Synaptic plasticity deficits in an experimental model of rett syndrome: long-term potentiation saturation and its pharmacological reversal

Neuroscience 180 (2011) 314 –321

SYNAPTIC PLASTICITY DEFICITS IN AN EXPERIMENTAL MODEL OF RETT SYNDROME: LONG-TERM POTENTIATION SATURATION AND ITS PHARMACOLOGICAL REVERSAL S.-M. WENG,a,b,c F. McLEOD,a M. E. S. BAILEYb AND S. R. COBBa*

tation, by onset of overt signs several months postnatally and by a constellation of clinical features (Neul et al., 2010). Distinctive aspects contributing to the diagnosis include developmental regression (with accompanying loss of hand skills, mobility skills and speech) and stereotypic hand movements. During regression social interaction deficits (with features reminiscent of autism) are common. Associated features, such as microcephaly, respiratory/autonomic abnormalities, seizures, scoliosis, growth deficits and early hypotonia, are very prevalent. RTT cases are usually the result of dominantly-acting, sporadic mutations in the X-linked gene MECP2, which encodes methyl-CpG-binding protein 2 (MeCP2) (Amir et al., 1999). MeCP2 is expressed quite widely throughout the body, with notably high expression in postnatal neurons (LaSalle et al., 2001; Shahbazian et al., 2002; Zhou et al., 2006). Most pathogenic mutations in MECP2 cause RTT in heterozygous females, while mutations leading to other phenotypic outcomes are also known (Moretti and Zoghbi, 2006). Boys inheriting a mutant MECP2 allele are much more severely affected, presenting with infantile encephalopathy and usually not surviving infancy. Since most MECP2 mutations leading to RTT involve loss of function of the mutant allele, RTT can be modelled using gene knockout mice that recapitulate many of the key clinical signs that characterise RTT in humans (Chen et al., 2001; Guy et al., 2001). In most of the models, the null males demonstrate apparently normal early development before the onset of overt signs at about 6 weeks of age, and progression is usually fairly aggressive, with death at about 16 –20 weeks. Heterozygous female mice have a more delayed onset of overt signs and much slower progression over a period of months followed by stabilisation. The neurobiological changes in the RTT brain are likely to be complex. At the cellular level, studies show rather subtle changes in neuronal electrical properties (Dani et al., 2005; Taneja et al., 2009; Kline et al., 2010) but more overt changes in synaptic function including reduced synaptic plasticity (Asaka et al., 2006; Moretti et al., 2006; Guy et al., 2007; Nelson et al., 2008) and changes in basal inhibitory and excitatory synaptic transmission (Dani et al., 2005; Medrihan et al., 2008; Nelson et al., 2008; D’Cruz et al., 2010; Kline et al., 2010; Maliszewska-Cyna et al., 2010). Anatomical studies have shown changes in synaptic connectivity and neuronal structure (Armstrong et al., 1995, 1998, 2005; Belichenko et al., 1997; Chao et al., 2007) whilst at the network level there are changes in

a Institute for Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK b

School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8QQ, UK

c

Department of Paediatrics, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan

Abstract—Rett syndrome (RTT), a disorder caused almost exclusively by mutations in the X-linked gene, MECP2, has a phenotype thought to be primarily of neurological origin. Disruption of Mecp2 in mice results in a prominent RTT-like phenotype. One of the consequences of MeCP2 absence in the brain is altered functional and structural plasticity. We aimed to characterize synaptic effects related to plasticity in the hippocampus further and establish whether plasticity defects are amenable to pharmacological reversal. Using male mice in which Mecp2 expression was prevented by a stop cassette, we assessed synaptic plasticity in area CA1 at different phenotypic stages, scoring the mice weekly for overt RTT-like signs. Strongly symptomatic Mecp2stop/y mice displayed reduced long-term potentiation (LTP, 40.2ⴞ1.6% of wild-type), post-tetanic potentiation (PTP, 45ⴞ18.8% of wildtype) and paired-pulse facilitation (PPF, 78ⴞ0.1% of wild type) (all P<0.05), the impairment increasing with symptom severity score. These plasticity impairments were absent in presymptomatic mice. Repeated high frequency stimulation revealed pronounced LTP saturation in symptomatic Mecp2stop/y mice, suggesting an LTP ‘ceiling’ effect. Bath application of the weak NMDA receptor blocker memantine (1 ␮M) resulted in partial restoration of a short-term plasticity component. These data support that idea that progressive functional synaptic impairment is a key feature in the RTT brain and demonstrate the potential for the pharmacological restoration of plasticity function. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: MECP2, Rett syndrome, memantine, LTP, plasticity.

