acidosis on intracellular pH in differentiating neural progenitor cells

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Research Report

Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells Tommy Nordström⁎, Linda C. Jansson, Lauri M. Louhivuori, Karl E.O. Åkerman Biomedicum Helsinki, Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland

A R T I C LE I N FO

AB S T R A C T

Article history:

The response of differentiating mouse neural progenitor cells, migrating out from

Accepted 20 April 2012

neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular

Available online 28 April 2012

acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pHi), that two main populations can be distinguished by exposing migrating

Keywords:

neural progenitor cells to low extracellular pH or by performing an acidifying ammonium

Neurosphere

prepulse. The cells dominating at the periphery of the neurosphere culture, which were

Stem cell

positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting

pH

pHi and could also easily be further acidified by lowering the extracellular pH. Moreover, in

Hypoxia

this population, a more profound acidification was seen when the cells were acidified using

Acidosis

the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pHi took place in this population. In contrast, depolarization caused an increase in pHi (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pHi (pH 6.9–7.1) also responded to acute hypoxia. During hypoxic treatment the resting pHi decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Sublethal ischemia causes significant alterations in brain development and may be an etiologic factor of neuropsychiatric disease (reviewed in Basovich, 2010). During recovery from ischemia, however, a significant increase in neurogenesis in the subventricular zone is observed suggesting the presence of an inherent mechanism for repair (Fagel et al., 2006). In fact short intermittent hypoxia increases

neurogenesis and cognitive functions (Zhu et al., 2010). Neural progenitor cells (NPC) migrate to the injured area in ischemia (Jin et al., 2005; Kelly et al., 2004; Kim et al., 2004) suggesting that chemo-attractant signals are provided by lesioned tissue. Neural progenitor cells can be expanded in cultures as so called neurospheres (Reynolds and Weiss, 1992). Although the neurosphere model is now widely used, the physiological properties of the neurosphere-derived cells have been only partially characterized. Upon induction of differentiation the

⁎ Corresponding author at: Biomedicum Helsinki, Institute of Biomedicine/Physiology, University of Helsinki, P.O. Box 63, FI-00014 Helsinki, Finland. Fax: + 358 9 19125302. E-mail address: [email protected] (Tommy. Nordström). 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.04.043

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cells migrate out from the sphere and have the ability to differentiate into functional neurons (Ding et al., 2006). The differentiation, viability and proliferation of NPCs are considerably enhanced in hypoxic conditions (Bürgers et al., 2008; Fagel et al., 2006; Santilli et al., 2010; Zhu et al., 2005a). Hypoxia induced factors (HIFs) have been suggested to provide signals for these cells (Zhu et al., 2005b). Ischemic conditions also cause a rapid decrease in extracellular pH, which causes activation of acid channels in surrounding cells (reviewed in Huang and McNamara, 2004). Moreover, two-pore-domain potassium channels are also sensitive to changes in pH (Morton et al., 2005). Depending on channel isoform they are either activated or inactivated during conditions of low extracellular or intracellular pH (Lesage and Lazdunski, 2000; Sandoz et al., 2009). To our knowledge, the mechanism by which neural progenitor cells respond to extracellular acid treatment (mimicking acidosis) has not been studied. Here we have investigated, by using BCECF to monitor pHi, how neural progenitor cells respond to extracellular or intracellular acidosis. The aim of this study was to monitor the responses of migrating neural progenitor cells to hypoxia, acidification, intracellular acidification and high potassium in order to obtain information concerning their reactivity to those changes which occur in ischemia.

2.

Results

2.1. Exposure of differentiating neural progenitor cells to acute hypoxia Neurosphere-derived cells were induced to differentiate by removing the mitogens basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Within a few hours after mitogen removal, differentiating cells started to migrate out from the sphere. The migrating cells form a monolayer, enabling the analysis of the spatial distribution of cellular responsiveness using fluorescent imaging procedures. Our earlier studies using Ca2 + imaging and immunocytochemistry on migrating neurosphere-derived cells suggest that there is a gradient of differentiation towards the outer edge of the migration layer and that two distinct main populations of cells coexist among the migrating cells based on their responsiveness to glutamate (Jansson et al., 2011; Kärkkäinen et al., 2009). The cells in the outer migration layers progressively differentiate, gain ionotropic glutamate receptors and stain for neuronal markers. Radial cell staining is seen at migration areas closer to the neurosphere. Cells, that had been allowed to differentiate for 5 days, were loaded with the pH indicator BCECF and the fluorescence of the cells in the migration zone was monitored (Fig. 1A). The basal pHi showed a variation from 6.4 to 7.3 (Fig. 1B). The lowest pHi values were seen at the outer edge in the periphery of the migration layer (6.79 ± 0.01, n = 225). In the more inner layer of migration (cells that had migrated less than 100 μm) the pHi was 7.00 ± 0.01, n = 225 (Fig. 1C). In order to test the response of migrating cells to acute hypoxia the BCECF loaded cells were perfused with HEPES buffered medium (HBM) pH 7.4 that had been deoxygenated by N2 treatment (N2 bubbled through HBM for 20 min at 37 °C).

Fig. 1 – Intracellular pH in differentiating neural progenitor cells. (A). Fluorescence microscope image of BCECF loaded differentiating neural progenitor cells. Area 2: inner cells and area 3: outer cells were selected for the pH and membrane potential measurements. (B). Scatter diagram showing intracellular pH in migrating neural progenitor cells. (C). Average pH (7.00 ± 0.01 n = 225) of inner (area 2) and outer (area 3) cells (6.79 ± 0.01 n = 225) (*p < 0.001).

