Growth of sympathetic nerve fibers in culture does not require extracellular calcium

Neuron,

Vol. 3, 733-743,

December,

1989, Copyright

0 1989 by Cell Press

Growth of Sympathetic Nerve Fibers in Culture Does Not Require Extracellular Calcium Robert B. Campenot Department of Anatomy Faculty of Medicine University of Alberta Edmonton, Alberta Canada T6G 2H7

and Dwight and

Cell

D. Draker Biology

Summary A compartmented culture system in which distal neurites from newborn rat sympathetic neurons entered a fluid environment separate from that bathing the cell bodies and proximal neurites was used to investigate effects of extracellular Ca2+ deprivation on nerve fiber growth. Neurites readily grew into, elongated for many days within, and regenerated after neuritotomy within distal compartments substantially deprived of Ca*+ (0 added CaZ+, 0.5-5 mM EGTA), provided Ca2+ was supplied to the cell bodies. The Ca2+-deprived neurites generally extended at rates 20X-35% slower than controls. Growth of neurites did, however, cease within 2 days when the cell bodies were deprived of Ca2+, and the neurites and cell bodies eventually degenerated. These results show that neither extracellular Ca2+ nor the influx of Ca2+ at or near the growth cone is required for sustained neurite growth. They also rule out the possibility that the promotion of neurite growth by nerve growth factor is mediated by the influx of extracellular Ca2+. Introduction Studies of vertebrate as well as invertebrate neurons suggest that the motile activity of neuronal growth cones is associated with an increase in their internal free Ca2+ (Connor, 1986; Cohan et al., 1987). This increase may originate from Ca*+ influx across the surface membrane of the growth cone (Mattson and Kater, 1987). However, several experimental treatments known to cause Ca*+ influx did not increase neurite growth, but depressed it (Haydon et al., 1984; Cohan and Kater, 1986; Cohan et al., 1987; Mattson and Kater, 1987; Mattson et al., 1988a, 1988b), and a reduction in extracellular Cal+ has been reported to produce an increase in neurite growth in some systems (Mattson and Kater, 1987; Bixby and Spitzer, 1984). A hypothesis offered to explain these conflicting results, many of which were obtained with different types of neurons, is that a neuronal growth cone produces optimal growth at a certain intracellular Ca*+ concentration, or “set point,” above or below which growth diminishes (Mattson and Kater, 1987; Kater et al., 1988). According to this scheme, some types of neurons have growth cones with baseline intracellular Ca*+ concentrations substantially below the optimum, and treatments causing Cal+ influx could increase the growth of

these neurons by bringing the intracellular concentration closer to the optimum. Conversely, some types of neurons are presumed to have growth cones with baseline intracellular Ca*+ concentrations substantially above the optimum, in which case treatments that reduce Ca*+ influx could increase neurite growth. Although this hypothesis helps to explain the data, the matter must be regarded as unsettled, since there are so many biochemical mechanisms that can mediate the effects of Ca*+. The situation is complicated further because cytoplasmic Ca2+ can originate not only from influx, but also from intracellular storage organelles (see Carofoli, 1987; Volpe et al., 1988). For these reasons we regard it likely that the influence of Ca*+ on neurite growth results from an interplay of many mechanisms with diverse functions acting in concert, and we suspect that the variety of results obtained arises from species and/or developmental differences in which mechanisms predominate. The experiments reported here were performed in an effort to separate out the different ways in which Ca*+ might influence nerve fiber growth. The approach employed compartmented cultures of rat sympathetic neurons, an experimental system with several advantages compared with those previously used. Cell bodies of rat sympathetic neurons resided in a proximal compartment and produced neurites that elongated to the left and right, penetrated silicone grease barriers, and entered separate fluid environments within distal compartments (Figure 1). As a result, these cultures permitted long-term observations of neurons while depriving either just distal neurites or just cell bodies and proximal neurites of extracellular Ca2+. In this way the long-term requirements for extracellular Ca*+ at the site of neurite growth as well as in the region of the cell body were separately investigated. It was also possible to determine whether Ca2+ influx was involved in mediating the growth-promoting effects of nerve growth factor (NGF).

Results Neurites Regenerate Readily in Distal Compartments Deprived of Ca2+, Provided Ca2+ Is Supplied in the Cell Body Compartment Sympathetic neurons from newborn rats were plated in center compartments of three-compartmented culture dishes (see Figure 1). On day 11.9 two cultures were neuritotomized left and right by means of a jet of sterile, distilled water delivered with a syringe (see Experimental Procedures). Right compartments were then given L15C02 medium without Ca*+ and with 0.5 mM ECTA added, and left and center compartments were given medium containing 1 mM Ca*+. Medium without Ca2+ added and containing ECTA will hereafter be termed “Caz+-deprived,! and medium with 1 mM Ca2+ added will be termed “Ca2+-supplied:’ In all experiments all

Neuron 734

a.

Comuartmented

Figure 1. Schematic Culture

culture

Scratches substratum

in

35 mm tissue

her

b.

Enlargement

compartment

of a single

a Compartmented

(a) illustrates an entire culture, and (b) is an enlargement of a single track. Cell bodies are located in the center compartmeni, and neurites extend to the left and right, under silicone grease barriers, and into the separate fluid environments of left and right companments. The tracks are formed on the floor of a 35 mm plastic tissue culture dish between a series of parallel scratches from which the dried collagen substratum has been scraped away. Each track is about 200 u.m wide. One culture can contain up to 20 such tracks occupied by neurons. Neurite extension along the tracks is measured with a stage micrometer out to about 5 mm into left and right compartments, where the neurites reach the ends of the tracks.

