Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1−/y mice

Abstract

Hypersensitivity in response to sensory stimuli and neocortical hyperexcitability are prominent features of Fragile X Syndrome (FXS) and autism spectrum disorders, but little is known about the dendritic mechanisms underlying these phenomena. We found that the primary somatosensory neocortex (S1) was hyperexcited in response to tactile sensory stimulation in Fmr1/y mice. This correlated with neuronal and dendritic hyperexcitability of S1 pyramidal neurons, which affect all major aspects of neuronal computation, from the integration of synaptic input to the generation of action potential output. Using dendritic electrophysiological recordings, calcium imaging, pharmacology, biochemistry and a computer model, we found that this defect was, at least in part, attributable to the reduction and dysfunction of dendritic h- and BKCa channels. We pharmacologically rescued several core hyperexcitability phenomena by targeting BKCa channels. Our results provide strong evidence pointing to the utility of BKCa channel openers for the treatment of the sensory hypersensitivity aspects of FXS.

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Figure 1: Accelerated spread of evoked neocortical activity in Fmr1−/y mice.
Figure 2: Increase in sensory stimulus evoked responses of neocortical pyramidal neurons in Fmr1−/y mice.
Figure 3: Increase in AP duration, ADP and firing output of neocortical pyramidal neurons of Fmr1−/y mice in vitro.
Figure 4: Altered dendritic properties of neocortical neurons of Fmr1−/y mice in vitro.
Figure 5: Enhanced synaptic summation and AP backpropagation in distal dendrites of Fmr1−/y neurons in vitro.
Figure 6: Decrease in h-channel expression in distal dendrites of Fmr1−/y neurons in vitro.
Figure 7: Computer simulation of the effect of BKCa channel reduction on dendritic excitability.
Figure 8: Rescue of dendritic hyperexcitability and sensory hypersensitivity by boosting BKCa channel activity.

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Acknowledgements

We thank B. Sakmann, P. Seeburg, the Max Planck Society and the Deutsche Forschungsgemeinschaft for initial support of this study. We thank J.-C. Delpech, S. Dos Santos Carvalho and W. Crusio for technical and scientific advice. Some data was acquired using equipment of the Bordeaux Imaging Center, the ESPCI ParisTech and Biochemistry Facility of the Bordeaux Neurocampus. This research was funded by FRAXA Research Foundation, INSERM, CNRS, Conseil de la Region d'Aquitaine, Fondation Jérôme Lejeune, Fédération pour la Recherche sur le Cerveau, Fondation pour la Recherche Médicale (SPF20130526794) and Labex-BRAIN (ANR-10-LABEX-43).

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Authors

Contributions

A.F. conceived the project. Y.Z. and A.B. collected the in vitro electrophysiological and calcium imaging data. G.B. carried out the in vivo electrophysiological experiments. A.B. and N.S. performed the western blot experiments. I.F. performed the VSD imaging experiments. S.P. and M.G. carried out the behavioral experiments. G.L. collected the NEURON modeling data. B.O. provided the second-generation Fmr1/y mouse line. J.R. assisted in data interpretation. A.F., M.G., A.B., Y.Z., G.B. and I.F. wrote the paper and all of the other authors provided feedback.

Corresponding author

Correspondence to Andreas Frick.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Input-output function of the apical dendrite in Fmr1–/y and WT neurons in vitro.

(a) Experimental design: Dendritic whole-cell recordings were performed near the major branch-point. (b) Dendritic voltage responses (top and middle traces; WT: Vm = – 62 mV; Fmr1–/y: Vm = – 61 mV) to suprathreshold current injections at the dendrite (bottom) for a WT and an Fmr1–/y neuron. (c) Average number of spikes as a function of the current injected (Fmr1–/y, n = 59; WT, n = 54; p < 0.05). Data are shown as means ± s.e.m. *p < 0.05 (Fmr1–/y compared to WT). Statistical significance was calculated by repeated measure two-way ANOVA (c).

Supplementary Figure 2 Enhanced summation of simulated EPSPs (sEPSPs) in distal dendrites of Fmr1–/y neurons in vitro.

(a) Experimental design: Dendritic whole-cell recordings were performed near the major branch-point. (b) A train of five sEPSPs at 50 Hz was evoked by current wave injections in a WT dendrite and an Fmr1–/y dendrite (WT: Vm = – 57 mV; Fmr1–/y: Vm = – 58 mV). (c) Average summation ratio of the 5th to 1st sEPSP amplitudes (Fmr1–/y: 1.33 ± 0.03, n = 9; WT: 1.17 ± 0.05, n = 6; p < 0.05). Data are shown as means ± s.e.m. *p < 0.05 (Fmr1–/y compared to WT). Statistical significance was calculated by unpaired Student's t-test (c).

Supplementary Figure 3 Dendritic calcium spikes in Fmr1–/y and WT neurons in vitro.

