Previous Article | Next Article 
The Journal of Neuroscience, April 15, 1999, 19(8):3007-3022
Cholinergic and GABAergic Inputs Drive Patterned Spontaneous
Motoneuron Activity before Target Contact
Louise D.
Milner and
Lynn T.
Landmesser
Department of Neurosciences, Case Western Reserve University,
School of Medicine, Cleveland, Ohio 44106
 |
ABSTRACT |
Patterned spontaneous electrical activity has been demonstrated in
a number of developing neural circuits and has been proposed to play a
role in refining connectivity once axons reach their targets. Using an
isolated spinal cord preparation, we have found that chick lumbosacral
motor axons exhibit highly regular bursts of activity from embryonic
day 4 (E4) (stage 24-25), shortly after they exit the
spinal cord and while still en route toward their target muscles.
Similar bursts could be evoked by stimulating descending pathways at
cervical or thoracic levels. Unlike older embryonic cord circuits, the
major excitatory transmitter driving activity was not glutamate but
acetylcholine, acting primarily though nicotinic non-
7
receptors. The circuit driving bursting was surprisingly robust and
plastic, because bursting was only transiently blocked by cholinergic
antagonists, and following recovery, was now driven by GABAergic
inputs. Permanent blockade of spontaneous activity was only achieved by
a combination of cholinergic antagonists and bicuculline, a
GABAA antagonist. The early occurrence of patterned motor
activity suggests that it could be playing a role in either peripheral
pathfinding or spinal cord circuit formation and maturation. Finally,
the characteristic differences in burst parameters already evident
between different motoneuron pools at E4 would require that the
combination of transcription factors responsible for specifying pool
identity to have acted even earlier.
Key words:
motoneurons; spontaneous neural activity; rhythmicity; nicotinic receptors; GABA; cholinergic inputs; spinal cord
interneurons; embryonic networks
 |
INTRODUCTION |
The nervous system consists of
complex neural circuits initially constructed during development by a
combination of intrinsic molecular cues that guide neurons to their
targets and activity-dependent cues that fine tune those connections
(for review, see Goodman and Shatz, 1993
; Katz and Shatz, 1996
). The
role of activity in circuit formation was first explored in the visual
system in which visual experience appeared to drive the fine tuning of
cortical connections (Hubel and Wiesel, 1970
). However, more recently, it has become clear that many developing circuits, including both visual and motor, are spontaneously active before environmental experience (Bekoff et al., 1975
; O'Donovan and Landmesser, 1987
; Galli
and Maffei, 1988
; Mooney et al., 1996
) and that such spontaneous activity can also refine connections (Shatz and Stryker 1988
; Ruthazer
and Stryker, 1996
; Weliky and Katz, 1997
; Penn et al., 1998
).
In the motor system, activity is important for modulating target
innervation. Both the extent of initial synaptogenesis (Ding et al.,
1983
; Dahm and Landmesser, 1991
) and the later refinement of
connections are regulated by activity (O'Brien et al., 1978
; Thompson,
1985
; Greensmith and Vrbova, 1991
). Activity is also necessary for
maturation of muscle targets, including proper secondary myogenesis
(Harris, 1981
; McLennan, 1983
; Fredette and Landmesser, 1991
). In the
chick, spontaneous EMG activity has been shown to exist as soon as
motor neurons make functional contacts with target muscles (Bekoff et
al., 1975
; O'Donovan and Landmesser, 1987
), resulting in spontaneous
recurrent episodes of limb movements (Hamburger and Balaban, 1963
).
Neither sensory nor supraspinal input is necessary for these movements
(Hamburger et al., 1966
), indicating that activity of neurons within
the spinal cord is sufficient to generate both the rhythmicity and
pattern of movement.
In chick, the pattern and frequency of spontaneous motoneuron bursting
episodes changes in a systematic way from initial target innervation
[stages 29-30; embryonic day 6 (E6)] until after the motoneuron cell
death period (stages 36-38; E10-E12) (O'Donovan and Landmesser,
1987
). Pharmacological characterization of the cord circuit at later
stages (E10-E12) has revealed that the excitatory transmitter
glutamate is primarily responsible for driving the frequency of
bursting episodes, whereas GABA, glycine, and ACh modify burst
structure (Barry and O'Donovan, 1987
; Sernagor et al., 1995
; Chub and
O'Donovan, 1998
). However, the circuit exhibits considerable
plasticity, for if glutamate transmission is blocked, spontaneous
bursting recovers but is now driven by GABAergic connections (Chub and
O'Donovan, 1998
).
Because the nature of the circuit driving the earliest spontaneous
activity occurring before target contact was unknown, we characterized
early activity by recording from muscle nerves that were growing toward
their targets. We show that motoneurons exhibit spontaneous recurrent
episodes of bursting activity as early as E4 (stages 24-25). Unlike
later stages, early activity appears to be driven primarily by
cholinergic circuits, with minor roles from GABA and glutamate. These
results indicate that, not only do motoneurons exhibit patterned
activity earlier than previously shown, but that the circuit
responsible undergoes a switch in transmitters during the second week
of gestation. The major changes in activity and pharmacology coincided
with target contact, suggesting that target influences may play a role
in circuit maturation.
 |
MATERIALS AND METHODS |
Nerve recordings. All recordings of spontaneous motor
nerve activity were made in White Leghorn chick embryos between stages 24 and 29.5 (Hamburger and Hamilton, 1951
). Nerves were recorded in an
in vitro spinal cord-hindlimb preparation as described
previously (Landmesser and O'Donovan, 1984
). Briefly, embryos were
removed from the egg, decapitated, eviscerated, and placed in cool
oxygenated Tyrode's solution. A ventral laminectomy was
performed to expose the spinal cord and to allow oxygen to diffuse to
the motoneurons. Muscle nerves were exposed by carefully removing the
skin and surrounding limb connective tissue with a fine tungsten
needle. After dissection, Tyrode's solution was warmed to 27°C for
the duration of the experiment. Nerves were recorded from using extra fine-tip suction electrodes pulled from polyethylene tubing (PE-190; Clay Adams, Parsippany, NJ). By applying a light suction, the tips of
growing muscle nerves were pulled into the electrode and a tight seal
was established (Fig.
1A). Activity was
recorded continuously on an analog tape (Vetter, Rebersburg, PA) and
was displayed on an oscilloscope (R5030; Tektronix, Beaverton, OR) and
chart recorder (Gould Inc, Cleveland, OH). In some cases, a single
electrical pulse was given to the spinal cord to induce bursting.
Stimuli were administered using a standard stimulator (S88; Grass,
Quincy, MA) that was isolated from ground with a stimulator isolation
unit (Grass PSIU6B).

View larger version (22K):
[in this window]
[in a new window]
|
Figure 1.
Motoneurons exhibit spontaneous episodes of
bursting as they grow toward their targets. A, Schematic
of the motor circuit in the chick lumbosacral spinal cord. Patterned
motor activity is generated by local circuits in the cord
(hypothetical CPG) and can be initiated by stimulating
descending inputs from the rostral cord. Nerve recordings from specific
muscle nerves were made using tight-fitting suction electrodes. In most
figures, we have paired records from an extensor motor pool on the
top trace with a flexor motor pool on the bottom
trace. B, Example of spontaneous activity from
the femorotibialis (top trace) and obturator
(bottom trace) muscle nerves from a stage 25 embryo.
