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Mutations in MECP2 cause Rett syndrome and some related forms of mental retardation and autism. Mecp2-null mice exhibit symptoms reminiscent of Rett Syndrome including deficits in learning. Previous reports demonstrated impaired LTP in slices of symptomatic Mecp2-null mice, and decreased excitatory neurotransmission, but the causal relationship between these phenomena is unclear. Reduced plasticity could lead to altered transmission, or reduced excitatory transmission could alter the ability to induce LTP. In order to help distinguish these possibilities we compared LTP induction and baseline synaptic transmission at synapses between Layer 5 cortical pyramidal neurons in slices of WT and Mecp2-null mice. Paired recordings reveal that LTP induction mechanisms are intact in Mecp2-null connections, even after the onset of symptoms. However fewer connections were found in Mecp2-null mice and individual connections were weaker. These data suggest that loss of MeCP2 function reduces excitatory synaptic connectivity and that this precedes deficits in plasticity.
Rett syndrome (RTT; MIM:312750) is a neurodevelopmental disorder usually caused by mutations in the X-linked gene, Methyl CpG binding Protein-2 (MECP2) (Amir et al., 1999; Chahrour and Zoghbi, 2007). Affected girls appear to develop normally for the first 6-18 months but then begin displaying symptoms that include profound impairment of learning and memory, motor abnormalities and autistic behavior (Hagberg, 1997). Affected boys can exhibit a similar range of symptoms, often with a rapid and severe course (Villard, 2007). Mice lacking functional Mecp2 (Mecp2-nulls) show neurological symptoms reminiscent of RTT (Chen et al., 2001; Guy et al., 2001). Hemizygous null males develop symptoms rapidly after 4 weeks, including severe motor and respiratory abnormalities, learning deficits and lethality (Stearns et al., 2007). Postnatal MeCP2 overexpression in forebrain neurons or re-activation of a silenced endogenous allele rescues many symptoms of Mecp2-null mice (Giacometti et al., 2007; Guy et al., 2007).
Previously, we observed reduced circuit activity in neocortical slices of Mecp2-null mice due to a shift in the balance between synaptic excitation and inhibition onto Layer 5 pyramidal neurons (Dani et al., 2005). However, these changes could not be completely accounted for by the subtle changes in frequency and amplitude of miniature excitatory post-synaptic currents (mEPSCs). In dissociated hippocampal neuron cultures and autaptic cultures from neonatal Mecp2-null mice larger reductions in mEPSC frequency and evoked EPSC amplitude have been reported (Nelson et al., 2006; Chao et al., 2007), but whether these changes involve changes in release probability or in the number of release sites seems to depend on preparation. Synaptic marker staining intensity is reduced in hippocampal slices of 2-week-old Mecp2-null mice, but not in symptomatic, 5-week-old Mecp2-null mice (Chao et al., 2007), leaving open the question of precisely which functional changes in synaptic properties occur in vivo.
Deficits in long-term potentiation (LTP) at neocortical and hippocampal synapses have been reported in Mecp2-mutant mice (Asaka et al., 2006; Moretti et al., 2006). However, LTP induction using extracellular stimulation is notoriously dependent on the strength of excitatory stimulation and on the balance between excitation and inhibition (Artola and Singer, 1987; Bear et al., 1992; Bliss and Collingridge, 1993; Sjöstrom et al., 2001). Moreover, the highly recurrent nature of neocortical circuits complicates interpretation of extracellular field potentials, and renders analysis of synapse-specific potentiation less straightforward than in some circuits. Determining whether an initial plasticity deficit alters the circuit, or whether a change in circuitry alters plasticity induction, may hold important mechanistic implications for how loss of Mecp2 disrupts forebrain function.
To directly address the mechanisms underlying altered synaptic transmission and plasticity in local neocortical circuits, we studied unitary synaptic connections between L5 thick tufted pyramidal neurons using quadruple whole-cell recordings in cortical slices from 2-4-week-old Mecp2-null and wild-type mice. We observed reduced connectivity and weaker synapses in slices of 4-week-old Mecp2-null mice, while comparable LTP could be induced in both conditions. Hence an early effect of Mecp2 mutation is to reduce cortical excitatory synapses through a mechanism distinct from blocking LTP.
