Local increases in intracellular calcium elicit local filopodial responses in helisoma neuronal growth cages

Neuron,

Vol. 9, 405-416,

September,

1992, Copyright

0 1992 by Cell Press

Local Increases in Intracellular Calcium Elicit local Filopodial Responses in Helisoma Neuronal Growth Cones Roger W. Davenport and Stanley B. Kater Program in Neuronal Growth and Development Department of Anatomy and Neurobiology Colorado State University Fort Collins, Colorado 80523

Summary Highly localized changes in intracellular Ca*+ concentration ([Caz+]J can be evoked in neuronal growth cones; these are followed by local changes in filopodia. Focally applied electric fields evoked spatially restricted, high magnitude increases in growth cone [Ca% The earliest and greatest increases were localized to small regions within a growth cone. Such fields also produced characteristic changes in the disposition of filopodia: both file podial length and number were significantly increased on the cathode side of growth cones. The requirement for extracellular Caz+ and the strong correlation between the evoked rise in [Ca*+]i and the changes in filopodia (r = 0.98) indicate that cathode stimulation results in local Ca*+ influx, leading to locally increased [Ca*+]i and local changes in filopodial behavior. Introduction Neuronal growth cones act as navigators during the pathfinding and target recognition critical for normal neuronal development. This role requires the growth cone to explore its environment in order to direct subsequent outgrowth of the forming axons and dendrites. Intracellular Ca*+ has been implicated as a messenger that can be used by neuronal growth cones to elicit major changes in growth cone morphology, such as growth cone collapse (Cohan and Kater, 1986; Haydon et al., 1984; Bandtlow et al., 1992) and orientation (Gundersen and Barrett, 1980; McCaig, 1989; for review see Kater and Mills, 1991). Additionally, intracellular Ca*+ has been shown to affect more subtle changes in growth cone morphology, such as local lamellipodial extension (Goldberg, 1988) and overall filopodial extension (Rehder and Kater, 1992). In the experiments by Rehder and Kater (1992), filopodial disposition was shown to be directly correlated with measured transient changes in intracellular Ca2+ concentration ([Ca*+]J. [Ca*+]i across the entire growth cone waschanged by elevation of extracellular K+concentration or by application of the Ca2+ ionophore A23187. Both filopodial length and filopodial number throughout the growth cone were strongly dependent on the magnitude of the induced Ca*+ transient. The net effect of the transient filopodial elongation seen in these experiments was the ability to contact localized environmental signals hours in advance of a control growth cone extending at a normal rate. Since filopodia have been regarded as potential environmental

sensors

for

the

growth

cone,

this

transient

elongation might strongly affect subsequent growth cone behaviors. Experimentally, global changes in neuronal [Ca*+], and morphology are reproduced readily. It seems reasonable, however, that in the normal function of the growth cone in vivo, more subtle growth cone behaviors occur. For example, the importance of single filopodia to the overall neuronal development of the grasshopper limb bud suggests a critical role for local changes in growth cone behavior. Within the grasshopper limb bud, individual filopodia can direct outgrowth of the pioneer neurons by singularly contacting guidance cues and initiating changes that completely alter the path of outgrowth (O’Connor et al., 1990). Bandtlow et al. (1990) describe another scenario in which individual filopodia strongly affect the status of the entire growth cone. Contact between a single filopodium of a dorsal root ganglion neuron and an oligodendrocyte can cause the collapse of the entire neuronal growth cone. These examples suggest that local growth cone behaviors, such as selective filopodial extension, can directly affect the final morphology of individual neurons and ultimately the resultant neuronal circuit. The present investigation asks whether neuronal growth cones from Helisoma, previously studied in the context of global changes in [Ca*+]i and growth cone behavior, can display local changes in [Ca*+]i and whether such local changes also result in local changes in filopodial disposition. To investigate directly a potential role for local [Ca*+]i in the control of filopodia in restricted regions of the growth cone, this investigation takes advantage of the high degree of experimental tractability afforded by a focal electric field stimulation paradigm. The simplicity of the experimental set up and the ability to quantify stimulus strength and to localize a stimulus spatially and temporally to discrete regions of the growth cone make this paradigm unique. With electric fields focally applied to the growth cones of buccal ganglion neurons from Helisoma, we demonstrate two highly localized responses: a rise in [Ca*]i that begins in a limited region of the growth cone nearest the cathode and then spreads across the growth cone, and the generation and elongation of filopodia on the side of the growth cone nearest the cathode, which coincides with the area of the initial increase in [Ca*+]i. Both responses are correlated with the magnitude of electric field and can be blocked by preventing influx of Ca*+. The time course, localization, strong correlation, and influx dependency of these responses suggest a regulatory roleof [Ca2+l, for local changes in growth cone morphology. Results Ca*+ Changes localized Growth Cones The production of an

within electric

Individual field

with

the

cathode

Neuron 406

Figure

1. Changes

in [Ca”],

in Helisoma

Neuronal

Growth

Cones

Evoked

by Focally

Applied

Electric

Fields

(A) The effect of electrode location and thus electric stimulation magnitude on changes in [Ca2+],. (A) The cathode source was located approximately equidistant from each growth cone, thus creating suprathreshold stimulation strengths in both (8.4 and 9.3 mV/um, left and right growth cones, respectively). A large increase in [CaZ+], occurred in both growth cones. (A’) The cathode source was placed closer to one growth cone, thereby creating unequal stimulation (11.2 and 4.1 mV/Pm). The induced [Ca2’], changes were well localized. (B) Time course of [Caz’], changes associated with a focally applied electric field. The cathode source (8.0 mV/gm) was located to the right of this growth cone at a distance of approximately 25 Pm. Note the homogeneous and low [Ca*‘], 1 min prior to (Pre) and 1 min after (Post) stimulation. The rise in [Ca”], occurred locally first, then spread across the growth cone. Note the transient peak (
Local Rise in [Cal] 407

A

Alters

C

s c 1500. F h 1000.

