Developmental changes in NT3 signalling via TrkA and TrkB in embryonic neurons.

Neurotrophins promote neuronal survival by signalling through Trk receptor tyrosine kinases: nerve growth factor signals through TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)4 through TrkB and NT3 through TrkC. Although studies in some, but not all, cell lines indicate that NT3 can also signal through TrkA and TrkB, it is not known if such signalling can occur in neurons. We show that NT3 can promote the in vitro survival of sensory and sympathetic neurons isolated from embryos that are homozygous for a null mutation in the trkC gene. During the mid-embryonic period, NT3 promoted the survival of as many trigeminal and nodose neurons as the preferred neurotrophins, NGF and BDNF. However, later in development, these neurons lost their ability to respond to NT3. NT3 also promoted the survival of almost all sympathetic neurons, but no decrease in effectiveness was observed during development. Trigeminal neurons from trkC-/- trkA-/- embryos did not respond to NT3 and nodose neurons from trkB-/- embryos likewise failed to respond to NT3. These results show that NT3 can signal through TrkA and TrkB in neurons at certain stages of development and may explain why the phenotype of NT3-/- mice is more severe than that of trkC-/- mice.     

Developmental changes of hypothalamic, pituitary and striatal tachykinins in response to testosterone: influence of prenatal melatonin.

Substance P (SP) and neurokinin A (NKA), members of the family of mammalian tachykinins, are involved in the regulation of many physiological functions and are widely distributed in mammalian tissues. In this report, the effects of prenatal melatonin on the postnatal developmental pattern of NKA, and SP, and on testosterone secretion were investigated. Also, tachykinin response to the administration of testosterone propionate (TP) was studied. The brain areas studied were medio-basal-hypothalamus, pituitary gland and striatum. Male rat offspring of control or melatonin treated mother rats were studied at different ages of the sexual development: infantile, juvenile or prepubertal periods, and pubertal period. Both groups received exogenous TP (control-offspring+TP and MEL-offspring+TP), or the vehicle (control-offspring+placebo and MEL-offspring+placebo). Hypothalamic concentrations of all peptides studied in control-offspring+placebo remained at low levels until the juvenile period, days 30-31 of age. After this age, increasing concentrations of these peptides were found, with peak values at puberty, 40-41 days of age, then declining until adulthood. In the MEL-offspring+placebo a different pattern of development was observed; hypothalamic concentrations of NKA and SP from the infantile period until the end of juvenile period were significantly higher than in control-offspring+placebo. TP administration exerted a more marked influence on MEL-offspring than on control-offspring and prevented the elevation in tachykinin concentrations associated with prenatal melatonin treatment. TP administration to control-offspring resulted in significantly reduced (P < 0.05) tachykinin concentration only at 40-41 days of age, and increased (P < 0.01) during infantile period as compared to control-offspring+placebo. Pituitary NKA concentrations were lower than in the hypothalamus. In control-offspring+placebo pituitary NKA levels did not show significant changes throughout sexual development. A different developmental pattern was observed in MEL-offspring+placebo, with significantly increased (P < 0.05) pituitary NKA concentrations at 35-36 days of age than in control-offspring+placebo. TP administration to control-offspring influenced pituitary NKA levels at the end of the infantile and pubertal periods, showing at both stages significantly higher (P < 0.05) NKA levels as compared to control-offspring+placebo. NKA levels in MEL-offspring+TP were only affected at 21-22 days of age, showing significantly increased (P < 0.01) values as compared to MEL-offspring+placebo. Striatal tachykinin concentrations in control-offspring did not undergo important modifications throughout sexual development, but during the prepubertal period they started to increase. Maternal melatonin and TP injections produced short-lived alterations during the infantile period. The results showed that prenatal melatonin delayed the postnatal testosterone secretion pattern until the end of the pubertal period and postnatal peptide secretion in brain structures. Consequently, all functions depending of the affected areas will in turn, be affected.     

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.     

