 |
 Previous Article | Next Article 
The Journal of Neuroscience, October 1, 1998, 18(19):7930-7940
Interstitial Branches Develop from Active Regions of the Axon
Demarcated by the Primary Growth Cone during Pausing Behaviors
Györgyi
Szebenyi1,
John L.
Callaway1,
Erik W.
Dent2, and
Katherine
Kalil1, 2
1 Department of Anatomy and 2 Neuroscience
Training Program, University of Wisconsin, Madison, Wisconsin 53706
 |
ABSTRACT |
Interstitial branches arise from the axon shaft, sometimes at great
distances behind the primary growth cone. After a waiting period that
can last for days after extension of the primary growth cone past the
target, branches elongate toward their targets. Delayed interstitial
branching is an important but little understood mechanism for target
innervation in the developing CNS of vertebrates. One possible
mechanism of collateral branch formation is that the axon shaft
responds to target-derived signals independent of the primary growth
cone. Another possibility is that the primary growth cone recognizes
the target and demarcates specific regions of the axon for future
branching. To address whether behaviors of the primary growth cone and
development of interstitial branches are related, we performed
high-resolution time-lapse imaging on dissociated sensorimotor cortical
neurons that branch interstitially in vivo. Imaging of
entire cortical neurons for periods of days revealed that the primary
growth cone pauses in regions in which axon branches later develop.
Pausing behaviors involve repeated cycles of collapse, retraction, and
extension during which growth cones enlarge and reorganize. Remnants of
reorganized growth cones are left behind on the axon shaft as active
filopodial or lamellar protrusions, and axon branches subsequently
emerge from these active regions of the axon shaft. In this study we
propose a new model to account for target innervation in
vivo by interstitial branching. Our model suggests that delayed
interstitial branching results directly from target recognition by the
primary growth cone.
Key words:
interstitial axonal branch; growth cone behavior; time-lapse imaging; cortical neuronal culture; target innervation; cortical development
| |
INTRODUCTION |
Cortical neurons extend a
single axon that branches in specific regions along its length so that
it can innervate multiple targets. Despite the importance of axonal
branches, little is known about the mechanisms underlying branching.
Studies on cultured peripheral neurons (Bray, 1973 ; Wessells and
Nuttall, 1978) showed that branches can form via bifurcation of the
terminal growth cone. However, in vivo studies on cortical
axons demonstrated that interstitial branches can form at great
distances behind the primary (terminal) growth cone and up to several
days after the growth cone has advanced beyond the target innervated by
the branch (O'Leary and Terashima, 1988; Kuang and Kalil, 1994).
Observations of retinal ganglion cell axons (Harris et al., 1987),
cultured hippocampal neurons (Dotti et al., 1988; Yu et al., 1994), and developing cortical axons in living brain slices (Halloran and Kalil,
1994; Bastmeyer and O'Leary, 1996) confirmed that branches form
principally as interstitial collaterals along the axon shaft. One
reasonable interpretation of these results is that the primary growth
cone does not play a significant role in delayed interstitial axon
branching (O'Leary and Koester, 1993; Kennedy and Tessier-Lavigne, 1995; Joosten, 1997; Castellani and Bolz, 1997).
However, growth cones are known to be highly responsive to
target-derived guidance cues (Goodman, 1996; Tessier-Lavigne and Goodman, 1996) that might also regulate branching behavior (Bolz and
Castellani, 1997). It is possible that as the terminal growth cone
advances, it leaves signals behind along discrete regions of the axon
shaft that orchestrate formation of interstitial branches. Support for
this view derives from studies on growth cone behaviors in brain slices
(Halloran and Kalil, 1994) and organotypic cultures (Yamamoto et al.,
1997) in which it was observed that regions of growth cone pausing
correlate with axon branching. During such pausing behavior, the growth
cone could in theory impart branching information to that specific
region of the axon shaft.
To test this hypothesis, in the present study we sought to determine
first whether growth cone pausing is consistently associated with axon
branching and second whether such behaviors by growth cones lead
directly to development of interstitial branches. We have used
high-resolution time-lapse imaging of living cortical neurons in
dissociated cultures to observe the entire length of developing axons
over periods of days. We followed the development of individual
branches from the initial behaviors of the primary growth cone through
the elongation of stable branches. This approach permitted an
unprecedented opportunity to relate the history of the primary growth
cone to subsequent branching along the axon shaft. The present study
documents that most axon branches arise from regions of growth cone
pausing. These results suggest a new model in which the primary growth
cone demarcates specific regions of the axon for development of
interstitial branches.
| |
MATERIALS AND METHODS |
Cell culture. All reagents were purchased from Life
Technologies (Grand Island, NY), unless specified. Cultures were
prepared from cortical tissue obtained from 3-d-old Syrian golden
hamsters (Mesocricetus auratus). The day of birth was
considered postnatal day 0. Pups were anesthetized on ice and
decapitated. The skull was removed, and the forebrain was transferred
to ice-cold dissection medium (Hibernate-A supplemented with B27, 0.3%
glucose, 1 mM L-glutamine, and 10 µM gentimycin sulfate). After the meninges were removed,
the sensorimotor cortex was dissected out. The cortex was minced into
small pieces in fresh dissection medium supplemented with the
excitotoxic amino acid blockers kynurenic acid (1 mM) and
aminophosphovalonic acid (APV, 50 µm) (both from Sigma, St. Louis,
MO). Cortical pieces were dissociated with 0.025% trypsin for 15 min
at 37°C in HBSS, 1 mM kynurenic acid, 50 µM
APV, 0.27 mM EDTA, and 0.05% DNase I (Sigma) with gentle
agitation every 5 min, followed by gentle trituration 4-6 times in
serum-containing media (SCM: 10% fetal bovine serum (Hyclone, Logan,
UT), 1× B27 supplement, 0.3% glucose, 1 mM
L-glutamine, and 10 µM gentimycin sulfate in
Neurobasal medium) supplemented with 1 mM kynurenic acid
and 50 µM APV. Undissociated pieces of tissue were
allowed to settle, the cell suspension was collected, and the
trituration was repeated twice with fresh SCM. Pooled cell suspensions
were centrifuged at 200 rpm for 5 min, resuspended in SCM, and counted on a hemocytometer.
