Neurochemically distinct circuitry regulates locus coeruleus activity during female social stress depending on coping style

Abstract

Stress-related psychiatric diseases are nearly twice as prevalent in women compared to men. We recently showed in male rats that the resident-intruder model of social stress differentially engages stress-related circuitry that regulates norepinephrine-containing neurons of the locus coeruleus (LC) depending on coping strategy as determined by the latency to assume a defeat posture. Here, we determined whether this social stress had similar effects in female rats. LC afferents were retrogradely labeled with Fluorogold (FG) and rats had one or five daily exposures to an aggressive resident. Sections through the nucleus paragigantocellularis (PGi), a source of enkephalin (ENK) afferents to the LC, and central nucleus of the amygdala (CeA), a source of corticotropin-releasing factor (CRF) afferents to the LC, were processed for immunocytochemical detection of c-fos, a marker of neuronal activity, FG and ENK or CRF. Like male rats, female rats defeated with a relatively short latency (SL) in response to a single resident-intruder exposure and showed significant c-fos activation of LC neurons, PGi-ENK LC afferents, and CeA-CRF-LC afferents. With repeated exposure, some rats exhibited a long latency to defeat (LL). LC neurons and CeA-CRF-LC afferents were activated in SL rats compared to control and LL, whereas PGi-ENK LC afferents were not. Conversely, in LL rats, PGi-ENK LC and CeA-CRF-LC afferents were activated compared to controls but not LC neurons. CRF type 1 receptor (CRF1) and µ-opioid receptor (MOR) expression levels in LC were decreased in LL rats. Finally, electron microscopy showed a relative increase in MOR on the plasma membrane of LL rats and a relative increase in CRF1 on the plasma membrane of SL rats. Together, these results suggest that as is the case for males, social stress engages divergent circuitry to regulate the LC in female rats depending on coping strategy, with a bias towards CRF influence in more subordinate rats and opioid influence in less subordinate rats.

Keywords: Central nucleus of the amygdala; Corticotropin-releasing factor; Enkephalin; Locus coeruleus; Nucleus paragigantocellularis; Social stress; µ-Opioid receptor and corticotropin-releasing factor receptor.

Figure 1.

Fluorogold injection into the locus coeruleus and retrograde labeling in the nucleus paragigatocellularis (PGi) and central nucleus of the amygdala (CeA). Ai-Ci. Schematic diagram adapted from the rat brain atlas (Paxinos and Watson, 1998) depicting the anteroposterior levels of the representative injection site (A). Following FG injection into the LC, a representative brightfield photomicrograph showing the FG retrograde labeling in the nucleus PGi (Bii at bregma - 12.30 mm and bregma - 13.68; Paxinos and Watson, 1998) and CeA (Cii at bregma - 2.12; Paxinos and Watson, 1998). The arrows indicate immunoperoxidase labeled cells. Scale bars: A, 50 μm; B, C, 25 μm.

Figure 2.

Low (B-D) and high (E-G) magnification confocal immunofluorescence photomicrographs showing FG and TH immunoreactivities in the LC B, E. FG immunolabeling was detected by fluorescein isothiocyanate (green). C, F. TH was detected by rhodamine isothiocyanate (red). D, G. Merged image. Arrows point to FG and TH-containing neurons. Arrowhead point to singly labeled FG-containing neuron. Corner arrows indicate dorsal (D) and lateral (L) orientation. 4V, fourth ventricle. Scale bar, B-D: 50 μm; E-G: 25 μm.

Figure 3:

Activation of locus coeruleus (LC) neurons following single exposure to resident-intruder stress. Representative sections from rats exposed to a single control manipulation (Control) and a single resident-resident intruder stress (Stress). Scatterplot showing the number of c-fos profiles in the LC after a single stress (n = 6) or control manipulation (n = 5) for individual control and stressed rats. Lines through the points indicate the group mean.

Figure 4.

High magnification confocal immunofluorescence photomicrographs showing c-fos and TH immunoreactivities in the LC (A-C). A. c-fos immunolabeling was detected by fluorescein isothiocyanate (green). B. TH was detected by rhodamine isothiocyanate (red). C. Merged image. Arrows point to c-fos within TH-containing neurons. Arrowheads point to singly labeled TH neurons. Corner arrows indicate dorsal (D) and lateral (L) orientation. Scale bar, 25 μm.

