Specific targeting of the GABA-A receptor α5 subtype by a selective inverse agonist restores cognitive deficits in Down syndrome mice

Abstract

An imbalance between inhibitory and excitatory neurotransmission has been proposed to contribute to altered brain function in individuals with Down syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and accordingly treatment with GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a genetic model for DS. However, GABA-A antagonists are also convulsant which preclude their use for therapeutic intervention in DS individuals. Here, we have evaluated safer strategies to release GABAergic inhibition using a GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype (α5IA). We demonstrate that α5IA restores learning and memory functions of Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks. Furthermore, we show that following behavioural stimulation, α5IA enhances learning-evoked immediate early gene products in specific brain regions involved in cognition. Importantly, acute and chronic treatments with α5IA do not induce any convulsant or anxiogenic effects that are associated with GABA-A antagonists or non-selective inverse agonists of the GABA-A-benzodiazepine receptors. Finally, chronic treatment with α5IA did not induce histological alterations in the brain, liver and kidney of mice. Our results suggest that non-convulsant α5-selective GABA-A inverse agonists could improve learning and memory deficits in DS individuals.

Figure 1.

Dose–response effect of α5IA. The optimal α5IA promnesic dose was determined in euploid mice trained in the DMTP task. (a) Schematic representation of the DMTP protocol. Training was performed during 7 consecutive days. Each day, mice underwent one acquisition trial (T1) and three retention trials (T2–T4); inter-trial interval was 60 seconds. The position of the platform was changed every day, but remained constant within each session. (b) Performance (distance to platform; mean ± SEM) of the mice between acquisition and retention trials. Data from the seven training days have been pooled. All mice showed a significant increase in behavioural accuracy within each session. While vehicle-treated mice and mice receiving 1 mg/kg of α5IA showed similar retention, mice that were treated with the 5 mg/kg dose of α5IA displayed a significantly higher retention performance in comparison to other groups (*p < 0.05, ANOVA with Fisher's post hoc comparisons).

Figure 2.

α5IA restores spatial learning in Ts65Dn mice. (a) Schematic representation of the MWM protocol (see the text for explanations; PT: probe trial). (b) Data on learning performance have been pooled into two blocks of 3 days. Vehicle-treated Ts65Dn mice demonstrated decreased learning index in comparison with the three other trained groups. This deficit was corrected by treatment with α5IA. (c) A hit was defined as reaching the platform before 90 seconds. Vehicle-treated Ts65Dn mice showed a clear delay in conditioning that was rescued after treatment with α5IA. (d) Only euploid mice showed a spatial bias for the platform target quadrant during probe trial. Impaired retention of the platform location in Ts65Dn mice was not rescued after drug treatment. For (b) and (d), horizontal dotted lines at 25% correspond to random performance. For (d), percentage of time spent in other quadrants (‘others’) calculated as the mean time spent in these three non-target quadrants. *p < 0.05: ANOVA with repeated measures and contrast analysis. #p < 0.05: paired Student's t-tests.

Figure 3.

α5IA relieves the use of inadequate behavioural navigating strategies in the Morris water maze. A robust effect of α5IA was observed on thigmotaxy (percentage of time spent performing thigmotaxy has been pooled over the six training sessions, mean ± SEM). This inadequate strategy to locate the platform in the water maze was strongly decreased following treatment with α5IA but more particularly in Ts65Dn mice and to a lesser (non-significant) extent in euploid mice, likely due to some ceiling effects as Ts65Dn mice displayed an overall increased basal level of thigmotaxy in comparison to euploid mice. *p < 0.05; **p < 0.001; ***p < 0.0001; ANOVA with repeated measures and contrast analysis.

Figure 4.

Spatial impairments in Ts65Dn mice are not due to visual deficits. Following evaluation of spatial memory in the Morris water maze (MWM), mice were trained in a visually guided navigation task (cued visible platform). Performance was assessed using an unbiased learning index (mean ± SEM, same as in Figure 2). Analysis indicated that behavioural accuracy to locate the visible platform increased across sessions with no effect of genotype or treatment. The horizontal dotted line at 25% represents level of performance due to random navigation in the pool. As illustrated, all trained groups performed largely above this level.

Figure 5.

α5IA alleviates recognition memory deficits in Ts65Dn mice and potentiates neuronal activity (a) Upper part: general protocol of the novel-object recognition (NOR) (see the text for explanations). Lower part: Learning index (see Table 1 for raw data). Under vehicle, Ts65Dn mice were found to be impaired. Following i.p. injection of α5IA (5 mg/kg), both euploid and Ts65Dn mice improved their NOR performance and the deficit of Ts65Dn mice was abolished. *p < 0.05; **p < 0.001; ***p < 0.0001; ANOVA with Fisher's post hoc comparisons. (b) Upper part: general protocol for assessing the levels of Fos after behavioural stimulation (see the text for explanations). Lower part: histograms depict the relative increase of Fos immunoreactivity in α5IA-treated mice normalized against values obtained for vehicle-treated littermates. In all brain regions sampled, except the dentate gyrus, a significant increase of Fos was observed after α5IA injection. ^#p < 0.05; ^###p < 0.001; two way ANOVA with repeated measures and contrast analysis. No differences between genotypes were observed.

Figure 6.

α5IA does not alter locomotor activity and anxiety of Ts65Dn and euploid mice in the open field. Effects of α5IA (5mg/kg) were evaluated on locomotion and anxiety in the open field. (a) Analysis of horizontal activity (travelled distances; mean ± SEM) did not show any effect of treatment, underscoring that a single α5IA injection did not modify the gross locomotor activity of both euploid and Ts65Dn mice. (b) To assess anxiety during the open field session, a periphery-to-centre exploration ratio was measured (P/C ratio; mean ± SEM). Analysis of this measure did not reveal any effects of Genotype or Treatment.

Figure 7.

α5IA does not induce anxiety-related behaviours. Anxiety was assessed in the standard elevated plus maze task, in both euploid and Ts65Dn mice under vehicle or α5IA (one single 15 mg/kg i.p. injection; left panel of the figure). Under vehicle condition, Ts65Dn mice showed a trend for hypoanxiety (increased time in open arms) in comparison to euploid mice. Acute treatment with α5IA did not modify the behaviour of euploid mice, but significantly reduced the time spent in open arms by Ts65Dn mice. This effect can be ascribed to a normalization of behaviour in the Ts65Dn mice. Semi-chronic injections of α5IA in euploid mice (5 mg/kg five times a week for 2 weeks; right panel of the figure) did not alter the anxiety levels. Horizontal dotted line indicates the baseline performance of mice acutely treated with vehicle. *p < 0.05, ANOVA with Fisher's post hoc comparisons.

Figure 8.

α5IA does not induce any histological lesions after chronic treatment. Following chronic treatment with α5IA (5 mg/kg; five injections/week for 5 weeks), different organs were ablated and processed for routine histopathological examination. As illustrated, haematoxylin–eosin staining did not reveal any significant macroscopic or microscopic tissue alterations in liver or kidney in any of the three experimental groups (non-injected, vehicle-injected or α5IA-treated mice). The same negative findings were observed following periodic acid-Schiff staining of the tissues (not illustrated). Examination of brain, hepatic and renal tissues under polarized light revealed the lack of abnormal crystals in mice receiving injections of α5IA. The size and distribution of urine crystals (not illustrated) appeared to be very similar in the different groups. Scale bar = 100 µm.