Right compartment Teflon divider

of

track

Cell bodies

Nerve /ibars

Scratch

in subs&turn

compartments were supplied with 1 pg of 7S NGF/ml and the center compartments containing the cell bodies were also supplied with 2.5% rat serum and 1 mg/ml ascorbic acid. The day after neuritotomy, regenerated neurites were present on all 30 tracks in left and right compartments, and regeneration continued throughout 6.7 days of observation. Figure 2 shows photomicrographs of neurites on a single track in this experiment, 3.8 days after neuritotomy. The fine filamentous and varicose appearance of the Ca*+-deprived neurites in the right compartment was typical of neurites on all tracks regenerated in a locally Ca*+-deprived environment. Figure 3 shows higher resolution photomicrographs of Ca*+-supplied and Ca*+-deprived neurites from a similar experiment in which the morphological differences show very clearly. l-he distance that neurites extended along each track was measured with a digital stage micrometer (see Experimental Procedures) on day 6.7 after neuritotomy. At this time, neurites extended 4.83 mm (SEM = 0.15 mm, n = 30) in Ca*+-supplied compartments and 3.78 mm (SEM = 0.10 mm, n = 30) in Ca*+-deprived compartments, a reduction of 22%. Whether neurites were of obviously greater, obviously lesser, or roughly equal density left versus right was determined visually for each track (see Experimental Procedures). On day 6.7 only 3% of tracks displayed neurites more dense in Ca*+-supplied compartments; 53% displayed neurites more dense in Ca*+-deprived compartments. Thus, it was evident that Ca2+-deprived neurites were not reduced in density compared with Ca2+-supplied neurites, and the former tended to be somewhat increased in density. The above result was replicated in 5 cultures and showed again that Ca*+-deprived neurites extended at a slower rate. Cultures were neuritotomized on day 6.8,

and 3 cultures were given Ca*“-deprived medium (0.5 mM EGTA) in right compartments and Ca*+-supplied medium in left and center compartments. Neurite extension is plotted in Figure 4a. Linear regression of data collected during the 3.2 days after neuritotomy showed that Ca*+-supplied neurites extended at a rate of 1.20 mm/day, whereas Ca *+-deprived neurites extended at 0.77 mm/day, 2 36% reduction. At 3.2 days after neuritotomy, 59% of the 37 tracks observed were judged of equal density left and right, 27% were more dense in the Ca*+-deprived compartments, and 74% were more dense in the Ca2+-supplied compartments. To determine whether elevating the EGTA concentration TO-fold might increase the effect of Ca*+ deprivation, the remaining 2 cultures were given Caz+-deprived medium with 5.0 mM EGTA in right compartments and Ca2+-supplied medium in left and center compartments (Figure 4b). The results were virtually identical, with Ca*+-supplied neurites extending at a rate of 1.14 mm/day and Ca*+-deprived ones extending at 0.78 mm/day, a reduction of 32%. At 3.3 days after neuritotomy, 45% of the 25 tracks observed were of equal density left and right, 36% were more dense in Ca*+-deprived compartments, and 7% were more dense in Ca2f-supplied compartments.

Neurites Grow Readily from a Ca*+-Supplied Center Compartment into a Ca2+-Deprived Side Compartment The ability of sympathetic neurites to cross into Ca*+deprived compartments was tested in 53 tracks in 4 cultures. In this experiment the compartmented dishes were initially set up with Ca*+-supplied medium in left compartments and Ca’+-deprived medium (0.5 mM EGTA) in right compartments. Neurons were then plated

Nerve 735

Figure

Fiber Growth

without

2. Phase-Contrast

Ca2+

Photomicrographs

of Rat Sympathetic

Neurons

on a Single Track

Regenerating

after

Neuritotomy

The panels are 1.5 mm long, and the scratches in the substratum that form the borders of the track are visible along the top and bottom of each panel. This culture had been maintained with Ca*+-supplied medium in all compartments until day 11.9 in culture, when neuritotomy was performed. The photomicrographs were taken 3.8 days later. (a) shows neurites that regenerated within the left compartment, which was given Ca%upplied medium after neuritotomy. The silicone grease is visible as the dark right edge of the panel. (b) shows the cell bodies clustered in the center compartment, which was also given Ca 2+-supplied medium after neuritotomy. The silicone grease is visible at the left and right edges. (c) shows neurites that regenerated within the right compartment, which was given Ca*+-deprived medium (0.5 mM ECTA) after neuritotomy. The silicone grease is visible at the left edge.

