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. 2000 Jun 1;525(Pt 2):549–564. doi: 10.1111/j.1469-7793.2000.t01-1-00549.x

Group I disynaptic excitation of cat hindlimb flexor and bifunctional motoneurones during fictive locomotion

J Quevedo 1, B Fedirchuk 1, S Gosgnach 1, D A McCrea 1
PMCID: PMC2269940  PMID: 10835053

Abstract

  1. The incidence of short latency excitation of motoneurones innervating flexor and bifunctional muscles evoked by group I intensity (≤ 2 × threshold) electrical stimulation of hindlimb muscle nerves was investigated during fictive locomotion in decerebrate cats. Intracellular recordings were made from hindlimb motoneurones in which action potentials were blocked by intracellular diffusion of a lidocaine (lignocaine) derivative (QX-314) and fictive locomotion was evoked by electrical stimulation of the midbrain.

  2. Few motoneurones (16%) received group I-evoked oligosynaptic excitation in the absence of fictive locomotion. During fictive locomotion 39/44 (89%) motoneurones innervating ankle, knee or hip flexor muscles and 18/28 (64%) motoneurones innervating bifunctional muscles received group I-evoked oligosynaptic EPSPs. In flexor motoneurones, locomotor-dependent excitation was present in both step cycle phases but largest during flexion. In bifunctional motoneurones, EPSPs were often largest at the transition between flexion and extension phases.

  3. Activation of homonymous afferents most consistently evoked the largest locomotor-dependent excitation (amplitude up to 4.6 mV), but in some cases stimulation of heteronymous flexor or bifunctional muscle nerves evoked large EPSPs. EPSP amplitude became maximal as stimulation intensity was increased to about twice threshold. This suggests that tendon organ afferents can evoke group I EPSPs during locomotion. The EPSPs resulting from brief, small stretches of extensor digitorum longus tendons indicate that group Ia muscle spindle afferents can also evoke the group I excitation of flexors. Stimulation of extensor group I afferents did not result in excitation of flexor motoneurones.

  4. The mean latency of locomotor-dependent group I excitation in flexor and bifunctional motoneurones was 1.64 ± 0.16 ms, indicating a path consisting of a single interneurone interposed between group I afferents and motoneurones innervating flexor and bifunctional muscles. This disynaptic excitation is analogous to that recorded in extensor motoneurones and evoked from extensor group I afferents during locomotion. Differences in the phase dependence and sources of group I excitation to flexor and extensor motoneurones during locomotion suggest the existence of separate groups of excitatory interneurones exciting flexor and extensor motoneurones.

  5. The wide distribution of group I disynaptic excitation in motoneurones innervating extensor, flexor and bifunctional muscles acting on hip, knee and ankle joints suggests that these pathways can play an important role in the reinforcement of ongoing locomotor activity throughout the limb.

In many preparations activation of hindlimb extensor group I muscle afferents evokes predominantly inhibitory reflexes at oligosynaptic (di- and trisynaptic) latencies in extensor motoneurones (reviewed in Jankowska, 1992). This non-reciprocal group I inhibition (Jankowska et al. 1981a) is considered as a feedback system whereby proprioceptive activity in tendon organ and primary muscle spindle afferents reflexly inhibits extensor motoneurones (reviewed in Jami, 1992). However, during both fictive (Conway et al. 1987; Guertin et al. 1995) and over-ground (Whelan & Pearson, 1997; Hiebert & Pearson, 1999) locomotion stimulation of the same afferents evokes an excitation of extensors (for review see Pearson, 1995; McCrea et al. 1995). The reorganization of extensor group I reflexes results in a proprioceptive feedback system that can increase extensor activity and provide additional weight support and forward propulsion during extension in both cats (Pearson et al. 1992; Pearson & Collins, 1993; Guertin et al. 1995; McCrea et al. 1995; Angel et al. 1996; Whelan & Pearson, 1997; Hiebert & Pearson, 1999) and man (Harkema et al. 1997; Stephens & Yang, 1999).

Intracellular recordings from extensor motoneurones during fictive locomotion have shown that locomotor-dependent reflex excitation involves the suppression of non-reciprocal inhibition (Gossard et al. 1994; Brownstone et al. 1994; McCrea et al. 1995; Angel et al. 1996) and the appearance of both disynaptic (McCrea et al. 1995; Angel et al. 1996) and longer latency reflexes (Gossard et al. 1994; Brownstone et al. 1994; Guertin et al. 1994). The disynaptic component of this reflex reversal from inhibition to excitation is particularly interesting because it involves the activation of previously unknown groups of excitatory interneurones contacting extensor motoneurones (McCrea et al. 1995; Angel et al. 1996; McCrea, 1998).

It has been suggested that sensory feedback from flexor group I afferents also plays an important role in the modulation of the locomotor step cycle (Kriellaars et al. 1994; Hiebert et al. 1996). During treadmill locomotion, stretching of hip or ankle flexor muscles can enhance ongoing flexor activity or during the stance phase reset the step cycle to flexion (Hiebert et al. 1996). These observations indicate the possibility that lengthening of hip and ankle flexor muscles during extension activates group I afferents that access elements of the locomotor spinal circuitry to regulate the transition from stance to swing (Kriellaars et al. 1994; Hiebert et al. 1996). Activation of sartorius group I afferents during the flexion phase of fictive locomotion prolongs flexor motoneurone activity (Perreault et al. 1995). Although the enhancement of flexor activity by flexor group I afferents probably plays a role during normal forward locomotion, it may be even more important under other locomotor tasks (Trank & Smith, 1996; Carlson-Kuhta et al. 1998; Smith et al. 1998).

In view of these observations, the question arises as to whether motoneurones innervating flexor muscles are, like extensor motoneurones, subject to group I disynaptic excitation during locomotion. Preliminary studies have described the group I disynaptic excitation of some flexor motoneurones during fictive locomotion (Quevedo et al. 1998a,b; see also Degtyarenko et al. 1998b). The present work investigates the distribution of locomotor-dependent group I disynaptic excitation in motoneurones innervating flexor muscles acting at different joints and in motoneurones showing complex patterns of depolarization during locomotion, i.e. motoneurones innervating bifunctional muscles. This study of group I reflex modulation will provide insight into the organization of the interneurones mediating disynaptic excitation during fictive locomotion.

METHODS

Experiments were carried out on 17 decerebrate and paralysed cats (2.1-4.5 kg). Guidelines set by the Canadian Council on Animal Care were followed throughout. In all preparations anaesthesia was induced and maintained for the duration of surgery with halothane in a mixture of oxygen (70 %) and nitrous oxide (30 %). Animals were given atropine (0.2 mg) and dexamethasone (4 mg). An intravenous solution of glucose and bicarbonate in saline was administered continuously throughout the experiment (5 ml h−1). A lethal injection of pentobarbital anaesthetic was administered at the end of the experiment. Some of the present results were obtained in the same preparations used to examine the depression of group Ia monosynaptic EPSPs during fictive locomotion (Gosgnach et al. 2000).

