Supplementary Material
Supplementary Methods
Behavioral studies
To decrease the chances that the behavioral responses were altered by the prior test history, the most invasive procedures were performed last. The studies were performed in the following order: motor test, rotarod test, open field test, plus maze, hole board test, Morris water maze and fear conditioning.
1. Motor tests
A battery of motor tests was performed following the procedure described by Martínez-Cué et al. [45]. The cerebellar and vestibular functions (visual placing reflex), grip strength, equilibrium, motor coordination and prehensile reflexes were evaluated.
2. Rotarod
Motor coordination was evaluated using a rotarod device (Ugo Basile, Comerio, Italy) following the same procedure described by Martínez-Cué et al. [45].
1. Open field
Exploratory behavior and anxiety were assessed using a square-shaped open field (55 cm x 55 cm, surrounded by a 25 cm-tall fence), which was divided into 25 equal squares. The mice were placed in the center of the field, and the number of vertical (rearing) activities and horizontal crossings (from square to square, subdivided into center vs. peripheral crossings) were scored in a single 5 min trial.
2. Plus maze
The elevated plus maze consisted of two closed arms (5 cm x 30 cm, with clear perplex walls 15 cm high) and two open arms (5 cm wide x 30 cm long) that were raised 40 cm from the floor. In a single 5 min trial, the mice were placed in the center of the maze, and the number of arm entries, the time spent in the open and closed arms and the number of stretch attend postures (SAP) and head dippings (HD) were registered.
3. Hole Board
The hole board is a wooden box (32 x 32 x 30 cm) with four holes. The floor is divided into nine 10 cm squares. In a single 5 min trial, the number of explorations, the time spent exploring each hole, and the overall activity in the apparatus were measured. A repetition index was also calculated (exploration of holes previously explored) as a function of the number of ABA alternations.
Statistical Analysis
The LTP data were analyzed using an RM MANOVA (‘time x karyotype x genotype’). The remaining data were analyzed using a two-way (‘karyotype’ x ‘genotype’) ANOVA. The mean values of each experimental group were compared using post hoc Bonferroni tests. All analyses were performed using SPSS for Windows version 22.0. (Armonk, New York, USA).
Supplementary Results
Behavioral tests
1. Motor tests
The number of functional copies of the Gabra5 gene did not affect the motor abilities, reflexes, equilibrium or motor coordination of the TS or CO mice (Supplementary table 2). The TS mice with the three genotypes displayed a reduced prehensile reflex and an altered motor coordination in the coat hanger test as they fell sooner from the coat hanger, performed a lower number of crossings and showed a larger latency of arrival to the end of the bar.
2. Rotarod
Consistent with the results of the coat hanger test in which no effect of the Gabra5 gene dosage was found on motor coordination, the three genotypes did not affect this variable in the TS or CO mice when assessed at different constant speeds (ANOVA ‘genotype’: 25 r.p.m. F(2,60)=0.41, p=0.66; 50 r.p.m. F(2,60)=0.20, p=0.81; Supplementary figure 1a) or during the acceleration cycle (F(2,60)=0.67, p=0.51; Supplementary figure 1b).
However, the TS mice with no, one or two copies of Gabra5 showed a deterioration in motor coordination at the highest constant speed (ANOVA ‘karyotype’: F(1,60)=14.26, p<0.001; ‘karyotype x genotype’ F(2,60)=0.027, p=0.97). This effect was not evident at the lower constant speeds (25 r.p.m. ANOVA ‘karyotype’: F(1,60)=0.59, p=0.44; ‘karyotype x genotype’ F(2,60)=0.24, p=0.78) or during the acceleration cycle (ANOVA ‘karyotype’: F(1,60)=1.66, p=0.20; ‘karyotype x genotype’ F(2,60)=1.17, p=0.31).
3. Open field
In the open field test, the TS mice appeared to be hyperactive (Supplementary figures 2a, b and c) since they traveled a longer distance in the periphery (ANOVA ‘karyotype’: F(1,60)=8.76, p=0.004) and the center (F(1,60)=8.65, p=0.005) and had a longer total distance (F(1,60)=11.06, p=0.001; Supplementary figure 2a), a larger number of entries into the center of the maze (F(1,60)=6.65, p=0.012, Supplementary figure 2b) and faster speeds (F(1,60)=11.09, p=0.001, Supplementary figure 2c). Although this tendency was lower in the heterozygous and homozygous TS mice, this effect was not significant, and no differences were found between the three genotypes in the TS or CO mice (distance periphery: ANOVA ‘genotype’ F(2,60) =0.97, p=0.93; ‘karyotype x genotype’: F(2,60)=1.40, p=0.25; distance center ANOVA ‘genotype’ F(2,60) =0.87, p=0.42; ‘karyotype x genotype’: F(2,60)=0.87, p=0.42; total distance: ANOVA ‘genotype’ F(2,60) =1.17, p=0.31; ‘karyotype x genotype’: F(2,60)=1.13, p=0.32; speed: ANOVA ‘genotype’ F(2,60) =1.17, p=0.31; ‘karyotype x genotype’: F(2,60)=1.11, p=0.33; number of entries: ANOVA ‘genotype’ F(2,60) =1.24, p=0.28; ‘karyotype x genotype’: F(2,60)=1.20, p=0.30).
