School of Biology, Gatty Marine Laboratory, University of St. Andrews, St. Andrews, Fife KY16 8LB, Scotland
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ABSTRACT |
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Reith, Carolyn A. and
Keith T. Sillar.
Development and Role of GABAA Receptor-Mediated
Synaptic Potentials During Swimming in Postembryonic Xenopus
laevis Tadpoles.
J. Neurophysiol. 82: 3175-3187, 1999.
We have investigated the
contribution of GABAA receptor activation to swimming in
Xenopus tadpoles during the first day of postembryonic
development. Around the time of hatching stage (37/8), bicuculline
(10-50 µM) causes a decrease in swim episode duration and cycle
period, suggesting that GABAA receptor activation
influences embryonic swimming. Twenty-four hours later, at stage 42, GABAA receptor activation plays a more pronounced role in
modulating larval swimming activity. Bicuculline causes short, intense
swim episodes with increased burst durations and decreased cycle
periods and rostrocaudal delays. Conversely, the allosteric agonist,
5-pregnan-3
-ol-20-one (1-10 µM) or the uptake inhibitor,
nipecotic acid (200 µM) cause slow swimming with reduced
burst durations and increased cycle periods. These effects
appear to be mainly the result of GABA release from the spinal
terminals of midhindbrain reticulospinal neurons but may also involve
spinal GABAergic neurons. Intracellular recordings were made using KCl
electrodes to reverse the sign and enhance the amplitude of
chloride-dependent inhibitory postsynaptic potentials (IPSPs).
Recordings from larval motoneurons in the presence of strychnine (1-5
µM), to block glycinergic IPSPs, provided no evidence for any
GABAergic component to midcycle inhibition. GABA potentials were
observed during episodes, but they were not phase-locked to the
swimming rhythm. Bicuculline (10-50 µM) abolished these sporadic
potentials and caused an apparent decrease in the level of tonic
depolarization during swimming activity and an increase in spike
height. Finally, in most larval preparations, GABA potentials were
observed at the termination of swimming. In combination with the other
evidence, our data suggest that midhindbrain reticulospinal neurons
become involved in an intrinsic pathway that can prematurely terminate
swim episodes. Thus during the first day of larval development,
endogenous activation of GABAA receptors plays an
increasingly important role in modulating locomotion, and GABAergic
neurons become involved in an intrinsic descending pathway for
terminating swim episodes.
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INTRODUCTION |
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Although much is known about glycine's role in
mediating reciprocal inhibition between antagonistic spinal motor pools
in a range of vertebrates (e.g., Alford and Williams
1987; Buchanan 1982
; Cohen and
Harris-Warrick 1984
; Dale 1985
; Soffe
1987
), the contribution of GABA to rhythm generation is less
clear. Nevertheless, the widespread distribution of GABA throughout the
brain and spinal cord suggests important functional roles for GABAergic
transmission during locomotor activity. In the lamprey, GABA has been
implicated in modulating burst rate and intersegmental coordination,
with GABAB receptors affecting the magnitude and
GABAA receptors reducing the variability of the
phase lag between segmental oscillators (Tégner et al.
1993
). In the neonatal rat, both GABAA
and GABAB receptor blockade increases swimming
frequency, but only GABAA receptor activation
affects burst amplitude (Cazalets et al. 1994
). More
recent experiments on the neonatal rat preparation showed that either
strychnine or bicuculline could abolish ipsilateral flexor/extensor
alternation and disrupt left-right coordination, suggesting that the
coactivation of GABAA and glycine receptors may
be important in coordinating activity between antagonistic motor pools
(Cowley and Schmidt 1995
).
During fictive swimming in immobilized Xenopus embryos
(stage 37/8) (Nieuwkoop and Faber 1956), ventral root
activity consists of simple biphasic impulses that alternate across the
body and progress caudally with a brief intersegmental delay (for
review, see Roberts 1990
). The left/right alternation
appears to be mediated solely by glycinergic spinal commissural
interneurons (Dale 1985
; Soffe 1987
).
There is no evidence that GABAA receptor
activation contributes significantly to rhythm generation at this early
stage because fictive swimming is apparently unaffected by either
bicuculline (40 µM) or curare (100 µM), two
GABAA receptor antagonists in this preparation
(Soffe 1987
). However, motoneurons do possess GABAA receptors (Soffe 1987
) and
GABA-immunoreactive neurons are present in the embryonic brain and
spinal cord (Roberts et al. 1987
). So far, a role for
only one population has been described. Gentle pressure applied to the
rostral cement gland causes swimming to prematurely terminate,
coincident with a train of bicuculline-sensitive potentials recorded
intracellularly in embryonic motoneurons (Boothby and Roberts
1992a
). Cement gland afferents are thought to activate GABAergic midhindbrain reticulospinal (mhr) neurons whose axons descend
and terminate within the spinal cord (Boothby and Roberts 1992b
). GABAB receptor agonists have been
shown to alter spike threshold and presynaptically modulate the
strength of reciprocal glycinergic inhibition, but the circumstances
under which these effects are brought into play during swimming are
unclear (Wall and Dale 1993
).
Twenty-four hours later (at stage 42) fictive swimming activity is more
complex and flexible (Sillar et al. 1991,
1992
). Given that GABA receptors play an important role
during locomotor activity in other vertebrate preparations
(Cazalets et al. 1994
; Tégner et al.
