Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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ABSTRACT |
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Saltiel, Philippe, Matthew C. Tresch, and Emilio Bizzi. Spinal cord modular organization and rhythm generation: a NMDA iontophoretic study in the frog. J. Neurophysiol. 80: 2323-2339, 1998. Previous work using electrical microstimulation has suggested the existence of modules subserving limb posture in the spinal cord. In this study, the question of modular organization was reinvestigated with the more selective method of chemical microstimulation. N-methyl-D-aspartate (NMDA) iontophoresis was applied to 229 sites of the lumbar spinal cord gray while monitoring the isometric force output of the ipsilateral hindlimb at the ankle. A force response was elicited from 69 sites. At 18 of these sites, tonic forces were generated and rhythmic forces at 44. In the case of tonic forces, their directions clustered along four orientations: lateral extension, rostral flexion, adduction, and caudal extension. For the entire set of forces (tonic and rhythmic), the same clusters of orientations were found with the addition of a cluster directed as a flexion toward the body. This distribution of force orientations was quite comparable to that obtained with electrical stimulation at the same sites. The map of tonic responses revealed a topographic organization; each type of force orientation was elicited from sites that grouped together in zones at distinct rostrocaudal and depth locations. In the case of rhythmic sequences of force orientations, some were distinctly more common, whereas others were rarely elicited by NMDA. Mapping of the most common rhythms showed that each was elicited from two or three regions of the cord. These regions were close in location to the tonic regions that produced those forces that represented components specific to that rhythm. There was an additional caudal region from which the different rhythms also could be elicited. Taken together, these results support the concept of a modular organization of the motor system in the frog's spinal cord and delineate the topography of these modules. They also suggest that these modules are used by the circuitry underlying rhythmic pattern generation by the spinal cord.
In previous experiments, the output of the frog spinal cord was investigated using electrical microstimulation of the lumbar gray. Before microstimulation, the isometric passive forces recorded at the ankle with the limb placed in different positions define a force field that converges to an equilibrium point of zero forces corresponding to the resting position of the limb. Bizzi et al. (1991) Preparation
Thirteen bullfrogs (Rana catesbeiana) were anesthetized with 1 ml of tricaine methanesulfonate subcutaneously, supplemented with ice. The cervicomedullary junction was exposed and spinalization carried out by suction at the level of the obex. The back muscles overlying vertebrae 4-6 were removed in preparation for laminectomy. Bipolar electrodes were implanted in 12 hindlimb muscles (rectus internus, adductor magnus, semitendinosus, sartorius, vastus internus, rectus anterior, vastus externus, iliopsoas, biceps femoris, semimembranosus, gastrocnemius, tibialis anterior) to record electromyographic (EMG) activity (to be reported in a separate paper). On the next day, after laminectomy of the fourth, fifth, and sixth vertebrae, the dura was opened with electrocautery and scissors to expose the dorsal surface of the right side of the lumbar spinal cord from above the seventh root to below the tenth root. The pia was opened with fine scissors, and a detailed drawing was made of the exposed spinal cord vasculature and dorsal root entry zones, which later served to document the points of entry of the micropipette. Experiments began once all surgery was completed, i.e., 2-3 days after spinalization. Frogs were kept refrigerated between surgical procedures and between experiments (usually 2-3 per frog). All procedures were approved by the animal care committee at our institution.
