Reduced seizure threshold and altered network oscillatory properties in a mouse model of Rett syndrome
Highlights
► We show altered seizure threshold in a mouse model of Rett syndrome. ► Network level hyperexcitability is also revealed in brain slices from Rett mice. ► This contrasts with normal intrinsic excitability at the level of single neurons.
Introduction
Rett syndrome (RTT), traditionally considered a neurodevelopmental disorder, mainly affects girls and is principally due to mutations in the x-linked gene methyl-CpG-binding protein 2 (MECP2) (Amir et al., 1999, Neul et al., 2010, Gadalla et al., 2011). The age of onset can vary with characteristic symptoms including loss of speech, reduced head growth, stereotypic hand movements, motor dysfunction and autistic-like features (Chahrour and Zoghbi, 2007, Neul et al., 2010). The development of epilepsy in ∼50–80% of RTT patients is another prominent phenotype (Hagberg et al., 2002, Glaze et al., 2010) with diverse seizure types ranging from complex partial to myoclonic seizures (Steffenburg et al., 2001, Kim et al., 2012). Epilepsies are thus common in RTT and have an age-related onset but with the severity of seizures appearing to fall in late adolescence (Steffenburg et al., 2001). Some authors report no significant clinical difference in seizures between patient genotypes (Cardoza et al., 2011) but a recent large scale study suggests that seizures may indeed vary by mutation type with T158M (74%) and R106W (78%) mutations being most frequently associated with epilepsy (Glaze et al., 2010). The occurrence of seizures is also associated with a greater overall clinical severity including impaired ambulation and communication. Abnormal electroencephalography (EEG) recordings are commonly detected in RTT patients including giant evoked somatosensory potentials (cortical hyperexcitability), epileptiform abnormalities and the occurrence of rhythmic slow theta activity (Glaze, 2005). Whilst the EEG is invariably abnormal at some stage, there is no characteristic or diagnostic EEG pattern for RTT (Glaze, 2005).
Whilst the majority (>95%) of classical RTT cases are due to mutations in the gene methyl-CpG-binding protein 2 (MECP2), the underlying function of MeCP2 protein and its regulation remain unclear (Gadalla et al., 2011, Guy et al., 2011). Many lines of mice have been developed in which Mecp2 has been deleted, silenced or mutated to mimic major human mutations and these mouse lines replicate many of the features observed in RTT patients (Chen et al., 2001, Guy et al., 2001, Guy et al., 2007, Shahbazian et al., 2002, Goffin et al., 2012) and provide valuable tools for investigating MeCP2-related function/dysfunctions. EEG recordings reveal Mecp2-null mice to display abnormal spontaneous rhythmic discharges of 6–9 Hz in the somatosensory cortex during wakefulness and altered theta frequency hippocampal rhythms (D’Cruz et al., 2010) with some similarities to those observed in RTT patients. In addition to background rhythms, recent studies have shown alterations in the amplitude and latency of event-related potentials (ERPs), brain activations that occur during certain behavioural tasks, in Mecp2-null mice suggesting alterations in the strength and timing of cognitive processes (Goffin et al., 2012). This study also reported an increased power of high gamma frequency (70–140 Hz) EEG in Mecp2-null mice compared to controls, an activity that can be observed in the EEG before and during seizures and perhaps indicative of a hyper-excitability phenotype (Goffin et al., 2012). Spontaneous myoclonic seizures have also been reported in mice expressing a truncated form of MeCP2 (Shahbazian et al., 2002).
Cellular level studies have indicated alterations in the balance between synaptic inhibition and excitation in cortical/hippocampal circuits of the Mecp2-null mouse (Dani et al., 2005). Features reported include reduced excitatory synaptic function (Dani et al., 2005, Asaka et al., 2006, Nelson et al., 2006), reduced synaptic plasticity (Asaka et al., 2006, Guy et al., 2007, Weng et al., 2011), altered connectivity in terms of excitatory synapses/spine number (Chao et al., 2007, Belichenko et al., 2009, Chapleau et al., 2009, Robinson et al., 2012) as well as reduced GABA levels/GABA release (Chao et al., 2010, Gadalla et al., 2012). In contrast, studies have shown a surprising absence of altered intrinsic properties in principal cells of the Mecp2-null neocortex and hippocampus (Dani et al., 2005, Zhang et al., 2008). Despite this, voltage-sensitive dye measures and multiunit recording in brain slices reveal hyper-excitability of phenotypes when viewed at the hippocampal network level (Calfa et al., 2011).
The aim of the current study was to investigate seizure threshold in a mouse model of RTT and to further characterize alterations in hippocampal network excitability resulting from MeCP2 deficiency.
Section snippets
Mecp2-stop mice
Heterozygous (Mecp2stop/+) female mice in which the endogenous Mecp2 allele is silenced by a targeted stop cassette (Mecp2tm2Bird, Jackson Laboratories stock No. 006849) were used as a breeding stock and backcrossed onto a C57BL6/J background by crossing with wild type (WT) C57BL6/J males (Harlan, UK). The genotype of the mice was determined by PCR (Guy et al., 2007). Unless otherwise stated, experiments were conducted using hemizygous (Mecp2stop/y) male mice and wild-type male littermates aged
Mecp2stop/y mice have a heightened sensitivity to kainate-induced seizures in vivo
Epilepsy is a prominent feature in RTT syndrome patients (Hagberg et al., 2002, Neul et al., 2010) and aberrant discharge patterns have been detected in EEG recordings from Mecp2-null mice (D’Cruz et al., 2010). To systematically examine the propensity of mice lacking MeCP2 to develop seizures, we challenged male Mecp2stop/y mice (functional Mecp2-KO) and their wild-type (WT) littermates with the convulsant drug kainic acid (25 mg/kg, IP) or with vehicle (saline). Mecp2stop/y mice were aged (6–10
Discussion
Epilepsy is a prominent feature of RTT patients (Hagberg et al., 2002, Glaze, 2005, Glaze et al., 2010) yet Mecp2−/y mice are reported to very rarely develop full electrographic seizures (Chao et al., 2010). The main finding of the current report was the demonstration that silencing/deletion of Mecp2 in mice resulted in a heightened susceptibility to seizures when challenged with a convulsant drug paradigm (kainic acid). Whilst not showing any overt seizure phenotype under homecage conditions,
Conclusion
In conclusion, we show that MeCP2-deficient mice show an underlying pro-seizure phenotype that can be revealed by pharmacological convulsant challenge. As such, application of this seizure challenge models may be beneficial in future studies testing novel pharmacological and genetic approach therapies in RTT (Cobb et al., 2010, Gadalla et al., 2011) to establish whether putative benefits extend into the epilepsy domain of the RTT-like phenotype.
Acknowledgements
We are grateful to the MRC (Grant G0800401) and the Rett Syndrome Association Scotland for generous support. F.M. was supported by a SULSA studentship.
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