Journal of Neuroscience Research 26: 168-180 (1990)
Calcium-Dependent Volume Reduction in Regenerating Ganglion Cell Axons In Vitro B.T. Edmonds and E. Koenig Department of Physiology, State University of New York at Buffalo
The effects of increasing [CaZ+lion volume regulatory behavior was investigated by phase-contrast videomicroscopy in immature axons regenerating from goldfish retinal explants in vitro. Elevating [Ca2+Ii by using EGTA-buffered, ionomycin-containing bathing media with either 2100 pM [Caz+l0or 1 pM [CaZ+],with N-methylglucamine substituted for Na+ caused axons to undergo a “syneresis.” The syneresis was characterized by a marked loss in volume and condensation of axoplasm, accompanied by a proliferation of lateral processes, which resulted ultimately in an arrest of visible particle transport. The random appearance of dynamic phase-lucent axial protrusions in the distal axon, apparently caused by microtubules, was a frequent early manifestation. Syneresis was also produced by increasing the tonicity of the Cortland saline with sorbitol or treating axons with either valinomycin or with permeant cyclic AMP analogs in normal Cortland saline. In the latter case, extracellular Ca2 was required. Preterminal axons showed an increase in phalloidin fluorescence after syneresis, suggesting polymerization and/or rearrangement of the actin cytoskeleton. Digitonin-permeabilized axonal field models, which maintained good morphology and particle transport, failed to develop a syneresis even when [Ca2+], was increased to 250 pM. Cytochalasin D did not interfere with the development of a syneresis, hut did suppress the proliferation of lateral processes. Syneresis could be blocked by high [K+],, putative antagonists of Ca2+activated K + channels, or by calmidazolium, a calmodulin antagonist. The experimental findings suggest that cytoskeletal changes associated with volume reduction in growing retinal ganglion cell axons are secondary to a loss of cell water and that calcium/ calmodulin-activated K channels very likely play a primary role in dehydration through the loss of K + and osmotically obligated water. +
+
Key words: volume regulation, immature axons, axonal cytoskeleton, potassium channels
INTRODUCTION Regulatory volume decrease (RVD), which many cells exhibit in response to hypotonic challenge, is associated with a cellular loss of KC1 (see Macknight, 1988; Grinstein and Dixon, 1989; Hoffmann and Simonsen, 1989). Calcium has been implicated in mediating RVD in some cases, possibly through Ca2’/calmodulin-regulated K f channels (Grinstein et al., 1982, 1984; Cala, 1983; Hoffmann et al., 1984, 1986; Sarkadi et al., 1985; Pierce et al., 1989). Recently, however, a role for an actin cytoskeleton in volume regulation has been postulated (Mills and Skiest, 1985; Mills, 1987), based on observations that cytochalasins, a class of F-actin binding agents (see Cooper, 1987), cause a reduction in cell volume (Mills and Lubin, 1986) concomitant with a loss of intracellular ions (Allen et al., 1986) and interfere with the RVD response during exposure to a hypotonic bathing medium (Foskett and Spring, 1985; Gilles et al., 1986). Furthermore, volume reduction in cultured Madin-Darby canine kidney (MDCK cells) cells induced by cyclic adenosine monophosphate (CAMP) analogs is accompanied by rearrangement of F-actin (Mills and Skiest, 1985; Mills and Lubin, 1986). Because calcium is capable of modifying the organization of the actin cytoskeleton in many cell types, presumably by operating through various calcium-sensitive, actin-binding proteins (Weeds, 1982; Pollard and Cooper, 1986), it is possible that cytoskeletal changes associated with cell volume changes could be mediated by calcium. While it is unclear how the cytoskeleton could function directly in the efferent pathway involved in regulation of cell volume, several potential modes include the following: 1) exertion of tension by a regulated contractile network located in the subplasmalemmal domain to reduce surface area in opposition to osmotic pressure changes induced by anisotonic conditions (Heubusch et al., 1985); 2) cytoskeletal regulation of the
Received October 3, 1989; revised December 11, 1989; accepted December 18, 1989. Address reprint requests to Dr. Edward Koenig, State University of New York at Buffalo, 313 Cary Hall, Buffalo, NY 14214.
