Contributions of the Reticulospinal System to the Postural Adjustments Occurring During Voluntary Gait Modifications

Stephen D. Prentice1 and Trevor Drew2

 1Department of Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3G1; and  2Department of Physiology, University of Montréal, Montreal, Quebec H3C 3J7, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Prentice, Stephen D. and Trevor Drew. Contributions of the Reticulospinal System to the Postural Adjustments Occurring During Voluntary Gait Modifications. J. Neurophysiol. 85: 679-698, 2001. To test the hypothesis that reticulospinal neurons (RSNs) are involved in the formation of the dynamic postural adjustments that accompany visually triggered, voluntary modifications of limb trajectory during locomotion, we recorded the activity of 400 cells (183 RSNs; 217 unidentified reticular cells) in the pontomedullary reticular formation (PMRF) during a locomotor task in which intact cats were required to step over an obstacle attached to a moving treadmill belt. Approximately one half of the RSNs (97/183, 53%) showed significant changes in cell activity as the cat stepped over the obstacle; most of these cells exhibited either single (26/97, 26.8%) or multiple (63/97, 65.0%) increases of activity. There was a range of discharge patterns that varied in the number, timing, and sequencing of the bursts of modified activity, although individual bursts in different cells tended to occur at similar phases of the gait cycle. Most modified cells, regardless of the number of bursts of increased discharge, or of the discharge activity of the cell during unobstructed, control, locomotion, discharged during the passage of the lead forelimb over the obstacle. Thus, 86.9% of the modified cells increased their discharge when the forelimb ipsilateral to the recording site was the first to pass over the obstacle, and 72.2% when the contralateral limb was the first. Approximately one quarter of the RSNs increased their discharge during the passage of each of the four limbs over the obstacle in both the lead (27.1%) and trail (27.9%) conditions. In general, in any one cell, the number and relative sequencing of the subsequent bursts (with respect to the lead forelimb) was maintained during both lead and trail conditions. Patterns of activity observed in unidentified cells were very similar to the RSN activity despite the diverse population of cells this unidentified group may represent. We suggest that the increased discharge that we observed in these reticular neurons reflects the integration of afferent activity from several sources, including the motor cortex, and that this increased discharge signals the timing and the relative magnitude of the postural patterns that accompany the voluntary gait modification. However, based on the characteristics of the patterns of neuronal activity in these cells, we further suggest that while individual RSNs probably contribute to the selection of different patterns of postural activity, the ultimate expression of the postural response may be determined by the excitability of the locomotor circuits within the spinal cord.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Voluntary limb movements that have the potential to destabilize posture are typically accompanied by appropriately scaled postural responses to offset the changes in equilibrium associated with the focal movement (Bouisset and Zattara 1981; Brown and Frank 1987; Frank and Earl 1990; Massion 1992; Rogers and Pai 1990; Zattara and Bouisset 1988). These postural adjustments are particularly important in walking (Jian et al. 1993; MacKinnon and Winter 1993; Prince et al. 1994; Winter et al. 1993; Yang et al. 1990), where the number of supporting limbs and their relative positioning changes throughout the task and the addition of a voluntary modification would require further postural adjustments. During gait modifications in the cat, phases of increased postural activity have been characterized by increases in the vertical ground reaction force (vGRF) as well as in the electromyographic (EMG) activity of extensor muscles during stance (Lavoie et al. 1995).

While the neural mechanisms responsible for the control of these modifications are not fully understood, there is considerable evidence to demonstrate the involvement of the motor cortex in modifying limb movements during voluntary gait modifications (Amos et al. 1990; Beloozerova and Sirota 1993; Drew 1988, 1993; Drew et al. 1996b; Widajewicz et al. 1994). The elements responsible for the coordination and control of the accompanying postural adjustments have been inferred from less direct evidence. Lesions of both the brain stem (Kuypers 1964; Lawrence and Kuypers 1968) and the ventrolateral quadrants of the spinal cord (Afelt 1974; Bem et al. 1995; Brustein and Rossignol 1998; Eidelberg et al. 1981; Gorska et al. 1990, 1993) result in deficits in the postural aspects of movement and thus demonstrate the potential of the pontomedullary reticular formation (PMRF) for this control. Similarly, impairment of the postural responses that accompany voluntary limb movements has been reported following injections of cholinergic agents into the PMRF (Luccarini et al. 1990; Sakamoto et al. 1991).

From a functional standpoint, stimulation within the pontine regions of the PMRF in the standing cat has been shown to either increase or decrease the generalized postural tone in all four limbs (Mori 1987, 1989), while microstimulation of the medullary regions of PMRF has been shown to evoke reciprocal responses among combinations of the four limbs during walking where the responses are integrated into the locomotor rhythm (Degtyarenko et al. 1993; Drew 1991; Drew and Rossignol 1984; Orlovsky 1972; Perreault et al. 1994). In addition, unit recording studies during locomotion have shown that many RSNs in the medullary and caudal pontine regions exhibit phasic modulation that is correlated to locomotor activity (Drew et al. 1986; Orlovsky 1970; Perreault et al. 1993; Shimamura and Kogure 1983; Shimamura et al. 1982).

More recently, the role of the PRMF in scaling postural adjustments has gained additional support from studies characterizing the corticoreticular pathway as an important link in the integration of posture and movement. Kably and Drew (1998b), for example, have demonstrated that many of those cortical neurons whose axon descended through the pyramidal tract and that exhibited changes in discharge activity during voluntary gait modifications also sent collaterals to the PMRF. Thus the PMRF seems well suited to coordinate the dynamic postural responses that accompany voluntary movements based on both the nature of the descending input it receives and its projections to the spinal cord.

The aim of the present research was to determine if the discharge patterns recorded from RSNs during voluntary gait modifications are supportive of their proposed role in mediating the increased postural demands introduced as the individual limbs pass over the obstacle. While it has already been established that RSNs are phasically modulated during unobstructed walking (see preceding text), the current investigation attempts to establish if and when these cells alter their discharge when stepping over an obstacle, and how these changes coincide with the changes in muscle activity which represent the postural adjustments associated with these gait modifications (Drew 1993; Lavoie et al. 1995; Widajewicz et al. 1994).

Preliminary results have been previously published in abstract form (Prentice and Drew 1995, 1999).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed on three cats (weight: 3.2, 5.0, and 6.8 kg) that were trained to walk and to step over obstacles attached to a moving treadmill belt (see Drew 1988, 1993 for details) At the completion of training, the following surgical procedures were performed under general anesthesia and in aseptic conditions. All procedures complied to the Canadian Medical Research Council's guidelines for animal care and were approved by the animal ethics committee at the University of Montréal.

Surgical procedures

Microwire electrodes (50 µm, TriML insulated stainless steel) were implanted into the ventral quadrant of the lumbar spinal cord at L2, ipsilateral to the recording site (Drew et al. 1986), both cerebral peduncles and, in two of the cats, bilaterally into the motor cortex. (The procedures used for the latter 2 sites will be presented in a subsequent paper detailing the cortical inputs to cells recorded in the PMRF.) A small stainless steel base-plate (10 × 6 mm ID), used to form a recording chamber, was positioned over the cerebellum, and 25 pairs of Teflon-insulated, stainless steel wires were implanted bilaterally into selected fore- and hindlimb muscles. Forelimb activity in this report is represented by the cleidobrachialis (ClB; shoulder protractor and elbow flexor) and triceps brachii, lateral head (TriL; elbow extensor) while the hindlimb representation is provided by the anterior head of sartorius (Srt; hip flexor) and the vastus lateralis (VL; knee extensor). Analgesics (Buprenex, 5 µg/kg) were administered for the 48 h following surgery. Antibiotics (penicillin in the form of Cephalex or Amoxil tablets) were given for at least 10 days following each surgical procedure.