Micropipette
A three-barrel and a single-barrel pipette were prepared from 1-mm glass capillary tubes using a vertical puller and their tips broken to 2.5 µm. The single barrel pipette was bent 15° under the heat of a coil, first at the shoulder level and then in the reverse direction at the shank level. The lower shanks of the two pipettes then were approximated fully and glued together with cyanoacrylate, the single-barrel tip protruding by 10-15 µm (Crossman et al. 1974 Experiment
The spinalized frog was immobilized on a moistened molded stand with body and pelvis clamps. A force sensor attached to the right ankle at the same vertical level as the hip measured the horizontal isometric forces Fx, Fy developed by the hindlimb in response to cord stimulation (Fig. 1A). One unit of force was equivalent to ~0.01 N, which corresponded to the resolution of our force transducer. The ankle was at the center of our force field grid designed to sample the leg workspace (Fig. 1B). At this location, it is generally possible to distinguish different vector orientations corresponding to the different kinds of force fields resulting from stimulation applied to different parts of the spinal cord (Giszter et al. 1993
Assessment of drug spread and neural substrate of NMDA action
We estimated the spread of NMDA by computing the radius of tissue exposed to suprathreshold concentrations of NMDA by the average time of onset of forces. We used 15 µM NMDA as a conservative estimate for the threshold concentration: 2.5-100 µM NMDA typically is bath-applied to in vitro whole cord or slice preparations (Alford and Grillner 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
found that after addition of active forces generated by microstimulation, the force field recorded at the ankle remained convergent in most cases, but its equilibrium point shifted to a point close to the periphery of the limb's workspace.
, 1995
; Giszter et al. 1993
).
; Poon 1980
), it was soon discovered that NMDA, which is not taken up by the very active glutamate transporters, is a more effective agent (Grillner et al. 1981
). In the past, experiments on the spinal cord using NMDA aimed at understanding central pattern generation, and NMDA either bathed the whole spinal cord (Grillner et al. 1988
) or was applied topically to the dorsal surface of spinal cord segments (Currie 1994
). To our knowledge, this is the first study directed at investigating the spinal cord output using focal intraspinal NMDA iontophoresis. This study provides results relevant both to the modularity of the spinal cord motor system and to the circuitry underlying central pattern generation.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Epoxy glue was added to consolidate the pipette assembly.
), Ringer for current balancing, and 2 M NaCl for stimulation.
).
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FIG. 1.
Apparatus and limb configuration. A: apparatus. Spine and pelvis were held clamped by restraints (latter not shown). Micropipette assembly was descended in the spinal cord for electrical stimulation and N-methyl-D-aspartate (NMDA) iontophoresis. Mechanical response to stimulation was recorded by the force sensor attached to the limb at the ankle. Limb configuration was constrained by the pelvis restraint and the force sensor. B: sketch of the locations in the workspace where recording of isometric forces has led to the recognition of a few types of convergent force fields in prior work with electrical stimulation. In the present study, the ankle was kept in the center of the grid where the orientation of the force vector best distinguishes between the different types of force fields, and points in the direction of the overall flow of the field to its area of convergence. A and B were modified from Giszter et al. (1993) .
100 nA, continued until EMG activity began or for a maximum of 30 s. The forces and EMGs were collected for 40 s beginning with the iontophoresis. Rarely, the 40-s records were begun later, 20-25 s after the onset of a repeated iontophoresis at a site that had responded late to a prior iontophoresis (4 sites). Movements of the contralateral hindlimb were not monitored.
; Barry and O'Donovan 1987
; Hochman et al. 1994a
,b
; Soffe 1996
); in dissociated cells, the EC50 (half the NMDA concentration that achieves a maximal effect) is 30 µM for the accumulation of cyclic guanosine monophosphate (Garthwaite 1985
) and 35 µM for the NMDA receptor current (Patneau and Mayer 1990
). The distribution of tissue concentrations of NMDA achieved by iontophoresis was computed using the equation for diffusion from a continuous point source (Carslaw and Jaeger 1959
; Curtis et al. 1960
; Nicholson et al. 1979
)
where C is the tissue NMDA concentration (moles/liter) at time t (s) and distance r (µm) from the pipette tip; n the ionic transference number, estimated at 0.3 for NMDA from values for other excitatory amino acids (Stone 1985); I the iontophoretic current (0.1 µA); Z = 1, the valency of NMDA; D the diffusion coefficient, estimated from the molecular weight of NMDA at 0.6*10
(1)
5 cm2/s at 25°C (Curtis et al. 1960
; Longsworth 1953
);
= 1.6 and
= 0.21, the tortuosity factor and extracellular space fraction respectively (Nicholson and Phillips 1981
; Nicholson and Syková 1998
); and erfc the complementary error function (Carslaw and Jaeger 1959
).
where Q is the ejected flow rate (moles/s) and the other parameters as in Eq. 1.
