JOURNAL OF NEUROTRAUMA
Volume 22, Number 2, 2005
© Mary Ann Liebert, Inc.
Cervical Spinal Cord Injury Upregulates Ventral
DAVID D. FULLER, TRACY L. BAKER-HERMAN, FRANCIS J. GOLDER,
NICHOLAS J. DOPERALSKI, JYOTI J. WATTERS, and GORDON S. MITCHELL
Following chronic C2spinal hemisection (C2HS), crossed spinal pathways to phrenic motoneurons
exhibit a slow, spontaneous increase in efficacy by a serotonin (5-HT)–dependent mechanism asso-
ciated with 5-HT2Areceptor activation. Further, the spontaneous appearance of cross-phrenic ac-
tivity following C2HS is accelerated and enhanced by exposure to chronic intermittent hypoxia (CIH).
We hypothesized that chronic C2HS would increase 5-HT and 5-HT2Areceptor expression in ven-
tral cervical spinal segments containing phrenic motoneurons. In addition, we hypothesized that
CIH exposure would further increase 5-HT and 5-HT2Areceptor density in this region. Control,
sham-operated, and C2HS Sprague-Dawley rats were studied following normoxia or CIH (11% O2-
air; 5-min intervals; nights 7–14 post-surgery). At 2 weeks post-surgery, ventral spinal gray matter
extending from C4and C5was isolated ipsilateral and contralateral to C2HS. Neither C2HS nor CIH
altered 5-HT concentration measured with an ELISA on either side of the spinal cord. However, 5-
HT2Areceptor expression assessed with immunoblots increased in ipsilateral gray matter following
C2HS, an effect independent of CIH. Immunocytochemistry revealed increased 5-HT2Areceptor ex-
pression on identified phrenic motoneurons (p ? 0.05), as well as in the surrounding gray matter.
Contralateral to injury, 5-HT2Areceptor expression was elevated in CIH, but not normoxic C2HS
rats (p ? 0.05). Our data are consistent with the hypothesis that spontaneous increase in 5-HT2Are-
ceptor expression on or near phrenic motoneurons contributes to strengthened crossed-spinal synap-
tic pathways to phrenic motoneurons following C2HS.
Key words: breathing; crossed phrenic phenomenon; plasticity; serotoninl; spinal hemisection
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin.
fective synaptic pathways to ipsilateral phrenic mo-
toneurons gradually increase in strength (Goshgarian,
2003; Nantwi et al., 1999; Golder et al., 2001a). These
FTER CERVICAL SPINAL HEMISECTION rostral to the
phrenic motor nucleus, existing but initially inef-
strengthened crossed phrenic synaptic pathways restore
phrenic nerve and hemidiaphragm function (Goshgarian,
2003), thereby contributing to inspiratory volume
(Golder et al., 2003). This functional recovery is time-
dependent, exhibiting progressive recovery over weeks-
to-months. For example, 2 weeks following C2spinal
hemisection (C2HS), small inspiratory bursts are ob-
served in the previously silent phrenic nerve during base-
line conditions in approximately 50% of anesthetized rats
(Fuller et al., 2003). However, by 8 weeks post-C2HS,
larger, more robust inspiratory bursts are observed in
80–100% of rats under similar conditions (Golder et al.,
2001a; Nantwi et al., 1999).
The mechanisms underlying spontaneous improve-
ment in crossed phrenic motor output following C2HS re-
main unclear, but serotonin (5-HT) plays an important
role (for review, see Mitchell and Johnson 2003; Gosh-
garian 2003). For example, pre-treatment with a serotonin
synthesis inhibitor (p-chlorophenylalanine) reduces
crossed phrenic inspiratory activity and prevents mor-
phological changes associated with strengthened crossed
phrenic synaptic pathways (Hadley et al., 1999a,b). Sim-
ilarly, pre-treatment with a serotonergic neurotoxin (5,7-
DHT) reduces crossed phrenic activity (Golder et al.,
2001a). Conversely, the serotonin precursor 5-hydroxy-
tryptophan (5-HTP) activates crossed phrenic pathways
after acute (Ling et al., 1994; Fuller et al., 2003) and
chronic C2HS (Zhou et al., 1999, 2000). Serotonin may
activate crossed phrenic pathways by binding to 5-HT2A
receptors located on phrenic motoneurons (Basura et al.,
2001, 2002; Zhou et al., 2001). For example, pharmaco-
logical activation of 5-HT2Areceptors activates crossed
phrenic pathways (Zhou et al., 2001), and these recep-
tors co-localize with phrenic motoneurons (Basura et al.,
2001). Activation of 5-HT2A receptors could increase
phrenic motoneuron excitability, leading to greater
phrenic motor output for a given amount of excitatory
bulbospinal synaptic drive (Mitchell et al., 2001; Feld-
man et al., 2003).
