Neuronal swelling and surface area regulation: elevated intracellular calcium is not a requirement

T. L. Herring1, I. M. Slotin2, J. M. Baltz2,3, and C. E. Morris2

1 Department of Biology and 3 Departments of Obstetrics and Gynecology and of Cellular and Molecular Medicine, University of Ottawa, and 2 Ottawa Loeb Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada K1Y 4E9

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Neurons are mechanically robust. During prolonged swelling, molluscan neurons can triple their apparent membrane area. They gain surface area and capacitance independent of extracellular Ca concentration ([Ca]e), but it is unknown if an increase in intracellular Ca concentration ([Ca]i) is necessary. If Ca for stimulating exocytosis is unnecessary, it is possible that swelling-induced membrane tension changes directly trigger surface area readjustments. If, however, Ca-mediated but not tension-mediated membrane recruitment is responsible for surface area increases, swelling neurons should sustain elevated levels of [Ca]i. The purpose of this investigation is to determine if the [Ca]i in swelling neurons attains levels high enough to promote exocytosis and if any such increase is required. Lymnaea neurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 µM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min) were done at either a standard [Ca]e or very low [Ca]e level (0.9 mM or 0.13 µM, respectively). In neither case did the [Ca]i increase to levels that drive exocytosis. We also monitored osmomechanically driven membrane dynamics [swelling, then formation and reversal of vacuole-like dilations (VLDs)] with the [Ca]i clamped below 40 nM via 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]i did not change with swelling, and VLD behavior was unaffected, consistent with tension-driven, [Ca]i-independent surface area adjustments. In addition, neurons with [Ca]i clamped at 0.1 µM via an ionophore could produce VLDs. We conclude that, under mechanical stress, neuronal membranes are compliant by virtue of surface area regulatory adjustments that operate independent of [Ca]i. The findings support the hypothesis that plasma membrane area is regulated in part by membrane tension.

membrane tension; cell volume; molluscan neuron

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IT IS WIDELY RECOGNIZED that cells regulate their volume in response to osmotic perturbations, metabolic loads, and cytomorphological changes. Much less consideration is given to the fact that cell surface area, too, must be continually regulated. Because surface area and cytoplasmic volume changes are not linearly related, we presume that regulatory mechanisms for surface area and for volume use distinct feedback systems. For surface area regulation, membrane tension changes may be the parameter that signals the need for changes in surface area (J. Dai, M. P. Sheetz, and C. E. Morris, unpublished data; Refs. 33, 42). This would constitute mechanosensitive membrane disposition.

Molluscan neurons are well suited for studying mechanosensitive membrane disposition because their large size facilitates the monitoring of many parameters. We showed that osmotically swelling Lymnaea neurons are astonishingly compliant, increasing their apparent surface area threefold over 1 h (40). Robustness under osmomechanical stress thus relies partly on an ability to recruit membrane during swelling. It is likely that this compliance is normally not used to deal with osmotic stresses but to accommodate cell morphology changes in response to varying mechanical stresses (4, 23, 39).

The existence of a mechanosensitive disposition of membrane between the cell surface and internal stores is suggested by various lines of evidence (Dai et al., unpublished data; Refs. 7, 13, 23, 33, 40, 42). Ca-mediated exocytosis (31) and endocytosis, well-known processes by which cells alter plasma membrane area, may be modulated by tension. However, both rely on finely coordinated cellular chemistry and cytoarchitecture, which could be compromised by stresses that create an urgent demand for surface area increase (e.g., abrupt exposure to anisosmotic media or to large mechanical perturbations). Area increases triggered directly by the increased membrane tension might be more fail-safe.

In osmotically perturbed molluscan neurons (see Fig. 1a), capacitance increases and decreases are accompanied by apparent changes in surface area (assuming a specific membrane capacitance of ~0.7-1 µF/cm2). Moreover, swelling and shrinking not only alter total plasma membrane area, they cause measurable membrane tension increases and decreases (Dai et al., unpublished data) and elicit visually dramatic rearrangements of membrane (33). Less than 1 min after they are returned to normal saline (NS), previously swollen neurons develop vacuole-like dilations (VLDs) that can be 10 µm across. In neurons made to reswell, the VLDs rapidly reverse (disappear), as if their membrane is drawn back to the general plasma membrane surface. Membrane tension variations in differentially adhesive regions of membrane are probably instrumental in VLD dynamics. VLDs are initially invaginations at the substrate, but some become fully internalized as true vacuoles (L. R. Mills and C. E. Morris, unpublished data). We interpret eventual VLD vacuolation as a sign of a particularly vigorous surface area regulation engendered by episodes of swelling and then shrinking.

Swelling neurons may recruit lumenless infoldings of membrane to the surface. Electron micrographs of cultured Aplysia neurons reveal such infoldings (12) that, by their ultrastructural appearance, are unlikely to contribute measurable capacitance except when opened out at the surface. Fejtl et al. (12) postulated that they constitute "hot spots" of reserve membrane for area increases.

