Axons With Highly Branched Terminal RegionsSuccessfully Regenerate Across Spinal MidlineTransections in the Adult Cat

Keith K. Fenrich* and P. Ken Rose

CIHR Group in Sensory-Motor Systems, Department of Physiology, Center for Neuroscience, Queen’s University, Kingston, ON, Canada

ABSTRACTWe recently reported that some, but not all, axotomized

propriospinal commissural interneurons (PCI) of the

adult mammal can regenerate through spinal midsagit-

tal transection injury sites (Fenrich and Rose [2009] J

Neurosci 29:12145–12158). In this model, regenerating

axons grow through a lesion site surrounded by a

dense deposition of chondroitin sulfate proteoglycans

(CSPG), which are typically inhibitory to regenerating

axons. However, the mechanisms that lead some regen-

erating axons to grow through spinal cord injury (SCI)

sites while others remain trapped in the CSPG zones or

retract to their soma remain unknown. As a first step

toward elucidating these mechanisms, here we show

that the ability of PCI axons to regenerate across a SCI

site depends on the branching patterns of their distal

terminals. Using 3D reconstruction techniques through

multiple serial sections and immunohistochemical analy-

ses, we found that at 7 days postinjury a majority of

PCI axons terminated in CSPG zones ipsilateral of the

spinal midline. Conversely, at 9 days postinjury some

PCI axons had regenerated across the midline, but

others terminated outside the CSPG zones near their

soma. Furthermore, we show that the most successful

regenerators were those with the most extensive

branching patterns, whereas those that terminated out-

side the CSPG zones had terminal regions indistinguish-

able from dystrophic terminals. Our results demonstrate

that the morphological characteristics of regenerating

axons play an important role in their ability to regener-

ate across SCI sites, and that the branching patterns of

some regenerating axons are more extensive and have

a far greater complexity than previously reported.

J. Comp. Neurol. 519:3240–3258, 2011.

VC 2011 Wiley-Liss, Inc.

INDEXING TERMS: axon regeneration; spinal interneuron; spinal cord injury; chondroitin sulfate proteoglycans; growth

cone; axon guidance

Regenerating axons in the adult mammalian central

nervous system (CNS) typically extend rapidly in intact

nervous tissue (Davies et al., 1999), but often cannot

cross traumatic injury sites (Ramon Y Cajal, 1959; Yiu

and He, 2006). This switch from growing to growth-inhib-

ited is coupled with a change in morphology of their distal

tips. For instance, the axons of transplanted adult dorsal

root ganglia (DRG) neurons typically follow a straight tra-

jectory and have a small and streamlined terminal swel-

ling while located within uninjured white matter (Davies

et al., 1999). However, within spinal cord injury (SCI)

sites, these terminal regions follow irregular paths and

have swollen endballs (Davies et al., 1999). Similar mor-

phologies have also been described for cut corticospinal

axons within glial scars (Li and Raisman, 1994, 1995).

Consistent with the terminal regions of regenerating

axons, during development the morphologies of axonal

growth cones vary depending on their local environment.

For example, the terminals of developing axons projecting

from the spinal cord to the periphery have filopodia and

lamellipodia at nerve plexus regions or follow a circuitous

path and have multiple branches and enlargements at

muscle regions, but are small with few varicosities in spi-

nal nerves (Tosney and Landmesser, 1985). Similarly,

axon terminals growing across the CNS midline at the

corpus callosum (Ozaki and Wahlsten, 1992; Halloran

Grant sponsor: Canadian Institutes for Health Research; Grant number:MOP-79299; Grant sponsor: Trevor C. Holland Fellowship; Dr. Robert JohnWilson Fellowship; Ontario Graduate Scholarship (all to K.K.F.).

*CORRESPONDENCE TO: Keith K. Fenrich, PhD, Department ofPhysiology, Queen’s University, Kingston, ON, Canada, K7L 3N6. E-mail:keith@biomed.queensu.ca

VC 2011 Wiley-Liss, Inc.

Received August 23, 2010; Revised December 15, 2010; Accepted May28, 2011

DOI 10.1002/cne.22686

Published online June 14, 2011 in Wiley Online Library (wileyonlinelibrary.com)

3240 The Journal of Comparative Neurology | Research in Systems Neuroscience 519:3240–3258 (2011)

RESEARCH ARTICLE

and Kalil, 1994) or optic chiasm (Mason and Wang, 1997)

have more branches and enlargements as they approach

and cross the midline compared to those that have al-

ready crossed, which are simple and flat.

Recent studies have shown that certain neurons in the

adult mammal can regenerate their cut axons through

SCI sites in the absence of therapeutic interventions. For

instance, Dray et al. (2009) found that mouse spinal DRG

axons can regenerate through pinprick SCIs. In addition,

we recently showed that axons of some axotomized pro-

priospinal commissural interneurons (PCI) can regenerate

through SCI sites in the adult cat (Fenrich and Rose,

2009). However, whether there are certain morphological

characteristics that distinguish between axons that cross

a lesion site and those that fail to cross a lesion site

remains unknown.

In the present study we used serial reconstruction

techniques to trace the path(s) taken by axons of individ-

ual PCIs after axotomy, from their origin at the soma to all

of their terminations. These reconstructions were used to

compare the morphological characteristics of axotomized

PCI axons and gauge their regenerative capacity. This

approach revealed that many axons form elaborate termi-

nal and preterminal arborizations whose full complexity

only became evident using serial reconstruction techni-

ques. The axons that had grown the furthest toward the

contralateral spinal cord had the most complex terminal

and preterminal arborizations. In contrast, the terminal

regions furthest from crossing the midline typically had

compact swollen terminals with multiple short protru-

sions and few arborizations. Our results suggest that the

branch-structure morphology of the terminal regions of

regenerating PCI axons is a key indicator of successful

penetration and navigation through SCI sites.

MATERIALS AND METHODS

SurgeriesAll surgical and animal care protocols for these experi-

ments were consistent with the guidelines established by

the Canadian Council of Animal Care and were approved

by the Queen’s University Animal Care Committee. These

experiments were performed on six adult cats (>10

months of age) weighing 2.8–3.5 kg. Animals were pre-

medicated with a cocktail of medetomidine (0.03 mg/

kg), ketamine (5.0 mg/kg), glycopyrrolate (0.005 mg/

kg), and hydromorphone (0.075 mg/kg) injected subcuta-

neously; deep anesthesia was maintained with either iso-

flurane (recovery experiments; 1–2%) or sodium pento-

barbital (terminal experiments; 10 mg/kg intravenously

[i.v.]; supplemental dosages, 5 mg/kg i.v.). The surgical

procedures, peripheral nerve stimulation protocols, and

postoperative care procedures have previously been

described (Fenrich et al., 2007; Fenrich and Rose, 2009).

Briefly, the animal’s head was placed in a stereotaxic

head-holder, the C2 and C3 vertebra were exposed and

the dorsal bone covering the C3 segment was removed.

The spinous process of the T2 or T3 vertebra was

exposed and the animal’s torso was further suspended

with spinal clamps attached to the exposed spinous pro-

cess. The nerves innervating the C3 biventer cervicis and

complexus (BCCM) muscles were isolated and mounted

on bipolar hook electrodes. The exposed dura was

resected and folded onto the lateral vertebral opening to

expose the dorsal surface of the C3 spinal cord.

Spinal midline transectionsThe spinal midline was located in two ways. First, we

identified the midline as a point equidistant from the

BCCM motoneuron nuclei located in the left and right

ventral horns. We recorded the field potentials of anti-

dromically activated BCCM motoneurons and found the

center of the motoneuron pool on both sides of the spinal

cord. The midpoint between these bilateral motoneuron

pools was considered the ‘‘ventral’’ midline. Second, we

removed the arachnoid and pia matter from the exposed

dorsal spinal cord. This served two purposes: first, to

reduce the amount of tissue drag and dimpling encoun-

tered during the lesion (see below); and second, to ex-

pose the dorsal median sulcus. The sulcus was used to

mark the ‘‘dorsal’’ midline of the spinal cord. In most

experiments the ventral and dorsal midpoints matched.

