Laboratory of Neural Control, Section on Developmental Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Wenner, Peter and Michael J. O'Donovan. Mechanisms That Initiate Spontaneous Network Activity in the Developing Chick Spinal Cord. J. Neurophysiol. 86: 1481-1498, 2001. Many developing networks exhibit a transient period of spontaneous activity that is believed to be important developmentally. Here we investigate the initiation of spontaneous episodes of rhythmic activity in the embryonic chick spinal cord. These episodes recur regularly and are separated by quiescent intervals of many minutes. We examined the role of motoneurons and their intraspinal synaptic targets (R-interneurons) in the initiation of these episodes. During the latter part of the inter-episode interval, we recorded spontaneous, transient ventral root depolarizations that were accompanied by small, spatially diffuse fluorescent signals from interneurons retrogradely labeled with a calcium-sensitive dye. A transient often could be resolved at episode onset and was accompanied by an intense pre-episode (~500 ms) motoneuronal discharge (particularly in adductor and sartorius) but not by interneuronal discharge monitored from the ventrolateral funiculus (VLF). An important role for this pre-episode motoneuron discharge was suggested by the finding that electrical stimulation of motor axons, sufficient to activate R-interneurons, could trigger episodes prematurely. This effect was mediated through activation of R-interneurons because it was prevented by pharmacological blockade of either the cholinergic motoneuronal inputs to R-interneurons or the GABAergic outputs from R-interneurons to other interneurons. Whole-cell recording from R-interneurons and imaging of calcium dye-labeled interneurons established that R-interneuron cell bodies were located dorsomedial to the lateral motor column (R-interneuron region). This region became active before other labeled interneurons when an episode was triggered by motor axon stimulation. At the beginning of a spontaneous episode, whole-cell recordings revealed that R-interneurons fired a high-frequency burst of spikes and optical recordings demonstrated that the R-interneuron region became active before other labeled interneurons. In the presence of cholinergic blockade, however, episode initiation slowed and the inter-episode interval lengthened. In addition, optical activity recorded from the R-interneuron region no longer led that of other labeled interneurons. Instead the initial activity occurred bilaterally in the region medial to the motor column and encompassing the central canal. These findings are consistent with the hypothesis that transient depolarizations and firing in motoneurons, originating from random fluctuations of interneuronal synaptic activity, activate R-interneurons, which then trigger the recruitment of the rest of the spinal interneuronal network. This unusual function for R-interneurons is likely to arise because the output of these interneurons is functionally excitatory during development.
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INTRODUCTION |
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Spontaneous activity is a characteristic feature of developing
circuits in virtually every part of the nervous system that has been
examined to date (Ben-Ari et al. 1989; Christie
et al. 1989
; Fortin et al. 1995
; Ho and
Waite 1999
; Itaya et al. 1995
; Landmesser
and O'Donovan 1984
; Lippe 1995
; Maffei
and Galli-Resta 1990
). This type of network-driven embryonic
activity is remarkably similar in tissues as diverse as the
hippocampus, retina, and spinal cord (see O'Donovan
1999
for review) and is manifest as recurrent depolarizing
events, during which cells within the network are synchronously
activated. During these events, intracellular calcium is elevated
(Garaschuk et al. 1998
; Kulik et al.
2000
; Leinekugel et al. 1995
; O'Donovan
et al. 1994
; Wong et al. 1995
), suggesting a
role in developmental or trophic processes. In the spinal cord,
spontaneous activity has been implicated in the development of limb
muscles, bones, and joints (Hall and Herring 1990
;
Persson 1983
; Toutant et al. 1979
), the
projections of cutaneous afferents to the dorsal horn (Mendelson
1994
), motoneuronal neurite outgrowth in culture
(Metzger et al. 1998
), and the maturation of motoneuron electrical properties in organotypic culture (Xie and
Ziskind-Conhaim 1995
).
Despite the presumed importance of this form of periodic
activity, very little is known about the mechanisms that regulate its
onset. In the developing hippocampus, it has been shown that spontaneously occurring giant depolarizing potentials (GDPs) are preceded by an increase in the frequency of spontaneously occurring synaptic events. Menendez de la Prida and Sanchez-Andres
(1999) showed that GDPs occurred 100-300 ms after the
frequency of excitatory postsynaptic potentials (EPSPs) in hippocampal
neurons exceeded a specific threshold. Between GDPs, they also observed
transient increases in EPSP frequency that were lower than those
occurring before a GDP. These findings suggest that developing networks experience transient increases of synaptic activity, presumably arising
from the coordinated firing of groups or clusters of interneurons and
that these events can trigger synchronized network activity when they
become large or frequent enough.
In the embryonic chick spinal cord, some progress has been made in
characterizing the basic mechanisms underlying spontaneous activity
(Chub and O'Donovan 1998; Fedirchuk et al.
1999
; Milner and Landmesser 1999
; Tabak
et al. 2000
). In this preparation, spontaneous bursting lasts
for ~1 min (referred to as an episode) and is followed by a period of
little or no network activity that persists for many minutes (referred
to as the inter-episode interval). Episodes are composed of many cycles
of discharge whose period progressively lengthens throughout the
episode (Landmesser and O'Donovan 1984
) and are
generated by combined action of GABAergic, glutamatergic, and
cholinergic inputs (Chub and O'Donovan 1998
; Sernagor et al. 1995
).
The mechanisms responsible for initiating spontaneous episodes are
unknown. Using optical recordings from the transversely cut face of the
spinal cord, it has been shown that the earliest activity at the
beginning of an episode occurred in and around the lateral motor column
and then evolved as a dorsomedial wave (O'Donovan et al.
1994). Consistent with a role for motoneurons in episode
initiation, Ritter et al. (1999)
showed that motoneuron firing began hundreds of milliseconds prior to the start of an episode,
before activity could be detected in interneurons. If motoneuron
activity is involved in episode initiation, we hypothesized that the
intraspinal neuronal targets of motoneuron recurrent collaterals might
mediate this process (Wenner and O'Donovan 1999
). These
recently identified interneurons (R-interneurons), which appear to be
the avian homologue of the mammalian Renshaw cell identified in the
adult cat (Eccles et al. 1954
; Renshaw
1946
), receive excitatory cholinergic input from motoneuron
collaterals and project depolarizing GABAergic synapses to motoneurons
and to other spinal interneurons (Wenner and O'Donovan
1999
).
In the present work, we have also investigated whether or not spinal
neurons including motoneurons and interneurons experience transient
increases of synaptic activity as has been reported in developing
hippocampal networks (Menendez de la Prida and Sanchez-Andres 1999) and if such transients are associated with episode
initiation. In addition, we have investigated the hypothesis that the
pre-episode motoneuron discharge triggers an episode of spontaneous
activity by exciting R-interneurons, which in turn excite the rest of
the interneuronal network. To examine these questions, we have compared the timing of electrical activity in motoneurons, R-interneurons, and
other spinal interneurons at episode onset. We have examined the
ability of motor axon stimulation to trigger an episode and have used
calcium imaging to visualize the recruitment of R-interneurons during
such stimulation and compare it with the recruitment pattern occurring
spontaneously. We have also investigated the effects of blocking the
synaptic connections to and from R-interneurons on the recruitment
patterns and timing of spontaneous activity. Some of this work has been
published in a conference proceeding (Wenner et al.
