Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis

Abstract

Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/-) mice) show deficits in learning and memory. Cognitive deficits in Tsc2(+/-) mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.

Figure 1

Tsc2 ^+/− mice show learning deficits in three hippocampus-dependent tasks. (a) Quadrant occupancy and target crossings during the probe trial given after completion of Morris water maze training (n = 12 mice per genotype; two-way ANOVA for quadrant occupancy with genotype as between-subjects factor and pool quadrant as within-subjects factor, genotype × pool quadrant interaction: F (3,88) = 4.763, P = 0.004; two-way ANOVA for target crossings with genotype as between-subjects factor and pool quadrant as within-subjects factor, effect of genotype: F (1,88) = 4.278, P = 0.0415). Pool quadrants: target quadrant (T), adjacent right (AR), adjacent left (AL), opposite quadrant (O). Dashed line marks chance performance in the Morris water maze. (b) Number of across-phase errors in eight-arm radial maze plotted against training session (n = 19 mice per genotype; one-way repeated-measures ANOVA with genotype as between-subjects factor: F(1,36) = 4.724, P = 0.0364). (c) Context discrimination: freezing scores before shock (baseline) and during the test in the training context (WT mice: n = 11 mice; Tsc2^+/− mice: n = 9 mice) or the novel context (WT mice: n = 10 mice; Tsc2^+/− mice: n = 9 mice). *P < 0.05, ***P < 0.001, n.s., not significant (P > 0.05). Data represent means ± s.e.m.

Figure 2

An E-LTP stimulation paradigm elicited L-LTP in Tsc2^+/− mice. Initial fEPSP slopes recorded from hippocampal slices are shown before (baseline) and following LTP induction (with a 1-s, 100-Hz tetanus) for the tetanized pathway and for a separate, untetanized pathway (control). Data are plotted in 2-min blocks for baseline and 8-min blocks for time after LTP induction (WT slices, n = 9 slices from 9 mice; Tsc2^+/− slices, n = 7 slices from 7 mice; one-way repeated-measures ANOVA with genotype as between-subjects factor, measure × genotype interaction: F (29,406) = 2.436, P < 0.0001; t-test, last 10 min of recording, P = 0.0328). Sample traces show responses (ten responses were averaged) during baseline and the last 10 min of recording, respectively. Scale: vertical bar, 1 mV; horizontal bar, 10 ms. Data represent means ± s.e.m.

Figure 3

Rapamycin reversed context discrimination and spatial learning deficits in Tsc2 ^+/− mice. (a) Context discrimination: freezing scores before shock (baseline) and during the test in the training context (vehicle-treated WT mice, n = 12; rapamycin-treated WT mice, n = 13; vehicle-treated Tsc2^+/− mice, n = 9; rapamycin-treated Tsc2^+/− mice, n = 9) or the novel context (vehicle-treated WT mice, n = 12; rapamycin-treated WT mice, n = 12; vehicle-treated Tsc2^+/− mice, n = 10; rapamycin-treated Tsc2^+/− mice, n = 11). (b) Quadrant occupancy and target crossings during the probe trial that was given after completion of Morris water maze training (vehicle-treated WT mice, n = 17; WT mice treated with 1 mg/kg rapamycin, n = 15; WT mice treated with 5 mg/kg rapamycin, n = 16; vehicle-treated Tsc2^+/− mice, n = 16; Tsc2^+/− mice treated with 1 mg/kg rapamycin, n = 15; Tsc2^+/− mice treated with 5 mg/kg rapamycin, n = 14; three-way ANOVA for quadrant occupancy with genotype and treatment as between-subjects factors and pool quadrant as within-subjects factor, genotype × treatment × quadrant interaction: F(6,348) = 2.168, P = 0.0456; three-way ANOVA for target crossings with genotype and treatment as between-subjects factors and pool quadrant as within-subjects factor, genotype × treatment × quadrant interaction: F(6,348) = 2.908, P = 0.0088). Dashed line marks chance performance in the Morris water maze. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent means ± s.e.m.

Figure 4

Rapamycin treatment rescues lethality, reduces abnormal brain enlargement and improves neurological findings in Tsc1^cc–αCaMKII-Cre mice. (a) Percentage of untreated and rapamycin-treated Tsc1^cc–αCaMKII-Cre mice that survived to the age of three months. (b) Brain weight at the age of 3 months (untreated Tsc1^cc–αCaMKII-Cre mice, n = 3 mice; rapamycin-treated Tsc1^cc–αCaMKII-Cre mice, n = 4 mice; t-test, P < 0.001). (c) Percentage of untreated and rapamycin-treated Tsc1^cc–αCaMKII-Cre mice that showed a pathological hindlimb clasping reflex at the age of 3 months. (d) Ambulatory distance in the open field assessed at the age of 3 months (untreated Tsc1^cc–αCaMKII-Cre mice, n = 3 mice; rapamycin-treated Tsc1^cc–αCaMKII-Cre mice, n = 7 mice; t-test, P < 0.001). ***P < 0.001. Data represent means ± s.e.m.