Localization of Mineralocorticoid Receptors at Mammalian Synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids

Preabsorbing antibodies with aldosterone or a peptide reduces or blocks visualization.

<p>MR-ir controls were established to ensure specificity of the antibodies. MA1-620 (A) and rMR1-18 1D5 (D) were visualized using SG chromogen. Tissue was incubated with 1 µMol aldosterone 30 min before addition of primary antibody. Aldosterone reduced observable chromogen visualization in neurons in the LA (B) when incubated with the antibody MA1-620. No observable difference was found when incubating aldosterone in tissue with the antibody rMR1-18 1D5 (E). Incubating the peptide used to generate the second antibody with the MA1-620 antibody (C) did not produce observable differences in chromogen visualization. Incubating the peptide in tissue with the antibody rMR1-18 1D5 completely blocked the antibody from binding to tissue (F). Scale bars = 200 µm for A–F, 5 µm for insets.</p

Quantitative analysis of labeling at postsynaptic density of asymmetrical synapses.

<p>MR-ir and GR-ir at asymmetrical synapses was determined using a relative measure of density of pixels at individual asymmetrical synapses. GR-ir and MR-ir labeled asymmetrical synapses had significantly greater immunoreactivity compared with primary antibody omitted control sections, suggesting the presence of the receptors at the synapse (A). No differences were found between the two MR-ir groups nor between the MR and GR groups. Two control analysis were utilized to ensure that the differences in intensity of labeling at the synapses were not due to the intensity of gray of images taken (B) nor to the size of the PSD (C). (*** depicts p<.001).</p

MR-ir in extra nuclear structures.

<p>A significant difference between MR-ir with both antibodies and primary antibody omitted control sections were observed in multiple extra nuclear structures. Although not observed as frequently as principal neuronal structures, GABAergic interneurons were observed to have immunoreactivity (see comparison with an unlabeled asymmetrical synapse) (A). Glial processes (B), dendritic membranes (C), Golgi apparatus (D), mitochondrial membranes (E), and presynaptic vesicles (F) also were observed to have immunoreactivity compared with respective primary antibody omitted controls. Scale bars = A–B 500 nm, C–F = 100 nm.</p

Immunoblots of MR and GR in amygdala (Amg), hippocampal (HC) and hypothalamic (Hyp) nuclei.

<p>As indicated by the representative immunoblots standardized to β-Actin (A) there was a significant difference in relative density of MR between brain nuclei (B). GR was equally distributed in all brain regions. (** denotes p's<.01).</p

GR and MR labeling at asymmetrical synapses.

<p>Asymmetrical synapses were examined for immunoreactivity since electrophysiological evidence has examined excitatory synaptic responses of MR and GR. Compared with primary antibody omitted control asymmetrical synapses (A), immunoreactivity was observed in the postsynaptic density of GR-ir LA neurons (B) and MR-ir (MA1–620; rMR1–18 1D5) LA neurons (C and D, respectively). MR-ir was also observed at presynaptic terminals of asymmetric synapses (D). Scale bar = 100 nm.</p