Cells were cultured on etched-grid coverslips (Bellco, Vineland, NJ)
coated with 0.5 mg/ml poly-D-lysine (Sigma) in borate buffer for 1 hr, rinsed 3 times with distilled water, and then coated
with 20 µg/ml laminin in Neurobasal medium at 37°C for at least 4 hr. Cells were plated in SCM at densities ranging from 500 to 1000 cells/cm2. After 1.5-2 hr, medium was changed to a
serum-free formulation (Neurobasal medium with B27 supplement, 0.3%
glucose, 1 mM L-glutamine, 55 µM
2-mercaptoethanol, and 10 µM gentimycin sulfate). Because FGF-2 has been reported to increase axon branching by hippocampal neurons (Aoyagi et al., 1994), some cultures were treated with 20 ng/ml
FGF-2 (Promega, Madison, WI), which was added to the serum-free medium
either 2 or 24 hr after plating. However, treated cells were not
included in the numerical data analysis. Cultures were maintained at
37°C in 5% CO2 for 4-6 d without feeding. These conditions resulted in neuronal cultures that remained viable for 5-7
d in the presence of very few glial cells (<5%).
Microscopy and digital imaging. To prevent changes in the pH
of the medium, cultures were sealed by attaching a 24 mm round coverslip to a 15 mm glass ring (Thomas Scientific, Swedesboro, NJ)
with silicone grease (Dow Corning, Midland, MI). Culture dishes were
placed on the stage of a Zeiss 35M inverted microscope (Carl Zeiss,
Thornwood, NY) that was warmed to ~37°C with an airstream incubator
(Nicholson Precision Instruments, Bethesda, MD). Cortical neurons were
viewed with a 20× (0.5 NA Neofluor) objective (with or without a 1.6×
optivar lens) under phase-contrast optics. Images were acquired with a
Micro-Max cooled CCD digital camera (Princeton Instruments, Trenton,
NJ) at full-chip resolution (1317 × 1035 pixels) and stored on
the hard drive of a Pentium 133 MHz computer (Datastor, Boulder, CO).
With 20× magnification it was possible to obtain high-resolution
digital images. The 20× objective and 6.8 µm2
pixel size of the CCD chip (Kodak KAF 1400) met the Nyquist criterion for sampling (Inoué 1986; Inoué and Spring 1997). The
microscope was equipped with a Uniblitz electronic shutter (Vincent
Associates, Rochester, NY) interposed in the light path to reduce
damaging illumination to the cells. The shutter was opened for 50-200
msec during image acquisition. For permanent storage, data were written to recordable CDs. The shutter for the light source and camera shutter
were controlled with Metamorph 3.0 software (Universal Imaging, West
Chester, PA).
In a given culture dish we typically chose 10-40 cortical neurons
4-26 hr after plating for long-term imaging. In total, the data
presented here came from ~30 separate culture dishes, derived from 18 separate cell platings. Images were acquired at intervals ranging from
3 min to several hours during imaging sessions lasting up to 6 d
after plating. Dishes were returned to the incubator if the imaging
interval was  1 hr. The etchings on the coverslips permitted us to
return to the same neurons in each recording session.
Data analysis. Branched and unbranched axon regions were
analyzed for growth cone behaviors in relationship to subsequent branching activity. Neurons with pyramidal morphologies and at least
one process 100 µm or longer were analyzed. Only processes longer
than 30 µm were considered as branches, because most shorter processes were transient. A single-branching region was defined as a 70 µm axon region that had one or several branches. We adopted this
criterion because typically growth cones observed during pausing
behaviors advanced and retracted over a distance of ~70 µm. Regions
of the axon closer than 50 µm to the cell body were excluded from
analysis, because it was not possible to distinguish between the axon
and minor processes in regions close to the cell body. Axon regions
were considered to be unbranched if no branch developed along a 70 µm
segment at any time during the life of the neuron. Regions classified
as unbranched had to remain unbranched for the lifetime of the neuron
(a minimum of 30 hr). We also required that the neuron had to survive
for at least 30 hr after formation of the analyzed axon region. No more
than two branched and two unbranched axonal regions per neuron were
analyzed. Our quantitative data were obtained from a total of 58 branched regions from 42 neurons and 27 unbranched regions from 21 neurons. Because cortical neurons became highly branched after 3-4 d
in culture, we obtained roughly twice as many branching regions as
unbranched regions.
Using the morphometric analysis tools of Metamorph software, we
measured axon length at different time points from stored digital
images and then calculated average growth cone velocities. We also
measured lengths of axon branches, the distance between branch points
and the cell body, and the distance between branch points and the tip
of the axon. In addition we measured growth cone areas. To correlate
activity along the axon shaft with development of branches, we assigned
scores ranging from 0 to 3. Activity level 0 refers to consolidated
cylindrical regions of the axon that had only transient filopodia that
showed no correlation with branching. Activity level 1 refers to axon
regions with stable filopodia at locations of future branch points.
Activity level 2 refers to axon regions with small lamellar expansions
that are typically asymmetric and consolidate into varicosities before branching. Activity level 3 refers to regions with larger, more complex
lamellar expansions that are symmetrically distributed on the axon
shaft and persist after branching occurs.
