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Stage-dependent spontaneous frog dorsal root ganglion neuritogenesis on polylysine in vitro.

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THE ANATOMICAL RECORD 228:220-224 (1990)
Stage-Dependent Spontaneous Frog Dorsal Root
Ganglion Neuritogenesis on Polylysine In Vitro
Institute for the Study of Developmental Disabilities (E.D.P., V.L.),Department of
Biological Sciences (E.D.P.),and the Committee on Neuroscience (E.D.P.), University of
Illinois, (MIC 627), Box 6998, Chicago, Illinois 60680
Dorsal root ganglia of larval frog extended elaborate neuritic arrays in vitro under minimal culture conditions in the absence of medium-supplemented stimulatory factors. The highly adhesive attachment substratum polylysine provided the necessary condition for exuberant outgrowth, the extent of
which was dependent on the developmental stage of the animal from which the
neural tissue was derived, a s was the capability for long-term survival. It appears
that an appropriate substratum can substitute for added growth factors in eliciting
robust and long-lived sensory neurites in vitro.
It is generally held that embryonic sensory neurons thetized in 70% ethyl alcohol followed by immersion in
do not extend substantial numbers of nerve fibers in Earle’s balanced salt solution (BSS)with 50 Fgiml genvitro in the absence of nerve growth factor (NGF; tamicin. The sterile culture medium a t pH 7.2-7.4 and
Harper and Thoenen, 1981; Levi-Montalcini, 1982) or 255-265 mOsmikg consisted of Eagle’s minimum esthe products of assorted target tissues for these neu- sential medium with Earle’s BSS (without NaHCO,)
rites (Steele and Hoffman, 1986; Crutcher, 1987). Fur- supplemented with 50 Fgiml gentamicin, 1ml of
thermore, the need for NGF or other exogenous factors 200 mM L-glutamine/lOO ml complete medium and
in order to sustain developing sensory neurons is well 10mM Hepes buffer. Cultures were maintained a t
documented (Grain et al., 1980; Calissano et al., 1984; 19°C and photographically recorded a t 1-2 day interCrutcher, 1987). The survival of adult rodent dorsal vals. Dorsal root ganglia cultured on reconstituted r a t
root ganglia (DRG) and their sensory neurons, on the tail collagen under the same conditions were devoid of
other hand, is reported to be dependent on growth fac- neuritic outgrowth at any developmental stage.
tors other than NGF (Grothe and Unsicker, 1987; DelRESULTS
ree et al., 1989). In contrast, Lindsay and Harmar
(1989) found that NGF regulates mRNAs for neuropeptides in adult sensory neurons, yet conditioned media
The vast majority (86%)of dorsal root ganglia exhibthat supported chick sympathetic ganglia and neonatal ited neurite outgrowth on polylysine within 48 hours,
mouse DRG did not enhance the survival of embryonic and all that were to extend neurites (99%) did so by
5-6 days. This is in contrast to a complete failure of
chick DRG, although NGF did (Varon e t al., 1981).
Since central nervous tissues demonstrate particu- DRG neurite outgrowth on type I collagen (although in
larly exuberant neuritic outgrowth when explanted a separate study collagen did support target-evoked
onto the polycationic substratum polylysine, we asked DRG neurite growth and sensory neurite extension in
if DRG would do the same in a defined culture medium the presence of NGF; Pollack et al., 1979). Outgrowth
to which neither NGF, target tissues, nor serum sup- characteristically was radial and distributed around
plements were added. The results overwhelmingly the explant (Fig. 1).Table 1 illustrates that the most
demonstrated that DRG neurons are quite competent substantial outgrowth occurred when the DRG were
to extend vigorous nerve fibers that are maintained in from stage XI animals, with 100% of these cultures
a healthy state for extended durations in a culture sys- extending neurites and 70% exhibiting maximum outtem to which no growth factors have been added. This growth. On either side of stage XI, that is, for younger
is, however, dependent on the developmental stage of or older animals, the DRG neuritic outgrowth response
was substantially reduced. Similarly, nerve fibers from
the animal from which the tissues were removed.
the stage XI DRG extended much further from the exMATERIALS AND METHODS
plant than did those from the other stages (Table 1,
Whole lumbosacral DRG from Rana pipiens tadpoles Fig. 1).
