Enhanced synaptic plasticity and spatial memory in female but not male FLRT2-haplodeficient mice

Abstract

The Fibronectin Leucine-Rich Transmembrane protein 2 (FLRT2) has been implicated in several hormone -and sex-dependent physiological and pathological processes (including chondrogenesis, menarche and breast cancer); is known to regulate developmental synapses formation, and is expressed in the hippocampus, a brain structure central for learning and memory. However, the role of FLRT2 in the adult hippocampus and its relevance in sex-dependent brain functions remains unknown. We here used adult single-allele FLRT2 knockout (FLRT2^+/-) mice and behavioral, electrophysiological, and molecular/biological assays to examine the effects of FLRT2 haplodeficiency on synaptic plasticity and hippocampus-dependent learning and memory. Female and male FLRT2^+/- mice presented morphological features (including body masses, brain shapes/weights, and brain macroscopic cytoarchitectonic organization), indistinguishable from their wild type counterparts. However, in vivo examinations unveiled enhanced hippocampus-dependent spatial memory recall in female FLRT2^+/- animals, concomitant with augmented hippocampal synaptic plasticity and decreased levels of the glutamate transporter EAAT2 and beta estrogen receptors. In contrast, male FLRT2^+/- animals exhibited deficient memory recall and decreased alpha estrogen receptor levels. These observations propose that FLRT2 can regulate memory functions in the adulthood in a sex-specific manner and might thus contribute to further research on the mechanisms linking sexual dimorphism and cognition.

Figure 1

FLRT2+/− mice do not present with gross, detectable phenotypical abnormalities. (A) Adult male or female FLRT2^+/− mice do not present with distinctive gross morphological abnormalities in body or brain structure. (B) Microphotographs of Nissl-stained coronal brain sections showed no obvious differences in shape and major cytoarchitectonic brain organization between wild type and FLRT2^+/− male or female mice. (C) Significant main effect of the sex was observed for body weights (p < 0.001, F_(1,20) = 44.07) with a consistent pattern between wild type and FLRT2^+/− mice. (D) No significant differences were observed in brain weights (ns, p > 0.05). (E) FLRT2^+/− males and females presented with significantly lower levels of FLRT2 mRNA in the hippocampus as compared to their respective wild type littermate controls, thus exhibiting a main effect of the genotype (p < 0.0001, F_(1,19) = 27.93). Results of the post hoc test are indicated in the graph by asterisks. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Number of subjects is indicated within bar charts. Data are displayed as mean ± SEM, sample sizes are indicated inside the bars.

Figure 2

Behavioral analysis of FLRT2^+/− mice shows sex-specific changes in memory recall. Open field test did not reveal significant changes in locomotor activity in male or female FLRT2^+/− mice compared to their corresponding wild type littermates, as determined by measuring (A) total distance traveled. When the average velocity during the test was examined, however, a significant main effect of sex (p = 0.004, F_(1,46) = 9.23) and a main effect of genotype (p = 0.009, F_(1,46) = 7.72) was apparent as indicated in (B) with data showing no significance differences between groups after post hoc analysis. (C) Examinations in the accelerating Rota-rod test showed a significant main effect of sex (p < 0.00001, F_(1,50) = 20.01) with no main effect of genotype on motor coordination. (D) No significant differences in spatial learning were detected between either male or female FLRT2^+/− mice when compared to the their respective wild type littermate controls as determined by escape latencies during training days in the Morris water maze (p > 0.05, n = 8–14). However, there was a statistically significant interaction between the effects of sex and genotype on long-term memory retention (E) during probe trial (p = 0.0001, F_(1,32) = 19.43, n = 8–14). (F) No significant differences in swimming speeds were observed (p > 0.5, n = 8–14). Latencies to reach the hidden platform and average swimming speeds displayed in the graph are averages of three trials per day. Results of the post hoc test are indicated in the graph by asterisks. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Data are displayed as mean ± SEM; sample sizes are indicated inside the bars.

