c-Fos mapping of brain regions activated by multi-modal and electric foot shock stress

Abstract

Real-world stressors are complex and multimodal, involving physical, psychological, and social dimensions. However, the brain networks that mediate stress responses to these stimuli need to be further studied. We used c-Fos mapping in mice to characterize brain circuits activated by exposure to a single episode of multimodal stress (MMS), and compared these to circuits activated by electric foot shocks (EFS). We focused on characterizing c-Fos activity in stress-relevant brain regions including the paraventricular nucleus (PVN) of the hypothalamus and the bed nucleus of the stria terminalis (BNST). We also assessed stress-induced activation of CRH-positive neurons in each of these structures. MMS and EFS activated an overlapping network of brain regions with a similar time course. c-Fos expression within the PVN and the BNST peaked 30-60 min after exposure to both MMS and EFS, and returned to baseline levels within 24 h. Quantification of c-Fos expression within BNST subregions revealed that while c-Fos expression peaked in all subregions 30-60 min after MMS and EFS exposure, the neuronal density of c-Fos expression was significantly higher in the dorsomedial and ventral BNST relative to the dorsolateral BNST. Our preliminary assessment indicated that a great majority of MMS or EFS-activated neurons in the PVN were CRH-positive (>87%); in contrast, about 6-35% of activated neurons in the BNST were CRH-positive. Our findings indicate that both MMS and EFS are effective at activating stress-relevant brain areas and support the use of MMS as an effective approach for studying multidimensional stress in animal models. The results also reveal that the PVN and BNST are part of a common neural circuit substrate involved in neural processing related to stress.

Keywords: BNST; CRH; Multimodal stress; PVN; c-Fos.

Fig. 1

Stress induction protocols using multimodal stress and electrical foot shock. (A) Multi-modal Stress (MMS) model: mice were exposed to 2 h of bright illumination, loud music and jostling of a shaker while being restrained in a 50 ml tube. The mice were perfused separately at time points of 30–60 min, 24 h and 1 week after stress. The non-stressed control group of mice was perfused with the 30–60 min post stress group. (B) Electrical Foot Shock (EFS) model: mice were exposed to electric shocks (0.4 mA, 1 s duration) with a random inter-trial interval of 15–45 s for 30 min (60 shocks total). The mice were perfused separately at time points of 30–60 min, 24 h, and 1 week after stress. The control group of mice was perfused with 30–60 min post stress group.

Fig. 2

MMS and EFS increase plasma corticosterone levels at 30–60 min after the completion of stress induction. The bar graph plots the average measurements of corticosterone levels in the unit of ng/ml for the non-stress controls (control for MMS, N = 14, control for EFS, N = 8) and the groups of 30–60 min (MMS N = 14, EFS N = 9), 24 h (MMS N = 16, EFS N = 7) and 1 week (MMS N = 10, EFS N = 7) after stress induction. There is a statistically significant difference across the time points (F = 7.80, p < 0.001), but not in the types of stress (F = 0.14, p = 0.71) (two-way ANOVA). There is no interaction effect (F = 0.096, p = 0.96). For MMS, the Student-Newman-Keuls post-hoc analysis indicates a significant difference (*) between 30–60 min post-stress and any other time point, 24 h post-stress (p = 0.012), 1 week post-stress (p = 0.013), and controls (p = 0.021). For EFS, the statistical analysis indicates a significant difference (*) between 30–60 min post-stress and any other time point, 24 h post-stress (p = 0.037), 1 week post-stress (p = 0.013), and controls (p = 0.018).

Fig. 3

Overall c-Fos activation patterns in different brain regions in response to the MMS. (A). Overview of coronal mouse sections from the mouse perfused at 30–60 min after MMS. The Fos staining is imaged using a fluorescent microscope. Boxed areas are enlarged in 3B (scale bar = 2 mm). (B). Enlarged photomicrographs illustrate c-Fos activation in various mouse brain structures related to stress modulation (scale bar = 50 μm). mPFC = medial prefrontal cortex, LS = lateral septal nucleus, BNST = bed nucleus of stria terminalis, MD = mediodorsal thalamic nucleus, PVN = paraventricular hypothalamic nucleus, BLA = basolateral amygdaloid nucleus, PAG = periaqueductal grey.

Fig. 4

Overall c-Fos activation patterns in different brain regions in response to EFS. (A). Overview of coronal mouse sections from the mouse perfused at 30–60 min after EFS. Boxed areas are enlarged in 4B (scale bar = 2 mm). (B). Enlarged photomicrographs illustrate c-Fos activation in various brain structures (scale bar =50 μm). mPFC=medial prefrontal cortex, LS=lateral septal nucleus, BNST=bed nucleus of stria terminalis, MD=mediodorsal thalamic nucleus, PVN=paraventricular hypothalamic nucleus, BLA=basolateral amygdaloid nucleus, PAG=periaqueductal grey.