I I I

Figure 3. Phase-Contrast Photomicrographs of Neurites of Rat Sympathetic Neurons on a Single Track Regenerating after Neuritotomy This culture had been maintained with CaZ+supplied medium in all compartments until day 14.9 in culture, when neuritotomy was performed. The photomicrographs were taken 2.0 days later. The panels are 640 pm long, and the scratches in the substratum that form the borders of the track are visible along the top and bottom of each panel. (a) shows neurites that regenerated within the left compartment, which was given Ca*+supplied medium after neuritotomy. The silicone grease is visible at the right edge of the

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(a) Neurite extension on 37 tracks in 3 cultures after neuritotomy on day 6.8 in culture is plotted. After neuritotomy, cultures were given medium supplied with 1 mM Ca l+ in left and center compartments and Ca*+-deprived medium (0.5 mM EGTA) in right compartments. The SEM (based on the number of tracks) was within the symbols in all cases. (b) Mean neurite extension on 25 tracks in 2 cultures treated identically to those in (a), except that after neuritotomy, right compartments were supplied with Ca*+-deprived medium containing 5 mM ECTA. Again, the SEM (based on the number of tracks) was within the symbols in all cases. (c) Percentage of tracks with neurites is plotted for two groups of cultures in which the center compartments were changed to Ca*+-deprived medium (0.5 mM ECTA) on day 0.9. Filled squares represent combined data from 49 tracks in left and right compartments of 3 cultures also given Caz+-deprived medium (0.5 mM ECTA) in left and right compartments on day 0.9. Open squares are combined data from 33 tracks in left and right compartments of 2 cultures given Ca2+- supplied medium in left and right compartments. (d) Percentage of tracks with neurites is plotted for 67 tracks in 4 cultures in which only right compartments were changed to Ca 2+-deprived medium (0.5 mM EGTA) on day 0.9 in culture. Left and center compartments were given Ca 2+-supplied medium. Open squares represent Caz+-supplied compartments, and filled squares represent @+-deprived compartments.

Cal+-supplied center compartments. On day 5.0, of the tracks contained neurites in left compartments and 94% contained neurites in right compartments. At this time, neurites had extended an average of 1.89 mm (SEM = 0.11, n = 50) into Ca2+-deprived compartments, 34% shorter than the 2.91 mm (SEM = 0.15, n = 49) that neurites extended into Cal+-supplied compartments. in the

92%

Similar results were obtained in another experiment involving the collection of a large amount of medium for atomic emission analysis of its total Ca2+ content (see

below). Thirtyone cultures were constructed with Ca’+deprived medium (5 mM EGTA) in right compartments and Ca2+-supplied medium in left compartments. On day 6 neurites were measured in all of the cultures. Neurites extended into Ca2+-supplied compartments on 98% of the 533 tracks, and they extended into Ca2’-deprived compartments on 97% of the tracks. The mean neurite extension in individual cultures ranged from 1.36 to 4.03 mm in Ca2+-supplied compartments and from 1.09 to 2.92 mm in Ca2+-deprived compartments. These experiments show that during the first few days

Nerve 737

Fiber Growth

without

Ca”

in culture, growth cones of sympathetic neurons from newborn rats readily crossed from a Ca*+-supplied environment into a Caz+-deprived environment. Once within the Ca*+-deprived compartment, neurites continued to grow, although they reached somewhat shorter distances along the tracks than their counterparts in opposite compartments supplied with Ca2+. Extracellular Ca*+ Is Required at the Cell Bodies for Growth and Survival of Sympathetic Neurites Three cultures were used to determine the effect on neurite growth of supplying only Ca2+-deprived medium in all three compartments. Ca*+-supplied medium had been initially provided in all compartments, but on day 0.9 the cultures were changed to Ca2+-deprived medium throughout (0.5 mM EGTA). They were examined periodically to determine what percentage of the 49 tracks in the left and right compartments contained neurites. This reached 39% on day 2.8, but decreased to 15% by day 6.7, as the neurites degenerated (Figure 4~). Another group of sister cultures were also given Caz+-deprived medium in center compartments on day 0.9, but left and right compartments were given Ca2+-supplied medium. The pattern of response was similar, with neurites present on 56% of tracks on day 2.9 and completely absent by day 6.7 (Figure 4~). Thus, the availability of Ca*+supplied medium to the neurites in the side compartments did not support their survival when the cell bodies and proximal neurites were deprived of Ca*+. A group of 4 sister cultures served as controls in this experiment. As above, Ca2+-supplied medium was initially provided in all compartments. But on day 0.9 after plating, only right compartments were changed to Ca2+-deprived medium (0.5 mM EGTA). Ca2+-supplied medium was continuously provided to left and center compartments. Sixty-seven tracks were observed, and neurites entered Caz+-deprived compartments just as readily as Ca*+-supplied compartments. By day 2.9 in culture, neurites were present on 97% of tracks in Ca*+deprived compartments and on 96% of tracks in Ca*+supplied compartments (Figure 4d). The depression of neurite growth and survival that resulted when Ca*+-deprived medium was provided to center compartments of newly plated neurons was also observed in older neurons. After maintenance for 11.9 days with Ca*+-supplied medium in all compartments, 2 cultures were neuritotomized left and right and given Ca*+-deprived medium in all compartments. One day later regenerating neurites were present in both left and right compartments on all but 2 of the 30 tracks, but neurites never extended very far, averaging just over 1 mm by 2.7 days after neuritotomy and then declining (Figure 5a). Debris indicative of neurite degeneration was noted on the left and right in all tracks in these cultures by day 9.7 after neuritotomy. A similar pattern was observed in 2 sister cultures with Ca2+-deprived cell bodies and proximal neurites, but given Ca2+-supplied medium in left and right compartments (Figure 5a). These regenerated to a distance twice as far as neurites in cultures deprived of Ca*+ in all

