Chemotactic Response of Nerve Fiber Elongation to Nerve Growth...

DEVELOPMENTAL BIOLOGY 66, 183-196 (1978)

Chemotactic Response of Nerve Fiber Elongation to Nerve Growth Factor

PAUL C. LETOURNEAU Department of Structural Biology, Sherman Fairchild Building, Stanford University School of Medicine,

Stanford, California 94305

Received March 1, 1978; accepted in revised form May 1, 1978

These studies seek to determine whether the locomotory behavior of the extending tip of growing nerve fibers exhibits chemotaxis to nerve growth factor (NGF). Sensory neurons from dissociated dorsal root gangha of chick embryos were cultured within semisolid agar matrices containing concentration gradients of NGF which diffused from an adjacent source. A preferential orientation of approximately 60% of the tips of nerve fibers and an enhanced extension of fibers up NGF gradients were observed. The initiation sites of nerve fibers on cell bodies were not consistently oriented by these NGF gradients. Orientation of nerve fiber extension toward the NGF source was observed in chambers with NGF sources ranging from 25 to 1000 rig/ml of /3- NGF and from 400 to 5000 rig/ml of 7 S NGF, but no orientation was observed when the concentration of the NGF source was 15000 rig/ml of fi-NGF. This oriented response apparently is not a concentration-dependent trophic response to NGF. The evidence supports the notion that chemotaxis is a regulatory factor in neuronal morphogenesis.

INTRODUCTION

A cardinal feature of the various cell mi- grations which occur during embryogenesis is their directionality, reproduced from one individual to the next (Trinkaus, 1976). This directionality may be attributed to intrinsic cellular properties or to extrinsic influences such as other cells and extracellular physicochemical features. In vitro studies have examined the influence of several factors on direction of cell movement. Contact inhibition of cell movement illus- trates a cell-cell interaction which confers directionality to cell locomotion (Aber- crombie, 1967). The adhesive interactions of cells with extracellular surfaces also influence cell migration, whether by contact guidance or by movements in response to regional variations in strength of cell-substratum adhesion (Carter, 1965; Harris, 1973; Rovensky and Slavnaya, 1974; Weiss, 1961). The notion that morphoge- netic cell movements exhibit chemotaxis, that is, orientation of migration relative to

a concentration gradient of soluble chemi- cals, arose from early investigations. How- ever, in few cases has evaluation of the actual locomotory behavior of cells supported the concept that chemotaxis ac- counts for directionality (Trinkaus, 1969).

Two bona fide examples of chemotaxis are the movements of motile bacteria relative to certain molecules and the aggregation of the amoebas of cellular slime molds (Adler, 1976; Bonner, 1947; Gerisch et al., 1975). Leukocytes from mammals are at- tracted by bacterial products and components of the complement series (Schiffman et al., 1975). Although this has obvious physiological value, the complex milieu of vertebrate organs does not favor elucida- tion of the precise role of chemotaxis in this or other vertebrate cell movements by in vivo experimentation.

In spite of past negative results concern- ing chemotaxis (Weiss and Taylor, 1944), recent reports state that extension of nerve fibers in vitro from ganglia explanted from embryos is directed toward pieces of tissues

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0012-1606/78/0661-0183$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

184 DEVELOPMENTAL BIOLOGY VOLUME 66,1978

innervated by these ganglia (Charnley et al., 1973; Chamley and Dowel, 1975; Charl- wood et al., 1972; Coughlin, 1975; Ebendal and Jacobson, 1977). In addition, the extension of fibers is enhanced on that side of an explanted peripheral ganglion which is clos- est to a capillary tube containing high concentrations of nerve growth factor, NGF (Charlwood et al., 1972; Ebendal and Ja- cobson, 1977). These results have prompted the hypothesis that the direction of nerve fiber extension is controlled by chemotactic responses. NGF is postulated to be an attractant in uiuo, as it is found in many tissues innervated by NGF-responsive neurons (Johnson et al., 1971; Levi-Montalcini, 1976; Murphy et aZ., 1977).