Osmotically induced capacitance changes and VLD dynamics occur with equal facility in normal and in very low (0.5 µM) extracellular Ca concentration ([Ca]e) (33, 40). Swelling neurons increase their capacitance even with ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in the cytosol (40). Although this does not rule out the possibility that swelling stimulates exocytosis, it suggests that membrane tension may trigger the surface area increases.

Vacuole-like membrane has been linked to cell surface area regulation in various other neuronal preparations (5, 9, 14) and in skeletal muscle (19). Cheng and Reese (5) showed that lumenless membranous disks close to the plasma membrane in chick growth cones are part of an internal membrane pool for augmenting growth cone surface area. Dailey and Bridgman (9) observed vacuole-like organelles that are confluent with the surface in rat superior cervical ganglion neurons; they appeared spontaneously and then disappeared several minutes later, as if they were participating in surface membrane recycling. On the basis of noninvasive capacitance measurements, Sukhorukov et al. (38) postulated that internal membrane reserves are instantaneously available to the plasma membrane when myeloma, hybridoma, and fibroblast cells swell osmotically. In frog muscle cells, Krolenko et al. (19) observed vacuolar dilations, which, like Lymnaea VLDs, formed in shrinking cells and readily reversed with swelling.

In this study, our focus is on events during swelling. Specifically, we ask if, over the time frame used to elicit VLDs, sustained swelling-induced intracellular Ca concentration ([Ca]i) increases occur that might be capable of driving exocytotic membrane area increases. We find that on average (but not in all neurons) swelling is accompanied by a small rise in cytosolic Ca but never to a level sufficient to stimulate exocytosis. We also show that neurons can swell and then shrink and make VLDs under various conditions of enforced low [Ca]i. Swelling neurons need not, we conclude, rely on Ca-driven exocytosis as a means of avoiding rupture.

Membrane tension therefore continues to be a good contender as a primary signal for swelling-induced recruitment of membrane from internal stores. Beyond enabling neurons to adjust during osmomechanical perturbations, tension-mediated membrane disposition could be important during passive stretch of neurites and during their growth and retraction.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cells, solutions, and flow-through chambers. Circumesophageal ganglia were dissected from adult snails (Lymnaea stagnalis) and placed immediately into NS containing (in mM) 50 NaCl, 1.6 KCl, 3.5 CaCl2 (or 0.5 µM CaCl2 for low-Ca experiments), 2.0 MgCl2, 5.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 5.0 glucose, pH adjusted to 7.6 with 1 M NaOH. Gentamicin was added to 50 µg/ml. Osmolarity was 126 mosM, as determined by the Advanced MicroOsmometer (model 3MO). Ganglia were digested for 30 min in reduced Ca (0.5 mM CaCl2) NS with 0.25% type XIV protease (Sigma, St. Louis, MO) and washed twice in NS.

Swelling solutions were made by simple dilution of NS. The Ca concentration of NS was varied, as noted, but usually contained 3.5 mM or 0.5 µM Ca, and the respective 0.25× NS solutions had 0.9 mM Ca or 0.13 µM Ca. The low (0.5 µM)-Ca NS was like NS except that 0.5 mM Ca and 1 mM EGTA (Sigma) were used, yielding a free Ca concentration of 0.5 µM (33).

Neurons were plated on large sterile coverslips (24 × 60 mm) and cultured for 2 days at room temperature as follows. Sterile silicone grease (Dow Corning) was pipetted by syringe into a large oval on the coverslip. NS was added, and neurons from one ganglion were teased out with forceps and allowed to settle and adhere to the untouched glass surface. Coverslips were kept moist and sterile in damp chambers.

Shortly before an experiment, 22 × 22-mm glass coverslips were pressed onto the grease ovals on the larger coverslips, leaving a 100- to 150-µm gap between coverslips. Excess solution and grease were removed, and small dots of molten soft wax (equal parts lanolin, paraffin wax, and petroleum jelly) were placed at the corners of the small coverslip to secure the gap. This created a flow-through chamber that was operated by adding solutions (150-250 µl) by pipette tip at one edge and wicking through the chamber using small strips of filter paper at the other open edge. Solution changes were completed in ~30 s. Experiments were carried out at room temperature.

We previously determined (Dai et al., unpublished data) that, with flow-through chambers, swelling for 4 min in 0.5× NS is sufficient to elicit VLDs in all neurons on return to NS and to generate measurable membrane tension changes. In the present experiments, we used even more extreme swelling conditions, namely 0.25× NS for 4 or 5 min (as noted), so that [Ca]i changes, if any, would be exaggerated.

For cells loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), in a final concentration of 0.3% dimethyl sulfoxide (DMSO) (Molecular Probes, Eugene, OR), 0.5 µM Ca-NS was used. In some experiments, cells were simultaneously loaded in BAPTA-AM and fura 2-AM; for these cases, 3.5 mM Ca-NS was used.

