Optical controlling reveals time-dependent roles for adult-born dentate granule cells

Abstract

Accumulating evidence suggests that global depletion of adult hippocampal neurogenesis influences its function and that the timing of the depletion affects the deficits. However, the behavioral roles of adult-born neurons during their establishment of projections to CA3 pyramidal neurons remain largely unknown. We used a combination of retroviral and optogenetic approaches to birth date and reversibly control a group of adult-born neurons in adult mice. Adult-born neurons formed functional synapses on CA3 pyramidal neurons as early as 2 weeks after birth, and this projection to the CA3 area became stable by 4 weeks in age. Newborn neurons at this age were more plastic than neurons at other stages. Notably, we found that reversibly silencing this cohort of ~4-week-old cells after training, but not cells of other ages, substantially disrupted retrieval of hippocampal memory. Our results identify a restricted time window for adult-born neurons essential in hippocampal memory retrieval.

Figure 1. Adult-born neurons form functional synapses on CA3 pyramidal neurons

(a) Upper: Experimental timeline. Lower: Image showing adult-born DGCs (4 wpi) and their axons (EGFP⁺). Scale bar: 50 μm. (b) Light pulses (473 nm, 5 ms) elicit action potentials in ChR2-EGFP-infected, but not neighboring (EGFP ) DGCs. Left: Image showing recorded DGCs filled with biocytin (white) in an acute brain slice (4 wpi). i, a ChR2-EGFP⁺ newborn neuron (green and white); ii, a non-infected neighbor (white). Scale bar: 50 μm. Right: Light induced action potentials in EGFP⁺ (i) but not in EGFP⁻(ii) DGC. (c) Optically-evoked EPSCs from adult-born DGCs. Left: Image showing ChR2-EGFP⁺ axonal terminals (green) of adult-born DGCs (4 wpi) and a recorded CA3 pyramidal neuron (white). Scale bar: 25μm. Right: a sample optically-evoked EPSCs recorded from this cell, subsequently blocked by 50 μM CNQX. (d) Axon integration (left, EGFP) and formation of functional synapses on CA3 neurons (right) at 1, 2, 3 and 4 wpi. Scale bar: 10 μm. (e) Amplitude of EPSCs at 1, 2, 3, 4, 6 and 8 wpi (p<0.05; n=5–15 cells, two-tailed unpaired t-test). (f) Latency of EPSCs at 2, 3, 4, 6 and 8 wpi. (g) Shown is a summary of EPSCs which were inhibited by 50μM L-AP4 (t=5.465, p=0.012; n=5 cells, two-tailed paired t-test) or 1μM LY354740 (t=3.891, p=0.027; n=6 cells, two-tailed paired t-test), and blocked by 50μM CNQX (t=5.835, p=0.007; n=15 cells, two-tailed paired t-test). All values represent mean±SEM (*, p<0.05).

Figure 2. Adult-born neurons at 4 weeks of age show enhanced plasticity at output synapses

(a) Experimental timeline. (b) Optically stimulating adult-born neurons produces fEPSPs in the CA3 area. Left, fEPSPs blocked by infusion of 50μM CNQX (but not saline). Right, optical stimulation (50 Hz pulses of 5 ms) reliably induced fEPSPs. Scale bars: 5 ms and 0.1 mV. (c) Theta-burst optical stimulation of adult-born neurons produces LTP at CA3 synapses in an age-dependent manner. Top row, examples of fEPSPs LTP from a single animal at 3, 4 or 8 wpi. Insets: Averaged traces of fEPSPs from 5 consecutive recordings before (1), immediately following (2), and after (3) LTP induction using TBS (blue arrow, Supplementary Fig. 2). Shown at the bottom is a summary of LTP from groups of animals, respectively. (d) Percentage of mice (3, 4 and 8 wpi) exhibiting reliable LTP. (e) Summary of the mean potentiation of fEPSPs amplitude 45–60 min following TBS from mice under control condition (3, 4 and 8 wpi with 6, 8 and 8 animals, respectively; t-test between groups: 3 vs. 4 wpi t=3.386, p=0.007; 8 vs. 4 wpi, t=4.483, p=0.002) or after application of mibefradil (25mg/kg, i.p.) (t-test between control and mibefradil conditions: 3 wpi, t=3.823, p=0.007, n=4; 4 wpi, t=5.656, p<0.001, n=5; 8 wpi, t=0.954, p=0.41, n=4). In each group, all animals tested with stable baseline were included. All values represent mean±SEM (both * and # mean p<0.05; n.s., p>0.05; n is the number of animals; two-tailed unpaired t-test).