Statistical analysis was performed using Excel 97 (Microsoft, Redmond,
WA) and Sigmaplot 4.0 (SPSS, Chicago, IL) software. Images were
processed with Metamorph 3.0 and Photoshop 4.0 (Adobe Systems, San
Jose, CA). Figures were prepared directly from digital files. Images
shown in the figures were modified using the unsharp mask filter and
brightness-contrast adjustment tools in Photoshop to enhance detail
and contrast.
| |
RESULTS |
Cortical neurons in culture develop axonal processes similar to
those in vivo
We selected neurons for analysis that resembled large pyramidal
projection neurons, because efferent axons from these neurons are known
to give rise to interstitial branches (O'Leary and Terashima, 1988;
Kuang and Kalil, 1994; Halloran and Kalil, 1994). To determine whether
cultured cortical neurons develop similarly to those in vivo, we imaged single neurons continuously over several days. By
imaging the entire neuron, it was possible to monitor the development of all of the processes simultaneously. As shown in Figure
1, neurons initially extended minor
processes equal in length and tipped by growth cones. Subsequently, one
of the processes elongated to become clearly distinguishable as the
axon. Axonal processes grew to an average length of 300-400 µm in
3 d. The other processes remained shorter than 50 µm during this
time frame (Figs. 1, 2). This sequence of
development is similar to that reported for cultured hippocampal
neurons (Dotti et al., 1988).
View larger version (97K):
[in this window]
[in a new window]
|
Figure 1.
Cortical neurons in culture develop processes
similar to those in vivo. In A and
B minor processes are easily distinguished from the
single long primary axon. Arrows indicate axon branches.
All of the branches are tipped by growth cones. The etched
grids on the coverslips serve as landmarks for identification
of the same cells at different times. Times shown indicate hours after
plating. The scale bar applies to all images in A and
B.
|
|
View larger version (44K):
[in this window]
[in a new window]
|
Figure 2.
Branches develop from cortical axons over an
extended time period. A, Representative images selected
from a series of 1320 time-lapse images acquired at 3 min intervals
over a period of 3 d. This neuron developed minor processes first
(5 hr), followed by elongation of the axon (20-35 hr) and extension of
interstitial branches (35-53 hr). The axon is labeled with an
asterisk. One axon branch (at right) was
already present when imaging began, but most of the branches extended
as a cluster from the region indicated by the arrows.
Times shown indicate hours after plating. B, Time course
of elongation of minor processes (squares), the axon
(circles), and axon branches (triangles),
excluding the initial branch of the neuron shown in A.
For minor processes and axon branches, total neurite length, not that
of a single neurite, is graphed. Note that before extension of branches
elongation of the axon has halted. No data were collected during the
gaps, but branches did elongate during these periods. C,
Frequency histogram summarizing the time course of branching on 42 axons. Bars show numbers of new branches
(n = 58) >30 µm that extended during each of the
time periods indicated. Most of the branches extended on the second and
third day in culture. Only branches with full recorded histories, from
the beginning of branch initiation, were included.
|
|
Interstitial branches began to extend from the axon shaft after the
axon was already distinct from the minor processes (Figs. 1, 2). Most
of the branches (57%) started to extend between 20-40 hr after
plating (Fig. 2C). Cortical axons typically had 4-5
branches, which in about half of all cases appeared in clusters of
2-5. Axons often exhibited numerous short filopodia-like processes under 30 µm in length. Most of these were transient and did not develop growth cones. However, all processes that grew longer than 30 µm formed growth cones at their tips (Fig.
3). These branches persisted for the
entire observation period (47 hr on average) and eventually grew to
lengths up to 668 µm, averaging 135 ± 126 µm. Thus, we define
true axon branches, as opposed to transient filopodia, as stable
processes longer than 30 µm. As in vivo, the axon segment
distal to the branch often either ceased extension (Fig.
2B) or, as shown in Figure 3, degenerated.
View larger version (72K):
[in this window]
[in a new window]
|
Figure 3.
Elongation of an interstitial branch is preceded
by the formation of a growth cone. These images illustrate transient
filopodial activity (0-48 min) leading to a stable branch over 30 µm
led by a growth cone (156 min). This sequence of images was selected
from 72 images acquired at 3 min intervals. Time 0 corresponds to 20 hr
after plating. As shown in the last frame, once the interstitial branch
is advancing forward, the distal segment of the primary axon
degenerates. P and D refer to proximal
and distal segments of the axon, respectively.
|
|
Primary axon growth cones pause in regions in which axon branches
later develop
Cortical growth cones in living brain slices of the corpus
callosum undergo complex pausing behaviors involving repeated bouts of
collapse, retraction, and forward extension in regions in which cortical axon branches subsequently develop. This contrasts with steady, rapid forward growth in regions of the callosum in which the
same axons do not branch (Halloran and Kalil, 1994). To determine whether specific growth cone behaviors are consistently associated with
extension of cortical axon branches, we imaged cortical axons and their
growth cones at varying intervals of 3-6 hr over periods of 4-5 d.
The sequence of images in Figure 4 shows
the entire history of a region of the axon, including changing
positions of the primary growth cone and development of axon branches.
During a 30 hr time period (45-75 hr) this primary growth cone
underwent repeated cycles of collapse, retraction, and extension
without any net forward extension. The distance over which these
behaviors occurred averaged 70 µm. We define these as growth
cone-pausing regions. In the next 45 hr several branches subsequently
developed on the axon within the region of growth cone pausing. From
observations of several hundred axon regions, 58 that met our criteria
(see Materials and Methods) were chosen for analysis of growth
cone pausing in relation to axon branching. Measurements of average rates of growth cone extension (Fig.
5A) showed that in branching regions average velocity (13 ± 12 µm) was less than half that measured for unbranched axon regions (29 ± 15 µm). These
differences in velocity were highly significant
(p < 0.01) and suggest a strong correlation
between growth cone pausing and axon branching. Observations of growth
cone behaviors at more frequent intervals (3-20 min) showed that
during pausing growth cones could remain in place while showing
filopodial and lamellar activity, or could extend, collapse, and
retract over the same 70 µm region of the axon. In general, slower
average velocities resulted from growth cone-pausing behaviors that
could last from 1-30 hr. Of 58 branching regions, 84% of the growth
cones underwent pausing. Pausing by 28% of these growth cones resulted
in no net forward advance in 3 hr. In the rest of the cases, growth
cones remained within the same 70 µm region of the axon during a
given 3 hr time period. In contrast, only in one case of 27 unbranched
regions (4%) did we observe growth cone pausing. We therefore conclude
that regions of axon branching are highly correlated with regions of
pausing by the primary growth cone.