1993
), it is conceivable that GABAergic neurons may become involved in the control of larval swimming. Thus in the present study
we focused on the possible contribution made by
GABAA receptor activation to swimming at larval
stage 42 compared with embryonic stage 37/8. Aspects of this work have
been published previously in abstract form (Reith and Sillar
1995
).
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METHODS |
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Experiments were carried out on stage 37/8 embryos and stage 42 larvae of the South African clawed frog, Xenopus laevis
(Nieuwkoop and Faber 1956), which were obtained by
induced breeding following injection of human chorionic gonadotrophin
(1,000 U/ml, Sigma) into pairs of adults from a laboratory colony.
Tadpoles were immobilized in the neuromuscular blocking agent,
-bungarotoxin (1.25 µM, Sigma) and then transferred to a
preparation bath (~2 ml volume) containing frog Ringer of the
following ion composition (in mM): 115 NaCl, 2.5 KCl, 2.5 NaHCO3, 10 HEPES, 1 MgCl2,
and 2 or 4 CaCl2 (2 mM for extracellular
experiments and 4 mM for intracellular experiments); buffered
to pH 7.4 with 1 N NaOH. The saline was continuously recirculated,
being gravity fed from a stock bottle containing 100 ml of saline and
returned by means of a peristaltic pump.
The animals were secured on their right side through the notocord,
using fine pins etched from tungsten wire, to the silicone elastomer
(Sylgard) surface of a rotating Perspex table located in the
preparation bath. After removal of the flank skin, on the left side,
from around the level of the anus to the otic capsule, extracellular
recordings of ventral root activity were made by placing glass suction
electrodes (~50 µM tip opening) in the exposed intermyotomal clefts
wherein lie the axons of spinal motoneurons. The position of each
electrode was noted as its distance in clefts numbered sequentially
from the otic capsule. Fictive swimming activity was initiated either
by dimming the illumination or by applying brief (0.5-1 ms) current
pulses (using a Digitimer DS2 isolated stimulator) to the tail skin via
a glass suction electrode. In three experiments a second stimulating
electrode was positioned on the cement gland of embryo preparations,
and similar stimuli were used to terminate swimming (cf. Boothby
and Roberts 1992a). For intracellular recordings, a rostral
section of myotomes was removed, using tungsten needles, to reveal the
underlying spinal cord. Recordings were then made using glass
microelectrodes pulled on a Campden Instruments moving coil
microelectrode puller (model 753) or a P2000 laser puller (Sutter
Instruments) from filamented borosilicate glass capillary tubes (1 mm
OD). The electrodes were filled with 2 M KCl and had resistances of
80-130 M
. KCl electrodes were chosen because the leakage of
chloride ions into the interior of these small neurons causes
chloride-dependent inhibitory postsynaptic potentials (IPSPs) to be
massively reversed in sign so that they become large and depolarizing.
This greatly facilitates the detection and analysis of GABAergic and
glycinergic IPSPs. In addition, depolarizing GABA IPSPs can be readily
distinguished from glycinergic ones on the basis of their duration and
their sensitivity to selective agonists and antagonists (Reith
and Sillar 1997
). Penetrations were made in the ventral portion
of the spinal cord using capacity overcompensation. The recorded cells
were rhythmically active during fictive swimming and were assumed to be
motoneurons because the ventral portion of the cord is known to consist
almost entirely of cells of that type (Roberts and Clarke
1982
). Electrophysiological data were recorded and stored onto
video tape using a VR100B digital recorded (Instrutech, New York,
NY) and analyzed off-line either manually or using the "Spike
2" analysis software package (CED, Cambridge, UK).
Pharmacological agents were bath applied to the perfusate by adding
known quantities to the stock bottle to achieve the desired final
concentration. The drugs used in this study were strychnine (1-5 µM,
Sigma), bicuculline (10-50 µM, Sigma), and
5-pregnan-3
-ol-20-one (5
3
; 1-5 µM, Sigma). The duration
of fictive swim episodes was averaged over five consecutive episodes
evoked at 5-min intervals recorded under each experimental condition
from five similar experiments. For each experiment, measurements were
made of rostral and caudal burst durations; cycle period, which was
measured from the onset of one burst to the onset of the next and
rostrocaudal delay, measured from the start of the rostral burst to the
start of the caudal burst. The first 500 ms of activity in each episode
was ignored to avoid possible influences arising directly from sensory stimulation. Averages were calculated, for each parameter, from a total
of 60 measurements (20 measurements from each of 3 different episodes
under each condition). In the case of spinalization experiments, where
episodes were <20 cycles long, more episodes were measured to obtain a
total of 60 cycles. Averages are given as means ± SE.
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RESULTS |
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Comparison of the effects of bicuculline on swimming at stage 37/8 and stage 42 activity
In Xenopus embryos (stage 37/8, Fig.
1A1) and
larvae (stage 42, Fig. 1B1), episodes of sustained swimming
activity lasting many seconds are initiated following a brief
electrical stimulus to the tail skin. At each stage the coordination of
swimming activity is similar, but larval swimming comprises much
longer, more complex ventral root bursts than those seen in the embryo
(Sillar et al. 1991). We compared the effects of
bicuculline (10-50 µM) on four parameters of fictive swimming
(episode length, rostral and caudal burst duration, cycle period, and
rostrocaudal delay) at these two developmental stages. At both stages,
bicuculline caused a reversible reduction in the duration of swim
episodes evoked in response to stimulation of the skin (Fig. 1,
A2 and B2). Figure 1, A3 and
B3, compares the effect of 50 µM bicuculline on episode lengths in five consecutive episodes from five different embryo and
five different larval experiments. Under control conditions the average
episode length was similar at each stage: 40.1 ± 7.1 s and
40.6 ± 7.8 s (mean ± SE) for the embryonic and larval
preparations, respectively. In the presence of 50 µM bicuculline, the
reduction in episode duration was much larger in larval preparations
than in embryos. In embryos, episodes decreased by ~50% to 21.7 ± 6.2 s, whereas the larval episodes in the presence of
bicuculline were reduced to only 3.4 ± 0.8 s, ~8% of
control durations. The effects of bicuculline were reversible because
returning to control saline increased the length of episodes again to
49.8 ± 13.6 s for embryo and 17.1 ± 3.2 s for
larval preparations. These data suggest that endogenous activation of
GABAA receptors has a marked influence on the
duration of swim episodes, with the effect becoming much more
pronounced by stage 42.
