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Department of Biology, Washington University, St. Louis, Missouri 63130
Submitted 6 August 2003; accepted in final form 4 September 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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). During some behaviors, conventional synergies occur, e.g., knee extensors are active during hip-extensor activity. During other behaviors, mixed synergies occur, e.g., knee extensors are active during hip-flexor activity. Berkowitz and Stein (1994b), Berkowitz (2001a,b, 2002), Brown (1911, 1914), Grillner (1981), Jankowska et al. (1967), and Lundberg (1981) discussed hypotheses for conventional synergies; Grillner, Berkowitz, and Stein also discussed mixed synergies. The complexity of behaviors produced by the spinal cord demonstrates that spinal circuitry has significant computational ability (Grillner 1981; Orlovsky et al. 1999; Rossignol 1996; Stein et al. 1997).
During normal rostral scratching, a mixed-synergy motor pattern generated by the spinal turtle, there is rhythmic hipflexor alternation between activity and quiescence; hip-extensor motor activity occurs during hip-flexor quiescence (Robertson et al. 1985). Knee-extensor motor activity occurs during the latter portion of each hip-flexor burst. During a spontaneously occurring variation, termed rostral scratching with hipextensor deletions, there is no hip-extensor activity and no hip-flexor quiescence (Robertson and Stein 1988; Stein and Daniels-McQueen 2002a, 2003; Stein et al. 1995, 1998; Stein and Grossman 1980). These motor patterns were observed with electromyographic recordings (EMGs) during actual scratching with a moving hindlimb and with electroneurographic recordings (ENGs) during fictive scratching with an immobilized hindlimb. Bakker and Crowe (1982) reported knee-flexor (iliofibularis) EMGs during actual normal rostral scratching; the end of knee-flexor EMG activity occurs near the start of knee-extensor EMG activity. We report here knee-flexor ENGs during fictive normal rostral scratching obtained in a D3-end preparation with a single complete transection of the spinal cord just posterior to the forelimb enlargement between the D2 and D3 spinal segments. These data were presented previously in an abstract (Stein and Daniels-McQueen 2003).
In our previous paper (Stein and Daniels-McQueen 2002a), we used single-unit recording techniques (Berkowitz and Stein 1994a; Currie and Stein 1990) to examine the properties of descending propriospinal interneurons that fired in distinct bursts during normal rostral scratching in the spinal turtle. These data were obtained in a D3–D10 preparation with two complete transections of the spinal cord: one transection between the D2 and D3 spinal segments, and the second transection in the hindlimb enlargement between the D10 and S1 spinal segments. Some of the units were hip-extensor–related interneurons that fired during hip-extensor motor activity and displayed no overlap with hip-flexor motor activity during normal scratching; these units were mainly quiet during rostral scratching with hip-extensor deletions. This result is consistent with a "modular" (Jordan 1991) organization of the turtle spinal cord: these hip-extensor interneurons belong to a hipextensor module active during hip-extensor motor activity and quiet during hip-extensor motor quiescence.
Other units described in our previous paper displayed 20–80% overlap with hip-flexor motor activity; these units usually were active during rostral scratching with hip-extensor deletions. We noted in our previous paper that additional measurements of some 20–80% overlap units may reveal relationships with the onset of knee-extensor activity. We report such measurements here. These new measurements were presented previously in an abstract (Stein and Daniels-McQueen 2002b). The strategy we describe is a step toward studying knee-related interneurons that may belong to knee-related spinal modules.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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1 h before surgery to induce hypothermic analgesia (Melby and Altman 1974). Each turtle was spinalized just caudal to the forelimb enlargement by a complete spinal transection midway between the D2 and D3 dorsal roots (Mortin and Stein 1990; Robertson et al. 1985; Stein and Daniels-McQueen 2002a; Stein and Grossman 1980). All procedures were approved by the Washington University Animal Studies Committee.
We used two types of experimental preparations. In the first type, termed "D3-end preparation" (n = 4), we recorded knee-flexor ENGs along with hip-flexor and two knee-extensor ENGs. In the second type, termed "D3–D10 preparation" (n = 3), we obtained single-unit recordings from descending propriospinal interneurons along with hip-flexor and two knee-extensor ENGs. Some analyses of data from this preparation were presented in a publication (Stein and Daniels-McQueen 2002a). We present here new analyses of a subset of these data.
