Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome

Abstract

Rett Syndrome (RTT) is a devastating neurological disorder that is caused by mutations in the MECP2 gene. Mecp2-mutant mice have been used as a model system to study the disease mechanism. Our previous work has suggested that MeCP2 malfunction in neurons is the primary cause of RTT in the mouse. However, the neurophysiological consequences of MeCP2 malfunction remain obscure. Using whole-cell patch-clamp recordings in cortical slices, we show that spontaneous activity of pyramidal neurons is reduced in Mecp2-mutant mice. This decrease is not caused by a change in the intrinsic properties of the recorded neurons. Instead, the balance between cortical excitation and inhibition is shifted to favor inhibition over excitation. Moreover, analysis of the miniature excitatory postsynaptic currents (mEPSCs)/inhibitory postsynaptic currents (mIPSCs) in the Mecp2-mutant cortex reveals a reduction in mEPSC amplitudes, without significant change in the average mIPSC amplitude or frequency. These findings provide the first detailed electrophysiological analysis of Mecp2-mutant mice and provide a framework for understanding the pathophysiology of the disease and tools for studying the underlying disease mechanisms.

Fig. 1.

Spontaneous firing of L5 pyramidal neurons in S1 is reduced in Mecp2 mutants, compared with WT controls. (A) Representative spontaneous firing recordings at room temperature (25 ± 1°C) from a WT (Left) and Mecp2-mutant cell (Right). (B) Average spontaneous firing rate of WT and mutants. The ≈4-fold difference in mean firing rate of WT and mutant was statistically significant (Student's t test, P < 0.001). (C) Recordings performed at closer to physiological temperature (31-33°C) also showed a decreased firing rate in mutants. (D and E) Smaller reductions in spontaneous firing rate at presymptomatic ages. Mean firing rate was reduced ≈2-fold in presymptomatic mice at P 14-P 16 (D), or at P 21-P 24 (E). Statistical significance is indicated by ^* for P < 0.05 and ^** for P < 0.01.

Fig. 2.

Intrinsic excitability of L5 neurons from Mecp2 mutants is unchanged. All recordings are from 4- to 5-week-old mice. Slices were continuously perfused in standard ACSF (at room temperature) containing APV, DNQX, and bicuculine. (A) Sample traces of spikes evoked by a depolarizing current step of 130 pA in a WT (Left) and a mutant (Right)L5 cell. (B) Mean firing rate vs. injected current amplitude (F-I curve). The mutant and WT F-I curves are almost identical. The mean first spike threshold (C) and input resistance (D) of mutant cells (n = 12) were not significantly different from WT cells (n = 12).

Fig. 3.

The balance between excitation and inhibition onto L5 pyramidal neurons is altered in Mecp2 mutants. Recordings were made in the presence of ongoing spontaneous activity. Total excitatory and inhibitory synaptic charge was calculated by integrating baseline subtracted spontaneous synaptic current. (A) Representative recordings of spontaneous excitatory postsynaptic currents (EPSCs) (recorded at the chloride-reversal potential) from L5 pyramidal neurons in slices from 4- to 5-week-old WT and mutant mice in modified ACSF. (B) The average excitatory charge is reduced in mutant mice (n = 12 for WT and mutants; P < 0.01, Student's t test) Representative recordings of spontaneous inhibitory postsynaptic currents (IPSCs) (recorded at the reversal potential for spontaneous EPSCs. (C) Representative recordings of spontaneous IPSCs (recorded at the reversal potential for spontaneous EPSCs. (D) The average inhibitory charge recorded from the same cells as in C is increased, compared with WT controls (n = 12 for WT and mutant, P < 0.05, t test).

Fig. 4.

Reduced amplitude of excitatory quantal transmission in L5 pyramids of 4- to 5-week-old Mecp2-mutant mice. mEPSCs recorded from L5 pyramidal cells in the presence of APV, bicuculine, and TTX. (A) Sample traces from WT and mutant cells voltage clamped at -70 mV. (B) Averaged mEPSC from 12 WT and 12 mutant cells. (C) mEPSC amplitude was reduced by 15%, and this difference was statistically significant (P < 0.01, t test). (D) Cumulative histograms of 300 events from each of 12 WT cells and 12 mutant cells showed a significant leftward shift in the mEPSC amplitude for mutant cells. (E) a small reduction in the mean mEPSC frequency was observed in mutants was not statistically significant (P = 0.12, t test).

Fig. 5.

Subtle differences in unitary IPSCs in L5 pyramids of 4- to 5-week-old Mecp2 mutant mice. mIPSCs recorded from L5 pyramidal cells in the presence of APV, DNQX, and TTX. (A) Sample traces from WT and mutant cells voltage-clamped at -70 mV. (B) Averaged mIPSC from 17 WT and 17 mutant cells. The slightly increased mIPSC amplitude in mutant cells was not statistically significant across cells (C) (P = 0.23, t test), although the cumulative histograms of events from WT and mutant cells (D) showed a skew toward larger amplitudes that was highly significant. There was no significant change in the mean mIPSC frequency (E) and a small but significant change in the mean decay time constant (Table 1).