Selected hindlimb nerves were dissected, sectioned and prepared for placement on conventional bipolar hook electrodes for either stimulation or recording. These included posterior biceps (PB), semitendinosus (St), semimembranosus and anterior biceps (SmAB), medial gastrocnemius (MG), lateral gastrocnemius and soleus (LGS), plantaris (Pl), flexor digitorum longus (FDL), flexor hallucis longus (FHL), tibial (TIB), peroneus longus (PerL), tibialis anterior (TA), extensor digitorum longus (EDL), extensor digitorum brevis (EDB), peroneus tertius and brevis (PerT + B) and superficial peroneal. Vasti, rectus femoris (RF) and sartorius (Sart, anterior and medial branches) were mounted in a triple ventral cuff electrode. In some experiments vasti and RF were mounted together (Quad, quadriceps). Selected contralateral nerves were used to monitor contralateral fictive locomotor activity. The obturator nerves and remaining branches of the sciatic and femoral nerves were sectioned bilaterally and tendons around the hip severed.

Following a L4-L6 laminectomy the animals were transferred to a recording frame. The brain was exposed by a craniotomy and a mechanical precollicular-postmamillary decerebration performed using a spatula. Both cortices and all tissue rostral to the transection were removed. Anaesthesia was then discontinued and the cat was paralysed with gallamine triethiodide (2-3 mg kg−1 h−1) and artificially ventilated. A bilateral pneumothorax was made to reduce movements of the spinal cord and tidal volume was adjusted to maintain expiratory CO2 at about 4 %. The blood pressure was monitored and decreases below 80 mmHg were counteracted by intravenous infusion of dextran. Mineral oil pools were made with the skin flaps to prevent drying of exposed tissues. Temperatures were maintained at 37°C by means of feedback-controlled radiant heat.

Following paralysis, fictive locomotion was elicited by unilateral or bilateral electrical stimulation of the mesencephalic locomotor region (MLR, 30–200 μA, 1 ms pulses, 12–18 Hz). To permit better comparison of the postsynaptic potentials (PSPs) evoked by different nerves, stimuli were delivered to up to three nerves separated by ∼20 ms (e.g. Figs 2, 4, 5 and 6). In one preparation fictive locomotion was induced by the administration of naloxone (100 μg kg−1) and clonidine (200 μg kg−1). Single shocks to peripheral nerves were delivered at 3–5 Hz independently of MLR stimulation. Intensity of electrical stimulation to the nerves is expressed in multiples of the threshold (T) of the most excitable fibres as recorded by a ball electrode placed on the cord dorsum. In one experiment the ipsilateral EDL nerve was left in continuity with the muscle and dissected free to allow electrical stimulation of the nerve or selective activation of muscle spindle afferents by brief stretches (1 ms, 30 μm) of the EDL tendon.

Figure 2. Group I excitatory EPSPs in EDL motoneurones are evoked by synergist but not by extensor afferents during both phases of fictive locomotion.

Figure 2

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A, averaged PSPs produced by stimulation of the EDL nerve at 2T (data from the same motoneurone illustrated in Fig. 1). Averages during non-locomoting (control) conditions (dotted trace) and during MLR-evoked fictive locomotion at 2T (continuous traces) and at 1.4T (dashed trace) are overlaid. In this and subsequent figures the letters E and F and associated numbers denote the phase of the step cycle from which the average was obtained (see panels D and E). The vertical dashed line in A and B indicates the minimum central latency (1.65 ms) of the PSP component following the monosynaptic EPSP measured during the last portion of flexion (F4) phase. The amplitude of the disynaptic EPSP was measured from the peak amplitude of the monosynaptic EPSP in the corresponding locomotor phase (horizontal dashed lines). B, average PSPs recorded in the same motoneurone and evoked by the stimulation of the EDB nerve at 2T. The minimum latency was 1.8 ms (F4, dashed vertical line). There were no oligosynaptic EPSPs before locomotion (dotted trace). C, stimulation of the LGS nerve at 2T produced no clear effects. D, cycle-normalized averages of LGS and Sart ENGs and of the EDL intracellular recording from 71 cycles of fictive locomotion. Averages were based on the LGS step cycle and extension and flexion phases (vertical dashed line) were normalized separately. E, the step cycle was divided into 6 bins (E1, E2 and F1-F4). Peak amplitudes of PSPs evoked by stimulation of EDL at 2T (▴ and ▪, mono- and oligosynaptic EPSP, respectively) and EDB at 2T (○, oligosynaptic EPSP) are shown. The number of sweeps used to calculate PSP averages ranged from 21 (F2) to 34 (E1). Control amplitudes of mono- and oligosynaptic EPSPs are shown at the left of panel E (C).

Figure 4. Group I excitatory postsynaptic potentials are expressed during locomotion induced by clonidine and naloxone.

Figure 4

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A, from left to right, ENGs recorded in the GS and Sart nerves (vertical orientation), and low gain (vertical orientation) and high gain (horizontal orientation) intracellular recordings from a PerL motoneurone (PerL intracell.) during fictive locomotion induced by clonidine and naloxone. PSPs were evoked by stimulation of EDL and PerL nerves at 2T during flexion (dots) and extension (dashes). B, average PSPs evoked by stimulation of the PerL nerve at 2T. The step cycle was divided into 6 bins (E1-E3 and F1-F3) based on the Sart ENG activity. C, average PSPs evoked by stimulation of the EDL nerve at 2T. In both cases disynaptic EPSPs, almost restricted to the F3 phase, had a minimum latency of 1.8 ms (vertical dashed line). B and C were obtained from 20 fictive locomotor step cycles.

Figure 5. Differential modulation of disynaptic EPSPs evoked by homonymous and extensor group I afferents in a motoneurone innervating the bifunctional muscle peroneus tertius and brevis (PerT + B).

Figure 5

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A, from top, LGS and EDL ENGs and low gain intracellular recordings from a PerT + B motoneurone. Upper traces in B, averaged, cycle-normalized LGS and EDL ENGs and PerT + B membrane potential during 87 MLR-evoked step cycles. The step cycle was divided into 6 bins (E1-E3 and F1-F3). Histograms in B, amplitudes of PSPs evoked by PerT + B (1.8T) and MG (2T) nerve stimulation. C, averaged PSPs evoked by stimulation of the homonymous (PerT + B) nerve at 1.8T. The minimum latency for disynaptic EPSPs was 1.8 ms (vertical dashed line). D, averaged PSPs evoked by 2T MG nerve stimulation. The latency of the EPSPs recorded during E2 was 1.4 ms.

Figure 6. Phase dependence of group I disynaptic EPSPs and IPSPs in a bifunctional motoneurone innervating the extensor digitorum brevis (EDB) muscle.

Figure 6

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A, from top, LGS and TA ENGs, and low gain intracellular recording from an EDB motoneurone. Upper traces in B, averaged LGS and TA ENGs and low gain EDB intracellular recording during 21 fictive locomotor step cycles. Histograms in B, PSP amplitudes evoked by the stimulation of EDB (1.3T), EDL (1.8T) and PerT + B (1.6T) nerves. C, averaged PSPs evoked by EDB nerve stimulation at 1.3T (latency 1.7 ms). The arrow indicates a later, possibly trisynaptic, component during E1. D, stimulation of the EDL nerve at 1.8T produced disynaptic IPSPs during flexion (hatched histograms in B) and disynaptic EPSPs (latency 1.8 ms) during E1. E, stimulation of the PerT + B nerve at 1.6T produced disynaptic EPSPs with a minimum latency of 1.7 ms.

