Egr3, a synaptic activity regulated transcription factor that is essential for learning and memory

Abstract

Learning and memory depend upon poorly defined synaptic and intracellular modifications that occur in activated neurons. Mitogen activated protein kinase-extracellular regulated kinase (MAPK-ERK) signaling and de novo protein synthesis are essential aspects of enduring memory formation, but the precise effector molecules of MAPK-ERK signaling in neurons are not well defined. Early growth response (Egr) transcriptional regulators are examples of MAPK-ERK regulated genes and Egr1 (zif268) has been widely recognized as essential for some aspects of learning and memory. Here we show that Egr3, a transcriptional regulator closely related to Egr1, is essential for normal hippocampal long-term potentiation (LTP) and for hippocampal and amygdala dependent learning and memory. In the absence of Egr3, the defects in learning and memory appear to be independent of Egr1 since Egr1 protein levels are not altered in amygdala, hippocampus or cortex. Moreover, unlike Egr1-deficient mice which have impairments in late phase hippocampal LTP and consolidation of some forms of long-term hippocampus- and amygdala-dependent memory, Egr3-deficient mice have profound defects in early- and late-phase hippocampal LTP, as well as short-term and long-term hippocampus- and amygdala-dependent learning and memory. Thus, Egr3 has an essential role in learning and memory processing that appears to be partly distinct from the role of Egr1.

Figure 1

Hippocampal development is normal in Egr3-deficient mice. Normal neuron distribution, synaptic terminal labeling and interneurons are identified in the hippocampus of (A, B, C, D) WT and (A′, B′, C′, D′) Egr3^−/− mice. Compared to (A) WT, the hippocampus appears structurally normal in (A′) Egr3^−/− brains. Similarly, (B, B′) NeuN immunohistochemistry to label neurons, (C, C′) synaptophysin (Syn) immunohistochemistry to label synaptic terminals and (D, D′) parvalbumin (Parv) immunohistochemistry to label interneurons shows no definitive differences in the hippocampus between WT and Egr3^−/−mice. [Scale bars: 200 μm]

Figure 2

Normal CA1 hippocampal neuron spine density and hippocampal neuron numbers in Egr3^−/− mice. (A) Golgi-cox staining shows normal CA1 pyramidal neuron morphology with typical primary apical dendrites (1°) and spine laden secondary dendrites (2°). Spine morphology on WT and Egr3^−/− secondary dendrites is similar (enlarged regions obtained from representative 2° dendrites similar to white outlined area), and spine density on CA1 pyramidal neuron secondary dendrites is not significantly different (p > 0.3) in WT and Egr3^−/− brains. (B) Neuron density in the (d) dorsal and (v) ventral blade of the dentate gyrus as well as in areas CA3 and CA1 are not significantly different in Egr3^−/− hippocampus relative to WT (p > 0.8, p >0.5, p > 0.5, p > 0.4, Student’s unpaired t-test, respectively). (results represent mean ± SEM with white bars = WT and black bars = Egr3^−/−) [Scale bars: 10 μm (A) and 200 μm (B)]

Figure 3

Biochemical analysis of glutamatergic signaling molecules in WT and Egr3^−/− hippocampus, amygdala and somatosensory cortex. (A) Glutamate receptor subunits, Erk1/2, and activated Erk1/2 (p-Erk1/2) protein levels were analyzed by Western blotting and quantitative densitometry in amygdala, hippocampus and somatosensory cortex. There were no significant differences identified for any of the proteins and brain regions examined in WT and Egr3^−/− mice (p > 0.1). (B) The level of Egr1 protein, a transcriptional regulator closely related to Egr3 and known to play an essential role in learning and memory, was not significantly different between WT and Egr3^−/− in amygdala, hippocampus and somatosensory cortex (p > 0.1 for each comparison). For Egr1 detection, the blots and densitometry results were derived from 5 minute ECL exposures to detect low basal levels of Egr1 in unstimulated (directly from home cage) WT and Egr3^−/− mice. (results represent mean ratios of Egr3^−/−/WT protein ± SEM with black bars = amygdala, white bars = hippocampus and gray bars = somatosensory cortex)

Figure 4

Normal basal synaptic transmission in Egr3^−/− hippocampal Schafer collateral synapses. (A) CA1 excitatory post synaptic potentials (EPSPs) elicited by stimulating WT and Egr3^−/− Schaffer collateral synapses with varying current pulse intensity (0.1 ms pulse width, 20–250 μA) were not significantly different (p > 0.5). (B) Paired-pulse facilitation (PPF) was not significantly different between WT and Egr3^−/− Schaffer collateral synapses using pulse intervals of either 20 ms, 50 ms or 100 ms (p> 0.8). (results represent mean ± SEM with WT = white bars and Egr3^−/− = black bars).

