The NO-cGMP-PKG signaling pathway coordinately regulates ERK and ERK-driven gene expression at pre- and postsynaptic sites following LTP-inducing stimulation of thalamo-amygdala synapses

Abstract

Long-term potentiation (LTP) at thalamic input synapses to the lateral nucleus of the amygdala (LA) has been proposed as a cellular mechanism of the formation of auditory fear memories. We have previously shown that signaling via ERK/MAPK in both the LA and the medial division of the medial geniculate nucleus/posterior intralaminar nucleus (MGm/PIN) is critical for LTP at thalamo-LA synapses. Here, we show that LTP-inducing stimulation of thalamo-LA inputs regulates the activation of ERK and the expression of ERK-driven immediate early genes (IEGs) in both the LA and MGm/PIN. Further, we show that pharmacological blockade of NMDAR-driven synaptic plasticity, NOS activation, or PKG signaling in the LA significantly impairs high-frequency stimulation-(HFS-) induced ERK activation and IEG expression in both regions, while blockade of extracellular NO signaling in the LA impairs HFS-induced ERK activation and IEG expression exclusively in the MGm/PIN. These findings suggest that NMDAR-driven synaptic plasticity and NO-cGMP-PKG signaling within the LA coordinately regulate ERK-driven gene expression in both the LA and the MGm/PIN following LTP induction at thalamo-LA synapses, and that synaptic plasticity in the LA promotes ERK-driven transcription in MGm/PIN neurons via NO-driven "retrograde signaling".

Figure 1

High-frequency stimulation of the MGm/PIN promotes ERK phosphorylation in LA at 5 min and in the MGm/PIN at 30 min after stimulation. (a) Placement of stimulation electrode and schematic representation of the experimental protocol. (b) Schematic representation of the HFS and LFS stimulation protocols. Anesthetized rats were given HFS or LFS and sacrificed at 5 min, 30 min or 60 min after stimulation. (c) Images of Western blots for phospho-ERK1/2 and associated GAPDH loading controls from LA (upper) and MGm/PIN (lower) samples after HFS or LFS. (d-e) Mean (±SEM) percent phospho-ERK1/2 immunoreactivity from LA punches taken from rats receiving HFS (left) or LFS (right) and sacrificed at 5 min (HFS: n = 6; LFS: n = 6), 30 min (HFS: n = 6; LFS: n = 8), or 60 min (n = 6). (f-g) Mean (±SEM) percent phospho-ERK1/2 immunoreactivity from MGm/PIN punches taken from rats receiving HFS (left) or LFS (right) and sacrificed at 5 min (HFS: n = 6; LFS: n = 6), 30 min (HFS: n = 5; LFS: n = 5), or 60 min (n = 6). For each figure, phospho-ERK1/2 levels have been normalized to total-ERK1/2 levels for each sample and counts on the ipsilateral (stimulated) side have been expressed as a percentage of those on the contralateral (nonstimulated) side. *P < .05 relative to the ipsilateral side N.S. = not significant.

Figure 2

High-frequency stimulation of the MGm/PIN promotes ERK-driven IEG expression in both the LA and MGm/PIN. (a) Placement of stimulation electrode and schematic of the experimental protocol. Rats were given HFS or LFS and sacrificed 2 hours after stimulation. (b) Images of Western blots for Arc/Arg3.1, c-Fos, EGR-1, and GAPDH from both LA (top) and MGm/PIN samples (bottom). (c) Mean (±SEM) percent IEG immunoreactivity from LA punches taken from rats given HFS (n = 9) or LFS (n = 9). (d) Mean (±SEM) percent IEG immunoreactivity from MGm/PIN punches taken from rats given HFS (n = 9) or LFS (n = 9). In each figure, IEG levels have been normalized to GAPDH for each sample, and IEG expression on the ipsilateral side has been expressed as a percentage of that on the contralateral side for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant.