1998
).
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METHODS |
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Physiology
Chick embryos were removed from the egg at E9-E11 (stage
35-37) and staged according to the criteria of Hamburger and
Hamilton (1951). The great majority of the experiments
were performed on E10 embryos; data in figures were obtained from E10
embryos unless stated otherwise. Embryos were decapitated and the
spinal cords were isolated as described previously (O'Donovan
1989
; O'Donovan and Landmesser 1987
) in
recirculating cold (15°C) Tyrode's solution [concentration (in mM):
139 NaCl, 3 KCl, 17 NaHCO3, 12 glucose, 3 CaCl2, and 1 MgCl2] in
accordance with National Institutes of Health guidelines. The spinal
cord was isolated together with certain muscle nerves (adductor = adductors and obturator; femorotibialis = external and medial
head, femorotibialis internal head, sartorius). After the dissection,
the solution was allowed to reach room temperature and left undisturbed
for
2 h (overnight preparations were left
12 h at 17°C). The
solution was then cooled to 17°C, dorsal pia was removed, and a
horizontal cut was made using a vibrating razor blade at the midpoint
of the dorsoventral axis, leaving equal dorsal and ventral halves from
about the last thoracic segment (T) 7 to lumbosacral segment (LS) 5. The ventral piece, with intact ventral roots and muscle nerves, was
then transferred to the recording chamber and the solution temperature
was increased to 27°C for the remainder of the experiment. Muscle
nerves (with cut dorsal roots) were drawn into suction electrodes for
recording and/or stimulating. Whole cell electrodes [4-8 M
, with a
K-gluconate solution concentration (in mM): 10 NaCl, 130 K-gluconate,
10 HEPES, 1.1 EGTA, 0.1 CaCl2, 1 MgCl2, and 1 Na2ATP] were
driven ventrally through the dorsal aspect of the ventral piece of
cord. The electrode was positioned directly over the R-interneuron
region dorsal to the medial part of the motor column (see Fig. 1).
Typically the electrode was driven 100-200 µm into the tissue before
recordings were obtained. In two cells, recordings were obtained while
driving the electrode through the transversely cut face as described
previously (Wenner and O'Donovan 1999
). All whole-cell
recordings were obtained using an Axoclamp 2B amplifier and custom
written data-acquisition software (Labview 4.0). Extracellular suction
electrode recordings were obtained from muscle nerves or from a slip of
the VLF (2-5 mm long) and were amplified 1,000 times and filtered at
DC-1 kHz (low-pass) or 200-3 kHz (high pass). VLF recordings have been shown to reflect the population activity of spinal neurons
(Ritter et al. 1999
). Cells were only accepted for
further study if their resting membrane potential was more negative
than
40 mV. Single-pulses and stimulus trains (20-50 Hz for 0.5 ms)
of 30 µA were delivered to muscle nerves to activate R-interneurons
(Wenner and O'Donovan 1999
). When testing whether
motoneuronal inputs could trigger an episode, we stimulated the ventral
root (to activate motoneurons antidromically). Although single shocks
could sometimes evoke an episode of activity, stimulus trains were more
effective and were used routinely.
R-interneurons were identified by the presence of short latency
synaptic input following stimulation of muscle nerves. Cells falling
into this category had latencies to the onset of the earliest synaptic
potential of 5 ms (see Wenner and O'Donovan 1999
).
Our previous work has shown that the great majority of R-interneurons produce a depolarizing potential in motoneurons temporally coupled to
the occurrence of a spike in the recorded interneuron. In the present
work, we used spike-triggered averaging to identify the source of
spiking in the adductor muscle nerve (see Fig. 2). For this purpose, we
recorded spontaneous spiking in an adductor motoneuron and averaged
traces acquired from the adductor muscle nerve time-locked to the
motoneuron spikes.
Optical recordings
To visualize the interneurons activated by stimulation of
motoneurons, we loaded a calcium dye (Ca-green1 dextran 10,000 MW; Molecular Probes) into ventrally located spinal interneurons. A section
of the ventrolateral cord (LS4-LS5 border), often including a portion
of the lateral motor column, was drawn into a suction electrode
containing ~20% wt/vol of the dye dissolved in distilled water
containing 0.2% Triton X-100 detergent (O'Donovan et al. 1993). This configuration was left overnight to allow
retrograde transport of the calcium-sensitive dye back to the
interneuronal cell bodies. After this loading period, the pia was
removed between adjacent roots and the cords were cut transversely
(vibrating razor blade) rostral and caudal to a particular ventral root
(LS2 or LS3) leaving a single segment slice of cord (~1 mm thick). The cord was then positioned in a recording chamber on the stage of an
inverted microscope (Nikon Diaphot). The rostral face of the slice was
viewed with epifluorescence illumination. Images were continuously
acquired to videotape using an intensified video camera (Stanford
Photonics) while the preparation produced spontaneous episodic activity
or while stimulus trains (20-50 Hz, 100-200 ms) were applied to the
ventral root to define the R-interneuron region. The tissue was
illuminated using a 75-W Xenon Arc lamp with an excitation filter of
450-490 nm, dichroic of 510 nm, and a barrier filter of 520 nm.
Various ND filters were used to reduce photodynamic damage. 1-3 mM KCl
was added to the circulating Tyrode's solution (increasing the
K+ concentration to 4-6 mM) to increase the
frequency of spontaneously occurring episodes.
Image analysis
During the experiment, video data (30 fps) were stored on S-VHS
tape (Sony SVO-9500 MD). Images were digitized off-line, frame by
frame, and processed on Metamorph software (Image Systems). To display
regions activated by stimulation or spontaneous activity, we
constructed difference images normalized to the background fluorescence
(F/F). These were generated by subtracting a
30-frame average obtained prior to the stimulus or spontaneous episode (background image) from consecutive frames during the activity. Resulting images were then divided by the background image. A 5 × 5 median filter was applied to the background and subtracted images to
remove noise. These images were displayed in false color and were
stretched to occupy the full 8-bit range (0-255) of the frame store.
To determine the R-interneuron region, 10 consecutive frames were
selected as those with the greatest intensity changes and averaged.
Quantification of fluorescence changes was performed on specific
regions of interest (ROI) as described previously (O'Donovan et
al. 1994
). Time series at the onset of an episode were
generated by averaging several episodes time-locked by the frame that
showed a 15% increase in fluorescence. Optical activity at the onset
of an episode was defined as the area in the first frame that showed an
increase of
2 SD over the background. Resolution of a slowly
developing diffuse signal at episode onset in the presence of
mecamylamine required a 3- to 10-frame average.