Low gain DC coupled and high gain AC coupled intracellular recordings of antidromically identified lumbar motoneurones were made using glass microelectrodes (1.6-2 μm) filled with 2 M potassium citrate. To block action potentials and facilitate analysis of locomotor-related postsynaptic potentials 100 mM QX-314 (N-[2,6-dimethylphenylcarbamonyl-methyl]triethylammonium bromide; Alamone Laboratories, Jerusalem, Israel) was added to the electrodes. PSPs evoked by the stimulation of group I afferents were recorded before and during locomotion.

In order to analyse modulation of PSPs, activity in integrated and rectified electroneurograms (ENGs) was used to divide the fictive locomotor step cycle into flexor and extensor phases. The normalized step cycle was typically divided into six time bins that were distributed throughout the step cycle according to the average duration of flexion and extension phases. In Fig. 2 for example the flexion phase was much longer than the extension phase and the cycle was divided into four bins in flexion and two in extension. The choice of six bins ensured a sufficient number of sweeps per average in all cells. The individual PSPs produced by stimulation of different nerves were assigned to the appropriate bin to calculate the mean of the intracellular and cord dorsum recordings. Because of fluctuations in the relative durations of the flexion and extension phases during some trials, flexion and extension were normalized independently to avoid misplacement of the corresponding PSPs. Any sweeps that spanned the flexion and extension phases were excluded from analysis, as were sweeps containing motoneurone action potentials. Since the amplitude of the monosynaptic EPSP is usually reduced during fictive locomotion (Gosgnach et al. 2000), the peak amplitude of the disynaptic component was measured from the peak amplitude of the monosynaptic EPSP recorded in the same part of the step cycle (see Fig. 2A). Latencies were measured in the averaged recordings from the peak of the first positive component in the cord dorsum potential (negativity up) to the onset of the intracellular recorded PSPs. Since latencies tended to be longer when PSPs were smaller, the minimum latency was measured in the bin with the largest averaged PSP (see Results). Intracellular recordings, the cord dorsum potential and rectified and integrated ENGs were sampled at 10, 5 and 0.5 kHz, respectively. Data capture and analysis were accomplished with software developed within the Winnipeg Spinal Cord Research Centre on Pentium PCs running QNX or Linux.

RESULTS

Under control conditions without MLR stimulation and fictive locomotion, the effects of group I strength (≤ 2T) stimulation of flexor nerves were examined in 28 flexor and 10 bifunctional motoneurones in decerebrate cats. In the absence of locomotion oligosynaptic EPSPs were evoked by 6 of the 53 nerves stimulated in 4 of the 28 flexor motoneurones, and by stimulation of 4 of 17 nerves in 2 of the 10 bifunctional motoneurones. During fictive locomotion, the presence of oligosynaptic group I EPSPs was examined in 44 flexor and 28 bifunctional motoneurones. As will be illustrated and summarized in the tables, stimulation of 63 of the 101 flexor nerves tested produced excitation in 39/44 (89 %) flexor motoneurones. Stimulation of 26/47 flexor or bifunctional muscles nerves produced disynaptic excitation in 18/28 (64 %) of the bifunctional motoneurones.

Group I disynaptic EPSPs in flexor motoneurones during fictive locomotion

The intracellular records from an EDL motoneurone in Fig. 1 show the effects of group I strength stimulation of the EDL nerve. Before fictive locomotion EDL stimulation evokes a monosynaptic EPSP (Fig. 1B, control). Figure 1A shows the alternating extensor (LGS) and flexor (Sart) ENG activity during fictive locomotion (shown vertically), as well as the low gain (vertical) and high gain (horizontal) intracellular recordings from this EDL motoneurone. Note the rhythmic depolarization of the motoneurone during the flexor phase of the step cycle. During fictive locomotion EDL nerve stimulation evokes not only a monosynaptic EPSP (open arrow) but also a longer latency excitation (filled arrow). EDL stimulation was delivered at 4 Hz throughout the bout of fictive locomotion during both the flexion (indicated by dots) and extension phases (dashes) of the step cycle and was not synchronized to the phase of the step cycle. The horizontal intracellular records in panel A are expanded in panel C and grouped into those evoked during flexion and extension. The longer latency components of the EPSP evoked by stimulating EDL are present in both phases but larger during flexion.

Figure 1. Latency and amplitude fluctuations of locomotor-dependent, group I disynaptic EPSPs evoked in an extensor digitorum longus (EDL) motoneurone.

Figure 1

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A, from left to right, vertical display of the rectified and integrated electroneurograms (ENG) from lateral gastrocnemius and soleus (LGS, extensor) and sartorius (Sart, flexor) nerves, low gain (vertical orientation) and high gain (horizontal orientation) intracellular recordings (EDL intracell.) during a sequence of MLR-evoked fictive locomotion. Stimulation of the EDL nerve with single shocks at 2T during flexion is indicated by dots and during extension by dashes. Open and filled arrows indicate the onset of mono- and oligosynaptic PSPs, respectively. The bottom trace shows the cord dorsum recording. B, expanded and amplified intracellular recordings show EDL-evoked PSPs recorded before locomotion (control). C, PSPs from A recorded during locomotion sorted into those evoked during flexion (dots) and extension (dashes). The vertical dotted line denotes the minimum central latency of locomotor-dependent EPSPs (1.65 ms; see traces identified with asterisks). Here and in subsequent figures, 0 in the time scale indicates the arrival of the afferent volley from which central latencies were measured.

The central latencies of the EPSPs were measured to provide an estimate of the number of interneurones interposed between group I afferents and motoneurones. The start of the time scale in Fig. 1C is aligned with the arrival of the afferent volley at the cord dorsum (bottom trace). The vertical dotted line in Fig. 1B and C indicates the minimum latency for the oligosynaptic EPSPs marked with asterisks. Latencies of the second component varied between 1.65 and 2.0 ms. Latencies between 1.1 and 1.8 ms were considered as being mediated disynaptically (see Discussion).

Phase dependence of group I disynaptic excitation in different species of motoneurones innervating flexor muscles

The variation of the amplitude of the longer latency EPSP during locomotion illustrated in Fig. 1 is explored further in Fig. 2. To address the possibility that group I EPSPs were modulated within the flexion or extension phases, the step cycle was first normalized using a longer period of fictive locomotion than that illustrated in Fig. 1. In this example 71 bursts of activity in the LGS ENG were used to produce a time normalization of the extension phase of the fictive step cycle. The period of inactivity of LGS ENG was then used to normalize the flexion phase. Figure 2D shows that the duration of extensor activity was about 1/4 as long as that of flexor activity. Accordingly, extension was divided into two bins (E1 and E2) and flexion into four bins (F1-4). High gain intracellular records occurring in these bins were sorted and averaged to produce the records in Fig. 2A–C and the graph in Fig. 2E.