Rett syndrome (RTT; MIM 312750) is a predominantly neurological disorder and a primary cause of severe mental retardation in girls with an incidence of approximately one in 10,000 female births (Neul et al., 2010). RTT is characterised by an almost complete female gender limi*Corresponding author. Tel: ⫹44-(0)141-330-2914. E-mail address: [email protected] (S. R. Cobb). Abbreviations: fEPSPs, field excitatory postsynaptic potentials; HFS, high frequency stimulation; LTP, long-term potentiation; MeCP2, methyl-CpG-binding protein 2; PPF, paired-pulse facilitation; PTP, posttetanic potentiation; RTT, Rett syndrome.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.01.061

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network excitability (Zhang et al., 2008; D’Cruz et al., 2010). It appears that although lack of functional Mecp2 results in a nervous system primed to malfunction at a critical point during postnatal brain development, function can be restored to a large degree once MeCP2 expression is restored to wild-type levels, raising expectations about the possibility of therapeutic intervention (Guy et al., 2007). There is increasing evidence that MeCP2 is an important regulator of neuronal plasticity and that synaptopathy is a major component of the Rett phenotype. Our aim in this study was to characterise hippocampal synaptic plasticity in the Mecp2-stop mouse model of RTT and to investigate the potential for pharmacological manipulation of plasticity deficits.

EXPERIMENTAL PROCEDURES Mecp2-stop mice Heterozygous female Mecp2-stop mice (Mecp2stop/⫹ genotype) in which one endogenous Mecp2 allele is silenced by a targeted STOP cassette (Guy et al., 2007) were obtained from the laboratory of Prof. Adrian Bird (University of Edinburgh, Edinburgh, UK). A local colony was established by breeding heterozygous Mecp2stop/⫹ females with wild-type males. All mice used in experiments were hemizygous Mecp2stop/y males and wild-type male littermates resulting from a breeding scheme involving at least six generations of backcross from a congenic C57BL/6 background onto a BALB/c background. Offspring genotype was determined by PCR as described in (Guy et al., 2007). Mice were housed in groups of one to three, maintained on a 12 h light/dark cycle and provided with food and water ad libitum. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and a project license with local ethical approval under the UK Scientific Procedures Act (1986).

Phenotype scoring Mice were scored for a number of signs of the Mecp2 knockout phenotype as previously described (Guy et al., 2007). These assessments probed the cardinal RTT-like features and were carried out weekly to generate a semi-quantitative measure of phenotype status. Each of six observable features including mobility, gait, hindlimb clasping, tremor*, breathing* and general condition* were scored on a 0 (no signs), 1 (mild signs) or 2 (severe signs) scale, and scores for each feature were added to yield a composite symptom score between 0 and 12. This score was also used to determine when animals should be subjected to humane culling. Any animal scoring 2 for any of the categories indicated* would routinely be culled. Thus animals referred to in the text (for reasons of brevity) as having “died” had been subject to culling according to these criteria.

Immunohistochemistry and immunoblotting Protein extracts for Western blot analysis were prepared from isolated hippocampus. Samples were homogenized in a lysis buffer (20 mM HEPES, PH⫽7.0, 10 mM KCl, 1 mM MgCl2, 0.5 mM DTT, 0.1% Triton X-100, 20% Glycerol, 2 mM PMSF, 5 mg/ml Aprotinin, 5 mg/ml Leupeptin) with a disposable pellet pestle. Protein concentration was quantified using the Bradford assay. Protein samples were mixed with 4⫻ lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Paisley, UK) and boiled for 5 min to shear genomic DNA. Equal masses of protein (20 ␮g) were electrophoresed through a 4 –12% Bis-Tris NuPAGE gradient gel and transferred by electroblotting onto PVDF membrane. Membranes