Definition of the R-interneuron region
We defined the R-interneuron region (in the cut transverse face
of the spinal cord) as the area that showed an increase in fluorescence
6 SD above the mean fluorescence of the same region under control
conditions when a stimulus train was applied to the ipsilateral ventral
root or a muscle nerve (Wenner and O'Donovan 1999). We
have argued that this optical signal is derived from the activity of
interneurons monosynaptically activated by motoneuron recurrent
collaterals. Because of the importance of this interpretation to the
present experiments, we present here additional data supporting this
conclusion. To address the possibility that activation of ventral root
afferents (Jiang et al. 1991
) might contribute to the
optical signals, we bath-applied glutamatergic antagonists [50 µM
AP5, 20 µM 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX)] and found
that neither the amplitude of the ventral root-evoked optical signal
(peak transient 128.2 ± 56.5% of the pre-drug signal n = 3) nor its location dorsomedial to the lateral
motor column were substantially changed (measured
15 min after
application of the drugs). We then established if the location of the
ventral root evoked optical signal changed when the outputs from
R-interneurons were blocked with the addition of a GABAergic antagonist
to the glutamatergic antagonists (Fig.
1C, 50 µM bicuculline and
AP5/CNQX) or in a separate experiment, a combination of a GABAergic and a glycinergic antagonist (strychnine, 1 µM). Although the
ventral-root-evoked ventral root response was abolished in the presence
of the drugs (Fig. 1D), we found that the optical response
still occurred in the same region as under control conditions. In the
presence of the drugs, the optical signal was more diffuse and less
intense than under control conditions. We hypothesize that the
reduction of the optical response occurred because bicuculline blocks
the reciprocal GABAergic connections between R-interneurons themselves or between R-interneurons and motoneurons (Wenner and O'Donovan 1999
). Alternatively, it may be that some component of
the signal is derived the activation of non-R-interneurons by the
GABAergic projections of R-interneurons that is lost in the presence of bicuculline. Whatever the reason for the decline of the signal, the
unchanging location of the signal in the presence of GABAergic and
glutamatergic blockade and its abolition following bath-application of
the cholinergic antagonist mecamylamine strongly supports the idea that
this region contains interneurons directly activated by cholinergic
motoneuron collaterals. Further support for this idea was obtained from
whole-cell recordings made from identified R-interneurons. In these
experiments, we recorded from identified R-interneurons in a horizontal
preparation of the ventral cord in which the dorsal half had been
removed. We found that stimulation of the adductor muscle nerve (dorsal
roots cut) not only produced a short latency synaptic response in
R-interneurons but also an antidromic field potential field potential
~2 ms before the onset of the evoked synaptic potential (data not
shown). This observation suggested that R-interneuron cell bodies were
close to those of the adductor motoneurons in the anterior segments
(LS1-LS3) where most of these recordings were made. Indeed, we found
that R-interneurons cell bodies were located ~40 µm dorsal to the
adductor motor nucleus. We reconstructed the location of both adductor
motoneurons and R-interneurons in the rostrocaudal and mediolateral
plane as shown in Fig. 1E. Mediolateral position was defined
using midline and lateral border of the spinal cord and expressed as a
percentage of the distance from each boundary. Rostrocaudal position
was obtained with respect to adjacent ventral roots and expressed as a
percentage of the distance from one root to another. The mediolateral
boundaries of the R-interneuron column mapped in this way could then be
superimposed on the optically imaged cut transverse face of the cord,
as shown in Fig. 1B. It can be seen that this boundary
coincides with that of the R-interneuron region estimated from the
optical recordings. Collectively, these results strongly suggest that
the region activated optically by ventral root stimulation in the
transverse plane contains the cell bodies of neurons directly activated
by motoneuron collaterals (R-interneurons).
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RESULTS |
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Inter-episode activity of motoneurons and interneurons
Motoneurons and spinal interneurons in the chick embryo spinal
cord are spontaneously activated in recurrent episodes, which typically
last from 30 to 90 s. The episodes are composed of depolarizing cycles that often become most evident toward the end of the episode as
the cycle period lengthens. These spontaneously occurring episodes are
separated by quiescent periods that vary from 10 to 20 min. During an episode there is a massive
recruitment of spinal networks in which motoneurons and interneurons
are activated synchronously (O'Donovan et al. 1994).
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To better understand the events leading to a spontaneous episode, we
examined the activity occurring during the inter-episode interval and
just before an episode. In the first 2-3 min after an episode, spinal
neurons were inactive. After this time, certain classes of motoneuron
(adductor and sartorius) began to discharge and their activity became
progressively more intense until the next episode occurred. Other
classes of motoneuron (e.g., femorotibialis) did not discharge during
the inter-episode interval. Figure 2A compares activity
recorded from the adductor muscle nerve and intracellularly from an
adductor motoneuron with the discharge recorded from interneurons
projecting into the VLF. Discharge begins in the motoneurons and the
muscle nerve record well before the occurrence of the episode, but
little or no discharge could be recorded from the VLF during this
period. After the episode, the motoneuron activity stopped.
Spike-triggered averaging from the adductor motoneuron spike revealed a
short-latency action potential in the muscle nerve (Fig.
2B), indicating that the muscle nerve discharge originates
from the firing of motoneurons. Inter-episode motoneuron discharge was
also recorded from the ventral roots and began ~5 min after the end
of an episode (Fig. 2C, ).
During the inter-episode interval, we also recorded transient
depolarizations from the ventral roots (Fig.
3, A-C) that often appeared
to be associated with the initiation of an episode (initial depolarization; Fig. 3C). These transient depolarizations
may be generated by interneuronal activity because optical recordings from interneurons in the cut transverse face of the cord revealed transient increases of fluorescence synchronized with the ventral root
depolarizations (Fig. 3B, asterisk). Alternatively, the
interneuronal fluorescence transients might originate from spontaneous
action-potential independent transmitter release (see
DISCUSSION). Although transients were evident two minutes
before an episode (Fig. 3A, a), they were not observed in
the two minutes after an episode (Fig. 3A, b). This finding
is consistent with the progressive inter-episode increase in the
amplitude of evoked and spontaneous synaptic potentials reported in
earlier work (Chub and O'Donovan 2001;
Fedirchuk et al. 1999
; Tabak et al.
2000
).
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About 500 ms before the onset of the episode, a depolarization was
observed in virtually all ventral root and muscle nerve recordings (see
also Ritter et al. 1999). This pre-episode or initial
depolarization had a similar time course to the depolarizing transients. This is shown by scaling and superimposing a transient on
the initial depolarization of the episode (Fig. 3C). These initial depolarizations were larger in amplitude (19.7 ± 7.0% of
the episode amplitude, 13 episodes measured in 5 experiments) than
transient depolarizations averaged in the 2 min before an episode
(9.4 ± 2.5% of the episode amplitude; 13 episodes measured in 5 experiments
P = 0.015, t-test). In some
motoneurons (e.g., sartorius and adductor), the initial depolarization
was accompanied by significant discharge (Fig. 3E). This
discharge was not observed in individual interneurons or in the VLF
(Ritter et al. 1999
).
Can motoneuron discharge trigger episodes?