Bursting activity occurred in episodes (bracket)
containing multiple bursts (arrows). At stage 25, episodes occurred regularly, ~1-2 min apart (interepisode
interval). C, The earliest activity was
recorded from the sciatic nerve at early stage 24, when each episode
contained a single burst and occurred every 3 min (top
trace). The two bottom traces on an expanded
time scale show that a burst evoked by stimulation of descending input
from more rostral cord levels (bottom trace,
arrow marks stimulus artifact) was similar to those
occurring spontaneously (middle trace).
CPG, Central pattern generator; SART,
sartorius; FEMORO, femorotibialis.
|
|
Drug treatments. Neurochemicals and receptor blockers were
bath applied in the in vitro spinal cord-hindlimb
preparation using a pump that superfused circulated oxygenated
Tyrode's solution over the preparation. Each drug was evaluated either
alone or in combination with other drugs for its effect on spontaneous bursting activity. The drugs were applied for a minimum of 20 min, and
their effect on spontaneous activity was evaluated by quantifying the
change in burst frequency, number of bursts per episode, and burst
structure. As shown in Figure 1B, episodes of
bursting activity consisting of one or more bursts (depending on the
stage) occurred every several minutes. The interepisode interval was
used to quantify the frequency of episodes, and the lengths of bursts
and of interburst intervals were also measured in some cases. When
comparing different drug treatments, 10 or more episodes were measured,
and the data were displayed as mean ± SE. Except where indicated,
a given drug treatment was performed on two or more embryos with
similar results, and, in most cases, drugs were washed until burst
parameters returned to control values (several minutes to 1-2 hr
depending on the drug).
A list of the drugs used includes the following: cholinergic receptor
blockers: d-tubocurarine (dTC), atropine, dihydro-B-erythroidine (DHBE),
-bungarotoxin, and methyllycoconitine (MLA); cholinergic agonists: nicotine and carbachol; acetylcholinesterase inhibitor: eserine; GABA receptor blockers: bicuculline and phaclofen; GABA agonist: muscimol; GABA reuptake blocker: nipecotic acid; glutamate receptor blockers: APV, CNQX, and kyurinate; glutamate agonists: glutamate and kainate; glutamate reuptake blocker: dihydrokainate (DHK); serotonin: 5-hydroxytryptamine; gap junction blockers: octanol
and carbenoxolone. The effects of low-calcium (0.2 mM)/high-magnesium (7 mM) Tyrode's solution or
normal calcium (2 mM)/high-magnesium (12 mM)
Tyrode's solution were tested to determine the contribution of
synaptic input on spontaneous activity.
 |
RESULTS |
Development of spontaneous bursting activity
Previous studies had inferred that chick limb motoneurons were
spontaneously active in ovo by E6 (stages 28.5-29)
because this activity resulted in recurrent episodes of hindlimb
movements (Hamburger and Balaban, 1963
). This was later confirmed by
recordings in ovo (Ripley and Provine, 1972
; Bekoff et
al., 1975
; Bekoff, 1976
) and in an in vitro spinal
cord-hindlimb preparation (Landmesser and O'Donovan, 1984
; O'Donovan
and Landmesser, 1987
). These studies showed that motoneurons exhibit
spontaneous bursts of activity and that muscle contractions and thus
limb movements do not occur in the absence of activation by
motoneurons. To determine whether motoneurons were spontaneously
bursting while growing toward their targets, we used an in
vitro spinal cord-hindlimb preparation to record from the growing
tips of muscle nerves with tight-fitting suction electrodes as
soon as they emerged from the plexus as individual entities (Fig.
1A,B).
As early as stage 24 (E4), when motor axons have just begun to bundle
together (Fig. 1A) after a period of defasciculation in the plexus region (Tang et al., 1994
), we found that they exhibit spontaneous recurrent episodes of patterned bursting, consisting of a
single short burst of ~500 msec every 3.1 ± 0.33 min (mean ± SE). Examples of such bursts (Fig. 1C) show that even at
this early stage, stimulation of the cervical cord (bottom
trace, arrow) is able to elicit a burst that is
very similar to those occurring spontaneously (middle
trace). Because of the difficulty in recording from the
relatively defasciculated motor axons at this stage, we characterized
early activity in more detail at stages 25-25.5, ~12 hr later, when
motor axons had just refasciculated into individual muscle nerves.
At this stage, motoneurons produced a single burst every 1-2 min (Fig.
2A,G).
Motor axons to flexor and extensor muscles burst simultaneously rather
than in the alternating manner characteristic of more mature patterns
of activity (O'Donovan and Landmesser, 1987
; O'Donovan, 1989
).
However, even at this early stage, different pools of motoneurons could
be distinguished by their unique pattern of activity. Specifically,
flexor motoneurons, such as the sartorius (Fig. 2A,
bottom trace), had shorter bursts than extensor motoneurons, such as the caudilioflexorius/ischioflexorius (Fig.
2A, top trace) or the femorotibialis. For
example, in one case, the mean burst duration (mean ± SE) of the
sartorius was 0.35 ± 0.038 versus 0.95 ± 0.046 sec. In two
other cases, the sartorius durations were 0.28 ± 0.024 and
0.20 ± 0.014 versus 1.05 ± 0.05 and 1.06 ± 0.03 sec
for the femorotibialis. In general, extensor pools, such as the
femorotibialis or ischioflexorius/caudilioflexorius, had burst
durations of ~1 sec, whereas flexor pools, such as the sartorius, had
burst durations between 200 and 350 msec. Throughout the figure
legends, the sartorius burst will be shown on the bottom trace paired with a femorotibialis burst on the top
trace, unless otherwise noted. As at stage 24, a single
stimulus to the rostral cord (cervical or thoracic) elicited a single
burst (Fig. 2E, arrow) that was very
similar to that occurring spontaneously (Fig. 2A).

View larger version (26K):
[in this window]
[in a new window]
|
Figure 2.
Developmental changes in bursting activity
recorded from muscle nerves from stages 25-28.5. A, At
stage 25, each episode consists of a single burst, and characteristic
differences between motoneuron pools are already apparent; the
ischioflexorius/caudilioflexorius nerve (top trace) has
longer duration bursts than the sartorius nerve (bottom
trace). Extensors (top trace) and flexors
(bottom trace) are activated synchronously.
B, At stage 25.5, a second burst routinely occurs in
each episode of activity, and differences in burst characteristics
between muscle nerves are more distinct. Top trace,
FEMORO nerve. Bottom trace, SART nerve.