Developmental changes in carbohydrate antigens in embryonic rat lens

The functions of glycosphingolipids, especially those containingthe     

Developmental changes in gangliosides in cultured cerebellar granule neurons

The content and composition of gangliosides in cultures enriched in granule neurones and in astrocytes from rat cerebellum (P6–8) showed marked differences; astrocytes contained less than 10% of the amount of granule neurones and the profile was dominated by simple gangliosides with lactosyl ceramide backbone, while gangliosides of the b series, which constitute about 40% in nerve cells, were virtually undetectable. Granule cell maturation was accompanied by a 16-fold increase in the ganglioside content during the initial 8 days in a serum-supplemented medium (S⁺), reaching a plateau much earlier and at a higher level than observed in the cerebellum in vivo. Developmental changes were characterized, as in vivo, by a pronounced decrease in the GD3 proportion and an increase in the b series of gangliosides. Compared with S⁺, adhesion among cells and fibres is different in a serum-free medium (S^–) in which the rise in cellular ganglioside content was less (30%) but the developmental changes in ganglioside profile were similar. However, in cultures in S^– only, GM3 was not detectable, while the distribution of GM1 and GD3 indicated that maturation is retarded relative to cells in S⁺. Surface exposure of gangliosides (studied by the periodate/[³H]borohydride method) was similar under the two culture conditions. There was an initial delay, especially in S^–, in the insertion of gangliosides into the plasma membrane, while the labelling of GD3 (the dominant ganglioside of immature granule cells) was very low compared with all the other species throughout the whole cultivation time.Special issue dedicated to Dr. Frederick E. Samson.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Excitability changes of cortical neurons during the postnatal period in rats exposed to prenatal hypobaric hypoxia

Pregnant rats were exposed to intermittent hypobaric hypoxia (at a simulated altitude of 7000 m or 5000 m) and the excitability of cortical neurons of their pups was tested. Stimulation of the sensorimotor cortex of rats prenatally exposed to hypoxia shortened the duration of cortical afterdischarges in 12-day-old rats, but did not change the excitability in 25-day-old animals. Shortening of the first afterdischarge in 35-day-old rats but the prolongation of the first afterdischarge in adult rats (as compared to the duration of cortical afterdischarges in rats not exposed to prenatal hypoxia) were registered. The possible mechanisms of different excitability of cortical neurons in rats prenatally exposed to hypobaric hypoxia are discussed.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Developmental changes in fat body and midgut chromosomes of Drosophila auraria

Changes in puffing activity of fat body (FB) and midgut (MG) chromosomes of Drosophila auraria during late larval and white prepupal development as well as after in vitro culture with or without ecdysterone were studied and compared with those of the salivary gland (SG). The Balbiani Rings characteristic of the SG chromosomes of D. auraria, are not formed in FB and MG. Most of the inverted tandem chromosomal duplications that have been found to be common to all three tissues showed differentiation of puffing activity of the bands considered to be homologous. The major early ecdysone puffs 73A and 73B (considered to be homologues of D. melanogaster puffs 74EF and 75B, respectively), together with other early ecdysone puffs were present in all three tissues. Clear intermoult and postintermoult puffs were not evident in FB and MG chromosomes. However, a small set of late ecdysone puffs could be scored in FB, while no late ecdysone puffs were abserved in MG. Other tissue-specific puffs were identified, but a very small number of them were limited to MG.by W. Beermann     

Developmental changes in mesenteric artery reactivity in embryonic and newly hatched chicks