Figure 3. Reversibly silencing 4 week-old adult-born neurons impairs hippocampal memory retrieval

(a) Optical stimulation (589 nm) silences Arch-EGFP expressing adult-born neurons. Scale bars: 100 ms and 30 mV. (b) Schematic drawing showing a mouse with implanted optrodes connected to an orange light source via optic fibers and an optic rotatory joint. (c) Time-line of watermaze experiment. (d) Animals were trained with no light. Shown is the training curve of latency to find the platform. (e) Optically inactivating a cohort of 4 week-old adult-born neurons impaired hippocampal memory retrieval. During the probe, mice in the “no light” condition spent significantly more time searching in the target quadrant (NE) compared to the other quadrants: (One-way repeated measures ANOVA F_3,39=7.139, p<0.001; NE>NW, SW, SE by paired t-test planned comparison, n=14), showing robust spatial memory. In contrast, mice in the “light” condition (inactivation) didn’t spend significantly more time in NE compared to other quadrants (F_3,39=0.9655, p=0.4187; p>0.05 NE vs. NW, SW, SE; n=14), showing a disruption of spatial memory (NE_{no light}>NE_light t=2.153, p=0.0253 by planned comparison). (f) Optical inactivation did not alter swim speed in hidden probe tests (P1 and P2, t=1.046, p=0.3145; n=14) and visible probe tests (P3 and P4, t=0.6464, p=0.2646; n=14). (g) Optical inactivation did not alter swim distance in hidden probe tests (P1 and P2, t=1.008, p=0.1660; n=14) and visible probe tests (P3 and P4, t=0.0173, p=0.09867; n=14). All values represent mean±SEM (*: p<0.05; one-way ANOVA or two-tailed t-test).

Figure 4. Temporary silencing of 4 week-old newborn neurons impairs expression of a fear conditioning memory

(a) Time-line of fear conditioning test. Adult mice were infused with a retroviral vector (Arch-EGFP), implanted with optrodes and trained in fear conditioning (single tone-shock pairing). Twenty-four hours after training, contextual fear memory was assessed. An additional tone test was performed in which mice were placed in a novel context and the tone replayed. Animals were divided into two groups and optical silencing was counterbalanced in contextual and tone freezing tests. (b) Silencing of adult-born DGCs at 4 wpi reduced freezing to the context compared to control (n=7, 7). (c) Optically inactivating adult-born DGCs at 4wpi reduced freezing to the context (first 2 minutes of the context test, t=2.239, p=0.0224; n=7, 7), but had no effect on tone fear memory (t=1.675, p=0.0599; n=7, 7). To avoid potential interference from with-in session extinction, we measured freezing time in the first 2 minutes. All values represent mean±SEM (*: p<0.05, two-tailed t-test).

Figure 5. Behavioral roles of adult-born DGCs is sensitive to their age

(a–b) Silencing adult-born DGCs at 2 or 8 wpi showed no significant effect on memory retrieval. Mice at 2 (a) or 8 (b) wpi spent more time searching in the target quadrant (NE) compared to the other quadrants in both “no light” (One-way repeated measures ANOVA 2 wpi: F_3,15=15.25, p<0.0001; 8 wpi: F_3,15=8.081, p=0.0019; NE>NW, SW, SE by paired t-test planned comparison in both; n=6,6) and “light” conditions (2 wpi: F_3,15=9.125, p=0.0011; 8 wpi: F_3,15=14.20, p=0.0001; NE>NW, SW, SE in both; n=6, 6). (c) Silencing adult-born DGCs impacted memory retrieval age-dependently. 4 wpi group showed significant less time searching in target quadrant in “light” condition (n=14) comparing to 2 (n=6, t=2.360, p=0.030), 8 wpi (n=6, t =2.922, p=0.0135), and EGFP 4 wpi (n=8, t=2.392, p=0.0286) by two-tailed unpaired t-test. (d) Silencing of adult-born DGCs at 2 or 8 wpi failed to affect fear memory retrieval. Percent freezing of Arch animals at 2 (t=0.2029, p=0.4246; n=5), 8 wpi (t=0.3824, p=0.3568; n=8) or EGFP at 4 wpi (t=0.4593, p=0.3326; n=6). Two-tailed paired t-test comparison was made between “no light” and “light” conditions. (e) Mibefradil prevented hippocampal memory retrieval of the animals at 4 wpi. Shown is a summary similar to those in (a) (Saline_{No light}: F_3,9=10.43, p=0.0027; NE>NW, SW, SE; Saline_Light: F_3,9=2.849, p=0.0975; Mibefradil_{No light}: F_3,9=1.173, p=0.3730; Mibefradil_Light: F_3,9=1.061, p=0.4128; n=4,4 for all). All values represent mean±SEM (*: p<0.05; n is the animal number, one-way ANOVA or two-tailed t-test).

Figure 6. Task-switching experiments showing that adult-born DGCs at 4 wpi are important for memory retrieval

(a) Upper: Timeline of watermaze and fear conditioning tests. Lower panel: At 4 wpi, watermaze trained animals under the “no light” condition searched selectively (ANOVA F_3,12=9.487, p=0.0017; NE>NW, SW, SE; n=5, 5; Refers the analysis to Figs. 3–5), whereas in the “light” condition they failed to search selectively (F_3,12=4.004, p=0.0345; NE vs. NW t=0.0702, p=0.43; NE vs. SW t=1.792, p=0.077). When trained in the contextual fear conditioning at 8 wpi, animals displayed a similar percent freezing (t=0.3600, p=0.3685). (b) Upper: Time-line of fear conditioning and watermaze tests. Similar experiments have been performed to those in (a), but the order of behavioral tests has been switched: first fear condition and second watermaze. Lower panel: animals showed a significant decrease in percent freezing (t=3.046, p=0.0191) when trained at 4 wpi. However, at 8wpi, both No light and Light groups showed intact watermaze memory (No light: F_3,12=14.48, p=0.0003; Light: F_3,12=9.644, p=0.0016; NE>NW, SW, SE; n=5, 5). All values represent mean±SEM (*: p<0.05; n is the number of animals, one-way ANOVA or two-tailed paired t-test).