Neuroscience

Adult-born granule cells modulate CA2 network activity during retrieval of developmental memories of the mother

Blake J. Laham
Isha R. Gore
Casey J. Brown
Elizabeth Gould author has email address

Princeton Neuroscience Institute, Princeton University, Princeton NJ 08544

https://doi.org/10.7554/eLife.90600.2

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Figures and data

Adult-born granule cells support retrieval of developmental remote memories of the mother.
(a-aiii) Confocal images of dentate gyrus granule cells, both mature (ZnT3+) and adult-born (3R-Tau+), and their robust projections to CA2. Confocal images of abGC 3R-Tau+ axons (arrows)
(b) near proximal dendrite of a CA2 PCP4+ pyramidal cell and (c) near proximal dendrite of a CA2 mCherry+ PV+ interneuron. (d) abGC fibers (3R-Tau+) in the CA2 are more abundant near proximal dendrites (n = 7; paired t test: t₆ = 3.163, p = 0.0195) of PV+ interneurons (mCherry+) than those of pyramidal cells (PCP4+). (e) Timeline for behavioral experiment. (f) Schematic demonstrating direct social interaction assay used at 3 experimental time points. (g) Ablation of abGCs abolishes difference in investigation time between mother and novel mother (CD1: n = 17; TK: n = 14; 2-way RM ANOVA: Genotype x Drug: F_2,58 = 5.352, p = 0.0074; Šídák’s test p = 0.0021). After removing VGCV from rodent chow and allowing adult neurogenesis to recovery over the course of 6 weeks, mice were able to discriminate between mother and novel mother again (p > 0.999). (h-hii) Confocal images of abGCs in the DG at 3 different drug time points. VGCV administration produces a dramatic loss of (h-hii) abGCs (3R-Tau+ cells) in DG (VGCV-: n = 8; VGCV+: n = 8; unpaired t test: t₁₄ = 6.588, p < 0.0001) and (i-iii) 3R-Tau+ fibers in CA2 (VGCV-: n = 8; VGCV+: n = 8; unpaired t test: t₁₄ = 3.488, p = 0.0036). 6 weeks after removing VGCV from the rodent chow, DG abGC number (j,k) undergoes a 70% recovery (CD1: n = 8; TK: n = 8; unpaired t test: t₁₄ = 3.218, p = 0.0062), and (l,m) 3R-Tau+ fibers present in CA2 undergo a complete recovery (CD1: n = 8; TK: n = 8; unpaired t test: t₁₄ = 0.7271, p = 0.4791). *p < 0.05, bars represent mean + SEM. Scale bars = 200 μm for a, h, i; 2 μm for b,c. ns = not significant

Inhibiting 4-6-week-old abGC projections to CA2 prevents discrimination between mothers and novel mothers.
(a) Schematic of breeding to produce Nestin-cre:Gi double transgenic offspring. (b) Timeline outlining tamoxifen administration, cannula implantation, and behavioral testing. (c) Schematic demonstrating direct social interaction testing. (d-dii) Confocal images depicting 4-6-week-old Gi-DREADD+ abGCs in the DG. (e) Inhibiting 4-6-week-old abGC projections to CA2 with CNO prevents discrimination between mother and novel mother (Nestin-cre: n = 31; Nestin-cre:Gi: n = 33; 2-way RM ANOVA: Genotype x Drug: F_1,62 = 4.042, p = 0.0487; Šídák’s test p = 0.0057). (f) Inhibiting 10-12-week-old abGC projections to CA2 does not influence discrimination between mother and novel mother (Nestin-cre: n = 21 (Veh), n = 17 (CNO; Nestin-cre:Gi: n = 18 (Veh), n = 15 (CNO); Mixed-effects model: Genotype x Drug: F_1,67 = 1.669, p = 0.2008. (g) Schematic for experiment assessing abGC contributions to social memory consolidation. (h) Systemic inhibition of abGCs during memory consolidation has no significant effect on social memory (Nestin-cre: n = 21; Nestin-cre:Gi: n = 18; 2-way RM ANOVA: Genotype x Drug: F_1,37 = 1.238, p = 0.2730). *p < 0.05, bars represent mean + SEM. ns = not significant. Scale bars in d = 200 μm

