Cage effects on synaptic plasticity and its modulation in a mouse model of fragile X syndrome

Abstract

Fragile X syndrome (FXS) is characterized by impairments in executive function including different types of learning and memory. Long-term potentiation (LTP), thought to underlie the formation of memories, has been studied in the Fmr1 mouse model of FXS. However, there have been many discrepancies in the literature with inconsistent use of littermate and non-littermate Fmr1 knockout (KO) and wild-type (WT) control mice. Here, the influence of the breeding strategy (cage effect) on short-term potentiation (STP), LTP, contextual fear conditioning (CFC), expression of N-methyl-d-aspartate receptor (NMDAR) subunits and the modulation of NMDARs, were examined. The largest deficits in STP, LTP and CFC were found in KO mice compared with non-littermate WT. However, the expression of NMDAR subunits was unchanged in this comparison. Rather, NMDAR subunit (GluN1, 2A, 2B) expression was sensitive to the cage effect, with decreased expression in both WT and KO littermates compared with non-littermates. Interestingly, an NMDAR-positive allosteric modulator, UBP714, was only effective in potentiating the induction of LTP in non-littermate KO mice and not the littermate KO mice. These results suggest that commonly studied phenotypes in Fmr1 KOs are sensitive to the cage effect and therefore the breeding strategy may contribute to discrepancies in the literature.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Keywords: CFC; Fmr1; LTP; NMDAR PAM; STP; littermate syndrome.

Figure 1.

Schematic of the breeding paradigm used to generate mice of each experimental genotype^{maternal genotype} and cage background (littermate vs non-littermate). FXS is an X-linked disorder and, therefore, male mice only inherit their X chromosome from their mothers, whereas female mice inherit one X chromosome from each of the parents. A pool of breeders was created by crossing (i) a HET mother and WT father, and (ii) a HET mother with a KO father (shared grandparents). Using parents from the pool of breeders, male WT and hemizygous KO (males have one X chromosome) subject mice (experimental animals) were obtained; their genotype notation is shown together with their mother’s genotype in superscript (subject genotype^{maternal genotype}). Non-littermate WT^WT mice were produced by crossing WT female and WT male mice, whereas for the non-littermate KO^KO mice, KO female mice were crossed with KO males. To obtain littermate WT and KOs, HET females were crossed with male WTs to produce WT^HET and KO^HET mice. These WT^WT, KO^KO, WT^HET and KO^HET mice were used for electrophysiological, behavioural and Western blotting experiments.

Figure 2.

Breeding in a littermate or non-littermate fashion determines the synaptic plasticity deficit outcome in Fmr1 KO mice. (a) Time course of synaptic responses (fEPSPs) in CA3-CA1 hippocampus synapses from non-littermate WT^WT (dark blue circles; n = 9) and KO^KO mice (light blue circles; n = 10). (b) Time course of fEPSPs in littermate WT^HET (dark green circles; n = 8) and KO^HET mice (light green circles; n = 11). Sample fEPSP traces (representative experiment) in the upper right sections of plots (a) and (b) are superimposed from (1) baseline, (2) STP and (3) LTP time points as indicated. Scale bar: 0.5 mV per 5 ms. (c) STP was affected by the genotype (p = 0.046), but not the cage effect (p = 0.269), with a significant interaction between the two variables (p = 0.026). Specifically, the extent of STP was significantly decreased in KO^KO and WT^HET mice compared with WT^WT mice (p = 0.004 and p = 0.039, respectively). There were no significant (ns) differences between slices from littermate KO^HET and WT^HET mice. (d) Neither genotype (p = 0.777) nor cage effect (p = 0.383; interaction, p = 0.596) influenced the decay time constant ( ${\displaystyle \tau }$ ) of STP. (e) The genotype (p = 0.003) but not the cage effect (p = 0.475; interaction, p = 0.158) had an effect on the level of LTP induced. Notably, there was only a significant deficit in non-littermate KO^KO mice compared with WT^WT mice (p = 0.002). Experiments were analysed with a two-way ANOVA with Fisher’s LSD post hoc test.