A B C

500. O-

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Elrctrk

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Bll

Filopodia

A B

2000,

a 6 E 9

Locally

0.0

1.0

2.0 3.0 Tlma (sac)

Fkld 4.0

5.0

6.0

600

b --~---‘--------------------^I

h

Ekctrlc Fkld

-i

0

;

10 1’5 Tim* (mln)

20

m

30

C 600 600 2 *a 01 200

,/F-

4-7400 I 0

2

Figure 2. Time Course Focal Electric Fields

4 Ekctrk

and

6 6 Fkld Magnltuda (mV/pm)

Magnitude

10

of [Ca2+], Responses

12

to

(A) The rapid increase in [Cap], and the subsequent development of an [Ca2+], gradient is shown. Plotted is the estimated [Ca2+], from the ratioed, fluorescent emission (f&f; 380 nm excitation) at three distinct locations (4.3 wrn apart) for the growth cone on the left in Figure IA’ (inset). The rate of rise was greatest at the site closest to the cathode. This gradient was maintained for x 5. (B) The long-term changes in [Caz’], (measured at the center of each growth cone; n = 36) associated with constant, sustained focal electric field stimulation. Estimated [Cap], (dual wavelength determination) increased rapidly to a peak value near micromolar concentrations within 30 s. From this peak, estimated [Ca”], decreased to a plateau level that remained significantly elevated (approximately Zfold; p < 0.001) for as long as the electric field was maintained. Growth cone [Ca2+]i returned to control values within 2 min following removal of the stimulus (p > 0.25). No significant rise in [Caz+l, occurred upon application of electric fields in Ca*+-free medium (dashed line; n = 6, p > 0.4). (C)The average peak rise in [Cati], is plotted against the applied field strength, over a 5-fold range of field strengths. A simple, linear fit to the data shows that a strong statistical correlation exists (r = 0.99).

source near fura-Zloaded growth cones produced an abrupt and dramatic increase in [Ca*+]i (Figure 1). As a result of the decay of field strength with distance (see Experimental Procedures), dramatic changes in [Ca*+]i could be induced in growth cones of a given neurite without affecting the [Ca*+]i of growth cones on separate neurites. Furthermore, the induced rise in [Ca*‘]i could be localized to individual growth cones emanating from the same neurite, separated by only a single branch point (see Figures IA and IA’). Changes in [Ca*+]i always occurred first on the side of the growth cone nearest the cathode source, where the extracellular field strength was greatest, then spread as a wave across the growth cone at an overall average rate of 2.1 f 0.3 pm/s (n = 14; Figure IB). The rate of this Ca*+ wave that spread across the growth cone upon stimulation, however, was dependent upon the electric field magnitude. The rate of the Ca*+ wave was directly correlated with the applied field strength (r = 0.94). In all experiments (n = 16), the wave of Ca*+ was localized to the growth cone and the most distal portion of the neurite. Gradients of [Ca”]i across the growth cone (Figure IB) occasionally were sustained throughout the duration of field application (7 of 19 cases). Often, however, such gradientswere not apparent after the initial rise in [Ca*+]i. An example of the distribution of [Ca*+], over time (Figure 2A) demonstrates that the gradient of [Ca*‘]i established across the growth cone can be readily observed after 2-3 s of stimulation. An extremely pronounced gradient, however, can be seen in the first 500 ms, as the result of the rapid rate of change in [Ca*+]i that occurs at the site closest to the cathode source. In all 19 experiments, the greatest change in [Ca’+]i was observed to be on the cathode side of the growth cone within the first 10 s of field application. To quantify the time course of the changes in [Ca*+]i evoked by focal electric fields, [Ca*+]i was measured (n = 36) at a location that was readily defined in all growth cones (the center of the growth cone proper). The center of the growth cone, however, was never the site of the greatest change in [Ca*+]i, and thus, the numbers presented and shown in Figure 26 are a minimal estimate of the peak change in [Ca*+]i. The most striking feature of the change in [Ca*‘]i over a period of 30 min was the extremely large transient in [Ca*+]i that occurred during the first 5 min of stimulation. The peak change in [Ca*+]i represented nearly a 6-fold increase over rest levels (pre: 128 f 5 nM; <30 s during stimulation: 720 f 139 nM, n = 36growth cones, p < 0.001). Despite a constant field strength, this high [Ca*‘]i was not maintained-[Ca*+]i began decreasing rapidly and reached a sustained and significant plateau by about 10 min (7.5 min during stimulation: 208 f 16 nM, n = 22, p < 0.001). The [Ca2’], remained significantly above rest levels as long as the field was maintained (p < 0.001). When the field was turned off, [Ca’+]i returned to pre levels within 2 min (post: 127 k 4 nM, n = 31, p > 0.25).

Neuron 408

The magnitude of the peak [Ca2+], change evoked by focally applied electric fields was dependent upon the applied field strength (Figure 2C). Measured at the center of the growth cone proper, the largest change in [Ca*+], was directly correlated with the applied field strength (r = 0.99) over a 5-fold range of field strengths. Electric fields smallerthan 1 mV/um did not induce changes in [Ca*+]i (data not shown); however, at 2 mV/um a 79% increase was induced (233 + 40 nM, n = 7, p < 0.01). Electric fields larger than 4 mV/um always produced at least a doubling of [Ca2+], (419 f 66 nM, n = 7, p < 0.01). Larger field strengths (-11 mV/pm) produced even larger changes in [Ca2+]i (635 + 132, n = 5, p < 0.05). To examine the possibility of even greater spatial localization of the Ca*+ second messenger signal, experiments required high temporal resolution, since from the point of initiation, Ca*+ waves spread rapidly to other parts of the growth cone (Figure 2A). In 16 experiments in which fura- fluorescence was monitored at video rates, all intracellular Ca2+ waves initiated at the region within individual growth cones closest to the cathode. The initial rise could be localized to an area less than 20% of the growth cone and typically reached values of several hundred nanomolar (Figure 3A). By moving the stimulus electrode to different regions on the same side of the same growth cone (Figure 3A), the initial peak [Ca*+]i response (a400 nM) could be spatially restricted to areas that were less than 15% of the growth cone. Thus, at the level of intracellular Ca*+, growth cones exhibit the ability to respond locally to environmental stimuli. To determine whether the evoked rise in [Ca2+l, is dependent upon extracellular Ca*+, growth cones were stimulated in Ca*+-free medium. Electric fields focally applied to growth cones in Ca2+-free medium did not alter [Ca’+]i (Figure 2B, dashed line; Figure 38). Six growth cones were stimulated in normal extracellular Ca*+ medium approximately45 min prior to stimulating in Ca *+-free medium. All growth cones significantly changed [Ca*+]i in normal medium (average peak [Ca”]i = 845 nM, p < 0.001 Mann-Whitney U test), while none showed any changes in [Ca*+], when in Ca*+-free medium (p > 0.4 for all time points). Four growth cones were again stimulated in normal medium (approximately 30 min after wash) to demonstrate that they retained the ability to change [Ca2+]i, and indeed all displayed significant changes (p < 0.01). Thus, the initial rise in [Ca*+], that occurs in response to focally applied electric fields requires the presence of an extracellular Ca2+ source. Morphological Changes localized within Individual Growth Cones Within minutes of onset, electric fields evoked a strikingorientation of filopodiatoward thecathode source (Figure 4). Many filopodia simultaneously appeared to elongate and bend or curve toward the cathode source. Other filopodia elongated and appeared to thicken, as was reported for growth cones of pioneer