However, in two experiments the medial-lateral location

of the ventral and dorsal midlines did not match (�150–

200 lm difference), thus indicating that the spinal cord

was slightly twisted. In these cases the midpoint between

the dorsal and ventral midlines was considered the

midline.

Lesions were performed using specially modified dou-

ble-edged scalpel blades. These blades were mounted on

a microdrive attached to the stereotaxic frame. The lesion

protocol has previously been described in detail (Fenrich

et al., 2007; Fenrich and Rose, 2009). Briefly, the blade

was aligned with the spinal midline on the dorsal surface

of the spinal cord at the caudal edge of the planned lesion

site. The blade was lowered 2.5 mm into the spinal mid-

line and then retracted to the surface. The blade was

advanced rostrally 0.5 mm and the spinal midline was

pierced using the same procedure as at the previous loca-

tion. In previous studies (Fenrich et al., 2007; Fenrich and

Rose, 2009) we used advances of 0.75 mm. The shorter

intervals used in the present study reduced spinal com-

pression and resulted in more effective lesions. These

rostral advances and piercing movements were continued

to the rostral edge of the lesion, where the blade was low-

ered 2.5 mm. While the blade was still lowered 2.5 mm,

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3241

the blade was moved caudally then rostrally then caudally

the entire length of the lesion. From a depth of 2.5 mm

and starting at the caudal edge of the lesion, the piercing

and advancing procedure was repeated except the blade

was lowered until a 5-mm depth indicator on the blade

was no longer visible at the dorsal surface of the spinal

cord. Once the rostral edge of the lesion was reached,

the blade was lowered so that the 5-mm depth indicator

marked on the blade was no longer visible. The blade was

then moved caudally then rostrally then caudally the

entire length of the lesion and removed from the spinal

cord. We have previously shown that this lesion protocol

effectively cuts all axons that cross the midline through-

out the entire length of the lesion (Fenrich and Rose,

2009). All lesions for this study were between 4 mm and

6 mm in length. Postoperative pain was controlled with

burenorphine (0.005–0.01 mg/kg s.q., every 6 hours or

as required) and Metacam (1 drop oral, per day as

required).

Neurobiotin stainingAll terminal experiments were performed at 7 days or 9

days postlesion. In these experiments the dorsal verte-

brae were removed to expose the C1 to C5 spinal cord.

The animal was suspended from a stereotaxic frame and

the nerves innervating the left C3 and C4 BCCM, C2 and

C3 splenius, and trapezius muscles were mounted on

hook stimulating electrodes. At 7 days postlesion dense

scar tissue had developed on the dorsal surface of the

spinal cord at the SCI site. Attempts to advance glass

micropipettes through this tissue caused the micropip-

ettes to break. The scar tissue was therefore removed

using curved spring scissors (Fine Science Tools, North

Vancouver, BC, Canada). All animals were paralyzed with

gallamine triethiodide (Sigma, St. Louis, MO; 2.5–5 mg/

kg/hr i.v.) and ventilated using a respirator.

The Neurobiotin injection protocols are identical to

those used for previous studies (Fenrich et al., 2007; Fen-

rich and Rose, 2009). Briefly, glass micropipette tips

were broken to achieve a tip diameter of �4 lm. Pipettes

were filled with 12% Neurobiotin (Vector, Burlington, ON,

Canada) in 0.5 M KCl and 0.1 M Tris-HCl buffer, pH 8.2.

All injections were made into the extracellular space of

the left ventral horn. For each track in which Neurobiotin

was injected, an injection was made at 200 lm dorsal of

the BCCM field potential and another at 800 lm dorsal of

the BCCM field potential. Injections were made with 5 lApositive pulses for 90 seconds with a 10-second on-off

duty cycle (Midgard Electronics, Canton, MA; model

CS3). These injection parameters yielded about 10–20

well-stained neurons per injection site. Not all of the

stained neurons were PCIs; however, only axotomized

PCIs were included in this study (see below).

The first injection track of each experiment was 1–2

mm caudal of the caudal edge of the midline lesion site.

Each subsequent injection track was 750 lm rostral of

the previous. Injections were made throughout the entire

rostral-caudal length of the midline lesion and 1–2 mm

rostral of the midline lesion. Following the final Neurobio-

tin injection, the tracer was allowed to spread for 8–10

hours prior to fixation.

Tissue preparation andimmunohistochemistry

All animals were administered Heparin (25,000 IU i.v.)

followed by a euthanizing dose of sodium pentobarbital.

The animals were perfused with 1 L of normal saline fol-

lowed by 4% paraformaldehyde in 0.1 M phosphate-

buffered saline (PBS) (pH 7.4) for 20 minutes. The spinal

cords were removed, postfixed overnight, and then trans-

ferred to 20% sucrose in PBS for several days prior to

sectioning.

Horizontal serial sections (50 lm thick) were cut using

a freezing microtome. Sections were placed in PBS with

30% sucrose and 30% ethylene glycol and stored at

�20�C for several weeks. Sections were removed from

the freezer and rinsed several times in PBS. Sections

were incubated in PBS with 0.1% NaBH4 for 25 minutes,

and then rinsed several times in PBS. To visualize the dis-

tribution of chondroitin sulfate proteoglycans (CSPG),

sections were incubated with CS-56 monoclonal antibod-

ies, which are reported to be specific for the glycosamino-

glycan (GAG) moieties of CSPGs (1:250; mAb CS-56;

Sigma; cat. no. C80735) for 2 nights at 4�C. These anti-

bodies were raised in mouse against ventral membranes

of chicken gizzard fibroblasts. These antibodies were

visualized with AMCA-conjugated secondary antibodies

(1:250; goat antimouse; IgM l-chain specific; Jackson

ImmunoResearch, Mississauga, ON, Canada). To visualize

Neurobiotin the sections were incubated in streptavidin

conjugated to Cy5 (1:100; Jackson ImmunoResearch).

Sections were mounted on subbed slides with Vecta-

shield mounting medium (Vector) and stored at 2–4�C.

Antibody characterizationTo characterize the specificity of the CS-65 monoclonal

antibodies in cats we first compared the pattern of stain-

ing along and immediately adjacent to spinal midline

transection sites in the cat with labeling at spinal cord

injury sites of other species as reported in previous stud-

ies (Davies et al., 1999; Kim et al., 2006). In the cat, at 7

and 9 days postmidline transection CS-56 antibodies

yielded densely stained regions along and immediately

adjacent to the transection sties, with less staining in the

adjacent gray matter, and very little staining in the lateral

K.K. Fenrich and P.K. Rose

3242 The Journal of Comparative Neurology |Research in Systems Neuroscience

white matter. The CS-56 staining patterns at and near the

spinal transection sites in the cat were nonuniform due to

densely labeled interconnected ‘‘tube-like’’ structures

(Fig. 1A,C). In zones between the tube-like structures the

CS-56 staining was less intense and more diffuse. These

staining patterns are very similar to the CS-56 staining

patterns at and near spinal cord injury sites in adult mice

and rats at 7 and 8 days post-SCI (Davies et al., 1999;

Kim et al., 2006). To further characterize the specificity of

the CS-56 antibody in injured cat spinal tissue, we

digested the GAG portion of the CSPG molecules in some

tissue sections using chondroitinase ABC (6 or 24 hours

incubation; 0.05 units/ml; Sigma; cat. no. C2905) before

continuing with the CS-56 incubation protocol (Munyer

and Schulte, 1991). For these control experiments we

used a pair of serial tissue sections from each animal:

one tissue section was incubated with chondroitinase

ABC prior to the CS-56 incubation protocol, whereas the

other tissue section was simultaneously incubated in

buffer alone prior to the CS-56 incubation protocol. We

used tissue sections that were immediately dorsal to

those used for the rest of this study. Digestion of the

GAGs eliminated the dense CS-56 staining at and near

SCI sites for both 7 and 9 day post-SCI experiments (Fig.