Stimulation of the homonymous nerve (EDL) with single shocks at 2T produced only a monosynaptic EPSP during non-locomoting conditions (dotted trace, Fig. 2A) with an average amplitude of 2.5 mV (leftmost triangle in Fig. 2E). During fictive locomotion the amplitude of this monosynaptic EPSP was reduced (Fig. 2A, ▴ in Fig. 2E). This reduction is typical of that found for composite monosynaptic Ia EPSPs during fictive locomotion (Gosgnach et al. 2000). During locomotion, longer latency EPSPs occurred on the falling portion of the monosynaptic EPSP. These were largest during the last portion of the flexion phase (F4), present during early extension (E1), and practically absent in the transition from E2 to F1 (▪ in Fig. 2E). Since the monosynaptic component varied during locomotion, the amplitude of the longer latency component was measured from the monosynaptic EPSP recorded in the same phase of the step cycle. For example, the longer latency component during F4 was 4.6 mV above the monosynaptic EPSP peak (Fig. 2A). Stimulation of the EDL nerve at 1.4T in the subsequent trial also produced disynaptic EPSPs (Fig. 2A, dashed trace; average during F4).

Twice threshold stimulation of the EDB nerve produced disynaptic EPSPs during fictive locomotion with a latency of 1.8 ms and no monosynaptic component (Fig. 2B). These EPSPs were modulated in a phase-dependent manner (○ in Fig. 2E) and were not present during control conditions (dotted line in Fig. 2B). EDB EPSPs were almost as large during early extension as late flexion. Stimulation of an extensor nerve, LGS, failed to produce disynaptic excitation (Fig. 2C). Figure 2E also shows that the modulation of mono- and disynaptic EPSPs during locomotion was different. The monosynaptic component was largest when the disynaptic EPSP was smallest (F1) and smallest when the disynaptic EPSP was largest (F4).

Distribution of group I disynaptic excitation of flexor motoneurones

During fictive locomotion extensor motoneurones innervating muscles operating at the hip, knee and ankle can receive group I disynaptic EPSPs from extensor afferent stimulation (Angel et al. 1996). Figure 3 shows the distribution of disynaptic excitation in flexor motoneurones innervating the ankle flexor muscles EDL and TA (Fig. 3A and B), and the knee (St) and hip (Sart) flexor muscles (Fig. 3C). Averaged intracellular records obtained during the flexion phase of fictive locomotion are shown as continuous lines and those obtained during extension as dashed lines. Non-locomotor, control records (dotted lines) and the arithmetic difference between the flexion and extension traces (continuous thin line above the cord dorsum records) are also shown. Electrical stimulation of homonymous muscle nerves (Fig. 3A and C) produced EPSPs with disynaptic latencies during fictive locomotion in all of these motoneurones. As in Figs 1 and 2 disynaptic EPSPs were largest during flexion but also present during extension.

Figure 3. Motoneurones innervating ankle, knee and hip flexor muscles receive disynaptic excitation from group Ia and Ib afferent fibres.

Figure 3

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Motoneurones from which intracellular records were obtained are indicated above and the nerves stimulated are indicated below each panel. Thick continuous traces, averaged intracellular recordings obtained during the flexion phase of MLR-evoked fictive locomotion. Dashed traces, recordings obtained during extension. Dotted traces, recordings before locomotion. Thin continuous traces show the arithmetic difference between records obtained during flexion and extension. A, electrical stimulation of the EDL nerve evokes EPSPs during flexion in both EDL and TA motoneurones. B, stretch of the EDL muscle tendon (30 μm, 1 ms) activates group Ia muscles afferents and evokes short latency excitation during flexion. C, electrical stimulation of Sart and St nerves evokes disynaptic excitation in knee (St) and hip (Sart) flexor motoneurones.

Figure 3A shows EPSPs produced by electrical stimulation of homonymous and heteronymous nerves in two ankle flexor motoneurones, recorded in different experiments. In the EDL motoneurone, stimulation of the homonymous nerve at 2T produced mono- and disynaptic EPSPs while stimulation of TA at 2T was without effect. In the TA motoneurone both EDL and TA nerve stimulation resulted in mono- and disynaptic EPSPs. Because the TA nerve was stimulated at 1.4T to avoid antidromic activation of the motoneurone, the amplitudes of EPSPs evoked by EDL and TA stimulation cannot be compared (compare the effects of 1.4T and 2T stimulation in Fig. 2A). Table 1 shows that EDL group I afferents were the most frequent source of heteronymous disynaptic EPSPs (see Discussion).

Table 1.

Motoneurones innervating flexor muscles receiving group I disynaptic EPSPs

During flexion During extension At rest
A. Motoneurones n EDL TA PerL EDB CP PerT+B Sart From ≥1 nerve From ≥1 nerve
EDL 13 12/13 3/10 0/2 2/2 — 2/2 1/3 11/13 0/8
TA 8 8/8 5/8 0/4 2/2 — 1/1 0/1 7/8 2/8
PerL 3 2/3 — 2/3 — — 2/3 — 3/3 0/1
EDB 1 1/1 — — 1/1 — — — 1/1 0/1
CP* 4 3/4 1/1 1/1 1/1 1/1 2/2 — 4/4 2/4
B. Motoneurones n St Sart RF Vasti PB EDL SmAB From ≥1 nerve From ≥1 nerve
St 2 1/2 0/1 — 0/1 0/1 1/1 1/2 1/2 0/2
Sart 6 — 6/6 1/1 0/1 0/1 0/3 — 3/6 0/2
Hip* 2 — 2/2 — 0/1 — — 0/1 0/2 0/2
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Disynaptic EPSPs during flexion in 39/44 motoneurones (89%); 63/101 nerves tested. Mean latencies were 1.66 ± 0.15 ms (minimum 1.3 ms; maximum 2.0 ms). Disynaptic EPSPs during extension in 37/44 motoneurones (84%). Disynaptic EPSPs at rest in 4/28 motoneurones (14%); 6/53 nerves tested. EDL, extensor digitorum longus; TA, tibialis anterior; PerL, peroneus longus; EDB, extensor digitorum brevis; CP, common peroneal; PerT+B, peroneus tertius and brevis; Sart, sartorius; St, semitendinosus; RF, rectus femoris; PB, posterior biceps; SmAB, semimembranosus and anterior biceps; Hip, hip flexor

*

not identified. Numbers in bold, stimulation of homonymous group I afferents.

Table 1 summarizes the incidence and latency of disynaptic EPSPs recorded in flexor motoneurones during fictive locomotion (monosynaptic EPSPs are not represented). Most (39/44, 89 %) of the motoneurones depolarized during flexion (13 EDL, 8 TA, 3 PerL, 1 EDB, 4 common peroneal, 6 Sart, 2 St, and 2 hip flexors) showed disynaptic EPSPs from at least one of the nerves tested during locomotion. In total 63 EPSPs were produced following stimulation of 101 nerves while recording from these 44 motoneurones. Minimum central latencies ranged from 1.3 to 2.0 ms with a mean of 1.66 ± 0.15 ms for the 63 EPSPs. The latency of 57 EPSPs was ≤ 1.8 ms (47 < 1.8 ms), suggesting a path consisting of a single excitatory interneurone interposed between group I afferents and motoneurones. Disynaptic EPSPs were larger during flexion, but except for two motoneurones, were also present in extension (see Table 1 and examples in Figs 2 and 3). As mentioned, only 14 % (4/28) of the flexor motoneurones tested exhibited group I disynaptic EPSPs in the absence of fictive locomotion.