Figure 5

Locomotor activity, shock threshold response, thermal nociception, non-associative learning and overtraining fear conditioning behavior compared between WT and Egr3^−/− mice. (A) Naïve WT and Egr3^−/− mice showed no significant differences in baseline activity (AC0) or in their activity response to audible tone while exploring context (AT0). However, they showed a highly significant increase in activity immediately after foot shock (AS) and there was no difference in the locomotor response to shock between WT and Egr3^−/− mice. (B) There was no significant difference between WT and Egr3^−/− mice in the shock intensity threshold required to elicit either involuntary flinching behavior or a vocalization response to the shock stimulus. (C) Similarly, there was no significant difference in thermal nociception as determined by hotplate testing and hindpaw lick latency in WT and Egr3^−/− mice. (D) Egr3^−/− mice showed normal habituation to exploratory activity in a non-associative learning and memory task. (E) In addition, they were capable of discriminating between two different contexts. WT and Egr3^−/− mice were subjected to weak contextual fear conditioning and after 24 hours, they showed significantly decreased activity as a consequence of fear conditioned freezing. When the mice were immediately exposed to a novel context, their locomotor activity was significantly increased back to levels that were not statistically different from WT and Egr3^−/− naïve mice. (F) To examine whether Egr3^−/− mice are capable of eliciting a normal freezing behavior and to determine whether they have any capacity to learn after highly reinforced training, WT and Egr3^−/− mice received 7 consecutive rounds of context-shock pairing on Day 1 and Day 2, and freezing behavior was scored. Egr3^−/− mice showed consistently less freezing behavior than WT mice after each reinforcement trial but by the end of training on Day 2, freezing behavior between WT and Egr3^−/− was maximal (100%) and not significantly different. (results represent mean ± SEM with WT = white bars and Egr3^−/− = black bars; n.s. = not significant, * = p < 0.001, Student’s unpaired t-test)

Figure 6

Examination of hippocampus and amygdala dependent memory in Egr3^−/− mice. (A) Naïve WT and Egr3^−/− mice showed very little freezing to context prior to training. However, 24 hours after weak training freezing to context was significantly increased in WT mice. By contrast, Egr3^−/− mice had no memory of the aversive shock 24 hours after weak training since there was no significant increase in freezing to context compared to naïve Egr3^−/− mice. Similarly, Egr3^−/− mice showed significantly less freezing to context than WT mice 0.5 and 24 hours after strong training. Whereas freezing to context remained high and did not significantly change between 0.5 and 24 hours after strong training in WT mice, freezing to context significantly dissipated between 0.5 and 24 hours in Egr3^−/− mice. (B) Habituated WT and Egr3^−/− mice spent equal time exploring two familiar (F) objects presented in the context (Train). WT mice showed a highly significant preference to explore a novel (N) object when it was substituted for a familiar object, whereas Egr3^−/− mice showed no such preference either 10 minutes or 24 hours after habituation training. Egr3^−/− mice apparently had no memory of the remaining familiar object after one of the familiar objects was replaced with a novel object either 10 minutes or 24 hours after training since in either case there was no significant preference to explore one object relative to the other. (C) Naïve WT and Egr3^−/− mice showed minimal freezing to tone. However, 24 hours after weak cued associative training both WT and Egr3^−/− mice showed significantly increased freezing to tone compared to naïve mice, but freezing to tone was significantly decreased in Egr3^−/− compared to WT mice. Similarly, 30 minutes and 24 hours after strong training, Egr3^−/− mice showed significantly less freezing to tone compared to WT mice. (results represent mean ± SEM with WT = white bars and Egr3^−/− = black bars; n.s. = not significant, * = p < 0.001, Student’s unpaired t-test).

Figure 7

Defective LTP in Schaffer collateral synapses in Egr3^−/− mice. LTP was reliably induced in Schaffer collateral synapses using either (A) TBS or (B) HFS in WT and Egr3^−/− hippocampal slices. However, LTP decreased in Egr3^−/− mice shortly after induction relative to WT and remained significantly decreased, at least up to 210 minutes after induction. (C) Representative EPSP traces at three time points after LTP induction by HFS. In WT slices, EPSP magnitude was markedly increased after LTP induction (thick line trace) relative to EPSP magnitude prior to LTP induction (thin line trace) 30, 60 and 210 minutes after HFS. In Egr3^−/− slices however, the CA1 EPSP magnitude progressively declined over the 30–210 minute interval after LTP induction by HFS. (D) LTP in Schaffer collateral synapses was significantly decreased in Egr3^−/− slices compared to WT at 30, 60 and 210 minutes post-LTP induction. Dashed line represents EPSP baseline (before HFS). (results represent mean ± SEM with WT = white and Egr3^−/− = black; A, average of 9 slices from 9 animals each genotype; B,D, average of 5 slices from 5 WT animals and average of 6 slices from 5 Egr3^−/− animals; * = p < 0.05, ** = p < 0.01, Student’s unpaired t-test). [Scale bars: 10 msec, 0.1 mv]