Stock solutions for the fura 2 calibration curve were 1) NS with 2 mM EGTA and zero Ca and 2) 1 M CaCl2. Five 1-ml aliquots of solution 1 were prepared. To the first, no Ca was added, therefore yielding a 0 µM Ca saline. To the second, 1.59 µl of solution 2 were added, yielding a free Ca concentration of 0.1 µM. To the third, fourth, and fifth aliquots, 1.77, 1.90, and 2.00 µl of solution 2, respectively, were added, yielding 0.2, 0.5, and 5.0 µM free Ca, as determined by Fabiato and Fabiato (11). The stability constant for EGTA at 20°C and pH 7.6 (3.819) was assumed.

Testing for VLD formation. Because the ratio imaging system did not allow simultaneous visualization of cell morphology (specifically, VLD formation and reversal) and measurement of [Ca]i, VLD dynamics were checked in parallel experiments, using the test solutions on fura 2-loaded, BAPTA-loaded, and/or Ca-permeabilized cells. The flow-through chambers and the solutions were the same as for the [Ca]i measurements, and fura 2 loading was identical (see below). What we took note of was whether VLD formation occurred, was reversible, and was repeatable at discrete locations (see Fig. 1, a and b), as in previous work (33) without fura 2. 


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Fig. 1.   a: Protocol and terminology for vacuole-like dilation (VLD) formation, reversal, and recovery. Repeated swell/shrink cycles cause VLDs to form and reverse repeatedly at the same sites. Neurons bearing VLDs undergo recovery (a process sensitive to the listed drugs) if left in normal saline (NS) (33). NEM, N-ethylmaleimide. b: A neuron sketched in dorsoventral section before swelling (control; asterisks indicate putative membrane stores), during swelling, and then on return to NS (shrunk), illustrating the topology of VLDs. Some pinch off as true vacuoles (22). c: VLDs produced in a Lymnaea neuron with normal and then with low extracellular Ca (see text). Control (i), swelling (ii) and shrinking (iii) in normal Ca media and swelling (iv) and shrinking (v) in low-Ca conditions are shown. Arrows show that VLDs reappear at discrete sites. Scale bar: 10 µm.

In these experiments, images were recorded with a Sony charge-coupled device (CCD) video camera and JVC BR-S601 MU video recorder, using an Olympus IMT-2 microscope fitted with Hoffmann modulation contrast optics (×40 objective). Subsequently, taped videoframes were digitized by a Data Translations 3155 frame grabber using Image Tool software. Digitized images were processed in Corel Photo Paint.

Fura 2 measurements of [Ca]i. About 2 h before an experiment, neurons in flow-through chambers were loaded for 45 min with 2 µM fura 2-AM (Molecular Probes) in 0.1% DMSO plus 0.02% Pluronic F-127, a dispersing agent. At 45 min and again at 75 min, chambers were washed with NS (5 × 50 µl), enabling neurons to rid themselves of untransformed fura 2-AM (intracellular fura 2 is generated when endogenous esterases cleave the AM) either by hydrolysis to fura 2 or by washout. A field with one or more neurons bearing a lamella was located (VLDs are easier to see in thin extensions than under the soma). The objective was focused not at the substrate but at a rounded region of the cell body, and the focal plane was maintained throughout. Unless noted, pairs of fluorescent images (at two excitation wavelengths, see below) of the field were generated every 30 s; each image was the average of 32 frames collected at 30 frames/s for a total duration of 1.07 s. A background image pair was taken at the end of each experiment in a field of view containing no neurons. Neurons exhibiting abnormally high resting [Ca]i were not used in the experiments.

[Ca]i was measured ratiometrically with a quantitative imaging fluorescence videomicroscopy system built around a Zeiss Axiovert inverted epifluorescence microscope. Fluorescence excitation was via a grating-type monochromator (Photon Technologies International, New Brunswick, NJ) with an electronic shutter (Uniblitz, Vincent Associates, Rochester, NY). Images were detected with a solid-state video camera (CCD72, Dage-MTI, Michigan City, IN) fitted with a Geniisys image intensifier (Dage-MTI). The emission wavelength was selected by a 510-nm bandpass filter. All of these were controlled by Isee and dsp/os software and imaging hardware (Inovision, Durham, NC) running on a Silicon Graphics Indigo2 (R4400, Extreme Graphics) Unix workstation (Silicon Graphics, Mountain View, CA). The excitation wavelengths were 350 nm (for detecting Ca-bound fura 2) and 380 nm (for Ca-free fura 2). For a circular area covering most of the soma of each neuron (preswelling), the ratio of the intensity at 350 nm divided by that at 380 nm was calculated pixel-by-pixel after background was subtracted using the Inovision Isee software, and the mean ratio was recorded. The quantitized areas were representative of the image of the entire ratio of the neurons, which were very homogeneous in appearance. Occasionally, however, it was evident from these images that a neuron had moved between data acquisition at 350 and 380 nm; striking crescents of low and high intensity appeared at opposite edges of the neuron. Data from these neurons were discarded.