In the D3-end preparation, the only spinal cord exposure was that required for the D2–D3 transection. In the D3–D10 preparation, a second spinal cord exposure was required for the complete transection that was made at the posterior end of the D10 segment (for details, see Stein and Daniel-McQueen 2002a).
In the D3-end preparation, we dissected the right knee-flexor nerve that innervates the iliofibularis muscle (Bakker and Crowe 1982; Crowe and Linnartz 1985; Walker 1973). The iliofibularis muscle originates dorsally at the posterior corner of the iliac crest and inserts via a tendon on the dorsal surface of the fibula (Walker 1973). Its insertion is about one-third of the distance from the knee to the mesotarsal joint. This arrangement gives the muscle a considerable mechanical advantage as a knee flexor; its dorsal origin on the ilium allows it to serve also as a hip abductor.
In the D3-end preparation, we obtained ENG recordings from the iliofibularis motor nerve, also termed the knee-flexor nerve. Iliofibularis motor neuron axons enter the muscle shortly after leaving the peroneal nerve. Their length is short and they are difficult to dissect. We focused our dissection on the main nerve bundle that enters the muscle near the muscle origin, at a point 10–20% of the distance from origin to insertion; we did not dissect small motor nerve bundles that entered the muscle at more distal locations. We obtained a length of the main motor nerve bundle suitable for ENG recordings by first locating intramuscular branches of motor axons deep within the muscle 50% of the distance from origin to insertion. We dissected these branches toward the origin of the muscle and freed up additional branches as the dissection proceeded centrally. After reaching the 20% point from the origin to the insertion, we obtained a group of intramuscular branches in a bundle; we used this bundle as a guide to dissect a length of the main knee-flexor nerve back to the peroneal nerve that was suitable for ENG recordings. We tied off the bundle of intramuscular branches distally with surgical thread (Ashaway Line and Twine, Ashaway, RI) and cut the bundle distal to the surgical knot.
In both the D3-end and the D3–D10 preparations, we also recorded ENGs from three other nerves to right hindlimb muscles (Walker 1973). Their dissection was described previously (Robertson et al. 1985; Stein and Grossman 1980): the hip-flexor nerve (VP-HP), which innervates the puboischiofemoralis internus, pars anteroventralis muscle; the monoarticular knee-extensor nerve (FT-KE), which innervates triceps femoris, pars femorotibialis (data not shown in Stein and Daniels-McQueen 2002a); and the right biarticular kneeextensor nerve (AM-KE), which innervates triceps femoris, pars ambiens (labeled knee extensor in Stein and Daniels-McQueen 2002a). Ambiens is a knee-extensor and a hip-adductor muscle (Walker 1973). Stein and Daniels-McQueen (2002a) studied 20 turtles using the D3–D10 preparation; we focused here on data from three of those turtles in which ENGs were obtained from all three of these nerves.
In both types of preparation, after surgery was completed the turtle was allowed to warm up to room temperature and was immobilized with gallamine at a dosage of 6–8 mg/kg body weight. Stein and Daniels-McQueen (2002a) describe methods for ENG and single-unit recordings. Fictive scratch motor patterns were elicited in most episodes by mechanical stimulation of sites in the right rostral-scratch receptive fields; in other episodes, bilateral stimulation of mirrorimage sites in the left and the right rostral-scratch receptive fields was used.
In the D3–D10 preparation of our previous study (Stein and Daniels-McQueen 2002a), we reported single-unit recordings of action potentials of descending propriospinal interneurons in the right dorsolateral funiculus at the posterior cut face of the D10 segment during rostral scratching. For each of the units discussed in that study, we measured 10 cycles of normal rostral scratching while recording from at least two ENG channels: hip-flexor and one other. In the present study, we considered only those units recorded in our previous study that had 25 cycles of normal rostral scratching while recording three ENG channels: hip-flexor, monoarticular knee-extensor, and biarticular knee-extensor.
We used double-referent measurement techniques (see Fig. 1; also see Berkowitz and Stein 1994b and Fig. 3 of Stein and Daniels-McQueen 2002a) to measure the onsets and offsets of bursts of motor activity (both types of preparation) and single-unit descending propriospinal interneuron activity (D3–D10 preparation). Stein and Daniels-McQueen (2002a) used the term ON-phase to designate the onset phase of each burst. In the D3–D10 preparation discussed in the present paper, we introduce the term ON-unit. Therefore, in the present paper, we elect to replace the term "ON-phase" with the term "startphase" to designate the onset phase of each burst. In our previous paper, we used the term OFF-phase to designate the offset phase of each burst. In the present paper, we introduce the term OFF-unit. Therefore, in the present paper, we elect to replace the term "OFF-phase" with the term "end-phase" to designate the offset phase of each burst.
