www.elsevier.com/locate/brainres
Brain Research 1013 (2004) 174–181
Research report
Levels of brain-derived neurotrophic factor and neurotrophin-4 in lumbar
motoneurons after low-thoracic spinal cord hemisection
Rosario Gulinoa, Salvatore Andrea Lombardoa, Antonino Casabonaa,Giampiero Leanzab, Vincenzo Perciavallea,*
aDepartment of Physiological Sciences, University of Catania, Viale Andrea Doria, 6-95125, Catania, ItalybDepartment of Physiology and Pathology, University of Trieste, Via Fleming, 22-34127, Trieste, Italy
Accepted 31 March 2004
Abstract
Neuroplasticity represents a common phenomenon after spinal cord (SC) injury or deafferentation that compensates for the loss of
modulatory inputs to the cord. Neurotrophins play a crucial role in cell survival and anatomical reorganization of damaged spinal cord, and
are known to exert an activity-dependent modulation of neuroplasticity. Little is known about their role in the earliest plastic events, probably
involving synaptic plasticity, which are responsible for the rapid recovery of hindlimb motility after hemisection, in the rat. In order to gain
further insight, we evaluated the changes in BDNF and NT-4 expression by lumbar motoneurons after low-thoracic spinal cord hemisection.
Early after lesion (30 min), the immunostaining density within lumbar motoneurons decreased markedly on both ipsilateral and contralateral
sides of the spinal cord. This reduction was statistically significant and was then followed by a significant recovery along the experimental
period (14 days), during which a substantial recovery of hindlimb motility was observed. Our data indicate that BDNF and NT-4 expression
could be modulated by activity of spinal circuitry and further support putative involvement of the endogenous neurotrophins in mechanisms
of spinal neuroplasticity.
D 2004 Elsevier B.V. All rights reserved.
Theme: Development and regeneration
Topic: Neurotrophic factors: expression and regulation
Keywords: Spinal cord hemisection; Motoneuron; Plasticity; Neuromuscular activity; Neurotrophin; Rat
1. Introduction Several studies suggested that neurotrophinsmay also play
Neurotrophins are a family of chemically related pro-
teins, which play an important role in development, survival
and maintenance of neuronal function in the peripheral and
central nervous system [12,13,19,25]. Brain-derived neuro-
trophic factor (BDNF) and neurotrophin-4 (NT-4) have a
wide variety of effects on spinal motoneurons. During
development and in the adult rat [12,25], they may represent
target-derived and activity-dependent trophic signals for
motoneurons by regulating several neural functions such
as fiber sprouting, electrical and metabolic properties and
cell size [5,8]. Recently, it has been demonstrated that
neurotrophins may act via autocrine–paracrine as well as
anterograde mechanisms, in an activity-dependent manner
[1,17,22].
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.03.055
* Corresponding author. Tel.: +39-95-339095; fax: +39-95-330645.
E-mail address: [email protected] (V. Perciavalle).
a crucial role in spinal cord (SC) plasticity. For example,
recent investigations showed neurotrophin expression in
motoneurons [2] indicating that endogenous BDNF and
NT-4 may be involved in functional changes induced by
exercise onmotor unit properties [6,7,21,26]. It is also known
that spinal injury or deafferentation induce plastic changes in
the spinal circuitry, which could compensate for the alteration
of modulatory inputs and promote functional recovery [4,23].
In addition to changes that occur at the lesion site, spinal and
supraspinal circuits undergo substantial reorganization by
two different mechanisms: (1) anatomical reorganization of
circuits and formation of new pathways by sprouting; (2)
modification of pre-existing circuits by modulation of syn-
aptic strength [4,23]. Anatomical reorganization requires
long time, so the earliest signs of functional recovery are
likely attributable tomodifications of excitability of the spinal
circuitry. Althoughmany studies have investigated the role of
neurotrophins in preventing neuronal death or promoting
R. Gulino et al. / Brain Research 1013 (2004) 174–181 175
anatomical reorganization after SC injury [14,16,18], the
involvement of endogenous neurotrophins in the dynamic
modulation of local circuitry remains to be elucidated.