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were cut to separate MeCP2 from GAPDH-containing regions and each segment placed in blocking solution containing 5% (w/v) dried skimmed milk powder (Marvel) diluted in Tris Buffered Saline (137 mM NaCl, 20 mM Tris–HCl) for 1 h and incubated overnight at 4 °C with rabbit anti-MeCP2 (Millipore, Watford, UK, 07-013; 1/1000 dilution) or rabbit anti-GAPDH (Santa Cruz, Heidelberg, Germany, SC-25778; 1/2000 dilution) primary antibodies, as appropriate. Goat anti-rabbit secondary antibody (Millipore, Watford, UK) conjugated with horseradish peroxidase was applied and results visualised using enhanced chemiluminescence (Thermo Scientific, Epsom, UK). For immunohistochemistry, mice were deeply anaesthetised with pentobarbital (Sagital, 150 mg/kg, i.p.) and fixed by intracardiac perfusion (4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.3). Coronal sections were cut at 60 ␮m thickness (Leica VT1000 microtome, Leica, UK) and washed prior to overnight incubation with anti-MeCP2 (Millipore, Watford, UK, 07-013; 1/500 dilution) in phosphate buffered saline (PBS) containing 0.3% Triton X-100. After washes in 0.1 M PB, sections were incubated for 3 h in 0.1 M PB with a fluorescent secondary antibody, goat anti-rabbit Alexa Fluor 488, at 1/500 dilution. Finally, sections were washed three times in PBS before being mounted under a coverslip in DAPI-containing Vectashield (Vector Laboratories, UK) for fluorescence detection by confocal microscopy (Radiance 2100, BioRad, UK).

Electrophysiology Mice were killed by cervical dislocation and brains transferred to ice-cold oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF; in mM): NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; MgSO4, 1; glucose, 10; CaCl2, 2. Transverse (parasaggital) hippocampal slices (400 ␮m thick) were prepared and transferred to an interface-type tissue chamber maintained at 32 °C, as described (McNair et al., 2006). Standard extracellular recording was used to monitor field excitatory postsynaptic potentials (fEPSPs) in the stratum radiatum of area CA1 in response to Schaffercollateral afferent stimulation (range⫽0 –30 mA, DS2A, Digitimer, UK). Both recording and stimulating electrodes were glass patch electrodes filled with ACSF and with a resistance of 1–5 M⍀. Recordings were made with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA) and signals further processed (100⫻ amplification, 5 kHz low pass filter) using a Brownlee model 440 signal processor (Brownlee Inc. CA, USA). Data were monitored on-line and fEPSP slopes re-analysed off-line using WinLTP software (Anderson and Collingridge, 2001). fEPSPs were evoked at a frequency of 0.05 Hz and long-term potentiation (LTP) was induced by high frequency stimulation (HFS; a single train of 100 stimuli at 100 Hz). Stimulation strength was set to approximately 50% of the maximal response. Most recordings used a stimulation protocol involving paired pulses 50 ms apart. Paired-pulse facilitation (PPF) was calculated as the slope ratio of second to first response for each paired stimulation event. Posttetanic potentiation (PTP) was calculated as the mean of the first three slope data points following HFS. LTP was measured at 40 – 60 min post-HFS and at 12–15 min post-HFS in the LTP saturation experiments. Data were gathered from recordings made from one to three slices per mouse brain and are expressed as mean⫾SEM of all the slices. Wild-type mice used as controls were age-matched to their respective experimental (i.e. symptomatic or presymptomatic) groups. Data from the PPF, PTP and LTP experiments were compared by a repeated-measures ANOVA with Tukey’s post hoc analysis (Minitab version 10) and statistical significance was accepted at P⬍0.05.

Drugs and drug application For in vitro experiments, 1 mg/ml memantine stock was made by dissolving memantine hydrochloride (Tocris Bioscience, Bristol,

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UK) in ACSF. This was subsequently added to the slice perfusion medium at 1 ␮M final concentration and pumped into the bath containing the slices being recorded. Recordings were commenced 30 – 60 min after the slices first became exposed to memantine. For in vivo dosing, memantine hydrochloride was dissolved in sterile water. Dosing was administered orally by gavage at a dose of 30 mg/kg. Mice were dosed daily over a 28 day period.

RESULTS Robust silencing of MeCP2 expression in the Mecp2-stop mouse model Western blot analysis confirmed the absence of detectable MeCP2 in the brains of the hemizygous Mecp2stop/y mice (Fig. 1A) used in this study. This conclusion was supported by the absence of detectable MeCP2 within the hippocampal formation of these mice by immunohistochemistry (Fig.