Start-phases of hip-flexor motor activity were defined as 0.00 and 1.00; end-phases of hip-flexor motor activity were defined as 0.50 (see Fig. 1; Berkowitz and Stein 1994b; also Fig. 3 of Stein and Daniels-McQueen 2002a). We used vector-averaging techniques (Batschelet 1981) to calculate mean phase ± angular deviation of start- and end-phases of knee-flexor motor bursts (D3-end preparation), knee-extensor motor bursts (both types of preparation), and single-unit bursts (D3–D10 preparation). We applied the Rayleigh test to determine whether the distribution of these phases was significantly different from a random distribution (Batschelet 1981).
In the data from the D3–D10 preparation described in the present paper, we focused on units whose mean start- or end-phases were close to the mean start-phase of knee-extensor motor-neuron bursts: we obtained additional measurements of units in our previous paper whose mean start- or end-phases were between 0.25 and 0.40. We used the labels ON-unit and OFF-unit to assist in our description of these units. A unit was designated an ON-unit if the unit's start-phase was between 0.25 and 0.40; a unit was designated an OFF-unit if the unit's end-phase was between 0.25 and 0.40. We presented 72 units in our previous paper: 13 were ON-units whose mean start-phase was between 0.25 and 0.40; 10 were OFF-units whose mean end-phase was between 0.25 and 0.40. Seven of 13 ON-units and 4 of 10 OFF-units met the additional criteria of 25 cycles of normal rostral scratching while recording from hip-flexor, monoarticular knee-extensor, and biarticular knee-extensor ENGs.
We used Mardia's circular–circular rank-correlation test (Batschelet 1981; Mardia 1975) to examine the statistical significance of correlation between two sets of phases. Bonferroni corrections were used to account for the increase in type I error that resulted from doing multiple tests. We report Mardia's r, a correlation coefficient for circular data; r is a mean vector length calculated from the ranked data. The best-fit least-squares line was plotted. We used the term "knee-related interneuron" to describe an ON- or an OFF-unit whose start- or end-phases, respectively, were positively correlated with knee-extensor start-phases.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Hip-flexor motor neurons rhythmically alternated between activity and quiescence during normal fictive rostral scratching in the D3-end spinal turtle (Fig. 1A). Rostral scratching is a mixed-synergy motor behavior (Earhart and Stein 2000a,b; Robertson et al. 1985). Start-phases of knee-extensor motor activity occurred near the middle of hip-flexor motor activity; end-phases of knee-extensor motor activity occurred in the early part of hip-flexor quiescence. Knee-flexor ENG motor activity occurred during knee-extensor quiescence; knee-flexor motor activity rhythmically alternated with knee-extensor motor activity (Fig. 1).
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). The start-phases of knee-extensor motor activities were marked with filled triangles: the biarticular knee-extensor had a start-phase of 0.27 and the monarticular knee-extensor had a start-phase of 0.36 in this cycle. The mean start- and end-phases of knee motor activity during 184 cycles of normal rostral scratching in the D3-end preparation are shown in Fig. 1B. All the mean start- and end-phases were statistically significant (P < 0.001, Rayleigh test). End-phases of knee-flexor and start-phases of knee-extensor activities were related: earlier knee-flexor end-phases were associated with earlier knee-extensor start-phases and later knee-flexor end-phases were associated with later knee-extensor start-phases. This was shown in the plot of knee-flexor end-phase as a function of biarticular knee-extensor start-phase (Fig. 2A). The dashed diagonal line at 45° in Fig. 2A represented graph locations at which knee-flexor end-phase equaled biarticular knee-extensor start-phase. Since most graph locations were near this 45° line (Fig. 2A), most knee-flexor end-phases were near biarticular knee-extensor start-phases. Biarticular knee-extensor start-phase was plotted as a function of monoarticular knee-extensor start-phases (Fig. 2B). All the graph locations were just below and to the right of the 45° dashed diagonal line (Fig. 2B): biarticular knee-extensor start-phase occurred just prior to monoarticular knee-extensor start-phase.