View larger version (136K):
[in this window]
[in a new window]
|
Figure 4.
Extension of axon branches occurs from regions of
the axon in which the primary growth cone undergoes pausing behaviors.
During 45-75 hr after plating, the primary growth cone (the
top growth cone in the figures) collapsed, retracted,
and reextended in the region indicated by the arrows.
The arrows in all the figures indicate the same
location. After the growth cone reextends (at 75 hr) it changes the
angle of its trajectory. In the pausing region of the axon small active
protrusions appear (82 hr) from which branches extend (97-120
hr).
|
|
View larger version (82K):
[in this window]
[in a new window]
|
Figure 5.
During prolonged pausing periods, growth cones
enlarge and reorganize. A, Graph comparing average
velocities of growth cones in 70 µm axon regions that later branched
(n = 58 regions from 42 neurons) versus regions
that remained unbranched (n = 27 regions from 21 neurons). B, Graph comparing average areas of growth
cones during forward extension (growing is defined as growth >25
µm/hr; n = 20) versus pausing (defined as growth
<5 µm/hr; n = 20). In A and
B error bars are SEs, and asterisks
indicate p < 0.01 in a two-tailed t
test. C, Representative example of a growth cone
undergoing morphological changes during an 18 hr pausing period. The
lamella of the growth cone gradually enlarges over time. At 12 hr the
primary growth cone is emerging from the distal tip of the growth cone.
By 18 hr the primary growth cone has resumed elongation, and a large
lamellar expansion remains behind on the axon shaft.
|
|
Growth cones enlarge and reorganize during pausing
What changes occur in primary axonal growth cones during pausing
behaviors before branch formation? First, we consistently observed that
growth cones by the end of the pausing period had become greatly
enlarged. Measurements of 40 growth cones confirmed this observation
and showed that on average growth cones by the end of pausing periods
of 1-20 hr were 5.4 times larger than growth cones that were advancing
(Fig. 5B). Pausing growth cones had an average area of
341 ± 48 µm2 (n = 20), and
advancing growth cones had an area of 64 ± 6 µm2 (n = 20). The largest growth
cones were those that had paused for over 15 hr. In addition to
increased size, growth cones also changed their shapes. The most
striking change was the increase in the size of the lamellipodium. As
shown in Figure 5C, the neck and the central region of the
growth cone did not change their positions during 18 hr, whereas the
lamellipodium elongated for ~50 µm and spread out laterally as
well. At 12 hr a new growth cone can be seen forming from the distal
part of the enlarged growth cone. By the end of the pausing period, the
new growth cone had emerged to lead the growing axon. The lamellipodium
remained behind as an expanded membranous region on the axon. In the
growth cone illustrated in Figure 5C, the newly organized
growth cone continued in the same trajectory as the original primary
growth cone, and the direction of axon elongation did not change.
However, we frequently observed that after growth cone pausing, axons
resumed forward extension in a new direction. Moreover, from large
expanded growth cones it frequently happened that several growth cones emerged, each oriented in a different direction.
Axon branches emerge from remnants of growth cones
Expanded regions on the axon shaft in growth cone-pausing regions
subsequently gave rise to interstitial axon branches. As shown in
Figure 6A, these axonal
regions closely resembled the form of the pausing growth cone.
Time-lapse imaging at intervals of 2-5 min revealed that these regions
also exhibited filopodial and lamellar activities. Levels of activity
along branched and unbranched regions of axons were scored according to
morphologies, which ranged from transient filopodial protrusions to
large lamellar expansions that resembled growth cone lamellipodia. The
majority (60%) of the 70 µm segments of axons that remained
unbranched for 2 d showed no history of activity. In contrast,
83% of axon segments that did develop interstitial branches had
filopodial or lamellar activity (Fig. 6B). Greater
levels of activity (i.e., formation of lamellar expansions) on the axon
were highly correlated with growth cone pausing (Fig. 6C).
The average activity level in branched regions of the axon was 3.4 times higher than in unbranched regions (Fig. 6D).
Larger lamellar expansions often gave rise to clusters of two to five
branches (Fig. 6E). Branches extended from the axons
at approximately right angles (Fig.
7A,B)
and at varying times after the end of the pausing period ranging from 0 hr to delays of up to 68 hr (Fig. 7C). The average delay
between the time when the primary growth cone resumed forward advance and an interstitial branch extended was 16 ± 15 hr. In 2 of 58 cases we observed both the branch and the axon elongating
simultaneously (Fig. 7A). In such cases the growth cone
appeared to bifurcate. We also measured distances between the branch
point and the axonal growth cone and found that on average branches
extended from the axon 182 ± 175 µm behind the primary growth
cone. In summary, results from our observations of dissociated cortical
cultures show that the overwhelming majority of the branches developed interstitially from expanded active regions of the axon, some at
relatively long distances (up to 1 mm) behind the primary axonal growth
cone (Fig. 7D).
View larger version (65K):
[in this window]
[in a new window]
|
Figure 6.
Levels of filopodial and lamellipodial activity
remaining on the axon shaft after growth cone reorganization correlate
with axon branching. A, Examples from four different
neurons, showing different levels of activity along the axon shaft
ranging from 0 (no visible activity), 1 (transient filopodial or
lamellar activity), 2 (small, lamellar expansions that consolidate into
varicosities) to 3 (large, more symmetrical lamellar expansions). Each
series of images shows the same region of the axon over time, beginning
in each case with the primary growth cone. B, Bar graphs
showing percentage of axon regions with activity levels of 0-3
(indicated at bottom right) that either branched or
remained unbranched. Note that most of the axon regions with no
activity remain unbranched, whereas most axon regions with high levels
of activity develop branches. C, Bar graph showing
average velocity of the growth cone that gave rise to different levels
of axon activity (0-3). Slower rates of growth cone advance correlate
with higher levels of axon activity. D, Bar graph
showing average levels of activity in unbranched and branched regions
of axon shafts. Asterisks indicate p < 0.01 in a two-tailed t test. E, Bar
graph showing average number of branches extending from axon regions
with different levels of activity. More branches extended from axon
regions with higher activity. In C-E,
numbers above the bars indicate 70 µm axon regions
analyzed. Error bars are SEs in each of the graphs.
|
|
View larger version (46K):
[in this window]
[in a new window]
|
Figure 7.
