Article Figures & Data
Figures
- Fig. 1.
A, Neophobia during first access to a novel solid food is attenuated on second exposure. Mice were given 10 min access to Nutri-Grain blueberry bar, and intakes were recorded. Ten minute intakes on the second are significantly higher, demonstrating attenuation of neophobia through familiarization. (*p < 0.05 by one-way ANOVA). B,Pairing of solid novel food with LiCl produces a conditioned taste aversion. Mice injected with LiCl after a 10 min access to blueberry bar consume significantly less than NaCl-injected when tested (***p < 0.001 by one-way ANOVA).
- Fig. 2.
Memory for a novel food and acquisition of a conditioned taste aversion is MAPK-dependent. LiCl, LiCl-DMSO, and LiCl-SL327 mice all received 10 min access to blueberry bar followed by injection of LiCl. NaCl mice received NaCl after blueberry bar. SL327 (100 mg/kg, s.c.) injected 1 hr before blueberry bar significantly attenuates conditioned taste aversion, as shown by test intakes. (*p < 0.05 relative to LiCl by ANOVA and Duncan interval).
- Fig. 3.
Effect of the histone deacetylase inhibitor trichostatin A on histone and non-nuclear protein acetylation in insular cortex slices. A, Two hour treatment with trichostatin A produced significant increases in acetylation of histones H4, H2A, but not H2B or H3. Acetylation of an unknown protein, termed p55AcK, was also increased by trichostatin A. Acetylation of two other proteins, p42AcK and p70AcK, was not altered by trichostatin A (*p < 0.05, **p < 0.01, ***p < 0.001 relative to controls by one-way ANOVA; y-axis: IOD, integrated optical density, i.e., pixel intensity × area). B,Representative blots showing acetylated lysine immunoreactivity after Western blots of histone extracts (left) or non-nuclear proteins (right).
- Fig. 4.
Effects of forskolin activation of PKA on MAPK activation and lysine acetylation. A, Forskolin produces a significant activation of both p42 and p44 isoforms of MAP kinase. The selective MEK inhibitor U0126 completely abolishes phospho-MAPK immunoreactivity (D) and is not plotted here. Two-way ANOVA revealed a significant main effect of forskolin on phosphorylation of both p42ERK (p < 0.001) and p44ERK (p < 0.05); there was also a significant main effect of U0126 (p < 0.001). B, Activation of the MAPK cascade elicits an increase in lysine acetylation of p42AcK that is blocked by U0126. Two-way ANOVA revealed a significant main effect of both forskolin (p < 0.05) and U0126 (p < 0.01). Post hoc Dunnett analysis revealed that forskolin control acetylation was significantly greater than forskolin U0126 (p < 0.05).C, Inhibition of the MAPK cascade by U0126 causes increased acetylation of p55AcK. Two-way ANOVA revealed a significant main effect of U0126 (p < 0.05) but not forskolin. D, Representative Western blots of phospho-MAPK (left) and acetylated lysine (right). Note the complete absence of phospho-MAPK immunoreactivity in U0126 slices.
- Fig. 5.
Acetylation of a 42 and 55 kDa protein, but not a 70 kDa protein, is altered by previous novel taste exposure. Mice that had been used to screen for solid food preferences were used for an initial screen for lysine acetylation in insular cortex.A, Western blot revealed three prominent acetylated proteins in both novel and naive mice. B, Lysine acetylation of both the 42 and 55 kDa proteins was shown to be increased after several previous exposures to novel foods (p < 0.05 by one-way ANOVA).
- Fig. 6.
Acetylation of p42AcK (A) and p55AcK (B) after novel and familiar taste. Novel mice were given a single 10 min access to blueberry bar (denoted by arrow at t = 0 hr), and time of killing is indicated by the x-axis. The data point att = 0 hr represents the control group of naive mice that did not receive novel food. Familiar mice received three previous exposures at 0, 24, and 48 hr (denoted by arrows) followed by an additional 48 hr before the fourth exposure att = 96 hr. The data point at t= 96 hr represents mice with three previous exposures only and is the control group for the familiar groups. All data are expressed as integrated optical density (area × pixel intensity) normalized to the t = 0 control mice. A, Lysine acetylation of p42AcK shows a biphasic response in the novel group and a persistent increase in the familiar group. Two-way ANOVA showed significant main effects of both group (novel vs familiar;p < 0.01) and time (p< 0.05). Post hoc Dunnett analysis revealed a significant increase in p42AcK acetylation in the novel group at 1, 2, 12, and 24 hr. Relative to t = 96 hr controls, there was no significant change in the familiar group.B, Lysine acetylation of p55AcK. Two-way ANOVA revealed significant effects of both group (novel vs familiar;p < 0.001) and time (p< 0.001). Post hoc Dunnett analysis revealed significant increases in acetylation of p55AcK at all time points up to 12 hr, but not at 24 and 48 hr. There was no significant change in acetylation of the familiar group at any time points relative tot = 96 hr. C, Representative immunoblots showing changes in acetylation of p42AcK and p55AcK. Note that there is no change in the acetylation of p70AcK.
- Fig. 7.
Phosphorylation of p42 and p44 MAP kinase after novel or familiar taste. A, B, Two-way ANOVA revealed significant main effects of both group (novel vs familiar, p < 0.001) and time (p < 0.001) for both MAPK isoforms.A, Post hoc Dunnett analysis of p42ERK revealed significant increases in phosphorylation at 1, 2, 6, 24, and 48 hr. B, p44 MAPK phosphorylation was significantly increased at 2, 6, and 48 hr. A, B, For both MAPK isoforms, phosphorylation was significantly increased at the 144 hr time point; this is 48 hr after the fourth and final exposure to the now-familiar taste. C, Total MAPK levels are unchanged in both the novel and familiar groups for both MAPK isoforms. Normalized p42 values are plotted against the left y-axis, and p44 values against the right y-axis. Two-way ANOVA revealed that there was no significant effect of either group (novel vs familiar) or time. D, E,Representative immunoblots showing phospho-MAPK and total MAPK, respectively.
- Fig. 8.
Phosphorylation of MAP kinase cascade elements MEK, Raf, and p90 RSK. A, phospho-MEK shows a biphasic response. Two-way ANOVA revealed a significant main effect of both group (novel vs familiar; p < 0.001) and time (p < 0.001). Post hocDunnett analysis revealed significant increases in phospho-MEK at 1, 2, 24, and 48 hr in the novel group but no significant effect of time in the familiar group. B, Phosphorylation of Raf does not show the biphasic response seen with p42AcK and phospho-MAPK. Two-way ANOVA revealed a significant main effect of both group (novel vs familiar; p < 0.05) and time (p < 0.001). phospho-Raf was significantly increased at 0.5, 1, 2, 6, and 12 hr, but then declined to baseline at 24 and 48 hr. Within the familiar group, phospho-Raf levels att = 144 hr (48 hr after the fourth taste exposure) were significantly depressed relative to the t = 96 hr control. C, phospho-p90RSK also shows a biphasic response to novel taste. Two-way ANOVA revealed significant main effects of both group (novel vs familiar; p < 0.001) and time (p < 0.001). Within the novel group, phosphorylation was significantly increased at all time points, although the plot shows the characteristic biphasic response. As with other members of the MAPK cascade, there was no significant effect of time within the familiar group. D,Representative immunoblots showing phospho-MEK, phospho-Raf, and phospho-p90RSK immunoreactivity.
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