Afferent Inputs Modulate the Activity of a Rhythmic Burst Generator in the Rat Disinhibited Spinal Cord In Vitro
E. Bracci,
M. Beato, and
A. Nistri
Biophysics Sector and Istituto Nazionale Fisica della Materia Unit, International School for Advanced Studies, 34013 Trieste, Italy
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ABSTRACT |
Bracci, E., M. Beato, and A. Nistri. Afferent inputs modulate the activity of a rhythmic burst generator in the rat disinhibited spinal cord in vitro. J. Neurophysiol. 77: 3157-3167, 1997. Application of strychnine and bicuculline to the isolated spinal cord of the neonatal rat induces spontaneous bursting of regular rhythmicity (cycle period ~30 s). This phenomenon is important because it shows that a spinal network, made up by excitatory connections only, generates a very reliable rhythmic pattern. To find out how signals from the periphery or higher centres might influence the operation of the rhythmogenic network, the present experiments examined whether synaptic inputs from dorsal root (DR) or ventrolateral (VL) afferent fibers could modulate this spontaneous rhythmicity. This issue was addressed with intracellular recording from motoneurons or extracellular recording from ventral roots after eliciting bursting with strychnine plus bicuculline. Single electrical shocks (0.1 ms; intensity 1-4 times threshold) applied to one DR reset spontaneous bursting without altering its period or duration. Repetitive stimulations at periods ranging from 20 to 2 s entrained bursts on a 1:1 basis. Burst duration was shorter at lower stimulation periods whereas burst amplitude was unchanged. The lowest stimulation period compatible with burst entrainment depended on stimulus strength. At stimulation periods <2-s entrainment was always lost and spontaneous bursts unexpectedly returned even if electrical pulses still elicited ventral root reflexes. Such spontaneous bursts had similar properties as those recorded in the absence of electrical pulses. Analogous results were obtained with VL stimulations. It is concluded that the spinal rhythmogenic network was highly susceptible to external synaptic inputs, which paced burst generation whereas burst duration was adapted to interstimulus interval. A scheme is provided to explain the modulatory role of synaptic inputs as well as the escape of bursting from fast stimulus entrainment in terms of a rhythmogenic network functionally separated from reflex pathways activated by DR or VL tracts.
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INTRODUCTION |
Pharmacological block of synaptic inhibition provides an experimental condition useful to investigate those properties of a neural network that rely upon its excitatory connections only. In several regions of the central nervous system (CNS), such a block induces spontaneous bursting. This phenomenon has been observed in vitro in the cerebral cortex in the presence of bicuculline (Hwa et al. 1991
), in the hippocampus after application of picrotoxin (Hablitz 1984
; Miles et al. 1984
) or tetanus toxin (Jordan and Jefferys 1992
), and more recently in the neonatal rat spinal cord when strychnine and bicuculline are co-applied to suppress glycine and
-aminobutyric acid-A (GABAA)-receptor-mediated inhibition, respectively (Bracci et al. 1996a
,b
). In the latter preparation, this bursting activity is of particular interest because it exhibits a highly regular rhythmic pattern, which arises spontaneously and lasts for many hours (Bracci et al. 1996a
), showing that a disinhibited network is able not only to generate bursts but also to control their duration and periodicity. Such a spontaneous rhythmic activity can be recorded from single motoneurons as well as from ventral roots (VR) and appears synchronously in all lumbar motoneuronal pools (Bracci et al. 1996a
,b
). This bursting was shown to be driven by a premotoneuronal network requiring glutamatergic transmission and to survive surgical ablation of the dorsal laminae (Bracci et al. 1996a
,b
).
An important question of functional significance is whether the rhythmogenic network responsible for this behavior is accessible to external synaptic inputs and to what extent such inputs are able to modulate its operation as one might expect for a network capable of expressing appropriate responses to behavioral requisites (for a review, see Marder and Calabrese 1996
). Because in the mammalian spinal cord rhythmogenic networks controlling patterned motor activity (locomotion, scratching, etc.) are modulated by sensory inputs and descending pathways (for discussion see Katz 1996
), it is important to examine whether the bicuculline- plus strychnine-induced rhythm is similarly susceptible to modulation by such afferent inputs. Hence, the present study addressed the following questions: can inputs from DR or VL descending fibers affect the disinhibited rhythm? Are the characteristics of bursting modified by afferent signals? Can such a rhythm be entrained by patterned afferent impulses and if so, can certain stimulation protocols result in uncoupling of bursting from spinal reflex activity? Addressing the latter issue also should clarify whether rhythmogenesis involves the whole spinal circuits deprived of inhibition or whether the rhythm is generated by some specialized areas (distinct from reflex pathways) and then relayed to motoneurons. This study is therefore expected to provide a minimal wiring scheme to account for interaction of DR and VL afferent inputs with the rhythm generating network.