1). Our results strongly suggest that CS-56 antibodies

specifically recognize the GAG chains of CSPGs in the cat

spinal cord. Considering the close similarity between the

CS-56 staining patterns in our study to the CS-56 staining

Figure 1. CS-56 antibodies specifically recognize CSPG GAG chains in the cat spinal cord. A,B: Fluorescent images showing CS-56 anti-

body labeling from equivalent regions along a spinal midline transection site from adjacent serial sections from a 7-day post-SCI experi-

ment. The tissue section shown in A was preincubated in buffer for 24 hours prior to staining with CS-56 antibodies, whereas the tissue

section shown in B was preincubated in chondroitinase ABC for 24 hours prior to staining with CS-56 antibodies. C,D: Fluorescent images

showing CS-56 antibody labeling from equivalent regions along a spinal midline transection site from adjacent serial sections from a 9-day

post-SCI experiment. The tissue section shown in C was preincubated in buffer for 6 hours prior to staining with CS-56 antibodies,

whereas the tissue section shown in D was preincubated in chondroitinase ABC for 6 hours prior to staining with CS-56 antibodies. All

images were acquired in one imaging session using the same microscope settings and subjected to the same brightness and contrast

adjustments. Scale bars ¼ 100 lm.

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3243

patterns in other spinal cord injury studies using the

same antibodies, and that incubation of dorsal tissue sec-

tions in chondroitinase ABC abolished CS-56 staining, we

consider the CS-56 staining observed in the present

study to be indicative of the distribution of CSPGs.

ReconstructionsAxons were reconstructed using a fluorescent micro-

scope (Olympus BX60) equipped with 40� (0.75 NA) and

60� (0.90) dry immersion lenses, a Neurolucida System

(MBF Biosciences, Williston, VT, v. 8.22), and a Retiga EXi

camera (QImaging, Surrey, BC, Canada). A reconstruction

is a 3D tracing of a neuron, or part of a neuron, through

serial histological sections in which each process is repre-

sented as a series of connected points. Each point has an

x- (mediolateral), y- (rostrocaudal), and z- (dorsoventral)

coordinate and a diameter. To begin a reconstruction,

image stacks of an axon terminal were acquired from one

tissue section. The axon terminal was reconstructed

using these image stacks. As the axon and reconstruction

progressed to processes found on adjacent tissue sec-

tions, image stacks of the axon were acquired from these

sections. The image stacks of each section was aligned

and stitched to the image stacks of the previous sections

so that by the end of the reconstruction process the

entire axon was stored as a series of stitched digital

image stacks. All reconstructions began at an axon termi-

nal and followed the trajectory of the terminal region

branches, as well as the parent axon back to the cell of

origin in the ventral horn. Unless noted, all axonal proc-

esses were followed to their termination, defined by an

abrupt loss of Neurobiotin.

Only axons that were well stained with Neurobiotin and

were sufficiently well isolated from other Neurobiotin-

stained processes were reconstructed. To restrict our

analysis only to axotomized PCI axons (Steward et al.,

2003), only axons that terminated either 1) within the

dense CSPG labeling of the lesion site, or 2) within the

gray matter adjacent to the dense CSPG staining were

chosen for reconstruction. All reconstructions were cor-

rected for tissue shrinkage in the z-axis.

The reconstruction techniques described above are

ideally suited for tracing tubular structures such as axons

and dendrites. In some cases the axon terminal region

consisted of one or more large swellings. The surface of

these swellings was often covered with smaller bulbous

appendages or formed convoluted shapes such as a ca-

lyx. These structures cannot be rendered using Neurolu-

cida. In these cases only the parent axon and the main

shaft of the axon was reconstructed as described above.

To visualize the structure of these axon terminal regions,

confocal image stacks were acquired (Leica TCS SP2 mul-

tiphoton microscope). Three-dimensional images of these

image stacks were rendered using ImagePro Plus (v. 6.3;

Media Cybernetics, Silver Springs, MD), and the contrast

and brightness were optimized. With the exception of the

CS-56 control illustrations (Fig. 1), these image acquisi-

tion techniques were used for all photomicrograph

illustrations.

Morphological quantification and statisticalanalysis

Quantitative analysis was performed on entire terminal

regions and their parent axon, or on the terminal regions

only. To analyze segments of axons and/or their terminal

regions, the segments of interest were extracted from the

reconstruction and analyzed individually. Quantitative

measures, such as the number of branch points, were col-

lected using NeuroExplorer (MBF Biosciences, v. 4.7).

Statistical significance was evaluated using nonpara-

metric tests, including the Two-Sample KS test, Kruskal–

Wallis test, Mann–Whitney U-test, and Fisher’s exact test;

and, where applicable, parametric tests including simple

linear regression models and Student’s t-test (Systat:

SPSS, Chicago, IL). The specific tests used for each evalu-

ation are described in the text.

RESULTS

Axonal growth from 7 to 9 dayspostaxotomy

At 7 days following a spinal C3 midsagittal transection,

the terminals of many axotomized PCI axons are located

in the ipsilateral spinal cord, and very few cross the

injured midline (Fenrich and Rose, 2009). In contrast, by

56–72 days postinjury many axotomized PCI axons had

crossed the midsagittal transection site and terminated

in the contralateral ventromedial funiculus. In the present

study we examined axotomized PCI axons at 7 and 9 days

following a spinal C3 midsagittal transection. Our goal

was to obtain two ‘‘snapshots’’ of the regenerative pro-

cess in close temporal succession. Comparisons of the

distribution of the leading edge of the advancing axon

terminals provide an estimate of the migration, stasis, or

retraction of PCI axons, as a population, as they

regenerate.

The axons and somata of 52 axotomized PCIs were

reconstructed (Fig. 2A) (n ¼ 22 at 7 days postinjury; n ¼30 at 9 days postinjury). In all experiments there was a

dense deposition of CSPGs along the midline, and

extended the same rostrocaudal distance as the lesion

(Fenrich and Rose, 2009). CSPGs are extracellular matrix

molecules that are typically inhibitory to the growth of

regenerating axons (Rudge and Silver, 1990; McKeon

et al., 1991; Bradbury et al., 2002), and are released by

reactive astrocytes that invade injury sites and contribute

K.K. Fenrich and P.K. Rose

3244 The Journal of Comparative Neurology |Research in Systems Neuroscience

to the formation of a glial scar following SCI (McKeon

et al., 1991; Silver and Miller, 2004). Considering their

role in axonal regeneration, we traced the border of the

dense CSPG zones every 3–5 tissue sections relative to

the location of the gray matter, midline, and the recon-

structed neurons. The CSPG dense zones extended later-

ally several hundred microns to the gray matter border or

within 100 lm of the gray matter border. All reconstruc-

tions began at the distal regions of the cut PCI axons ei-

ther within or adjacent to the dense CSPG zone and con-

tinued to their cell body. All cell bodies were located

within the ventral horn adjacent to the dense CSPG label-

ing and in close proximity to a Neurobiotin injection site.

The distal regions of many cut axons were located within

the CSPG zones (Fig. 2B) and their proximal regions

located in the adjacent gray matter (Fig. 2C). Closer to

the soma the axons had a uniform diameter and some-

times had very short, fine, right-angled branches (Fig.

2D). Similar to uninjured axons, the cut axons emerged

from a soma or proximal dendrite and had characteristics,

such as a thin diameter, typical of an initial segment (Fig.

2E). Similar to previous studies (Fenrich et al., 2007; Fen-

rich and Rose, 2009), all somata were located in the ven-

tral horn and within a few hundred microns of an injection

site. In the mediolateral axis, the majority of somata were

located near the center third of the ventral horn, with only

a few located near the medial or lateral edge of the ven-

tral horn.

To compare the migration of axon terminals toward the

contralateral spinal cord at 7 days and 9 days postaxot-

omy, the mediolateral distance from the leading process

(i.e., the process furthest toward the contralateral spinal

cord) of each axon to the midline was measured (Fig. 3A).

At 9 days postinjury, cut PCI axons had grown an average

of 180 lm further toward the contralateral spinal cord,

relative to the midline, compared to cut PCI axons from 7

day postinjury experiments (P < 0.01, Two-Sample KS

test). In addition, more axons had regenerated across the

midline at 9 days compared to 7 days postinjury.

