Hippocampal interneurons are direct targets for circulating glucocorticoids

Abstract

The hippocampus has become a significant target of stress research in recent years because of its role in cognitive functioning, neuropathology, and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite the pervasive impact of stress on psychiatric and neurological disease, many of the circuit- and cell-dependent mechanisms giving rise to the limbic regulation of the stress response remain unknown. Hippocampal excitatory neurons generally express high levels of glucocorticoid receptors (GRs) and are therefore positioned to respond directly to serum glucocorticoids. These neurons are, in turn, regulated by neighboring interneurons, subtypes of which have been shown to respond to stress exposure. However, GR expression among hippocampal interneurons is not well characterized. To determine whether key interneuron populations are direct targets for glucocorticoid action, we used two transgenic mouse lines to label parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons. GR immunostaining of labeled interneurons was characterized within the dorsal and ventral dentate hilus, dentate cell body layer, and CA1 and CA3 stratum oriens and stratum pyramidale. While nearly all hippocampal SST+ interneurons expressed GR across all regions, GR labeling of PV+ interneurons showed considerable subregion variability. The percentage of PV+, GR+ cells was highest in the CA3 stratum pyramidale and lowest in the CA1 stratum oriens, with other regions showing intermediate levels of expression. Together, these findings indicate that, under baseline conditions, hippocampal SST+ interneurons are a ubiquitous glucocorticoid target, while only distinct populations of PV+ interneurons are direct targets. This anatomical diversity suggests functional differences in the regulation of stress-dependent hippocampal responses.

Keywords: glucocorticoid receptor (GR); hippocampus; interneurons; parvalbumin (PV); somatostatin (SST); stress.

Figure 1.. Transgenic labeling of interneurons.

A & B: Flippase-mediated recombination led to robust eGFP (488) labeling of PV (A) and SST (B) interneurons in the hippocampus, while flippase-negative control mice lacked eGFP expression. Scale bar = 250 μm. C & D: Confocal images of dentate gyri in SST-eGFP tissue immunostained with GR antibodies (C) and a no-primary control, for which the GR antibody was excluded (D). Scale bar = 25 μm. E & F: Fluorescence intensity profiles for 405 (nuclear blue), 488 (eGFP) and 647 (GR) channels along the length (Mm from left to right) of the horizontal yellow lines shown in C and D. GR immunostained tissue shows corresponding peaks for nuclear blue, eGFP and GR immunoreactivity (E), while the GR peak (red line) is absent from the no-primary control (F). Fluorescence intensity from no-primary controls was also used to determine background fluorescence and establish a threshold (2x background) for defining GR-positive from GR-negative cells.

Figure 2.. eGFP and PV co-expression in the hippocampus of PV;eGFP reporter mice.

A) Representative micrographs of dorsal and ventral CA1, CA3 and dentate gyrus (DG). Solid arrowheads highlight co-expressing cells throughout the stratum oriens (SO), stratum pyramidale (PYR), dentate granule cell body layer (DGC-L) and hilus (H). Scale bar = 100 μm. B) Sensitivity of eGFP for PV (eGFP+PV+/all PV+). C) Specificity of eGFP for PV (eGFP+PV+/all eGFP+). D) Cell counts (n) for each condition. Bars represent mean±SEM.

Figure 3.. eGFP and SST co-expression in the hippocampus of SST;eGFP reporter mice.

A) Representative micrographs of dorsal and ventral CA1, CA3 and dentate gyrus (DG). Solid arrowheads highlight co-expressing cells, while empty arrows indicate non-co-expressing cells throughout the stratum oriens (SO), stratum pyramidale (PYR), dentate granule cell body layer (DGC-L) and hilus (H). Scale bar = 100 μm. B) Sensitivity of eGFP for SST (eGFP+SST+/all SST+). C) Specificity of eGFP for SST (eGFP+SST+/all eGFP+). D) Cell counts (n) for each condition. Bars represent mean±SEM.

Figure 4.. Hippocampal PV+ interneuron GR expression is heterogeneous.

A) Representative micrographs of PV+ interneuron GR expression in dorsal and ventral CA1, CA3 and dentate gyrus (DG). Solid arrows highlight co-expressing cells, while empty arrows indicate non-co-expressing cells throughout the stratum oriens (SO), stratum pyramidale (PYR), dentate granule cell body layer (DGC-L) and hilus (H). Scale bar = 100 μm. B) Percentage of PV+ interneurons which express GR by dorsal (white) and ventral (red) hippocampal subregion. *p<0.05, **p<0.01, ***p<0.001, main effect by two-way RM ANOVA with Bonferroni post-hoc pairwise comparison. C) Percentage of PV+ interneurons which express GR, presented by cell layer within CA1, CA3 and DG. D) Cell counts (n) for each condition. *p<0.05, **p<0.01, ***p<0.001 by Student’s t test or Mann-Whitney Rank Sum test. Breaks in the x-axis indicate groups of data which were not statistically compared. Bars represent mean±SEM.

Figure 5.. Hippocampal SST+ interneurons ubiquitously express GR.

A) Representative micrographs of SST+ interneuron GR expression in dorsal and ventral CAI, CA3 and dentate gyrus (DG). Solid arrowheads highlight co-expressing cells throughout the stratum oriens (SO), stratum pyramidale (PYR), dentate granule cell body layer (DGC-L) and hilus (H). Scale bar = 100 μm. B) Percentage of PV+ interneurons which express GR by dorsal (white) and ventral (red) hippocampal subregion. C) Percentage of SST+ interneurons which express GR, presented by cell layer within CAI, CA3 and DG. D) Cell counts (n) for each condition. Bars represent mean±SEM.

Figure 6.. Biological selectivity of GR primary antibody.

A) Experimental approach and timeline for viral-mediated GR deletion representing Cre-mediated deletion of exon 3 from Nr3c1. B) Representative micrographs demonstrating preservation of GR immunoreactivity in a virus-injected control (GR^wt/wt) mouse and loss of GR from mCherry-expressing dentate granule cells in the GR knockout or GRKO (GR^fl/fl) mouse. Scale bar = 25 μm.