Alterations of Both Dendrite Morphology and Weaker Electrical Responsiveness in the Cortex of Hip Area Occur Before Rearrangement of the Motor Map in Neonatal White Matter Injury Model

Abstract

Hypoxia-ischemia (H-I) in rats at postnatal day 3 causes disorganization of oligodendrocyte development in layers II/III of the sensorimotor cortex without apparent neuronal loss, and shows mild hindlimb dysfunction with imbalanced motor coordination. However, the mechanisms by which mild motor dysfunction is induced without loss of cortical neurons are currently unclear. To reveal the mechanisms underlying mild motor dysfunction in neonatal H-I model, electrical responsiveness and dendrite morphology in the sensorimotor cortex were investigated at 10 weeks of age. Responses to intracortical microstimulation (ICMS) revealed that the cortical motor map was significantly changed in this model. The cortical area related to hip joint movement was reduced, and the area related to trunk movement was increased. Sholl analysis in Golgi staining revealed that layer I-III neurons on the H-I side had more dendrite branches compared with the contralateral side. To investigate whether changes in the motor map and morphology appeared at earlier stages, ICMS and Sholl analysis were also performed at 5 weeks of age. The minimal ICMS current to evoke twitches of the hip area was higher on the H-I side, while the motor map was unchanged. Golgi staining revealed more dendrite branches in layer I-III neurons on the H-I side. These results revealed that alterations of both dendrite morphology and ICMS threshold of the hip area occurred before the rearrangement of the motor map in the neonatal H-I model. They also suggest that altered dendritic morphology and altered ICMS responsiveness may be related to mild motor dysfunction in this model.

Keywords: cortical layer I-III; dendritic branches; golgi staining; hip area; hypoxia-ischemia in premature infants; intracortical microstimulation (ICMS); white matter injury.

Figure 1

Cortical motor map by ICMS at 10 weeks of age. We stimulated the sensorimotor cortex per 0.5 mm at 1.0–3.0 mm right and 1.0–3.0 mm posterior from the bregma. Each grid shows a cortical map of each rat cortex in control group (n = 8) and neonatal WMI group (n = 8). Blocks in the grids show the portion of the rat body in which twitching was observed (hip joint, knee joint, trunk, and forelimb), and the current threshold is presented in each grid. Cortical maps are shown as a 5-colored code in the grid: twitching of the hip joint (red), knee joint (yellow), foot joint (light blue), and trunk (green), and non-responsive square (gray).

Figure 2

Electrophysiological analysis of sensorimotor cortex by ICMS at 10 weeks of age. (A) A summary of the motor maps in Figure 1 is shown as a heat map that represents the occurrence rate of each stimulated spot: the number of animals in which the movement was elicited was taken over the number of times that spot was stimulated in each group (n = 8 for each group). Each number in the grid shows the rate of responsive individuals in each portion, displayed as monochrome heat maps. Note that the rate for the hip area decreased in the WMI model, whereas the rate for the trunk area increased. (B) The number of grids associated with the hip area was significantly reduced, while that of the trunk area significantly increased in neonatal WMI model animals. (C) The minimal current intensity (threshold) to evoke muscle twitch was unaltered between the groups with regard to both the hip and trunk area. **p < 0.01 by Mann–Whitney U-tests. Data are presented as mean ± SEM.

Figure 3

Dendrite morphological changes in the motor cortex at 10 weeks of age. (A) We performed Golgi staining on brain slices, focusing on the hindlimb motor cortex. We particularly focused on layers I-III, in which histological changes were observed in our previous study. (B) To evaluate dendrites projecting from a cell body, a total of 135 Golgi-positive neurons in both ipsilateral and contralateral sensorimotor cortex were assessed by Sholl analysis in the neonatal WMI model (n = 5) at 10 weeks of age. Sholl analysis revealed that the number of cross sections increased over 40 μm from cell bodies on the right (ipsilateral H-I) side of the cortex. (C) Dendrite projections of 27 Golgi-positive neurons in the sham-operated right sensorimotor cortex were similar to those in the left cortex, which was equivalent to the left (contralateral control) side of the WMI group in B. **p < 0.01 by Mann-Whitney U-tests. Scale bar, 100 μm in lower figures. Data are presented as mean ± SEM.

Figure 4

Motor map and dendrite expansion at 5 weeks of age. (A) A summary of the motor maps in Supplemental Figure 1 is shown as a heat map. Although map size at 5 weeks of age appears to be smaller than that at 10 weeks of age, the size of the hip area of the WMI group (n = 11) was unchanged compared with control animals (n = 9). (B) The number of blocks in the hip area was unaltered between groups. (C) The threshold for eliciting hip joint twitch was significantly higher in the WMI group. (D) Twenty-seven Golgi-positive neurons per animal were assessed by Sholl analysis in both ipsilateral and contralateral sensorimotor cortex of neonatal WMI model (n = 4) at 5 weeks of age. Sholl analysis revealed that the number of cross sections increased over 60 μm from cell bodies on the right side of the cortex, even at 5 weeks of age. *p < 0.05 and **p < 0.01 by Mann–Whitney U-tests. Data are presented as mean ± SEM.