The presence of intense pre-episode discharge in some motoneurons
raised the possibility that it might be causally involved in triggering
an episode. To test the ability of ventral root stimulation to trigger
episodes, stimulus trains (50 Hz, 10 pulses) were delivered every 2 min
during the inter-episode interval, while recording spontaneous episodes
from another ventral root. The experiments were performed in two
segment preparations (LS2-LS3 or LS3-LS4) in which spontaneous
activity occurred at regular intervals (Fig.
4). Because episodes occur spontaneously,
it was important to ensure that the ventral root stimulus actually
triggered an episode and was not simply coincident with a spontaneously occurring episode. We adopted two criteria for this purpose. The first
was that the latency from the stimulus to episode onset should be <700
ms. We chose this comparatively long latency because the time it takes
for a stimulus to trigger an episode can be several hundred ms
depending on the stimulus intensity (Ritter et al.
1999) and when in the in the inter-episode interval the stimulus is presented. The second was that the stimulus should cause
the episode to occur significantly prematurely. For this purpose, we
first obtained control recordings of spontaneous episodes to establish
the control inter-episode interval. Stimulus trains (10 pulses at 50 Hz) were then delivered to one of the ventral roots every 2 min while
recording from an adjacent ventral root.
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An example of this type of experiment is illustrated in Fig. 4. In this experiment, the control inter-episode interval was 11 min 37 s ± 19 s measured from three inter-episode intervals. We found that ventral root stimuli were capable of evoking episodes prematurely at 8 min 20 s and 8 min after the previous episode (70.3% of the normal interval) and that the latency from the stimulus to episode onset was 433 ms. We also found that the stimulus was only effective when presented in the last 1/4 of the interval. In four experiments, eight episodes were evoked by such stimuli at 73.0 ± 7.8% (P < 0.01, paired t-test) of the normal interval of spontaneously occurring episodes and with an average latency of 427 ± 76 ms from the onset of the stimulus.
In the next set of experiments, we investigated the mechanism of the
ventral root triggering of the episode. Previous work had shown that
motoneurons make direct cholinergic connections with a class of
interneuron located dorsomedial to the lateral motor column
(R-interneurons, see Fig. 1) (see also Wenner and O'Donovan
1999). R-interneurons project depolarizing GABAergic (and
possibly glycinergic) connections onto motoneurons and other interneurons. To establish if R-interneuron activation mediated the
activation of episodes by ventral root stimulation, we examined the
ability of ventral root simulation to trigger episodes in the presence
of mecamylamine (to block the nicotinic cholinergic inputs to
motoneurons) or in the presence of bicuculline and strychnine (to block
the depolarizing outputs of R-interneurons to other spinal neurons).
In the presence 50 µM mecamylamine (applied for >30 min; Fig.
4B), we found that the ventral root-evoked ventral root
response was significantly reduced but not abolished during a train of stimuli (10 pulses at 50 Hz; Fig. 4B, right,
). In addition, the presence of the drug reduced the frequency of
spontaneous episodes but did not block them (inter-episode interval in
mecamylamine 20 min 4 s ± 250 s vs. 12 min 44 s ± 102 s in control, P < 0.05). Despite the
persistence of a small synaptic response in the adjacent ventral root,
we found that the ventral root stimulus was ineffective at triggering
an episode. This was true even when the stimulus was presented in the
last 10% of the inter-episode interval when network excitability
(defined as the ability of an external stimulus to trigger an episode)
was at its highest (average time between stimulus and episode = 62 s, range 9-107 s, determined from 11 spontaneous episodes in 4 experiments). The shortest time between the ventral root stimulus and
the next episode was 9 s (Fig. 4B,
,
right). The failure of ventral root stimulation to trigger episodes in the presence of mecamylamine suggests that the effect requires a functional synaptic connection between motoneurons and
R-interneurons and is not due to some other nonspecific effect of
activating motoneurons (e.g., K+ release,
electrical coupling). This result also makes it very unlikely that the
ventral root activation of an episode under control conditions was due
to the stimulation of afferents in the ventral root.
We also repeated the stimulus protocol in the presence of bath-applied
bicuculline (50 µM alone) or together with the glycine antagonist
strychnine (1 µM). We found that the drugs abolished the ventral root
evoked responses in motoneurons, suggesting that the R-interneuronal
output was effectively blocked (Fig. 4C, , right). In contrast to control conditions, episodes were
never evoked by the ventral root stimulus at short latency
(average = 67 s, range 28-112 s, 6 episodes, 2 experiments)
even when the stimuli were presented in the last part of the
inter-episode interval when network excitability was high (Fig.
4C). The shortest time between the stimulus and the next
episode was 28 s (Fig. 4C,
, right). In
one other experiment, 10 µM bicuculline and 1 µM strychnine significantly reduced but did not abolish the ventral root evoked ventral root response. In this experiment, two of two episodes occurred
independently of the stimulus trains.
These results show that motoneuron stimulation can trigger an episode
during the last quarter of the inter-episode interval when the network
is most excitable (Fedirchuk et al. 1999;
Tabak-Sznajder et al. 2000
) and that the effect is
likely to be mediated by the synaptic excitation of R-interneurons.
The R-interneuron-motoneuron loop can be substantially activated while the rest of the spinal network is relatively inactive
To understand better the activation of the network by the ventral
root stimulus and the role of R-interneurons in this process, we
obtained whole cell recordings from 24 R-interneurons in multi-segment, spontaneously active cords while stimulating ventral roots or muscle
nerves (dorsal roots cut). R-interneurons were identified by the
presence of short-latency (4.6 ± 0.5 ms, range 3.9-5.3 ms,
n = 21, in 3 additional cells the latency was <5 ms
but the stimulus artifact prevented a precise measurement) monosynaptic potentials following stimulation of the ventral roots or a muscle nerve
with the dorsal roots cut (Wenner et al. 1999).
Stimulus trains were delivered to one muscle nerve while recording intracellularly from an R-interneuron and extracellulary from another muscle nerve or from the VLF. When the stimulus train was delivered early in the inter-episode interval (Fig. 5, left), the intracellular membrane potential of the R-interneuron and the slow potential recorded from the adductor muscle nerve both rose to a peak within 200-300 ms but then decayed back to baseline within a second. Despite the failure of the stimulus to evoke an episode, a substantial depolarization of R-interneurons and motoneurons occurred whose amplitude could approach that of an episode (compare Fig. 5A, right and left). Despite the positive-feedback nature of this connection and the substantial recruitment of motoneurons, the stimulus did not trigger an episode. These large amplitude depolarizations were not simply aborted episodes because recordings from the VLF, a monitor of interneuronal activity, revealed only weak depolarizations that were much smaller than those accompanying an episode (Fig. 5B). Therefore the R-interneuron-motoneuronal circuit could be significantly activated while other interneurons were relatively inactive.
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The large amplitude of the R-interneuron and motoneuron depolarizations probably occurs because of the positive-feedback excitatory interconnections between motoneurons and interneurons. To illustrate this, recordings from several experiments were averaged to compare the timing and form of the rectified integrated muscle nerve discharge and the membrane potential trajectory of R-interneurons (Fig. 5C). They show that shortly after the second stimulus (Fig. 5C, bottom, *) in the muscle nerve train, asynchronous motoneuron discharge is recorded from the muscle nerve, presumably derived from the activated R-interneurons. This additional evoked discharge will further excite R-interneurons and probably accounts for their substantial depolarization.
