MeCP2 is required for normal development of GABAergic circuits in the thalamus

Abstract

Methyl-CpG binding protein 2 (MeCP2) is highly expressed in neurons in the vertebrate brain, and mutations of the gene encoding MeCP2 cause the neurodevelopmental disorder Rett syndrome. This study examines the role of MeCP2 in the development and function of thalamic GABAergic circuits. Whole cell recordings were carried out in excitatory neurons of the ventrobasal complex (VB) of the thalamus and in inhibitory neurons of the reticular thalamic nucleus (RTN) in acute brain slices from mice aged P6 through P23. At P14-P16, the number of quantal GABAergic events was decreased in VB neurons but increased in RTN neurons of Mecp2-null mice, without any change in the amplitude or kinetics of quantal events. There was no difference between mutant and wild-type mice in paired-pulse ratios of evoked GABAergic responses in the VB or the RTN. On the other hand, unitary responses evoked by minimal stimulation were decreased in the VB but increased in the RTN of mutants. Similar changes in the frequency of quantal events were observed at P21-P23 in both the VB and RTN. At P6, however, quantal GABAergic transmission was altered only in the VB not the RTN. Immunostaining of vesicular GABA transporter showed opposite changes in the number of GABAergic synaptic terminals in the VB and RTN of Mecp2-null mice at P18-P20. The loss of MeCP2 had no significant effect on intrinsic properties of RTN neurons recorded at P15-P17. Our findings suggest that MeCP2 differentially regulates the development of GABAergic synapses in excitatory and inhibitory neurons in the thalamus.

Fig. 1.

GABAergic transmission in the thalamus. A: schematic view of GABAergic circuits in the ventrobasal complex (VB) and reticular thalamic nucleus (RTN). GABAergic neurons in the RTN provide the main inhibitory input to VB neurons and are interconnected through GABAergic synapses. B and C: spontaneous inhibitory postsynaptic currents (IPSCs) recorded in a VB (B) and RTN (C) neuron from wild-type (WT) mice at P14 in the absence (top traces in B and C) and presence of the specific GABA_A antagonist SR95531 (bottom traces in B and C).

Fig. 2.

Opposite changes in quantal GABAergic transmission in the VB and RTN of Mecp2-null mice at P14–P16. A–E: data obtained from VB neurons. A: miniature IPSCs (mIPSCs) recorded from VB neurons of a WT (top trace) and a Mecp2-null (bottom trace) mouse. B and C: cumulative distributions of the interevent interval (B) and peak amplitude of mIPSCs (C) for WT (black) and Mecp2-null (gray) VB neurons. For each group, the distribution was established using 200 consecutively detected events from each cell. The inset in C shows the averaged mIPSCs from VB neurons of WT (black) and Mecp2-null (gray) mice. D and E: the mean frequency (D) and mean peak amplitude (E) of mIPSCs from WT (empty) and Mecp2-null (gray) VB neurons. F–J: data obtained from RTN neurons. F: mIPSCs recorded from RTN neurons of a WT (top trace) and a Mecp2-null (bottom trace) mouse. G and H: cumulative distributions of the interevent interval (G) and peak amplitude of mIPSCs (H) for WT (black) and Mecp2-null (gray) RTN neurons. For each group, the distribution was established using 150 consecutively detected events from each cell. The inset in H shows the averaged mIPSCs from RTN neurons of WT (black) and Mecp2-null (gray) mice. I and J: the mean frequency (I) and mean peak amplitude (J) of mIPSCs from WT (empty) and Mecp2-null (gray) mice.

Fig. 3.

Paired-pulse ratio of evoked IPSCs in VB (A–C) and RTN (D and E) neurons. A: paired-pulse responses in VB neurons from a WT (top trace) and Mecp2-null mouse (bottom trace) at P15. In both cases, 10 consecutive responses were shown. B: plot of paired-pulse ratios obtained at 50 and 100 ms interstimulus intervals in VB neurons from WT (empty) and Mecp2-null (hatched) mice at P14–P16. C: cumulative distributions of the maximal responses obtained in VB neurons from WT (black) and Mecp2-null (gray) mice. D: 10 consecutive paired-pulse responses recorded from a WT (top trace) and a mutant RTN neuron (bottom trace) at P14. E: plot of paired-pulse ratios obtained at 50 and 100 ms interstimulus intervals in RTN neurons from WT (empty) and Mecp2-null (hatched) mice at P14–P16.

