels.

Expression of the fibroblast growth factor family and their receptor family genes during mouse brain development

The fibroblast growth factor (FGF) family is composed of nine members and four genes encode protein tyrosine kinase receptors for them. To gain insight into the involvement of FGFs and their receptors in the development of nervous system, their expression in brains of perinatal and adult mice was examined by semi-quantitative reverse-transcription-linked polymerase chain reactions and in situ hybridization. Although all the genes, with the exception of FGF-4, were found to be expressed, FGF-3, FGF-6, FGF-7 and FGF-8 genes demonstrated higher expression in the late embryonic stages than in postnatal stages, suggesting that these members are involved in the late stages of brain development. In contrast, expression of FGF-1 and FGF-5 increased after birth. Interestingly, FGF-6 expression in perinatal mice was restricted to the central nervous system and skeltal muscles, with intense signals in the developing cerebrum in embryos but in cerebellum in 5-day-old neonates. Furthermore, FGF-receptor (FGFR)-4, a cognate receptor for FGF-6, demonstrated similar spatiotemporal expression, suggesting that FGF-6 and FGFR-4 plays significant roles in the maturation of nervous system as a ligand-receptor system. The results indicate that individual member of the fibroblast growth factor and their receptor family are expressed either sequentially or simultaneously in brain development, strongly suggesting their involvement in the regulation of a variety of developmental processes of brain, i.e., proliferation and migration of neuronal progenitor cells, neuron and glia differentiation, neurite extensions, and synapse formations.

Nitric oxide and antioxidant status in glucose and oxygen deprived neonatal and adult rat brain synaptosomes.

Nitric oxide (NO.) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO., as reflected by nitrite (NO2-) + nitrate (NO3-) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO2- + NO3- production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO2- + NO3- from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO. from nerve terminals. The ratios of antioxidant capacity to NO. production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.

Anti-G M3 (II 3Neu5Ac-lactosylceramide) ganglioside antibody labels human fetal Purkinje neurons during the critical stage of cerebellar development

The ganglioside G M3 (II 3Neu5Ac-lactosylceramide) represents a minor ganglioside in normal human brain compared to major gangliosides with gangliotetraose-backbone. In this study the presence of G M3 in three 23 and 24 weeks of gestation old human cerebella was demonstrated by immunostaining extracted gangliosides on thin-layer chromatography plate as well as by immunohistochemical analysis of cerebellar cryosections. During this stage of brain development G M3 was found to be dominantly expressed on cells corresponding to Purkinje neurons. Delipidation of histological sections with chloroform/methanol caused significant reduction of anti-G M3 immunostaining, thus confirming the prevalent ganglioside nature of this antigen. These results give evidence that (1) G M3 ganglioside is associated with a specific subset of human fetal cerebellar neurons during the critical developmental stage, and (2) a definite ganglioside in general is distributed to a specific subset of cells in normal human brain.

Influence of iron deficiency and lead treatment on behavior and cerebellar and hippocampal polyamine levels in neonatal rats.

Effect of lead exposure and iron-deficiency on polyamine levels in neuronal and glial cells of cerebellum and hippocampus was investigated in weaned rats. Lactating dams with one day old litters were given 0.2% (w/v) lead acetate in drinking water from postnatal day one to twenty one and maintained on an iron-deficient diet. There was an overall reduction of putrescine, spermidine and spermine in neuronal and glial cells of cerebellum and hippocampus consequent to lead exposure and iron-deficiency alone. Lead exposure and iron-deficiency together did not potentiate the polyamine levels in neuronal and glial cells of cerebellum and hippocampus uniformly. However, the enhanced lowering of putrescine in the hippocampal glia, spermidine in cerebellar neuronal and spermine in both neuronal and glial cells of cerebellum during the critical stage of brain development may result in stunted neuronal growth and sprouting in lead exposed and iron-deficient animals. The behavioral alterations as observed in the present study may be due to impaired neuronal development resulting from a depressed polyamine pathway and which could be attributed to cognitive deficits in growing children.