(2)
; Nicholson 1985
)
where Cf is the APV concentration of the ejected droplet, b the "effective" radius of the droplet equal to the radius of the ejected droplet divided by (
(3)
)1/3 (
= 0.21, the extracellular space fraction), D the diffusion coefficient of APV estimated at 0.5287*10
5 cm2/s from its molecular weight, erf the error function, and the other parameters as in Eq. 1.
50 µM APV concentration during the time period that corresponds to the average latency of onset of forces produced by unantagonized NMDA iontophoresis. An APV concentration of 50 µM is considered sufficient to block high NMDA concentrations, such as those achieved near the pipette tip (Davies et al. 1981
; Garthwaite 1985
). Because APV has a rapid offset (and onset) of action (Mayer et al. 1988
), NMDA iontophoresis was begun shortly, i.e., 20 s after the APV application.
, 1994
) was ejected during 4 s, resulting in an estimated radius of TTX blockade of 300 µm (Eq. 3), i.e., the radius with a TTX concentration >1 µM (Hochman et al. 1994a
,b
; Larkum et al. 1996
; Schwindt and Crill 1997
; Skydsgaard and Hounsgaard 1994
). NMDA iontophoresis was repeated 3 min after the application of TTX.
Analysis of forces
For each force record, the initial passive force was subtracted to yield the active force. The force angle was computed from Fx, Fy with the following notation: 0° rostral, ±180° caudal, 90° lateral, 90° adduction, and its magnitude was
The time courses of the force angle and magnitude then were plotted.
). We determined, using a randomization test (Fisher 1993
) whether this mean difference was significantly smaller than expected if the responses from NMDA and electrical stimulation at a site were unrelated.
. The essential idea of this method is to find the optimal fit of the observed distribution to a mixture of a given number of modes and then examine the goodness of fit of this mixture to the observed data: if the given mixture is a poor fit to the observed distribution, then it is concluded that there is at least one more mode in the data.
; Mardia 1972
), described by parameters specifying its mean and concentration (analogous to the mean and variance of the normal distribution). A mixture of von Mises distributions is described by the set of means and concentrations for all of the modes as well as by weighting parameters, which describe how much of the data are produced by each mode.
. Once an optimal fit of a mixture of n von Mises distributions to the data was found, the goodness of fit of this mixture was calculated using the U2 statistic for circular data (Mardia 1972
), analogous to the
2 statistic for linear data. We assessed the significance of this U2 statistic using a bootstrap procedure (Hsu et al. 1986
). This procedure gave the probability that the data were actually produced by the optimal mixture of n modes. A significantly low probability would suggest that the data were likely not to have been produced by the mixture, indicating that there is at least one more mode in the data.
), also can be used to categorize the data: each data point is assigned to the mode with the nearest mean. K-means will update its mixture model, therefore differently than EM, and the two methods may produce slightly different patterns of results. We therefore compared the results obtained from each method to one another and to our visual inspection of the data.
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RESULTS |
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Focal nature and reproducibility of NMDA iontophoresis
We applied NMDA iontophoretically at 229 sites of 85 different spinal cord penetrations in 13 frogs. These sites were essentially all responsive to electrical stimulation (215/224 tested sites).
Assessment of drug spread
Additional evidence for a limited spread of NMDA is found using Eq. 1 (see METHODS): at 22.5 s after the onset of NMDA iontophoresis, which is the average latency for the onset of the forces, the NMDA tissue concentration is >15 µM only at distances <270 µm from the pipette tip. At two sites responding to NMDA iontophoresis at the average latency of 22.5 s, pressure application of 4 mM NMDA as droplets of 46-62 µm radius ejected during 35-33 s closely reproduced the forces and EMGs elicited by NMDA iontophoresis, albeit at a longer latency of 32 s and to a similar or greater intensity. Using Eq. 2 (see METHODS), we find that at 32 s, the pressure-ejected NMDA would have reached a tissue concentration >15 µM only at distances <240-285 µm from the pipette tip. At four sites silent to NMDA iontophoresis, pressure-ejection of similar or greater volumes of 4 mM NMDA (55-104 µm radius during 60 s) also produced no effect. Thus the two methods of NMDA application gave similar results both in terms of identifying active and silent sites and in the estimate of spread of NMDA. In our frogs, a sphere of 270-µm radius represents 0.88% of the volume of an average lumbar cord segment (1,000-µm radius and 3,000-µm length).
