Extent and Role of Multisegmental Coupling in the Lamprey Spinal Locomotor Pattern Generator

William L. Miller and Karen A. Sigvardt

Center for Neuroscience and Department of Neurology, University of California, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Miller, William L. and Karen A. Sigvardt. Extent and Role of Multisegmental Coupling in the Lamprey Spinal Locomotor Pattern Generator. J. Neurophysiol. 83: 465-476, 2000. Timing of oscillatory activity along the longitudinal body axis is critical for locomotion in the lamprey and other elongated animals. In the lamprey spinal locomotor central pattern generator (CPG), intersegmental coordination is thought to arise from the pattern of extensive connections made by propriospinal interneurons. However, the mechanisms responsible for intersegmental coordination remain unknown, in large part because of the difficulty in obtaining quantitative information on these multisegmental fibers. System-level experiments were performed on isolated 50-segment preparations of spinal cord of adult silver lampreys, Ichthyomyzon unicuspis, to determine the dependence of CPG performance on multisegmental coupling. Coupling was manipulated through use of an experiment chamber with movable partitions, which allowed separate application of solution to rostral, middle, and caudal regions of the spinal cord preparation. During control trials, fictive locomotion, induced by bath application of D-glutamate in all three regions, was recorded extracellularly from ventral roots. Local synaptic activity in a variable number of middle segments was subsequently blocked with a low-Ca2+, high-Mn2+ saline solution in the middle compartment, whereas conduction in axons spanning the middle segments was unaffected. Spectral analysis was used to assess the effects of blocking propriospinal coupling on intersegmental phase lag, rhythm frequency, correlation, and variability. Significant correlation and a stable phase lag between the rostral and caudal regions of the spinal cord preparation were maintained during block of as many as 16 and sometimes 20 intervening segments. However, the mean value of this rostrocaudal phase decreased with increasing number of blocked segments from the control value of approximately 1% per segment. By contrast, phase lags within the rostral and caudal end regions remained unaffected. The cycle frequency in the rostral and caudal regions decreased with the number of blocked middle segments and tended to diverge when a large number of middle segments was blocked. The variability in cycle frequency and intersegmental phase both increased with increasing number of blocked segments. In addition, a number of differences were noted in the properties of the motor output of the rostral and caudal regions of the spinal cord. The results indicate that the maximal functional length of propriospinal coupling fibers is at least 16-20 segments in I. unicuspis, whereas intersegmental phase lags are controlled at a local level and are not dependent on extended multisegmental coupling. Other possible roles for multisegmental coupling are discussed.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Networks of oscillatory neurons perform diverse functions in sensory processing and motor control and are also likely to underlie many cognitive tasks (Gray 1993, 1994; Jacklet 1989; Kahn et al. 1997; Ritz and Sejnowski 1997; Steriade 1997). Among the most well-studied classes of oscillatory networks are the central pattern generators (CPGs) that control rhythmic motor behaviors (Calabrese 1995; Cohen et al. 1988, 1992; Harris-Warrick et al. 1992; Marder 1998; Marder and Calabrese 1996). CPGs serve two primary motor functions: the generation of periodic commands to the individual muscles directly responsible for rhythmic behavior and the coordination of proper timing of contractions among all the muscles subserving the behavior. In elongated swimming animals such as the lamprey, efficient undulatory locomotion requires appropriate phasing of muscular contraction from segment to segment along the animal, and these intersegmental phase relationships must hold through a range of swimming speeds (Wallén and Williams 1984). Although a CPG is necessarily responsive to descending and sensory input in the intact animal, the defining feature of CPGs is their ability to generate behaviorally appropriate output, including appropriate phase relationships, under experimental isolation from extrinsic input (Graham-Brown 1911; Grillner and Zangger 1974, 1979; Selverston et al. 1976; Sherrington 1906; Wilson 1961). Thus, intersegmental coordination is generated by the CPG itself, through the pattern of connectivity among the participating neurons (Grillner and Wallén 1985; Marder and Calabrese 1996; Skinner and Mulloney 1998).

In the lamprey, a primitive fish, undulatory locomotion is controlled by a CPG distributed throughout the spinal cord (Cohen and Wallén 1980; Poon 1980; Wallén and Williams 1984; reviewed in Sigvardt 1989). The neuronal mechanisms by which frequency and intersegmental phase are generated and controlled are not yet understood, although many studies have shown that these parameters can be influenced by a wide range of receptor agonists, modulators, and cellular dynamical properties and inputs from mechanoreceptors (Buchanan 1993, 1996; Grillner et al. 1981, 1991; Hagevik and McClellan 1994; Harris-Warrick and Cohen 1985; Kemnitz et al. 1995; Matsushima and Grillner 1992a,b; McClellan and Sigvardt 1988; McPherson et al. 1994; Sigvardt and Williams 1996; Tegnér et al. 1993; Williams et al. 1990). A key difficulty in understanding the origin and control of frequency and phase has been determining the role played by the extensive array of propriospinal neurons. Some of these are phasically active, and many project to targets a few to as many as 30 or more segments away from the cell body in the ascending and descending directions in the lamprey spinal cord (Buchanan 1982; Buchanan and Cohen 1982; Buchanan and Grillner 1988; Buchanan and McPherson 1995; Buchanan et al. 1989, 1996; Cohen 1987a; Rovainen 1985; reviewed in Buchanan 1996). Potential roles for these propriospinal coupling fibers have emerged from a combination of system-level experiments and theoretical analyses of coupled oscillator systems (reviewed in Cohen and Kiemel 1993). Some or all of the propriospinal neurons may serve a role in intersegmental coordination, as has been suggested when localized regions of lamprey spinal cord are either blocked or lesioned (Cohen 1987a; Cohen et al. 1989; McClellan 1994; Rovainen 1985). However, the specific connectivity of most of these projecting neurons and the extent of their functional influence are not known and are difficult to test experimentally. Therefore, theoretical and simulation studies of lamprey-like CPG systems have mainly included only nearest-neighbor coupling (Buchanan 1992; Cohen et al. 1982, 1992; Grillner et al. 1988; Kopell and Ermentrout 1986, 1988, 1990; Kopell et al. 1991; Wallén et al. 1992), whereas a few have explored small subsets of the myriad possible configurations of longer (multisegmental) propriospinal coupling (Hellgren et al. 1992; Kopell et al. 1990; Ullström et al. 1998; Wadden et al. 1997; Williams 1992a,b).