Male Wild type (WT) controls and Mecp2-null mice were obtained by crossing females heterozygous for the Mecp21lox allele (Chen et al., 2001) and male C57BL/6 (Jackson Laboratories, Bar Harbor, ME, USA). Mothers of all animals used in this study were progeny of back-crossings with C57BL/6 mice over > 6 generations. All procedures for animal handling and sacrificing were approved by the Animal Care and Use Committee of Brandeis University.
The artificial cerebrospinal fluid (ACSF) used for brain slicing contained (in mM): NaCl 125, KCl 2.5, MgCl2.6H2O 3, NaHPO4 1.25, NaHCO3 25, CaCl2.2H2O 1, Dextrose: 14. For incubation of slices and for recordings ACSF was modified as follows (in mM): MgCl2.6H2O 1, CaCl2.2H2O 2. ACSF was continuously bubbled with a 95:5 mixture of O2:CO2. For whole cell patch clamp recordings, pipette internal solution contained (in mM): KCl 20; K-Gluconate 100; HEPES 10; Biocytin 0.2 %(w/v); MgATP 4; Na-GTP 0.3, Na2-Phosphocreatinine 10.
Anesthetized animals were decapitated, and the brain was removed under cold ACSF. 300 μm thick coronal brain slices were obtained from 2-4-week-old animals on a Leica VT100S (Leica Microsystems, Deerfield, IL, USA). Slices were incubated initially at 35°C for 15 minutes and then kept at room temperature.
Slices were continuously perfused with oxygenated ACSF maintained at 31-33° C with a TH324B in-line solution heater and controller (Warner Instruments, Hamden, CT, USA). Somatic whole-cell patch clamp recordings were obtained by patching up to 4 visually identified thick-tufted Layer 5 (L5) pyramidal neurons in the primary somatosensory cortex. Patch pipettes were pulled from borosilicate capillaries (World Precision Instruments, Sarasota, FL, USA) on a Sutter P-97 Flaming/Brown puller (Sutter Instruments, Novato, CA). The somas of cells targeted for patching were typically within 50μm of each other. Following breakthrough of cell membrane, resting membrane properties and access resistance were checked. Identity of cell type was confirmed by testing for a non-adapting action potential (AP) firing type (Hattox and Nelson, 2007) and post-hoc staining for biocytin. Data, acquired with Multiclamp 700A and 700B amplifiers (Molecular Devices, Sunnyvale, CA), were low pass filtered at 3 KHz and digitized at 10 KHz. Recordings were acquired and analyzed using Igor Pro Software (Wavemetrics, Lake Oswego, OR) and in-house programs.
Trains of 5ms long depolarizing current pulses (0.7-1.2 nA) at 20Hz elicited timed presynaptic action potentials, and were repeated at 0.056 Hz, throughout the recording (except during LTP induction). Monosynaptic connections were identified when the offline average of at least 10 repetitions in any candidate postsynaptic neuron showed excitatory postsynaptic potentials (EPSPs) time locked with the train of presynaptic action potentials, and if the EPSP amplitude in the averaged waveform was at least 100μV. Thus, most monosynaptic connections could be easily identified (Supplemental Figure 1), but those with extremely high failure rates or low initial strength were most likely excluded.
After acquiring at least 30 baseline synaptic responses, long term potentiation (LTP) was induced with trains of 5 precisely timed action potentials at 40Hz, repeated 20 times at 0.1 Hz, such that each post-synaptic AP followed the pre-synaptic AP by 8-10ms. This spike timing relationship between pre- and post-synaptic firing is known to induce LTP at neocortical excitatory synapses (Markram et al., 1997a; Feldman, 2000; Sjöstrom et al., 2001). Recordings were included for LTP analysis only if pre-and postsynaptic cells remained stable for at least 35 minutes from the start of the baseline period, typically up to 50 minutes (70 minutes in some cases). Neurons were discarded from analysis if a >25% change in resting membrane potential (Vm), series or input resistance (Rs or Rin) was observed.