Immediately following C2HS, it is expected that the
serotonergic innervation of the ipsilateral spinal cord will
decrease due to interruption of descending serotonergic
fibers (Saruhashi et al., 1996). However, serotonergic in-
nervation of the spinal cord ipsilateral to hemisection pro-
gressively increases with time, restoring or even exceed-
ing normal levels of serotonergic innervation (Saruhashi
et al., 1996, F.J. Golder and G.S. Mitchell, unpublished
data). Because serotonin is associated with enhanced
crossed spinal synaptic pathways to phrenic motoneu-
rons, restored or even enhanced serotonergic function fol-
lowing spinal hemisection may be necessary to reveal the
crossed phrenic pathway in a time-dependent manner.
Thus, we hypothesized that 5-HT concentration and/or 5-
HT2Areceptor density would be elevated in regions of
the spinal cord associated with the phrenic motor nucleus
following chronic C2HS.
Within minutes to hours after C2HS, crossed phrenic
pathways can be activated by increasing respiratory drive
(e.g., asphyxia; Goshgarian, 2003) or by pharmacologi-
cal manipulations (e.g., 5-HTP; Ling et al., 1994). How-
ever, these are transient effects that reflect recruitment or
neuromodulation in an existing, but functionally ineffec-
tive neural pathway (Mitchell and Johnson, 2003). Cer-
tain chronic treatments may cause enduring effects (i.e.,
plasticity) that lead to enhanced crossed phrenic output
(Nantwi et al., 2003; Fuller et al., 2003). We previously
reported that chronic intermittent hypoxia (CIH) en-
hances the efficacy of crossed spinal synaptic pathways
to phrenic motoneurons after C2HS (Fuller et al., 2003).
Because CIH strengthens phrenic motor output via a sero-
tonergic mechanism in spinally intact rats (Ling et al.,
2001), at least partially via 5-HT2Areceptors, we postu-
lated that increased serotonin and/or 5-HT2Areceptor ex-
pression may be necessary to account for the effects of
CIH on crossed phrenic pathways following chronic
C2HS (Fuller et al. 2003). Thus, our second hypothesis
was that 5-HT and 5-HT2Areceptor levels in spinal re-
gions associated with the phrenic motor nucleus would
be further elevated in rats receiving both C2HS and CIH
(versus C2HS alone).
MATERIALS AND METHODS
Procedures were approved by the Animal Care and Use
Committee of the University of Wisconsin School of Vet-
erinary Medicine. Initial experiments were performed on
male Sprague-Dawley rats (age 3–5 months; n ? 55;
Charles River Laboratories, Kingston, NY). These rats
were divided into six experimental groups: (1) control
(i.e., unoperated) exposed to normoxia (n ? 9), (2) con-
trol ? CIH (n ? 11), (3) sham-operated exposed to nor-
moxia (n ? 8), (4) sham-operated ? CIH (n ? 9), (5)
C2HS exposed to normoxia (n ? 10), and (6) C2HS ?
CIH (n ? 8). All CIH exposures took place on nights
7–14 post-surgery or the equivalent time period in un-
operated rats. Additional immunocytochemistry experi-
ments were performed on four Sprague-Dawley rats.
Isoflurane anesthesia was induced in a closed cham-
ber and maintained (2–3%) via a nose cone. Rats were
treated with an analgesic (buprenorphine, 0.1 mg/kg), and
anti-inflammatory (carprofen, 4 mg/kg) and antibiotic
(enrofloxacin, 5 mg/kg) drugs. Following C2laminec-
tomy and durotomy, the spinal cord was hemisected cau-
dal to the C2dorsal roots with a microscalpel. To ensure
the hemisection was complete, an ?1-mm gap was cre-
ated at the incision site using a blunt-tipped 25-gauge
needle connected to a suction pump. Wounds were su-
tured, and the rats were carefully monitored post-hemi-
section. Buprenorphine (0.1 mg/kg), carprofen (4 mg/kg),
and enrofloxacin (5 mg/kg) were administered as needed.
FULLER ET AL.
The identical surgical approach was used for sham
surgery, but the spinal cord was not hemisected.
At the time that spinal tissues were harvested for
ELISA and Western blots, rostral spinal segments
(C1–C3) containing the lesion were removed and placed
in 4% paraformaldehyde solution. These tissues were
paraffin embedded, sectioned (5 ?m), slide mounted, cre-
syl violet–stained, and examined with light microscopy.