However, these results on nerve growth are open to other interpretation. In no case was a concentration gradient of an attractant described. Besides the general neces- sity of this demonstration to a study of chemotaxis, it is particularly cogent to studies which involve NGF. Since nerve growth factor is reported to have multiple trophic influences on cultured sensory and sympathetic neurons in a dose-dependent manner (Angeletti et al., 1965; Chun and Patterson, 1977; Greene, 1977a,b; Herrup and Shooter, 1973; Levi-Montalcini et al., 1972; Mizel and Bamburg, 1976), it is difficult to distin- guish between a trophic response and a chemotactic response of nerve fiber growth without description of the NGF gradient plus evaluation of response over a range of NGF concentrations. The reports of directed growth of nerve fibers toward target tissues rely heavily upon end results to support the contention that neurite extension exhibits chemotaxis. Similar end results could occur if fiber growth were ran- domly directed, but fibers adhered differ- ently to different tissues, or preferentially survived, if connected to one tissue but not another. Alternatively, a gradient of NGF or attractant might influence the point of origin on the cell soma of a neurite, and, even if subsequent elongation were ran- domly directed, the reported result could

be observed, because of a preferential orientation of the initiation site. The use of whole ganglia further complicates elucida- tion of the influence of concentration gradients upon the initiation of neurites and the process of neurite elongation. The possibility of directive influences contributed by the dense packing of neurons within ganglia, the close association of neurons with Schwann cells and satellite cells (Le- tourneau, 1975a; Luduefia, 1973), and the interactions of nerve fibers with each other and with non-neuronal cells (Dunn, 1971; Ebendal, 1976; Letourneau, 1975a,b) pre- clude unambiguous evaluation of a single element of locomotory behavior, such as chemotaxis.

In an attempt to clarify these problems the studies reported here assess the locomotory behavior of the growing tips of nerve fibers extending from single dissociated neurons in the presence of concentration gradients of nerve growth factor. In order to reduce turbulence that might de- stroy a gradient and also to observe unam- biguously the neurites extended by single cells the neurons were cultured in semisolid matrices of agar and culture media. The results indicate that the extension of nerve fibers by sensory neurons from chick embryos does exhibit chemotaxis.

MATERIALS AND METHODS

Media and solutions. Tissue culture medium was Ham’s F12 (GIBCO) buffered by bicarbonate and supplemented with 50 U/ml of penicillin and streptomycin plus 10% by volume of fetal calf serum (desig- nated F12 SlO). Ganglia were rinsed before dissociation in Ca”, Mg2+-free Hanks’ salt solution buffered at pH 7.4 with sodium bicarbonate. Bactotrypsin (Difco) was used for trypsin dissociation of sensory ganglia. Agar flakes (Difco, Noble agar) were dis- solved in Hanks salts to make a stock solution of 3% agar. The agar was liquified by boiling and held at 60°C until added to culture media. P-NGF, 7 S NGF, and 1251- labeled NGF were generous gifts from Dr.

PAUL C. LETOURNEAU Chemotaxis in Nerve Elongation 185

Eric Shooter. NGF was kept frozen in a stock solution of 0.02% acetic acid containing 1 mg/ml of bovine serum albumin. The concentration of NGF in stock solutions varied from 1 to 2500 ng/pl.

Culture chamber. Chambers for the culture of sensory neurons in gradients of NGF were made by glueing together two 25 x 75- mm glass slices on three edges with silicone cement (Dow-Corning); see Fig. 1. A space of 2 mm separated the slides. The cement was air-dried overnight. The chambers were washed three times with 70% alcohol, then three times with sterile water, and were left filled with water overnight.

Dissociation of dorsal root ganglia. Dor- sal root ganglia were dissected from 8-day chicken embryos and dissociated into single cells by incubation in Ca’+, M8+-free Hanks’ salts containing 0.2% Bactotrypsin (Difco), as described in Letourneau (1975a). After centrifugation to remove the trypsin, the cells were resuspended in F12 SlO warmed to 4O”C, and 1 part 3% agar was added to 8 parts culture medium with cells to obtain a final agar concentration of 0.33%. In many cases a low concentration of NGF was included with the cells to promote initial cell survival, as explained under Re- sults. This suspension was rapidly mixed and pipetted into the culture chamber as described in the following paragraph.

