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Previous Article | Next Article
Volume 16, Number 18, Issue of September 15, 1996 pp. 5593-5602
Copyright ©1996 Society for NeuroscienceReduced Growth Cone Motility in Cultured Neurons from Drosophila Memory Mutants with a Defective cAMP Cascade
Yun-Taik Kim and Chun-Fang Wu Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
ABSTRACT
Drosophila memory mutants dunce (dnc) and rutabaga (rut) are known to have altered intracellular cAMP levels, nerve terminal growth, and plasticity of synaptic transmission. Because the growth cone is responsible for neurite outgrowth and synaptogenesis, video microscopy was used to examine growth cone morphology and behavior of mutant neurons in larval CNS cultures. We found that growth cone exploratory movement was nearly arrested by both mutations, even though they change cAMP levels in opposite directions. The dnc phenotype could be mimicked by normal neurons when perfused with dibutyryl cAMP (db-cAMP) or forskolin. In contrast, rut growth cones became active when perfused with db-cAMP. Furthermore, motility was also restored by counterbalancing the effects of the two genes in double mutants, indicating that dynamic control of growth cone motility in developing Drosophila neurons requires optimal cAMP levels within an operational range. These findings represent the first demonstration of altered growth cone properties in learning and memory mutants and establish in a natural setting the role of cAMP in growth cone motility and neuronal plasticity.
Key words: dnc; rut; growth cone; lamellipodia; neuronal plasticity; learning; memory; cAMP; Drosophila; CNS; culture
INTRODUCTION
The Drosophila mutants dnc and rut were isolated because of their learning deficiencies (Dudai et al., 1976; Aceves-Pina et al., 1983
The above phenotypic abnormalities illustrate the close relationship between developmental and physiological plasticity. Because the cellular mechanisms regulating neurite branching and synaptic contacts in Drosophila have not been well established, analysis of the neuronal growth process in these mutants may provide insight into the role of cAMP in developmental plasticity. We examined cultured dnc and rut neurons dissociated from the larval CNS. This culture system is suitable for pharmacological studies on isolated neurons (Wu et al., 1983
Preliminary reports of part of this work have been published previously (Kim and Wu, 1991b
MATERIALS AND METHODS
Animal stocks. The wild-type strain Canton-Special (CS), three different alleles of the dnc locus, dnc1, dncM11, dncM14, one allele of the rut gene, rut1, double mutants dnc1 rut1 and dncM14 rut1, and rut1/Df(1)KA9 of Drosophila melanogaster were raised on standard Drosophila medium at 22°C. KA9 carries a small X-chromosome deletion uncovering rut. Male dncM11 and dncM14 were used in the experiments and maintained in a balanced stock with attached X-chromosomes because of their female sterility (Mohler, 1977Dissociated larval CNS cell culture. Dissociated neurons from the wild-type strain CS were used for normal controls throughout the experiments. The procedure for culturing neurons from Drosophila larvae was identical to that reported previously (Kim and Wu, 1991a
Video microscopy. Within 2 d of culture, growth cones with flat veil-like lamellipodia in type III neurons (Wu et al., 1983
Pharmacology. Dibutyryl cAMP (db-cAMP) (Sigma) was dissolved in Drosophila normal saline (Wu et al., 1983
Analysis of growth cone morphology and behavior. The method to analyze growth cone morphology and motility was essentially the same as reported previously (Soll et al., 1988
area/perimeter2), have been described previously (Soll et al., 1988
RESULTS
Abnormal growth cone motility in dnc and rut neurons
It is important to examine multiple dnc and rut alleles so that any abnormalities observed could be attributed to the mutational effects of these genes. We studied several independent isolates of the two genes, including dnc1, dncM11, dncM14, rut1, and rut1/Df(1)KA9. (KA9 is a deletion uncovering rut.)The larval CNS culture consists of several types of neurons at different stages of differentiation (Wu et al., 1983