Calibration of fura 2, a free Ca concentration indicator, in Lymnaea neurons. Instead of calibrating by only determining emission ratios for extremely low (zero Ca) and extremely high (saturating) Ca levels, as is normally done (15), we calibrated fura 2 in neuronal cytoplasm (Fig. 2) over a range of extreme and intermediate concentrations. Thirty minutes before the experiment, 20 µM of the Ca ionophore 4-bromo-A-23187 (a nonfluorescent form) in 0.6% DMSO was added to the series of calibration solutions. Neurons were exposed to the 0 µM Ca-NS-A-23187 solution for 25 min. Fluorescent image pairs were taken, and then neurons were exposed for 10 min each to successively higher Ca-A-23187 solutions, with image pairs obtained at the end of each incubation. Background fluorescent images were subtracted before analysis. The calibration curve for [Ca]i was constructed by plotting the fluorescence ratios vs. free Ca concentration and fitting to an arbitrary polynomial equation using a nonlinear least squares fit. In essence, this is a computer-assisted "by eye" fit. The fitted line was used to read off interpolated Ca concentrations. To verify the accuracy of our in vivo calibration, the process was repeated months apart by different workers, using newly made solutions. The in vivo half-maximal ratios for fura 2 from the calibration curves of the two workers were at Ca concentrations of 0.13 and 0.14 µM.


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Fig. 2.   Intracellular Ca concentration ([Ca]i) calibration curve(s) for Lymnaea neurons loaded with 2 µM fura 2, permeabilized to Ca using 4-bromo-A-23187 and equilibrated to various Ca solutions. Two different symbols (bullet  and star ) represent curves generated several months apart by 2 workers. Half-maximal ratio values were obtained at Ca concentrations of 0.13 and 0.14 µM from the 2 curves. Inset: ratio images of a sample neuron in NS (a) and 0 (b), 0.1 (c), 0.2 (d), 0.5 (e), and 5.0 (f) µM Ca. Scale bar: 20 µm.

Results are presented as mean values ± SE (n = number of experiments). Paired t-tests were used to test for statistical significance of changes in [Ca]i between experimental conditions. A P value of 0.05 was used to indicate statistical significance.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

VLDs: a phenomenon that occurs at submicromolar [Ca]e. Figure 1c demonstrates that VLDs were elicited in the same neuron with both normal and low-Ca media (see METHODS regarding osmotic dilutions) and, furthermore, confirms that [Ca]e does not affect the ability of neurons to form VLDs. Osmotic perturbations (swell, shrink, swell, i.e., 0.25× NS, 1× NS, 0.25× NS) were first done with normal Ca and then repeated with low-Ca solutions. VLDs formed when swollen neurons shrank (in NS) and reversed during the second swell (0.25× NS). During the low-Ca repeat runs, VLDs formed at the same sites as happened previously, reversed with a second swelling, and, finally, reformed when a second reshrinking occurred (in low-Ca NS).

Clearly, the VLD dynamics (formation, reversal, and reformation at discrete sites) are not dependent on [Ca]e levels but, even when swelling is stimulated in 0.13 µM [Ca]e, free [Ca]i may become elevated as a result of swelling-induced release of Ca from intracellular stores. The following experiments address this possibility.

Does [Ca]i change during swelling and shrinking? Fura 2-loaded cells were used to measure [Ca]i during swelling. In preliminary trials using Hoffman modulation optics (which emphasize membranous structures), we confirmed that fura 2-loaded neurons retain their ability to repeatedly make VLDs, as above. For the Ca-monitoring experiments, inspection of the cells by transmitted light at the end of the experiment usually revealed the VLDs that had formed, but the optics were not set up for transmitted light, so during fluorescence image acquisition only fluorescence imaging was done.

As shown in Fig. 3a, [Ca]i for Lymnaea neurons in NS before any osmotic insult was 96 ± 9 nM (n = 19), which is in keeping with values for other molluscan neurons [40 nM in Helix aspersa (17), 90 nM in Helix pomatia (18), and 10-100 nM in Helix pomatia (25)]. Neurons were swollen in [Ca]e of 0.9 mM (Fig. 3a). Of 19 neurons tested, 11 showed a [Ca]i increase, 4 did not change, and 4 decreased; the average [Ca]i for a swollen neuron was 105 ± 11 nM, amounting overall to an 11 ± 0.5% increase over normal [Ca]i (P = 0.016, n = 19, within-neuron paired t-tests). After 5 min of swelling, neurons were returned to NS. At this point, they shrank and made VLDs and their average [Ca]i was 100 ± 10 nM, with three increased, seven decreased, and nine unchanged (a 9 ± 0.3% decrease but not significant, P = 0.08). Pairwise t-tests (n = 19) also showed that [Ca]i values in NS before swelling and in NS after the swelling episodes were statistically indistinguishable (P = 0.05). Thus, with [Ca]e at 0.9-3.5 mM during osmotic perturbations, there were at best marginal increases in intracellular Ca.