Addition of NGF, agar, and cells to culture chambers. Polyethylene tubing was inserted into the bottom of the chamber,

FIG. 1. The chamber in which neurons were cultured with gradients of NGF. Two glass slides were glued together along the edges, leaving one side open for introduction of agar solutions. There was no agar- free space between the NGF source and the cell matrix; however, an interface was visible between the two compartments of the matrix.

and 0.45 ml of a solution containing F12 SlO, 0.33% agar, and NGF was pipetted through the tubing into the bottom of the chamber. The tubing was withdrawn and the chamber was set upright to form a horizontal surface (the future interface) at the top of the NGF-containing agar matrix (this is named NGF source in the following text). When this matrix had become semisolid, 1.8 ml of a suspension of 3 X lo5 cells in warm F12 SlO plus 0.33% agar and any other components was added to the chamber (this will be called the cell matrix). The chamber was set horizontally and incu- bated at 37°C in a 5% COZ humidified incubator (Fig. 1).

Assessment of orientation of nerve fiber elongation. The influence of concentration gradients of NGF upon the extension of neurites was assessed in several ways. All observations were done with a Wild M40 inverted microscope equipped for phase contrast. One type of measurement was a determination of the point of origin on the cell soma of neurites relative to the NGF source. An eyepiece containing an ocular grid was rotated to align one set of grid lines perpendicular to the direction of the NGF gradient. After centering one of these lines on a nerve cell body, a neurite initiation point was scored as being toward or away from the NGF source, depending on its position on the spherical cell body relative to the grid line passing through the center of the cell.

A second type of measurement involved the orientation of the tips of nerve fibers. This was determined as being toward or away from the NGF source, also using the ocular grid. The tip was defined as the terminal 15 pm of the nerve fiber, and care was taken to score the orientation of the tip of the fiber only and not thin filopodia which are transient structures extended from the tip. A fiber was not scored unless it was longer than 15 pm, so as to reduce confusion with thin filopodia extended from a cell soma. All neurites within successive microscope fields were scored as the cham-


ber was scanned perpendicular to the direction of. the gradient. The diameter of a microscope field with a 10x objective and 10X ocular was 1.3 mm.

Another measurement was determination of whether the tip of a nerve fiber was situated up the NGF gradient or down the gradient relative to the initiation point of the fiber on the cell soma. This was done by orienting one set of orthogonal grid lines within the eyepiece perpendicular to the direction of the NGF gradient and position- ing the initiation point of a neurite along one of these lines. The tip of the neurite was then scored as being either on the side of the line toward the NGF source or alternatively on the side away from the NGF source. This determination was made without regard to the orientation of the nerve tip itself, which was determined as described in the previous paragraph.

The final determination involved measurement of the net distance traveled from the cell body by a growth cone either toward the NGF source or away from the source. This was defined as the distance, measured parallel to the concentration gradient, between the position of the initiation point and the nerve tip, as depicted in Fig. 2.

RESULTS

Demonstration of Concentration Gra- dients of NGF

In order to show that gradients of NGF are established in these cultures, chambers were prepared containing lz51-labeled p- NGF, of specific activity 55 cpm/pg, mixed with unlabeled P-NGF to adjust the specific activity of the mixture in the NGF source to lo6 cpm/ml. Chambers were prepared with NGF concentrations of 100 and 1000 rig/ml in the NGF source and were incu- bated for 24 to 72 hr in a CO2 incubator at 37°C. After incubation the agar was frozen by placing the chamber on dry ice, the chamber was cut open, the frozen agar matrix was cut into 2-mm slices with a tube gel slicer, and the slices were counted in a

FIG. 2. Illustration of how the distance between a nerve tip and its origin was measured relative to the direction of an NGF gradient. For illustrative purpose, assume the direction of the gradient is horizontal. Rather than measure the actual lengths of these fibers, the lines A, B, and C were recorded.

Beckman gamma counter. The graphs presented in Fig. 3 illustrate

that gradients of NGF are present after 24 hr of incubation and remain for at least 72 hr. It was noted that the gradient is shal- lower at 72 hr compared to 24 hr. Also, there is a change in slope at the distal end of the gradient, further from the NGF source. This may reflect the presence of a small fraction of the 1251 radioactivity in the form of free iodide ion which was not separated from NGF following iodination. The average diffusion rate of the smaller iodide ion is over 10 times greater than that of a P-NGF molecule. Hence, the distribution of 1251 at the distal end of the chambers may be the more mobile iodide ion, present as a minor component of the total radioactivity.