When the stimuli were delivered during the last quarter of the
inter-episode interval (Fig. 5, right), the intracellular
membrane potential of the R-interneuron and the slow potential recorded from the adductor muscle nerve both rose to a peak within 200-300 ms
(Fig. 5A), followed immediately by a full episode (at )
accompanied by large depolarizing potentials in the VLF (Fig.
5B, right).
Imaging the recruitment of interneurons following ventral root stimuli
Based on the pharmacological and electrophysiological experiments
described in the preceding text, we hypothesized that a motor nerve
stimulus applied in the latter part of the inter-episode interval would
first activate R-interneurons, which would in turn activate the rest of
the interneuronal network. To test this idea directly, we used calcium
imaging to compare the onset of optical activity in the R-interneuron
region with that of other interneurons retrogradely labeled by the
application of calcium-green dextran to the ventrolateral cord 1-2
segments caudal to the site that was imaged (O'Donovan et al.
1993; Wenner and O'Donovan 1999
). These and
subsequent imaging experiments were performed on a single isolated
segment of the cord (typically LS3) to minimize the possibility that
the initiating activity would occur deep in the tissue, remote from the
cut face.
We first established the location of the R-interneuron region
stimulating the ventral root early in the inter-episode interval. This
activated a region dorsomedial to the lateral motor column that we have
argued is the location of synaptically activated R-interneurons (see
Fig. 1) (see also Wenner and O'Donovan 1999). When the
ventral root was stimulated in the last quarter of the interval, the
optical activity was first observed in the R-interneuron region but
then expanded to include many of the labeled interneurons ipsi- and
contralaterally, as an episode was triggered (Fig.
6A) and the rhythmic cycling
activity was observed (Fig. 6B). This finding suggested that
R-interneurons were among the first spinal neurons to become active
following the stimulus and that other interneurons were recruited
subsequently. This pattern of recruitment was observed in 3/3
additional preparations. The earliest optical activity was observed in
the R-interneuron region for ~120 ms (4 frames; range 3-5) before
the episode was triggered (Fig. 6). This result, together with the
pharmacological evidence indicating that ventral-root evoked episodes
are blocked in the presence of cholinergic and GABAergic antagonists,
strongly supports the hypothesis that motoneuron activity
can activate the interneuronal network through the
activation of R-interneurons.
|
R-interneuron activity during spontaneous episodes
The next set of experiments was designed to establish if motoneuron activity and subsequent R-interneuron firing initiate spontaneously occurring episodes. For this purpose, we first obtained whole cell recordings from identified R-interneurons during spontaneously occurring episodes to determine their patterns of activity and to establish if these patterns were consistent with a role in episode initiation. We then used calcium imaging to establish if the R-interneuron region is activated before other labeled interneurons at the onset of spontaneously occurring episodes. Finally, we report the effects of cholinergic blockade on the pattern of activity and the sequencing of interneuronal recruitment at episode onset.
Whole cell recordings of R-interneuron activity during spontaneous episodes
During spontaneous episodes, R-interneurons received a
depolarizing synaptic drive that was similar in form to the population potentials recorded from muscle nerves (Fig.
7A). Consistent with a role in
the initiation of the activity, they fired an initial burst of spikes
(Fig. 7A, *) at the onset of the episode; 18 of 24 cells
fired a burst of action potentials [63 ± 27 (SD) Hz], while the
remaining 6 cells fired a single spike at the onset of the episode. The
pattern of R-interneuron discharge during the episode was most clear
when cycling was distinct (Fig. 7A, right). In
all of the cells (24/24), firing also occurred at the beginning of each
cycle. Figure 7B compares the timing of the initial burst of
spikes recorded from 11 R-interneurons (from 9 experiments) at episode
onset with the slow potential recorded from the sartorius muscle nerve
averaged from the same nine experiments. The onset of spiking in
R-interneurons occurred in 7/11 cells before the peak of the averaged
depolarization recorded from the sartorius muscle nerve at an
appropriate time to initiate activity in other interneurons. For
technical reasons, we did not record from VLF because we were
interested in comparing the electrical activity recorded from the
muscle nerves with that of R-interneurons. However, in a previous
study, we noted that the peak discharge of the sartorius nerve occurred
before the onset of firing in a small sample of non-R-interneurons
(Ritter et al. 1999) consistent with our hypothesis that
R-interneurons trigger activity in the rest of the network.
|
Optical recordings of R-interneuron activity at the onset of spontaneous episodes
In the next set of experiments, we tested the hypothesis that R-interneurons become active before other spinal interneurons at episode onset, by comparing the activity of the R-interneuron region with that of other interneurons. The cells were labeled with calcium-sensitive dyes as described in the preceding text. Single-segment slices were used so that the optical activity would not simply reflect the spread of activity from a distant initiation site. Our strategy was to visualize many labeled interneurons simultaneously at the onset of spontaneously occurring episodes and establish which region became active first. We recognize the limitations of this experiment because not all interneurons will be labeled. However, if the R-interneuron region does not become active before other labeled interneurons, it will refute the hypothesis.
Figure 8 illustrates the optical signals
originating from labeled interneurons visualized in the cut transverse
face of a cord segment at the beginning of a spontaneous episode. In
these experiments, we first defined the R-interneuron region by ventral root stimulation applied during a period of low network excitability as
described previously (Fig. 8A). This region was marked, and then spontaneous episodes were monitored. Figure 8C
illustrates the initial video frames at the onset of a spontaneous
episode subtracted and normalized to the background fluorescence
(F/F, averaged from 4 spontaneous episodes
where activity began on the side ipsilateral to the application of the
dye). It can be seen that the earliest activity begins in the
R-interneuron region (Fig. 8C,
) from where it spreads to
the contralateral cell group near the central canal (Fig.
8C,
) to encompass the labeled cells on both sides of the
cord as an episode is triggered (Figs. 8 and
9A). In some instances, the
spread to the contralateral side occurred very early in the progression
of the optical activity (Fig. 8C). To establish the
stability of the R-interneuron recruitment pattern, we compared several
spontaneously occurring episodes in a single preparation. In each case
the R-interneuron region was the first to become active (Fig.
8B). In 2/2 other preparations, four or more episodes per
preparation were imaged and in each case the R-interneuron region was
the first region activated at episode onset.
|
|
Figure 8, D-H, shows the pattern of recruitment observed at episode onset across 5 different preparations. As can be seen from the figure, in each of these experiments (Fig. 8, D-H1) the optical activity was initiated in the R-interneuron region at episode onset. In one unusual experiment, optical activity occurred within the R-interneuron region as in the other experiments (Fig. 8H1). However, when a second episode occurred prematurely 1 min later, the earliest activity began outside this region (Fig. 8H2). This was the only occasion that we observed this pattern in a single segment preparation. Collectively, therefore, these data demonstrate the consistency of the onset pattern both within individual preparations and between different preparations.
These optical results are consistent with the whole-cell recordings from individual R-interneurons and provide additional evidence that R-interneurons are among the first interneurons recruited at the onset of spontaneous episodes.
