Functional maturation of isolated neural progenitor cells from the adult rat hippocampus

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximate to -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca2+ concentration, which was dependent on extracellular Ca2+ and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na+ channels, low- and high-voltage-activated Ca2+ channels and delayed-rectifier K+ channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs

Monitoring Cl- movement in single cells exposed to hypotonic solution

Self-referencing ion--selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 microm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (VN) measured in 100 and 10 mM NaCl (deltaVN = 57 +/- 2 mV), but was slightly greater than ideal when comparing 1 and 10 mM NaCl (deltaVN = 70 +/- 3 mV). The response was also linear in the presence of 1 mM glutamate, gluconate, or acetate, 10 microM tamoxifen, or 0.1, 1, or 10 mM HEPES at pH 7.0. The ISE was approximately 3 orders of magnitude more selective for Cl- over glutamate or gluconate but less than 2 orders of magnitude move selective for Clover bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 microM 5-nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 +/- 5 mOsm) solution. The increase in efflux peaked 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 microM tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell leve

Differential regulation of K+ channels in Arabidopsis epidermal and stelar root cells.

The patch clamp technique was applied to protoplasts isolated from the epidermis and pericycle of Arabidopsis roots and their plasma membrane currents investigated. In the whole cell configuration, all protoplasts from the epidermis exhibited depolarization-activated time-dependent outwardly rectifying (OR) currents whereas OR currents were present in only 50% of cells from the pericycle. The properties of the OR currents in the epidermis and pericycle were compared with respect to their selectivity, pharmacology and gating. The time-dependent activation kinetics, selectivity and sensitivity to extracellular tetraethyl ammonium of the OR current in each cell type were not significantly different. The reversal potential (Erev) of the OR currents indicated that they were primarily due to the movement of K+. However, the gating properties of the OR currents from the epidermis differed markedly from those exhibited in the pericycle. Although both cell types displayed OR currents with voltage-dependent gating modulated in a potassium-dependent fashion [i.e. the activation threshold (V0.5) was displaced to more positive voltages as extracellular K+ increased], the OR currents in the epidermis also displayed voltage-independent gating by extracellular K+ which dramatically regulated current density. In the present study, reducing extracellular K+ activity from 40 to 0.87 mm reduced the OR current density in epidermal cells by approximately 80%. The chord conductance of the OR current saturated as a function of extracellular K+ and could be fitted with a Michaelis–Menten function to yield a binding constant (Km) of 10.5 mm. The ability of other monovalent cations to substitute for K+-gating of the OR currents was also investigated and shown to exhibit a relative sequence of K+ ≥ Rb+ > Cs+ > Na+ ≥ Li+ (Eisenmann sequence IV) with respect to efficacy of gating. Furthermore, single channel recordings demonstrated that channel activity rather than the single channel conductance was modulated by extracellular K+. In contrast, OR current density in the pericycle was largely independent of extracellular K+. It is suggested that the contrasting gating properties of the K+ channels in the epidermis and pericycle reflect their different physiological roles, particularly with respect to their role in K+ (nutrient) transport from the soil solution to the shoot

Asymmetric block of a monovalent cation-selective channel of rabbit cardiac sarcoplasmic reticulum by succinyl choline

SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents*. (DOC 123 kb