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, 28 (20), 5178-88

Circuit and Plasticity Defects in the Developing Somatosensory Cortex of FMR1 Knock-Out Mice

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Circuit and Plasticity Defects in the Developing Somatosensory Cortex of FMR1 Knock-Out Mice

Ingrid Bureau et al. J Neurosci.

Abstract

Silencing of the Fmr1 gene causes fragile X syndrome. Although defects in synaptic plasticity in the cerebral cortex have been linked to cognitive impairments in Fmr1 knock-out (ko) mice, the specific cortical circuits affected in the syndrome are unknown. Here, we investigated the development of excitatory projections in the barrel cortex of Fmr1 ko mice. In 2-week-old Fmr1 ko mice, a major ascending projection connecting layer 4 (L4) to L3 (L4-->L3), was defective in multiple and independent ways: its strength was reduced, caused by a lower connection probability; the axonal arbors of L4 cells were spatially diffuse in L2/3; the L4-->L3 projection did not show experience-dependent plasticity. By 3 weeks, the strength of the L4-->L3 projection was similar to that of wild type. Our data indicate that Fmr1 shapes sensory cortical circuits during a developmental critical period.

Figures

Figure 1.
Figure 1.
Laser-scanning photostimulation by glutamate uncaging to map circuitry in brain slices from wild-type and Fmr1 knock-out mice. A, Examples of synaptic input maps for individual L3 cells from a wild-type (left) and a Fmr1 ko (right) mouse. The pixel values encode the mean amplitudes of EPSCs measured over 100 ms after the stimulus (see B). The black pixels are sites where glutamate uncaging evoked direct responses in the recorded cells that polluted synaptic responses. The white circles mark the cell body position of the recorded cells. The dashed lines indicate three barrels. The red box in L4 corresponds to the traces shown in B. B, Examples of EPSCs evoked by glutamate uncaging in L4 (corresponding to red box in A). The arrowheads indicate time of the photostimulus (duration, 1 ms). The horizontal lines indicate time windows used for the analysis (duration, 100 ms). C, Synaptic input maps from wild-type (left) and Fmr1 ko (right) mice averaged over L3 cells. D, Vertical profile (75 μm bins) of synaptic inputs in the wild-type (open symbol) and Fmr1 ko (solid symbol) mice. Inset, The gray bar indicates the region of analysis. E, Horizontal profile (75 μm bins) of L4 inputs in the wild-type (open symbol) and Fmr1 ko (solid symbol) mice. Inset, The gray bar indicates the region of analysis. Note that we limited analysis to the top two-thirds of L4 because previous data indicates that L5A neurons are excited on photostimulation in the bottom one-third of L4 (Bureau et al., 2006). F, Summary of interlaminar connectivity in wild-type (open bars) and Fmr1 ko (solid bars) mice. The regions of analysis for L4 (green), L5A (blue), and L5B (orange) inputs are shown in the inset. The asterisk indicates significant difference (p < 0.05). Error bars indicate SEM.
Figure 2.
Figure 2.
The cell density and photoexcitability in L4 are normal in Fmr1 ko mice. A, Across-row barrel cortex brain slice stained with the nuclear marker NeuN. The black horizontal lines indicate the position of barrels. B, Nuclei from L4 cells labeled with NeuN. C, L4 neuronal density in wild-type and Fmr1 ko mice. D, Photostimulation-evoked APs (left) and excitation profile (right) of a L4 cell recorded in loose-patch mode. Glutamate uncaging elicited APs only around the soma (blue circle). The bottom trace corresponds to an AP in the map. The arrowhead indicates time of the photostimulus. E, Excitation profiles averaged across L4 cells in wild-type and Fmr1 ko mice. F, Total number of APs elicited over the 8 × 8 pixel uncaging grid in L4 cells. Error bars indicate SEM.
Figure 3.
Figure 3.