Fig. 4.

Unitary IPSCs in VB and RTN neurons. A: IPSCs and failures of 60 consecutive trials at minimal stimulation intensities in WT (left) and mutant (right) VB neurons. B: cumulative distributions of IPSC amplitude obtained using minimal stimulation in VB neurons from WT (black) and Mecp2-null (gray). C: IPSCs and failures of 50 consecutive trials in a WT (left) and mutant (right) RTN neuron. D: cumulative distributions of IPSC amplitude obtained using minimal stimulation in RTN neurons from WT (black) and Mecp2-null (gray). B and D, cumulative distributions were established using 30 consecutive responses from each of the recorded cells.

Fig. 5.

Quantal GABAergic transmission in VB (A–E) and RTN (F–J) neurons at P6. A: mIPSCs recorded from VB neurons of a WT (top trace) and a Mecp2-null (bottom trace) mouse at P6. B and C: cumulative distributions of the interevent interval (B) and peak amplitude (C) obtained from WT (black) and Mecp2-null (gray) neurons. D and E, plots of the mean frequency (D) and mean peak amplitude (E) of mIPSCs from VB neurons of WT (black) and Mecp2-null (gray) mice at P6. F: mIPSCs recorded from RTN neurons of a WT (top trace) and a Mecp2-null (bottom trace) mouse at P6. G and H: cumulative distributions of the interevent interval (G) and peak amplitude (H) obtained from WT (black) and Mecp2-null (gray) neurons. I and J: plots of the mean frequency (I) and mean peak amplitude (J) of mIPSCs from RTN neurons of WT (black) and Mecp2-null (gray) mice at P6.

Fig. 6.

Changes in quantal GABAergic transmission in VB (A–E) and RTN (F–J) neurons of Mecp2-null mice at weaning. A: mIPSCs recorded from VB neurons of a WT (top trace) and a Mecp2-null (bottom trace) mouse at P21. B and C: cumulative distributions of the interevent interval (B) and peak amplitude (C) obtained from WT (black) and Mecp2-null (gray) neurons. D and E: plots of the mean frequency (D) and mean peak amplitude (E) of mIPSCs from VB neurons of WT (black) and Mecp2-null (gray) mice at P21–P23. F: mIPSCs recorded from RTN neurons of a WT (top trace) and a Mecp2-null (bottom trace) mouse at P21. G and H: cumulative distributions of the interevent interval (G) and peak amplitude (H) obtained from WT (black) and Mecp2-null (gray) RTN neurons. I and J: plots of the mean frequency (I) and mean peak amplitude (J) of mIPSCs from RTN neurons of WT (black) and Mecp2-null (gray) mice at P21–P23.

Fig. 7.

Changes in the number of GABAergic terminals in the VB and RTN of Mecp2-null mice. A and B: immunostaining for vesicular GABA transporter (VGAT) in the VB (A) and RTN (B) of WT and Mecp2-null mice at P18. Scale bar, 20 μm. For all 4 images in A and B, contrast was increased by 20%. C: histogram of the density of VGAT-positive puncta in the VB and RTN of WT (empty) and Mecp2-null (gray) mice at P18–P20. D: double immunostaining for VGAT (left) and GAD67 (middle) in the VB indicating that VGAT puncta are GAD67 positive (right). Scale bar, 5 μm. E: double immunostaining for VGAT (left) and neuronal nuclear antigen A60 (NeuN) (middle) in the RTN of a mutant mouse. VGAT is present in the cytoplasm. Scale bar, 5 μm. Images in D and E were taken with a ×63 objective and a ×6 optical zoom and merged using ImageJ.