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; Mardia 1975).
These fictive rostral scratching data in the D3-end preparation demonstrate that, during the middle of hip-flexor motor activity, there are strong relationships between the end of knee-flexor and the start of knee-extensor motor activities as well as between the start of monoarticular and biarticular knee-extensor motor activities (Figs. 1 and 2). In the next section, we describe additional analyses of knee-extensor motor activity in a D3–D10 preparation initially presented by Stein and Daniels-McQueen (2002a). Knee-flexor motor activity was not recorded in the D3–D10 preparation.
Knee-extensor motor activities during fictive rostral scratching in the D3–D10 preparation
We report biarticular and monoarticular knee-extensor motor activities during fictive rostral scratching in the D3–D10 preparation using a subset of data from Stein and Daniels-McQueen (2002a). We examined the start-phases of the biarticular and the monoarticular knee-extensors during 238 cycles of normal rostral scratching. Mean start-phase ± angular deviation were 0.26 ± 0.07 for the biarticular knee-extensor (P < 0.001, Rayleigh test) and were 0.33 ± 0.05 for the monoarticular knee-extensor (P < 0.001, Rayleigh test). See the filled bars in Fig. 7B for a graph of the mean start- and end-phases ± angular deviations of these knee-extensor motor bursts in the D3–D10 preparation.
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Biarticular knee-extensor start-phases were positively correlated with monoarticular knee-extensor start-phases (P < 0.0001, r = 0.63, Mardia's circular–circular rank correlation; Batschelet 1981; Mardia 1975). The relationship between the start-phases of these two knee-extensor motor bursts in the D3–D10 preparation (Fig. 3) was similar to that in the D3-end preparation (Fig. 2B).
Unit recordings from knee-related descending propriospinal interneurons in the D3–D10 preparation
The ENG data during fictive rostral scratching indicate an important transition in knee-related motor activity that occurred near the middle of hip-flexor motor activity (Fig. 1). We wish to characterize spinal interneurons with activities related to this transition. As a step in this characterization, we examined descending propriospinal interneurons whose bursts either began (ON-units) or ended (OFF-units) near the onset of knee-extensor motor activity during fictive rostral scratching. We studied the following relationships: between the start-phases of ON-unit activity and the start-phases of knee-extensor motor activity, between the end-phases of OFF-unit activity and the start-phases of knee-extensor motor activity, and between the start-phases of ON-unit activity and the end-phases of OFF-unit activity. We made these measurements on a subset of interneuronal data initially described by Stein and Daniels-McQueen (2002a).
KNEE-RELATED ON-UNITS WITH START-PHASES NEAR KNEE-EXTENSOR MOTOR START-PHASES. An example of a descending propriospinal interneuron whose start-phases were near the onsets of knee-extensor motor bursts is shown in Fig. 4A; we term such a unit an "ON-unit." We recorded from this ON-unit during 30 cycles of normal rostral scratching. Mean start-phase ± angular deviation for this ON-unit were 0.37 ± 0.04 (P < 0.001, Rayleigh test). The start-phases of this ON-unit were positively correlated with monoarticular knee-extensor start-phases (P < 0.0001, r = 0.75, Mardia's circular–circular rank correlation; Fig. 4B) and with biarticular knee-extensor start-phases (P < 0.0001, r = 0.74, Mardia's circular–circular rank correlation).
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) that were recorded during 25 cycles of normal rostral scratching along with hip-flexor, biarticular knee-extensor, and monoarticular knee-extensor ENGs. The start- and end-phases of these seven ON-units were significantly different from a random distribution (P < 0.001, Rayleigh test; Stein and Daniels-McQueen 2002a). For five of these seven ON-units, there was a significant positive correlation between the ON-unit start-phase and monoarticular knee-extensor start-phase (P < 0.004 for 1 unit with r = 0.42; P < 0.001 for 2 units with r = 0.53 and r = 0.56; P < 0.0001 for 2 units with r = 0.65 and r = 0.75; Mardia's circular–circular rank correlation). See the filled bars in Fig. 7A that display the mean start- and end-phases ± angular deviations of these five ON-units. For three of these five ON-units, there was a significant positive correlation between the ON-unit start-phase and the biarticular knee-extensor start-phase (P < 0.004 for 1 unit with r = 0.47; P < 0.0001 for 2 units with r = 0.53 and r = 0.74; Mardia's circular–circular rank correlation). KNEE-RELATED OFF-UNITS WITH END-PHASES NEAR KNEE-EXTENSOR START-PHASES. An example of a descending propriospinal interneuron whose end-phases were near the onsets of knee-extensor motor bursts is shown in Fig. 5A; we term such a unit an "OFF-unit." We recorded from this OFF-unit during 44 cycles of normal rostral scratching. Mean end-phase ± angular deviation for this OFF-unit were 0.27 ± 0.07 (P < 0.001, Rayleigh test). The end-phases of this OFF-unit were positively correlated with monoarticular knee-extensor start-phases (P < 0.0001, r = 0.58, Mardia's circular–circular rank correlation; Fig. 5B) and with biarticular knee-extensor start-phases (P < 0.0001, r = 0.56, Mardia's circular–circular rank correlation).
