Endogenous GluR1-containing AMPA receptors translocate to asymmetric synapses in the lateral amygdala during the early phase of fear memory formation: an electron microscopic immunocytochemical study.

Schematic of the fear conditioning paradigm for the paired and unpaired rats.

Light micrographs depicting the dorsal tip of the lateral amygdala (LAd) within adult rat brain sections. B,D: Position of trapezoidal mesas chosen for ultrathin sectioning; photographed with a 5′ objective. A,B: Section immunolabeled for the GluR1 subunit of AMPA receptors (AMPARs). C,D: Section immunolabeled for the GluR2/3 subunits of AMPARs. GluR1 labeling is relatively weak within the LAd, compared with the immediately surrounding regions, which are the caudate-putamen nucleus (CP) and cortex (Ctx) dorsally, the amygdalostriatal transition area (AST) and central amygdala (CeA) medially, and the basal nucleus of the amygdala (BA) ventrally. Scale bar = 0.5 mm in A–D.

Categorization of postembedding immunogold labeling (PEG) in relation to the postsynaptic density (PSD; gray fill) of asymmetric synapses. PEG particles positioned at the cleft, over the PSD or near the PSD (<60 nm from the postsynaptic membrane surface) were categorized as “postsynaptic.” PEG particles at positions removed from the postsynaptic membrane surface by more than 60 nm were all categorized as “nonsynaptic.” The “nonsynaptic” PEG particles included those residing in the spine neck, sometimes as much as a micrometer removed from the PSD. PEG particles residing along the plasma membrane or in the intracellular cytoplasm were included in the nonsynaptic category. The total number of PEG particles residing postsynaptically and nonsynaptically was categorized as “total spinous” for spine profiles.

Representative electron micrographs from the dorsal LA of rats that received no stimuli (i.e., naïve), paired, and unpaired presentation of stimuli.A–C: Postembedding gold (PEG) labeling for the GluR1 subunit of AMPARs. D: PEG labeling for the GluR2/3 subunits of AMPARs. Black arrows across all panels indicate synaptic PEG labeling; white arrows indicate nonsynaptic labeling within spine heads (B,C) and spine neck (D). Asterisks point to PSDs of unlabeled synapses. Scale bar = 500 nm in D (applies to A–D).

Preadsorption control reduces background GluR1 and GluR2/3- immunolabeling in the vicinity of synapses. Preadsorption control was performed to determine the degree to which the postembedding gold (PEG) labeling results from nonspecific binding of the primary antibody to ultrathin sections. A: Preincubation of the control peptide (CP) with the GluR1 antibody decreased the frequency of PEG occurring in the vicinity of spines to 7%. B: Similarly, preincubation of the control peptide with the GluR2/3 antibody also reduced the frequency of PEG labeling within spines down to 5%. C: The number of PEG particles occurring within spines was predominantly zero for grids incubated with the GluR1 antibody, even in the absence of the control peptide, but the frequency increased further in the presence of the control peptide. D: Following preincubation of the GluR2/3 antibody with the control peptide, the frequency of spines containing no PEG particle rose threefold, leaving 5% of the spines labeled by one PEG particle and none labeled by more than one PEG particle.

The proportion of spines immunoreactive for GluR1 increases following fear conditioning, whereas GluR2/3 immunoreactivity remains unchanged. GluR1 immunoreactivity was analyzed by sampling approximately 200 spines from the dorsal tip of the LA of each of four naïve animals, together with four animals that received the paired presentation of a tone with foot-shock (paired) and three animals that received the foot shock preceding the tone (unpaired). A: The proportion of spines immunolabeled for GluR1 at the synaptic junction or nonsynaptically was not significantly different across the three treatment groups. B: The proportion of spines immunolabeled for GluR1 at nonsynaptic sites within spines was also not significantly different. C: However, analysis of those spines immunolabeled specifically at the synaptic junction was significantly different among the treatment groups (P < 0.05). Fisher’s LSD post hoc comparisons revealed a significant difference between the paired and naïve groups. GluR2/3 immunoreactivity was analyzed by sampling 120 synapses per animal from the same region of the LA of four naïve, four paired, and four unpaired animals. D–F: In contrast to the GluR1 pattern, neither the paired nor unpaired conditioning evoked an increase in the proportion of spines labeled (D), the proportion of spines labeled at nonsynaptic sites (E) or the proportion of spines labeled specifically at synaptic sites (F). Bars represent mean and SEM. *, P < 0.05.

GluR1 immunoreactivity is increased by fear conditioning at axo-shaft synapses, but GluR2/3 immunoreactivity is unchanged. A: The proportion of axo-shaft synapses immunolabeled for GluR1 increased for the paired group of animals, relative to axo-shaft synapses of the naïve and unpaired groups of animals (P < 0.001). B: The increase was also evident for GluR1 immunoreactivity at the axo-shaft synapses (P < 0.0001), reflected by the increase of PEG counts at the axo-shaft synaptic junction of the paired group, relative to the PEG counts of naïves. PEG counts at the synaptic junction of the unpaired group decreased, relative to the PEG counts of naïves. For both graphs, N indicates the number of animals analyzed. C,D: Identical analysis of sections immunolabeled for GluR2/3 revealed no effect of the treatment on the proportion of synaptic synapses labeled (C) or the PEG counts at shaft synapses (D). **, P < 0.005; ***, P < 0.0005 using Fisher’s post hoc LSD comparisons.