Neural substrate of NMDA responses
We applied TTX focally at active sites and examined the response to repeated NMDA iontophoresis. The estimated radius of TTX blockade was 300 µm (see METHODS), therefore sparing the motoneuron somas/axons which are located >500 µm away from our deepest iontophoretic sites (see DISCUSSION). At all four sites that we could test in two frogs, the response to NMDA iontophoresis was abolished completely by TTX (with Ringer alone, 2 sites showed no change, 1 potentiation, 1 slight depression). The latter site is shown in Fig. 3. It actually had responded first to a brief iontophoresis of 6 s with a rhythm beginning very early at 4.1 s, but, after a Ringer droplet, had failed to respond again to an equally brief iontophoresis. Because these NMDA applications had been unusually brief, we continued to study this site. Repeated NMDA iontophoresis for 28 s produced a similar rhythm (Fig. 3A). After a Ringer droplet, the effect of repeated NMDA iontophoresis, although a bit weaker, was mostly preserved with a qualitatively similar EMG pattern, latency of onset and rhythm frequency (Fig. 3B). After the TTX droplet, however, NMDA iontophoresis had absolutely no effect (Fig. 3C). By contrast, withdrawal to cutaneous pinch of the ipsilateral foot remained easy to elicit and entirely normal, as judged by EMGs.
General features of NMDA responses
In this paper, we report on 69 active sites (66 of which produced forces in response to a 1st NMDA iontophoresis and 3 of which elicited only EMGs to a first iontophoresis but produced forces to repeated iontophoresis).
Comparison between NMDA and electrical stimulation
We compared the orientations of the chemically and electrically evoked forces at each of the 69 NMDA-responsive sites. This was not straightforward because NMDA commonly produced a sequence of forces as did electrical stimulation although less frequently. Moreover electrical stimulation often was tested at several intensities, sometimes with a change in the orientation of the force. We therefore compared in the force sequences the first NMDA-evoked force and the first electrically evoked force, selecting for the latter the one closest in amplitude to the chemically evoked force. The distribution of the absolute difference in orientation between these two responses at the same site is shown in Fig. 5. Although there is a wide variation in angular differences between the two responses, with large differences not being uncommon, this difference was small at the majority of sites. The mean difference was 49°, which was significantly less than the one expected (68°) if there was no relationship between the two responses (randomization test, P < 0.001). Thus there was a relationship, though not very strong, between the force orientation elicited by NMDA and by electrical stimulation at the same site.
Clusters of force orientations
Next we examined how the force orientations elicited by NMDA and by electrical stimulation were distributed for the entire set of 69 NMDA-responsive sites. The distribution for the NMDA-elicited stable forces, obtained by parsing the records as indicated in Fig. 4, is shown in the circular histogram of Fig. 6. It is clear that some force orientations happened at greater frequency than others. Specifically, a careful visual inspection of Fig. 6 suggests that the force orientations clustered in five preferred directions. These clusters are labeled as lateral extension (LE), rostral flexion (RF), flexion to body (FB), adduction (ADD), and caudal extension (CE), a terminology based on the direction the ankle would move if it were free to do so (Giszter et al. 1993
Comparison of tonic and rhythmic forces
An important finding was that only a subset of the NMDA-responsive sites produced a simple tonic response. Tonic forces were elicited from 18 sites in 10 frogs (an example was shown in Fig. 4A). Their orientations are plotted in Fig. 9. They included LE, RF, ADD, and CE, but it is noteworthy that no tonic FB was obtained.
