Circuit level defects in the developing neocortex of Fragile X mice

Abstract

Subtle alterations in how cortical network dynamics are modulated by different behavioral states could disrupt normal brain function and underlie symptoms of neuropsychiatric disorders, including Fragile X syndrome (FXS). Using two-photon calcium imaging and electrophysiology, we recorded spontaneous neuronal ensemble activity in mouse somatosensory cortex. Unanesthetized Fmr1(-/-) mice exhibited abnormally high synchrony of neocortical network activity, especially during the first two postnatal weeks. Neuronal firing rates were threefold higher in Fmr1(-/-) mice than in wild-type mice during whole-cell recordings manifesting Up/Down states (slow-wave sleep, quiet wakefulness), probably as a result of a higher firing probability during Up states. Combined electroencephalography and calcium imaging experiments confirmed that neurons in mutant mice had abnormally high firing and synchrony during sleep. We conclude that cortical networks in FXS are hyperexcitable in a brain state-dependent manner during a critical period for experience-dependent plasticity. These state-dependent network defects could explain the intellectual, sleep and sensory integration dysfunctions associated with FXS.

Figure 1. Delayed network desynchronization in the neocortex of unanesthetized Fmr1^–/– mice

(a) Typical field of view of L2/3 neurons (green) stained with OGB-1 AM and imaged with in vivo two-photon microscopy. Sulforhodamine 101 was used to stain glia (yellow). Sum intensity projection (xyt) of a representative calcium imaging movie (3 min, 3.9 Hz) from a P15 mouse. (b) Automated detection of neuronal cell bodies obtained through algorithmic segmentation of the image shown in a. (c) Raw ΔF/F calcium traces of 6 different L2/3 neurons from representative movies at P9-11, P14-16 and P30-40. Gray arrows and vertical dashed lines represent times of synchronous firing. (d) Mean correlation coefficients for all cell pairs vs. distance separating cell pairs in unanesthetized WT mice at different ages (n = 12, 9, 8 mice at P9-11, P14-16, and P30-40, respectively). The largest difference in correlation coefficients between P9-11 and P14-16 occurred for cell pairs <100 μm apart (Bonferroni corrected *p < 0.0001, two-way ANOVA). (e) Mean correlation coefficients for all cell pairs within 100 μm of each other for WT (black) and Fmr1^–/– (red) mice at different ages. For Fmr1^–/– mice, n = 9, 10, 8 at P9-11, P14-16, and P30-40, respectively. Both age and genotype significantly affected correlation coefficients (*p < 0.05 after Bonferroni correction, two-way ANOVA). The difference in correlation between WT and Fmr1^–/– was largest at P14-16 (*p = 0.039, t-test). Error bars indicate the standard error of the mean (s.e.m.). (f) Mean correlation coefficients vs. distance for WT and Fmr1^–/– mice at P14-16. Dashed lines indicate s.e.m. boundaries.

Figure 2. A higher proportion of L2/3 neurons are recruited to peaks of synchrony in unanesthetized Fmr1^–/– mice

(a) Raster plot showing identified spiking events in L2/3 cells from a calcium movie in an unanesthetized WT mouse at P15 (same as Fig. 1a, b). Events shown in red were identified as having participated in a peak of synchrony (see Methods). The bottom trace shows the cumulative fraction of active cells over time. (b) Mean frequency of peaks of synchrony in WT and Fmr1^–/– mice at P14-16. (c) Mean fraction of active L2/3 cells at the maximum point of each peak of synchrony in WT and Fmr1^–/– mice at P14-16 (*p = 0.023, t-test).

Figure 3. Elevated firing rates in unanesthetized Fmr1^–/– mice during Up/Down states

(a) Cartoon of in vivo patch-clamp recordings of L2/3 neurons in barrel cortex of unanesthetized mice. (b) Representative examples of whole-cell recordings (3 min long) in different behavioral brain states of the animal: Fast oscillatory activity (FOA; consistent with active wakefulness or REM sleep) and Up/Down states (consistent with slow-wave sleep or quiet wakefulness). The bottom trace shows a transition from Up & Down states to FOA. Note the movement artifact (arrow) when the animal transitions between the two different brain states. The segments highlighted in yellow are expanded in panels c and d. (c, d) Traces with membrane potential (V_m) fluctuations typical of Up/Down states were algorithmically detected. Shown here are typical examples of FOA (e) and Up/Down state (f) recordings that were automatically sorted with this approach. Note that the latter exhibit a bimodal V_m distribution (insets). (e) Firing rates for WT and Fmr1^–/– mice during recordings in FOA vs. Up/Down brain states. *p < 0.05 after Bonferroni correction, 2-way ANOVA. (f) Fraction of time spent in FOA vs. Up/Down states for WT and Fmr1^–/– mice. There were no significant differences between genotypes.

