Mitogen-activated protein kinase in the amygdala plays a critical role in lithium chloride-induced taste aversion learning

Abstract

The intracellular mitogen-activated protein kinase (MAPK) pathway in the brain is necessary for the formation of a variety of memories including conditioned taste aversion (CTA) learning. However, the functional role of MAPK activation in the amygdala during lithium chloride (LiCl)-induced CTA learning has not been established. In the present study, we investigated if local microinjection of SL327, a MAPK kinase inhibitor, into the rat amygdala could alleviate LiCl-induced CTA learning. Our results revealed that acute administration of a high dose of LiCl (0.15M, 12 ml/kg, i.p.) rapidly increased the level of phosphorylated MAPK (pMAPK)-positive cells in the central nucleus of the amygdala (CeA) and nucleus of the solitary tract (NTS) of rats as measured by immunohistochemistry. Local microinjection of SL327 (1 μg/0.5 μl/hemisphere) into the CeA 10 min before LiCl administration decreased both the strength of LiCl-induced CTA paired with 0.125% saccharin and the level of LiCl-induced pMAPK-positive cells in the CeA, but not in the NTS. Our data suggest that the intracellular signaling cascade of the MAPK pathway in the CeA plays a critical role in the processing of visceral information induced by LiCl for CTA learning.

Figure 1

Photomicrographs of pMAPK immunohistochemistry in the CeA after LiCl administration. Rats were sacrificed 10, 30, 60 or 180 min after LiCl (0.15 M, 12 ml/kg, i.p.). LiCl greatly increased the number of pMAPK-positive cells at 10–60 min; at 3 h after LiCl the number of pMAPK-positive cells returned to the level of NaCl-injected rats (not shown). Scale bar, 500 µm.

Figure 2

Photomicrographs of pMAPK immunohistochemistry in the NTS after LiCl administration. As in the CeA, LiCl greatly increased the number of pMAPK-positive cells in the medial intermediate and subpostremal NTS at 10–60 min, which by 3 h returned to the level of NaCl-injected rats. st, solitary tract; iNTS, medial intermediate NTS; IV, fourth ventricle. Scale bar, 500 µm.

Figure 3

Quantification of pMAPK-positive cells in the CeA and NTS at 10 min, 30 min, 1 h and 3 h following LiCl (0.15 M, 12 ml/kg, i.p.) or NaCl. LiCl greatly increased the number of pMAPK-positive cells in the CeA (A) and NTS (B) from 10 to 60 min compared to the NaCl-injected groups. * p <0.005 vs. NaCl.

Figure 4

Diagram showing the location of bilateral SL327 microinjection sites within the CeA. Black dots indicate the location of the tip of guide cannulae for rats whose data were used in analyses in experiment 2 and 3 (coronal rat brain sections, spanning −2.30 mm through −3.14 mm relative to bregma; modified from Paxinos and Watson, 1997). rf, rhinal fissure; ec, external capsule; ot, optic tract.

Figure 5

A. Timeline for Experiment 2. Water-restricted rats were given 10-min access to 0.125% saccharin, then injected bilaterally with either SL327 (1 µg/0.5 µl/hemisphere in 50% DMSO in saline) or vehicle into the CeA, followed by a systemic injection of LiCl or NaCl (0.15 M, 12 ml/kg, i.p.) The day after conditioning 2-bottle preference tests were begun to assess CTA. B. Saccharin preference scores measured by 24-h, 2-bottle preference tests during CTA extinction. Rats injected with vehicle or SL327 before NaCl (Veh-NaCl and SL327-NaCl group) showed a high preference for saccharin. Rats injected with vehicle before LiCl (Veh-LiCl group) showed a significantly lower preference for saccharin across all 16 test days compared to the Vehicle-NaCl group. Rats injected with SL327 before LiCl initially showed a significantly lower saccharin preference compared to the Veh-NaCl group, but showed a significantly higher saccharin preference compared to the Veh-LiCl group from test day 9 onwards such that their CTA rapidly extinguished. * p <0.05 SL327-LiCl vs. Veh-NaCl, † p < 0.05 SL327-LiCl vs. Veh-LiCl.

Figure 6

Photomicrograph of cannula placement and pMAPK immunohistochemistry in the CeA after vehicle injection into the CeA and systemic LiCl injection. B is magnification of inset in A. rf, rhinal fissure; ec, external capsule; ot, optic tract. Scale bars, 1 mm.

Figure 7

Quantification of pMAPK-positive cells in the CeA and NTS. Rats were injected with SL327 or vehicle into the CeA 10 min before LiCl or NaCl. Both LiCl-injected groups (Veh-LiCl and SL327-LiCl) showed a significant increase in the number of pMAPK-positive cells in the CeA and NTS compared to the NaCl-injected group (Veh-NaCl). Injection of SL327 into the CeA significantly decreased the number of LiCl-induced pMAPK-positive cells in the CeA, but not in the NTS. * p <0.005 vs. Veh-NaCl, † p < 0.05 vs. Veh-LiCl.