Postnatal Developmental And Cytobiological Study On Oligodendrocyte Precursor Cells Of Rats

The cellular composition of the vertebrate central nervous system (CNS) is traditionally thought of as consisting of neurons, astrocytes, oligodendrocytes, and microglia. Recently, a novel glial cell type has been characterized by expression of the NG2 chondroitin sulphate proteoglycan and named as oligodendrocyte precursor cell (OPC). They are thought to belong to the oligodendrocyte lineage, but do not express proteins characteristic of mature myelinating oligodendrocytes. In addition to differentiating into myelinating oligodendrocytes, OPC has been proved to be able to generate neurons and astrocytes in culture, suggesting that NG2+ OPCs in the CNS may possess stem cell-like characteristics, including multipotentiality in vitro and in vivo. NG2+ progenitors migrate throughout the developing CNS at a stage before axons have fully matured and before myelination begins. Although the majority of NG2+ cells generated during CNS development do give rise to oligodendrocytes, a significant proportion of NG2+ cells do not differentiate into myelin-forming oligodendrocytes but remain in the mature CNS with an immature phenotype. The persistence of numerous OPCs in the mature CNS has raised questions about their identities, relation to other type of CNS cells, and functions besides their progenitor role. Several lines of evidence suggest that NG2+ cells in the adult CNS represent a population of reactive glial cells. Despite of their abundance as a progenitor population and potential importance in maintenance and repair of neurological function, the developmental ontogeny of OPCs remains controversial. It was proposed that NG2 positive cells in the CNS parenchyma comprise a unique population of glia, distinct from oligodendrocytes and astrocytes. However, there was insufficient developmental evidence supporting that proposal yet.OPCs can form direct synapses with glutamatergic and GABAergic neurons. They receive presynaptic input from neurons and respond to neurotransmitters released at synapses. As in neuron-neuron synapses, neuron-OPC synapses exhibit paired pulse facilitation and activity dependent potentiation similar to LTP in neurons, but it is not clear how activation of presynaptic neuron leads to activation of these cellular functions at different locations and developmental stages in vivo.For this reason, we investigated the expression patterns and localization of NG2 positive OPCs in the normal adult CNS by immunohistochemistry. And then, we observed the cytological features of OPCs in perinatal hippocampus and diversities after hypoxia-ischemic brain damage. Finally, it was undertaken to determine the morphological and electrophysiological features of these cells during different postnatal developmental stages. The main results are as follows:1. OPCs are widely distributed in several areas of adult CNS. The morphological heterogeneity of OPCs in hippocampus, grey matter and white matter of cerebral cortex was specially noted. In the grey matter, they were more densely distributed in non-neuronal layers with the classical stellate morphology. Processes of grey matter OPCs radiated in all directions from soma. The length of the processes mainly ranged from 10 to 40μm (average 25.6μm). In contrast, those in white matter had elongate cell bodies (small and round in cross section) with parallel processes extending predominantly from the two poles and passing along the nerve axis. The length of processes ranged from 20 to 50μm (average of 32.3μm), which is longer than OPCs in grey matter. Moreover, OPCs soma in white matter occupied the significantly small cell surface area than that in grey matter. No significant differences in numerical cell density were found between white matter and gray matter.2. OPCs express instinct electrophysiological features different from astrocytes and neurons. OPCs located in the stratum radiatum region of area CA1 exhibited small Na~+ currents, large A-type and delayed rectifier K+ currents. Cells with these properties marked with intracellular Lucifer yellow had a stellate morphology, with thin, highly branched processes that extended from a small cell body. Like neurons, NG2 cells expressed voltage-gated Na~+ channels. Under physiological conditions, these Na~+ channels produced a small inflection on the rising phase of membrane potential responsed following depolarizing current injection. However, the peak amplitude of the Na~+ current was smaller than that observed in neurons under similar conditions, indicating that they expressed only a fraction of the Na~+ channels. The small number of these channels and the comparatively large K+ conductance present prevent NG2 cells from generating action potentials. Despite the higher membrane resistance of NG2 cells, their high