Phosphorylation of ERK/MAP kinase is required for long-term potentiation in anatomically restricted regions of the lateral amygdala in vivo

Abstract

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term potentiation (LTP) in the amygdala, in vitro. The present experiments evaluated the role of the ERK/MAPK signaling cascade in LTP at thalamo-LA input synapses, in vivo. We first show that ERK/MAPK is transiently activated/phosphorylated in the LA at 5 min, but not 15 or 60 min, after high-frequency, but not low-frequency, stimulation of the auditory thalamus. ERK activation induced by LTP-inducing stimulation was anatomically restricted to the same regions of the LA previously shown to exhibit ERK regulation following fear conditioning. We next show that intra-LA infusion of U0126, an inhibitor of ERK/MAPK activation, impairs LTP at thalamo-LA input synapses. Collectively, results demonstrate that ERK/MAPK activation is necessary for synaptic plasticity in anatomically defined regions of the LA, in vivo.

Figure 1.

Regulation of ERK/MAPK in the LA after LTP-inducing stimulation, in vivo. (A) Anesthetized rats were given 100-Hz high-frequency stimulation (HFS) through an electrode implanted into MGm/PIN. (B) Significant activation of ERK1 (phospho-ERK1; top) and ERK2 (phospho-ERK2; bottom) was observed within 5 min after LTP- inducing stimulation but not at t = 15 or 60 min after LTP. Data are shown for the amygdala ipsilateral (black bars) and contralateral (gray bars) to the stimulation (n = 5/group). *P < 0.05 relative to the contralateral side. In each experiment, levels of phospho-ERK were normalized to total ERK for each sample. Representative blots can be seen in the inset above. (C) Observed increase in ERK/MAPK activation following LTP-inducing stimulation is not due to a change in the total amount of either ERK1 (top) or ERK2 (bottom). Representative blots can be seen in the inset above. (D) Anatomical localization of cells expressing activated ERK/MAPK at 5 and 15 min after LTP-inducing stimulation at different anterior–posterior levels in the LA. Note the strong activation of ERK in the more ventral portions of the LAd, as well as the LAvl and amygdala–striatal transition zone (AST). This segregation of pERK label persists at all A–P levels. (E) Cell counts of pERK-labeled cells taken at the three rostrocaudal levels represented in D. *P < 0.05 relative to the contralateral side. (F) Higher-level magnification images (10×, 20×, and 40×, respectively) of activated ERK/MAPK in the LA after LTP-inducing stimulation. (G) Stimulation electrode placements in the MGm/PIN.

Figure 2.

Regulation of ERK/MAPK in the LA after LTP-inducing stimulation is specific to HFS. (A) Low-frequency stimulation (LFS) protocol. Rats received the same number of pulses (300) over the same time period (2 min) but at lower frequency (2.5 Hz). (B) Confirmation that HFS, but not LFS, induces significant LTP at thalamo-LA synapses. On the left, both groups (HFS, LFS) are represented across the entire 60-min recording session (HFS, n = 5; LFS, n = 4). Representative traces for HFS and LFS can be seen in the inset (scale, 0.2 mV by 5 msec). On the right, data are presented as percent change from baseline in the field potential amplitude during “STP,” defined as the first 10 min after induction, and “LTP,” defined as the last 10 min of the recording session. *P < 0.05 relative to the HFS group. (C) Representative blots of phospho-ERK and total ERK in both HFS and LFS conditions. (D) HFS, but not LFS, of the MGm/PIN leads to significant elevations in activated ERK1 (top) and ERK2 (bottom) in the LA. *P < 0.05 relative to the contralateral side (n = 4/group). (E) No change in total ERK1 (top) or ERK2 (bottom) was observed. (F) Representative photomicrographs from the LA after HFS (top) or LFS (bottom). Note the complete lack of ERK activation in the LA of the LFS animal.

Figure 3.

Impaired LTP at thalamic inputs, in vivo, by a localized infusion of an ERK/MAPK inhibitor in the LA. (A) Anesthetized rats with a stimulation electrode implanted into the auditory thalamus and a recording cannula/electrode in the LA received HFS of the MGm/PIN 30 min after intra-amygdala infusion of either vehicle (50% DMSO; n = 8) or the MEK inhibitor U0126 (1 μg; 0.5 μL; n = 7). Recordings from the LA were made for 1 h following LTP induction. (B) Representative field potentials in the LA evoked by MGm/PIN stimulation in vehicle and U0126 groups before and after LTP induction. Scale, 0.4 mV by 5 msec. (C,D) Infusion of U0126 (1 μg; gray circles/bars) impaired LTP at thalamic inputs to the LA, relative to vehicle-treated animals (black circles/bars). Here, “STP” is defined as the first 10 min after induction, while “LTP” is defined as the last 10 min of the recording session. *P < 0.05 relative to vehicle. (E) Cannula and recording electrode tip placements in or near the LA. Cannula tips are represented by the black circles; recording tips are represented by the gray circles.