Figure 3.

The cage effect determines fear memory deficit in Fmr1 KO mice. At 24 h after contextual fear conditioning, mice were placed back into the conditioning chambers and their immobility (freezing) during the 5 min test was determined. The non-littermate KO^KO mice (open blue bar; n = 28) demonstrated deficits in freezing compared with WT^WT mice (filled blue bar; n = 23; p = 0.049). However, KO^HET mice (open green bar; n = 15) immobility was similar to their littermate WT^HET mice (filled green bar; n = 17; p = 0.840). Experiments were analysed with a two-way ANOVA with Fisher’s LSD post hoc test.

Figure 4.

Differential expression of NMDAR subunits in the CA1 area of the hippocampus from littermate versus non-littermate Fmr1 KO mice. (a) Representative blots of NMDAR GluN1, GluN2A and GluN2B subunits from hippocampus of WT^WT, WT^HET, KO^HET and KO^KO mice. Quantification of each targeted protein was normalized to total protein and expressed as a per cent of WT^WT (see the electronic supplementary material, figure S1 for full blots). (b) The cage effect (p = 0.001), but not genotype (p = 0.786; interaction, p = 0.983) influenced the expression of GluN1 subunits. WT^HET (filled green bar; n = 9) and KO^HET mice (open green bar; n = 9) had significantly decreased expression of GluN1 compared with WT^WT (filled blue bar; n = 9; p = 0.012) and KO^KO mice (open blue bar; n = 9; p = 0.012), respectively. (c) The decreased GluN2A expression in Fmr1 KO mice was a result of the cage effect (p < 0.001) and not genotype (p = 0.269; interaction, p = 0.615). GluN2A expression in WT^HET mice (n = 9) was decreased compared with WT^WT mice (n = 9; p = 0.015) and expression in KO^HET mice (n = 8) was decreased compared with KO^KO mice (n = 9; p = 0.003). (d) GluN2B expression was not affected by the genotype (p = 0.515) but was affected by the cage effect (p = 0.001; interaction, p = 0.434). Specifically, expression of GluN2B was reduced in WT^HET mice (n = 9) compared with WT^WT mice (n = 9; p = 0.043) and in KO^HET mice (n = 8) compared with KO^KO mice (n = 9; p = 0.004). Experiments were analysed with a two-way ANOVA with Fisher’s LSD post hoc test.

Figure 5.

LTP in non-littermate KO^KO but not littermate KO^HET, is sensitive to a NMDAR PAM. (a) Time course of fEPSPs in hippocampal slices from KO^KO mice, under vehicle (blue circles; n = 7) or UBP714 (100 μM; pink circles; n = 7) conditions. (b) Time course of fEPSPs in KO^HET slices, under vehicle (green circles; n = 9) or UBP714 (100 μM; pink circles; n = 10) conditions. Sample fEPSP traces (upper right section of plots in (a) and (b) are superimposed from (1) baseline, (2) STP and (3) LTP periods as shown. Scale bar: 0.5 mV per 5 ms. (c) Neither UBP714 application (p = 0.360), nor cage effect (p = 0.513) influenced STP (interaction, p = 0.211) in the two types of KO mice. (d) Similarly, the τ of STP was unaffected by the cage effect (p = 0.590) or UBP714 application (p = 0.097; interaction: p = 0.832). (e) Treatment with UBP714 had a significant effect on LTP (p = 0.035), with a significant interaction between UBP714 treatment and the cage effect (p = 0.004). The cage effect itself had no significant effect on LTP (p = 0.587). Specifically, the application of UBP714 to non-littermate KO^KO hippocampal slices significantly enhanced LTP (p = 0.001), while in the KO^HET slices UBP714 had no effect (p = 0.464). Notably, the level of LTP in KO^KO + UBP714 was significantly greater than in KO^HET + UBP714 slices (p = 0.012). Experiments were analysed with a two-way ANOVA with Fisher’s LSD post hoc test.