Branches extend from active regions of the axon
after varying delays after the primary growth cone has resumed forward
advance. A, Series of images showing an example of a
branch (arrow) extending at a right angle simultaneously
with forward advance of the primary growth cone. This gives the
appearance of growth cone bifurcation. B, Series of
images showing an example of a branch (arrow) extending
after a delay of 47 hr after the primary growth cone has resumed
forward advance. By 23 hr the primary growth cone has emerged from the
growth cone that is still pausing at 22 hr. By 69 hr the branch
(arrow) is extending interstitially from the active
region on the axon. C, Frequency histogram showing
numbers of branches that extended after varying delays after the
primary growth cone had resumed forward advance. D,
Frequency histogram showing numbers of branches that extended from the
axon at varying distances behind the primary growth cone.
|
|
| |
DISCUSSION |
This study demonstrates how branches form on developing cortical
axons. Our results show that primary growth cones of cortical neurons
undergo prolonged pausing behaviors during which they extend and
retract repeatedly without forward advance. During these pausing
periods growth cones greatly enlarge and reorganize to give rise to
several new processes. Typically, the distal part of the enlarged
growth cone becomes the primary axonal growth cone and extends forward,
whereas the rest of the growth cone remains behind on the axon shaft in
the form of filopodial or lamellar activity. After delays of hours or
days after the primary growth cone resumes forward advance,
interstitial branches tipped by growth cones extend at right angles
from these active regions of the axon. When these delays are short,
branching appears to occur by growth cone bifurcation. Therefore,
growth cone bifurcation and interstitial branching differ only in the
time course in which the branch elongates. The present results suggest
a novel mechanism whereby the primary growth cone demarcates future
branch points by pausing, reorganizing, and leaving behind remnants
from which interstitial branches arise. Our model predicts that
in vivo it is the growth cone that recognizes targets to be
innervated by delayed interstitial branches.
The similarities between the development of cortical axons in our
cultures and in situ validate the relevance of our results as a model for branching in vivo. Postnatal cortical neurons
in culture resemble efferent pyramidal neurons whose axons develop interstitial branches to the spinal cord (Kuang and Kalil, 1994) and
the contralateral cortex (Norris and Kalil, 1992) beginning at several
days postnatal. Further, the number (two to five) and clustering of
branches was similar to patterns of axon branching in vivo
(O'Leary and Terashima, 1988; Kuang and Kalil, 1994) and in
situ (Bastmeyer and O'Leary, 1996). The majority of axon branches extended at right angles from the axon at 0-1 mm behind the primary growth cone, similar to positions of axon collaterals in
vivo (O'Leary and Terashima, 1988; Kuang and Kalil, 1994) and
in situ (Halloran and Kalil, 1994). In addition, delays in
extension of axon branches on cultured neurons (ranging from hours to
days) correspond to delayed interstitial branching in vivo
(O'Leary and Terashima, 1988). Concomitant with the elongation of axon branches in vivo, the primary axon often degenerates as we
have observed in culture (O'Leary et al., 1990). Taken together,
similarities in numbers, location, and time course of axon branching
suggest that our culture system provides a valid model for branch
formation in vivo. Although interstitial branching has been
documented previously in slice preparations, neither the resolution nor
the time periods of observations permitted an understanding of how new
branches form. Thus for the first time, the present study demonstrates that axon branches arise from regions of growth cone pausing.
Because the present study was performed on isolated neurons in the
absence of targets, it is unclear why cortical axons develop collateral
branches. One possibility is that growth cone pausing and axon
branching occur in response to exogenous membrane-bound and soluble
cues, which are known to influence growth cone behaviors (Zheng et al.,
1996; Gallo and Pollack 1997) and axon branching (Heffner et al., 1990;
Roskies and O'Leary 1994; Hubener et al., 1995; Sato et al., 1994).
These exogenous cues may include discontinuities in the substrate, such
as landmark etchings on the coverslips, cellular debris, and soluble
factors. Growth cones did contact etchings on the coverslips, which may
have influenced their behavior. Nevertheless, growth cone pausing and
axon branching took place on both etched and unetched regions of the
coverslips. We have observed that membrane particles induce pausing
behaviors by axonal growth cones and subsequent extension of branches
toward the debris (data not shown). We have also found that several
peptide growth factors stimulate branching of cortical neurons in
culture (our unpublished observations). Some aspects of
branching may be intrinsic to cortical neurons (Aigner et al., 1995;
Caroni, 1997; Benowitz and Routtenberg, 1997) because it is known that
different neuronal types have distinct axonal projection patterns
(Koester and O'Leary, 1993). These characteristics, as we and others
(Dent and Meiri, 1992; Aigner and Caroni, 1995) have shown, are
retained in culture. Regardless of the factors that promote branching
in our cultures, our results provide a reliable history of how axon
branches develop.
In previous studies, it was not possible to determine directly the
relationship between the behaviors of the axonal growth cone and
branching. Nevertheless, our results are consistent with previous
observations of growth cone behaviors and branching activity along the
axon shaft. Accumulating evidence from time-lapse imaging in intact
preparations of the vertebrate CNS has shown that growth cones exhibit
complex morphologies and behaviors in decision regions in which growth
cones change direction (Godement et al., 1994; Mason and Wang, 1997),
enter target regions (Kaethner and Stuermer, 1992), or approach targets
(Halloran and Kalil, 1994). Growth cones at the optic chiasm develop
large complex morphologies during pausing behaviors (Sretavan and
Reichardt, 1993; Mason and Wang, 1997) and in the corpus callosum,
growth cones beneath cortical targets have elaborate morphologies
during pausing behaviors involving repeated cycles of collapse,
withdrawal, and reextension (Halloran and Kalil, 1994). Development of
interstitial branches from these same regions of callosal axons
suggests a relationship between growth cone pausing and axon branching.