Growth of DRG Neurons in Agar and Re- sponses to NGF Gradients

The extension of nerve fibers from sensory neurons suspended in an agar matrix containing NGF has been described by Strassman et al. (1973). As depicted in Fig. 4, nerve fibers extended into the agar matrix are crooked and often depart greatly from the initial direction of fiber growth, even in the absence of a gradient in NGF concentration. The fraction of neurons


ol 2 4 6 8 IO 12 14 16 IS 20 22 24

mm from NGF source

38

0’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 2 4 6 8 IO I2 I4 16 I8 20 22

mm from NGF source

FIG. 3. Concentration gradients of “?-labeled /3-NGF established in these agar matrices. The radioactivity within a 2-mm-thick slice of the cell matrix is expressed as a percentage of the radioactivity within slices of the NGF source and is plotted as a function of the distance of the slice from the NGF source-cell matrix boundary. (A) From chambers with an NGF source of 100 rig/ml of j?-NGF; (B) f rom chambers with an NGF source of 1000 rig/ml of /3-NGF. (A) 24-hr incubation; (0) 72-hr incubation.

which extend neurites and the rate of neurite elongation within agar is much lower than for neurons cultured on plastic or polyornithine-coated plastic in liquid media (Letourneau, 1975a; Ludueria, 1973). The fraction of neurons which extended fibers into agar was not determined here because neurons cannot be distinguished from the unspread non-neuronal cells in the agar (Strassman et al., 1973). As shown in Figs. 5A-8A the number of fibers present contin- ued to increase after 24 hr and through 72 hr.

If NGF was not added to that portion of the matrix containing cells when the chamber was set up, fewer fibers were found at the distal end of the chamber than proxi- mally nearer the NGF source. This was most evident when low concentrations of NGF were present in the source, 106 rig/ml of P-NGF or 400 rig/ml of 7 S NGF (Fig. 6a). Apparently, many of the neurons farther from the NGF source die or lose the

capacity to extend neurites, because they are not furnished a minimum amount of NGF in the first few hours of in vitro culture as NGF diffuses from the source. When 0.7 rig/ml of /3-NGF or 5 rig/ml of 7 S NGF was added to the agar mixture containing neurons at the beginning of culture, this decrease in fibers with distance from the NGF source was much reduced (Figs. 7A and 8A).

Orientation of Nerve Fiber Tips

Figures 5B-8B illustrate results of the determination of the orientation of nerve tips. The percentage of nerve tips oriented toward the NGF source is expressed as a function of distance from the source. Table 1 presents the same data plus those from other experiments; however, the determinations made at different distances from the NGF source are pooled. As seen in Table 1, if NGF is added to the cell matrix in the same concentration as in the NGF

188 DEVELOPMENTAL BIOLOGY VOLUME 66,19X3

FIG. 4. Camera lucida drawings of sensory neurons cultured within an agar matrix. These nerve fibers are extended in three dimensions. X 400.

source, approximately 50% of the nerve tips source, suggesting the long-range stability are oriented toward the NGF source. This of these matrices. At the high NGF source indicated that the extension of neurites is concentration of 15 pg/ml there was no random in these chambers in the absence orientation of fiber tips toward the NGF of a NGF gradient. When a wide range of source. This is similar to the observed ab- concentration gradients of NGF was made, sence of bacterial chemotaxis at saturating however, growth cones tended to point to- levels of chemoattractant (Mesibov et al., ward the NGF source (Figs. 5B-8B and 1973). Table 1). Approximately 60% of nerve tips Nerve tips do not show a preferential were oriented up the gradient in a series of orientation up a gradient of another protein chambers containing between 25 and 1000 diffusing from the source matrix. As shown rig/ml of /?-NGF and from 400 to 5000 in Table 1, the orientation of nerve tips is rig/ml of 7 S NGF in the NGF source. This random when the source matrix contains 5, orientation toward the NGF source was 50, or 2000 rig/ml of the egg white protein, observed as far as 23 mm from the NGF ovalbumin.

120-

PAUL C. LETOURNEAU Chemotaxis in Nerve Elongation

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189

. I I I I 1 I I I I LLULLU- L----U- 2 4 6 6 IO 12 14 16 I6 2 4 6 6 IO I2 14 I6 18 2 4 6 6 10 12 I4 I6 16

dittancs (mm1 distance hm) distance (mm)

FIGS. 5-8. (A) The number of tips counted in an equivalent volume per row; (B) the percentage of neurite tips directed toward the NGF source; and (C) the percentage of initiation sites directed toward the NGF source, all plotted as a function of distance from the NGF source at (0) 24, (0) 48, and (A) 72 hr.