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METHODS |
Experiments were performed on spinal cord preparations isolated from neonatal rats (2-13 days old) under urethan anesthesia (6 ml ip of 10% wt/vol solution) according to previously described methods (Ballerini et al. 1995
, 1997
; Fisher and Nistri 1993
). Spinal cords (from midthoracic region to cauda equina) were fixed to the bottom of a small bath (at room temperature) and continuously superfused (7.5 ml/min) with Krebs solution of the following composition (in mM): 113 NaCl, 4.5 KCl, 1 MgCl27H2O, 2 CaCl2, 1 NaH2PO4, 25 NaHCO3, and 11 glucose, gassed with 95% O2-5% CO2, pH 7.4. Drugs were applied via the bathing solution at the concentrations mentioned in the text. Lumbar (L4 or L5) motoneurons (Fulton and Walton 1986
) were identified functionally by all-or-none firing after antidromic stimulation (0.1 ms; near-threshold intensity) of a VR. For intracellular recording, 3 M KCl-filled microelectrodes (25-60 M
) under current-clamp conditions (either in bridge or discontinuous current-clamp mode) were used. VR recordings (predominantly from L5, although L3 or L4 also were used) were performed via miniature suction electrodes (containing an Ag/AgCl pellet) filled with Krebs solution. DR or VR stimulations were delivered via a miniature bipolar suction electrode. VL descending tracts were stimulated via a bipolar tungsten electrode consisting of two filaments insulated except at their tips (0.026 mm diam) and placed in contact with the spinal cord lateral surface at the level of the low thoracic cord (T11-T13). For VL and DR stimulations, stimulus duration was 0.1 ms, whereas intensity varied between 1 and 4.5 times threshold (×T) to recruit low-threshold fibers (see Kiehn et al. 1992
). Threshold was defined as the minimum intensity that, in standard solution (without strychnine and bicuculline), elicited a synaptic response in a motoneuron or in the ipsilateral ventral root [on average T = 1.8 ± 0.5 V (mean ± SD) for dorsal root, n = 28 and T = 18 ± 4 V for ventrolateral stimulations, n = 11]. Tests were carried out to monitor the afferent volley induced by DR stimulation. For this purpose, a 3 M NaCl-filled microelectrode was inserted at the site of entry of the dorsal root at a distance of 3.5 ± 0.5 mm from the stimulating electrode. In the range 1-4.5 × T, the afferent volley appeared as a biphasic waveform with the earliest detectable peak occurring at 0.5 ms from the start of the stimulus artifact (any faster components could not be resolved from the artifact). Such a peak corresponded to fiber activity with 6.3-m/s conduction velocity and was associated with ventral root responses having latencies <4 ms. Repetitive stimuli at a given period usually were applied for 5 min (though in some cases for
15 min). Ventral root responses (which comprised electrotonically conducted slow potentials and spike activity) were recorded with DC coupled (30-kHz low-pass filter) amplification, stored on video tape for further analyisis, digitized at 1-10 kHz with pClamp program (Axon Instruments; version 6.2), and displayed on a Gould RS3400 chart recorder. Details concerning the definition of bursts and their measurements (duration and cycle period) are as previously reported (Bracci et al. 1996a
,b
). The coefficient of variation for the cycle period (CVp) is defined as the ratio between the standard deviation of the cycle period and the mean of the cycle period and converted into percent values. The coefficient of variation of burst duration (CVd) was calculated in a similar manner. Data are expressed as means ± SD. Statistical differences were analyzed by using the Student's t-test. Two groups of data were considered not significantly different when P > 0.05.
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RESULTS |
Experiments were performed on 35 spinal cord preparations in the presence of strychnine (1 µM) and bicuculline (20 µM) to block glycine and GABAA receptors, respectively (see Bracci et al. 1996b
). Under these conditions, a regular pattern of rhythmic bursts developed spontaneously on lumbar motoneurons (Bracci et al. 1996a
) with an average cycle period of 27.3 ± 7.5 s and average burst duration 7.3 ± 4.1 s.
Characteristics of intracellularly recorded bursts
The effects of DR stimulations delivered during spontaneous bursting first were studied with intracellular recording from motoneurons. An example of this type of experiment is illustrated in Fig. 1A: under current-clamp conditions, a L5 motoneuron displayed the typical bursting pattern (top) with spontaneous bursts comprising large amplitude intraburst oscillations and action potentials, (Fig. 1A, bottom left), where part of the record is displayed at higher speed. The interburst period was characterized by synaptic quiescence. The arrow in Fig. 1A, top, indicates the time when a single stimulus (1.5 × T) was delivered to the ipsilateral L5 dorsal root. Such a stimulation evoked a bursting event similar to the spontaneous ones and comprising an equivalent intraburst oscillatory structure, as also illustrated on a faster time scale in Fig. 1A, bottom right. The dorsal root stimulation did not disrupt the regular pattern induced by strychnine and bicuculline but merely reset it, because the interval between the stimulus and the onset of the subsequent spontaneous burst was similar to the spontaneous cycle period, which then resumed its regular rhythm.

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| FIG. 1.
Effect of dorsal root (DR) stimulation on spontaneous bursting recorded intracellularly from lumbar motoneuron. A, top: spontaneous bursts occurring in presence of strychnine and bicuculline are reset by a single DR pulse [1.5 times threshold (×T); applied at time indicated ( )], which induces a bursting edpisode. Note that, after this event, bursting recommences with similar period as before. Bottom: expanded timebase trace of 2 individual bursts either arising spontaneously (left, immediately before electrical pulse) or evoked by DR stimulus (right). Note similar structure and duration of evoked and spontaneous events. Resting potential, 71 mV. B: scatter plot of interval between end of preceding spontaneous burst and stimulus (abscissa) vs. interval between stimulus and first spontaneous burst (ordinate, data are normalized with respect to mean of interburst interval for spontaneous bursting in each cell, n = 5). Horizontal line is regression line fitted to the data. C: different cell than in A; fast timebase records of responses elicited by DR pulses of varying intensity (indicated in terms of T values) and comprising initial synaptic potential followed by burst with latency progressively reduced up to fusion with early synaptic response (bottom, right). Resting potential, 74 mV.
