Developmental Brain Research, 4 (1982) 361-364 361 Elsevier Biomedical Press

Target tissue control of nerve fiber growth rate and periodicity in vitro

WILLIAM L. MUHLACH and EMANUEL D. POLLACK

Institute for the Study of Developmental Disabilities and Department of Biological Sciences, University of lllinois, 1640 West Roosevelt Road, Chicago, IL 60608 (U.S.A.)

(Accepted March 1st, 1982)

Key words: spinal cord - - dorsal root ganglion - - neurite growth - - neurite growth rate - - growth cycles - - effect of targets

Spinal cord and dorsal root ganglion nerve fibers of tadpoles elongate in vitro at an increased rate in the presence of target tissue or target-conditioned medium. These neurites extend in a cyclic fashion with the period between the occurrence of maximal growth rates being under target influence. Rate parameters may be an important feature in the understanding of directed nerve growth during development and regeneration.

Target tissues for elongating nerve fibers (neuri- tes) in tissue culture have been demonstrated to influence neurite growth and survival 1~,16,2°. One dynamic characteristic of actively extending nerve fibers that has had spurious attention is that of growth rate parameters, and particularly of influen- ces exerted by nerve targets on growth rate. Analysis of nerve fiber growth rates may assist in explaining target-regulated phenomena of developing and rege- nerating nerves. Time-lapse films of extending nerve fibers derived from frog (Rana pipiens) tadpole spinal cord and dorsal root ganglion (DRG) explants demonstrate that: (i) target tissue presence greatly accelerates the rate of nerve fiber extension; (ii) activity at the nerve growth cone is increased in the presence of target tissue; (iii) effects of the appropriate target tissue are similar regardless of the origin of the nerve fibers; and (iv) the extension rate of actively advancing neurites is cyclic with periodi- city being a function of target tissue presence.

Spinal cord, D R G and limb target tissues were explanted from tadpoles at developmental stages of optimal growth interactions14,1L Spinal cord explants of immature stage V tadpoles 19 were co- cultured with largely undifferentiated stage V mesenchymal limb tissue, and stage XI D R G was co-cultured with stage XI muscular limb tissue

according to previously published methods 10. Twenty-four hour time-lapse cinephotomicrography revealed that the mesenchymal limb target elicited an increased rate of spinal cord neurite extension that was 4.25 times greater than that of control cord cultures lacking the limb tissue. An increase of similar magnitude occurred for the D RG neurites grown in the presence of the more mature limb target (Table I). In both instances, the level of filopodial and lamellipodial activity at the growth cone was markedly enhanced, although quantifi- cation of this observation has not been performed. Thus, the effects of the appropriate target tissue on neurite growth rates of central as well as peripheral nerve fibers is similar. The presence of target tissue was the critical variable in achieving a more rapid rate of growth in comparison to controls. Interes- tingly, the mean growth rate and relative increases in rates over the control rates were very similar for both sets of neurites. Neuritic outgrowth from spinal cord co-cultured with inappropriate target tissue, i.e. liver, heart or older limb tissue, was previously shown to be the same as control outgrowth lz.

Previous studies have provided evidence of a limb mesenchyme-released factor that is detectable through its effects in conditioned medium that had

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TABLE I

Rate of neurite extension (l~m/24 h) from spinal cord and dorsal root ganglion explants in vitro with respect to target tissue presence and type of substrate

Values represent mean growth rates expressed in/~m/24 h 4- S.E. of the mean (S.E.M.). Neural and target tissue from R. pipiens tadpoles were prepared and cultured in a defined medium (Eagle's Minimum Essential Medium with Earle's salts) in Sykes-Moore culture chambers following established methods 10. Eleven cultures ranging from 5 to 13 days in vitro were used in the time-lapse analysis with (n) representing the number of fibers analyzed in each group. Cultures were photographed on an inverted microscope under differential interference and phase-contrast optics with a 16 mm camera for periods of 24 h at a time-lapse rate of 1-3 frames/ min. Advancement of each growing fiber in the field was recorded by charting the growth cone position from time-lapse film at intervals throughout the 24-h period. From these charts the extension rate per interval was calculated for each fiber. Statistical com- parisons by Student's t-test showed that all groups were significantly different from controls (P < 0.01 ) with the exception of cultures on polylysine substrate (see text). Tadpole staging is according to Taylor-Kollros 1°.

Substrate Collagen Poly-DL-lysine (l mg/ml) Collagen

Control Stage V spinal cord (7) Stage V spinal cord (6) Stage XI dorsal root ganglion (4)

120 ± 20 1 2 0 ± 20 80 ~= 30 Experimental Stage V cord + stage Stage V cord + limb- Stage V cord ~ stage Stage XI DRG + stage

co-culture V limb (60) conditioned medium (10) V limb (7) XI limb (10) 510 i 40 280 ± 40 300 ± 100 490 ± 100

been transferred to untreated spinal cord cultures 16. When such limb-conditioned medium was provided to naive spinal cord cultures, the rate of neurite extension was significantly accelerated to a mean value intermediate to those of the control cultures and the cord-target co-cultures (Table I). This effect is probably dosage-dependent and is an aspect under investigation. Although a few reports have presen- ted data as to rates of nerve fiber extension under several conditionsl,2,4,5,9,n,18,21, this is to our

knowledge the first that implicates the target tissues as a regulating factor in the rate of neurite out- growth. This finding suggests an interaction-depen- dent growth rate as an important consideration in

the critical timing of nerve-target relationships in situ.