In general, no obvious differences in growth cone morphology (Fig. 1) and frequency of occurrence (Table 1) were found between normal and mutant cultures. (There seems to be a small increase in the frequency of occurrence of dnc1 rut1 neurons with lamellipodial growth cones, but the smaller sample size for this genotype shown in Table 1 precludes a firm conclusion.) In a series of systematic surveys designed for random sampling in 16- to 48-hr-old cultures, 33.3% of type III monopolar neurons in normal cultures displayed extended lamellipodia at the tip of the neurite, whereas dnc1, rut1, and rut1/Df(1)KA9 cultures showed 33.6%, 31.6%, and 34.3%, respectively (Table 1). A population of these lamellipodia appeared lighter and thinner in phase optics (60.7%, 63.9%, 62.3%, and 64.7% in normal, dnc1, rut1, and rut1/Df(1)KA9, respectively). The rest of the growth cones appeared darker and thicker and were nearly completely immobile (Fig. 1c,f,i). As reported previously, these morphological features are associated with more advanced stages of development (Hadley et al., 1985
Fig. 1. Neurons with growth cones in cultures from normal, dnc1, and rut1 larval CNS. Approximately 60-65% of growth cones in both mutant and normal cultures showed lighter images (a, b, d, e, g, h) in phase-contrast optics. The remaining growth cones appeared thicker and darker (c, f, i). The morphology of phase-light growth cones of dnc1 and rut1 neurons was indistinguishable from that of normal neurons. The phase-dark growth cones of both dnc1 and rut1 neurons appeared darker than those in normal neurons. Cultured neurons from the wild-type strain CS were used as normal controls in this and the following figures (see Materials and Methods).
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Table 1. Occurrence and optical properties of growth cones in normal and mutant cultures
Genotype Number of cells (cultures) examined Cells with growth cone Growth cone phase quality vs activity Light (act/inact) Dark (act/inact) Normal 183 (8) 33.3% 60.7% (23/14)* 39.3% (0/24) dnc1 214 (8) 33.6% 63.9% (0/31) 36.1% (0/14) rut1 193 (7) 31.6% 62.3% (0/38) 37.7% (0/23) 99 (5) 34.3% 64.7% (0/22) 35.3% (0/12) dnc1 rut1 52 (4) 48.1% 80.0% (15/5)* 20.0% (0/5) Growth cones are defined as nerve terminals with flattened veil-like lamellipodia, which were frequently accompanied by filopodia. Only growth cones of type III monopolar neurons were included in the analysis. Note active motility (*) found in only normal and dnc rut double-mutant cultures. dnc rut cultures appeared to have higher frequency of active, phase-light growth cones. act, Active; inact, inactive.
Table 2. Growth cone morphology and motility of normal and mutant neurons in culture
Genotype Growth cone morphology Growth cone motility Filopodia Lamellipodia n No/GC ± SEM Length ± SEM Area ± SEM Roundness ± SEM Index ± SD n Normal 3.1 ± 0.2 5.0 ± 0.3 18.3 ± 1.0 0.38 ± 0.02 53 0.36 ± 0.09 5 dnc1 3.1 ± 0.2 4.7 ± 0.3 17.7 ± 1.7 0.40 ± 0.16 58 0.10 ± 0.03* 4 dncM11 3.5 ± 0.3 5.2 ± 0.4 16.2 ± 2.5 0.37 ± 0.02 39 0.09 ± 0.03* 4 dncM14 3.3 ± 0.7 4.6 ± 0.5 15.5 ± 1.8 0.29 ± 0.09 10 0.07 ± 0.03* 4 rut1 2.9 ± 0.2 5.2 ± 0.4 13.4 ± 0.9 0.41 ± 0.02 60 0.07 ± 0.03* 4 dnc1 rut1 3.2 ± 0.6 4.9 ± 0.7 18.2 ± 2.0 ND 20 0.36 ± 0.08 5 Nerve growth cones were observed between 16 and 48 hr after plating on uncoated glass coverslips. Filopodial length is given in micrometers and lamellipodial area in micrometers squared. For roundness and motility index, see text. The data include only growth cones with phase-light lamellipodia in type III monopolar neurons. *p < 0.001. ND, Not determined. Despite the similarity in morphology of the phase-light growth cones, striking differences in the motility of growth cone lamellipodia were evident when normal and mutant neurons were compared (Fig. 2; compare Fig. 8). In normal cultures, ~62% (23/37 in the above survey; see Table 1) of lamellipodia showed high motility, readily detectable at 5 sec intervals. Ruffle movement of lamellipodia and filopodial extension and retraction from their edges were observed at each 5 sec interval in normal cultures (Fig. 2, top). In contrast, growth cones of dnc alleles and rut neurons were relatively stable, showing greatly reduced motility (Fig. 2 for dnc1, dncM11, and rut1; compare Fig. 8 for dncM14 and rut1/KA9). Lamellipodial ruffling was not detected in dnc and rut growth cones during several 5 sec intervals. Only minor changes of lamellipodial shape could be detected after longer intervals of quiescence.