In the present study, the expression of BDNF and NT-4 in
lumbar motoneurons was investigated at various time inter-
vals after low-thoracic SC hemisection. This way we could
evaluate the basal production of these neurotrophins and
observe the time course of their expression immediately
after the disruption of spinal and supraspinal pathways and
during the spontaneous post-lesion recovery of hindlimb
motility, that normally starts within few days after surgery.
2. Materials and methods
2.1. Animals and treatment
Young adult male rats (Wistar, 200–250 g, n = 22) were
used in this study. They were given free access to water and
food and were housed under standard conditions of humidity
and temperature with 12-h light/dark cycle. Animal care and
handling were carried out in accordance with guidelines
issued by the Committee of Experimental Animals of Uni-
versity of Catania; all efforts were made to minimize animal
suffering and to reduce the number of animal used. Sixteen
animals were deeply anaesthetised with chloral hydrate (400
mg/kg, Fluka, Germany) andmounted on a stereotaxic device
(David Kopf Instruments, USA).We stabilized the column by
suspending it with vertebral clamps and drilled a hole on the
dorsal surface of the ninth thoracic vertebra (corresponding to
spinal segments T10–T11). Then, we removed the dura
mater from the dorsal surface of SC and transected the right
side of the cord by using a microsurgical knife attached to the
arm of the stereotaxic frame, under visual guidance via an
operating microscope. Care was taken in order to avoid
damage to the median blood vessels. This operation has the
advantage of producing consistent lesion, with virtually no
bleeding and causes minimal if any local operative discom-
fort. The hemisection site was filled with sterile gelfoam and
muscles and skin were sutured. Three other animals were
operated but not hemisected and they were used as sham
operated controls, which were euthanized 4 h after surgery.
Hemisected rats were divided in four experimental groups
(four animals each), which were sacrificed 30 min, 4 h, 12
h (short-term) and 14 days after lesion (long-term), respec-
tively. Long-term animals were allowed to recover sponta-
neously after injury, without administration of any
treatments. Finally, three intact animals served as normal
controls. All rats were sacrificed by intracardiac perfusion,
using saline followed by cold 4% paraformaldehyde in 0.1 M
phosphate buffer (PB).
2.2. Immunohistochemistry
After perfusion, the thoracic-lumbar parts of SCs were
removed, postfixed for 2 h in the same fixative and
cryoprotected by overnight immersion in 0.1 M PB con-
taining 20% sucrose. Two series of SC horizontal sections
(40 Am) were cut on a freezing microtome and used for free-
floating BDNF and NT-4 immunostaining. Sections were
rinsed in 0.05 M Tris buffer saline (TBS) and soaked in TBS
containing 3% H2O2 and 10% methanol, for 10 min, in
order to quench the endogenous peroxidase activity. Sec-
tions were then preincubated at room temperature for 1 h in
TBS containing 5% normal goat serum and 0.3% Triton.
After pre-incubation, the two series of sections were incu-
bated at 4 jC for 1 week with either anti-BDNF (1:1000;
Chemicon, USA, Cat. No. AB1534) or anti-NT-4 (1:1000;
Chemicon, Cat. No. AB1781) rabbit polyclonal antibodies.
This was followed by incubation with a biotinylated goat
anti-rabbit antibody (1:200; Vector Laboratories, USA) for 2
h, and by reaction with Vectastain ABC complex (Vector
Laboratories). BDNF or NT-4 immunoreactivity (IR) was
visualised as a brown colour by using diaminobenzidine and
H2O2 as substrate. All sections belonging to control and
injured animals were incubated in the same antibody and
DAB solutions, under identical incubation time and temper-
ature conditions.
Control of immunostaining specificity was performed by
omitting the primary antibody or by substitution of the anti-
rabbit secondary antibody. These controls did not exhibit
any specific immunostaining.
A cresyl violet staining was performed on alternate
sections from the normal group and the 14 days group.
This staining allowed us to count the number of motoneuron
profiles and verify whether the hemisection caused moto-
neuronal depletion due to neuronal death or only a down-
regulation of the marker.
2.3. Morphometrical analyses
The sections were examined using a Zeiss light micro-
scope coupled with a computer assisted video camera
(Sony). The Scion Image software (ScionCorp., NIH,
USA) was used to count immunopositive motoneuron
profiles and for densitometric and morphometric analyses.