The hemisection was considered anatomically complete
if there was no evidence of healthy, intact neural tissue
in the ipsilateral half of the spinal cord at the site of in-
jury (a representative C2HS is shown in Fig. 1C). Based
on these guidelines, the majority of rats displayed an
anatomically complete lesion. However, in three animals,
histological exam indicated that a portion of the ipsilat-
eral ventral funiculus remained intact. The 5-HT data
from one of these animals were excluded after Dixon’s
test indicated that the data were significant outliers (Sokal
and Rohlf, 1994). Since data from the remaining two rats
were indistinguishable from other rats in their respective
groups, they were included in the data presentation and
The particular CIH pattern was chosen because it has
been shown to accelerate cross-phrenic motor recovery
(Fuller et al., 2003). Cages were placed in a Plexiglas
chamber that, between 6 p.m. and 6 a.m., was flushed at
a flow rate of 2 L/min/rat with an air/O2/N2 mixture to
achieve quasi-square wave (45 sec equilibration) inter-
mittent poikilocapnic hypoxia (5 min hypoxia [FIO2?
0.11]/5 min normoxia; Ling et al., 2001). Between 6 a.m.
and 6 p.m., the chamber was flushed with air at the same
flow rate. Chamber temperature was 22–24°C.
Tissue Preparation and Analyses
Tissues were harvested 14 days post-surgery or at the
equivalent time point in control rats. Anesthesia was in-
duced with isoflurane in a closed chamber and maintained
by sodium pentobarbital (i.p. 40–60 mg/kg). To increase
the efficiency of tissue collection, ?10 mL of blood was
drawn from the inferior vena cava. Rats were then euth-
anized via cardiac injection of sodium pentobarbital
(150–200 mg/kg) and briefly (3 sec) submerged in liquid
nitrogen. The fourth and fifth cervical spinal segments
were removed, cut transversely into 2-mm sections, and
placed in ice-cold saline. White matter was carefully re-
moved using a microscalpel and dissecting microscope,
and the remaining gray matter was divided into quadrants
(described relative to the C2HS injury; Fig. 1); data from
ventral ipsilateral and ventral contralateral tissues are re-
Tissue samples were stored at ?80°C prior to ho-
mogenization in lysis buffer solution [20 mM Tris (pH ?
8.0), 137 mM NaCl, 1% Tergitol (Type NP-40), 10%
glycerol, 20 ?g/mL aprotinin, 2 ?g/mL leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM sodium meta-
vanadate]. The homogenates were acidified to a pH of
?3.0 using 25 ?L of 1 N HCl, incubated for 15 min at
room temperature, and neutralized with 25 ?L of 1 N
SPINAL SEROTONIN RECEPTORS AFTER CERVICAL INJURY
depicting the site of spinal hemisection and bulbospinal path-
ways to phrenic motoneurons. Crossed phrenic pathways pro-
ject across the spinal midline caudal to C2. (B) Diagram illus-
trating how the spinal cord ventral gray matter was parceled
into quadrants ipsilateral and contralateral to the C2spinal hemi-
section. (C) Representative photomicrograph of a 5-?m trans-
verse section of the second cervical spinal segment demon-
strating the completeness of the hemisection injury. Note that
no spinal tissue is preserved on the side of injury while the con-
tralateral spinal cord remains intact. LF, lateral funiculus; VF,
ventral funiculus; DH, dorsal horn; VH, ventral horn.
(A) Diagram of the medulla and cervical spinal cord
NaOH to a pH of ?7.6. Each sample was then centrifuged
for 15 min at 12,000 ? g; the supernatant was then ana-
lyzed. Total protein was quantified using a commercially
available kit (Pierce Biotechnology Inc., Rockford IL).
5-HT was quantified using an ELISA kit per manufac-
turer’s instructions (IBL Inc., Hamburg, Germany).
5-HT2Areceptor concentration was determined by im-
munoblotting. Samples were treated with 10? SDS-
PAGE sample buffer (100 mM Tris, 7.5 mM EDTA, 100
mM DTT, 10% SDS, 30% glycerol and 1% bromophe-
nol blue), boiled for 4–5 min, and 25 ?g protein was
loaded into each well. Proteins were separated by 10%
SDS-PAGE gels and then transferred to Immobilon
polyvinylidene difluoride (PVDF) membranes (Millipore
Corp., Bedford, MA). Membranes were blocked in 5%
non-fat milk/TBST (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.05% Tween 20) at 37°C for 30 min and then
probed for 1 h at 37°C with anti-5 HT2Aantibody (BD
Biosciences, San Diego, CA) diluted 1:1000 in 5%
milk/TBST. Membranes were washed 3 ? 5 min in
TBST and subsequently probed with anti-mouse antibody
conjugated to horseradish peroxidase (1:4000; Santa Cruz
Biotechnology, Santa Cruz, CA). The immunoreactive
bands were visualized using SuperSignal West Pico
Chemiluminescent Substrate (Pierce Biotechnology,
Rockford, IL) and the Autochemi detection system (UVP,
Inc, Upland, CA). To ensure equal protein loading, blots
were probed with anti-GRB2 (1:2500; Santa Cruz
Biotechnology, Santa Cruz, CA) or stripped at 55°C for
30 min (stripping buffer ? 67.5 mM Tris, pH 6.8, 2%
SDS and 0.7% ?-mercaptoethanol), reblocked in 5%
milk/TBST, and probed with anti-?-tubulin (1:5000;
Santa Cruz Biotechnology, Santa Cruz, CA).