compartments, and they declined relatively less. Debris indicative of neurite degeneration was present on the left and right in all 30 tracks in these cultures by day 9.7 after neuritotomy. Thus, although provision of Ca*+-supplied medium to distal regenerating neurites ameliorated to some extent the effect of Ca2+-deprived medium supplied to cell bodies and proximal neurites, it did not completely prevent the degeneration of the distal neurites. To control for nonspecific effects of the 0.5 mM EGTA present in Ca*+-deprived medium, another experiment was performed with 4 cultures. All cultures were neuritotomized on day 7.0; 2 cultures were given Ca*+-deprived medium in all three compartments; and 2 were given medium with 2 mM Ca2+ and 0.5 mM EGTA in all three compartments, i.e., enough Ca2+ to bind all of the EGTA at 211 stoichiometry and leave at least 1 mM free Ca2+. As in the experiments described above, neurites in Ca*+-deprived cultures initially regenerated slightly and then substantially degenerated (Figure 5b). In the cultures given 2 mM Ca*+ and 0.5 mM EGTA regeneration appeared normal. Thus, any nonspecific effects of EGTA could not account for the depressive effects observed when Ca*+-deprived medium was supplied to the cell bodies and proximal neurites. In all of the cultures in which cell bodies were deprived of Ca2+, the neurons appeared to have begun degenerating by the end of the observations. Therefore, to test the effects of Ca2+-deprived medium on cell survival while better visualizing the cell bodies, noncompartmented cultures were set out in 24 well plates. On day 5.9, 3 cultures were given Ca*+-deprived medium (0.5 mM EGTA), and 3 cultures were given Ca*+-supplied medium. To control for nonspecific effects of EGTA, another group of 3 cultures was given medium containing 2 mM Ca*+ and 0.5 mM EGTA. Ten days later neurites of the Ca*+-deprived neurons appeared disintegrated, and the cell bodies were shrunken and indistinct (Figure 6a), whereas both the Ca2+-supplied cultures (Figure 6b) and the EGTA control cultures (Figure 6c) contained healthy looking neurons showing no signs of degeneration. These results suggest that the cessation of neurite growth and neurite degeneration observed when cell bodies and proximal neurites were deprived of extracellular Ca*+ reflect a general deterioration of the neurons. Measurements of Extracellular Ca2+ in Side Compartments after Neurite Growth To determine what levels of free Ca*+ were actually present during the growth of neurites in Ca*+-deprived compartments, total Ca*+ was measured using atomic emission spectroscopy. To obtain enough medium, 31 culture dishes were constructed with Ca*+-supplied medium in left and center compartments and Ca*+-deprived medium (5 mM EGTA) in right compartments. On day 6 neurites extended in Caz+-supplied compartments and Ca*+deprived compartments on virtually all of the tracks in all of the cultures (data presented above). Medium was harvested from the cultures on day 6.0

a.

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mM EGTA

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Figure 6. Phase-Contrast Photomicrographs of Rat Sympathetic Neurons in 24 Well Plates on Day 15.7 in Culture

01

2 Days

34 since

Figure 5. Effects of Ca 2f Deprivation Regeneration

5

On day 5.9 the cultures were changed from Ca+.upplied medium to either (a) Ca2’-deprived medium (0.5 mM ECTA), (b) 2 mM Ca*+ (0.5 mM ECTA), or (c) controls retained at 1 mM Ca*+ (no EGTA). The panels are 415 pm long.

6

neuritotomy of the Cell Bodies

on Neurite

(a) Neurons were in culture 11.9 days with Ca*+-supplied medium in all compartments. They were then neuritotomized, and 2 cultures (30 tracks) were given Ca Z+-deprived medium (0.5 mM EGTA) in all compartments (filled squares). Two other cultures (30 tracks) were also given Ca2+-deprived medium (0.5 mM ECTA) in center compartments, but were given Ca%upplied medium in left and right compartments. Unless shown, error bars fall within the symbols. fb) Control for possible nonspecific effects of EGTA on neurite extension. Neurons were in culture 7.0 days with Ca2’-supplied medium in all compartments. They were then neuritotomized. Filled squares represent combined left and right data (n = 58) from medium (0.5 mM ECTA) in all three 2 cultures given Ca 2+-deprived compartments after neuritotomy. Open squares represent combined left and right data (n = 70) from 2 cultures given medium with 2 mM Ca*+ and 0.5 mM EGTA in all three compartments. Error bars fall within the symbols in all cases.

and replaced with fresh medium. Atomic emission of the harvested medium from the Ca2+-deprived compartments detected 22 uM total Ca2+. On day 9.9 medium was harvested from the cultures again, and the total Ca2+ in the medium from Ca2+-deprived compartments was 15 PM. Ca2+ was measured in 2 replicate samples of fresh, Ca*+-deprived medium from the same batch as that given to the cultures, and the values obtained were 10 uM and 12 PM. These results suggest that during the first 6 days, Ca2+ accumulated in the Ca2+-deprived compartments, increasing the concentration by about 1.7 PM/day. During days 6.0-9.9, the concentration in-

