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Mapping contingency awareness in fear conditioning
C.J. Han

Abstract. The goal of this project is to employ two types of Pavlovian conditioning: trace and delay, to investigate the awareness of the contingency of the conditioned stimulus (CS) and unconditioned stimulus (US). We have successfully established the behavioral and molecular paradigms over the past year and are currently collecting data of the effects of the anterior cingulate cortex lesion and the immediate early gene c-fos expression patterns in the mouse brain.

In the two types of Pavlovian conditioning, trace conditioning is different from delay conditioning in that there is a temporal gap between the end of the conditioned stimulus (CS) and the start of the unconditioned stimulus (US). It has been shown in humans that trace, but not delay, conditioning requires awareness of the CS-US contingency and is dependent on the integrity of the hippocampus (Clark & Squire, 1998).

1. Establishment of trace vs. delay fear conditioning paradigm in mice. We have successfully established a paradigm for trace vs. delay fear conditioning in mice (Fig. 1). In the delay paradigm, the US (footshock) is delivered concurrently with the termination of the CS (tone), while in the trace paradigm it is delivered after a "trace interval" of 18 sec. Testing is carried out 3 days later, in a different room and cage, in order to assess the efficacy of cue (tone) conditioning in the absence of interference from context conditioning.

As shown in Figure 2, successful trace as well as delay conditioning has been achieved. Freezing was scored from videotapes of the testing session by a blind observer, every 2 sec. Percent freezing was derived by dividing the number of observations scored as freezing by the total number of observations. Freezing percentages were calculated for the 3-min baseline period, the three trials of 1-min CS (tone) presentation and the three trials of 1-min inter-trace intervals (ITI). The final tone and ITI freezing were the average across the three trials. Single-factor ANOVA was used to test the significance of difference among groups.


2. Quantitative immediate early gene (IEG) analysis. We have developed a procedure for non-isotopic detection of IEG mRNAs by in situ hybridization on thick (120 µM) coronal brain sections (Fig. 3). This procedure allows 3 different kinds of quantitative analysis: 1) automatic detection of stained cell profiles, 2) bias-free stereological measurements of cell numbers with an optical fractionator, and 3) 3D serial reconstruction. Using a motorized stage and Neurolucida

software, low-magnification views are assembled from individual 6X fields, retaining high-resolution data (Fig. 3). Positive cell profiles can be automatically extracted from these images using a threshholding program, quantified and assembled to produce 3-D views (Fig. 4). These data provide an overview that can be used to select specific regions for accurate cell counting using unbiased stereology.

3. Selective lesioning of extrahippocampal regions important in trace but not delay conditioning. We have performed preliminary lesions in rats of the anterior cingulate cortex (ACC), a region implicated in attention and awareness in humans. Figure 5A shows a section through a specimen

lesioned by injection of the excitoxin NMDA. Behavioral analysis of such rats showed a selective impairment of trace but not delay conditioning (Fig. 5B). We have also performed preliminary experiments to assess the extent to which trace but not delay conditioning is impaired by a sham surgical procedure, in mice. These data indicate that sham lesions over the ACC or

area V1 (visual cortex) cause no impairment of trace conditioning (Fig. 6). These preliminary results indicate that our surgical procedure is feasible to perform specific excitotoxic lesioning experiments in mice to assess the requirement in trace conditioning for extra-hippocampal areas identified by the IEG analysis. We are currently conducting the experiments of comparing the ACC lesion effects on trace and delay conditioning.

Reference
Classical conditioning and brain systems: The role of awareness. Clark RE, Squire LR (1998) Science 280:77-81.