Associatively learned representations of taste outcomes activate taste-encoding neural ensembles in gustatory cortex

Abstract

Through learning processes, cues associated with emotionally salient reinforcing outcomes can come to act as substitutes for the reinforcer itself. According to one account of this phenomenon, the predictive cue associatively elicits a representation of the expected outcome by reactivating cells responsible for encoding features of the primary reinforcer. We tested this hypothesis by examining the role of neural ensembles in gustatory cortex (GC) during receipt of gustatory stimuli (sucrose and water) and cues associated with those stimuli using the immediate early genes (IEGs) Arc and Homer1a. Because these plasticity-related IEGs are expressed in the neuronal nucleus 5 and 30 min, respectively, after salient events, we examined how individual neurons encoded these stimuli in two separate behavioral epochs. In experiment 1, we showed that tasting identical sucrose solutions, but not tasteless water, in the two epochs increased both IEG activity and the degree of overlap between neural ensembles in GC. In experiment 2, odor cues associated with sucrose, but not water, evoked potentiation of IEG activity in GC similar to sucrose itself. Surprisingly, lesions of the basolateral amygdala had minimal effects on associative encoding in GC. Finally, these associatively driven representations of sucrose appeared to be outcome specific, as neural ensembles that were activated by the sucrose-associated cue were also activated by sucrose itself. This degree of overlap between associative and primary taste activity at the ensemble level suggests that GC neurons encode important information about anticipated outcomes. Such representations may provide outcome-specific information for guiding goal-directed behavior.

Figure 1.

A, Design for experiments 1 and 2. In both experiments, animals were run in two epochs, each 5 min long, separated by a 20 min interepoch interval. Orange circles are 1 min bouts of fluid exposure. Events in epoch 1 (left) that drive IEG expression will be visible as H1a nuclear mRNA at the time of killing (sacrifice), while events in epoch 2 (right) that drive IEG expression will be visible as nuclear Arc mRNA. Thus, cells active in both epochs will be double labeled for both Arc and H1a. B, Sample of fluorescent in situ hybridized tissue. Blue regions are Nissl-stained neurons, green dots (indicated by pale arrows) show H1a expression, red dots (indicated by red arrows) show Arc expression. Cells that are double labeled for both genes are indicated by double vertical arrows. Sample image taken at 40× magnification.

Figure 2.

Experiment 1 IEG results. Rats in the SAME condition were given identical tastants in both epochs, either 10% sucrose (n = 7) or sweet cereal (n = 1), and showed similar rates of IEG expression. Similarly, rats in the WATER condition (n = 4) received unflavored water in both conditions and showed significantly lower rates of IEG expression. In the DIFFERENT condition, rats (n = 5) received sucrose in one epoch and water in the other. All rats in this condition showed greater activity in the sucrose epoch than in the water epoch, regardless of order presented. Colored circles show individual rates for each subject. Red circles indicate averages for rats that experienced sucrose in the first epoch, while blue circles are for the rats that experienced water in the first epoch. The identity of the tastant, rather than the order of sucrose presentation, drives differential IEG expression in GC. Left bars (SAME and WATER conditions) are for epoch 1, right bars for epoch 2; for the DIFFERENT condition, the left bars are the average of the sucrose presentations and the right bars are the average for the water presentations. Gray shading indicates that sucrose was presented in that epoch, white indicates water presentation. **p < 0.05 for sucrose greater than water in DIFFERENT condition. Error bars are ±SEM between subjects.

Figure 3.

Experiment 1 double labeling results. Data on the left show the absolute rate of double labeling, expressed as a percentage of double labeled cells out of all cells counted. Using this metric, there were significantly more double labeled cells in the SAME group compared with both the DIFFERENT and WATER groups, although there was no difference between DIFFERENT and WATER. This suggests that similar ensembles in GC are reactivated when animals taste identical tastants, but not when they experience either different tastants or tasteless water. Individual data points for each subject are shown by the colored circles overlying each bar. Rats that received sucrose in the first epoch are indicated by red circles and rats that experienced water first are indicated by blue circles. **p < 0.005 compared with the SAME condition.

Figure 4.

Histology on BLA sections for O1 versus O2 subjects. A, Sham-lesioned animal shows intact (BLA) and lateral (LA) subregions of the BLA nucleus. Note large pyramidal cells in both subregions. B, Neurotoxic NMDA injections damage cells in both BLA and LA. In both subregions, there is noted cell shrinkage and gliosis observable throughout the region. However, this damage did not encroach significantly into adjacent cortical or subcortical regions. White arrows indicate terminal location of needles used to inject neurotoxin.

Figure 5.

Composite drawings of BLA lesions. Individual lesions are in transparent light gray such that darker regions indicate regions of greater overlap between lesions. Numbers at right indicate mm from bregma; lesions extended from −2.0 to −3.8 mm posterior to bregma. Images adapted from Paxinos and Watson, 1997.

Figure 6.

Experiment 2 IEG results. A, In the O1 versus O2 condition, rats were trained with pairings of odor 1 (O1) with sucrose and odor 2 (O2) with water. During test, all rats received water paired with O1 in one epoch and O2 in the other epoch. Despite tasting the same water in both epoch, rats in both sham-lesioned (5 of 5) and BLA-lesioned (4 of 4) conditions showed significantly greater IEG activity in O1 epoch compared with the O2 epoch (**p < 0.05 greater for O1 compared with O2 IEG expression). As in experiment 1, order had no effect on IEG rates. Red circles are data from subjects that received O1 in epoch 1, and blue circles are for subjects that received O2 in epoch 1. BLA lesions had a moderate effect on overall IEG expression rates (p < 0.05), but did not affect the ability for O1 to potentiate IEG expression over O2. B, In the O1 versus sucrose condition, rats received the same training as in O1 versus O2, but on the day of test received water paired with O1 in one epoch (same as in O1 vs O2), and unscented sucrose in the other epoch. Rats showed similarly elevated rates of IEG expression in the O1 epoch, and indeed, this activity was nearly identical to receipt of sucrose itself in the other epoch. These data suggest that in GC, a representation of a taste outcome is as effective at driving GC IEG expression as tastants themselves.

Figure 7.

Experiment 2 double labeling results. In the O1 versus O2 condition, rats consumed water in both test sessions, but received O1 in one epoch and O2 in the other. Consonant with findings in experiment 1, this led to low levels of double labeling in both sham controls and BLA-lesioned subjects. Further, there was a significant decrease in the rate of double labeling between sham and BLA-lesioned subjects in the O1 versus O2 condition, p < 0.05. However, subsequent analysis using a normalized E1RR index showed that this was simply due to lower overall IEG expression rates in the BLA-lesioned condition. In contrast, in the O1 versus sucrose condition, rats that consumed sucrose in one epoch and water+O1 in the other showed significantly greater rates of double labeling than in either of the O1 versus O2 conditions (*p < 0.01 less double labeling than O1 vs sucrose; **p < 0.01 less double labeling than the O1 vs sucrose condition and O1 vs O2 Sham). Importantly, rats in the O1 versus sucrose condition actually consumed two different solutions—water and sucrose—across epochs. However, O1 was able to activate the same ensembles as tasting sucrose alone, as suggested by double labeling indexes that are numerically similar to that found in the SAME condition in experiment 1 (Fig. 3).