FIG. 5. NGF source: 1606 rig/ml of /3-NGF. Cell matrix: no NGF.

:i-::_-; :if;:,;i, CJ/;;;l?;;;

2 4 6 6 IO 12 I4 16 distance (mm) distance (mm) distance (mm)

FIG. 6. NGF source: 106 rig/ml of /3-NGF. Cell matrix: no NGF.

This response of nerve tips is certainly sensitive to NGF, as it involves orientation relative to nanogram levels of NGF in the presence of milligram levels of serum proteins. The sensitivity of this response to NGF was further illustrated by adding a high concentration of insulin to the cell matrix and source matrix, besides the NGF added to the source matrix. NGF and insulin possess some structural similarities and may interact with similar cell surface receptors (Frazier et al., 1972, 1974). As seen in Table 1, the presence of 1 pg/ml of insulin does not eliminate orientation of nerve tips toward an NGF source with a concentration of 25 rig/ml.

Orientation of nerve tips toward the NGF source was observed even at the distal portion of chambers containing a source con-

centration of 25 rig/ml. At these distances from the source the NGF concentration range was 2.5 rig/ml and less (Fig. 8b), which is 10-l’ M! This displays a greater sensitivity to chemoattractant than other chemotactic responses. For example, the threshold concentration of n-formyl methi- onyl phenylalanine for leukocyte chemotaxis is lo-’ M (Mesibov et al., 1973; Schiff- man et al., 1975).

Orientation of Initiation Points

As seen in Figs. 5C-8C and in Table 1, it is unclear whether the point of origin of nerve fibers was affected by NGF gradients. Measurements at 24 hr when the gradient is steeper may indicate that initiation points are oriented toward the NGF source (Table 1). However, when the orientation

190 DEVELOPMENTAL BIOLOGY

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FIG. 7. NGF source: 100 rig/ml of P-NGF. Cell matrix: 0.7 rig/ml of /3-NGF.

of initiation points was determined at 72 hr the results appeared similar to those of cultures with no NGF gradient.

Position of Nerve Tip Relative to Initiation Site

If the orientation of a nerve tip actually is indicative of the direction of neurite extension, then one would conclude from Ta- ble 1 and Figs. 5B-8B that nerve tips should tend to be situated up an NGF gradient relative to their initiation points on cell bodies. This conclusion was tested by the second determination of neurite response to NGF gradients. Table 2 presents measurements in chambers with an NGF source of P-NGF concentration 25 rig/ml. Sixty percent or more of nerve tips had moved up the NGF gradient relative to their initiation noints.

point. This distance was measured as shown in Fig. 2. The data in Table 3 show that tips of fibers extended up the gradient tend to be farther from their initiation point, along a line parallel to the gradient, than tips of fibers extended down the gradient. Although 62% of the total population of fiber tips were observed to be extended toward the NGF source after 46 hr, those tips which had been extended more than 160 pm from their initiation points were even more predominantly oriented toward the NGF source. It was not determined whether this difference in distance from initiation point to tip was due to a greater length of those fibers extended up the gradient or a more directed orientation of the fibers along the gradient.

NGF Dose Dependency of Elongation Rate

The final determination of the influence The length of fiber extended up a gra- of NGF gradients on nerve fiber elongation dient may exceed the length extended down was measurement of the distance traveled a gradient because of dose-dependent by a nerve tip up or down the gradient trophic effects on the rate of axon elonga- relative to the position of the initiation tion at different concentrations of NGF. In


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FIG. 8. NGF source: 25 rig/ml of P-NGF. Cell matrix: 0.7 rig/ml of /3-NGF.

order to test this idea, sensory neurons were plated in tissue culture dishes with F12 SlO (as in Letourneau, 1975a) containing NGF concentrations between 0.1 and 1000 rig/ml, which represent the range of concentrations present in these chambers. As seen in Table 4 the average length of nerve fibers after 22 hr of culture was similar at all NGF concentrations tested. This suggests that the rate of nerve fiber elongation is not enhanced by elevation of NGF concentrations above 0.1 rig/ml.

