Zif268/Egr1 gain of function facilitates hippocampal synaptic plasticity and long-term spatial recognition memory

Abstract

It is well established that Zif268/Egr1, a member of the Egr family of transcription factors, is critical for the consolidation of several forms of memory; however, it is as yet uncertain whether increasing expression of Zif268 in neurons can facilitate memory formation. Here, we used an inducible transgenic mouse model to specifically induce Zif268 overexpression in forebrain neurons and examined the effect on recognition memory and hippocampal synaptic transmission and plasticity. We found that Zif268 overexpression during the establishment of memory for objects did not change the ability to form a long-term memory of objects, but enhanced the capacity to form a long-term memory of the spatial location of objects. This enhancement was paralleled by increased long-term potentiation in the dentate gyrus of the hippocampus and by increased activity-dependent expression of Zif268 and selected Zif268 target genes. These results provide novel evidence that transcriptional mechanisms engaging Zif268 contribute to determining the strength of newly encoded memories.

Keywords: Egr1; conditional mutant mouse; dentate gyrus; long-term potentiation; recognition memory; transcription factor.

Figure 1.

Zif268 overexpression in the brain of the transgenic mice. (a) Coronal brain sections of Dox-induced expression of ß-galactosidase in WT and Zif268-overexpressing mice. Note the strong expression of lacZ in CA1, CA3 and dentate gyrus (DG) areas of the hippocampus (Hip), in neocortex (Ctx), medial caudate putamen (CPu), amygdala (AM) and piriform cortex (Pir). Bottom right image shows sparse lacZ expression in perirhinal (PRh) and lateral entorhinal (Lent) cortices. S2, somatosensory cortex; Pir, piriform cortex; LV, lateral ventricle. Scale bars, top: 2000 μm, bottom: 500 μm. (b) Photomicrographs showing higher Zif268 staining in CA1, CA3 and DG of the hippocampus in Zif268-overexpressing mice. Scale bar, 50 μm. (c) The density of Zif268⁺ cells was significantly increased in the DG of Zif268-overexpressing mice compared with WT mice. *p < 0.05. (Online version in colour.)

Figure 2.

Recognition memory in Zif268-overexpressing mice. (a) Schematic of the object recognition memory paradigm and retention performance of WT and Zif268-overexpressing (Zif268-over) mice 1 and 3 days after training. The histograms represent the recognition index expressed as the per cent time spent exploring the novel object over the total time of objects exploration. Both WT and Zif268-overexpressing mice spent significantly more time exploring the novel object at the 1 day (n = 13 and n = 9, respectively) and 3 days (n = 12 and n = 9, respectively) retention delays. (b) Schematic of the object–place recognition memory paradigm and retention performance of WT and Zif268-overexpressing mice 30 min, 1 and 3 days after training. Both WT (n = 11) and Zif268-overexpressing mice (n = 9) showed preferential exploration of the displaced object at the 30 min delay (left histograms). WT mice no longer showed a preference for the displaced object at the 1 day (n = 20) or the 3 days (n = 19) retention tests with the training protocol used in this study, whereas Zif268-overexpressing mice still spent significantly more time exploring the displaced object at both the 1 day (n = 16) and 3 days (n = 16) retention delays, indicating that Zif268 overexpression facilitates the formation of long-term object–place recognition memory. The horizontal line represents equal exploration of the familiar and novel (a) or displaced (b) objects. *p < 0.05, **p < 0.01, ***p < 0.005 compared with chance.

Figure 3.

Synaptic transmission and plasticity in the dentate gyrus of Zif268-overexpressing mice. (a) The graph plots stimulus–response relationships at MPP–granule cell synapses using a range of stimulation intensities (0–600 µA) in anaesthetized WT and Zif268-overexpressing mice (Zif268-over). Each data point is an average of fEPSP slope values from three responses (abscissa in the log scale). No significant change in the I/O relationship was observed between WT (open circles, n = 6) and Zif268-overexpressing mice (filled circles, n = 5). (b,c) Dentate gyrus LTP in WT and Zif268-overexpressing mice. (b) Time course of LTP induced at MPP–granule cell synapses (WT: open circles, n = 6; Zif268-over: filled circles, n = 5). Responses were recorded for 30 min before and 90 min after the tetanus (arrow) to the MPP. Each data point is an average of 10 consecutive responses recorded over 5 min. All mice showed significant potentiation of fEPSP slope after tetanic stimulation. Inserts are sample waveforms from a WT (a) and a Zif268-overexpressing mouse (b) recorded before (grey line) and 10 min after tetanic stimulation (black line). Scale bars, 1.5 mV; 10 ms. (c) Per cent potentiation of the fEPSP over three 30 min periods post-tetanus. LTP in Zif268-overexpressing mice was of a higher magnitude and lasted longer than in WT mice. Significant difference from baseline: *p < 0.05; **p < 0.01, ***p < 0.005.

Figure 4.

LTP-induced expression of Zif268, synapsin II and the proteasome 20S β-subunit PSMB9 in Zif268-overexpressing mice. (a) Zif268, (b) synapsin II and (c) PSMB9 protein expression was measured by western blotting from the dentate gyrus taken 3 h after induction of LTP (LTP side) and from the contralateral (non-stimulated) side (Cont). Data for each protein and genotype are normalized to the reference protein β-actin and expressed as a per cent change relative to protein levels in the control side of WT mice. There was a trend towards an increase in Zif268, but not synapsin II or PSMB9 expression after LTP in WT mice, and a higher and significant increase of all three proteins after LTP in Zif268-overexpressing mice (Zif268-over). Sample western blots of each protein and actin are represented above the histograms. *p < 0.05.