Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors.

Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.

Signaling through EAAT-1/GLAST in cultured Bergmann glia cells.

Glutamate, the major excitatory amino acid, activates a wide variety of signal transduction cascades. Synaptic plasticity relies on activity-dependent differential protein expression. Ionotropic and metabotropic glutamate receptors have been critically involved in long-term synaptic changes, although recent findings suggest that the electrogenic Na(+)-dependent glutamate transporters, responsible of its removal from the synaptic cleft, participate in glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells albeit most of the glutamate uptake occurs in the glial compartment. Within the cerebellum, Bergmann glial cells are close to glutamatergic synapses and participate actively in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of Bergmann glia glutamate transporters as signaling entities. To this end, primary cultures of chick cerebellar Bergmann glial cells were exposed to d-aspartate (D-Asp) and other transporter ligands and the serine 2448 phosphorylation pattern of the master regulator of protein synthesis, namely the mammalian target of rapamycin (mTOR), determined. An increase in mTOR phosphorylation and activity was detected. The signaling cascade included Ca(2+) influx, activation of the phosphatidylinositol 3-kinase and protein kinase B. Furthermore, transporter signaling resulted also in an increase in activator protein-1 (AP-1) binding to DNA and the up-regulation of the transcription of an AP-1 driven gene construct. These results add a novel mediator of the glutamate effects at the translational and transcriptional levels and further strengthen the notion of the critical involvement of glia cells in synaptic function.

Glycogen stores are impaired in hypothalamic nuclei of rats malnourished during early life.

Perinatal nutrition has persistent influences on neural development and cognition. In humans and other animals, protein malnutrition during the perinatal period causes permanent changes, inducing to adulthood metabolic syndrome. Feeding is mainly modulated by neural and hormonal inputs to the hypothalamus. Hypothalamic glycogen stores are a source of glucose in high energetic demands, as during development of neural circuits. As some hypothalamic circuits are formed during lactation, we studied the effects of malnutrition, during the first 10 days of lactation, on glycogen stores in hypothalamic nuclei involved in the control of energy metabolism. Female pregnant rats were fed ad libitum with a normal protein diet (22% protein). After delivery, each dam was kept with 6 male pups. During the first 10 days of lactation, dams from the experimental group received a protein-free diet and the control group a normoprotein diet. By post-natal day 10 (P10), glycogen stores were very high in the arcuate nucleus and median eminence of control group. Glycogen stores decreased during development. In P20 control animals, glycogen stores were lower when compared to P10 control animals. Animals submitted to malnutrition presented a staining even lower than control ones. After P45, it was difficult to determine differences between control and diet groups because glycogen stores were reduced. We also showed that tanycytes were the cells presenting glycogen stores. Our data reinforce the concept that maternal nutritional state during lactation may be critical for neurodevelopment since it resulted in a low hypothalamic glycogen store, which may be critical for establishment of neuronal circuitry.

Correlative microscopy of cerebellar Bergmann glial cells.

Double fluorescent labelling of rat cerebellar cortex using antibody to glial fibrillary acidic protein (GFAP) and Alexa fluor conjugates for secondary detection for confocal laser scanning microscope (CLSM), field emission scanning electron microscopy (FESEM) of Rhesus monkey cerebellar cortex, ultrathin sectioning and freeze-etching replica method for transmission electron microscopy of mouse cerebellar cortex have been examined in an attempt to obtain a new and more accurate view of three-dimensional image of Bergmann glial cells (BGC) and their topographic relations in the molecular layer. Intense immunopositive GFAP green staining was observed in the BGC and glial limiting layer. Secondary antibody conjugated with Alexa fluor 488 and Alexa fluor 668-1B4 stained in red capillary endothelial cells and microglial cells. BGC morphology revealed the existence of several cell types or subpopulations of BGC. Bergmann glial fibers, in palisade arrangement, branch and rebranch forming a complex glial network in the molecular layer. Field emission SEM and freeze-fracture SEM method show the SE-I image of high mass dense Bergmann glial cytoplasm ensheathing like a veil the Purkinje cell (PC) soma and dendritric arborization. Bergmann glial fibers appeared completely surrounding individual parallel fibers or parallel fiber bundles, terminal climbing fiber collaterals, basket and stellate cells and capillaries. Freeze-etching direct replicas showed the typical orthogonal arrangement of intramembrane particles, corresponding to the large repertoire of BGC receptors. The study reveals three-dimensional Bergmann glial cells heterogeneity and the complex network formed by Bergmann glial cells in the molecular layer.

NMDA receptors in cultured radial glia.

The expression of the NMDA subtype of glutamate receptors was investigated by Western blot analysis and RT-PCR in cultured chick Bergmann and Müller glial cells. Using subunit-specific antibodies directed to the carboxy terminus of the rat NMDAR2A/B we detected the expression of the NMDAR2 subunit in both kinds of culture. The functional subunit of the NMDA receptor, NMDAR1, was detected by means of RT-PCR. These results, together with our previous functional characterization of NMDA receptors in radial glia, provide conclusive evidence for the expression of functional NMDA receptor/channels in Bergmann and Muller glia cells. Our findings strengthen the notion of a modulatory role of glial cells in synaptic transmission.