We report the results of system-level experiments on the isolated spinal cord of the silver lamprey, Ichthyomyzon unicuspis, in which systematic manipulation of multisegmental coupling through localized synaptic block leads to important and revealing changes in CPG output properties and localization of function. Several earlier closely related studies on the isolated lamprey spinal cord served as important guides in this work. Rovainen (1985) used a combination of electrical stimulation and partial localized blocking techniques to determine the presence of ascending and descending multisegmental coupling fibers. Through localized block of synapses with GABA or glycine, that study suggested that the rhythmic activity in one region of spinal cord could influence the activity in segments as distant as 20 segments away through propriospinal coupling. Cohen (1987a) combined specific lateral and medial lesioning with localized synaptic block to define putative functional coupling subsystems associated with particular fiber tracts. In that study, a phase lag was maintained between two end regions of spinal cord when synaptic activity in a region of as many as 10 intervening segments was blocked with a saline solution in which Mn2+ completely replaced Ca2+. Others have investigated functional coupling with similar techniques in the combined brain stem-spinal cord preparation (e.g., Hagevik and McClellan 1994 for the larval sea lamprey). However, results from these studies are difficult to compare with those in which the isolated spinal cord was used, given the extensive reentrant connectivity between the brain stem and spinal cord.

Although earlier studies of coupling in the isolated lamprey spinal cord suggested a functional role for multisegmental propriospinal projections, little quantitative analysis was performed concerning the effects the experimental manipulations had on the various properties of fictive locomotion. In this study, we used frequency-domain time-series analysis together with ensemble statistical analysis to study the effects of manipulating coupling on motor rhythm frequency, intersegmental phase, and their variability, and to investigate the functional length of intersegmental coupling. Preliminary results of these experiments have appeared (Miller and Sigvardt 1995, 1996; Sigvardt and Miller 1998).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental procedure

Sixteen adult silver lampreys from the upper Mississippi River were obtained from Dennis and Sandy Mason (Marquette, IA) and maintained in a dark aquarium at 3°C. Methods of dissection and maintenance of the in vitro spinal cord preparation were similar to those reported previously (Sigvardt and Williams 1996). Approximately 50 segments (44-51 segments) of spinal cord with the notocord were removed from each animal from the level of the caudalmost gill hole to the cloaca. The preparation was pinned at five-segment intervals through the notocord to a Sylgard (Dow Corning, Midland MI)-lined experiment chamber and bathed in cooled (8-11°C) physiological saline (Wickelgren 1977). Three isolated bathing compartments of adjustable size were created in the experimental chamber using two 1/8-in.-thick Plexiglas partitions with central notches that fit around the spinal cord preparation; the interfaces between the partitions, chamber walls and preparation were sealed with petroleum jelly (Fig. 1A; Cohen 1987a; Rovainen 1985; Sigvardt and Williams 1996). Partition seal integrity was checked visually on a regular basis throughout each experiment.



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Fig. 1. Example of effect of blocking on the motor rhythm. A: experimental setup. Three isolated bathing compartments were established through use of movable plastic partitions (vertical bars). During control trials, all three compartments contained D-glutamate in physiological saline. During blocking trials, the solution in the middle compartment was replaced with high-manganese, low-calcium saline to eliminate local synaptic activity, but not transmission of action potentials in neuronal projections spanning the blocked region. The size of the middle compartment ranged from 4 to 20 segments. Numbered arrows: relative placement of extracellular electrodes along the spinal cord. Named pairs of electrodes (rostral, caudal, rostrocaudal) were used in the data analysis. Electrode 3 in the middle compartment was used for ensuring that there was no output motor rhythm in the middle compartment during blocking trials and for ensuring full recovery of the motor rhythm after blocking trials. B: a control trial for one preparation. Fictive locomotion was induced by 0.3 mM D-glutamate in the bathing solution in all three compartments. Top and middle: raw extracellular electrode recordings from electrodes 2 and 4 (corresponding ventral roots are indicated in parentheses); bottom: corresponding extracted spike trains in combined traces. Dashed lines represent the threshold for spike detection in each channel. C: immediately subsequent trial in the same animal, in which synaptic activity was blocked locally in 19 middle segments by replacing the D-glutamate solution in the middle compartment with low-calcium, high-manganese saline solution. Raw and extracted spike trains are from the same electrodes as in B.

For electrode and partition placement, and later analysis, distances between points on the spinal cord were measured in units of myotome segments, which are visibly delineated by dark bands on the tissue covering the notochord. Lateral pairs of motor roots typically exit the spinal cord ventrally once per segment. In these animals, the length of a segment (along the rostral-caudal axis) is ~2 mm in rostral segments, decreasing to ~1 mm in length in the caudal segments.