To localize changes in 5-HT2Areceptor expression fol-
lowing C2HS and to confirm that such changes occur in
identified phrenic motoneurons, four additional rats were
studied (two control and two C2HS). One week follow-
ing C2HS, ipsilateral phrenic motoneurons were back-la-
beled as follows. Rats were anesthetized as described
above, and the phrenic nerve on the side of injury (left
phrenic nerve in controls) was exposed and the nerve
sheath injected with the fluorescent tracer Dextran Texas-
red (2 ?L, 50 mg ? mL?1; absorption 595 nm; Molecu-
lar Probes, Eugene, OR). One week later the rats were
anesthetized with isoflurane in a closed chamber, and the
ascending aorta was perfused with 300 mL of heparinized
saline (4 IU ? mL?1) followed by 500 mL of 4% parafor-
maldehyde and 0.1% glutaraldehyde (pH 7.35–7.40). The
cervical spinal cord was removed and post-fixed for 1 h,
and then stored in 0.1M phosphate buffer solution with
0.02% sodium azide at 4°C. Prior to microtome section-
ing, the C4spinal segment was cryoprotected with 20%
sucrose and 5% glycerol in 0.1M phosphate buffer solu-
tion. Forty-micron transverse sections were then cut with
a freezing microtome.
We used a custom antibody targeted against the rat 5-
HT2Areceptor (Brownfield et al., 1998). The antibody
was generated by constructing a multiple antigenic pep-
tide as the immunogen. The antiserum was purified by
affinity chromatography. Details of primary antisera pro-
duction, isolation, and characterization have been re-
ported elsewhere (Brownfield et al., 1998). A dilution se-
ries (1:250; 1:500; 1:1000, serotonin 2Aantisera) was
performed with control and injured spinal sections to de-
termine the concentration of antisera that provided the
best contrast in immunoreactivity between control and
injured tissue. All tissue sections were processed at the
same time. Sections were washed, incubated in 3% hy-
drogen peroxide for 30 min, and rewashed. Sections were
blocked with 3% normal goat serum for 30 min and in-
cubated overnight in primary serotonin 2Areceptor anti-
sera made in rabbit (1:500) with 1% normal goat serum.
Sections were then washed, incubated in biotinylated goat
anti-rabbit IgG (1:300; Vector, Burlingame, CA),
washed, and then incubated in ABC solution (1:166; Vec-
tastain Elite Kit). Finally, sections were reacted with
0.04% 3,3?-diaminobenzidine with 0.1% nickel ammo-
nium sulfate, mounted, dehydrated, and coverslipped.
Light microscopy images of the C4ventral horn on
both sides of the cord were captured at 4?, 40?, and
200? magnification with a digital color camera (SPOT
II, Diagnostics Instruments, Sterling Heights, MI). Dur-
ing high magnification, back-labeled phrenic motoneu-
rons were first examined under fluorescent microscopy
to position the field over the phrenic motor nucleus. 5-
HT2A receptor immunoreactivity was then examined
without moving the microscope stage. Spinal 5-HT2Are-
ceptor immunoreactivity was quantified by placing a cir-
cular area of interest over the phrenic nucleus (see Fig.
5). The size and position of the area of interest was stan-
dardized across sections. An image was then captured
(200? magnification), and the pixel area occupied by 5-
HT2A receptor–positive elements was measured using
imaging software (Image-Pro Plus version 18.104.22.168, Sil-
ver Spring, MD). Equivalent adjustments to tone scale,
gamma, and sharpness were made across all images.
The 5-HT concentration was expressed relative to the
total protein concentration in each tissue sample. To re-
FULLER ET AL.
duce concerns regarding variability across ELISA mea-
surements, the relative changes in 5-HT following sham
surgery, C2HS, or CIH were expressed as a percent
change from control, unoperated, and normoxic rats. A
two-way analysis of variance (surgical treatment [con-
trol, sham, C2HS]; oxygen profile [normoxia, CIH]) with
the Student Neuman Keuls post hoc test (Sigma Stat, Jan-
del Scientific, St. Louis, MO) was used to detect statis-
tically significant treatment effects for each variable.
