The upright posture improves plantar stepping and alters responses to serotonergic drugs in spinal rats

Abstract

Recent studies on the restoration of locomotion after spinal cord injury have employed robotic means of positioning rats above a treadmill such that the animals are held in an upright posture and engage in bipedal locomotor activity. However, the impact of the upright posture alone, which alters hindlimb loading, an important variable in locomotor control, has not been examined. Here we compared the locomotor capabilities of chronic spinal rats when placed in the horizontal and upright postures. Hindlimb locomotor movements induced by exteroceptive stimulation (tail pinching) were monitored with video and EMG recordings. We found that the upright posture alone significantly improved plantar stepping. Locomotor trials using anaesthesia of the paws and air stepping demonstrated that the cutaneous receptors of the paws are responsible for the improved plantar stepping observed when the animals are placed in the upright posture.We also tested the effectiveness of serotonergic drugs that facilitate locomotor activity in spinal rats in both the horizontal and upright postures. Quipazine and (±)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) improved locomotion in the horizontal posture but in the upright posture either interfered with or had no effect on plantar walking. Combined treatment with quipazine and 8-OH-DPAT at lower doses dramatically improved locomotor activity in both postures and mitigated the need to activate the locomotor CPG with exteroceptive stimulation. Our results suggest that afferent input from the paw facilitates the spinal CPG for locomotion. These potent effects of afferent input from the paw should be taken into account when interpreting the results obtained with rats in an upright posture and when designing interventions for restoration of locomotion after spinal cord injury.

Figure 1. EMG analysis of locomotor patterns of intact rats (n = 5) in horizontal and upright postures

A, plantar paw placement and weight supported stepping is evident in the video frames of the same animal in the two postures. B, well-coordinated EMG patterns in the soleus (Sol) and tibialis anterior (TA) muscles bilaterally are illustrated for the two postures. Rectified and filtered EMG records are normalized to the step cycle, taking the onset of activity in the right TA as the onset of the cycle and show left–right and flexor–extensor coordination over the same step cycles displayed in the raw EMG records. l, left; r, right. C, the relationships between the step cycle durations and burst durations for the left and right TA and Sol muscles. F, bar graph showing the slopes (±SD) of this relationship. The darker green bar is the mean slope from the horizontal posture trials in left soleus muscle (l Sol), while the lighter green bar represents the trials in the upright posture. Dark (horizontal) and light (upright) grey represent the slopes of right soleus muscle (r Sol), and dark and light blue and red represent left tibilalis anterior muscle (l TA) and right tibialis anterior muscle (r TA), respectively. D and E, polar plots showing the relationships between the onset of r TA activity and either the contralateral TA or the ipsilateral extensor (r Sol). The 0 position on the polar plot corresponds to the onset of activity in the right TA muscle, and the positions of the filled circles (orange) indicate the times of onset of activity in the left TA (interlimb coordination, D) or the onset of activity in the right Sol (intralimb coordination, E). The black bars in D demonstrate the average time of onset (angle, a) of activity in the contralateral l TA for each animal, and the length of each bar is a measure of the strength of the relationship (correlation, r) between the right TA and left TA times of onset. The positions of the red bars represent the mean angle and their lengths represent correlation. The polar plots in E show the same relationships for intralimb coordination, with the times of onset of ipsilateral Sol activity plotted in relation to the times of onset of the ipsilateral r TA. The bar graph in G demonstrates the means (±SD) of the angle of the left–right TA (l-r) and of the right Sol – right TA (r S-T) relationships (relative timing of the onsets for the two muscles represented in each polar plot in the two postures, a). The dark green bars represent the angle in the horizontal posture (a in H) and the light green represent the angles in the upright posture (a in U). The bar graph in H shows the means (±SD) of the correlation coefficient (r) between the times of onset of activity in both left–right and flexor–extensor EMG comparisons for the two postures. The black bars represent r in the horizontal posture (r in H) and the grey bars represent r in the upright posture (r in U). Student’t test: *P < 0.05, **P < 0.005, P < 0.0005.

Figure 2. Spinal rats placed in the upright posture (n = 6) display near-normal locomotor patterns

A, the same rat in the horizontal posture (upper panel) and the upright posture (lower panel) is depicted. C, the relationships between the step cycle durations and burst durations for the left and right TA and Sol muscles. F, bar graph showing that the slopes of this relationship differ significantly between the horizontal and upright posture trials (Student's paired t test). D and E, polar plots illustrating the interlimb (D) and intralimb (E) coordination changes that occur when the animals is placed in the upright posture (lower panel) compared to the horizontal posture (upper panel). G, bar graph showing the mean angle (a, ±SD) of polar plots for the left–right TA (l-r) and for the right Sol–right TA (r S-T) relationships. H, bar graph demonstrating the mean correlation coefficients (r, ±SD) for the left–right TA (l-r) and for the right Sol–right TA (r S-T) relationships, indicating a highly significant increase in r for inter- and intralimb coordination in the upright posture trials. Student’t test: *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 3. Changes in mean ± SD cycle duration, peak amplitude, burst duration and burst area established on the basis of EMG recordings from soleus (Sol) and tibialis anterior (TA) muscles of both hindlimbs during locomotion in the horizontal and upright postures in different experimental conditions

