Centre National de la Recherche Scientifique, Laboratoire de
Neurobiologie et Mouvements, 13402 Marseille Cedex 9, France
 |
INTRODUCTION |
Serotonin (5-HT) is a widely distributed
neuromodulator that has a role in numerous functions in adulthood
(Vogt 1982
) and that modulates rhythmic activities of
the locomotor (Cazalets et al. 1992
;
Sqalli-Houssaini et al. 1993
) and the respiratory (Hilaire and Duron 1999
) networks at birth. Moreover, as
suggested by its early expression and its role in the control of cell
division, differentiation, growth, and synaptogenesis (Lauder
1993
; Lauder and Krebs 1978
; Levitt et
al. 1997
; Whitaker-Azmitia 1991
), 5-HT may
affect CNS maturation (Cases et al. 1996
;
Chubakov et al. 1986
; Mooney et al. 1998
;
Yan et al. 1997a
,b
). Therefore, besides its role in
modulating the locomotor network, 5-HT might also play a role in its
maturation, as suggested by the transient difficulties in swimming
abilities observed at birth in rats prenatally treated with
para-chlorophenylalanine (Nakajima et al. 1998
).
In the MAOA-deficient (Tg8) neonatal mouse, which was created from the
C3H/HeJ (C3H) strain by disrupting the gene encoding monoamine oxidase
A (MAOA), the enzyme that degrades 5-HT, the lack of MAOA activity
results in 5-HT levels 10 times larger than those in C3H neonates
(Cases et al. 1995
; Lajard et al. 1999
). To determine whether MAOA deficiency affects locomotor network maturation, we used in vivo and in vitro approaches to compare the
locomotor activity produced by Tg8 neonates with that produced by C3H
neonates. Both results suggest a transient delay in the maturation of
the Tg8 locomotor network, which provides increasing evidence for a
possible role of 5-HT in mammalian CNS maturation.
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METHODS |
In the in vivo experiments, the swimming behavior of 35 C3H and
24 Tg8 pups was observed daily from postnatal day 0 to postnatal day 7 (P0-P7). Each pup was gently immersed in a warmth-regulated water bath
(37 ± 1°C) for a 30 s trial during which its motor activity was observed and classified in one of three swimming patterns,
H0, H1, or H2, depending on whether it used 0, 1, or 2 hindlimbs for
swimming (see RESULTS). The observer did not know which
strain was being observed. In addition, seven Tg8 and six C3H mice from
two different litters of each strain were again tested from P13 to P15.
Results were analyzed with statistical software SYSTAT in
multidimensional contingency tables as the occurrence of the H0, H1,
and H2 swimming patterns versus the strains and the classes of age. The
multidimensional contingency tables were treated as log-linear models
(Fienberg 1980
).
The in vitro experiments were performed as reported previously for
newborn rats (Cazalets et al. 1992
;
Sqalli-Houssaini et al. 1993
). Newborn mice (5 C3H and 5 Tg8 pups at P0-P5 and 3 Tg8 pups at P11) were decapitated under ether
anesthesia and their spinal cords were dissected and cut at the level
of T8. The motor output from the lumbar ventral roots was recorded,
amplified, and stored. Up to three spinal cords were pooled in the same
experimental chamber so that simultaneous recordings could be
performed. The spinal cords were continuously superfused with
oxygenated saline into which drugs could be added (Sigma Chemicals).
The mean period (± SE) of ventral root rhythmic activity was estimated
from 60 cycles, and differences between the means were taken as
significant at P < 0.05 (unpaired
t-test).
 |
RESULTS |
Rodents are immature right after birth and exhibit a very poor
motor repertoire. Locomotor activity can be observed, however, when
postural constraints are removed [Cazalets et al. 1990
(swimming experiments); McEwen et al. 1997
(air-stepping
experiments)]. Behavioral data were collected in vivo using mouse
swimming activity as a criterion of locomotor network maturation.
