Cell Endfeet Research Articles

Müller cell zinc transporter-3 labeling suggests a role in outer retina zinc homeostasis.

Labeling for zinc transporter protein-3 (ZnT-3), which can be found localized to glutamatergic vesicles elsewhere in the nervous system, has revealed an unexpectedly high concentration of this transporter protein in the outer limiting membrane region of the murine retina, a region that contains the mitochondria-rich portion of photoreceptor inner segments and is not involved with vesicle release. Having suggested the possibility that Müller cell apical villi forming the outer limiting membrane may be associated with the labeling observed, we used immunohistochemical techniques to look for ZnT-3 labeling of Müller cells isolated from rat and mouse retinas. With DAB labeling, rat Müller cell apical villi, soma, and endfeet exhibited ZnT-3 reactivity. FITC label and confocal analysis revealed that ZnT-3 protein appeared throughout the length of mouse Müller cells. We conclude from these observations that the dense labeling for ZnT-3 in the photoreceptor inner segment region of murine retinal slices is due to labeling of ZnT-3 protein associated with Müller cell apical villi. Based on these findings we suggest that Müller cells utilize ZnT-3 to regulate retinal zinc homeostasis and that this role is important to mitochondrial function in the photoreceptor inner segments.

Müller Cell Zinc Transporter-3 Labeling Suggests a Role in Outer Retina Zinc Homeostasis

Labeling for zinc transporter protein-3 (ZnT-3), which can be found localized to glutamatergic vesicles elsewhere in the nervous system, has revealed an unexpectedly high concentration of this transporter protein in the outer limiting membrane region of the murine retina, a region that contains the mitochondria-rich portion of photoreceptor inner segments and is not involved with vesicle release. Having suggested the possibility that Müller cell apical villi forming the outer limiting membrane may be associated with the labeling observed, we used immunohistochemical techniques to look for ZnT-3 labeling of Müller cells isolated from rat and mouse retinas. With DAB labeling, rat Müller cell apical villi, soma, and endfeet exhibited ZnT-3 reactivity. FITC label and confocal analysis revealed that ZnT-3 protein appeared throughout the length of mouse Müller cells. We conclude from these observations that the dense labeling for ZnT-3 in the photoreceptor inner segment region of murine retinal slices is due to labeling of ZnT-3 protein associated with Müller cell apical villi. Based on these findings we suggest that Müller cells utilize ZnT-3 to regulate retinal zinc homeostasis and that this role is important to mitochondrial function in the photoreceptor inner segments.

Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS α- and β-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.

EAAT1 and d-serine expression are early features of human retinal development

In the developing central nervous system (CNS), the activation of N-methyl- d-aspartate (NMDA) receptors is probably an important regulator of processes such as synaptogenesis and neurite growth. NMDA receptor activation is dependent upon the homeostasis of glutamate and the presence of co-agonists such as d-serine. We have investigated the expression of the glutamate transporter excitatory amino acid transporter-1 (EAAT1 or GLAST) as the key regulator of retinal extracellular glutamate levels, and the ontogeny of d-serine expression in the developing human retina. The expression of EAAT1 and d-serine was compared to the temporal and spatial distribution of the synaptic vesicle marker synaptophysin and the synaptic vesicle glutamate transporter vGLUT1. We also examined the co-expression of EAAT1 and cellular retinaldehyde-binding protein (CRALBP), and the co-expression of EAAT1 and d-serine. Human retinae aged 10–20 weeks' gestation (WG) were prepared for immunocytochemistry or for Western blotting. Expression of EAAT1 was evident at 10 WG in cell bodies, processes and end-feet of radial glia-like cells at all retinal eccentricities. d-Serine immunolabelling was also evident in radial glia-like cells by 12 WG. In contrast, immunoreactivity for synaptophysin only started to appear in the central retina at 12 WG whilst immunoreactivity for vGLUT was slightly later. EAAT1 and d-serine were co-localised to the same cell population. In addition, EAAT1 and CRALBP were also co-localised to the same cell population of radial glia-like cells, suggesting that the EAAT1 and d-serine-positive cells may be Müller cells. This study shows that key potential modifiers of NMDA receptor activity are present before synaptic vesicle proteins are evident and may thus play a role in shaping synaptogenesis in the developing human retina.