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Comparable data were obtained from five intracellular experiments (in 4 different preparations) in which single DR stimulations were delivered at different phases of the cycle (during the quiescent period). Dorsal root stimulation reset the spontaneous rhythm regardless of its timing after the end of the previous spontaneous burst. To assess quantitatively this phenomenon, the interval between the onset of an evoked burst and the onset of the following spontaneous one was plotted as a function of the time between the end of the preceding spontaneous burst and the stimulus (Fig. 1B;n = 5). The slope coefficient of the regression line of these data was not significantly different from 0. Furthermore, the average interval between the stimulus and the onset ofthe next spontaneous burst (25.3 ± 3.3 s) was not significantly different from the average spontaneous cycle period (28.2 ± 2.3 s; measured for >3 min before stimulation). CVp or CVd values measured during 3 min before stimulation were not significantly different from corresponding values measured during 3 min after stimulation (CVp = 17 ± 6% and 15 ± 5%; CVd = 10 ± 4% and 12 ± 4%).
The sensitivity of bursts to stimulation strength (range between 1 × T and 4 × T) was next tested. In the presence of strychnine and bicuculline, the threshold for postsynaptic potentials (PSPs) did not change with respect to the one found in control solution; nevertheless, even a PSP at or just above threshold was always followed (after a certain latency) by a burst. This is exemplified in Fig. 1C in which the fast traces display the short-latency PSP and the initial component of the bursting episode. Although amplitude, duration, or structure of evoked bursts did not depend on pulse intensity (data not shown), the burst time-to-peak (from stimulus artifact to initial peak of evoked burst) was related strongly to the stimulus strength as illustrated by the intracellular record in Fig. 1C (different preparation from Fig. 1A). A single 1.1 × T stimulation (note downward deflection representing stimulus artifact and delivered after
15-s quiescence) elicited, first, a short-latency (<10 ms) small-amplitude PSP (amplitude = 2.1 mV), which was followed by a burst peaking at 265 ms. Under the same conditions, increasing stimulation intensity to 1.3 or1.7 × T decreased the burst time-to-peak to 206 or 129 ms, respectively, whereas the PSP grew in amplitude from 11.2 to 21.3 mV without latency change. With 3.4 × T stimulation, the burst peaked at 94 ms so that it became indistinguishable from the PSP. On the other hand, single or repetitive stimulations of one (or more) VR at intensity
10 × T applied during quiescent periods did not trigger or affect bursting. This phenomenon is illustrated in the example of Fig. 2A in which a train of three VR stimuli (4 × T for firing) failed to modify bursting when delivered midcycle between bursts or immediately before the onset of a burst. Each VR pulse however produced a full antidromic spike (see Fig. 2B; similar data were obtained in 5 cells).

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| FIG. 2.
Antidromic spikes do not affect spontaneous bursting. A: intracellular records of spontaneous bursting induced by strychnine and bicuculline. Arrowed ventral roots (VRs) indicate antidromic stimuli (4 × T stimulation) to L5 ventral root, which always evoked action potentials. Artifacts are clipped by sampling frequency. *, responses shown as individual action potential at faster time base in B. Membrane potential during quiescent periods was 65 mV. Note that regardless of timing of antidromic firing during bursting cycle there was no alteration in spontaneous bursting activity.
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Repetitive stimulation protocols often led to unstable recording conditions that made unsuitable the use of the intracellular technique for this type of study. Because rhythmic bursting in the disinhibited spinal cord takes place simultaneously within motoneuron pools, ventral root recordings are adequate to detect bursts and intraburst oscillations with all their essential characteristics (Bracci et al. 1996b
). Bursting entrainment was investigated further by means of this technique, which additionally allowed monitoring responses from more than one VR.
Entrainment at different frequencies
Entrainment of bursting was defined as the ability of a periodic external stimulus to elicit bursts on a 1:1 basis. The effects of repetitive DR stimuli delivered at various intervals were first tested. Figure 3 illustrates one example in which left L5 VR activity was recorded during stimulation (at different periods) of the ipsilateral L5 DR. Spontaneous bursting (Fig. 3, top) took place with a cycle period of 25.4 ± 1.8 s and a burst duration of 2.8 ± 0.5 s. Individual bursts comprised several intraburst oscillations, as shown in the expanded trace (Fig. 3, right) of the arrowed event (left). An example of entrainment is shown in Fig. 3, middle. In this case, stimuli were delivered at 10-s intervals and 3 × T intensity. Each stimulus was followed by an evoked burst similar to the spontaneous ones but significantly (P < 0.01) reduced in duration (on average 2.1 ± 0.3 vs. 2.8 ± 0.5 s, see also expanded trace on right). For this preparation, the minimum period at which entrainment was possible was 2 s (not shown). Figure 3, bottom, shows that, although1-s period stimulation failed to elicit bursting, spontaneous bursting activity (with frequency similar to the one observed before stimulations) rather unexpectedly returned in the presence of DR pulses (the latter are seen as fast upward deflections). When this type of stimulation ceased, spontaneous bursting persisted with an apparently unperturbed time course. The expanded trace in Fig. 3, bottom right, shows that at 1-s intervals, DR stimuli, despite the absence of bursting entrainment, still elicited depolarizing reflexes (lasting
200 ms) as indicated by the time-locked responses that still were present even during spontaneous bursts and seemed to be influenced minimally by the underlying burst. At the same time, bursts maintained a time course similar to the one recorded in the absence of stimulations and were not time-locked with the electrical stimuli. Similar results were obtained from 25 preparations using in each case stimuli of fixed intensity (3 × T) delivered to one L5 DR while recording from the ipsilateral L5 VR. The lowest interval of stimulation able to support entrainment was in the range of 2-5 s (see histogram of Fig. 4B) with an average value of 2.8 ± 0.8 s. Spontaneous bursting, which recommenced despite the 1-s stimulus interval, occurred with a period of 21.1 ± 6.8 s, a value not significantly different from the one in the absence of stimuli (24.7 ± 4.5 s) although the CVp was larger (24.5 ± 10.8 vs. 10.4 ± 8.9%) during such a stimulation.

