Restraint-induced fra-2 and c-fos expression in the rat forebrain: relationship to stress duration

Abstract

The protein product of the fra-2 gene (Fra-2), a fos-family member, can compete with Fos protein for participation in activating protein-1 (AP-1) transcription factor complexes and each protein can contribute different transactivational consequences to an AP-1 complex. To date, there is limited characterization of fra-2 mRNA expression in the rat forebrain. We examined basal and restraint-induced mRNA expression (in situ hybridization) of fra-2 in the rat forebrain and compared its temporal-spatial pattern to c-fos. In contrast to the very low basal expression of c-fos, fra-2 basal expression was moderately high throughout cortex and some subcortical structures, including prominent basal expression in the hypothalamic paraventricular nucleus (PVN). Restraint-induced fra-2 expression was quantified in the prefrontal cortex (PFC), lateral septum (LS) and PVN. Maximal fra-2 gene induction in the PFC and LS was delayed (60 min) after restraint onset with respect to c-fos (15 min), whereas in the PVN, fra-2 mRNA increased within 15 min of restraint. Additionally we compared c-fos and fra-2 gene expression in rats given shorter or longer restraint durations, but equal total time from stress onset to sample collection, to determine the extent to which the kinetics of gene induction matched that of a hypothalamic-pituitary-adrenal axis hormone response. Rats given 45 min recovery after 15 min restraint showed less c-fos expression in the PVN, less fra-2 expression in the prelimbic and infralimbic PFC, and no difference in the LS compared with rats restrained for 60 min. Thus, the expression of both genes was sensitive to stressor duration, but this sensitivity varied with brain region. Differential basal and stress-induced expression patterns of the fra-2 and c-fos genes are likely to have important functional consequences for AP-1 transcription factor dependent regulation of neural plasticity.

Figure 1

ACTH and CORT plasma levels after different periods of restraint. ACTH (top) and CORT (bottom) were measured in the plasma of rats at 0, 15, 60, or 180 min of restraint stress (●). Two additional data points represent plasma hormone levels of rats given a recovery period post-restraint but equal total time from restraint onset to death. One group was given 15 min of restraint followed by 45 min of recovery (△), and another group was given 60 min of restraint followed by 120 min of recovery (▲).⋆, significantly different from home-cage control (0 min); Fisher’s LSD, P<0.05. #, significantly different from the group given equal total time from restraint onset to death; independent samples t-test, P<0.05.

Figure 2

Basal c-fos and fra-2 mRNA expression in the rat forebrain. Top row illustrates neuroanatomical subregions of coronal sections collected at the rostral-caudal level of the prefrontal cortex (PFC), lateral septum (LS), and hypothalamic paraventricular nucleus (PVN) (adapted from Paxinos and Watson (Paxinos G, 1998)). Numbers indicate regions of interest for densitometry analysis: 1 = prelimbic PFC, 2 = infralimbic PFC, 3 = ventral orbital PFC, 4 = LS, 5 = PVN. Letters indicate additional brain regions with relatively high basal fra-2 mRNA: a = piriform cortex, b = nucleus accumbens shell, c = endopiriform nucleus, d = supraoptic nucleus, e = hippocampus, f = dentate gyrus. The bottom row presents representative autoradiogram images of c-fos expression (left half of each coronal section) and fra-2 expression (right side of each coronal section) for comparison purposes.

Figure 3

c-fos and fra-2 mRNA relative levels in the prefrontal cortex after different periods of restraint. c-fos (top) and fra-2 (bottom) expression were measured in the PL (left), IL (middle), and VO (right) of the prefrontal cortex of rats at 0, 15, 60, or 180 min of restraint stress (●). Two additional data points represent mRNA levels of rats given a recovery period post-restraint but equal total time from restraint onset to death. One group was given 15 min of restraint followed by 45 min of recovery (△), and another group was given 60 min of restraint followed by 120 min of recovery (▲).⋆, significantly different from home-cage control (0 min); Fisher’s LSD, P<0.05. #, significantly different from the group given equal total time from restraint onset to death; independent samples t-test, P<0.05.

Figure 4

Representative autoradiograms of stress-induced c-fos and fra-2 gene expression in the prefrontal cortex. Rats were exposed to 0, 15, 60, or 180 min of restraint. c-fos expression (top) is evident throughout the PL, IL, and VO by 15 min of restraint and has diminished by 180 min of restraint. fra-2 expression (bottom) significantly increases above control levels in the PL, IL, and VO after 60 min of restraint, and diminishes by 180 min of restraint.

Figure 5

c-fos and fra-2 mRNA relative levels in the lateral septum (LS) after different periods of restraint. c-fos (top) and fra-2 (bottom) expression were measured in the LS of rats after 0, 15, 60, or 180 min of restraint stress (●). Two additional data points represent mRNA levels of rats given a recovery period post-restraint but equal total time from restraint onset to death. One group was given 15 min of restraint followed by 45 min of recovery (△), and another group was given 60 min of restraint followed by 120 min of recovery (▲).⋆, significantly different from home-cage control (0 min); Fisher’s LSD, P<0.05. #, significantly different from the group given equal total time from restraint onset to death; independent samples t-test, P<0.05.

Figure 6

Representative autoradiograms of stress-induced c-fos and fra-2 gene expression in the lateral septum. Rats were exposed to 0, 15, 60, or 180 min of restraint. c-fos expression (top) is evident by 15 min of restraint and has diminished by 180 min of restraint. fra-2 expression (bottom) significantly increases above control levels after 60 min of restraint, and diminishes by 180 min of restraint.

Figure 7

c-fos and fra-2 mRNA relative levels in the hypothalamic paraventricular nucleus (PVN) after different periods of restraint. c-fos (top) and fra-2 (bottom) expression were measured in the PVN of rats at 0, 15, 60, or 180 min of restraint stress (●). Two additional data points represent mRNA levels of rats given a recovery period post-restraint but equal total time from restraint onset to death. One group was given 15 min of restraint followed by 45 min of recovery (△), and another group was given 60 min of restraint followed by 120 min of recovery (▲).⋆, significantly different from home-cage control (0 min); Fisher’s LSD, P<0.05. #, significantly different from the group given equal total time from restraint onset to death; independent samples t-test, P<0.05.

Figure 8

Representative autoradiograms of stress-induced c-fos and fra-2 gene expression in the hypothalamic paraventricular nucleus. Rats were exposed to 0, 15, 60, or 180 min of restraint. c-fos expression (top) is evident by 15 min of restraint and has diminished by 180 min of restraint. fra-2 expression (bottom) is evident even in home-cage control animals (0 min restraint). fra-2 expression significantly increases above control levels within 15 min of restraint, and diminishes by 180 min of restraint.