Network hyperexcitability in hippocampal slices from Mecp2 mutant mice revealed by voltage-sensitive dye imaging

Abstract

Dysfunctions of neuronal and network excitability have emerged as common features in disorders associated with intellectual disabilities, autism, and seizure activity, all common clinical manifestations of Rett syndrome (RTT), a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Here, we evaluated the consequences of Mecp2 mutation on hippocampal network excitability, as well as synapse structure and function using a combination of imaging and electrophysiological approaches in acute slices. Imaging the amplitude and spatiotemporal spread of neuronal depolarizations with voltage-sensitive dyes (VSD) revealed that the CA1 and CA3 regions of hippocampal slices from symptomatic male Mecp2 mutant mice are highly hyperexcitable. However, only the density of docked synaptic vesicles and the rate of release from the readily releasable pool are impaired in Mecp2 mutant mice, while synapse density and morphology are unaffected. The differences in network excitability were not observed in surgically isolated CA1 minislices, and blockade of GABAergic inhibition enhanced VSD signals to the same extent in Mecp2 mutant and wild-type mice, suggesting that network excitability originates in area CA3. Indeed, extracellular multiunit recordings revealed a higher level of spontaneous firing of CA3 pyramidal neurons in slices from symptomatic Mecp2 mutant mice. The neuromodulator adenosine reduced the amplitude and spatiotemporal spread of VSD signals evoked in CA1 of Mecp2 mutant slices to wild-type levels, suggesting its potential use as an anticonvulsant in RTT individuals. The present results suggest that hyperactive CA3 pyramidal neurons contribute to hippocampal dysfunction and possibly to limbic seizures observed in Mecp2 mutant mice and RTT individuals.

Fig. 1.

Voltage-sensitive dye (VSD) imaging revealed pronounced hyperexcitability in area CA1 of acute hippocampal slices from symptomatic Mecp2 mutant mice. A: dorsal hippocampal slice with a stimulation electrode in CA1 stratum radiatum to stimulate afferent Schaffer collaterals. Note the red LEDs marking the hexagonal photodiode array. B: representative examples of the spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in slices from a symptomatic Mecp2 mutant mouse (right) and a wild-type littermate (left). Arrow points to the location of the stimulation electrode. In this and Figs. 2–8, normalized changes in RH-414 fluorescence (ΔF/F) are represented with a pseudocolor scale (warmer colors indicate larger depolarizations). C: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in slices from symptomatic Mecp2 mutant, presymptomatic Mecp2 mutant, and age-matched wild-type littermate controls. Data are means ± SD. aCSF, artificial cerebrospinal fluid; P, postnatal day. #P < 0.05, symptomatic vs. wild-type; *P < 0.05, presymptomatic vs. symptomatic Mecp2 mutant, young wild-type vs. older wild-type.

Fig. 2.

Basal excitatory synaptic transmission and short-term plasticity at CA3-CA1 synapses. A: input-output relationship between stimulus intensity and slope of field excitatory postsynaptic potentials (fEPSPs) evoked in area CA1 by Schaffer collateral stimulation. Fitted slopes (m) of the linear portion of these input-output curves were used for statistical comparisons. Insets show representative traces of field EPSPs in Mecp2 mutant and wild-type slices (dotted line represents the slope of the fEPSP used for measurements). B: paired-pulse stimulation at different interstimulus intervals (ISIs; 10, 20, 50, and 100 ms). *P < 0.0001, repeated-measures one-way ANOVA; P < 0.05, post hoc Newman-Keuls test. Data means ± SE. Inset: representative traces of field EPSPs at 50- and 250-ms interpulse intervals in a slice from a wild-type mouse.

Fig. 3.

Quantitative analysis of synapse ultrastructure in hippocampal area CA1. A: representative electron micrographs of asymmetric synapses on dendritic spines in a symptomatic Mecp2 mutant and a wild-type littermate. B: representative electron micrographs of symmetric synapses on dendritic shafts in a symptomatic Mecp2 mutant and a wild-type littermate. Arrowheads indicate the edges of the postsynaptic density, and docked vesicles are marked with white circles. Bar graphs show the number of synapses normalized per 10 μm² of neuropil, the number of docked synaptic vesicles normalized per active zone length, and the number of synaptic vesicles in the reserve pool normalized per terminal area. Data are means ± SD. *P < 0.05, symptomatic vs. wild-type.

Fig. 4.