Observations in organotypic thalamocortical cocultures (Yamamoto et
al., 1997) relating growth cone-stopping behaviors to induction of axon
branches provides further support for this view. However, the spatial
and temporal resolution of the methods used in these studies was not
sufficient to demonstrate in detail how growth cone behaviors resulted
in axon branching.
Results of the present study show that formation of interstitial
branches is highly correlated with growth cone-pausing behaviors. However, in agreement with other studies, pausing behaviors do not
always lead to axon branching. For example, stopping behaviors by
geniculocortical axons in organotypic cocultures (Yamamoto et al.,
1997) could result either in no branching or transient branching.
Moreover, axon branches could emerge without stopping behaviors by the
growth cone. In intact preparations of the developing visual system
(Kaethner and Stuermer, 1992; Sretevan and Reichardt, 1993; Godement et
al., 1994; Mason and Wang, 1997) growth cones were shown to pause for
varying time periods, undergoing shape changes without forward advance.
In tectal targets, growth cone pausing was associated with local
exploration and transient branching followed by extension in a
preferred direction (Kaethner and Stuermer, 1992). Growth cones pausing
within the optic chiasm developed branches oriented in different
directions before their choice of a new axis of growth (Godement et
al., 1994). Although retinal axons do not actually establish persistent
branches at the chiasm, growth cone pausing was typically associated
with transient branches leading to shifts in direction of growth. Thus,
in agreement with results of the present study, growth cone pausing in
the developing visual system has been generally associated with new
trajectories of axonal growth.
Our results are also consistent with other in vivo and
in vitro studies of activity along the shafts of cortical
axons in relation to branching. A number of in vivo studies
have identified swellings or varicosities along corticospinal axons (de
Kort et al., 1985; Bastmeyer et al., 1998), some of which appear at
branch points. On cultured hippocampal neurons, branching activity in the form of short filopodial protrusions was observed before extension of axon branches (Yu et al., 1994). Branching activity on cortical axons was observed in situ in the corpus callosum beneath
cortical targets in which axons exhibited surges of membrane activity
(Halloran and Kalil, 1994). Branches to cortical targets frequently
developed in these regions of the callosum. Corticospinal axons
overlying pontine targets exhibited dynamic behaviors, including
formation of varicosities and filopodia-like protrusions (Bastmeyer
and O'Leary, 1996). Some of these filopodia were stable over several hours and could develop into a branch. Our results show that active regions of the axon shaft give rise to branches with a high
probability. However, we also found that unless processes grew to a
length of at least 30 µm and developed a growth cone they did not
become a stable branch. Consistent with previous results, most shorter processes tended to be transient. The present results not only show
that activity along the axon shaft precedes axon branching, but also
that activity along the axon shaft is often a remnant of the primary
growth cone.
Our results demonstrate the relation of the primary growth cone to the
development of branches. Nevertheless, a number of questions remain for
future studies. First, what changes occur when the growth cone
undergoes pausing behaviors, enlarges, and reorganizes? Growth cone
pausing, perhaps in response to stop signals (Baird et al., 1992), may
allow the growth cone to explore and sample cues in target regions.
During enlargement, the growth cone may be concentrating and storing
signaling molecules as well as materials for growth of new processes.
During axon extension, the growth cone is continuously consolidating
into new axon (Goldberg and Burmeister, 1986; Aletta and Greene, 1988).
During growth cone pausing, the cytoskeleton of the axon may be
transformed so it does not consolidate but remains dynamic at branch
points (Yu et al., 1994; Tanaka and Sabry, 1995). Although we have
shown that branches can arise from growth cone remnants, it would be of
interest to know whether active regions of the axon shaft are particularly responsive to branching signals or whether branches can be
induced on any region of the axon (Bray et al., 1978; Williams et al.,
1995; Ziv and Spira, 1997). A related question is why in
vivo branches arise at precise locations from axons specific to a
given target in pathways such as the corticospinal tract (O'Leary and
Terashima, 1988; Kuang and Kalil, 1994). Our results suggest that
growth cone target specificity underlies the specificity of axon
branching. However, in addition to signals for induction of branches at
specific locations, other signals may be required for stabilization of
branches that subsequently innervate targets.
We have provided a detailed account of how growth cone behaviors lead
to axon branching in culture, which suggests a new model for target
innervation by interstitial axon branches in vivo (Fig. 8). According to this model, during
growth cone pausing in target regions, the growth cone enlarges and
reorganizes. After the primary growth cone resumes forward advance,
parts of the growth cone remain as filopodial or lamellar activity
along the axon shaft. Branches typically extend from these active
membrane regions after various delays to innervate targets. This model
is attractive because it suggests that interstitial branching results
directly from growth cone target recognition.
View larger version (7K):
[in this window]
[in a new window]
|
Figure 8.
The in vitro results suggest the
following model for interstitial branching by cortical neurons
in vivo. Schematics represent different stages in axon
branching. A, Growth cone of an efferent cortical axon
is advancing along a pathway toward its target, indicated by the
circle. B, In response to target-derived signals, the
primary growth cone pauses in the vicinity of the target for extended
time periods and enlarges. C, After the primary growth
cone has resumed forward advance, remnants of the reorganized growth
cone appear as filopodial or lamellar activity along the axon shaft.
D, A branch from these active membrane regions extends
toward the target after various time delays. Typically, branches extend
interstitially some distance behind the primary growth cone. However,
when the primary growth cone and the branch extend simultaneously,
branching appears to occur by bifurcation (data not shown).
|
|
| |
FOOTNOTES |
Received April 6, 1998; revised June 19, 1998; accepted July 13, 1998.
This work was supported by National Institutes of Health Grant NS14428
to K.K. and a predoctoral training grant award (GM07507) to E.W.D. We
thank Dr. P. W. Baas for helpful comments on this manuscript.
