Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke

Abstract

Stroke is a leading cause of disability, but no pharmacological therapy is currently available for promoting recovery. The brain region adjacent to stroke damage-the peri-infarct zone-is critical for rehabilitation, as it shows heightened neuroplasticity, allowing sensorimotor functions to re-map from damaged areas. Thus, understanding the neuronal properties constraining this plasticity is important for the development of new treatments. Here we show that after a stroke in mice, tonic neuronal inhibition is increased in the peri-infarct zone. This increased tonic inhibition is mediated by extrasynaptic GABA(A) receptors and is caused by an impairment in GABA (γ-aminobutyric acid) transporter (GAT-3/GAT-4) function. To counteract the heightened inhibition, we administered in vivo a benzodiazepine inverse agonist specific for α5-subunit-containing extrasynaptic GABA(A) receptors at a delay after stroke. This treatment produced an early and sustained recovery of motor function. Genetically lowering the number of α5- or δ-subunit-containing GABA(A) receptors responsible for tonic inhibition also proved beneficial for recovery after stroke, consistent with the therapeutic potential of diminishing extrasynaptic GABA(A) receptor function. Together, our results identify new pharmacological targets and provide the rationale for a novel strategy to promote recovery after stroke and possibly other brain injuries.

Figure 1. Elevated tonic inhibition in peri-infarct cortex

a, Images showing the peri-infarct recording site. Whole-cell patch-clamp recordings were made from post-stroke brain slices, within 200μm of infarct (top left), from layer-2/3 (top right) pyramidal neurons (bottom panels). b, Box-plot (boxes: 25–75%, whiskers:10–90%, lines: median) showing significantly elevated tonic inhibition in peri-infarct cortex (asterisk: P<0.05; see Supplementary Fig. 2 for additional analyses). c,d, Representative traces showing tonic inhibitory currents in control and peri-infarct neurons, respectively. Tonic currents were revealed by the shift in holding currents after blocking all GABA_ARs with gabazine (>100μM). Cells were voltage-clamped at +10mV.

Figure 2. Post-stroke impairment in GABA transport and effect of blocking α5-GABA_ARs

a, Blocking GAT-1 (NO-711) produced a higher % increase in I_tonic after stroke; combined blockade of GAT-1 and GAT-3/4 (NO-711 + SNAP-5114) produced a substantial I_tonic increase in controls but only an increase equivalent to blocking GAT-1 alone after stroke. b,c, I_tonic in sequential drug applications. Note the lack of response to SNAP-5114 application in the post-stroke slice. d, L655,708 reduced I_tonic. e, L655,708 significantly decreased post-stroke I_tonic. f, Drug treatment reverted post-stroke I_tonic to near-control level (asterisk: P<0.05; n.s.: no significance, bar graphs represent mean ± s.e.m.).

Figure 3. Behavioral recovery after stroke with L655,708 treatment and in Gabra5^−/−and Gabrd^−/− animals

a–c, L655,708 treatment starting from 3-days post-stroke resulted in a dose-dependent improvement in functional recovery post-stroke. d–f, Gabra5^−/− and Gabrd^−/− mice also showed decreased motor deficits post-stroke. Functional recovery was assessed with forelimb (a, d) and hindlimb foot-faults (b, e), and on the forelimb asymmetry (c, f). Low-dose L655,708 = 200μg/kg/day per animal; high-dose L655,708 = 400μg/kg/day per animal. Data are ± s.e.m. *** = P≤0.001 stroke + vehicle vs Sham; + = P≤0.05, ## = P≤0.01, # = P≤0.001 vs stroke + vehicle.

Figure 4. Inflection point in L655,708 treatment effect on infarct size

Representative Nissl stained sections at 7-days post-stroke from stroke + vehicle-treatment (a), stroke + L655,708-treatment starting at the time of stroke (b) and stroke + L655,708-treatment starting from 3-days post-insult (c). Quantification of the stroke volume is shown in panel (d). Data are mean ± s.e.m. for n=4 per group, * = P≤0.05.