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Brain Sci
2021 Mar 02;113:. doi: 10.3390/brainsci11030315.
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Targeting GABAC Receptors Improves Post-Stroke Motor Recovery.
van Nieuwenhuijzen PS
,
Parker K
,
Liao V
,
Houlton J
,
Kim HL
,
Johnston GAR
,
Hanrahan JR
,
Chebib M
,
Clarkson AN
.
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Ischemic stroke remains a leading cause of disability worldwide, with limited treatment options available. This study investigates GABAC receptors as novel pharmacological targets for stroke recovery. The expression of ρ1 and
ρ
2 mRNA in mice were determined in peri-infarct tissue following photothrombotic motor cortex stroke. (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (R)-4-ACPBPA and (S)-4-ACPBPA were assessed using 2-elecotrode voltage electrophysiology in Xenopus laevis oocytes. Stroke mice were treated for 4 weeks with either vehicle, the α5-selective negative allosteric modulator, L655,708, or the ρ1/2 antagonists, (R)-4-ACPBPA and (S)-4-ACPBPA respectively from 3 days post-stroke. Infarct size and expression levels of GAT3 and reactive astrogliosis were determined using histochemistry and immunohistochemistry respectively, and motor function was assessed using both the grid-walking and cylinder tasks. After stroke, significant increases in ρ1 and ρ2 mRNAs were observed on day 3, with ρ2 showing a further increase on day 7. (R)- and (S)-4-ACPBPA are both potent antagonists at ρ2 and only weak inhibitors of α5β2γ2 receptors. Treatment with either L655,708, (S)-4-ACPBPA (ρ1/2 antagonist; 5 mM only), or (R)-4-ACPBPA (ρ2 antagonist; 2.5 and 5 mM) from 3 days after stroke resulted in a significant improvement in motor recovery on the grid-walking task, with L655,708 and (R)-4-ACPBPA also showing an improvement in the cylinder task. Infarct size was unaffected, and only (R)-4-ACPBPA significantly increased peri-infarct GAT3 expression and decreased the level of reactive astrogliosis. Importantly, inhibiting GABAC receptors affords significant improvement in motor function after stroke. Targeting the ρ-subunit could provide a novel delayed treatment option for stroke recovery.
Figure 1
Temporal changes in α5, ρ1, and ρ2-subunit expression were assessed in the peri-infarct region using real time qPCR after photothrombotic stroke to the motor cortex. (A) GABAA α5 expression, (B) GABAC ρ1 expression, and (C) GABAC ρ2 expression was assessed in stroke (red) and sham (blue) animals. * p < 0.05, ** p < 0.01, *** p < 0.001 (n = 5 for sham and 1, 3, 7, 14 and 28-days post-stroke). Data expressed as box plot (boxes, 25–75%; whiskers, minimum and maximum; lines, median). GABA: γ-aminobutyric acid.
Figure 2
Dose–response curves of GABA (☐—open black boxes) in the presence of (A) 10 µM (▽—green triangle), 30 µM (△—red triangle), and 100 µM (◯—blue circle) (S)-4-ACPBPA and (B) 10 µM (▽—green triangle), 30 µM (△—red triangle), and 60 µM (◯—blue circle) (R)-4-ACPBPA at human recombinant ρ2 GABAC receptors expressed in xenopus oocytes. (C) Representative responses of α5β2γ2 receptors expressed in xenopus oocytes to GABA (10 µM) alone and GABA (10 µM) with inhibitors (R) and (S)-4-ACPBPA (10 and 100 µM). (R) and (S)-4-ACPBPA (10 µM) inhibited GABA (10 µM) response by 2.5 ± 1.7 and 5.5 ± 2.5%, respectively, whilst (R) and (S)-4-ACPBPA (100 µM) inhibited GABA (10 µM) response by 36 ± 0.8 and 37 ± 0.6%, respectively. Data are expressed are mean ± SEM (n = 6 oocytes). (R)-4-ACPBPA: (R)-4-amino-cyclopent-1-enyl butylphosphinic acid, (S)-4-ACPBPA: (S)-4-amino-cyclopent-1-enyl butylphosphinic acid.
Figure 3
Behavioral recovery was assessed pre-op (before) and 7, 14, 21, 28, and 42 days after a photothrombotic stroke. NAM, 5 mM; L655,708, and the GABAC ρ1 antagonist, 2.5 and 5 mM (S)-4-ACPBPA. Motor function was assessed by analyses of (A) foot-faults and (B) forelimb asymmetry in the grid-walking and cylinder tasks, respectively. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to stroke + vehicle. Data are expressed as mean ± SD for n = 10/treatment group.
Figure 4
Behavioral recovery was assessed pre-op (before) and 7, 14, 21, 28, and 42 days after a photothrombotic stroke in the presence of the GABAA α5 NAM, 5 mM L655,708, and the GABAC ρ2 antagonist, 2.5 and 5 mM (R)-4-ACPBPA. Motor function was assessed by analyses of (A) foot-faults and (B) forelimb asymmetry in the grid-walking and cylinder tasks, respectively. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to stroke + vehicle. Data are expressed as mean ± SD for n = 10 / treatment group.
Figure 5
Assessment of infarct volume was carried out by quantifying cresyl violet stained sections generated 42 days post-stroke. Data expressed as box plot (boxes, 25–75%; whiskers, minimum and maximum; lines, median) for an n = 6/treatment group.
Figure 6
Changes in GABA transporter type 3 (GAT3) and glial fibrillary acidic protein (GFAP) expression were assessed 42 days post-stroke. (A) Representative photomicrographs showing GAT3 (green) and GFAP (red) labeling from stroke + vehicle (VEH), Stroke + L655,708 (L655), stroke + S-4-ACPBPA (S-), and stroke + (R)-4-ACPBPA (R-) High magnification images of each of the treatment groups from right next to the stroke border. Normalized fluorescent intensity measurements for GAT3 (B) and GFAP (C) were obtained from peri-infarct regions 400 µm from the stroke border. + p < 0.05, ++ p < 0.01. Data expressed as box plot (boxes, 25–75%; whiskers, minimum and maximum; lines, median) for an n = 6/treatment group. The scale bar in the insert = 100 um, whereas the scale bar shown in the main photomicrograph = 400 um.
Figure 7
Behavioral recovery was assessed pre-op (before) and 7, 14, 21, 28, and 42 days after a photothrombotic stroke in the presence of the GABAA α5 NAM, 5 mM L655,708, the GABAC ρ2 antagonist, 5 mM (R)-4-ACPBPA, or a combined 5 mM L655,708 and 5 mM (R)-4-ACPBPA. Motor function was assessed by analyses of (A) foot-faults and (B) forelimb asymmetry in the grid-walking and cylinder tasks, respectively. ## p < 0.01, ### p < 0.001, compared to stroke + vehicle; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared to sham + L655,708 + (R)-4-ACPBPA; $$ p < 0.01, $$$ p < 0.001, compared to stroke + L655,708. Data are expressed as mean ± SD for n = 8 / treatment group.
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