Correspondence should be addressed to Katherine Kalil, University of
Wisconsin, Department of Anatomy, 1300 University Avenue, Madison, WI
53706.
Dr. Szebenyi's present address: Department of Cell Biology and
Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9111.
| |
REFERENCES |
-
Aigner L,
Caroni P
(1995)
Absence of persistent spreading, branching, and adhesion in GAP-43-depleted growth cones.
J Cell Biol
128:647-660[Abstract/Free Full Text].
-
Aigner L,
Arber S,
Kapfhammer JP,
Laux T,
Schneider C,
Botteri F,
Brenner HR,
Caroni P
(1995)
Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.
Cell
83:269-278[ISI][Medline].
-
Aletta JM,
Greene LA
(1988)
Growth cone configuration and advance: a time-lapse study using video-enhanced differential interference contrast microscopy.
J Neurosci
8:1425-1435[Abstract].
-
Aoyagi A,
Nishikawa K,
Saito H,
Abe K
(1994)
Characterization of basic fibroblast growth factor-mediated acceleration of axonal branching in cultured rat hippocampal neurons.
Brain Res
661:117-126[ISI][Medline].
-
Baird DH,
Baptista CA,
Wang LC,
Mason CA
(1992)
Specificity of a target cell-derived stop signal for afferent axonal growth.
J Neurobiol
23:579-591[ISI][Medline].
-
Bastmeyer M,
O'Leary DDM
(1996)
Dynamics of target recognition by interstitial axon branching along developing cortical axons.
J Neurosci
16:1450-1459[Abstract/Free Full Text].
-
Bastmeyer M,
Daston MM,
Possel H,
O'Leary DDM
(1998)
Collateral branch formation related to cellular structures in the axon tract during corticopontine target recognition.
J Comp Neurol
392:1-18[ISI][Medline].
-
Benowitz LI,
Routtenberg A
(1997)
GAP-43: an intrinsic determinant of neuronal development and plasticity.
Trends Neurosci
20:84-91[ISI][Medline].
-
Bolz J,
Castellani V
(1997)
How do wiring molecules specify cortical connections?
Cell Tissue Res
290:307-314[ISI][Medline].
-
Bray D
(1973)
Branching patterns of individual sympathetic neurons in culture.
J Cell Biol
56:702-712[Abstract/Free Full Text].
-
Bray D,
Thomas C,
Shaw G
(1978)
Growth cone formation in cultures of sensory neurons.
Proc Natl Acad Sci USA
75:5226-5229[Abstract/Free Full Text].
-
Caroni P
(1997)
Intrinsic neuronal determinants that promote axonal sprouting and elongation.
Bioessays
19:767-775[ISI][Medline].
-
Castellani V,
Bolz J
(1997)
Membrane-associated molecules regulate the formation of layer-specific cortical circuits.
Proc Natl Acad Sci USA
94:7030-7035[Abstract/Free Full Text].
-
de Kort EJ,
Gribnau AA,
van Aanholt HT,
Nieuwenhuys R
(1985)
On the development of the pyramidal tract in the rat. I. The morphology of the growth zone.
Anat Embryol
172:195-204[Medline].
-
Dent EW,
Meiri KF
(1992)
GAP-43 phosphorylation is dynamically regulated in individual growth cones.
J Neurobiol
23:1037-1053[ISI][Medline].
-
Dotti CG,
Sullivan CA,
Banker GA
(1988)
The establishment of polarity by hippocampal neurons in culture.
J Neurosci
8:1454-1468[Abstract].
-
Gallo G,
Pollack ED
(1997)
Temporal regulation of growth cone lamellar protrusion and the influence of target tissue.
J Neurobiol
33:929-944[ISI][Medline].
-
Godement P,
Wang LC,
Mason CA
(1994)
Retinal axon divergence in the optic chiasm: dynamics of growth cone behavior at the midline.
J Neurosci
14:7024-7039[Abstract].
-
Goldberg DJ,
Burmeister DW
(1986)
Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy.
J Cell Biol
103:1921-1931[Abstract/Free Full Text].
-
Goodman CS
(1996)
Mechanisms and molecules that control growth cone guidance.
Annu Rev Neurosci
19:341-377[ISI][Medline].
-
Halloran MC,
Kalil K
(1994)
Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy.
J Neurosci
14:2161-2177[Abstract].
-
Harris WA,
Holt CE,
Bonhoeffer F
(1987)
Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse study of single fibers in vivo.
Development
101:123-133[Abstract].
-
Heffner CD,
Lumsden AG,
O'Leary DDM
(1990)
Target control of collateral extension and directional axon growth in the mammalian brain.
Science
247:217-220[Abstract/Free Full Text].
-
Hubener M,
Gotz M,
Klostermann S,
Bolz J
(1995)
Guidance of thalamocortical axons by growth-promoting molecules in developing rat cerebral cortex.
Eur J Neurosci
7:1963-1972[ISI][Medline].
-
Inoué S
(1986)
In: Video microscopy. New York: Plenum.
-
Inoué S,
Spring K
(1997)
In: Video microscopy, 2nd edition. New York: Plenum.
-
Joosten EA
(1997)
Corticospinal tract regrowth.
Prog Neurobiol
53:1-25[ISI][Medline].
-
Kaethner RJ,
Stuermer CA
(1992)
Dynamics of terminal arbor formation and target approach of retinotectal axons in living zebrafish embryos: a time-lapse study of single axons.
J Neurosci
12:3257-3271[Abstract].
-
Kennedy TE,
Tessier-Lavigne M
(1995)
Guidance and induction of branch formation in developing axons by target-derived diffusible factors.
Curr Opin Neurobiol
5:83-90[Medline].
-
Koester SE,
O'Leary DDM
(1993)
Connectional distinction between callosal and subcortically projecting cortical neurons is determined prior to axon extension.
Dev Biol
160:1-14[ISI][Medline].
-
Kuang RZ,
Kalil K
(1994)
Development of specificity in corticospinal connections by axon collaterals branching selectively into appropriate spinal targets.