To examine the growth of PCI axons relative to the

dense CSPG labeling, we determined the proportion of

PCI axons that had penetrated the dense CSPG zones at

7 and 9 days postinjury. Each axon was categorized

according to the mediolateral location of its leading

Figure 2. Some regenerating axons progress further toward the contralateral spinal cord than others at 7 and 9 days postinjury. A: Recon-

struction of a PCI that terminated within the CSPG-rich lesion site ipsilateral of the midline (cell 71-10 in Table 2). Black, axons; dark gray,

somata. B–E: Confocal image stacks showing parts of the representative Neurobiotin stained PCI axon and CSPG labeling shown in A. The

dashed boxes in A indicate the location of the images in B–E. B: The distal terminals of the axon within the zone with dense CSPG label-

ing. C: The axon at the border of the CSPG zone. D: An intermediate segment of the axon. E: The soma and initial segment (between

arrowheads). Scale bars ¼ 100 lm in A; 25 lm in B–E.

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3245

Figure 3. PCI axons grow further toward the contralateral spinal cord and the CSPG zone shrinks from 7 to 9 days postinjury. A: Cumula-

tive histograms showing the mediolateral progress of PCI axons toward the contralateral spinal cord relative to the midline at 7 and 9

days postinjury. B: Schematic description of the mediolateral regions used to classify the leading edges of regenerating axon terminals.

The schematic shows examples of an axon that would be classified as ‘‘crossed the midline’’ (top), and an axon that would be classified

as ‘‘at the CSPG border’’ (bottom). C: Bar graphs showing the proportion of axons whose leading edge terminated across the midline,

within the CSPG zone, at the CSPG border, and outside the CSPG zone at 7 and 9 days postaxotomy. D: Cumulative histograms showing

the mediolateral distance from the soma of each cell to the leading edge of its axon at 7 and 9 days postinjury. E: Cumulative histograms

showing the mediolateral distance from the soma of each cell to the midline at 7 and 9 days postinjury. F: Cumulative histograms showing

the mediolateral distance from the midline to the border of the zones with dense CSPG labeling at the rostrocaudal level of the leading

terminal of reconstructed PCI axons at 7 and 9 days postinjury.

K.K. Fenrich and P.K. Rose

3246 The Journal of Comparative Neurology |Research in Systems Neuroscience

process relative to the midline and CSPG labeling. The

categories were: a) crossed the midline; b) within the

CSPG zone, but not across the midline; c) within 50 lm of

the border of the CSPG zone; and d) outside the CSPG

zone in the ipsilateral spinal cord (Fig. 3B). Unexpectedly,

the proportion of axons that terminated at the CSPG bor-

der or outside the CSPG zone was greater at 9 days post-

injury (n ¼ 57%) compared to 7 days postinjury (n ¼ 32%)

(Fig. 3C). This change in distribution was most noticeable

outside of the CSPG zone (n ¼ 9% at 7 days to 30% at 9

days). Together, these findings, in combination with the

findings that PCI axons had migrated further toward the

contralateral spinal cord by 9 days raised the questions:

1) does the rate of axon growth change between 7 and 9

days; and/or 2) does the size of the CSPG zone change

between 7 to 9 days postinjury?

To test whether some axons stall or retract from 7 to 9

days postinjury, the mediolateral distance from the soma

to the leading process was measured for each cell. At 9

days postinjury, cut PCI axons had grown an average of

222 lm further from their soma toward the contralateral

spinal cord compared to cut PCI axons at 7 days postin-

jury (Fig. 3D) (P < 0.01, Two-Sample KS test). This pro-

gression away from their soma is consistent with the pro-

gression toward and across the midline from 7 to 9 days

postinjury (cf. Fig. 3A). If, however, some axons were

stalled or had retracted from 7 to 9 days postinjury, we

would expect these axons to be at the same mediolateral

distance or closer to their soma at 9 days compared to 7

days postinjury. Such an outcome would result in a

change in the slope of the distribution of the mediolateral

distances between their somata and leading edges. To

test if this is the case, we compared the slopes of the

simple linear regressions of these distributions at 7 days

(slope ¼ 0.141; R2 ¼ 0.96) and 9 days (slope ¼ 0.138;

R2 ¼ 0.89) postinjury and found no differences (P ¼0.446; Student’s t-test). However, the increased medio-

lateral distance from somata to the leading edge of their

axons at 9 days postinjury may be because the popula-

tion of PCIs examined at 7 days postinjury were located

more medially than the PCIs sampled from 9 day experi-

ments. To test whether this is the case, the mediolateral

distance from the soma to the midline was measured for

each cell. For the 7-day postinjury experiments the

somata were located an average of 42 lm further from

the midline compared to 9 days postinjury, but these dif-

ferences were not significant (Fig. 3E) (P > 0.05, Two-

Sample KS test), indicating that the PCIs from the 7 and

9 day experiments occupy the same mediolateral

regions. Moreover, although the differences in the aver-

age mediolateral distances from soma to midline are not

significant between 7- and 9-day experiments, the 42-lmshift away from the midline for 7-day experiments

accounts for the discrepancy between the average

mediolateral axon progression as measured relative to

the midline (180 lm; Fig. 3A) and somata (222 lm; Fig.

3D). Collectively, these data indicate that PCI axons con-

tinued to grow toward the contralateral spinal cord from

7 to 9 days postinjury.

To determine whether the size of the CSPG zone

changes from 7 to 9 days postinjury, the mediolateral dis-

tance from the midline to the ipsilateral CSPG border was

measured for all PCI axons at the rostrocaudal level of

their leading edge. At 7 days postinjury the width of the

dense CSPG labeling from the midline to the ipsilateral

border was an average of 301 lm wider than at 9 days

postinjury (Fig. 3F) (P < 0.01, Two-Sample KS test).

These data show that between 7 and 9 days postinjury

the CSPG zone retracts a greater distance toward the

midline than the average mediolateral growth of PCI

axons toward the contralateral spinal cord.

Collectively, these data show that PCI axons continue

to grow toward the contralateral spinal cord from 7 to 9

days postinjury, and the mediolateral extent of the CSPG

zone diminishes more than the average distance of axo-

nal growth toward the contralateral spinal cord in the

same time period (see Fig. 9). These results suggest that

some of the terminal regions located outside of the CSPG

zone at 9 days postinjury may have been located within

the CSPG zones at 7 days postinjury. Furthermore, our

data suggest that this relocation from within the CSPG

zone at 7 days to outside of the CSPG zone at 9 days may

be due to retraction of the CSPG zone toward the midline

rather than a retraction of the axons toward their somata.

Taken together, these data suggest that growing PCI

axons can be divided into two groups: those that grow to-

ward the contralateral spinal cord as fast as or faster

than the retraction of the CSPG zone, and those that are

unable to grow toward the contralateral spinal cord as

fast as the retraction of the CSPG zone. We therefore

asked the question: do the morphological characteristics

of terminal regions located in the CSPG zones and across

the injured midline differ from terminal regions located at

the CSPG border or outside of the CSPG zones? However,

to answer this question we must first define what consti-

tutes the terminal regions of cut PCI axons and describe

their morphologies.

Terminal regions of regenerating PCI axonshave a wide range of morphologiesDefinition of terminal regions

In the preceding section we used the location of the

leading process of PCI axons to describe the morphology

of the axons en route to the contralateral ventromedial

funiculus. However, this parameter does not describe the

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3247

complex structural features of the terminal region of PCI

axons at 7 and 9 days following axotomy. For this purpose

we defined an axon’s terminal region as the distal seg-

ments of the axon with many branches and/or swellings

that emerged from a parent axon. In many cases the

boundary between the terminal region and parent axon

was obvious. However, in some cases the transition was

gradual and the start of the terminal zone was difficult to

identify. As a consequence, the proximal segment of the

terminal region was defined as a 50-lm long segment

with no swellings or branches. There was one exception

to this definition. If the axon entered the CSPG zone, all

axon segments located within the CSPG zone were con-

sidered part of its terminal region. Together, this means

that some terminal regions were outside of CSPG zones,

some were largely restricted to CSPG zones, while others

began outside of CSPG zones but also had segments

located within CSPG zones.

