Activation of Anterior Cingulate Cortex Extracellular Signal-Regulated Kinase-1 and -2 (ERK1/2) Regulates Acetic Acid-Induced, Pain-Related Anxiety in Adult Female Mice

Abstract

In visceral pain, anxiety and pain occur simultaneously, but the etiogenesis of this effect is not yet well-described. The anterior cingulate cortex (ACC) is known to be associated with the affective response to noxious stimuli. The aim of the current study is to define the role of ACC extracellular signal-regulated (ERK)-1 and-2 (ERK1/2) activity in the development of pain-related anxiety/depression and the nocifensive response in acetic acid (AA)-elicited visceral pain. The model of visceral pain was created by intraperitoneal (ip) injection of AA to female Kunming mice. We found that AA injection resulted in a dynamic, bilateral ERK1/2 activation pattern in the ACC. Inhibition of ERK1/2 activation 2 hr after AA injection by subcutaneous (sc) injection of the mitogen-activating extracellular kinase (MEK) inhibitor, SL327, had no effect on the nocifensive responses, but did attenuate anxiety-like behavior, as determined by elevated plus-maze and open-field testing results. These data suggest that AA-induced visceral pain activates expression of ACC ERK1/2, which regulates visceral pain-related anxiety, but not the nocifensive response.

Keywords: anterior cingulate cortex (ACC); anxiety-like behavior; extracellular signal-regulated kinase (ERK); visceral pain.

Fig. 1

Time course of ERK1/2 activation in the ACC after AA injection. A, B: Representative photomicrographs showing p-ERK1/2 immunoreactivity in the ACC from coronal brain sections in control group (A) or at 10 min (B), 30 min (C), 1 hr (D) and 2 hr (E) after AA injection. F: Quantitative analysis of number of p-ERK1/2 positive cells in both sides of ACC at indicated times points (n=5) for each group per indicated time. Bar=200 µm. ACC, anterior cingulate cortex; p-ERK, phosphorylated extracellular signal-regulated kinase. *** p<0.001 versus control group. * p<0.05 versus control group.

Fig. 2

Effect of AA injection on p-ERK1/2 expression in the ACC by Western blot. A: Representative Western blot of p-ERK1/2, total-ERK1/2, and GAPDH in the ACC after AA injection. B, C: Semiquantitative analysis of p-ERK1/2 (B) and total-ERK1/2 (C) relative to GAPDH. AA injection induces an increase in the intensity of the band for p-ERK1/2. Increased p-ERK1/2 expression appears at 10 min after AA injection and returns to baseline level 30 min after injection. At 1 hr after injection, p-ERK1/2 expression is increased again, and returns to baseline level 2 hr after injection (n=6 for each group). *** p<0.001 versus control, * p<0.05 versus control. GAPDH is the loading control. ACC, anterior cingulate cortex; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3

Localization of p-ERK1/2 and GAD67 in the ACC 10 min after AA injection. A, B: Representative photomicrographs of the double labeling of p-ERK1/2 (green, A) with GAD (red, B). Arrows (A) indicate p-ERK1/2 immunoreactive cells. Arrowheads (B) indicate GAD67 immunoreactive cells. C: Merged pictures of A and B. Bar=50 µm. GAD, glutamic acid decarboxylase.

Fig. 4

Effect of SL327 on abdominal contractions of mouse after AA injection. SL327 was administered by sc injection 30 min before AA injection. Abdominal contractions were recorded 1 hr after AA injection. There was no significant difference in the SL327-treated group compared with vehicle group (n=8 for each group). SL327=alpha-[Amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile.

Fig. 5

Effect of SL327 on anxiety-like behavior after AA injection. A: The percentage of time spent in the open arm in EPM test in vehicle and SL327 groups. B: Open-field test shows increases in time spent in the inner square after SL327 injection (n=8 for each group). SL327=alpha-[Amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile. * p<0.05 versus the vehicle group, *** p<0.001 versus vehicle group.