Glycinergic Inhibition Contributes to the Generation of Rostral Scratch Motor Patterns in the Turtle Spinal Cord

Effects of strychnine and glycine on the burst frequency of fictive rostral scratch motor patterns during unilateral stimulation

Superfusion of strychnine over spinal segments D7–D8 or D7–D10 (in and near the anterior hindlimb enlargement) increased the burst frequency of fictive rostral scratch motor patterns, elicited by electrical or mechanical stimulation of the rostral scratch receptive field on either the right or left side. Experiments were carried out in eleven “D3–end” preparations, two “D3–D10” preparations, and one “D3–D9” preparation. D3–end preparations had intact spinal cords posterior to the D2–D3 transection site; D3–D10 and D3–D9 preparations had complete spinal transections within the drug-superfusion region, at the posterior end of segment D10 or D9, respectively. In the experiment illustrated in Figure 2, we electrically stimulated a site in the left rostral scratch receptive field of a D3–end preparation. Superfusion of 50 μmstrychnine over the D7–D10 segments increased the frequency of HF ENG bursts during the period of stimulation and decreased burst durations (Fig. 2B), compared with control (Fig.2A). These effects were reversed after the spinal cord was washed with normal physiological saline (Fig.2C).

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Fig. 2.

Strychnine increased the burst frequency of fictive rostral scratch motor patterns and increased the amplitude of contralateral motor output during unilateral stimulation of a site (SP2.5) in the left rostral scratch receptive field. Motor output was recorded bilaterally from hindlimb muscle nerves in Experiment 10, a D3–end preparation. D3–end preparations had intact spinal cords posterior to the D2–D3 transection site. Stimulation consisted of trains of 33 electrical pulses (10 V, 1 msec pulses) delivered at 320 msec interpulse intervals. Recordings were obtained from a knee extensor (KE), a hip flexor (HF), and a hip extensor (HE) muscle nerve. A, Control response obtained while spinal segments D7–D10 of the anterior hindlimb enlargement were bathed in normal saline. B, After 1.25 hr, superfusion of the exposed spinal segments with 50 μm strychnine. Note the appearance of right-side cHF bursts and the increased amplitude of right HE bursts compared with the control.  Arrows in B indicate the timing of cHF deletions: cycles that lacked cHF bursts.C, After 7 hr + 25 min wash with normal saline.

We quantified the effects of strychnine on HF burst frequencies in six D3–end preparations during rostral scratch motor patterns (Table 1). These preparations were chosen for analysis because they exhibited robust rostral scratch responses on both the right and left sides during electrical shell stimulation under control conditions (before strychnine application). In the present study, we defined a “trial” as a series of stimulations on one side (right or left) for each of the three conditions (control, strychnine, and wash) in a given experiment. In three D3–end preparations (Experiments 8, 9, and 11), left-side trials were not included in the analysis because of frequent “HE deletion” cycles after strychnine superfusion, making it impossible to identify clear HF burst onsets. HE deletions (formerly termed “B-phase deletions”) are scratch cycles that exhibit consecutive HF bursts without clear quiescent periods separating them and without corresponding HE bursts (Robertson et al., 1985; Stein et al., 1995). Table 1 shows that treatment with 50 μm strychnine produced a statistically significant increase in rostral scratch burst frequency in 7 of the 9 remaining trials, ranging from 118.2 to 194.7% relative to control averages. A comparable increase in burst frequency was also observed in D3–D10 and D3–D9 preparations during unilateral stimulation (data not shown). Two of these preparations (Experiments 12 and 14) exhibited a reversible increase in burst frequency without HE deletions or other signs of a breakdown in the motor pattern, even after ≥2.5 hr of strychnine superfusion. The remaining D3–D10 preparation (Experiment 13) exhibited an increased burst frequency along with frequent HE deletions. In addition, continued exposure to strychnine (≥2 hr) in this preparation caused the motor pattern to break down completely.