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| FIG. 3.
Effects of repetitive stimuli applied to L5 DR (at various periods) on bursting recorded from L5 VR. Top: spontaneous bursts occurring in presence of strychnine and bicuculline. , event displayed (at right) at 10 times faster speed. Note intraburst oscillatory structure. Middle: 1:1 entrainment of bursts by DR pulses (10-s period); , event displayed (at right) at faster speed. Evoked bursts are of shorter duration while retaining oscillatory structure. Bottom: failure to entrain bursts by DR stimuli at 1-s periods (these are indicated by sharp, evenly spaced upward deflections): spontaneous bursts return despite electrical pulses and persist after end of electrical stimulation; right, shows, at faster speed, reflex activity induced by 1-s period pulses and 1 independent spontaneous burst during which evoked synaptic responses continue to be present.
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| FIG. 4.
Effects of high period stimulation of DR fibers on bursting induced by strychnine and bicuculline. A, top: right L5 VR record of spontaneous bursts; middle: electrical pulses (30-s period) are applied repetitively to ipsilateral L5 DR to coincide with early decay phase of spontaneous bursts. Electrically evoked bursts are preceded by stimulus artifacts. Note that this low period of stimulation fails to elicit 1:1 entrainment because spontaneous events coexist with evoked ones. Bottom: similar results were obtained at 40-s stimulation periods. B: distribution of the lowest stimulation period capable of supporting entrainment over 25 preparations. C: distribution of largest period of stimulation capable of supporting entrainment over 15 preparations.
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We next explored the longest stimulus interval able to induce 1:1 entrainment of spontaneous bursting. An example of this approach is illustrated in Fig. 4A in which a spontaneous cycle period of 26.6 ± 1.5 s (recorded from right L5 VR) was first observed (Fig. 4, top). When DR stimulation was delivered at 30-s intervals (see Fig. 4A, middle), each pulse evoked a burst followed by a spontaneous one that preceded the next evoked event. Figure 4A, bottom, shows comparable data with an interstimulus interval of 40 s. Thus in neither case the stimulation period was able to entrain bursting. In this preparation, the maximum period for one-to-one entrainment was 20 s, corresponding to a situationin which bursts never appeared spontaneously during stimulation. This maximum stimulus period for entrainment was lower than the period for spontaneous bursting. Valuesfor the largest interval of stimulation are illustrated byFig. 4C (n = 15 preparations), with an average value of19.5 ± 3.8 s.
Collectively, these data show that entrainment was possible over a relatively wide range of stimulation periods (typically between 2 and 20 s).
Side-to-side coordination during DR entrainment
In the absence of synaptic inhibition, spontaneous rhythmic bursts and intraburst oscillations take place simultaneously on both sides of the cord (Bracci et al. 1996a
). To verify whether such a synchronicity was preserved during repetitive DR stimulation, we recorded bilaterally from pairs of homologous VRs while stimulating one DR at the same segmental level. An example is illustrated in Fig. 5. In this case, stimulations (2 × T) were applied to left L5 DR while recording from left and right L5 VRs. During the 5-s stimulation periods, evoked bursts comprised three to five intraburst oscillations, which appeared simultaneously on both sides of the cord (Fig. 5, top). During the 1-s stimulation periods (Fig. 5, bottom), bursts could not be entrained (see also Fig. 3) and appeared simultaneously on both sides. Under these conditions, DR stimulation evoked a depolarizing reflex from the ipsilateral VR but was almost ineffective on the contralateral VR, as indeed observed even before adding strychnine and bicuculline (records not shown). Similar results were obtained with bilateral recording from L5, L4,or L3 VRs in four preparations. These data suggest strong, bilateral coupling of the rhythm generating network because bursts could not be restricted to one side only even by DR stimuli strongly asymmetrical in their ability to generate PSPs.

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| FIG. 5.
Bilateral recording of bursting activity at 2 different stimulation periods. Top: simultaneous recording from left (l) and right (r) L5 VRs showing 1:1 entrainment of bursting by left L5 DR stimuli applied every 5 s. Bottom: with similar protocol but lower stimulation period (1 s) burst entrainment fails whereas electrical pulses elicit reflexes detected only from lL5 VR. Note that spontaneous bursts and their intraburst oscillations appear simultaneously in both VRs.