Experimental procedures

Recordings were made using conventional glass-coated tungsten electrodes (impedance, 0.5-2 MOmega ) introduced into the brainstem via the cerebellum (see Drew et al. 1986, 1996a). Each isolated, single unit was tested to determine if it was a RSN by stimulating through the microwire electrodes in the spinal cord. A cell was classified as a RSN if an antidromic potential was evoked at a constant latency and if this potential could be negated, at an appropriate latency, by spontaneous orthodromic firing in a collision test (Lipski 1981). Neuronal activity was then recorded for a period of 3-5 min as the cat stepped over the obstacle. At the end of each recording session, the cat was placed on the experimenter's lap, and we recorded the movements evoked by microstimulation (<= 35 µA, 33-ms train of 0.2-ms pulses at 330 Hz) through the recording electrode at 0.5-mm intervals throughout the dorsoventral extent of the brain stem. Small lesions (10-30 µA, DC) were introduced in selected penetrations to assist in the histological reconstruction.

Histological procedures

After the final recording session, the cats were anesthetized (pentobarbital sodium, Somnotol, 40 mg/kg ip) and perfused per aortum. The motor cortex and brain stem were removed along with a section of the lumbar spinal cord. The specimens were sectioned (40 µm) in the sagittal (motor cortex and brain stem) or transverse (spinal cord) planes and stained using cresyl violet. Electrode penetrations and microwire sites were marked on tracings taken from each brain stem section that were then calibrated to the appropriate section from the atlas of Berman (1968; see Drew et al. 1986).

Data analysis

The cell activity was converted to digital pulses through amplitude discrimination and sampled along with the muscle activity at a frequency of 1 kHz on a microcomputer for further analysis. The onset and offset of muscle activity was determined using customized software and used to classify each step cycle as a control step or the step before, over, or after the obstacle. Control steps were defined as cycles occurring at least two steps before the passage of the lead forelimb over the obstacle. Steps related to the obstacle were further classified as to those in which the left limb (ipsilateral) to the recorded cell was the first (lead condition) or the second (trail condition) to pass over the obstacle. Once classified, the cycles were averaged by synchronizing all cycles to the onset of the cleidobrachialis (ClB) muscle of the limb ipsilateral to the recording site and then normalizing the duration to 256 bins based on the average step cycle time (see Drew and Doucet 1991; Udo et al. 1982). Comparisons between control walking and steps over the obstacle were made by displaying together the ensemble-averages for both conditions across three consecutive steps as shown in Fig. 4B. A confidence interval (P < 0.01, based on the SE) of the activity during the control cycles was calculated, and any differences in the cell discharge over the obstacle that exceeded this boundary for more than 25 consecutive bins were deemed significant (Drew 1993). Bursts representing significant changes in cell activity during steps over the obstacle were identified and characterized based on the relative timing of the peak difference in cell activity. The latencies of these peaks were calculated from the onset of the ipsilateral ClB muscle and expressed as a percentage of the step cycle.

We defined eight periods to categorize the individual bursts of increased activity in each cell that occurred during the gait modifications. The selection of these particular periods was designed to allow us to relate the timing and sequencing of the modifications of unit activity to the periods of modified postural activity that occurred as the different limbs stepped over the obstacle. Four of the periods (LFL, left forelimb; RFL, right forelimb; LHL, left hindlimb; RHL, right hindlimb), are identified in Fig. 1 by the appropriate abbreviations, and the light shading identifies, approximately, the swing phase when the respective limb passed over the obstacle. Essentially, the LFL and RFL phases were bound by the duration of the respective ClB activity, and the LHL and RHL phases were defined by the duration of the corresponding Srt activity. Inclusion within each of these phases required the peak of the increased unit discharge to lie within these boundaries. However, the definitions of both RFL and LHL phases required additional specification to account for the overlap between the swing phases of these two limbs (see Fig. 1). To be included within the RFL phase, the increase in unit discharge had to be completed before the peak activation of the left Srt. Inclusion within the LHL phase required that the peak increase in unit discharge had to occur after the peak of the right ClB activity.



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Fig. 1. Ensemble averages of the electromyographic (EMG) activity observed in the representative flexors and extensors of all 4 limbs are displayed for control walking and the lead condition over the obstacle. Three consecutive cycles of each condition are plotted with each cycle being synchronized to the onset of the left cleidobrachialis (ClB) activity. The central period indicates the time that the cat begins to step over the obstacle. The arrows at the bottom indicate the approximate mid-point of the swing phase of the respective limbs as they passed over the obstacle. The thick traces indicate the EMG activity during the steps over the obstacle while the thin traces represent the steps during unobstructed control walking. A confidence interval (P < 0.01) for the control walking activity is also displayed as dotted lines. Phases representing the period when each limb passed over the obstacle have been marked using light shaded boxes indicating the boundaries as associated with the respective flexor muscle. The darker boxes define 3 periods of transition between subsequent limbs passing over the obstacle (see METHODS for details). Srt, sartorius, anterior head; TriL, triceps brachii, lateral head; VL, vastus lateralis. L (l) and R (r) are used to designate muscles on the left and right side, ipsilateral and contralateral to the recording site, respectively. LFL, left forelimb; LHL, left hindlimb; RFL, right forelimb; RHL, right hindlimb.

We also defined three phases to represent the transitions between each subsequent limb passing over the obstacle. These are represented by the three areas of dark shading in Fig. 1. The transition between the left and right forelimbs (LFL/RFL) was defined as the period occurring during the time of the last 10% of the left ClB activity and the first 10% of the right ClB activity. In a similar fashion, the transition between the left and right hindlimb (LHL/RHL) was defined as the period between the activation of the left and right Srt. The nature of the transition between the right forelimb and the left hindlimb (RFL/LHL) required a more sophisticated criterion representing two possible types of patterns: the increase in discharge peaked within the activation of the right ClB and continued into the LHL phase or the bust began in the RFL phase but peaked during the activation of the left Srt.

Although there were no changes in vGRF prior to the passage of the LFL over the obstacle (Lavoie et al. 1995), we did observe a large number of cells that increased their discharge frequency before the gait modification (detailed in RESULTS). We, therefore identified an eighth category (early; not illustrated in Fig. 1) that identified those neurons that exhibited changes in discharge activity that began more than 200 ms before the onset of activity of the left ClB.

Overall, therefore using the categories defined in the preceding paragraphs, the eight identified periods that were used in the analysis were, in order: early, LFL, LFL/RFL, RFL, RFL/LHL, LHL, LHL/RHL, and RHL.

Similar periods were used to categorize the bursts of increased activity of each cell during the trail condition. The order in which the limbs pass over the obstacle changes during the trail condition is reversed, such that the right forelimb is the first to step over the obstacle followed by the left forelimb, right hindlimb, and left hindlimb. The definition of the phases described above were adapted for the trail condition by simply interchanging the right and left limbs in each definition such that the order of the phases would be early, RFL, RFL/LFL, LFL, LFL/RHL, RHL, RHL/LHL, and LHL.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EMG activity during the gait modifications

An example of the changes in EMG activity that occurred in the fore- and hindlimbs during steps over the obstacle in the lead condition is presented in Fig. 1. While the changes in muscle activity during these gait modification have been detailed in previous studies (Drew 1993; Lavoie et al. 1995; Widajewicz et al. 1994), the results shown here are intended to highlight those changes specific to the muscles recorded and to provide the appropriate context to evaluate changes in the cell activity observed during these modifications.

When the left forelimb led over the obstacle, as for the example in Fig. 1, the changes in forelimb muscle activity began with a substantial increase in the amplitude and duration of the left ClB (selected to represent the increase in muscle activity responsible for transporting the limb over the obstacle) (see Drew 1993). Toward the end of the swing phase and in particular during the transition from left to right swing, there were increases in the level of activity of both the left and right TriL. These changes in postural activity, together with the increased level of activity in the left VL (lVL) at this time, have been suggested to contribute to the elevation of the body in preparation for the passage of the trail forelimb (see Lavoie et al. 1995).