Electrical stimulation of homonymous group I afferents produced disynaptic EPSPs in motoneurones innervating ankle (mainly EDL, TA and PerL), hip (Sart) and knee (St) flexor muscles (Table 1, numbers in bold). Stimulation of heteronymous group I afferents also produced disynaptic EPSPs in ankle flexor motoneurones (Table 1). For example, EDL motoneurones received disynaptic EPSPs following stimulation of TA, EDB, PerT + B, and Sart group I afferents. Group I disynaptic excitation was evoked from afferents to motoneurones across the joints in only one EDL motoneurone (from the Sart nerve) and in one St motoneurone (from EDL nerve).

Group I disynaptic excitation is generated by activation of muscle spindle and tendon organ afferents

As illustrated in Fig. 2A, increasing stimulus intensity from 1.4T to 2T resulted in larger disynaptic excitation during locomotion. Because of the problem of antidromic activation of motoneurones, the effect of higher intensity stimulation on disynaptic EPSP amplitude was not examined systematically. Given this caveat, maximum EPSP amplitudes in this study occurred with stimulation intensities around 2T, i.e. at intensities maximal for activation of group I afferents. No evidence for an additional contribution to EPSP amplitude from activation of group II afferents was found (not illustrated). Since electrical stimulation of peripheral nerves at 2T recruits both Ia and Ib afferents (Jack et al. 1978), selective activation of group Ia muscle spindle afferents was produced by applying brief stretches (1 ms, 30 μm) of the EDL muscle tendons (nerve left in continuity with the muscle). Stretch-evoked EPSPs were recorded in all three motoneurones tested (two EDL and one TA) and are illustrated in Fig. 3B. Similar to the results obtained with electrical stimulation of the nerves in the same motoneurones (not illustrated), small muscle stretches produced disynaptic EPSPs during fictive locomotion that were larger during the flexion phase. The latencies of these disynaptic EPSPs were 2.0 ms in the EDL and 1.8 ms in the TA motoneurone and slightly longer than those evoked by electrical stimulation. Because of the asynchronous activation of spindle afferents, stretch-evoked EPSPs with latencies as long as 2.2 ms can be considered as being evoked disynaptically (Jankowska et al. 1981b). These observations suggest that activation of muscle spindle afferents alone is sufficient to produce disynaptic EPSPs in flexor motoneurones during fictive locomotion. As in the case of locomotor-dependent group I excitation of extensor motoneurones (McCrea et al. 1995; Angel et al. 1996) our interpretation is that both group Ia and group Ib afferent fibres can activate the interneurones responsible for the group I excitation of flexor motoneurones during locomotion.

Expression of disynaptic EPSPs during locomotion evoked without MLR stimulation

Stimulation of the MLR produces oligosynaptic EPSPs in flexor and extensor motoneurones, probably through activation of at least one interposed spinal interneurone (Shefchyk & Jordan, 1985; Degtyarenko et al. 1998a). The possibility arises that the group I EPSPs reported during fictive locomotion require a facilitatory influence from the MLR for their expression. Thus the presence of disynaptic EPSPs was examined in one preparation in which fictive locomotion was produced by intravenous administration of clonidine and naloxone. Figure 4A shows, from left to right, ENGs from GS and Sart nerves and the low and high gain intracellular records from a PerL motoneurone during drug-induced fictive locomotion. Averaged PSP records show that stimulation of the PerL (Fig. 4B) and of the heteronymous EDL (Fig. 4C) nerves produced disynaptic EPSPs (latencies of 1.8 ms) that were mainly restricted to the late flexion (F3) phase. Disynaptic EPSPs were observed in six motoneurones (3 Sart, 2 EDL and 1 PerL). Thus the expression of the disynaptic excitation in flexor motoneurones during locomotion does not require MLR stimulation.

Locomotor modulation of group I disynaptic excitation in motoneurones innervating bifunctional muscles

Motoneurones innervating bifunctional muscles and exhibiting depolarization during both phases of fictive locomotion, usually in the phase transitions of the step cycle, were considered as bifunctional motoneurones (see Perret & Cabelguen, 1980). Figure 5A shows the membrane potential during fictive locomotion of a motoneurone innervating either the PerT or the PerB muscle. This motoneurone was rhythmically depolarized during the transition from flexion to extension and during the transition from extension to flexion (lower traces, Fig. 5A and B). The normalized step cycle (upper traces, Fig. 5B) was based on 87 LGS step cycles. As in the case of flexor motoneurones, group I disynaptic EPSPs were absent at rest (dotted lines in Fig. 5C and D). Figure 5C shows large disynaptic EPSPs (latency 1.8 ms) evoked by stimulation of the PerT + B nerves during both the early extension (E1) and late flexion (F3) phases of locomotion. Disynaptic EPSP modulation within the step cycle is also shown in the histogram in Fig. 5B. Stimulation of the MG nerve at 2T produced disynaptic EPSPs during phase E2 (latency of 1.4 ms) and IPSPs during E1 and F3. The difference in the modulation of the PSPs evoked by MG and PerT + B stimulation (recorded in the same trial) is also seen in the histograms in Fig. 5B. PerT + B stimulation-evoked disynaptic EPSPs were largest in the same phase in which MG stimulation evoked inhibition. Another PerT + B motoneurone showed a similar modulation of homonymous and MG PSPs. PerT + B motoneurones also received group I disynaptic EPSPs from flexor group I afferents, 2/3 from EDL and 1/2 from TA (see Table 2). These disynaptic EPSPs were expressed in both phases but were largest during flexion.

Table 2.

Motoneurones innervating bifunctional muscles receiving group I disynaptic EPSPs

During flexion and extension At rest
A. Motoneurones n PerT+B EDB MG EDL TA PerL Pl From ≥1 nerve
PerT+B 8 8/8 0/1 2/2 2/3 1/2 0/1 0/2 1/6
EDB 2 2/2 2/2 — 0/1 0/1 — 0/1 —
B. Motoneurones n Quad Vasti RF Sart LGS Pl St From ≥1 nerve
Quad 3 3/3 — — — — 0/2 — —
Vasti 2 — 2/2 0/1 0/1 0/1 0/1 — 0/2
RF 3 — 1/3 2/3 1/2 — 0/1 0/1 1/2
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Disynaptic EPSPs in 18/28 motoneurones (64%); 26/47 nerves tested. Mean latencies were 1.60 ± 0.16 ms (minimum 1.3 ms; maximum 1.9 ms). Disynaptic EPSPs in 2/10 motoneurones (20%); 4/17 nerves tested. Pl, plantaris; Quad, quadriceps: Vasti and RF were mounted together; LGS, lateral gastrocnemius and soleus; for other abbreviations, see Table 1.