1B). In contrast, in wild-type mice strong MeCP2 signal was detected from a similar amount of protein in the Western analysis and strong expression was apparent in the majority of cell nuclei in the hippocampal formation including nearly all principal cells of the hippocampus and dentate gyrus. Thus the functional silencing of Mecp2 in this knockout model was robust. Onset and progression of the phenotype in Mecp2stop/y mice The hemizygous male mice used here developed overt signs during postnatal development that mirrored those reported (Guy et al., 2007). In order to select appropriate time points for the electrophysiological experiments we monitored the onset and progression of the observable phenotype using the semi-quantitative observational scoring system developed previously (Guy et al., 2007).

Fig. 1. Mecp2stop/y mice show deficits in LTP and reduced paired-pulse facilitation upon onset of neurological signs. (A) Western blot of protein extract from hippocampus of wild-type and hemizygous male mice. Samples from each of two mice were loaded in adjacent lanes. GAPDH detection was carried out as a loading control. (B) Fluorescence micrographs of dentate gyrus from wild-type (I, II) and from hemizygous (III, IV) male mice immunostained for MeCP2 (II, IV) and co-stained with DAPI (I, III) to reveal nuclei. (C) Time plot showing onset and progression of phenotypic signs in Mecp2-stop mice (n⫽15). Scores for wild-type mice (n⫽10) are also plotted for comparison. Note that hemizygous Mecp2stop/y mice develop overt signs from around 5 wk of age, with the severity score increasing over the subsequent 10 –12 wk. The curve for the hemizygous mice is censored at the top end due to the humane culling policy in operation (see Experimental procedures). (D) Stimulus intensity plot showing fEPSP slope (mean⫾SEM; n⫽6 –10 slices, four to nine mice in each group) in response to increasing stimulus strength for wild-type (●) and Mecp2stop/y samples. Hemizygous males were used at two developmental time points, presymptomatic (severity score ⬍2, blue symbols), and symptomatic (mean severity score 5.3⫾0.2; generally at between 12 and 15 wk of age, orange symbols). (E) Demonstration of LTP in wild-type and hemizygous male mice. EPSP slope is plotted against time after HFS stimulation (large arrow; time 0), as a percentage of the averaged pre-HFS baseline slope for each slice used. Insets show representative fEPSPs before (small arrow; 1) and following (small arrow; 2) HFS stimulation for each of the three comparison groups. Arrowheads indicate point of stimulus. (F) Paired-pulse facilitation recorded in the same experiments as in (E). Paired-pulse ratios are plotted as mean⫾SEM (n⫽6 –10 slices, four to nine mice per group). The slope ratio time course is plotted relative to the delivery of HFS stimulation (time⫽0). Insets show representative fEPSPs in response to paired stimuli (arrowheads) for the presymptomatic and symptomatic comparison groups. Graph to right shows pooled paired-pulse facilitation data showing significant deficit in slices from symptomatic mice but similar paired pulse ratios in presymptomatic Mecp2-stop mice and wild-type mice sampled at 4 –5 wk (first WT bar) and at 12–15 wk (second WT bar) showing absence of age-depended changes in paired-pulse response. Scale bars in (E, F): vertical, 0.2 mV; horizontal, 10 ms. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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Hemizygous male mice showed no overt phenotype for the first 4 –5 postnatal weeks and were indistinguishable from their wild-type littermates. Thereafter, Mecp2stop/y mice developed a range of signs including hypoactivity, hindlimb clasping, gait disturbance, tremor, breathing disturbances and general signs of deterioration including hunched and ungroomed appearance. Scoring revealed onset usually between 4 and 6 weeks with the aggregate score then increasing rapidly over the next few weeks (Fig. 1C). In contrast, wild type animals did not develop signs and scored 0 for the duration of the study. Synaptic plasticity defects in strongly symptomatic mice Synaptic plasticity was investigated in acute hippocampal slices from hemizygous male Mecp2stop/y mice and littermate controls. Assessment of evoked synaptic transmission at the Schaffer-collateral-to-CA1 pyramidal cell synapse using conventional field recording revealed no significant differences between genotype or phenotype groups with respect to baseline fEPSP slope (Fig. 1D, P⫽0.42, repeated-measures ANOVA) or presynaptic volley amplitude (not shown, P⫽0.61, n⫽30 slices from 12 to 15 mice per group). Assessment of LTP revealed that HFS produced a robust and enduring potentiation of the fEPSP slope in slices from wild-type mice (152.7⫾5.3% of baseline measured at 1 h following HFS; n⫽16 slices from 13 mice, Fig. 1E). The two groups of wild-type mice used as age-matched controls for the presymptomatic (4 weeks old, n⫽6 slices from four mice) and symptomatic (12–15 weeks old, n⫽10 slices from nine mice) groups of Mecp2stop/y mice were themselves compared for age-related differences in LTP and no such differences were observed (P⫽0.45, t-test), so data from these two groups have been pooled in subsequent analyses. Presymptomatic Mecp2stop/y mice showed a robust LTP (155.2⫾10.4% of baseline at 1 h, n⫽6 slices from four mice) that was similar in magnitude to that evoked in wild-type controls (P⫽0.35). In contrast, slices obtained from symptomatic Mecp2stop/y mice (mean severity score 5.3⫾0.2) showed a significantly lower LTP (115.7⫾3.2% of baseline at 1 h, (n⫽10 slices from nine mice), which represents approximately 40% of the wild-type value) than both the wild-type and presymptomatic groups (F(2,28)⫽12.14; P⬍0.001 for both post hoc pairwise comparisons). PTP also differed between groups (F(2,25)⫽3.492; P⫽0.046), with symptomatic Mecp2stop/y mice exhibiting PTP values that were 45⫾18% of those observed in wild-type mice (wt⫽ 216⫾46.2% of baseline, stop⫽97.8⫾40.8%; post hoc Tukey test P⫽0.036, Fig. 2), while presymptomatic mice did not differ from wild-type. To establish a measure of presynaptic function, PPF was evaluated (Fig. 1F). Tested by repeated-measures ANOVA, there were significant differences between the symptomatic Mecp2stop/y group and the other two (wild-type and presymptomatic Mecp2stop/y) groups (F(2,29)⫽16.978; P⬍0.001). As with LTP, PPF did not show any age-related differences in wild-type mice. The paired pulse ratio was also found to be of similar magnitude in presymptomatic Mecp2stop/y mice