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Fig. 3.   a: Fura 2 measurements of [Ca]i in Lymnaea neurons at rest (0-5 min) and as they swell (5-10 min) in 0.25× NS and then reshrink (10-20 min) in NS. Concentrations above traces are mean [Ca]i used for 19 neurons during each phase; individual values are the average of readings taken every 30 s. Unlabeled sections between each phase indicate when solution changes were made; one 30-s time point for [Ca]i was skipped during each change. For one trace (dashed line), representative ratio images for each phase are displayed (top). Because the contrast between background and cell in the images was low (albeit readily visible on a computer monitor), cell perimeter has been outlined by hand. Circles are regions from which ratios were averaged. When reshrunken cells were checked with transmitted light at the end of the experiment, VLDs were typically visible in the lamellar region. Scale bar: 22 µm. b: [Ca]i of Lymnaea neurons during the first 1.5 min of swelling. For the first 10 s, neurons were in NS and then 0.25× NS was wicked through chamber (~15 s). Through the whole procedure, image pairs were taken every 2 s. For runs at this speed, it was not possible to average image frames during real-time data acquisition (see METHODS), so ratios are calculated from pairs of single video frames (1/30 s each).

In the above experiments, we did not monitor [Ca]i during the solution changes, so it was possible that a fast transient change was missed. To assess the likelihood of overlooked [Ca]i transients, we followed three neurons at 2-s intervals for ~1.5 min during solution changes (NS to 0.25× NS, with 3.5 mM Ca in NS). Care was taken not to mechanically disturb the chamber as solution was wicked off. As Fig. 3b shows, swelling neurons experienced no transient change in [Ca]i during the first 1.5 min of swelling, even though there was abundant external Ca. High frequency monitoring of this initial stage (which required two workers) was not, therefore, done on a routine basis.

Trials were also run with [Ca]e at 0.5 and 0.13 µM for 1× NS and 0.25× NS, respectively, collecting data as in Fig. 3a (Fig. 4). For these experiments, neurons were preexposed to 0.5 µM Ca NS for 5 min before the "control" part of the experiment (0-5 min in Fig. 4). The [Ca]i value before swelling was 53 ± 7 nM (n = 11), whereas in swollen neurons it was 61 ± 10 nM and in NS after swelling it was 54 ± 8 nM. Of the 11 neurons tested, 6 showed a [Ca]i increase and 5 were unchanged. Overall, this resulted in a significant 11 ± 0.4% increase in [Ca]i during swelling (P = 0.04, within-neuron paired t-tests) followed by a significant (P = 0.02) fall during shrinking, with 7 decreasing and 4 unchanged. Again there was no difference between pre- and postswelling [Ca]i (P = 0.27).


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Fig. 4.   Fura 2 measurements of [Ca]i of Lymnaea neurons in a low extracellular Ca concentration ([Ca]e) medium (0.5 µM Ca) before swelling (0-5 min), during swelling (5-10 min), and during shrinkage (10-20 min). As in Fig. 3a, concentrations above traces are mean values used for 11 neurons during each phase and dashed trace corresponds to the neuron displayed in each phase at top. A cell outline was generated as in Fig. 3; because swelling and shrinking occurred mostly in a vertical direction for this cell, little cross-sectional change is evident. Scale bar: 22 µm.

In summary, we detected ~10% increases in [Ca]i with swelling under both conditions of [Ca]e. This amounted to increases from 96 to 105 nM Ca and from 53 to 61 nM Ca in the normal and low extracellular Ca conditions, respectively.

VLDs form with [Ca]i clamped to 0.1 µM using a Ca ionophore. Having determined (Fig. 3a) that resting [Ca]i (preswell, 3.5 mM [Ca]e) for these neurons was ~0.1 µM, we clamped [Ca]i at equilibrium with this level of external Ca and then tested the neurons' ability to form VLDs in response to osmotic perturbations. Under these conditions, [Ca]i should be unable to fluctuate above 0.1 µM Ca during perturbations.

After a brief wash in 0.1 µM Ca-NS, neurons were incubated for 25 min in 0.1 µM Ca NS containing 20 µM of the Ca ionophore, 4-bromo-A-23187 (Molecular Probes) dissolved in 0.6% DMSO. While doing the fura 2 calibration procedures, we found that Ca clamping with this ionophore requires the ionophore to be continuously present in the medium. In the continued presence of the ionophore, therefore, neurons were swollen in 0.25× NS for 4 min and then reshrunk in NS (note that for 0.1 µM Ca-NS this results in 0.025 µM Ca during swelling). The osmotic perturbation protocol was repeated to test if VLDs could reverse and then reform. Only about one-third of the neurons remained healthy during the 25-min preincubation in 0.1 µM [Ca]e. The other two-thirds retracted processes and/or became grainy. Nevertheless, those neurons with a tolerably normal appearance after the low-Ca preincubation withstood the osmotic perturbation cycles and formed and then reversed VLDs. Figure 5 shows two such neurons with VLDs in a lamellar region. The important point here is that VLDs can form in neurons that are prevented from experiencing a Ca perturbation during the swelling stimulus.


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Fig. 5.   Examples of VLDs elicited in neurons with [Ca]i clamped at a [Ca]e of 0.1 µM by A-23187. Scale bar: 10 µm.