In order to ensure consistency in the analyses, all sections
were stained at the same time. Also, we used the same
illumination level, microscope and digital camera settings
throughout the image capturing session. Three SC sections
from each animal were used for these analyses and all
sections were separately examined by two investigators
uninformed about the experimental procedures performed
on the animals. These sections were cut horizontally through
the ventral horn in a region corresponding to the dorsolateral
part of lamina IX and to the segments L4–L5 (Fig. 1). This
motoneuron pool is also known as retrodorsolateral nucleus
(RDLN) and contains motoneurons that innervate the hin-
dlimb via the sciatic nerve [20,27]. Only unambiguous
immunopositive profiles characterized by evident nucleus
and well-defined motoneuronal features were considered.
Average soma diameter and optical density (OD) were
Fig. 1. Schematic representation of the SC area where the motoneurons
included in the study were located. (A) The rectangle represents the area
from which the horizontal sections were collected. Note that, when the SC
is placed horizontally on its ventral surface, the RDLN is present at the
same level along L4 and L5 segments, thus almost all horizontal sections
show this motoneuronal pool for all the length of measured area. (B)
Example of an horizontal section showing the longitudinal extent of these
motoneuronal pools.
R. Gulino et al. / Brain Research 1013 (2004) 174–181176
measured from motoneuron bodies outlined manually by the
experimenter from greyscale images (400� magnification),
using a protocol modified from Skup et al. [26]. The OD
measured in the grey matter of the control sections obtained
by omitting the primary antibody was used as background
staining and subtracted from each measurement. The immu-
nopositivity of inter-perikaryal grey matter was evaluated by
measuring the average OD of three circular areas (3 cm
diameter at 400� magnification) randomly selected within
each SC side, at L4–L5 level.
2.4. Statistical analysis
Quantitative variations of the number of motoneuron
profiles and of the intra-motoneuron OD with respect to
the time-point after lesion and to the SC side were evaluated
by means of a two-way ANOVA and Bonferroni’s post-hoc
test. Student’s t-test were also used within each animal
group to compare the left and right SC sides. All analyses
were performed by using SYSTAT software package.
3. Results
All rats survived surgery throughout the experimental
period. Spinal cords were inspected after removal in order to
verify the position and extent of the hemisection. We found
that all cords were hemisected correctly at low thoracic level
except for two animals, belonging to the 14 days and 4
h groups, respectively. These two animals were excluded
from the study due to incomplete lesion. The extent of the
lesion in each animal was assessed by reconstruction from
40-Am-thick serial longitudinal sections taken through the
entire dorso-ventral aspect of the spinal cord at the level of
the lesion. Hemisections were considered complete when
they included the ipsilateral dorsal column and corticospinal
tract, and did not encroach on the contralateral side for more
than 10% of its width, as assessed on digitized images by
the Scion Image software. Only individuals with complete
hemisections were considered for the study.
Hemisected animals exhibited a pronounced paralysis of
the right hindlimb. The group of rats that were allowed to
survive for 14 days showed visible improvement of hin-
dlimb support and stepping ability within 3–4 days after
injury. This recovery was also more evident at the end of the
survival period.
In normal animals, a number of large multipolar profiles
showed intense BDNF-like and NT-4-like IR (Fig. 2A and B,
normal). Dark BDNF-like IR was also observed inside and
around the cell nuclei (Fig. 2A, normal, arrows) and within
dendritic arborizations and axons (Fig. 2A, arrow heads).
These profiles were located in the ventral portion of SC
corresponding to RDLN and were classifiable as motoneur-
ons. Smaller cells, such as interneurons or glial cells, also
showed some staining.
The number of BDNF-like and NT-4-like positive pro-
files as well as their staining density did not differ between
intact and sham lesioned rats (data not shown); thus, these
animals were pooled in a single normal group.
Both BDNF-like and NT-4-like IR of inter-perikaryal
grey matter was found to be constant among groups and
between sides of the SC (data not shown).
To evaluate the modification of neurotrophin expression
during the post surgery period, we analysed the variations of
the number of immunopositive profiles and the changes of
intra-motoneuronal OD 30 min, 4 h, 12 h and 14 days after
lesion.