C, Stage 28 bursting patterns are similar to those at
stage 25.5. Top trace, FEMORO nerve; bottom
trace, SART nerve. D, At stage 28.5, bursting
characteristics changed more dramatically, with a third or fourth burst
occurring in each episode and with significant increases in burst
duration of both extensors (top trace, FEMORO nerve) and
flexors (bottom trace, SART nerve). E,
Bursting can be elicited at stage 25 by stimulation of the rostral
spinal cord (arrow marks stimulus). F, In
the presence of low-calcium/high-magnesium Tyrode's solution,
spontaneous activity is eliminated, and stimulation of the rostral cord
(top trace) does not elicit a burst
(arrows mark stimulus artifacts). However, direct
stimulation of the motoneurons causes a compound action potential with
a short latency (bottom trace, arrow
marks stimulus artifact) but does not elicit a burst. G,
Graph showing that the frequency of spontaneous bursting episodes,
plotted as the interval between episodes in minutes, at different
stages declines with increasing developmental age. Frequency
measurements shown are from the embryos in A-D. Each
bar represents the data from a single embryo,
representative of that stage (mean ± SE). Traces
from A, E, and F are from
the same embryo. SART, Sartorius; FEMORO, femorotibialis. Calibration:
A-E, 2 sec; F, top, 2 sec; F, bottom, 5 msec.
|
|
To determine whether this early activity was generated by a network of
synaptically connected neurons or alternatively represented pacemaker-like activity of electrically coupled neurons, we bathed the
embryo in Tyrode's solution with altered calcium
(Ca2+) and magnesium (Mg2+)
levels (0.2 mM Ca2+/7 mM
Mg2+ or 2 mM Ca2+/12
mM Mg2+) to selectively block chemical
transmission. Under these conditions, spontaneous bursts of activity
ceased, although spontaneous unit activity was still present (data not
shown). Low-calcium solution also inhibited our ability to elicit
bursting by stimulation of the rostral cord (Fig. 2F,
top trace, arrows mark stimulus artifacts), indicating that descending input drives the spinal circuit via chemical
synapses. However, by positioning the stimulating electrode over the
lateral motor column at the appropriate level, it was possible to
directly activate the motoneurons, producing a large compound action
potential in the muscle nerve (Fig. 2F, bottom trace, arrow marks stimulus). Such stimulation,
however, did not elicit bursts. Blockade of bursting was reversed by
returning to normal Tyrode's solution (3 mM
Ca2+/1 mM Mg2+).
These results suggest that early bursting activity is generated by a
synaptically connected network of neurons in the spinal cord and can be
driven by descending input from more rostral cord levels.
Within a few hours of development (stage 25.5), two bursts of activity
began to occur in each spontaneous episode (Fig. 2B), indicating that the circuit was now capable of generating more than one
cycle of activity per episode, although the interburst interval varied
considerably between episodes. In addition, the differences between the
patterns of activity in different nerves became more pronounced (Fig.
2, compare A, B). However, the frequency of
episodes remained similar to that in early stage 25 embryos (Fig.
2G).
By stage 28 (E6), when motor axons first contact their targets
(Landmesser, 1978
), the frequency of spontaneous episodes had slowed to
approximately one every 3-5 min (Fig. 2G). Each episode usually contained two bursts of activity spaced more regularly apart
than at younger stages, but other characteristics of the bursting
pattern remained similar (Fig. 2C). However, at stage 28.5, both the pattern and frequency of spontaneous activity changed more
dramatically. Episodes now occurred every 6-7 min and contained multiple (two to four) bursts (Fig.
2D,G). For the first time, burst
duration began to increase, and, in some cases, flexors and extensors
began to fire out of phase for a portion of each burst.
As summarized in Figure 2G, at all stages studied, the
frequency of spontaneous bursting episodes occurred with great
regularity, although the interepisode interval increased with
increasing age. Interestingly, the greatest change in frequency and
bursting characteristics occurred shortly after initial target contact,
but whether target contact is necessary for these changes will require
additional experiments.
Pharmacology of the developing circuit driving
spontaneous bursting
The mechanisms underlying spontaneous rhythm generation in the
spinal cord are not fully understood. At later stages of development (E10-E12), spontaneous activity of chick lumbosacral motoneurons appears to be driven primarily by excitatory connections, especially glutamate (Barry and O'Donovan, 1987
; Chub and O'Donovan, 1998
). Glutamate receptor blockers APV and CNQX were shown to shut off spontaneous bursting activity when bath applied to an in
vitro spinal cord-hind limb preparation (Chub and O'Donovan,
1998
). Inhibitory connections, on the other hand, were shown to be
important for generating the structure of bursts, especially the
alternation of flexors and extensors (Sernagor et al., 1995
). However,
this system exhibits considerable plasticity in that "inhibitory"
circuits (GABA and glycine) were able to drive normal spontaneous
activity after chronic blockade of excitatory (glutamate and ACh)
transmission (Chub and O'Donovan, 1998
). To determine whether the
earliest chick circuit displays similar pharmacology, we bath applied
either blockers or agonists of neurotransmitter receptors and recorded the subsequent changes in spontaneous motor output.
Glutamatergic transmission is not required for normal
bursting activity
The excitatory amino acid glutamate has been shown to stimulate
patterned bursts of activity in a number of locomotor circuits, including chick. In many systems, the effect of glutamate can be
mimicked by NMDA, an agonist of NMDA-type glutamate receptors, suggesting that glutamate drives activity in large part through activating NMDA receptors (Barry and O'Donovan, 1987
; Kudo and Yamada,
1987
; Hernandez et al., 1991
; Soffe, 1996
).
To determine whether NMDA receptors are also important in the early
chick cord, we bath applied NMDA (30 µM) to stages 25 and
28 isolated cord preparations while recording from specific muscle
nerves (Fig. 3). Similar to observations
in older (stage 36) chick cords (Barry and O'Donovan, 1987
), NMDA
elicited rhythmic bursting activity that continued for the duration
(several minutes) of drug application (Fig.
3B,D). At stage 25, bursts induced
by NMDA (Fig. 3B) were well formed and resembled control
spontaneous bursts (Fig. 3A) in pattern and duration (Fig.
3, right traces are expanded time base records of portions
of the left traces). Flexors and extensors still burst
synchronously, but more unit activity occurred between bursts (Fig.
3B, asterisk) than in control. After extended
drug application, burst duration began to lengthen and look more ragged
(Fig. 3B, right trace, last
burst). This effect of NMDA was completely blocked in the
presence of the NMDA receptor blocker APV (100 µM) (Fig.
3C). At stage 28 (Fig. 3D), NMDA-induced bursts
occurred even more frequently than at stage 25 (Fig. 3B),
and this effect was also blocked by 100 µM APV (data not
shown).

View larger version (25K):
[in this window]
[in a new window]
|
Figure 3.
Activation of NMDA receptors elicits rhythmic
bursting but is unnecessary for the generation of spontaneous bursting
episodes. Traces on the left are shown at
a slow time scale to demonstrate the effect of drug treatments on the
frequency of bursting. Corresponding traces on the
right show a subset of bursts on an expanded time scale
to show details of the burst structure. Unlike control bursting
(A), application of 30 µM NMDA at
stage 25 (B, arrow) elicits multiple
bursts of activity that continue for the duration of drug treatment.
Initially, NMDA-induced bursts are well formed (B,
right, left two bursts) and resemble
controls (A, right). After prolonged NMDA
exposure, unit activity increased (B,
left, asterisk), and bursts increased in
duration and looked more ragged (B,
right, far right burst).
C, Pretreatment with 100 µM APV does not
alter frequency or structure of spontaneous bursts (left
and right) but does block the ability of NMDA to
induce bursting (arrows mark application of NMDA).
A-C are from the same embryo. Top traces
in each pair, FEMORO nerve. Bottom traces in each pair,
obturator nerve. D, At stage 28, NMDA application
induced more frequent bursting than at stage 25 (B). NMDA bursts were also well formed
(D, right). In D,
top traces are FEMORO nerves, and bottom
traces are SART nerves. FEMORO, Femorotibialis; SART,
sartorius. Calibration: A-D, left, 20 sec; right, 4 sec.
|
|
Despite the presence of activatable NMDA receptors in these early
circuits, application of APV alone did not affect the frequency (Fig.
