Spinal cord-transected mice learn to step in response to quipazine treatment and robotic training

Abstract

In the present study, concurrent treatment with robotic step training and a serotonin agonist, quipazine, generated significant recovery of locomotor function in complete spinal cord-transected mice (T7-T9) that otherwise could not step. The extent of recovery achieved when these treatments were combined exceeded that obtained when either treatment was applied independently. We quantitatively analyzed the stepping characteristics of spinal mice after alternatively administering no training, manual training, robotic training, quipazine treatment, or a combination of robotic training with quipazine treatment, to examine the mechanisms by which training and quipazine treatment promote functional recovery. Using fast Fourier transform and principal components analysis, significant improvements in the step rhythm, step shape consistency, and number of weight-bearing steps were observed in robotically trained compared with manually trained or nontrained mice. In contrast, manual training had no effect on stepping performance, yielding no improvement compared with nontrained mice. Daily bolus quipazine treatment acutely improved the step shape consistency and number of steps executed by both robotically trained and nontrained mice, but these improvements did not persist after quipazine was withdrawn. At the dosage used (0.5 mg/kg body weight), quipazine appeared to facilitate, rather than directly generate, stepping, by enabling the spinal cord neural circuitry to process specific patterns of sensory information associated with weight-bearing stepping. Via this mechanism, quipazine treatment enhanced kinematically appropriate robotic training. When administered intermittently during an extended period of robotic training, quipazine revealed training-induced stepping improvements that were masked in the absence of the pharmacological treatment.

Figure 1.

Experimental timeline. The sequence of experiments conducted on the step trained (blue bars) and nontrained (red bars) groups are shown for experiments I–IV (A) and experiment V (C). Test dates are indicated by the vertical dashed lines. Yellow stripes denote the periods during which quipazine was administered as a single bolus daily. Time course plots of the aggregate stepping performance of the trained (blue line) and nontrained (red line) mice are shown for experiments I–IV (B) and experiment V (D). Aggregate stepping scores were determined as a qualitative weighting of the three stepping measurements used in the study (number of steps, step rhythm, and step shape consistency) and were normalized against the best stepping observed during each group of experiments (denoted as 100%). Number of steps performed was the predominant factor in determining the aggregate score. During the periods labeled as “Suspension,” the nontrained mice were placed in the harness with their hindlimbs unloaded for 15 min/d. This amount of unloading is extremely unlikely to have adversely affected stepping ability. Open circles, open squares, and filled triangles denote the time points used for examining the effects of manual training, quipazine treatment, and robotic training, respectively, in experiments I–III. It is important to note that the baseline scores against which these treatments were compared are time independent and represent the maximal performance that can be expected for nontrained, untreated mice. Open diamonds denote the time points compared in the longitudinal experiment IV. Double daggers denote the time point studied in the parallel experiment V.

Figure 2.

Step trajectories. Step trajectories of robotically trained mice showed consistent and rhythmic patterns. Trajectories recorded during the best 12 s periods of treadmill stepping of a representative nontrained mouse (A) and a representative robotically trained mouse (B) are shown. Ankle velocity is implicitly represented by the spacing of the data points. The rostral and caudal orientations of the mouse are noted, as well as the general regions of touchdown and toe-off.

Figure 3.

Fast Fourier transform analysis. FFT analysis provides information about step rhythm. Twelve seconds of horizontal ankle motion during stepping are shown for a nontrained mouse given quipazine that stepped arrhythmically (A) and for a trained mouse given quipazine that stepped rhythmically (B). The corresponding frequency spectrum and the FWHM of the peak at the primary stepping frequency for A and B are shown in C and D, respectively. Lower values of FWHM correspond to more rhythmic stepping. For successful spinal stepping on a treadmill at 3 cm/s, the peak corresponding to the primary stepping frequency lies between 0.4 and 1.4 Hz (D, crosshatched region). C_n represents the number of incidences during a test session that the mouse stepped at a particular frequency.

Figure 4.

Principal component analysis. The first principal component from a PCA identifies the most representative step pattern executed by a mouse. A mouse with a high PCA percentage for its first principal component stepped with a more consistent step shape than a mouse with a lower PCA percentage score. Plots of several x trajectories (dashed lines) and their corresponding first principal component (solid lines) are shown for a quipazine-treated nontrained mouse (A) and for a quipazine-treated trained mouse (B). The corresponding PCA percentages, 67.9% (A) and 97.6% (B), respectively, indicate that quipazine facilitated greater improvement in spatial stepping consistency in trained than nontrained mice. Note also from the different scales of the x-displacement (ordinate) axis that trained mice generally took much longer steps than nontrained mice.

Figure 5.

Progression of locomotor performance attributable to administration and withdrawal of quipazine during continued robotic training. After the initial period of robotic training, which ended at P79, the number of steps (A) and the step shape consistency (B) of the mice continued to improve when robotic training and quipazine treatment were used together (P91) and then decreased when quipazine was withdrawn (P105a). This suggests that the improved performance observed after the combination treatment was primarily mediated by quipazine and, hence, that the net performance was attributable to an interaction effect between quipazine and robotic training. Moreover, an additional bolus treatment with quipazine (P105b) immediately generated stepping that was significantly better than that exhibited at P91, despite the fact that quipazine had not been administered during the preceding 9 d. This finding suggests that the acute quipazine treatment improved stepping by facilitating effects of chronic robotic training that were masked in the absence of drug treatment. Unlike the number of steps and step shape consistency, the step rhythm (C) improved steadily throughout the course of robotic training, as depicted by the plot of inverse FFT score. This is consistent with the results of experiments II and III, which indicated that robotic training has a greater effect on step rhythm than quipazine. RT, Robotically trained; +Q, treated with quipazine; -Q, not treated with quipazine.