Two clear populations could be distinguished based on their response to hypoxic treatment. Hypoxic treatment induced a decrease in resting pHi (by 0.15 pH units) in 55 ± 1.8% of the cells (n = 82, N = 3). At reperfusion with normoxic HBM pH 7.4, a prompt recovery in pHi could be observed (Fig. 2A). The migrating cells that responded to acute hypoxia had an initial resting pHi between 6.9 and 7.15 and were predominantly

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membrane potential as calculated on the whole population was −75.93 ± 1.59 mV, n = 146, N = 3. However, a more detailed analysis on inner and outer cells showed that, the resting membrane potential in the inner population was markedly more depolarized (RMP − 65.5 ± 1.80 mV) as compared to the outer cells (RMP −81.40 ± 1.72 mV). A similar value from the outer cells was determined using patch clamp (−82.1 ± 0.6 mV, n = 5). A typical DiBAC4(3) recording of one representative experiment is shown in Fig. 2C. The trace shown is an average of 50 randomly selected single cells. Removal of oxygen induced a hyperpolarization and a prompt recovery in the resting membrane potential was seen during the reoxygenation process.

2.2. Immunostaining of differentiated neural progenitor cells showing the localization of radial glial cells and neurons We performed immunostaining to identify further the putative two cell populations monitored above. Cells differentiated for 5 days after plating were stained with the nuclear stain 4′,6diamidino-2-phenylindole (DAPI), glial glutamate-aspartate transporter marker (GLAST) and the neuronal specific, microtubule associated protein-2 (MAP-2) antibody. The staining of a dense region of radial processes with GLAST is shown in Fig. 3A, overlayed with DAPI staining of nuclei in Fig. 3B. As shown, staining of radial cells is concentrated in the inner migration layers, closer to the mother neurosphere body while the nuclei were seen also in the outer layers of the migration, outside the radial processes (Fig. 3B). Fig. 3C shows staining with MAP-2 antibody to visualize neuronal cells and in Fig. 3D overlayed with GLAST. Note the MAP-2 staining outside the layer of radial processes. To further characterize the neuronal population cells were costained with MAP-2, DAPI and NeuN, a marker of neuronal nuclei as well as calbindin, a marker of early neurons. Note that all the nuclei in the outermost layer were NeuN positive (stained red) while many nuclei closer to the neurosphere were NeuN negative (Figs. 3E, F). The co-staining of NeuN positive cells with calbindin, a marker of early neurons (Fig. 3E) and MAP-2 staining (Fig. 3F) of neuronal processes reveal the neuronal morphology of the outer cells. Fig. 2 – Effect of acute hypoxia on pHi and membrane potential. (A). pHi change in BCECF loaded neural progenitor cells (astroglial cells) exposed to hypoxia. (B). Histogram showing ΔpH (initial pH–pH at 600 s) of area 2 and area 3 cells (*p < 0.001). (C). Membrane potential change in neural progenitor cells exposed to acute hypoxia. Average signal of 50 randomly selected cells is shown.

located in the inner layer of the neurosphere culture. Thus the pH of the outer population of migrating cells (located in area 3 in Fig. 1A) did not show any major change in pH in response to hypoxia while in the inner layer (located in area 2 in Fig. 1A) with a higher pHi showed an acidification (Fig. 2B). In parallel experiments the membrane potential of individual migrating cells was monitored using the voltachromic dye DiBAC4(3). In contrast to the pH experiments showing effects on a subpopulation of cells, a measurable hyperpolarization of the membrane by 5.16 ± 0.29 mV took place in the whole population when exposed to acute hypoxia. The average

2.3. Exposing migrating neural progenitor cells to low extracellular pH To study how pHi is affected by extracellular acidosis in migrating progenitor cells, we exposed BCECF loaded neural progenitor cells to low extracellular pH. A typical response of 50 individual cells randomly selected from one coverslip is illustrated in Fig. 4. Perfusion of the cells with HBM pH 6.0 induced a marked intracellular acidification in a fraction of the cells (34.7% of the cells in this experiment). In the outer cell population pHi decreased from pH 6.84 ± 0.01 down to 6.36 ± 0.01 within 3.5 min. To further demonstrate this response to acidosis, the cell population was re-perfused with HBM pH 7.4 followed by HBM pH 4.0. During this treatment a faster and more pronounced acidification was observed (pHi decreased from 6.74 ± 0.07 to 5.85 ± 0.29 within 2 min). When the extracellular pH, after the acid treatment period, was switched back to 7.4, a clear recovery in the cytoplasmic pH (0.11 pH/min) could be observed. In line with the data in Fig. 1, two populations

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Fig. 3 – Immunostaining of cells in neurosphere culture. (A) GLAST (green) staining of radial processes. (B) GLAST (green) overlayed with DAPI (blue) staining. (C) Microtubule associated protein 2, (MAP-2) (red) neuronal staining. (D) Overlapping images of MAP-2 (red) and GLAST (green). (E) Overlapping images of DAPI (blue), neuron-specific nuclear protein (NeuN) (red) and calbindin staining (green). (F) Overlapping images of DAPI (blue), NeuN (red) and (MAP-2) (green) neuronal staining. Scale bar = 50 μm.

of cells could be identified. The cell population at the outer periphery of the migration layer with a lower initial resting pHi showed a more robust response to acidification as compared to the inner cell layer. Summary results of 3 independent experiments are shown in Table 1.