Imaging of synaptic vesicle recycling with FM1–43 in acute slices by multiphoton excitation microscopy. A: representative example of the CA1 region of a wild-type slice stained with FM1–43. Note colored regions of interest over distal (s. radiatum) and proximal (s. pyramidale) FM1–43 puncta. Plots are a representative example of FM1–43 bleaching during a 5-min-long image sequence in the absence of afferent stimulation both as raw fluorescence intensity (F) in arbitrary units (AU; top) and as ΔF/F ratio (bottom) for comparison with ΔF/F traces of afferent stimulation-evoked FM1–43 destaining shown in B. B: representative examples of the time course of FM1–43 destaining from the sucrose-loaded readily releasable pool (RRP) evoked by afferent Shaffer collateral fiber stimulation in a wild-type slice (top) and a Mecp2 mutant slice (bottom). Horizontal bar denotes the onset and duration of afferent stimulation, and gray area denotes the 9-frame baseline period for calculation of ΔF/F. C: time course of activity-dependent FM1–43 destaining from distal puncta within CA1 s. radiatum. D: time course of activity-dependent FM1–43 destaining from proximal puncta within CA1 s. pyramidale. The inverse of the time constant of single exponential fittings to the first 11 frames of ΔF/F destaining traces was used for statistical comparisons. Note that the sucrose-loaded RRP of vesicles destained with a faster rate than the total recycling pool (TRP) loaded by hyperkalemic solutions. Data are means ± SD. *P < 0.05, symptomatic vs. wild-type.

Fig. 5.

Surgically isolating CA3 from CA1 in Mecp2 mutant slices prevented hyperexcitability in CA1, where GABAergic inhibition is intact. A: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in the presence of bicuculline in minislices from symptomatic Mecp2 mutant and wild-type littermate controls. Data are means ± SD. *P < 0.0005, aCSF vs. bicuculline in wild-type minislices by ANOVA; **P < 0.0005, aCSF vs. bicuculline in Mecp2 mutant minislices by ANOVA. B: representative examples of the spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in minislices from a wild-type mouse (top) and a symptomatic Mecp2 mutant littermate (bottom), before and after the application of the GABA_A inhibitor bicuculline (10 μM). Arrow points to the location of the stimulating electrode. C: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in minislices from symptomatic Mecp2 mutant and wild-type littermate controls. Data are means ± SD. *P < 0.05, Student's t-test of Mecp2 mutant minislices vs. wild-type minislices. D: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in wild-type slices. Data are expressed as means ± SD. *P < 0.05, Student's t-test of wild-type intact slices vs. wild-type minislices. E: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in Mecp2 mutant slices. Data are means ± SD. *P < 0.05, Student's t-test of Mecp2 mutant intact slices vs. Mecp2 mutant minislices.

Fig. 6.

Spontaneous neuronal firing is higher in CA3 of Mecp2 mutant mice than in wild-type littermates. A: representative traces of extracellular recordings from CA3 s. pyramidale of wild-type (top) and Mecp2 mutant slices (bottom). Insets: spontaneous multiunit spikes at higher temporal resolution. B: frequency of spontaneous multiunit spikes. Extracellular K⁺ concentration was raised to confirm the neuronal origin of spikes and to show the heightened susceptibility of Mecp2 mutant slices to membrane depolarization. Data are means ± SD. #P < 0.0001, wild type or Mecp2 symptomatic in 3.5 m K⁺ vs. 5 mM K⁺ by ANOVA; *P < 0.0001, symptomatic vs. wild-type in 3.5 m K⁺ by ANOVA; **P < 0.0001, symptomatic vs. wild-type in 5 mM K⁺ by ANOVA.

Fig. 7.

VSD signals evoked in CA3 and CA1 by mossy fiber stimulation show frequency-dependent facilitation and were enhanced in Mecp2 mutant slices. A: representative examples of the spatiotemporal pattern of VSD signals evoked in area CA3 (red arrow) and CA1 (white arrow) by stimulation of afferent mossy fibers in slices from a symptomatic Mecp2 mutant mouse and a wild-type littermate. Asterisk shows the location of the stimulating electrode. Ventral hippocampal slices with stimulation electrodes in CA3 s. lucidum to stimulate afferent mossy fibers. Note the red LEDs marking the hexagonal photodiode array. B: amplitude and spatiotemporal pattern of VSD signals evoked in area CA3 by mossy fiber stimulation. C: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by mossy fiber stimulation. Data are means ± SD. *P < 0.05, Student's t-test of symptomatic vs. wild-type.

Fig. 8.

Adenosine prevents hyperexcitability in area CA1 of Mecp2 mutant slices. A: representative examples of the spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in slices from a symptomatic Mecp2 mutant mouse and a wild-type littermate before and after the application of adenosine (50 μM). Arrow points to the location of the stimulation electrode. B: amplitude and spatiotemporal pattern of VSD signals evoked in area CA1 by stimulation of afferent Schaffer collaterals in the presence of adenosine in slices from symptomatic Mecp2 mutant and wild-type littermate controls. Data are means ± SD. *P < 0.005, aCSF vs. adenosine in wild-type slices by ANOVA; **P < 0.005, aCSF vs. adenosine in Mecp2 mutant slices by ANOVA; #P < 0.05 Mecp2 mutant vs. wild-type slices in aCSF by ANOVA.