Fig. 2. Time-lapse video micrographs of growth cones from normal, dnc1, dncM11, and rut1 neurons. Video-enhanced phase-contrast images of growth cones were photographed from the video monitor screen. The first three frames for each growth cone were captured at 5 sec intervals and the last frame at 5 min after the first frame. Note the active lamellipodial ruffling and filopodial sweeping in the normal growth cone (top). Growth cones of rut1 and two alleles of dnc showed greatly reduced motility (bottom three rows). Scale bar, 5 µm.
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Fig. 8. Time-lapse video micrographs of dncM14, rut1/KA9, and dnc1 rut1 growth cones. Growth cones were photographed and displayed in the same way as in Figure 2. Note that active lamellipodial ruffling and filopodial sweeping were restored in the dnc1 rut1 double-mutant growth cone (bottom). Growth cones of both dncM14 and rut1/KA9 (heterozygote of rut1 over KA9, a chromosome bearing a deficiency of the rut locus) showed greatly reduced motility (upper two rows). Same magnification as in Figure 2.
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This can be visualized better with difference pictures (Kim and Wu, 1991a
Fig. 3. Difference pictures of growth cones of normal, dnc, rut, and double-mutant neurons. Areas in black, which represent lamellipodial expansion and retraction, were generated by superimposing two consecutive frames of a growth cone lamellipodium 5 sec apart. Note the reduced motility in the single mutants and the larger displacement of lamellipodia seen in both normal and dnc rut double-mutant growth cones.
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The dynamic changes of growth cones were quantified further by using a motility index. The area changes in difference pictures at 5 sec intervals were determined according to the following ratio: (area of expansion + area of retraction)/(area of earlier image + area of later image) (Kim and Wu, 1991a
Fig. 4. Motility index in normal, dnc, rut, and double-mutant growth cones plotted over time. The motility index was determined on the basis of consecutive difference pictures at 5 sec intervals (see Fig. 3). For each genotype, the sample included a total of 57-115 difference pictures based on three to five growth cones. Data points for individual growth cones are connected. The single mutants had similar motility indices, which were significantly lower than those of normal and double-mutant neurons (p < 0.001, Student's t test). Segments of two different time scales are presented to show the short-term and long-term changes.
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In contrast, throughout this study, we never found actively ruffling lamellipodia (558 cells in 24 cultures) in dnc or rut cultures, but observed only slow expansion and retraction (Fig. 3) with a motility index comparable to the that of inactive lamellipodia in normal cultures. The average motility index of lamellipodia was 0.10, 0.09, 0.07, and 0.07 for dnc1, dncM11, dncM14, and rut1, respectively (Fig. 4 and Table 2). Figure 4 shows examples of the time course for the motility index in growth cones from normal, dncM14, and rut1. Motility indices were obtained during a period of 12-13 successive 5 sec intervals and from several later frames collected at the same intervals for up to 12 min. The defects observed in the mutant strains are most likely attributable to the dnc and rut loci, because consistent phenotypes were seen in different independent isolates.