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Bicuculline had only a relatively small effect on other parameters of embryonic swimming activity (n = 5). Excerpts of swimming activity before and after the addition of bicuculline are shown for one typical experiment in Fig. 2A, where the only obvious change is a small decrease in cycle periods (Fig. 2, A1 cf. A2). The data graphed in Fig. 2A3 show that cycle periods were indeed significantly (P < 0.001) reduced from 53.9 ± 0.8 ms to 48.5 ± 0.6 ms, 10 min after the bath application of 50 µM bicuculline, whereas burst durations and rostrocaudal delays were not significantly affected (t-test, P > 0.01, not shown). Thus at stage 37/8, the frequency of fictive swimming was the only other parameter to be affected significantly after GABAA receptor blockade in each of five experiments (t-test, P < 0.01). When the data from these five experiments are pooled, embryo cycle periods decreased on average by 9.7% (range 7.0-12.6%). This change, accompanied with the decrease in swim episodes suggests that GABAA receptor activation plays some role in controlling the output of the central pattern generator for embryonic swimming. More specifically, the fact that blockade of GABAA receptor-mediated inhibition reduced the duration of swim episodes implies that the maintenance of rhythmic activity is somehow facilitated by endogenous GABAergic influences.
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In contrast to the situation just described for embryos, by larval
stage 42, the marked decrease in swim episodes by bicuculline (Fig. 1,
B2 and B3) is accompanied by significant changes
in all parameters of the swimming motor pattern. The example in Fig. 2B clearly shows that bath application of 50 µM
bicuculline results in fast, intense activity. Like the embryo, cycle
periods decreased, from 52.8 ± 0.6 ms to 45.6 ± 1.1 ms
(Fig. 2B3). This decrease in cycle periods under bicuculline
was significant in 10 of 11 preparations examined (t-tests;
P < 0.01) where on average cycle period were reduced
by 11.6% (range 5.5-27.8%). In addition, rostral burst durations
increased by 15.6% from 14.7 ± 0.4 ms to 17.0 ± 0.5 ms
(Fig. 2C1), whereas caudal burst durations also increased by
>20%, from 12.8 ± 0.3 ms to 15.6 ± 0.4 ms (Fig.
2C2). In pooled data from 9 of 11 experiments where
bicuculline significantly increased burst durations the average effect
was 18% (range 6.3-54%). In addition, rostrocaudal delays decreased
from 8.0 ± 0.2 ms to 4.9 ± 0.3 ms under bicuculline (Fig.
2C3). In contrast to the situation in embryos, rostrocaudal
delays in larvae vary with cycle periods so that long cycle periods are
accompanied by long delays and vice versa (Tunstall and Sillar
1993). Hence the decrease in delays under bicuculline could be
produced simply as a consequence of the decrease in cycle periods.
Figure 2D shows plots of rostrocaudal delays against cycle
periods, over whole episodes before and during GABAA receptor blockade. It can be seen that
bicuculline causes short cycle periods that are associated with
rostrocaudal delays shorter than those measured under control
conditions. However, in the range of cycle periods that overlap in
control conditions and under bicuculline (40-80 ms), delays under
bicuculline declined as a proportion of cycle period from 13.8 ± 0.4% (n = 104 cycles) to 10.7 ± 0.6%
(n = 47 cycles). Thus GABA may affect intersegmental coordination in ways other than purely through changing cycle periods.
The preceding results suggest that the endogenous activation of
GABAA receptors has a profound influence on
larval swimming activity by directly affecting the excitability of the
central pattern generator. If this is the case and the results are not due to some nonspecific effect of bicuculline, then agents that potentiate GABAA receptor activation would be
expected to have the opposite effect to bicuculline on larval activity.
We therefore used the neurosteroid, 5-pregnan-3
-ol-20-one
(5
3
), a potent allosteric modulator of the
GABAA receptor. We have previously shown that
5
3
is a specific agonist at the GABAA
receptor in Xenopus tadpoles, which enhances responses to
endogenous GABA release in this preparation (Reith and Sillar
1997
). 5
3
(1-10 µM) had consistent and significant
effects on larval ventral root activity (n = 5), which
are illustrated in Fig. 3. 5
3
(5 µM) caused a decrease in episode length as shown in the example in Fig. 3A, where episode length was reduced from 48.0 ± 15.0 s (A1) to 16.1 ± 6.4 s (A2).
