Specific and differential activation of mitogen-activated protein kinase cascades by unfamiliar taste in the insular cortex of the behaving rat

Abstract

Rats were given to drink an unfamiliar taste solution under conditions that result in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1-2 (ERK1-2) in the cortical homogenate was significantly activated within <30 min of drinking the taste solution, without alteration in the total level of the ERK1-2 proteins. The activity subsided to basal levels within <60 min. In contrast, ERK1-2 was not activated when the taste was made familiar. The effect of the unfamiliar taste was specific to the insular cortex. Jun N-terminal kinase 1-2 (JNK1-2) was activated by drinking the taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remained unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, was activated with a time course similar to that of ERK1-2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuated long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It was concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory.

Fig. 1.

The effect of gustatory experience on activation of ERK1–2 in the rat insular cortex. A, Activated ERK1–2 (dpERK1–2). B, Total ERK1–2. Three types of experimental groups were used. In group 1, rats were allowed to drink water. In group 2, rats were allowed to drink a solution of saccharin, a taste that they had never encountered before (the unfamiliar taste). In group 3, rats were allowed to drink the saccharin solution, which in this case was made familiar earlier by allowing them to drink it once for 10 min 3 d before presenting it again in the experiment (see Materials and Methods). Several sets of experiments were conducted in different combinations of the groups. Each graph depicts a “magnitude ratio,” which is the ratio of the combined intensity of the 42 + 44 kDa bands in one experimental group over another (group_i/group_j) as a function of time after the offset of drinking. A,Filled circles, Data from a set of experiments depicting group₂/group₁, i.e., the ratio of dpERK1–2 in the insular cortex of rats allowed to drink unfamiliar saccharin solution over that in rats allowed to drink water.A, Open circles, Data from another set of experiments depicting group₂/group₃, i.e., the ratio of dpERK1–2 in the insular cortex of rats allowed to drink unfamiliar saccharin over that in rats allowed to drink familiar saccharin.Inset, A representative blot of dpERK1–2 att = 30 min after drinking water (W), unfamiliar saccharin (S), or familiar saccharin (SF), and quantification of experiments similar to those depicted in the blot (activation in the water group, 1.0;n = 9–11 each). The results show that the effect of taste experience on the activation of ERK1–2 in the insular cortex is attributable to the unfamiliarity of the taste. B, Same as in A but depicting the results for total ERK1–2. (In this and all other figures, values are mean ± SEM, and *p < 0.05.)

Fig. 2.

The level of activated ERK1–2 and total ERK1–2 in various brain regions 30 min after the exposure to the unfamiliar taste. A, Activated ERK1–2. B, Total ERK1–2. IC, Insular cortex; OL, occipital lobe; OB, olfactory bulb; PC, piriform cortex; CB, cerebellum. n = 6 in each group.

Fig. 3.

Impairment of CTA by local microinjection of the reversible MEK inhibitor PD098059 into the insular cortex. AI values are plotted versus time after training. Each group of animals was tested only once at the indicated time. Filled circles, Rats microinjected with 1 μl of 30 μm PD089059 per hemisphere into the insular cortex 30 min before CTA training;open circles, rats microinjected with 1 μl of ACSF plus 1% DMSO (n = 6 each). Inset, Spatial and temporal specificity of the inhibitor effect.A, Control animals. B, PD098059 microinjected 2 mm above the stereotaxic coordinates used for injection into the insular cortex. C, PD098059 microinjected into the insular cortex 30 min before the first memory test.n = 8 in each group.

Fig. 4.

Inhibition of taste-dependent ERK1–2 activation by PD098059 in the insular cortex. A, A representative blot of samples of insular cortex of animals subjected to different manipulations. Water, Animals drinking 10 ml of water;Sacch, animals exposed to 10 ml of saccharin;PD+Sacch, animals injected with PD098059 30 min before the exposure to saccharin. Rats were killed 30 min after each treatment, and the samples were blotted with anti-diphospho ERK1–2 antibodies. B, Summary of data from experiments like those in A. Open bar, Animals exposed to water; filled bar, animals exposed to saccharin;shaded bar, animals microinjected into the insular cortex with PD098059 and 30 min later presented with saccharin.n = 4 per group.

Fig. 5.

Activation of Elk-1 in the insular cortex as a function of time after sampling a saccharin solution as an unfamiliar taste. Magnitude ratio is expressed as pElk-1 (saccharin)/pElk-1 (water) (filled circles), Elk-1 (saccharin)/Elk-1 (water) (open circles), or pElk-1 (saccharin)/pElk-1 (familiar saccharin) (filled square,t = 30 min). In the latter case, the saccharin solution was made familiar by preexposure, as detailed in the legend to Figure 1. n = 8–12 per time point.

Fig. 6.

Activation of JNK1–2 in the insular cortex as a function of time after sampling a novel taste. Magnitude ratio is expressed as dpJNK (saccharin)/dpJNK (water) or JNK (saccharin)/JNK (water), respectively (combined values for both bands in each case).Inset, A representative blot at t = 60 min after drinking. Filled circles, Diphospho-JNK;open circles, total JNK. n = 9–12 per time point.

Fig. 7.

The effect of gustatory experience on Akt and p38MAPK in the insular cortex. A, The level of phospho-Akt (pAkt) and total Akt as a function of time after drinking saccharin or water. Filled circles, pAkt; open circles, total Akt. Inset, A representative blot at t = 30 min after drinking.B, The level of diphospho-p38MAPK (dp-p38MAPK) and total p38MAPK as a function of time after drinking saccharin or water. Filled circles, dp-p38MAPK; open circles, total p38MAPK.Inset, A representative blot at t = 30 min after drinking. In both A and B,n = 11–15 per time point.