Individuals L4/L3 connections have normal synaptic strength in the Fmr1 ko. A, Example of L3 responses evoked by minimal photostimulations in L4 in a wild-type (top) and Fmr1 ko (bottom) animal. The vertical dashed line indicates time of the photostimulus. The arrowheads indicate UPSCs evoked by the stimulation of distinct presynaptic L4 cells (one color per presynaptic cell). B, Amplitude of UPSCs evoked in a wild-type (top) and Fmr1 ko (bottom) animal as a function of latency. In the wild type, two clusters of UPSCs (red and blue) evoked by distinct presynaptic L4 cells had different amplitudes and latencies. Spontaneous EPSCs (black) had different latencies and/or amplitudes than clusters of UPSCs. The cells are the same as in A. C, Histogram of amplitude for clusters of UPSCs (red and blue) evoked in a wild-type (top) and Fmr1 ko (bottom) animal. Noise amplitude is shown in gray. The cells are the same as in A. D, Cumulative distribution of UPSC mean amplitudes in wild-type (gray) and Fmr1 ko (black) mice.
Figure 4.
Figure 4.
L4 cell axonal arbors in L2/3 are spatially diffuse in Fmr1 ko mice. A, A L4 cell axonal arbor (left; soma shown in red) from a wild-type animal and the spatial distribution of the axonal length density for the same cell (right). The pixel values encode the axonal length in a pixel of 50 × 50 μm2 of the arbor reconstruction projected in two dimensions. B, Same as in A for a Fmr1 ko. C, Average distribution of the axonal length density for L4 cells from wild-type mice. The L4 cell arbors were aligned by soma position (white circle). D, Same as in C for Fmr1 ko mice. E, Horizontal profile (50 μm bins) of L4 cell axonal length density in L2/3. The region of analysis is shown in inset (shaded area). Error bars indicate SEM.
Figure 5.
Figure 5.
Experience-dependent plasticity is blocked at selected intracortical projections in Fmr1 ko mice. A, Averaged synaptic input maps from L3 cells for sensory deprived wild-type (left) and Fmr1 ko (right) mice. The horizontal dashed line is the L5A/L5B boundary. B, Difference map was made by subtracting the averaged control from the deprived map for wild-type (left) and Fmr1 ko (right) mice. C, Horizontal profile of L4 synaptic input in control condition (open symbol) and after sensory deprivation (close symbol) in wild-type mice (left) and Fmr1 ko (right) mice. D, Same as in C for L5A synaptic input. Error bars indicate SEM.
Figure 6.
Figure 6.
Sensory deprivation depresses the L4/L3 synapses in wild-type and Fmr1 ko mice. A, Example of L3 responses evoked by the minimal photostimulation of a single L4 cell in a deprived wild-type (top) and a deprived Fmr1 ko (bottom) animal. The arrowheads indicate UPSCs evoked by the stimulation of distinct presynaptic L4 cells (one color per presynaptic cell). B, Amplitude of UPSCs evoked in a deprived wild-type (top) and deprived Fmr1 ko (bottom) animal as a function of latency. Clusters of UPSCs are shown in blue and red. Spontaneous EPSCs are shown in black. The cells are the same as in A. C, Histogram of amplitude for clusters of UPSCs (red and blue) evoked in a wild-type (top) and Fmr1 ko (bottom) animal. Noise amplitude is shown in gray. The cells are the same as in A and B. D, Cumulative distribution of UPSC mean amplitudes in wild-type (gray) and Fmr1 ko (black) mice in control conditions (dashed lines) and after sensory deprivation (solid lines).
Figure 7.
Figure 7.
The L4→L3 circuit phenotype of the Fmr1 ko is developmentally transient. A, Averaged synaptic input maps from L3 cells in P19–P22 wild-type (left) and Fmr1 ko (right) mice. B, Horizontal profile of L4 synaptic inputs in wild-type and Fmr1 ko mice. C, Average distribution of the axonal length density for L4 cells from P20–P22 wild-type (left) and Fmr1 ko (right) mice. D, Horizontal profile of L4 cell axonal length density in L2/3. Error bars indicate SEM.
Figure 8.
Figure 8.
Phenotypes of the developing L4→L3 projections in the barrel cortex of Fmr1 ko mice. L4 cell, Star; L3 pyramidal cell, triangle; axon, line; synaptic connection, black circle. The lines are functional connections. The dashed symbols indicate not-connected L4 neurons.

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