Figure S6.

Long-term impact on the CSA region after lesion closure, related to Figure 6

(A) P28 coronal hemisections of brains immunostained with the pan axonal neurofilament marker SMI-312 and myelin basic protein (MBP) and Hoechst reveal alterations in the CSA region in Csf1r^{ΔFIRE/ΔFIRE} mice. Axonal tracts of the amygdalar capsule, located between the neocortex and the basolateral nucleus of the amygdala (BLA), are disorganized in mutants (solid arrowheads) compared with controls (open arrowheads), highlighting long-term morphological consequences of microglial absence and early life CSA lesions, even after lesion closure (n_controls = 4, n_CSF1RFire = 4, from at least two distinct litters).

(B) P20 coronal hemisections of brains immunostained with the pan axonal neurofilament marker SMI-312 and myelin basic protein (MBP) (left) or with MBP and FOXP2 (right) reveal alterations in the CSA region of mice prenatally exposed to PLX3397 (solid arrowheads) compared with controls (open arrowheads). Axonal tracts of the amygdalar capsule and associated FOXP2-positive inhibitory interneurons are disorganized, highlighting long-term morphological consequences of early microglial absence and transient CSA lesions (n_{controls-SMI/MBP} = 9; n_{PLX3397-SMI/MBP} = 7; n_{controls-MBP/Foxp2} = 5; n_{PLX3397-MBP/Foxp2} = 5; from at least two distinct litters for each condition).

(C) Schematic representation of the experimental approach (left) used to record in P60 slices both excitatory post-synaptic currents (EPSCs) and inhibitory post-synaptic currents (IPSCs) from BLA pyramidal neurons in response to stimulation of the amygdalar capsule. Importantly, amygdalar capsule stimulation was designed to trigger EPSCs with an amplitude between −150 and −350 pA in both mutants and PLX3397-prenatally exposed mice, and IPSCs were subsequently measured by changing the holding potential in order to evaluate the inhibition/excitation ratio (I/E). EPSCs and IPSCs amplitude and I/E ratio (right) show an altered balance in PLX3397-exposed embryos, compared with controls at P60 (n_controls = 18 cells from 5 animals and at least 2 distinct litters, n_PLX3397 = 21 cells, from 5 animals and at least 2 distinct litters).

Scale bars, 500 μm (A and B, low magnification) and 100 μm (B, high magnification). Graphs show means ± SEM. Mann-Whitney U tests were performed for statistical comparison, ^∗∗∗∗p < 0.0001.

Am, amygdala; ampl, amplitude; BLA, basolateral nucleus of the amygdala; CSA, cortico-striato-amygdalar boundary; EPSCs, excitatory post-synaptic currents; I/E, inhibition/excitation ratio; IPSCs, inhibitory post-synaptic currents; Ncx, neocortex.