DISCUSSION

The following are the results of these experiments. (1) Concentration gradients of NGF are established in these agar matrices. (2) The tips of neurites tend to point up the concentration gradient of NGF. (3) More nerve fibers are extended up the gradient from their point of origin than down the gradient. (4) The average distance which nerve fibers are extended up the gradient from their point of origin exceeds the average distance extended down the gradient. (5) The response is sensitive to NGF in the presence of excess proteins and occurs at

concentrations as low as or lower than the thresholds for other types of chemotaxis.

Is this truly a chemotactic response? Does it involve the locomotory activities of the growing tips of nerve fibers? Is it a response only to the concentration gradient of NGF and not a function of the trophic effects of NGF? Establishment of cultures of dissociated cells, suspended in agar, sim- plifies assessment of the locomotory behavior of nerve tips relative to an NGF gradient. This demonstrated orientation of nerve tips toward the NGF source and the enhanced extension of fibers up a gradient, compared to down a gradient, are consistent with a chemotactic response of the locomotory activities associated with nerve fiber elongation. The initiation point of a fiber does not necessarily determine the ultimate directions of elongation of the fiber, as seen in tracings of neurons (Fig. 4) and from the difference in the influence of NGF gradients on the point of origin of neurites on cell bodies versus the behavior of the elongating nerve tips.

The possibility that this oriented response is due to differential trophic effects


TABLE 1

ORIENTATION OF NERVE FIBER TIPS AND INITIATION POINTS RELATIVE TO NGF SOURCE" Addition to NGF source

(w/ml) Addition to ceII matrix

(w/d) Time (W

Tips oriented to- Initiation points ward NGF toward NGF

(%) (%)

1. /3-NGF: 15,009 /3-NGF: 0.7 24 51 (912)b - 48 50 (546) -

2. /3-NGF: 1,000 0 21 54 (117) 43 (107)h 72 60 (428) 49 (274)

3. fi-NGF: 100 0 22 57 (77) 56 (77) 72 60 (263) 51 (183)

4. B-NGF: 100 /3-NGF: 0.7 25 59 (140) 50 (125) 72 60 (1,616) 53 (1,132)

5. /T-NGF: 25 /3-NGF: 0.7 23 61 (204) 57 (185) 72 60 (2,229) 52 (1,697)

6. &NGF: 25 fi-NGF: 0.7 48 61 (218) - Insulin: 1,000 Insulin 1,000 72 59 (484) -

7. P-NGF: 25 P-NGF: 25 24 50 w4 49 (472) 48 50 (492) 50 (363)

8. OvaIbumin: 5 /?-NGF: 0.7 24 48 (127) - /3-NGF: 0.7 70 50 (350) -

9. OvaIbumin 50 /3-NGF: 0.7 24 51 (246) - /3-NGF: 0.7 70 50 (535) -

10. OvaIbumin 2,000 ,&NGF: 0.7 24 52 (138) - P-NGF: 0.7 70 51 (429) -

11. 7 S NGF: 5,000 0 24 56 (121) 58 (95) 70 60 (305) 50 (195)

12. 7 S NGF: 400 0 24 53 (104) 46 (85) 72 60 (591) 51 (343)

13. 7 S NGF: 250 7SNGF:250 24 52 (137) 46 (100) 48 51 (257) 49 (170)

a Sensory neurons were cultured in agar matrices containing NGF plus other additions to the NGF source and to the ceII matrix at levels specified above. Determinations were made as described under Materials and Methods. The data include neurons situated at the boundary with the NGF source to distances of 12-23 mm from the NGF source.

b Value in parentheses is number of tips (points).

at different concentrations of NGF should which exceed the levels at which NGF stim- be eliminated. The stimulation by NGF of ulates neurite formation by chick neurons neurite formation by dissociated chick em- in a dose-dependent manner (Greene, bryo neurons can be expressed in a 1977a,b; Mobley et al., 1977). Apparently, dose-response curve with a plateau in re- there is no evidence of dose-dependent sponse observed above 1 rig/ml of /3-NGF trophic responses by dissociated chick neu- (Greene, 1977a,b; Mobley et al., 1977). rons to NGF in the concentration ranges There is no dose-dependent increase in vol- where the chemotactic response reported ume of nerve cell body above 0.5 rig/ml of here occurs. The survival, growth, and dif- P-NGF (Greene, 1977a), and Table 4 indi- ferentiation of cultured neonatal rat sym- cates there is no dose-dependent stimula- pathetic neurons are stimulated by NGF in tion of the rate of neurite elongation above a dose-dependent manner (Chun and Pat- 0.1 rig/ml of P-NGF in the first 22 hr of terson, 1977). Saturation of these NGF ef- culture. The preferential orientation of neu- fects were achieved at doses higher than rite elongation relative to NGF gradients those for chick neurons, however, the rat occurs over a wide range of NGF concen- neurons were cultured for much longer pe- trations (25-1000 rig/ml of P-NGF), all of riods than the chick neurons. This question


would be answered unequivocably if NGF could be modified to eliminate its trophic activity while retaining its chemotactic properties.

