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

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 GABAA receptor function. We found that hampering GABAA 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.

Rose Bengal (0.2 ml of a 10mg/ml solution in PBS; Sigma Aldrich) was injected intraperitoneally.
After 5 min, the brain was illuminated through the intact skull for 15 min using a cold light source (ZEISS CL 6000) linked to a 20X objective that was positioned 0.5 mm anterior and 1.75 mm lateral from Bregma (i.e. in correspondence with the caudal forelimb area; Tennant et al. 2011;Vallone et al. 2016). Sham animals underwent scalp incision and Rose Bengal injection but no light irradiation. At the end of the surgery, the skin was sutured and mice were allowed to awaken from anaesthesia.

Intracortical Microstimulation (ICMS)
Animals were anesthetized using a ketamine (100 mg/kg) and xylazine (10mg/kg) cocktail. A stable level of anaesthesia was maintained delivering 1/10 of the starting dose every 30 minutes. Animals were placed in a stereotaxic apparatus, the skull was exposed and a craniotomy (extending 3 mm and 4mm in the medio-lateral and antero-posterior direction, respectively) was performed in the ipsilesional hemisphere.
The cortex was stimulated through a tungsten microelectrode (1 MΩ, FHC, USA), inserted slowly into the brain at 700 µm depth for each stimulation point, following a grid with nodes spaced 250 µm. The ground electrode was placed under the skin of the neck. As reported in Tennant et al., 2011, at each penetration site, a 40 ms train of 13 cathodic current pulses (0.2 ms duty cycle) was delivered at 350 Hz from an electrically isolated, constant current stimulator (World Precision Instruments Inc., USA) guided by an electronic board (National Instruments Corp, USA). The amplitude of the pulses was increased from a minimum of 20 μA to a maximum of 60 μA (with steps of 10 μA). Movements of several body parts were collected by a second experimenter, blinded to the stimulation coordinates in the grid. At the end of the ICMS procedure, the animal was sacrificed and the brain dissected for histology.

Data Analysis
Data collected during ICMS were analyzed through a custom made algorithm developed in Matlab To improve the visual resolution, the matrix was 10-fold upsampled and Gaussian filtered.
Contralateral forelimb (FL), contralateral hindlimb (HL) and tail (TL) showed a high and widespread activation after stroke (see Results). Thus we merged their BP maps to indentify the common activation areas before and after lesion. To study the maximum of the activation, the three BP maps computed with a 60 μA current amplitude were used. The maps were then thresholded in PA by considering a 50% threshold value, and finally overlapped. A colorimetric index was used to classify all of the possible cases observed (FL, FL+HL, FL+Tail, TL, HL+TL, FL+HL+TL). To smooth such maps, borders were manually drawn on the basis of matlab computed maps.
Since the distance between adjacent sites was 250 μm, the areas of 250 x 250 µm 2 centred at the each site were considered to calculate the Percentage of Responding Area (i.e. percentage of stimulated area which elicits the BP movement, for a specific amplitude of stimulation current) of the whole maps: where Kis the number of responsive sites and Total Area is defined as × (250 ) 2 , where NS is number of total stimulated sites. We also quantified how each cortical site changes its forelimb preference after the ischemic injury.
We were interested in those sites that maximized the difference in forelimb activation probability in sham and stroke animals, i.e. those sites that had a high (or low) probability to evoke a forelimb movement in the sham group and a low (or high) probability to elicit forelimb movement after stroke. Thus, we defined a novel parameter, the Transition Index (TI), which shows the shamstroke difference in forelimb activation probability. In order to quantify the TI amplitude, which shows the extent of the sham-stroke change, the average probability matrices of forelimb activation where N=240), the amplitude of the TI was defined as an Euclidean distance: = √ ( ( ℎ ) ) 2 + ( ( ) ) 2 where ΔP (sham) j = (fPA j -nfPA j ) for sham animals, whereas ΔP (stroke) j = (fPA j -nfPA j ) for stroke animals. Accordingly to this definition the TI value is defined within a range of ±√2. Since we focused only on sites that inverted their ΔP after stroke (i.e. ΔP (sham) and ΔP (stroke) have different signs),we did not consider cases with ΔP (sham) and ΔP (stroke) with the same sign, thus arbitrarily setting the TI of these sites to zero. In the other cases, the sign of TI (sTI) that shows the direction of the change, was assigned accordingly as follow: (i) when ΔP (sham) >0 and ΔP (stroke) <0 then sTI was defined positive (i.e. loss of forelimb movements); (ii) when ΔP (sham) <0 and ΔP (stroke) >0 then sTI was defined negative (i.e. gain of forelimb).

Antibodies for immunohistochemistry
In the paper we used following antibodies and concentrations: NeuN