2.4. Effect of high-potassium depolarizing extracellular medium on pHi To test for the electrogenic characteristics of pH regulation in the two populations we next studied the effect of depolarization on BCECF loaded neural progenitor cells in depolarizing conditions. When BCECF loaded neural progenitor cells were exposed to extracellular bicarbonate free HBM containing 30 mM potassium chloride (isosmotic replacement of Na+ ions

by K+) an increase in pHi could be observed in 66% of the cells (99/150 cells N = 3). A typical response of 50 individual cells is shown in Fig. 5A. The cells that responded to the depolarizing stimuli were located closer to the mother neurosphere body while the cells in the periphery did not respond to the depolarizing conditions (Fig. 5B). In this figure it can also be seen that no change in pHi took place in the cells having initial resting pH <6.8. Moreover, it can be seen that the inner cells having higher resting pHi responded more rapidly to depolarizing conditions. Overall, when high extracellular potassium was present, pHi increased from 6.9 ± 0.03 to 7.47 ± 0.02 within 5 min (n = 99, N = 3). When higher concentrations, 50 or 70 mM of K+ was used, pHi increased in the inner cell population to 7.41 ± 0.03 and 7.42 ± 0.03 respectively. Similarly, cells in the outermost layer did not respond to this treatment.

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Fig. 4 – Effect of an extracellular acidosis on pHi in neural progenitor cells. Representative pHi records obtained from BCECF loaded individual neural progenitor cells (n = 50) exposed to low extracellular pH (pH 6.0 and pH 4.0). Where indicated by an arrow, neural progenitor cells were perfused with bicarbonate free HBM pH 7.4, pH 6.0 or pH 4.0. A cell population that responded more strongly to extracellular acidification could be observed.

2.5. Response to intracellular pH changes using the ammonium chloride prepulse technique Intracellular pH regulation has previously been studied in various cell types by using the ammonium chloride pre-pulse technique. One representative experiment out of 4 conducted is shown in Fig. 6A. When the migrating progenitor cells were perfused with HBM containing 20 mM ammonium chloride the pHi increased in the whole cell population from 6.78 ± 0.02 to 7.37 ± 0.01. However, in the continuous presence of ammonium

Fig. 5 – Effect of membrane depolarization on pHi. (A). Representative pHi records obtained from BCECF loaded individual neural progenitor cells (n = 50) exposed to depolarizing concentration of extracellular potassium (30 mM K+). Two cell populations having different initial resting pH could be observed. (B). The cell population located at the edge of the neurosphere culture (marked by an asterisk) had initial lower resting pH and did not alkalinize upon depolarizing potassium treatment.

Table 1 – Effect of extracellular acidosis on pHi. Conditions (pH)

Outer cells

Inner cells

Buffer pH 7.4 From buffer pH 7.4 to pH 6.0 Rate of pH decrease in pH 6.0 Rate of pH recovery in pH 7.4 From buffer pH 7.4 to pH 4.0 Rate of pH decrease in pH 4.0 Rate of pH recovery in pH 7.4

6.84 ± 0.01 (67) * ΔpH 0.48 ± 0.01*

6.93 ± 0.01 (82) ΔpH 0.22 ± 0.01

0.25 ± 0.01*

0.09 ± 0.02

0.09 ± 0.02*

0.03 ± 0.01

ΔpH 1.12 ± 0.03*

ΔpH 0.40 ± 0.02

1.07 ± 0.06*

0.31 ± 0.05

0.18 ± 0.03*

0.03 ± 0.01

Values are means ± S.E.M., with number of cells in parentheses (*p < 0.05). The result shown is based on 3 separate experiments. In each experiment, 2 sequential acid treatments were performed on same coverslip. The rate of decrease or recovery is given as pH units/min (pH decrease calculated within the first 30 s and rate of recovery within the first 120 s (slope of linear fit)).

chloride, pHi started immediately to decline (0.88 pH/min) down to 6.68± 0.01 in the outer cell population probably due to influx of ammonium ions. Removal of the ammonium chloride induced a more profound acidification in the outer cell subpopulation (for cell location see Fig. 6B). The pH declined to 5.67 ± 0.01 after ammonium chloride removal. A recovery from the acid load could also be observed in this population (initial recovery rate, 0.15 pH units/min). The population in the inner migration layers having higher initial resting pHi showed a distinct response to ammonium chloride prepulses. In this population the cytoplasmic pH initially increased normally due to ammonia (NH3) influx. However, the pHi remained on a higher level when NH4Cl was present indicating that no putative ammonium ion influx took place. After removal of the NH4Cl, very little acidification was seen compared to that seen in the outer layer. The pHi remained on a higher value, pH 6.40 ± 0.03. Results of 4 independent experiments are summarized in Table 2.

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Table 2 – NH4Cl induced changes in pHi. Parameter

Outer cells

Inner cells

Rate of induced decrease in pHi induced decrease in pHi Rate of in the presence of 100 μM Ba2 + Nadir pH after NH+4 removal ΔpH after NH+4 removal

0.42 ± 0.15* 0.1 ± 0.06

0.11 ± 0.02 0.06 ± 0.02

5.96 ± 0.13* 0.66 ± 0.13

6.60 ± 0.08 0.47 ± 0.12

NH+4 NH+4

Values are means ± S.E.M. (*p < 0.05). The result shown is based on 4 separate experiments, n = 200. The rate of decrease is given as pH units/min (pH decrease calculated within the first 30 s (slope of linear fit)).

100 μM Ba2 + was present. When NH4Cl was removed, only a mild cytoplasmic acidification response was seen. In the presence of Ba2 + pHi declined in the outer cell population to 6.33 ± 0.09 after NH4Cl washout. A secondary NH4Cl prepulse was performed on the same cells, but now in the absence of Ba2 +, resulted in a typical pH response (Fig. 7B). Under these conditions, the outer cell population, allowed NH4+ influx during the NH4Cl treatment and was also strongly acidified. In this experiment pH declined to 5.65 ± 0.06 in the outer cell population when NH4Cl was removed (summary results of 4 exp. shown in Table 2).