(a) Experimental design: Dendritic whole-cell recordings were performed near the major branch-point. (b) Example traces of dendritic calcium spikes evoked by current wave injections in control and their abolition following bath-application of the voltage-gated Ca2+ channel blockers NiCl2 (100 µM) and CdCl2 (500 µM) in a WT dendrite (Vm = – 62 mV). (c) Example traces of dendritic calcium spikes in a WT dendrite and an Fmr1–/y dendrite (WT: Vm = – 62 mV; Fmr1–/y: Vm = – 59 mV). (d) Average voltage-time integral of dendritic calcium spikes (Fmr1–/y, 2.58 ± 0.14 mV*s, n = 4; WT, 1.91 ± 0.05 mV*s, n = 4; p < 0.01). Data are shown as means ± s.e.m. **p < 0.01 (Fmr1–/y compared to WT). Statistical significance was calculated by unpaired Student's t-test (d).

Supplementary Figure 4 Hyperexcitability of L5 pyramidal neurons in vivo.

(a) Experimental design: Whole-cell recordings were made from L5 pyramidal neurons in the primary somatosensory cortex of anaesthetized Fmr1–/y and WT mice. Example of a recorded L5 pyramidal neuron (biocytin, red fluorescence) and neuronal cell bodies (DAPI, blue). Arrowheads mark the cell body in L5 and the trajectory of the apical dendritic arbor throughout all the layers towards the pia. Scale bar: 100 µm. (b) Sample traces of trains of three APs at 120 Hz were superimposed and aligned to the last AP of each train (APs are truncated). The difference in ADP amplitude is indicated by an arrow, and the inset illustrates the difference in the AP half-width (solid line). (c) The amplitude of the ADP at 40, 120, and 170 Hz was significantly greater for Fmr1–/y neurons compared to WT neurons (Fmr1–/y: n = 9; WT: n = 9; p < 0.001). (d) The ratio of the half-width of the 3rd and 1st APs at 40, 120, and 170 Hz was significantly greater for Fmr1–/y neurons (Fmr1–/y: n = 9; WT: n = 9; p < 0.001). Data are shown as means ± s.e.m. Statistical significance was calculated by repeated measure two-way ANOVA (c-d).

Supplementary Figure 5 Dendritic cell-attached voltage-clamp recordings of Ih.

(a) Experimental design. Dendritic cell-attached voltage-clamp recordings of Ih were performed near the major branch-point. (b) Examples of Ih measurements and blockade of currents with Cs+ (3 mM, green trace), ZD7288 (100 µM, blue trace), or both drugs (orange trace). Traces are individual measurements.

Supplementary Figure 6 Full-length pictures of the blots presented in Figure 6.

Representative Western blot from Fmr1–/y and WT somatosensory cortex extracts for the membrane-bound fraction of HCN1 and HCN2, and for GAPDH.

Supplementary Figure 7 Effect of h-channel blocker ZD7288 on EPSP summation and resonance frequency in Fmr1–/y and WT neurons.

(a) Experimental design: Synaptic input was evoked by extracellular L1 stimulation while dendritic whole-cell recordings were performed in the distal dendrites of L5B pyramidal neurons. (b) Dendritic voltage traces for EPSPs summation before and after 10 min application of ZD7288 (100 µM) in an Fmr1–/y and a WT neuron. (c) Average increase in the EPSP summation ratio following ZD7288 application (Fmr1–/y: 31 ± 9 %, n = 9; WT: 132 ± 14 %, n = 10). (d) Dendritic voltage responses (top and middle) to the ZAP20 stimulus before (left column) and after (right column) ZD7288 application in an Fmr1–/y and a WT neuron. (e) Impedance amplitude as a function of frequency calculated from corresponding color-coded traces in (d). (f) Average resonance frequency at a holding potential of – 65 mV following ZD7288 application (Fmr1–/y: 0.45 ± 0.14 Hz, n = 10; WT: 0.76 ± 0.15 Hz, n = 10). Data are shown as mean ± s.e.m. ***p < 0.001, *p < 0.05 (ZD7288 compared to baseline). Statistical significance was calculated by repeated measure two-way ANOVA with Bonferroni’s multiple comparisons test (c, f).

Supplementary Figure 8 Effect of h-channel blocker ZD7288 on subthreshold membrane properties of Fmr1–/y and WT neurons.