When the trailing, right, forelimb was brought over the obstacle, there was a marked increase in the activity of the shoulder retractor, Teres major (not illustrated here) (see Drew 1993) and an accompanying increase in the magnitude of the shoulder flexion (Lavoie et al. 1995). However, the subsequent shoulder and elbow protraction was similar to that observed during the unobstructed steps, and, as illustrated in Fig. 1, there was therefore little change in the level of activity in the right ClB (rClB). There was increased postural support during this time, however, that was primarily reflected in the continued increase in the level as well as the duration of the activity of the left TriL. In addition, the right hindlimb swing was shortened as evidenced by the decreased duration of the right Srt (rSrt) and there was a correspondingly premature burst of activity in the right VL (rVL).

As the hindlimbs passed over the obstacle, the hip flexor, Srt, showed similar changes in the lead and trail condition to those described for the ClB. Thus there was a large increase in the left Srt activity in the lead condition, which, like the ClB in the forelimb, acts to flex the limb above and over the obstacle (see Widajewicz et al. 1994). In the trail condition, there was little change in the right Srt, although there were marked increase in the activity of the knee flexor, semitendinosus, which served to raise the trail hindlimb above the height of the obstacle (not illustrated) (see Widajewicz et al. 1994). The transition between the left and right hindlimb swing phases was accompanied by bilateral and sequential increases in the knee extensor (VL) activity which occurred as the hindlimb support changed from right to left in much the same way that the TriL muscles increased in the forelimb. It has been demonstrated that the increases in vGRF at this time act to elevate the hindlimbs as they straddle the obstacle (Lavoie et al. 1995).

Overall, the changes in the level, duration, and timing of the extensor muscle activity during these gait modifications serve as a valid representation of the complex postural adjustments that occur as each limb, in turn, is brought over the obstacle.

Unit activity during gait modifications

DATABASE. A total of 400 cells (183 RSNs; 217 unidentified reticular cells) in three cats were successfully recorded from the PMRF as the animals stepped over an obstacle attached to the treadmill. The location of each of these cells within the brainstem is illustrated in Fig. 2. Cell recordings were obtained throughout the depth of the brain stem and within a region that ranged from stereotaxic coordinates, anteroposterior (AP) -1.2 to -10.5 according to the atlas of Berman (1968). With the exception of three neurons, all cells were recorded within 2.1 mm of the midline. The majority of the cells in cats RS14 and RS18 were recorded within the more rostral aspects of the nucleus reticularis gigantocellularis (NRGc); the recordings in RS16 included, in addition, a large number of cells within the nucleus pontis caudalis (NRPc) and nucleus pontis oralis (NRPo). The recorded RSNs and unidentified cells were mostly intermingled throughout the recording region.



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Fig. 2. Locations of 180/183 reticulospinal (RSNs, ) and all 217 unidentified neurons (open circle ) recorded during locomotion for the three cats, RS14, RS16, and RS18 (A-C, respectively) used in these studies. All plots are made on standardized tracings (laterality 1.2 mm from the mid-line) of sagittal brain stem sections taken from Berman (1968). Note that there are less points than cells because several neurons were recorded in very close proximity. The approximate locations of the 4 major reticular nuclei found in the medial reticular formation, according to Brodal (1957), are illustrated by the dotted outlines. 7G, genu of the facial nerve; IO, inferior olive; NRGc, nucleus reticularis gigantocellularis; NRMc, nucleus reticularis magnocellularis; NRPc, nucleus reticularis pontis caudalis; NRPo, nucleus reticularis pontis oralis; PH, nucleus praepositus hypoglossi; TB, trapezoid body.

The conduction velocities of 173 of the 183 identified RSNs recorded during locomotion, based on the distance measured between AP-7 and the spinal implant in each cat, are illustrated in Fig. 3. The majority of these RSNs had fast conducting axons (mean = 98 m/s). The bias toward the recording of large neurons during these experiments is, in part, due to the difficulty in recording smaller cells for extended periods during the walking trials.



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Fig. 3. Conduction velocities for 173 of the identified RSNs recorded during locomotion. The average velocity for the population was 98 m/s. There were 10 cells for which the latency was not noted.

GENERAL CHARACTERISTICS OF CELLS IN THE DATABASE DURING LOCOMOTION. Based on the activity of the cells when the leg ipsilateral to the recording site (left limb) was the first to step over the obstacle, we defined four classes of discharge patterns. As detailed in Table 1, a large proportion of both RSNs and unidentified neurons significantly changed their level of discharge activity when the cat stepped over the obstacle (modified) as compared with the control steps between the obstacles. Most cells exhibited either single or multiple increases in cell discharge activity during this step (see following text), although a small number of neurons decreased their level of activity (inhibited). The next most common group of neurons were those that showed no change in activity when the cat stepped over the obstacle with the lead limb (unmodified). Note that as detailed later, whether a cell was modified or not during the gait modification was independent of its pattern of discharge during control locomotion. The two other classes of neurons were composed of those cells that were either completely unrelated to the locomotor rhythm (i.e., they did not show a consistent discharge in each step cycle) or did not discharge at all, either during the control steps or those over the obstacle (silent). These latter cells discharged neither during locomotion nor during any spontaneous movements that the cat made.


                              
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Table 1. Discharge characteristics of neurons recorded during locomotion: lead condition

RSN ACTIVITY DURING THE LEAD CONDITION. An example of a RSN that changed its pattern of discharge activity during the voluntary gait modifications is illustrated in Fig. 4. This neuron responded to antidromic activation of the lumbar spinal cord at a latency of 2.8 ms (Fig. 4C), representing a conduction velocity of 102 m/s, and had a receptive field that covered both sides of the face and the entire ipsilateral (left) surface of the body, with little or no input from the contralateral trunk or limbs (Fig. 4D). During unobstructed walking, the activity of this neuron was phasically modulated, increasing twice per step cycle (Fig. 4, A and B). Examination of raster displays triggered on the onset of the period of activity in each of the recorded muscles (not illustrated) suggested that these two periods of activity temporally covaried with the period of activity of the ipsilateral (left) and contralateral (right) Srt. During the steps over the obstacle, there were significant changes in cell activity that occurred as multiple bursts of increased discharge frequency distributed across the time when the different limbs passed over the obstacle (Fig. 4B). Using the definitions given in METHODS and illustrated in Fig. 1, this RSN was determined to exhibit significantly increased activity during four different phases during the gait modification: LFL, LFL/RFL, LHL, and RHL. In addition, in this and in a few other neurons, there was also a later burst of activity that occurred in the step following the passage of the RHL over the obstacle. Although changes in activity at this time were not analyzed, they were not unexpected as the pattern of EMG activity in this step was also sometimes different from that observed in the control cycles.



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Fig. 4. A: example of the discharge of a RSN and the EMG activity of selected flexor muscles from all 4 limbs during the lead obstacle condition. B: ensemble averages of the unit and EMG activity displayed for control walking and the lead condition over the obstacle. Three consecutive cycles of each condition are plotted as described in Fig. 1. The thick line represents the cell discharge during steps over the obstacle and the thin line indicates the activity during unobstructed walking. C: RSN identification as illustrated by the collision of spontaneous action potentials (asterisks) and antidromic potentials evoked from spinal cord stimulation. In this display, the filled asterisk indicates spontaneous action potentials that were used to trigger the stimulus at a delay less than the antidromic latency; there was, therefore a collision of the spontaneous and antidromic potential. The open asterisk identifies spontaneous action potentials that were used to trigger the stimulus at a delay greater than the antidromic latency; the potentials, therefore did not collide. The vertical dashed line indicates stimulus onset. D: shading indicates cutaneous receptive field (RF) of the cell.