4F) or the pattern
(Fig. 3, compare A, C) of spontaneous activity at
either stages 25 or 28. Thus, although NMDA receptors are present and
their activation evokes rhythmic stereotyped bursts of activity, they
are not necessary for the normal generation of bursting at these early
stages.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 4.
Activation of non-NMDA receptors by kainate
induces bursting activity but is not necessary for the generation of
spontaneous bursting episodes. Traces are from stage 25 embryos treated with kainate (B, E), with
(D, E) or without (B) glutamate
receptor blockers. Addition of 10 µM kainate
(B) induces a series of bursts that resemble
control bursts (A). Extended application causes
an increase in unit activity, especially marked in extensor motoneurons
(asterisk). Top traces in
A, B, FEMORO nerve. Bottom
traces in A, B, SART nerve. In
another embryo, application of a cocktail of glutamate receptor
blockers APV (100 µM), CNQX (50 µM), and
kyurinate (1 mM) had no significant effect on bursting
(compare with control in C) but did prevent kainate
(E, arrow) from eliciting bursts. The
burst in E that follows kainate
application was the second in a spontaneous episode and was not induced
by kainate. C-E are from one experiment. Top
traces, FEMORO nerve; bottom traces, obturator
nerve. Calibration: 2 sec. F, Bar graph showing the
effect of glutamate drugs on frequency of spontaneous bursting
episodes. Glutamate blockers APV, CNQX, and kyurinate have no
significant effect on frequency of spontaneous bursting at stages 25 or
28. Similarly, DHK, a glutamate uptake inhibitor, had no effect on
bursting at stage 25. Three separate experiments are shown, two at
stage 25 and one at stage 28 (mean ± SE). C,
Control. APV, 100 µM. CNQX, used at 50 µM
at stage 25, 20 µM at stage 28. Kyurinate, 1 mM. DHK, 200 µM.
|
|
Endogenously released glutamate could also be acting via kainate-AMPA
receptors, and bath application of 10 µm kainate was able to induce
bursting activity (Fig. 4B). The pattern of activity induced by kainate differed from that induced by NMDA in two ways. First, kainate induced only a few bursts of activity rather than the
continuous bursting elicited by NMDA. Second, these bursts were then
followed by a significant increase in background unit activity,
especially in extensor motoneurons (Fig. 4B,
asterisk), and the cessation of further bursting activity.
Background activity remained pronounced during drug application but
returned to baseline within 1-2 min after the onset of a wash in
normal Tyrode's solution (data not shown). Bursting activity also
returned shortly after the beginning of the rinse. To determine whether
kainate receptors were necessary for normal spontaneous bursting, we
treated embryos with 20 µM CNQX, a selective kainate
receptor blocker. CNQX did not affect the frequency of bursting at
either stages 25 or 28 (Fig. 4F). However, in some
cases, it did induce a slight inhibitory period in extensor bursts
shortly after burst onset, which subsequently disappeared after washout
of the drug (data not shown).
Because it remained possible that glutamate was acting through both
NMDA and non-NMDA glutamate receptors and that blocking alone was
insufficient to alter normal bursting, we blocked both with a
combination of APV (100 µM), CNQX (20 µM),
and in one case, kyurinate (1 mM). As seen in Figures 4 and
5, this treatment did not significantly
alter either the frequency of bursting (Fig. 4F) or
the burst shape (Fig. 4, compare C, D). However,
it did block the ability of kainate to induce multiple bursts (Fig.
4E, arrow marks addition of kainate to
embryo pretreated with APV, CNQX, and kyurinate).

View larger version (27K):
[in this window]
[in a new window]
|
Figure 5.
Effect of GABA agonists and antagonists on the
pattern of spontaneous bursting. A, Bicuculline (50 µM) has little effect on the structure or pattern of
bursting at stage 25 (top trace in each pair, SART
nerve; bottom trace in each pair, obturator nerve).
However, at stage 28 (B), bicuculline prevented
the generation of multiple bursts in an episode and altered the burst
structure, which became triangular in shape (top traces
in each pair, FEMORO nerves; bottom traces in each pair,
SART nerves). C, At stage 25, episodes in the presence
of nipecotic acid (1 mM) contained only a single burst,
which was, however, of similar duration to control bursts (top
traces in each pair, FEMORO nerves; bottom
traces in each pair, obturator nerves). D, At
stage 28, although nipecotic acid prevented spontaneous bursting (see
Fig. 6B, open circles), a burst
could still be elicited by stimulation of the rostral spinal cord (Fig.
5D, arrow, bottom traces).
However, in contrast to stage 25 (C), burst
structure was markedly altered and considerably lengthened. In
addition, flexor pools, such as the sartorius (bottom
trace in each pair), exhibited a long period of quiescence
after initial activation, after which they resumed bursting. Such a
quiescent period did not occur in extensor pools (top
trace in each pair, FEMORO). E, In the presence
of 5 µM muscimol, a GABAA agonist,
stimulation of rostral cord (top trace,
arrow marks stimulus artifact) no longer elicits a burst
in the FEMORO nerve. However, a compound action potential in this nerve
was produced by stimulation directly over the motor pool in LS3
(second trace, asterisk). FEMORO
motoneurons could also be activated synaptically by cord stimulation of
LS1 (third trace, asterisk). This
synaptic response could be blocked by a train of stimuli at 1 Hz
(fourth trace, arrow marks
stimulus) and in other experiments by low-Ca+2
Tyrode's solution (data not shown). FEMORO,
Femorotibialis; SART, sartorius. Arrows
mark stimulus artifacts in each trace where applicable.
Calibration: A-D, 2 sec; E, 5 msec.
|
|
Finally, as an additional test of whether endogenously released
glutamate was involved in locomotor activity, we bath applied DHK, which blocks the reuptake of glutamate at the synapse. We found that DHK (200 µM) had no effect on the frequency
(Fig. 4F) or pattern (data not shown) of spontaneous activity.
Together, these results demonstrate that although glutamate receptors
are present in the spinal cord at early stages (E4-E5), they are not
important for the initiation of rhythmic bursting. However, because
CNQX induced a slight inhibitory period in extensor but not flexor
bursts and treatment with the agonist kainate increased spontaneous
background activity in extensors more than flexors, glutamate, acting
through kainate-AMPA receptors, may play a minor role in the circuit
responsible for generating burst shape, especially in extensor motor
pools. Finally, because 1 mM glutamate was still able to
induce several bursts, even in the presence of APV (100 µM), CNQX (20 µM), and kyurinate (1 mM) (data not shown), glutamate receptors not blocked by
these antagonists may be present. Nevertheless, in striking contrast to
stage 36 (Chub and O'Donovan, 1998
), blocking glutamate receptors at
early stages with APV and CNQX did not have a significant effect on the
generation of rhythmic bursting activity.
Role of GABA in spontaneous bursting episodes
At stage 36, blockade of inhibitory transmission with the
GABAA receptor blocker bicuculline and the glycine receptor
blocker strychnine results in a reduction in the frequency and
regularity of spontaneous episodes, as well as a loss of
flexor-extensor alternation (Sernagor et al., 1995
; Chub and
O'Donovan, 1998
).
To test whether endogenous GABA circuits are also important for the
frequency or pattern of early spontaneous activity, we first treated
stages 25-29 embryos with the GABAA receptor antagonist bicuculline. Bath application of bicuculline (50 µM)
tended to slow the frequency of bursting episodes at both stages 25 and 28, although some variability was observed, with frequency in some
cases increasing and in other cases decreasing or remaining unchanged
(Fig. 6A). In all
cases, however, the frequency of bursting episodes in the presence of
bicuculline occurred at ~5-7 min intervals, regardless of the
starting rate of control activity. Together, these observations
indicate that by stage 25, GABA circuits are present and can modulate
patterned activity.

