Activity-dependent death of transient Cajal-Retzius neurons is required for functional cortical wiring

Abstract

Programmed cell death and early activity contribute to the emergence of functional cortical circuits. While most neuronal populations are scaled-down by death, some subpopulations are entirely eliminated, raising the question of the importance of such demise for cortical wiring. Here, we addressed this issue by focusing on Cajal-Retzius neurons (CRs), key players in cortical development that are eliminated in postnatal mice in part via Bax-dependent apoptosis. Using Bax-conditional mutants and CR hyperpolarization, we show that the survival of electrically active subsets of CRs triggers an increase in both dendrite complexity and spine density of upper layer pyramidal neurons, leading to an excitation/inhibition imbalance. The survival of these CRs is induced by hyperpolarization, highlighting an interplay between early activity and neuronal elimination. Taken together, our study reveals a novel activity-dependent programmed cell death process required for the removal of transient immature neurons and the proper wiring of functional cortical circuits.

Keywords: Cajal-Retzius neurons; apoptosis; cortical development; developmental biology; excitation/inhibition ratio; mouse; neuronal activity; neuroscience; transient.

Figure 1.. Hyperpolarization induces the survival of SE-CRs.

(A) Confocal images of cortical sections from P7, P15 and P25 ΔNp73^cre/+;R26^mT/+ controls (left) and ΔNp73^cre/+;R26^Kir2.1/+ mutants (right) stained for DsRed (red) and Hoechst (blue). Arrowheads indicate DsRed⁺ CRs at P15 and P25. (B) Quantification of CR density (CRs/mm³) at the pial surface in the somatosensory (S1) cortex (P7: n = 6 for controls and n = 4 for mutants, p=0.716; P15: n = 4 for controls and n = 10 for mutants, p=0.001; P25: n = 3 for controls and n = 8 for mutants, p=0.006). (C) Confocal images of cortical sections from P7, P15 and P25 Wnt3a^cre/+;R26^mT/+ controls (left) and Wnt3a^cre/+;R26^Kir2.1/+ mutants (right) stained for DsRed (red) and Hoechst (blue). Arrowheads indicate DsRed⁺ CRs at P15 and P25. For simplicity, arrowheads were not displayed at P7, as there are too many CRs at this stage. (D) Quantification of CR density (CRs/mm³) at the pial surface in the somatosensory (S1) cortex (P7: n = 5 for controls and n = 6 for mutants, p=0.2251; P15: n = 4 for controls and n = 3 for mutants, p=0.771; P25: n = 3 for controls and n = 3 for mutants, p=0.813). Mann-Whitney U Test. Scale bar represents 200 μm. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 1—figure supplement 1—source data 1.

Figure 1—figure supplement 1.. Electrophysiological properties and morphology of rescued CRs in ΔNp73^cre/+; R26^Kir2.1/+ mice.

(A) Schematic representation of the electrophysiological recordings from ΔNp73^cre/+ CRs. (B) Biocytin-filled CRs in ΔNp73^cre/+;R26^mT/+ at P17 and ΔNp73^cre/+;R26^Kir2.1/+ at P15 and P28 displayed a similar characteristic morphology of CRs. (C) Electrophysiological properties of control and rescued CRs expressing Kir2.1. Note that rescued CRs expressing Kir2.1 are hyperpolarized (resting potential, V_rest) at both P15-P17 (n = 16 for controls and n = 17 for mutants, p=0.0001) and P25-29 (n = 11 for mutants, p=0.002; Kruskal-Wallis test followed by a Bonferroni multiple comparison). Moreover, Kir2.1 expression modifies input resistance (Rin), without showing modifications of the amplitudes and durations of the first and second spikes or the afterhyperpolarization (AHP) (n = 12 for controls and n = 15 for mutants at P15-P17 and n = 7–8 for mutants at P25-P29; p<0.0001 for both ages; ANOVA test followed by a Bonferroni multiple comparison). (D) Quantifications of soma diameter of rescued ΔNp73^cre/+;R26^Kir2.1/+ CRs at P15-P17 and P25-P29 compared to control ΔNp73^cre/+;R26^mT/+ CRs at P13-P17 (n = 8 for controls and n = 8 for mutants at P15-P17, p=0.075; n = 9 for mutants at P25-P29, p=0.004, compared to P13-P17 controls; one-way ANOVA test followed by a Bonferroni multiple comparison). (E) Quantifications of filament length of ΔNp73^cre/+;R26^Kir2.1/+ CRs at P15-P17 and P25-P29 compared to control ΔNp73^cre/+;R26^mT/+ CRs at P13-P17 (n = 8 for controls and n = 7 for mutants at P15-P17, p=0.636; n = 8 for mutants at P25-P29, p=0.964, compared to P13-P17 controls). (F) Merged and single channel confocal images of P7 and P25 mutant and control brains stained for DsRed (red), Reelin (Reln, green) and Hoechst (blue), showing that DsRed positive cells express Reln. Scale bars represent 100 μm. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 1—figure supplement 1—source data 1.