The distribution of apolipoprotein E in mouse olfactory epithelium

Previous studies from our laboratory suggest that apolipoprotein (apoE), a lipid transporting protein, facilitates olfactory nerve regeneration. We have shown that apoE is enriched in the olfactory nerve and around the glomeruli of the olfactory bulb (OB). The studies reported herein were undertaken to identify possible sources of apoE in the olfactory epithelium (OE). Immunoblotting results revealed apoE expression in the OE of wild-type (WT) mice, but not in apoE deficient/knockout (KO) mice. Immunohistochemical studies revealed that the perikarya and processes of sustentacular (Sus) cells expressed apoE-like immunoreactivity. Minimal neuronal apoE immunostaining was seen, although apoE was observed in the interstial spaces between olfactory receptor neurons (ORN). Substantial apoE-like immunoreactivity was localized to the endfeet and terminal process of Sus cells surrounding the basal cells. Double labeling immunocytochemical studies confirmed that the cell bodies and endfeet of Sus cells expressed high levels of apoE. The endothelial cells of blood vessels were intensely stained for apoE in the lamina propria. Cells forming Bowman's gland also immunostained for apoE. The apoE staining in the nerve fascicles was less intense, but was uniformly distributed throughout the core of the nerve bundles. Heavily stained cells, probably ensheathing glia, surrounded the nerve fascicles. These results revealed that apoE is expressed in the adult OE and lamina propria at strategic locations where it could facilitate the differentiation, maturation and axonal growth of the ORN, perhaps by recycling lipids from degenerating ORN for use by growing axons.

Behavior of in vitro cultured ameboid microglial cells migrating on Müller cell end‐feet in the quail embryo retina

Ameboid microglial cells migrate tangentially on the vitreal part of quail embryo retinas by crawling on Müller cell end-feet (MCEF) to which they adhere. These microglial cells can be cultured immediately after dissection of the eye and isolation of sheets containing the inner limiting membrane (ILM) covered by a carpet of MCEF (ILM/MCEF sheets), to which the cells remain adhered. Morphological changes of microglial cells cultured on ILM/MCEF sheets for 4 days were characterized in this study. During the first minutes in vitro, lamellipodia-bearing bipolar microglial cells became rounded in shape. From 1 to 24 h in vitro (hiv), microglial cells swept and phagocytosed the MCEF on which they were initially adhered, becoming directly adhered on the ILM. MCEF sweep was dependent on active cell motility, as shown by inhibition of sweep after cytochalasin D treatment. From 24 hiv on, after MCEF phagocytosis, microglial cells became more flattened, increasing the surface area of their adhesion to substrate, and expressed the beta1 subunit of integrins on their membrane. Morphological evidence suggested that microglial cells migrated for short distances on ILM/MCEF sheets, leaving tracks produced by their strong adhesion to the substrate. The simplicity of the isolation method, the immediate availability of cultured microglial cells, and the presence of multiple functional processes (phagocytosis, migration, upregulation of surface molecules, etc.) make cultures of microglial cells on ILM/MCEF sheets a valuable model system for in vitro experimental investigation of microglial cell functions.

EVALUATION OF THE INTERNAL LIMITING MEMBRANE AFTER CONVENTIONAL PEELING DURING MACULAR HOLE SURGERY

We evaluated the histologic features of the internal limiting membrane (ILM) of the retina removed during macular hole surgery without indocyanine green staining. Our investigation focused on the presence or absence of retinal structures adherent to the retinal surface of the ILM. Because only tiny retinal cellular fragments were observed especially in ILM folds, we conclude that conventional ILM peeling can be performed safely with a cleavage plane between the retinal surface of the ILM and Müller cell endfeet.

Differential effect of α-syntrophin knockout on aquaporin-4 and Kir4.1 expression in retinal macroglial cells in mice

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that α-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in α-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the α-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that α-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and α-syntrophin null mice exhibit strong β1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that β1 syntrophin might be involved in the anchoring of Kir4.1 and the α-syntrophin independent pool of aquaporin-4.

Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat

The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5 μl intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500 μ m concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500 μ m concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85 sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.