0 1990 Wiley-Liss, Inc.
Volume Reduction in Growing Axons
number and activity of transport units in the plasma membrane (Christensen, 1987; Mills, 1987; Sachs); and 3) cytoskeleton-mediated insertion of vesicles containing transport systems into the plasma membrane (Loo et al., 1983; Foskett and Spring, 1985). Indirectly, however, actin filaments could also operate in the afferent pathway, and, indeed, have been postulated to be in-parallel with cytoskeletal elements that couple membrane tension with cationic stretch receptor channels (Sachs, 1987). We have utilized goldfish ganglion cell (RGC) axons regenerating in retinal explant culture as a model to investigate volume regulation. These axons have certain structural and functional features that lend themselves to probing involvement of the cytoskeleton in volume regulation. Videomicroscopy reveals that, as immature axons in which tubulin and actin are predominant polypeptides (Koenig and Adams, 1982), they exhibit a striking structural plasticity associated with rapid bulk redistribution of axoplasmic masses in the form of varicosities and smaller phase-dense inclusions (Koenig et al., 1985; Edmonds and Koenig, 1987). We shall refer to such structures collectively as axoplasmic varicose aggregates (AVAs). Fusion, fission, and translocation of AVAs reflect a structural plasticity associated with the temporary storage and bulk redistribution of axoplasm (Koenig et al., 1985; Edmonds and Koenig, 1987). On the basis of electron microscopy and immunocytochemistry, AVAs contain an abundance of tubulovesicular smooth endoplasmic reticulum (SER) embedded in an actin-based cytomatrix. In the present report, time-lapse, phase-contrast videomicroscopy has been used to monitor dynamic structural changes in volume and intracellular transport of RGC axons in response to changes in [Ca2+1, brought about by utilizing a calcium ionophore and buffering [Ca2+],, with EGTA. Our findings show that apparent elevation of [Ca2+]],leads to a marked loss of axonal volume as reflected in a decrease in girth, an apparent condensation of axoplasm and reduction in visible particle transport activity. Cytochalasin D does not affect the loss of axonal volume. The experimental evidence is consistent with the hypothesis that Ca2+/calmodulin-dependent K + channels play a predominant role in regulation of volume reduction in these immature axons and that accompanying cytoskeletal changes are secondary events.
MATERIALS AND METHODS Biochemicals The following were purchased from Sigma (St. Louis, MO): cytochalasin D, poly-I-lysine, 5-fluorodeoxyuridine, gentamycin sulfate, uridine, methyl cellulose, N-methyl-d-glucamine, sodium and calcium salts of glu-
169
conic acid, sorbitol, valinomycin, digitonin, ionomycin, A23 187, tetraethylammonium bromide (TEA), and dibutyryladenosine 3'-5' cyclic monophosphate (dbcAMP). Calmidazolium was purchased from Boehringer Mannheim. Quinine and 8-bromo-CAMP were gifts of Dr. Michael Duffey; 3,3'-diethylthiadicarbocyanine bromide (diS-C,-(5)) was a gift of Dr. James Goldinger.