At birth, the intestine becomes the sole site for nutrient absorption requiring a dramatic increase in blood flow. The vascular changes accompanying this transition have been partly characterized in mammals. We investigated, using wire myography, the developmental changes in chick mesenteric artery (MA) reactivity. Rings of the MA from 15-day (E15) and 19-day (E19) chicken embryos (total incubation 21 days) as well as non-fed 0–3-h-old (NH3h) and first-fed 1-day-old (NH1d) newly hatched chicks contracted in response to KCl, norepinephrine (NE), U46619, and endothelin (ET)-1 and relaxed in response to acetylcholine (ACh), sodium nitroprusside (SNP), and forskolin indicating the presence of electro- and pharmaco-mechanical coupling as well as cGMP- and cAMP-mediated relaxation. In ovo development and transition to ex ovo life was accompanied by alterations in the response of the MAs, but a different developmental trajectory was observed for each reactivity pathway tested. Thus, the contractile efficacy of KCl underwent a linear increase (E15 < E19 < NH3h < NH1d). The efficacy of NE and U46619 increased in ovo, but not ex ovo (E15 < E19 = NH3h = NH1d) and the efficacy of ET-1 peaked at E19 (E15 < E19 > NH3h = NH1d). The relaxations elicited by ACh (endothelium-dependent), SNP, and forskolin did not undergo significant developmental changes. In conclusion, the ability of chick MAs to constrict in response to pharmacological stimuli increases during the embryonic period, but no dramatic changes are induced by hatching or the first feeding. Maturation of vasodilator mechanisms precedes that of vasoconstrictor mechanisms. Alterations of the delicate balance between vasoconstrictors and vasodilators may play an important role in perinatal intestinal diseases.     

Developmental changes in rat brain monoamine metabolism and β-adrenoceptor subtypes after chronic prenatal exposure to propranolol

The aim of this study was to investigate whether a chronic prenatal β-blockade can alter the maturation of the noradrenergic system in the rat brain. Pregnant female and adult male rats were treated for 10 days with the β-antagonist propranolol dissolved in the drinking water (40–50 mg/kg/day). Direct and long-term effects on β-adrenoceptors and monoamine metabolism in various rat brain regions were determined.

After the prenatal treatment the propranolol level in the foetal brain was 0.9 μg/g, while in the adult brain 2.0 μg/g was present. The foetal β1-receptors were significantly up-regulated by propranolol (200%), whereas the β2-receptor number remained unaltered. On postnatal days 4 and 21 the number of both β-subtypes was the same as that of controls. Noradrenaline, its metabolite 3-methoxy-4-hydroxyphenylglycol and their ratio were unaltered directly after the prenatal treatment. In the PN 21 offspring, however, the metabolite level had increased in the frontal cortex (+17%) and hippocampus (+32%), and the ratio in the hippocampus (37%) and medulla pons (+34%). Prenatal treatment also induced a significant increase of the 5-hydroxyindolacetic acid·5-hydroxytryptamine ratio (+15%) in the medulla pons at GD 21. No direct or lasting effects were found on dopamine metabolism. Propranolol treatment of adult rats gave no direct changes in monoamine metabolism.

We concluded that chronic prenatal propranolol exposure (a) reversibly up-regulates foetal β1-adrenoceptors, and (b) increases the NA activity in the brain in later life.     

Developmental changes of synapsin I subcellular localization in rat cerebellar neurons

Synapsin I, one of the major synaptic proteins, is thought to associate with synaptic vesicles and to play a regulatory role in neurotransmitter release. In mature neurons, it is concentrated almost exclusively in presynaptic nerve endings. Here, we studied the subcellular localization of synapsin I during the development of rat cerebellar cortices by immunocytochemistry, using anti-synapsin I antibodies and found that during the development of rat cerebellar cortices it tentatively exists in the dendritic growth cones of immature internal granule cells and in the axonal growth cones of mossy fibers as well as mature presynaptic endings. Also, we found that synapsin I, in the axonal and dendritic growth cones does not necessarily associate with vesicles, but rather with fuzzy filamentous structures in the cytoplasm. In search of the structure of synapsin I in vivo, we employed the quick-freeze, deep-etch technique after immunogold labeling. Synapsin I seems to thereby connect synaptic vesicles or anchor them to cytoskeletons in presynaptic endings.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Developmental expression of survivin during embryonic submandibular salivary gland development

Background  

The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival.     