2.7. Effects of butyric acid on neural progenitor cell resting pHi

Fig. 6 – Effect of an ammonium prepulse on pHi in neural progenitor cells. (A). Representative pHi records obtained from BCECF loaded individual neural progenitor cells (n = 50) exposed to an ammonium prepulse. Where indicated by an arrow, neural progenitor cells were perfused with bicarbonate free HBM pH 7.4 supplemented with 20 mM ammonium chloride. After 2 min perfusion, the ammonium chloride was rapidly perfused away and pHi was monitored for an additional 4 min. (B). Fluorescence image (490 nm) of BCECF loaded neural progenitor cells. The cells marked by an asterisk responded more strongly to 20 mM ammonium chloride giving an initial transient alkalinization followed by a deeper acidification in the cytosol after removal of the ammonium chloride. These cells were predominantly located at the edge of the neurosphere.

Having observed that the outer and inner cell population had different resting pH and responded differently to an ammonium prepulse, we next decided to control for the BCECF fluorescence properties in the cell population. To do this we used butyric acid. Butyric acid was chosen since this acid has higher pKa value (pKa 4.82) than lactic acid (pKa 3.79) and the undissociated form present can rapidly diffuse through the cell membrane and induce intracellular acidification. In this set of experiments, the resting pHi of the outer cells was 6.70 ± 0.01, n = 66 and 7.0 ± 0.01, n = 83 in the inner cells located closer to the neurosphere body. Addition of 5 mM butyric acid to the perfusion solution induced a rapid decrease in the resting pHi in the whole cell population down to pH 6.11 ± 0.01, n = 149, N = 3. Immediately after withdrawal of butyrate, a rapid recovery to normal pH values was seen in all cells. A typical response on 50 randomly selected individual cells from one coverslip is shown in Fig. 8.

2.8. Effects of prolonged extracellular acidosis on neural progenitor cell migration and survival 2.6. Effect of barium on ammonium influx and intracellular acidification To further study the mechanisms for the transient alkalinization response to NH4Cl in the outer layer, we next performed experiments in the presence of barium. Ba2 +-sensitive inward rectifying K+ channels have a high NH4+ conductance and the 100:1 ratio of NH4+/NH3 at pH 7.4 would therefore result in more efficient ammonium ion (NH4+) loading into cells having NH4+ permeable K+ channels (Nagaraja and Brookes, 1998). As shown in Fig. 7A, the two cell population could not be separated in an ammonium prepulse experiment when

Our short-time experiments showed that an acute extracellular acidosis had a strong impact on intracellular pH in a subfraction of cells in the neurosphere culture that were identified as neurons by NeuN immunostaining and more specifically as migrating neurons by MAP-2 and calbindin immunofluorescent staining. The next question we wanted to address was whether long-term incubation at low pH also affects neural stem cell differentiation. Surprisingly, exposing cells to pH 6.8 and pH 6.5 for 24 h had minor effects on the cell migration process as compared to control cells kept at normal pH 7.4 (Figs. 9A, B, C). However, as shown in Fig. 9D, lowering

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Fig. 7 – Effect of barium on ammonium chloride induced change in pHi in neural progenitor cells. (A). Representative pHi records obtained from BCECF loaded individual neural progenitor cells (n = 50) exposed to 20 mM ammonium chloride in the presence of 100 μM barium chloride (Ba2 + present throughout the experiment shown in (A)). When ammonium chloride had been present for 2 min, the cells were perfused with HBM pH 7.4 and change in the intracellular pH was monitored for an additional 3 min. (B). When pHi had recovered completely, the same cells were reperfused with HBM pH 7.4 and again exposed to 20 mM ammonium chloride. After washout of the ammonium chloride, two populations could clearly be distinguished.

the extracellular pH to 6.2 for 24 h markedly reduced the radial glial cell migration and the total number of MAP-2 positive neuronal cells (Fig. 9G). Incubating the cells for 72 h in pH 6.2 caused cell death (Fig. 9F) while migrating neurons could still be observed in the cultures kept at pH 6.5 (Figs. 9E and H).

3.

Discussion

Intracellular pH regulation within a narrow range is known to be critical during early embryonic development and for cells in order to maintain their energy metabolism, intracellular ionic signaling, differentiation, quiescence and proliferation (Musgrove et al., 1987; Pouysségur et al., 1985; Taylor and