While complex responses similar to this cell were observed in a number of RSNs that changed their activity over the obstacle, there was a range of discharge patterns that varied in the number, timing, and sequencing of the bursts of modified activity. Figure 5 illustrates some examples of the different patterns of unit discharge observed in RSNs during the lead obstacle condition, while Fig. 6, A-C, classifies the discharge patterns of the illustrated cells, and the rest of the database, using the method illustrated in Fig. 1.



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Fig. 5. Examples of different discharge patterns of RSNs recorded during the lead condition (A-E). The cell activity, antidromic identification of each cell and the RF are displayed as described in Fig. 4. For the RFs, the shading on the figurines indicates that the cells were activated by cutaneous stimulation or light touching, the bars with arrows in A and D indicate that movement of the head was required to activate the cell, the question mark (?) in B and E indicates that the neurons were tested for a receptive field but that none could be identified. Note that in C, 3 different RSNS with similar characteristics are shown. Representative flexor muscle activity (F) of the left fore- and hindlimb are displayed as in Fig. 1. The shaded rectangles identify peaks in the different cells that occurred at similar times during the gait modification (see text).



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Fig. 6. Classification of bursts of increased activity in RSNs during the different phases (see definition of phases in METHODS) of the lead and trail condition. A: the distribution of bursts in RSNs that exhibited a single phase of increased activity. B: the distribution of bursts in RSNs that exhibited only multiple periods of increased discharge. C: the distribution of all RSN bursts including both single and multiple increases in activity during steps over the obstacle. The light shaded portion of each bar in B and C represents the phase in which the RSNs exhibited their initial period of increased activity. D-F: same organization for trail data. All data are potted as a percentage of the total number of periods of increased activity observed across all RSNs.

The simplest type of modification of activity observed in these RSNs was a single period of increased or decreased activity during the gait modification (see Table 1). Such a pattern of modification was observed in 34/97 (35.1%) RSNs. Of these RSNs, only 8/97 showed a significant decrease in their discharge activity during the gait modification (see e.g., Fig. 5E) and will not be considered further. Of the 26/89 (29.2%) RSNs that showed a single increase in their activity during the task, most discharged either prior to (Fig. 5A), or during (Fig. 5B) the passage of the left, lead, (ipsilateral) forelimb (LFL) over the obstacle. Indeed, as summarized in the histogram of Fig. 6A, the majority of the RSNs that showed a single period of increased activity (21/26) discharged either during the passage of the LFL or just prior to this time (early). The population of RSNs, discharging exclusively during the passage of the LFL, comprised 24.7% of the total population of modified RSNs.

The majority (63/89, 70.8%) of the modified RSNs exhibited more than one period of increased activity during the voluntary gait modification. In most of these cases (42/63), the RSNs showed an initial increase in their discharge activity as the lead limb passed over the obstacle (LFL), and then subsequent periods of activity later on as the remaining legs passed over (Figs. 5C and 6B). In nearly all of the other cases, the initial increases in activity appeared either before the passage of the LFL (Early) or just after it (LFL/RFL), as in the example illustrated in Fig. 5D. Although the three examples illustrated in Fig. 5C demonstrate that the number and amplitude of these bursts of increased activity varied substantially among cells, it was, nevertheless, noticeable that the peak discharge of certain bursts occurred during the same phase in many of these cells. For example, the initial peak of increased activity of the cell illustrated in Fig. 5D, occurring in the LFL/RFL transition phase, was also seen in two of the cells shown in Fig. 5C (1st, light shaded bar). Similarly, the second and third bursts of the cell shown in Fig. 5D are also seen in several of the other cells (2nd and 3rd shaded bars). It appears that despite the wide variation in the number and sequencing of bursts, there is a strong similarity in the phasing of many of these periods of increased activity. Moreover, as can be seen from inspection of Fig. 6B, there is little evidence that there is any tendency for subsequent bursts of activity to occur preferentially in one period as opposed to any other.

Overall, as illustrated in Fig. 6C, it can be seen that the RSNs recorded during the lead condition had a strong tendency to increase their discharges during the LFL phase irrespective of whether the increased discharge was limited to a single phase (Fig. 6A) or was accompanied by subsequent bursts of increased activity (Fig. 6B). Among the multi-burst cells, the most common activity was an exclusive increase linked to the passage of the LFL and RFL over the obstacle (15.3%). The next most frequent patterns were increased activity linked to the LFL, RFL, and LHL (8.2%). However, more than a quarter of the neurons (27.1%) showed periods of increased activity related to all four limbs.

RELATIONSHIP TO EXTENSOR MUSCLE ACTIVITY. Inspection of the exemplar EMG activity illustrated in Fig. 1 suggests that the pattern of extensor muscle activity associated with each one of the eight phases that we defined is quite different. Correspondingly, therefore, each of the periods of increased cellular discharge in neurons with multiple bursts of activity is associated with a distinctly different pattern of modified muscle activity. This is explicitly illustrated in Fig. 7 for the neuron shown in Fig. 4. For example, the first period of increased activity occurs during the passage of the left forelimb and is associated with increased activity in the ClB. At this time during the gait modification, however, there are very few, if any, changes in extensor muscle activity. During the next period of increased activity, which occurs during the LFL/RFL transition period, there is increased activity, to varying degrees, in the level of the extensor muscle activity of each of the four limbs, while during the third period of increased activity (LHL), there are prominent increases of activity only in the iVL and the coVL. Conversely, while there is increased activity in the iTriL throughout the stance phase of the left forelimb, there is no correspondingly long increase in RSN activity at this time. Similar results were seen for the other RSNs. In general, there was no one distinct and consistent pattern of increased activity in the recorded flexor and extensor muscles that matched the periods of increased activity observed in the neurons.



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Fig. 7. Relationship between the change in RSN activity and the change in extensor muscle activity during the gait modification. The averaged activity during the control cycles has been subtracted from all traces to show the difference between the discharge and EMG activity when the cat steps over the obstacle and the activity during control locomotion. The dotted lines indicate the confidence level of the activity in the control situation. Unit activity that was significantly different from control (see METHODS), and which occurred during one of the 8 defined periods is highlighted by the shaded rectangles. The unit is the same as that illustrated in Fig. 4.

RSN ACTIVITY DURING THE TRAIL CONDITION. Altogether, 83/89 RSNs with increased discharge activity during the lead condition were also recorded in the trail condition. Of these, 65/83 (78.3%) also showed increased activity during the trail condition. In addition 4/8 RSNs that were inhibited during the lead condition showed increased activity in the trail condition as did 6/32 (18.8%) other RSNs that were unmodified in the lead condition.

Figure 8 compares the discharge of the same cell shown in Fig. 4 during both lead and trail conditions. In both Fig. 8 A and B, the traces are synchronized to the onset of activity in the left ipsilateral ClB. In the lead condition (Fig. 8A), passage of the RFL is subsequent to that of the LFL (activity in the rClB follows that in the lClB) and, therefore, occurs during the second half of the middle cycle, whereas in the trail condition (Fig. 8B), activity in the RFL precedes that in the LFL (rClB precedes lClB) and is, therefore, displayed in the first cycle. In essence, the order of the legs is reversed in the two conditions. Inspection of the cell discharge during the trail condition showed that the pattern of activity was similar to that observed in the lead condition but that it was temporally shifted with respect to the onset of activity in the lClB. On the other hand, comparison of the discharge pattern with respect to the onset activity of the ClB of the lead limb shows strong similarities between the two patterns. For example, in the lead condition, a large burst of increased cell discharge (burst 1) was observed at the onset of the passage of the left forelimb over the obstacle while during the trail condition, a similar burst was seen at the same time during the passage of the right forelimb over the obstacle. The burst occurring during the LFL/RFL transition period in the lead condition (burst 2) likewise had its analogue during the RFL/LFL transition period in the trail condition. Similar arguments may be made for bursts 3 and 4 with respect to the periods of activity in the hindlimb muscles. Thus it appears that this cell may participate in a similar manner in the modifications associated with both the lead and trail conditions, despite the fact that the changes in muscle activity associated with these two conditions are quite different (see Figs. 1 and 8). In other words, these data support the results presented in Fig. 7 that suggest that cells do not appear to be involved in encoding changes in muscle activity limited to a single specific limb or to a single specific postural pattern but may assist in encoding a variety of different modifications involving various combinations of limbs.