Topography of NMDA responses
Figure 10 shows for each force component the relative frequency with which it was seen among the different rhythmic forces elicited from each spinal cord segment. A one-way analysis of variance performed on that data showed a highly significant relationship between rhythmic force component orientation and the rostrocaudal location of the rhythmic site [F(4,347) = 12.65, P < 0.001]. In particular, a clear difference is seen between LE and CE in Fig. 10, and this was confirmed by a post hoc t-test [Scheffé F(4,347) = 9.46, P < 0.001]. Segments 9,10 were the most likely to produce a LE rhythmic component, whereas segments 7,8 were the most likely to produce a CE rhythmic component. For ADD, FB, and RF, the distributions of relative frequency of occurrence along the cord in Fig. 10 were intermediate between those for LE and CE, i.e., they were less caudally biased than for LE and less rostrally biased than for CE; however, these distributions did not show clear peaks, but were rather widespread.
The most important result of this paper is that the spinal cord produces a limited number of discrete motor outputs, identified as five preferential directions of the isometric force vector recorded at the ankle in a single limb position. These are: lateral extension, rostral flexion, flexion to body, adduction, and caudal extension (Fig. 6). This confirms the earlier report of a small number of convergent force field types with equilibrium points in the lateral, rostral, flexed to body, adducted, and caudal locations of the workspace produced by electrical stimulation of the frog spinal cord (Bizzi et al. 1991 We thank Dr. S. Rossignol for helpful comments on the manuscript and Dr. J. Crandall for the histology processing.
This work was supported by Office of Naval Research Grant ONR:N00014/90/J/1946 and National Institute of Neurological Disorders and Stroke Grant (NS-09343) to E. Bizzi and a Howard Hughes Medical Institute predoctoral fellowship to M. C. Tresch.
Address for reprint requests: E. Bizzi, Dept. of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Ave., E25-526, Cambridge, MA 02139. Received 12 January 1998; accepted in final form 20 July 1998.
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FIG. 2.
Effect of repeated NMDA iontophoresis at 1 site and responses at other sites in the same track. Tested depths from the cord dorsum were 673 µm (A), 977 µm (B), 1,127 µm (C), and 1,427 µm (D) as shown (inset); the order of tested depths was B, C, D, and A. At depth B, the iontophoresis was repeated after an interval of 21 min. For each trial, NMDA iontophoresis started at time 0 and stopped at 30 s, and every 75th sample of the active isometric force vector is plotted to show its time course (in this and subsequent figures, the 1st 60 ms of force, i.e., resting force, has been subtracted to yield the active force). The force traces are oriented with respect to the frog as shown in the bottom inset. All forces are drawn in the same scale (top right).
; Stone 1986
), was never achieved beyond a radius of 270-225 µm with the higher and lower concentration APV droplets.
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FIG. 3.
Effect of a focal application of tetrodotoxin (TTX) at a site responding to NMDA iontophoresis. A: electromyographic responses recorded in 12 muscles of the ipsilateral leg in response to NMDA iontophoresis applied for the duration of the horizontal bar. B: NMDA iontophoresis repeated 3 min after focal application of a 100-µm-radius droplet of Ringer ejected over 4 s at the iontophoretic site. C: NMDA iontophoresis repeated 3 min after focal application of a 100 µm radius droplet of TTX 20 µM ejected over 4 s at the iontophoretic site. Interval between successive NMDA iontophoreses was 11 min. Electromyograms (EMGs) are labeled. RI, rectus internus; AM, adductor magnus; SM, semimembranosus; ST, semitendinosus; IP, iliopsoas; VI, vastus internus; RA, rectus anterior; GA, gastrocnemius; TA, tibialis anterior; BF, biceps femoris; SA, sartorius; VE, vastus externus.
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FIG. 4.
Tonic and rhythmic effects of NMDA. In each example, NMDA iontophoresis was applied for the duration of the horizontal bar, and every 75th sample of the active force vector is shown (top) with its magnitude scaled as indicated. Angle of that force vector was plotted over time, with the convention for the force orientation as shown (inset). In each record, the shaded regions identify the stable forces. A: example of a tonic effect. B: example of a rhythmic effect. A and B are from 2 different frogs.
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FIG. 5.
Distribution of the absolute difference in orientation between the 1st electrically evoked force and the 1st NMDA-evoked force at each NMDA-responsive site (n = 69).