Figure 4. Higher probability of neuronal firing during Up states in Fmr1^–/– mice

(a) Sample traces during Up/Down states from in vivo whole cell recordings of L2/3 neurons in unanesthetized WT (top) and Fmr1^–/– mice (bottom). The highlighted segments are expanded in panel b. (b) Expanded traces of Up & Down segments in WT (top) and Fmr1^–/– mice (bottom). (c, d) Mean duration (c) and frequency (d) of Up states in WT and Fmr1^–/– mice. Note that not all recordings (or neurons) showed segments that were classified as Up/Down states (n = 5 and 6 recording segments with Up/Down states in WT and Fmr1^–/– mice, respectively). There were no significant differences between genotypes. (e) Mean Up-Down voltage step (in mV) in WT and Fmr1^–/– mice. The trend toward higher step in mutant mice was not significant (p = 0.21, t-test). (f) Mean number of action potentials per active Up state (i.e., Up states with 1 or more spikes) in WT and Fmr1^–/– mice. p = 0.07, t-test. (g) Mean firing probability during any given Up state (active or silent) in WT and Fmr1^–/– mice. **p < 0.01, t-test.

Figure 5. Fmr1^–/– mice exhibit abnormal modulation of neuronal activity in different brain states

(a) Cartoon of the mouse skull showing the typical montage of EEG electrodes with respect to the cranial window (red circle) in the combined EEG and calcium imaging experiments. (b) Representative examples of calcium traces for individual cells during EEG recordings in unanesthetized WT mice at P14-16 manifesting low (left) or high(right) L/H power, which is a measure of the behavioral state of the animal. Low and high L/H power is consistent with active wakefulness and sleep, respectively. Shown are typical calcium traces of 5 cells (black) and the EEG (blue; 20 Hz boxcar filter). (c) Plot of mean estimated firing rates (extrapolated from calcium traces) and L/H power for WT and Fmr1^–/– mice at P14-16. Each square and circle represents a different 3 min EEG/calcium imaging recording. Arrows point to the WT animals shown as examples in b. Lines represent linear-regression fits; statistics were done using an F-test. (d) Plot of mean correlation coefficients and L/H power for WT and Fmr1^–/– mice at P15-16. (e, f) Mean estimated firing rates (e) and correlation coefficients (f) for EEG recordings with L/H power > 200 (which corresponds to sleep; Ref. 25). *p = 0.023 (d) and *p = 0.039 (f) (t-test). (g) L/H power for WT and Fmr1^–/– mice (p > 0.05, t-test).

Figure 6. The synchrony of L2/3 cortical pyramidal neurons is not modulated by anesthesia in Fmr1^–/– mice

(a, b) Typical examples of calcium traces for 5 cells in WT mice (a) and Fmr1^–/– mice (b) during recordings at P14-16 without anesthesia (left) and under isoflurane anesthesia (right). The vertical dashed lines represent times of synchronous firing during recordings under anesthesia. Note also the higher synchrony of calcium transients in the Fmr1^–/– mouse compared to the WT mouse. (c, d) Mean correlation coefficients for all cell pairs (< 100 μm) in (c) WT mice (0.24 ± 0.02 vs. 0.17 ± 0.01, n = 9 and 9 mice, respectively; p = 0.0045, t-test) and (d) Fmr1^–/– mice (0.21 ± 0.01 vs. 0.18 ± 0.01, n = 10 and 10 mice, respectively, p = 0.14, t-test), at P14-16. Calcium imaging was done under no anesthesia vs. with isoflurane anesthesia (1-2%, inh; n = 5 WT and 7 Fmr1^–/– mice).