Adult-born neurons influence CA2 SWR changes associated with discrimination between novel mother and mother
(a)Timeline of experiment demonstrating tamoxifen injection schedule and electrode implantation in CA2. (b) Confocal image demonstrating accuracy of electrode placement. (c) Schematic of electrophysiological recordings during baseline and social trials. (d) Example SWR raw trace (top) and filtered trace (bottom). (e) CA2 SWR frequency is increased during social interactions, regardless of whether the stimulus is the novel mother or mother (Veh: n = 14; CNO: n = 14; 2-way RM ANOVA: Stimulus: F_1,13 = 46.40, p < 0.0001). (f) Inhibiting 4-6-week-old abGCs with CNO prevents characteristic SWR production patterns present during exposure to novel mothers vs mothers (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,110 = 18.6529, p < 0.0001; Tukey’s test p < 0.0001). (g) Inhibiting 4-6-week-old abGCs significantly reduces social trial SWR production (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,228 = 16.2465, p < 0.0001; Tukey’s test p = 0.0018). (h) Inhibiting 4-6-week-old abGCs increases SWR peak amplitude during social interaction trials (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,238 = 3.69, p = 0.0559) and (j) baseline (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,237 = 6.0322, p = 0.01477; Tukey’s test p = 0.0133). (i) Changes in baseline SWR production did not reach significance (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,238 = 3.5101, p = 0.0622). (k) At the 10-12 wk abGC time point, CA2 SWR frequency is increased during social interactions, regardless of stimulus (Veh: n = 9; CNO: n = 11; Mixed-effects model: Stimulus: F_1,10 = 133.1, p < 0.0001).
(l) Inhibiting 10-12-week-old abGCs has no influence on characteristic SWR production patterns present during exposure to the novel mother vs mother (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 7 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,82 = 1.7431, p = 0.1904), nor on (m) SWR production during social trials (Nestin-cre: n = 4 (Veh), n = 5 (CNO) Nestin-cre:Gi: n = 8 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,170 = 0.4496, p = 0.503), or (o) during baseline recording (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 8 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,170 = 2.0552, p = 0.15352). (n) Inhibiting 10-12-week-old abGCs has no influence on SWR peak amplitude social interaction trials (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 8 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,18 = 0.9744, p = 0.3250) or (p) during baseline (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 8 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,172 = 0.540, p = 0.8165), *p < 0.05, bars represent mean + SEM. Scale bar in b = 200 μm. ns = not significant.

Adult-born neurons influence CA2 PAC changes associated with retrieval of developmental memories of the mother
(a) Example trace of raw LFP (black), 4-12 Hz theta oscillation (magenta), and 50-100 Hz gamma oscillation (green). (b,c) Graphs demonstrating mid gamma amplitude modulation across theta phases in Nestin-cre:Gi mice. (d) Inhibiting 4-6-week-old abGCs abolishes the increase in theta-mid gamma PAC present during exposure to the mother (Nestin-cre: n = 7; Nestin-cre:Gi: n = 8; Mixed-effects model: Genotype x Drug: F_1,103 = 4.6729, p = 0.03296; Šídák’s test p = 0.0219). e,f) Graphs demonstrating mid gamma amplitude modulation across theta phases in Nestin-cre:Gi mice. (g) Inhibiting 10-12-week-old abGCs has no influence on theta-mid gamma PAC during exposure to the mother (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 8 (Veh), n = 7 (CNO); Mixed-effects model: Genotype x Drug: F_1,18 = 0.2616, p = 0.6153).

Total investigation times during social interaction.
a) Total investigation times of the mother and a novel mother were assessed for CD1 and GFAP-TK mice before (VGCV-), during (VGCV+), and after VGCV administration (VGCV-Recovery) (CD1: n = 17; TK: n = 14; RM ANOVA: Genotype x Drug: F_6,116 = 1.672, p = 0.1339; Drug: F_1.773,102.9 = 5.515, p = 0.0072). b) Total investigation times of the mother and a novel mother were quantified for Nestin-cre mice and Nestin-cre:Gi mice with a 4-6-week-old population of inhibitory DREADD+ abGCs (Nestin-cre: n = 31; Nestin-cre:Gi: n = 33; Mixed-effects ANOVA: Genotype x Drug x Stimulus: F_1,56 = 4.290, p = 0.0430; Tukey’s test: Nestin-cre Veh Mother vs Novel p = 0.0004, Nestin-cre CNO Mother vs Novel p = 0.0274, Nestin-cre:Gi Veh Mother vs Novel p = 0.0326, Nestin-cre:Gi CNO Mother vs Novel p = 0.9904). c) Total investigation times of the mother and a novel mother were quantified for Nestin-cre mice and Nestin-cre:Gi mice with a 10-12-week-old population of inhibitory DREADD+ abGCs (Nestin-cre: n = 21; Nestin-cre:Gi: n = 17; Mixed-effects ANOVA: Genotype x Drug x Stimulus: F_1,12 = 1.204, p = 0.2941; Drug: F_1,40 = 60.46, p < 0.0001).

VGCV or CNO administration does not alter locomotion.
(a) GFAP-TK mice display normal locomotion during social interaction after 6 weeks of VGCV administration (CD1: n = 17; TK: n = 14; 3-way ANOVA: Genotype x Drug x Stimulus: F_1,29 = 0.01998, p = 0.8886). CNO administration has no effect on locomotion during (b) baseline (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; 2-way RM ANOVA: Genotype x Drug: F_1,14 = 0.02291, p = 0.8818) or (c) social stimulus trials (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; 3-way ANOVA: Genotype x Drug x Stimulus: F_1,14 = 0.06126, p = 0.8081) in Nestin-cre and Nestin-cre:Gi mice. A significant 2-way interaction was observed during social trials, but all post hoc tests failed to reach significance (2-way ANOVA: Genotype x Drug: F_1,14 = 7.180, p = 0.0180). Bars represent mean + SEM. ns = not significant.