Subsequent application of 20 µM bicuculline further reduced episodes
to an average of 4.3 ± 1.4 s (A3). Thus like the
experiments described earlier for bicuculline alone (i.e., Fig. 1),
bicuculline in the presence of steroid still caused episodes to
decrease in duration rather than reversing the effect of the steroid
(see DISCUSSION). In contrast to the antagonist, however, 5
3
caused the swimming pattern to become slow and weak (Fig. 3,
2 cf. B1) an effect that was reversed by the
subsequent bath application of bicuculline (not shown). The steroid
significantly (P < 0.001) decreased the average
rostral burst duration from 15.0 ± 0.4 ms to 8.3 ± 0.3 ms
(Fig. 3C1) and the average caudal burst duration from
11.4 ± 0.3 ms to 8.6 ± 0.2 ms (Fig. 3C2). The
frequency of swimming was also significantly reduced, with average
cycle periods increasing from 49.6 ± 0.7 ms to 67.8 ± 0.7 ms (Fig. 3C3). This was accompanied by an increase in the magnitude of the rostrocaudal delays from 10.4 ± 0.3 ms to
20.0 ± 0.2 ms (Fig. 3C4). Plots of delay against cycle
period showed that, although shorter cycle periods with shorter delays
were only seen under control conditions, at similar cycle periods, the
steroid still increased delays to a greater extent than those seen
under control (Fig. 3D). Again, as suggested by bicuculline experiments described in Fig. 2, delays appear to be directly affected
by GABA neurotransmission. Similar experiments were also carried out
with the GABA uptake inhibitor, nipecotic acid (200 µM). Figure 3,
E1 and E2, shows that nipecotic acid (200 µM)
produces a slow, weak swimming rhythm, similar to that recorded in the presence of steroid (n = 5). Moreover, in the majority
of experiments (3 of 5), nipecotic acid, like 5
3
, reduced episode
lengths (not illustrated).
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Because the actions of bicuculline, nipecotic acid, and 53
all
rely on an endogenous source of GABA to exert their effects, the main
conclusion from these initial ventral root experiments is that by stage
42, an intrinsic GABAergic system is capable of significantly
influencing the output of the central pattern generator for swimming.
The main effect of GABAA receptor activation is
to reduce the frequency, intensity, and duration of swimming, although,
paradoxically, the block of GABAA receptors also
produces short swim episodes (see DISCUSSION).
Effects of bicuculline and 53
in the presence of strychnine
To determine more precisely the role of synaptic inhibition mediated by GABAA receptor activation alone during rhythmic swimming activity, the next set of experiments was carried out after first blocking glycinergic inhibition with strychnine. Blocking both types of inhibition (with 1-5 µM strychnine and 20-50 µM bicuculline) revealed another developmental difference between embryonic and larval preparations. Figure 4 shows an example of the effects of simultaneously blocking glycine and GABAA receptors at each stage. Following bath application of 5 µM strychnine to the embryo, episode length was largely unaffected, although there was a slight increase in swimming frequency (Fig. 4, A2 cf. A1 and A5). Subsequent bath application of 50 µM bicuculline in the presence of strychnine elicited an intense nonrhythmic burst before the onset of each swim episode (Fig. 4A3). This suggests that GABAA receptors are normally activated in response to skin stimulation. The sustained swimming activity that followed the initial nonrhythmic burst showed a further increase in frequency (Fig. 4, A3 and A5). The effects of strychnine are not so easily reversible as those of bicuculline, but returning to control saline did abolish the burst of activity at the beginning of the episode and reduce the swim frequency (Fig. 4, A4 and A5).
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By stage 42, a very different picture emerges as shown in Fig.
4B. Following the bath application of either strychnine
alone (1-5 µM, Fig. 4B2) or bicuculline (20-50 µM,
e.g., Fig. 2B2), rhythmic ventral root activity is still
recorded. Like bicuculline, strychnine causes an increase in burst
durations and a decrease in cycle periods (Fig. 4, 2 cf.
B1 and B5) but with an increase rather than a
decrease in episode length (Fig. 41 cf. Fig.
1B2). However, following the coapplication of bicuculline
and strychnine (B3) essentially all rhythmic activity is
abolished, and only an intense nonrhythmic burst is normally recorded
on the ventral roots. In a few cases this nonrhythmic burst terminated
with a few cycles of rhythm (e.g., arrowed in Fig. 4B3).
Although the activity immediately after electrical stimulation of the
skin is nonrhythmic, a rostrocaudal delay, like that seen during
swimming, was still observed (not shown), indicating that the motor
system is not engaging in fictive struggling; this alternative motor pattern is characterized by a marked increase in burst durations but is
accompanied by a reversal of the intersegmental delay to caudorostral
(Soffe 1991). Rhythmic activity returned following wash
in control saline (4, B4 and B5). Thus by stage
42, a certain amount of inhibition seems to be required for the central
pattern generator to produce rhythmic ventral root activity.
Enhancing GABA inhibition, in the presence of strychnine, with
53
, resulted in similar effects to those already reported for the
steroid alone (see above). Figure
5 shows an example of the consistent
effects of the steroid in the absence of glycinergic inhibition
(n = 5). Application of 5
3
in the presence of
strychnine weakens swimming (Figs. 5, A3 cf.
A2 and A1). Although 2 µM strychnine increased rostral burst durations from 13.8 ± 0.3 ms to 20.7 ± 0.6 ms, 5 µM 5
3
in the presence of strychnine decreased
average burst durations to 12.1 ± 0.4 ms (Fig.
5B1). Similarly, caudal burst durations increased from
10.1 ± 0.3 ms to 15.2 ± 0.5 ms under strychnine and
decreased to 8.3 ± 0.2 ms after the bath application of 5
3
(Fig. 5B2). This decrease in the intensity of bursts by
the steroid was paralleled by a slowing of the rhythm because cycle
periods increased from 42.0 ± 0.6 ms in strychnine to 53.2 ± 0.5 ms in the presence of both agents (Fig. 5B3).
