Olfactory fear conditioning induces field potential potentiation in rat olfactory cortex and amygdala

Abstract

The widely used Pavlovian fear-conditioning paradigms used for studying the neurobiology of learning and memory have mainly used auditory cues as conditioned stimuli (CS). The present work assessed the neural network involved in olfactory fear conditioning, using olfactory bulb stimulation-induced field potential signal (EFP) as a marker of plasticity in the olfactory pathway. Training consisted of a single training session including six pairings of an odor CS with a mild foot-shock unconditioned stimulus (US). Twenty-four hours later, the animals were tested for retention of the CS as assessed by the amount of freezing exhibited in the presence of the learned odor. Behavioral data showed that trained animals exhibited a significantly higher level of freezing in response to the CS than control animals. In the same animals, EFPs were recorded in parallel in the anterior piriform cortex (aPC), posterior piriform cortex (pPC), cortical nucleus of the amygdala (CoA), and basolateral nucleus of the amygdala (BLA) following electrical stimulation of the olfactory bulb. Specifically, EFPs recorded before (baseline) and after (during the retention test) training revealed that trained animals exhibited a lasting increase (present before and during presentation of the CS) in EFP amplitude in CoA, which is the first amygdaloid target of olfactory information. In addition, a transient increase was observed in pPC and BLA during presentation of the CS. These data indicate that the olfactory and auditory fear-conditioning neural networks have both similarities and differences, and suggest that the fear-related behaviors in each sensory system may have at least some distinct characteristics.

Figure 1.

Percentage of freezing (Mean ± SEM) for each minute of the CS-test session, in the different experimental groups. The bottom of the figure summarizes the training procedures used in the different groups. (A) Group 1, trained animals (n = 6, •) and control animals (n = 6, ○). The animals were trained and tested with Isoamylacetate. (B) Group 2, Test 1, trained animals (n = 6, •) and control animals (n = 6, ○). The animals were trained with Isoamylacetate and tested with a novel odor, Eugenol. (C) Group 2, Test 2, trained animals (n = 6, •) and control animals (n = 6, ○). Twenty-four hours after Test 1, the animals were tested with Isoamylacetate CS. (D) Group 2, trained animals, Test 1 vs. Test 2. Comparison of the freezing response to the Isoamylacetate CS (n =6, •) and to Eugenol (n =6, ○). (^*) Significant difference between the two sessions (p < 0.05).

Figure 2.

Mean (±SEM) variation in EFPs amplitude measured in control (A, n = 8) and trained (B, n = 9) animals, in the four recording sites, during the CS-test session. Changes are expressed in percentage of variations compared with the amplitude of the baseline obtained before conditioning. EFPs collected during the first 2 min of the session (Pre-odor period) and during the 6 min of CS odor presentation (Odor period) were pooled separately. (aPC) Anterior piriform cortex; (pPC) posterior piriform cortex; (CoA) cortical nucleus of the amygdala; (BLA) basolateral amygdala. (⋄) Significant difference between Pre-odor and Odor periods (p < 0.05). (^*) Significant difference compared with baseline signals (p < 0.05).

Figure 3.

Mean (±SEM) variation in EFPs amplitude of signals collected in the four recording sites, in trained (n = 9) and control (n = 8) animals, during the 8-min CS-test session. The EFPs collected during each minute of the session were averaged and analyzed separately. Changes are expressed as percent variations from the baseline signals collected before training. (aPC) Anterior piriform cortex; (pPC) posterior piriform cortex; (CoA) cortical nucleus of the amygdala; (BLA) basolateral amygdala. (^*) Significant difference compared with baseline signals (p < 0.05). (⋄) Significant difference with at least 1 of the first 2 min (p < 0.05).

Figure 4.

Schematic representation of the implanted electrodes and recorded evoked field-potential (EFPs) signals. (A) A bipolar stimulation electrode was implanted in the mitral cell layer of the olfactory bulb (OB). Four monopolar recording electrodes were respectively implanted in the anterior piriform cortex (aPC), posterior piriform cortex (pPC), cortical nucleus of the amygdala (CoA), and basolateral amygdala (BLA). (B) An example of EFPs induced in the four recording sites in response to electrical stimulation of the OB. The amplitude (A_mV) of the evoked main component was measured in each site, as indicated on the figure. (St) Stimulation artifact. (C) Schematic drawing representing recording electrodes' tip placements. Numbers on the right indicate position of the coronal sections in millimeter relative to bregma (Paxinos and Watson 1998).