Figure 1 shows that swimming pattern
development is different in C3H and Tg8 strains. At birth (P0-P1),
most of the Tg8 pups presented the H0 pattern (no rhythmic hindlimb
movements); they either floated motionlessly or with smooth
uncoordinated leg movements, or they presented alternated movements of
the forelimbs while their hindlimbs were extended backward. In the same
age class, 23% of the C3H pups already presented the H1 pattern; they
showed alternated rhythmic forelimb movements while one hindlimb (and
only one) moved in synchrony with the contralateral forelimb. The
second hindlimb was maintained extended backward. In the P2-P3 age
class, 75% of the Tg8 pups still presented the H0 pattern whereas 46% of the C3H pups were displaying the H1 pattern and 24% already showed
the H2 pattern, wherein they swam with all four legs and presented
rhythmic and alternated movements of their forelimbs and hindlimbs. In
the P4-P5 age class, 21% of the Tg8 neonates still exhibited the H0
pattern, 36% acquired the H1 pattern, and 43% acquired the H2
pattern. Most of the C3H pups (85%), however, already displayed the H2
pattern. By P6-P7, almost all the pups of the two strains showed the
H2 pattern. Finally, the adult swimming pattern, wherein alternated
rhythmic hindlimb movements are evident while immobile forelimbs are
extended forward, was present by P14 in both strains. To swim, rats and
mice first use only their forelimbs, then both forelimbs and hindlimbs,
and finally only hindlimbs. In C3H and Tg8 pups, statistical treatment
revealed that acquisition of the swimming pattern was age- and
strain-dependent (P < 0.001); Tg8 pups were less
able to use their hindlimbs for swimming than C3H pups up to P6-P7.

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Fig. 1.
Developmental changes of juvenile swimming patterns in control (C3H)
and monoamine oxidase A (MAOA)-deficient (Tg8) pups. For C3H
(A) and Tg8 (B) neonatal mice, the
histograms represent frequency of occurrence of H0, H1, and H2 swimming
patterns (depending on whether pups swam with 0, 1, or 2 hindlimbs) vs.
postnatal day ages (white bars, P0-P1; light gray bars, P2-P3; gray
bars, P4-P5; black bars, P6-P7). Frequency of occurrence of a given
swimming pattern at a given age is expressed as a percentage of the
total number of observations for the given age class. Statistical
comparison of A and B values showed
delayed ability of Tg8 mice to swim with their hindlimbs.
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In vitro experiments were carried out on spinal cord preparations
similar to the one used to analyze locomotor-like activity in neonatal
rats (Cazalets et al. 1992
; Sqalli-Houssaini et
al. 1993
). As in rats, the mouse isolated locomotor network was
quiescent when the spinal cord was superfused with normal saline (no
activity in ventral roots), but could be activated by adding drugs to
the saline, which led to rhythmic motor command of the hindlimbs (Fig. 2). In C3H preparations
(n = 5), 5-HT (5 × 10
5 M)
induced alternating rhythmic bursts of action potentials in the left
and right ventral roots (Fig. 2A1; mean burst
period ± SE, 7.1 ± 0.5 s). In contrast, 5-HT (from
10
6 to 10
3 M) never induced rhythmic or
tonic activity in Tg8 preparations from P1 to P7 (n = 5) (Fig. 2B1). To show that the lack of activity in
the Tg8 spinal cord during 5-HT bath application was not caused by a
general disruption of all neuronal activity, we tested the action of
other transmitters. N-methyl-D, L-aspartate
(NMA, 2 × 10
5 M), an
N-methyl-D-aspartate (NMDA) receptor agonist
known to induce locomotor-like activity in the newborn rat
(Cazalets et al. 1992
), also induced rhythmic bursting
in all the tested C3H preparations (Fig. 2A2;
n = 5) and in three-fifths of the Tg8 preparations
(Fig. 2B2). In the two remaining Tg8 preparations, NMA
only induced tonic discharges in ventral roots. The burst period of the
NMA-induced activity, although irregular, was significantly shorter in
C3H (1.6 ± 0.6 s) than in Tg8 preparations (2.6 ± 0.1 s). In C3H mouse (Fig. 2A3), as in rat
preparations (Sqalli-Houssaini et al. 1993
), adding 5-HT
(5 × 10
5 M) to the NMA-containing saline modified
and improved the motor rhythm. The burst period of the NMA-induced
activity was significantly lengthened (2.5 ± 0.1 s,
n = 5, Fig. 2A3). In contrast, in
three Tg8 preparations, adding 5-HT had no effect over NMA alone (Fig. 2B3, 2.4 ± 0.1 s). Because MAOA deficiency
affects both noradrenaline and 5-HT levels, we tested noradrenaline
effects. In both Tg8 (n = 5) and C3H
(n = 5) preparations, noradrenaline
(10
5 M) (Fig. 2, A4-B4)
induced slow rhythmic discharges in ventral roots. 5-HT (5 × 10
5) was applied in three Tg8 preparations at P11 and
induced ventral root tonic discharges in each preparation. The response
was observed only once at the first trial, however, and after washout
with normal saline could not be elicited again. This suggests that at
least a partial recovery occurred with age for the 5-HT response in
Tg8. Because of the viability of the preparation at this age, however,
it could not be concluded if recovery was complete or not.