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Fig. 8. Cell discharge from a single RSN during both lead (A) and trail (B) conditions. The cell activity and muscle activation patterns are displayed for 3 consecutive cycles, as in Fig. 4. The thick line in each condition represents the cell discharge during steps over the obstacle and the thin line indicates the activity during unobstructed walking. It is important to note that the 3 cycles displayed for the trail condition (B) were also synchronized and labeled with respect to the left ClB, and thus the middle cycle in B represents the step when the trailing (left) forelimb passes over the obstacle. The periods when the individual limbs passed over the obstacle are indicated by the arrows at the bottom of each figure. Note the difference in limb order during the trail condition. The first 4 bursts of increased activity in each condition are identified on the cell trace in each condition.

Similar findings are illustrated by the additional examples shown in Fig. 9 where these cells also showed a tendency to preserve the number of bursts and for the bursts to be similarly sequenced with respect to the relative order of the individual limbs passing over the obstacle. In each case, bursts occurring during periods of activity in the lead (left) limb in the trail condition can be identified at similar times during the period of activity of the lead (right) limb in the trail condition. In most cases, the number of bursts was maintained across conditions (burst 2a in Fig. 9A being an exception) although the relative amplitude of analogous bursts may be different. For example, in both Fig. 9 B and C, the bursts occurring during the LHL/RHL transition period in the lead condition (bursts 3 and 2, respectively) occurred during the RHL/LHL transition period in the trail condition but were larger in the trail condition than in the lead condition. These changes in phase were also true for cells that discharged prior to the gait modification; for example, the RSN illustrated in Fig. 9D increased its activity before the onset of activity in the lClB in the lead condition but before the onset of activity in the rClB in the trail condition.



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Fig. 9. Further comparison of discharge patterns of RSNs recorded during the lead and trail conditions and arranged as in Fig. 8. A-D: examples of RSNs that exhibited different patterns of increased discharge in the lead condition. The discharge pattern for each cell is displayed along with representative muscle activity (E) from all 4 limbs both the lead and trail conditions as described in Fig. 4. Numbers above each trace indicate bursts of activity in the lead and trail condition for each cell.

An analysis of the overall frequency of occurrence of the different peaks across the population of cells in the trail condition is illustrated in Fig. 6, D-F. As in the lead condition (see Fig. 6), there was a clear tendency for cells discharging in a single burst to be active during the period of activity of the lead forelimb (Fig. 6D) although in the trail condition this obviously corresponded to the RFL instead of the LFL. Overall 13.9% of the modified RSNs discharged only during the passage of the RFL during the trail condition. Similarly the initial period of activity of cells that discharged in multiple bursts also occurred most frequently just before or during the passage of the lead limb (RFL) over the obstacle. Nevertheless, as can be appreciated from comparing the lightly shaded areas in Fig. 6, C and F, there was a greater tendency (76/83, 91.6%) for the initial burst of activity in the lead condition (including single and multiple burst activity) to occur during the early, LFL, and LFL/RFL phases during the lead condition than during the analogous phases during the trail steps (68/89, 76.4%). The most common patterns in the lead condition were also seen in the trail condition, although with the limbs reversed, i.e., RFL and LFL (10.1%) and RFL, LFL, and RHL (7.6%). Again, a large proportion of RSNs (27.9%) discharged in relation to the passage of all four limbs.

Taking together all of those neurons that showed an increase at any one particular part of the step cycle, irrespective of the overall pattern, fully 51.2 of the cells were active during the modified periods of both the left and right forelimb during the lead condition and 48.1% during the trail condition. Similarly during both lead and trail conditions, there was approximate equal probability that any given cell would influence the activity of at least three of the limbs. One quarter of the cells showed increased activity during the passage of all four limbs.

ACTIVITY OF UNIDENTIFIED RETICULAR CELLS DURING THE OBSTACLE CONDITIONS. The activity of unidentified reticular cells was very similar to the RSNs during the obstacle conditions and exhibited the same wide range of response patterns, including 92/217 (42.4%) neurons that modified their discharge activity during the lead condition (Table 1). Apart from a slightly increased number of neurons that were inhibited during the modified cycle, the most noticeable difference between the two populations was the larger proportion of neurons showing a single period of increased activity during the gait cycle. As detailed in Table 1, while there were almost twice as many RSNs displaying multiple bursts as compared with single bursts, among the unidentified neurons, the proportions was almost equal.

As for the RSNs, most of those neurons with a single increase in discharge activity were active in relationship to the gait modification of the LFL in the lead condition (an example of such an unidentified neuron can be seen in Fig. 10A). Overall the patterns of activity observed among the unidentified cells were very similar to those described for the RSNs and are not further detailed. However, it should be noted that a smaller percentage of unidentified cells, compared with RSNs, discharged during the passage of each of the four limbs over the obstacles in both the lead (10 vs. 27.1%) and the trail (17.1 vs. 27.9%) conditions.



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Fig. 10. Comparison of cell activity during the lead and control condition for 3 cells (A-C) whose discharge patterns were strongly associated with flexor muscle activity during control walking. The ensemble averages of cell discharge along with the activity from the associated flexor muscle for 3 consecutive strides are displayed on the left. The thick line represents the activity during the lead obstacle condition, and the thin lines indicate the activity during control walking. The figures in the right column include postevent histograms and raster plots in which the cell activity during control walking has been aligned to the onset of the associated flexor muscle. In the raster displays, the solid vertical line represents the onset of muscle activity, the 1st set of staggered vertical lines represents the offset and the 2nd set of staggered vertical lines represent the onset of the muscle in the next cycle. The raster order has been arranged such that the trials with the longest duration of muscle activity are displayed at the top.

RELATIONSHIP BETWEEN CELL DISCHARGE ACTIVITY DURING THE LEAD AND CONTROL CONDITIONS. As in our previous studies (Drew et al. 1986, 1996a; Matsuyama and Drew 2000), we classified the cells recorded in this study into three groups based on their discharge activity during the periods of control locomotion. These three groups included those neurons that exhibited a phasic discharge pattern that was temporally linked to the periods of activity in the flexor or extensor muscles of any given limb, or pairs of limbs (EMG-related), those that discharged rhythmically in each step cycle but whose discharge was not temporally linked to the periods of onset or offset of any of the recorded limb EMGs (locomotor-related), and those that discharged tonically or that discharged irregularly (unrelated). Among those RSNs whose control discharge was classified as EMG-related and whose activity increased during the steps over the obstacle, the discharge of 42/55 (76.4%) was temporally linked to the period of activity of one or more of the flexor muscles during the control locomotion. Within this flexor-related subpopulation, 30/42 (71.4%) were best related to the periods of EMG activity of the hindlimb flexors, either ipsilateral or contralateral to the recording site. Within the unidentified population, a similar percentage (37/53, 69.8%) of the EMG-related neurons was related to flexor muscle activity, but within this flexor-related subpopulation, only 15/37 (40.5%) of the neurons were related to hindlimb flexors; the majority were thus best related to the activity of the forelimb flexors.

