Corticosterone rapidly increases thorns of CA3 neurons via synaptic/extranuclear glucocorticoid receptor in rat hippocampus

Abstract

Modulation of synapses under acute stress is attracting much attention. Exposure to acute stress induces corticosterone (CORT) secretion from the adrenal cortex, resulting in rapid increase of CORT levels in plasma and the hippocampus. We tried to test whether rapid CORT effects involve activation of essential kinases as non-genomic processes. We demonstrated rapid effects (~1 h) of CORT on the density of thorns, by imaging Lucifer Yellow-injected neurons in adult male rat hippocampal slices. Thorns of thorny excrescences of CA3 hippocampal neurons are post-synaptic regions whose presynaptic partners are mossy fiber terminals. The application of CORT at 100, 500, and 1000 nM induced a rapid increase in the density of thorns in the stratum lucidum of CA3 pyramidal neurons. Co-administration of RU486, an antagonist of glucocorticoid receptor (GR), abolished the effect of CORT. Blocking a single kinase, including MAPK, PKA, or PKC, suppressed CORT-induced enhancement of thorn-genesis. On the other hand, GSK-3β was not involved in the signaling of thorn-genesis. Blocking AMPA receptors suppressed the CORT effect. Expression of CA3 synaptic/extranuclear GR was demonstrated by immunogold electron microscopic analysis. From these results, stress levels of CORT (100-1000 nM) might drive the rapid thorn-genesis via synaptic/extranuclear GR and multiple kinase pathways, although a role of nuclear GRs cannot be completely excluded.

Keywords: corticosterone; hippocampus; kinase; spine; stress; thorn.

FIGURE 1

Changes in the density of thorns by CORT in hippocampal slices. Maximal intensity projections onto XY plane from z-series confocal micrographs, showing thorns along the primary dendrites of hippocampal CA3 pyramidal neurons. Left image shows a traced whole image of Lucifer Yellow-injected CA3 neuron. Right images show thorns (red arrowheads) without drug-treatments (Control) or thorns after 1 μM CORT treatments (CORT) for 1 h. Bar 10 μm.

FIGURE 2

Time dependency and dose dependency of CORT effects on the thorn density of CA3 neurons. Thorns were analyzed along the primary and secondary dendrites of pyramidal neurons in the stratum lucidum of CA3 neurons. (A) The time dependency of CORT effects on the thorn density in CA3 neurons, after 0.5 h treatment (0.5 h), 1 h treatment (1 h), and 2 h treatment (2 h) in ACSF with 1 μM CORT. As a control, no treatment with CORT (0 h) is shown. (B) Dose dependency of CORT treatments on the thorn density. A 1 h treatment in ACSF without CORT (0 nM), with 10 nM CORT (10 nM), with 30 nM CORT (30 nM), with 100 nM CORT (100 nM), with 500 nM CORT (500 nM), and with 1 μM CORT (1 μM). Vertical axis is the average number of thorns per 1 μm of dendrite. Results are reported as mean ± SEM. The significance of CORT or drug effect was examined using the Tukey–Kramer post hoc multiple comparisons test when one way ANOVA tests yielded P < 0.05. The significance yielded **P < 0.01 *P < 0.05, **P < 0.01 to 0 h and 0 nM. For each drug treatment, we investigated 3 rats, 6 slices, 12 neurons, 12 dendrites, and 1400–1800 thorns.

FIGURE 3

Effects of blockers of receptors on CORT-induced changes in the thorn density. A 1 h treatment in ACSF without drugs (Control), with 1 μM CORT (CORT), with 1 and 10 μM RU486 (CORT + RU), with 1 μM CORT and 20 μM CNQX (CORT + CNQX), and with 1 μM CORT and 50 μM MK-801 (CORT + MK). Vertical axis is the average number of thorns per 1 μm of dendrite. Results are reported as mean ± SEM. The significance of CORT or drug effect was examined using the Tukey–Kramer post hoc multiple comparisons test when one way ANOVA tests yielded P < 0.05. *P < 0.05, **P < 0.01 vs. Control. ^#P < 0.05 vs. CORT. For each drug treatment, we investigated 3 rats, 6 slices, 12 neurons, 12 dendrites, and 1400–1800 thorns.

FIGURE 4

Suppression effects by kinase inhibitors on CORT-induced changes in the density of thorns.(A) A 1 h treatment in ACSF without drugs (Control), with 1 μM CORT (CORT), with 1 μM CORT and 20 μM PD98059 (MAPK inhibitor; CORT + PD), with 1 μM CORT and 10 μM H-89 (PKA inhibitor; CORT + H89), with 1 μM CORT and 10 μM chelerythrine (PKC inhibitor; CORT + Chel), and with 1 μM CORT and 10 μM GSK-3β Inhibitor VIII (CORT + I8). (B) No effect of kinase inhibitors alone on the density of thorns in CA3 neurons. Abbreviations are the same as (A). Vertical axis is the average number of thorns per 1 μm of dendrite. Results are reported as mean ± SEM. The significance of CORT or drug effect was examined using the Tukey–Kramer post hoc multiple comparisons test when one way ANOVA tests yielded P < 0.05. ^**P < 0.01 vs. Control. ^#P < 0.05, ^##P < 0.01 vs. CORT. For each drug treatment, we investigated 3 rats, 6 slices, 12 neurons, 12 dendrites, and 1600–1800 thorns.

FIGURE 5

Immunoelectron microscopic analysis of the distribution of GR within the mossy fiber synapses, dendrites in stratum lucidum and nuclei of pyramidal cells in CA3 region. Gold particles (arrowheads), specifically indicating the presence of GR, were localized in the pre- and postsynaptic regions (A). In dendrites, gold particles were often found in the cytoplasmic space (B). Gold particles were also localized in the nuclei (C). A search for immuno-gold labeled GR proteins was performed at least 30 synapses at CA3 region from more than 100 independent images. A 1:3000 dilution of IgG was used to prevent non-specific labeling. Pre, presynaptic region; Post, post synaptic region; Den, dendrite; Nuc, nucleus. Scale bar, 500 nm.

FIGURE 6

Model illustration. (A) Schematic illustration of CORT-driven multiple kinase pathways. Upon binding of CORT, GR induces the sequential activation of PKA, PKC, MEK, and Erk MAPK. Erk MAPK regulates phosphorylation of actin-related proteins such as cortactin, resulting in actin reorganization. (B) Schematic illustration of CORT-induced rapid thorn-genesis via multiple kinase pathways.