Neural stem/progenitor cells derived from the embryonic dorsal telencephalon of D6/GFP mice differentiate primarily into neurons after transplantation into a cortical lesion.

D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na(+)-currents and K (A)- and K (DR) currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.

Quantification of astrocyte volume changes during ischemia in situ reveals two populations of astrocytes in the cortex of GFAP/EGFP mice.

Energy depletion during ischemia leads to disturbed ionic homeostasis and accumulation of neuroactive substances in the extracellular space, subsequently leading to volume changes in astrocytes. Confocal microscopy combined with 3D reconstruction was used to quantify ischemia-induced astrocyte volume changes in cortical slices of GFAP/EGFP transgenic mice. Twenty-minutes of oxygen-glucose deprivation (OGD) or oxygen-glucose deprivation combined with acidification (OGD(pH 6.8)) revealed the presence of two distinct astrocytic populations, the first showing a large volume increase (HR astrocytes) and the second displaying a small volume increase (LR astrocytes). In addition, changes in resting membrane potential (V(m)), measured by the patch-clamp technique, supported the existence of two astrocytic populations responding differently to ischemia. Although one group markedly depolarized during OGD or OGD(pH 6.8), only small changes in V(m) toward more negative values were observed in the second group. Conversely, acidification (ACF(pH 6.8)) led to a uniform volume decrease in all astrocytes, accompanied by only a small depolarization. Interestingly, two differently responding populations were not detected during acidification. Differences in the expression of inwardly rectifying potassium channels (Kir4.1), glial fibrillary acidic protein (GFAP), and taurine levels in cortical astrocytes were detected using immunohistochemical methods. We conclude that two distinct populations of astrocytes are present in the cortex of GFAP/EGFP mice, based on volume and V(m) changes during exposure to OGD or OGD(pH 6.8). Immunohistochemical analysis suggests that the diverse expression of Kir4.1 channels and GFAP as well as differences in the accumulation of taurine might contribute to the distinct ability of astrocytes to regulate their volume.

Three-dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ.

Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.

Transplantation of embryonic neuroectodermal progenitor cells into the site of a photochemical lesion: immunohistochemical and electrophysiological analysis.

GFP labeled/NE-4C neural progenitor cells cloned from primary neuroectodermal cultures of p53- mouse embryos give rise to neurons when exposed to retinoic acid in vitro. To study their survival and differentiation in vivo, cells were transplanted into the cortex of 6-week-old rats, 1 week after the induction of a photochemical lesion or into noninjured cortex. The electrophysiological properties of GFP/NE-4C cells were studied in vitro (8-10 days after differentiation induction) and 4 weeks after transplantation using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. After transplantation into a photochemical lesion, a large number of cells survived, some of which expressed the astrocytic marker GFAP. GFP/GFAP-positive cells, with an average resting membrane potential (Vrest) of -71.9 mV, displayed passive time- and voltage-independent K+ currents and, additionally, voltage-dependent A-type K+ currents (KA) and/or delayed outwardly rectifying K+ currents (KDR). Numerous GFP-positive cells expressed NeuN, betaIII-tubulin, or 68 kD neurofilaments. GFP/betaIII-tubulin-positive cells, with an average Vrest of -61.6 mV, were characterized by the expression of KA and KDR currents and tetrodotoxin-sensitive Na+ currents. GFP/NE-4C cells also gave rise to oligodendrocytes, based on the detection of oligodendrocyte-specific markers. Our results indicate that GFP/NE-4C neural progenitors transplanted into the site of a photochemical lesion give rise to neurons and astrocytes with membrane properties comparable to those transplanted into noninjured cortex. Therefore, GFP/NE-4C cells provide a suitable model for studying neuro- and gliogenesis in vivo. Further, our results suggest that embryonic neuroectodermal progenitor cells may hold considerable promise for the repair of ischemic brain lesions.