The density of immunoblot bands (5-HT2Areceptor,
GRB2, and ?-tubulin) was quantified using a computer-
ized imaging system (LabWorks; UVP, Inc., Upland,
CA). 5-HT2Areceptor density was normalized to GRB2
or ?-tubulin, and then expressed as a percentage of con-
trol rats. A t-test was used to determine if 5-HT2Are-
ceptor density was elevated in the treatment groups (ver-
sus control, unoperated, normoxic rats). A one-way
analysis of variance was used to detect significant dif-
ferences in 5-HT2Areceptor expression among treatment
groups. For the immunohistochemistry data, the Wilcoxin
signed rank test was used to compare 5-HT2Areceptor
expression between C2HS and control, unoperated, nor-
moxic rats. Data were considered statistically significant
if p ? 0.05.
Prior to surgery, there were no significant differences
in body mass between rat groups (385–417 g). As ex-
pected, C2HS significantly reduced body weight relative
to pre-injury (?38 ? 9 g; p ? 0.001). CIH also reduced
weight gain over the course of the CIH exposure; this im-
pairment in weight gain was similar among treatment
groups. At the time that spinal tissues were harvested,
control ? CIH, sham ? CIH, and C2HS ? CIH rats
weighed 28 ? 7, 10 ? 9, and 12 ? 9 g less than the cor-
responding values during normoxia (p ? 0.015). The av-
erage 5-HT concentration in the ventral spinal cord of
control, unoperated, normoxic rats was 3.2 ? 0.5 ng per
?g of total protein.
Ipsilateral Ventral Spinal Cord
5-HT concentration in ipsilateral gray matter of C2HS
or sham rats was not different than control, unoperated
rats (Fig. 2). In addition, CIH did not alter serotonin ex-
pression relative to control in any of the treatment groups
(Fig. 2). In contrast, 5-HT2A receptor expression as-
sessed with immunoblots was increased following C2HS
but not sham surgery (Figs. 3 and 4). Injured rats ex-
posed to CIH also had elevated 5-HT2Areceptor ex-
pression in ipsilateral gray matter but this was not sig-
nificantly different than C2HS alone (Figs. 3 and 4). As
shown in Figure 5, immunocytochemistry revealed in-
creased 5-HT2Areceptor staining on labeled phrenic mo-
toneurons (p ? 0.05 vs. control) and throughout the sur-
rounding gray matter following C2HS. In control rats,
5-HT2A immunostaining was heavily localized to the
phrenic motor nucleus (Fig. 5) similar to previous re-
ports (Doly et al., 2004).
Contralateral Ventral Spinal Cord
The 5-HT concentration in ventral C4–C5gray matter
contralateral to C2HS was not different than values ob-
tained in control or sham rats (Fig. 2). Further, C2HS did
not alter 5-HT2Areceptor expression in contralateral gray
matter as determined by immunoblotting (Figs. 3 and 4).
This finding was confirmed by the immunostaining ex-
periments in three of four rats. However, one rat had ev-
idence of increased 5-HT2A receptor immunostaining
throughout the contralateral gray matter. CIH signifi-
cantly increased contralateral 5-HT2Areceptor expression
SPINAL SEROTONIN RECEPTORS AFTER CERVICAL INJURY
matter segments encompassing the ipsilateral (top panel) and
contralateral (bottom panel) phrenic motor nuclei were not al-
tered by spinal hemisection (C2HS), sham surgery, or chronic
intermittent hypoxia (CIH). Ipsilateral and contralateral de-
scribe the spinal segments relative to injury (Fig. 1).
Serotonin concentration in ventral cervical spinal gray
(immunoblots) in the C2HS but not sham or control
groups (Figs. 3 and 4).
Increased 5-HT2A receptor expression in ipsilateral
ventral spinal segments associated with the phrenic mo-
tor nucleus following chronic C2HS may confer greater
capacity to elicit serotonin-dependent plasticity follow-
ing spinal injury. 5-HT2Areceptor expression was in-
creased in the ipsilateral ventral gray matter (im-
munoblots) and was confirmed to occur on labeled
phrenic motoneurons (immunohistochemistry), although
this localization was not exclusive. After C2HS, CIH in-
creased 5-HT2Areceptor expression in contralateral (but
not ipsilateral) ventral gray matter near the phrenic mo-
tor nucleus. In contrast, neither C2HS nor CIH had dis-
cernable effects on 5-HT concentration. Our correlative
results suggest that 5-HT2Areceptor upregulation on or
near phrenic motoneurons has the potential to contribute
to spontaneous functional recovery of phrenic motor out-
put ipsilateral to C2HS (Nantwi et al., 1999; Golder et
al., 2001a). Further, increased 5-HT2Areceptor expres-
sion may enable or amplify CIH effects on crossed spinal
synaptic pathways to phrenic motoneurons following
C2HS (Fuller et al., 2003), since intermittent hypoxia elic-
its spinal plasticity via 5-HT2Areceptor activation (Fuller
et al., 2001; Mitchell et al., 2001).