General observationsTables 1 and 2 summarize the branching structure,

length, and surface area of the reconstructed PCI axon

terminal regions at 7 days and 9 days postinjury, respec-

tively. Terminal regions were classified into two catego-

ries, ‘‘arboreal’’ and/or ‘‘compact,’’ based on their

morphological characteristics. Arboreal terminal regions

had thin processes that extended from thicker shafts,

easily discernible branch points, and swellings that were

smooth and elliptical. Compact terminal regions had dis-

tinctive large swellings with irregular 3D shapes (i.e., not

smooth and/or elliptical). For instance, some of these

formed thin veils in the shape of a calyx, while others had

a pitted or multilobed globular surface. Compact terminal

regions with these features were called ‘‘irregular.’’ Other

compact terminal regions had many short fine filopodia-

like branches that emerged from a large terminal or en

passant swelling. These compact terminal regions were

called ‘‘filopodial.’’ A small number of axons had terminal

regions that consisted of two or more zones, with features

in one zone consistent with one classification, whereas

the other zones had features consistent with a different

classification. These terminal regions are described as

hybrids with the most distal zone listed first (e.g., Arboreal

þ Compact; Tables 1 and 2). Since the parts of the axon

that had advanced the farthest en route to the contralat-

eral spinal cord were, from the perspective of replacing

lost connections, the closest to meeting this objective,

the classification of the distal zone was used to assign

axons with hybrid terminal regions to a specific class.

Arboreal terminal regionsAs summarized in Tables 1 and 2, 38 terminal regions

had arboreal features. All arboreal terminal regions had

the same basic structural components. The parent axon

gave rise to short branches and/or longer branches that

divided several times to form more elaborate branching

structures. Although the parent axons with arboreal ter-

minal regions typically followed a relatively straight path

toward the contralateral spinal cord, several took a more

tortuous route. In contrast, the terminal and preterminal

branches often followed a meandering path. These termi-

nal branches were sometimes in close proximity to the

densely CSPG stained tube-like structures located at and

near the lesion site, but most terminals were located in

zones with less dense CSPG labeling between the tube-

like structures. Branch points often occurred at swellings,

and some swellings gave rise to more than one branch.

All arboreal terminal regions had en passant varicosities,

and most branches ended with a terminal swelling. Both

en passant varicosities and terminal swellings tended to

be smooth and elliptical.

Although all arboreal terminal regions had similar char-

acteristics, the number of branches and swellings per ter-

minal region varied greatly. For instance, some terminal

TABLE 1.

Comparison of Branch Structure and Size of

Reconstructed Terminal Regions at 7 Days Postinjury

Cell

Morphological

classification1

Total

no. of

branches

Total

length

(lm)2

Surface

area

(lm2)2

66-4 Arboreal 11 1091.3 5418.666-5 Compact

(Irregular)12 1709.6 1202.8

73-6 Arboreal 12 447.7 2621.666-3 Arboreal 15 493.6 1833.966-2 Arboreal 16 703 2770.466-1 Arboreal 23 1090.2 7878.266-8 Arboreal 24 1062.5 4009.873-1 Arboreal þ

Compact(Irregular)

36 1117.9 4283

73-5 Arboreal 36 1007.7 2885.966-7 Arboreal 44 2005.7 6529.166-12 Arboreal 44 1161 4866.966-6 Arboreal 48 1652.5 5441.173-3 Arboreal 56 2314.8 7032.873-2 Arboreal 79 3868.3 10144.467-1 Arboreal 81 2676.9 6113.767-5 Arboreal 88 3338.8 11200.367-7 Arboreal 93 2861.8 6125.167-3 Arboreal 286 6612.3 19373.766-13 Arboreal þ

Compact(Irregular)

382 16322.5 45191.7

66-10 Compact(Irregular þFilopodial)

NA NA NA

66-11 Filopodial NA NA NA73-4 Filopodial NA NA NA

1See Results for description of morphological classifications.2Includes the root axon and all branches.

K.K. Fenrich and P.K. Rose

3248 The Journal of Comparative Neurology |Research in Systems Neuroscience

regions had only 2 or 3 branches, whereas others had

over 100 branches. To more accurately describe the ar-

boreal terminal regions, they were further categorized

based on the number of branch points as ‘‘simple,’’ ‘‘inter-

mediate,’’ and ‘‘complex.’’ The criteria for each of these

categories was arbitrarily set at 20 or fewer branch

points, between 21 and 100 branch points, and greater

than 100 branch points, respectively.

Fifteen of the arboreal terminal regions were classified

as ‘‘simple.’’ The parent axons followed a fairly straight

path, and had only a few branch points (from 2 to 20) and

swellings (Fig. 4). The branches of these terminal regions

were short and had few second- and third-order

branches. The branches belonging to seven of the simple

terminal regions were largely restricted to the most distal

regions of the axon, whereas the branches that formed

the arbor in the remaining eight simple terminal regions

also originated from more proximal regions of the axon

(cf. Fig. 4A,B, respectively).

Eighteen of the arboreal terminal regions were classi-

fied as ‘‘intermediate,’’ and had between 22 and 93

branch points. Most of the parent shafts of these terminal

regions were quite tortuous, forming ‘‘S’’ trajectories or

loops and only a few followed a relatively direct route to-

ward the contralateral spinal cord (cf. Fig. 5A,B, respec-

tively). Turns and loops occurred primarily at either the

border of the gray and white matter or as the axon

entered the CSPG zone (Fig. 5A). Many of the branches of

intermediate arboreal terminal regions were short with a

few swellings, similar to the simple arboreal terminal

regions. However, some intermediate arboreal terminal

regions also had much longer branches that gave rise to

several levels of higher-order branches that often had

multiple swellings, and occasionally followed a tortuous

trajectory. The branches were largely restricted to the

most distal parts of 15 of the terminal regions (Fig. 5A). In

the remaining three intermediate arboreal terminal

regions, most branches were restricted to a distal cluster,

but there was a second smaller cluster of branches more

proximally (Fig. 5B).

Four arboreal terminal regions had highly intricate

structures, each with several hundred branch points and

many swellings. These arboreal terminal regions were

classified as ‘‘complex.’’ Two of these terminal regions

were reconstructed in their entirety (Fig. 6), whereas only

the parent axon and some of the simpler branches were

reconstructed for the remaining two. These terminal

regions were not reconstructed in their entirety because

their branches were tightly intertwined and could not be

traced without fear of linking segments of branches incor-

rectly. However, by stacking images of aligned serial sec-

tions we were able to visualize the 3D structure of these

complex arboreal terminal regions and identify all of the

branch points (Table 2). Despite the high branching den-

sity, the size of these terminal regions and the character-

istics of their swellings were similar to terminal regions

that were reconstructed completely. All four of the com-

plex arboreal terminal regions had many fine branches

that projected over several tens to hundreds of microns

TABLE 2.

Comparison of Branch Structure and Size of

Reconstructed Terminal Regions at 9 Days Postinjury

Cell

Morphological

classification1

Total

no. of

branches

Total

length

(lm)2

Surface

area

(lm2)2

71-19 Arboreal 2 158 168474-2 Arboreal 3 469 154371-4 Arboreal 4 304 143571-5 Arboreal 4 334 189371-17 Compact

(Irregular)4 182 3191

71-18 Arboreal 4 203 102571-21 Arboreal 5 525 184771-20 Compact

(Irregular)7 323 5025

71-16 Compact(Irregular) þArboreal

8 404 2280

71-11 Arboreal 10 242 199975-2 Compact

(Irregular)13 1025 11050

71-22 Compact(Irregular)

14 379 3832

71-3 Arboreal 15 413 196071-15 Arboreal 17 809 519271-10 Arboreal 19 553 178775-3 Arboreal 20 821 432871-14 Arboreal þ

Compact(Irregular)

22 660 3041

71-9 Arboreal 26 1547 1035275-5 Arboreal 32 1497 431871-2 Arboreal 36 1711 672771-8 Arboreal 43 1662 468371-13 Compact

(Irregular)49 1385 8735

74-5 Arboreal 61 2187 607974-3 Arboreal 3273 NA NA75-1 Arboreal 3613 NA NA71-6 Compact

(Filopodial)NA NA NA

71-7 Compact(Filopodial)

NA NA NA

74-1 Compact(Filopodial þIrregular)

NA NA NA

74-4 Compact(Filopodial)

NA NA NA

75-4 Compact(Filopodial þIrregular)

NA NA NA

1See Results for description of morphological classifications.2Includes the root axon and all branches.3Terminal region not reconstructed in entirety; branch points were

counted through serial sections; see Results section for description of

counting method.