Calcium Increases in Retinal Glial Cells Evoked by Light-Induced Neuronal Activity

Electrical stimulation of neurons in brain slices evokes increases in cytoplasmic Ca(2+) in neighboring astrocytes. The present study tests whether similar neuron-to-glial signaling occurs in the isolated rat retina in response to light stimulation. Results demonstrate that Müller cells, the principal retinal glial cells, generate transient increases in Ca(2+) under constant illumination. A flickering light stimulus increases the occurrence of these Ca(2+) transients. Antidromic activation of ganglion cell axons also increases the generation of Müller cell Ca(2+) transients. The increases in Ca(2+) transients evoked by light and antidromic stimulation are blocked by the purinergic antagonist suramin and by TTX. The addition of adenosine greatly potentiates the response to light, with light ON evoking large Ca(2+) increases in Müller cells. Suramin, apyrase (an ATP-hydrolyzing enzyme), and TTX substantially reduce the adenosine-potentiated response. NMDA, metabotropic glutamate, GABA(B), and muscarinic receptor antagonists, in contrast, are mainly ineffective in blocking the response. Light-evoked Ca(2+) responses begin in Müller cell processes within the inner plexiform (synaptic) layer of the retina and then spread into cell endfeet at the inner retinal surface. These results represent the first demonstration that Ca(2+) increases in CNS glia can be evoked by a natural stimulus (light flashes). The results suggest that neuron-to-glia signaling in the retina is mediated by neuronal release of ATP, most likely from amacrine and/or ganglion cells, and that the response is augmented under pathological conditions when adenosine levels increase.

Transcorneal Electrical Stimulation Rescues Axotomized Retinal Ganglion Cells by Activating Endogenous Retinal IGF-1 System

To investigate the effect of transcorneal electrical stimulation (TES) on the survival of axotomized RGCs and the mechanism underlying the TES-induced neuroprotection in vivo. Adult male Wistar rats received TES after optic nerve (ON) transection. Seven days after the ON transection, the density of the surviving RGCs was determined, to evaluate the neuroprotective effect of TES. The levels of the mRNA and protein of insulin-like growth factor (IGF)-1 in the retina after TES were determined by RT-PCR and Northern and Western blot analyses. The localization of IGF-1 protein in the retina was examined by immunohistochemistry. TES after ON transection increased the survival of axotomized RGCs in vivo, and the degree of rescue depended on the strength of the electric charge. RT-PCR and Northern and Western blot analyses revealed a gradual upregulation of intrinsic IGF-1 in the retina after TES. Immunohistochemical analysis showed that IGF-1 immunoreactivity was localized initially in the endfeet of Muller cells and then diffused into the inner retina. TES can rescue the axotomized RGCs by increasing the level of IGF-1 production by Muller cells. These findings provide a new therapeutic approach to prevent or delay the degeneration of retinal neurons without the administration of exogenous neurotrophic factors.

Basement Membrane–Dependent Survival of Retinal Ganglion Cells

The present study investigates the role of the inner limiting membrane (ILM) in the survival of ganglion cells (GCs) in embryonic chick and mouse retina. In chick embryos, the ILM was enzymatically removed at embryonic day (E)5 and E7 in ovo, and GCs were counted over the following 7 days of incubation. In mice, the ILM ruptured early in development due to a targeted mutation of laminin-gamma1. The number of surviving GCs in late embryonic homozygous mutant mice was compared to GC counts in heterozygotic and nonmutant siblings. The survival of GCs was also assayed in vitro, using the ILM as a substrate. When the ILM was removed in E5 chick embryos, almost all GCs underwent apoptosis by E10. GC apoptosis was prevented by reconstituting the ILM shortly after its disruption. Apoptosis of retinal GCs also occurred in mouse embryos with a fragmented ILM. ILM disruption in both chick and mice not only affected GC survival but also led to the retraction of the end-feet processes of radial cells from the GC layer. In vitro, GCs thrived better on substrates of radial cell end feet than on plain ILM, indicating that the contact with the end feet is more important than the presence of the ILM for GC survival. The radial cell end feet are the natural substrate for GCs and are essential for their survival. The importance of the ILM lies in its function to anchor the radial end feet to the vitreal surface of the retina.