creased by about 1.4 PM/day. The 20% difference between these values is probably within experimental error. To permit evaluation of the accuracy of the Ca2+ measurements, serial dilutions had been made, one with Ca2+ added to culture medium and one with Ca2+ added to water. These were measured concurrently with the experimental samples and provided two calibration curves (Figure 7). In water the measured Ca2+ accurately reflected the amount added. With Ca2+ of 1 uM or less added to culture medium, measured values were consistently in the lo-15 uM range, thus confirming this as about the amount of Ca*+ contained in fresh, Ca*+-deprived medium. At 10 uM added Ca*‘, values in 2 replicate samples were 20 PM and 25 PM, about the same as that obtained for the medium in which neurites had grown during the first 6 days. The only departure from expectation occurred at 1 mM added Ca2+, at which the measured value in medium was consistently about half the added Ca2’. This did not occur with 1 mM Ca2+ added to water and could have resulted because the medium was thickened with methylcellulose and a very small volume was aspirated to measure this relatively high Ca*+ concentration. A relatively large dead volume might have occurred and resulted in an underestimate. In conclusion, the measurements of total Ca2+ by atomic emission spectra clearly indicated that the fresh Ca2+deprived medium contained about lo-12 ELM Ca2+.

7~3’9”’ Fiber Growth

without

min

6”’

water

q in culture

medium

deprivation was obtained in experiments showing that when Ca*+-deprived medium (0.5 mM EGTA) was supplied to the cell body-containing compartment, neurite growth ceased in the side compartments after a few days and the neurites degenerated even when the side compartments were given Ca*+-supplied medium (see above).

Discussion Influx of Extracellular Cone Is Not Required

-‘. .Ol

.l

Added

1

Ca”

10

100

1000

(PM)

Figure 7. Calibration Curves Relating Known Amounts of Ca” Added to Culture Medium or Water and Total Ca2+ Measured by Atomic Emission Spectroscopy Filled squares are values obtained with CaC12 in glass-distilled, Milli-Q filtered water and show a reliable calibration throughout the range tested. Open squares are values obtained with CaCI, added to culture medium. In the low range measured values level off at about 10 PM, indicating this as the amount of Ca*+ present in freshly prepared, Ca*+-deprived medium. The measured value for 1000 uM added Ca2+ is low (note log-log scale), an artifact that may have resulted from the high viscosity of the methylcellulosecontaining medium.

When Ca*+-deprived medium was incubated in side compartments, enough Ca2+ was transferred from center compartments during 6 days to raise the Ca2+ concentration to about 22 uM. The source of the Ca*+ added to right compartments was not the collagen substratum. Two samples of collagen solution from the same batch used to make the substratum of these cultures yielded values of 1.8 and 2.3 uM Ca*+. Taking into account the volume used per culture and the surface area of the side compartment, the Ca2+ concentration of medium would have increased less than 0.03 PM if all of the Ca*+ in the substratum were released into it. Thus the source of the Ca*+ appearing in right compartments must have been leakage or diffusion across the barrier from center compartments or release from the neurites. Using 22 PM Ca*+ as an estimate of the highest concentration occurring in the Cal+-deprived medium during an experiment, the free Ca*+ at pH 7 was calculated from the concentration of EGTA and the dissociation constant (4.9 x lo-b), using custom software on a Macintosh personal computer. In medium with 0.5 mM EGTA, the calculated maximum free Ca2+ was 9.4 nM, and in medium with 5 mM EGTA, the calculated maximum free Ca2+ was 0.90 nM. Thus at the EGTA concentrations used, the extracellular Ca*+ was 1 or 2 orders of magnitude below typical values of intracellular Ca*+. Biological confirmation of the effectiveness of Ca*+

CaZ+ locally at the Growth for Neurite Growth

Recent studies suggest a role for Ca2+ in controlling nerve fiber growth. Furaimaging in cultured embryonic neurons from the rat diencephalon have shown intracellular free Ca*+ to be elevated into the 200-1000 nM range in spontaneously motile growth cones compared with 3080 nM in nonmotile growth cones and cell bodies (Connor, 1986). Spontaneously motile growth cones of neurons from the snail, Helisoma, also showed increased intracellular free Ca2+ compared with nonmotile growth cones (Cohan et al., 1987). Since the application of Ca*+ channel blocking ions (10m4 M l-as+, TO-* M Co*+, and 10e4 M Cd*+) to Helisoma neurons caused a reduction in neurite growth, a role for Ca*+ influx in regulating neurite growth has been suggested (Mattson and Kater, 1987). However, in several experimental systems,. treatments known to cause Ca2+ influx did not increase neurite growth, but depressed it. Dendritic growth was depressed in hippocampal pyramidal neurons in response to glutamate, to Ca2+ ionophore A23187, and to elevated K+ (Mattson et al., 1988a); in certain Helisoma neurons serotonin and electrical activity raised intracellular free Ca2+ in growth cones into the 300-500 nM range, but depressed neurite elongation (Haydon et al., 1984; Cohan and Kater, 1986; Cohan et al., 1987); and exposure to Ca*+ ionophore A23187 also depressed neurite growth in Helisoma neurons (Mattson and Kater, 1987). Experiments that specifically address whether or not the effects of Ca*+ influx are exerted locally at the growth cone have also been reported: Ca*+ ionophore A23187 was applied topically via a micropipette to growth cones of hippocampal neurons (Mattson et al., 1988b) or was bath-applied to Helisoma growth cones that had been excised from the parent neurite (Mattson and Kater, 1987). In both cases growth ceased, suggesting that the depressive effects of Ca*+ resulted from its influx into the growth cone. These experiments raise a problem because some results suggest that Ca*+ influx promotes neurite growth and others suggest that it depresses growth. An explanation offered to resolve the apparent contradiction is that a neuronal growth cone might grow optimally at a certain intracellular Ca2+ concentration, or set point, and at Ca*+ concentrations above or below this optimum, growth might be diminished or cease altogether (Mattson and Kater, 1987; Kater et al., 1988). It is interesting in this connection that in intact Helisoma neurons and in excised growth cones, the Ca*+ channel blocking ions La3+, Co*+, and Cd*+ were reported to increase