3.1. Changes of the number of positive profiles
In all groups, the average soma diameters for BDNF-like
or NT-4-like IR profiles were distributed bimodally, with an
apparent boundary at about 23 Am (data not shown). The
Fig. 2. High magnification (150� ) photomicrographs showing examples of BDNF-like and NT-4-like immunoreactivity. (A) The cytoplasmic BDNF IR
appears very intense in the motoneurons belonging to normal animals, and a large and dense body is evident inside and around the nuclei (normal, arrows);
dendritic arborizations and axons are also greatly stained (arrow heads). Both the cytoplasmic and nuclear staining density appear reduced after hemisection,
but they gradually recovered until reaching near-normal levels of optical density, 14 days after hemisection (14 days). (B) The NT-4 IR is uniformly distributed
within cytoplasm and there are not any accumulations of NT-4 IR around the neuronal nuclei (normal, arrows). The lesion determined a small reduction of
optical density that returned to normal levels 12 h after lesion. Axons and dendrites appear weakly stained (normal, arrow heads).
R. Gulino et al. / Brain Research 1013 (2004) 174–181 177
profiles with average soma diameter ranging between 11
and 23 Am were presumed to be g-motoneurons, whereas
larger ones were considered a-motoneurons [10]. The mean
soma diameter of both populations of motoneurons was not
apparently affected by the hemisection, since no significant
difference was observed between left and right sides or
between groups (data not shown).
No changes in the total number of motoneuron profiles
were seen after hemisection by using Cresyl violet staining
(data not shown). Therefore, the different number of
Table 1
Number of immunopositive motoneuron profiles
BDNF+motoneuron profiles NT-4 +motoneuron profiles
Contralateral Ipsilateral % of normal Contralateral Ipsilateral % of normal
Gamma Alpha Gamma Alpha Gamma Alpha Gamma Alpha Gamma Alpha Gamma Alpha
Normal 14F 1 32F 3 13F 1 32F 4 100F 7 100F 8 14F 1 36F 3 14F 1 35F 4 100F 6 100F 8
30 min 9F 1 27F 3 9F 2 27F 3 67F 9 84F 8 14F 2 35F 2 14F 1 30F 3 98F 9 92F 6
4 h 7F 1 29F 2 8F 1 23F 1 57F 3 81F 8 14F 1 32F 3 13F 2 31F 2 95F 8 89F 5
12 h 11F 2 30F 3 9F 1 28F 4 73F 15 90F 15 11F1 30F 3 13F 1 30F 4 83F 15 85F 15
14 days 12F 2 28F 3 11F1 31F 2 83F 9 91F 6 12F 2 30F 3 11F1 29F 3 80F 12 84F 10
The profiles number is reported as mean number per sectionF S.E.M.
Bold text indicates significant difference from normal controls ( P< 0.05).
R. Gulino et al. / Brain Research 1013 (2004) 174–181178
immunoreactive neurons we observed in the lesioned
animals could be entirely attributable to changes in neuro-
trophin expression.
Fig. 3. Effect of hemisection on BDNF-like (A) and NT-4-like (B) immunoreactivit
density of the contralateral side of normal group was arbitrarily considered as 100%
after hemisection. Then, the expression of BDNF and NT-4 partially recovered u
indicates significant differences from normal controls, while (y) indicates significanrepresent the standard error of the mean (S.E.M.).
Table 1 summarizes the mean number of BDNF-like and
NT-4-like IR profiles per section in all groups. The most
dramatic changes were seen in the number of BDNF-like IR
y, evaluated by measuring the intracellular optical density. The mean optical
. Both BDNF-like and NT-4-like immunoreactivity immediately decreased
ntil reaching near normal levels of optical density (see Section 3). The (*)
t differences between ipsilateral and contralateral side of SC. The error bars
R. Gulino et al. / Brain Research 1013 (2004) 174–181 179
profiles. In particular, the number of positive g-motoneuron
profiles appeared reduced by 33%, 43%, 27% and 17% at 30
min, 4 h, 12 h and 14 days after lesion, respectively
(P= 0.002), with no significant side difference (P= 0.456)
or group by side interaction (P= 0.871). Pairwise compar-
isons showed significant reduction at 30 min (P= 0.042) and
4 h after lesion (P= 0.002), whereas the other groups did not
differ significantly from normal controls (P>0.05). On the
other hand, the number of IR a-motoneuron profiles was less
affected by hemisection, showing only a maximal decrease of
19%, with no significant group or side differences. T-statistics
confirmed that there was no significant side difference in each
group for both the populations of motoneurons (P>0.05).