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) that were recorded during 25 cycles of normal rostral scratching along with hip-flexor, biarticular knee-extensor, and monoarticular knee-extensor ENGs. The start- and end-phases of these four OFF-units were significantly different from a random distribution (P < 0.001, Rayleigh test; Stein and Daniels-McQueen 2002a). For three of these four units, there was a significant positive correlation between the OFF-unit end-phase and monoarticular knee-extensor start-phase (P < 0.0001 with r = 0.58, r = 0.61, and r = 0.73; Mardia's circular–circular rank correlation). See the unfilled bars in Fig. 7A that display the mean start- and end-phases ± angular deviations of these three OFF-units. For two of these three OFF-units, there was a significant positive correlation between the OFF-unit end-phase and biarticular knee-extensor start-phase (P < 0.0001 with r = 0.56 and r = 0.70, Mardia's circular–circular rank correlation). SIMULTANEOUS RECORDINGS FROM KNEE-RELATED OFF-UNIT AND ON-UNIT INTERNEURONS. An example of a simultaneous recording of an ON- and an OFF-unit whose start- and end-phases, respectively, were near the start-phases of knee-extensor motor bursts is shown in Fig. 6A. We recorded from this ON-unit/OFF-unit pair during 43 cycles of normal rostral scratching. Mean start-phase ± angular deviation for this ON-unit were 0.31 ± 0.07 (P < 0.001, Rayleigh test). Mean end-phase ± angular deviation for this OFF-unit were 0.33 ± 0.06 (P < 0.001, Rayleigh test). Start-phases of this ON-unit were positively correlated with end-phases of this OFF-unit (P < 0.011, r = 0.36, Mardia's circular–circular rank correlation; Fig. 6B). We obtained simultaneous recordings from one other ON-unit/ OFF-unit pair. For that pair, there was also a positive correlation between the ON-unit start-phases and the OFF-unit end-phases (P < 0.0004, r = 0.56, Mardia's circular–circular rank correlation). These data demonstrate that the end-phases of some OFF-units were strongly related to the start-phases of some ON-units.
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) categorized each unit according to the percentage of overlap of hip-flexor motor activity with each unit's burst (based on the mean start- and end-phases of each unit). For the eight units studied here, the overlap percentages of the five ON-units ranged from 21 to 44% and the overlap percentages of the three OFF-units ranged from 46 to 68%. Units with overlap percentages in these ranges usually were active during cycles of rostral scratching with hip-extensor deletions (see Figs. 5 and 6 as well as the second, third, and fourth bars of Fig. 9 of Stein and Daniels-McQueen 2002a).
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) recorded from these five ON- and three OFF-units during rostral scratching with hip-extensor deletions. During a total of 30 deletion cycles, each ON-unit was active. In 24 of these deletion cycles, the ON-unit fired in a burst; in the other 6 deletion cycles, the ON-unit fired continuously. During a total of 19 deletion cycles, each OFF-unit fired in a burst. During rostral scratching with hip-extensor deletions, the activity of these knee-related ON- and OFF-units contrasts with the quiescence of hip-extensor-related interneurons (Stein and Daniels-McQueen 2002a). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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The iliofibularis muscle is a knee-flexor in amphibians, reptiles, and birds as is its mammalian homologue, tenuissimus (Laidlaw et al. 1995; Lance-Jones 1979). Turtle iliofibularis has complex histochemistry and is highly compartmentalized (Laidlaw et al. 1995; Mutungi and Johnston 1987). We describe here the first ENG recordings in the turtle from the knee-flexor nerve (Fig. 1) that innervates iliofibularis (Stein and Daniels-McQueen 2003). The cell bodies of iliofibularis motor neurons are located in the middle three segments of the hindlimb enlargement of the turtle spinal cord (Ruigrok and Crowe 1984); their axons run peripherally in the peroneal nerve. During embryonic development, iliofibularis (Ruigrok and Crowe 1984) and tenuissimus (Lance-Jones 1979) are formed from the dorsal muscle mass. Tenuissimus is present in adult cats; tenuissimus transiently appears during embryonic development in mice but it is not present in adult mice (Lance-Jones 1979). During rhythmic motor behaviors, EMG recordings have been obtained from iliofibularis in amphibians (Ashley-Ross and Lauder 1997), reptiles (Bakker and Crowe 1982; Crowe and Linnartz 1985; Gatesy 1997), and birds (Gatesy 1999; Johnston and Bekoff 1996) and from tenuissimus in cats (Pratt et al. 1991).