Figure 3

High-frequency stimulation of the MGm/PIN promotes increased immunolabeling of ERK-driven IEGs in the LA (a) Placement of stimulation electrode. (b) Schematic of experimental protocol. Rats were given HFS or LFS and sacrificed 2 hours after stimulation. (c) Schematic of the amygdala at Bregma −3.2. (d) Mean (±SEM) percent Arc/Arg3.1 immunoreactive cells in the LA from rats receiving HFS (n = 6) or LFS (n = 6). (e) Photomicrographs showing Arc/Arg3.1-labeled cells from rats receiving HFS (left) or LFS (right). (f) Mean (±SEM) percent EGR-1 immunoreactive cells in the LA from rats receiving HFS (n = 6) or LFS (n = 6). (g) Photomicrographs showing EGR-1-labeled cells from rats receiving HFS (left) or LFS (right). (h) Mean (±SEM) percent c-Fos immunoreactive cells in the LA from rats receiving HFS (n = 6) or LFS (n = 6). (i) Photomicrographs showing c-Fos-labeled cells from rats receiving HFS (left) or LFS (right). In each experiment, ipsilateral cell counts have been expressed as a percentage of contralateral cell counts for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant. LAd = dorsal division of the lateral amygdala; LAv l = ventrolateral division of the lateral amygdala; LAvm = ventromedial division of the lateral amygdala; CE = central amygdala; B = basal amygdala; AST = amygdala-striatal transition zone.

Figure 4

High-frequency stimulation of the MGm/PIN promotes increased immunolabeling of ERK-driven IEGs in the MGm/PIN. (a) Placement of stimulation electrode. (b) Schematic representation of experimental protocol. Rats were given HFS or LFS and sacrificed 2 hours after stimulation. (c) Schematic representation of the auditory thalamus at Bregma –5.6. (d) Mean (±SEM) percent Arc/Arg3.1 immunoreactive cells in the MGm/PIN from rats receiving HFS (n = 6) or LFS (n = 6). (e) Photomicrographs showing Arc/Arg3.1-labeled cells from rats receiving HFS (left) or LFS (right). (f) Mean (±SEM) percent EGR-1 immunoreactive cells in the MGm/PIN from rats receiving HFS (n = 6) or LFS (n = 6). (g) Photomicrographs showing EGR-1-labeled cells from rats receiving HFS (left) or LFS (right). (h) Mean (±SEM) percent c-Fos immunoreactive cells in the MGm/PIN from rats receiving HFS (n = 6) or LFS (n = 6). (i) Photomicrographs showing c-Fos-labeled cells from rats receiving HFS (left) or LFS (right). In each experiment, ipsilateral cell counts have been expressed as a percentage of contralateral cell counts for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant. MGm = medial division of the medial geniculate nucleus; MGv = ventral division of the medial geniculate nucleus; PIN = posterior intralaminar nucleus.

Figure 5

Pharmacological blockade of NMDAR-driven synaptic plasticity or NOS activation in the LA impairs ERK activation in both the LA and MGm/PIN following HFS, while blockade of extracellular NO impairs ERK activation in the MGm/PIN but not LA. (a) Placement of stimulation electrode and infusion cannula. (b) Schematic representation of the experimental protocol. Rats were given intra-LA infusion of the vehicle or drug (1 μg/side) followed 30 min later by HFS of the MGm/PIN. Rats were sacrificed at 5 min (for the LA group) or 30 min (for the MGm/PIN group) following stimulation. (c) Images of Western blots for phospho-ERK1/2 and associated GAPDH controls from LA (top) and MGm/PIN (bottom) samples. (d) Mean (±SEM) percent phospho-ERK1/2 immunoreactivity from LA punches taken from rats given intra-LA infusions of 50% DMSO (vehicle; n = 5), 1 μg/side ifenprodil (n = 5), 7-Ni (n = 6) or c-PTIO (n = 5). (e) Mean (±SEM) percent phospho-ERK1/2 immunoreactivity from MGm/PIN punches taken from rats given intra-LA infusions of 50% DMSO (vehicle; n = 5), 1 μg/side ifenprodil (n = 6), 7-Ni (n = 6) or c-PTIO (n = 5). For each figure, phospho-ERK1/2 levels have been normalized to total-ERK1/2 levels for each sample and counts on the ipsilateral side have been expressed as a percentage of those on the contralateral side.*P < .05 relative to the ipsilateral side N.S. = not significant