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Dependence of burst duration on stimulation period
Because each preparation could be entrained by DR stimulation within a wide range of periods, the questionthen arises whether within this range burst duration and intraburst structure were also affected. This issue was explored in experiments like the one depicted in Fig. 6A. This preparation could be entrained between 15- and 2-s stimulation intervals (intensity = 3 × T), and records pertaining to 10, 5 and 2 stimulation periods are shown in Fig. 6A, top to bottom. During the 10-s stimulus periods (Fig. 6A, top), evoked bursts lasted 4.1 ± 0.4 s and comprised 9-13 intraburst oscillations (note that fast downward deflections are spike activity of ventral roots). During the 5-s stimulus periods, burst duration was 2.1 ± 0.2 s with four to seven intraburst oscillations. During the 2-s stimulus periods, burst duration was reduced to 1.1 ± 0.1 s and bursts comprised only three discernible oscillations. These changes in burst duration were statistically significant (P < 0.01) although they were not accompanied by significant changes in burst amplitude. Analogous results were obtained from 15 preparations. The average dependence of burst duration on stimulation period is plotted in Fig. 6B, where data are normalized with respect to the burst duration measured, in each experiment, for 10-s stimulation periods. It is apparent that burst duration decreased over a fivefold range when stimulation period was decreased, thus allowing the network to return earlier to the quiescent state when temporally closer stimuli were delivered.

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| FIG. 6.
Dependence of burst duration on stimulation period. A, top: bursts entrained by 10-s period of DR stimulation. Biphasic stimulus artefacts at beginning of each burst are closely preceded by short, upward calibration steps (0.5 mV). Fast downward deflections during burst oscillations are summated spike responses of motoneurons. Middle: halving stimulation period produces a reduction in burst duration of ~50%. Bottom:2-s period of stimulation still produces 1:1 entrainment of bursts, which become short event of ~1-s duration but retain their oscillatory structure. B: graph of stimulation period (log scale; s) vs. burst duration normalized with respect to response observed in each preparation with 10-s periods of stimulation. Data are from 25 preparations.
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Dependence of entrainment on stimulation strength
We also investigated whether the range of the stimulation period that supported entrainment depended on the stimulus strength. An example of this type of experiment is illustrated in Fig. 7. Figure 7A shows left L5 VR responses induced by single pulses applied to the ipsilateral L5 DR at 1 × T or4 × T in control solution before application of strychnine and bicuculline. Although 1 × T stimulation elicited a slight depolarizing response (0.12-mV amplitude), 4 × T stimulation not only produced a larger depolarization (0.24 mV) but also a fast, large-amplitude biphasic response presumably due to motoneuron action potentials. After establishing spontaneous bursting with strychnine and bicuculline (not shown), both stimulus strengths (at a constant 10-s period) were able to entrain bursts (Fig. 7B) that possessed similar amplitude, duration, and intraburst oscillatory structure, as also illustrated in the expanded traces of Fig. 7C. Like the case of a single DR stimulus (see Fig. 1B), only the time-to-peak of bursts depended on stimulation strength, as shown by the fast time scale tracings of Fig. 7D: with 1 × T stimulation, the initial peak was reached after 70-80 ms, whereas with 4 × T stimulation, it was reached after 25-35 ms. In the latter case, a fast biphasic response similar to that observed in control solution also was present.

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| FIG. 7.
Effects of changes in stimulus intensity on ability to entrain bursts. A: in standard solution (without addition of strychnine and bicuculline), a just threshold (1 × T; left) or 4 × T (right) pulse to L5 DR evokes small synaptic response or larger event with spike activity (see biphasic event during early part of response). Responses are averages of 5 recordings in each instance. B: in presence of strychnine and bicuculline, 1 × T or4 × T stimulation (10-s period) produces 1:1 entrainment of bursting. C: faster records of event marked in B ( ) indicate similar oscillatory structure of evoked bursts. Voltage calibration refers to B and C. D: on an even faster timebase, early component of evoked bursts depicted in C is displayed. Note that stronger pulse elicits response of shorter latency and with earlier spike activity.
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When the stimulation period was decreased, major differences between the effects of 1 × T and 4 × T stimulations emerged. These are illustrated in Fig. 8A (same preparation as in Fig. 7). In Fig. 8A, top, 1 × T stimuli (2-s period) could not entrain rhythmic activity, even if each stimulation generated a depolarizing reflex (duration
200 ms). Figure 8A, bottom, shows the effect of increasing stimulation intensity to 4 × T: in this case, entrainment at the 2-s period was possible as each pulse evoked a burst (lasting on average 1.25 ± 0.05 s). These results are summarized in Fig. 8B inwhich the lowest limit for the stimulation period capable of burst entrainment was plotted against values for stimulation intensity (1-4.5 × T range). It appears that with 1 × T intensity, entrainment was possible only at periods
10 s; between 1 × T and 2.5 × T values the lowest period at which entrainment was observed decreased from 10 to 2 s and reached a steady state level for intensities >2.5 × T. Conversely, changes in stimulus strength did not alter the largest value of stimulus period able to support entrainment (data not shown). Similar results were obtained in two other preparations. These results indicate that during closely spaced pulses the efficacy of DR pathways in triggering a burst episode was critically dependent on the stimulation intensity.

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| FIG. 8.
Different stimulus intensity shows differential ability to induce burst entrainment. A, top: at 2-s stimulation periods, a weak pulse intensity (1 × T before addition of strychnine and bicuculline) fails to support burst entrainment; a spontaneous burst develops during repetitive electrical stimulation (large downward deflections are spike activity of motoneurons); bottom: with same stimulation period as above, increase in pulse strength (4 × T) supports 1:1 entrainment of bursts. B: graph of stimulus intensity (expressed as multiples of threshold, T, obtained before adding strychnine and bicuculline) of DR electrical pulses vs. the lowest period of stimulation that allows burst entrainment. All traces are from same preparation shown in Fig. 7.