A phenomenon of periodicity in the rate of neurite extension is also apparent from the time- lapse data (Table II). In order to detect this perio- dicity it was necessary to determine the changing rates of extension at numerous points in time over the 24 h of filming for each neurite in the microscope field. Individual nerve fibers did not grow continu- ously throughout the 24-h period; instead, they had periods of growth ranging from 1 to 6 h in duration. During these times there was a characteristic growth rate fluctuation showing a periodicity that was influenced by the presence of target tissue (Figs. 1

TABLE 11

Characteristics of the target-dependent periodic growth cycle of stage V spinal cord neurites

The difference between the growth peak intervals of control (n -- 5) and experimental (n -- 29) groups was highly significant by Student's t-test; P = 2.43 x 10 -6.

Mean interval between peaks of maximal growth (h :~ S.E.M.)

Mean maximal growth rate with range (l*m/24 h ± S.E.M.)

Mean minimal growth rate (btm/24 h ± S.E.M.)

Control Stage V spinal cord 4.5 £ 0.34 500 ± 160 50 ~- 20

200-1200 Experimental

Stage V spinal cord + stage V 1.7 £ 0.17 1450 ± 130 limb co-culture 400-3400

280 ~ 40

A

#

E ,9- 1500

1000

500 . "~'~

z o " / N . , * \ . • tu 0 2 4 6 8 10

TIME (hours)

Fig. 1. Representation of the mean periodic activity during the growth phase for stage V spinal cord neurites ( A - ' - A ) and stage V spinal cord neurites in the presence of stage V limb ( H ) . Mean maximal and minimal growth rates and the mean interval between maximal rates were used to construct the growth cycle. The time period between growth cycles is not known although in several instances individual neurites in the presence of target tissue went through more than one growth cycle in the same time period in which control neurites went through only one cycle (also see Fig. 2).

2000

# E 1500 3-

W

1000 I

500 W

W Z

0 2 4 6 8 10

TIME (hours)

Fig. 2. Growth rate curves for two individual spinal cord neurites demonstrating response to target tissue presence ( e ~ - - - - O ) in comparison to target tissue absence ( A n - A ) . Note the shortened cycles with increased growth rates in the presence of the limb target.

and 2). Neurites extending from control spinal cord explants attained maximal rates of growth at mean intervals of 4.5 h. In the presence of the limb tissue, the interval between maximal growth rates was sharply reduced to 1.7 h. The range of rates attained

363

by individual neurites reflects this difference between the two groups (Table II). In addition, when spinal cord was co-cultured with target tissue, both the mean maximal and minimal neurite growth rates were increased over control rates, 3.0 and 5.6 times, respectively. Similar cycling can be detected for D R G neurites and for spinal cord neurites in the presence of mesenchymal limb-conditioned medium. The periodicity and maximal rates attained are apparently not simple circadian rhythms but functions of the nerve environment. As in prior reports 13,16 increased neurite fasciculation occurred

in the presence of target tissue. In contrast to the results of studies reporting

substratum-dependent nerve fiber growth rates s,9, we found that the control neuritic outgrowth from stage V spinal cord cultures occurred at the same rate on both collagen and poly-DL-lysine substrata, even though the total amount of nerve fiber out- growth was increased on the polylysine. Inclusion of limb tissue in cultures on polylysine substratum increased the mean neurite growth rate by 2.5 times, which was not highly significant (Student's t-test; P ---- 0.1). Since polylysine elicits increased out- growth without increasing the rate of neurite exten- sion, and target tissue and target-conditioned medium stimulate both increased outgrowth and increased rate of neurite growth, we propose that at least two separate controls may interact to regulate nerve fiber extension. The first is of a chemotactic nature mediating the relationship between the nerve fiber and its attachment substratum 6. Such an inter- action that establishes optimal conditions for neurite elongation could account for enhanced and directed growth of nerve fibers. Second, the stimu- lation of metabolic activity of the neuron by a target factor could result in positive chemokinesis 3 as shown by increased growth cone activity, increased rate of neurite extension, and decreased time inter- val between maximal growth rates.

It thus appears that one primary response of nerve fibers to their targets in vitro is related to parameters of growth rate. How the target regulates the accele- ration of nerve fiber extension and its associated periodic activity remains under investigation. Parallels to bacterial 7 and leukocyte 17 migratory activity may provide clues in understanding the mechanisms involved.

364

We thank G. Procento for technical assistance

and G. Dombrowski for computer analysis of data.

This study was supported by Gran t NS13814 from

the Nat ional Insti tute of Health.

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