Rostral scratching is a mixed-synergy motor behavior
Central pattern generators in the spinal cord produce sequences of motor activity that drive alternation between flexion and extension of each hindlimb joint during rhythmic motor behaviors (Stein and Smith 1997). Rostral scratching in the turtle is a mixed-synergy motor pattern: knee-extensor motor activity occurs mainly during the latter portion of hip-flexor motor activity. This combination of synergies is produced during fictive rostral scratching in the immobilized turtle as well as during actual rostral scratching in the turtle with hindlimb movement (Bakker and Crowe 1982; Earhart and Stein 2000a,b; Robertson et al. 1985). During actual rostral scratching, this mixed synergy contributes to the successful rub of the foot against a site located in the midbody region of the turtle: the foot can rub against the stimulated site only when the hip is flexed and the knee is extended (Mortin et al. 1985).
KNEE-FLEXOR MOTOR PATTERNS. We present here recordings of knee-flexor motor activity during fictive rostral scratching (Fig. 1). Knee-flexor motor activity began during hip-flexor motor quiescence and ended during hip-flexor motor activity. The end-phases of knee-flexor motor activity were positively correlated with the start-phases of knee-extensor motor activities (Fig. 2A). The strong relationship between knee-flexor end-phases and knee-extensor start-phases that we observed during fictive rostral scratching is similar to their relationship during actual rostral scratching in the turtle (Bakker and Crowe 1982; Crowe and Linnartz 1985). The accurate timing of the transition from knee-flexor to knee-extensor motor activity is a key feature of the rostral scratch motor pattern.
START-PHASES OF KNEE-EXTENSOR MOTOR ACTIVITIES DURING HIP-FLEXOR MOTOR ACTIVITY. We characterized knee-flexor motor activity in a D3-end preparation with a single transection of the spinal cord between the D2 and D3 spinal segments. In the D3-end preparation, we observed that the start-phases of biarticular knee-extensor motor activities occurred just prior to the start-phases of monoarticular knee-extensor motor activities (Fig. 2B). We characterized descending propriospinal interneuron activities during fictive rostral scratching in a D3–D10 preparation with two spinal transections; the anterior transection between the D2 and D3 spinal segments and the posterior transection between the D10 and S1 spinal segements. We did not record knee-flexor motor activity in the experiments with the D3–D10 preparation; we did observe, however, that biarticular knee-extensor start-phases occurred just prior to monoarticular knee-extensor start-phases (Fig. 3). Thus the D3–D10 preparation displayed an important characteristic observed in the D3-end preparation. We predict that future recordings of knee-flexor motor activities during fictive rostral scratching in the D3–D10 preparation will reveal knee-flexor end-phases near knee-extensor start-phases.
Knee-related descending propriospinal interneurons during fictive rostral scratching
Our analyses of motor-neuron activities during fictive rostral scratching indicated a transition from knee-flexor to knee-extensor motor activities during hip-flexor motor activity (Figs. 1 and 2). We wish to determine whether there is a set of knee-related interneurons whose activities are correlated with this transition. We suggest that interneurons whose start-phases (ON-units) or end-phases (OFF-units) are near to knee-extensor motor activity start-phases are candidate knee-related interneurons. We described ON- and OFF-units whose start- and end-phases, respectively, were near to and positively correlated with knee-extensor start-phases (Figs. 4, 5, 6, 7). Our observations of these positive correlations are a step in the identification of knee-related interneurons. Future experiments that examine additional features of interneurons with these characteristics will be important in determining their role in motor pattern generation.