Each bar is the mean of 4–6 spinal rats and 5 experiments from intact rats taken from 10–30 consecutive steps in both hindlimbs during rhythmic movements in a particular experimental condition. The means in A are expressed as the percentage of that obtained during locomotor trials in the horizontal posture. The means in B are expressed as the percentage of the values obtained during trials in the upright posture. 100% represents the mean value obtained in the control situation for each analysed index (white bar). The black bar on the left represents results obtained during locomotor trials of intact rats in the upright posture in comparison to the horizontal posture (A) and in the horizontal posture in comparison to the upright posture (B). For example, in the case of the cycle duration index, the mean cycle duration of intact rats in the upright posture is longer than that obtained during locomotor trials in the horizontal posture. Similarly, the grey bars represent the means obtained in spinal rats, showing that the cycle duration increases significantly when going from the horizontal to the upright posture (A), and decreases when going from upright to the horizontal posture (B). The orange bar represents the means of each index for air-stepping. C and D, pharmacological treatments induce different effects on locomotor movements recorded in the horizontal and upright postures. The values after treatment are expressed as a percentage of the values measured from locomotor trials taken before the pharmacological treatment. Abbreviations: U, upright posture; H, horizontal posture; air step, air-stepping; Quip, quipazine (0.1–0.25 mg kg⁻¹i.p.); 8-OH, 8-OH-DPAT (0.1–0.4 mg kg⁻¹i.p.); Lido, lidocaine injections into the hindpaw bilaterally (0.05 ml, one medial and one lateral in each paw); BLD, both Quip and 8-OH-DPAT, low dose (0.1 mg kg⁻¹). Wilcoxon's non-parametric test: *P < 0.05, **P < 0.02, ***P < 0.01.

Figure 4. Removal of load receptor inputs during air stepping disorganizes the locomotor rhythm induced by tail pinching

A–D, hindlimb movement during air stepping (n = 6, lower panels) significantly differed from the hindlimb movement obtained during walking in the upright posture as illustrated in the EMG recordings (A) and in the relationship between the soleus EMG burst and cycle duration (B and E) as well as in the inter- and intralimb coordination (upper panels in C and D). F and G, bar graphs showing the mean angle of polar plots (a, ±SD) for the left–right TA (l-r) and for the right Sol–right TA (r S-T) relationships and the mean of correlation coefficients (r, ±SD) for the left–right TA (l-r) and for the right Sol–right TA (r S-T) relationships, indicating a highly significant decrease in r for inter- and intralimb coordination in air stepping trials. The remainder of this figure is as described in the legend to Fig. 1.

Figure 5. Anaesthesia of plantar surface of the paw eliminates plantar stepping produced by tail pinching in the upright posture

A–D, lidocaine (n = 4) disrupts locomotion in the upright posture (A–C), as shown in EMG recordings (A) and by the alteration in the burst duration–cycle duration relationship (B and E), as well as the disruption of inter- (C) and intra-limb (D) coordination (upper panels, pre-drug; lower panels, after lidocaine application). F and G, bar graphs showing the mean angle of polar plots (a, ±SD) for the left–right TA (l-r) and for the right Sol–right TA (r S-T) relationships and the mean of correlation coefficients (r, ±SD) for the left–right TA (l-r) and for the right Sol–right TA (r S-T) relationships, indicating a highly significant decrease in r for inter- and intralimb coordination in the upright posture trials after lidocaine application. The remainder of this figure is as described in the Fig. 1 legend.

Figure 6. Quipazine improves hindlimb movement induced by tail pinching of the rats in the horizontal posture

The individual frame in A was taken from the rhythmic movements observed with the animal in the horizontal posture after administration of quipazine (0.25 mg kg⁻¹i.p.). The pre-drug condition for this same animal is shown in Fig. 2A–E (upper panels in C, D and E). I–P, quipazine disrupts locomotion in the upright posture. The photograph in I shows a single frame taken from the rhythmic movements after administration of quipazine (0.25 mg kg⁻¹i.p.). The pre-drug situation for this same animal is shown in Fig. 2A–E (lower panels in G, D and E). The remainder of this figure is as described in the Fig. 1 legend.

Figure 7. 8-OH-DPAT improves hindlimb movement of the rats in the horizontal posture

8-OH-DPAT, a 5-HT₇/5-HT_1A agonist (0.4 mg kg⁻¹i.p.), improves locomotor coordination in chronic spinal rats (n = 5) induced to walk on a treadmill with tail pinching. A and B, the drug improves plantar paw placement (A), and produces a much more regular and well-coordinated EMG pattern in the soleus (Sol) and tibialis anterior (TA) muscles bilaterally (B). In B, rectified and filtered records are overlaid, based upon the onset of activity in r TA, showing the improved left–right and flexor–extensor coordination after the drug. C and F, the relationship between burst duration and step cycle duration (C) is also improved to be more like locomotion in intact animals, with significant changes in the slopes of the extensor muscle records (F). D and E, polar plots showing the relationships between the onset of r TA activity and either the contralateral TA or the ipsilateral extensor (r Sol). H, bar graph demonstrating significant increases in the correlation (r) between the times of onset of activity in both left–right and flexor–extensor comparisons. G, the angles (a) representing the relative timing of the onsets for the two muscles represented in each polar plot were not significantly changed. I–P, 8-OH-DPAT does not significantly alter locomotion induced by tail pinching when the rat is in the upright posture. Further details are as described in Fig. 1 legend.

Figure 8. Combined treatment with low doses of quipazine and 8-OH-DPAT optimizes spinal stepping and mitigates the need for exteroceptive stimulation

A and B, EMG activity during hindlimb movement induced by tail pinching in the same spinal rat is shown in the horizontal posture (A) and the upright posture (B) pre-drug (upper panel), after quipazine (Quip, middle panel) and after a subsequent dose of 8-OH-DPAT (Quip + 8-OH-DPAT, lower panel). C, consecutive frames of a video recording from a rat walking in the upright posture after quipazine (0.1 mg kg⁻¹, i.p.) and 8-OH-DPAT (0.1 mg kg⁻¹, i.p.) treatment, showing well-coordinated plantar stepping without tail stimulation.