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Entrainment with VL stimulation
To analyze whether entrainment of the spontaneous rhythm in the disinhibited cord was restricted to DR stimulations, we also investigated the effects of VL stimulations. In control solution, these pulses (0.1 ms) evoked short latency (3-4 ms) synaptic responses comprising a depolarizing component (with superimposed spike activity when stimulus intensity was >3 × T) decaying biexponentially. These responses are similar to those previously reported for motoneuronal excitatory PSPs (EPSPs) evoked by VL stimulation (see for example Elliott and Wallis 1993
). To avoid excessive stimulation, pulse intensity was usually fixed at 3 × T. In the presence of bicuculline and strychnine, a single VL stimulus evoked a burst similar to the spontaneous ones and reset the spontaneous rhythm (data not shown) in the same fashion as previously found with single pulses applied to a DR (see Fig. 1). These observations prompted testing an extended range of VL stimulus periods to find out their entrainment ability. Figure 9 depicts the effect of three different stimulating periods on a preparation with stable spontaneous bursting (average cycle duration, 26.0 ± 5.2 s; burst duration, 3.2 ± 0.8 s; recorded from left L5 VR). Figure 9, top, shows that pulses at 10-s intervals fully entrained bursting on an 1:1 basis whereas the evoked burst duration (1.9 ± 0.3 s) was significantly reduced with respect to the spontaneous one (P < 0.01). One individual burst (marked by vertical arrow) is displayed on the right at expanded time scale to indicate that burst structure was preserved. Fig. 9, middle, shows that a train of 5-s interval stimuli induced entrainment and significantly reduced (P < 0.01) burst duration to 1.0 ± 0.2 s (see also faster right-hand trace of recording marked by arrow). Figure 9, bottom, represents the effect of 1-s interval stimuli: in this case the stimulation did not elicit a burst, but only a polysynaptic reflex (lasting 640 ± 80 ms) similar to the one recorded in the absence of strychnine and bicuculline (duration = 480 ± 70 ms). Despite the continuous application of VL pulses at 1-s intervals, spontaneous bursts still occurred (although at slower rate in this example). The arrowed sample trace of Fig. 9 is shown on the right at a faster speed, which allows to observe that the burst started just between two stimuli.

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| FIG. 9.
Effects of ventrolateral (VL) stimulation on bursting induced by strychnine and bicuculline. Top: 1:1 entrainment of bursting by 10-s periods of stimulation. Faster record of event indicated ( ) is shown (right). Middle: 1:1 entrainment persists with VL stimulation at 5-s periods. Evoked bursts are further reduced in duration. Events corresponding to arrow are displayed on a faster timebase on the right. Bottom: failure of entrainment by 1-s period stimulation (large upward deflections) while spontaneous bursts re-emerge. Burst marked ( ) is also shown on a faster timebase (right); note that this event is independent from evoked synaptic responses (time-locked with stimulus artifacts). Recordings from left L5 VR.
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Similar data were obtained from eight preparations, in which the minimum period of VL stimulation (at 3 ×T intensity) able to support entrainment was 2-5 s (average = 2.9 ± 1.0 s). In four cases in which VL stimulation at 1-s period was tested, it induced ventral root responses similar in shape and duration (123 ± 34%) to the ones obtained before strychnine and bicuculline. During such a stimulation, spontaneous bursts developed with an average cycle period of 31.2 ± 11.8 s, not significantly different from that measured in the absence of stimuli (26.3 ±6.8 s), even if in the former conditions the CVp was larger, namely 20.0 ± 7.3% versus 11.7 ± 7.4%. In seven preparations, the largest interval of stimulation able to support 1:1 entrainment was found to be on average 20.2 ± 2.9 s.
Like the case of DR stimulations, decreasing the stimulus interval of VL pulses reduced burst duration: in fact, the normalized duration of evoked bursts was changed from1.5 ± 0.3 to 0.25 ± 0.12 when the stimulus interval was reduced from 20 to 2 s (normalization was carried out with respect to data with 10-s stimulation periods).
In two further preparations, we investigated whether the range of stimulation periods for which entrainment was possible depended on pulse strength. Results were similar to the ones obtained with DR stimulations (cf. Fig. 8). In both cases, the lowest stimulation period that caused entrainment was 2 s when pulse strength was
3 × T whereas with1 × T stimulation intensity the lowest stimulation period was 10 s.
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DISCUSSION |
The principal finding of the present study on the rat disinhibited spinal cord is that DR or VL fiber stimulation entrained spontaneous bursting within a wide range of periods (usually 2-20 s). Decreasing the stimulus interval to <2 s led to loss of entrainment with unexpected return of spontaneous bursts (identical to those found in the absence of stimulation) superimposed with electrically evoked reflexes. These data suggest the existence of a rhythm-generating network distinct from polysynaptic reflex pathways and readily modulated by afferent synaptic inputs.