TABLE 2

EFFECT OF NGF GRADIENT ON POSITION OF NERVE TIP RELATIVE TO INITIATION POINT”

Experiment Time (hr)

Tips situated toward NGF source

@) 1 25 60 (f4Qb

46 62 (87) 2 24 61 (109)

72 fx @Jo)

a Sensory neurons were cultured in agar chambers with 25 rig/ml of fi-NGF as NGF source concentration and 0.7 rig/ml of P-NGF added to the cell matrix. After 24-72 hr in vitro the position of the growing tips of nerve fibers relative to their initiation point was scored as toward or away from the NGF source as described under Materials and Methods. All neurons were scored within a number of microscope fields situated between 1 and 15 mm from the NGF source.

b Value in parentheses is number of tips.

TABLE 3

RELATIONSHIP BETWEEN DIRECTION OF NEURITE EXTENSION AND DISTANCE EXTENDED PARALLEL TO

NGF GRADIENT“

Time Distance extended (hr)

Percentage of parabel to gradient group situated up

b.4 gradient

25 O-40 48 (42)b 25 40-80 50 (18) 25 80-120 a2 (11) 25 >120 76 (17)

Total 60 (88)

46 S-80 58 (33) 46 80-160 48 (27) 46 160-240 73 (11) 46 >240 88 (1’3

Total 62 (87)

a Sensory neurons were cultured in a chamber containing 25 rig/ml of fi-NGF in the NGF source and 0.7 rig/ml of /3-NGF in the cell matrix. The distance, parallel to the NGF gradient, between the tip of the nerve fiber and its origin on the cell soma was measured for a sample of cells (see Fig. 2). Measurements were made after 25 and 46 l-u of incubation.

’ Value in parentheses is number of tips counted.

TABLE 4

EFFECTS OF NGF CONCENTRATION ON NEURITE LENGTH AND LENGTH OF NEURITE/NEURON AFTER

22 HR IN VITRO”

NGF concert- Length of fiber Length of tration fiber/neuron (w/ml)

(pm) h-4

0.1 69 f 47 (77)* 102 f 64 (52) 0.5 79 + 52 (69) 108 + 66 (51) 1.0 71 -t 52 (78) 107 + 72 (51)

10 72 f 56 (74) 101 + 63 (53) 100 66 f 48 (74) 93 + 51 (52)

1000 73 f 71 (92) 112 f 88 (60)

’ Sensory neurons were plated in duplicate 35mm tissue culture dishes containing F12 SlO, supplemented with /3-NGF at levels between 0.1 and 1000 rig/ml. After 22 hr of incubation neurite lengths were measured for all neurons within several microscope fields from equivalent areas of the dishes. The mean length of individual fibers and the mean total length of fiber/cell are presented above.

b Mean +- SD. Value in parentheses is number of fibers (fiber/neuron) measured.

It is unclear why the magnitude of this oriented response of neurite elongation is not greater; approximately 60% of the fibers oriented up the gradient versus 50% if there were no chemotaxis. It may be that the rate of change of NGF concentration, that is, steepness, in these in vitro gradients is less than what may exist in vivo and may be more difficult to detect. These in vitro gradients are determined solely by diffusion from the source, while concentration gradients in vivo could be modified after release of attractant from a target source, through further synthesis, inactivation, or degradation of the attractant by local cell populations and microenvironments. Alter- natively, a fraction of the neurons in these cultures may be incapable of chemotaxis, either naturally or because of restrictions imposed by the in vitro environment. The ventrolateral neurons of sensory ganglia, which are thought not to respond to NGF, may not be sensitive to NGF gradients. Dissociation with trypsin may have com- promised the chemotactic ability of sensory neurons. Chemotaxis might also be reduced because of the slow rate of neurite extension in agar. The data in Table 3 suggest that