Figures 2 and 3 show an increase in disynaptic EPSP amplitude as the motoneurone becomes more depolarized during the step cycle. This could result from either a cycle-dependent modulation of the excitability of interneurones responsible for disynaptic excitation or a voltage-dependent facilitation of the EPSP. The example in Fig. 5 shows a reduction in disynaptic EPSP amplitude in late extension (E3) as the motoneurone was further depolarized. Similar observations of the separation of motoneurone depolarization and EPSP amplitude have been reported for locomotor-related group I EPSPs evoked from extensor nerves (Angel et al. 1996).

Figure 6 provides an example with a different EPSP modulation in a motoneurone innervating the bifunctional muscle EDB. As illustrated in Fig. 6A and B (lower traces), this motoneurone slowly depolarizes during flexion, becoming maximally depolarized during early extension. Averaged traces and amplitude histograms (based on 21 normalized step cycles) of the effects of 1.3T stimulation of the homonymous (EDB) nerve are illustrated in Fig. 6C and the top histogram in B, respectively. EDB stimulation produces group I disynaptic EPSPs (minimum latency 1.7 ms) in all parts of the step cycle with the largest effects during E1 and the smallest during E2. An additional, probably trisynaptic, component appears during early flexion (arrow, E1 in Fig. 6C). EDL stimulation (Fig. 6D and middle histogram in B) produces disynaptic IPSPs throughout flexion but excitation during E1. The minimum latency of these EPSPs is 2.0 ms. Stimulation of the PerT + B nerve evokes disynaptic EPSPs (minimum latency of 1.7 ms) that are smaller during late extension (Fig. 6E and lower histogram in B). Comparison of the histograms in Fig. 6 illustrates the independent modulation of the patterns of disynaptic EPSPs evoked from different nerves recorded at the same time.

Table 2 summarizes results from bifunctional motoneurones. During fictive locomotion stimulation of 47 nerves resulted in 26 EPSPs recorded in 18/28 motoneurones (64 %). The average latency was 1.60 ± 0.16 ms (range 1.3-2.0 ms; n= 22) with 20 observations ≤ 1.8 ms. As illustrated in Figs 5 and 6, disynaptic EPSP amplitude modulation throughout the locomotor cycle was complex but EPSPs were often larger during the transitional phases between flexion and extension. As in the case of flexor motoneurones, the muscle origin of the group I afferents evoking disynaptic EPSPs in bifunctional motoneurones was mainly from homonymous and close synergists.

DISCUSSION

The principal finding of this study is that the majority of hindlimb motoneurones innervating flexor and bifunctional muscles are subject to group I-evoked disynaptic reflex excitation during locomotion. While occurring in both phases, disynaptic EPSPs are usually larger during flexion and are infrequent in the absence of locomotion. While not all combinations of nerves and motoneurones were investigated for the presence of group I disynaptic excitation, these results extend the demonstration of disynaptic excitation of EDL motoneurones (Degtyarenko et al. 1998b) to flexor motoneurones innervating the hip, knee and ankle and to bifunctional motoneurones (Tables 1 and 2). The sources of this locomotor-dependent excitation are both primary muscle spindle and tendon organ afferents from homonymous or close synergist muscle nerves. An assessment of the relative contribution of group Ia or Ib afferents to disynaptic excitation, however, must await further study since only the effectiveness of EDL Ia afferents was tested.

Combining observations from flexor and bifunctional motoneurones, the mean latency of the locomotor-dependent EPSPs was 1.64 ms, with the majority evoked at < 1.8 ms. Since the latencies of monosynaptic Ia EPSPs are 0.6-1 ms, the 1.6 ms latency indicates a disynaptic pathway between group I afferents and flexor or bifunctional motoneurones (e.g. Jankowska et al. 1981a; Degtyarenko et al. 1998b). Whether the EPSPs with slightly longer (1.8-2.0 ms) latencies are also evoked disynaptically remains to be determined. Figure 1 shows fluctuations in EPSP latency with the larger amplitude EPSPs having shorter latencies. Presumably such fluctuations reflect changes in the excitability of the interposed interneurones and it has been argued (Degtyarenko et al. 1998b) that latencies of < 2.2 ms may also reflect transmission through a disynaptic pathway. The possibility that some EPSPs were evoked trisynaptically or that an additional trisynaptic excitation contributed to the disynaptic depolarization cannot, however, be ruled out.

Ventral roots were left intact in these experiments in order to monitor fictive locomotor activity in the peripheral nerves. This necessitated reducing peripheral nerve stimulation intensities to below the threshold for antidromic activation of the impaled motoneurone in order to minimize contamination of the group I-evoked EPSPs by antidromic M spikes and recurrent inhibition. Thus the maximum amplitude of disynaptic excitation was not determined in most motoneurones. Despite this limitation, some disynaptic EPSPs were as large as 4.6 mV (Fig. 2A). Furthermore in some portions of the step cycle, disynaptic EPSPs were larger than the monosynaptic EPSP recorded in the same motoneurone (e.g. Figs 2A, 3A and 4). This indicates that the disynaptic excitation of flexor and bifunctional motoneurones may be a significant source of reflex excitation of motoneurones during locomotion.

As outlined in the Introduction, the locomotor-dependent disynaptic excitation of hindlimb extensor motoneurones is well documented. While not presented in Results, the presence of disynaptic EPSPs in extensors was seen in the same preparations in which the disynaptic excitation of flexors and bifunctional motoneurones was found. Thus during fictive locomotion in decerebrate cats, all of the motoneurone pools that have been examined are subject to a disynaptic excitation upon stimulation of group I afferents in homonymous or close synergist muscle nerves. The demonstration in the cat of disynaptic group I excitation in flexors (present results) and extensors (Angel et al. 1996) during fictive locomotion without MLR stimulation as well as short latency group I excitation during walking in man (Stephens & Yang, 1996) suggests that it is the activity of the locomotor circuitry and not the MLR stimulation per se that permits expression of group I disynaptic EPSPs.

Disynaptic reflex excitation from low threshold muscle afferents is also a feature during other mammalian motor tasks. For example, during inspiration muscle spindle afferents from external intercostal muscles can evoke disynaptic EPSPs that are absent during expiration (Kirwood & Sears, 1982). Activation of group I afferents also produces disynaptic excitation in extensor and flexor motoneurones during fictive scratching (Degtyarenko et al. 1998b; Perreault et al. 1999a) and fictive weight support (Perreault et al. 1999a). During mastication, stimulation of low threshold orofacial sensory inputs increases the activity of masseter motoneurones, presumably through disynaptic excitatory pathways (Westberg et al. 1998). It would thus appear that, in the cat, sensory evoked disynaptic excitation from muscle afferents can play a role in the reinforcement of ongoing motoneurone activity during respiration, mastication, scratching and locomotion.