). Figure 7 shows the stable force angles obtained with electrical stimulation at the same 69 sites. It can be seen that the two distributions of force orientations, one from NMDA application, the other from electrical stimulation, are similar in their main features. In particular, similar clusters of preferred force orientations appear to be present in Figs. 6 and 7.
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FIG. 6.
Orientations of the NMDA-elicited forces (n = 388) obtained in 13 frogs from 69 spinal cord sites, that responded to NMDA. Circular histogram of 7.5° binwidth, oriented with respect to the frog as shown (bottom left inset). Number of responses in each bin is displayed. Clusters of force orientations are labeled. LE, lateral extension; RF, rostral flexion; FB, flexion to body; ADD, adduction; CE, caudal extension. A similar histogram was obtained when plotting directly the responses automatically detected by the computer algorithm without any correction (bottom right inset).
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FIG. 7.
Orientations of the forces elicited by electrical stimulation (n = 134) at the 69 NMDA-responsive sites of Fig. 6. Circular histogram of 7.5° binwidth. Number of responses in each bin is displayed. Clusters of force orientations are labeled as in Fig. 6.
) (see METHODS). There was strong evidence to suggest the existence of at least five modes in the distribution (P < 0.01, 0.05, 0.05 for n = 2, 3, and 4 modes, respectively). A test for the existence of six modes failed to reach significance, although the probability value approached a trend (P = 0.12). This analysis therefore allows us to conclude that there are at least five modes of force orientation within the responses produced by NMDA.
; Hartigan and Wong 1979
) (see METHODS). For five modes, the two methods produced different patterns of classification. K-means divided the data into five clusters corresponding to the LE, CE, ADD, FB, and RF clusters (Fig. 8B) identified from our own visual inspection as described above. EM, on the other hand (Fig. 8A, left), appeared to identify the LE, CE, and ADD clusters but did not achieve separation between RF and FB and found an additional cluster centered at ~45°. When the number of clusters was increased to six, however, both methods produced a very similar classification. Both EM (Fig. 8A, right) and k-means (data not shown) found essentially the same six clusters: the five main ones identified by visual inspection plus the cluster near 45°. The exact divisions between the six modes were not identical, but the main classes were the same. Given that the statistical analysis described earlier allowed us to conclude the presence of only five modes in the data and that k-means identified the same five modes we found from visual inspection of the data in Fig. 6 and from other analyses (see for instance Figs. 7 and 15), in the rest of this section, we present results based on a classification of the data according to the divisions identified by k-means with five modes. We also have analyzed the results obtained using the divisions identified by EM with five or six modes as well by k-means with six modes, and although the details are slightly altered in each case, the basic pattern of results we report here are the same.
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FIG. 8.
Clustering analyses. A: distribution of stable force orientations elicited by NMDA (reproduced in B) was modeled as a mixture of 5 or 6 von Mises probability distributions, which are shown plotted on a circle, with the usual orientation convention relative to the frog. P values below evaluate the null hypothesis that the NMDA directional data could have been generated by a mixture of a smaller number ( 5) probability distributions. B is the number of repetitions of the bootstrap procedure. B: partitioning of the NMDA-elicited forces into 5 clusters by the k-means algorithm.
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FIG. 15.
Seventy-three weakest forces (2 < mag < 4) have been removed from the 388 NMDA-elicited forces of Fig. 6. Remaining responses have been divided in 2 groups according to force magnitude (mag), and their orientations are displayed in circular histograms. Left: forces with 4 mag
30 (n = 185). Magnitude range of these forces is similar to that of the tonic responses. Right: forces with mag >30 (n = 130).
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FIG. 9.
Orientations of the tonic force responses elicited by NMDA at 18 of the 69 sites of Fig. 6. Circular histogram of 7.5° binwidth. Abbreviations for labels as in Fig. 6. No tonic FB was obtained.
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FIG. 10.
