Early Postnatal Brain Development Research Articles

Molecular Cloning of a Novel mRNA (Neuronatin) That Is Highly Expressed in Neonatal Mammalian Brain

As differential gene expression governs the progression of development into senescence, we attempted to define the genes that are selectively expressed during postnatal brain development. A cDNA fragment selectively expressed in neonatal rat brain was identified by differential display and used to screen a cDNA library prepared from the same mRNA sample. The full length cDNA, neuronatin, was 1195bp long and coded for a novel protein of 81 amino acids. The cDNA detected an mRNA species of similar size that was highly expressed in rat neonatal and human fetal brain. The deduced protein exhibited a hydrophobic N-terminal and hydrophilic C-terminal, suggesting that it is membrane bound and might function in signal transduction. The selective expression of this novel mRNA in late fetal and early postnatal brain development, and loss of expression in adulthood and senescence, suggests that downregulation of neuronatin may be involved in terminal brain differentiation.

Regulation of neurotrophin receptor expression during embryonic and postnatal development

Members of the NGF family of proteins act as neurotrophic agents for defined populations of peripheral and central neurons during embryonic and postnatal development. We have studied the presence of receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 and -4/5 (NT-3, NT-4/5) by cross-linking radioiodinated neurotrophins to specific cell surface receptors. We have identified neurotrophin receptors representing full-length TrkB and TrkC and their truncated forms (lacking a functional cytoplasmic kinase domain) in neuronal as well as in non-neuronal tissues. During chicken embryonic and early postnatal brain development, expression of full-length TrkB and TrkC proteins preceded the onset of the truncated forms of these receptors. A similar pattern was also observed in mouse embryonic and early postnatal brain. The relative levels of neurotrophin receptors in the basal forebrain and in the hippocampus did not change significantly with age in mice. High levels of receptors for the three neurotrophins were detected in the nigrostriatal system. Full-length TrkB and TrkC receptors were found in chicken and rat embryonic ventral spinal cord, as well as on purified motoneurons. Again, truncated TrkB appeared significantly later than the full-length form on spinal motoneurons. In chicken embryonic retina and optic tectum we detected full-length TrkB and TrkC; however, the optic tectum also expressed large amounts of the truncated form of TrkB. TrkC but not TrkB was detected in chicken embryonic skeletal muscle, suggesting that NT-3 may have a novel function in this tissue. The presence of neurotrophin receptors in a wide variety of embryonic and postnatal tissues underlines the significant role of BDNF, NT-3, and NT-4/5 in embryonic and postnatal development. The regulation of the ratio of full-length versus truncated neurotrophin receptors may play an important role in the development, maturation, and maintenance of various neuronal networks.

Spatiotemporal expression of SCIP and myelin genes in rat brain during early postnatal development.

The expression of the SCIP and myelin genes (PLP, BP and MAG) was quantitated by Northern blot analysis during early postnatal rat brain development. The SCIP gene was already profoundly upregulated on day 1 postpartum. The SCIP message level in the forebrain was 4- and 17-fold higher than the message level in the cerebellum and brainstem, respectively. By day 20, the SCIP gene expression declined in the forebrain to approximately 5% of the day-1 level, and became undetectable in the brainstem and cerebellum. The myelin gene expression sharply upregulated at approximately day 7-8, and in general, was highest in the brainstem, and lowest in the forebrain. The results demonstrate that the expression of the SCIP gene in the oligodendrocytic lineage cells is relatively low, whereas other cellular elements feature very active expression of the gene during the early postnatal development. Additionally, the variation in the relative ratios of the myelin messages among the brain regions indicates that the levels of expression of the myelin genes are not tightly synchronized.

Midbrain dopamine neurons regulate preprotachykinin-A mRNA expression in the rat forebrain during development

Intracerebroventricular 6-hydroxydopamine injections were performed at postnatal days 3 and 6 in animals pretreated with the norepinephrine uptakeblocker desimipramine in order to generate a selective lesion of dopamine neurons. In situ hybridization was then used to analyze preprotachykinin-A (PPT-A) mRNA expression in the lesioned as well as in saline-injected control animals. The midbrain dopaminergic lesion caused a 22–25% increase in the level of PPT-A mRNA in cingulate cortex and frontoparietal cortex when analysed at 2 weeks of age, compared to saline-injected control animals. In contrast, the lesion caused no change in PPT-A mRNA expression in the neonatal caudate-putamen. These results indicate that dopamine neurons downregulate the expression of PPT-A mRNA specifically in cingulate cortex and frontoparietal cortex during early postnatal brain development. In the adult rat forebrain, lesioned at P3 and P6, no change in the level of PPT-A mRNA was seen in cingulate cortex and frontoparietal cortex. However, a 29% decrease in PPT-A mRNA was seen in the lateral caudate-putamen with no significant change in neurons of medial caudate-putamen. Thus, dopamine neurons appears to exert a region specific influence on PPT-A mRNA expression during brain development.

Changes in adrenoceptors and monoamine metabolism in neonatal and adult rat brain after postnatal exposure to the antihypertensive labetalol.