D-Glutamate (0.2 mM-0.5 mM) was bath applied to induce stable fictive locomotion (Cohen and Wallén 1980; Poon 1980). Once chosen, the concentration of D-glutamate solution was not changed during the experiment. During control trials, all three compartments contained the D-glutamate solution. During "blocking" trials, the control solution in the middle compartment was replaced with a low-Ca2+, high-Mn2+ saline solution containing (in mM) 91 NaCl, 2.1 KCl, 0.26 CaCl2, 3.6 MnCl2, and 20 NaHCO3, adjusted to pH 7.4, to eliminate local synaptic activity (Berry and Pentreath 1976; cf. Cohen 1987a) while retaining transmission of action potentials along any long propriospinal axons spanning the blocked region. The length of the blocked region (i.e., middle compartment) was varied between 4, 8, 12, 16, and 20 segments (±1-2 segments). Blocking trials could last up to approximately a half-hour, depending on the cycle frequency (a minimum number of ventral root bursts were required to be recorded; see later description). Recovery of the rhythm in the middle compartment after blocking, particularly after long blocking sessions, often required multiple washes with normal physiological saline followed by slow titration of D-glutamate to its final concentration. A subsequent control trial was always performed before the next blocking trial. To control for possible time effects, the sequence of blocked region lengths was changed for each animal to satisfy a Latin square design as much as possible.

Fictive locomotion, measured as ventral root activity, was recorded en passant with five suction electrodes placed near ventral roots exiting from the right side of the spinal cord, in segments 6 and 12 (rostral compartment), 25 (middle compartment), and 37 and 43 (caudal compartment). The placement of electrodes varied somewhat from these positions for each animal, depending on preparation length and quality of the recording at any particular root. The electrode numbering convention shown in Fig. 1A is referred to throughout. For each trial, ~100-200 (typically 150) bursts of ventral root activity were recorded for subsequent analysis. Electrode signals were amplified, filtered to remove frequencies outside the range 0.1-1.0 KHz, and recorded on magnetic tape. Electrode signals were digitized at 5 kHz into a 486-based computer (model ISC-64 DAQ board, ComputerScope EGAA version 3.5, RC Electronics, Santa Barbara, CA). Action potential occurrence times (spike trains) were extracted from the digitized signals after setting individual rise and fall thresholds for each data channel (dashed lines, Fig. 1, B and C). We estimate that ~10% of the extracted "spikes" were actually compound action potentials. Such misassignments of spike events would result in underestimation of the number of spikes and spike frequency within bursts. However, neither measure was used in the present analysis. Moreover, our frequency-domain analysis (see following section) was restricted to frequencies in the range of the bursting rate, a regimen that is well separated from the within-burst spike frequency range in the power spectrum.

Data analysis

Most studies of the lamprey locomotor system have used "burst analysis" methods in which start and end times of ventral root bursts are identified and used to compute frequency and intersegmental phase lag (e.g., Hagevik and McClellan 1994; Mellen et al. 1995; Sigvardt and Williams 1996). In the present experiments, extensive blocking often led to instability in the motor pattern in many trials, leading to difficulty in choosing start and end times for bursts. We instead used a Fourier (frequency-domain) analysis method that works directly on spike event times (Rosenberg et al. 1989), allowing us to measure frequency, intersegmental phase lags, correlation, and variability in motor activity without needing to identify bursts (Miller and Sigvardt 1998). Spectral analysis provides information on the variables of interest over a range of frequencies, thus facilitating insight into the "state" of the locomotor CPG system during the experimental treatments. A brief explanation of frequency-domain analysis follows. A complete discussion of our adaptation of this method to lamprey locomotor CPG data of the kind presented here can be found in Miller and Sigvardt (1998). All spectral analysis calculations were performed with routines developed in our laboratory using MatLab (version 4.2c0.1, Mathworks, Natick, MA) on a Silicon Graphics Indigo workstation (Miller 1997, Miller and Sigvardt 1998).

In Fourier analysis, the power spectrum provides a means of determining the important frequencies in a signal. Similarly, the cross-spectrum indicates the important shared frequencies between two signals. The cycle frequency of fictive locomotion was determined for the rostral and caudal ends from the main peak of the cross-spectrum for the pair of spike trains recorded from the electrodes in each end compartment (e.g., Fig. 2A1). To obtain a better estimate of the true cross-spectrum, we used the common approach of "window averaging": the spikes from two electrode recordings were first divided into a series of eight equal-time windows, a cross-spectrum was calculated for the spike trains in each window, and the resultant eight spectra were averaged. To minimize the small distorting effects of abrupt window edges on the Fourier transform, spikes in each window were "tapered" before spectral calculation by multiplication with a cosine bell function so that spike amplitudes (initially set to 1.0) were maximal at the window center and approached zero at the window edges (see Miller and Sigvardt 1998). The relative variability in each of the rostral and caudal cycle frequencies was estimated by measuring the width of the main cross-spectral peak at the quarter-maximal level (bandwidth, e.g., Fig. 2A1, horizontal line). A similar calculation of the cross-spectrum between electrodes 2 and 4 (Fig. 2B1) gave the rostrocaudal shared frequency.