Effects of cholinergic blockade on interneuronal recruitment at episode onset
The results described in the preceding text are consistent with a role for R-interneurons in episode initiation. To investigate this hypothesis further, we applied cholinergic antagonists (3 experiments) and compared the timing of electrical activity recorded from ventral roots and optical activity recorded from labeled interneurons. In two experiments, mecamylamine and atropine were bath applied together. In another experiment, mecamylamine was first added to the bath solution alone and then atropine was added later. The results were similar for both conditions. In two of these experiments, interneurons were labeled bilaterally to allow comparison of the recruitment patterns on each side of the cord.
Following bath application of nicotinic and muscarinic cholinergic antagonists (50 µM mecamylamine, 2 µM atropine) to block the recurrent input from motoneurons to R-interneurons, the pattern of interneuronal recruitment changed. We found that interneurons located medial to the motor column on both sides of the cord, sometimes including the R-interneuron region, became active synchronously at episode onset. In addition, the rise time of the optical and electrical signals slowed in the presence of the drugs (Fig. 9B).
These differences in the spatial pattern of recruitment can be seen
more clearly in the averaged records shown in Fig. 9C. In
this experiment, three successive frames were averaged under control
conditions to show the location of the initial optical activity at
episode onset. In Fig. 9C, right, a similar average was
performed when the activity began in the presence of cholinergic blockade (mecamylamine and atropine). In this case, 15 frames could be
averaged because episode initiation occurred more slowly and emphasizes
the differences in the spatial distribution of activity under the two
conditions. This finding raises the possibility that the region around
the central canal is important in the initiation of activity in the
absence of a functional motoneuron to R-interneuron connection. This
medial region has also been implicated in rhythmogenesis in the
neonatal rat spinal cord (Kjaerulff and Kiehn 1996;
Kjaerulff et al. 1994
).
In addition to these changes in recruitment pattern at episode
onset, we also found that network excitability was decreased under
cholinergic blockade. This was manifest in three ways. First, as we
have mentioned, episode initiation was substantially slower in the
presence of the drugs. Under control conditions, the average rise time
(10-90%) of the optical signals in the R-interneuron region of the
initiating side (measured in 10 episodes, 3 experiments) was 179.7 ± 50.3 ms, and this slowed to 298.1 ± 119.8 ms (measured in 8 episodes, 3 experiments). Second, the interval between episodes in
single segment preparations lengthened by 82.5 ± 38.6%
(n = 4 experiments, 13 control intervals, 8 drug
intervals) in the presence of the drugs. This lengthening of the
interval may also explain why episodes are longer in the presence of
cholinergic antagonists (Ritter et al. 1999)
because previous work has shown that longer intervals are
accompanied by longer episodes (Tabak et al. 2000
). As
illustrated in Fig. 10A, the
inter-episode interval lengthened after application of the cholinergic
antagonists as described previously for glutamatergic blockade
(Barry and O'Donovan 1987
; Chub and O'Donovan
1998
; Tabak et al. 2000
). Finally, the amplitude
of the spontaneous transients recorded from the ventral roots and
optically from labeled interneurons increased over the control values
(Fig. 10B). Under cholinergic blockade, the initial depolarizations were 166.5 ± 23.3% of their control value
(n = 5 experiments, 8 episodes). The increased
amplitude of these initial depolarizations is consistent with the idea
that the threshold for episode initiation has increased in the presence
of the drugs. Indeed, we also found that the amplitude of the transient
ventral root depolarizations occurring in a 2-min pre-episode measuring period, was significantly larger in the presence of the drugs than in
control (153 ± 37% of the control level, P < 0.01, paired t-test, 76 transients in drugs; 44 in control
measured in 5 experiments, Fig. 10B).
|
The increased amplitude of the transients may be related to the lengthening of the inter-episode interval that accompanied cholinergic blockade. We showed earlier (Fig. 3) these transients are depressed after an episode, suggesting that their appearance later in the inter-episode interval reflects the progressive increase in the amplitude of evoked and spontaneous synaptic potentials during the inter-episode interval. Because the inter-episode intervals are longer under cholinergic blockade, the recovery time is also longer, which may account for the larger amplitude of the events. Although we do not have causal evidence that the initial depolarizations actually initiate an episode, the increased amplitude of these initial depolarizations under cholinergic blockade is consistent with the idea that larger transients are required to initiate an episode in the absence of a functional motoneuron to R-interneuron connection.
We quantified changes in the timing of optical activity at episode onset by measuring the fluorescence changes within the R-interneuron and another region medial to the lateral motor column (non-R-interneuron region, see Fig. 11, inset) in each video frame. These were then compared with the simultaneously recorded ventral root potentials (Fig. 11). The rise of the fluorescence in the R-interneuron region on the initiating side started before that in the non-R-interneuron region on the same side in every episode we examined (12 episodes in 3 experiments; Fig. 11A). The average delay of the optical signal measured from the R- to non-R-interneuron region on the initiating side of the cord was 52 ± 10 ms (range 14-116 ms). In these and in subsequent experiments, we measured the delay when the fluorescence change reached 5% because at higher levels the optical signal in both regions was contaminated by the fluorescence increases of cells outside these regions. In 4/12 episodes, the next region to become active was the non-R-interneuron region on the contralateral side of the cord (rather than the ipsilateral non-R-interneuron region). The delay to the activity of this contralateral region was only 17 ± 3 ms (measured at 5% fluorescence) and may suggest the existence of a contralateral connection to this medial group of cells.
|
If motoneurons activate R-interneurons that then recruit the rest of the interneuronal network into an episode, then blocking the recurrent excitation of the R-interneuron population should change the timing of recruitment in the R- and non-R-interneuron regions. We therefore investigated the effects of cholinergic blockade on the timing of optical and ventral root activity. In the presence of the nicotinic cholinergic antagonists mecamylamine or a combination of mecamylamine and the muscarinic antagonist atropine, the sequence of recruitment changed (Fig. 11B). We found that the earliest optical activity began in the non-R-interneuron region in 8/10 episodes (3 experiments). The average delay between activity in the non-R-interneuron region and the next active region was 124 ± 31 ms (range 6-266 ms, 8 episodes/3 experiments).
Collectively, the results show that cholinergic blockade changes the way the network is recruited in single segment preparations and are consistent with our hypothesis that under normal conditions the pre-episode motoneuron discharge activates R-interneurons. Furthermore, the increase of the inter-episode interval in cholinergic blockade and the larger initial depolarizations suggest that, in the absence of the facilitating action of motoneuron discharge on R-interneurons, it takes longer for the interneuronal network excitability to achieve a level that can sustain an episode.
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DISCUSSION |
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In this paper, we have investigated the mechanisms involved in triggering spontaneous episodes in developing spinal networks of the chick embryo. We have found that motoneurons and interneurons experience spontaneous transient depolarizations that can be observed 2 min before an episode and appear to be responsible for the pre-episode discharge of motoneurons. Such transients are not detectable in the 2 min immediately after an episode. These motoneuronal depolarizations and discharge can trigger network activity through activation of the intra-spinal target of motoneurons (R-interneurons). This result suggests a special importance for transient fluctuations of spontaneous synaptic activity and the output elements (motoneurons) of spinal networks in the initiation of spontaneous activity during development.