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3249

in the x, y, and z axes. Higher-order branches were tortu-

ous and often intertwined with one another. Most of the

branch points of these terminal regions were located in

one main cluster, and the majority of the remaining

branches were located in one or two smaller clusters. The

main cluster of branches was located at the most distal

zone of two of these terminal regions (Fig. 6A). Con-

versely, the main cluster of branches was located more

proximally, with relatively simple clusters located more

distally, in the other two complex arboreal terminal

regions (Fig. 6B).

Compact terminal regionsAs summarized in Tables 1 and 2, 18 terminal regions

had compact features. Compact terminal regions had

large irregular swellings with complex contours and/or a

very dense array of fine branches.

Thirteen compact terminal regions were classified as

‘‘irregular.’’ All had very large swellings, a pitted surface

(Fig. 7A,B), and/or had bulbous appendages or flaps that

projected from their surface (Fig. 7C). In addition, the

main shaft of some of these terminal regions formed one

or more thin veiled structures connected on either end by

small diameter cylindrical shafts (Fig. 7A,B). These veiled

structures were distended in one plane and thin in the

perpendicular plane, and thus had the appearance of a

calyx.

Despite their complex surface characteristics, most of

the features of irregular terminal regions could be cap-

tured by our reconstruction techniques with two small

modifications. First, most of the wave-like folds and flaps

were reconstructed as swellings unless they protruded

longer than the diameter of the parent shaft, in which

case they were reconstructed as a branch. Second, the

calyx-like structures were represented as large swellings

with a diameter equal to the width of the calyx.

Compact terminal regions with many delicate, short

branches were classified as ‘‘filopodial’’ (Fig. 7D). These

branches ranged in length from a few microns to many

tens of microns. These branches were usually straight,

with few or no higher order branches, and often termi-

nated in a small swelling. Because the filopodia-like

Figure 4. Some PCI axons have simple arboreal terminal regions with few branches and swellings. A,B: Reconstructions of axotomized PCI

axons with simple arboreal terminal regions (black, axon) at 9 days postinjury (A) (cell 74-2; see Table 2), and 7 days postinjury (B) (cell 66-

2; see Table 1). Regions distal (left) of the asterisks indicate the location of the terminal regions of the axon. Scale bars ¼ 50 lm.

K.K. Fenrich and P.K. Rose

3250 The Journal of Comparative Neurology |Research in Systems Neuroscience

branches were very delicate and numerous, filopodial ter-

minal regions could not be reconstructed in their entirety.

Instead, we reconstructed the axon that gave rise to the

filopodial terminal region, all of the larger branches, and

all of the swellings, but not the filopodial branches.

Eight of the compact terminal regions had morphologi-

cal characteristics that were classified as ‘‘filopodial.’’

Large swellings with filopodia-like branches were located

on both the proximal segments of the axon that gave rise

to the filopodial terminal region as well as at the most dis-

tal terminals.

Terminal regions of regenerating PCI axonschange between 7 and 9 days postinjury

We next compared the proportion of each class of ter-

minal region observed at 7 and 9 days postinjury (Tables

1, 2). At both time intervals most of the terminal regions

have arboreal characteristics, but a larger proportion of

the terminal regions at 7 days postinjury are arboreal (n

¼ 18 of 22) compared to terminal regions at 9 days post-

injury (n¼ 19 of 30). At 9 days postinjury nearly a third of

the terminal regions had compact features (n ¼ 11 of

30), whereas at 7 days postinjury few terminal regions

had compact features (n¼ 4 of 22).

To test whether there were differences in the complex-

ity of terminal region branching structures, we compared

the number of branch points per arboreal terminal region

between 7-day and 9-day experiments (Fig. 8). At 7 days

postinjury the number of branch points per terminal

region is greater than at 9 days postinjury (P < 0.05,

Two-Sample KS test). Similar results were obtained when

irregular terminals were included in the analysis (data not

shown). Taken together, this decrease in branch number

is consistent with the finding that a majority of the termi-

nal regions from 7-day experiments fall within the

Figure 5. Some PCI axons have intermediate arboreal terminal regions with many tens of branches and swellings. A,B: Reconstructions of

two axotomized PCI axons with intermediate arboreal terminal regions (black, axon) at 7 days postinjury (A) (cell 66-6; see Table 1), and 9

days postinjury (B) (cell 75-5; see Table 2). Regions distal (left) of the asterisks indicate the location of the terminal regions of the axon.

Scale bars ¼ 50 lm.

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3251

Figure 6. Some PCI axons have complex arboreal terminal regions with over 100 branches and swellings. A,B: Reconstructions of two

axotomized PCI axons with complex arboreal terminal regions (black, axon) at 7 days postinjury. A: The terminal region of the axon belong-

ing to cell 66-13 (see Table 1). B: The terminal region of the axon belonging to cell 67-3 (see Table 1). Regions distal (left) of the asterisks

indicate the location of the terminal regions of the axon. Scale bars ¼ 50 lm.

K.K. Fenrich and P.K. Rose

3252 The Journal of Comparative Neurology |Research in Systems Neuroscience

intermediate arboreal category, whereas a majority of the

terminal regions from 9-day experiments fall within the

simple arboreal category.

Axons with highly branched arborealterminal regions progress further toward thecontralateral spinal cord than axons withcompact terminal regions

It is conceivable that the axons with arboreal terminals

belong to PCIs whose somata are located in different

regions of the ventral horn than the somata of PCIs whose

axons have compact terminal regions. To test whether

this is the case we measured the mediolateral distance

from the soma to midline for each PCI, and found no dif-

ferences between PCIs whose axons had arboreal termi-

nal regions and PCIs whose axons had compact terminal

regions (data not shown). In addition, all Neurobiotin-

stained PCI somata were restricted to a small zone in the

dorsoventral axis since all were in close proximity to Neu-

robiotin injection sites located in the ventral horn

between 200 and 800 lm dorsal of the motoneuron

pools. Furthermore, we found several examples of closely

adjacent PCI somata whose axons had either arboreal ter-

minal regions or compact terminal regions. Taken to-

gether, these data suggest that the morphology of PCI

Figure 7. Some PCI axons have compact terminal regions that are swollen, have complex membrane features, and are restricted to small

regions. Confocal image stacks showing irregular terminal regions (A-C) and a filopodial terminal region (D) at 9 days postinjury. A,B: The

same axon in adjacent serial sections. The part of the axon shown in A is distal to B, and the star in A shows the continuation point of

the axon from the star in B. Arrows in B,C indicate the continuation points of the axon in adjacent tissue sections. In all images the mid-

line is to the left and the somata are located to the right. Scale bars ¼ 20 lm.

Figure 8. PCI axons with arboreal terminal regions have more

branch points at 7 days postaxotomy compared to 9 days postax-

otomy. Cumulative histograms showing the number of branch

points per axonal terminal region at 7 and 9 days postaxotomy.

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3253

axon terminals is not dependent on the location of their

somata in the ventral horn.

Given that PCI axons can be divided into two classes

based on their progression toward the contralateral cord,

and that terminal regions can be classified as either arbo-

real or compact, we returned to our question: Do the mor-

phological characteristics of terminal regions located in

the CSPG zones and across the midline differ from termi-

nal regions located at the border or outside of the dense

CSPG zones?

