Reducing GABAA-mediated inhibition improves forelimb motor function after focal cortical stroke in mice

Abstract

A deeper understanding of post-stroke plasticity is critical to devise more effective pharmacological and rehabilitative treatments. The GABAergic system is one of the key modulators of neuronal plasticity, and plays an important role in the control of "critical periods" during brain development. Here, we report a key role for GABAergic inhibition in functional restoration following ischemia in the adult mouse forelimb motor cortex. After stroke, the majority of cortical sites in peri-infarct areas evoked simultaneous movements of forelimb, hindlimb and tail, consistent with a loss of inhibitory signalling. Accordingly, we found a delayed decrease in several GABAergic markers that accompanied cortical reorganization. To test whether reductions in GABAergic signalling were causally involved in motor improvements, we treated animals during an early post-stroke period with a benzodiazepine inverse agonist, which impairs GABA_A receptor function. We found that hampering GABA_A signalling led to significant restoration of function in general motor tests (i.e., gridwalk and pellet reaching tasks), with no significant impact on the kinematics of reaching movements. Improvements were persistent as they remained detectable about three weeks after treatment. These data demonstrate a key role for GABAergic inhibition in limiting motor improvements after cortical stroke.

Figure 1. Plasticity of motor maps after stroke.

Average motor maps of sham (n = 9) and ischemic animals (n = 6) obtained by the intracortical microstimulation (ICMS) technique following a grid of stimulation site of 250 μm (see coordinates from bregma and Fig. 2a). For each pixel in the maps, a color code indicates the probability (high – red; low – blue) to elicit movement of a body part (contralateral forelimb in (a,b); contralateral hindlimb in (d,e); tail in (g,h) after stimulation with a train of cathodal pulses (current, 30 μA). The quantitative analysis was performed in healthy regions rostral and caudal to the stroke, excluding the infarct zone, which is indicated by the shaded areas (black dots indicate the center of the lesion). (c,f,i) Quantification of the percentage of perilesional motor areas eliciting forelimb (c), hindlimb (f) and tail responses (i) in sham and stroke animals. After stroke, there is a significant decrease in the cortical area that evokes forelimb responses (t-test, p < 0.05), with a corresponding expansion of hindlimb motor maps (t-test, p < 0.05). Tail representations are not altered by the infarct (t-test, p = 0.551). Data are mean ± SE.

Figure 2. Loss of movement selectivity following a cortical infarct.

(a) Scheme of the average map showing major changes in forelimb (FL), hindlimb (HL) and tail (TL) representation after stroke. Colors shows the preferential activation of different regions, showing the specificity of each zone (single vs multiple movements). Dotted circle indicates the average position of our ischemic lesion and white dots represent the grid of stimulation used in ICMS experiments. (b) Surface area eliciting movement of forelimb (FL) alone, forelimb + hindlimb (HL) or tail (TL), or the three body parts together at a stimulation current of 30 μA in sham (black, n = 9) and ischemic animals (red, n = 6). The quantitative analysis was performed in regions rostral and caudal to the infarct (see Fig. 1). Following stroke, very few sites retain their selectivity for forelimb movements, while most cortical locations elicit simultaneous movement of forelimb, hindlimb and tail (t-test, p < 0.05). Data are mean ± SE. (c) Map of the Transition Index (TI; see Methods) showing for each pixel the tendency to gain (blue) or lose (red) forelimb movement after stroke in our experimental sample. The colorimetric index is defined within a range of (see Methods).

Figure 3. Reduced density of PNNs in peri-infarct areas.

(a) Number of cells surrounded by PNNs in 200 μm wide cortical columns, lateral (black bars) and medial (grey bars) to the ischemic lesion, 7 (n = 5) and 30 days post injury (n = 8). Similar cortical regions were also sampled in controls (n = 8). PNN density is significantly reduced at 30 but not 7 days after stroke (one Way ANOVA, post hoc Holm-Sidak test, ***p < 0.001). Data are mean ± SE. (b) Representative images acquired from coronal sections of the motor cortex from sham and stroke mice at 7 and 30 days. Column width = 200 μm.