The six groups of TS or CO animals did not significantly differ in their vertical activity (ANOVA ‘karyotype’ F(1,60)=1.26, p=0.26; ‘genotype’: F(2,60)=0.48, p=0.61; ‘karyotype x genotype’: F(2,60)=1.56, p=0.21, Supplementary figure 2d).
4. Plus maze
TS mice with three genotypes were also hyperactive in the plus maze. These animals traveled a longer distance in the open (ANOVA ‘karyotype’: F(1,60)=18.52, p<0.001) and closed arms (F(1,60)=5.57, p=0.21) and the center of the maze (F(1,60)=11.79, p=0.001); therefore, they traveled a longer total distance during the entire test (F(1,60)=17.36, p<0.001; Supplementary figure 3a).
However, the gene dosage of Gabra5 did not affect the activity of the TS or CO mice (ANOVA ‘genotype’: open arms F(2,60)=0.51, p=0.60; closed arms F(2,60)=0.44, p=0.67, center F(2,60)=0.51, p=0.60, total distance F(2,60)=0.22, p=0.80; ANOVA ‘karyotype x genotype’: open arms F(2,60)=1.60, p=0.20; closed arms F(2,60)=0.27, p=0.76, center F(2,60)=0.16, p=0.85, total distance F(2,60)=0.37, p=0.68).
The TS mice also made more entries into the open arms of the maze but not in the closed arms or the total number of entries (ANOVA ‘karyotype’ open arms F(1,60)=4.18, p=0.045; closed arms F(1,60)=0.12, p=0.72; total number of arm entries F(1,60)=2.36, p=0.12; Supplementary figure 3b). No significant differences were found in the number of arm entries between animals carrying different numbers of copies of the Gabra5 gene (ANOVA ‘genotype’ open arms F(1,60)=1.32, p=0.27; closed arms F(1,60)=0.39, p=0.67; total number of arm entries F(1,60)=1.19, p=0.31; (ANOVA ‘karyotype x genotype’ open arms F(1,60)=0.12, p=0.88; closed arms F(1,60)=2.61, p=0.081; total number of arm entries F(1,60)=1.20, p=0.30).
TS and CO mice with the three genotypes did not differ in most of the motor or cognitive manifestations of anxiety. No significant differences were found in the amount of time spent freezing at the beginning of the tests between the different groups of animals (ANOVA ‘karyotype’: F(1,60)=0.01, p=0.95; ‘genotype’: F(2,60)=0.70, p=0.50; ‘karyotype x genotype’: F(2,60)=2.34, p=0.10; Supplementary figure 3c). In addition, all animals performed a similar number of risk assessment behaviors (ANOVA ‘karyotype’: F(1,60)=2.92, p=0.09; ‘genotype’: F(2,60)=0.93, p=0.40; ‘karyotype x genotype’: F(2,60)=0.29, p=0.74; Supplementary figure 3e).
Regarding the motor component of anxiety that is evaluated by the plus maze (i.e., percentage of time in the open arms, Supplementary figure 3d), the TS mice tended to spend more time in the open arms than their littermate controls, and the Gabra5 gene dosage did not modify this tendency in the TS or CO mice (ANOVA ‘karyotype’: F(1,60)=4.45, p=0.03; ‘genotype’: F(2,60)=1.13, p=0.32; ‘karyotype x genotype’: F(2,60)=0.87, p=0.42). Although an increased time in the open arms has been traditionally attributed to reduced anxiety, this effect is likely related to the hyperactivity and inability to attend to relevant stimuli that are characteristic of TS mice (see discussion).
5. Hole Board
The results of the hole board test corroborate the hyperactive phenotype displayed by the TS mice. These animals traveled a longer distance at a faster speed than the CO mice (Supplementary table 3), and these effects were not corrected in the heterozygous or homozygous animals.
Although the number of repetitions of recently explored holes (ABA) is an index of attention, no significant differences were found between the 6 groups of mice in their performance. However, the time spent exploring the holes was reduced in the TS mice, which also suggests that their attention is lower than that of CO mice.
Weight
TS mice with the three genotypes weighed less than the CO mice (ANOVA ‘karyotype’: F(1,54)= 28.6, p<0.01; Supplementary figure 4). In addition, the homozygous Gabra5 KO TS and CO mice displayed a smaller weight during their first five months of life (ANOVA ‘genotype’: F(1,54)=3.23, p=0.047; ANOVA ‘karyotype x genotype’: F(1,54)=0.75, p=0.47). This effect was particularly evident in the CO -/- mice compared with the CO +/- and CO +/+ mice.