Pharmacological characteristics of spinal bursting
Application of either strychnine or bicuculline to the neonatal rat spinal cord does not elicit regular spontaneous bursting but random discharges or uncoordinated paroxysmal activity, respectively. Only when these two agents are applied in combination, persistent clocklike bursting activity isinvariably observed (Bracci et al. 1996a
). Spontaneous bursting requires the activation of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatereceptors because 6-cyano-7-nitroquinoxaline-2,3-dioneeliminates bursts whereas N-methyl-D-aspartate (NMDA) receptor antagonists either suppress bursting or slow them down without abolishing their late components (Bracci et al. 1996a
). This situation is somewhat different from the hippocampal bursting induced by block of GABAA receptors (glycine is not a major inhibitory transmitter in hippocampus) because, in this preparation, although bursting requires AMPA receptors, NMDA receptor antagonism only impairs the late phase of bursts (Brady and Swann 1986
; Traub et al. 1992
). It seems thus likely that the glutamate receptor mechanisms subserving bursting in hippocampus and spinal cord play dissimilar roles in the genesis and maintenance of bursting activity. Although in the hippocampus, the CA3 region is considered to play a critical role in triggering synchronized discharges due to the presence of recurrent excitatory synapses (Taylor et al. 1995
; Traub et al. 1992
), in the spinal cord, lesion experiments have shown that the ventral horn area is sufficient to support rhythmic bursting (Bracci et al. 1996b
). The present study relied on data emerging from entrainment experiments to propose a wiring diagram for the spinal oscillatory system that complements data about localization of the rhythmogenic networks.
Properties of entrainment of spinal bursting
DR stimulus intensities just above threshold for short-latency synaptic responses were sufficient to elicit a burst virtually identical to spontaneous ones. Single pulses merely reset the bursting pattern, which then resumed its normal activity; in this case, increasing pulse intensity from 1 to4 × T progressively reduced burst latency with no change in burst amplitude or structure. Because a single juxta-threshold DR stimulus delivered during the quiescent period generated a spontaneous-like burst, it appears that the rhythm-generating network was not in an absolutely refractory state during the interburst interval and that a relatively small synaptic input could reactivate burst generation. These properties are typical of an autoregenerative mechanism that needs a small triggering input to start its operation that is then self-sustained for a given time. An analogy may be drawn from the explosive bursting behavior observed in the hippocampus after block of GABAA receptor mediated inhibition by picrotoxin (Traub et al. 1992
); in this case, the event is thought to be initiated by the firing of a small number of neurons and to spread through the network via recurrent excitatory collaterals. In the disinhibited spinal cord, activation by afferent fibers of a cluster of spinal interneurons was perhaps sufficient to initiate self-sustaining excitation throughout the circuitry. Shorter latencies produced by increasing stimulus strength might be explained by the larger number of neurons recruited by stronger afferent input activation, resulting in a faster build-up of mutual excitation through the network.
With repetitive stimulation at periods comparable with (or larger than) the spontaneous cycle period, a spontaneous burst regularly developed after each evoked one at the same interval observed in the absence of stimulation. This finding suggests that a burst (spontaneous or evoked) activated a mechanism that kept the network silent for ~30 s (but was readily overcome by afferent synaptic inputs). Bursting events were always simultaneously observed on both sides of the spinal cord. Because longitudinal hemisection of the spinal cord preserved rhythmic activity in right and left halves (Bracci et al. 1996b
), the present experiments show that, although each hemicord possessed an autonomous rhythm generating network, these networks were strictly coupled in the intact, disinhibited spinal cord.
Lowering the stimulation period (at constant intensity) entrained bursts that became progressively shorter without changes in their amplitude. This property demonstrates the plasticity of the network that enables it to follow relatively fast external inputs on a 1:1 basis without altering the burst peak amplitude. This condition is different from the behavior of the organotypic spinal cultures in which evoked bursts are consistently smaller in amplitude than spontaneous ones (Streit 1993
). It is interesting that bursts never fused together to generate an ictal-like sustained depolarization as described for feline spinal motoneurons in vivo in which, unlike rat motoneurons in vitro (Bracci et al. 1996a
), intrinsic postsynaptic conductances also play a major role (Schwindt and Crill 1984
). In the neonatal rat spinal cord in vitro, when the pulse period was <2 s, the scenario suddenly changed because polysynaptic reflexes were still present in VRs, entrainment ceased and spontaneous bursting (independent from stimulation) resumed. These data were obtained with fixed stimulus intensity through the range of stimulation periods. In the range 1-2.5 × T, bursts evoked by strong pulses retained essentially the same structure as those elicited by weak pulses although they remained entrained even at much lower stimulus periods. In other words, an increase in stimulus intensity reduced the ability of bursts to escape entrainment. Further increases in stimulus strength did not change entrainment. It is noteworthy that comparable data also were observed when stimulating the VL tract, indicating that the possibility to entrain bursting was not a peculiarity of DR fibers.
Scheme to account for rhythmogenesis in the disinhibited spinal cord
The current data can be explained by postulating the existence of afferent polysynaptic pathways (subserving reflex activity recorded from motoneurons) distinct from a spontaneously active rhythmogenic network which for simplicity will be termed rhythm generator (RG) according to the model of Sernagor et al. (1995)
. The presence of a distinct RG can be inferred from the present data showing spontaneous bursting recommencing independent from polysynaptic reflex activity at low stimulation periods. Hence, it is proposed that in the absence of inhibition rhythmogenesis was not a common property of the whole spinal network. Selective lesions recently have shown that in the disinhibited spinal cord the ventral horn area is sufficient to produce rhythmic activity (Bracci et al. 1996b
) and therefore can be postulated to contain the RG. Furthermore, the observation that antidromic activation of one or more ventral root was unable to elicit a burst or to affect spontaneous bursting time course, suggests that motoneurons, rather than being involved actively in rhythmogenesis, played the role of output elements of the RG and were unable to backpropagate excitatory signals sufficient to trigger burst generation.