A ‘pairing’ protocol was also used to induce LTP (Malinow, 1991; Chen et al., 1999). An extracellular stimulating electrode consisting of an ACSF-filled pipette (0.7-2MΩ) was placed 10-50μm from the patched L5 pyramidal neuron soma. Fibers passing through layer 5 were stimulated with bipolar current pulses (10-70μA; 0.1-0.5s) using a linear stimulus isolator (A395, World Precision Instruments, Sarasota, FL, USA). Evoked EPSCs were measured while holding the postsynaptic neuron at −70mV in voltage clamp. Only those responses that did not have a component that reversed at −45mV/−40mV were recorded further to test for LTP induction. During each induction trial presynaptic stimulation at 2Hz was paired with postsynaptic depolarization to 0 mV for 5 seconds, and then repeated 20 times at 0.1Hz. GABAergic inhibition was not blocked.
Synaptic strength (mean EPSP amplitude) and failure rate were calculated for the 1st EPSP amplitude in the train during the pre-induction/baseline period. EPSP waveform properties were quantified from the average waveform for the first EPSP in the train. Rise time was defined as the time from 20-80% of peak amplitude, latency was defined as the time between the peak of the presynaptic AP and the onset of the EPSP (defined at 5% of peak), and decay time constant was obtained by fitting a single exponential to the decaying phase of the EPSP. Short-term plasticity was assessed as the paired pulse ratio (PPR) computed as follows:
The connection probability (CP) was expressed as the ratio of the number of identified synaptic connections to the number of synaptic connections tested. Difference in CP was tested by a χ2 Test for a 2×2 contingency table, in which rows consisted of the numbers of connected and unconnected pairs and columns consisted of the genotype (WT and Mecp2-null).
All bar plots represent mean values ± Standard Error of the Mean (SEM).
Quadruple whole cell recordings from L5 pyramidal neurons (Fig. 1 A) were performed to study monosynaptic connections in slices from both WT and Mecp2-null animals. Evoked excitatory postsynaptic potentials (EPSPs) were identified as depolarizing responses time locked to pre-synaptic spikes (Fig 1. B). A set of baseline EPSPs were used to study properties of evoked synaptic transmission in WT and Mecp2-null conditions. LTP was induced using a spike timing dependent plasticity (STDP) protocol (see Methods and Fig. 1C.). A connection was considered potentiated if the mean post-induction EPSP amplitude (from 30 consecutive trials averaged around the 30th minute, from start of recording) was >120% of the mean baseline EPSP amplitude, and if this increase was statistically significant by a t-test. Input resistance and resting membrane potential were monitored throughout the recording (Fig. 1D center and bottom graphs, respectively).