neurons in an in vivo preparation of the grasshopper limb bud (O’Connor et al., 1990). Two parameters affected by electric fields were quantified: filopodial length and number (Figure 5). Prior to field application, both filopodial length and number varied, but with no consistent pattern. During this control period with the pipette in position, no sustained differences were observed between the two sides of the growth cones. Within minutes of field application, however, both filopodial length and number began to increase on the cathode side of the growth cones. Within 5 min, the first time point quantified, filopodia began elongating (127% + 9%, n = 19, p < 0.01; Figure 5A). Filopodia reached a maximum of almost 2-fold greater than average control lengths by 25 min (195% + 39%, n = 16, p < 0.01). Filopodia on the cathode side remained elongated for the duration of the experiment (up to 60 min; p < 0.05 at all time points). Filopodia on the opposite side of the growth cone statistically did not change length (p > 0.16 at all time points). In addition to filopodial elongation, the number of filopodia on the cathode side increased significantly within 5 min of electric field stimulation (123% + 9%, n = 21, p < 0.05; Figure 5B). This effect was transient; filopodial numbers decreased across the entire growth cone after about 20 min of constant stimulation. At 35 min, the number of filopodia on the cathode side had returned to control values (101% f 13%, n = 8) and the filopodial number on the anode side had dropped significantly from control values (68% * 9%, p < 0.01). Thus, while the increase in filopodial number on the cathode side of the growth cone remained for only about 20 min, the difference in filopodial numbers between the two sides of the growth cone was maintained for more than 30 min. After 45 min of field stimulation, however, filopodial numbers on both the cathode and the anode side were not significantly different from control values (p > 0.06). The magnitude of the changes in filopodial length and number evoked by focally applied electric fields was dependent upon the applied field strength (Figure 5C). The increases in both filopodial length and numberweredirectlycorrelatedwith theappliedfield strength over a Ifold range of field strengths (r = 0.91 and r = 0.98, respectively). No significant changes in filopodia on the opposite sides of growth cones were induced. Electric fields smaller than 4 mVIt.rm did not induce changes in filopodial length or number (107% + IO%, 100% f 8%, respectively, p > 0.52). Electric fields of approximately 4 mV/f.rrn did increase significantly the average length of filopodia on the cathode side (128% + 6%, p < 0.005), but not the average number of filopodia (p > 0.15). With larger field strengths (-12 mV/um), both filopodial length and number were significantly increased (138% +_ 8%, 173% f 26%, respectively, p < 0.05). To examine the possibility of even greater spatial localization of the behavioral responses, the spatial relationship between initial filopodial elongation and

Local Rise in [CC] 409

Locally

Figure 3. Localization of Augmented [Ca2’],

Alters

Filopodia

of the Initiation Site of Augmented [CaZ+], in Response induced by Focal Electric Fields on Extracellular CaZ+

to Highly

Localized

Electric

Fields

and the Dependence

(A) A cathode source (4.2 mV/pm) was placed close to this growth cone at a position to the left (A) and, several minutes later, at a position - 30 pm to the right (A’). [Caz’], rose most rapidly at the site within the growth cone closest to the micropipette (cathode). (B) Extracellular Ca*+ dependence of field-induced [Ca”], increases. Electric field stimulation was repeated sequentially on the same growth cone: defined Helisoma medium (B); Caz+-free medium (B’); and after reintroduction to defined medium (87. Field strengths were 8.0,9.2, and 5.9 mV/um, respectively. Dual wavelength images shown were collected before, 2 s during, and 1 min after stimulation. Bar, 10 urn (A); 20 urn (B).

Figure

4. Focal

Electric

Fields

Alter

Growth

Cone

Morphology

Representative examples from time-lapse, phase-contrast images show growth cones before and after the application of a sustained (A) and a brief (B) electric field. (A) The left image was taken 1 min prior to the onset of an electric field; the right was taken at 11 min during the sustained field of strength 1.8 mV/um (the stimulating pipette tip is visible on the right margin in [Al). (B) The left image was taken 1 min prior to the onset of an electric field (5 min duration); the right was taken 10 min later (4 min after the stimulation was terminated; field strength = 4.6 mV/pm). Filopodial elongation is readily observed in (A) and (B’); however, when all growth cones were examined during sustained stimulation, a statistically significant increase in filopodial number was also observed on the cathode side (see Figure 58). Bars, 20 pm (A); IO urn (B).

cathode source was determined. In 12 of 13 experiments, the earliest filopodial elongation (420 min) occurred by filopodia that were located closest to the cathode. The initial response could be localized to

less than 20% of the growth cone perimeter (Figure 6). By moving the stimulus electrode to different regions on the same side of the same growth cone (Figure 6), the initial filopodial response(elongation greater than