Rostrocaudal delays similarly increased from 14.7 ± 0.3 ms to
18.6 ± 0.3 ms (Fig. 5B4). These effects of the
steroid in the presence of strychnine are consistent with there being a
prominent GABAA receptor-mediated input during fictive
swimming, which helps to maintain swimming activity even in the absence
of glycinergic inhibition. The next topic to be investigated addresses
the location of the GABAergic neurons that influence swimming.
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Possible role for the midhindbrain reticulospinal neurons during larval swimming activity
Of the eight populations of neurons that show GABA
immunoreactivity at stage 37/8 (Roberts et al. 1987),
two populations are located in the spinal cord, three populations in
the hindbrain, and the remaining three populations are located in the
mid- and forebrain. The most caudal of the hindbrain neurons are the
mhr neurons that lie ~200 µm caudal to the otic capsule.
The vestibular complex neurons are located at the level of the otic
capsule, and the third, rostral hindbrain population lie just rostral
to the entry of the trigeminal nerve. Immunocytochemical studies at
larval stage 42 indicate that the same populations of GABAergic neurons
are present, and no new populations have developed compared with the
situation at stage 37/8 (Reith 1996
). Transecting the brain at the level of the otic capsule ("transected" preparation, Fig. 6A)
should therefore leave only the mhr neurons and two populations of
spinal GABA neurons intact. The essential components of the rhythm
generator for swimming are located in the spinal cord, so at this level
of transection, larval preparations can still sustain episodes of
swimming activity. Figure 6B shows activity from a
transected preparation before and after the addition of bicuculline.
Bicuculline (50 µM) caused the usual reversible decrease in episode
length, which in the example in Fig. 6B decreased from an average of 21.9 ± 3.0 s in control to 1.0 ± 0.01 s in the presence of bicuculline and then increased again to
8.3 ± 0.5 s after returning to control saline. The
histograms in Fig. 6C also confirm that the antagonist
still causes the typical increase in burst durations (Fig. 6,
C1 and C2) and decrease in cycle periods
and rostrocaudal delays (Fig. 6, C3 and
C4). These data suggest that the population of neurons
that plays a role in modulating larval swimming is located below the
level of the otic capsule, and therefore either the mhr or the spinal
populations (or both) are involved.
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Spinalizing preparations at the level of the fifth postotic myotome
("spinalized" preparation, Fig. 6A) removes the
influence of all but the two populations of spinal GABAergic neurons.
At this level of spinalization, however, the preparation can only produce very short bouts of swimming presumably because a proportion of
the descending excitatory interneuron pool has also been removed (cf.
Roberts and Alford 1986). Hence, in these experiments,
low doses (10-20 µM) of
N-methyl-D-aspartate (NMDA), insufficient to
trigger swimming on their own, were applied to raise the levels of
excitation and thus increase episode durations following skin stimulation. Bicuculline was found to have only small and inconsistent effects on burst durations and rostrocaudal delay that were often not
reversible. As in the intact and transected preparations, however,
there was a tendency for the length of episodes to be reversibly
reduced by bicuculline (5 of 7 experiments). An example of the effect
on episode length is shown in Fig. 6D, where bicuculline was washed on to the preparation twice (only the 1st application is
shown) and on each occasion caused a decrease in episode length. The
average episode length in control was 2.2 ± 0.1 s, reduced to 1.1 ± 0.05 s in 50 µM bicuculline and then increased to
3.7 ± 0.7 s after returning to control saline. Reapplying
the bicuculline reduced episode lengths again to 1.3 ± 0.07 s (not illustrated). The other most consistent change in the ventral
root activity of the spinalized animal was a small but significant
decrease in cycle period (5 of 7 experiments), which decreased from
67.8 ± 1.4 ms in control to 62.7 ± 1.2 ms in 50 µM
bicuculline and increased again to 70.0 ± 0.9 ms after washing
off the bicuculline (Fig. 6E3). However, although the
decrease in cycle period was significant, it was not as marked as the
effect of bicuculline on the swimming frequency of intact or otic
capsule transected preparations (Figs. 2B and
6C3). The other parameters of swimming were not
consistently or significantly changed by bicuculline in the spinalized
preparation (Fig. 6, E1, E2, and E4).
These results suggest that there is only a small contribution to
swimming from the spinal GABA populations, although if these neurons
were normally excited by descending systems then this will be removed following spinalization. It is notable that episode length and cycle
period are the same two parameters that are affected by bicuculline at
stage 37/8, suggesting that embryo swimming might be influenced by
spinal GABAergic neurons.
The main conclusion, therefore from these spinalization experiments is
that by stage 42, an important GABA input to the spinal swimming
circuitry comes from a population of neurons located in the caudal
hindbrain, most probably the mhr neurons described by Roberts et
al. (1987).
GABAergic input during rhythm generation
Thus far we have described a developmental increase in the
contribution of GABAergic transmission to swimming during early postembryonic development. The next question we addressed was when
during swimming are GABAA receptors activated?