Relative frequencies with which each force component occurred among the rhythmic forces elicited from the different spinal cord segments. Numbers of NMDA-elicited rhythmic forces from segments 7 to 10 were n = 27, 147, 118, and 60 as indicated in the spinal cord diagram. These rhythmic forces were generated from n = 5, 16, 9, and 12 rhythmic sites located in segments 7-10, respectively. Of the 44 rhythms in Fig. 12, 2 are missing here (1 from segment 11 and 1 of unknown topography).
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FIG. 11.
Topography of the sites producing tonic forces and of the sites producing strong rhythmic forces. A: topography of the 18 spinal cord sites where NMDA elicited a tonic response (10 frogs). Depth is vertical with the dorsal and ventral surfaces at depths of 0 and 2,000 µm, respectively. Rostrocaudally, dorsal roots DR6-DR10 delimit the positions of segments 7-10. Note the different scales for the dorsoventral and rostrocaudal axes. All sites were located 150-350 µm from the midline. Four different kinds of tonic responses are indicated by the different letter symbols. Outlines have been drawn to suggest how these tonic responses define a coarse topography. c, caudal extension; r, rostral flexion; a, adduction; l, lateral extension. B: topography of NMDA-elicited rhythmic forces of the same orientations than the tonic forces and of magnitude >40, in relation to the zones eliciting tonic responses. Force labels as in Fig. 11A. Superimposed labels indicate that >1 kind of force orientation with magnitude >40 was obtained at the site. For example, there are 3 sites below DR8 that gave both r and l responses of magnitude >40. As an aid to the reader, the suggested topography of the tonic zones has been reproduced in dotted outlines in Fig. 11B to facilitate comparison with the topography of the strong rhythmic forces.
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FIG. 12.
Rhythmic responses elicited by NMDA at 44 spinal cord sites (13 frogs). Number of sites at which each rhythm with the indicated force components was obtained is indicated. Besides these 44 rhythmic sites and the 18 tonic sites, the other 7 NMDA-responsive sites included 4 sites with single phasic responses and 3 sites that did not fit any category essentially because they produced a force changing sufficiently in angle not to qualify as a tonic response, yet remaining within the same cluster of force orientation.
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FIG. 13.
Well-developed rhythms of the 3 most common combinations of forces elicited by NMDA iontophoresis in the frog's spinal cord (see Fig. 12). A: RF,FB rhythm. B: LE,RF,FB rhythm. C: ADD,CE,FB rhythm. Component forces of these rhythms are identified in the figures ( ). A and B are rhythms from the same frog as the rhythm of Fig. 4B. C is a rhythm from the same frog as the tonic response of Fig. 4A.
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FIG. 14.
Topography of the spinal cord sites where NMDA elicited a tonic response or 1 of the 3 most common rhythms. A: topography of the 4 different kinds of tonic responses as in Fig. 11A. B: sites eliciting the RF,FB rhythm. C: sites eliciting the LE,RF,FB rhythm and the LE,RF and LE,FB subsets. D: sites eliciting the ADD,CE,FB rhythm and the indicated subsets.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
).
).
50 µM concentration of APV, adequate to antagonize the >100 µM concentrations of NMDA produced by iontophoresis at such distances, was sufficient to abolish the response to NMDA.
) and now confirmed with NMDA, support the concept of a modular organization of the spinal cord motor circuitry (Bizzi et al. 1995
).
), and there is some evidence, mostly indirect, that motoneurons have dendritic NMDA receptors (Durand 1991
; Moore et al. 1995
; Petralia et al. 1994
; Skydsgaard and Hounsgaard 1994
; see, however, Robinson and Ellenberger 1997
).