Are VLD dynamics possible when [Ca]i is held at very low levels by BAPTA? To clamp the [Ca]i to a lower level, cells were loaded with a membrane-permeant fast Ca chelator, BAPTA, by incubating them in 30 µM BAPTA-AM in 0.3% DMSO for 1 or 3 h. The ester (i.e., the AM form) freely crosses the lipid bilayer (6); in the cytoplasm, esterases cleave the ester, trapping BAPTA in the cytoplasm. Although the cytoplasmic BAPTA concentration is not known because loading is not an equilibration process, intracellular BAPTA concentration ([BAPTA]i) should have been at least as high at 3 h as at 1 h. At 1 or 3 h, the neurons were swollen in low-Ca 0.25× NS (0.13 µM Ca) for 4 min and then reshrunk in NS with 0.5 µM Ca. All neurons formed VLDs, which were then reversed and reformed twice more with the same osmotic shock solutions. Figure 6a illustrates VLDs made by this swell/shrink protocol in a control neuron, and b and c show neurons preloaded with BAPTA for 1 and 3 h (respectively) and treated in the same way.


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Fig. 6.   VLD formation in neurons loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) before a swell/shrink perturbation in low-Ca media. Neurons were swollen in 0.25× NS (top images) and subsequently in NS (bottom images), showing VLDs. a: A control cell. b: Cell loaded with BAPTA-acetoxymethyl ester (AM) for 1 h. c: Cell loaded with BAPTA-AM for 3 h. Scale bars: 10 µm. d: Fura 2 was used to measure [Ca]i in Lymnaea neurons that were loaded with BAPTA. With the use of fura 2, no changes in [Ca]i from control levels (0-5 min) in neurons subjected to swelling (5-10 min) or shrinking (10-15 min) are shown. This method also shows that BAPTA-AM loading is effective, buffering [Ca]i to less than one-half the resting [Ca]i measured in NS when no exogenous Ca chelator is present in the cytoplasm (cf. Fig. 3).

Neurons were also loaded simultaneously with BAPTA-AM and fura 2-AM to measure the neuronal [Ca]i when swelling and shrinking were allowed, using the same data collection protocol as in Fig. 3a. To allow for adequate cellular processing of both esters, the following loading procedure was used: BAPTA-AM and fura 2-AM (in a final concentration of 0.3% DMSO) were applied for 45 min and then cells were washed in NS and left to rest 30 min, washed again in NS, and allowed to rest another 45 min, and then experiments were begun. Experiments were completed over the next 60 min, so that a maximum of 3 h elapsed since the beginning of load time. As Fig. 6d shows, BAPTA effectively buffered neuronal [Ca]i to 34.6 ± 1 nM (n = 6) and, when swelling occurred, no change in [Ca]i was evident. Because the BAPTA was loaded for only 45 min in these experiments, we assume that [Ca]i levels were at least as low after the previously mentioned 1- and 3-h incubations.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Resting [Ca]i in Lymnaea neurons was about one-half the normal level when [Ca]e was changed to 0.5 µM instead of the normal 3.5 mM (Figs. 3a and 4). Evidently, neuronal Ca homeostasis mechanisms cannot effectively counteract extremely low extracellular Ca levels by drawing on internal Ca stores. Because submicromolar external Ca would not be experienced in vivo, this is not surprising. It is, moreover, advantageous here because the induced low [Ca]i facilitates the study of putatively [Ca]i-independent events.

Swelling coincided with a small average increase in [Ca]i for media of both low and high [Ca]e. Although they are statistically significant, these increases are not physiologically significant in the context of the Ca levels normally thought to drive exocytosis (see below). The increases (from 96 to 105 nM and from 53 to 61 nM for the 0.9 mM and 0.13 µM [Ca]e + 0.25× NS swelling solutions, respectively) amount to absolute changes of <10 nM. Moreover, as seen in individual [Ca]i traces (see Figs. 3b and 4), some neurons experienced either no increase or even a decrease during swelling. The between-neurons variation in baseline [Ca]i is easily in the 10 nM range, as is the noise level for some neurons. This noise is probably cellular rather than instrumentational, since BAPTA-loaded neurons (Fig. 6a) were markedly quieter.

Movement is inherent in swelling and shrinking neurons. Swelling is most dramatic in the first minute after any osmotic perturbation in Lymnaea neurons (24), as in any cell; movement artifacts would have been most problematic as solution changes were implemented, and in most experiments [Ca]i was not monitored over this period. Spatially, movement artifacts are most pronounced at the cell perimeter; as mentioned (METHODS), we discarded data if such artifacts were evident. The large size of Lymnaea neurons (~20-80 µm diameter) should enhance the reliability of our measurements because, the larger a cell, the smaller its perimeter-to-cross-sectional ratio. Moreover, measurements were not made of the entire cytoplasm but made from a circular region slightly less than the minimum diameter of the soma, established for the control condition and then used throughout. This largely obviated problems at the perimeter of the cell related to inflation and deflation. In future experiments, it would be desirable to monitor the immediate submembrane region, but movements here will always introduce error. In any case, we know that transmembrane influxes of Ca are not needed for cells to produce VLDs or to increase capacitance as they swell (33, 40), so a sustained Ca release into the bulk of the cytoplasm from internal stores is what we were trying to detect.