Effects of db-cAMP and forskolin on normal growth cones
Because dnc and rut affect the enzymes that regulate cAMP metabolism, it is of interest to determine whether acute application of pharmacological agents that perturb cAMP regulation in normal neurons would mimic the dnc and rut phenotype. The membrane-permeable cAMP analog db-cAMP and the drug forskolin, which stimulates AC, have been used widely in perfusion experiments. We found that motile lamellipodia in normal Drosophila neurons were very sensitive to db-cAMP. Figure 5 shows a series of time-lapse images observed during db-cAMP perfusion. The growth cone displayed high motility before the onset of perfusion (Fig. 5a,b) and was not affected by perfusion of normal medium (up to 200 µl/min in laminar flow; Fig. 5c,d). Within 2 min of 25 µ db-cAMP perfusion, the rapid lamellipodial movement declined gradually (Fig. 5e,f). Even though the morphology of the thin lamellipodia remained intact in the presence of 25 µ db-cAMP, a brief (4 min) exposure had a long-lasting effect on motility (Fig. 5g,h). In fact, high level motility did not recover within 2 hr after returning to normal medium. Only slow extension and retraction of lamellipodia (comparable to dnc growth cones shown in Fig. 2) were observed. Even 10 µ db-cAMP affects lamellipodial motility, and only partial recovery was obtained after a brief exposure of 4 min (data not shown). In contrast, a higher concentration of db-cAMP (50 µ) not only rapidly inhibited lamellipodial motility but also transformed growth cones into thick, club-shaped endings within 6 min (Fig. 5i,j).
Fig. 5. Arrest of growth cone motility in normal neurons by perfusion with db-cAMP. The growth cone of a normal neuron displayed high motility (arrowheads) before the onset of perfusion (a, b) and was not affected (arrowheads) by perfusion of normal medium (c, d). Within 2 min of perfusion with 25 µ db-cAMP, growth cone motility was severely retarded (e, f). Long after returning to normal medium, the arrest was not reversed (g, h; see text). Shown for a different growth cone (i, j), a higher concentration of db-cAMP (50 µ) caused not only motility arrest but also retraction of lamellipodia. For a-h, the onset of the first normal medium, 25 µ db-cAMP, and second normal medium perfusion was at 30 sec, 4.5 min, and 8.5 min, respectively. For i and j, 50 µ db-cAMP perfusion started at time 0.
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We also found that 50 µ forskolin had an effect similar to 25 µ db-cAMP (Fig. 6). A series of time-lapse images of a growth cone from a forskolin perfusion experiment in normal culture is shown in Figure 6. A high level of growth cone motility was maintained during a perfusion with standard medium (Fig. 6a,b). Shortly after switching to a medium containing 50 µ forskolin, however, lamellipodial movement decreased gradually and filopodia retracted. After 2 min of perfusion, lamellipodial ruffling was inhibited (Fig. 6c,d). Minor changes in lamellipodial shape could be detected only over longer time periods.
Fig. 6. Reversible arrest of growth cone motility in normal neuron by forskolin. Active ruffling of lamellipodia (arrowheads) during normal medium perfusion (a, b) was arrested soon after perfusion with medium containing 50 µ forskolin, a drug stimulating AC (c, d). Lowered growth cone motility by forskolin is similar to that seen in dnc neurons (see Fig. 2). Motility resumed (arrowhead) after wash by perfusion with normal medium (e, f). The onset of the first normal medium, 50 µ forskolin, and second normal medium was at2 min, 2 min, and 6 min, respectively.
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The motility was still suppressed long after reperfusion with normal medium (Fig. 6e,f). After 4 min of wash, slow expansion of lamellipodia was barely detectable (Fig. 6f); however, lamellipodial movement was not restored to normal level during a 2 hr observation period on returning to normal medium (not shown). Throughout this study, consistent arrest of motility by both db-cAMP and forskolin was observed in 33 growth cones from 25 independent cultures.
