Reducing GABAA α5 receptor-mediated inhibition rescues functional and neuromorphological deficits in a mouse model of down syndrome

Abstract

Down syndrome (DS) is associated with neurological complications, including cognitive deficits that lead to impairment in intellectual functioning. Increased GABA-mediated inhibition has been proposed as a mechanism underlying deficient cognition in the Ts65Dn (TS) mouse model of DS. We show that chronic treatment of these mice with RO4938581 (3-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine), a selective GABA(A) α5 negative allosteric modulator (NAM), rescued their deficits in spatial learning and memory, hippocampal synaptic plasticity, and adult neurogenesis. We also show that RO4938581 normalized the high density of GABAergic synapse markers in the molecular layer of the hippocampus of TS mice. In addition, RO4938581 treatment suppressed the hyperactivity observed in TS mice without inducing anxiety or altering their motor abilities. These data demonstrate that reducing GABAergic inhibition with RO4938581 can reverse functional and neuromorphological deficits of TS mice by facilitating brain plasticity and support the potential therapeutic use of selective GABA(A) α5 NAMs to treat cognitive dysfunction in DS.

Figures

Figure 1.

RO4938581 improved spatial learning and memory of TS mice. a, Schematic drawing of the Morris water-maze test design. Chronic treatment with RO4938581 improved performance of TS mice in the acquisition and probe sessions of the Morris water maze. In addition, RO4938581 treatment reduced thigmotactic behavior in TS and CO mice. Data are presented as means ± SEM of the latency to reach the platform during the 12 acquisition (b) and cued (d) sessions, the percentage of time spent in the periphery of the maze during the acquisition sessions (c), the percentage of time in the four quadrants during the probe trial (e), the number of entries in the trained quadrant (f), and the number of crossings over the platform position (g) by the four groups of mice. *p < 0.05, **p < 0.01, ***p < 0.001, TS versus CO; #p < 0.05, ##p < 0.01, ###p < 0.001, vehicle versus RO4938581, Bonferroni's tests after significant ANOVAS (RM-ANOVAs; for statistics, see Table 3).

Figure 2.

RO4938581 rescued LTP in TS mice. Time courses of the initial slope of fEPSPs recorded from the apical dendritic layer of the CA1 region in hippocampal slices after stimulation of the Schaffer collateral–commissural pathway at 30 s intervals. After 20 min of stable baseline recording, a theta burst stimulus induced robust LTP in hippocampal slices of vehicle-treated CO but not TS mice. Long-term treatment with RO4938581 resulted in enhanced LTP that reached statistical significance in slices of TS but not CO mice. The traces on top are superimposed, representative fEPSPs recorded before issuing the stimulus and at 60 min after stimulation for each genotype. Calibration: 10 ms, 1.0 mV. Data are presented as means ± SEM from vehicle-treated CO (n = 11 slices from 11different mice), vehicle-treated TS (n = 12 slices from 12 different mice), RO4938581-treated CO (n = 9 slices from 9 different mice), and RO4938581-treated TS (n = 10 slices from 10 different mice) mice, respectively (for statistics, see Table 4).

Figure 3.

RO4938581 rescued neuronal proliferation in the hippocampus of TS mice. a, Representative microscope images of coimmunostaining of DAPI (labeling all cell nuclei) and Ki-67 (proliferating cells) in the DG region of hippocampus of vehicle- and RO4938581-treated TS and CO mice. Arrowheads indicate Ki-67-positive cells. Scale bar, 50 μm. b, Means ± SEM of the density of Ki67-positive cells in vehicle- and RO4938581-treated TS and CO mice. **p < 0.01 TS versus CO; #p < 0.05, ##p < 0.01 vehicle- versus RO4938581-treated mice; Bonferroni's tests after significant ANOVAs.

Figure 4.

RO4938581 rescued granular cell density in the hippocampus of TS mice. a, Representative microscope images of DAPI immunostaining in the GCL of the hippocampal DG of vehicle- and RO4938581-treated TS and CO mice. Scale bar, 5 μm. b, Means ± SEM of the density of DAPI-positive cells in the GCL of vehicle- and RO4938581-treated TS and CO mice (a). #p < 0.05 vehicle- versus RO4938581-treated mice; Bonferroni's tests after significant ANOVAs.

Figure 5.

RO4938581 normalized the density of GABAergic synapse markers in the hippocampus of TS mice. a, Representative confocal microscope images of GAD65, GAD67, VGAT, and gephyrin immunostaining in the inner molecular layer of the hippocampal DG, lining the most external layer of granule neuron in the GCL of vehicle- and RO4938581-treated TS and CO mice. b, Means ± SEM of the percentage area occupied by GAD65-, GAD67-, VGAT-, and gephyrin-positive boutons in the hippocampus of vehicle- and RO4938581-treated TS and CO mice. *p < 0.05, **p < 0.01, TS versus CO; #p < 0.05, ##p < 0.01, vehicle- versus RO4938581-treated mice; Bonferroni's tests after significant ANOVAs.

Figure 6.

RO4938581 administration did not induce anxiety in the open field. Data are expressed as mean ± SEM of the number of crossings performed by RO4938581- and vehicle-treated TS and CO mice in the center, periphery and of total activity in the open field. **p < 0.01, TS versus CO; #p < 0.05, RO498581 versus vehicle; Bonferroni's tests after significant ANOVAs (for statistics, see Table 5).

Figure 7.

RO4938581 administration reduced hyperactivity of TS mice without inducing anxiety-like behavior in the plus maze. Data are expressed as mean ± SEM of the number of entries in the open and closed arms and number of total entries (a), the initial freezing time (b), the percentage of time spent in the open arms (c), and the number of risk assessment behaviors (d) in the plus maze. *p < 0.05, **p < 0.01, CO versus TS; #p < 0.05, RO498581 versus vehicle; Bonferroni's tests after significant ANOVAs (for statistics, see Table 5).

Figure 8.

Inhibition of [3H]RO0154513 in vivo binding by RO4938581 (20 mg/kg) in TS and CO mice. a, Representative ex vivo autoradiographical images of sagittal brain sections of CO and TS mice injected with [3H]RO0154513 and pretreated with vehicle or RO4938581. b, Inhibition of specific radioligand binding by RO4938581 (20 mg/kg) in the hippocampus of CO and TS mice. Data are expressed as the hippocampus/cerebellum ratio of radioligand binding assuming that binding in the cerebellum is mainly nonspecific.