Intracellular recordings from ventrally positioned spinal neurons,
presumed motoneurons, were made to determine the timing of GABAergic
IPSPs. To facilitate this goal, glycinergic transmission was first
blocked with strychnine, and 2 M KCl was used as the microelectrode
electrolyte because this has the effect of reversing and enhancing
chloride dependent potentials, including GABA-mediated IPSPs
(Reith and Sillar 1997). Figure
7 shows an episode of swimming recorded
from a larval motoneuron (top traces, MN) with an
accompanying ventral root monitor (bottom traces, vr) before
strychnine application (A1). The expanded trace
(A2) shows more clearly the alternating pattern of on-cycle
excitation, leading to action potentials, in time with the ipsilateral
ventral root, and depolarizing (reversed) midcycle inhibition,
occurring when neurons on the opposite side of the cord are active. The
phase of the midcycle glycinergic inhibition was measured as a marker
to determine whether or not a midcycle component remained after the
bath application of strychnine. Measurements were made of the
time from the peak of the on-cycle spike to the start of the midcycle
IPSP divided by the time to the peak of the subsequent on-cycle spike
(i.e., y/x, Fig. 7A2). The onset of the IPSPs was chosen
because this will closely reflect the timing of the action potentials
in contralateral rhythmic neurons. The plot of midcycle IPSP phase
values over a range of cycle periods (Fig. 7A2) shows
little variation in the onset of the strychnine-sensitive IPSPs that
occurred at phase values of ~0.5 over a whole episode of rhythmic
activity. One feature of swimming that was present in control but
becomes more apparent in the presence of strychnine (Fig.
7B1) is that, although midcycle inhibition is largely
abolished, sporadic depolarizing potentials occur in some cycles of
activity. When these potentials occur, they are relatively long in
duration and cause a decrease in input resistance, as evidenced by the
decrease in spike height (asterisks in Fig. 7B1). These
potentials were considered to be GABAergic because of 1)
their long duration compared with glycinergic IPSPs (Reith and
Sillar 1997
), 2) their duration was enhanced by
5
3
, and 3) they were blocked by bicuculline (see
Fig. 8). Episodes of swimming, recorded
in the presence of strychnine, were analyzed to determine whether these
GABA potentials occurred at any particular phase of the swim cycle. The
expanded traces in Fig. 7B2 show excerpts of activity
from four parts of an episode in which there are 1) no
apparent underlying GABA potentials, 2) GABA potentials that appear to fall midcycle and GABA potentials occurring in either
3) the falling or 4) the rising phase of
the on-cycle excitation. This suggests that the GABA potentials are
variable in their timing within a cycle of activity, and indeed the
phase plot, measured from six episodes under strychnine, confirms that
GABA potentials can occur at any point during a cycle (Fig.
7B3). Note, however, that GABAergic IPSPs occurring
on-cycle, during the excitatory phase of swimming, would be excluded
from this type of analysis (see DISCUSSION). It seems
likely that the mhr neurons are responsible for generating these
GABAergic IPSPs, although the possibility that they are produced by
spinal neurons normally activated by descending excitatory pathways
cannot be completely discounted.
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Termination of swimming by GABAergic inhibition
The idea that GABAergic mhr neurons become involved in the
descending control of larval fictive swimming is supported by the fact
that episodes of larval swimming can terminate with a barrage of
depolarizing IPSPs in motoneurons. These IPSPs were observed in at
least some and occasionally in all episodes recorded in two-thirds of
larval preparations (24 of 36; e.g., Fig. 8, B1 and
B2). This phenomenon has never been documented in recordings from embryonic motoneurons (e.g., Reith and Sillar 1997;
Roberts et al. 1984
; Sillar and Roberts
1993) and was not observed in 14 preparations examined in the
present study either where embryonic swimming episodes end with the
membrane potential smoothly declining to its resting level (Fig.
8A1). Interestingly, however, the self-termination of
larval swimming episodes (Fig. 8, B1 and
B2) closely resembles the termination of embryonic
swimming following cement gland stimulation (Fig. 8A2)
(see Boothby and Roberts 1992a
; Reith and Sillar
1997
), in that trains of depolarizing IPSPs are evident. We
sought confirmation that the potentials coinciding with the end of the
majority of larval swim episodes result from GABAA
receptor-mediated inhibition. First, they are long compared with
glycinergic IPSPs (Reith and Sillar 1997
), and second,
they are still observed at the end of episodes recorded in the presence
of 5 µM strychnine (Fig. 8C1, n = 18). Third, 10 µM 5
3
massively enhanced the duration of the
potentials at the termination of swimming to the point that they
summated, greatly increasing the time for the membrane potential to
return to control levels (Fig. 8C2,
n = 4). Fourth, bicuculline completely abolished
these IPSPs (Fig. 8C3, n = 12).
These observations further support the idea that GABAA
receptor-mediated potentials in spinal neurons are used to terminate
larval swim episodes.
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DISCUSSION |
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The results presented in this paper show that between stage 37/8 and 42, the endogenous synaptic activation of GABAA receptors plays an increasingly important role in controlling rhythmic swimming activity in Xenopus tadpoles. Over the first 24 h of larval development, the effect of GABAA receptor blockade changes from affecting only swim duration and frequency to modulating all swimming parameters.
Paradoxically, both removing and enhancing GABAergic
inhibition decreases the duration of swim episodes, indicating that
GABA transmission also plays a role in maintaining rhythm generation. Presumably, any reduction in inhibition will lead to higher levels of
excitability in the network that might prevent or prematurely terminate
activity. This could occur if, for example, the increased excitability
triggered active membrane properties in spinal neurons that then lead
to strong hyperpolarizing conductances that suppressed network
activity. Oscillatory membrane properties are present in spinal cord
neurons of Xenopus larvae following activation of NMDA
receptors, and they are enhanced following block of inhibitory amino
acid receptors (Reith et al. 1998;
Scrymgeour-Wedderburn et al. 1997
). These bistable
membrane properties are common in larval neurons but rare in embryonic
ones, providing a plausible explanation for differences in the effects
of coapplying strychnine and bicuculline in larvae where rhythm
generation is abolished and in embryos where rhythm generation
persists. Alternatively, it could be argued that bicuculline at high
concentrations may be having some direct nonspecific effect, and it has
been reported that at higher concentrations bicuculline blocks
apamin-sensitive calcium-dependent potassium channels in adult
mammalian thalamic reticular neurons (Debarbieux et al.