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| FIG. 10.
Idealized scheme of minimal synaptic connections able to account for spontaneous bursting and its entrainment in the presence of strychnine and bicuculline. It is postulated that VL tracts project to motoneurons (MN; via mono- and polysynaptic pathways), to rhythm generator (RG) and to an unidentified pool of excitatory interneurons (INT). Similar arrangements are presumed to exist for DR afferents activated by relatively low-intensity, short-duration electrical shocks. - - -, more hypothetical nature of those projections that are postulated only as one possible explanation to account for similar loss of entrainment at low periods of stimulation observed with DR or VL stimulation. Scheme should be considered simply as an operational diagram for the most parsimonious explanation of spontaneous bursting and entrainment. Note that, due to pharmacological block of Cl -mediated inhibition, inhibitory interneurons are omitted from scheme.
|
|
An idealized diagram of simplified connections is shown in Fig. 10 from which inhibitory pathways are omitted because, in the present experimental model, Cl
-mediated inhibition was suppressed pharmacologically (Bracci et al. 1996b
). According to this scheme, the DR and VL afferents, via mono or polysynaptic pathways involving interneurons, are distributed to the motoneuron pool as well as to the RG. This arrangement can thus account for burst entrainment and resetting by electrical stimulation of DR or VL fibers. Because closely spaced stimuli to DR or VL afferents could not entrain bursts, they therefore were deemed unable to drive RG, whose activity was expressed as reemergence of spontaneous bursts with superimposed reflexes.
The question then arises why entrainment failed at periods <2 s. This might be due to either an intrinsic inability of RG to follow fast stimuli or a decreased strength of the synaptic inputs impinging on RG: in the latter case, the afferent input would be unable to bring RG neurons to spike threshold and thus to trigger burst generation. Such a decrease might be explained by postulating frequency-dependent depression of the synapses linking fiber afferents to RG. As far as the monosynaptic DR-evoked reflex is concerned, synaptic depression is known to occur at stimulation intervals <60 s (Lev-Tov and Pinco 1992
). Indirect support for a role of synaptic depression in the present experiments comes from the observation that the lowest entrainment period was further reduced when the stimulus strength was raised, thus presumably recruiting a larger number of fibers that compensated for diminished synaptic efficacy. VL-evoked monosynaptic EPSPs of motoneurons do not however display frequency-dependent depression (Pinco and Lev-Tov 1994
). Hence, either VL fibers impinging on RG have properties different from those reaching motoneurons or part of VL and DR afferents impinge on a common interneuronal target (see postulated projection indicated by dashed lines in Fig. 10) that in turn sends frequency-sensitive connections to RG. The latter hypothesis is supported by the finding that VL descending tracts share some common targets with DR afferents both in cat (Maxwell and Jankowska 1996
) and in rat (Antal et al. 1996
) spinal cord.
Possible implication of burst modulation by synaptic inputs
Although the neonatal rat usually displays poorly coordinated motor activity during complex behaviors such as swimming (Cazalets et al. 1990), in the isolated spinal cord of the same age, a rhythmic pattern of motoneuron firing can be elicited by exogenously applied agents like serotonin or NMDA (for a review, see Rossignol and Dubuc 1994
). This pattern is similar to the one observed during locomotion of the adult animal and is thus termed fictive locomotion. The rhythm observed in the presence of strychnine and bicuculline is slower than fictive locomotion and does not display left-right alternation typical of that pattern (Cowley and Schmidt 1995
). It is, however, noteworthy that serotonin or NMDA speeds up rhythmic bursting induced by strychnine and bicuculline to the same range of frequencies of fictive locomotion (Bracci et al. 1996a
,b
). The latter observation suggests the possibility that disinhibited bursting and fictive locomotion rely on a similar rhythmogenic mechanism, although further studies will be necessary to clarify this issue.
In the case of fictive locomotion, afferent fibers stimulation affect the rhythmic pattern to a lesser degree than in the disinhibited spinal cord: rather than single pulses, a short train of DR stimuli (or activation of extensor afferents) is needed to reset the cycle phase of fictive locomotion and can only entrain it within a very narrow range of frequencies (Kiehn et al. 1992
). Furthermore, bilateral DR or VL afferent stimulation has only slight effect on fictive locomotion frequency (Magnuson et al. 1995
; Sqalli-Houssaini et al. 1993
). The present study suggests that lack of synaptic inhibition might have facilitated modulation of intrinsic rhythmic patterns by afferent signals, thus providing a possible framework for the integration of excitatory inputs with rhythmogenic activity within the spinal cord. One might suppose that in the presence of normal synaptic inhibition either afferent inputs to RG are subjected to inhibitory control, or the operation of RG is less prone to modulation by afferent signals.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Laura Ballerini for participating in intracellular experiments.
This work was supported by grants to A. Nistri from European Union (network grant of the Human and Capital Mobility Programme), Istituto Nazionale di Fisica della Materia, Consiglio Nazionale delle Ricerche, and Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica.
 |
FOOTNOTES |
Address for reprint requests: E. Bracci, SISSA, Via Beirut 2-4, 34013 Italy.
Received 12 November 1996; accepted in final form 5 February 1997 .
 |
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