Local

Rise in [CaZ’] Locally

Alters

Filopodia

411

-60

-40

-20

-60

-40

-20

0 Time (mln)

io

40

60

20

40

60

0.50 0 Time (mln)

075 0

2

4

6

6

10

12

Electric Field Magnitude (mV/pm)

Figure 5. Time Course and Magnitude to Focally Applied Electric Fields

of Filopodial

Responses

Changes in filopodial length (A) and number(B) are shown. Filopodial extension and initiation toward a cathode current source begins within minutes after the current is turned on (field strengths = 0.75-17 mV/um). Values shown represent averages (mean f SEM) for all growth cones relative to their respective average values collected during the control period. (A) Filopodial extension on the cathode side reached a maximum at about 25 min (195% of control values; n = 16, p < 0.01) and remained elongated during continuous stimulation (p < 0.05). Filopodia on the anode side of growth cones statistically were unaffected by this stimulus (p > 0.16). (B) New filopodia were generated on the cathode side of growth cones within minutes (123% of control values at 5 and 15 min; n = 21 and 19, respectively, p < 0.05), but filopodia were then transiently lost over the entire growth cone at about 35 min (cathode facing filopodia numbers: 101% of control, n = 8; anode: 6896, n = 8, p < 0.01). (C) The average change in filopodial length and number is plotted against the applied field strength, over a 5-fold range of field strengths. A simple, linear fit to the data shows that a statistical correlation exists for both filopodial length and number (r = 0.91 and r = 0.98, respectively).

60%) could be spatially restricted to less than 12% of the growth cone perimeter. Thus, even at the level of dynamic growth cone behaviors, growth cones exhibited the ability to restrict and localize their response spatially to environmental stimuli. Since the initial rise in [Ca2+]i that occurred in response to focally applied electric fields was dependent upon extracellular Ca2+, growth cones were again stimulated in CaZ+-free medium. Growth cones in Ca2+-free medium appeared qualitatively similar to growth cones in defined and conditioned medium for at least the first 2 hr. Electric fields focally applied to growth cones in Ca2+-free medium did not alter either filopodial length or number (Table 1). Application of electric fields to growth cones in Ca2+-free medium did not elicit the generation or the elongation of filopodia (p > 0.70). Only the cathode side of growth cones in normal extracellular Ca2+ medium was affected by focally applied electric fields (Table I). Thus, the change in filopodial disposition that occurs in response to focally applied electric fields requires the presence of an extracellular Ca2+ source. Persistent Morphological Changes Induced by Brief Field Stimulation The largest changes in [Ca2+], that occurred in response to focally applied electric fields occurred during the first 5-10 min after stimulus onset (Figure 2B). During this period, [Ca”]i rose to approximately 7 times the rest levels; after this period, [Ca2+], remained at a relatively constant elevation of twice rest levels. To determine whether this peak change in [Ca2’], was sufficient to evoke changes in filopodia, or whether both the peak and the sustained plateau rise in [Ca2+]i were necessary, a series of growth cones were assayed for filopodial changes after a brief (3-5 min) stimulation. As with sustained stimulation, an attraction of filopodia toward the cathode source occurred within the first few minutes (Figure 4B). Filopodial measurements were made during the 20 min period following the onset of the electric field to test for sustained changes in filopodial disposition. During this period, filopodia on the cathode side of growth cones significantlyelongated(128% f 7%,p 0.3). In contrast to the evoked changes in filopodial lengths, no significant increase in filopodial number on the cathode side was sustained following the brief field stimulation (98% f 5%, p > 0.7). Thus, the large peak change in [Ca’+]i appears to be sufficient to change filopodial length, but at these field strengths, a sustained change in [Ca2+], is necessary to sustain a significant change in filopodial number. Statistical Correlation between Changes in [Ca2+k and in Filopodial Disposition Electric fields of similar strength were used to induce large changes in [Ca2+], and in filopodial disposition. The responses were well correlated with the magnitude of the applied electric field (Figure 2C; Figure

Neuron 412

5C). To examine further the relationship between the magnitude of the rise in [Ca*‘]i and the change in filopodia, these data were analyzed for a statistical correlation. At given field strengths, the peak [Ca2+], obtained was plotted against the average increase in filopodial length and number (Figure 7). Using a simple linear fit to the data, correlation coefficients between peak [Ca2+], and filopodial length and number were both r = 0.98. Thus, a strong correlation exists between the evoked changes in [Ca*‘]i and both filopodial length and number. Discussion

10 min

Pre

To present a precisely localized and quantified stimulus to active neuronal growth cones, electric fields werefocallyapplied to Helisomagrowth cones. Quantitative morphological analyses confirmed and extended the observations of others concerning the changes in filopodial disposition in an applied electric field (Pate1 and Poo, 1984; McCaig, 1989). Direct measurements of [Ca2+]r during field stimulation demonstrated the involvement of the second messenger that previously had been implicated in both generalized filopodial changes (Rehder and Kater, 1992; Haydon et al., 1987) and filopodial responses to focally applied electric fields (McCaig, 1989). The strong statistical relationships found in the present paper argue for a significant role for intracellular Ca2+ in the highly localized responses of neuronal growth cones to their environment. Causal Relationship between [Ca*+]i and Filopodiai Disposition Multiple lines of evidence suggest that changes in [Ca2’], indeed are causal to the changes in growth cone morphology. -Induced changes in [Ca2+]r occurred prior to and with a lower threshold than the induced changes in filopodia (see Figure 2 and Figure 4). These are necessary conditions to demonstrate causality between an intracellular signal and a morphological response. The rise in [Ca*+]r occurred within milliseconds, but remained highly elevated (greater than twice rest lev-

Figure 6. Filopodial Electric Fields

Elongation

in Response

to Highly

Localized

The initial filopodial elongation toward a focally applied cathode source occurs from filopodia located closest to the cathode source (see Figure 3A). This elongation is readily observed upon the background changes in filopodial length that occur in all growth cones. A cathode source (11.5 mV/nm) was placed close to this growth cone: at a position to the left (A) and, several minutes later, at a position - 30 nm to the right (B). During field stimulation, filopodia closest to the cathode source elongated, while other filopodia remained unchanged. The schematic at the bottom of each series highlights all filopodia that elongated greater than 20% during the respective stimulation. Bar, 20 pm.