At each stage for the DRG cultures, there seemed to
a t stages V, IX, XI, and XV (early through late-midbe
a limit to the neurite outgrowth zone that surrounds
larvae; stages of Taylor and Kollros, 1946) were explanted onto poly-DL-lysine-coated coverglasses ( 1 mg/ the DRG, this being greatest for stage XI. In actuality,
ml incubated a t room temperature for 24 hours) in individual nerve fibers were often substantially longer
Sykes-Moore chambers with defined medium lacking
NGF, target tissue, embryonic extracts, or serum supplements a s described previously (Pollack and Koves,
1975; Muhlach and Pollack, 1978).Tadpoles were anesReceived October 23, 1989; accepted March 8, 1990.
Fig. 1. Typical representations of the DRG demonstrate the lower amount of neuritic outgrowth from
a stage V DRG (A, x 100)as compared to that from a stage XI DRG (B, x 100). Note that the nonneuronal cells among the neurites follow, rather than precede, the nerve fibers, nor do they form an
underlying substratum.
TABLE 1. Characteristics of DRG neuritic outgrowth on polylysine
Stage of DRG (n)
V (6)
Percent exhibiting
No outgrowth
Minimal outgrowth
Moderate outgrowth
Maximum outgrowth
Mean survival (days; i S.E.M.)
Outgrowth zone relative to diameter of DRG
2 1.8
than the outgrowth zone since they usually engaged in
extensive curving, particularly as they neared the zone
border. Occasionally, individual neurites elongated far
beyond the denser outgrowth zone, but invariably they
turned back into the mass of neurites. Although nonneuronal cells have been observed among the neurites,
they migrated from the DRG only after the neurites
had extended and were never found distal to the neurite terminals. Interestingly, DRG neurites a t stages
XI and XV expressed numerous microprocesses, providing a step-ladder appearance to the fibers that
seemed to anchor neurites to the substratum (Fig. 2B).
These processes did not differentiate further into neuritic branches.
IX (6)
XI (17)
X v (6)
29 2 2.1
52 i 3.8
57 5 11.4
Neurite Survival
Coupled with the issue of nerve fiber growth is that
of survivability of the neuron and nerve fiber. Neuritic
degeneration, identified by beading and disruption of
neurites (Fig. 2A), always was noted first in the distal
regions of the outgrowth zone, although this may have
reflected the difficulty in identifying degenerating fibers in the densely population region most proximal to
the explant. When the DRG no longer exhibited any
outgrowth, explants were largely devoid of neurons as
seen in histological section.
The neurites of the younger DRG explants (stages V
and IX) as a group survived in culture for less than
Fig. 2. Degenerating neurites that demark the time a t which DRG
survival is considered terminated can be identified by their beaded,
disrupted appearance (A, x 630). Viable neurites on polylysine are
characterized by microspike-like extensions (arrowheads in B, x 630)
that give a ladder-like appearance to the fibers and may assist in
anchoring the neurite to the substratum.
one-half the time of the older DRG neurites (stages XI
and XV) as indicated by the onset of degeneration (25.5
vs. 54.5 days, respectively; P = 2.8 x
Histological examination demonstrated that organotypic features of the DRG were retained in vitro (Fig. 3). Thus
it appears t h a t the attachment surface on which these
neural tissues were cultured had a survival-promoting
influence. But clearly the inherent survival capability
of the DRG is based on developmental considerations
as well.
In contrast to the generally held view that the developing DRG will not extend substantial neurites in vitro
in the absence of added NGF or target tissue products,
we have demonstrated for the first time that frog tadpole DRG cultured in a minimal medium on a polylysine attachment surface can form elaborate and longlived neuritic arrays. Other than developmental stage
dependency, there appeared to be no hinderance in
neurite extension under the conditions of this culture
system. The fact t h a t the stage XI DRG produced the
densest neuritic networks coincides with other findings
from our laboratory (Pollack and Muhlach, 1982), indicating that, a t least on collagen, the DRG is most
responsive to the presence of its target tissues, i.e., the
limb or spinal cord, a t stage XI, a time when target
Fig. 3. Cross-section of a stage XI DRG a t 14 days in vitro exhibits
organotypic organization with clearly visible large sensory neurons.
Some necrotic cells can be seen, but whether these represent a selective degeneration of specific cell types or are those engaging in the
naturally occurring neuron death characteristic of this stage is not
known. x400.
innervation is underway a s is naturally occurring neuronal death (see Bibb, 1988). Although it would appear
that target tissue is important in regulating nerve fiber growth (Peterson and Crain, 1981; Pollack et al.,
1981; Varon and Adler, 1981) i t is now apparent that
“spontaneous” growth may require only a suitable attachment surface to support DRG neurite extension.