Although for some cells the modification of activity during the gait modifications could be described as a simple modification of the underlying background activity during the control steps (see Fig. 10A), for most cells the changes could not be so simply predicted on the basis of the control activity (see e.g., Figs. 4, 5C, and 10B). As shown in Table 2, in general terms there was a much greater tendency for both RSNs and unidentified cells that were unmodified during the gait modification to have a tonic or irregular discharge during control locomotion (57.3%) than those that were modified (17.5%). Correspondingly a much higher proportion of neurons (57.7%) whose discharge patterns were modified during the gait modification had phasic (EMG-related) activity patterns during the control locomotion. Nevertheless it is emphasized that all three types of discharge activity during the control steps were observed for both modified and unmodified cells and that some very-well-modulated cells during control locomotion showed no changes during the gait modification (e.g., Fig. 10C), while other cells that discharged tonically during the control steps increased their activity during the gait modifications.


                              
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Table 2. General relationships of pattern of activity during the gait modification compared to that observed during control locomotion

Overall there was little relationship between the type of activity observed during the control steps and during the steps over the obstacle. Indeed given the preponderance of both RSNs and unidentified neurons that showed an initial increase of activity during the passage of the LFL over the obstacle, this could hardly be otherwise. Thus of those neurons that showed a single increase of activity during the passage of the LFL over the obstacle in the lead condition, only 9/34 cells (including RSNs and unidentified neurons) were phasically modulated in phase with lClB activity during control locomotion. Of the other cells (25/34), 12/34 were phasically modulated during the period of activity of a different EMG (including 8/34 with hindlimb flexor muscles), 7/34 were locomotor-related and 6/34 were unrelated to locomotion.

Examined from the other perspective, of 35 RSNs and unidentified neurons whose discharge was related to the lClB during control steps, 9/35 showed a single increase in activity during the passage of the LFL during the modified steps, and 11/35 showed a multiple burst with the initial burst during the passage of the LFL. Of 50 neurons that were best related to the lSrt during the control steps, 7/50 showed a single increase in activity during the passage of the LFL and 15/50 discharged in multiple bursts with the initial burst during the passage of the forelimb (see e.g., Fig. 10B). Thus while a total of 20/35 (57.1%) of the neurons active in phase with the activity of the lClB during control locomotion showed an increase in activity during passage of the LFL over the obstacle, so did 22/50 (44%) of the neurons whose discharge was temporally linked to the time of activity of the lSrt during control locomotion.

Thus while there does appear to be some link between the phasic modulation during control walking and the changes observed during the lead obstacle condition, it appears to be more complex than a simple association with those muscles whose activity is best correlated to the phasic cell activity.

Inter-nuclear differences

Because previous studies have shown differences in the afferent and efferent projections of different regions of the PMRF (Kuypers 1958; Magni and Willis 1964; Matsuyama and Drew 1997; Newman et al. 1989; Peterson et al. 1975; Rho et al. 1997), we examined whether there were any differences in the types of discharge patterns that were found in different reticular nuclei. The results of this analysis are documented in Table 3. In general, we found that approximately equal percentages of modified and unmodified RSNs were found within the NRGc, NRMc, and NRPc, suggesting some homogeneity, at least of generalized locomotor function, within these areas. However, there was a larger percentage of silent RSNs within the NRPc and an even larger percentage within the NRPo where they made up half of the, admittedly small, sample recorded from a single cat. Correspondingly the number of modified neurons in the NRPo was the smallest of the four regions. Within the modified population, there were proportionately more neurons showing a single increase in their discharge frequency during the task within the NRPc and proportionately less within the NRMc. Given the small numbers, no further attempt was made to determine if there was any tendency toward one pattern of activity or another within these areas.


                              
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Table 3. Discharge characteristics of RSNs in different divisions of the PMRF

Receptive fields

Receptive fields could be determined for 97/132 (73.5%) of the RSNs that were tested. Of the 35 RSNs that were unresponsive to passive manipulation of the body, 23/35 did not discharge during locomotion and were classified as silent. Excluding the silent neurons from consideration, 97/109 (89%) discharged to passive manipulation of the body. Roughly equal proportions of modified (90%) and unmodified neurons (86%) had a somatic receptive field. It is, therefore unlikely that the receptive field of the cell was the determining factor as to whether a cell would be modified or not in the task.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The findings of this study address the contributions of the reticulospinal system to the production of postural adjustments during voluntary gait modifications. The results indicate that the majority of RSNs demonstrated complex changes in discharge frequency during voluntary gait modifications during steps over the obstacle. Most cells exhibited multiple bursts of increased activity where the timing of each burst corresponded, approximately, to the passage of each limb over the obstacle. We propose that the characteristics of these cells within the PMRF are not compatible with a role in specifying the details of the postural responses that accompany the voluntary gait modifications but are compatible with a more general role in signaling the timing and the magnitude of these responses.

Database and general considerations

The population of cells making up the database used in these experiments was recorded from a similar region of the brain stem as examined in previous studies that have recorded RSN activity during unobstructed walking in both the intact (Drew et al. 1986; Matsuyama and Drew 2000), thalamic (Orlovsky 1970; Shimamura and Kogure 1983; Shimamura et al. 1982), and fictive (Perreault et al. 1993) preparations. Although slightly different regions of the PMRF were sampled in each cat, there was overlap between the regions studied in different cats, particularly for the NRGc, and there were no obvious differences in the characteristics of the cells recorded from different animals, with the exception of the regional differences detailed in Table 3. The similarity in the responses of cells from different animals can also be appreciated from inspection of Fig. 5. In addition, we made no effort to preselect cells based on any particular characteristic; each cell that we encountered was recorded during locomotion, provided that the recording of the action potential was stable. As such, the recording volume represents, as far as possible, an unbiased sample of the PMRF with respect to both RSNs and unidentified neurons, with the caveat that most of the recordings were probably from neurons with large soma, as indicated by Fig. 3.

The postural patterns that were generated as the cat stepped over the obstacle were represented in this study by recordings of EMG activity. As we have shown previously, at least for overground locomotion, changes in the EMG activity of the extensor muscles give a good indication of changes in vertical GRFs (Lavoie et al. 1995). Overall, as the cat passes over the obstacle, there are four periods of modified postural activity associated with the passage of each of the limbs together with separate periods of modified postural activity associated with the three periods of double support that occur in between (see Figs. 1 and 7). Each of these periods requires a specific pattern of modified activity in the supporting limbs to take into account the different geometry of the body. In the discussion that follows, we will suggest that these changes in posture are signaled by the neuronal discharge that we recorded from the reticular neurons.