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Fig. 2. Spectral analysis of the trials shown in Fig. 1. A1 and A2: rostral (thin line) and caudal (heavy line) cross-spectra, calculated from spike trains from electrode pairs 1 and 2, and 4 and 5, respectively (refer to Fig. 1A) as described in METHODS). To facilitate comparison, each spectrum was normalized to its spectral power at zero frequency (not shown). Circles denote main peak values corresponding to the cycle frequency (bursting rate) of fictive locomotion in each end compartment (control: 0.86 Hz, rostral and caudal; blocked: 0.26 Hz, rostral, and 0.4 Hz, caudal). Horizontal bars indicate the width of the main peak at the quarter-maximal level (bandwidth), a relative measure of variability in cycle frequency (only caudal bandwidth is shown for clarity; control: 0.17 Hz; blocked: 0.33 Hz). B1 and B2: rostrocaudal cross-spectrum, calculated from electrodes 2 and 4. B2: circled value of the rostrocaudal cross spectrum denotes a preferred shared frequency between the rostral and caudal regions of spinal cord, although the individual rostral and caudal preferred frequencies differ (circled values in A2). The rostrocaudal cross-spectrum was used in the calculation of rostrocaudal phase and coherence spectra (see C and E). C1 and C2: rostrocaudal phase spectrum. The mean phase lag at the peak shared frequency was calculated from the points in the shaded region in each plot, which was defined by the bandwidth of the peak of the rostral-caudal cross-spectrum (B). These mean values were divided by the segmental distance between the electrodes (21 segments in this case) to obtain the rostrocaudal phase lag per segment at the cycle period (i.e., fraction of the cycle period per segment; control: 0.0190; blocked: 0.0123). D1 and D2: rostrocaudal SD of phase spectra (same electrode pair as in B and F). Circled minima are within the shaded regions shown in C1 and C2 and represent the relative variability in the phase lag at the shared cycle frequency in the same units as the phase spectrum (control: 0.025 rad/2 pi; blocked: 0.113 rad/2 pi). E1 and E2: rostrocaudal coherence spectrum, which can range in value between 0 and 1. Circles denote the peak values (control: 0.95; blocked: 0.60). Dashed horizontal line: 99% confidence limit upper bound (see METHODS). Heavy line in E2: rostrocaudal coherence in the 19-segment blocking trial; thin line: coherence spectrum after the spinal cord was transected at ventral root 25 in the center of the middle compartment.

The lamprey locomotor CPG generates delays in segmental activation in the caudal direction that result in a tailward wave of lateral curvature on the animal's body during forward swimming. The intersegmental delay scales with cycle period so that their ratio (phase lag) is held constant to approximately 1% per segment, independent of cycle frequency (Wallén and Williams 1984). In the present experiments, the phase lag between the rostral and caudal ends of the preparation was determined as follows. First, the phase spectrum was calculated as the arctangent of imaginary over real parts of the window-averaged cross-spectra (e.g., Fig. 2B1; see Miller and Sigvardt 1998). Each phase lag was then determined as the mean value of the phase spectrum over a small range of frequencies centered on the cycle frequency (Fig. 2C1). This value was subsequently divided by the number of segments separating the electrodes to obtain the phase lag per segment. The rostral and caudal intersegmental phase lags were similarly determined from the cross-spectra of the rostral and caudal electrode pairs, respectively.

The variability in intersegmental phase lag at the cycle frequency was estimated as follows. First, a phase spectrum was calculated for each of the individual eight windows used to obtain the window-averaged cross-spectrum above. The standard deviation of the resultant eight phase values at each frequency was then calculated to produce a standard deviation of phase (sdPhase) spectrum. The minimum value of the sdPhase spectrum always occurred at (or within one point of) the cycle frequency and was taken as a relative measure of the variability in the phase lag at the cycle frequency (e.g., Fig. 2D1).

In previous blocking studies (Cohen 1987a; Rovainen 1985), histograms of time delays in ventral root bursts in different segments were used both to determine intersegmental phase lag and to assess whether rostral-caudal coordination remained on blocking intervening segments. Spectral analysis allows a separate assessment of intersegmental phase lag (via the phase spectrum) and the presence of correlated activity between two regions, via the coherence spectrum (e.g., Fig. 2E1). Coherence is defined as the squared cross-spectrum divided by the product of the power spectra of the two individual signals, and is thus akin to cross-correlation in the time-domain (Shumway 1988, chapt. 2; refer to Miller and Sigvardt 1998). The maximal coherence value of 1 at a given frequency indicates perfect correlation between the two signals at that frequency, whereas the minimal coherence value of 0 at a given frequency indicates that there is no correlation between the two signals at that frequency. Significance of a peak in the coherence was tested against a 99% confidence limit according to a formula that depends only on the number of windows used in calculating the spectra (Rosenberg et al. 1989):
<IT>C</IT><SUB>I</SUB> = 1 − (1 − &agr;)<SUP>1/(<IT>L</IT>−1)</SUP><SUB>,</SUB>
where alpha  = 0.99 and L = 8 windows in the present analysis. The presence of significant rostral-caudal coherence in the frequency band centered on the cycle frequency was taken as evidence of the presence of physical coupling between rostral and caudal segments during the blocking trials. Because the test line is set at the 99% level, approximately 1% of the points in the coherence spectrum may randomly appear as "false positives" above this line. By the same probability argument, in most such cases the immediate neighbors on either side of such false positives can be expected to occur below the test line. In the present analysis we used this objective criterion to eliminate borderline cases of one-point-wide peaks from consideration in determining significant coherence.

Blocking was deemed effective if all rhythmic neural activity in the electrode in the middle compartment was eliminated. During most recording sessions, a few events were recorded from the electrode in the middle compartment (electrode 3); most of these events were attributable to external (nonbiological) electrical noise. However, in a few trials, some tonic activity or a few burst events were recorded, indicating a failure of the blocking treatment. Such trials were either dropped from the analysis or reperformed. In addition, for all blocking trials in which any events were recorded from the middle compartment electrode, the coherence between the signals from the middle electrode and an electrode in each end compartment was tested for significance. If significant coherence was detected, the trial was not included in data analysis.