Body Mass Effects
Loss of body mass is common following high cervi-
cal spinal injury and may reflect inactivity-induced mus-
cle atrophy. Although a predictable consequence of se-
vere spinal injury, it is unclear what (if any) impact the
altered metabolic state and associated loss of body mass
in C2HS rats may have had on our results. However, since
CIH-exposed rats also lose mass, weight loss per se is
not sufficient to cause 5-HT receptor upregulation in the
cervical spinal cord. The effect of CIH on body mass was
unexpected based on prior reports (Ling et al., 2001). Al-
though CIH prevents the normal, time-dependent gain in
FULLER ET AL.
munoblots. In ipsilateral gray matter (top panel), 5-HT2Arecep-
tor expression was elevated following C2HS, and this increase
was maintained in CIH treated rats. In contralateral tissue (bot-
tom panel), 5-HT2Areceptor expression appears to be enhanced
in C2HS and sham rats exposed to CIH, although this effect was
only statistically significant in the C2HS group (Fig. 4).
Representative 5-HT2Areceptor and ?-tubulin im-
vical spinal gray matter harvested from segments encompass-
ing the ipsilateral and contralateral phrenic motor nuclei. 5-
HT2Areceptor expression is expressed relative to the density
seen in control, unoperated rats. In ipsilateral tissue (top panel),
5-HT2Areceptor expression was significantly elevated follow-
ing C2HS, and this increase was maintained (but not further in-
creased) after CIH. In contralateral tissue (bottom panel), only
C2HS rats exposed to CIH showed an increase in 5-HT2Are-
ceptor expression. *Significantly greater than control, unoper-
ated, normoxic rats.
Average 5-HT2Areceptor expression in ventral cer-
body mass, its impact was similar in all treatment groups
(e.g., C2HS versus sham versus control). Thus, body mass
effects were independent from changes in 5-HT2Are-
Time-Dependent Enhancement of
Crossed Phrenic Activity
A major goal in spinal cord injury research is to iden-
tify mechanisms that improve the function of spared mo-
tor pathways following injury (Ramer et al., 2000). In
this regard, C2HS is an ideal experimental model because
time-dependent plasticity post-C2HS strengthens exist-
ing, but functionally ineffective spinal synaptic pathways
to a well defined (respiratory) motor output. Hours to
days post-hemisection, inspiratory motor output ipsilat-
eral to C2HS occurs only during intense chemoreceptor
stimulation (e.g., asphyxia), or after pharmacological
treatments that increase respiratory drive (Nantwi and
Goshgarian, 2002) or serotonergic function (Zhou et al.,
2001; Fuller et al., 2003). However, by 2 weeks post-in-
jury, small but quantifiable crossed phrenic inspiratory
bursts can be detected during baseline conditions
(PaCO2? 40 mm Hg, PaO2? 100 mm Hg) in 50%
(Fuller et al., 2003) to 100% of rats (F.J. Golder and G.S.
Mitchell, unpublished data). This spontaneous recovery
grows progressively, such that most rats exhibit robust
inspiratory bursts in ipsilateral phrenic or hemidiaphrag-
matic recordings at 1–2 months after C2HS (Nantwi et
al., 1999; Golder et al., 2001a). However, the contribu-
tion of these pathways to inspiratory tidal volume remains
small (Golder et al., 2003).
Mechanisms of Spontaneous Phrenic
The spontaneous increase in crossed phrenic motor
output following C2HS is associated with morphological
plasticity in the phrenic motor nucleus (Sperry and Gosh-
garian, 1993; Tai and Goshgarian, 1996; Tai et al., 1997;
Mantilla and Sieck, 2002). Hemisection rapidly increases
the number of dendro-dendritic appositions and synapti-
cally active zones in the ipsilateral phrenic motor nucleus;
these changes could facilitate transmission of excitatory
descending synaptic inputs to phrenic motoneurons (for
review, see Goshgarian, 2003). Moreover, the surface
area of phrenic motoneuron cell bodies ipsilateral to
C2HS is significantly decreased 2 weeks post-injury
(Mantilla and Sieck, 2002). Decreased phrenic motoneu-
ron size suggests increased excitability following C2HS,
potentially enhancing crossed phrenic activity for a given
amount of descending excitatory synaptic input.
Considerable evidence indicates a prominent role for
5-HT in spontaneous recovery of crossed phrenic activ-
ity after C2HS. The rapid morphological changes that fol-
low C2HS are blunted when animals are given a sero-
tonin synthesis inhibitor prior to hemisection (Hadley et
al., 1999). Furthermore, treatments that systemically al-
SPINAL SEROTONIN RECEPTORS AFTER CERVICAL INJURY
gray matter from a control, uninjured rat (A) and a chronic C2HS rat (B). The gray matter in B is from the side of the spinal cord
ipsilateral to the C2HS injury. The area encompassing the phrenic motor nucleus is enclosed in the dashed circle and is shown
at higher magnification (original magnification, ?400) in the inset of each panel. In the control rat, 5-HT2Aimmunostaining is
heaviest in the phrenic motor nucleus (A); 5-HT2Aimmunostaining is enhanced in the phrenic motor nucleus and throughout C4
gray matter after chronic C2HS (B).