RSN activity during gait modifications

Individual RSNs showed phasic increases of activity at different times of the step cycle. Nevertheless, it was noteworthy that in the lead condition, more cells (86.9%) tended to modify their discharge either before or as the leading forelimb (LFL) passed over the obstacle than when any of the remaining limbs followed over the obstacle. This was true both for cells that exhibited a single period of increased activity as well as those that exhibited multiple bursts of increased activity (Fig. 6, A-C). This is a particularly striking finding since all of the RSNs that we recorded had axons that projected as far as the lumbar spinal cord and some of these cells, during unobstructed locomotion, showed phasic activity that was temporally related to the periods of activity in the hindlimb flexor muscles (see e.g., Fig. 4). While it is logical to consider that the increased activity at these times might modify hindlimb muscle activity, it should be remembered that there was no significant change in either the vGRFs (Lavoie et al. 1995) or the activity patterns of the hindlimb extensor muscles that we sampled (Figs. 1 and 7), either before or during the passage of the LFL. It is therefore hard to argue that this period of increased discharge is encoding changes in hindlimb activity. It is possible, however, that these cells might be modifying nuchal and forelimb muscle activity at these times as many RSNs that project to lumbar levels also innervate the cervical spinal cord (Peterson et al. 1975). For example, RSNs that discharge before the gait modification may be involved in modifying neck EMG activity as the cat orients its head to examine the approaching obstacle, while cells that discharge during the passage of the LFL over the obstacle might participate in the production of the changes in flexor activity observed in muscles such as the ClB during this period. Cells that discharge during the latter part of the swing period might also participate in the changes in the level and timing of the extensor muscle activity that are observed in both forelimbs and the ipsilateral hindlimb at this time. Such a role for the increased discharge observed at these times would be compatible with our previous findings (Drew and Rossignol 1990a,b) that microstimulation in these areas of the PMRF evoke strong twitch responses in both neck and forelimb muscles. However, why these neurons project to the lumbar spinal cord but have no apparent effect on hindlimb musculature is less clear. One possibility is that changes in hindlimb muscles at these periods of the gait modification are too subtle to be detected by our current analytical methods. A more likely possibility, however, is that the efficacy of the synaptic responses from these reticulospinal neurons at different levels of the spinal cord is contingent on the context of the movement. From this viewpoint, it is possible that whether or not the discharge in the reticulospinal neurons produces an effect at the lumbar level depends on descending signals in other descending pathways, or in propriospinal pathways. For example, excitability in the hindlimb interneuronal pathways may be increased, or gated in, by corticospinal pathways controlling hindlimb movement or by those that signal aspects of both the fore- and hindlimb gait modifications (see Widajewicz et al. 1994).

While the presence of activity occurring just before or during the passage of the LFL over the obstacle dominated, increased activity during subsequent phases was also prevalent with approximately half of the modified RSNs increasing their activity during the passage of at least three of the limbs over the obstacles and a considerable number of the modified RSNs (27.1%) increasing their discharge during the passage of each of the four limbs. This suggests that individual RSNs have the potential to modify locomotor activity in more than one phase of the gait modification. Furthermore, it should be remembered that many of the neurons that fired before or during the passage of the LFL also discharged later on in the step cycle, at times when there was no increased activity in the lClB and when there was unlikely to be a need for the cat to orient its head in any directed manner. This suggests that the increased periods of discharge occurring at different times in the gait cycle might not all have the same motor effect and that there is no single pattern of muscle activity in the limbs that occurs coincidentally with each period of increased discharge (see Fig. 7). In addition, it must be considered that many of the modified RSNs also featured similar complex changes in the trail condition. As with the lead condition, these increases were more frequent during the period when the initial forelimb, in this case the right (RFL), passed over the obstacle and, in many cases, individual cells discharged in phase with the LFL in the lead condition but in phase with the RFL in the trail condition (see Fig. 8). Taking the lead and the trail data together, the results suggest that some individual RSNs not only have the potential to modify the pattern of muscle activity at multiple times during the gait modification but also may modify this participation as the order of the passage of the limbs over the obstacle changes.

Two major possibilities may account for these neuronal properties. The first is that these increases in discharge frequency are not causally implicated in producing the postural patterns in the limbs that accompany the voluntary gait modifications. For example, such cells might be involved in producing modifications in unrecorded axial muscles. The second is that different motor patterns in the limbs are, indeed, expressed by each period of increased discharge activity. Although we have no direct evidence that the cells that we are recording are involved in modifying both flexor and extensor EMG activity in the limbs, there are several compelling reasons to believe that this must be so for at least some, if not a majority, of the cells, including: 1) the large proportion of neurons that showed these patterns of activity (Table 1), 2) the fact that all of these RSNs had an axon that descended to the spinal cord, 3) the fact that the discharge frequency of many of these was modulated in phase with the activity of flexor or extensor muscles during the control locomotion, and 4) the fact that microstimulation in most loci from which cells were recorded evoked responses in flexors and extensors of at least two limbs as well as in neck musculature (not reported here but see Drew and Rossignol 1990a,b). In addition, the characteristics of the regions from which we recorded neuronal activity, encompassing primarily the NRGc and the NRPc, are compatible with their proposed contribution in forming complex postural responses during voluntary gait modifications. For example, both of these regions have been demonstrated both anatomically and physiologically to widely innervate both the cervical and lumbar enlargements (Peterson et al. 1975) where they facilitate flexor and extensor motor neurons of fore- and hindlimb and axial muscles (Anderson et al. 1972; Grillner et al. 1971; Peterson 1979; Peterson et al. 1978, 1979; Wilson and Yoshida 1969). In addition, the axons of individual RSNs have been demonstrated to branch widely within the gray matter and, in some cases, to be distributed both ipsilaterally and contralaterally (Matsuyama et al. 1988, 1997). Thus overall we believe it probable that most of the RSNs that we recorded have the potential to modify the pattern of activity in multiple limb muscles and to facilitate the type of changes that are required to modify posture during locomotion.

Nevertheless, given the complex and dynamic nature of the postural patterns that occur during the task and the lack of any clear relationship with those patterns (see Fig. 7), it is difficult to imagine that each period of increased activity in a RSN is explicitly encoding the complicated changes in EMG activity that occur during each period of modified postural activity. Rather we suggest that RSNs provide a more general signal that signals the timing and the magnitude of the postural activity that is required, while the ultimate expression of the postural response may depend on the phasic excitability of the spinal locomotor circuits on which these RSNs project.

As we suggested in the preceding text, one putative mechanism that might determine whether a given period of increased activity modifies the activity of any given group of muscles would be the context of the movement and the activity in other descending pathways. For example, there is convergence from the motor cortex and the PMRF onto common interneurons in the lumbar spinal cord (Floeter et al. 1993), and it is possible that the corticospinal signal is required to bring these interneurons to a sufficient level of excitability before the reticulospinal signal may exert an influence. Thus the effect of each period of multiple activity in the RSNs will be dependent on the presence of supplementary descending (and propriospinal) inputs. In addition, as we have previously suggested (Drew 1991; Drew and Rossignol 1984; Drew et al. 1986, 1996a), it is probable that the ultimate expression of the reticulospinal signal will also depend on the phasic level of excitability induced by the intrinsic rhythmicity of the spinal central pattern generator. In brief, we suggest that the signal descending along the reticulospinal axon modulates the activity in interneurons contacting both flexor and extensor motoneurons and that the result of that modulation is the sum product of the intensity of the descending signal and the level of polarization of the interneurons and motoneurons that it contacts. Put simply, activity will be increased in interneurons and motoneurons that are in a depolarized state and unchanged in neurons that are in a hyperpolarized state.

The combination of these two mechanisms, contextual gating and phasic changes in the level of excitability of the spinal interneuronal patterns, might provide the neuronal substrate to explain how multiple periods of increased activity in a single cell could participate in different postural patterns, both with respect to the passage of different limbs over the obstacle (see Fig. 7) and with respect to the differences observed in the lead and trail conditions (see Fig. 8). Such a mechanism would provide a basis whereby single neurons may influence a large number of muscles in a functionally appropriate manner. In some RSNs with simple discharge patterns and restricted termination patterns, changes in activity may occur in a restricted number of muscles, perhaps in a single limb, allowing for differential control of activity. At the other extreme, RSNs with complicated discharge patterns and with widely branching axons may have an influence on the activity on a large number of muscles in several limbs and might be activated under a variety of different circumstances.