Retina1 Explant Preparation Goldfish retinal explants were prepared as described elsewhere (Koenig and Adams, 1982; Koenig et al., 1985) and were used for experimental observations after 3-5 days in culture. Briefly, 2-4 weeks after crushing the optic nerve, the retina was isolated, chopped into squares (0.65 X 0.65 mm), plated out onto polylysine-coated No. 1.5 circular coverslips, and cultured in L-15 (Gibco, Grand Island, NY) medium supplemented with 10% fetal calf serum (Flow, Indianapolis, IN), 0.02 M Hepes, 0.1 mM 5-fluorodeoxyuridine, 0.1 mg/ml gentamycin sulfate, 0.2 mM uridine, and 0.6% methyl cellulose. Explants were cultured in humid air atmosphere at 27°C. Videomicroscopy For viewing, the circular coverslip containing retinal explants was either mounted in a Dvorak-Stotler chamber (Nicolson Precision Instruments, Gaithersburg, MD) or was inverted over a 35 x 50 mm No. 2 coverslip supported by 0.5-1 mm thick spacers polymerized and trimmed from Silastic medical elastomer (Dow, Midland, MI). The standard bathing medium was a modified Cortland physiological fish saline (Koenig and Adams, 1982) composed of (in mM): 132 NaCl, 5 KCl, 1.6 MgCI,, 1.8 CaCI,, 5.5 glucose, 20 Hepes, adjusted to pH 7.2 with Tris. All experiments were conducted at 22-24°C. Axonal fields were viewed under phase-contrast microscopy (Olympus BHS microscope) with a X 100 oil immersion planapochromat objective (Zeiss, N.A. = 1.25) combined with an achromat condenser (Olympus, N.A. = 1.4) oiled to the bottom coverslip. The microscope stage was isolated from external vibration by a Vibraplane air-suspension tabletop platform (Kinetic Systems, Inc.). The phase image was displayed on a video monitor (Sanyo) using a DAGE NC-67M video camera with a Newvicon tube (DAGE-MTI, Inc.) mounted on a trinocular head of the microscope. Experiments were recorded in a time-lapse mode with a video recorder (TLC 2001; GYYR, Inc., Anaheim, CA), where time was compressed by a factor of 12. Still photographs were taken from the monitor screen using a Polaroid CU-5 Land camera type 665 positivehegative Polaroid film or directly through the microscope with an attached 35 mm photomicrographic system (Olympus PM- 1OAD).
170
Edmonds and Koenig
Analysis of Particle Transport Frequency Evaluation of changes in organelle transport activity has been described in detail elsewhere (Edmonds and Koenig, submitted) and was performed as follows. During video playback, a line transecting a single axon or fascicle was drawn on the screen of the video monitor. The number of bidirectionally mobile AVAs, particles, and mitochondria crossing the line within multiple 1 min intervals of elapsed time of the video record ( 2 5 min) was counted before and after a given experimental treatment. A “transport change index” (TCI) ratio of particle transport frequency was computed as follows:
TCI
frequency of motile organelles after =
frequency of motile organelles before
A ratio of I .O indicated no change, while a ratio
Calcium-Dependent Volume Reduction in Regenerating Ganglion Cell Axons In Vitro B.T. Edmonds and E. Koenig Department of Physiology, State University of New York at Buffalo
The effects of increasing [CaZ+lion volume regulatory behavior was investigated by phase-contrast videomicroscopy in immature axons regenerating from goldfish retinal explants in vitro. Elevating [Ca2+Ii by using EGTA-buffered, ionomycin-containing bathing media with either 2100 pM [Caz+l0or 1 pM [CaZ+],with N-methylglucamine substituted for Na+ caused axons to undergo a “syneresis.” The syneresis was characterized by a marked loss in volume and condensation of axoplasm, accompanied by a proliferation of lateral processes, which resulted ultimately in an arrest of visible particle transport. The random appearance of dynamic phase-lucent axial protrusions in the distal axon, apparently caused by microtubules, was a frequent early manifestation. Syneresis was also produced by increasing the tonicity of the Cortland saline with sorbitol or treating axons with either valinomycin or with permeant cyclic AMP analogs in normal Cortland saline. In the latter case, extracellular Ca2 was required. Preterminal axons showed an increase in phalloidin fluorescence after syneresis, suggesting polymerization and/or rearrangement of the actin cytoskeleton. Digitonin-permeabilized axonal field models, which maintained good morphology and particle transport, failed to develop a syneresis even when [Ca2+], was increased to 250 pM. Cytochalasin D did not interfere with the development of a syneresis, hut did suppress the proliferation of lateral processes. Syneresis could be blocked by high [K+],, putative antagonists of Ca2+activated K + channels, or by calmidazolium, a calmodulin antagonist. The experimental findings suggest that cytoskeletal changes associated with volume reduction in growing retinal ganglion cell axons are secondary to a loss of cell water and that calcium/ calmodulin-activated K channels very likely play a primary role in dehydration through the loss of K + and osmotically obligated water. +