As shown in Table 1, the post-lesion decrease of the
number of NT-4-like IR profiles was smaller than the
changes observed for BDNF positive profiles, and both g-
and a-motoneurons displayed no significant differences
between groups or sides.
3.2. Changes of intracellular OD
The post-surgery time course of intracellular OD changes
showed a similar pattern for BDNF and NT-4 staining. Since
the populations of g- anda-motoneurons showed comparable
modifications in the immunoreactivity throughout the exper-
imental period, they were collected in a single group and
analysed together. At 30 min, 4 h, 12 h and 14 days post-
lesion, the intracellular BDNF staining density was reduced
by 38%, 30%, 21% and 16%, respectively (Fig. 3A), whereas
the intracellular NT-4 staining density was reduced by 19%,
27%, 10% and 8% (Fig. 3B). There was a significant effect of
the group for both neurotrophins expression changes
(P= 0.000) reflecting the reduction of OD throughout the
post surgery period. The effect of side was not significant and
the side by group interaction showed significant changes only
for NT-4-like immunoreactivity (P= 0.009). The nuclear
BDNF-like labelling was dramatically reduced immediately
after injury, but it returned to normal levels after 14 days (Fig.
2A, arrows).
The reduction of BDNF-like IR (Figs. 2A and 3A) was
stronger than that of NT-4 (Figs. 2B and 3B) but pairwise
comparisons revealed significant differences (P= 0.000)
between all group pairs for both neurotrophins. Thus, all
lesioned groups were significantly different from normal
group and, throughout the recovery period, the groups were
different from each other (except for the comparison be-
tween the 14 days and the 12 h group of NT-4 OD).
The recovery of neurotrophins staining density was
equally evident on both lesioned and unoperated side (t-
test, P>0.05), except for the 14 days group, which showed
ipsilateral mean density values significantly lower than the
contralateral (t-test: P < 0.05) (Fig. 3).
Taken together, these results point out that the spinal cord
hemisection elicited a rapid down-regulation of BDNF and
NT-4 expression within lumbar motoneurons. This reduc-
tion was more evident for BDNF-like IR and in particular
within the populations of g-motoneurons, which showed a
reduction of both number of positive profiles and their
intracellular OD. After the initial reduction, the levels of
endogenous BDNF and NT-4 expression recovered progres-
sively throughout the experimental period, although this
recovery appeared incomplete, especially in the ipsilateral
SC side.
4. Discussion
In this study, we evaluated the basal expression of BDNF
and NT-4 by lumbar motoneurons belonging to RDLN and
the modifications of their expression levels following SC
hemisection and along the survival period, during which we
observed a spontaneous, although incomplete, recovery of
hindlimb function.
We found that spinal cord hemisection determined a
down-regulation of neurotrophins expression in the soma
of lumbar motoneurons. BDNF down-regulation was more
evident and longer-lasting than NT-4 reduction. In fact, the
number of BDNF-like positive profiles and their intracellu-
lar OD were more markedly affected by hemisection than
those of NT-4-like IR profiles.
The reduction of neurotrophin expression occurred im-
mediately after interruption of the spinal pathways, suggest-
ing a local expression of these proteins whose magnitude
might be related to the level of motoneuron activity. In fact,
as the flaccid paralysis of hindlimb muscles indicated,
motoneuron activity was dramatically reduced after lesion.
The fact that the down-regulation of BDNF was more
extensive in gamma rather than alpha motoneurons further
supports the notion of activity-dependent expression of
neurotrophins. In fact, the g-motoneurons are known to
have, in normal conditions, high excitability and typical
tonic activity; so the reduced downstream discharge induced
by hemisection would reduce mostly the discharge of g-
motoneurons rather than the activity of more phasic neurons
such as the large a-motoneurons.
The issue of a possible intraspinal source of neuro-
trophins we propose, has recently been addressed by Buck
et al. [2] who demonstrated a good correspondence of
protein and mRNA expression in adult rat motoneurons.
Thus, the reduction of BDNF and NT-4 immunostaining in
lumbar motoneurons is likely to reflect, at least in part, a
down-regulation of their synthesis by these cells, even
though a reduced retrograde transport of neurotrophins to
the soma cannot be entirely excluded [28].