Previously we observed an early reduction in circuit excitability of 2-3-week-old Mecp2-null mice (Dani et al., 2005). Therefore we first studied monosynaptic pyramid-pyramid connections in slices from presymptomatic (post-natal day, P16-P21) Mecp2-null mice and WT controls. Synaptic connections in both WT and Mecp2-null mice could be successfully potentiated using the STDP protocol (Fig. 2). Averages of normalized EPSPs from all connections, for which LTP was attempted, revealed no significant differences in plasticity between WT and Mecp2-null mice (Fig 2 B,C). In order to determine whether or not LTP induction was impaired later in development, during the onset of primary symptoms in male Mecp2-null mice, we performed LTP experiments in slices obtained from 4-week-old (P26-P29) WT and Mecp2-null mice (Supplemental Figure 2: Sample LTP experiments). A few recordings in older animals were attempted, but analysis of LTP proved impractical due to the increased difficulty of finding and maintaining stable paired recordings in slices from older animals. There were also no significant differences in the magnitude of LTP in slices obtained from 4 weeks old WT and Mecp2-null animals (Fig.2D,E; WT: 157±23%; Mecp2-null: 136±12%, p = 0.43, Student's t-test). Analysis of changes in the inverse square of the coefficient of variation (CV−2) relative to changes in mean EPSP amplitude can provide information about relative changes in quantal content (usually presynaptic) or quantal amplitude (often postsynaptic) (Malinow and Tsien, 1990; Faber and Korn, 1991; Larkman et al., 1992; Sjöstrom et al., 2003, 2007). We have shown previously that two apparently pre- and postsynaptic forms of LTP coexist at L5 synapses in rat visual cortex, and that this leads to changes in short-term plasticity and CV that vary between synaptic connections, but are concordant at any one connection (Sjöstrom et al., 2007). Such an analysis for WT and Mecp2-null connections did not reveal major differences in the locus of LTP expression between WT and Mecp2-null connections at any age (Supplemental Figure 3). To ensure that the inability to see differences in LTP induction was not specific to the STDP induction protocol, we also used a ‘pairing’ protocol in which extracellular stimulation was used to activate excitatory inputs during strong depolarization of the postsynaptic neuron (Fig.3 A, B). This protocol is known to produce robust strengthening of excitatory synapses (Malinow, 1991; Chen et al., 1999). Consistent with prior observations, pairing induced LTP had a much greater amplitude than that observed following the STDP protocol (Malinow, 1991; Markram et al., 1997a; Chen et al., 1999; Sjöstrom et al., 2001), however, no significant difference in LTP existed between synapses from WT and Mecp2-null slices using this protocol either (Fig. 3 C). Thus, even during the early symptomatic phase of the mouse disorder, the signaling mechanisms required for long-term strengthening of synapses seem to be intact in Mecp2-nulls, at least for synapses on to L5 neurons.
Differences in the properties of evoked synaptic transmission between neurons from WT and Mecp2-null slices were analyzed from baseline synaptic responses acquired before LTP induction. There were no differences in the mean amplitude of the first EPSP (Fig. 4 B, WT: 0.67±0.1 mV; n=36; Mecp2-null: 0.58±0.08 mV, n=24; p=0.46, 2-tailed, unpaired Student's t-test) or the rate of failures (Fig. 4 C, % Failures, WT: 14.9±3.0%; Mecp2-null: 18.8±2.8%), between the two conditions. The average latency, rise time and decay time constants for the first EPSP in the train were comparable between the two genotypes (Supplemental Table T1). There were also no significant differences in the short-term plasticity of these synapses as quantified by the PPR for a train of 3 pulses (Fig 4. D). A train of 5 APs was delivered to a subset of these connections, but again, no significant differences were observed (Supplemental Figure 4). The CV−2, which is proportional to quantal content assuming a binomial model of synaptic transmission, was not significantly different for any of the three stimuli in the train (Fig 4. E, CV−2). Thus at L5 pyramid-pyramid connections, there are no major differences in the properties of synaptic transmission in slices obtained from pre-symptomatic, 16-21 day old animals.
Since more dramatic effects in spontaneous firing rates in L5 pyramids accompanied by reduced excitatory synaptic drive were previously observed in slices from 4-5-week-old Mecp2-null mice, we analyzed properties of baseline synaptic transmission in 4-week-old (P26-P29) animals (Fig. 5A: sample traces of WT and Mecp2-null monosynaptic connections). The mean EPSP amplitude of L5 pyramid-pyramid synapses in Mecp2-nulls was reduced by nearly 45% compared to WT (Fig. 5B; WT: 0.66±.13 mV, n=19; Mecp2-null: 0.36±0.03 mV, n=18, p<0.05, 2-tailed unpaired Student's t-test). The apparent increase in the mean failure rate of Mecp2-null synapses was not statistically significant (Fig. 5C; % Failures, WT: 19.9±3.6%, n=19; Mecp2-null: 27.2±4.2%, n =18; p=0.19, 2-tailed Student's t-test). Mean EPSP latency, rise time and decay kinetics were also not significantly different between WT and Mecp2-null synapses (Supplemental Table T1). As in younger animals, no significant changes were observed in short-term plasticity with repeated stimulation at 20Hz (Fig. 5D, WT: n=19; Mecp2-null: N=18, p=0.52, one way ANOVA). However, WT synapses had significantly higher CV−2 during the first 2 EPSPs in the train compared to Mecp2-null synapses. In addition, consistent with short-term depression of quantal content, CV−2 decreased on average during a train of EPSPs for WT synapses, but for Mecp2-null synapses it did not change significantly (Fig. 5E: CV−2 for WT (N=18) and Mecp2-null (N=18) synapses ; P<0.05, two-factor ANOVA). Thus, the reduced excitatory synaptic strength in Mecp2-null mice at an early symptomatic phase could be partially explained by reduced quantal content at these synapses.