Fig. 5

Overall c-Fos activation patterns in the control mouse brain. (A). Overview of coronal mouse sections of home cage (non-stressed) control. Boxed areas are enlarged in 5B (scale bar = 2 mm). (B). Enlarged photomicrographs illustrate c-Fos activation in the various brain structures (scale bar = 50 μm). mPFC = medial prefrontal cortex, LS = lateral septal nucleus, BNST = bed nucleus of stria terminalis, MD = mediodorsal thalamic nucleus, PVN = paraventricular hypothalamic nucleus, BLA = basolateral amygdaloid nucleus, PAG = periaqueductal grey.

Fig. 6

c-Fos activated neurons in the PVN of the hypothalamus following MMS and EFS. (A). C-Fos activated neurons in the PVN are labeled by Fos immunostaining (green) in the non-stress control and stressed mice of 30–60 min, 24 h and 1 week after MMS or EFS (scale bar = 200 μm). (B). The bar graphs show average measurements of c-Fos activated neurons in the units of neurons/mm2 for non-stress controls (wild type: MMS N = 4, EFS N = 6), and groups of 30–60 min (wild type and CRH-cre;Ai9: MMS N = 7, EFS N = 8), 24 h (wild type: MMS N = 6, EFS N = 6), and 1 week after stress (wild type: MMS N = 4, EFS N = 4). There is a statistically significant difference across the time points (F = 110.12, p < 0.001), but not for the types of stress induction (F = 0.21, p = 0.65) (two-way ANOVA). There is no interaction effect (F = 0.53, p = 0.66). For MMS, the Student-Newman-Keuls post-hoc analysis indicates a significant difference (***) between 30–60 min post-stress and any other time point, 24 h post-stress (p < 0.001), 1 week post-stress (p < 0.001), and controls (p < 0.001). For EFS, the statistical analysis indicates a significant difference (+++) between 30–60 min post-stress and any other time point, 24 h (p < 0.001), 1 week (p < 0.001), and controls (p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

c-Fos activated neurons in the bed nucleus of the stria terminalis (BNST) following MMS and EFS. (A–H). C-Fos activated neurons in the BNST are labeled by Fos immunostaining (green) in the non-stress control and stressed mice of 30–60 min, 24 h and 1 week after MMS or EFS (scale bar = 250 μm). (I) Mean densities of c-Fos activated neurons in the medial BNST, lateral BNST and ventral BNST for multi modal Stress (MMS) (N = 4–8 mice per time point). The total number of Fos immunopositive neurons was measured in all the subregions of BNST, then normalized by the area size to obtain the neural density in the units of neurons/mm2. There were significant effects of both time course (F = 93.00, p < 0.001) and BNST sub-regions (F = 12.52, p < 0.001) as well as an interaction effect (F = 5.14, p < 0.001). *** indicates that there was significantly (p < 0.001) more c-Fos activation at 30–60 min post-stress compared to all other time points within each BNST sub-region. +++ indicates that within the 30–60 min post-stress time point, both dmBNST and vBNST have significantly (p < 0.001) more density of c-Fos activated neurons than dlBNST (Student-Newman-Keuls post-hock analysis). (J). Quantification (N = 4–8 mice per time point) of density of c-Fos activated neurons in medial BNST, lateral BNST and ventral BNST for Electric Foot Shock (EFS). The total number of c-Fos activation neurons was counted in all the subregions of BNST: medial BNST, lateral BNST and ventral BNST. The density of c-Fos activated neurons is measured in the units of neurons/mm2. There were significant effects of both time course (F = 56.04, p < 0.001) and BNST sub-regions (F = 5.79, p = 0.005) as well as an interaction effect (F = 2.49, p = 0.031). *** indicates that there was significantly (p < 0.001) more c-Fos activation at 30–60 min post-stress compared to all other time points within dmBNST and vBNST sub-regions. ** indicates that there was significantly (p < 0.01) more c-Fos activation at 30–60 min post-stress compared to all other time points within the dlBNST sub-region. +++ indicates that within the 30–60 min post-stress time point, dmBNST has significantly (p < 0.001) more density of c-Fos activated neurons than dlBNST. ++ indicates that within the 30–60 min post-stress time point, vBNST has significantly (p = 0.003) more density of c-Fos activated neurons than dlBNST. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

MMS and EFS activate corticotropin-releasing hormone (CRH) neurons in the BNST and PVN. (A–E), confocal images showing Fos immunopositive (green) and genetically labeled CRH (red) neurons in the BNST control group (A) and 30–60 min post-stress in response to MMS (B, C, D) and EFS (E) (scale bar = 100 μm). (C–D), enlarged photomicrographs from B. Co-localization of CRH and Fos immunopositive neurons are indicated with arrowheads (scale bar = 100 μm). (F), photomicrograph showing the co-localization of CRH and Fos-positive neurons in the PVN (scale bar = 100 μm) of a control mouse. (G), percentage of neurons expressing c-Fos that also expressed CRH in the control group. (H), photomicrograph showing the co-localization of CRH and Fos-positive neurons in the PVN (scale bar = 100 μm) at 30–60 min after MMS. (I), percentage of neurons expressing c-Fos that also expressed CRH at 30–60 min after MMS. (J), photomicrograph showing the co-localization of CRH and Fos-positive neurons in the PVN (scale bar = 100 μm) at 30–60 min after EFS. (K), percentage of neurons expressing c-Fos that also expressed CRH 30–60 min after EFS.