The main statistical results over all animals were obtained by performing repeated-measures analysis of variance (ANOVA) (Statistica 4.5 for Windows, StatSoft, Tulsa, OK) with two repeated-measures factors: number of blocked segments (four levels corresponding to 4, 8, 12, and 16 blocked segments), and a factor with two levels corresponding to control and blocked values. Separate analyses were performed on rostral, caudal, and rostrocaudal values. To test for interactions between the rostral and caudal values, combined ANOVAs were performed with an additional repeated-measures factor with two levels corresponding to rostral and caudal recordings.

The variance in the measured quantities across all animals tended to increase with the number of blocked segments, potentially leading to violations of the compound symmetry assumption in the univariate ANOVA method. Therefore univariate P values were corrected using the method of Greenhouse and Geisser (1959); in addition, multivariate ANOVA (MANOVA) was performed for comparison, because this method does not depend on the presence of compound symmetry in the data (O'Brien and Kaiser 1985). In 5 of the 16 animals studied, the 20-segment blocking treatment was either not performed (3 animals) or did not have adequate motor rhythm for analysis. The 20-segment blocking category was consequently not included in the ANOVA analysis, but the results are presented in the figures.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in the motor pattern quantified by spectral analysis

A typical example of the effect of blocking on the motor rhythm in one preparation is presented in Fig. 1. During the control trial (Fig. 1B), when all compartments contained D-glutamate in saline, the motor rhythm was characterized by highly regular bursts of action potentials in the ventral roots. The characteristic rostrocaudal phase lag can be clearly seen by comparing the spike trains for electrodes 2 and 4 (Fig. 1B, lower combined traces). When 19 middle segments were subsequently blocked with a low-calcium, high-manganese saline solution (Fig. 1C), the motor activity in each end compartment slowed and became much more variable. In addition, the phase lag between the activity in the rostral and caudal compartments appeared to be changed and more variable (Fig. 1C, lower combined traces).

Graphic display of frequency-domain spectra is a highly effective means of visualizing and quantifying these experimentally induced changes in the motor pattern. As shown in Fig. 2A1, the highly regular, single-cycle frequency was captured in the sharp, overlapped main peaks of the rostral and caudal cross-spectra with little power appearing in neighboring frequencies. By contrast, during blocking (Fig. 2A2) the main cross-spectral peaks occurred at lower frequencies relative to the control frequency and at different frequencies relative to each other. The increased rhythm variability during blocking evident in Fig. 1C is represented as lower and broadened peak regions relative to the controls (compare the horizontal bandwidth bars in Fig. 2, A1 and A2). The rostrocaudal phase lag was reduced in the blocking trial relative to the control (compare the horizontal bars in Fig. 2, C1 and C2); the bars are centered on the peak frequency of the rostrocaudal cross-spectrum (Fig. 2, B1 and B2). Increased variability in phase during blocking was captured in the increased value of the sdPhase spectrum at the shared frequency (compare the circled points in Fig. 2, D1 and D2). Finally, blocking in this example resulted in a severely decreased value of the coherence relative to the control value at the shared cycle frequency (compare the circled points of the coherence spectra in Fig. 2, E1 and E2, heavy line), indicating a reduced level of correlation between the rostral and caudal motor rhythms. Nevertheless, the peak coherence in the blocking trial remained above the significance test line (dashed line in Fig. 2, E1 and E2), suggesting the presence of physical coupling fibers that span the blocked region. When the spinal cord was subsequently transected in the middle of the blocked region (Fig. 2E2, thin line), the coherence fell to below significance.

The length of functional coupling

In control trials for all animals, nearly maximal coherence was typically measured in the activity in the rostral and caudal regions of the preparations (Fig. 3A), as well as in the activity between the rostral and caudal ends (Fig. 3B). Blocking had a negligible effect on coherence in local rostral and caudal activity in most preparations (Fig. 3A).



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Fig. 3. Local correlation in fictive locomotion did not change, whereas rostrocaudal correlation decreased during blocking. A: local rostral (electrodes 1 and 2) and caudal (electrodes 4 and 5) coherence. There was no change in the local coherence with blocking, according to a linear regression of the data for blocking over 4-12 segments; rostral: P = 0.098; caudal: P = 0.12, and P = 0.045 for blocking over 16-20 segments. B: rostrocaudal coherence (electrodes 2 and 4). P values shown for the 4- and 8-segment blocking categories indicate the level of significance for difference between blocked and corresponding control values (repeated-measures ANOVA, see METHODS). Dashed lines: 99% confidence interval upper bound (same as in Fig. 2, E1 and E2). In this and subsequent figures, n = 16, except for 20 blocked segments (n = 11).

By contrast, an increasing number of blocked middle segments resulted in significant and increasing reduction of the peak rostral-caudal coherence (Fig. 3B; P values in the figure). Because we controlled for possible time effects (see METHODS) a systematic decrease in rostral-caudal coherence was related only to the number of blocked middle segments. Despite reductions in amplitude, rostrocaudal coherence remained statistically significant in most preparations during blocking of 16 segments. With the exception of two cases, all preparations showed significant coherence in the neighborhood of the shared cycle frequency. When 20 segments were blocked, peak values of rostrocaudal coherence remained above the test line in many preparations. However, inspection of the individual spectra for this treatment revealed many borderline cases with one-point-wide peaks. When these cases are eliminated (see rationale in METHODS), only in 7 of 11 preparations can the coherence be considered significant. In addition, two other cases in the 20-segment blocking category had not been analyzed because of inadequate rhythmicity in the preparation (see METHODS). Thus, the detrimental effect of blocking 20 middle segments on rostrocaudal coherence was more profound than is revealed in Fig. 3B. Nevertheless, these results indicate that propriospinal projections as long as 16 segments in most preparations, and 20 segments in some preparations, were capable of maintaining some correlated activity between distant regions of the spinal cord during fictive locomotion, despite the removal of a significant amount of intervening segment-to-segment coupling pathways and in the absence of descending and sensory input.