Local Rise in [Ca”] 413

Table

1. Filopodial

Locally

Alters

Orientation

Filopodia

in Focally

Applied

Electric

Fields

Is Dependent

upon

the

% of Control Average

Media Normal

media

CaL+-free

Data erage ap < bp <

(n = 21)

Filopodial

Anode

Length Number Length Number

(n = IO)

from individual growth cones control values. 0.001; paired Student’s t test. 0.03; paired Student’s t test.

averaged

105 94 114 108 during

field

stimulation

els) for more than 5 min. The initial filopodial changes occurred in less than 5 min. -The initial changes in both [Ca*+]i and filopodia consistently were localized to the cathode side of the growth cone. Each demonstrated a graded response across the growth cone that could be qualitatively (Figure 1; Figure 4) and quantitatively (Figure 2A; Figures 4A and 4B) recorded. These Ca*+ and filopodial responses could be spatially mapped, i.e., the cathode side of growth cones showed the greatest change in [Ca’+]i and in filopodial length and number. -Induced changes in [Ca*+]i and in filopodia could be spatially restricted to a similar degree (<20% of the growth cone) and to a similar location (the site closest to the cathode source) even when repeating stimulation at different sites on the same growth cone (Figure 3; Figure 6). -Preventing the change in [Ca*+], by removing extracellular Ca*+ from the media blocked the change in filopodia (Table 1). Filopodia remained active in the CaZ+-free medium, but did not orient in applied electric fields. Thus, the influx of Ca2+ and not the absence

1.80 *.:/

1

0

400 200 Peak[Ca++li

Figure 7. Statistical Correlation in [Ca2+], and in Filopodia

600 (nM)

between

[ '.50

800

the

Induced

Changes

The average increases in filopodial length and number on the cathode side of growth cones are plotted against the average peak [Caz+], evoked by electric fields of similar magnitude and duration. Simple, linear fits to the data show a strong correlation between a rise in [Ca2+], and a local change in filopodial length and number (r = 0.98 for each).

(from

f f * +

Presence

Ca’+

Period

Side

Cathode 145 122 102 107

3 6 10 7

5 min through

of Extracellular

55 min)

* f * f

is compared

Side 9" 9b 5 7 with

respective

av-

of extracelIularCa*+ is implicated asa necessarycondition for induced filopodial changes. -Using a stimulus duration sufficient to cause the greatest change in [Ca*+]i and the greatest gradient of [Ca2+]i caused a significant and long-lasting change in filopodia. This is predicted if indeed the peak [Ca2’], and/or the induced gradient of [Ca2+], are causal to the local filopodial changes. -For sustained stimulation, the peak [Ca*+], reached during the rapid, large, and spatially localized rise in [Ca*+]i was well correlated with the local changes in both filopodial length and number (both with correlation coefficients of r = 0.98; Figure 7). The strong correlation suggests a physiological relationship between the induced rise in [Ca2+]i and the induced changes in filopodial disposition. Taken together, the data strongly implicate a causal relationship between local [Ca2+]i and local morphological responses of growth cones. Field-induced Changes within Neuronal Growth Cones Previous investigators have demonstrated the growth cone’s ability to alter filopodial disposition locally and have also suspected changes in [Ca*+]i as a causal second messenger. Several investigators have focally applied electric fields to orient neurite outgrowth of embryonic neurons from the dissociated neural tube of Xenopus (Pate1 and Poo, 1984; Patel, 1986; McCaig, 1986, 1989). McCaig (1986) demonstrated an increase in filopodial number only on the cathode side of the growth cones concomitant with orientation changes. With inorganic Ca I+ channel blockers, the change in filopodial distribution on Xenopus growth cones was prevented (McCaig, 1989). McCaig and others have suggested that electric fields may alter [Ca*‘]i (see Robinson, 1985, and McCaig and Rajnicek, 1991, for reviews). The only second messenger definitively demonstrated to orient Xenopus growth cones, however, is CAMP (extracellularly applied to growth cones; Poo and Quillan, 1992). These results indicate that in response to electric fields, Xenopus and Helisoma neurons could use different messenger systems, or alternatively, each could use both systems, but to different degrees.

The present results demonstrate the ability of focally applied electric fields to alter [Ca’+]i in neuronal growth cones. Influx from extracellular sources was necessary to induce this rise in [Ca*+]i. Voltagedependent Ca*+ channels likely played a major role in this electrically triggered influx. In the present experiments, field strengths that were just above threshold to evoke [Ca’+]i changes were -2 mV/pm. A theoretical, isopotential cell of diameter 20 urn and much less than the length constant would be predicted to depolarize about 20 mV (Cooper and Schliwa, 1985). While we do not know whether growth cones maintain isopotentiality, the electric field strengths used in the present study likely are sufficient to change the membrane potential of at least the cathode side of the growth cone. Achange in membrane polarization may well activate high voltage-activated Ca*+ currents known to exist on growth cones in this preparation (Haydon and Zoran, 1989). A recent report (Bedlack et al., 1992) clearly demonstrates the ability of electric fields applied across the entire culture dish to alter [Ca*+], and membrane potential of NIE-115 mouse neuroblastoma cells. Thus it is likely that in both the present study and the study by Bedlack and colleagues, electric fields induce a depolarization of the membrane, opening voltage-dependent Ca*+ channels, which are responsible at least in part for the rapid and large increase in [Ca2+],. In the present study, preventing the influx of Ca*+ by bathing cells in a Ca*+-free medium completely blocked the induced rise in [Ca*+],. Thus, an influx of Ca*+ is necessary for at least part of the large rise in [Ca*+]i observed within the first minute of stimulation. It remains possible, however, that only a small influx is necessary to induce a large efflux of Ca2+from intracellular stores (Fabiato, 1985; Callewaert et al., 1988; Thayer et al., 1988). Indeed, Ca*+ waves observed in several preparations have been attributed to Ca2+induced Ca*+ release from intracellular stores (see reviews by Jaffe, 1991, and Berridge and Irvine, 1989). The average Ca*+ wave rate reported here (2.1 f 0.3 pm/s), however, is relatively slow and can be correlated with the field strength applied (r = 0.94). No step increase in rate was found at any field strength. A step increase might have indicated a threshold for release from intracellular stores. Furthermore, no recurrent Ca*+ waves were ever observed after the initial wave, as has been observed with many Ca2+-induced Ca*+ release waves (Alkon and Rasmussen, 1988; Lechleiter and Clapham, 1992). Thus, a parsimonious explanation for the observed Ca*+ changes is that as field strength was increased, a greater influx occurred through voltage-dependent Ca2+ channels, which led to a greater, passive diffusion rate. Additionally, the sustained elevation in [Ca*+]i was maintained as long as the field was maintained, suggesting that influxwas at least partially responsible for both the initial large rise and the sustained elevation in [Ca*+]i. These results highlight the functional role that clustering of Ca2’ channels (Ross et al., 1988; Roberts et al., 1990;