Mechanisms of chloride influx during KCl-induced swelling in the chicken retina

An increase in extracellular KCl ([KCl]o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl o-induced retinal swelling were investigated in the present study, with emphasis on the Cl- transport mechanisms. Increasing [KCl]o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]o was biphasic, with one component from 5 to 35 mM [KCl]o and another from 35 to 100 mM. As Cl- omission prevented swelling in all conditions, the effect of cotransporter or Cl- channel blockers was examined to investigate the mechanisms of Cl- influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]o 25 mM (K25), and by 14-17% at [KCl]o 54 mM (K54). The Cl- channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90-95% at K54 (NPPB IC50 60.7 microM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC50 173.9 microM). Na+ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl- influx at K25 and of Cl- channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Muller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.

Staining and Peeling of the Internal Limiting Membrane in the Cat Eye

Purpose: To investigate the cat vitreomacular interface using trypan blue (TB) and indocyanine green (ICG) and to determine the validity of the cat model in terms of staining and peeling of the internal limiting membrane (ILM). Methods: Lensectomy and vitrectomy were performed in four eyes of two cats. The ILM of two eyes was stained with TB (0.15%). ILM peeling was performed in one eye. Two eyes were stained with ICG (0.5%). One eye was illuminated for 3 min. Light and transmission electron microscopy and confocal microscopy were performed. Results: Clinically, both dyes stained the cat ILM similar to human ILM. TB staining resulted in a normal ultrastructure and antigenity of the retina. ILM peeling was associated with intraretinal bleeding. There were fragments of Müller cells adherent to the retinal side of the ILM, and Müller cell endfeet were ruptured and avulsed. ICG staining of the ILM followed by illumination caused severe inner retinal damage. ICG without illumination resulted in focal ILM detachments associated with tearing of Müller cell endfeet. Conclusions: The cat can be used as a model to study the effect of TB and ICG on the central area of the cat retina, as previous results from clinical and experimental postmortem settings in human eyes were confirmed in the current study. Peeling of the ILM as a sheet as performed in human macular surgery is not feasible. Differences in the ultrastructure of the ILM and a strong adhesion of the ILM to Müller cell endfeet may account for this observation.

Spatial pattern of nonmuscle myosin-II distribution during the development of the Drosophila compound eye and implications for retinal morphogenesis

Nonmuscle myosin-II is a motor protein that drives cell movement and changes in cell shape during tissue and organ development. This study has determined the dynamic changes in myosin-II distribution during Drosophila compound eye morphogenesis. In photoreceptor neurons, myosin-II is undetectable at the apical domain throughout the first half of pupal life, at which time this membrane domain is involuted into the epithelium and progresses toward the retinal floor. Myosin-II is deployed at the apical surface at about 60% of pupal development, once the developing rhabdomeres reach the retinal floor. Subsequently, myosin-II becomes restricted to two stripes at the sides of the developing rhabdomere, adopting its final position within the visual cells R1–6; here, myosin-II is associated with a set of actin filaments that extend alongside the rhabdomeres. At the midpupal stage, myosin-II is also incorporated into stress-fiber-like arrays within the basal endfeet of the pigment cells that then change their shape. This spatiotemporal pattern of myosin-II localization and the morphological defects observed in the eyes of a myosin-II mutant suggest that the myosin-II/F-actin system is involved in the alignment of the rhabdomeres within the retina and in the flattening of the retinal floor. The observation that the myosin-II/F-actin arrays are incomplete or disorganized in R7/R8 and in rhodopsin-1-null R1–6 suggests further that the establishment and stability of this cytoskeletal system depend on rhodopsin-1 expression.

Peeling of the basal membrane in the human retina: Ultrastructural effects

Purpose To study the effects of internal limiting membrane (ILM) peeling, usually performed in cases of macular hole formation, on the structural integrity of the human retina. Design Experimental study. Material Two human donor eyes. Methods A human donor retina was subjected to ILM peeling shortly after enucleation, fixed, and studied by electron microscopy. Main outcome measures Electron microscopic findings. Results Within the peeled area, and even at its margins, a substantial number of Müller (glial) cell endfeet and vitreal Müller cell processes was severely damaged. However, many glial cells remained intact, and the cell bodies of the injured cells showed no signs of destruction. Conclusions We have shown that ILM peeling results in minor, but demonstrable, damage of the adjacent retina. Clinical studies are required to demonstrate unequivocally that ILM peeling does not induce functional deficits rather than improvement of visual function.