Our goal in these experiments was to investigate the effects of maximal blockade of spinal glycine receptors on rostral scratch motor patterns; therefore, we did not systematically test the effects of low strychnine concentrations. In our first several experiments, however, we tested a series of concentrations ranging from 5 to 50 μm. Concentrations of 5–20 μm had little or no visible effect on rostral scratch motor patterns, even after 1–2 hr superfusion. In contrast, 50 μm strychnine produced obvious effects in most preparations within 20 min. Previous work in our laboratory suggested that 50 μm strychnine, superfused over the turtle spinal cord in vivo, blocked glycine receptors but not GABA receptors (Currie and Lee, 1996). In this earlier study, pretreatment of the cord with 50 μmstrychnine did not prevent exogenous GABA (2 mm) from reducing the integrated ENG amplitudes of fictive flexion reflexes, but it did prevent exogenous glycine (2 or 5 mm) from reducing flexion reflex amplitudes. Strychnine concentrations of up to 50 μm were also found to significantly reduce glycine- but not GABA-induced depolarizations in motor neurons of hemisected neonatal rat spinal cords in vitro (Wu et al., 1992) (L. Ziskind-Conhaim, personal communication). Nevertheless, the low sensitivity of our preparations to strychnine calls for caution, because it is known that smaller concentrations (1–10 μm) are capable of blocking various ion channels in isolated cell preparations (Shapiro et al., 1974; Oyama et al., 1988) and in the exposed spinal cords of Xenopus embryos (Dale, 1995). The relatively high strychnine concentration required in our experiments may be caused by several factors, including (1) the diffusion barrier presented by the thickness of the spinal hindlimb enlargement (3–4 mm diameter), (2) the intact vascular circulation within the exposed spinal segments, and (3) the fact that only the dorsal surface of the cord was stripped of pial meninges and in direct contact with superfused drug solutions.

In contrast to strychnine, glycine superfusion over the anterior hindlimb enlargement reduced rostral scratch burst frequencies relative to controls. Although we did not routinely compare the effects of strychnine and glycine, in two D3–end preparations we found that glycine superfusion over the D7–D10 spinal segments reversibly lowered burst frequency. In these experiments, mechanical (Experiment 7) or electrical (Experiment 8) stimulation of the right SP2 site elicited control rostral scratch motor patterns with an average HF burst frequency of 0.48 Hz. The HF frequencies after glycine superfusion changed as follows (percentage of control average ± SD): Experiment 7: (control) 100.0 ± 8.8, (glycine, 5 mm) 38.7 ± 25.7, (wash) 90.1 ± 18.4; Experiment 8: (control) 100.0 ± 11.8, (glycine, 2 mm) 51.8 ± 13.5, (wash) 103.5 ± 9.9 (five to seven episodes in each condition). Figure 3 shows the effect of glycine on fictive rostral scratch motor patterns in Experiment 8, which was reversed after the spinal cord was washed with normal physiological saline. Glycine effects and recoveries after washing were significant in both experiments at p < 0.005, using the Mann–WhitneyU test.

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Fig. 3.

Glycine decreased the frequency and amplitude of rostral scratch ENG bursts. Motor output was recorded from the KE, HF, and HE nerves on the right side in Experiment 8, a D3–end preparation. Stimulation consisted of trains of 33 electrical pulses (10 V, 1 msec pulses) delivered at 320 msec interpulse intervals to a site (SP2.5) in the right rostral scratch receptive field. A, Control response obtained while spinal segments D7–D10 were bathed in normal saline. B, After 35 min superfusion of the exposed spinal segments with 2 mm glycine. C, After 5 hr + 45 min wash with normal saline.

Effects of strychnine on the amplitude of contralateral ENG bursts during unilateral stimulation

Strychnine increased the amplitude of contralateral motor output during unilaterally evoked fictive rostral scratch motor patterns. During control responses, we observed low-amplitude ENG activity in contralateral hindlimb nerves alternating with ipsilateral ENG bursts, similar to what has been described previously for the rostral and pocket scratch in turtles (Currie and Stein, 1989; Berkowitz and Stein, 1994a,b; Stein et al., 1995). Figure 2A shows a typical example of contralateral motor output before strychnine. Stimulation of a site in the left rostral scratch receptive field elicited rostral scratch motor output in all three of the left (ipsilateral) hindlimb nerves and weak rhythmic discharge in the right (contralateral) HE nerve that alternated with left HE bursts. No activity was observed in the right HF or KE nerves. After strychnine superfusion over spinal segments D7–D10, the amplitudes of right HE bursts were greatly increased and rhythmic bursting appeared de novo in the right HF nerve (Fig. 2B). These effects were reversed after the cord was washed for several hours with normal saline (Fig. 2C). Note the appearance of three “contralateral HF (cHF) deletion” cycles during strychnine treatment in Figure 2B. We define cHF deletions (not described previously) as scratch cycles that exhibit consecutive HE bursts contralateral to the stimulus, without clear quiescent periods separating them and without associated contralateral HF bursts. It is particularly interesting that these cHF deletions occurred without corresponding ipsilateral HE deletions. This suggests a dissociation of the network components that activate ipsilateral HE and contralateral HF motor pools, which otherwise appeared to be intimately coupled. The remaining cycles in Figure 2B exhibited 1:1 alternation between right and left HE discharge and between right and left HF discharge. We observed cHF deletions in this one experiment only; other preparations consistently exhibited 1:1 alternation of right and left hip agonists after strychnine treatment.