Figure 2.. Pure GABAergic sPSCs and ePSCs in rescued CRs.

(A) Spontaneous PSCs (sPSCs) recorded in rescued CRs from ΔNp73^cre/+;R26^Kir2.1/+ at P27 (blue) and ΔNp73^cre/+;Bax^lox/lox mutants at P26 (green), respectively. (B) Plots of the frequency and amplitude of sPSCs (n = 11 for ΔNp73^cre/+; R26^Kir2.1/+ and n = 11 for ΔNp73^cre/+; Bax^lox/lox mice at P24-29; frequency: p=0.552, amplitude: p=0.580, Student T Test). Rise time is 2.10 ± 0.42 ms vs 1.02 ± 0.20 ms and decay time 34.26 ± 6.39 ms vs 29.14 ± 3.56 ms for ΔNp73^cre/+;R26^Kir2.1/+ and ΔNp73^cre/+;Bax^lox/lox mice, respectively. (C) Mean evoked PSCs (ePSCs) for rescued CRs respectively from a ΔNp73^cre/+;R26^Kir2.1/+ mutant at P29 (blue) and a ΔNp73^cre/+;Bax^lox/lox mutant at P26 (green) upon stimulation of LI neuronal fibers (stimulation time, arrowhead) in control conditions (top), with SR95531 (middle) and SR95531 in Mg²⁺-free solution (bottom). Note that ePSCs completely disappeared after bath application of SR95531. (D) Amplitudes of ePSCs in control conditions, with SR95531 and with SR95531 in Mg²⁺-free solution (ΔNp73^cre/+;R26^Kir2.1/+ mice at P24-29: n_control = 10, n_SR95531 = 8 and n_SR95531/_Mg2+free=8; ΔNp73^cre/+;Bax^lox/lox: n_control = 8, n_SR95531 = 5 and n_SR95531/_Mg2+free=5; Kruskal-Wallis test followed by a Bonferroni multiple comparison when comparing the three conditions for each mutant; Student T test for comparison of control ePSCs between ΔNp73^cre/+;R26^Kir2.1/+ and ΔNp73^cre/+;Bax^lox/lox mutants, p=0.638). To detect CRs in ΔNp73^cre/+;Bax^lox/lox mutants the R26^mT/+ reporter line was used. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 2—figure supplement 1—source data 1.

Figure 2—figure supplement 1.. Pure GABAergic sPSCs and ePSCs in control CRs during early postnatal development.