Fig. 2. Synaptic plasticity deficits in the symptomatic Mecp2stop/y mouse is associated with a saturation of LTP. (A) Time plot showing mean fEPSP slope upon repeated high frequency stimulation of afferents in hippocampal slices prepared from wild-type (score 0) and increasingly symptomatic (symptom score range 2– 6) Mecp2stop/y mice. Inset shows representative traces from a wild-type (top) and symptomatic Mecp2-stop (bottom) mice. Scale bar 0.25 mV, 5 ms. Stimulus (artefact removed for clarity) is indicated by inverted arrow head. Traces represent baseline and sweeps at 15 min following HFS 1, 2, 3 and 4. (B) Bar plot showing 15 min LTP (mean⫾SEM, n⫽7–10 slices, four to eight mice per group) following each consecutive HFS, normalised to the preceding pre-HFS baseline. Note that fEPSP slope shows a robust further LTP enhancement in response to second and subsequent HFS in wild-type mice but a reduced propensity to produce further LTP enhancement in slices from symptomatic Mecp2stop/y mice. (C) Bar plot showing level of post-tetanic potentiation (PTP; mean⫾SEM, n⫽7–10 slices, four to eight mice per group) following HFS in wild-type littermate controls (score 0) and progressively more symptomatic Mecp2stop/y mice. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

(1.85⫾0.04) and wild-type controls (1.84⫾0.02). In contrast, symptomatic Mecp2stop/y mice showed a significant reduction in baseline paired pulse ratio (ratio⫽1.43⫾0.02; post hoc Tukey test P⬍0.001) compared to age-matched wild-type controls. PPF analysis over the duration of the LTP experiments (Fig. 1F) revealed that the symptomatic mice showed a consistent deficit relative to both other groups (before and after the induction of LTP), with the exception of a brief period immediately following HFS (coinciding with the peak of the post-tetanic potentiation) during which all groups showed a similar reduced peak ratio. Abnormal LTP saturation characteristics in Mecp2stop/y mice Further assessment of plasticity defects in the Mecp2stop/y mice was conducted by delivering repeated episodes of HFS (carried out in separate experiments to those described above) to investigate possible saturation effects