Effects of db-cAMP on rut growth cones
The above results indicate that abnormally high cAMP levels could lead to the arrest of growth cone motility; however, the observed retardation of growth cone motility in rut neurons (Figs. 2, 3, 4) suggests that an abnormally low level of cAMP synthesis could also cause a similar effect. To test whether restoration of an optimal level of cAMP could bring about normal growth cone motility, we applied db-cAMP and forskolin to rut growth cones in culture at the time of plating. Indeed, motility of growth cones was rescued, and clear lamellipodial movement could be observed in rut cultures chronically incubated with 1.5-2.5 µ db-cAMP for 40-56 hr. Twenty-two of 50 growth cones subjected to continuous observation for 2-10 min in 10 independent rut cultures showed active lamellipodial movement, which was readily detectable in 10 sec intervals (Fig. 7a-d). Ruffling movements of lamellipodia and extension and retraction of filopodia were evident. Most importantly, the recovery of growth cone motility assisted by db-cAMP in rut cultures seemed to require an optimal range of concentrations. When rut neurons were incubated with 5 µ or higher concentrations of db-cAMP, no clear recovery in motility was observed. Instead, growth cones showed phase-dark images. Thus, too high concentrations of db-cAMP when applied chronically to rut growth cones resulted in phenotypes resembling the effect of forskolin or high concentrations of db-cAMP on normal growth cones.
Fig. 7. Rescue of growth cone motility in rut neurons by db-cAMP. Growth cone motility in the rut neuron was restored, and clear lamellipodial movement could be observed when incubated with 2.5 µ db-cAMP from the time of plating for 24 hr. Ruffling movement of lamellipodia (arrowheads) was readily detectable at 10 sec intervals (a-c), and considerable changes in growth cone shape were seen after a longer period (d, 75 sec). e-l show the perfusion experiment for another growth cone. The rut growth cone did not respond to perfusion with normal medium (e, f). Within 1 min of 10 µ db-cAMP perfusion, rut growth cone became motile with rapid lamellipodial expansion (g, h) and filopodial sweeping (i, j). Motility subsided and the growth cone returned to the original state after the wash with normal medium perfusion (k, l).
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Another set of experiments used perfusion of db-cAMP to determine the response time of rut growth cones. Significantly, immobile rut lamellipodia responded rapidly to acute perfusion of db-cAMP-containing medium (~5.0-12.5 µ). Visible lamellipodial expansion began within 1 min after the onset of db-cAMP perfusion (Fig. 7g,h). The response reached the plateau within 2-3 min, showing lamellipodial ruffling and filopodial sweeping (Fig. 7i,j). On returning to normal perfusion medium, the growth cone activity subsided within 1 min and gradually retracted to the original immobile state (Fig. 7k,l).
In contrast to the results of db-cAMP, forskolin at concentrations between 5 and 12.5 µ never brought about active growth cone motility in rut cultures in chronic incubation or acute perfusion experiments. This is consistent with the previous observation that AC in rut adult homogenates fails to respond to forskolin stimulation (Dudai et al., 1985
Counterbalancing effects of dnc and rut on growth cone motility
The dnc gene codes for cAMP PDE, which hydrolyzes cAMP, whereas the rut locus codes for a Ca2+/calmodulin-sensitive AC that synthesizes cAMP. Even though the dnc and rut genes have an opposite effect on cAMP metabolism, as we described above, mutations of either gene result in greatly retarded motility of growth cone. With these data, it is desirable to obtain an independent line of evidence showing that an optimum level of cAMP is required to maintain normal growth cone motility. One key experiment, made possible by the use of genetics, was to pair the two mutations dnc and rut in a natural setting of double-mutant neurons and to look for compensation for the effect of abnormal cAMP levels caused by each single mutation.We examined the dnc rut double-mutant growth cones in culture. The morphology of dnc rut growth cones was almost the same as normal for several morphometric parameters, including number and length of filopodia and area of lamellipodia (Table 2). Interestingly, the motility of dnc rut growth cones was active enough to be detected in time-lapse observations with 5 sec intervals (Fig. 8, bottom). The recovery of motility by the counterbalancing effects of dnc and rut was clearly evident in difference pictures (Fig. 3, bottom) and the motility index over time (Fig. 4). The motility index of the double mutant was 0.36 + 0.08, which appears identical to that of normal cultures (0.36 + 0.09; see Table 2).