Silver et al., 1990) or regional Ca2+ buffering capacity (Simon and Llinb, 1985; Adler et al., 1991) could play in neuronal development. local Changes in [Ca*+]i and Growth Cone Morphology The present study demonstrates that a locally applied extracellular signal leads to a local change in a second messenger sufficient to alter locally the morphology of a neuronal growth cone. The significance of these observations is not limited to electric field stimulation. Presumably any stimulus that alters [Ca*+]i in a restricted region of the neuronal growth cone may accordinglyaffectfilopodiaquitelocally.Asimilarscenario was suggested by Silver et al. (1990) as a possible physiological role for the “hot spots” of Ca*+ changes observed in response to depolarization. Smith and Jahr (1992) demonstrated a potential role for local Ca2+ signaling beyond the neuronal growth cone: local application of glutamate onto hippocampal axons produced regional morphological responses. This neurotransmitter is known to increase [Ca*+]i in growth cones and cell bodies of hippocampal neurons (Mattson et al., 1988), suggesting that changes in [Ca*‘]r may also underlie this response. Thus, the present results highlight the degree of spatial localization of intracellular Ca*+ signaling and the resultant local morphological responses that are possible in neuronal growth cones. Gradients of [Ca2+]i were postulated to guide polarized eosinophils in culture by focally applied chemotactic stimuli (Brundage et al., 1991). Growth cones may indeed integrate local signals and respond preferentially to the site that experiences the first or the greatest change in [Ca*+],, or even the greatest rate of change in [Ca*+]i. The rate of rise in the region of greatest field strength can be extremely large, reaching several hundred nanomolarwithin 30 ms. Gelsolin and many other Ca*+-dependent, actin-binding proteins likely would be affected strongly by such changes in [Ca*‘], (Forscher, 1989). The kinetics or localization of particular cytoskeletal components could explain the integration of signals performed by the growth cone and the resultant localization of the morphological responses. The local extension of filopodia from an individual growth cone could dramatically affect the final morphology of individual neurons and thus the resultant circuit. Filopodia have been suggested to act as both the sensory unit (Sperry, 1963; Bentley and Keshishian, 1982) and the directional motor unit (Nakai and Kawasaki, 1959; Bray, 1982; Goldberg, 1988; O’Connor et al., 1990; Heidemann et al., 199O)of neuronal growth cones. These roles are not mutually exclusive, however. Local filopodial extension would be expected to affect growth cone guidance strongly in either case. If the function of filopodia is to sense the environment, then a local increase in filopodial length and number will increase the area of environment surveyed. If the function of filopodia is to serve as the scaffolding for

Local

Rise in [Ca”]

Locally

Alters

Filopodia

415

process extension, then a local increase in filopodial length and number will direct the subsequent outgrowth of the entire process. Thus, the demonstration that local changes can occur in the growth cone intracellular Ca2+ second messenger system and that these local changes elicit local changes in filopodial disposition suggests a role for intracellular Ca*+ signals in subtle aspects of the development of the nervous system. Experimental

Procedures

Cell Culture Isolated, identified neurons from buccal ganglia of adult pond snails (Helisoma trivolvis, inbred laboratory stock) were prepared according to the methods of Wong et al. (1981). Briefly, buccal ganglia were dissected, and identified neurons 85 and B19 were removed and plated individually onto glass coverslips coated with poly-clysine (Sigma). Defined growth medium consisted of half-strength Leibovitz L-15 from which all inorganic salts were removed (GIBCO, special order). Salts were added to this medium: 40 mM NaCI, 1.7 mM KCI, 1.5 mM MgCb, 4.1 mM CaCb, 5 mM HEPES, 50 pg of gentamycin per ml, 0.15 pg of glutamine per ml (pH 7.3). To promote outgrowth, cells were maintained prior to experimentation in a conditioned medium (Wong et al., 1981,19B4). Conditioned medium was produced by incubating 2 Helisoma brains per ml of the defined medium for 3 days, followed by removal of the brains and filtration of the medium (0.22 pm pore size; Costar). Cultures were kept in a humidified chamber at room temperature for 15-24 hr prior to experimentation. Experiments were carried out at room temperatureineitherdefinedmediumoraCa~-freemediumcontaining the salt concentrations for defined medium except MgCI, was substituted for CaCI? and 0.5 mM ECTA was added. Medium changes were performed by carefully removing all medium from the dish, except for a center well that contained about 200 pl of medium, and adding the new medium. Cells were washed a minimum of 4 times in the new medium. Growth cones in Ca2+free medium were qualitatively similar to growth cones in defined or conditioned medium for at least the first 120 min. To control against possible long-term deleterious effects of Ca*+free medium, these experiments were confined to (90 min (control periods were shortened to 30 min). Electric Field Stimulation Small, focally applied electric fields were produced according to methods of Pate1 and Poo (1984). Glass micropipettes were fire polished to tip diameters between 2 and 5 pm and filled with 7% agarose (type 7; Sigma) in either defined medium or Ca2+-free medium to match the culture conditions. Constant DC currents between 2 and 15 pA were passed between the micropipette (cathode) and an agarose bridge (7% type 7) located approximately l-2 cm away (anode). The agarose bridge (3-5 cm in length) connected culture medium and saline within a second dish that contained a Ag/AgCI ground electrode. Currents were maintained by a constant current stimulator and monitored continuously during experiments. Micropipettes were positioned approximately 15-25 pm from growth cones at 4S”-90° to the direction of outgrowth. Current density at a distance r from the pipette is calculated from: J = l/2& (Pate1 and Poo, 19B4), assuming the culture chamber acts as a perfect insulator. An electric field strength is derived from thecurrent density and the resistivity of the culture medium (225 Dcm). Approximate electric field strengths ranged between 0.75 and 17 mV/pm. Measurements of electric field strengths, however, are imprecise due to the different geometries of individual micropipettes and the dynamics of growth cones that affect the distance from the growth cone to pipette. Following a control period with the micropipette in position and passing no current, constant current was passed, maintaining the micropipette as a cathode source. In most morphological experiments, the field was maintained for the dura-