resting membrane potential indicated that their membranes were largely permeable to K~+ at rest. Injection of positive current into NG2 cells elicited nonlinear, time-dependent changes in membrane potential unlike the passive behavior of astrocyte membranes. The predominant voltage-gated conductance responsible for these changes in membrane potential was carried by K~+. Depolarizing voltage steps from the resting potential elicited both rapidly inactivating and sustained K~+ currents. These currents consisted of conventional"A-type''(KA) and"delayed rectifier''type K~+ currents that are antagonized by 4-aminopyridine and tetraethylammonium, respectively. In addition to these outward K~+ currents, NG2 cells also exhibited inward K~+ currents to a variable degree. These currents were likely to result from the inwardly rectifying K~+ channels (Kir). Kir channels stabilized the membrane potential near the K~+ equilibrium potential, and maybe involved in the accumulation, buffering, or siphoning of K~+ released during neuronal activity. Other than these three electrophysiological glial profiles, a typical time dependent inactivation of symmetric K~+ tail currents following the withdrawal of voltage steps were recorded, which may participate in rest membrane potential regulation. When comparing different brain regions, the significant differences were for RMP, Rm and Cm between cells in the CA1 region of the hippocampus and the white matter of cerebral cortex.3. OPCs in hippocampus of perinatal rats after hypoxia-ischemic brain damage for 2 hours showed more active membrane depolarization reacts than control group. Passive membrane properties for each cell were also determined with the increase of whole-cell membrane capacitance and membrane resistance, but the decrease of resting membrane potential. Both transient currents and inward rectifier currents were previously increased in OPCs of HIBD, while delayed rectifying currents decreased. To obtain a more quantitative assessment of current expression by these cells, in voltage-clamp mode a series of stimulation protocols evoked the inward and outward current in OPCs. Current traces showed qualitatively similar electrophysiological profiles in these cells, but with three notable differences: the sodium current in cells with voltage-gated Na~+ channels was significantly larger, increase of"A-type"potassium currents, and showed a larger K~+ tail current. In addition to the alternation of current amplitude, the voltage of half-maximum activation (Vmid), and the slope factor (k), which were fitted by Voltage-Charge Boltzmann equation, showed more excitable and the increase of activation of OPCs in HIBD. Na~+ to K~+ conductance ratio was also increased mainly own to the increase and activation of sodium currents. However, such changes in ion channel expression have not been obviously recorded in astrocytes and neurons in hippocampus.4. NG2 immunopositive OPCs were continuously distributed in cerebral cortex and hippocampus during different postnatal developmental stages. These cells rapidly increase in number over the postnatal 7 days and migrate extensively to populate with abundant processes both in developing cortex and hippocampus. The morphology of OPCs exhibits extremely complex changes with the long distance primary process gradually increased the distribution number from neonatal to adult CNS. Although, in current-clamp mode, some OPCs in the early developmental stage show fluctuations in their basal membrane potential, no clear postsynaptic events were measurable. OPCs in the stratum radiatum region of area CA1 in adult (P50) hippocampus have typical voltage-gated currents exhibited large A-type and delayed rectifier K~+ currents, small inward currents which was sensitized to TTX meaning Na~+ currents, and did not fire action potentials. Membrane capacitance (Cm) of OPCs increased during postnatal development. The Na-to-K conductance ratio proved to be increased in OPCs from P0 to P50, for depolarization produced large Na~+ inward currents. These results showed that OPCs in adult were more excitable than those in perinatal stage. But low Na-to-K conductance ratio indicated that they still belonged to nonexcitable cells.Overall, in the present study, several lines of evidence indicated that a population of NG2 immunopositive OPCs in the CNS can be considered as another separate macroglial cell type:⑴extremely morphological complex changes from neonatal to adult stage;⑵the increase of long and multiple branches processes from neonatal to adult stage;⑶the increase of membrane capacitance and Na-to-K conductance ratio during postnatal developmental stages;⑷the distinct cytological properties in perinatal hippocampus and during hypoxia-ischemic brain damage;⑸the morphological and electrophysiological features obviously different from neuron and astrocyte.