1998
). Because Xenopus neurons do possess a
calcium-dependent potassium current, albeit with exceptionally slow
kinetics and which does not produce a clear slow afterhyperpolarization
(Wall and Dale 1995
), it is possible that the action of
bicuculline at 50 µM could be nonspecific (Wall and Dale
1995
). However, a shortening of episodes was seen at concentrations as low as 10-20 µM, which would not be expected to
act nonspecifically. The maximum shortening of episode lengths was
above 20 µM, but the precise concentration could vary between preparations. The marginally higher concentrations used here compared with previous studies (e.g., Soffe 1987
) were probably
necessary because for our extracellular experiments the spinal cord was not exposed and drug access problems could become relevant. In addition, using apamin on embryo preparations resulted in an increase in the length of swim episodes (Wall and Dale 1995
), and
therefore any nonspecific effect on the calcium dependent potassium
current by bicuculline would be expected to have the opposite effect to that observed in this present study.
How do the results of the present study compare with those obtained in
other vertebrate locomotor systems? For the most part such comparisons
are made difficult by the fact that rhythm generation in other systems,
such as the lamprey and the neonatal rat, is usually induced by
activating the network with endogenous excitants such as NMDA.
Nevertheless, during fictive swimming evoked by tail skin stimulation
in the lamprey, simultaneous block of GABAA and
glycine receptors causes an increase in burst durations at the
beginning of an episode to the point where there is no clear interburst
interval, as in Xenopus. However, in contrast to
Xenopus larvae, rhythmic activity still persists after the
initial burst and an increase in episode length is observed
(Alford et al. 1990). The location of the GABAergic
systems that impinge on the locomotor network may also explain
interspecies differences. The results of our spinalization and brain
stem transection experiments suggest that the main GABA input to
Xenopus swimming lies between the level of the otic capsule
and the fifth postotic cleft, therefore implicating a role for mhr
neurons (Boothby and Roberts 1992b
; Roberts et
al. 1987
). In contrast, for both the lamprey (Alford et
al. 1990
) and the neonatal rat (Cazalets et al.
1994
), GABAergic neurons that regulate locomotor activity must
be spinal in origin because significant effects of bicuculline are
observed in the isolated spinal cord preparation. In the lamprey,
immunocytochemical studies indicate that the GABAergic input to
swimming derives from a population of multipolar neurons with local
arborizations extending only to nearby spinal segments (Brodin
et al. 1990
). In the neonatal rat preparation it has been
established that there are GABAergic projections from the lower brain
stem to the spinal cord, which could modulate swimming (Holstege
et al. 1991
), but electrophysiological evidence supports the
view that spinal GABAergic neurons alone can profoundly affect
locomotor activity (Cazalets et al. 1994
). In this
study, although the effects of bicuculline on spinal larval
preparations were inconsistent, there was a tendency for episodes to
decrease and cycle periods to increase. It is notable that the same two
parameters are affected by bicuculline during swimming activity in
intact embryonic preparations. Perhaps spinal GABA neurons exert a
small influence on episode durations and cycle periods at both stages
of development, whereas the mhr neurons only influence swimming at
larval stages.
The present study has focused on the role of
GABAA receptors in the control of swimming, but
if GABA neurons are active during fictive locomotion as we suggest,
then it is likely that GABAB receptors are also
activated and play a role. Indeed, studies on other vertebrates have
suggested GABA transmission influences locomotor activity via
coactivation of GABAA and
GABAB receptors, which modulate different aspects
of the locomotor rhythm (Cazalets et al. 1994;
Tégner et al. 1993
). The role of
GABAB receptor activation on Xenopus
embryonic swimming rhythm has been investigated previously (Wall
and Dale 1993
). The GABAB agonist,
baclofen, causes a decrease in the reliability of action potentials and in the reliability of midcycle inhibition, both of which contribute to
the observed decrease in ventral root amplitude and episode length.
However, blockade of these receptors had little effect on swimming,
making it unclear whether endogenous GABAB
receptors are normally activated during embryonic swimming (Wall
and Dale 1993
). Similarly in Xenopus larvae,
preliminary experiments using the GABAB
antagonist 2-hydroxysaclofen failed to detect any obvious effects
on the swimming rhythm (unpublished observations).
When are GABAergic neurons active during swimming, and how does
GABAA receptor activation modulate locomotor
rhythm generation? In the lamprey it has been suggested that
GABAA receptor activation is important in the
repolarization of spinal neurons at the end of the burst in each cycle
(Alford et al. 1990), whereas in the neonatal rat, there
is evidence that both GABAA and glycine receptor activation may be important in mediating reciprocal left-right and
flexor-extensor coordination (Cowley and Schmidt 1995
).
