Learning and memory and synaptic plasticity are impaired in a mouse model of Rett syndrome

Abstract

Loss-of-function mutations or abnormal expression of the X-linked gene encoding methyl CpG binding protein 2 (MeCP2) cause a spectrum of postnatal neurodevelopmental disorders including Rett syndrome (RTT), nonsyndromic mental retardation, learning disability, and autism. Mice expressing a truncated allele of Mecp2 (Mecp2(308)) reproduce the motor and social behavior abnormalities of RTT; however, it is not known whether learning deficits are present in these animals. We investigated learning and memory, neuronal morphology, and synaptic function in Mecp2(308) mice. Hippocampus-dependent spatial memory, contextual fear memory, and social memory were significantly impaired in Mecp2(308) mutant males (Mecp2(308/Y)). The morphology of dendritic arborizations, the biochemical composition of synaptosomes and postsynaptic densities, and brain-derived neurotrophic factor expression were not altered in these mice. However, reduced postsynaptic density cross-sectional length was identified in asymmetric synapses of area CA1 of the hippocampus. In the hippocampus of symptomatic Mecp2(308/Y) mice, Schaffer-collateral synapses exhibited enhanced basal synaptic transmission and decreased paired-pulse facilitation, suggesting that neurotransmitter release was enhanced. Schaffer-collateral long-term potentiation (LTP) was impaired. LTP was also reduced in the motor and sensory regions of the neocortex. Finally, very early symptomatic Mecp2(308/Y) mice had increased basal synaptic transmission and deficits in the induction of long-term depression. These data demonstrate a requirement for MeCP2 in learning and memory and suggest that functional and ultrastructural synaptic dysfunction is an early event in the pathogenesis of RTT.

Figure 1.

Mecp2^308/Y mice display learning and memory deficits. A–C, Abnormalities of spatial memory in the Morris water maze. A, Mecp2^308/Y mice donot display reduced swimming speed compared with control littermates. B, Mecp2^308/Y mice took significantly longer than control littermates in locating a submerged hidden platform (p < 0.001), and there was a significant interaction between genotype and day of training (p < 0.01). C, During the probe trial, whereas control mice spent significantly longer in the quadrant in which the platform was previously located, Mecp2^308/Y mice spent similar amounts of time in all four sectors (p = 0.001 for the interaction of genotype and quadrant). Post hoc analysis showed significant genotype differences for the trained quadrant (p < 0.01). D, E, Contextual fear conditioning is impaired in Mecp2^308/Y mice. D, In control experiments, there were no significant differences in the threshold current required to elicit stereotyped behavioral responses in 20-week-old wild-type and mutant mice. E, Using a training protocol consisting of a single foot shock of 0.5 mA for 2 sin a contextual fear conditioning cage, Mecp2^308/Y mice showed a significant reduction in the amount of freezing compared with wild-type littermates (p = 0.023). F, Deficits of hippocampus-dependent social recognition in Mecp2^308/Y mice. Naive Mecp2^308/Y mice spent less time than wild-type controls interacting with unfamiliar juvenile males (p < 0.001). Exposure to the same partners after a 24 h delay was associated with a significant reduction in interaction time in controls (p < 0.01) but not in mutant mice. Values represent mean ± SEM. The asterisks indicate significant geno type differences (*p<0.05;**p<0.01;***p<0.001). Error bars indicate SEM.

Figure 2.

Neuronal dendritic arborizations are not significantly altered in Mecp2^308/Y mice. A–D, Sholl analysis of basal and apical dendrites of pyramidal neurons in frontal cortex layer III and layer V. A, B, Number of basal (A) and apical (B) dendrites at increasing distance from the soma of pyramidal neurons in frontal cortex layer III. C, D, Number of basal (C) and apical (D) dendritesatan increasing distance from the soma of pyramidal neuron sin frontal cortex layer V. Values on the x-axis represent individual concentric 20μm rings centered on the cell body and at an increasing distance. Analysis of 30 neurons per cortical layer and genotype failed to reveal significant abnormalities in the morphology of neuronal processes of 12- to 15-month-old Mecp2^308/Y mice. Values represent mean ± SEM.

Figure 3.