Group I disynaptic EPSPs from homonymous and synergist muscle nerve afferents are not expressed on flexor motoneurones in anaesthetized and decerebrate preparations at rest

The ability of extensor group I afferents to evoke oligosynaptic excitation of flexor and bifunctional (mainly PB or St) motoneurones is well documented in non-locomoting preparations (Eccles et al. 1957b; Jankowska et al. 1981b; Harrison et al. 1983). The ability of flexor afferents to do the same has not been thoroughly investigated. As reported here, there is little disynaptic excitation of flexor and bifunctional motoneurones in the absence of fictive locomotion in decerebrate, spinal cord intact preparations. The extent to which disynaptic excitation evoked under resting conditions is mediated by the same interneurones during locomotion remains unknown. EPSPs were readily evoked during fictive locomotion and often in both phases of the step cycle. Mechanisms that could account for the emergence of disynaptic excitation during locomotion include: (1) a voltage-dependent excitation that is only seen as the motoneurone is depolarized, (2) an increase in synaptic transmission from primary afferents to interneurones during locomotion, (3) an excitatory input from the locomotor circuitry to the interposed interneurones during locomotion, and (4) a release of the interneurones from tonic inhibition.

In many cases the amplitude of the disynaptic EPSPs in flexor motoneurones increased with the depolarization of the motoneurone in which it was recorded (e.g. EDL-evoked EPSPs in Fig. 2). This could arise from a voltage dependency of the depolarization that would be seen as an apparent lack of excitation during control conditions and in those parts of the step cycle without motoneurone depolarization. Records from bifunctional and some flexor motoneurones suggest that this is an unlikely explanation for the modulation of disynaptic EPSP amplitude. EPSPs were also present during the extension phase when many motoneurones were most hyperpolarized or minimally depolarized (Figs 2, 5 and 6). These observations are contrary to the suggestions (Burke, 1999) that the amplitude of disynaptic EPSPs in flexors during fictive locomotion increases in parallel with the depolarization of the motoneurone. Stimulation of extensor group I afferents evokes not only a disynaptic but also a longer latency excitation of extensor motoneurones (Gossard et al. 1994). The longer latency disynaptic excitation of extensors is voltage dependent and enhanced by motoneurone depolarization (Brownstone et al. 1994; McCrea et al. 1997), while the disynaptic excitation appears not to vary with motoneurone membrane potential (Angel et al. 1996). The expression of disynaptic excitation of flexors and bifunctional motoneurones at both depolarized and hyperpolarized motoneurone membrane potentials is thus similar to the disynaptic excitation of extensors.

An increase of transmitter release during locomotion from primary afferents to the interneurones responsible for disynaptic excitation also seems an unlikely explanation of the emergence of disynaptic excitation during locomotion. On the contrary, available evidence points to a decrease in synaptic transmission from group I afferents during fictive locomotion. This evidence includes the depression of monosynaptic group I field potentials recorded in both the ventral horn (Gosgnach et al. 2000) and intermediate laminae (Perreault et al. 1999b) as well as the increase in group I afferent fibre excitability (Dueñas & Rudomin, 1988) and the rhythmic depolarization of group I afferents (Gossard et al. 1991; Gossard, 1996) during fictive locomotion. Furthermore, monosynaptic group Ia EPSPs recorded in the same motoneurones in which disynaptic EPSPs are expressed are often depressed during fictive locomotion (e.g. Fig. 2; data presented in Gosgnach et al. 2000). This depression most probably results from a presynaptic inhibitory process (Gosgnach et al. 2000). While a differential presynaptic regulation of a subset of group I terminals remains a possibility (Quevedo et al. 1997), it seems more likely that the emergence of the disynaptic excitation of flexors and bifunctional motoneurones during locomotion results from an increase in excitability of specific sets of interneurones. We are in complete agreement with the notion (Burke, 1999) that disynaptic EPSP modulation results from cyclic changes in the excitability of spinal interneurones.

If the emergence of disynaptic excitation results from changes in the excitability of interneurones then the question is whether there is a tonic inhibition of the interneurones in the absence of locomotion or whether a strong excitatory input from the locomotor circuitry is a prerequisite for the recruitment of interneurones by group I afferents. The present experiments cannot directly address this issue but the complex patterns of EPSP modulation during locomotion indicate a complex regulation of interneurone excitability. The phase-dependent expression of disynaptic EPSPs and the depression of monosynaptic EPSPs during locomotion offers the opportunity for a rich regulation of group I-evoked reflexes during locomotion.

Interneuronal organization of the disynaptic excitation of motoneurones during locomotion

Figure 7 presents a schematic diagram of the possible organization of interneurones mediating the disynaptic excitation of extensor (E), flexor (F) and bifunctional (Bi) motoneurones (indicated by open diamonds) during fictive locomotion. Neither non-reciprocal group I reflex pathways operating in the absence of locomotion, nor the suppression of the group I non-reciprocal inhibition of extensors during locomotion (Gossard et al. 1994; McCrea et al. 1995; Angel et al. 1996) are included. During locomotion there is an emergence of group I-evoked disynaptic excitation of motoneurones. This is depicted in Fig. 7 by a disinhibition; an inhibition of inhibitory interneurones (filled circles) projecting to the excitatory interneurones interposed in group I reflex pathways (open circles labelled 1 and 2). Such disinhibition offers a reasonable explanation of the failure to evoke disynaptic excitation without locomotion but has not been demonstrated. During locomotion there is an excitation from the central pattern generator for locomotion to the excitatory interneurone populations that allows a phasic expression of disynaptic EPSPs. Because disynaptic group I EPSPs are evoked in extensor motoneurones only during extension (McCrea et al. 1995; Angel et al. 1996), only the extensor portion of the spinal locomotor circuitry contacts interneurone population 1. The present results show that flexor group I afferents evoke a disynaptic excitation of flexors that is larger during the flexion phase of fictive locomotion (see also Degtyarenko et al. 1998b). The weaker excitation of interneurone population 2 from the extensor portion of the locomotor circuitry is shown as a dotted line. Figure 7 shows several differences in the organization of group I disynaptic excitation of flexor and extensor motoneurones. The two interneurone populations differ in their projections, the afferents from which they receive excitation and their regulation during locomotion. It remains to be determined if the disynaptic excitation of bifunctional motoneurones results from activity in both populations of interneurones, as shown, or from activation of a third population of interneurones. Finally, ankle extensor afferents frequently evoke disynaptic excitation of motoneurones operating at other joints while this was uncommon for flexor-evoked disynaptic excitation.

Figure 7. Scheme of interneuronal pathways mediating group I disynaptic excitation in extensor, flexor and bifunctional motoneurones during fictive locomotion.

Figure 7

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The spinal locomotor circuitry is illustrated by mutually inhibiting extensor (E) and flexor (F) half-centres. For simplification pathways mediating non-reciprocal group I reflexes and Ia reciprocal inhibition are not illustrated. Disynaptic excitation in extensor, flexor and bifunctional motoneurones is mediated by at least two populations of interneurones (1 and 2). Interneurones of population 1 are monosynaptically activated by extensor group I afferents and interneurones of population 2 by flexor and bifunctional group I afferents. Both sets of interneurones are subject to an unknown tonic inhibitory mechanism operating at rest (represented by the filled circles). Activity of population 1 interneurones is regulated by the extensor locomotor circuitry, and activity of population 2 interneurones is regulated by both the flexor and the extensor circuitry. It is assumed that the extensor and flexor locomotor half-centres exert this control by cyclic excitatory inputs and, perhaps, by a reduction of tonic inhibition of the interneurones. See text for details.