Considering the autocrine–paracrine action mechanism of
neurotrophins, as well as their possible anterograde and
retrograde transport, the changes in neurotrophin expression
we observed might have altered trophic and modulatory
signals for the motoneuron itself, for target cells and for other
spinal and supraspinal neurons [1,2,17,22,24,30]. In partic-
ular, BDNF is involved in the synaptic plasticity [7,19],
neuroprotection and sprouting of spared pathways after
R. Gulino et al. / Brain Research 1013 (2004) 174–181180
injury [14,16,18]. On this basis, it is reasonable to think that
the modifications of endogenous neurotrophins we reported
here may be linked to the plastic phenomena responsible for
functional reorganization after SC injury. A result consistent
with this view has been described by Johnson et al. [15], who
found increased levels of BDNF and NT-3 in the ventral horn
of cervical and thoracic spinal cord following dorsal rhyzot-
omy that eliminated diaphragmatic afferents, which are
primarily inhibitory to phrenic motor output. The presence
of BDNF-like IR associated with the nucleus of the moto-
neurons and its post-lesion modifications (Fig. 2A, arrows)
may represent an additional support for the role of this
neurotrophin in plasticity. Several authors, in fact, have
reported the possibility of such forms of BDNF entering the
neuronal nuclei, where it could influence transcription direct-
ly [28,29]. Although the role of BDNF in neuroplasticity is
much more documented, similar hypotheses have been pro-
posed for the involvement of NT-4 [5,24,26]. For example,
exercise activity elicited an up-regulation of NT-4 in spinal
motoneurons and glia [26], as well as in the muscle [5],
suggesting an activity-dependent trophic and modulatory
action on neuromuscular system [5,24,26].
The down-regulation of neurotrophins was followed by a
significant recovery of expression, which seemed to parallel
the improvement of support and locomotion. However, the
neurotrophins recovery was incomplete, probably due to a
severe impairment of the local circuitry, which, conversely,
could have affected behavioural recovery. In this respect,
exogenous administrations of BDNF or extensive behav-
ioural training could be helpful to improve restorative
processes [4,7,14,16,23].
Surprisingly, the hemisection-induced reduction of neu-
rotrophins IR in the lumbar SC was equally distributed on
both sides and, only at the longest time point (14 days), we
found a more robust recovery in the intact compared to the
lesioned side. Several hypotheses could be proposed to
account for this finding. It is known, for instance, that,
although descending supraspinal pathways terminate mainly
ipsilaterally, a significant part of these projections may
decussate in the SC [3,11]. Therefore, the hemisection is
likely to partially affect the contralateral side as well.
Moreover, within few hours after hemisection, a variety of
dynamic processes in the spinal circuitry, such as modifi-
cations of synaptic efficacy and spinal excitability or reor-
ganization of pattern generators [23,30], could compensate
for the reduced neuronal activity and may thus sustain
minimal levels of expression also in the affected side. These
processes could be sufficient to promote the recovery of
neurotrophins expression and the functional retrieval ob-
served within 2 weeks after lesion, whereas a more robust
functional recovery requires an anatomical reorganization of
spinal circuitries, perhaps through compensatory sprouting
of the spared pathways [4,16,23,30]. During functional
recovery, animals relied on the contralateral hindlimb to
support the body for posture and locomotion, and this
behaviour should determine higher levels of activity in the
contralateral pool of motoneurons. This increased activity
might likely account for the higher levels of IR in the
contralateral side 14 days post-lesion. Moreover, the retro-
grade transport of neurotrophins from the neuromuscular
junction could be also reduced on the lesioned side, because
of the reduced muscle activity [5,7].
Together, these experimental results provide new eviden-
ces about the involvement of neurotrophins (mainly BDNF)
in the modulation of spinal motor circuitry, but suggest that
the overall effect on this system could be very complex.
Similar evidences has been obtained from the dorsal horn
neurons, in a model of chronic spinal cord hemisection,
where BDNF seems to exert modulatory effects on noci-
ceptive processing. In this model, the effect of BDNF did
not differ between sides and showed an intricate mechanism
of action [9].