Hodson, 1984). In murine models, neurosphere-derived neural progenitor cells migrate towards ischemic brain areas and differentiate into functional neurons (Jin et al., 2005; Kelly et al., 2004; Kim et al., 2004; Olstorn et al., 2011). Ischemic conditions in general are thought to be harmful. The mechanism of damage is thought to be due to hypoxiainduced disturbances of energy metabolism, and consequent release of glutamate as well as acidosis, which cause excitotoxic cell death through ionotropic glutamate receptors or activation of acid-sensing ion channels (ASICs) (Huang and McNamara 2004; Szydlowska and Tymianski, 2010). Migrating progenitors must thus possess mechanisms which render them resistant to ischemic conditions since hypoxic conditions and glutamate have been shown to enhance progenitor differentiation (Sclett, 2006; Zhu et al., 2005a). The response of migrating progenitors to acidosis is not very well known. Our results identify two different populations of cells with distinct properties with respect to their responsiveness to hypoxia, changes in extra- and intracellular pH as well as changes in membrane potential. The difference in pH regulation was seen as a graded reduction in intracellular pH from the neurosphere edge towards the outer migration layers. Radial glial cells dominate in the inner cell layers since there was a strong immunocytochemical signal for GLAST. On the other hand, all the cells in the outermost layer (which had migrated a longer distance) showed a positive signal for neuronal markers, MAP-2, calbindin, and the neuron specific nuclear marker NeuN. Neuroblasts are born as daughter cells upon radial glial cell division and migrate along radial glial processes. Our results suggest that these newly born neurons progressively lose their “radial glial type” of pH regulation and gain a “neuronal type” pH regulation, seen in the outermost layer of cells, which also express ionotropic receptors and voltage gated channels (Jansson et al., 2011; Kärkkäinen et al., 2009). The inner layer of cells responded with an intracellular acidification to hypoxia. Hypoxia has been shown to induce pHi decrease in brain cells, in particular astrocytes, and it has been hypothesized that astroglial cells play critical roles in neuronal development and the fine tuning of the microenvironment in the brain (Bondarenko and Chesler, 2001; Fujiwara et al., 1992; Ransom, 2000). In line with this, we found that hypoxic treatment specifically induced a decrease in resting pHi in the inner migrating cells. The difference in the responsiveness of the two layers is probably due to differences in the metabolism of the two cell types and their pH regulation. Both layers showed a hyperpolarization to hypoxia. Hypoxia induced hyperpolarization has previously been observed in neurons (Fujimura et al., 1997; Fujiwara et al., 1987; Hyllienmark and Brismar, 1999). The hyperpolarization seen in hippocampal neurons during hypoxic treatment has been attributed to the activation of ATP sensitive K+ channels (KATP channels) by hypoxia (Godfraind and Krnjević, 1993). An effect through KATP channels in our experiments is also likely since addition of 100 μM tolbutamide, a KATP channel blocker, induced a depolarization in the majority of cells. Activation of neuronal KATP channels is thought to play a neuroprotective role during tissue hypoxia or ischemia (Ballanyi, 2004, review). The resulting membrane hyperpolarization blunts excitotoxic

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Fig. 8 – Effect of butyric acid on pHi. Representative pHi records obtained from BCECF loaded individual neural progenitor cells (n = 50) exposed to 5 mM butyric acid.

responses to excitatory agonist and reduces the overall cellular reactivity. This would in part explain the resistance of these cells to ischemic conditions. Exposing the cells to acute extracellular acidosis revealed that the outermost, but not the inner layer of cells, rapidly acidified suggesting that their cell membranes are permeable to protons. The lower resting pHi in this population might thus reflect an ongoing proton leak over the cell membrane. Thus at more negative resting membrane potentials than the EH +, protons tend to leak into cells. The difference in EK + − EH + is −44.48 mV and it is possible that the acute extracellular acidification to pH 6.0 is low enough to force proton influx over the cell membrane. However if there is an electrogenic pathway for proton influx in operation one would expect an effect of changes in membrane potential on intracellular pH which could not be observed in this cell population. This would suggest that an electroneutral pathway is responsible for this response to extracellular acidification. A similar response to extracellular acidification as seen here has also been observed in ventricular myocytes (Kopito et al., 1989; Sun et al., 1996). The acid loading observed in these cells was due to a novel chloride/bicarbonate exchanger (AE3) which is also expressed in neurons (Becker et al., 2003). Lowering extracellular pH activates the forward mode of the exchanger. Although we used a nominally bicarbonate free buffer solution, such solutions usually contain 100–200 μM bicarbonate derived from atmospheric carbon dioxide (Deitmer and Schneider, 1995). While extracellular acidosis may be harmful (Duan et al., 2011) there are several reports suggesting that lowering of intracellular pH has a protective effect. Lowering intracellular pH reduces bursts of activity through ionotropic glutamate receptors (Bonnet et al., 1998). N-Methyl-D-aspartate (NMDA) receptors become inactivated at low pH (Saybasili, 1998) and acidosis associated with hypoxia-ischemia (Giffard et al., 1990; Tang et al., 1990). Likewise, acidification can directly block voltage-dependent Ca2 + channels (Chen et al., 1996). Furthermore, several in vitro studies have suggested that moderate extracellular acidosis might protect central neurons

from excitotoxic injury (Chu et al., 2003; Kaku et al., 1993; Sapolsky et al., 1996). Two-pore-domain potassium channels, TREK1 and TREK2, are activated by low intracellular pH and have a neuroprotective role (Franks and Honoré, 2004; Honoré et al., 2002; Huang and Yu, 2008). As these channels are expressed in neural progenitor cells (Xi et al., 2011) they probably protect these cells during acidic conditions. Thus, despite the fact that intracellular pH was lowered in neurons during acute acidosis we found that the neural stem cell differentiation could still take place when extracellular pH was lowered to pH 6.5. However, an abrupt inhibition of cell migration and marked cell death took place in the neurosphere culture when extracellular pH was lowered to pH 6.2. To our knowledge, this effect has not been earlier reported on neural stem cells. Our findings are in line with earlier reports showing that brain tissue pH, during severe cerebral ischemia, might locally decrease to 6.2–6.0 and cause irreversible damage (Nemoto and Frinak, 1981). A role for iron catalyzed free radical formation has been postulated to mediate the cell death during these extreme low pH levels (Lipscomb et al., 1998; Siesjö, 1988). Extracellular acidosis has been shown to decrease cell proliferation and migration in a variety of nonneuronal cell systems (Faff and Nolte, 2000; Perdikis et al., 1998; Simchowitz and Cragoe, 1986). The two cell populations could also be distinguished by measuring membrane potential. The resting membrane potential in the outermost layer was −81.40 ± 1.72 mV. The hyperpolarized membrane potential observed in the outer neuron like population is in agreement with earlier electrophysiological studies performed on neural progenitor cells isolated from mice forebrain subventricular zone (RMP around −80 to −82 mV) (Yasuda et al., 2008). Values as low as −90 mV, have been measured in NPCs isolated from adult rat hippocampus (Hogg et al., 2004). In contrast, more depolarized RMPs (− 34 to −41 mV) have also been reported in neural stem cell cultures (Cai et al., 2004; Piper et al., 2000). The resting membrane potential seems to vary dependent on the neural stem cell system used (stem cells from different brain regions or species, experiments with acutely prepared brain slices or