Our data do not rule out a minor release of Ca from stores in swelling neurons, but comparing the swelling-induced increases of [Ca]i in Lymnaea to the [Ca]i increases in various cells undergoing exocytosis puts the swelling-induced changes in perspective. Eosinophil [Ca]i rose from 240 nM to 1.5 µM on exocytotic stimulation with guanosine 5'-O-(3-thiotriphosphate) (29), a difference of ~1,300 nM. In squid giant nerve terminals, presynaptic [Ca]i rose from 100 to 1,000 nM during trains of 250 action potentials (2), a 10-fold increase or, in absolute terms, an increase of 900 nM. Neher and Augustine (26) measured the [Ca]i in chromaffin cells at rest to be ~100 nM, and, once depolarized, this measurement was found to be ~550 nM at the plasma membrane and 500 nM deep in the cytoplasm. In the same type of preparation, Penner and Neher (32) found levels in stimulated cells in the range of 400-1,500 nM. Others have confirmed these results, showing that the [Ca]i at rest ranges from 10-100 nM and [Ca]i, when depolarization occurs, rises to 300-1,000 nM (3).

Even in normal [Ca]e, the largest swelling-related [Ca]i increases in Lymnaea neurons should not have been able to drive Ca-dependent exocytosis. Nevertheless, membrane capacitance and area increases in swelling Lymnaea neurons (40) and volume increases in swelling Aplysia neurons (12) indicate that the membrane is recruited by swelling. A simple possibility is that lumenless membrane folds or mechanically accessible membranous cisternae (12) are drawn into the plasma membrane by elevated tension. We have measured membrane tension in swelling neurons using plasma membrane tethers pulled by optical tweezers (Dai et al., unpublished data); Lymnaea neurons swelling in a 50% medium experience a tension transient that relaxes to a sustained 0.12 mN/m, compared with 0.04 mN/m before swelling. This is consistent with acquisition of new membrane in response to swelling tension. The sustained threefold increase in tension is not dangerously high, since 0.12 mN/m is about two orders of magnitude below membrane lytic tension in Lymnaea neurons. In fact, 10-12 mN/m has been determined as the lytic pressure in many biological membrane preparations (27).

Nichol and Hutter (28) showed that [Ca]i of 2 µM but not 0.8 µM is mechanically treacherous; a sharp decline in membrane strength occurs between these concentrations. Because the "danger zone" of [Ca]i overlaps that of neurons undergoing Ca-driven exocytosis, it may be imprudent for a mechanically stressed neuron to rely on Ca-driven exocytosis as a means of increasing membrane area. The small swelling-induced Ca concentration elevations in Lymnaea neurons (even in the higher external Ca concentrations) are well below the danger range.

The specific capacitance of Lymnaea neurons before swelling is consistent with a relatively nonwrinkled membrane surface (40), and fluorescent aqueous phase markers examined by confocal microscopy (33) reveal no dye-accessible infoldings. Thus, to prevent rupture, major options for a swelling neuron are 1) to retract processes and/or 2) to recruit new membrane. Brief swelling episodes elicit VLDs yet seldom cause retraction of processes, so we assume recruitment of membrane occurs. In cell culture, some cell types (20, 41) generate discrete spherical blebs as they swell. In contrast, swelling neuronal cells, including Lymnaea neurons and rat hippocampal neuron cultures (Dai et al., unpublished data), seldom bleb. Neurons made unhealthy by overdigestion during isolation, by age in culture, or by Ca-loading generate large discrete blebs (unpublished observations) consistent with the availability of membrane in this form. Perhaps the subplasmalemmal reserves discussed in Fejtl et al. (12) are the source of bleb membrane in weakened neurons. However, even when swelling is stimulated in near-distilled water (40), healthy neurons remain bleb-free. Contractile resistance to hydrostatic stress may help protract the duration of swelling and capacitance increase (40). Overall, neurons seem to maintain good control of membrane reserves, playing them out only as needed to keep membrane tension low (Dai et al., unpublished data). When stress is excessive, rupture, not blebbing, results. In initial trials for the present work, healthy Lymnaea neurons exploded as distilled water abruptly washed through the double coverslip chambers; previously (33), stepwise exchanges to distilled water were used, presumably allowing sufficient time for augmentation of membrane area.

Any reserve membrane contiguous with the plasma membrane (5, 9, 19, 38) could play a role in mechanosensitive membrane disposition. In addition, cells undergo constitutive exocytosis and endocytosis; a swelling-induced increase in exocytosis and/or decrease in endocytosis would result in net recruitment.

Holding [Ca]i constant does not prevent VLDs. Control neurons and BAPTA-loaded neurons were similar in appearance and produced VLDs. BAPTA liberated intracellularly has low membrane permeability, and so [BAPTA]i may have continued to increase during 3 h of loading. Experiments with neurons loaded for 45 min with BAPTA plus fura 2 showed that [Ca]i was buffered below 40 nM and that no [Ca]i increase was elicited by swelling. Therefore, we conclude that <40 nM [Ca]i did not impair the ability of neurons to swell without rupturing or to form VLDs repeatedly at the same sites. Morán et al. (21) also showed that BAPTA plus fura 2-loaded cerebellar granule neurons exposed to 50% medium for 90 s maintain a fixed [Ca]i; no tendency for low-Ca neurons to rupture was reported.