neurite elongation when applied at concentrations about an order of magnitude lower than the concentrations that suppressed neurite growth (Mattson and Kater, 1987). This could have resulted from a partial block of Caz+ influx, possibly bringing intracellular Ca’+ down from a supraoptimal concentration closer to the hypothetical set point (Mattson and Kater, 1987). Consistent with this hypothesis was the observation that neurite growth in Helisoma neurons also increased in response to a reduction in the concentration of Caz+ supplied in the culture medium (Mattson and Kater, 1987). Interestingly, results of the present experiments have shown that although distal neurites grew long distances for many days when locally deprived of Ca2+, deprivation of Ca2+ in the ceil body-containing compartment led to cessation of neurite growth and the disintegration of neurites. This suggests, not surprisingly, that Ca2+ deprivation can affect the neuron through multiple mechanisms, in this case localized in different neuronal regions. But since there are so many mechanisms that can mediate the effects of Ca2+, it is likely that several mechanisms are present together in any given region of the neuron. Thus, we think that if there is an optimal intracellular Ca2+ concentration for growth, this reflects a balance between many mechanisms with diverse functions affecting neurite growth and survival, rather than a single or even a few mechanisms functioning primarily in growth control. Therefore, although the concept of a Ca2+ set point could be meaningful in the context of a specified biochemical mechanism, as a phenomenological description, it may simply indicate that to function reasonably well, the growth cone must have an intracellular Caz+ concentration within certain limits. Above or below these limits, various functions could break down, resulting in nonspecific deleterious effects upon growth and survival. Examples of possible mechanisms by which Ca2+ might influence neuronal growth include Ca2+-dependent phosphorylation mediated by protein kinase C (Katz et al., 1985; Meiri et al., 1986; Hall et al., 1988), calmodulinmediated phosphorylation (Katz et al., 1985), calmodulin-mediated actin and myosin binding (Adelstein, 1982), Ca2f effects upon microtubules (Schliwa et al., 1981) and microfilaments (Adelstein and Eisenberg, 19801, and Ca2+-dependent fusion of synaptic vesicles with the surface membrane (Katz and Miledi, 1965, 1967). Any of these actions of Ca2+, or actions yet undiscovered, could play a role in the effects of Ca2+ on nerve growth, and a mechanism relevant during one time in development or relevant to one type of growth cone could possibly be irrelevant during a different time or to another type of growth cone. The fact that cytoplasmic Ca2+ can originate either from the extracellular environment or from intracellular storage organelles (see Carafoli, 1987; Volpe et al., 1988) adds another source for possible variation between growth cone types. A few studies have indicated that, at least in the short term, neurite growth from a variety of neuron types can occur with Ca2+ substantially depleted from the extracellular environment. Dissociated embryonic frog spi-

nal neurons cultured in Ca*+-deprived medium (1 mM EGTA) for up to 22 hr still produced neurites, and the rate of neurite elongation was about 3 times that of controls (Bixby and Spitzer, 1984). Although Helisoma neurons displayed a 68% reduction in elongation rate in response to Ca*+-deprived medium (1 mM EGTA), the elongation did not cease (Mattson and Kater, 1987). The present results are consistent with these reports of a lack of requirement for extracellular Ca2+ for neurite growth. Our experiments have clearly shown that local extracellular Ca2+ is not required for sustained neurite growth and survival. Although no studies demonstrating Ca2+ channels in the growth cones of rat sympathetic neurons have been reported, they presumably contain them, since their cell bodies have been shown to possess L-type and N-type Ca 2+ channels (Hirning et al., l988), and Ca2+ channels have been demonstrated in mouse neuroblastoma cell growth cones (Anglister et al., 1982) and in growth cones of frog sympathetic neurons (Lipscombe et al., 1988). In our experiments the extracellular free Ca2’ concentration in distal compartments containing elongating neurites was maintained at about l-10 nM, which is most likely 1 or 2 orders of magnitude below the concentration inside the growth cone. With the extracellular Ca2+ concentration so low, the influx of Ca2+ through Ca2+ channels in the growth cone could not have been sufficient to influence neurite growth. Thus the present results indicate that influx of Ca2+ is not required for growth cones to engage in sustained locomotory behavior or for sustained neurite elongation. In this connection it has recently been suggested that Ca2+ influx regulates neurite growth in cultured chick dorsai root ganglion neurons by controlling the assembly and disassembly of actin filaments and microtubules (Lankford and Letourneau, 1989). The present results clearly show that in rat sympathetic neurons, sustained neurite growth can occur without regulation by Ca2+ influx. Studies of the role of extracellular Ca2+ in the formation of veils in Aplysia growth cones has shown that veil formation ceased in response to short-term extracelluiar Ca2+ depletion and resumed upon local micropipette application of medium supplied with Ca*” (Goldberg, 1988). This indicates that extracellular Ca2” is required for veil formation at the leading edges of Aplysia growth cones. Although this might appear to conflict with OUI results, growth data were not presented and evidence from other syst.ems suggests that neurite elongation can continue under conditions that suppress formation of fiiopodia and lamellipodia (Marsh and Letourneau, 7984; Letourneau, 1985). Also, Helisorna growth cones showed a marked reduction in both filopodial numbers and lamellipodial area in response to low concentrations of Ca2+ channel blocking ions (e.g., 10-s M !anthanum), whereas neurite eiongation actually increased (Mattson and Kater, 1987). Thus, although observations suggest that in the Aplysia neurons veil membrane passes back over the growth cone to form the axonal membrane (Goldberg and Burmeister, 1986), the evidence presented does not rule out the possibility that under Ca*+-

