Rostrocaudal Progression in the Development of Periodic
Spontaneous Activity in Fetal Rat Spinal Motor Circuits In Vitro
Kiyomi
Nakayama,1,2
Hiroshi
Nishimaru,1
Makito
Iizuka,2
Shigeru
Ozaki,1 and
Norio
Kudo1
1Department of Physiology, Institute of
Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8575; and 2Center for Medical Sciences,
Ibaraki Prefectural University of Health Sciences, Ami, Ibaraki
300-0394, Japan
 |
ABSTRACT |
Nakayama, Kiyomi,
Hiroshi Nishimaru,
Makito Iizuka,
Shigeru Ozaki, and
Norio Kudo.
Rostrocaudal progression in the development of periodic spontaneous
activity in fetal rat spinal motor circuits in vitro. Developmental changes in the periodic spontaneous bursts in cervical and lumbar ventral roots (VRs) were investigated using isolated spinal
cord preparations obtained from rat fetuses at embryonic days (E) 13.5-18.5. Spontaneous bursts were
observed in the cervical VR at E13.5-17.5, and in the
lumbar VR at E14.5-17.5. Bursts occurrence in the cervical
and lumbar VRs was correlated in a 1:1 fashion at
E14.5-16.5. The bursts in the cervical VR preceded those in the lumbar VR at E14.5, but the latter came to precede the
former by E16.5. The interval between spontaneous bursts in
the lumbar VR was greatly prolonged after spinal cord transection at
the midthoracic level at E14.5, whereas that in the cervical
VR became significantly longer at E14.5-16.5. These results
suggest that the dominant neuronal circuit initiating the spontaneous
bursts shifts from cervical to lumbar region during this period. Bath application of a glutamate receptor antagonist, kynurenate (4 mM), had
little effect on the spontaneous bursts in either cervical or lumbar
VRs at E14.5-15.5. At E16.5, kynurenate
abolished the spontaneous bursts in the cervical VR. Concomitant
application of kynurenate and strychnine (5 µM), a glycine receptor
antagonist, abolished all spontaneous bursts, suggesting that the major
transmitter mediating the spontaneous bursts changes from glycine to
glutamate in the cervical region by E16.5, but not in the
lumbar region during this period.
 |
INTRODUCTION |
In rats, periodic spontaneous motoneuronal
activity is observed in the lumbar spinal cord during the last week
before birth, a time that coincides with the period during which
formation of the spinal neuronal networks accelerates (Kudo and
Yamada 1987
; Ozaki et al. 1996
). This
spontaneous activity is likely to be generated at this time as a result
of the temporary excitatory effects of glycine and
-aminobutyric
acid (GABA) (Nishimaru et al. 1996
), suggesting that the
spontaneous activity might be playing an important role in the
activity-dependent development of the neuronal network. Moreover, this
activity may be responsible for the spontaneous limb movements observed
at the same period in utero (Narayanan et al. 1971
).
However, the developmental profile and the nature of the correlation
between forelimb and hindlimb activity both remain unclear. Our present
results demonstrate that as early as embryonic day 14.5 (E14.5) independent neuronal mechanisms in the cervical and
lumbar regions serve to evoke periodic spontaneous motoneuronal
activity and that these mechanisms are highly interactive. Parts of
this study have been published in abstract form (Ozaki et al.
1990
).
 |
METHODS |
Isolated spinal cord preparations from rat fetuses aged
E13.5-18.5 were obtained as previously described
(Nishimaru et al. 1996
). The ventral roots (VRs) from
the C6-C8 and L2-L5
segments were cut and incorporated into glass suction electrodes. The
preparation was placed in an experimental chamber perfused with normal
Krebs solution (composition in mM: 118.4 NaCl, 4.69 KCl, 2.52 CaCl2, 1.25 MgSO4, 25.0 NaHCO3,
1.18 KH2PO4, and 11.1 glucose) saturated with
95% O2-5% CO2 (pH 7.3-7.4 at room
temperature). In 17 preparations, the isolated spinal cord was further
transected at the thoracic level during the course of the experiment.