View larger version (12K):
[in this window]
[in a new window]
|
Figure 6.
The effect of GABA antagonists and uptake
inhibitors on the frequency of bursting episodes. A, The
GABAA antagonist bicuculline altered the frequency of
bursting episodes at both stages 25 and 28, in some cases decreasing
and in others increasing the interval between episodes. The bar graphs
show the results from three separate embryos, and data are presented as
mean ± SE interval. B, The effect of 1 mM nipecotic acid (a GABA uptake inhibitor) on the interval
between bursting episodes at stages 25 (filled
circles) and 28 (open circles). In both cases,
activity was blocked, but it resumed after 25 min at stage 25, whereas
at stage 28, only one spontaneous episode occurred (open
circle) during prolonged nipecotic acid exposure. Such
"single" spontaneous bursts that occasionally occurred during 1 or
more hour of drug application may actually have been set off by
extrinsic stimuli. Because bursts could still be evoked by stimulation
of descending input in nipecotic acid, it is possible that this single
burst might have resulted from some stimulated activation of the cord
generator and may not reflect spontaneous activity.
Arrow and dashed line show addition of
nipecotic acid to the bath. The x symbol marks an
episode that was induced by electrical stimulation of the rostral
spinal cord of the stage 28 embryo.
|
|
In contrast to its effect on frequency, bicuculline at stage 25 did not
appreciably affect burst structure (Fig. 5A). However, by
stage 28, bicuculline altered both the number and structure of bursts
in each episode (Fig. 5B). First, it prevented the
generation of multiple bursts; regardless of the number of bursts
generated in control Tyrode's solution, only one burst per episode
occurred in the presence of bicuculline (Fig. 5B). Second,
unlike controls, bursts appeared triangular in shape and were longer in duration.
To confirm that GABA was being released during early activity, we
treated embryos with nipecotic acid (Fig. 5C,D),
which blocks the reuptake of GABA and should increase the amount of
transmitter at GABAergic synapses. We reasoned that if GABA is playing
a role in spontaneous activity then blocking GABA reuptake should
interfere with the normal pattern or frequency of bursting episodes.
Results in Figures 5, C and D, and
6B confirm that endogenous GABA is being released
during spontaneous activity. Specifically, at stage 25, bath
application of nipecotic acid (1 mM) caused an initial cessation of spontaneous bursting episodes (Fig. 6B,
filled circles), but bursting returned after 15 min at a
slightly lower frequency. In addition, only one burst occurred during
each episode, and it was slightly shorter in duration than control
bursts (Fig. 5C).
In contrast to stage 25, the loss of spontaneous activity induced by
nipecotic acid (1 mM) at stage 28 remained for the duration of drug treatment (Fig. 6B, open circles).
However, as shown in Figure 5D, stimulation of the rostral
cord could still elicit a single burst, in contrast to the multiple
bursts characteristic of stage 28 controls. Bursts in the presence of
nipecotic acid were also much longer than control (30 vs 1 sec), and
flexor and extensor motoneurons responded differently, with flexors
exhibiting a long inhibitory period after the onset of the burst (Fig.
5D, bottom, bottom trace), whereas
extensors did not (Fig. 5D, bottom, top
trace). These results suggest that GABAergic connections
influencing flexor and extensor bursts differ or that flexor and
extensor motoneurons respond differently to exogenously released GABA. The blocking effect of nipecotic acid on spontaneous activity was
reversed quickly (within 3 min) after return to normal Tyrode's solution, and bursting rebounded at an accelerated rate (interval between episodes, 40 sec vs 4-5 min in controls) for the first hour of
wash before returning to control levels (data not shown). Together,
these observations show that GABA can modulate motoneuron activity even
at stage 25 and that its role in pattern generation increases and
changes with increasing embryonic age.
To confirm that GABAA receptors were present in the cord at
stages 25 and 28, we treated embryos with muscimol, a GABAA
agonist. At both stages, muscimol blocked spontaneous bursting, and
activity did not resume until washout of the drug (data not shown).
Muscimol treatment differed from nipecotic acid treatment in that
muscimol sometimes stimulated a series of bursts before shutting off
activity (data not shown). This was followed by a transient increase in background unit activity lasting several minutes, after which spontaneous bursting ceased. At this point, bursts could no longer be
evoked by electrical stimulation to the rostral cord (Fig. 5E, top trace). However, stimulation directly
over the lumbar lateral cord, although not eliciting a burst, directly
activated motoneurons, producing a large short latency compound action
potential in the muscle nerve (Fig. 5E, second
trace, asterisk). Motoneurons could also be activated
synaptically by stimulating the cord more medially. Stimulation at this
site evoked a compound action potential (Fig. 5E,
third trace, asterisk) of 6 msec longer latency
than that evoked by direct stimulation. Consistent with synaptic
activation, this compound action potential recorded from the muscle
nerve was blocked by 1 Hz stimulation (a frequency that blocks early synapses) (Fig. 5E, bottom trace) and by low
Ca2+/high Mg2+ Tyrode's solution
(data not shown). In contrast, the compound action potential elicited
by direct stimulation of the motoneurons was not blocked at 20 Hz
stimulation nor by low Ca2+ Tyrode's solution (data
not shown). The effects of muscimol were similar throughout stages
25-28, indicating that GABAA receptors are present at
these stages and that their indiscriminate activation can disrupt
spontaneous activity more severely than GABAA receptor antagonists or GABA reuptake blockers.
Despite the similarity in the effect of muscimol at stages 25 and 28, the change in sensitivity to GABAA receptor blockers over
this period indicates that the circuit responsible for generating rhythmic activity is changing and that GABA receptors are being used
differently at these two stages in the developing locomotor circuit.
Cholinergic input strongly modulates early
spontaneous activity
As shown above, neither blockade of glutamate nor GABA receptors
with specific antagonists blocked recurrent spontaneous bursting activity in motoneurons, indicating that other transmitters must be
involved in driving spontaneous activity. At later stages, both ACh and
glycine have been shown to affect patterned activity (Sernagor et al.,
1995
), although they appear to play a minor role. Glycine, an
inhibitory neurotransmitter, is not believed to be expressed by neurons
in the cord until later stages of development (Berki et al., 1995
).
Acetylcholine, however, is the major excitatory transmitter in
motoneurons, and if released centrally by motoneurons or interneurons,
could provide excitatory drive to the network responsible for
spontaneous bursting.
To test this hypothesis, we bath applied dTC, a nicotinic ACh
receptor (nAChR) antagonist, at stages 25-29. At all stages tested,
dTC (5 × 10
6 M) transiently
stopped spontaneous bursting activity (Fig.
7A), although bursting
episodes could still be elicited by electrical stimulation of the
descending input (data not shown). Interestingly, spontaneous bursting
resumed after 10-25 min in the presence of drug, but at a lower
frequency than control. The number and structure of bursts were also
altered with the amplitude and number of bursts being increased in both
flexor and extensor motoneurons (Fig. 7B,C). Extensors and flexors were
differentially affected by dTC, with the burst duration of extensors
being increased (Fig. 7C, top trace), whereas
that of flexors was decreased (Fig. 7C, bottom trace). Furthermore, dTC appeared to increase the frequency of background unit activity in extensors more than flexors (data not
shown), suggesting that cholinergic connectivity to extensors and
flexors differs.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 7.