At present there is no information available about the nature or location of interneurones producing disynaptic excitation of flexor or bifunctional motoneurones. By examining the sources and targets of disynaptic excitation and the phasic modulation of these EPSPs, this study is the first step towards finding the responsible interneurones. Recent work (McCrea, 1998) suggests that those exciting extensors (population 1) are located in intermediate laminae and close to the motoneurones to which they project.

Putative functional role of the group I disynaptic excitation of flexor and bifunctional motoneurones during locomotion

The importance of feedback from extensor group I afferents in enhancing extensor motoneurone activity during locomotion is well described. For example, a recent estimate suggests that 50–70 % of the ankle extensor force generation during treadmill locomotion in the cat results from excitatory group I reflexes (Hiebert & Pearson, 1999). Similar results have been obtained in man (Stephens & Yang, 1999). The disynaptic excitation of extensors is probably an important component of this reflex excitation (McCrea et al. 1995; Angel et al. 1996). The present demonstration of the disynaptic excitation of most flexor and bifunctional motoneurones suggests that it may also be an important feature of their reflex regulation during locomotion.

Hip, knee and ankle extensor muscle activity during locomotion begins and ends at similar times during locomotion in a variety of locomotor behaviours (Abraham & Loeb, 1985; Buford & Smith, 1990; Perell et al. 1993; Pratt et al. 1996; Trank & Smith, 1996; Carlson-Kuhta et al. 1998; Smith et al. 1998). A consistent finding concerning the disynaptic group I excitation of extensors has been the failure to evoke EPSPs during the flexion phase of locomotion (Schomburg & Behrends, 1978; McCrea et al. 1995; Angel et al. 1996). Thus there is a gating of the disynaptic excitation of extensors so that reflex excitation is only permitted when the motoneurones are depolarized (i.e. during extension). This makes functional sense since increased muscle stretch or tension during stance would reinforce the antigravity actions of extensors and help maintain weight support. The functional importance of the group I-evoked reinforcement of flexor activity is not as readily apparent. Because flexor muscles should be relatively unloaded during normal forward locomotion, there should be little need for afferent feedback to reinforce their activity. Furthermore, why can flexor motoneurones receive disynaptic EPSPs during both the flexion and extension phases?

In contrast to the extensors, the timing of the activity of flexor and bifunctional muscles seems to depend on the particular type of locomotion (Abraham & Loeb, 1985; Buford & Smith, 1990; Perell et al. 1993; Pratt et al. 1996; Trank & Smith, 1996; Carlson-Kuhta et al. 1998; Smith et al. 1998). For example in cats walking downslope, flexor EMG activity at the hip (iliopsoas), knee (St) and ankle (TA, EDL) begins during late extension and increases throughout flexion (Smith et al. 1998). Because group I disynaptic EPSPs produced by stimulation of the EDL nerve, like monosynaptic EPSPs (see also Eccles et al. 1957a), were the most frequent source of disynaptic excitation of ankle flexor motoneurones (Figs 1, 2A, 3A and B, 4C, 6D and Table 1) we will consider EDL for discussion of the potential significance of the disynaptic excitation of flexors during locomotion.

We propose that group I disynaptic reflex pathways are available during both flexion and extension to support ongoing locomotor activity in flexor and bifunctional muscles according to the demands of the particular locomotor behaviour. In awake, unrestrained cats EDL activity is delayed from the onset of flexion and can overlap with extension (Engberg & Lundberg, 1969; Abraham & Loeb, 1985; Trank & Smith, 1996). During backward locomotion EDL activity overlaps even more with extensor activity (Trank et al. 1996). As the EDL muscle has a flexor action on the ankle and an extensor action at the toes, the reinforcement of ongoing activity through disynaptic pathways may be very relevant during digitigrade locomotion. Reflex reinforcement of EDL activity during the early stance would assure smooth stepping by producing an increased extension (dorsiflexion) of the toes. Consider also the situation when the hindlimb becomes stuck or is grabbed by a predator during the extension phase of locomotion. Maintaining the excitability of the group I-activated interneurones during extension would permit increases in flexor muscle length or tendon force during extension to produce useful co-contractions of flexors and extensors. Thus the ability to evoke disynaptic excitation of EDL motoneurones during both phases of the step cycle may be a reflection of the variable activity of EDL during real locomotion. Enhancement of EDL activity also seems to be relevant during scratching since group I disynaptic EPSPs in EDL motoneurones are about twice as large during fictive scratching as during fictive locomotion (Degtyarenko et al. 1998b).

In view of the powerful enhancement of flexion produced by stretching the hip flexor muscles inserting on the lesser trochanter (psoas major and iliacus; Hiebert et al. 1996), it would have been interesting to examine the presence of disynaptic EPSPs in motoneurones innervating this muscle. Unfortunately, electrical stimulation of the nerves to psoas major and iliacus was not attempted. However, all motoneurones of the other hip flexor studied, Sart, showed group I-evoked disynaptic EPSPs upon stimulation of the homonymous nerve (Fig. 3C and Table 1). In half of the Sart motoneurones group I-evoked disynaptic EPSPs were also evoked during extension. While we have observed prominent Sart ENG activity only during the flexion phase of fictive locomotion, during treadmill locomotion, the anterior Sart muscle displays activity both during flexion and extension (Hoffer et al. 1987).

In many fictive locomotor preparations we have also observed different patterns of ENG activity in some flexor and bifunctional nerves (e.g. PB, St and EDL). For example there is a delay in the onset of EDL activity during flexion and an overlap with extension. The fact that fictive locomotor preparations are able to display locomotor patterns more complicated than a simple alteration of flexion and extension suggests that these patterns of activity are intrinsic to the spinal locomotor circuitry and shows that the central drive to motoneurones can be quite detailed (Grillner & Zangger, 1975). Perhaps then, the presence of disynaptic EPSPs in flexor and bifunctional motoneurones during both phases of fictive locomotion reflects both the more complex flexor and bifunctional motoneurone activity and, in the absence of detailed sensory feedback and descending control, only a crude regulation of these pathways.

Compared to the organization of group I reflexes to hindlimb extensor motoneurones, much less is known of the organization of group I reflex pathways from flexor afferents to flexor and bifunctional motoneurones. The present results show that most of these motoneurones are also subject to a powerful group I disynaptic excitation and stress the need for more studies on the interneurones responsible for these actions.

Acknowledgments

This study was supported by the Medical Research Council of Canada. The skilful assistance of Sharon McCartney is gratefully appreciated. We also thank Professor Susan Shefchyk for her valuable comments on the manuscript. Brent Fedirchuk was supported by the Manitoba Division of the Canadian Paraplegic Association. Jorge Quevedo was supported by the Rick Hansen Man in Motion Legacy Fund and by the Manitoba Neurotrauma Initiative.

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