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(Fig. 2A). In wild-type mice synaptic tone increased over four successive rounds of HFS at 15 min intervals, each successive increase in EPSP slope being smaller than the previous one such that a plateau is being approached by 1 h (LTP⫽194⫾17%, 274⫾21%, 277⫾19% and 300⫾19% of baseline for four consecutive HFS). In contrast, strongly symptomatic Mecp2stop/y mice showed, in addition to their deficit in the response to the initial HFS, a profound inability to generate further LTP following subsequent HFS episodes and even a modest depression in the most symptomatic mice (Fig. 2A, B; LTP⫽133⫾13%, 141⫾19%, 129⫾16% and 122⫾15% of baseline for four consecutive HFS). Slices obtained from mildly symptomatic (phenotype score of 2– 4) mice, which did not display overt LTP deficits after the first HFS over 15 min, did reveal an intermediate but significant additional LTP deficit in response to subsequent episodes of HFS (Fig. 2A, B). Analysis of PTP in these experiments (Fig. 2C) revealed a similar impairment in symptomatic mice (mean wild-type PTP⫽268⫾49% of baseline, n⫽10 slices from eight mice; mean symptomatic (score 5⫹) Mecp2stop/y PTP⫽176⫾31% of baseline, n⫽7 slices from four mice) that tended to increase with increasing symptom score (P⫽0.074 in a linear regression of PTP vs. aggregate symptom score, n⫽29; wild-type mice were treated as having a symptom score of 1 for this test to mimic linearity; symptomatic mice with scores of 5 and 6 were pooled for this analysis). Memantine partially reverses synaptic deficits in hippocampal slices from Mecp2stop/y mice Previous reports have suggested that disease-related LTP deficits, saturation and ceiling effects may be due to increased tonic basal activation of postsynaptic NMDA receptors and are amenable to reversal by the weak NMDA receptor-blocking drug, memantine (Frankiewicz and Parsons, 1999). We therefore repeated the LTP saturation experiments in slices pre-incubated and bathed in 1 ␮M memantine. In control slices from wild-type mice, application of memantine did not produce any significant change in LTP, PTP or LTP saturation (Fig. 3). In contrast, in slices from strongly symptomatic (mean score⫽5.3⫾0.2, n⫽7 slices from four mice) Mecp2stop/y mice memantine produced a significant enhancement of both PTP and 15 min LTP (i.e. following the first HFS), restoring these parameters to wild-type levels (⫹memantine: 100% of wild-type 15 min LTP, 104% of wild-type PTP; untreated: 69% of wildtype LTP and 61% of wild-type PTP for untreated Mecp2stop/y samples). In response to subsequent episodes of HFS, memantine-treated slices from Mecp2stop/y mice showed enhanced PTP and short term plasticity but the potentiation rapidly decayed to the preceding baseline level within the 15 min inter-HFS period. Memantine does not affect symptom onset or lethality in Mecp2stop/y mice The precise underlying pathophysiology of RTT is likely to be complex but may involve a significant ‘neuroplasticity’ element (Cobb et al., 2010). Indeed, genetic rescue of symptoms by delayed activation of Mecp2 in mice has

Fig. 3. Partial reversal of plasticity deficits in symptomatic Mecp2stop/y mice by memantine. Time plot showing reduced hippocampal LTP and PTP and elevated synaptic saturation in slices from symptomatic Mecp2stop/y mice (orange ●, seven slices from four mice) compared to wild-type littermate controls (black ●, 10 slices from eight mice). LTP/ PTP reduction and LTP saturation is partially reversed in Mecp2stop/y slices incubated in 1 ␮M memantine (orange Œ, 10 slices from five mice). In contrast, memantine did not affect LTP, PTP or LTP saturation in slices obtained from wild-type mice (black Œ, eight slices from five mice). Arrows indicate points of afferent HFS. The dotted line indicates the mean LTP level 15 min after the first HFS in memantinetreated Mecp2stop/y mice for easier comparison with subsequent 15 min post-HFS time points. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

been shown to be associated with a reversal of LTP defects (Guy et al., 2007). In light of the apparent ability of memantine to produce a partial reversal of plasticity deficits in vitro (Fig. 3) and improve symptoms in other neurodevelopmental models (Rueda et al., 2010), we examined whether memantine administered systemically could effect any changes in the expression or progression of the phenotype in the Mecp2stop/y mice. Memantine administered daily from the onset of signs in Mecp2stop/y mice for a 4 week period revealed no significant difference in severity or progression from vehicle-treated Mecp2stop/y mice (Fig. 4A, n⫽8 mice). As expected, wild type mice scored 0 throughout the study and memantine administration did not affect this score. Mecp2stop/y mice had reduced survival, but again this was not altered by memantine treatment (Fig. 4B).