The drugs kynurenate (Sigma) and strychnine (Wako Chemicals) were
applied by addition to the perfusate. Electrical activity in the VRs
was amplified using AC-coupled amplifiers (gain: 10 k, band-pass
filter: 15 Hz to 3 kHz; Nihon Kohden). Parts of the records were
integrated with a time constant of 0.5 s. The burst duration and
the interval between spontaneous bursts were measured over 10-20
cycles and are given as means ± SD. The significance of
differences was determined using a Student's t-test.
 |
RESULTS |
Motoneuronal activity was recorded in cervical and lumbar VRs
using isolated spinal cords from E13.5-18.5 rat fetuses
(n = 67). Periodic spontaneous bursts could be recorded
in the cervical VR as early as E13.5 (n = 4;
Fig. 1Aa), but they were first
recorded in the lumbar VR at E14.5. Such activity showed a
1:1 correlation between cervical and lumbar VRs in all preparations
examined at E14.5 (n = 16) and at
E15.5 (n = 16), and in 19 of 22 preparations at E16.5 (Fig. 1, Ab and Ac),
indicating a degree of temporal synchronization between the two
regions. In fact, the activity in the cervical VR tended to precede
that in the lumbar VR at E14.5 (Fig. 1, Ba and
Bd). However, by E16.5, most bursts of VR activity in the lumbar region preceded the corresponding burst in the
cervical region (Fig. 1, Bc and Bd). The temporal
relationship between the bursts in cervical and lumbar VRs was less
consistent at E15.5 (Fig. 1, Bb and
Bd). The mean delay between the activity in the cervical VR
and that in the lumbar VR was 1.6 ± 1.1 (SD) s at
E14.5 (n = 6), 0.5 ± 1.6 s at
E15.5 (n = 6) and
2.4 ± 1.8 s
at E16.5 (n = 6). Some of the bursts in the
lumbar VR failed to entrain bursts in the cervical VR in 3 of 22 preparations at E16.5 and in 3 of 5 preparations at
E17.5 (Fig. 1Ad). In two preparations at
E17.5 and in all preparations at E18.5
(n = 4; Fig. 1Ae), no periodic spontaneous
bursts were observed in either VR. These results suggest 1)
that neuronal connections exist to coactivate the motoneurons
periodically in these two limb-innervating regions during this period
of development and 2) the spontaneous burst starts from the
rostral region at early stages and from the caudal region at later
stages.

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Fig. 1.
Developmental changes in spontaneous bursts in cervical and lumbar
ventral roots (VRs). A: integrated records of
spontaneous bursts in the cervical (top trace) and
lumbar (bottom trace) VRs at different ages.
B: faster sweeps of single spontaneous bursts in the
cervical and lumbar VRs at E14.5 (a),
E15.5 (b), and E16.5
(c). The time lag (d) represents the time
difference between the onset of cervical burst activity and the onset
of lumbar burst activity. Each symbol indicates the mean time lag in an
individual preparation at E14.5 ( ),
E15.5 ( ), or E16.5
( ), with the vertical bar indicating SD.
|
|
To examine the relative ability of the cervical and lumbar spinal cord
to initiate spontaneous bursts, the spinal cord was transected at the
midthoracic level (T7-T8) at
E14.5-16.5. Spontaneous bursts could still be
observed in both cervical and lumbar VRs after this transection in all
preparations (Fig. 2). The effects of the
transection on burst interval and burst duration are summarized in
Table 1. The interval between spontaneous
bursts was significantly (P < 0.05) prolonged in the
cervical and lumbar VRs by such transection at E14.5 (Fig.
2A; Table 1). Moreover, the interval between spontaneous bursts became longer in the lumbar VR than in the cervical VR (P < 0.05). At E15.5, the transection had
differential effects: it prolonged the burst interval in the cervical
VR, but did only slightly and nonsignificantly affected the lumbar VR
burst interval (Fig. 2B; Table 1). After transection at
E16.5, the interval between the spontaneous bursts recorded
from the cervical VR was highly variable, and the mean interval value
was significantly increased (P < 0.05, Fig.
2C; Table 1). At this stage, the spontaneous bursts recorded
from the lumbar VR were not affected at all.

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Fig. 2.
Effects of transection of the spinal cord at the
T7-T8 segment. Spontaneous bursts before
(a) and after (b) such transection at
E14.5 (A), E15.5
(B), and E16.5 (C).
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Table 1.
Interval and burst duration for spontaneous bursts recorded before and
after spinal cord transection at T7-T8
|
|
The duration of the individual bursts was little affected by
transection at E14.5-15.5 (Table 1). At
E16.5, before transection the correlated spontaneous bursts
had a variable time course with some having more than two peaks and a
long duration of >40 s (Fig. 2C). However, such bursts were
found only in the cervical VR after the transection, whereas the burst
duration became less variable in the lumbar VR. The results of the
transection experiment indicate that both the cervical and lumbar
regions of the spinal cord are capable of generating spontaneous
activity during this period of development.
