Expression of voltage-dependent calcium channels in the embryonic rat midbrain.

The diversity of expression of high-voltage activated voltage-dependent calcium channels (VDCC) was investigated with whole-cell voltage-clamp recordings from dissociated embryonic rat ventral mesencephalic cells over a 7-day culture period. Cell phenotype was identified post-recording by fluorescent immunocytochemistry as tyrosine hydroxylase positive (TH+) or glutamic acid decarboxylase positive (GAD+). Both TH+ and GAD+ cells displayed high-threshold calcium (Ca(2+)) currents activated by depolarisations positive to -60 mV. In both cell types, pharmacological dissection using selective VDCC inhibitors, omega-agatoxin IVA (Aga IVA), omega-conotoxin GVIA (GVIA) and nifedipine demonstrated the existence of P/Q-, N- and L-type VDCC, respectively. The remaining residual current could be blocked by cadmium. It was found that the contribution to the whole-cell current by the N-type channel was greater in TH+ cells than GAD+ cells at each time point examined, whilst the contribution to the whole-cell current by the L-type channel was greater in GAD+ cells than TH+ cells. However, over the 7-day culture period, the expression of VDCC types in both cell phenotypes changed in a similar fashion, with the contribution to the whole-cell current from the N-type current decreasing, and the contribution from the R-type current increasing. Our data could provide new insights into a range of neurodevelopmental mechanisms related to Ca(2+) homeostasis in developing mesencephalic neurons.

Effects of acetylcholinesterase and butyrylcholinesterase on cell survival, neurite outgrowth, and voltage-dependent calcium currents of embryonic ventral mesencephalic neurons.

The aim of this study was to investigate the effect of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) on cell survival, neurite outgrowth and voltage-dependent calcium currents in developing rat ventral mesencephalic (VM) neurons. Both BuChE and AChE have been shown to promote neurite outgrowth in postnatnal preparations. However, the effect of these substances has never been investigated on rat embryonic VM cells, which are used in animal models of foetal transplantation as a treatment for Parkinson's disease. The effects of incubation with BuChE and tetrameric (G(4))- or monomeric (G(1))-AChE on cell survival and neurite outgrowth were characterised over a 7-day period on dopaminergic cells within embryonic VM cultures. The acute effects of these treatments on voltage-dependent calcium currents from embryonic VM cells were then investigated using whole-cell voltage-clamp recordings. The chronic effect of modulating voltage-dependent calcium channels was subsequently explored using the selective calcium channel antagonists omega-agatoxin IVA, omega-conotoxin GVIA, and nifedipine. The results presented here demonstrate firstly trophic effects of BuChE and G(4)- and G(1)-AChE upon dopaminergic neurite outgrowth, secondly that BuChE and G(4)- and G(1)-AChE have an inhibitory effect on voltage-dependent calcium currents, and finally that selective voltage-dependent calcium channel inhibitors also have trophic effects upon dopaminergic neurite outgrowth.

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 approximately -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 Ca(2+) concentration, which was dependent on extracellular Ca(2+) 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 Ca(2+) 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.