(A) Spontaneous PSCs (sPSCs) recorded in a control CR from ΔNp73^cre/+;R26^mt/+ at P10. (B) Plots of the frequency and amplitude of sPSCs (n = 8) (p=0.0025 and p=0.0022 for the frequency of sPSCs of controls compared to rescued CRs of both ΔNp73^cre/+;R26^Kir2.1/+ and ΔNp73^cre/+;Bax^lox/lox;R26^mT/+ mice (Figure 2A); one-way ANOVA followed by a Bonferroni post hoc test). (C) Mean evoked PSCs (ePSCs) for a control CR at P9-11 upon stimulation of LI neuronal fibers in control conditions (top), with SR95531 (middle) and SR95531 in Mg²⁺-free solution (bottom). Note that ePSCs completely disappeared after bath application of SR95531. Stimulation artefacts were blanked for visibility. The stimulation time is indicated (arrowheads). (D) Amplitudes of ePSCs in control conditions (p=0.0027 and p=0.0067 for the amplitude of ePSCs in controls compared to rescued CRs of ΔNp73^cre/+;R26^Kir2.1/+ and ΔNp73^cre/+;Bax^lox/lox;R26^mT/+mice, (Figure 2D); one-way ANOVA followed by a Bonferroni post hoc test), in the presence of SR95531 and with SR95531 in Mg²⁺-free solution (n_control = 8, n_SR95531 = 7 and n_SR95531/_Mg2+free=7; Kruskal-Wallis test followed by a Bonferroni multiple comparison). (E) Confocal images of a control CR at P10 in ΔNp73^cre/+;Tau^GFP/+ mice expressing GFP (green), Gephyrin (red) and contacted by GABAergic GAD65/67-positive presynaptic terminals (blue; objective 93×; stack of 109 Z sections, each 0.07 µm, n = 11). Arrowheads show GABAergic synapses onto the CR. Note the partial co-localization of GAD65/67 and Gephyrin on the GFP⁺ membrane of the CR (Inset). Scale bars: 5 µm and 1 µm (inset). (F) Confocal images of a Layer I CR in the cortex and non-CR in the hypothalamus at P10 stained for DAPI (blue), KCC2 (red) and GFP (green) in ΔNp73^cre/+; Tau^GFP/+ mice (objective 93×; single plane of 0.07 µm, n = 6). Note the low level of KCC2 expression in control CR (upper panels) compared to non-CR of ΔNp73^cre/+;Tau^GFP/+ mice (bottom panels). Scale bar represents 5 µm. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 2—figure supplement 1—source data 1.

Figure 3.. Increased dendritic branches in LII/LIII pyramidal neurons of ΔNp73^cre/+;Bax^lox/lox mutants.

(A) Representative examples of LII/III pyramidal neurons filled with biocytin in control (P25) and ΔNp73^cre/+;Bax^lox/lox mutant (P24) somatosensory cortex. (B) Quantification of the number of dentritic branches in control and ΔNp73^cre/+;Bax^lox/lox mutant LII/III pyramidal neurons, expressed as a percentage of dendritic branches relative to the mean of controls (n = 7 for controls and n = 8 for mutants at P23-28 p=0.0182 for apical dendrites and p=0.014 for basal dendrites; Mann-Whitney U Test). (C) Sholl analysis for the apical and basal dendrites in control and ΔNp73^cre/+;Bax^lox/lox mutants showing an increased cell complexity between 180 and 240 µm (p-value=0.04, 0.027, 0.0007 and 0.005, respectively) and 60 and 80 µm (p value=0.027 and 0.019, respectively) from the soma, respectively (n = 7 for controls and n = 8 for mutants). Multiple T-test. Scale bar represents 100 μm. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 3—figure supplement 1—source data 1.

Figure 3—figure supplement 1.. Morphological reconstruction of LII/LIII pyramidal neurons in ΔNp73^cre/+;R26^Kir2.1/+ mutants.

(A) Representative examples of somatosensory cortex LII/III pyramidal neurons filled with biocytin in control (P24) and ΔNp73^cre/+;R26^Kir2.1/+ (P27) mice. (B) Quantification of number of dendritic branches of LII/III pyramidal neurons in controls and ΔNp73^cre/+;R26^Kir2.1/+ mice, expressed as a percentage relative to the mean of controls (apical dendrites n = 7 for controls and n = 9 for mutants at P23-P29, p=0.550; basal dendrites n = 7 for controls and n = 8 for mutants at P23-P29, p=0.199; Mann-Whitney U Test). (C) Sholl analysis for the apical and basal dendrites in control and ΔNp73^cre/+;R26^Kir2.1/+ mutant mice showing no significant change in cell complexity with respect to the distance from the soma, (apical dendrites n = 7 for controls and n = 9 for mutants; basal dendrites n = 7 for controls and n = 8 for mutants). Multiple T-test. Scale bar represents 100 μm. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 3—figure supplement 1—source data 1.

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.