We examined the effect of the broad-spectrum glutamate receptor
antagonist, kynurenate (4 mM), on the correlated spontaneous bursts in
the cervical and lumbar VRs at E14.5-16.5. Bath application of this antagonist failed to abolish the spontaneous bursts recorded from the cervical and lumbar VRs at E14.5 (n = 7; Fig. 3A) and E15.5 (n = 5). In contrast, at
E16.5 the spontaneous bursts in the cervical VR were
abolished by kynurenate in 11 of 12 preparations. The spontaneous
bursts reappeared after 7-25 min (while kynurenate was still present)
in 7 of 11 preparations (Fig. 3B). In the lumbar VR,
however, the spontaneous bursts persisted in the presence of kynurenate
in all 12 preparations examined. Spontaneous bursts with variable time
course and long duration were not observed in the lumbar VR when
glutamate receptors were blocked. Bath application of kynurenate
together with the glycine receptor antagonist, strychnine (5 µM),
blocked the spontaneous bursts in the cervical and lumbar VRs at all
embryonic days examined (n = 5 at E14.5;
n = 4 at E15.5; n = 8 at
E16.5; Fig. 3, A and B). These results
indicate that glutamate becomes the dominant transmitter responsible
for generating spontaneous bursts in the cervical spinal cord by
E16.5, while glutamate becomes dominant in the lumbar spinal
cord at E17.5 (Nishimaru et al. 1996
). It is
thus likely that neuronal circuits generating periodic spontaneous
bursts develop earlier in the rostral than in the caudal parts of the
spinal cord.

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Fig. 3.
Effects of kynurenate and strychnine on spontaneous bursts. VR
discharges during bath application of 4 mM kynurenate (represented by a
white horizontal bar) or 4 mM kynurenate plus 5 µM strychnine
(represented by a black horizontal bar) at E14.5
(A) and E16.5 (B). Records
obtained after 30 min of washing are shown on the
right.
|
|
 |
DISCUSSION |
The present study has revealed correlated spontaneous burst
activity in the cervical and lumbar VRs in rat fetuses at and after
E14.5. Periodic spontaneous body movements having a close correlation between forelimb and hindlimb are observed during corresponding developmental stages in mouse fetuses, i.e., at and after
E12.5 (Suzue 1996
). The duration of each
episode and the interval between episodes, as well as the delay between
the onset of forelimb and hindlimb movements, are in a similar range to
the corresponding values obtained for the spontaneous burst activity
recorded in the present study. This is consistent with the idea that
the neuronal activity described in this study may generate the periodic
spontaneous body movement shown by the fetus in utero.
The present study shows that the location of the neuronal circuit
initiating the periodic spontaneous bursts shifts during development
from the cervical to the lumbar region in the fetal rat spinal cord, as
is the case for the neurogenesis of motoneurons (Nornes and Das
1974
) and for the formation of cutaneous reflexes (Narayanan et al. 1971
). The results of this study
suggest that at E14.5 the major excitatory drive for such
neuronal circuits is likely to be provided by glycine-mediated synapses
throughout the whole spinal cord, although spontaneous bursts are
typically initiated in the cervical region. At E16.5,
however, the dominant excitatory transmitter has become glutamate in
the cervical region, although it is still glycine in the lumbar region.
The ability to initiate the spontaneous motor activity was greater in
the lumbar spinal cord at this stage. Thus it is suggested that the immature neuronal circuits, which contain mainly glycine-mediated excitatory synapses, are more excitable than those in the region in
which glutamate has become the dominant excitatory transmitter. These
immature neuronal circuits may promote the rostrocaudal development of
spinal neuronal networks, perhaps by supporting neuronal differentiation.
A recent study in the neonatal rat spinal cord has shown that
strychnine can antagonize the action of GABA (Jonas et al.
1998
). However, GABAA receptors are likely to be
less involved in the excitatory pathways underlying the present type of
spontaneous activity because concomitant application of kynurenate and
bicuculline (20-40 µM), a GABAA receptor antagonist,
does not abolish the spontaneous bursts in the cervical
(n = 4 at E14.5; K. Nakayama, unpublished
observation) or lumbar cord (Nishimaru et al. 1996
).
 |
ACKNOWLEDGMENTS |
The authors thank A. Ohgami for technical assistance.
This study was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science and Culture of Japan
(project No. 10156205; to N. Kudo) and by the Human Frontier Science
Program (N. Kudo).
 |
FOOTNOTES |
Address for reprint requests: H. Nishimaru, Dept. of Physiology,
Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba,
Ibaraki 305-8575, Japan.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Received 14 December 1998; accepted in final form 22 January 1999.
 |
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