To test whether arboreal and compact terminal regions

are distributed differently along the mediolateral axis,

each axon was categorized according to the mediolateral

location of its leading process relative to the midline and

CSPG labeling as described above (see Fig. 3B). At 7 days

postinjury most arboreal axon terminals (n ¼ 14 of 18)

were located in the CSPG zone, including one that had

crossed the midline (Fig. 9A). At 9 days postinjury the ar-

boreal terminals were more widely distributed across the

mediolateral axis (Fig. 9B). In addition, we also found that

only arboreal terminals had grown across the midline

(Fig. 9A,B). Conversely, at both postaxotomy intervals the

compact terminals were largely restricted to the border

of the CSPG zones or outside of the CSPG zones (n ¼ 2/

3 at 7 days; n ¼ 9/11 at 9 days). When the mediolateral

distribution data from the 7 and 9 day postinjury experi-

ments were combined, a majority of the arboreal terminal

regions were located across the midline or in the CSPG

zone (n ¼ 25 of 37), whereas a majority of the compact

terminals were located outside the CSPG zone or at the

border of the CSPG zone (n ¼ 12 of 15) (P < 0.001 Fish-

er’s exact test). Collectively, the biased mediolateral dis-

tributions of terminal regions suggest that terminal region

morphology is dependent on its location relative to

CSPGs, and that arboreal terminal regions are more adept

at penetrating the CSPG labeling and crossing the injured

midline than compact terminal regions.

Considering that only arboreal terminal regions

crossed the midline, and there is a wide range of branch-

ing complexities between arboreal terminal regions, we

Figure 9. PCI axons with highly branched arboreal terminal regions are more successful regenerators than axons with simpler arboreal

and compact terminal regions. A,B: Bar graphs showing the number of axons with arboreal and compact terminal regions whose leading

edge terminated across the midline, within the CSPG zone, at the CSPG border, and outside the CSPG zone at 7 days (A) and 9 days (B)

postaxotomy. C,D: Bar graphs showing the average number of branch points per arboreal terminal region at 7 days (C) 9 days (D) postax-

otomy, grouped according to the mediolateral location of their leading edge. Values represent means 6 SEM (*P < 0.05, Kruskal–Wallis

one-way ANOVA and post-hoc analysis using Mann–Whitney U-test).

K.K. Fenrich and P.K. Rose

3254 The Journal of Comparative Neurology |Research in Systems Neuroscience

next asked whether the terminal regions that crossed the

midline had any distinguishing features. To test whether

arboreal terminal region complexity is dependent on their

mediolateral location, arboreal terminal regions were

grouped according to their mediolateral location, as

described above, and the number of branches per termi-

nal region was compared between groups. At 7 days post-

axotomy there was only one axon that had regenerated

across the midline, and only two arboreal terminal regions

that terminated outside the CSPG zone, thus statistical

analysis was not performed (Fig. 9C). A similar distribu-

tion was observed at 9 days postinjury (Fig. 9D). How-

ever, at 9 days postinjury terminal regions that crossed

the midline had significantly more branch points than ter-

minal regions in all other zones (P < 0.05, Kruskal–Wallis

one-way analysis of variance [ANOVA] and post-hoc

analysis using Mann–Whitney U-test) (Fig. 9D).

Taken together, these results show that axons with

highly branched arboreal terminal regions are more suc-

cessful at regenerating through CSPG zones and across

the lesioned midline compared to axons with simpler ar-

boreal terminals regions or compact terminal regions at 7

and 9 days postaxotomy.

DISCUSSION

After SCI, most cut axons fail to regenerate across the

injury site (Ramon Y Cajal, 1959; Yiu and He, 2006). Cut

PCI axons, however, can regenerate across SCI sites fol-

lowing a midsagittal transection of the spinal cord (Fen-

rich and Rose, 2009). Our present study was designed to

compare the morphology of cut PCI axons after a SCI,

and to evaluate whether certain morphological character-

istics are indicative of successful regeneration across the

injured midline. Here we show that at 7 and 9 days post-

injury the terminal regions of cut PCI axons have a wide

range of morphological characteristics, and the axons

with highly branched arboreal terminal regions grow fur-

ther toward the contralateral cord and crossed the

injured midline compared to axons with compact terminal

regions.

Growth within and outside of zones withdense CSPG labeling (arboreal vs. compact)

At later postinjury times (4 to 10 weeks) some cut PCI

axons have regenerated across the CSPG-rich SCI site,

whereas others remain within the dense CSPG zones ipsi-

lateral of the lesioned midline or have retracted to their

soma (Fenrich et al., 2007; Fenrich and Rose, 2009). At 7

days postmidline transection we found that most axon

terminal regions (n ¼ 26 of 27) were located within or

before the CSPG zones (Fenrich and Rose, 2009). In this

previous study we also observed one axon terminal that

had regenerated across the midline, through the CSPG-

rich zone, and terminated in the contralateral white mat-

ter. With the goal of capturing a population of axon termi-

nals that were before the CSPG zones, within the CSPG

zones, and had just crossed the CSPG zones, we

repeated the 7-day experiments for the present study.

Contrary to our hypothesis, we found that at 7 days post-

injury a majority of the axon terminals examined were

located in CSPG zones adjacent to the lesion site and had

not crossed the midline. To develop a more comprehen-

sive picture of regenerating axons across spinal midline

transection sites and capture more axons that had

crossed the midline, we examined axon terminal morphol-

ogy at 9 days postinjury. By 9 days postinjury more axons

had crossed the midline, but more also terminated out-

side the CSPG zones in the ipsilateral gray matter, and

none had successfully regenerated through the CSPG

zone to the contralateral gray matter. Together, these

data suggest that our previous observation of a single

axon that had crossed the midline and escaped the con-

tralateral CSPG zone at 7 days is a very rare event and

that growth across the lesion site is slower (about 90

lm/day) than we first suspected.

In the adult CNS the rate of axonal growth of trans-

planted adult DRG neurons in both uninjured and degen-

erating spinal white matter has been measured at �1

mm/day (Davies et al., 1999). However, the rate of

growth of these axons slows substantially near SCI sites,

and these axons eventually stop progressing and remain

trapped within the glial scar of SCI sites (Davies et al.,

1999). Similarly, in the regenerating peripheral nervous

system (PNS), cut axons grow at a much slower rate

across a nerve scar (about 0.25 mm/day) than in distal

nerve segments (about 0.4–3 mm/day) (Guttemann

et al., 1942; Al Majed et al., 2000). Our findings show

that regenerating PCI axons grow at a much slower rate

toward the contralateral spinal cord than DRG axons in

uninjured or degenerating white matter or regenerating

PNS axons. However, since transplanted DRGs do not

regenerate through CSPG-rich zones, whereas some PCI

axons do successfully regenerate through CSPG-rich

zones, the present findings indicate that the overall

regeneration rate of PCI axons through CSPG-rich zones

is faster than DRG axons through CSPG-rich zones. This

finding does not exclude the likely possibility that regen-

erating PCI axons grow faster once they reach the white

matter tracts of the contralateral spinal cord, but whether

this is the case remains to be tested.

Although it is not possible to follow the same axon ter-

minals from day 7 to day 9, our results suggest that some

axons terminating in the CSPG zones near the midline at

7 days continue to regenerate across the midline by 9

days, whereas others fail to maintain their presence

Morphology of regenerating interneuron axons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3255

within the retracting CSPG zone by 9 days (Fig. 10). This

change in mediolateral distribution of terminal regions is

associated with a change in their morphology and a

change in the mediolateral distribution of CSPGs. This

change in morphology is most apparent by the increased

number of compact terminal regions at the border of the

CSPG zone and outside the CSPG zone at 9 days com-

pared to 7 days postinjury (Fig. 10). This prevalence for

compact terminals to be located outside or at the border

of the CSPG zones suggests that compact terminal mor-

phology may be dependent on the density and/or distri-

bution of CSPGs surrounding the terminal.