Blocking nicotinic receptors alters both the
frequency and pattern of spontaneous bursting activity.
A, Graph plotting intervals (in minutes) between
bursting episodes before and after the addition of 5 µM
dTC, a general blocker of nicotinic receptors. At both stages 25 (filled diamonds) and 28 (open
diamonds), dTC caused the cessation of spontaneous activity for
a period of time; however, after prolonged exposure, bursting returned
at a lower frequency (arrow and dashed
line mark the addition of dTC). B-D, Effects of
dTC on structure of bursting. Low doses (0.5 µM) of dTC
did not block spontaneous bursting (data not shown) but increased the
number of bursts per episode and altered the burst duration and
amplitude (C, compare to control in B).
Extensor bursts tended to increase in duration (top
trace), whereas flexors decreased in duration (bottom
trace). D, At high doses (10 µM),
dTC blocked spontaneous activity for over 20 min, but after activity
resumed bursts were enhanced in both amplitude and duration in
extensors (top trace) and reduced slightly in duration
in flexors (bottom trace). Interestingly, at high doses,
there was a reduction in the number of bursts, some episodes only
containing one burst as opposed to the normal two in controls
(B). B-D are from a single stage
28 embryo. Top traces, FEMORO nerves; bottom
traces, SART nerves. FEMORO, Femorotibialis; SART,
sartorius.
|
|
The response to dTC changed in a dose-dependent manner. At stage 28, low concentrations of dTC (5 × 10
7
M) did not shut off bursting but caused primarily an
increase in the number of bursts per episode, with a slight change in
the burst duration (Fig. 7C). At higher concentrations, dTC
(2-5 × 10
6 M) blocked
spontaneous bursting for up to 40 min and had a more profound effect on
burst duration. At the highest concentrations (1 × 10
5 M), dTC blocked spontaneous
activity for longer periods and continued to enhance burst amplitude
and alter its duration (Fig. 7D). However, it also decreased
the number of bursts per episode. Together, these results indicate
that, at early developmental stages, nAChRs play a major role in
regulating both the frequency and pattern of spontaneous motor activity.
To further test this hypothesis, we treated embryos with eserine, an
acetylcholinesterase inhibitor, to block the degradation of
endogenously released ACh. Eserine should thus enhance the action of
any ACh released. Consistent with this, as shown in Figure
8, eserine had the opposite effect of
cholinergic antagonists, such as dTC, on spontaneous bursting. First,
eserine markedly increased the frequency of spontaneous bursting at
both stages 25 and 28 (Fig. 8A). Second, whereas
eserine did not affect burst structure at stage 25 (Fig.
8C), at stage 28, its affect on burst structure was quite
profound (Fig. 8B). With each successive burst, presumably as ACh accumulated in the synapse, the two short bursts occurring at this stage were gradually replaced by a single burst of
much longer duration (~20 sec compared with 1 sec in controls; see
also Fig. 8B, bottom,
traces on expanded time scale). It is interesting to note
that the effect of eserine on burst shape and duration for flexors
(sartorius; bottom trace of each pair) and extensors
(femorotibialis; top trace of each pair) differed; flexor bursts tended to contain a long inhibitory period shortly after
burst onset, whereas extensor bursts did not. These effects of eserine
were blocked by pretreatment with dTC and atropine (data not shown),
nicotinic and muscarinic receptor blockers, respectively, as would be
expected if eserine were accentuating the effect of endogenously
released ACh. The effect of enhancing the activity of endogenously
released ACh (via eserine) on burst shape and duration was very similar
to that produced by enhancing the effect of endogenously released GABA
(via nipecotic acid) (compare Figs. 6F,
8B). This might arise if one effect of ACh was to
facilitate the release of GABA, a possibility considered further in
Discussion.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 8.
The acetylcholinesterase inhibitor eserine
increases the frequency of spontaneous episodes and alters burst
structure. A, Graph showing that eserine (1 µM) increases the frequency of spontaneous episodes at
both stages 25 (filled diamonds) and 28 (open diamonds). After prolonged exposure, episode
frequency decreased in the presence of eserine, presumably as a result
of ACh receptor desensitization. B, Eserine also altered
the pattern of bursting in each episode. After eserine treatment,
bursts became considerably elongated in duration (compare control
bursting on left with bursting in eserine on
right). In addition, flexors (bottom
traces) were characterized by a period of quiescence shortly
after onset of activity, whereas extensors (top traces)
were not. Bottom panels in B show
representative bursts on an expanded time scale. C,
Although eserine increased bursting frequency in both stages 25 and 28 embryos (A), it did not alter bursting pattern at
stage 25 (C, compare control on left with
eserine on right). B, Top
traces, FEMORO nerve recordings; bottom traces,
SART nerve recordings. D, Top traces,
Ischioflexorius nerve recordings; bottom traces, SART
nerve recordings. FEMORO, Femorotibialis; SART, sartorius.
|
|
Acetylcholine acts primarily through non-
7 nicotinic receptors
to modulate bursting
Nicotinic receptors blocked by dTC are known to be a heterogeneous
population composed of different subunit types (for review, see Role
and Berg, 1996
; Colquhoun and Patrick, 1997
). The composition of
subunits determines the kinetics, pharmacology, and ion specificity of
the receptor. Two basic types of nicotinic AChRs have been described;
homomeric
7 receptors and multimeric receptors that do not contain
the
7 subunit but one of several other
subunits.
7 receptors
differ from nicotinic receptors containing other
subunits in that
they contain five subunits of the same type, are more permeable to
calcium, and are specifically blocked by
-bungarotoxin. However,
more recent evidence indicates that
7 subunits may also participate
in heteromeric receptors (Yu and Role, 1998
). In the present study, we
have simply distinguished between receptors that are blocked by
classical
7 blockers, such as
-bungarotoxin and low doses of
MLA, and those that are blocked by non-
7 blockers, such as
DHBE. At later stages of development in the chick (stage 36), blockade
of
7 receptors with
-bungarotoxin has been shown to shut off
activity (Landmesser and Szente, 1986
). Thus, we were interested in
whether early activity was similarly dependent on
7 receptor activation.
To distinguish between the effects of
7 and other nicotinic receptor
subtypes, we treated embryos with more specific receptor blockers. The
7 blockers
-bungarotoxin (3 µg/ml) and MLA (10 nM)
had no significant effect on either the frequency of spontaneous bursting episodes (Fig. 9A) or
the shape of bursts within an episode at either stages 25 or 28 (Fig.
9, compare B, C). MLA, however, did increase the
number of bursts per episode at stage 25 (data not shown) but not at
stage 28. In contrast to these relatively minor effects, application of
1 µM DHBE, which blocks a variety of nicotinic receptors
containing
subunits other than
7, decreased the frequency of
episodes at both stages 25 and 28 (Fig. 9A) and, like dTC,
resulted in changes in burst shape, including an increase in burst
amplitude and a change in burst duration (Fig. 9, compare B,
D). DHBE also increased the number of bursts per episode at stage 28 but decreased the number at stage 25. Together, these results
indicate that ACh is acting primarily through non-
7 nicotinic receptors to modulate both the frequency and pattern of spontaneous bursting activity.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 9.