Localization and developmental changes in the neuron-specific cyclin-dependent kinase 5 activator (p35 nck5a) in the rat brain

Mammalian brains contain a cdc2-like protein kinase which is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a brain-specific regulatory subunit with a molecular weight of 35,000, named p35 nck5a. Cdk5 has been identified as the major phosphorylating enzyme of tau protein in the brain. In this study, we examined the temporal and spatial expression patterns of p35 nck5a in the developing rat brain. Northern blot analysis showed that p35 nck5a messenger RNA expression was low in the brain of 12-day postcoitum rats, and increased to a much higher level from 18 days postcoitum to two weeks after birth, and then declined at three weeks after birth. These developmental changes in p35 nck5a expression correlated with the changes in Cdk5-associated kinase activity during brain development. These data suggest that p35 nck5a is the specific activator for Cdk5 in the brain. Immunohistochemical and in situ hybridization studies demonstrated the presence of p35 nck5a protein in postmitotic neurons but not in glial cells at all stages of brain development, indicating that p35 nck5a is a neuron-specific protein. In the adult brain, the protein was rich in cell bodies and dendrites, and only very low amounts were detected in axons. In fetal and neonatal brains, however, axonal pathways such as the corpus callosum and external capsule were also stained with anti-p35 nck5a antibody. Our findings suggest that p35 nck5a is neuron specific, and a specific activator for Cdk5, and the subcellular localization of the two is strictly regulated depending on brain development. Neuronal Cdc2-like kinase may play key roles in neuronal maturation, synaptic formation, and neuronal plasticity.

Chronic malnutrition caused by a corn-based diet lowers the threshold for pentylenetetrazol-induced seizures in rats.

The incidence of epilepsy is high in developing countries where malnutrition is prevalent. Although malnutrition is not a direct cause of seizures, chronic malnutrition may predispose the brain to seizures. In large undernourished human groups from Latin America, the most common sources of food are corn and corn derivatives. We used a rat model of chronic malnutrition, in which corn tortillas were the only solid food intake, to study the possible influence of malnutrition at late stages of brain development on the dynamics of experimental seizures induced by pentylenetetrazole (PTZ). The threshold and does of PTZ required to produce seizures were greatly reduced in malnourished rats. The model of malnutrition used in the study imitates a form of malnutrition common among large numbers of humans. Our results suggest that chronic malnutrition early in life induces changes that lower the seizure threshold and leave the brain more susceptible to seizures. Whether this observation relates to the high incidence of epilepsy in underdeveloped countries remains to be determined.

Timing of puberty and EEG coherence during photic stimulation

Selective elimination of a large proportion of cortical synapses constitutes the last major stage in brain development which is linked to sexual maturation. Various neurodevelopmental theories have proposed an association between schizophrenia and late brain maturational events. Based on epidemiological and psychopharmacological data, Saugstad hypothesised a relationship between timing of puberty, synaptic density and psychosis proneness. Early puberty should result in an earlier end to synaptic pruning, a larger number of surviving connections and an increased risk for affective psychosis. In contrast, in late maturers prolonged synapse elimination should lead to lower synaptic density which constitutes a diathesis for schizophrenia. We used EEG coherence as a neurophysiological measure of cortico-cortical connectivity. Late maturers were expected to show higher coherence, especially for short-distance connections where lower synaptic density should lead to decreased local differentiation. EEG was recorded from 12 bipolar derivations in 19 early and 19 late maturing subjects of both sexes under conditions of rest and photic stimulation at different flicker frequencies. Whereas there were no coherence differences between early and late maturers during rest, both groups differed during flicker stimulation. As hypothesised, late maturers were found to have higher short-distance coherence. Late maturers were also found to have higher intrahemispheric long-distance coherence. These findings were restricted to δ, α and β2 bands and were more pronounced in males. Our findings are interpreted in terms of a structural difference in neuronal connectivity between extreme early and late maturers.

Developmental processes and the pathophysiology of mental retardation

Some of the known causes of mental retardation (MR) include genetic abnormalities, exposure to toxins, many types of infections, malnutrition, brain trauma, and even extreme postnatal neglect. The developing brain is vulnerable to these insults during all the identifiable stages of brain development. Gastrulation leads to the formation of the three germ layers, and neurulation leads to the formation of the central and peripheral nervous systems. Cells formed in these nervous systems migrate from their site of origin to their final position in the brain and periphery. They then differentiate into structures and cells with the final functional properties of nerve cells during prenatal and postnatal activity-dependent periods. All reported causes of MR also produce other types of impairments, which may include physical, neurologic, or psychiatric manifestations and disabilities. The authors suggest that if the biological basis of normal learning, memory, and adaptive behavior can be reduced to cellular events, regulation of synaptic strength would be the most fundamental basis of neural plasticity and learning. Thus, understanding conditions or events that impair or delay development of brain mechanisms for plasticity, learning, and memory is an important first step in understanding the conditions that prevent their development. This article focuses on the particular aspects of neuroembryology, differentiation, and synaptic modification that are particularly vulnerable to developmental insults that cause MR. The most effective types of behavioral and other interventions in the future may be improved by knowledge of the rules that regulate the development of brain plasticity mechanisms in experimental animals. © 1997 Wiley-Liss, Inc.