Propriospinal coupling and the maintenance of intersegmental phase lags

We next examined whether the multisegmental coupling was required for the generation and maintenance of intersegmental phase lags. We did not include the 20-segment blocking category in our analysis of phase lag because there were relatively few cases of significant rostrocaudal correlation for this treatment (see description of coherence results). When the correlation between two signals is not significant, it is not appropriate to calculate a phase between the two.

Both rostral and caudal local intersegmental phase lags remained unchanged relative to control values during blocking of as many as 16 segments (Fig. 4A). During blocking, the segments in the rostral and caudal compartments did not receive input from propriospinal neurons originating in the middle segments (i.e., distances from a few to as many as 25 segments away). Thus, the absence of change in the values of local intersegmental phase lags indicates that the maintenance of intersegmental phase lag was not dependent on information conveyed by propriospinal coupling fibers of medium to long lengths.



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Fig. 4. Local intersegmental phase lag did not change, whereas rostrocaudal phase lag decreased during blocking. A: local rostral and caudal intersegmental phase lags, obtained from the phase spectra of electrode pairs 1 and 2, and 4 and 5, respectively. There was no change in either the rostral or caudal phase lags during blocking (P > 0.37, repeated-measures ANOVA). Caudal phase lags were significantly larger than rostral phase lags during both control and blocking trials (1.32 and 0.71% per segment, respectively; P < 0.0002). B: rostrocaudal phase lag obtained from the phase spectra of electrodes 2 and 4. P value shown in B indicates a significant difference from the corresponding control values (repeated measures ANOVA). All values are mean ± SD.

In contrast with the local phase lags, the phase lags between the two ends (rostrocaudal phase lags) decreased significantly relative to control values when as few as four middle segments were blocked (Fig. 4B). Rostrocaudal phase lags continued to decrease systematically with increased number of blocked segments to nearly half the control value at 16 blocked segments. The rostrocaudal phase decrease was exhibited in all but two preparations during block of 4 and 8 segments and all preparations during block of 12 and 16 segments. Thus, the medium to long fibers, although able to maintain correlated activity between two separated regions of spinal cord (Fig. 3B), were not able to maintain behaviorally appropriate (i.e., ~1% per segment) values of phase lag.

Motor rhythm frequency and variability changed during blocking

In addition to inducing reductions in rostrocaudal coherence and phase lags, blocking led to significant changes in the cycle frequency of fictive locomotion and in the variability in both frequency and intersegmental phase. Control values of cycle frequency differed from preparation to preparation (typically in the range of 0.5-1.0 Hz). Cycle frequency decreased linearly from control values with increasing number of blocked middle segments (Fig. 5A). In addition, the occurrence of a difference in peak frequency between the rostral and caudal regions increased with increased number of blocked segments. In approximately half the preparations, the rostral and caudal ends of the spinal cord took on different peak frequencies of more than 0.05 Hz when 16 or more middle segments were blocked (Fig. 5B). Variability in both rostral and caudal cycle frequency increased from control values with increasing numbers of blocked middle segments when >= 12 segments were blocked (Fig. 6A; P values in legend). Similarly, variability in rostral, caudal, and rostrocaudal intersegmental phase lags increased from control values with increasing number of blocked segments (Fig. 6B). Figure 6B also shows that the control rostrocaudal phase variability was greater than those for either end region and increased more dramatically than the local variability under increased number of blocked segments.



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Fig. 5. Rostral and caudal cycle frequencies decrease and diverge during blocking. A: cycle frequency decreases with increased number of blocked segments. Cycle frequency was obtained from the cross-spectra of electrode pairs 1 and 2 (rostral) and 4 and 5 (caudal). Values are expressed as difference from control because of the large interanimal variability in control cycle period (range: 0.4-1.0 Hz, median: 0.7 Hz). Slope of the linear regression line through all rostral and caudal data = -0.02 Hz/segment (slopes of individual rostral and caudal regressions: -0.019 Hz/segment and -0.013 Hz/segment, respectively; P < 10-6 for all regressions). B: increased occurrence of a difference in preferred rostral and caudal peak frequencies with increased number of blocked segments.



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Fig. 6. Variability in cycle frequency and intersegmental phase lags increased during blocking. A: variability in the cycle frequency. Rostral and caudal values of bandwidth of the peak cycle frequency were not significantly different from each other over all animals during control trials (P < 0.09) or blocking trials (P > 0.3). Peak frequency bandwidth during blocking differed significantly from control values when >= 12 segments were blocked (rostral: P < 0.004; caudal: P < 0.017, for 12 blocked segments). B: variability in intersegmental phase lags. Baseline variability in the caudal phase lags was greater than that in the rostral phase lags during control trials (P < 0.017). Rostral and caudal mean phase variability increased together until 16 segments were blocked, at which point the increase in the caudal variability was significantly greater than that for the rostral end (P < 0.027). Variability in rostrocaudal phase lags was greater than both rostral and caudal phase variability (P < 0.037) and increased at a greater rate during increased blocking. The value for rostrocaudal phase variability for 20 blocked segments is not shown, because there were far fewer valid values for rostrocaudal coherence and phase in this category.

