Figure 4. Spine density and evoked synaptic activity recorded in LII/LIII pyramidal neurons in both ΔNp73^cre/+;Bax^lox/lox and ΔNp73^cre/+;R26^Kir2.1/+ mutants.

(A, C) Representative confocal images showing spines in apical and basal dendritic segments of controls (left) and ΔNp73^cre/+;Bax^lox/lox (A, right) and ΔNp73^cre/+;R26^Kir2.1/+ mutants (C, right) at P24-25. (B, D) Quantification of the spine density (number of spines/µm) in apical and basal dendrites in LII/LIII pyramidal neurons for both controls and mutants from the same litters (for ΔNp73^cre/+;Bax^lox/lox apical and basal dendrites: n = 8 for controls and n = 12 for mutants at P23-28, p=0.012 for apical dendrites and p=0.0014 for basal dendrites; for ΔNp73^cre/+;R26^Kir2.1/+ apical dendrites: n = 10 for controls and n = 6 for mutants at P23-P29, p=0.166; basal dendrites: n = 9 for controls and n = 7 for mutants, p=0.652; Mann-Whitney U Test). Scale bar represents 5 μm. (E, G) Pyramidal neurons recorded in voltage-clamp at −70 mV and 0 mV in control at P26 (E, left) and P24 (G, left) and in a ΔNp73^cre/+;Bax^lox/lox mutant at P23 (E, right) and a ΔNp73^cre/+;R26^Kir2.1/+ mutant at P28 (G, right) during the extracellular stimulation of LII/III fibers as indicated (E, inset). Stimulation artefacts were blanked for visibility. The stimulation time is indicated (arrowheads). (F, H) Plots of E/I ratio calculated from eEPSCs and eIPSCs in controls and ΔNp73^cre/+;Bax^lox/lox mutants (F) and ΔNp73^cre/+; R26^Kir2.1/+ mutants (H) (for ΔNp73^cre/+;Bax^lox/lox: n = 10 for controls and n = 14 for mutants, p=0.031, Student T Test; for ΔNp73^cre/+;R26^Kir2.1/+: n = 8 for controls and n = 7 for mutants, p=0.612; Mann-Whitney U Test). Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 4—figure supplement 1—source data 1.

Figure 4—figure supplement 1. Evoked and spontaneous EPSCs and IPSCs of LII/LIII pyramidal neurons in ΔNp73^cre/+;Bax^lox/lox and ΔNp73^cre/+;R26^Kir2.1/+ mutants.

(A, B) Plots of the mean amplitude of eEPSCs (left) and eIPSCs (right) evoked by extracellular stimulation for the same pyramidal neurons of Figure 4 held at −70 mV and 0 mV, respectively, in controls and ΔNp73^cre/+;Bax^lox/lox (A) and ΔNp73^cre/+;R26^Kir2.1/+ (B) mutants. Note the significant increase in the mean amplitude of eEPSCs for ΔNp73^cre/+;Bax^lox/lox (p=0.042) but not for ΔNp73^cre/+;R26^Kir2.1/+ mutants (p=0.902). eIPSCs remained unchanged. (C) sEPSCs recorded in pyramidal neurons held at −70 mV in a control at P26 (black) and a ΔNp73^cre/+;Bax^lox/lox mutant at P23 (green). (D, E) Plots of the mean frequencies of sEPSCs in ΔNp73^cre/+;Bax^lox/lox (D) and ΔNp73^cre/+;R26^Kir2.1/+ (E) mutants at P23-26 and P23-28, respectively. Note the significant increase in the mean frequency of sEPSCs for ΔNp73^cre/+;Bax^lox/lox but not for ΔNp73^cre/+;R26^Kir2.1/+ mutants (n = 9 for controls and n = 11 for ΔNp73^cre/+;Bax^lox/loxp=0.038, Student T test; n = 7 for controls and n = 8 for ΔNp73^cre/+;R26^Kir2.1/+ mice p=0.942, Mann-Whitney U test). For the same cells, mean sEPSC amplitudes: −17.5 ± 2.2 pA for controls vs −19.2 ± 1.4 pA for ΔNp73^cre/+;Bax^lox/lox mice (p=0.501; Student T test) and −12.20 ± 0.81 pA for controls vs –14.30 ± 1.95 pA for ΔNp73^cre/+;R26^Kir2.1/+ mice (p=0.9551; Mann-Whitney U test). (F) Current-clamp recording of a ChR2-expressing rescued CR upon light stimulation in a ΔNp73^cre/+;Bax^lox/lox;ChR2^lox/+ mouse (see diagram). Note that photoactivation (blue pulses, 1 ms) evoked action potentials in response to every light pulse of a 5 Hz-light train of 10 s (bottom left). Similar results were obtained with a 5 Hz-light train of 30 s (n = 7) and in the presence of ionotropic receptor antagonists 10 µM NBQX, 50 µM AP5 and 10 µM SR95351 (n = 2). Average percentage of success to elicit action potentials with light trains of 10 s delivered from 2 to 20 Hz (right). Note the decreased number of action potentials triggered by photoactivation from 10 Hz (n = 7; Kruskal-Wallis test followed by a Bonferroni multiple comparison). (G) Simultaneous Layer II/III extracellular recording and whole-cell recording of a Layer I interneuron localized nearby a ChR2-expressing rescued CR (see diagram, top left). The firing of the interneuron in response to 800 ms depolarizing and hyperpolarizing steps is shown (inset, top right). A 5 Hz-light train of 10 s did not induce Layer II/III LFPs or postsynaptic currents in the recorded interneuron held at −70 mV (bottom left). Similarly, no responses were observed during extracellular recordings of Layer I (n = 3), Layer II/III (n = 5) and Layer V (n = 3) or during whole-cell recordings of Layer I interneuron (n = 3) in normal conditions or in the presence of 0 mM Mg²⁺, 3 mM Ca²⁺ and 4AP (n = 3). Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 4—figure supplement 1—source data 1.