In Xenopus larvae, GABAA receptor
activation does not appear to contribute to the midcycle inhibition,
which was completely abolished by strychnine. This finding largely
rules out the possibilities that GABAergic neurons are rhythmically
active in time with the swimming rhythm or that glycinergic commissural
interneurons co-release GABA, as has been shown recently in rat spinal
cord neurons (Jonas et al. 1998
). Nevertheless,
bicuculline and neurosteroid-sensitive GABA IPSPs occurred sporadically
throughout episodes of rhythmic activity. Although these IPSPs
were not locked to any particular phase of the swimming rhythm, their
frequency of occurrence tended to be higher nearer the starts and ends
of swim episodes. Moreover, the GABA IPSPs are relatively long in
duration (~200 ms) when compared with the cycle periods attained
during fictive swimming (~50-100 ms) so a single IPSP could
contribute inhibition lasting more than a single cycle. As a result,
successive IPSPs could summate to provide a tonic, low-level,
depolarizing inhibition that lasts throughout each episode. In support
of this idea, it is notable in Fig. 8C that the neurosteroid
enhances the level of tonic depolarization recorded under strychnine,
whereas bicuculline reduces it to below control (strychnine) levels.
Our results allow us to propose an important role for descending
GABAergic inhibition in the intrinsic termination of larval swimming.
In Xenopus embryos, activation of the descending GABAergic mhr pathway does not appear to be deployed in terminating swimming unless the cement gland is stimulated. Instead, episodes of embryonic swimming display a slow decline in frequency and then terminate spontaneously. Two mechanisms for the intrinsic termination of embryonic swimming have recently been proposed. First, swimming may
terminate due to an increase in the activation of a
KCa current resulting from a buildup of
intracellular calcium during an episode. This in turn would decrease
the input resistance and membrane time constant of neurons in the
locomotor network thereby increasing their firing threshold during an
episode to the point where they stop firing and swimming terminates
(Wall and Dale 1995). Second, the changing balance
between levels of extracellular ATP and adenosine may underlie the
rundown of the motor pattern. Early in an episode, levels of
extracellular ATP (presumably released from an unknown member of the
swimming network), rise, but adenosine levels (formed by the breakdown
of ATP by ectonucleotidases) are low. ATP causes a reduction in
voltage-gated K+ currents, reduces spike
threshold, and increases the overall excitability of the network. As
the episode proceeds, rising levels of adenosine block voltage-gated
Ca2+ currents to decrease excitability in the
network eventually reaching a level at which swimming can no longer be
sustained (Dale and Gilday 1996
). From a behavioral
viewpoint, both of these mechanisms for terminating embryonic swimming,
which are intrinsic properties of the spinal rhythm generating network,
could be considered as a last resort. They will ensure that in the
absence of any extrinsic signal (cement gland stimulation, for example)
swimming will eventually cease, but they are inappropriate for
terminating swimming mid-episode. It has been suggested that the
termination of swimming in embryos could result from a buildup in the
potency of inhibitory transmission (Wall and Dale 1995
),
although there is currently no evidence for such a mechanism. However,
our evidence suggests larval swimming can be terminated by a transient
increase in GABA release from mhr neurons. In two-thirds of
preparations, the ends of episodes of swimming activity coincide with a
barrage of GABA IPSPs, similar to those observed at the termination of
embryonic swim episodes following stimulation of the cement gland (Fig.
9A2) (Reith and Sillar
1997
). The mhr neurons in embryos are known to be
rhythmically inhibited in time with the swimming pattern
(Boothby and Roberts 1992a
). Cement gland stimulation
causes sufficient excitation of the mhr neurons that they overcome the
inhibition, fire a burst of action potentials, and so trigger the end
of an episode of swimming. However, the reliability of the cement gland
stopping pathway steadily declines from stage 37/8 onward and
completely disappears by stage 45 (Boothby and Roberts
1992b
). By this time the cement gland itself has completely
degenerated. Thus the mhr neurons are in the process of being
disconnected from their excitatory afferent input from the cement
gland. This situation raises the important question of the fate of the
mhr neurons during larval development. Numerous hypothetical
possibilities relate to the general problem of what happens to a set of
neurons when their function at one stage of development disappears at
later stages. First, the neurons could degenerate. Second, the neurons
could be retained for a different function. Third, the neurons could be
deployed in the same role but become activated via new or different routes. For the mhr neurons we propose that the third possibility applies. Immunocytochemical studies indicate that the mhr neurons are
present at stage 42 in similar numbers to stage 37/8 (Reith 1996
), and the electrophysiological experiments of the present study suggest that they continue to function in the same role, namely
in the termination of locomotor activity. The important distinction
between the function of the mhr neurons at the two stages is that early
in development they are activated exclusively by afferents of the
cement gland, whereas at later stages they are activated by alternative
central pathways that have yet to be discovered. This developmental
transition in the function of the descending GABA pathway provides the
tadpole with a method of terminating swimming "at will." Because
embryos spend much of their time hanging motionless from their cement
glands while larval animals become free swimming, the change in the
function of the mhr neurons may be more appropriate to the larval lifestyle.
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ACKNOWLEDGMENTS |
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We thank Dr. John Simmers, Dr. Beatrice Casasnovas, and S. Merrywest for valuable comments on the manuscript and D. McLean for providing the anatomic drawing of the larval preparation (Fig. 6A).
This work was supported by a Royal Society 1983 University Research Fellowship to K. T. Sillar and a grant from the Wellcome Trust. C. A. Reith was supported in part by a Biotechnology and Biological Sciences Research Council studentship.
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FOOTNOTES |
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Address reprint requests to K. T. Sillar.
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 10 June 1999; accepted in final form 3 August 1999.
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REFERENCES |
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