Rostral-caudal differences

An open question regarding the neural architecture of the lamprey locomotor CPG concerns the degree of uniformity of the interneuronal coupling along the spinal cord. Among the general types of rhythmically active lamprey spinal neurons, many are found in different percentages in different parts of the spinal cord and send projections asymmetrically in the ascending and descending directions (Buchanan 1982; Buchanan et al. 1989). Although rostral-caudal differences were not a focus of the present study, in the course of performing the present experiments, we noted a number of differences in the behavior of the rostral and caudal ends of the preparation.

During control trials, the rostrocaudal intersegmental phase lags took on values of ~1% per segment (Fig. 4B, 1.06 ± 0.02% per segment). However, the rostral and caudal control intersegmental phase lags were significantly different from each other: rostral phase lags tended to be <1% per segment, whereas caudal phase lags tended to be >1% per segment (Fig. 4A).

The preferred frequency of fictive locomotion in the rostral and caudal end regions tended increasingly to diverge during increased blocking (Fig. 5A). However, the frequency at the rostral end was not found to be either systematically less than or greater than that at the caudal end (P = 0.153). This finding supports the view established in earlier studies (see Cohen 1987b) that the preferred frequencies of the individual segments do not vary in a systematic way along the spinal cord.

Variability in the rostral and caudal cycle frequencies was not significantly different in control trials (Fig. 6A). However, blocking appeared to detrimentally affect motor output in the caudal end of the spinal cord more than in the rostral end. In five animals, it was difficult to detect a peak cycle frequency in the caudal cross-spectrum at 16 and 20 blocked segments, and in two additional animals, caudal activity was virtually eliminated at 20 blocked segments, whereas rostral rhythmicity continued to be maintained. The variability in caudal phase lags was higher on average than the variability in rostral phase lags during control trials (Fig. 6B; P < 0.017), and this trend continued during blocking. In addition, the variability in the caudal phase lags increased substantially at 16 blocked segments to approximately twice its value at 12 blocked segments (P < 0.027), whereas the variability in rostral phase lags did not change. The relatively higher caudal values of phase variability at 16 blocked segments were also observed in the tendency in several preparations to show a reduction in local caudal coherence (Fig. 3A). These results suggest that the rostral regions of the CPG are somewhat more intrinsically stable than more caudal regions (cf. Cohen et al. 1995).

Effects of rhythm variability and frequency difference on coherence

The coherence spectrum is known to be sensitive to noise in the input signals if the noise associated with the two signals is not correlated. Decreases in correlation of rostral and caudal activity measured with the rostrocaudal coherence may thus in part reflect increases in noncorrelated variability in the activity within the rostral and caudal end regions. A comparison of rostrocaudal coherence against rostral frequency variability (bandwidth) when 16 segments are blocked suggests that our coherence measurements were not affected to a great degree by increased variability (Fig. 7A). A comparison of rostrocaudal coherence against rostral-caudal cycle frequency difference when 16 segments are blocked suggests that our coherence measurements were also not affected by differences in preferred cycle frequency (Fig. 7B). These relationships among coherence, frequency variability, and small frequency differences were reproduced in simple simulations of two reciprocally coupled oscillators (Sigvardt and Miller 1998).



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Fig. 7. Neither variability in cycle frequency nor difference between rostral and caudal cycle frequency affected the peak value of rostrocaudal coherence during blocking of middle segments. A: rostrocaudal coherence compared with variability in rostral cycle frequency (difference from control). B: rostrocaudal coherence compared with difference in preferred rostral and caudal cycle frequency. In both A and B, 16 middle segments are blocked

Effects of rhythm variability and frequency difference on intersegmental phase lags

A potential source for the decrease in phase lags between the rostral and caudal regions was the appearance in many preparations of a difference in preferred rostral and caudal cycle frequencies during block of a relatively large number of middle segments (Fig. 5B; e.g., Fig. 2A2). A number of previous experimental results have shown that imposition of frequency differences in different parts of the spinal cord can lead to changes in the intervening phase lags (Matsushima and Grillner 1992a; Sigvardt and Williams 1996; Tegnér et al. 1993; cf. Hagevik and McClellan 1994). Such imposed changes were predicted by several oscillator-based models (Hagevik and McClellan 1994; Kopell and Ermentrout 1986, 1988, 1990; Matsushima and Grillner 1992a; also see Pearce and Friesen 1988; Tunstall and Roberts 1991, 1994). Figure 8 compares rostrocaudal phase lags with rostral-caudal difference in preferred frequency for blocking of 4-16 segments. As can be seen in the figure, rostral and caudal frequencies did not typically diverge when as many as eight segments were blocked (also see Fig. 5B), and no relationship between rostrocaudal phase lags and frequency difference was evident when <= 12 segments were blocked. When 16 segments were blocked, a significant relationship appeared between rostrocaudal phase lag and difference in preferred rostral and caudal frequencies, which suggests that during extensive blocking, rostrocaudal phase lag is susceptible to differences in rostral and caudal frequency. However, because rostrocaudal phase lag decreased significantly when just four segments were blocked (Fig. 4B), differences in preferred rostral and caudal cycle frequency can account only partially for the reduction in rostrocaudal phase lag in these experiments.



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Fig. 8. Difference in rostral and caudal peak frequency compared with rostrocaudal phase lags, over a range of 4-16 blocked middle segments. There were few cases of a difference in rostral and caudal frequency when 4-12 segments were blocked, and no apparent relationship between frequency difference and rostrocaudal intersegmental phase lag in these trials. When 16 middle segments were blocked, a significant relationship between frequency difference and rostrocaudal intersegmental phase lag appeared across all preparations in the study (linear regression and P value shown on plot).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Multisegmental propriospinal coupling and intersegmental phase lags

The present system-level experiments provide new information on the contribution of propriospinal projections to intersegmental phase lags and other properties of fictive locomotion in the silver lamprey spinal cord. We show that the concurrent activity in rostral and caudal regions of spinal cord remained correlated when local synaptic activity in 4 to <= 16-20 intervening segments was blocked, indicating that propriospinal projections are available to provide functional coupling over these distances in I. unicuspis. The functional length of these projections is consistent with other studies that have attempted to estimate the length of propriospinal projections by paired recordings (Buchanan 1982; Buchanan et al. 1989) and anatomic methods (Buchanan, unpublished observations).