Acetylcholine acts primarily through non- 7
nicotinic receptors to regulate spontaneous bursting. A,
Specific 7 blockers -bungarotoxin (BT, 3 µg/ml)
and methyllycoconitine (MLA, 10 nM) have
little or no effect on burst frequency (graph shows mean ± SE);
DHBE and dTC are statistically different from control;
p = 0.001. However, dihydro-B-erythroidine
(DHBE, 1 µM), which blocks
non- 7-containing nicotinic receptors, shuts off bursting
transiently, much like dTC, which binds both 7 and non- 7
receptors. B-D, 7 blockers have no significant
effect on burst shape (compare bursts with 10 nM MLA in
C with controls in B) However, 1 µM DHBE (D) increased the amplitude
and number of bursts per episode and also increased burst duration.
Top traces, Caudilioflexorius nerve; bottom
traces, sartorius nerve. Calibration: 2 sec.
|
|
Low and high doses of nicotine affect spontaneous
bursting differently
In other parts of the nervous system, ACh has been shown to act
via many different receptor subtypes, which have different affinities
for agonists, antagonists, and differing rates of desensitization (McGehe and Role, 1995
; Colquhoun and Patrick, 1997
; Fenster et al.,
1997
). Some are located presynaptically and modulate the release of
transmitters, including GABA (for review, see Role and Berg, 1996
;
McMahon et al., 1994
; McGehee et al., 1995
; Lena and Changeux, 1997
;
Guo et al., 1998
), whereas others are located postysnaptically and
mediate classical fast transmission (McGehee et al., 1995
; Role and
Berg, 1996
; Roerig et al., 1997
). Although preliminary, the effects of
nicotine that we observed on spontaneous motoneuron bursting suggest
that in the early embryonic cord ACh is probably acting at several
different sites via different receptor subtypes.
We found that high doses of nicotine (10 µM) induced a
series of bursts, followed by an increase in spontaneous unit activity (Fig. 10A). As this
unit activity returned to baseline, spontaneous bursting ceased for at
least 1 hr (the duration of the drug application). This may have
resulted from ACh receptor desensitization, which is common in many
receptor subtypes especially at high agonist concentrations (Fenster et
al., 1997
). In contrast, much lower doses of nicotine (0.1 µM) blocked spontaneous bursting without first inducing
bursts or obvious activation of motoneurons. In both cases, a normally
formed burst could still be elicited by stimulation of descending
input, suggesting that the effects of nicotine in blocking spontaneous
bursting are upstream of the circuit that actually generates the burst.
The blockade of bursting produced by low nicotine (0.1 µM) could be prevented by previous incubation with DHBE
(1 µM) but not by
-bungarotoxin (3 µg/ml) (Fig.
10B), indicating that these effects are mediated by
non-
7 receptors. The nicotine-induced blockade of bursting could
also be prevented by previous incubation with bicuculline. Together, these results are consistent with ACh being able to act via
high-affinity receptors to enhance GABA release. Given the complexity
of the circuit and the likelihood that ACh is acting at multiple sites, the precise cellular mechanisms for the observed effects of nicotine remain unclear. However, the doses of nicotine that block spontaneous patterned activity of this developing spinal circuit are levels that
would be expected to occur in the fetus after maternal cigarette smoking (Lambers and Clark, 1996
).

View larger version (24K):
[in this window]
[in a new window]
|
Figure 10.
Effect of nicotine on spontaneous bursting.
A, At stage 28, high doses of nicotine (10 µM) induce a series of bursts (bottom
traces, arrow marks addition of nicotine),
followed by a transient increase in unit activity
(asterisk). Top traces show bursting
induced by high nicotine on an expanded time scale to demonstrate
bursting characteristics. B, In contrast, low
concentrations of nicotine (0.1 µM) cause the cessation
of spontaneous bursting. Pretreatment of embryos for 30 min with DHBE
(1 µM), but not -bungarotoxin (3 µg/ml), prevent the
blockade of activity after low-nicotine treatment. In addition,
pretreatment with bicuculline (50 µM) to block
GABAA receptors also prevents activity blockade by low
nicotine. FEMORO, Femorotibialis; SART,
sartorius.
|
|
Early spontaneous bursting may be driven by either cholinergic or
GABAergic circuits
Thus far, we have shown that both cholinergic and GABAergic, but
not glutamatergic, neurons are important for early spontaneous motoneuron bursting in the chick. However, in all cases in which the
action of a neurotransmitter was blocked by receptor antagonists, spontaneous bursting activity recovered in the continued presence of
the drug(s), presumably now driven by a different transmitter system.
At stage 36, for example, recovery of spontaneous activity can occur
after blockade of excitatory transmission (Chub and O'Donovan, 1998
).
In this situation, activity is now driven by the inhibitory
transmitters (GABA and glycine), which can be depolarizing at early
developmental stages (Cherubini et al., 1991
; Owens et al., 1996
;
Rohrbough and Spitzer, 1996
). However, simultaneous blockade of
glutamate and GABA receptors resulted in complete activity blockade,
reversible only by drug removal (Chub and O'Donovan, 1998
).
To determine which transmitter systems were capable of driving
spontaneous activity at stage 25, we treated embryos with combinations of drugs. As shown for one embryo in Figure
11A, blockade of both GABA and glutamate receptors had no effect on spontaneous activity, in
contrast to its effect at stage 36 (Chub and O'Donovan, 1998
). Addition of dTC to these drugs resulted in a transient blockade of
activity. However, activity resumed, and it was not until the addition
of atropine to block muscarinic ACh receptors that spontaneous bursting
activity ceased completely. In this case, bursting recovered quickly,
within 3 min, after the drugs were rinsed out in normal Tyrode's
solution (Fig. 11A).

View larger version (17K):
[in this window]
[in a new window]
|
Figure 11.
Spontaneous activity at stage 25 does not occur
after blockade of both excitatory and inhibitory connections. Embryos
were treated with a combination of receptor blockers to determine which
transmitter systems were critical for spontaneous bursting. In each
graph, arrows mark the addition of each receptor
blocker. A, Blockade of GABA receptors with a
combination of phaclofen (Ph, 50 µM) and
bicuculline (Bic, 50 µM) has no effect on
the frequency of spontaneous episodes. Addition of glutamate receptor
blockers APV [50 µM and increasing to 100 µM (APV+)] and CNQX (20 µM)
to the GABA receptor blockers also has no effect on activity. However,
dTC (5 µM) causes a transient cessation of spontaneous
activity for 25 min, followed by a recovery of bursting. Bursting
ceases completely after atropine (1 µM) is added to the
cocktail. Bursting resumes quickly (within 3 min) to near control
frequency after washing in normal Tyrode's solution
(arrow marks onset of wash at hour 6 of the experiment).
The x symbols represent activity induced by electrical
stimulation of the rostral cord, an example being shown in
C. Such stimuli, which were used to determine
whether descending input was able to elicit bursts under
various drug applications, did not affect subsequent bursting or lack
thereof. B, Shows that atropine (1 µM) has
no effect on burst frequency when applied alone (Atr,
arrow), but addition of dTC (5 µM) to
atropine shuts off activity transiently, and bursting returns in the
continued presence of both drugs. This resumed activity may be mediated
by GABA, because the addition of bicuculline (50 µM) to
the cocktail shuts off activity completely. One burst did occur after
~1 hr, but no other bursts occurred. C, Although no
spontaneous activity occurred in the presence of GABAergic,
glutamatergic, and cholinergic receptor blockers shown in
A, bursting could be elicited by a single electrical
stimulus to the rostral cord (C, arrow
marks the stimulus artifact). Each stimulus evoked a single well formed
burst.
|
|
To determine which of the transmitter systems was the most critical for
spontaneous bursting, we next treated with a combination of cholinergic
and GABAergic drugs. We found that spontaneous bursting activity was
blocked completely in the presence of the cholinergic blockers atropine
and dTC and the GABAA blocker bicuculline (Fig.