tion of a given experiment; in a separate set of experiments the field was maintained for a limited 3-5 min period. In the latter experiments the pipette was maintained in position before, during, and after passing constant DC current to minimize possible artifacts from tip movement. Quantitative Analysis Phase-contrast imagesobtained with a4Ox objective(Nikon, Ph3 DL) were recorded via a silicon-intensified target camera (RCA TC1030) and digitized to a Macintosh II fx (Data Translation, Quick Capture frame grabber board). With the Image software package (National Institutes of Health), the number and average length of filopodia on either side of each growth cone were determined for every image obtained. Averaged at 5 min intervals, individual growth cone data were then grouped and summarized (mean f SEM; Figure 5). A paired Student’s t test was made between the average values obtained during the entire control period (filopodial number or average length) and the average values obtained during the experimental period (Table 1). Statistical correlations were determined between the electric field strength and the mean change in filopodial length and number (Figure SC). Cal+ measurements were obtained from growth cones whose cell bodies were injected with furapentapotassium salt (10 mM in HzO; Molecular Probes) approximately 1 hr prior to experimentation. Fluorescence measurements were obtained with a 40x objective (Nikon, Fluor 1.30x oil) via an intensified CCD camera (Quantex) linked to an image-processing system (QX7210, Quantex). Images obtained at two excitation wavelengths (350 and 380 nm interference filters) were each averaged for 16 frames, and fluorescence emission was filtered with a 495 nm long pass filter. Neutral density filters were used in the excitation pathway to minimize bleaching of the dye and damage to wellloaded cells. The two images taken at 350 and 380 nm were ratioed on a pixel-by-pixel basis, and [Ca*+] measurements were estimated according to Grynkiewicz et al. (1985). The values determined for the imaging system in use are R,,, = 0.48, R mm = 13.25, FJF, = IO, Ko = 224 nM. Estimated Ca2+ concentrations are reported as mean * SEM (Figure 2). A statistical correlation was determined between the electric field strength and the average peak change in [Cal’], (Figure 2C) and between the average peak change in [Cal’], and the mean filopodial changes (Figure 7). Changes in estimated [Ca*+kare shown by a Student’s t test comparison between the average estimated [Ca2+], at individual time points relative to the average estimated [Ca2+], during the control period. A series of high speed experiments was performed (Figure 2A; Figure 3A). A sequence of images was obtained at video rate with a single excitation wavelength (3BO nm). Images obtained during the stimulus were ratioed on a pixel-by-pixel basis to an image obtained prior to stimulation (f&f) and mapped to a pseudocolor look up table. An approximate calibration of these single wavelength ratios to known [CaZ’], values was made by obtaining a standard dual wavelength image within 3 s of acquiring the video rate images. The [CaZ+], could then be approximated from the dual wavelength image and corresponding calibration. Acknowledgments We thank Dr. V. Rehder for detailed discussions during the time of experimentation and manuscript preparation that directly impacted the focus of this work, Dr. P. Dou and D. Giddings for technical assistance, and Drs. C. S. Cohan, P. B. Cuthrie, and M. F. Schmidt for their critical comments on the manuscript. Portions of this work were presented in abstract form at the 1991 annual meeting of the Society for Neuroscience. This work was supported by National Institutes of Health grant NS246B3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

31, 1992;

revised

June

22, 1992.

Neuron 416

References

Kater, S., and Mills, L. (1991). Regulation by calcium. J. Neurosci. II, 891-899.

Adler, E. M., Augustine, G. J., Duffy, S. N., and Charlton, M. P. (1991). Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse. J. Neurosci. II, 1496-1507.

Lechleiter, J. D., and nisms of intracellular Cell 69, 283-294.

Alkon, D. L., and Rasmussen, H. (1988). A spatial-temporal of cell activation. Science 239, 998-1004. Bandtlow, C., Zachleder, T., and Schwab, drocytesarrest neuritegrowth by contact 10, 3837-3848.

model

M. E. (1990). Oligodeninhibition. J. Neurosci.

Bedlack, R. S., Wei, M.-d., and Loew, L. M. (1992). Localized brane depolarizations and localized calcium influx during tric field-guided neurite growth. Neuron, this issue. Bentley, pioneer

memelec-

D., and Keshishian, H. (1982). Pathfinding by peripheral neurons in grasshoppers. Science 278, 1082-1088.

Berridge, M. J., and Irvine, R. F. (1989). cell signalling. Nature 347, 197-205.

lnositol

phosphates

Bray, D. (1982). Filopodial contraction and growth cone ance. In Cell Behavior, R. Bellair, A. Curtis, and C. Dunn, (Cambridge: Cambridge University Press), pp. 298-318.

and guideds.

cone

D. E. (1992). Molecular excitability in X. laevis

behavior mechaoocytes.

Mattson, M. P., Guthrie, P. B., and Kater, S. B. (1988). Intracellular messengers in the generation and degeneration of hippocampal neuroarchitecture. J. Neurosci. Res. 27, 447-464. McCaig, C. D. (1986). growth and the effects 55-69.

