Activation of extracellular signal-regulated kinases in social behavior circuits during resident-intruder aggression tests

Abstract

Using a variety of experimental methods, a network of brain areas regulating aggressive behaviors has been identified in several groups of vertebrates. However, aggressive behavior expressed in different contexts is associated with different patterns of activity across hypothalamic and limbic brain regions. Previous studies in rodents demonstrated that short day photoperiods reliably increase both male and female aggression versus long day photoperiods. Here we used immunohistochemistry and western blots to examine the effect of photoperiod on phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK) in male California mice (Peromyscus californicus) during resident-intruder tests. Phosphorylated ERK (pERK) can alter neuronal activity in the short term and in the long term acts as a transcription factor. In the posterior bed nucleus of the stria terminalis (BNST) males tested in aggression tests had more pERK positive cells when housed in short days but not long days. This result was replicated in western blot analyses from microdissected BNST samples. In the medial amygdala (MEA), immunostaining and western analyses showed that pERK expression also was generally increased in short days. Immunostaining was also used to examine phosphorylation of cyclic AMP response element binding protein (CREB). CREB can be phosphorylated by pERK as well as other kinases and functions primarily as a transcription factor. Intriguingly, aggressive interactions reduced the number of cells stained positive for phosphorylated CREB in the infralimbic cortex, ventral lateral septum and MEA. This effect was observed in mice housed in long days but not short days. Overall, these data suggest that different (but overlapping) networks of aggressive behavior operate under different environmental conditions.

Figure 1

Immunostaining of phosphorylated ERK and CREB in P. californicus. Positive staining for pERK (A) was eliminated by pre-incubation with the pERK immunizing peptide (B) but not with a nonphosphorylated ERK peptide (C). Positive staining for pCREB (D) was eliminated by omission of the primary pCREB antibody (E). Histology for a 1 mm diameter micropunch sample of the bed nucleus of the stria terminalis (F). This section is stained for pCREB (blue black) and counterstained with eosin (pink). Abbreviations: lv, lateral ventricle; cc, corpus callosum; f, fornix; ac, anterior commissure; bed nucleus of the stria terminalis, bnst. Scale bar in panels A-E 20 μm. Scale bar in panel F = 0.5 mm.

Figure 2

Effect of photoperiod on number of bites and attack latency in resident intruder tests. * p < 0.05, ** p < 0.01.

Figure 3

Photomicrographs of phosphorylated extracellular-related kinase (pERK) immunoreactivity in the posterior BNST (A-D) and posterodorsal MEA (E-F) from California mice tested in control or resident intruder tests. In both the posterior BNST (I) and posterodorsal MEA (J), aggression testing caused a significant increase in the number of pERK positive cells in short day mice but not long day mice. For animals tested in aggression tests, there is a significant positive correlation between the number of pERK cells in the posterior BNST and bites during aggression tests (K, p < 0.05) and a nonsignificant trend in the posterodorsal MEA (L,, p = 0.09). Abbreviations: f, fornix; ac, anterior commissure; ot, optic tract. * P < 0.05 planned comparison vs control. Scale bars = 0.2 mm.

Figure 4

Photomicrographs of phosphorylated extracellular related kinase (pERK) immunoreactive cells the BLA from California mice housed in long days (A) or short days (B). Short day mice had more pERK positive cells than long day mice (C). The number of pERK positive cells was negatively correlated with attack latency (D). * P < 0.05 effect of photoperiod. Abbreviations: ec external capsule. Scale bars = 0.2 mm.

Figure 5

Photomicrographs of phosphorylated CREB immunoreactive cells in the IFL (A), VLS (B), and posterodorsal MEA (C). Abbreviations: cc, corpus callosum; lv lateral ventricle; ac, anterior commissure; ot, optic tract. Scale bars = 0.2 mm.

Figure 6

Western blot analyses of pERK and total ERK from punch samples of the BNST (A,B), MEA (C,D), and VMH (E, F). In the BNST aggression testing increased pERK expression in short day mice but not long day mice (A). In the MEA pERK expression was increased in short day mice compared to long day mice (C). In the VMH there was a nonsignificant trend (p = 0.1) for increased pERK expression in aggression-tested mice (E). There was no effect of photoperiod or aggression testing on total ERK expression in any brain area (B, D, F). * P < 0.05 planned comparison vs. control, † P < 0.05 effect of photoperiod.

Figure 7

Representative images from western blot analysis of protein extracted from BNST punch samples. A = aggression, C = control.