Gephyrin S305 phosphorylation controls fear memory stability and engram

The effect of S305A Gephyrin mutant on the size of the cellular ensemble during spatial exploration suggests that it may also affect memory function. To test whether Gephyrin S305 phosphorylation in excitatory neurons regulates contextual fear memory formation and cellular engram function, we bilaterally infected WT mice and direct the expression of either S305A, S305D or WT Gephyrin to excitatory neurons. Mice were exposed to a

CamKII_Cre x Dio_TdTomato x Dio_Gephyrin-GFP WT Dio_Gephyrin-GFP S305A

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Stefanelli et al., Figure 3bis

A B C

contextual fear conditioning and tested for memory recall 1 day and 1 week later (Figure 28A). During contextual memory recall 1 day and 1 week later, S305A infected mice showed increased freezing levels compared to control group (Figure 28B, Training t(27) = 0.03, p = 0.99, non significant (ns), Bonferroni post hoc; 1 day t(27) = 3.20, *p < 0.05, Bonferroni post hoc; 1 week t(27) = 3.98, **p < 0.01, Bonferroni post hoc, 5, 6 mice per group). The increased fear memory response in S305A expressing animals after 1 week correlated with a larger cellular engram size as shown by the number of c-fos activated cells immediately after the recall session (Figure 28C-D, t(10) = 3.92, **p < 0.01, 6 mice per group). On the other hand, freezing levels of S305D infected mice did not differ from control group in memory recall in both 1 day and 1 week recall sessions (Figure 28E, Training t(36) = 0.21, p = 0.99, non significant (ns), Bonferroni post hoc; 1 day t(36) = 2.38, p = 0.07, ns, Bonferroni post hoc; 1 week t(36) = 1.33, p = 0.58, ns, Bonferroni post hoc, 6, 8 mice per group). The size of the cellular engram in S305D mutant expressing mice after 1 week recall was similar to the control group (Figure 28F-G, t(11) = 0.34, p = 0.74, ns, 6,8 mice per group).

Figure 28. GABAergic synapse plasticity controls fear memory stability and engram size.

A. WT, SST-cre or PV-cre mice were infected bilaterally in the Dentate Gyrus (DG) of the dorsal hippocampus with a mix of viral vectors. 4 weeks later, mice were exposed to contextual fear conditioning and tested for memory retrieval 1 day and 1 week later. 1 hour after the last test mice were perfused for c-fos analysis. B. WT mice expressing S305A Gephyrin mutant in Granule cells (GCs) (Gephyrin-GFP S305A, red bar) showed increased freezing levels with respect to WT Gephyrin (Gephyrin-GFP WT, white bar) injected mice, Training p = 0.99, non significant (ns); 1 Day *p < 0.05; 1 Week **p < 0.01, two-way ANOVA, Bonferroni post hoc, 6, 5 mice per group. C. WT mice expressing S305A Gephyrin mutant in GCs showed a larger population of c-fos+ GCs induced by 1 week memory recall session, t(10) = 3.92, **p < 0.01, 6 mice per group. D. Illustration of hippocampal slices containing the DG upper blade labelled with c-fos (red) used to evaluate neuronal activation in WT mice expressing either WT Gephyrin or S305A Gephyrin mutant after 1 week recall session. Scale bar: 25 µm E. WT mice expressing S305D Gephyrin mutant in GCs (Gephyrin-GFP S305D, blue bar) showed similar freezing levels with respect to WT Gephyrin (Gephyrin-(Gephyrin-GFP WT, white bar) injected mice, Training p = 0.99, non significant (ns); 1 Day p = 0.07, ns; 1 Week, p = 0.57, ns, two-way ANOVA, Bonferroni post hoc, 8, 6 mice per group. F. WT mice expressing S305D Gephyrin mutant or WT Gephyrin in GCs, showed similar levels of c-fos+ GCs induced by 1 week memory recall session, t(11) = 0.34, p = 0.74, 8, 5 mice per group. G. Illustration of hippocampal slices containing the DG upper blade labelled with c-fos (red) to evaluate neuronal activation in WT mice expressing either WT Gephyrin or S305D Gephyrin mutant after 1 week recall session. Scale bar: 25 µm. H. SST-cre mice expressing S305A Gephyrin mutant in Somatostatin interneurons (SST) (Gephyrin-GFP S305A, red bar) showed reduced freezing levels with respect to WT Gephyrin (Gephyrin-GFP WT, white bar) injected mice, Training p = 0.99, non significant (ns); 1 Day **p < 0.01; 1 Week **p < 0.01, two-way ANOVA, Bonferroni post hoc, 7 mice per

cFos

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group. I. SST-cre mice expressing S305A Gephyrin mutant in SST showed a smaller population of c-fos+

GCs induced by 1 week memory recall session, t(11) = 2.25, *p < 0.05, 7, 6 mice per group. J. PV-cre mice expressing S305A Gephyrin mutant in Parvalbumin interneurons (PV) (Gephyrin-GFP S305A, red bar) showed similar freezing levels with respect to WT Gephyrin (Gephyrin-GFP WT, white bar) injected mice, Training p = 0.99, non significant (ns); 1 Day p = 0.99, ns; 1 Week p = 0.99, ns, two-way ANOVA, Bonferroni post hoc, 9, 8 mice per group. K. PV-cre mice expressing S305A Gephyrin mutant in PV showed a similar levels of c-fos+ GCs induced by 1 week memory recall session, t(15) = 0.24, p = 0.81, 8, 9 mice per group.

These experiments indicate that blockade of Gephyrin S305 phosphorylation in excitatory neurons of the DG increases the cellular engram size and memory stability.

SST+ and PV+ INs of the DG are known to participate in the mechanisms that allow memory formation and retrieval in the hippocampus (Lovett-Barron et al., 2014; Stefanelli et al., 2016; Yuan et al., 2017). For this reason, we tested the effect of blocking S305 phosphorylation in SST-cre and PV-cre mice in the contextual fear conditioning paradigm as previously described (Figure 28A). SST-cre mice infected with S305A showed decreased freezing levels at 1 day and 1 week recall session with a concomitant decrease in the number of c-fos+ GCs (Figure 28H-I, Training t(36) = 0.27, p = 0.99, ns, Bonferroni post hoc; 1 day t(36) = 3.78, **p < 0.01, Bonferroni post hoc; 1 week t(36) = 3.18, **p <

0.01, Bonferroni post hoc, 7 mice per group; c-fos t(11) = 2.52, *p < 0.05, 6, 7 mice per group). Infection of S305A in PV-cre mice did not produce apparent differences in memory recall in both 1 day and 1 week recall sessions (Figure 28J, Training t(42) = 0.75, p = 0.99, ns, Bonferroni post hoc; 1 day t(42) = 0.87, p = 0.99, Bonferroni post hoc; 1 week t(42) = 0.23, p = 0.99, Bonferroni post hoc, 8, 9 mice per group). In PV-cre mice injected with S305A mutant, the number of activated c-fos+ GCs at 1 week memory recall was similar to control group (Figure 28K, t(15) = 0.24, p = 0.81, 8, 9 mice per group). These results indicate that blockade of Gephyrin S305 phosphorylation in SST+ but not in PV+ INs of the DG regulates the size of the cellular engram and memory stability.

DISCUSSION