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with cells in culture), culture conditions, degree of differentiation (days in culture) and method used to trigger cell differentiation (Belleau and Warren, 2000; Cai et al., 2004). In

general, proliferating precursor cells are more depolarized than differentiated cells (Sundelacruz et al., 2009). The outer layer cells were unaffected by depolarization (by increasing

Fig. 9 – Effect of prolonged extracellular acidosis on neural progenitor cell migration and survival. Impact of extracellular pH, (A) pH 7.4, (B) pH 6.8, (C) pH 6.5 and (D) pH 6.2 on neural stem cell migration. Neurospheres were cultured at indicated pH for 24 h in a 5% CO2 incubator, and then fixed and stained with glial cell marker GLAST (green) and microtubule associated protein 2, (MAP-2) (red) neuronal marker (overlapping images shown). Higher magnification (63×) light microscopy pictures showing the morphology of cells kept for 72 h at pH 6.5 (E) and 6.2 (F). Histograms showing the number of MAP-2 positive neuronal cells migrating out of the neurospheres plated on poly-DL-ornithine-coated coverslips and incubated for 24 h (G) or 72 h (H) at pH 7.4, 6.8, 6.5 and 6.2. Scale bar = 20 μm. Average number of migrating cells from 3 spheres/treatment ± S.E.M. is shown.

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extracellular K+) while the inner cell layer cells were promptly alkalinized by depolarization. The most likely explanation for this response in the inner cell population is that K+ induced depolarization drives the electrogenic Na+/HCO3− cotransport in the reverse mode (HCO3 influx). Glial-type cells are known to express a voltage-dependent Na+/HCO3− cotransporter that can acidify or alkalinize the cytoplasmic pH depending on the extracellular milieu. Likewise, a voltage-dependent Na+/HCO3− cotransporter has been described in cultured mature rat cerebellar oligodendrocytes (Boussouf et al., 1997). In line with our findings, K+ induced depolarization has previously been shown to induce alkalinization in mouse cerebral astrocytes (Brookes and Turner, 1994), rat hippocampal astrocytes (Pappas and Ransom, 1994), myocytes (Yamamoto et al., 2005) and proximal tubule cells (Siebens and Boron, 1989). Another explanation for our findings could be that voltagegated proton channels are present in the inner cell population. Voltage-dependent proton channels preferentially function in proton extrusion and were first identified in snail neurons and evidence for the presence of voltage-dependent proton channels has since then been found in a variety of cell types (Thomas and Meech, 1982; reviewed by DeCoursey, 2003). The human proton channel gene has recently been discovered and cloned (Ramsey et al., 2006; Sasaki et al., 2006). Thus, in cell types where a proton channel is present, and the membrane potential can strongly depolarize and become more positive than the H+ equilibrium potential, a proton efflux from the cells can take place. In our hands, the resting membrane potential in the inner cell fraction was more depolarized, and hence K+ induced depolarization could drive the membrane potential in the radial glial cell population to more positive values than the calculated EH + equilibrium potential at pHo 7.4, pHi 6.9–7.0 = − 30.77–24.61 mV. This could result in proton extrusion and increase in pHi. More recently, a role for Hv1 proton channels has also been implicated in the motility of cells, which are adapted to low pH, such as neutrophils and sperm (El Chemaly et al., 2010; Lishko et al., 2010). The response to ammonium chloride also showed a significant spatial difference. The alkalinization of the outer cell layer, when exposed to ammonium chloride, was transient while the inner layer of cells showed a sustained alkalinization. Similar transient effects of NH4+ have earlier been described in other cell systems (Boron and De Weer, 1976; Heitzmann et al., 2000; Marcaggi and Coles, 2001) and have been interpreted to be due to the accumulation of charged NH4+ ions in the cell. After NH4+ washout, a significant acidification and a slow time dependent recovery from the acid load could be observed in the outer cell population while the pHi in the inner layers was instantly lowered to a new level. This also suggests that the difference is due the response of the cells to NH4+ ions. The different behavior of the inner and outer layer cells in response to extracellular acidosis or NH4+ induced pH changes was not due to uneven BCECF loading, calibration artifacts or different fluorescent properties of BCECF in the two cell populations. This can clearly be seen in the control experiment where butyric acid was used. When the weak acid butyrate was added, both populations were promptly acidified to the same level. Moreover, no differences in the acidification rate or recovery,

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from acidification after butyrate removal, could be observed between the cellular layers. In our hands, the ammonium chloride induced effects could also be seen with 5 mM ammonium chloride, a concentration that has been measured during hyperammonemia in the brain (Swain et al., 1992). In general, elevated levels of NH3/NH4+ in the brain due to metabolic disorders are known to be extremely toxic to both astrocytes and neurons. In our experiments using neural progenitor cells, NH4+ accumulation appeared to take place predominantly in the outer neuron layer. Several mechanisms have been described which may mediate NH4+ accumulation into cells. Inward rectifying Kir potassium channels have a significant NH4+ conductance (Choe et al., 2000; Nagaraja and Brookes, 1998). Kir channels are expressed early in neuronal development and they are essential in the function of neural progenitors (Karschin and Karschin, 1997; Yasuda et al., 2008). Due to inhibition of the acidification by 100 μM Ba2 + Kir potassium channels are the most likely candidates for driving NH4+ accumulation in our cells. Other potential mechanisms for NH4+ accumulation in neuronal cells might be the neuron-specific K+-Cl− cotransporter (KCC2), (Bergeron et al., 2003; Liu et al., 2003; Titz et al., 2006) and Na+,K+-ATPase (Kelly and Rose, 2010). In summary, we demonstrate by using BCECF to monitor pHi, that two main populations, which show considerable differences in their reactivity to hypoxia, extracellular acidosis and response to NH4+, can clearly be distinguished by exposing neural progenitor cells to low extracellular pH in the absence of bicarbonate or by performing an acidifying ammonium treatment. Our results indicate that neural stem cells tolerate quite well prolonged low extracellular pH values down to pH 6.5 while pH 6.2 totally inhibit neural stem cell development by causing cell death. Moreover our immunostaining data demonstrate that live pH measurements using BCECF can be used to identify and select specific cell populations in the neurosphere culture for further e.g. electrophysiological studies.