The microprocesses that had extended from the neurite
shaft may be indicative of enhanced neurite-to-surface
adhesion, which is known to be a facilitator of axonal
growth and branching (Letourneau, 1975).
An overriding consideration, however, is that a developmental-time restriction is incurred by these sensory neurons in their ability to extend nerve fibers.
Although a strict neuronal maturation element might
come into play, this would not totally account for the
reduced ability of the oldest DRG to extend neurites.
On the other hand, the explant itself might be the
source of a growth-promoter that establishes a substratum-bound gradient controlling the extent of the outgrowth. The DRG contains, in addition to the sensory
neurons, both glial and satellite cells that have been
suggested a s the source of trophic factors directed to
embryonic (Varon et al., 1981) and adult (Grothe and
Unsicker, 1987; Delree et al., 1989) sensory neurons.
Evidence for substratum-bound growth factors has
been proposed for several in vitro systems (Collins,
1978; Varon and Adler, 1981; Gundersen, 1985; Muhlach, 1988). Furthermore, if a time-related system for
the occurrence of factor production and response should
be regulated within the DRG a s for NGF in the skin
(Davies et al., 19871, then the present results might be
readily explained.
Dora1 root ganglia placed on collagen failed to extend
neurites in the absence of added NGF or target tissues
(Pollack et al., 1979, 1980). While i t is possible t h a t
those fibers extended on polylysine were from a growth
factor-independent population of neurons, then similar
neurites should have been observed on collagen. Similarly, if the explant itself were the source of NGF1 or
other DRG-directed growth factors, then outgrowth
should have occurred on collagen a s well unless, however, it were not a n appropriate surface for the requisite binding of a putative factor. Yet NGF effectively
promoted DRG neurite growth on collagen (Pollack e t
al., 1979). Recent results from our laboratory suggest
that both collagen and polylysine are adept in binding
specific proteins found in target-conditioned medium
that stimulates and maintains spinal cord neurites
(Kaminski and Pollack, in preparation). Michler et al.
(1989) have shown that serum components that mediate DRG neurite extension are those that bind to the
substratum, but as is often the case in comparable
studies, NGF was a constituent of the growth medium.
Several studies have shown that sensory neurites
may be responsive to non-NGF growth factors (Ebendal, 1979; Ebendal et al., 1983; Lumsden and Davies,
1983; Johnson and Yip, 1985; Davies et al., 1986) with
the overall implication that exogenous (to the DRG)
chemical factors are required for the processes described here. Dissociated adult rodent sensory neurons
do not require NGF for survival in long-term cultures,
although the regeneration of their axons is stimulated
by NGF (Lindsay, 1988). Recently, Rydel and Greene
(1988) reported that cyclic AMP analogs can promote
both sensory and sympathetic neuronal survival and
neurite outgrowth independent of NGF. These reports
together with the present results suggest that a number of alternatives to a system of NGF primacy may
The specific relationship of the substratum to the
prolonged survival of the DRG also can be considered
in the same respects a s for neurite growth, i.e., stagedependent response to the substratum and DRG-originated trophic factors. Naturally occurring neuron
death is a well-described aspect of DRG development
(Hamburger and Levi-Montalcini, 1949; Bibb, 1978;
'Although the focus of this study is on DRG neurite growth without
added NGF, preliminary results in our laboratory show that neuritic
outgrowth is not inhibited from stage XI DRGs that have been explanted into an excess of anti-NGF. However, when anti-NGF is
added to existing cultures some neurite degeneration occurs even
though new neurites continue to be established. This suggests that
any NGF intrinsic to the DRG acts largely in a survival capacity
rather than in neurite initiation, although we cannot completely
eliminate a dual neuronal population hypothesis at this time.
Carr and Simpson, 1978) and as it pertains to the DRG
is considered to be related to the availability of trophic
factors during specific developmental periods, at least
for certain neuron types (Levi-Montalcini, 1982; Hulsebosch et al., 1987; Barde, 1989). It is therefore of interest that the sole use of the polylysine attachment surface ensured the long-term survival of the DRG
neurites and their associated cell bodies within the explant.
Since spinal sensory neurons develop and persist in
a n aggregate with other cell types, analysis of the developing DRG in both organotypic explant and dissociated modalities should help to resolve how a n appropriate surface can subserve both growth and survival
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spontaneous, dorsal, stage, ganglion, dependence, roots, polylysine, neuritogenesis, frog, vitro
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