Developing brain as a target of toxicity.

The human brain forms over an unusually long period compared to other organs. While most of the basic structure is laid down before birth, neuron proliferation and migration continue in the postnatal period. The blood-brain barrier is not fully developed until the middle of the first year of life. The number of synaptic connections between neurons reaches a peak around age two and is then trimmed back by about half. Similarly, there is great postnatal activity in the development of receptors and transmitter systems as well as in the production of myelin. Many of the toxic agents known to damage the developing brain interfere with one or more of these developmental processes. Those with antimitotic action, such as X-ray and methyl mercury, have distinctly different effects on structure depending on which neurons are forming at the time of exposure. Vulnerability to agents that interfere with cell production decreases rapidly over the early postnatal period. Other toxic substances, such as psychoactive drugs and agents that alter hormone levels, are especially hazardous during synaptogenesis and the development of transmitter systems, and thus continue to be damaging for years after birth. Still other toxic substances such as lead, seem to have their greatest effects during even later stages of brain development, perhaps by interfering with the trimming back of connections. Guidelines designed to protect human populations from developmental neurotoxicity need to take into account the changing sensitivity of the brain as it passes through different developmental stages, as well as the fundamental differences in the effects of toxicants on the mature and the developing brain.

Regulation of myelin basic protein gene transcription: identification of a distal cis-acting regulatory element.

The myelin basic protein (MBP) gene contains sequences located upstream of its transcription start site which play a key role in glial-specific transcription of the MBP promoter. Earlier analysis of the 320 bp upstream regulatory sequence of MBP has revealed multiple cis-acting regulatory motifs which differentially regulate transcription of a heterologous promoter fused to a reporter gene in glial and nonglial cells. In the present study, we have focused on a region designated MB3, which is located between -93 to -130 nucleotides with respect to the RNA start site, and contains a binding site for the NF1/CTF family of transcription activators. Results from DNase I footprint protection analysis of nuclear proteins prepared from mouse brain revealed a major region within the MB3 regulatory element that specifically interacts with the proteins derived from mouse brain at various stages of brain development. Using synthetic oligonucleotides spanning the protected region, we show that the double-stranded MB3 sequence interacts with nuclear proteins from mouse brain and forms specific major C1 and a minor C2 complex. Methylation interference experiments have allowed the identification of the G-residues within nucleotides -100 to -108, named MB3a, which are distinct from the NF1/CTF of MB3 that contact with nuclear proteins to form the major C1 complex. Results from band shift studies revealed assembly of the C1 complex upon incubation of MB3 DNA with the nuclear proteins from various cells of glial origin. Site-directed mutagenesis experiments revealed that the identified G-residues for DNA-protein interaction are important to confer transcriptional activity to this domain in transiently transfected glial cells.

Effects of MK-801 and phenytoin on flurothyl-induced seizures during development.

We determined the effects of the N-methyl-D-aspartate (NMDA) receptor blocker MK-801 (0.05, 0.1, and 0.5 mg/kg intraperitoneally, i.p.) and phenytoin (PHT, 5, 10, and 20 mg/kg i.p.) on flurothyl-induced clonic and tonic-clonic seizures in 9-, 15-, 30-, and 60-day-old male rats. Both agents had seizure-, age-, and dose-specific effects. The highest dose of MK-801 was anticonvulsant against clonic flurothyl-induced seizures only in 9- and 60-day-old rats, but suppressed tonic-clonic seizures in all ages. The lowest dose of MK-801 (0.05 mg/kg) produced significant anticonvulsant effects only in 15 day old rats. PHT did not have any effect on clonic seizures throughout development. Both doses of PHT (10 and 20 mg/kg) were anticonvulsant against tonic-clonic seizures in adult rats but not in any other age group. The results indicate that NMDA receptors play an important role in tonic-clonic flurothyl-induced seizures throughout development (especially in 15-day-old rats) and that the anticonvulsant effects of PHT may vary at different stages of brain development.

