Mapping of olfactory memory circuits: region-specific c-fos activation after odor-reward associative learning or after its retrieval

Abstract

Although there is growing knowledge about intracellular mechanisms underlying neuronal plasticity and memory consolidation and reconsolidation after retrieval, information concerning the interaction among brain areas during formation and retrieval of memory is relatively sparse and fragmented. Addressing this question requires simultaneous monitoring of activity in multiple brain regions during learning, the post-acquisition consolidation period, and retrieval and subsequent reconsolidation. Immunoreaction to the immediate early gene c-fos is a powerful tool to mark neuronal activation of specific populations of neurons. Using this method, we are able to report, for the first time, post-training activation of a network of closely related brain regions, particularly in the frontal cortex and the basolateral amygdala (BLA), that is specific to the learning of an odor-reward association. On the other hand, retrieval of a well-established associative memory trace does not seem to differentially activate the same regions. The amygdala, in particular, is not engaged after retrieval, whereas the lateral habenula (LHab) shows strong activation that is restricted to animals having previously learned the association. Although intracellular mechanisms may be similar during consolidation and reconsolidation, this study indicates that different brain circuits are involved in the two processes, at least with respect to a rapidly learned olfactory task.

Figure 1

Odor task discrimination acquisition. Training was performed in a single session in five trials. On the first trial only, the reward was visible (almond, top left). The spatial configuration of the sponges was changed between trials and the reinforcement was always associated with the same odor. Trial × trial number of errors (nose poke to the nontarget odor) (mean ± SEM) are shown on the graph. Similar results were obtained for latencies. This training procedure results in a long-term memory trace lasting at least 1 wk.

Figure 2

(a) Schematic diagram of a section taken from Paxinos and Watson (1986) showing the sampling regions within the prelimbic cortex (PLC) used for quantifying the number of marked cells. A computerized microscope stage that recorded X-Y coordinates ensured sampling from exactly the same place for each rat (see text). Immunoreactivity in the PLC is shown in naïve (b), unpaired yoked controls (c), and trained animals (d). See text for treatment of each group. There are two visible marked cells in the naive rat (b), relatively sparse marking in the yoked control (c), and much denser marking in the trained rats (d). Areas were delineated at the magnification shown in the figure; cells were counted at a higher magnification (×40).

Figure 3

Frontal regions: Mean number of c-fos immunoreactive cells per 0.1 mm² in frontal regions of unpaired controls and trained rats after training (left) or after retrieval test (right). In the PLC and ventrolateral orbital cortex (VLO), there was a significant increase in c-fos immunoreactivity in trained rats relative to controls after training (**prelimbic: P < .005; *VLO: P = .03).

Figure 4

Amygdala: Mean number of c-fos immunoreactive cells per 0.1 mm² in the basolateral amygdala (BLA) and the central amygdala (CeA) of unpaired controls and trained rats after training (left) or after retrieval test (right). In the BLA, there was a significant increase in c-fos immunoreactivity in trained animals relative to controls after training. (**P < .005). There was not much difference in CeA after initial learning or retrieval and no effect of training in either condition.

Figure 5

Habenula: Mean number of c-fos immunoreactive cells per 0.1 mm² in the lateral habenula (LHab) of unpaired controls and trained rats after training (left) or after a retrieval test (right). This was the only region where there was a significant increase in c-fos immunoreactivity in trained animals relative to controls after a retrieval test (**P = .03). There was no difference between trained and controls after the training session.

Figure 6

c-fos expression after learning or after retrieval in the LHab. (a) Section from a trained rat killed after the retrieval test. There are an abundant number of marked cells limited to the LHab. Note the absence of any c-fos immunoreactivity in the medial habenula. (b) Section from a yoked unpaired control rat after a ‘retrieval‘ trial showing just a few scattered marked cells. (c) Section from a rat killed after initial training showing fewer and more scattered immunoreactive cells than in Figure 3a (scale bar, 100 μm).