Figure 4. Reduced density of GABAergic interneurons in peri-infarct areas.

(a,b) Number of PV-positive (a) and SOM-positive cells (b) in 200 μm wide cortical columns, lateral (black bars) and medial (grey bars) to the ischemic lesion, 7 (PV n = 7; SOM n = 6) and 30 days post injury (PV n = 8; SOM n = 4). Compared to controls (PV n = 7; SOM n = 6), density of PV-+ neurons is significantly dampened at 30 days (one Way ANOVA, post hoc Holm-Sidak test, p < 0.05). SOM-+ cells are significantly reduced at 7 days and decrease further at 30 days. Data are mean ± SE. *p < 0.05; **p < 0.01. (c,d) Representative low magnification images of PV (c) and SOM immunostaining (d) in the motor cortex. Images were taken from sham animals and from the perilesional areas of mice at 7 and 30 days post-infarct. Column width = 200 μm.

Figure 5. Late reduction of V-GAT-positive inhibitory terminals in perilesional cortex.

(a,b) Mean fluorescence intensity of inhibitory, V-GAT-positive profiles in the neuropil (a) and in puncta rings surrounding the soma of pyramidal neurons (b). Measures were taken in superficial (sup) and deep layers, medial and lateral to the infarct. A consistent reduction in fluorescence is observed 30 but not 7 days after injury as compared to sham controls (one way ANOVA followed by Holm-Sidak test, p < 0.05). Data are mean ± SE. *p < 0.05; **p < 0.01. (c) Representative V-GAT immunoreactivity in the motor cortex of sham animals and in perilesional tissues (7 and 30 days after the infarct). Note decreased staining at 30 days. Number of animals is as follows: control n = 5, stroke 7 days n = 5, stroke 30 days n = 4. Scale bar = 20 μm.

Figure 6. Quantitative analysis of V-GluT1 immunostaining.

(a,b) Mean fluorescence intensity of excitatory, V-GluT1-positive presynaptic structures in the neuropil (a) and in puncta rings surrounding the soma of pyramidal neurons (b). Measures were taken in superficial (sup) and deep layers, medial and lateral to the infarct. There is no significant variation in staining in stroke animals as compared to sham controls (one Way ANOVA, followed by Holm-Sidak test, p > 0.52). Data are mean ± SE. (c) Representative V-GluT1 staining in cortical sections from sham controls and stroke mice, 7 and 30 days following injury. Number of animals is as follows: control n = 6, stroke 7days n = 6, stroke 30 days n = 5. Scale bar = 20 μm.

Figure 7. Long-lasting improvements in general motor outcome following DMCM treatment in stroke mice.

Data are shown as percentage of the initial deficit, i.e. the difference in the fraction of foot faults/incorrect graspings between day 2 and baseline. Then, performances at day 9 and 30 have been normalized as a percentage of the initial deficit. Statistical analysis was performed on raw data and differences refer to day 2. (a) The number of foot faults in the gridwalk task decreases immediately after DMCM treatment and persists up to 30 days (two way RM ANOVA followed by Tukey test, p < 0.05; n = 10). Conversely, no significant improvements are detected in controls (p > 0.87; n = 3). (b) Performance in the single-pellet retrieval task. The fraction of incorrect graspings decreases after DMCM treatment, which rescues approximately 70% of the initial deficit 30 days post lesion (two way RM ANOVA, followed by Tukey test, p < 0.01). Data are mean ± SE.*p < 0.05; **p < 0.01.

Figure 8. Kinematic analysis of pellet reaching indicates compensatory adjustments in DMCM-treated stroke mice.

Data are expressed as percentage of the initial deficit. Post-stroke variation in the length of the whole trajectory (ArcLen; a) and in the smoothness of movement (b) during successful pellet retrievals in DMCM- (n = 3) and vehicle-treated stroke mice (n = 3). No significant differences are detected between the two groups (two way RM ANOVA, p > 0.24).