The connection probability (CP) at both developmental time points was computed for all potential connections tested in WT and Mecp2-null slices. Similar to previous estimates for synapses between thick-tufted, L5 pyramidal neurons in rat and mouse somatosensory and visual cortices (Markram et al., 1997b; Song et al., 2005; Krieger et al., 2007), the rate of finding paired connections was ~10% in 2-4-weeks old WT mice. Although CP was lower in 2-3-week-old Mecp2-null mice compared to WT (Table 1), this difference was not significant (P>0.25; 2-sided χ2 contingency test). CP in slices from 4-week-old Mecp2-null mice was found to be nearly half of that in WT slices (Table 1), and this difference was statistically significant (P<0.05; χ2 Test ). This suggests that one primary effect of Mecp2 mutation is to reduce excitatory connectivity within local cortical microcircuits.
In order to determine which properties of baseline excitatory synaptic transmission and synaptic plasticity are altered due to the loss of MeCP2 function, we recorded from pairs of synaptically connected pyramidal neurons, prior to and during the onset of symptoms in Mecp2-null mice. Our results reveal a progressive reduction in excitatory connectivity in the neocortex, reminiscent of that described at hippocampal autapses in vitro (Chao et al., 2007). On the other hand, our results also suggest that a reduced ability to induce LTP at cortical synapses is likely to be a late consequence of loss of MeCP2, and may be secondary to changes in circuit structure.
Despite the fact that L5 neocortical pyramidal neurons in Mecp2-nulls show reduced spontaneous activity (Dani et al., 2005) and reduced number and strength of local recurrent excitatory synapses (Fig. 5, and Table 1), LTP induction and expression appear to be intact at these synapses when postsynaptic depolarization is provided directly. This is true at 2-3 weeks when subtle changes in network excitability can first be detected (Dani et al., 2005) and remains true at the onset of symptoms at approximately 4 weeks (Fig. 2), when more marked changes in synaptic strength and number are already apparent. This shows that the signaling pathways mediating long term strengthening of excitatory synapses are unlikely to be initial targets of MeCP2, and that loss of LTP is unlikely to explain the reduced cortical connectivity. Prior studies using extracellular stimulation and recordings have reported deficits in the induction of LTP at hippocampal synapses of symptomatic Mecp2-null mice (Asaka et al., 2006) and Mecp2308 mice (Moretti et al., 2006). Reduced LTP of cortical field EPSPs in Layer 2/3 was also observed in symptomatic 35-40-week-old Mecp2308 mice (Moretti et al., 2006). Although it remains conceivable that there are early changes specific to L2/3 synapses, morphological abnormalities such as simplified dendritic arbors in L5 neurons parallel similar changes in many other excitatory neurons in the forebrain, including L2/3 neurons (Belichenko et al., 1994; Kishi and Macklis, 2004). Moreover, since defects in synaptic transmission and connectivity can be seen earlier in development (Nelson et al., 2006; Chao et al., 2007), reduced plasticity could be a late secondary effect of circuit miswiring.
