Fig. 2

Loss of 4EHP in excitatory neurons exaggerates hippocampal mGluR-LTD and impairs social behavior. a, b Confirmation of loss of 4EHP expression in the prefrontal cortex and hippocampus, respectively, of 4EHP-eKO (flx/flx) versus 4EHP-WT (+ / +) mice using western blot. GAPDH was used as a loading control. c, d Confirmation of loss of 4EHP expression in excitatory neurons in the prefrontal cortex and hippocampus, respectively, of 4EHP-eKO versus 4EHP-WT mice using immunofluorescence microscopy. 4EHP expression is colored in red and Hoechst-stained nucleus in blue. Scale bar represents 20 µm. e Schematic representation of stimulating (left) and recording (right) electrode position for measuring DHPG-induced long-term depression (mGluR-LTD) in the CA1 hippocampus. The red fibers represent CA3 pyramidal projections to the CA1 (Schaffer collaterals). f Field excitatory postsynaptic potential (fEPSP) recordings of CA1 pyramidal neurons during mGluR-LTD. Baseline was recorded for 30 min prior to adding mGluR1/5 agonist, DHPG (100 µM), to slices for 10 min. LTD was recorded for 90 min. The inset is the average of all fEPSPs at time a and b for each genotype, n = 8 per group. g Average of the last 10 min of recording. h Schematic representation of the three-chamber social preference and social novelty test. Mice were first habituated to the apparatus for 10 min. Two cages (mouse holding devices) were then placed in opposite corners of opposing chambers; one cage was empty (E) and one contained a conspecific stranger mouse (S1). After 10 min, a novel stranger mouse (S2) was added to E for the social novelty test lasting 10 min. i The amount of time the test mouse spent sniffing either S1 or E. j The amount of time the test mouse spent sniffing either S1 or S2. k Schematic representation of the direct (reciprocal) social interaction test. Test mice were first habituated to a clean home cage for 5 min. A novel stranger mouse was then added, and mice could freely interact for 10 min. l Nose-to-anogenital sniffing time of the stranger mouse by the test mouse. m Total interaction time including nose-to-nose sniffing, nose-to-anogenital sniffing, following, chasing, mounting, and fighting. Reciprocal interaction of the stranger mouse to test mouse was also included. Data are presented as mean ± s.e.m.; *p < 0.05, **p < 0.01, ****p < 0.0001, N.S., not significant; calculated by unpaired t-test or 2-way ANOVA with Bonferroni multiple comparisons test. Sample size is located within bar graphs. Eif4e2 is the mouse gene encoding 4EHP