Figure 5:

Activation of locus coeruleus (LC)-projecting enkephalin neurons in the nucleus PGi (A) and LC-projecting CRF neurons in the CeA (B) A. Representative high magnification immunofluorescence photomicrographs from a long latency (LL) rat showing LC-projecting neurons in the PGi. c-fos is labeled in blue, FG immunolabeling is labeled in green and ENK-immunolabeling is labeled in red. The merged image shows all labels. Arrows point to the same triple-labeled neuron in all images. Scale bar, 10 μm. B. Representative high magnification immunofluorescence photomicrographs from a SL rat showing LC-projecting neurons in the CRF. c-fos is labeled in blue, FG immunolabeling is labeled in green and CRF-immunolabeling is labeled in red. The merged image shows all labels. C. Scatterplot showing triple labeled cells on Day 1 (Single stress: n = 6; Control: n = 5) and Day 5 (Control: n = 5; SL: n = 6; LL: n = 3) in the PGi and CeA, respectively. Lines through the points indicate the group mean. Arrows point to the same triple-labeled neuron in all images. Scale bar, 10 μm. Scatter plot on the left showing the number of triple-labeled cells that were also immunolabeled with ENK and CRF, respectively for individual control and defeat group following 1 day of social stress exposure. Middle scatter plot showing the number of triple-labeled cells that were also immunolabeled with ENK for individual control, long latency (SL) and long latency (LL) following repeated social stress exposure. Scatter plot on the right showing the number of triple-labeled cells that were also immunolabeled with CRF for individual control, SL and LL following repeated social stress exposure.

Figure 6:

Activation of locus coeruleus (LC) neurons following repeated exposure to resident-intruder stress. Representative brightfield photomicrographs from a control rat exposed (Control) to five-day repeated manipulations (SL). Scatterplot showing the number of c-fos profiles in the LC after the fifth stress or control manipulation for individual control, SL and LL rats. Lines through the points indicate the group mean. (Control n = 5; SL n = 6; LL n = 3).

Figure 7:

Correlation studies on the number of c-fos in LC (Panel A), triple labeled neurons in the PGi (Panel B) and triple labeled neurons in the CeA (Panel C) with respect to the latency to defeat. The number of c-fos in LC showed a significant positive correlation with the defeat latency. A positive correlation was also observed on the number of triple labeled neurons in the PGi.

Figure 8:

Western blot analysis of CRF receptor 1 (CRF1) and mu opioid receptor (MOR) in the locus coeruleus in control rats or rats that were exposed to repeated social stress and were classified as short (SL) or long latency (LL). Scatter plot showing the mean ratio of the integrated intensity of each band of CRF1 or MOR protein to the corresponding GAPDH band from the same sample. * p < 0.05 vs SL **; p < 0.001 vs SL and control. (Control n = 5; SL n = 6; LL n = 5).

Figure 9:

Correlation studies on the MOR and CRF1 protein expression levels in LC (Panels A and B) and MOR and CRF1 cytoplasmic:total ratio in LC (Panels C and D) with respect to the latency to defeat. A positive correlation was observed between the MOR protein expression level and latency to defeat. Similar, positive correlation was observed between CRF1 protein expression level and latency to defeat. While the MOR cytoplasmic:total ratio was negatively correlated to defeat latency, the CRF1 cytoplasmic:total ratio was positive correlated to defeat latency.

Figure 10:

Electron microscopic evidence for repeated social stress-induced re-distribution of mu-opioid receptor (MOR) and CRF receptor 1 (CRF1) in the rat LC. Left panels show a dendrite from a control rat that contains immunogold-silver labeling for MOR along the plasmalemma is depicted by arrowhead. MOR within the cytoplasmic compartment is depicted by arrows. Left panel-middle shows MOR labeling in a dendrite from a SL rat. Immunogold-silver labeling for MOR (arrows) can be predominantly seen within the cytoplasmic compartment, although some MOR immunogold-silver particles are localized along the plasma membrane (arrowhead). Left panels illustrate that immunogold-silver labeling for MOR shifts from plasmalemma to the cytoplasmic compartment in SL rat. Right panels show immunogold-silver labeling for CRF1. Right panel-middle shows that CRF1 labeling shifts from cytoplasmic compartment to plasmalemmal localization in SL rat. Right panel-bottom shows CRF1 within the cytoplasmic compartment in LL rats. Scale bars, 0.5 μm

Figure 11

: Quantification of the ratio of cytoplasmic to total MOR and CRF1 labeling in the LC dendrites of female rats following social defeat exposure. Bars represent the mean ratio of cytoplasmic to total ratio of MOR and CRFr in control, SL and LL rats. Values are means ± SEM of 3–6 rats per group (Control n = 6; SL n = 6 and LL n = 3). *P < 0.001 compared with other groups.