4.

Experimental procedures

4.1.

Cell culture and cell differentiation

Neural progenitor cells were generated as previously described (Castrén et al., 2005; Clarke et al., 2000). Cells were scraped from the anterior portion of the lateral wall of the lateral ventricles of E14 embryonic mouse brains. Dissociated cells were plated in DMEM/F-12 culture medium (Gibco) containing 2 mM L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Sigma-Aldrich), B27 supplement (Gibco), 20 ng/ml epidermal growth factor (EGF, PeproTech EC Ltd, London, UK), and 10 ng/ml basic fibroblast growth factor (bFGF, PeproTech EC Ltd, London, UK) and maintained in a humidified 5% CO2/95% air incubator at 37 °C. Within 5–7 days the cells grew as free-floating aggregates termed neurospheres and were passaged after mechanical dissociation every 7–9 days. The culture media supplemented with growth factors were refreshed 2–3 times per week. For neuronal differentiation neurospheres were plated on polyDL-ornithine (Sigma-Aldrich) coated culture plates in the

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absence of mitogens. Growth factor withdrawal induces spontaneous migration of cells from the neurospheres and subsequent differentiation. The differentiated neural progenitor cells selected for pH and membrane potential measurements were single cells migrating outside the neurosphere body towards the periphery. The total cell number in a typical neurosphere cluster varied between 300 and 1000 cells. 4.2.

Materials

2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) and Fura-2 AM were from Invitrogen (Molecular Probes). All other chemicals used in the study were purchased from Sigma‐Aldrich (Helsinki, Finland). 4.3.

Solutions

The HEPES buffered medium (HBM) consisted of (in mM) 137 NaCl, 5 KCl, 2 CaCl2, 0.44 KH2PO4, 10 glucose, 10 HEPES, and 1.2 MgCl2. The pH was adjusted to 7.4 with NaOH. HBM containing 30 mM KCl was prepared by the isosmotic replacement of NaCl with KCl. All experiments were performed under normal atmospheric conditions (21% O2, 0.038% CO2 in air). 4.4.

Preparation of hypoxic solution

Hypoxic HEPES buffered medium (HBM) was prepared by bubbling 100% N2 gas directly into the solution for a minimum of 20 min at 37 °C. When O2 had been removed (dissolved O2 <0.5 mg/l) the 50 ml tube was capped and the perfusion tube was connected to a valve hole at center of the cap. A Jenway Dissolved Oxygen Meter having a Clark type polarographic sensor was used to measure the oxygen concentration in the solution. A pH of 7.4 was maintained in the buffer solution after bubbling with N2 gas. 4.5.

Measurement of intracellular pH

Intracellular pH was measured in neural progenitor cells migrating outside the neurosphere (Fig. 1B, areas 2 and 3) by using the cell permeable probe 2′,7′-bis(carboxyethyl)-5,6carboxyfluorescein acetoxymethyl ester (BCECF-AM) and a dual-wavelength InCytIm2 fluorescence imaging system (Intracellular Imaging Inc., Cincinnati, OH, USA). For the experiments, cells cultured and differentiated for 5 days on 25 mm round coverslips were loaded with 4 μM BCECF-AM in HBM for 30 min at 37 °C. After the dye loading step, the cells were washed 3 times with HBM, placed in the measuring chamber and transferred to the heat controlled chamber holder on the microscope (Nikon, TMS, 20× objective). During the experiment, the cells were perfused (1 ml/min) with warm (37 °C) HBM pH 7.4. To control for local pH variations within the neurosphere culture we also performed experiments with extracellular BCECF free acid in the perfusion buffer. With the cell density of the differentiated cells used in our measurements and the perfusion flow rate used (1 ml/min), no spatial differences in local extracellular pH could be observed in the neurosphere culture (data not shown). The cells were excited alternately with 490 nm and 440 nm wavelength light for

80 ms (rate of data capture 34/min). Data were analyzed with the Incyt 4.5 software and further processed with Origin 6.0 (OriginLab Corp., Northampton, MA) software. The BCECF fluorescence emission ratios (490/440) of the 510 nm fluorescence were turned into pH values by using a standard curve (Nordström et al., 1995). Briefly, single cells located outside the neurosphere body (cells in areas 2 and 3 in Fig. 1B) were equilibrated in K+ medium (140 mM) of varying pH (5.0–8.5) in the presence of 5 μM nigericin and calibration curves were constructed by plotting the extracellular pH against the corresponding fluorescence ratio. A linear relation between the 490/440 nm fluorescence ratio and pH was observed between pH 6.0 and 8.0. Intracellular pH was studied by exposing the cells to low pH extracellular medium or by using the ammonium prepulse technique. In this technique, the non-charged ammonia (in equilibrium with ammonium ion) enters cells and is intracellularly protoned to ammonium (increase in cytoplasmic pH can be observed). After washout, the reverse reaction takes place. Ammonium dissociates into ammonia and protons. Ammonia rapidly leaves the cells and the protons that simultaneously are released cause a rapid acidification in the cytosol. 4.6.