In theory, the ideal experiment is to clamp [Ca]i and [Ca]e to the resting [Ca]i level (0.1 µM, see Fig. 3a) and then attempt to elicit VLDs so that [Ca]i during swelling and subsequent VLD formation is known and invariant. However, Ca ionophores used to produce the clamp do not limit their effects to the plasma membrane. Ca released from intracellular stores (16) during A-23187 treatment probably caused the general cellular trauma noted, since, in BAPTA experiments, low [Ca]i per se was benign. A-23187 may also have sufficiently increased the proton permeability of plasma membrane and/or endomembranes to alter the intracellular pH. The granular appearance of A-23187-treated Lymnaea neurons echoes that of rat peripheral nerve (35) and dog spinal neurons (10). Neurons that survived and withstood the first osmotic perturbations were fragile, and recurrent shocks resulted in rounding up. Emery and Lucas (10) noted, as did we, varying degrees of damage with A-23187. As A-23187 releases Ca from stores, the Ca may stimulate exocytosis. BAPTA does not stimulate release of Ca from stores. Thus, even though we assume that A-23187-treated neurons swelled and then made VLDs with their cytoplasm clamped to [Ca]i of 0.1 µM, the ionophore clearly had unwanted side effects and we consider the BAPTA experiments a better indication that neurons can swell (presumably recruiting membrane) and subsequently form VLDs with no change in [Ca]i. In particular, the VLD formation experiments in low [Ca]e with BAPTA-AM-loaded neurons rule out the possibility that an undetected, fast, swelling-evoked Ca transient produced a net increase in plasma membrane area, either through Ca-mediated stimulation of exocytosis or through Ca-mediated inhibition of endocytic plasma membrane recycling.

[Ca]i and swelling in other preparations. In neurons, resting [Ca]i falls between 10 and 300 nM (17). This range easily encompasses all our findings, even those for swollen neurons. We did, however, reject at the outset, as potentially unhealthy, cells whose initial [Ca]i was abnormally high. O'Connor and Kimelberg (30) measured [Ca]i in swollen astrocytes using fura 2; [Ca]i increased rapidly to 600 nM in ~25 s, then decreased to 250 nM (over 5 min). The same pattern was seen without extracellular Ca. The astrocyte Ca transient is thought to be a component of the signaling pathway for regulatory volume decrease (RVD).

Like many vertebrate and invertebrate neurons (1, 12), Lymnaea neurons do not exhibit RVD (24). In contrast, Pasantes-Morales and co-workers (34), using 0.2-0.5× medium, observed partial RVD in isolated neurons. Nevertheless, in their most recent work, they show that RVD is not dependent on [Ca]i. Cerebellar granule cells were loaded with fura 2, and osmotic changes were made at high time resolution. In 8 of 10 cells, swelling-induced changes were absent or inconsequential (21). BAPTA buffered [Ca]i to levels comparable to what we observed and abolished any hint of swelling-induced effects. Swelling did not detectably release Ca from internal stores. In a [Ca]e of 0.13 µM, the swelling-associated increase of [Ca]i (from 53 to 61 nM) of 0.008 µM in Lymnaea neurons could have been supplied by the external medium or by stores.

The diminutive increases in Lymnaea neurons do not reflect insensitivity of the imaging system; in the same setup, we (36) showed that mouse zygotes (which exhibit RVD) loaded with fura 2 experience small swelling-induced increases in [Ca]i. As in the neurons, BAPTA clamped [Ca]i to a lower level.

In summary, swelling does not elicit in Lymnaea neurons a sustained increase in [Ca]i that could support membrane recruitment via Ca-driven exocytosis. Swelling-induced increases in surface area and capacitance may, therefore, be driven directly by elevated membrane tension. For a neuron changing shape in a manner that demands increased surface area, tension-induced recruitment could locally augment plasma membrane area independent of any cytoplasmic signal. In vivo, both Ca-mediated (31, 37) surface area regulation and a separate tension-mediated form of regulation may operate. Coexisting tension- and Ca-dependent membrane recruitment may be widespread; plant protoplasts exhibit both Ca-mediated and (with ~30 nM [Ca]i; Ref. 42) pressure-stimulated capacitance increases, and it is suggested that the membrane pools involved are different.

    ACKNOWLEDGEMENTS

This work was supported by grants to C. E. Morris from the Heart and Stroke Foundation of Ontario and Natural Sciences and Engineering Research Council of Canada and by a grant to J. M. Baltz from the Medical Research Council (MRC), Canada. J. M. Baltz is an MRC Scholar.

    FOOTNOTES

Address for reprint requests: C. E. Morris, Neurosciences, Loeb Institute, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9.

Received 24 July 1997; accepted in final form 10 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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