Bandtlow, C. E., Schmidt, M. F., Hassinger, T. D., Schwab, M. E., and Kater, S. B. (1992). Intracellular calcium mediates growth cone collapse evoked by the CNS myelin-associated neurite growth inhibitor NI-35. Science, in press.

Clapham, calcium

of growth

McCaig, C. D. (1989). frog nerve orientation 93, 723-730. McCaig, growth

Dynamic aspects of amphibian neurite of an applied electric field. J. Physiol. 375, Studies on the mechanism in a small applied electric

of embryonic field. J. Cell Sci.

C. D., and Rajnicek, A. M. (1991). Electrical fields, nerve and nerve regeneration. Exp. Physiol. 76, 473-494.

Nakai, J., and Kawasaki, Y. (1959). Studies of the mechanism determining the course of nerve fibers in tissue culture. Z. Zellforschung. 57, 108-122. O’Connor, T. P., Duerr, J. S., and Bentley, growth cone steering decisions mediated contacts in situ. J. Neurosci. IO, 3935-3946.

D. (1990). Pioneer by single filopodial

Patel, N. B. (1986). Reversible inhibition of neurite growth by focal electric currents. In Ionic Currents in Development, R. Nuccitelli, ed. (New York: Alan R. Liss, Inc.), pp. 271-278.

Brundage, R. A., Fogarty, K. E., Tuft, R. A., and Fay, F. S. (1991). Calcium gradients underlying polarization and chemotaxis of eosinophils. Science 254, 703-706.

Patel, N. B., and Poo, M.-M. of neurite growth by pulsed 4, 2939-2947.

Callewaert, C., Cleemann, L., and Morad, M. (1988). Epinephrine enhances Ca” current-regulated Ca*+ release and Ca2+ reuptake in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA 85,20092013.

Poo, M.-M., and Quillan, M. (1992). Growth orientation and transmitter secretion by nerve growth cones. In The Nerve Growth Cone, P. Letourneau, S. Kater, and E. Macagno, eds. (New York: Raven Press, Ltd.), pp. 231-236.

Cohan, C. S., and Kater, S. B. (1986). Suppression gation and growth cone motility by electrical 232, 1638-1640.

Rehder, V., and Kater, S. (1992). Regulation of neuronal growth cone filopodia by intracellular calcium. J. Neurosci. 72, 31753186.

Cooper, M. S., and Schliwa, trols of tissue cell locomotion Res. 13, 223-244.

of neurite elonactivity. Science

M. (1985). Electrical and ionic conin DC electric fields. J. Neurosci.

Fabiato, A. (1985). Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac Purkinjecel1.J. Gen. Physiol. 85, 247-289. Forscher, P. (1989). Calcium and of cytoskeletal dynamics. Trends

polyphosphoinositide control Neurosci. 12, 468-474.

Goldberg, D. J. (1988). Local role of Ca’+ in formation growth cones. J. Neurosci. 8, 2596-2605.

of veils

in

Crynkiewicz, C., Poenie, M., and Tsien, R. (1985). A new generation of Cal+ indicators with greatly improved fluorescence prop erties. J. Biol. Chem. 268, 3440-3450. Gundersen, R. W., and Barrett, J. N. (1980). Characterization the turning response of dorsal root neurites towards growth factor. 1. Cell Biol. 87, 546-554. Haydon, P. G., and Zoran, M. J. (1989). Formation of chemical connections: evoked acetylcholine growth cones and neurites of specific identified ron 2, 1483-1490. Haydon, P. G., McCobb, D. P., and Kater, selectively inhibits growth cone dynamics of specific identified neurons of Helisoma.

of nerve

and modulation release from neurons. Neu-

S. B. (1984). Serotonin and synaptogenesis Science226,561-564.

Haydon, P. C., McCobb, D. P., and Kater, S. B. (1987). The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin. J. Neurobiol. 78, 197-215. Heidemann, S. R., Lamoureux, P., and Buxbaum, Growth cone behavior and production of traction Biol. 777, 1949-1957.

R. E. (1990). force. J. Cell

Jaffe, L. F. (1991). The path of calcium in cytosolic calcium oscillations: a unifying hypothesis. Proc. Natl. Acad. Sci. USA 88,98839887.

(1984). Perturbation and focal electric

of the direction fields. J. Neurosci.

Roberts, W. M., Jacobs, R. A., and Hudspeth,A. J. (1990). Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J. Neurosci. IO, 3664-3684. Robinson, K. R. (1985). The response review. J. Cell Biol. 707, 2023-2027.

of cells

to electrical

field:

a

Ross, W. N., Arechiga, H., and Nicholls, J. (1988). Influence of substratum on thedistribution of calcium channels in identified leech neurons in culture. Proc. Natl. Acad. Sci. USA 85, 4075407a. Silver, R. A., Lamb, A. G., and Bolsover, S. R. (1990). Calcium hotspots caused by L-channel clustering promote morphological changes in neuronal growth cones. Nature 343, 751-754. Simon, S. M., and Llinls, R. R. (1985). Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release. Biophys. J. 48, 485-498. Smith, S., and Jahr, C. (1992). Rapid induction of filopodial sprouting by application of glutamate to hippocampal neurons. In The Nerve Growth Cone, P. Letourneau, S. Kater, and E. Macagno, eds. (New York: Raven Press, Ltd.), pp. 19-26. Sperry, R. W. (1963). Chemoaffinity fiber patterns and connections. 703-710.

in the orderly growth of nerve Proc. Natl. Acad. Sci. USA 50,

Thayer, S., Hirning, L., and Miller, R. (1988). The role of caffeinesensitivecalcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro. Mol. Pharmacol. 34, 664-673. Wong, R. G., Hadley, R. D., Kater, S. B., and Hauser, C. C. (1981). Neurite outgrowth in molluscan organ and cell cultures: the role of conditioning factor(s). J. Neurosci. I, 1008-1021. Wong, R. C., Barker, D. L., Kater, S. B., and Bodnar, D.A. (1984). Nerve growth promoting factor produced in culture media conditioned by specific CNS tissues of the snail Helisoma. Brain Res. 292, 81-91.