4-6-week-old abGCs influence multiple features of CA2 SWRs.
(a) Schematic of electrophysiological recordings during baseline and social trials during the 4-6-week-old abGC timepoint. (b) Silencing 4-6-week-old abGCs reduces average CA2 SWR integral (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,236 = 12.83, p = 0.0004017; Tukey’s test p = 0.0099). (c) Silencing 4-6-week-old abGCs reduces average CA2 SWR duration (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,223 = 24.077, p < 0.0001; Tukey’s test p < 0.0001). (d) Silencing 10-12-week-old abGCs has no effect on average CA2 SWR integral but a significant interaction is observed (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 8 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,172 = 4.0061, p = 0.0469; Tukey’s test p = 0.7457). (e) Silencing 10-12-week-old abGCs does not reduce average CA2 SWR duration but a significant interaction is observed (Nestin-cre: n = 4 (Veh), n = 5 (CNO); Nestin-cre:Gi: n = 8 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,172 = 8.5609, p = 0.003898; Tukey’s test p = 0.1439). *p < 0.05, bars represent mean + SEM. Ns = not significant.

Normalized SWR generation.
(a) Silencing 4-6-week-old abGCs alters CA2 SWR generation (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Linear mixed-effects model: Genotype x Drug x Stimulus: F_1,234 = 13.3621, p = 0.000317; Tukey’s test: Nestin-cre Veh Mother vs Novel p = 0.0042, Nestin-cre CNO Mother vs Novel p < 0.0001, Nestin-cre:Gi Veh Mother vs Novel p = 0.0003, Nestin-cre:Gi CNO Mother vs Novel p = 0.7190). (b) Silencing 10-12-week-old abGCs has no influence on CA2 SWR generation (Nestin-cre: n = 5; Nestin-cre:Gi: n = 7; Linear mixed-effects model: Genotype x Drug x Stimulus: F_1,161.844 = 2.1093, p = 0.14834; Stimulus: F_1,161.844 = 33.0013, p < 0.0001). Bars represent mean + SEM.

Nonsocial stimuli suppress CA2 SWR frequency.
(a) Schematic of electrophysiological recordings during baseline and exposure to nonsocial object (plastic toy animal) trials during the 4-6-week-old abGC timepoint. (b) Nonsocial stimuli slightly inhibit CA2 SWR frequency (Veh: n = 14; CNO: n = 16; 2-way RM ANOVA: Stimulus: F_1,28 = 7.760, p = 0.0095). (c) Silencing 4-6-week-old abGCs has no influence on CA2 SWR frequency during object exposure (Nestin-cre: n = 5 (Veh), n = 7 (CNO); Nestin-cre:Gi: n = 8 Veh), n = 9 (CNO); Mixed-effects model: Genotype x Drug: F_1,95 = 0.7761, p = 0.3806), (d) average integral (Nestin-cre: n = 5, n = 7 (CNO); Nestin-cre:Gi: n = 9 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,108 = 0.1653, p = 0.2027). (e) Silencing 4-6-week-old abGCs reduces average SWR duration during object trials (Nestin-cre: n = 5 (Veh), n = 7 (CNO); Nestin-cre:Gi: n = 9 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,106 = 7.9118, p = 0.005849; Tukey’s test p = 0.0094). (f) Silencing 4-6-week-old abGCs has no effect on SWR peak z-score (Nestin-cre: n = 5 (Veh), n = 7 (CNO); Nestin-cre:Gi: n = 9 (Veh and CNO); Mixed-effects model: Genotype x Drug: F_1,111 = 0.0003, p = 0.9874). *p < 0.05, bars represent mean + SEM. ns = not significant.

4-6-week-old abGC inhibition does not influence theta and gamma power during social interaction.
(a,ai,c,ci,e,ei) Normalized power spectra graphs (Social stimulus/baseline). (b) Silencing 4-6-week-old abGCs has no influence on theta power (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,14 = 0.2303, p = 0.6387). (d) Silencing 4-6-week-old abGCs has no influence on low gamma power (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,14 = 0.7979, p = 0.3868). (f) Silencing 4-6-week-old abGCs has no influence on mid gamma power (Nestin-cre: n = 7; Nestin-cre:Gi: n = 9; Mixed-effects model: Genotype x Drug: F_1,28 = 0.3702, p = 0.5478). Bars represent mean +/-SEM for a-ai,c-ci,e-ei; + SEM for b,d,e. ns = not significant.

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