Christof J. Schwiening

Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells

BACKGROUND Neural stem cells are a potential source of cells for drug screening or cell-based treatments for neurodegenerative diseases. However, ethical and practical considerations limit the availability of neural stem cells derived from human embryonic tissue. An alternative source of human neural stem cells is needed; a source that is readily accessible, easily expanded, and reliably induced to a neural fate. METHODS Dermis isolated from biopsy samples of adult human skin was cultured and expanded in the presence of the mitogens epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF 2), and then by serum. We used immunocytochemical techniques, clonal analysis, and physiological characterisation to assess neural differentiation after the treatment of expanded cells with novel induction media. FINDINGS Initial characterisation of skin samples confirmed the absence of nestin, a neural precursor marker. Sequential culture in EGF and FGF 2 followed by adherent expansion in serum, and re-exposure to mitogens in substrate-free conditions resulted in large numbers of nestin-positive/musashi-positive neural precursors. Subsequent exposure of these precursors to hippocampal-astrocyte-derived signals resulted in cells of neuronal morphology that had stable expression of markers of neuronal differentiation (neurofilament, beta tubulin). We also show the presence of voltage-dependent calcium transients, and demonstrate monoclonal neural potential. INTERPRETATION We describe the isolation and characterisation of cells derived from adult human dermis that can be expanded for extended periods of time in vitro, while retaining inducible neural potential. The generation of almost limitless numbers of neural precursors from a readily accessible autologous adult human source provides a platform for further experimental studies and has potential therapeutic implications.

Calcium–hydrogen exchange by the plasma membrane Ca-ATPase of voltage-clamped snail neurons

The submicromolar levels of free Ca2+ ions in animal cells are believed to be maintained in the long term by two different plasma membrane transport mechanisms. These are Na–Ca exchange, driven by the sodium gradient, and a Na-independent Ca pump, driven by ATP. There is good evidence from red blood cells, and indirect evidence from other non-neuronal preparations, that the Ca-ATPase exchanges internal Ca2+ for external H +. Although Ca extrusion from nerve cells is inhibited by high external pH , there as yet is no evidence for the counter-transport of H+. We have used both pH- and calcium-sensitive microelectrodes on the cell surface, and the Ca indicator fura-2 intracellularily, to investigate how snail neurons regulate cytoplasmic free Ca2+. We now report that in snail neurons the recovery of intracellular Ca2+ after an increase coincides with both the expected increase in surface Ca2+ and a decrease in surface H+. Recovery of intracellular Ca and the changes in surface pH and Ca are all blocked by intracellular vanadate. We conclude that snail neurons regulate intracellular Ca mainly by a Ca–H ATPase, and suggest that this Ca–H exchange may account for many of the reported extracellular pH changes seen with neuronal excitation.

Activation of TRP Channels by Protons and Phosphoinositide Depletion in Drosophila Photoreceptors

BACKGROUND Phototransduction in microvillar photoreceptors is mediated via G protein-coupled phospholipase C (PLC), but how PLC activation leads to the opening of the light-sensitive TRPC channels (TRP and TRPL) remains unresolved. In Drosophila, InsP(3) appears not to be involved, and recent studies have implicated lipid products of PLC activity, e.g., diacylglycerol, its metabolites, or the reduction in PIP(2). The fact that hydrolysis of the phosphodiester bond in PIP(2) by PLC also releases a proton is seldom recognized and has neither been measured in vivo nor implicated previously in a signaling context. RESULTS Following depletion of PIP(2) and other phosphoinositides by a variety of experimental manipulations, the light-sensitive channels in Drosophila photoreceptors become remarkably sensitive to rapid and reversible activation by the lipophilic protonophore 2-4 dinitrophenol in a pH-dependent manner. We further show that light induces a rapid (<10 ms) acidification originating in the microvilli, which is eliminated in mutants of PLC, and that heterologously expressed TRPL channels are activated by acidification of the cytosolic surface of inside-out patches. CONCLUSIONS Our results indicate that a combination of phosphoinositide depletion and acidification of the membrane/boundary layer is sufficient to activate the light-sensitive channels. Together with the demonstration of light-induced, PLC-dependent acidification, this suggests that excitation in Drosophila photoreceptors may be mediated by PLCs dual action of phosphoinositide depletion and proton release.