neurites which have extended greater distances show stronger indication of chemotaxis. The tips of nerve fibers will adhere to and grow along previously extended “pi- oneering” fibers in a process termed fasic- ulation (Lopresti et al., 1973; Nornes and Das, 1972; Weiss, 1941). If these “pioneer- ing” fibers were capable of chemotaxis, neurites which fasciculated along these “pi- oneers” would not need chemotaxis to extend up a gradient. In a similar vein, nu- merous other influences within an embryo may be coupled and integrated with a chemotactic response similar to that recorded in these artifical matrices, so nearly all fibers extend in the same direction. Dif- ferences in cell packing and associations within ganglia, interactions of nerve fibers with extracellular surfaces, and contact interactions of nerve fibers and their growth cones are all reported to influence nerve fiber elongation. Finally, it is difficult to compare the degree of this oriented response to other in vitro studies of chemotaxis, because they report chemotactic response in terms of cell number only and not as a percentage of the total population involved (Gallin and Rosenthal, 1974; Mesi- bov et al., 1973, Schiffman et al., 1975). The 60% orientation of neurites toward NGF reported here is similar in degree to the chemotactic response of leukocytes to bacteria (Ramsey, 1972).

Studies of chemotaxis in bacteria indicate how chemotaxis might operate. The presence of chemoattractant is detected by binding sites on the bacterial surface, and gradients are sensed by comparison of the degree of saturation of receptors with attractant at successive time points. Move- ment relative to a gradient is achieved by modulation of the tumble frequency during bacterial swimming (Adler, 1976).

Brief speculation can be made about the mechanism of chemotaxis in nerve fiber extension. Cell surface receptors for NGF are reported on cells from sympathetic and sensory ganglia. However, the reported affinities of these receptors do not agree and

are probably very dependent upon the tech- niques of measurement (Banerjee et al., 1973; Frazier et al., 1974; Herrup and Shooter, 1973; Sutter et al., 1977). The fact that orientation of neurites does not occur when a high source level of NGF is used (see Table 1) is consistent with the possibility that NGF is detected for chemotaxis by a saturable receptor, (Mesibov et al., 1973), such as the NGF receptors refer- enced above. There is other evidence which suggests that nerve tips possess NGF receptors (Campenot, 1977; Hendry et al., 1974; Stockel et al., 1974).

How do neurons detect a gradient of NGF? The “bacterial” method of temporal comparison is possible, but a spatial comparison at different points along a neurite which extends a hundred micrometers or more can also be considered. To answer this question we must know the distribution and affinities of NGF receptors on cultured neurons. The information about a gradient must be transmitted to the locomotory ap- paratus in some way. One suggestion has involved alterations in ion transport which follow binding of a chemotactic factor (Nac- cache et al., 1977).

The locomotory activities which produce neurite elongation might be modulated at several points to effect chemotaxis toward NGF. The extension of microspikes from the nerve tip is the first event of the locomotory cycle and might be influenced by chemical gradients (Gerisch et al., 1975; Ramsey, 1972); the adhesive interactions of microspikes and the nerve tip with other surfaces might be locally affected (Letour- neau, 1975b, 1977; Schubert and Whitlock, 1977); or the assembly of microtubules or other neurite structures may be regulated to produce directed growth of neurites (Hier et aZ., 1972; Menesini Chen et al., 1977; Yamada and Wessells, 1971).

In summary, these experiments indicate that the direction of nerve fiber elongation is influenced by concentration gradients of NGF. This response is very sensitive to NGF; it occurs in the presence of 10% serum

PAUL C. LETOIJRNEAU Chemotaxis in Nerve Elongation 195

and has a lower threshold than several other chemotactic responses. Trophic effects of NGF do not seem to be involved in the response. These data support the contention that this is a valid example of chemotaxis. A number of tissues in young mammals contain NGF at levels at which chemotaxis is demonstrated in these studies (Johnson et al., 1971). One function of NGF in these tissues may be as a chemoattractant to guide the elongation of nerve fibers toward synaptic targets during embryogenesis.

The author thanks Dr. Norman Wessells for sug- gestions to improve the manuscript and acknowledges helpful discussions with Dr. Arne Sutter. This work was supported by an institutional grant from the American Cancer Society and by Special Grant No. 869 from the California Division, Inc., of the American Cancer Society.

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