Alternatively, reduced plasticity may reflect the procedure used to induce it. Earlier studies of plasticity in Mecp2-mutant mice employed protocols that rely on extracellular stimulation to achieve sufficient postsynaptic depolarization and calcium influx during LTP induction. In these protocols, synaptic plasticity is highly cooperative, requiring activation of multiple excitatory inputs to produce sufficient postsynaptic depolarization to allow the NMDA receptor-mediated Ca2+ influx required for potentiation (Bliss and Collingridge, 1993; Kirkwood and Bear, 1994; Debanne et al., 1996; Sjöstrom et al., 2001). Thus, progressively weaker synapses and sparser connectivity in late symptomatic stages of Mecp2-null mice may impair LTP induction when postsynaptic depolarization or firing is not directly controlled. Using two different methods for LTP induction – STDP and a ‘pairing protocol’ – we observed comparable LTP induction at L5 excitatory synapses of Mecp2-null and WT mice when postsynaptic depolarization was provided. Furthermore, since local cortical circuits are made up of highly recurrent excitatory and inhibitory inputs, extracellular stimulation, even in the presence of low doses of GABAergic antagonists, often evokes a mixture of excitation and inhibition. GABAergic inhibition is known to potently block LTP at cortical excitatory synapses (Artola and Singer, 1987; Bear et al., 1992). Hence a shift in the balance between excitatory and inhibitory synaptic drives onto L5 pyramidal neurons to favor inhibition (Dani et al., 2005) could also contribute to impaired LTP induction following extracellular stimulation. During paired recording this circuit-level “gate” on plasticity is presumably circumvented by directly providing postsynaptic firing during synaptic activation.
Previous studies have reported reduced spontaneous and evoked excitatory neurotransmission in cultured hippocampal pyramidal neurons from Mecp2-null mice, but have differed on whether this was due to changes in release properties of individual synapses (Nelson et al., 2006), or changes in synapse number (Chao et al., 2007). Moreover, it was not known whether or not changes observed at hippocampal synapses also occur in the neocortex or if observations made in neuronal cultures were recapitulated in vivo. The latter point is important because targets of MeCP2, like BDNF, may be regulated differently in culture and in vivo due to very different prevailing activity patterns (Chang et al., 2006). Our results are consistent with a reduction in the number of recurrent excitatory synapses in the neocortex of symptomatic mice similar to that reported for autaptic hippocampal synapses in culture (Chao et al., 2007). This would explain the nearly two-fold reduction in connection probability observed (Table 1). It could also account for the nearly 2-fold decrease in mean EPSP amplitude observed at individual connections (Fig. 5B). However, we cannot rule out an additional change in the release properties of individual synapses, since each connection typically consists of multiple synaptic contacts (Markram et al., 1997b). The physiological evidence for reduced excitatory connectivity is consistent with anatomical evidence for reduced dendritic branching and reduced numbers of dendritic spines on cortical pyramidal neurons, especially in Layers 2/3 and 5, reported in both Rett patients and Mecp2-null mice (Belichenko et al., 1994; Armstrong, 2002; Zoghbi, 2003; Kishi and Macklis, 2004; Fukuda et al., 2005; Belichenko et al., 2009). Despite the reduced (~50%) connectivity between L5 pyramids, mEPSCs recorded in the same cell-type show only a mild reduction in frequency (Dani et al., 2005). However, mEPSC measurements are quite variable (CV for mEPSC frequency, WT: 45%; Mecp2-null: 42%, from Dani et al., 2005) and represent population measures of all excitatory inputs from other layers and cell types. Thus, if L5 pyramidal neurons comprise only a small fraction of inputs onto neighboring L5 pyramids, even a 50% reduction in local connectivity may not cause a sufficiently large difference in mEPSC frequency, if other excitatory inputs are unaltered. Additional studies will be required to test if other excitatory inputs onto L5 pyramids (for e.g. L2/3-to-L5 inputs) are altered in Mecp2-nulls.