Depolarization‐induced pH microdomains and their relationship to calcium transients in isolated snail neurones

Neuronal electrical activity causes only modest changes in global intracellular pH (pHi). We have measured regional pHi differences in isolated patch‐clamped neurones during depolarization, using confocal imaging of 8‐hydroxypyrene‐1,3,6‐trisulfonic acid (HPTS) fluorescence. The pHi shifts in the soma were as expected; however, substantially larger shifts occurred in other regions. These regional differences were still observed in the presence of CO2‐HCO3−, they decayed over many seconds and were associated with changes in calcium concentration. Lamellipodial HPTS fluorescence fell by 8.7 ± 1.3 % (n= 9; ∼0.1 pH unit acidification) following a 1 s depolarization to 0 mV; this was more than 4‐fold greater than the relative shift seen in the soma. Depolarization to +40 mV for 1 s caused a 46.7 ± 7.0 % increase (n= 11; ∼0.4 pH unit alkalinization) in HPTS fluorescence in the lamellipodia, more than 6‐fold that seen in the soma. Application of 5 % CO2‐20 mm HCO3− did not significantly reduce the size of the +40 mV‐evoked local pH shifts despite carbonic anhydrase activity. The pHi gradient between regions ∼50 μm apart, resulting from acid shifts, took 10.3 ± 3.1 s (n= 6) to decay by 50 %, whereas the pHi gradient resulting from alkaline shifts took only 3.7 ± 1.4 s (n= 12) to decay by 50 %. The regional rates of acidification and calcium recovery were closely related, suggesting that the acidic pH microdomains resulted from Ca2+‐H+ pump activity. The alkaline pH microdomains were blocked by zinc and resulted from proton channel opening. It is likely that the microdomains result from transmembrane acid fluxes in areas with different surface area to volume ratios. Such neuronal pH microdomains are likely to have consequences for local receptor, channel and enzyme function in restricted regions.

Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells

Our aim was to test the hypothesis that depolarization‐induced intracellular pH (pHi) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH‐sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole‐cell patch‐clamped rat cerebellar Purkinje cells. In the absence of added CO2‐HCO3−, depolarization to +20 mV for 1 s caused large (≈0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (≈0.06 pH units) and slower. The pHi shifts were smaller in the presence of 5 % CO2‐25 mm HCO3−‐buffered saline (≈0.08 pH units in the dendrites and ≈0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 μM) doubled the size of the dendritic acid shifts in the presence of CO2‐HCO3−, indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mm) inhibited the depolarization‐evoked acid shifts. We investigated more physiological pHi changes by evoking modest bursts of action potentials (≈10 s duration) in CO2‐HCO3−‐buffered saline. Such neuronal firing induced an acidification of ≈0.11 pH units in the fine dendritic regions, but only ≈0.03 pH units in the soma. There was considerable variation in the size of the pHi shifts between cells, with dendritic acid shifts as large as 0.2‐0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pHi changes (pH microdomains) might act as important signals in synaptic function.

Protons Released During Pancreatic Acinar Cell Secretion Acidify the Lumen and Contribute to Pancreatitis in Mice

BACKGROUND & AIMS Secretory granules are acidic; cell secretion will therefore lead to extracellular acidification. We propose that during secretion, protons co-released with proteins from secretory granules of pancreatic acinar cells acidify the restricted extracellular space of the pancreatic lumen to regulate normal physiological and pathophysiological functions in this organ METHODS Extracellular changes in pH were quantified in real time using 2-photon microscopy analysis of pancreatic tissue fragments from mouse models of acute pancreatitis (mice given physiological concentrations [10 -20 pM] of cholecystokinin or high concentrations of [100 nM] cerulein). The effects of extracellular changes in pH on cell behavior and structures were measured. RESULTS With physiological stimulation, secretory granule fusion (exocytosis) caused acidification of the pancreatic lumen. Acidifications specifically affected intracellular calcium responses and accelerated the rate of recovery from agonist-evoked calcium signals. Protons therefore appear to function as negative-feedback, extracellular messengers during coupling of cell stimuli with secretion. At high concentrations of cerulein, large increases in secretory activity were associated with extreme, prolonged acidification of the luminal space. These pathological changes in pH led to disruption of intercellular junctional coupling, measured by movement of occludin and E-cadherin. CONCLUSIONS By measuring changes in extracellular pH in pancreas of mice, we observed that luminal acidification resulted from exocytosis of zymogen granules from acinar cells. This process is part of normal organ function but could contribute to the tissue damage in cases of acute pancreatitis.

