Functional Maturation Of Neurons Research Articles

Development and activity-dependent expression of neuronal marker proteins in organotypic cultures of rat visual cortex

We are interested in activity-dependent mechanisms which govern the structural and functional maturation of neurons in the visual cortex. We have asked whether the expression of neuronal markers microtubule-associated proteins tau, MAP-2, synaptophysin (p38), and the growth-associated protein GAP-43 are dependent on cortical afferents or spontaneous activity. As a model system we have employed organotypic monocultures of rat visual cortex (OTCs, isolated from subcortical structures) in comparison with visual cortex in vivo (innervated by thalamic and other afferents) at different postnatal ages. We know from previous work that the OTCs, like the cortex in vivo, display a high rate of spontaneously generated action potentials. Therefore, as a third objective, we have analysed OTCs grown as monocultures under chronic blockade of spontaneous action potentials. Protein expression was detected by protein blots and/or immunohistochemistry. The proteins examined in this study are expressed in OTCs, even when grown under activity blockade. However, the pattern of expression differs from the cortex in vivo. Tau is expressed much weaker in OTCs than in cortex in vivo. The expression of the major band of about 50 kDa increases over time in vivo and in OTCs. Smaller isoforms of tau are dramatically downregulated, and larger (adult) isoforms do not appear within 35 days in vitro (DIV). Under activity blockade the expression of tau reaches a maximum by 21 DIV and decreases dramatically, so that the protein is hardly detectable by 47 DIV. MAP-2-immunoreactive proteins are localized in somata and dendrites, but also persist in axons. The expression in OTCs of p38 and GAP-43 correlates well with the expression observed in vivo. Synaptophysin (p38) occurs with a similar time course and amount in OTCs as in cortex in vivo. Synaptic boutons appear in all layers, and specialized terminal elements have been observed. Activity blockade slightly affects the p38 expression, although the late postnatal decline in p38 immunoreattivity observed on protein blots from cortex in vivo and in normal OTCs appears more accentuated in activity-blocked OTCs. The GAP-43 expression is prominent from birth onwards in vivo and in OTCs. However, in normal OTCs GAP-43 is not declining as it is in vivo, although it is downregulated in activity-blocked OTCs. As a major finding we report that neuronal markers which are normally expressed in immature neurons and axons during the period of differentiation and structural plasticity are continuously expressed in OTCs, suggesting that a monocultured cortex retains the ability for growth and structural changes longer than the cortex in vivo.

Distribution of Ca2+ and Na+ conductances during neuronal differentiation of chick DRG precursor cells

The distribution of Ca2+ and Na+ conductances on neuronal precursor cells was investigated during differentiation. Ionic conductances on the soma or on the growth cone were isolated by superfusing all other parts of the cells with sucrose. Conductances on the neuritic shaft were detected as additional conductances after removing sucrose from the neuritis shaft. Neuronal precursor cells were isolated from chick dorsal root ganglia by selectively killing differentiated neurons. Cultured precursor cells differentiated into morphological and functional mature neurons. Functionally undifferentiated precursor cells (during the first 10 hr in culture) expressed only low-voltage-activated (LVA) Ca2+ currents. High-voltage-activated (HVA) Ca2+ and Na+ currents appeared delayed after more than 10 hr in culture. Voltage-dependent conductances, if expressed by a cell, were present on all parts of the surface membrane at all stages of differentiation. LVA Ca2+ conductances were well represented on the growth cone as well as on the soma in functionally undifferentiated precursor cells. During differentiation of precursor cells, LVA Ca2+ and HVA Ca2+ as well as Na+ conductances were expressed on the somatic membrane, on the neuritic shaft, and on the growth cone. These results demonstrate the expression of Ca2+ channels on growth cones during differentiation.