In addition to the changes in distribution and morphol-

ogy, we also found that axons with highly branched termi-

nal regions had crossed the lesioned midline with a higher

prevalence than axons with simple arboreal or compact

terminal regions. Whether these axons with highly

branched arboreal terminals are the ones that will form

functional synaptic connections in the contralateral cord

given more time is unknown. We also cannot rule out the

possibility that the compact terminals can grow toward

and cross the midline at later timepoints. However, this

later scenario seems unlikely given their close morpho-

logical similarities with descriptions of stalled and retract-

ing terminal regions of other axons that had failed to grow

across SCI sites (Ramon Y Cajal, 1959; Risling et al.,

1983; Davies et al., 1999; Tom et al., 2004).

Collectively, our findings show that the morphology of

regenerating axon terminals can change dramatically with

time and/or their location within the injured spinal cord.

Moreover, these findings strongly suggest that a more

comprehensive examination of axon terminal morphology

at earlier and later timepoints may provide important in-

formation about the relationships between axon terminal

morphology, their locations relative to injury sites, and

postinjury time.

Arboreal branching patternsTo our knowledge, this is the first study to show regen-

erating axon terminal regions with such complex arboreal

branching patterns. Arboreal branching patterns have

previously been described for axon terminals approaching

or within SCI sites (Ramon Y Cajal, 1959; Linda et al.,

1992; Li and Raisman, 1994, 1995; Davies et al., 1999;

Dray et al., 2009). However, the terminal regions

described in these studies resemble the simple arboreal

terminal regions of regenerating PCI axons. The paucity

of complex arboreal terminal regions in these other stud-

ies may be due to two possibilities.

First, these other studies examined only the most distal

parts of the axon due to the extensive overlap of terminal

zones and axons in the immediate region (Dray et al.,

2009), described only axon terminals within single tissue

sections (Ramon Y Cajal, 1959; Li and Raisman, 1994,

1995; Davies et al., 1999), or focused primarily on the

proximal portion of the axon terminals (Linda et al.,

1992). These approaches therefore may have excluded

parts of the terminal regions. For this study, we recon-

structed regenerating axons from their soma to all of their

terminations by following each axon through serial tissue

sections. Using this approach we found that no individual

tissue section revealed the true complexity of the highly

branched arboreal terminal regions and the detailed

branching patterns of these terminal regions was only

apparent following reconstruction.

Second, with the exception of the work by Dray et al.

(2009) on DRG axons and the work by Linda et al. (1992)

on motoneuron axons, none of the axons examined in

previous studies can successfully regenerate through SCI

sites. Our study examined axons that spontaneously

regenerate across SCI sites in response to axotomy.

Although we cannot preclude the possibility that highly

branched arboreal terminal regions are unique to PCI

axons, our results suggest that this morphological pheno-

type may be part of an important strategy used by cut PCI

axons to regenerate across SCI sites.

Taken together, these observations raise the question:

What features of the highly branched arboreal terminal

Figure 10. Schematic representation of the CSPG environment

and axon growth/morphology changes from 7 days to 9 days

postaxotomy (black, axons; dark gray, somata). At 7 days most

axons terminate within the CSPG zone (upper axons). Between 7

and 9 days PCI axons continue to grow toward the contralateral

spinal cord and the mediolateral extent of the CSPG zone shrinks.

At 9 days some axons terminate outside of the CSPG zone; many

of these axons have compact terminal regions (middle axon). In

addition, at 9 days some axons have remained within the CSPG

zone and have regenerated across the midline; these axons have

highly branched arboreal terminals (lower axon).

K.K. Fenrich and P.K. Rose

3256 The Journal of Comparative Neurology |Research in Systems Neuroscience

regions allow them to navigate through CSPG-rich injury

sites?

Growth permissive vs. growth inhibitoryzones

In a previous study we postulated that PCI axons may

regenerate through CSPG-rich SCI sites using one, or a

combination of the following three mechanisms: PCI axons

1) are not inhibited by CSPGs; 2) have an unusually high

capacity for growth; or 3) are able to find the least inhibi-

tory path through zones with dense CSPG labeling (Fenrich

and Rose, 2009). In order for the third possibility to be true

the elongating terminal regions of PCI axons would have to

acquire information about its environment to identify the

path of least inhibition. Consistent with this idea, in our

present study we found that the axon terminals that had

regenerated the furthest toward and into the contralateral

spinal cord were those that were highly branched and were

thus well designed to sample large regions of the injured

spinal cord. Moreover, we observed that some of the termi-

nal and preterminal branches of these highly branched

axons were in close proximity to tube-like structures that

were densely stained for CSPGs, whereas other branches

were located in zones with less dense CSPG staining. These

observations suggest that at least some of the branches of

these axons are less likely to be inhibited by CSPGs than

other branches of the same axon. Whether this ability of

PCI axons to simultaneously sample large regions of the

injured spinal cord is also associated with a reduced

responsiveness to inhibitory molecules and/or with an

increased regenerative capacity remains to be determined.

One other possible reason why PCI axons can regener-

ate across midline lesions may be because the lesion site

and surrounding regions are permissive rather than inhibi-

tory to axonal regeneration. This idea stems from the find-

ings that in addition to releasing CSPGs, some astrocytes

can also support the growth of regenerating axons by pro-

ducing growth factors such as nerve growth factor (Goss

et al., 1998) and growth supportive substrates such as

laminin (Liesi, 1985; Liesi and Kauppila, 2002). The inhibi-

tory components of CSPGs are sulfated glycosaminogly-

can (GAG) chains attached to a protein core (Bradbury

et al., 2002; Rhodes and Fawcett, 2004). For this study

we stained CSPGs using CS-56 monoclonal antibodies

that specifically recognize the inhibitory GAG chains of

CSPGs (Avnur and Geiger, 1984). Thus, PCI axons must

traverse zones that contain molecules that are typically

inhibitory to regenerating axons. Nevertheless, we cannot

discount the possibility that there is a preferential upreg-

ulation of permissive factors that overcome the inhibitory

effects of CSPGs. This scenario is unlikely, since previous

studies have found that only astrocytes distal from SCI

sites are permissive to growth, and astrocytes within the

lesion core are inhibitory (Mansour et al., 1990; Davies

et al., 1999). Previous data have shown that the spinal

regions with dense CSPGs correspond to the regions with

reactive astrocytes and the lesion core (Davies et al.,

1999; Silver and Miller, 2004). For our study we found

high levels of CSPGs along, and surrounding, the midsa-

gittal lesion sites, indicating that these regions likely con-

tain predominantly inhibitory astrocytes.

Collectively, although we cannot discount the possibil-

ity that spinal midsagittal lesion sites contain some

growth-supporting substrates or growth-promoting cues,

CSPGs present an environment that prevents the growth

of some, but not all axons.

Significance for understandingregenerative mechanisms

This study provides compelling evidence that extensive

arboreal branching patterns are a common feature of PCI

axons that can successfully regenerate through SCI sites

and cross the injured midline at 7 and 9 days postinjury.

These structural correlates are the first step toward

understanding the mechanisms that allow cut PCI axons

to regenerate across spinal injury sites. Considering the

structural complexity of these terminal regions, with

some branches having progressed further toward the

contralateral spinal cord than others, or some closer to a

dense pocket of CSPG than others, one might hypothe-

size that the regenerative capacity of any one branch is

partially independent of the other branches. Such a sce-

nario implies that the molecular mechanisms governing

the regenerative capacity of an axon, as a whole, may be

conditional on the regenerative capacity of any given

branch at the terminal region of the axon. Together, these

interpretations suggest that understanding the molecular

mechanisms leading to successful regeneration must be

examined and interpreted in the context of their location

within the complex arbores of the axon terminal regions

rather than on a whole-cell basis.

ACKNOWLEDGMENTS

We thank Rob Maratta and Monica Neuber-Hess for

help with the CS-56 antibody control experiments. We

also thank Rebecca Cranham and Shauna Ketcheson for

help with animal care, surgeries, and data acquisition,

and Monica Neuber-Hess for help in conducting the

experiments.

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3258 The Journal of Comparative Neurology |Research in Systems Neuroscience