Nerve 741

Fiber Growth

without

Ca*’

deprived conditions, membrane Aplysia neurites via a different their growth to continue.

might route,

be supplied thus permitting

to

Fusion of Vesicles Supplying Surface Membrane during Neurite Growth May Not Require the Influx of Ca2+ into the Neurite The expansion of surface membrane that occurs during nerve fiber elongation is presumably supplied by fusion of intracellular vesicles with the surface membrane (see Pfenninger, 1982). Since vesicle fusion also occurs at nerve terminals during synaptic transmission, where it is triggered by the influx of Ca*+ (Katz and Miledi, 1965, 1967), it is reasonable to hypothesize that fusion of vesicles supplying surface membrane during growth might occur at the growth cone and also require the influx of extracellular Ca*+. Supporting this contention are results from several experimental systems suggesting that surface membrane is inserted at or near the growth cone (Bray, 1970; Koda and Partlow, 1976; Pfenninger and Maylie-Pfenninger, 1981; Griffin et al., 1981). However, in the present experiments distal neurites grew long distances within Ca*+-deprived compartments provided Ca*+ was supplied in the cell body-containing compartment. This means that either the fusion of vesicles supplying their membrane occurred without local Ca2+ influx or that vesicle fusion did not occur at or near the growth cone, but occurred proximally in the Caz+-supplied center compartment. The present results favor the hypothesis that fusion of vesicles can occur without Ca2+ influx, since neurites elongated for a few days after all compartments were given Ca2+-deprived medium. Whether Ca2+ influx can facilitate membrane expansion and where the membrane expansion actually occurs cannot be decided from the present results. The data do not rule out the possibility that vesicle fusion occurring in one locality could supply membrane for neurite growth at a remote site.

NGF Does Not Promote Causing Ca*+ Influx

Neurite

Growth

by

It was previously observed that neurites of newborn rat sympathetic neurons were virtually unable to grow from an NGFsupplied center compartment into a side compartment to which no NGF had been added (Campenot, 1977). The present experiments show that when all compartments are supplied with NGF, sympathetic neurites compartment into a readily cross from a Ca 2+-supplied Ca*+-deprived one. Had a hypothetical Ca*+ influx in response to NGF been responsible for neurite growth, neurites would have been excluded from growing into Ca2+-deprived compartments. Previous experiments have also shown that the density of neurites regenerating from rat sympathetic neurons is highly dependent on the local NGF concentration. Neurites in side compartments supplied with relatively low concentrations of NGF showed a consistent and marked reduction in density compared with opposite side controls given relatively high concentrations of NGF (Campenot, 1982). The present experiments show that neurites readily re-

generated after neuritotomy in Caz+-deprived side compartments and displayed equivalent or increased density compared with neurites in opposite side compartments given Ca*+-supplied medium. Had a hypothetical Ca2+ influx been responsible for the promotion of neurite growth by NGF, neurite density would have been substantially reduced in the Ca2+-deprived compartments. Thus, the present experiments provide strong evidence that influx of Ca*+ at or near the growth cone is not a necessary step in the NGF in rat sympathetic

promotion neurons.

of neurite

Ca2+ Is Not Required to the Substratum

for Adhesion

growth

of Growth

by

Cones

Ca2+ is a well-known mediater of cell-cell adhesion (Takeichi, 1988); in the case of neurons, this occurs via a cell adhesion molecule, N-cadherin (Tomaselli et al., 1988). A possible role of divalent cations in adhesion of neurites to laminin has also been indicated (Ignatius and Reichardt, 1988). Although results suggest that in the present experiments neurite growth occurred without extracellular Ca*+ mediating the adhesion of neurites or their growth cones to the collagen substratum, the possibility that Ca*+-dependent adhesion molecules may have functioned through binding of Mg*+, present in the medium at 1.46 mM, cannot be ruled out. Although rat sympathetic neurites grew well in Ca*+deprived medium, there were differences between Ca*+deprived and Ca*+-supplied neurites. Ca*+-deprived neurites elongated at rates generally 20%-35% less than those of Ca*+-supplied neurites and displayed a finer filamentous appearance interrupted by numerous varicosities. It is not known whether this resulted from intracellular or extracellular effects of Ca2+ deprivation, but in our experience, this appearance is frequently associated with culture conditions that produce better adhesion. For example, the use of laminin instead of collagen as culture substratum produces a finer filamentous appearance (unpublished data). It is possible that the finer filaments may have resulted in part from reduced fasciculation. This could indicate the absence of Ca*+mediated adhesion between neurites. We have no information that leads to speculation of how local Ca*+ deficiency produced more varicose neurites.