The length of PSDs at asymmetric synapses in area CA1 of the hippocampus is reduced in Mecp2^308/Y mice. A, Micrograph of a representative region of area CA1. The inset highlights the region enlarged in B. Scale bar, 1 μm. B, Enlarged image of the inset in A showing a presynaptic terminal juxtaposed to a postsynaptic terminal. C, Wild-type and Mecp2^308/Y mice have similar densities of PSDs in area CA1 of the hippocampus. D, The number of docked and undocked synaptic vesicles in area CA1 is not significantly different between wild-type and Mecp2^308/Y mice. E, The average length of PSDs is significantly reduced (p < 0.05) in area CA1 of Mecp2^308/Y mice. F, Box plot of PSD length in area CA1 of wild-type and Mecp2^308/Y mice depicting the median and interquartile ranges. Values in C–E represent mean ± SEM. The asterisk in E indicates a significant genotype difference (*p < 0.05). Error bars indicate SEM.

Figure 4.

Synaptic transmission is enhanced in Mecp2^308/Y mice. Input–output relationships and paired-pulse facilitation was assessed in Mecp2^308/Y and littermate mice at 18–22 weeks of age. A, Synaptic transmission as assessed by measuring the relationship of the rising slope of the fEPSP (postsynaptic depolarization) versus the rising slope of the fiber volley (presynaptic depolarization) over various stimulus intensities was significantly enhanced in Mecp2^308/Y mice relative to littermates. Inset, Representative traces show wt (solid line) and Mecp2^308/Y (dashed line) responses when maximal stimulus intensities were applied. Calibration: 1 mV, 5 ms. Note the identical slopes of the fiber volley, but the Mecp2^308/Y slice exhibits a dramatically larger fEPSP slope. B, Paired-pulse facilitation was significantly attenuated in Mecp2^308/Y mice relative to littermates, indicating neurotransmitter release was enhanced. Error bars indicate SEM.

Figure 5.

Long-term potentiation is impaired in area CA1 of the hippocampus ofMecp2^308/Y mice. Two different forms of LTP were assessed in Mecp2³⁰⁸ and littermate animals at 18–22 weeks of age. A, LTP induced with two 100 Hz tetani (1 s, 20 s apart) was significantly impaired in Mecp2^308/Y mice relative to littermate controls. B, LTP induced with theta-burst stimulation wassignificantlyimpairedinMecp2^308/Y mice relative to littermate controls. At wo-way ANOVA with repeated measures was used to analyze both data sets throughout the duration of the whole experiment. Representative traces are shown to the right of summary data. Calibration: 2 mV, 5 ms. Error bars indicate SEM.

Figure 6.

Long-term depression is impaired in area CA1 of the hippocampus of Mecp2^308/Y mice. Two different forms of LTD were assessed in Mecp2^308/Y mice and littermates at 4–7 weeks of age. A, Input–output was significantly enhanced in Mecp2^308/Y mice relative to littermate controls, indicating that the enhancement in basal synaptic connectivity was present at an early age. B, LTD induced by the group I mGluR agonist DHPG (50μm) was unaffected by the Mecp2³⁰⁸ mutation. C, LTD induced by 900 paired stimuli (50 msinterstimulus interval, 1 Hz) in the presence of AP-5 (50 μm) was significantly impaired in Mecp2^308/Y mice relative to littermate controls. Representative traces are shown above summary data. Calibration: 1 mV, 5 ms. Error bars indicate SEM.

Figure 7.

Long-term potentiation is impaired in primary motor and sensory cortex of Mecp2^308/Y mice. A, Cumulative plot of the input–output curves (field potential recruitment recorded in layer II/III of M1 motor cortex) shows normal basal synaptic transmission in Mecp2^308/Y mice compared with control littermates (p > 0.05). B, M1-LTP is impaired in Mecp2^308/Y mice compared with control littermates (p = 0.01). C, Cumulative plot of the input–output curves (field potential recruitment recorded in layer II/III of S1 sensory cortex) shows normal basal synaptic transmission in Mecp2^308/Y mice compared with control littermates (p > 0.05). D, S1-LTP is impaired in Mecp2^308/Y mice compared with control littermates (p = 0.02). Values represent mean ± SEM.