Figure 3

Loss of a single FLRT2 allele alters synaptic plasticity in female but not male mice. (A) Analysis of basal synaptic transmission (input/output curves) in CA3-Schaffer collateral-CA1 synapses. Main significant effect of voltage (p < 0.0001, F_{(7, 385)} = 1885, n = 11–18), groups (p = 0.002, F_{(3, 55)} = 6.0, n = 11–18) and interaction (p < 0.0001, F_{(21, 385)} = 4.8) were observed. Post hoc analysis showed significant difference between male and female wild type mice (p = 0.02, n = 11–18) and between female wild type and female FLRT2^+/− mice (p = 0.002, n = 11–18). Inset cartoon on top represents the approximate positioning of the stimulating and recording electrode. (B) Representative traces of field excitatory postsynaptic potential recorded from animals from respective sex and genotype as indicated. (C) Temporal courses of averaged slopes of fEPSP showed a main significant effect of time (p < 0.0001, F_{(90, 4140)} = 32.6, n = 11–13), a significant main effect between groups (p = 0.03, F_{(3, 46)} = 3.6, n = 11–13) and a significant interaction (p < 0.0001, F_{(270, 4140)} = 1.6). Post hoc analysis revealed statistically significant differences between male and female wild type mice (p = 0.03, n = 11–13) and between female wild type and female FLRT2^+/− mice (p = 0.04, n = 11–13). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Data are displayed as mean ± SEM.

Figure 4

FLRT2 haplodeficiency results in reduced hippocampal levels of EAAT2 and ERβ only in females. (A) Representative pictures of western blot assays (upper panel) and bar charts (below) for densitometric analyses. A significant main effect of the genotype on the protein levels of EAAT2 (p = 0.02, F_{(1, 20)} = 7.394, n = 5–7) and a significant interaction between effects of the genotype and sex (p < 0.0001, F_{(1, 20)} = 30.94) was observed. Blot images were cropped for comparisons; full-length blots are available in the supplementary data. (B) No differences between groups were observed for VGLUT1 levels (p > 0.5, n = 5–7). Blot images were cropped for comparisons; full-length blots are available in the supplementary data. (C) A strong effect of sex (p < 0.0001, F_{(1, 17)} = 31.98, n = 6) and a strong main effect of the genotype (p < 0.0001, F_{(1, 17)} = 33.32, n = 6) on the hippocampal ERα mRNA levels, as well as a significant interaction between two factors (p = 0.003, F_{(1, 17)} = 12.8, n = 6) were observed. (D) qRT-PCR revealed only a significant effect of genotype on the ERβ mRNA levels (p = 0.004, F_{(1, 16)} = 11.32, n = 6). Results of the post hoc test are indicated in the graph by asterisks. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Data are displayed as mean ± SEM; sample sizes are indicated inside the bars.

Figure 5

FLRT2 haplodeficiency affects apical dendritic complexity in hippocampal CA1 pyramidal neurons of female mice. Representative photomicrographs of brain sections showing dendritic arborizations of neurons from female FLRT2^+/+ (A), upper left panel and FLRT2^+/− mice (B), upper left panel as visualized by Golgi-Cox staining. Representative Neurolucida digital neuronal reconstructions are shown in the right panels (in A,B). Higher magnification (100 × oil objective) photomicrographs of tertiary apical branches are also depicted (lower left panels in A and B; Scale bar = 5.8 µm). (C–F) Quantification of the area of hippocampal CA1 pyramidal neuronal somata; total length of apical dendritic tree and number of ends and number of nodes, respectively. (G) Sholl analyses of the total number of dendritic intersections within each shell (as examined using a two-way ANOVA) showed a significant effect of the genotype (p < 0.0001) and a significant effect of radius (p < 0.0001) on the number of intersections. (H) Quantification Stubby, Mushroom, Thin and Long thin dendritic spines in CA1 pyramidal neurons from female FLRT2^+/+ and FLRT2^+/− mice. Data are represented as mean ± SEM; sample sizes are indicated inside the bars. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.