5. Conclusion
Our data shows that neurotrophins expression by lumbar
motoneurons rapidly decreased after low-thoracic spinal
cord hemisection and recovered significantly along the
experimental period. These findings suggest that the expres-
sion of neurotrophins, particularly BDNF, may depend upon
the activity of spinal circuitries and integrity of spinal and
supraspinal pathways. We postulate that neurotrophins
could be involved in the plasticity of neuromuscular sys-
tems, including the anatomical and functional reorganization
of the motor circuitry responsible for the recovery of support
and stepping abilities following SC injury. In particular, the
rapid modifications of their expression after injury could
indicate an involvement in processes, such as synaptic
plasticity, which should be partially responsible for the
earliest signs of recovery of hindlimb motility after low-
thoracic SC hemisection. Further studies are needed to
elucidate the complex role of these neurotrophins in this
model of neuroplasticity.
Acknowledgements
We thank Gianfranco Bosco for his kind contribution to
the final editing of the manuscript. This research was
partially supported by Ministero dell’Istruzione, dell’Uni-
versita e della Ricerca (M.I.U.R.).
References
[1] C.A. Altar, N. Cai, T. Bliven, M. Juhasz, J.M. Conner, A.L. Ache-
son, R.M. Lindsay, S.J. Wiegand, Anterograde transport of brain-
derived neurotrophic factor and its role in the brain, Nature 389
(1997) 856–859.
[2] C.R. Buck, K.L. Seburn, T.C. Cope, Neurotrophin expression by
spinal motoneurons in adult and developing rats, J. Comp. Neurol.
416 (2000) 309–318.
R. Gulino et al. / Brain Research 1013 (2004) 174–181 181
[3] J.W. Commissiong, Evidence that noradrenergic coerulospinal projec-
tion decussates at the spinal level, Brain Res. 212 (1981) 145–151.
[4] R.D. de Leon, R.R. Roy, V.R. Edgerton, Is the recovery of stepping
following spinal cord injury mediated by modifying existing neural
pathways or by generating new pathways? A perspective, Phys. Ther.
81 (2001) 1904–1911.
[5] H. Funakoshi, N. Belluardo, E. Arenas, Y. Yamamoto, A. Casabona,
H. Persson, C.F. Ibanez, Muscle-derived neurotrophin-4 as an activ-
ity-dependent trophic signal for adult motor neurons, Science 268
(1995) 1495–1499.
[6] F. Gomez-Pinilla, Z. Ying, P. Opazo, R.R. Roy, V.R. Edgerton, Dif-
ferential regulation by exercise of BDNF and NT-3 in rat spinal cord
and skeletal muscle, Eur. J. Neurosci. 13 (2001) 1078–1084.
[7] F. Gomez-Pinilla, Z. Ying, R.R. Roy, R. Molteni, V.R. Edgerton,
Voluntary exercise induces a BDNF-mediated mechanism that pro-
mote neuroplasticity, J. Neurophysiol. 88 (2002) 2187–2195.
[8] M. Gonzalez , W.F. Collins III, Modulation of motoneuron excitabil-
ity by brain-derived neurotrophic factor, J. Neurophysiol. 77 (1997)
502–506.
[9] B.C. Hains, W.D. Willis, C.E. Hulsebosch, Differential electrophysi-
ological effects of brain-derived neurotrophic factor on dorsal horn
neurons following chronic spinal cord hemisection injury in the rat,
Neurosci. Lett. 320 (2002) 125–128.
[10] K. Hashizume, K. Kanda, R.E. Burke, Medial gastrocnemius motor
nucleus in the rat: age-related changes in the number and size of
motoneurons, J. Comp. Neurol. 269 (1988) 425–430.
[11] J.C. Holstege, H.G.J.M. Kuypers, Brainstem projections to spinal
motoneurons: an update, Neuroscience 23 (1987) 809–821.
[12] E.J. Huang, L.F. Reichardt, Neurotrophins: roles in neuronal devel-
opment and function, Annu. Rev. Neurosci. 24 (2001) 677–736.
[13] P.J. Isackson, Trophic factor response to neuronal stimuli or injury,
Curr. Opin. Neurobiol. 5 (1995) 350–357.
[14] L.B. Jakeman, P. Wei, Z. Guan, B.T. Stokes, Brain-derived neuro-
trophic factor stimulates hindlimb stepping and sprouting of choliner-
gic fibers after spinal cord injury, Exp. Neurol. 154 (1998) 170–184.