Source of the modulation

Implicit in the preceding discussion is the point that the activity that we recorded in many of these RSNs does not specify the details of the postural patterns that are to be produced. Instead we suggest that the increased RSN activity represents a more general signal representing the timing and the magnitude of the postural changes that should accompany the gait modification and that these periods of increased activity are determined by the afferent input from those structures involved in specifying the voluntary gait modification. Although the PMRF receives afferent inputs from a number of diverse sources that are known to be involved in motor control, including the cerebellum (Eccles et al. 1975; Homma et al. 1995), it seems probable that a large part of this afferent signal arises in the motor cortex. In this respect, it should be noted that cells within the cat PMRF have a rich afferent input from the motor cortex and that, in many cases, these afferent inputs arise as collaterals from corticospinal axons (Canedo and Lamas 1993; Kably and Drew 1998a; Keizer and Kuypers 1984; Kuypers 1958; Lamas et al. 1994; Matsuyama and Drew 1997; Rho et al. 1997). In addition, individual RSNs have been shown to receive input bilaterally from the motor cortex (Berrevoets and Kuypers 1975; Kably and Drew 1998a; Magni and Willis 1964; Matsuyama and Drew 1997; Pilyavsky 1975; Rho et al. 1997; Rossi and Brodal 1956) as well as from the fore- and hindlimb representations (unpublished observations), although the input from the hindlimbs, in terms of density, is weaker than that from the forelimbs (Matsuyama and Drew 1997; Rho et al. 1997). Moreover, Kably and Drew (1998b) have provided evidence showing that among those motor cortical cells that increased their discharge activity during passage of the contralateral fore- or hindlimb over an obstacle was a substantial population that sent an axon both to the spinal cord and to the PMRF. They suggested that such neurons might be responsible for ensuring that postural responses are appropriately integrated with the voluntary movement.

The results obtained in this study are in general agreement with this proposal, showing as they do that RSNs within the PMRF, including many that receive cortical input (unpublished observations), increase their discharge activity as the cat steps over the obstacle. We suggest that this increased discharge activity reflects the descending command for movement, mediated in part by the motor cortex. More specifically, on the basis of the electrophysiological and anatomical evidence presented in the preceding paragraph, we suggest that the number and phase of the bursts in any given RSN is determined by the discharge patterns and the level of convergence of motor cortical cells. For example, in the case of the cell illustrated in Fig. 4, we suggest that it would receive convergent input from four separate populations of cortical neurons, namely those within the forelimb and hindlimb representations of both the left and right motor cortex. Each of these populations would be the cause of one of the periods of increased activity observed in the illustrated RSN. In this manner, the RSNs would receive descending signals providing information about the timing and the magnitude of the voluntary movements being undertaken by each limb that could be used to appropriately scale the postural responses to be produced by the PMRF.

Unmodified cells

Our sample of RSNs also included a number of neurons (Table 1) that did not change their activity during the gait modification even though their axon projected to the lumbar spinal cord. Moreover almost one-quarter of the RSNs was completely silent under all circumstances (see also Matsuyama and Drew 2000), suggesting that these cells might represent a population of RSNs with different functions than the majority. In this respect, it is interesting to speculate that these silent neurons might form a subpopulation that become active during sleep and whose function might be to promote the inhibition of motoneuronal activity during REM sleep (Chase and Morales 1990; Fung et al. 1982; Siegel 1979). Such a speculation is supported by the finding that silent neurons were more prevalent in the NRPo, which has been linked to the control of posture in waking cats and to the regulation of atonia in sleeping cats (López-Rodríguez et al. 1995; Mori 1987, 1989; Pereda et al. 1990; Siegel 1979; Takakusaki et al. 1993).

Leaving aside the silent neurons, only a small population of neurons was unrelated to the gait modification. These neurons included cells with receptive fields on the vibrissae that might be activated to change motor activity in response to specific sensory cues during exploratory behavior. The other class of cells comprised those that were active during the control steps between the obstacles but showed no significant change in activity during the gait modification. Although these cells included a large portion of cells whose activity was irregular or tonic during the control walking and that might, therefore, be expected to be less active during the gait modification, there was also a substantial population that was rhythmically active (see Fig. 10C) and might, therefore, be expected to increase their activity. Possibly, such cells might signal the onset and offset of the modifications of muscle activity that are required as opposed to the level, and/or may become active only at increased levels of activity.

Unidentified neurons

The database of unidentified reticular neurons probably included a very heterogeneous group because we were unable to characterize the projection of these cells. Moreover it is important to realize that a certain number of the unidentified cells may indeed be RSNs that could not be positively identified because they either projected exclusively to the forelimb regions or the lumbar stimulation failed to stimulate any hindlimb projections. Apart from the RSNs, the group of unidentified cells could have also included cells that do not project to the spinal cord and may be related to locomotor activity through involvement in afferent feedback from the spinal cord or in local circuits within the reticular formation. Indeed it was notable that the unidentified cells included a greater proportion of unrelated and unmodified cells as well as fewer modified cells confirming that many of these unidentified cells are unlikely to participate in the locomotor modifications.

However, a considerable number of unidentified cells did exhibit activity patterns during lead and trail conditions that were very similar to those observed in the RSNs. These modified but unidentified cells also featured a range of responses including single and multiple bursts of increased activity with individual cells exhibiting increases as the different limbs passed over the obstacles. Similar to the RSNs, unidentified cells with either single or multiple periods of increased activity had a strong tendency to increase their discharge early on in the gait modification when the lead forelimb was passing over the obstacle. However, while only 26/89 (29.2%) of the RSNs showed a single period of increased activity, 38/77 (49.4%) of the unidentified neurons did so. Of these latter cells, 27/38 discharged either during the passage of the LFL over the obstacle or during the LFL/RFL transition period, suggesting a greater relationship with the forelimbs than for the RSNs. This would be consistent with the possibility that many of these cells might be forelimb RSNs that do not project to the lumbar spinal cord. This suggestion would also be supported by the fact that the cell activity recorded from the EMG-related, unidentified neurons during control walking was more frequently related to forelimb flexor activity. The similarity of the behavior of these cells to RSNs during these modifications may suggest that similar mechanisms could be acting in the forelimb exclusive RSNs; however, confirmation of this is impossible given our inability to characterize the different types of unidentified reticular cells.

Conclusions

We have previously hypothesized that RSNs found within the PMRF may be responsible in part for production of the dynamic postural adjustments that accompany voluntary movement modifications, and we have further proposed that the corticoreticulospinal pathways may be responsible for selection and/or triggering of the appropriate postural response to accompany these voluntary modifications. The evidence presented in this paper suggests that the discharge patterns of these cells during gait modifications are compatible with this proposed organization. However, the complex patterns of activity that we observed in many of these RSNs, together with the relative shift in the phase of activity in the lead and trail conditions, suggest that most individual RSNs are unlikely to be specifically controlling modifications of EMG activity related to discrete phases of the gait modification. Rather we suggest that the discharge patterns that we observed in these RSNs reflect the cortical afferent inputs that provide corollary information about the movement that is being made. We suggest that these inputs define more the timing and the magnitude of the postural response that is to be made than the details of the postural patterns that are to be produced. We propose that the final expression of the appropriate postural responses would be determined by the anatomical projection pattern of the activated neurons, by the context of the movement, and by the state of the spinal interneuronal pools on which any given RSN projects. The first of these three mechanisms would determine which muscle groups may be potentially modified by the output and thus define the specificity of the modification, whereas the latter two mechanisms would determine the sign and the efficacy of the resulting modification. The sum result of this organization would be to permit complex and dynamic changes of postural activity that are coordinated within the gait cycle so as to produce a pattern of postural activity appropriate to the requirements of the situation.


    ACKNOWLEDGMENTS

We thank N. de Sylva and M. Bourdeau for technical assistance during these experiments. We also thank J. Lavoie for histological assistance and G. Gauthier for producing some of the illustrations. Drs. S. Rossignol and A. Smith are thanked for providing helpful comments on the manuscript.

This work was supported by a grant from the Medical Research Council. S. D. Prentice was supported by the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.


    FOOTNOTES

Address for reprint requests: T. Drew, Dept. of Physiology, University of Montréal, PO Box 6128, Station "centre-ville," Montreal, Quebec H3C 3J7, Canada (E-mail: drewt{at}ere.umontreal.ca).

Received 6 June 2000; accepted in final form 1 November 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society