(a) Experimental design: Dendritic whole-cell recordings were performed in the distal dendrites of L5B pyramidal neurons. (b) Dendritic voltage responses (top; WT: Vm = – 62 mV; Fmr1–/y: Vm = – 61 mV) to subthreshold current injections (bottom) before and after 10 min application of the h-channel blocker ZD7288 (100 µM) in a WT (left) and an Fmr1–/y (right) neuron. (c) Average values of input resistance in control and following ZD7288 application (Fmr1–/y-control 74.5 ± 11.1 MΩ, Fmr1–/y-ZD7288 120.1 ± 10.1 MΩ, n = 13; WT-control 51.6 ± 3.6 MΩ, WT-ZD7288 122.7 ± 8.0 MΩ, n = 21). (d) Average sag ratio values in control and following ZD7288 application (Fmr1–/y-control 24.4 ± 3.0 %, Fmr1–/y-ZD7288 1.4 ± 0.2 %, n = 13; WT-control 34.6 ± 2.4 %, WT-ZD7288 1.2 ± 0.2 %, n = 22). The sag response was abolished after blocking Ih (e) Average membrane time constant values before and after Ih blockade (Fmr1–/y-control 13.2 ± 2.6 ms, Fmr1–/y-ZD7288 30.7 ± 4.0 ms, n = 11; WT-control 8.7 ± 1.3 ms, WT-ZD7288 26.9 ± 2.6 ms, n = 11). Data are shown as mean ± s.e.m. ***p < 0.001 (ZD7288 compared to baseline). Statistical significance was calculated by repeated measure two-way ANOVA with Bonferroni’s multiple comparisons test (c–e).

Supplementary Figure 9 Effect of h-channel blocker ZD7288 on firing properties of Fmr1–/y and WT neurons.

(a) Experimental design: Dendritic whole-cell recordings were performed in the distal dendrites of L5B pyramidal neurons. (b) Dendritic voltage responses to suprathreshold current injections into the dendrite (bottom) in control (top traces) and after ZD7288 application (middle traces) in a WT (left) and an Fmr1–/y (right) neuron. (c) Average number of dendritic spikes as a function of the current injected before (solid line) and after (dashed line) blocking h-channels (ZD7288: Fmr1–/y, n = 12; WT, n =16; p = 0.005). Data are shown as mean ± s.e.m. Statistical significance was calculated by repeated measure two-way ANOVA.

Supplementary Figure 10 Reduced effect of Ih blocker ZD7288 on dendritic calcium signaling in Fmr1–/y neurons as compared to WT neurons.

(a) Experimental design: Ca2+ transients evoked by back-propagating APs were measured using 2-photon laser scanning microscopy in line-scan mode at the major apical branch-point as relative changes in Fluo-5F (green) fluorescence (normalized to Alexa-594 (red) fluorescence). (b) Dendritic calcium traces in response to a somatic burst of three APs at 100 Hz before and after ZD7288 (20 µM). (c) Average effect of ZD7288 on the amplitude of Ca2+ transients evoked by trains of three bAPs at 100 Hz (Fmr1–/y: 7.5 ± 21.3 %, n = 12; WT: 207.2 ± 61.7 %, n = 10). Data are shown as mean ± s.e.m. ***p < 0.001 (ZD7288 compared to baseline). Statistical significance was calculated by repeated measure two-way ANOVA with Bonferroni’s multiple comparisons test.

Supplementary Figure 11 Rescue of action potential duration by boosting BKCa channel activity.

(a) Experimental design: Ca2+ transients evoked by back-propagating APs were measured using 2-photon laser scanning microscopy in line-scan mode at the major apical branch-point as relative changes in Fluo-5F (green) fluorescence (normalized to Alexa-594 (red) fluorescence). (b) Dendritic calcium traces in response to a somatic burst of three APs at 100 Hz before and after ZD7288 (20 µM). (c) Average effect of ZD7288 on the amplitude of Ca2+ transients evoked by trains of three bAPs at 100 Hz (Fmr1–/y: 7.5 ± 21.3 %, n = 12; WT: 207.2 ± 61.7 %, n = 10). Data are shown as mean ± s.e.m. ***p < 0.001 (ZD7288 compared to baseline). Statistical significance was calculated by repeated measure two-way ANOVA with Bonferroni’s multiple comparisons test.

Supplementary Figure 12 Hypersensitivity to auditory stimuli.

(a) Experimental design: Mice were placed in a restraining tube positioned over a motion-sensitive platform inside a sound proof recording chamber. The whole-body startle response (of Fmr1–/y or WT mice) to brief auditory stimuli at 71 (+ 6), 77 (+ 12), 83 (+ 18), and 89 (+ 24) dB over a white background noise (65 dB) was measured. (b) The startle responses of Fmr1–/y mice were significantly increased compared to WT mice (n = 10 mice per genotype, p < 0.01). This effect was observed at all stimulus intensities. Data are shown as the mean ± s.e.m. **p < 0.01 (Fmr1–/y versus WT mice). Statistical analysis was performed using a 2 x 4 (genotype x stimulus level) ANOVA.

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Zhang, Y., Bonnan, A., Bony, G. et al. Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1−/y mice . Nat Neurosci 17, 1701–1709 (2014). https://doi.org/10.1038/nn.3864

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