+
Key words: volume regulation, immature axons, axonal cytoskeleton, potassium channels
INTRODUCTION Regulatory volume decrease (RVD), which many cells exhibit in response to hypotonic challenge, is associated with a cellular loss of KC1 (see Macknight, 1988; Grinstein and Dixon, 1989; Hoffmann and Simonsen, 1989). Calcium has been implicated in mediating RVD in some cases, possibly through Ca2’/calmodulin-regulated K f channels (Grinstein et al., 1982, 1984; Cala, 1983; Hoffmann et al., 1984, 1986; Sarkadi et al., 1985; Pierce et al., 1989). Recently, however, a role for an actin cytoskeleton in volume regulation has been postulated (Mills and Skiest, 1985; Mills, 1987), based on observations that cytochalasins, a class of F-actin binding agents (see Cooper, 1987), cause a reduction in cell volume (Mills and Lubin, 1986) concomitant with a loss of intracellular ions (Allen et al., 1986) and interfere with the RVD response during exposure to a hypotonic bathing medium (Foskett and Spring, 1985; Gilles et al., 1986). Furthermore, volume reduction in cultured Madin-Darby canine kidney (MDCK cells) cells induced by cyclic adenosine monophosphate (CAMP) analogs is accompanied by rearrangement of F-actin (Mills and Skiest, 1985; Mills and Lubin, 1986). Because calcium is capable of modifying the organization of the actin cytoskeleton in many cell types, presumably by operating through various calcium-sensitive, actin-binding proteins (Weeds, 1982; Pollard and Cooper, 1986), it is possible that cytoskeletal changes associated with cell volume changes could be mediated by calcium. While it is unclear how the cytoskeleton could function directly in the efferent pathway involved in regulation of cell volume, several potential modes include the following: 1) exertion of tension by a regulated contractile network located in the subplasmalemmal domain to reduce surface area in opposition to osmotic pressure changes induced by anisotonic conditions (Heubusch et al., 1985); 2) cytoskeletal regulation of the
Received October 3, 1989; revised December 11, 1989; accepted December 18, 1989. Address reprint requests to Dr. Edward Koenig, State University of New York at Buffalo, 313 Cary Hall, Buffalo, NY 14214.
0 1990 Wiley-Liss, Inc.
Volume Reduction in Growing Axons
number and activity of transport units in the plasma membrane (Christensen, 1987; Mills, 1987; Sachs); and 3) cytoskeleton-mediated insertion of vesicles containing transport systems into the plasma membrane (Loo et al., 1983; Foskett and Spring, 1985). Indirectly, however, actin filaments could also operate in the afferent pathway, and, indeed, have been postulated to be in-parallel with cytoskeletal elements that couple membrane tension with cationic stretch receptor channels (Sachs, 1987). We have utilized goldfish ganglion cell (RGC) axons regenerating in retinal explant culture as a model to investigate volume regulation. These axons have certain structural and functional features that lend themselves to probing involvement of the cytoskeleton in volume regulation. Videomicroscopy reveals that, as immature axons in which tubulin and actin are predominant polypeptides (Koenig and Adams, 1982), they exhibit a striking structural plasticity associated with rapid bulk redistribution of axoplasmic masses in the form of varicosities and smaller phase-dense inclusions (Koenig et al., 1985; Edmonds and Koenig, 1987). We shall refer to such structures collectively as axoplasmic varicose aggregates (AVAs). Fusion, fission, and translocation of AVAs reflect a structural plasticity associated with the temporary storage and bulk redistribution of axoplasm (Koenig et al., 1985; Edmonds and Koenig, 1987). On the basis of electron microscopy and immunocytochemistry, AVAs contain an abundance of tubulovesicular smooth endoplasmic reticulum (SER) embedded in an actin-based cytomatrix. In the present report, time-lapse, phase-contrast videomicroscopy has been used to monitor dynamic structural changes in volume and intracellular transport of RGC axons in response to changes in [Ca2+1, brought about by utilizing a calcium ionophore and buffering [Ca2+],, with EGTA. Our findings show that apparent elevation of [Ca2+]],leads to a marked loss of axonal volume as reflected in a decrease in girth, an apparent condensation of axoplasm and reduction in visible particle transport activity. Cytochalasin D does not affect the loss of axonal volume. The experimental evidence is consistent with the hypothesis that Ca2+/calmodulin-dependent K + channels play a predominant role in regulation of volume reduction in these immature axons and that accompanying cytoskeletal changes are secondary events.