Hip-extensor-related units belong to a population different from that of the knee-related units
Our previous study (Stein and Daniels-McQueen 2002a) reported single-unit recordings of descending propriospinal interneurons during normal rostral scratching and during rostral scratching with hip-extensor deletions. During normal rostral scratching, we characterized hip-extensor–related interneurons whose activity did not overlap with hip-flexor motor activity; this population of units was mainly quiet during rostral scratching with hip-extensor deletions (Figs. 4 and 9 of Stein and Daniels-McQueen 2002a). We proposed that this population is part of a hip-extensor "module" (Jordan 1991): the population is active during hip-extensor motor activity and is quiescent when there is no hip-extensor motor activity.
We also described interneurons in our previous study that, during normal rostral scratching, were active during a portion of each hip-flexor motor burst, e.g., 21–80% overlap interneurons. During rostral scratching with hip-extensor deletions, these intermediate–overlap interneurons were mainly active (see Figs. 5, 6, and 9 of Stein and Daniels-McQueen 2002a). In the present paper, we focused on a subset of interneurons with 21–80% overlap, those ON- and OFF-units with start- and end-phases, respectively, near the start-phases of knee-extensor activity. During rostral scratching with hip-extensor deletions, these knee-related ON- and OFF-units were mainly active and, in contrast, hip-extensor module interneurons were mainly quiet. Therefore these knee-related units belong to a population different from that of the hip-extensor module.
Possible roles of knee-related descending propriospinal interneurons during normal rostral scratching
The start-phases of ON-units and the end-phases of OFF-units were positively correlated with the start-phases of knee-extensor motor activity during normal rostral scratching (Figs. 4, 5, 6, 7). We suggest that knee-related ON- and OFF-units may play a role in timing knee motor neuron activity: some of these units may be premotor interneurons that directly synapse on motor neurons.
Based on the timing of both unit and motor-neuron activity, it is possible that some ON-unit premotor interneurons may either inhibit knee-flexor motor activity or excite knee-extensor motor activity. In the terminology of Grillner's (1981) Unit-Burst-Generator (UBG) Hypothesis, ON-unit premotor interneurons may be designated knee-extensor interneurons and members of a knee-extensor module. In Grillner's terminology, the present paper has shown that the knee-extensor module is a population different from the hip-extensor module; this is not consistent with the Half-Center Hypothesis of Brown and Lundberg (Brown 1911, 1914; Jankowska et al. 1967; Lundberg 1981) in which all extensor interneurons are grouped into a single module, the extensor half-center. In the terminology of the Berkowitz-Stein (1994b) Broad-Tuning Hypothesis, it is possible that ON-unit premotor interneurons may be rostrally tuned interneurons that excite both knee-extensor and hipflexor motor neurons.
Some OFF-unit premotor interneurons may either inhibit knee-extensor motor activity or excite knee-flexor motor activity. In the terminology of Grillner's (1981) UBG Hypothesis, OFF-unit premotor interneurons may be designated knee-flexor interneurons and members of the knee-flexor module. Berkowitz and Stein (1994b) did not discuss knee-flexor motor activity.
For two OFF-/ON-unit pairs, OFF-unit end-phases were positively correlated with ON-unit start-phases (Fig. 6). This is consistent with possible reciprocal inhibition between some OFF-units and some ON-units. In the terminology of Grillner's (1981) UBG Hypothesis, it is possible there may be a reciprocal inhibitory relationship between members of the knee-flexor module and the knee-extensor module.
Predictions that premotor ON-units excite and premotor OFF-units inhibit knee-extensor motor neurons are consistent with synaptic potentials recorded during rostral scratching from knee-extensor motor neurons (Robertson and Stein 1988). Knee-extensor motor neurons fire action potentials in response to excitatory postsynaptic potentials during the latter portion of hip-flexor motor activity in rostral scratching. Knee-extensor motor neurons are silent in response to inhibitory postsynaptic potentials during most of hip-flexor quiescence and the early portion of hip-flexor motor activity in rostral scratching. To date, there are no reports of synaptic potentials obtained from knee-flexor motor neurons during rostral scratching.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Address for reprint requests and other correspondence: P.S.G. Stein, Department of Biology, Washington University, St. Louis, MO 63130 (E-mail: stein{at}biology.wustl.edu).
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