DISCUSSION The current study demonstrates robust evidence for deficits in both short- and long-term forms of hippocampal synaptic plasticity in the brain of hemizygous mice in which Mecp2 expression has been prevented by targeted STOP cassette. These findings are in agreement with reports of deficits in previous studies conducted on Mecp2-null mice (Asaka et al., 2006) and in mice expressing a truncated form of MeCP2 (Moretti et al., 2006). An important observation is that hippocampal LTP is not impaired in mice during the first weeks of postnatal development but rather the deficits appear with the onset of overt RTT-like symptoms. These data mirror earlier studies conducted in hemizygous Mecp2-null mice (Asaka et al., 2006) and are consonant with studies in heterozygous female Mecp2-stop mice, which develop symptoms much later (at 6 –9 months) (Guy et al., 2007). These findings add to the

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Fig. 4. Memantine does not affect progression of Mecp2stop/y phenotype. (A) Plot showing phenotypic severity progression in wild-type (grey/black symbols) and Mecp2stop/y (orange symbols) mice treated with memantine (oral dosing 30 mg/kg, open symbols) or vehicle (closed symbols). (B) Survival curves plotted for the same four groups of mice (see Experimental procedures). Note that vertical steps indicate deaths due to culling under the humane culling policy (i.e. determined by symptom score) rather than natural lifespan in the Mecp2stop/y mice. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

growing consensus that the absence of functional MeCP2 impairs functional synaptic plasticity in the maturing nervous system. In contrast, those studies reporting the absence of deficits have mainly been confined to experiments conducted using relatively immature tissues from presumably asymptomatic mice (Dani and Nelson, 2009). However, an important finding of the current study is that deficits in synaptic plasticity can be observed in very mildly symptomatic male mice (from ⬃5 weeks postnatal) by adopting appropriate plasticity induction paradigms that assess cumulative synaptic potentiation (Fig. 2). With symptom progression, these subtle abnormalities in synaptic plasticity become more overt and can be detected by a single HFS protocol for LTP induction. These data hint at synaptic plasticity deficits being an early indicator of the disease process although the precise cellular and synaptic dysfunctions and their mechanistic link with MeCP2 deficiency remain obscure. In addition to long-term potentiation, we have shown that short-term forms of plasticity, including post-tetanic potentiation as well as paired-pulse facilitation, are also impaired in Mecp2stop/y mice upon symptom onset. As with LTP, these deficits become more pronounced with symptom progression. Whilst this is the first report addressing short-term plasticity in the Mecp2stop/y mouse, similar observations have been made in Mecp2-null mice (Asaka et al., 2006) and the Mecp2308 line (Moretti et al., 2006). Both PTP and PPF are believed to represent plasticity processes with largely presynaptic loci of expression (Zucker and Regehr, 2002). In contrast, sustained LTP at 15 and especially 60 min following high frequency stimulation has been shown to be mainly of postsynaptic origin at the Schaffer collateral-to-CA1 pyramidal cell synapse (Nicoll, 2003). It is conspicuous that the deficits in the two forms of plasticity (LTP and PTP) show strong parallelism. It is however unclear from the current data whether this relationship can be explained by some overlap in the underlying mechanisms or whether the reduction in LTP is a direct consequence of reduced PTP mediated via a predominantly presynaptic dysfunction. However, our previous