Supplementary Discussion
To evaluate the putative adverse effects of reducing the density of α5 GABAA receptors, we performed a behavioral characterization of TS and CO mice with different Gabra5 genotypes.
Although fine motor control remains perturbed in DS individuals throughout life [114, 115] and DS motor alterations are well documented in some mouse models [116], whether TS animals present altered motor activity is controversial despite their pronounced alterations in cerebellar morphology. Some studies have found that these animals have altered motor coordination [117, 118], while others report normal [80, 119, 120] or enhanced [121] motor coordination. The discrepancies found between different studies might be due to differences in experimental protocols or the ages of the animals. Although previous studies from our laboratory failed to find motor alterations in TS animals using the same protocols (sensorimotor and rotarod tests) as used in the present work [80, 119], here, we found that TS mice with different gene dosages of Gabra5 displayed a reduced prehensile reflex and altered motor coordination in the coat hanger and rotarod tests. These differences are likely due to differences in genetic backgrounds in the previous (BL6C3H) and present (BL6C3H*C57BL6) studies.
The number of copies of Gabra5 did not affect the motor abilities, reflexes, equilibrium or motor coordination of TS or CO mice, which is in agreement with the lack of motor side effects found following the administration of different α5 selective NAMs to these animals [25, 40, 44, 45]. These results might be due to the fact that motor abilities, motor control and motor learning are mainly controlled by the cerebellum, a structure in which the expression of GABAAα5 receptors is extremely low [49, 122, 123].
One of the most consistent anomalies in the behavior of TS mice is the hyperactivity that is displayed in different situations that provoke caution or the lack of movement in euploid animals, which is thought to be due to an inability to attend to relevant stimuli [124-127]. In this study, we also found that the TS mice displayed an increased activity in the open field, plus maze and hole board tests. This hyperactivity was not corrected by reducing the number of functional copies of the Gabra5 gene, which is inconsistent with previous reports in which the administration of RO4938581 corrected the hyperactivity [45]. In addition, no significant differences were found in the number of repetitions of recently explored holes (ABA), which is an index of attention, performed by the six groups of mice, although RO4938581 administration improved this type of attention in TS mice. This effect may be partially due to the different genetic background of the animals (BL6C3HF1 in the TS and CO mice and C57BL6 in the Gabra5 KO mice). Thus, the present results demonstrate that genetically reducing the density of α5 GABAA receptors did not rescue the hyperactivity or the altered attention in TS mice.
In the present study, reducing the dosage of this gene did not induce any motor or cognitive components of anxiety in the Plus maze or Open Field tests in the TS or CO mice, which is consistent with reports regarding the effects of the chronic administration of α5-selective NAMs and the analysis of the Gabra5 KO mice [25, 44, 45, 128-131]. These results provide further support for the specificity of the α5 subunit of the GABAA receptor in cognitive processes and the lack of involvement of this subunit in other physiological or pathological processes that depend on this receptor.
The viability of the TS mice was considerably reduced. This result, which has been described in other studies, is been attributed to the pre- and peri-natal death of these mice due to congenital heart diseases [66, 67]. Homozygous Gabra5 KO TS mice showed a very low viability (they represented approximately 3% of the animals born). Although this effect did not reach statistical significance, it led to a difficulty in obtaining enough animals from this karyotype and genotype to perform some of the experiments. The reason for the low viability of this group of animals remains unclear and should be investigated. When animals from both genotypes were analyzed separately, the number of TS and CO mice with two or no functional copies of this gene was approximately half of that of animals (TS or CO) carrying a single copy of Gabra5, which is consistent with the expected Mendelian distribution.
In addition, the body weight of the TS mice during the first 5 months of life was lower than that of the CO mice, which is consistent with previous reports [80]. Gabra5 -/- mice of both genotypes displayed a significantly lower weight than the +/- or +/+ TS and CO mice. This low body mass might have affected the viability of the animals, although, as demonstrated by the behavioral characterization, it did not exert any harm in their motor abilities or anxiety. Thus, it is unlikely that the reduced body weight played a role in the ability to perform the cognitive tests.
Supplementary references
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115. Latash ML, Kang N, Patterson D (2002) Finger coordination in persons with Down syndrome: atypical patterns of coordination and the effects of practice. Exp Brain Res 146: 345–355.
116. Altafaj X, Dierssen M, Baamonde C, Martí E, Visa J, Guimerà J, Oset M, González JR, Flórez J, Fillat C, Estivill X (2001) Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A (minibrain), a murine model of Down's syndrome. Hum Mol Genet 10:1915-23.