We further show, however, that these medium to long coupling fibers are not able to maintain behaviorally appropriate values of intersegmental phase lags between the separated rostral and caudal regions. Instead, rostrocaudal phase lags decreased from control values of ~1% per segment as the number of intervening blocked segments was increased. This decrease occurred even when only four intervening segments were blocked. By contrast, local intersegmental phase lags within the rostral and caudal regions were not changed by the loss of coupling input from distant segments during the blocking treatments. Our results thus indicate that phase lags are controlled at a local level, and that medium to long coupling fibers are neither necessary nor sufficient for the maintenance of appropriate intersegmental phase lags. Local control of phase was also indicated in a previous study in which the length of the spinal cord was systematically reduced from 50 to 15 segments without producing a change in the value of intersegmental phase lags (Miller 1997; Miller et al. 1995). We can thus define a "control domain" for the maintenance of intersegmental phase as the number of segments minimally required for generation and maintenance of appropriate intersegmental phase lags. Taken together, the results of the present experiments and the length-reduction experiments indicate that the phase control domain is likely to be no greater than 10-15 segments, and may be as small as 4-6 segments in the lamprey spinal cord. Although intersegmental phase lags have been routinely measured in spinal cord preparations as short as ~12 segments (e.g., Matsushima and Grillner 1992a) and in some instances in shorter preparations (Sigvardt, unpublished results), a quantitative assessment of phase lag values with length for short preparations has not yet been described.

Origin of the changes in frequency and variability during blocking

Among the most dramatic changes in the motor pattern during blocking was the systematic reduction in the cycle frequency (Fig. 5A). As the number of blocked segments increased from 4 to 20, the number of active segments in each end compartment decreased from 23 to 15. The decrease in cycle frequency with increased blocking may thus be the result of a systematic decrease in the total synaptic activity in the preparation. However, in other experiments in which the lamprey spinal cord was systematically reduced in length by physical removal of segments from one end while the concentration of D-glutamate was held constant, no corresponding decrease in cycle frequency was measured (Miller 1997; Miller et al. 1995). It seems likely that the decrease in cycle frequency during increased blocking in the present experiments was related somehow to changes in the relative complement of available shorter and longer intersegmental coupling in the preparation.

Variability in both frequency and intersegmental phase increased as a result of blocking, both in the local rostral and caudal activity, and rostrocaudally (Fig. 6). Although decreased total synaptic activity due to blocking was ruled out as a cause of the decrease in cycle frequency, it remains possible that this factor contributed to the increased variability of fictive locomotion. This relationship could be tested by increasing the concentration of D-glutamate in the end compartments during the blocking trials to try to recover more stable rhythmicity.

Alternatively, the increased variability in activity within each end region during blocking may have been caused by increasingly disruptive input from the opposite end via the medium and long coupling fibers. Local activity in the end regions may have been relatively more influenced by propriospinal input from distant segments during blocking trials than during control trials, because input from nearer middle segments was eliminated. The input from the opposite end of the preparation would be less appropriate and thus potentially more disruptive if, for instance, the preferred frequency of fictive locomotion was different at each end during blocking. Indeed, our results indicated an increased occurrence of a difference in preferred peak frequency between the rostral and caudal ends with an increase in the number of blocked segments (Fig. 5B).

Moreover, it is possible that the input from distant segments is always relatively inappropriate (in the context of control of frequency and intersegmental phase), but that errors associated with long-distance input are normally tempered by the presence of input from more nearby segments. Such a view is supported by our finding that longer coupling alone was not able to maintain appropriate values of intersegmental phase lag between the rostral and caudal ends when even just a few intervening segments were blocked (Fig. 4B). Removal of the full complement of coupling between the rostral and caudal ends during blocking may thus have affected the mechanism responsible for maintaining phase constancy, so that the rostrocaudal delays no longer scaled appropriately with cycle period. Because the delay information is provided only by the longer coupling during blocking, one implication is that the longer multisegmental coupling fibers are not "tuned" to provide 1% per segment phase information over medium to long distances (Kiemel 1990).

In the present study, CPG output was monitored during system-level manipulation of coupling to gain useful insight into the contribution of multisegmental propriospinal connections to system performance. Such an indirect approach to building an understanding of the functional architecture of the lamprey locomotor CPG has proven essential, because, on the one hand, a complete cellular-level description of coupling remains difficult to obtain, whereas on the other hand, the complicated nature of the known connectivity leads to many theoretical hypotheses about coupling and function that must be sorted according to objective, data-driven criteria. Additional quantitative information on propriospinal coupling and continued theoretical synthesis will both be required before a satisfactory ensemble understanding of the relationship between coupling and function is obtained for this system.


    ACKNOWLEDGMENTS

We thank N. Kopell for many helpful discussions of this work and J. Birmingham and J. Golowasch for comments on earlier versions of the manuscript.

This work was supported by National Institutes of Health Grant MH-10570 to W. L. Miller and NS-22360 and MH-47150 to K. A. Sigvardt.


    FOOTNOTES

Address for reprint requests: K. A. Sigvardt, Center for Neuroscience, University of California, 1544 Newton Court, Davis, CA 95616.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 March 1999; accepted in final form 14 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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