DISCUSSION
Growth cones in learning mutants
This is the first demonstration of altered growth cone properties in learning and memory mutants. We exploited a larval CNS culture system (Wu et al., 1983In addition, dnc and rut mutations also led to a darker appearance of phase-dark lamellipodia, which generally show little motility and are thought to be associated with neurons of older ages in normal cultures (Kim and Wu, 1991a
Reduced lamellipodial motility in dnc and rut neurons was not correlated directly to rate of neurite extension and neuronal growth. Although the growth rate of mutant neurons has not been determined using time-lapse recording, sampling of neuritic length in a large number of cultures of different ages indicated that the neurite extension rate (Kim and Wu, 1987
Modulation of synaptic efficacy as the physiological basis for learning has long been subjected to intensive investigation in several preparations (Kandel and Schwartz, 1982
Our findings show that the cellular machinery regulating growth cone behavior is demonstrably different in cultures of dnc and rut neurons. Defects in the same cellular machinery may influence neuronal development in vivo and contribute to the altered morphological plasticity observed previously in Drosophila. Both adult dnc and rut flies are known to have fewer fiber numbers in the mushroom bodies, which are involved in olfactory learning (Balling et al., 1987
Optimal levels of cAMP in growth cone regulation
Our findings suggest that normal lamellipodial motility in developing Drosophila neurons requires optimal cAMP levels with an operational range of dynamic control, as summarized below. First, either increased or reduced cAMP beyond a normal range in dnc and rut (Byers et al., 1981The exact mechanisms responsible for the restoration of growth cone motility in the double mutants await further studies: notably, certain phenotypes could be restored completely or partially, whereas others could be worsened in the double mutants. It is worth noting that even though the direction of concentration change in cAMP is opposite in the two mutants, dnc and rut growth cones display similar behavioral abnormalities, their neurons show similar aberrant excitability patterns (Zhao and Wu, 1994
Explanations for the apparent complexity in the actions of and interactions between rut and dnc may include several well established cellular mechanisms. It is known that many enzymes are compartmentalized and colocalized with downstream targets to mediate local physiological events. Some functional consequences of cAMP regulation may depend on its dynamic changes rather than its steady-state levels. Furthermore, compensatory interactions among second messenger systems may increase the complexity in the outcome of the cAMP regulation.
Several entry points to initiating studies of these problems have been suggested by previous observations. Rearrangement of actin filaments and microtubules has been implicated in the dynamics of lamellipodia and filopodia (Bray, 1991
cAMP, neuronal plasticity, and learning
It has been shown that genes encoding different forms of AC are differentially expressed in various regions of the vertebrate CNS (Mons and Cooper, 1995In other invertebrate systems, including Aplysia, the role of cAMP in several simple forms of learning has been well established (Byrne and Kandel, 1996
It will be of importance to determine whether the altered growth cone properties observed in dnc and rut cultures could be correlated to abnormal exploring behavior and growth pattern of developing neurons in vivo. Preparations for direct time-lapse observations in the developing Drosophila embryo exist in which growth cone motility and neurite growth may be observed continuously in situ (Halpern et al., 1991
The current culture system allows the first analysis of the defects in dnc and rut growth cones. Further development of this analysis may exploit mutations of additional genes and include phenomena related to interactions among different cell populations. High-density cultures can be initiated to study interactions among neurons of different genotypes or the process of synapse formation between different neuronal types, which may be vitally marked by using the green fluorescent protein reporter gene (Yeh et al., 1995
FOOTNOTES
Received March 19, 1996; revised June 25, 1996; accepted June 26, 1996.
This work was supported by National Institutes of Health grants to C.-F.W. and by a KOSEF-Korea Grant to Y.-T.K. We thank Mr. Peter Taft for technical assistance in data collection for Figures 5, 6, 7, 8, and Dr. Jeff E. Engel for comments on this manuscript. Some of the image processing was performed in the Cell Motility Core Facilities in the Department of Biological Sciences, University of Iowa.
Correspondence should be addressed to Dr. Chun-Fang Wu, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.
Dr. Kim's present address: Department of Life Science, Sogang University, One Sinsu Dong, Seoul 121-742, Korea.
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