Development of hypothalamic control over thyrotropin secretion during human prenatal life]

It was shown in the tissue culture experiments that the human hypophysis secreted autonomously the thyrotrophin during the last three fourths of the prenatal life. The intensity of secretion is the highest in the end of the first third of this period, then it decreases, but during the last third it increases reliably again. During the second half of development the level of thyrotrophin in female foetuses in reliably higher than in the male ones. In the beginning of the second third of prenatal life, the hypothalamic factors decrease the autonomous thyrotrophin secretion twice in foetuses of both the sexes. In the end of the second third, sexual differences appear in their effect; they decrease reliably the autonomous thyrotrophin secretion in the male foetuses, whereas no such effect is observed in the female ones. The stimulating effect of the hypothalamic thyrotrophin releasing hormone manifests itself during the last third of prenatal life in foetuses of both the sexes. During the second half of prenatal life, the thyrotrophin concentration in blood of female foetuses is also reliably higher than in male foetuses. There is a positive correlation in female foetuses between the thyrotrophin concentration in blood and the level of hypophysial secretion under the effect of hypothalamic factors. Thyrotrophin is found in the cranial fluid of foetuses. In some cases its concentration in the cranial fluid is higher than in the blood. No correlation was found between the levels of the hormone in fluid and blood in female foetuses; a positive correlation was found in male foteuses.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Developmental changes in phosphorylation state of neurofilament proteins in the chick embryonic optic nerve

Developmental changes in the phosphorylation state of neurofilament proteins (NFPs) in the chick embryonic optic nerve were histochemically and biochemically studied using monoclonal antibody (MAb) 82E10 specific to the highly phosphorylated components of high (180K)- and middle (160K)-molecular-weight subunits of neurofilament (NF) in the chicken. Cross sections of developing embryonic optic nerve were studied by enzyme immunohistochemistry using this MAb. The staining pattern showed marked changes with the developmental stage. In 6-day embryos (E6) the entire cross section was stained, whereas in E10 only about a ventroposterior half of the cross section was stained. In E14 nearly the entire area of the cross section became unstained. Thereafter, the immunoreactivity reappeared and gradually increased, such that in E20 the entire cross section became immunopositive again. Electrophoretic and immunoblot analyses were made on optic nerves dissected out of embryos of various stages. The 82E10 immunoreactivity at the position of NF-M underwent a transient loss in E14 in parallel with the time course of histochemical change. Two-dimensional gels stained for protein further showed that the highly phosphorylated form of NF-M is transiently lost from embryonic optic nerve in E14, while the less phosphorylated form persists throughout the embryonic developmental stages. In order to understand the orderly loss of the 82E10 immunoreactivity in relation to retinotopic and chronotopic organizations of the fibers in the embryonic optic nerve, retinal injection of a fluorescent dye DiI as an anterograde tracing marker for selected fibers was utilized. An ordered arrangement of the fibers was present within the embryonic optic pathway, suggesting that the orderly loss of the 82E10 immunoreactivity in the embryonic optic nerve reflects the chronological order of the optic axons. These changes in the phosphorylation state of NFPs in the embryonic optic nerve presumably reflect dynamic changes of the neuronal cytoskeleton at certain stages during development.     

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.     

Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development

Somatic sensation relies on the transduction of physical stimuli into electrical signals by sensory neurons of the dorsal root ganglia. Little is known about how and when during development different types of sensory neurons acquire transduction competence. We directly investigated the emergence of electrical excitability and mechanosensitivity of embryonic and postnatal mouse sensory neurons. We show that sensory neurons acquire mechanotransduction competence coincident with peripheral target innervation. Mechanotransduction competence arises in different sensory lineages in waves, coordinated by distinct developmental mechanisms. Sensory neurons that are mechanoreceptors or proprioceptors acquire mature mechanotransduction indistinguishable from the adult already at E13. This process is independent of neurotrophin‐3 and may be driven by a genetic program. In contrast, most nociceptive (pain sensing) sensory neurons acquire mechanosensitive competence as a result of exposure to target‐derived nerve growth factor. The highly regulated process of mechanosensory acquisition unveiled here, reveals new strategies to identify molecules required for sensory neuron mechanotransduction.