In all 11 D3–end preparations, strychnine produced either a de novo appearance (n = 5) or increased amplitude (n = 6) of contralateral HF discharge during unilateral stimulation. The increased amplitude of contralateral HE activity was much less reliable, however, being obvious in only two preparations (both with a D7–D10 strychnine superfusion). A possible explanation for this result may be that HF motor neurons have cell bodies located in spinal segments D8 and D9, entirely within the D7–D10 drug-soak region (n = 3) and partly within the D7–D8 drug-soak region (n = 8). In contrast, the cell bodies of HE motor neurons are located mainly in segments D10–S2 (Ruigrok and Crowe, 1984), largely outside of even the D7–D10 drug-soak region.

Strychnine-induced paroxysmal (seizure) activity

All of the turtles in this study exhibited some episodic seizure-like motor discharge after prolonged application (>60 min) of strychnine to the spinal hindlimb enlargement. Although this activity was generally weak and intermittent, it displayed a tendency to increase in frequency and amplitude with prolonged strychnine exposure. In no case did it become so frequent and intense that it interfered with the observation of fictive scratch responses. The shape, amplitude, and frequency of seizure bursts varied considerably across and within experiments, but always began with activity in the right or left HF motor pool. Figure 4 shows an episode of several seizure bursts in one animal. Weak discharges were often confined to the HF motor pool on one side (e.g., Fig. 4A, bursts1 and 2), whereas stronger activity spread from the HF to other motor pools on the same side (data not shown), and the most intense discharges were bilateral, spreading rapidly from one side to the other in either direction (Fig. 4A, bursts3 and 4, B,C). A few preparations (n = 3) exhibited rhythmic, mainly bilateral discharges after >2 hr of strychnine exposure; this activity superficially resembled the strychnine-induced bilateral motor activity that has been observed in neonatal mouse (Droge and Tao, 1993) and rat (Cowley and Schmidt, 1995) spinal cords in vitro.

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Fig. 4.

Seizure discharges observed after prolonged exposure to strychnine. Bilateral recordings were obtained from the KE, HF, and HE nerves after spinal segments D7–D10 were superfused with 50 μm strychnine for 68 min in Experiment 6.A, Seizure bursts (unfilled triangles, numbered 1–4). B, Time-expansion of burst 3 from A, showing left-to-right spread of motor activity. C, Time-expansion of burst 4 fromA, showing right-to-left spread of motor activity.

Effect of strychnine on the shape of rostral scratch HF bursts

The shape of the observed HF ENG bursts during the fictive rostral scratch was typically fusiform (i.e., spindle-shaped) and similar to results that have been described previously (Robertson and Stein, 1988). At the beginning of the HF burst, only motor neurons with small extracellular action potentials were recruited and there was low-amplitude ENG activity. Motor neurons with medium-sized extracellular action potentials were recruited later in the burst, with the largest HF motor neurons recruited just after 50% of the burst duration. At the end of the burst, there was an orderly derecruitment of motor neurons, with the smallest motor neurons shutting off last. In the present experiments, we found that strychnine treatment caused the onsets of rostral scratch HF bursts to become more abrupt in some preparations. This effect is illustrated in Figure5A,B, which shows raw and processed recordings of rostral scratch motor output from the ipsilateral HF nerve in Experiment 1, before and after strychnine was applied to spinal segments D7–D8. In Figure 5C, we superimposed the second bursts from Figure 5, A and B, and normalized them to the same duration to show that HF motor neurons with medium-to-large extracellular spikes were recruited earlier in the burst after strychnine treatment. Similar results were obtained when other bursts from this experiment or other experiments were compared before and after strychnine (data not shown). These results suggest that the onsets of HF ENG bursts became more abrupt after strychnine, because larger units were recruited earlier in the burst. This result is consistent with intracellular recordings, which showed that some HF motor neurons receive synaptic inhibition during the early part of the HF ENG burst (on-cycle inhibition) that overrides an underlying excitatory drive and delays their firing onsets (Robertson and Stein, 1988). Our data suggest that this on-cycle inhibition is at least partly glycinergic.

Figure 5D shows double-normalized averages of the rectified–integrated HF ENG bursts from the scratch episodes shown inA and B during the period of stimulation. Strychnine increased the average slope of integrated bursts over the initial 25–30% of burst duration. We used double-normalized slopes to quantify changes in the shape of ENG bursts so that the results would be independent of burst duration and amplitude. In Table 2, we quantified changes in slope for six different preparations (same trials used in Table 1) by calculating the average double-normalized linear regressions for the first 25% of burst duration in control, strychnine, and wash conditions (see Materials and Methods). Strychnine produced a statistically significant increase in the average slope for five of nine trials, ranging from 133.3 to 193.0% of control values.