MATERIALS AND METHODS Biochemicals The following were purchased from Sigma (St. Louis, MO): cytochalasin D, poly-I-lysine, 5-fluorodeoxyuridine, gentamycin sulfate, uridine, methyl cellulose, N-methyl-d-glucamine, sodium and calcium salts of glu-
169
conic acid, sorbitol, valinomycin, digitonin, ionomycin, A23 187, tetraethylammonium bromide (TEA), and dibutyryladenosine 3'-5' cyclic monophosphate (dbcAMP). Calmidazolium was purchased from Boehringer Mannheim. Quinine and 8-bromo-CAMP were gifts of Dr. Michael Duffey; 3,3'-diethylthiadicarbocyanine bromide (diS-C,-(5)) was a gift of Dr. James Goldinger.
Retina1 Explant Preparation Goldfish retinal explants were prepared as described elsewhere (Koenig and Adams, 1982; Koenig et al., 1985) and were used for experimental observations after 3-5 days in culture. Briefly, 2-4 weeks after crushing the optic nerve, the retina was isolated, chopped into squares (0.65 X 0.65 mm), plated out onto polylysine-coated No. 1.5 circular coverslips, and cultured in L-15 (Gibco, Grand Island, NY) medium supplemented with 10% fetal calf serum (Flow, Indianapolis, IN), 0.02 M Hepes, 0.1 mM 5-fluorodeoxyuridine, 0.1 mg/ml gentamycin sulfate, 0.2 mM uridine, and 0.6% methyl cellulose. Explants were cultured in humid air atmosphere at 27°C. Videomicroscopy For viewing, the circular coverslip containing retinal explants was either mounted in a Dvorak-Stotler chamber (Nicolson Precision Instruments, Gaithersburg, MD) or was inverted over a 35 x 50 mm No. 2 coverslip supported by 0.5-1 mm thick spacers polymerized and trimmed from Silastic medical elastomer (Dow, Midland, MI). The standard bathing medium was a modified Cortland physiological fish saline (Koenig and Adams, 1982) composed of (in mM): 132 NaCl, 5 KCl, 1.6 MgCI,, 1.8 CaCI,, 5.5 glucose, 20 Hepes, adjusted to pH 7.2 with Tris. All experiments were conducted at 22-24°C. Axonal fields were viewed under phase-contrast microscopy (Olympus BHS microscope) with a X 100 oil immersion planapochromat objective (Zeiss, N.A. = 1.25) combined with an achromat condenser (Olympus, N.A. = 1.4) oiled to the bottom coverslip. The microscope stage was isolated from external vibration by a Vibraplane air-suspension tabletop platform (Kinetic Systems, Inc.). The phase image was displayed on a video monitor (Sanyo) using a DAGE NC-67M video camera with a Newvicon tube (DAGE-MTI, Inc.) mounted on a trinocular head of the microscope. Experiments were recorded in a time-lapse mode with a video recorder (TLC 2001; GYYR, Inc., Anaheim, CA), where time was compressed by a factor of 12. Still photographs were taken from the monitor screen using a Polaroid CU-5 Land camera type 665 positivehegative Polaroid film or directly through the microscope with an attached 35 mm photomicrographic system (Olympus PM- 1OAD).
170
Edmonds and Koenig
Analysis of Particle Transport Frequency Evaluation of changes in organelle transport activity has been described in detail elsewhere (Edmonds and Koenig, submitted) and was performed as follows. During video playback, a line transecting a single axon or fascicle was drawn on the screen of the video monitor. The number of bidirectionally mobile AVAs, particles, and mitochondria crossing the line within multiple 1 min intervals of elapsed time of the video record ( 2 5 min) was counted before and after a given experimental treatment. A “transport change index” (TCI) ratio of particle transport frequency was computed as follows:
TCI
frequency of motile organelles after =
frequency of motile organelles before
A ratio of I .O indicated no change, while a ratio