Figure 6

Pharmacological blockade of NMDAR-driven synaptic plasticity impairs ERK-driven IEG expression in both the LA and MGm/PIN following HFS. (a) Placement of stimulation electrode and infusion cannula. (b) Schematic representation of experimental protocol. Rats were given intra-LA infusion of the vehicle or 1 μg ifenprodil followed 30 min later by HFS of the MGm/PIN. Rats were sacrificed 2 hours after stimulation. (c) Images of Western blots for Arc/Arg3.1, c-Fos, EGR-1 and GAPDH from both LA (top) and MGm/PIN (bottom) samples. (d) Mean (±SEM) percent Arc/Arg3.1, c-Fos and EGR-1 immunoreactivity from LA punches taken from rats given intra-LA infusion of 2% HBC-saline (vehicle; n = 8) or 1 μg/side ifenprodil (n = 8). (e) Mean (±SEM) percent Arc/Arg3.1, c-Fos and EGR-1 immunoreactivity from MGm/PIN punches taken from rats given intra-LA infusion of 2% HBC-saline (vehicle; n = 8) or 1 μg/side ifenprodil (n = 8). In each figure, IEG levels have been normalized to GAPDH for each sample, and IEG expression on the ipsilateral side has been expressed as a percentage of that on the contralateral side for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant.

Figure 7

Pharmacological blockade of NOS activation in the LA impairs ERK-driven IEG expression in both LA and MGm/PIN following HFS. (a) Images of Western blots for Arc/Arg3.1, c-Fos, EGR-1 and GAPDH from both LA (top) and MGm/PIN (bottom) samples. (b) Mean (±SEM) percent Arc/Arg3.1, c-Fos and EGR-1 immunoreactivity from LA punches taken from rats given intra-LA infusion of 50% DMSO (vehicle; n = 8) or 1 μg/side 7-Ni (n = 8). (c) Mean (±SEM) percent Arc/Arg3.1, c-Fos and EGR-1 immunoreactivity from MGm/PIN punches taken from rats given intra-LA infusion of 50% DMSO (vehicle; n = 8) or 1 μg/side 7-Ni (n = 8). In each figure, IEG levels have been normalized to GAPDH for each sample, and IEG expression on the ipsilateral side has been expressed as a percentage of that on the contralateral side for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant.

Figure 8

Pharmacological blockade of extracellular NO in the LA impairs ERK-driven IEG expression in the MGm/PIN, but not the LA, following HFS. (a) Images of Western blots for Arc/Arg3.1, c-Fos, EGR-1, and GAPDH from both LA (top) and MGm/PIN (bottom) samples. (b) Mean (±SEM) percent Arc/Arg3.1, c-Fos, and EGR-1 immunoreactivity from LA punches taken from rats given intra-LA infusion of 50% DMSO (vehicle; n = 8) or 1 μg/side c-PTIO (n = 8). (C) Mean (±SEM) percent Arc/Arg3.1, c-Fos and EGR-1 immunoreactivity from MGm/PIN punches taken from rats given intra-LA infusion of 50% DMSO (vehicle; n = 8) or 1 μg/side c-PTIO (n = 8). In each figure, IEG levels have been normalized to GAPDH for each sample, and IEG expression on the ipsilateral side has been expressed as a percentage of that on the contralateral side for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant.

Figure 9

Pharmacological blockade of PKG in the LA impairs ERK-driven IEG expression in both LA and MGm/PIN following HFS. (a) Images of Western blots for Arc/Arg3.1, c-Fos, EGR-1, and GAPDH from both LA (top) and MGm/PIN (bottom) samples. (b) Mean (±SEM) percent Arc/Arg3.1, c-Fos, and EGR-1 immunoreactivity from LA punches taken from rats given intra-LA infusion of ACSF (vehicle; n = 8) or 1 μg/side Rp-8-Br-PET-cGMPS (n = 8). (c) Mean (±SEM) percent Arc/Arg3.1, c-Fos and EGR-1 immunoreactivity from MGm/PIN punches taken from rats given intra-LA infusion of ACSF (vehicle; n = 8) or 1 μg/side Rp-8-Br-PET-cGMPS (n = 8). In each figure, IEG levels have been normalized to GAPDH for each sample, and IEG expression on the ipsilateral side has been expressed as a percentage of that on the contralateral side for each rat. *P < .05 relative to the ipsilateral side N.S. = not significant.

Figure 10

A model of the biochemical mechanisms underlying LTP at thalamo-LA synapses. See text for details