GABA agonist induced changes in ultrastructure and GABA receptor expression in cerebellar granule cells is linked to hyperpolarization of the neurons

GABA has been shown to exert a neurotrophic like activity by enhancing the morphological and functional maturation of neurons. Mechanisms involved in this effect of GABA are largely unknown but since GABA has been shown to mediate a hyperpolarizing action on neurons it can be assumed that this action might be important. In order to investigate this possibility, the ability to mimic the trophic actions of GABA of different agents known to influence the membrane potential or the GABA gated chloride channels was studied. Hence, GABA receptor expression as well as the ultrastructure of cerebellar granule cells were monitored after exposure of the cells in culture to either bromide, valinomycin or picrotoxin. It was found that cells which at early developmental stages (4 days in culture) were exposed to bromide or valinomycin expressed low affinity GABA receptors similar to cells treated with the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). This was in contrast to untreated cultures or cultures treated with both THIP and picrotoxin which expressed only high affinity GABA receptors and no low affinity receptors. In cultures at more mature stages (14 days in culture) bromide treatment did not lead to formation of low affinity GABA receptors. Studies of the ultrastructure of the cells (4-day-old cultures) showed that exposure to bromide or valinomycin mimicked the ability of THIP to enhance the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus, vesicles and coated vesicles. Again, in 14-day-old cultures treatment with bromide had no effect on the ultrastructure. Exposure of the cells to picrotoxin significantly reduced the ability of THIP to induce the increase in the density of the organelles mentioned above. Altogether the results strongly suggest that the hyper-polarizing action of GABA via the chloride channels coupled to high affinity GABA receptors is the permissive signal for initiation of further reactions involved in the trophic and differentiating actions of GABA.

Postembryonic neurogenesis in the CNS of the tobacco hornworm, Manduca sexta. II. Hormonal control of imaginal nest cell degeneration and differentiation during metamorphosis

The nervous system of an adult moth is comprised of retained larval neurons that are remodeled during metamorphosis and a set of new adult specific neurons. The new neurons arise from a stereotyped array of stem cells (neuroblasts) that divide during larval life to generate nests of up to 100 arrested postmitotic immature neurons, the imaginal nest (IN) cells. At the onset of metamorphosis, some of the IN cells die while the remainder differentiate into mature functional neurons. Metamorphosis in insects is regulated by 2 classes of hormones, the ecdysteroids and the juvenile hormones. The transition from larva to pupa requires the disappearance of juvenile hormones followed by 2 releases of ecdysteroids: a small "commitment peak" and a larger "prepupal peak." Through a series of endocrine manipulations, we demonstrate that the death and differentiation observed among the abdominal IN cells at metamorphosis are both influenced by these hormonal cues. If the abdomen was isolated from the hormonal sources in the anterior half of the larva before the onset of metamorphosis, death and differentiation of the IN cells were prevented. Infusion of ecdysteroids into such abdomens, to mimic the prepupal peak, resulted in the IN cells showing the same fate as seen in control animals during the early phases of metamorphosis. The response of the IN cells to the small commitment peak of ecdysteroids was heterogeneous. Exposure to this small peak of steroids caused some cells to become committed to resume their development, making them resistant to juvenile hormone application.(ABSTRACT TRUNCATED AT 250 WORDS)

Pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.

Pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.

Developmental profile of neuron-specific (NSE) and non-neuronal (NNE) enolase

Neurons and glia of mature brain can be distinguished by their isoenzyme content of the glycolytic enzyme enolase. Neurons contain neuron-specific enolase (NSE) and glial cells have non-neuronal enolase (NNE). Measurement of each isoenzyme by specific radioimmunoassay during the course of brain development in rat shows that NSE levels are very low in embryonic brain and increase at atime coincident with the morphological and functional maturation of neurons. NNE levels are high in embryonic brain and decrease when NSE first appears, followed by a gradual increase to adult levels. NSE levels rise at a slower rate in brain areas known to develop over a more protracted period (forebrain, cerebellum) compared to areas that develop more rapidly (brain stem). The data are consistent with a hypothesized switch from NNE to NSE during neuronal development. In E 60 and E 100f monkey brain tissue NSE/NNE ratios are higher in regions containing older neurons. This suggests that a similar switch from NNE to NSE also occurs during neuronal development in monkey.