, 1997
). However, the channels responsible for this amplification appear to be located on the axosomatic membrane or the proximal dendrites (Seamans et al. 1997
; Stuart and Sakmann 1995
) rather than on the distal dendrites themselves, hence outside the level at which motoneurons were exposed to our focal applications of TTX. Moreover in motoneurons there is indirect evidence that amplification of dendritic EPSPs may rely on TTX-resistant Ca2+ mechanisms (Hounsgaard and Kiehn 1993
; Lee and Heckman 1996
; Svirskis and Hounsgaard 1997
). In addition, two investigations suggest that blockade of putative TTX-sensitive Na+ channels in motoneuron dendrites at the site of NMDA iontophoresis does not account for the abolishing effect of focal TTX: 1) large membrane voltage oscillations can be induced in motoneurons of rat spinal cord slices by bath-application of NMDA in the presence of bath-applied TTX (Hochman et al. 1994a
); and 2) in turtle spinal cord slices, NMDA focally applied in the white matter to distal dendrites of motoneurons produced a depolarization but no spikes recorded at the motoneuron soma level even with more vigorous iontophoresis than ours. However, NMDA iontophoresis amplified the response caused by glutamate applied to the same dendrite into a larger depolarization or even to spikes or oscillatory regenerative events, and this amplification mechanism was TTX resistant (Skydsgaard and Hounsgaard 1994
).
), this constitutes further evidence that the effects of NMDA in our experiments were due to interneuron rather than direct motoneuron activation.
). NMDA given systemically or intrathecally in vivo or bathing the spinal cord in vitro has been shown to activate CPGs (Dale and Roberts 1984
; Douglas et al. 1993
; Grillner et al. 1981
; McClellan and Farel 1985
). In our study, NMDA was microapplied focally and, at locations where it was effective, most commonly produced a rhythmic sequence where the forces belonged to only two or three different orientations. In addition, some rhythms were distinctly more common than others (Fig. 12). These orderly features about the rhythmic effects and the fact that they were obtained with NMDA, together suggest that they represented activation of organized rhythmic circuitry, i.e., that we locally accessed circuitries belonging to different CPGs and activated them with NMDA. We can only state this as a suggestion because the limb was not deafferented to remove peripheral feedback and because we have no behavioral correlate. Nevertheless it is interesting that in a recent investigation where NMDA was bath-applied to transverse cord slices in the presence of TTX, a subgroup of cells recorded ventrolaterally to the central canal that responded with rhythmic oscillations rather than simple depolarization was interpreted to be part of a CPG (Hochman et al. 1994a
).
; Ho and O'Donovan 1993
; Kjaerulff and Kiehn 1996
; Mortin and Stein 1989
) or that the CPG (for locomotion) is localized strictly to the uppermost two lumbar segments (Cazalets et al. 1995
). On the other hand, a patchy motor organization of the spinal cord has been suggested in another context by Székely (1967) when he found on systematic microstimulation of the spinal cord in a grid-like pattern that a muscle would be activated at a certain locus in combination with some other muscles, then not be activated at the next locus, then reappear at a still farther locus in combination with a different set of muscles.
has proposed unit CPGs that might be responsible for oscillations at a single joint. The patterns of rhythmic forces that we saw were in general not those expected for such oscillations (e.g., ADD,LE or RF,CE, which would correspond to oscillations about the knee and hip joints respectively were rather rare; see Fig. 12). Our suggestion that the smaller units would be modules producing tonic forces is closer to that of Mortin and Stein (1989)
. They examined how the electroneurographic patterns of the three different forms of fictive scratch reflex in the turtle changed as segments were being progressively removed from the spinal cord. From this, they concluded that the same group of interneurons would be responsible for the hip protraction phase in all three scratch forms, whereas a separate group of interneurons with a different location would underlie the hip retraction phase in each case.
), but one may ask whether it could occur through purely spinal mechanisms and whether it could have another function. Spinal CPGs do not only "pick" force orientations but also organize them in time. A speculation would be that the tonic modules might be providing the force orientations, whereas coactivation might be more intimately related to timing. For example, all scratch forms in the turtle have strictly alternating hip protractor and retractor bursts, but it is the timing of coactivation with the knee extensor burst that crucially distinguishes between them (Stein 1983
). There has been much recent work suggesting the importance of synchrony in sensory systems (König et al. 1996
), but it is likely to be equally important in motor systems (Farmer 1998
).
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References
)2-amino-5-phosphonovalerate and (
)2-amino-7-phosphonoheptanoate as antagonists of N-methylaspartate and quinolinic acids and repetitive spikes in rat hippocampal slices.
Brain Res.
381: 195-198, 1986.[Medline]
0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society