Effects of strychnine on the timing of intralimb flexor and extensor bursts during unilateral stimulation

Fictive locomotion studies in mammalian preparations have shown that strychnine blockade of spinal glycine receptors can change the coordination between flexor and extensor ENG bursts within a limb from an alternating to a synchronized relationship (Kriellaars et al., 1988; Noga et al., 1993; Cowley and Schmidt , 1995). In the present study, most preparations (10 of 14) continued to express normal alternation between HF and HE ENG activity after strychnine treatment (up to 2.5 hr). Synchronous bursting of HF and HE nerves was never observed in any preparation; however, four preparations (three D3–end and one D3–D10) exhibited an increased number of HE deletion cycles after strychnine, which in one case progressed over time to a near-complete disruption of the motor pattern (Fig. 6). The D3–D10 preparation shown in Figure 6 had strychnine superfused over the terminal D7–D10 segments, which led to an increased burst frequency (Fig. 6A–C) and increased number of HE deletion cycles over the first 35 min of treatment (Fig.6C). By 50 min (Fig. 6D), the first 10 sec of the episode was mainly tonic discharge in the HF and KE nerves. After 2.25 hr (Fig. 6E), there was a near-total loss of rhythmicity, with tonic activity in all three nerves. Note that some organized cycles still developed near the end of the responses in Figure 6D,E, after periods of tonic or weakly modulated discharge. A time-expansion of this transition from tonic to rhythmic activity is illustrated in Figure 6F, showing that once rhythmicity developed, it still expressed the normal HF–HE alternation and the normal KE timing within the hip cycle (compare Fig. 6, A and F). Thus, in the present experiments, scratch rhythm andpattern generation seemed to be tightly linked and were not clearly separated via widespread blockade of glycinergic transmission.

Effects of strychnine on right-left coordination during bilateral stimulation

Simultaneous stimulation of sites in the right and left rostral scratch receptive fields produces bilateral rostral scratch motor patterns in which agonist motor pools (e.g., HFs) display alternating activity on the right and left sides (Stein et al., 1995). In the present study, we tested the hypothesis that glycinergic transmission was necessary to maintain an alternating coordination between right and left HF bursts during bilateral scratch motor patterns. Figure7 shows data from two D3–end preparations, two D3–D10 preparations, and one D3–D9 preparation. We used circular statistics (Batschelet, 1981) to analyze phase measurements. Strychnine produced a highly significant increase in the burst frequency of bilateral scratch motor patterns in all five experiments (Fig. 7A). The effect on interlimb coordination was less obvious, however, because the most consistent observation was an increased variability in the right-left phase relationship (Fig. 7B). This effect was apparent as larger angular deviations in the average onset and offset phase values of every experiment (left HF relative to right HF activity cycle). These increased deviations were largest in D3–D10 and D3–D9 preparations, in which the strychnine had improved access to all of the relevant motor circuitry. Despite the greater variability, however, right and left HF bursts continued to alternate in strychnine, even after >2 hr of superfusion. In fact, four of five experiments exhibited less overlap between the average left HF burstonset and right HF activity after application of strychnine, whereas three of five experiments also exhibited less overlap between left HF burst offsets and right HF activity.

Figure 8 further illustrates the persistence of right-left alternation in the presence of strychnine. In this D3–D10 preparation (same experiment as shown in Fig. 6), control unilateral stimulation of the right or left side evoked a normal rostral scratch motor pattern in the ipsilateral hindlimb nerves (Fig.8A1,B1). Unilateral stimulation of the right side also elicited weak bursting from the left (contralateral) HE nerve. Bilateral stimulation in the control elicited brisk rostral scratch motor output from both the right- and left-side hindlimb nerves (Fig.8C1), with alternating discharge in the right and left HF nerves (Fig. 8D1), similar to what has been described previously (Stein et al., 1995). After >2 hr of strychnine superfusion, unilateral stimulation on the right or left side elicited mostly tonic discharge (Fig. 8A2) or disorganized, choppy “packets” of activity (Fig. 8B2; also see Fig. 3 in Cowley and Schmidt, 1995) in ipsilateral and contralateral nerves. In contrast, bilateral stimulation in strychnine reestablished rostral scratch rhythmicity on both sides (Fig. 8C2). Bilateral responses still displayed strict alternation between right and left HF bursts (Fig. 8D2). Thus, spinal scratch circuits maintained an alternating right-left coordination even when unilaterally evoked motor patterns were completely disrupted by strychnine. This result provides further support for the hypothesis that alternating interlimb coordination during rostral scratch motor patterns is not critically dependent on glycinergic inhibition.