Contextual fear conditioning (FC) is a paradigm used to model the processes of learning and memory formation. The mechanism by which fear memories are formed is further elucidated by studying the amygdala, which plays a central role in the perception and processing of FC input. While the route by which information travels through this and nearby regions is fairly well understood, the specific cell types involved in such information transfer have yet to be determined.
One 2017 study reported the proportion of excitatory neurons activated by later recall of fear memory induced by contextual FC one day prior. In the present experiment, the proportion of excitatory neurons activated in response to the initial processing of FC is determined via immunohistochemistry and compared to those reported to activate during recall. We hypothesize that activity (represented by c-Fos expression within cells) will be greater among excitatory neurons (represented by CamKII-α expression) in animals activated by the conditioning process itself than those activated by recall.
This information will validate and lend further insight to how the amygdala functions as a processing and storage unit for fear memory.
Adult (12-13 weeks old) mice (n=8, female=5) were housed singly under a 12/12 h light/dark cycle at 22℃. Animals were handled by the experimenter for approx. 1 min for 8 days leading up to the experiment. FC was conducted in the Behavioral Testing Core (BTC) at UCLA. Experimental mice (n=4) were given 2 mins to explore the context before receiving a 0.75mA 2-sec foot-shock, after which 2 additional foot-shocks were given 1 min apart. All mice were in FC boxes for a total of 5 mins before returning to home cages for 60 mins holding. Mice were then anesthetized with isoflurane and transcardially perfused with 1x PBS followed by 4% paraformaldehyde. Brains were extracted and post-fixed in 4% PFA for between 3 and 18 hours according to time of perfusion. Brains were then transferred to 10%, 20%, and 30% sucrose on consecutive days, after which they were flash-frozen until cryostat sectioning. IHC was performed on consecutive 40µm sections (bregma: -1.22 to -2.18). Following washing and blocking (0.1M PBS with 0.3% Triton X-100 containing 6% normal goat serum), sections were incubated with primary antibodies [rabbit anti-c-Fos (1:500), mouse anti-CaMKII-α (1:500), and chicken anti-NeuN (1:500)] overnight at 4℃. Sections were later incubated with corresponding fluorescently-labeled secondary antibodies and stored in the dark at RT until viewing. One 40µm section per mouse was imaged using a Zeiss LSM 710 confocal microscope. Two distinct regions of the amygdala were imaged at 40x per animal. The experimenter was blinded to condition while tracing excitatory neuron ROIs. The average intensity of green light (marking c-Fos) was calculated per ROI in ImageJ, representing the activity level of excitatory neurons within each region.
Of the eight control images counted, 210 cells were marked positive for CaMKII-α with a mean c-Fos intensity of 970.4. Of the eight experimental images counted, 250 cells were marked positive for CaMKII-α with a mean c-Fos intensity of 1403.3. A one-tailed two-sample t-test shows a highly statistically significant difference in mean c-Fos intensity (p-value of 2.8617E-12).
- Excitatory neurons within the amygdala of fear-conditioned mice show higher levels of activity when compared to mice that did not undergo FC (p<0.01).
- At an arbitrary threshold value of 3500 representing an “activated” neuron, preliminary results suggest that only 3.7% of excitatory neurons, on average, were activated in response to FC. This percentage is lower than the ~5% reported to be activated by FC recall in a previously published study. This may be the result of differing threshold values or regional differences, or could suggest a larger role for the amygdala in recall versus the initial processing of context-associated fear.