The reduced probability of finding synaptic connections between pairs of L5 pyramidal neurons in Mecp2-null mice was more apparent in slices from symptomatic, 4-week-old Mecp2 -mice than in slices from younger mice. Connection probability of L5 pyramids in WT mice is constant over early postnatal development in our experiments (Table 1), and parallels similar observations in the rat somatosensory cortex (Frick et al., 2007). This suggests that cellular mechanisms involved in the maintenance of synaptic contacts during early postnatal development are perturbed in Mecp2-null mice. Recently it has been shown that activity dependent regulation of MeCP2 function is important for dendritic patterning and spine morphology (Zhou et al., 2006) and perturbation of MeCP2 levels can lead to altered activity dependent structural synaptic plasticity. Moreover, results from cell-type-specific gene expression profiling experiments indicate that a major class of dysregulated genes are involved in cell adhesion and synapse formation/maintenance (Sugino et al 2009, manuscript in preparation).
Recurrent excitatory connectivity is critical for the amplification and processing of input signals to the neocortex (Douglas and Martin, 2004). Thus, loss of recurrent excitatory connections may cause aberrant intracortical processing of sensory-motor information and impaired output from Layer 5 neurons to sub-cortical targets, many of which are crucial for motor control. Similar to the dendritic anomalies found in many regions of the forebrain of RTT patients and Mecp2-mutant mice (Armstrong, 2002; Zoghbi, 2003; Kishi and Macklis, 2004), loss of connectivity between L5 cortical pyramidal neurons reported here, is likely to be found in other forebrain regions. Therefore such changes in circuit wiring could underlie several of the motor, cognitive and learning deficits observed in Rett syndrome and related mental retardation disorders. On the other hand, however, studies of neocortical pyramidal neuron networks in the valproate model of autism in rats, have revealed hyper-, rather than hypo-connectivity, although individual connections between pyramids were weaker, disynaptic inhibitory connections were stronger, and pyramidal neurons were less excitable (Markram et al., 2007; Rinaldi et al., 2008). Increased LTP was also observed at Layer 2/3 synapses in this model of autism (Rinaldi et al., 2007). Our results showing hypo-connectivity and intact LTP in pyramidal neuron networks suggest there may be important mechanistic differences underlying circuit miswiring in Rett syndrome, a monogenic autism spectrum disorder, and the valproate model for autism.
Fine scale, cell-type-specific changes in local circuit organization in the forebrain may be a common mechanism underlying several mental retardation disorders that lack overt signs of neurodegeneration or developmental malformations. In hippocampal slices from Ts65Dn mice (a model for Down Syndrome), pharmacological blockade of inhibitory inputs has been shown to reverse a deficit in NMDA-receptor dependent LTP induction at hippocampal synapses (Kleschevnikov et al., 2004). Furthermore, study of monosynaptic connections between hippocampal CA3 neurons of Ts65Dn mice showed intact LTP induction mechanisms but alterations in excitatory and inhibitory synaptic connectivity (Hanson et al., 2007). Similarly, in a mouse model of Fragile X syndrome, weaker interlaminar inputs (L4-L3) due to reduced connection probability and more diffuse axonal arborizations have been transiently observed, probably during a developmentally regulated critical period (Bureau et al., 2008). Thus shifts in the excitation-inhibition balance caused by altered connectivity in forebrain circuits could be one underlying cause for learning and cognitive disabilities in several childhood mental retardation disorders including Rett Syndrome. However it remains to be determined whether or not these changes in connectivity are primary, cell autonomous changes, or reflect secondary effects in response to altered function of other cell types. Furthermore, deciphering the underlying changes in gene expression caused by Mecp2 mutation and primary targets of MeCP2 will be crucial to understanding the molecular mechanisms underlying altered neuronal connectivity in Rett syndrome.
We thank Zhe Meng and Hao Fan for help with genotyping, as well as Gina Turrigiano, Arianna Maffei, Chris Hempel, Ken Sugino, and Praveen Taneja for helpful comments. This work was supported by grants from NINDS, the McKnight Foundation and International Rett Syndrome Research Foundation.