1. The purpose of the present study was to investigate the acute (single injection), direct (chronic treatment) and the long-lasting effects after exposure to the alpha 1/beta-adrenoceptor antagonist labetalol during rat brain development on adrenoceptors and monoamine metabolism. 2. In 10-day-old rat pups, subcutaneously administered labetalol (10 mg kg-1) passed the blood-brain barrier, reaching a level of 2.1 micrograms g-1 tissue in the brain 90 min after injection. 3. Chronic labetalol treatment (10 mg kg-1, s.c., twice daily) during the first 10 days of life significantly increased alpha 1-adrenoceptor binding in the hypothalamus (+39%), but not in the occipital cortex. 4. This chronic postnatal labetalol treatment did not result in long-lasting changes in alpha 1- and beta-receptors measured on day 60. 5. A single labetalol injection (10 mg kg-1, s.c.) on postnatal day 10 significantly increased noradrenaline (NA) metabolism in all brain regions tested (+25 to 105%), but had no effects on 5-hydroxytryptamine (5-HT) or dopamine metabolism. 6. Chronic labetalol treatment between postnatal (PN) days 1 and 10 also increased NA metabolism on PN 10 (3-methoxy-4-hydroxyphenylglycol (MHPG)/NA, +20 to 100%), suggesting that tolerance to the acute effect of labetalol did not occur. A slight increase in 5-HT metabolism (20%) was induced by the chronic labetalol treatment in the hippocampus and meso-limbic system. 7. In general, long-lasting effects on NA metabolism could not be detected on day 60 more than one month after the treatment. However, 5-HT metabolism was significantly increased in all four brain regions measured (+20 to 70%). 8. We conclude that chronic labetalol exposure during early postnatal rat brain development does not cause long-lasting changes in beta-receptor number or NA metabolism, but appears to be critical for the rate of 5-HT metabolism in later life.

Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

Expression of Small Cytoplasmic Transcripts of the Rat Identifier Element In Vivo and in Cultured Cells†

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

Polysomes during early postnatal development of brain in the rat.

We have attempted to show eventual modifications in the brain protein synthesis apparatus of rat during the first three weeks after birth. Through this time we noted a steady decrease (about 60%) in the free polysomes, when expressed relative to tissue weight. This decrease does not correlate with changes in the polysome profile, indicating that no loss in the efficiency of protein synthesis was involved. Translation in a reticulocyte lysate also failed to reveal differences.

Expression of Myelin Basic Protein Genes in Several Dysmyelinating Mouse Mutants During Early Postnatal Brain Development

Northern blot and "dot" blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qk/+, jp/+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and "dot" blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.

Essential Fatty Acid Deficiency: Effects of Cross-Fostering Mice at Birth on Myelin Levels and Composition

Female mice were assigned an essential fatty acid (EFA)-deficient diet or a control diet at mating, and litters were cross-fostered at birth to produce three groups of animals: pups fed a control diet prenatally and deficient diet postnatally (C → D); a deficient diet prenatally and a control diet postnatally (D → C); and a control diet throughout life (C → C). The yield of myelin, the developmental pattern of the major proteins, and the proportion of major lipids were examined in the purified myelin of the three groups at 3, 6, and 9 weeks. Myelin yield was lower at 9 weeks in both the (C → D) and (D → C) groups compared to controls. There was an alteration in the ratios of the proteins and major lipid classes in myelin from the (C → D) animals at 9 weeks, whereas the ratios of these components were normal in the (D → C) animals at this age. However, at three weeks the lipid composition of the myelin isolated from (D → C) animals was abnormal. The results suggest that postnatal EFA deficiency results in hypomyelination in mice and that the myelin formed is of abnormal composition during early postnatal brain development. Prenatal EFA deficiency results in less severe hypomyelination with only the earliest myelin formed being of abnormal lipid composition.

RNA metabolism in isolated mouse brain nuclei during early postnatal development.

Abstract—RNA metabolism in isolated brain nuclei has been shown to be dramatically altered during early postnatal brain development. The present study involved an examination of the RNA products synthesized by nuclei at various stages of postnatal neural maturation. In all cases, the majority of the RNA appeared to be heterodisperse, non‐ribosomal and non‐tRNA in nature. In comparison to the RNA isolated from nuclei of neonatal tissue, the RNA from nuclei of 12‐day and 30‐day‐old mouse brain was found to be of smaller molecular weight. Despite the heterodisperse nature of these RNA molecules, the addition of α‐amanitin did not completely inhibit nuclear synthesis.An investigation of RNA synthesis in isolated neuronal and glial cell nuclei revealed that nucleic acid metabolism in these respective cell populations had different and distinct developmental patterns. Preparations enriched with glial cell nuclei were found to be most active at birth and then decreased in activity (3–4‐fold) during neural maturation. On the other hand, the rate of RNA synthesis in fractions enriched in neuronal cell nuclei was observed to increase dramatically in activity (4–5‐fold) until 14 days of age. From 14 days of age until adulthood, RNA synthetic activity remained essentially the same.