Measurement of membrane potential

Changes in the resting membrane potential of mice neural progenitor cells differentiated for 5 days (migrating cells located outside the neurosphere in areas 2 and 3 in Fig. 1A) were monitored using the potentiometric bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)), an anionic probe that, exhibits enhanced fluorescence when the cell membrane is depolarized (increased intracellular fluorescence do to dye influx). Conversely, hyperpolarization of the membrane potential leads to efflux of the probe and a decrease in fluorescence intensity. Cells cultured on 25 mm round coverslips were washed 3 times with HBM pH 7.4, placed in the measuring chamber and transferred to the heat controlled chamber holder on the microscope (Nikon, TMS inverted microscope, 20× objective). For the experiment, 500 nM DiBAC4(3) was added to the perfusion solution (HBM pH 7.4) and allowed to equilibrate across the cell membrane for 25 min (1 ml/min) before the data acquisition process was started. The cells were excited with 490 nm wavelength light for 80 ms (rate of data capture 25/min) and the emitted fluorescence was captured at 530 nm. The data collected were analyzed with the Incyt 4.5 software and further processed with Origin 6.0 (OriginLab Corp., Northampton, MA) software. At the end of each experiment, the cells were treated with depolarizing (20, 40 or 140 mM) K+-solutions containing 500 nM DiBAC4(3) and the fluorescence signal was monitored. Neural progenitor cells express K+ channels that are open at rest (mainly Kir channels that are permeable to K+) and the Nernst equation can be used to calculate membrane potential (Yasuda et al., 2008). Membrane potential (E) was therefore calculated using the Nernst equation assuming an intracellular potassium concentration of 140 mM and a temperature of 37 °C. E = 2.303 × RT/zF × log10 ([K+]e/[K+]i). Resting membrane potential in the outermost layer was controlled by using whole-cell patch-clamp technique (Hamill et al., 1981). Patch electrodes (model PG-150T, Harvard

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Apparatus, Kent, UK) were prepared with a PC-10 puller and flamed polished with microforge MF-900 (Narishige, London, UK) to a resistance of 3–4 MΩ. For current clamp experiments a potassium based internal solution containing (in mM): 140 K+-Aspartate, 2 MgCl2, 5 NaCl, 10 HEPES, 0.1 EGTA, 2 NaATP, and 0.5 NaGTP, pH 7.23–7.25. Data acquisition was done with an Axoclamp-2A amplifier and a Digidata interface board 1320E (Axon Instruments, Berkshire, UK). The signal was fed to a PC running pClamp8.1 software (Axon Instruments). Cells were compensated for pipette capacitance. For current clamp recordings data was digitally sampled at 5 kHz and filtered at 1 kHz. Liquid junction potential was corrected offline. 4.7. Immunocytochemical analysis of differentiated neural progenitor cells Immunocytochemical studies were carried out on neural progenitor cells differentiated for 5 days after plating. Cells plated on poly-DL-ornithine coated glass coverslips were fixed for immunocytochemical analysis using 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4) for 20 min at room temperature (RT) and washed twice with PBS. The cells were permeabilized and blocked for 60 min at RT using PBS containing 0.1% Triton X-100, 1% bovine serum albumin (BSA, Sigma-Aldrich), and 10% normal goat serum (NGS). Primary antibodies were applied overnight at 4 °C in PBS containing 0.1% Triton X-100, 1% BSA and 1% NGS, washed three times, and incubated with fluorescent secondary antibodies in PBS containing 1% BSA for 60 min at RT in the dark. Cells were then washed three times with PBS and mounted using Vectashield mounting media containing 4′,6diamidino-2-phenylindole (DAPI) for nuclear staining (Vector Laboratories, Inc., Burlingame, CA). The primary antibody used to stain radial glial cells was rabbit anti-EAAT1 (excitatory amino acid transporter 1, also known as glial glutamateaspartate transporter or GLAST, ab416, Abcam, Cambridge, UK). The primary antibodies used to stain neural cells were mouse anti-microtubule-associated protein (MAP)-2 (MAB364), rabbit anti-MAP-2 (AB5622), mouse anti-NeuN (MAB377) and rabbit anti-Calbindin D-28K (AB1778) (all from Chemicon (Millipore), Espoo, Finland). The secondary antibodies used were Alexa Fluor 568 goat anti-mouse IgG (A11004), and Alexa Fluor 488 goat anti-rabbit IgG (A11008) (both from Molecular Probes, Invitrogen, Life Technologies Ltd., Paisley, UK). For negative controls primary antibodies were omitted, resulting in the disappearance of all staining. Stained cells were viewed and photographed using an Olympus AX70 Provis microscope equipped with fluorescence optics and a PCO CCD camera. 4.8.

Statistics

Data are presented as means ± S.E.M. (n = number of cells, N = number of independent experiments). In each experiment 50 individual cells, located from the neurosphere body edge to the outer edge of the culture were randomly chosen. In order to demonstrate the responsiveness (individual traces) and localization (cells marked by an asterisk on the image) of the cells that responded/not responded to the treatments, data from one representative experiment is shown. Each experimental condition was repeated at least three times with

21

independent neural progenitor cell preparations. In all experiments, the initial pHi recovery rate was calculated (dpHi/dt, pH U/min) during the first 2 min of the recovery curve through linear regression analysis. Student's unpaired t-test was used to compare differences between the two cell populations. A p-value of < 0.05 was taken as statistically significant.

Acknowledgments This study was supported by grants from Magnus Ehrnrooth Foundation, Swedish Cultural Foundation in Finland, Academy of Finland, Sigrid Jusélius Foundation and the Finnish Medical Society (Finska Läkaresällskapet). We are grateful for the laboratory assistance provided by Jarno Hörhä.

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