studies on heterozygous Mecp2stop/y mice showed clear deficits in LTP following theta burst stimulation, in which pronounced PTP was absent (Guy et al., 2007). As such, it appears that both presynaptic and postsynaptic elements contribute to the deficits in hippocampal synaptic plasticity in mice with no expression of Mecp2. Whilst the exact mechanisms underlying the LTP deficits are difficult to assess from the extracellular recording data, one possibility is that there exists an overall reduction in plasticity per se with no change in basal synaptic strength. The input-output data (Fig. 1D) would be consistent with this interpretation. However, another possibility is that the apparent reduced LTP is due to synapses operating close to a maximal ceiling. Indeed, the inability to produce cumulative LTP in response to repeated high frequency afferent stimulation in slices from the mutant male mice supports the possibility that neurotransmission at the Schaffer collateral-to-CA1 pyramidal cell synapses in these mice operates in a state that is closer to saturation than in presymptomatic or wild-type mice. It is possible that this relates to the reduced synapse size as reported in the Mecp2308 model (Moretti et al., 2006), or to a reduction in the prevalence of NMDA receptor subunits within each synapse (Maliszewska-Cyna et al., 2010). In the latter case, this could be associated with consequent effects on glutamate receptor recycling and recruitment to synapses or effects on the levels of other synaptic proteins (Asaka et al., 2006; Moretti et al., 2006; Chao et al., 2007; Maezawa and Jin, 2010). Mecp2-null mice show a reduced number of synapses/spines and thus another possibility is a heightened synaptic tone within the context of a network that is more sparsely connected (Belichenko et al., 2009a,b; Chapleau et al., 2009). A similar saturation phenomenon has been described in mice in which NMDA receptor subunit combinations are altered and which show concomitant reductions in contextural learning (Kiyama et al., 1998). LTP saturation effects can be overcome by memantine, a drug prescribed to Alzheimer disease patients (Frankiewicz and Parsons, 1999). Our findings were that memantine applied to mutant

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mouse hippocampal slices at clinically-relevant concentrations (Frankiewicz and Parsons, 1999; Minkeviciene et al., 2004) resulted in heightened synaptic potentiation following high frequency stimulation (Fig. 3). In particular, memantine had a marked effect on the response to the first high frequency stimulation (elevated LTP). However, the effect of memantine on the subsequent episodes of HFS was limited to a transient boost of short-term plasticity which typically decayed to baseline within a 15 min period. This was a specific action targeting the MeCP2-related pathophysiology, as memantine had no effect on basal evoked synaptic transmission or control slices subjected to the LTP saturation paradigm. In contrast to the clear actions of memantine on hippocampal synaptic transmission in vitro in the disease state, the repeated dosing of mice with memantine did not change either the progression of symptoms or the survival of Mecp2-stop mice. Memantine has been used successfully to improve aspects of hippocampus-dependent dysfunction in a transgenic model of Alzheimer disease (Minkeviciene et al., 2004) including LTP deficits at the Schaffer-collateral-to-CA1-pyramidal cell synapse (Martinez-Coria et al., 2010) and has been shown recently to normalise phenotypic factors such as cognitive features and synaptic marker levels in a mouse model of Down syndrome (Rueda et al., 2010). The absence of any detectable improvement in RTT-like phenotype suggests that this drug may not hold promise in treating the full range of symptoms of RTT. However, males are very severely affected by loss of MeCP2 and it remains to be tested whether memantine can be beneficial in heterozygous females, which display a milder and more stable phenotype. In addition, the plasticity impairments we have demonstrated here ex vivo may manifest in a range of subtle cognitive and behavioural aspects of the Rett-like phenotype rather than affecting the observable signs and lethality. Thus, assessing the potential for memantine to improve the phenotype in vivo may require appropriate targeted outcome assays. Male Mecp2Stop/y mice have severe motor deficits and are difficult to screen in cognitive assays. Our phenotypic assessment of RTT-like signs does not involve a specific measure of hippocampal dysfunction and it is therefore difficult to draw comparisons between the data obtained here in vitro and in vivo. Abnormal synaptic plasticity is a prominent feature in RTT and a range of related ‘neurodevelopmental’ disorders. Here we have shown that synaptic plasticity shows progressive impairment in a mouse model of Rett syndrome. The ability to promote the restoration of normal plasticity remains an attractive approach in targeting the neurological dysfunction. The current findings suggest that memantine has the ability to target synaptic function in disease models without overt alteration in normal (control) synaptic tissues. It is possible that this ability to discriminate may be of benefit in the female Mecp2-mutant brain where neurons exist in a mosaic network in which there is evidence for cell autonomous (for MeCP2 status) synaptic changes (Belichenko et al., 2009a). However, the results in male mice presented in this report indicate that memantine

is ineffective in altering the disease progression in the male brain. Acknowledgments—We are grateful to the MRC (award G0800401) and Rett Syndrome Association Scotland (RSA-S) for support. SMW is funded by a studentship from the Taiwan government. We thank Imre Vida for comments on the manuscript.

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(Accepted 29 January 2011) (Available online 4 February 2011)