Representative photomicrographs (original magnification, ?40) depicting 5-HT2Areceptor immunostaining in C4spinal
ter serotonin availability or function produce results con-
sistent with a facilatory role for 5-HT in modulating
crossed phrenic activity (Mitchell and Johnson, 2003;
Goshgarian, 2003). Systemic administration of the specific
5-HT2A/2C-receptor agonist 2,5-dimethoxy-4-iodoam-
phetamine hydrochloride (DOI) induces crossed phrenic
activity following C2HS, an effect blocked by the 5-HT2A
receptor antagonist ketanserin (Zhou et al., 2001).
Neurophysiological and immunhistochemical data in-
dicate that 5-HT acts (at least in part) within the spinal
cord, possibly directly on phrenic motor neurons. Short
latency (?0.7 msec) spinally evoked compound action
potentials due to activation of crossed phrenic pathways
are revealed after systemic delivery of the serotonin pre-
cursor 5-hydroxy tryptophan, and this effect is antago-
nized by the 5-HT receptor antagonist methysergide
(Ling et al., 1994). The latency of the response indicates
that a mono- or paucisynpatic (spinal) input to phrenic
motoneurons is revealed by 5-HT. 5-HT2Areceptors co-
localize with phrenic motoneurons, indicating that this
receptor has the potential to modulate crossed phrenic ac-
tivity (Basura et al., 2001; Doly et al., 2004).
Because spinal 5-HT regulates crossed phrenic activ-
ity, several investigations have examined spinal seroton-
ergic innervation of the phrenic motor nucleus following
C2HS (Tai et al., 1997; Basura et al., 2001; Golder et al.,
2001a). The number of 5-HT immunoreactive terminals
in the phrenic motor nucleus, the number and length of
synaptic contacts per 5-HT terminal, and the number of
multiple synapses are increased 1 month post-C2HS (Tai
et al., 1997). Such changes may enhance the capacity for
serotonergic modulation of ipsilateral phrenic motor neu-
rons, similar to effects previously reported following cer-
vical dorsal rhizotomy (Kinkead et al., 1998; Fuller et al.,
2002a). Since cervical spinal 5-HT concentration in the
region of the phrenic motor nucleus is not significantly
different from control at 2 weeks (present study) or 2
months post-injury (Golder et al., 2001a), changes in
spinal 5-HT concentration per se cannot explain time-de-
pendent increases in crossed phrenic motor output fol-
lowing C2HS. Indeed, 5-HT immunoreactivity near
phrenic motoneurons does not change appreciably be-
tween 1 and 2 months post-C2HS despite increases in
crossed phrenic activity during this time (Golder et al.,
2001a). Our data suggest that increased spinal 5-HT2A
receptor expression (versus increases in spinal 5-HT con-
centration) is more likely to contribute to time-dependent
enhancement of crossed phrenic activity post-C2HS.
5-HT2AReceptors and Spinal Plasticity following
Episodic hypoxia leads to a long-lasting, 5-HT–de-
pendent enhancement of respiratory motor output termed
long-term facilitation (LTF; for review, see Mitchell et
al., 2001). While LTF can be evoked in different respi-
ratory motor outputs (e.g., hypoglossal, phrenic, inter-
costals), a growing body of evidence indicates that
phrenic LTF is initiated via activation of spinal 5-HT2A
receptors (Baker-Herman et al., 2002). Serotonergic ter-
minals are present in the phrenic motor nucleus (Mc-
Crimmon et al., 1995), 5-HT2Areceptors are present on
phrenic motoneurons (Basura et al., 2001), and hypoxia
elicits spinal serotonin release within the phrenic motor
nucleus (Kinkead et al., 2001). Furthermore, short latency
spinally evoked phrenic compound action potentials are
augmented following the induction of LTF, suggesting
an increase in spinal synaptic efficacy (Fuller et al.,
2002b). Activation of 5-HT2Areceptors is necessary to
initiate (but not maintain) phrenic LTF, because the 5-
HT2A receptor antagonist ketanserin prevents phrenic
LTF when given before (but not after) episodic hypoxia
(Fuller et al., 2001). Collectively, these observations sup-
port the hypothesis that phrenic LTF, a model of spinal
synaptic plasticity, results (in large part) from mecha-
nisms associated with activation of 5-HT2Areceptors on
phrenic motoneurons. The present data suggest that sim-
ilar serotonin-dependent mechanisms may strengthen
crossed phrenic pathways following C2HS.