What causes the transient depolarizations and firing in motoneurons?
Transient depolarizations were recorded in the ventral roots and in the VLF 2 min before, but not after, spontaneous episodes. These transient synaptic events may be responsible for the motoneuronal firing and depolarization that were recorded immediately before an episode. The firing was particularly prominent in the adductor and sartorius motor nerves although it was seen to various degrees in all of the muscle nerves we examined. The prominence of the discharge in sartorius and adductor probably occurred because some of these motoneurons were already firing just before the episode. Both classes of motoneuron began to fire at ~1/3 of the inter-episode interval and continued to fire until an episode occurred. The initial depolarizations in motoneurons were accompanied by spatially diffuse calcium transients in interneurons labeled with calcium green but, surprisingly, not by discharge recorded from the VLF. What, then, is the origin of these inter-episode depolarizing transients that appear to eventually trigger an episode?
We propose two possible sources for the transients that are not mutually exclusive. The first possibility is that the motoneuronal depolarizations originate from the spiking of premotor interneurons. In support of this idea is the observation that the transient ventral root depolarizations were accompanied by similar transients in the VLF, particularly after cholinergic blockade when the amplitude of the transients was maximal. In addition, calcium transients synchronized with the motoneuronal depolarizations were recorded from interneurons retrogradely labeled with calcium green. The coincidence of the transients in motoneurons and interneurons suggests that both may originate from the firing of premotor interneurons. Such interneurons are likely to be few in number and are probably scattered throughout the gray matter because the calcium transients accompanying the depolarizations were of low intensity and spatially diffuse. However, we have been unable to resolve any significant interneuronal spiking during the inter-episode transient depolarizations or accompanying the initial depolarizations when motoneurons fire briskly. Of course, it is possible that we have not observed such activity because the relevant interneurons are few in number.
An alternative possibility is that action potential-independent quantal
release of transmitter might be responsible for the transient
depolarizations of motoneurons and interneurons when, by chance,
release from several sources is briefly synchronized. Recordings from
voltage-clamped spinal neurons have shown that there is a progressive
increase in the amplitude and possibly the frequency of spontaneously
occurring synaptic currents during the inter-episode interval and that
these events are activity-independent (Chub and O'Donovan
2001). It is possible that the spatially diffuse nature of
interneuronal optical signals accompanying the motoneuronal depolarizations reflects pre- or postsynaptic calcium transients associated with stochastic increases of transmitter release. According to this idea, motoneurons fire during the transients because they are
either already firing or closer to spike threshold than interneurons. In future experiments, it should be possible to establish if the spatially diffuse interneuronal calcium transients require action potentials by determining if they occur in the presence of TTX.
Definition of the R-interneuron region
During the initial depolarization, motoneurons began to fire
intensely ~500 ms before the onset of an episode. We propose that
this firing activates R-interneurons, which then excite the rest of the
network. Some of the evidence for this hypothesis is derived from the
optical recordings that show that the earliest interneuronal activity
following ventral root stimulation, or at the onset of a spontaneous
episode, occurs in a region dorsomedial to the lateral motor column
that we have defined as the R-interneuron region (Wenner and
O'Donovan 1999). For this reason, we now consider additional
evidence that this region contains interneurons monosynaptically activated by motoneuron recurrent collaterals. First, reconstruction of
the mediolateral positions of physiologically identified R-interneurons in the LS1 and LS2 segments demonstrated that they were located dorsal
to, and overlapped mediolaterally with, the adductor motor nucleus.
This location, which was established electrophysiologically, coincided
with the R-interneuron region (Fig. 1). Second, ventral root
stimulation activated the same region in the presence of the
glutamatergic antagonists AP5 and CNQX, the GABA antagonist bicuculline, and the glycinergic antagonist strychnine. When coupled with the observation that ventral root activation of the R-interneuron region is abolished by cholinergic antagonists (Wenner et al. 1999
), these findings make it very unlikely that the region is activated exclusively through polysynaptic projections from motoneuron collaterals or by the activation of glutamatergic, ventral root afferents. Although glutamatergic ventral root afferents have been
identified in the rat spinal cord (Jiang et al. 1991
),
they have not been described in the chick embryo. Chu-Wang and
Oppenheim (1978)
have argued that the number of axons in the
ventral root corresponds to the number of cell bodies in the lateral
and medial motor columns, suggesting the presence of few, if any,
ventral root afferents at this stage of development. Finally, in
horizontally cut ventral-half preparations of the cord (see
METHODS), we have observed an optical signal in the
R-interneuron region with a single shock applied to the ventral root,
again making it unlikely that the region was activated polysynaptically
(unpublished observations). Thus while it is possible that the optical
signal following ventral root stimulation (in the absence of drugs) may
contain a contribution from non-R-interneurons, our evidence strongly
indicates that neurons monosynaptically activated by motoneuron
collaterals are located in this region.
Role of R-interneurons in the initiation of spontaneous episodes
Several lines of evidence implicate R-interneurons in episode
initiation. First, stimulation of the ventral roots or muscle nerves
was capable of triggering an episode when the stimulus was delivered in
the last quarter of the inter-episode interval when network
excitability was highest. This effect was mediated by R-interneuron
activation because it persisted in the presence of glutamatergic
blockade and was prevented by blockade of either the cholinergic
motoneuronal inputs to R-interneurons or the GABAergic output from
R-interneurons. Second, whole-cell recordings from physiologically
identified R-interneurons revealed that their initial spiking at
episode onset occurred ~50 ms before the peak depolarization recorded
from the sartorius muscle nerve. Although we did not compare the timing
of R-interneurons and non-R-interneurons, we can estimate their
relative timing indirectly from an earlier study. Ritter et al.
(1999) showed that the peak discharge of the sartorius nerve
occurred before the onset of firing in a small sample of
non-R-interneurons. Future experiments will be necessary to more
directly compare the timing of initial activity in R-interneurons versus other interneurons. Third, optical recordings from spinal cord
segments, containing interneurons labeled with calcium green dextran,
revealed that the earliest interneuronal fluorescence change at episode
onset began within the R-interneuron region and then spread to other
interneurons. Finally, we found that blocking the recurrent cholinergic
connection from motoneurons decreased the occurrence of spontaneous
episodes and altered the sequence in which interneurons were recruited.
Instead of the R-interneuron region leading the activity, we observed
an early signal experienced bilaterally in the medial part of the cord. Further, we found that in the presence of cholinergic antagonists, the
inter-episode interval lengthened, and the recruitment of motoneurons
and interneurons was substantially slowed at episode onset. In
addition, the initial depolarizations and corresponding optical
transients observed at episode onset were larger than under control
conditions, consistent with an increase in the threshold for episode initiation.