[15] R.A. Johnson, A.J. Okragly, M. Haak-Frendscho, G.S. Mitchell, Cer-
vical dorsal rhizotomy increases brain-derived neurotrophic factor and
neurotrophin-3 expression in the ventral spinal cord, J. Neurosci. 20
RC77 (2000) 1–5.
[16] D. Kim, T. Schallert, Y. Liu, T. Browarak, N. Nayeri, A. Tessler, I.
Fischer, M. Murray, Transplantation of genetically modified fibroblast
expressing BDNF in adult rats with a subtotal hemisection improves
specific motor and sensory functions, Neurorehabilitation Neural Re-
pair 15 (2001) 141–150.
[17] K. Kohara, A. Kitamura, M. Morishima, T. Tsumoto, Activity-depen-
dent transfer of brain-derived neurotrophic factor to postsynaptic neu-
rons, Science 291 (2001) 2419–2423.
[18] Y. Liu, B.T. Himes, M. Murray, A. Tessler, I. Fischer, Grafts of
BDNF-producing fibroblast rescue axotomized rubrospinal neurons
and prevent their atrophy, Exp. Neurol. 178 (2002) 150–164.
[19] A.K. McAllister, L.C. Katz, D.C. Lo, Neurotrophins and synaptic
plasticity, Annu. Rev. Neurosci. 22 (1999) 295–318.
[20] C. Molander, Q. Xu, G. Grant, The cytoarchitectonic organization of
the spinal cord in the rat: I. The lower thoracic and lumbosacral cord,
J. Comp. Neurol. 230 (1984) 133–141.
[21] J.B. Munson, R.C. Foehring, L.M. Mendell, T. Gordon, Fast-to-slow
conversion following chronic low-frequency activation of medial gas-
trocnemius muscle in cats: II. Motoneuron properties, J. Neurophysiol.
77 (1997) 2605–2615.
[22] H. Nawa, N. Takei, BDNF as an anterophin; a novel neurotrophic
relationship between brain neurons, Trends Neurosci. 24 (2001)
683–684.
[23] O. Raineteau, M.E. Schwab, Plasticity of motor systems after incom-
plete spinal cord injury, Nat. Rev., Neurosci. 2 (2001) 263–273.
[24] I.A. Scarinsbrick, P.J. Isackson, A.J. Windebank, Differential expres-
sion of brain-derived neurotrophic factor, neurotrophin-3, and neuro-
trophin-4/5 in the adult rat spinal cord: regulation by glutamate
receptor agonist kainic acid, J. Neurosci. 18 (1999) 7757–7769.
[25] M. Sendtner, G. Pei, M. Beck, U. Schweizer, S. Wiese, Developmen-
tal motoneuron cell death and neurotrophic factors, Cell Tissue Res.
301 (2000) 71–84.
[26] M. Skup, A. Dwornik, M. Macias, D. Sulejczak, M. Wiater, J.
Czarkowska-Bauch, Long-term locomotor training up-regulates
TrkBFL receptor-like proteins, brain-derived neurotrophic factor,
and neurotrophin 4 with different topographies of expression in
oligodendroglia and neurons in the spinal cord, Exp. Neurol. 176
(2002) 289–307.
[27] J.E. Swett, R.P. Wikholm, R.H.I. Blanks, A.L. Swett, L.C. Conley,
Motoneurons of the rat sciatic nerve, Exp. Neurol. 93 (1986) 227–252.
[28] F.L. Watson, H.M. Heerssen, D.B. Moheban, M.Z. Lin, C.M. Sauva-
geot, A. Bhattacharyya, S.L. Pomeroy, R.A. Segal, Rapid nuclear
responses to target-derived neurotrophins require retrograde transport
of ligand-receptor complex, J. Neurosci. 19 (1999) 7889–7900.
[29] C. Wetmore, Y. Cao, R.F. Pettersson, L. Olson, Brain-derived neuro-
trophic factor: subcellular compartmentalization and interneuronal
transfer as visualized with anti-peptide antibodies, Proc. Natl. Acad.
Sci. 88 (1991) 9843–9847.
[30] J.R. Wolpaw, A.M. Tennissen, Activity-dependent spinal cord
plasticity in health and disease, Annu. Rev. Neurosci. 24 (2001)
807–843.