Chronic Intermittent Hypoxia
CIH induces central neural plasticity in respiratory
control (for review, see Mitchell et al., 2001). In spinally
intact rats, CIH augments phrenic responses during hy-
poxia and enhances phrenic LTF following additional hy-
poxic episodes (Ling et al., 2001). Since these effects are
reversed by methysergide, a non-selective 5-HT receptor
antagonist, serotonin receptor activation is necessary for
CIH-induced respiratory plasticity (Ling et al., 2001).
Since there were no changes in 5-HT or 5-HT2Arecep-
tor expression in control, unoperated rats following CIH
in this study, the mechanism of CIH-induced plasticity
in spinally intact rats does not appear to involve a per-
sistent upregulation of 5-HT or 5-HT2Areceptors. Con-
sistent with this observation, the selective 5-HT2 recep-
tor antagonist ketanserin does not abolish CIH-induced
respiratory plasticity, suggesting an involvement of
non–5-HT2 serotonin receptors (Ling et al., 2001).
Following C2HS, CIH enhances crossed phrenic
synaptic pathways (Fuller et al., 2003). For example,
CIH-treated rats exhibit substantially greater inspiratory
phrenic activity below hemisection at baseline conditions
and during chemoreceptor activation. Furthermore, short
latency (?0.7 msec) phrenic potentials evoked by con-
tralateral spinal stimulation (reflecting activation of
crossed phrenic pathways) are amplified following CIH,
indicating increased efficacy of a mono- or paucisynap-
FULLER ET AL.
tic spinal synapse (Fuller et al., 2003). CIH-induced in-
creases in spinal 5-HT concentration or 5-HT2Areceptor
levels cannot explain enhanced crossed phrenic pathways
following CIH (Figs. 2–4). However, increased 5-HT2A
receptor levels due to C2HS alone may establish neces-
sary preconditions that enable CIH to strengthen crossed
phrenic pathways. Consistent with this interpretation,
pre-treatment with CIH (i.e., before C2HS) had no dis-
cernable effect on crossed phrenic pathways (Fuller et al.,
2003). We postulate that, once 5-HT2Areceptors have
been upregulated by C2HS, intermittent activation during
hypoxic episodes leads to greater plasticity than is pos-
sible in control animals. This intriguing hypothesis re-
quires further investigation.
Contralateral Effects of Cervical Hemisection
Unilateral spinal injury (e.g., C2HS) can induce plas-
ticity in contralateral motor pools. For example, after
spinal hemisection, animals regain posture and locomo-
tor skills via increased reliance on contralateral motor ac-
tivity (Muir et al., 1998). Between 1 and 2 months post-
C2HS, anesthetized rats show a reduction in contralateral
phrenic burst amplitude during hypercapnia in associa-
tion with progressive increases in motor output ipsilat-
eral to injury (Golder et al., 2001b). On the contralateral
side, neither 5-HT concentration nor 5-HT2Areceptor ex-
pression were changed after C2HS in normoxic rats. Sur-
prisingly, 5-HT2Areceptor density was increased in the
contralateral spinal cord in C2HS rats exposed to CIH.
Since no differences were found in contralateral phrenic
motor output in C2HS rats after CIH (Fuller et al., 2003),
the significance of increased 5-HT2Areceptors contralat-
eral to C2HS is unclear. Regardless, these data indicate
the potential for contralateral phrenic plasticity (e.g., en-
hanced LTF) following CIH in rats with C2HS.
In most spinal cord injury patients, partial locomotor
and respiratory recovery is observed over time following
the injury (Mansel and Norman 1990). Although this
recovery almost certainly arises from spared axonal path-
ways (Ramer et al., 2000), the cellular and synaptic mech-
anisms underlying spontaneous improvement of locomo-
tor and respiratory function are unclear. Our data suggest
that spontaneous upregulation of spinal 5-HT2Areceptors
(both on and around phrenic motoneurons) contributes to
respiratory motor recovery following spinal injury.
This work was partially funded by NIH grants HL
69064 and 65383. D.D. Fuller was supported by a Parker
B. Francis fellowship in pulmonary research. T.L. Baker-
Herman was supported by NIH Training Grant HL 07654.
F.J. Golder was supported by a grant from the Christo-
pher Reeve Paralysis Foundation. We thank Dr. M.S.
Brownfield for supplying the 5-HT2Aantibody used in
the immunocytochemistry experiments, and Dr. Mary
Behan and Laura Peterson for assistance with histologi-
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Address reprint requests to:
David D. Fuller, Ph.D.
Department of Physical Therapy
University of Florida
Health Science Center
PO Box 100154
Gainesville, FL 32610-0154
SPINAL SEROTONIN RECEPTORS AFTER CERVICAL INJURY