We propose that the reduction of network excitability in the presence
of cholinergic antagonists occurs, in part, because the facilitating
influence of the initiating motoneuron discharge on R-interneurons is
removed. As a result, the remainder of the interneuronal network has to
achieve a higher level of excitability to sustain episodes. We believe
in this condition, other interneurons, possibly in the region around
the central canal, are involved in the recruitment of the rest of the
spinal network. It is conceivable, however, that cholinergic
antagonists could depress network excitability independently of their
effects on the motoneuron to R-interneuron connection. Milner et
al. (1999) showed that cholinergic blockade reduced episode
frequency in chick embryos at E4-5, when the motoneuron/R-interneuron pathway is unlikely to be functional (unpublished observations). While
it is reasonable to believe that the effects of cholinergic blockade
differ between the two ages for developmental reasons, such a result
indicates that caution is required in attributing all of the effects of
cholinergic blockade on network excitability to an interruption of the
motoneuron/R-interneuron circuit.
Comparison with previous studies
In the neonatal hippocampus, spontaneous activity is expressed as
network-driven GDPs, which are similar to the spontaneous episodes
generated by the spinal cord. Menendez de la Prida and Sanchez-Andres (1999) used intracellular recording from
hippocampal pyramidal cells to investigate the mechanisms leading to
the initiation of a GDP. They found that some pyramidal cells in the
CA3 region fired during the interval between GDPs and then stopped
after a GDP. During the inter-GDP interval, the firing and the
underlying EPSPs exhibited transient increases that reached a maximum
100-300 ms before a GDP. The occurrence of a GDP could be predicted
when the underlying EPSP frequency exceeded a critical threshold,
suggesting that, like the spinal cord, the transients were involved in
triggering a GDP.
The spontaneous activity of hippocampal pyramidal cells described by
Menendez de la Prida and Sanchez-Andres shares many similarities to
that of the motoneurons we have described in the present work. This
raises the possibility that pyramidal cellsthe output neurons of the
hippocampus
might also play an important role in GDP initiation. However, a recent study using paired intracellular recordings from
different cell types in the neonatal hippocampus has argued that GDP
initiation does not depend on a specific neuronal population but
involves the co-operation between several cell classes (Menendez de la Prida and Sanchez-Andres 2000
). Nevertheless, these
authors did not directly attempt to record the initial activity within the slice or if such activity was reproducibly initiated by a particular cell class. It is generally assumed in both the hippocampus and in other developing networks that activity is initiated within a
small group of cells and then rapidly propagates to invade the rest of
the network. How the rest of the network is invaded will depend on the
details and connectivity of the particular network. Unfortunately,
activity does not originate from a fixed site so that capturing the
cellular details of this process is very difficult. High-resolution
optical imaging is one approach to this problem, but it is often
difficult to visualize a large field sufficient to capture the
initiation site and achieve cellular resolution. Calcium imaging has
been used to visualize activity within the developing hippocampus
(Canepari et al. 2000
; Leinekugel et al. 1995
), but no studies have focused on the recruitment of
different cell types at the initiation site. However, it might be
possible to approach this question in the hippocampus by imaging small pieces of tissue or islands that continue to generate spontaneous GDPs
(Kazipov et al. 1997
).
Instead of attempting to visualize the whole slice to capture the initiation site, the alternative is to record the activity of cellular populations. In the spinal cord, this is possible because the axons of motoneurons and some interneurons are accessible for recordings in the ventral roots and the VLF, respectively. These recordings allow us to monitor the earliest active members of the population at episode onset. Unfortunately, in a structure like the hippocampus this type of population recording is not possible.
Optical imaging to visualize initiation might prove more profitable in
the retina than in the hippocampus. This is because the retina is
essentially a planar structure that generates spontaneous waves that
propagate across the retinal surface (Feller et al. 1996; Meister et al. 1991
). It has been
postulated that retinal waves are initiated in the amacrine layer,
which is presynaptic to the output ganglion cells. According to a
recent model, waves are initiated when a critical number of amacrine
cells become coactive (Feller et al. 1997
). This model
predicts that amacrine activity should be more common than ganglion
cell activity, although some recent experimental evidence argues
against this conclusion (Zhou 1998
).
Functional significance of episode initiation by motoneurons and R-interneurons
We have argued that spontaneous episodes in the chick cord
are triggered by the motoneuron/R-interneuron circuit but have also
shown that spontaneous activity still occurs when this circuit is
interrupted pharmacologically. What then is the functional significance
of this particular mode of triggering, if network activity can still
occur in its absence? In previous work, we have proposed that the
recovery after pharmacological blockade occurs because of a progressive
increase in the strength of the remaining network connections, in
particular, GABAergic connections (Chub and O'Donovan
1998; Tabak-Sznajder et al. 2000
). Once
spontaneous activity has recovered in the presence of cholinergic
blockade, we propose that the distribution of activity and synaptic
strength across the population of active neurons has changed compared
with control conditions. We believe that some neurons (e.g., GABAergic neurons) now contribute comparatively more to network activity than
under control conditions. For example, preliminary results have
suggested that GABAergic synaptic potentials increase in amplitude
during recovery from excitatory blockade and remain elevated when the
inter-episode interval stabilizes (Tabak et al. 2000
).
The precise mechanisms of this increase are not understood but may
involve a change in intracellular chloride homeostasis (Chub and
O'Donovan 2000
). At present, the developmental consequences of
these changes in the distribution of activity and synaptic efficacy
within developing networks are unknown. However, it seems reasonable to
suppose that alterations of activity that occur by blocking the
recurrent connection and therefore changing the normal recruitment
pattern (i.e., lengthening inter-episode interval) may have important
developmental effects on neurons and their synaptic targets.
A second functional question to arise from our results is their
significance in terms of the adult function of R-interneurons. In adult
animals, the homologue of the R-interneuron is the Renshaw cell.
Studies in the adult cat have shown the Renshaw cell receives motoneuron input but inhibits its synaptic targets (Eccles et al. 1954). Although the precise function of Renshaw cells is
unknown, it is important to stress that the inhibitory output of adult Renshaw cells ensures that their function will be very different from
that during development (see Noga et al. 1987
) when the
output of the R-interneurons can be functionally excitatory. It is
highly unlikely therefore that Renshaw cells would initiate rhythmic activity in the adult spinal cord.
However, after blockade of the motoneuron/R-interneuron pathway,
the initial activity at episode onset occurred bilaterally in the
medial part of the cord in the vicinity of the central canal. This
region has been implicated in rhythmogenesis in the rat spinal cord
(Kjaerulff and Kiehn 1996; Kjaerulff et al.
1994
). The results of the recurrent blockade suggest that this
medial region may have a similar importance when the output of
R-interneurons becomes functionally inhibitory in the developing chick.
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ACKNOWLEDGMENTS |
---|
We are grateful to R. Burke, N. Chub, C. McBain, and J. Tabak for comments on the manuscript.
This study was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program.
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FOOTNOTES |
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Address for reprint requests: P. Wenner, NIH, NINDS, Lab. of Neural Control, Bldg. 49, Rm. 3A50, 49 Convent Dr., Bethesda, MD 20892-4455.
Received 18 December 2000; accepted in final form 12 April 2001.
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REFERENCES |
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