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Fig. 2.
Lack of serotonin (5-HT) responses in Tg8 isolated spinal cord.
A: in C3H isolated spinal cords, all the bath-applied
transmitters [A2, 2 × 10 5 M
N-methyl-D, L-aspartate (NMA);
A4, 10 5 M noradrenaline (NA)], including
5-HT (A1, 5 × 10 5 M), elicited
characteristic rhythmic activities in lumbar ventral roots that
alternated between right and left sides. In addition, 5-HT (5 × 10 5 M) affected NMA-induced activity
(A2-A3). B: in Tg8
isolated spinal cords, applications of 2 × 10 5 M
NMA (B2) and 10 5 M NA (B4)
elicited characteristic rhythmic activities in lumbar ventral roots,
but 5-HT applications (5 × 10 5 M) did not induce
rhythmic bursts (B1) and did not affect the NMA-induced
activity (B2-B3). The two spinal cords
in A1 and B1 were recorded simultaneously
in the same bath. The recordings in
B2-B4 were obtained from a single spinal
cord different from B1. Horizontal scale bar, 5 s.
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DISCUSSION |
Both in vivo and in vitro results suggest that MAOA
deficiency transiently delays locomotor network maturation, although it cannot be unambiguously established that the the same neuronal changes
underlie the perturbations observed in vivo and in vitro. Tg8 neonates
swim without their hindlimbs for a longer period than C3H neonates, but
both strains acquire the adult hindlimb swimming pattern by P14. The in
vitro study performed in parallel may partly explain the delayed onset
of hindlimb use in Tg8 swimming. Whereas 5-HT induced and modulated
locomotor-like activity in C3H neonates, it had no effects in Tg8
neonates. MAOA deficiency in Tg8 pups increases both noradrenaline and
5-HT levels by ~50% and 900%, respectively (Cases et al.
1995
; Lajard et al. 1999
). It seems unlikely
that this small noradrenaline change could be responsible for the
maturational differences we observed, although this possibility cannot
be totally rejected because noradrenaline effects persisted in Tg8
preparations. The substantial 5-HT excess in Tg8 pups, which has
already been shown to be responsible for maturational differences in
both the thalamocortical projections (Cases et al. 1996
)
and the central respiratory network (Bou et al.
1998a
,b
), is more likely to be involved. This perinatal 5-HT excess may have induced alterations in 1) the maturation of
the locomotor network itself, 2) the maturation of the
descending central projections, and/or 3) the expression of
spinal 5-HT receptors. As of now, none of these three possibilities can
be rejected. A role of 5-HT in the locomotor network development was
already suggested (Nakajima et al. 1998
) because 5-HT
synthesis blockade by prenatal treatment perturbs both 5-HT spinal
innervation and swimming movements in neonatal rats, with recovery
observed by P20. It is noteworthy that decrease (see Nakajima et
al. 1998
) and increase (present study) in 5-HT levels both
affect the maturation of the rodent locomotor network, suggesting that
normal 5-HT metabolism during gestation is a requisite for normal
maturation of mammalian central networks. Future experiments will be
performed to determine if the delay in maturation can be relieved with
5-HT antagonists administered during development.
The authors thank E. De Maeyer and I. Seif (Orsay, France) for a
gift of C3H and Tg8 mice. We gratefully acknowledge Professor Albert
Berger (Seattle, WA) for a valuable review of the manuscript.
Address for reprint requests: J.-R. Cazalets, CNRS, Laboratoire de
Neurobiologie et Mouvements, 31 Chemin Joseph Aiguier, 13402 Marseille
Cedex 9, France.
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