The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones

1 We have investigated the effects of changing intracellular pH on intracellular free calcium concentration ([Ca2+]i) in voltage‐clamped neurones of the snail Helix aspersa. Intracellular pH (pHi) was measured using the fluorescent dye 8‐hydroxypyrene‐1,3,6‐trisulphonic acid (HPTS) and changed using weak acids and weak bases. Changes in [Ca2+]i were recorded using either fura‐2 or calcium‐sensitive microelectrodes. 2 Acidification of the neurones with 5 mM or 20 mM propionate (≈0.2 or 0.3 pH units acidification, respectively) caused a small reduction in resting [Ca2+]i of 5 ± 2 nM (n = 4) and 7 ± 16 nM (n = 4), respectively. The removal of the 20 mM propionate after ≈40 min superfusion resulted in an alkalinization of ≈0.35 pH units and an accompanying rise in resting [Ca2+]i of 31 ± 9 nM (n = 4, P < 0.05). The removal of 5 mM propionate did not significantly affect [Ca2+]i. 3 Alkalinizations of ≈0.2‐0.4 pH units of Helix neurones induced by superfusion with 3 mM concentrations of the weak bases trimethylamine (TMA), ammonium chloride (NH4Cl) and procaine were accompanied by significant (P < 0.05) increases in resting [Ca2+]i of 42 ± 4 nM (n = 26), 30 ± 7 nM (n = 5) and 36 ± 4 nM (n = 3), respectively. The effect of TMA (0.5‐6 mM) on [Ca2+]i was dose dependent with an increase in [Ca2+]i during pHi increases of less than 0.1 pH units (0.5 mM TMA). 4 Superfusion of neurones with zero calcium (1 mM EGTA) Ringer solution inhibited depolarization‐induced calcium increases but not the calcium increase produced by the first exposure to TMA (3 mM). In the prolonged absence of extracellular calcium (≈50 min) TMA‐induced calcium rises were decreased by 64 ± 10% compared to those seen in the presence of external calcium (P < 0.05). 5 The calcium rise induced by TMA (3 mM) was reduced by 60 ± 5% following a 10 min period of superfusion with caffeine (10 mM) to deplete the endoplasmic reticulum (ER) stores of calcium (P < 0.05). 6 Cyclopiazonic acid (10‐30 μM CPA), an inhibitor of the ER calcium pump, inhibited the calcium rise produced by TMA (3 mM) and NH4Cl (3 mM) by 61 ± 4% compared to controls (P < 0.05). 7 These data are consistent with physiological intracellular alkaline shifts stimulating release of calcium, or inhibiting re‐uptake of calcium by an intracellular store. The calcium increase was much reduced following application of caffeine, treatment with CPA or prolonged removal of external calcium. Hence the ER was likely to be the source of mobilized calcium.

Astrocytes promote neurogenesis from oligodendrocyte precursor cells

The oligodendrocyte precursor cell (OPC) has until recently been regarded as a lineage‐restricted precursor cell. Considerable interest has been generated by reports suggesting that OPCs may possess a wider differentiation potential than previously assumed and thus be considered a multipotential stem cell. This study examined the neuronal differentiation potential of rat, postnatal cortical OPCs in response to extracellular cues in vitro and in vivo. OPCs did not exhibit intrinsic neuronal potential and were restricted to oligodendrocyte lineage potential following treatment with the neural precursor mitogen fibroblast growth factor 2. In contrast, a postnatal hippocampal astrocyte‐derived signal(s) is sufficient to induce functional neuronal differentiation of cortical OPCs in vitro in population and single cell studies. Co‐treatment with Noggin, a bone morphogenetic protein antagonist, did not attenuate neuronal differentiation. Following transplantation to the adult rat hippocampus, cortical OPCs expressed doublecortin, a neuroblast‐associated marker. The present findings show that hippocampal, astrocyte‐derived signals can induce the neuronal differentiation of OPCs through a Noggin‐independent mechanism.

Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions

This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (Em) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of Em despite cell shrinkage, which was attributable to elevation of intracellular [Cl−] above electrochemical equilibrium through Na+–Cl− and Na+−K+−2Cl− cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope xz‐scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl−‐free, normal and Na+‐free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady‐state resting potentials (Em). Fibres in isotonic Cl−‐free, normal and Na+‐free Ringer solutions showed similar Em values consistent with previously reported permeability ratios PNa/PK(0.03–0.05) and PCl/PK (∼2.0) and intracellular [Na+], [K+] and [Cl−]. Increased extracellular osmolarities produced hyperpolarizing shifts in Em in fibres studied in Cl−‐free Ringer solution consistent with the Goldman‐Hodgkin‐Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such Em shifts, suggesting a Cl−‐dependent stabilization of membrane potential. This stabilization of Em was abolished by withdrawing extracellular Na+ or by the combined presence of the Na+–Cl− cotransporter (NCC) inhibitor chlorothiazide (10 μm) and the Na+−K+−2Cl− cotransporter (NKCC) inhibitor bumetanide (10 μm), or the Na+−K+‐ATPase inhibitor ouabain (1 or 10 μm) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl− equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl−]i above its electrochemical equilibrium. Such splinting of [Cl−]i in combination with the high PCl/PK of skeletal muscle stabilizes Em despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where PCl/PK is low, the same cotransporters would instead permit RVIs but at the expense of a capacity to stabilize Em.

COMPARISON OF SIMULTANEOUS PH MEASUREMENTS MADE WITH 8-HYDROXYPYRENE-1,3,6-TRISULPHONIC ACID (HPTS) AND PH-SENSITIVE MICROELECTRODES IN SNAIL NEURONES

Abstract We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pHi) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H+ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50–7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pHi values, 7.16±0.05 (n=10) and 7.17±0.06 (n=9) respectively in nominally CO2/HCO3–-free saline. At both extremes of the pH range there were small discrepancies. At acidic pHi, 6.87±0.09 (n=5), the intracellular HPTS measurement differed by –0.08±0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pHi,7.32±0.10 (n=5), HPTS measurements produced pH values that differed by +0.07±0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77±15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 µM to 2 mM, did not block activity-dependent pHi changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00–8.00.