J Comp Neurol
344:270-282[ISI][Medline].
-
Mason CA,
Wang LC
(1997)
Growth cone form is behavior-specific and, consequently, position-specific along the retinal axon pathway.
J Neurosci
17:1086-1100[Abstract/Free Full Text].
-
Norris CR,
Kalil K
(1992)
Development of callosal connections in the sensorimotor cortex of the hamster.
J Comp Neurol
326:121-132[ISI][Medline].
-
O'Leary DDM,
Bicknese AR,
De Carlos JA,
Heffner CD,
Koester SE,
Kutka LJ,
Terashima T
(1990)
Target selection by cortical axons: alternative mechanisms to establish axonal connections in the developing brain.
Cold Spring Harb Symp Quant Biol
55:453-468[Medline].
-
O'Leary DDM,
Koester SE
(1993)
Development of projection neuron types, axon pathways, and patterned connections of the mammalian cortex.
Neuron
10:991-1006[ISI][Medline].
-
O'Leary DDM,
Terashima T
(1988)
Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and "waiting periods".
Neuron
1:901-910[ISI][Medline].
-
Roskies AL,
O'Leary DDM
(1994)
Control of topographic retinal axon branching by inhibitory membrane-bound molecules.
Science
265:799-803[Abstract/Free Full Text].
-
Sato M,
Lopez-Mascaraque L,
Heffner CD,
O'Leary DDM
(1994)
Action of a diffusible target-derived chemoattractant on cortical axon branch induction and directed growth.
Neuron
13:791-803[ISI][Medline].
-
Sretavan DW,
Reichardt LF
(1993)
Time-lapse video analysis of retinal ganglion cell axon pathfinding at the mammalian optic chiasm: growth cone guidance using intrinsic chiasm cues.
Neuron
10:761-77[ISI][Medline].
-
Tanaka E,
Sabry J
(1995)
Making the connection: cytoskeletal rearrangements during growth cone guidance.
Cell
83:171-176[ISI][Medline].
-
Tessier-Lavigne M,
Goodman CS
(1996)
The molecular biology of axon guidance.
Science
274:1123-1133[Abstract/Free Full Text].
-
Wessells NK,
Nuttall RP
(1978)
Normal branching, induced branching, and steering of cultured parasympathetic motor neurons.
Exp Cell Res
115:111-122[ISI][Medline].
-
Williams CV,
Davenport RW,
Dou P,
Kater SB
(1995)
Developmental regulation of plasticity along neurite shafts.
J Neurobiol
27:127-140[ISI][Medline].
-
Yamamoto N,
Higashi S,
Toyama K
(1997)
Stop and branch behaviors of geniculocortical axons: a time-lapse study in organotypic cultures.
J Neurosci
17:3653-3663[Abstract/Free Full Text].
-
Yu W,
Ahmad FJ,
Baas PW
(1994)
Microtubule fragmentation and partitioning in the axon during collateral branch formation.
J Neurosci
14:5872-5884[Abstract].
-
Zheng JQ,
Poo MM,
Connor JA
(1996)
Calcium and chemotropic turning of nerve growth cones.
Perspect Dev Neurobiol
4:205-213[ISI][Medline].
-
Ziv NE,
Spira ME
(1997)
Localized and transient elevations of intracellular Ca2+ induce the dedifferentiation of axonal segments into growth cones.
J Neurosci
17:3568-3579[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18197930-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
Y. Hayano and N. Yamamoto
Activity-Dependent Thalamocortical Axon Branching
Neuroscientist,
August 1, 2008;
14(4):
359 - 368.
[Abstract]
[PDF]
|
|
|
|
|
|
|
M. J. Galazo, V. Martinez-Cerdeno, C. Porrero, and F. Clasca
Embryonic and Postnatal Development of the Layer I-Directed ("Matrix") Thalamocortical System in the Rat
Cereb Cortex,
February 1, 2008;
18(2):
344 - 363.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Carter, L. Demizieux, R. B. Campenot, D. E. Vance, and J. E. Vance
Phosphatidylcholine Biosynthesis via CTP:Phosphocholine Cytidylyltransferase 2 Facilitates Neurite Outgrowth and Branching
J. Biol. Chem.,
January 4, 2008;
283(1):
202 - 212.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kakimoto, H. Katoh, and M. Negishi
Regulation of Neuronal Morphology by Toca-1, an F-BAR/EFC Protein That Induces Plasma Membrane Invagination
J. Biol. Chem.,
September 29, 2006;
281(39):
29042 - 29053.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Tang and K. Kalil
Netrin-1 Induces Axon Branching in Developing Cortical Neurons by Frequency-Dependent Calcium Signaling Pathways
J. Neurosci.,
July 13, 2005;
25(28):
6702 - 6715.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. M. Y. Moresco, S. Donaldson, A. Williamson, and A. J. Koleske
Integrin-Mediated Dendrite Branch Maintenance Requires Abelson (Abl) Family Kinases
J. Neurosci.,
June 29, 2005;
25(26):
6105 - 6118.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Uesaka, S. Hirai, T. Maruyama, E. S. Ruthazer, and N. Yamamoto
Activity Dependence of Cortical Axon Branch Formation: A Morphological and Electrophysiological Study Using Organotypic Slice Cultures
J. Neurosci.,
January 5, 2005;
25(1):
1 - 9.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Bouquet, S. Soares, Y. von Boxberg, M. Ravaille-Veron, F. Propst, and F. Nothias
Microtubule-Associated Protein 1B Controls Directionality of Growth Cone Migration and Axonal Branching in Regeneration of Adult Dorsal Root Ganglia Neurons
J. Neurosci.,
August 11, 2004;
24(32):
7204 - 7213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kakimoto, H. Katoh, and M. Negishi
Identification of Splicing Variants of Rapostlin, a Novel Rnd2 Effector that Interacts with Neural Wiskott-Aldrich Syndrome Protein and Induces Neurite Branching
J. Biol. Chem.,
April 2, 2004;
279(14):
14104 - 14110.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