Possible Role of Extracellular of Nerve Growth

Ca*+ in the Promotion

A remarkable feature of the present experiments little effect local Ca2+ deprivation had on neurite

is how growth.

Nerve fibers from cultured rat sympathetic neurons elongated long distances over many days wn compartments locally deprived of Ca2+. These experiments do not rule out the possibility that cytoplasmic Ca*+ plays a role in nerve growth, but they do indicate that any Ca*+-dependent growth mechanisms operative within the rat sympathetic growth cones and distal neurites must have been supplied with Ca*+ from internal stores, rather than from influx across the plasma membrane. It remains possible that in other systems Ca*+ influx into growth cones is a more important regulator of growth.

It is in fact possible that the source of cytoplasmic Ca2+ to regulate growth could be the local extracellular environment of the growth cone in the case of some types of neurons, intracellular stores in the case of other types, and some combination of the two in yet other types of neurons. Indeed it is even possible that neurons could shift between regulation by extracellular Ca2+ and regulation by intracellular stores under different conditions or at different times in their developmental histories.

Experimental

side compartmeni and recording whether the neurites emerging into the left compartment were obviously more dense or less dense than the neurites in the right compartment. When densities were not obviously different they were recorded as equal. Although only quasi-quantitative, this track by track record of comparative density can reliably detect large and consistent neurite density differences when they OCCIII between compartments (see Campenot, 1982). The neurite extension measurements and density estimates required about 15 min per culture. Ca2+ in culture medium was measured by atomic emission spectroscopy trometer. provided

using a Leco Plasmarray inductively coupled plasma specThe Chemistry Department of the University of Alberta this as a service.

Procedures

Principal neurons from superior cervical ganglia of newborn rats (Sprague-Dawleys supplied by the University of Alberta Farm) were dissociated and plated into three-compartmented culture dishes at a density of about 2 ganglia per dish. The general culturing procedures and formulations were as previously reported (Campenot, 19821, essentially following those described by Hawrot and Patterson (1979). Briefly, cultures were maintained in a 5% COz atmosphere at 37°C. L15 medium without antrbiotics (GIBCO Laboratories, Grand Island, additives including

NY) was bicarbonate

supplemented with and methylcellulose.

the prescribed This medium

contains 1 mM Ca’+ and was used for general culture maintenance. Ca*+-deprived medium was L15 medium made from scratch accordrng to the recipe supplied by ClJ3C0, but with Cal+ omitted. Ingredients were Sigma culture grade when available. For Ca%upplied medium, we used either this medium with CaCI, added to achieve a 1 mM final concentration, or the CIBCO medium. Ca’+-deprived medium used in the experiments had either 0.5 mM EGTA or 5 mM ECTA added. Rat serum (2.5%) prepared in the laboratory and ascorbic acid (1 mgiml) were supplied only in medium given to center compartments that contained the cell bodies. Unless otherwise indicated, medium given to all compartments included 1 pg of 75 NGFiml (see Chun and Patterson, 19771, generously supplred by Dr. Richard Murphy. Medium bathing the cultures was routinely changed every 3-6 days. Nonneuronal cells were virtually eliminated by an initial exposure to medium containing 10 PM cytosine arabinoside. Collagen extracted from rat tail tendons was dried onto tissue culture dishes to provide a culture substratum (see Hawrot and Patterson, 1979). For noncompartmented cultures, we used Falcon 24 well tissue culture plates in which the surface area of individual cultures was 2 cm2 and each was supplied with about 0.5 ml of medium. To make compartmented cultures (see Figure l), a series of collagen tracts was formed between parallel scratches made in the floors of 35 mm Falcon tissue culture dishes (Campenot, 1979). A Teflon divider was then seated to the surface as previously described (Campenot, 1979). Neurons were plated by depositing about 100 pl of methylcellulose-thickened culture medium containing a suspension of neurons in the center compartment of each dish by means of a 1 ml disposable syringe with a 1.5 inch, 22 gauge needle. The neurons were allowed to settle onto the substratum overnight, and the next day the outer perimeter of the dish was given about 1.5 ml of medium. The neurons came to rest on the substratum of collagen tracks, and neurites elongated on these tracks, crossing under the silicone grease barriers to enter left and right compartments. in some experiments, cuitures were neuritotomized and regeneration was observed. Neuritotomy was performed by washing away the neurites rn left and right compartments with a jet of sterile distilled water delivered with a syringe through a 22 gauge needle. The water was aspirated, and the wash was repeated twice. Then fresh culture medium was added. This procedure reliably removed virtually all visible traces of neurites. Neurite extension on individual collagen tracks was measured with a digital micrometer (One Sokki) mounted so as to measure stage movements of the Nikon Diaphot inverted microscope outfitted with phase-contrast optics. Left versus right neurite densities were compared by observing the initial 0.9 mm of the track (one microscopic field at 200x) where the neurites emerge into each

Acknowledgments This work was supported by an establishment grant from the Alberta Heritage Foundation for Medical Research. We thank Rrchard Murphy for supplying the NGF, Robert Swindlehurst for performing the atomic emission spectra analysis of Ca2+, Narcisse Oulette for writing software for calculating the free Ca*’ in ECTA-contaming solutions,

and

Received

March

Gregory

Morrison

10, 1989;

for

revised

photographic

August

work.

3, 1989

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