Concomitant distribution shift of glial GABA transporter and S100 calcium-binding proteins in the rat retina after kainate-induced excitotoxic injury

The goal of this study was to elucidate the involvement of neuronal and glial calcium-binding proteins in the stimulation of γ-aminobutyric acid (GABA) transport system by kainate-induced excitotoxicity in the rat retina. We used immunohistochemical method to assess the localization of GABA reuptake and calcium-binding proteins. After systemic administration of kainate, the neuronal GABA transporter does not show an association with calbindin D-28K. However, the localization of the GAT-3 transport system in Müller glial cells is closely correlated with the S100 proteins interacting with glial fibrillary acidic protein (GFAP) in response to kainate injury. Furthermore, we demonstrate that kainate-mediated excitotoxicity induced concomitant distribution shift of glial GABA transporter, S100 proteins and GFAP in the distal processes and endfeet of glial cells during the first 48 h.

Concomitant distribution shift of glial GABA transporter and S100 calcium-binding proteins in the rat retina after kainate-induced excitotoxic injury

The goal of this study was to elucidate the involvement of neuronal and glial calcium-binding proteins in the stimulation of γ-aminobutyric acid (GABA) transport system by kainate-induced excitotoxicity in the rat retina. We used immunohistochemical method to assess the localization of GABA reuptake and calcium-binding proteins. After systemic administration of kainate, the neuronal GABA transporter does not show an association with calbindin D-28K. However, the localization of the GAT-3 transport system in Müller glial cells is closely correlated with the S100 proteins interacting with glial fibrillary acidic protein (GFAP) in response to kainate injury. Furthermore, we demonstrate that kainate-mediated excitotoxicity induced concomitant distribution shift of glial GABA transporter, S100 proteins and GFAP in the distal processes and endfeet of glial cells during the first 48 h.

Expression of kynurenine aminotransferases in the rat retina during development

The study investigates the cellular expression of kynurenine aminotransferases (KAT I and II) in the rat retina during development. At P1 (the day of birth) and P7 (the 7th day after birth), KAT I expression was observed in the inner plexiform layer (IPL), the fiber layer (FL), and in vertically running processes in the ganglion cell layer (GCL) (but not in the cell bodies). At P14 (the 14th day after birth) a strong KAT I immunoreactivity was observed in Müller cell endfeet. KAT II was expressed in the IPL, the FL, and in cells in the GCL at P1 and P7. From P14 on, KAT II expression in the IPL decreased. Double labeling revealed that KAT I was expressed in Müller cell endfeet, whilst KAT II both on retinal ganglion cells (RGC) and Müller cell endfeet. In conclusion, KAT I and II are present in the rat retina during development. The heterogeneity of the KAT developmental profiles possibly reflects a neuromodulatory role in the retinal differentiation.

Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes

Based on the expression of glial fibrillary acidic protein (GFAP), a recent hypothesis considered stem or progenitor cells in the adult hippocampus to be a type of astrocyte. In a complementary approach, we used transgenic mice expressing green fluorescent protein (GFP) under the promoter for nestin, an intermediate filament present in progenitor cells, to demonstrate astrocytic features in nestin-GFP-positive cells. Morphologically, two subpopulations of nestin-GFP-positive cells were distinguishable; one had an elaborate tree of processes in the granule cell layer and expression of GFAP (but not of S100β, another astrocytic marker). Electron microscopy revealed vascular end feet of nestin-positive cells, further supporting astrocytic differentiation. Electrophysiological examination of nestin-GFP-positive cells on acutely isolated hippocampal slices showed passive current characteristics of astrocytes in one subset of cells. Among the nestin-GFP-expressing cells with lacking astrocytic features, two cell types could be identified electrophysiologically: cells with delayed-rectifying potassium currents and a very small number of cells with sodium currents, potentially representing signs of the earliest steps of neuronal differentiation.