11B). These results indicate that spontaneous
activity can be driven by either cholinergic or GABAergic circuits
acting through GABAA receptors and that simultaneous
blockade of both systems eliminates spontaneous activity.
Descending input
Despite our ability to block spontaneous activity with cocktails
of receptor blockers, we were unable to block the ability of rostral
cord stimulation to elicit a bursting episode. For example, in the
experiment shown in Figure 11A, we were still able to
evoke a burst by electrical stimulation of the rostral cord (x
symbols indicate stimulated bursts), even when spontaneous activity was completely shut off (Fig. 11C, arrow
marks stimulus). Two mechanisms could explain this finding. Descending
input could use a different transmitter, such as norepinephrine,
serotonin, or dopamine, to activate local circuits in the lumbosacral
spinal cord that generate bursts. Alternatively, the drugs used might not fully block all GABA, glutamate, and ACh receptors, possibly because of different affinities for embryonic forms of the
receptors. This finding also suggests the possibility that another
transmitter is involved in the local pattern generator circuit, because
blockade of GABA, glutamate, and ACh receptors did not eliminate the
ability of descending input, to stimulate a well formed burst of
motoneuron activity (Fig. 11C). Another possibility, that
electrical coupling between networks of motoneurons could produce
bursts when activated by descending input, will be considered next.
Potential role for electrical coupling in spontaneous
burst generation
Our observations have clearly demonstrated the importance of
chemical transmission in allowing patterned spontaneous bursting by
early lumbosacral motoneuron pools. They do not, however, preclude a
role for electrical coupling between motoneurons or interneurons in
generating the spontaneous bursting episodes. Electrical coupling has
been demonstrated between somatic motoneurons in Xenopus
embryos (Perrins and Roberts 1995a
,b
) and in neonatal rats (Walton and Navarette, 1991
) and between preganglionic motoneurons (Logan et al.,
1996
) in rat lumbar cord. Several of our observations, although
preliminary, are consistent with a role for electrical coupling via gap
junctions in spontaneous burst generation.
Octanol (3 mM), which blocks gap junction-mediated
electrical coupling and which reduces the frequency of spontaneous
Ca2+ transients in embryonic chick retina (Catsicas
et al., 1998
), reversibly abolished spontaneous bursting of stage 25 motoneurons for as long as it was present (data not shown). We were
also unable to elicit a burst by stimulating descending input;
stimulation of the lumbar cord also did not elicit a burst but did
directly activate the motoneuron pool producing a large compound action potential similar to that shown in Figure 2F. Because
in addition to blocking gap junctions octanol is known to increase
inactivation of sodium channels (Elliot and Elliot, 1989
), we also
tested a more selective gap junction blocker, carbenoxolone (Draguhn et al., 1998
; Leslie et al., 1998
). At 100 µM, carbenoxolone
also reversibly blocked spontaneous bursting of stage 25 motoneurons after ~50 min (data not shown). Before block of spontaneous bursting, burst amplitude was increased. After washout, bursting recovered within
25 min to predrug levels. These observations suggest that motoneurons
and/or interneurons may be electrically coupled at these stages and
that electrical coupling may act in combination with synaptic drive to
produce spontaneous bursting.
 |
DISCUSSION |
Our major finding is that as early as E4 (stages 24-25), 1-1.5 d
after being born (Hollyday and Hamburger, 1977
) and while their axons
are sorting into muscle-specific fascicles at the limb base, chick
lumbosacral motoneurons exhibit regular bursts of electrical activity
that are primarily driven by chemical synapses. At later developmental
stages (E10-E12), spontaneous activity in this circuit is driven by a
network of local neurons, including interneurons and possibly
motoneurons, which are interconnected via excitatory synapses (Sernagor
et al., 1995
: Chub and O'Donovan, 1998
). When excitation reaches some
threshold, the network is activated and drives the motoneurons in an
episode of bursting (Chub and O'Donovan, 1998
). This model can also
account for many of our observations. However, in contrast to later
stages when glutamate provides the main excitatory drive (Chub and
O'Donovan, 1998
), at early stages, this is provided by endogenously
released ACh acting for the most part via nicotinic non-
7 receptors.
The circuit we have pharmacologically characterized has many
similarities to the circuit that drives spontaneous waves of electrical
activity in the developing retinas of both ferret (Meister et al.,
1991
; Wong et al., 1998
; Feller et al., 1996
) and chick (Catsicas et al., 1998
). Thus, the properties of the early cord circuit
will first be compared with spontaneous activity in other developing
circuits. Next, we will consider possible roles for such early activity
in developing spinal cord. Finally, the current view of how motoneuron
subtype identity is specified (Tsuchida et al., 1994
; Sockanathan and
Jessell, 1998
) will be considered in light of the very early
differences in bursting activity found between different motoneuron pools.
Similarities in early circuits that generate
spontaneous activity
Unlike the local domains of spontaneous activity in developing
visual cortex that are propagated by second messenger spread through
gap junctions (Kandler and Katz, 1998
), both the waves of electrical
activity that spread across developing retina (Meister et al., 1991
;
Penn et al., 1994
; Feller et al., 1996
; Catsicas et al., 1998
; Wong et
al., 1998
) and the spontaneous bursting activity in spinal cord (Chub
and O'Donovan, 1998
; present results) require chemical transmission.
Although some degree of electrical coupling may be needed for effective
generation of spontaneous bursts (Penn et al., 1994
; Catsicas et al.,
1998
: Wong et al., 1998
; present results), early spontaneous activity
in the ferret (Feller et al., 1996
; Penn et al., 1998
) and chick
(Catsicas et al., 1998
) retina and in E4-E6 chick spinal cord (present
results) is strongly modulated by nicotinic transmission. Spontaneous
bursting in E11-E12 mouse spinal cord also requires nicotinic
transmission (S. Banerjee and L. T. Landmesser, unpublished
observations). Thus, nicotinic modulation of spontaneous
activity in early developing circuits may be widespread (Role and Berg,
1996
). In ferret retina (Feller et al., 1996
: Penn et al. 1998
) and
early chick spinal cord, ACh affects spontaneous activity primarily
through nicotinic receptors that are not blocked by
7 antagonists.
Nevertheless, the fact that blockade of
7 receptors in stage 25 cords resulted in an extra burst per episode indicates that these
receptors are being activated during early activity and could be
playing various roles, including regulation of gene expression (Spitzer
et al., 1993
; Fields et al., 1997
) via calcium influx (Rathouz et al., 1995
).
What is the source of the ACh that drives activity in early cord
circuits? The chick circuit is contained entirely within the ventral
cord (Ho and O'Donovan, 1993
), which at E4-E5 contains only
motoneurons and a small population of interneurons. ACh, released from
motoneuron collaterals, could provide excitatory drive to both
interneurons and motoneurons. In embryonic Xenopus cord,
motoneurons synapse on interneurons and other motoneurons and
contribute to the excitatory drive underlying fictive swimming (Perrins
and Roberts, 1994
, 1995a
,b
). Alternatively, more diffuse, paracrine-like release of ACh from motoneurons could be responsible given t