Adaptations in glutamate and glycine content within the lumbar spinal cord are associated with the generation of novel gait patterns in rats following neonatal spinal cord transection

Abstract

After spinal cord transection, the generation of stepping depends on neurotransmitter systems entirely contained within the local lumbar spinal cord. Glutamate and glycine likely play important roles, but surprisingly little is known about how the content of these two key neurotransmitters changes to achieve weight-bearing stepping after spinal cord injury. We studied the levels of glutamate and glycine in the lumbar spinal cord of spinally transected rats. Rats (n = 48) received spinal cord transection at 5 days of age, and 4 weeks later half were trained to step using a robotic treadmill system and the remaining half were untrained controls. Analyses of glutamate and glycine content via high-performance liquid chromatography showed training significantly raised the levels of both neurotransmitters in the lumbar spinal cord beyond normal. The levels of both neurotransmitters were significantly correlated with the ability to perform independent stepping during training. Glutamate and glycine levels were not significantly different between Untrained and Normal rats or between Trained and Untrained rats. There was a trend for higher expression of VGLUT1 and GLYT2 around motor neurons in Trained versus Untrained rats based on immunohistochemical analyses. Training improved the ability to generate stepping at a range of weight support levels, but normal stepping characteristics were not restored. These findings suggested that the remodeling of the lumbar spinal circuitry in Trained spinally transected rats involved adaptations in the glutamatergic and glycinergic neurotransmitter systems. These adaptations may contribute to the generation of novel gait patterns following complete spinal cord transection.

Figure 1.

Experimental design (see Materials and Methods). Trained and Untrained rats that received neonatal spinally cord transection were used in three experiments: (1) HPLC to measure glutamate and glycine levels; (2) immunohistochemical experiments for VGLUT1 and GLYT2 expression; and (3) hindlimb kinematic analyses. Asterisk (*) denotes that Normal rats were also included for the HPLC experiment (n = 4) and for the kinematic analyses experiment (n = 9).

Figure 2.

HPLC-measured glutamate and glycine levels in the lumbar spinal cord. A, Glutamate and glycine levels in Normal (gray), Untrained ST rats (white) and Trained ST rats (black). Asterisks ** and * indicate significant difference from Normal at p < 0.01 and p < 0.05 levels, respectively. B, C, The correlation of glutamate (B) and glycine (C) levels in the lumbar spinal cord and locomotor score during training (see Table 1 for details) are shown. D, Correlation of glutamate (squares) and glycine (triangles) levels in the cortex and locomotor score during training. Pearson correlation coefficients are shown on each plot in B, C, and D. Asterisks ** indicate significant correlation at p < 0.01 level.

Figure 3.

Expression of VGLUT1 and GLYT2 in lumbar spinal cord. A, VGLUT1 (green) expression in the ventral horn of the lumbar spinal cord in a Trained rat. Motor neurons were labeled with HSP27 (red). B, C, VGLUT1 expression (green) around HSP27-labeled motor neurons is shown in a representative Untrained (B) and Trained (C) ST rat, respectively. D, GLYT2 (green) expression in the ventral horn of the lumbar spinal cord in a Trained rat. Motor neurons were labeled with HSP27 (red). E, F, GLYT2 expression (green) around HSP27-labeled motor neurons is shown in a representative Untrained (E) and Trained (F) ST rat respectively.

Figure 4.

Comparison of hindlimb treadmill stepping in Untrained and Trained ST rats. A, C, Horizontal ankle movements recorded by the robotic device and the corresponding ankle trajectory are shown for representative Untrained (A) and Trained (C) rats. Arrows in the horizontal ankle movement indicate forward movements detected by the robotic device. Black arrows indicate weight-bearing steps whereas white arrows indicate nonweight-bearing movements. The vertical and horizontal calibration bars in the ankle trajectory are both 20 mm. The data were collected during 30 s of testing at 95% weight support at Week 4. B, D, The percentage of steps that were weight bearing (WB) during testing at Week 0 (B) and Week 4 (D) is shown for Untrained (white) and Trained (black) rats. The data are from tests at 95%, 85%, and 75% weight support levels. The data shown are averages (n = 11 for each group). Asterisks (*) indicates significant difference between Trained and Untrained (p < 0.05). The numbers 75 and 85 on the 95% bar indicate significant difference between 95% and the 75% and 85% body weight support levels, respectively (p < 0.05).

Figure 5.

Average ankle trajectories during hindlimb treadmill stepping in Normal and ST rats. A, B, The average trajectory of the ankle for the Untrained (thin line) and Trained (thick line) ST rats is shown for Week 0 (A) and Week 4 (B), respectively. Normal ankle trajectory is also shown (dashed line). The average trajectory for each group was calculated from 11 Untrained, 11 Trained, and 9 Normal rats (during 30 s of stepping at 85% weight support for each rat). Bar plots comparing kinematic characteristics between Untrained (UT, white) and Trained (T, black) are shown. Averages are shown (n = 11 Untrained; n = 11 Trained) and are expressed as a percentage of Normal values (100%). Area, Length, and Height of step cycle trajectory are shown. Lt-Rt is a measure of the alternating movements between the left (Lt) and right (Rt) hindlimbs. CP is cycle period. The asterisks ** and * indicate significant difference from Normal at the p < 0.01 and p < 0.05 levels, respectively. Significant differences between Untrained and Trained groups are indicated by the lines and asterisks (*) above the bars (p < 0.05).