An immunohistochemical and in situ hybridization study of insulin-like growth factor I within fetal neuron cell cultures

Fetal neuron cell cultures (NCC) from 22 day gestation and 18 day gestation fetal rabbit brain were studied for the presence of insulin-like growth factor I (IGF I). The 22 day gestation NCC were incubated in an IGF I free/insulin free/serum free medium. The 18 day gestation NCC were incubated in: (1) IGF I free/insulin free/serum free medium, (2) IGF I containing medium (100 ng)/serum free medium, and (3) serum containing medium. The 22 day gestation NCC survived in the IGF I free/insulin free/serum free medium. Furthermore, IGF I was detected in the medium by RIA from day one to day ten of incubation. In contrast, the 18 day gestation NCC did not survive in the IGF I free/insulin free/serum medium, but survived in the serum medium. When the 18 day gestation NCC were incubated in the serum free medium containing 100 ng IGF I the cells survived for a period of 2–3 days. Immunoreactive IGF I was found within the 22 day gestation NCC incubated in the IGF I free/insulin free/serum free medium and 18 day gestation NCC in serum medium. Likewise, IGF I mRNA was found only within the 22 day gestation NCC. Internalization studies of IGF I have shown that the peptide was internalized from the medium by the two different gestational age NCC's studied. IGF I receptors were found in both 22 day gestation and 18 day gestation NCC. In conclusion IGF I may promote cell survival in early stages of brain development, and may be of exogenous origin. In contrast the 22 day gestation NCC are capable of producing and secreting IGF I, and indeed appear to respond to this growth factor in an autocrine fashion.

Differential expression of alpha- and beta-thyroid hormone receptor genes in the developing rat brain under hypothyroidism.

One of the most alarming observations of iodine deficiency disorders (IDD) is neurological cretinism which is caused due to lack of T3 during the crucial stages of brain development. We have developed an iodine-deficient rat model to study the pattern of T3 receptor genes expression in the brain during postnatal development under iodine deficiency. We observed increased expression of c-erbA-alpha 2 and -beta 1 T3 receptor transcripts while c-erbA-alpha 1 remains unchanged in the brain of the iodine-deficient neonates compared with their euthyroid counterparts. Up-regulation of the beta 1 form in the developing brain under iodine deficiency is suggestive of an adaptive process coming into play to protect it from the damages that are inflicted due to hypothyroidism. Though the significance of the increased alpha 2 form in the iodine-deficient rat brain has not yet been ascertained, it can be conjectured that it acts as a homeostatic control over 'functional receptor' (namely beta 1) under developmental hypothyroidism.

Developmental neurotoxicity of ethanol: further evidence for an involvement of muscarinic receptor-stimulated phosphoinositide hydrolysis

Various lines of evidence suggest that muscarinic receptor-stimulated phosphoinositide hydrolysis during postnatal development in the rat brain may play a relevant role in glial cell proliferation and neuronal differentiation. We have previously shown that administration of ethanol to developing rats during the brain growth spurt causes microencephaly and selectively decreases muscarinic receptor-stimulated phosphoinositide hydrolysis. In the present study we have investigated the sensitivity of the phosphoinositide system coupled to muscarinic receptors to ethanol inhibition during distinct stages of the brain growth spurt. Different groups of rats were treated for 3 days with ethanol (4 g/kg per day) on postnatal days 2–4 (initial), 6–8 or 10–12 (peak), 13–15 (final stage of the brain growth spurt). The results show that the period of maximal sensitivity to ethanol of muscarinic receptor-stimulated phosphoinositide hydrolysis coincides with the peak of the brain growth spurt and with the period of maximal efficacy of muscarinic receptor agonists to induce inositol phosphates accumulation. Interestingly, only when muscarinic receptor-stimulated phosphoinositide hydrolysis was inhibited, a significant reduction of brain weight was observed. The close parallel between inhibition of this second messenger response and reduction of brain weight suggests that the phosphoinositide system coupled to muscarinic receptors may represent a target for the neurotoxic effects of ethanol during this stage of brain development.

Brain development, infant communication, and empathy disorders: Intrinsic factors in child mental health

AbstractDisorders of emotion, communication, and learning in early childhood are considered in light of evidence on human brain growth from embryo stages. We cite microbehavioral evidence indicating that infants are born able to express the internal activity of their brains, including dynamic “motive states” that drive learning. Infant expressions stimulate the development of imitative and reciprocal relations with corresponding dynamic brain states of caregivers. The infant's mind must have an “innate self-with-other representation” of the inter-mind correspondence and reciprocity of feelings that can be generated with an adult.Primordial motive systems appear in subcortical and limbic systems of the embryo before the cerebral cortex. These are presumed to continue to guide the growth of a child's brain after birth. We propose that an “intrinsic motive formation” is assembled prenatally and is ready at birth to share emotion with caregivers for regulation of the child's cortical development, on which cultural cognition and learning depend.The intrinsic potentiality for “intersubjectivity” can be disorganized if the epigenetic program for the infant's brain fails. Indeed, many psychological disorders of childhood can be traced to faults in early stages of brain development when core motive systems form.