Dynamics of stress-induced c-fos expression in the rat prelimbic cortex: lessons from intronic and mature RNA and protein analyses

Abstract

Despite the extensive use of c-fos as a marker of stress-induced neuronal activation, key aspects regarding its dynamics of expression remain poorly characterized. In the present study, we assessed in the prelimbic cortex of adult male rats the immediate transcriptional response of c-fos by measuring the heteronuclear (hn)RNA and mature (m)RNA expression by double fluorescent in situ hybridization as well as the c-Fos protein using immunofluorescence (FOS). We quantified in three different experiments the number of c-fos hnRNA+, mRNA+ and FOS+ neurons under basal conditions, immediately after different periods of immobilization stress (IMO), and after a recovery period. Our results indicate that stress induced a large increase in the number of positive neurons for all markers analyzed, each displaying a different time course. Moreover, our findings indicate that measuring the intensity of signal per neuron also provides relevant information. In addition, we report an increased number of FOS+ neurons after only 8-15 min of IMO, suggesting a surprisingly fast initiation of protein translation. Finally, the maturation from c-fos hnRNA+ to mRNA+ might depend on the duration and/or intensity of stress-induced activation. Our findings contribute to a better understanding of the dynamics of stress-induced c-fos expression and underscore the importance of examining multiple molecular components when using c-fos as a proxy of neuronal activation.

Keywords: Immobilization stress; Neuronal activation; Prelimbic cortex; Stress duration; c-fos expression.

Fig. 1

Experimental design. Schematic representation of the experimental groups for the study of c-fos RNA expression and FOS protein after different times of immobilization stress (IMO). Grey represents no stress exposure (home cage conditions). Animals were euthanized by intracardiac perfusion, and their brains were obtained for histological analyses.

Fig. 2

Representative images of c-fos RNA and FOS protein staining in the PrL after IMO exposure or in basal conditions. Confocal images of double fluorescent in situ hybridization (dFISH) and simple immunofluorescence (IF) assays for c-fos hnRNA and mRNA (upper panels, red and green signal, respectively) and FOS protein (lower panels, magenta signal). The white square highlights a magnified view of the dotted region for greater detail. Rats were exposed to 8 min immobilization stress (IMO) and euthanized either immediately after (middle) or following a 38-min recovery period (right) or under basal conditions (left). Scale bar = 20 μm.

Fig. 3

c-fos response to 8 min immobilization (IMO) stress. Data represented as mean and SEM (n = 4/group). Rats were euthanized under basal conditions or after exposure to 8 min IMO stress, either immediately after or following a 38-min recovery period. Panel A shows the number of hnRNA+ neurons per mm2. Panel B shows the number of mRNA+ neurons per mm2 (left), the total intensity of mRNA signal (middle) and the intensity of signal per neuron (right). Panel C shows the number of FOS+ neurons per mm2 (left), the total intensity of FOS signal (middle) and the intensity of signal per neuron (right). ∗p < 0.05, ∗∗∗p < 0.001 vs basal; ^♦ p < 0.05, ^♦♦♦ p < 0.001 vs IMO8.

Fig. 4

c-fos response to 15 min immobilization (IMO) stress. Data represented as mean and SEM (n = 3–4/group). Rats were euthanized under basal conditions or after 15 min IMO stress, either immediately after or following a 105-min recovery period. Panel A shows the number of hnRNA+ neurons per mm2. Panel B shows the number of mRNA+ neurons per mm2 (left), the total intensity of mRNA signal (middle) and the intensity of signal per neuron (right). Panel C shows the number of FOS+ neurons per mm2 (left), the total FOS signal and the integrated signal per neuron. ∗p < 0.05, ∗∗∗p < 0.001 vs basal; ^♦♦♦ p < 0.001 vs IMO15.

Fig. 5

FOS protein levels in response to 90 min immobilization (IMO) compared with 30 min IMO followed by a 60 min-recovery period. Data represented as mean and SEM (n = 4–5/group). Panel A shows the number of FOS+ neurons per mm2. Panel B shows the total intensity of FOS signal per neuron. ∗p < 0.05, ∗∗p < 0.01 vs IMO90.

Fig. 6

Schematic diagram of the molecular mechanisms regulating stress-induced c-fos expression in the PrL. After stress exposure, calcium influx through N-methyl-D-aspartate receptors (NMDAR) and voltage-gated calcium channels (VGCC) leads to the activation of calcium-regulated signaling proteins, including extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and calcium-calmodulin dependent kinases (CAMK). Furthermore, increases in cyclic adenosine monophosphate (cAMP) activate protein kinase A (PKA). ERK/MAPK activate the ribosomal S6 kinase (RSK), which phosphorylates the cAMP-response element-binding protein (CREB) that is also the target of CAMKIV and PKA. CREB, together with CREB binding protein (CBP) binds to the calcium/cAMP response element (Ca/CRE) in the c-fos promoter, a regulatory element critical for activity-dependent c-fos transcription. ERK/MAPK activation also phosphorylates Elk-1, which binds to the serum response factor (SRF) that, in turn, binds to the serum response element (SRE). These processes induce the rapid transcription of c-fos intronic RNA in the nucleus. The intronic RNA undergoes splicing to produce mature RNA, which is then exported to the cytoplasm for translation of FOS protein. Our findings indicate that the transition from intronic to mature RNA in the PrL seems to require sustained transcriptional activity (indicated by the dashed red arrow), which is directly associated to stress duration. Part of the scheme is based on Kovács (1998); Cruz et al. (2015).