Modulation of CA2 neuronal activity increases behavioral responses to fear conditioning in female mice

Abstract

Activity of hippocampal pyramidal cells is critical for certain forms of learning and memory, and work from our lab and others has shown that CA2 neuronal activity is required for social cognition and behavior. Silencing of CA2 neurons in mice impairs social memory, and mice lacking Regulator of G-Protein Signaling 14 (RGS14), a protein that is highly enriched in CA2 neurons, learn faster than wild types in the Morris water maze spatial memory test. Although the enhanced spatial learning abilities of the RGS14 KO mice suggest a role for CA2 neurons in at least one hippocampus-dependent behavior, the role of CA2 neurons in fear conditioning, which requires activity of hippocampus, amygdala, and possibly prefrontal cortex is unknown. In this study, we expressed excitatory or inhibitory DREADDs in CA2 neurons and administered CNO before the shock-tone-context pairing. On subsequent days, we measured freezing behavior in the same context but without the tone (contextual fear) or in a new context but in the presence of the tone (cued fear). We found that increasing CA2 neuronal activity with excitatory DREADDs during training resulted in increased freezing during the cued fear tests in males and females. Surprisingly, we found that only females showed increased freezing during the contextual fear memory tests. Using inhibitory DREADDs, we found that inhibiting CA2 neuronal activity during the training phase also resulted in increased freezing in females during the subsequent contextual fear memory test. Finally, we tested fear conditioning in RGS14 KO mice and found that female KO mice had increased freezing on the cued fear memory test. These three separate lines of evidence suggest that CA2 neurons are actively involved in both intra- and extra-hippocampal brain processes and function to influence fear memory. Finally, the intriguing and consistent findings of enhanced fear conditioning only among females is strongly suggestive of a sexual dimorphism in CA2-linked circuits.

Keywords: Amygdala; DREADD; Fear conditioning; Hippocampal CA2; RGS14; hM3Dq; hM4Di.

Figure 1.

Contextual and cued fear conditioning in hM3Dq mice. A, hM3Dq-mCherry expression colocalizes with PCP4, a marker of CA2 neurons. B, Schematic of the fear conditioning paradigm. C, Percent of time spent freezing during training. Freezing was measured during the baseline period (before any tone-shock pairing) and during each of the three conditioned stimulus (CS) tone-shock pairings. A shock (unconditioned stimulus, US) was delivered during the final 2 sec of each 30-sec CS tone presentation. The schematic diagram shows the relative time-course of tone CS and shock US presentation. Freezing during each CS presentation was measured for the entire tone presentation, which included the 2-sec shock. D, Freezing for each mouse during the first tone CS-shock presentation. Freezing was significantly increased in Cre+ mice. E-H, Contextual (E-F) and cued (G-H) fear results from Test 1. E, Mean freezing across time for each genotype showed main effects of genotype and time. A main effect of sex was also detected, as well as an interaction between sex and genotype, prompting us to analyze each sex independently. F, Mean percent of time freezing across all five minutes of the contextual fear test. Cre+ female, but not male, mice showed enhanced freezing behavior relative to Cre− mice. G, Freezing for each minute of the cued fear test. Analysis of freezing for all five minutes of the test showed main effects of genotype and time and an interaction between genotype and time. H, Percent of time freezing during the pre-cue period and the first minute of the tone presentation in the cued fear memory test for male and female mice. *p<0.05, **p<0.01, ****p<0.0001. Lines show means and SEM, filled dots represent individual male animals, open circles represent individual females. Scale bar represents 300 μm in A. See also Fig. S1-3, S5.

Figure 2.

Long-term retention of contextual (A-B) and cued (C-D) fear conditioning two weeks after training in hM3Dq mice. Data correspond to Test 2 from Fig. 1B. A, Mean percent of time freezing for all five minutes of the contextual fear retention test. Cre+ mice trended toward more freezing than Cre− mice. Freezing for all animals did not vary as a function of time in the context. B, Mean freezing across time for each genotype for males and females. C, Freezing for each minute of the cued fear test. Cre+ mice showed more freezing than Cre− mice. D, Percent of time freezing during the pre-cue period and the first minute of the tone presentation in the cued fear memory test for male and female mice. **p<0.01, ****p<0.0001. Lines show means and SEM, filled dots represent individual males, open circles represent individual females. See also Fig. S4-S5.

Figure 3.

Contextual and cued fear conditioning in hM4Di mice. A-B, hM4Di-mCherry expression colocalizes with PCP4, a marker of CA2 neurons. C, Percent of time spent freezing during the baseline period before any tone-shock pairing, and freezing during each of the three conditioned stimulus (CS) tone-shock pairings. D, Mean freezing across time upon re-exposure to the context 24 hours after training. A main effect of genotype and of time and an interaction between time and genotype were found. A main effect of sex was also detected. E, Average freezing over the five-minute context fear test. Males showed no difference in freezing across genotype, but among females, Cre+ mice showed significantly more freezing than Cre− mice. F, Freezing for each minute of the cued fear test. G, Percent of time freezing during the pre-cue period and the first minute of the tone presentation in the cued fear memory test. Lines show means and SEM, filled dots represent individual males, open circles represent individual females. Result of post hoc tests shown on graph. *p<0.05, ****p<0.0001. Scale bar represents 1 mm in A, 300 μm in B. See also Fig. S6, S8.

Figure 4.

Long-term retention of contextual (A-B) and cued (C-D) fear conditioning in hM3Di mice two weeks after training. Data correspond to Test 2 from Fig. 1B. A, Mean contextual freezing across time for each genotype. B, Mean percent of time freezing across all five minutes of the contextual fear test for males and females. C, Freezing across each minute of the cued fear test. D, Percent of time freezing during the pre-cue period and the first minute of the tone presentation in the cued fear test. Lines show means and SEM, filled dots represent individual males, open circles represent individual females. ****p<0.0001. See also Fig. S7, S8.

Figure 5.

Contextual and cued fear conditioning in RGS14 KO and WT mice. A-B, RGS14 protein staining in a whole-brain coronal section (A) and in the hippocampus, showing staining in CA2 and the fasciola cinerea (B). C, Western blot analysis showing protein expression of RGS14 and beta-actin loading control in RGS14 KO and WT control mice. D, Percent of time spent freezing during the baseline period before any tone-shock pairing, and freezing during each of the three conditioned stimulus (CS) tone-shock pairings. E, Mean freezing across time for each genotype in the context fear test. F, Mean percent of time freezing across all five minutes of the contextual fear test for each of males and females. G, Freezing across each minute of the cued fear test. H, Percent of time freezing during the pre-cue period and the first minute of the tone presentation in the cued fear memory test for males and females. Lines show means and SEM, filled dots represent individual males, open circles represent individual females. Scale bar represents 1 mm in A, 300 μm in B. ****p<0.0001. See also Fig. S9, S11.

Figure 6.

Long-term retention of contextual (A-B) and cued (C-D) fear conditioning in RGS14 KO and WT mice two weeks after training. Data correspond to Test 2 from Fig. 1B. A, Mean freezing across time for each genotype in the test of context fear. B, Mean percent of time freezing during all five minutes of the context fear test for males and females. C, Freezing for each minute of the cued fear test. D, Percent of time freezing during the pre-cue period and the first minute of the tone presentation in the cued fear test for male and female mice. Females showed significantly greater freezing during the first minute of the cue presentation. Lines show means and SEM, filled dots represent individual males, open circles represent individual females. *p<0.05, ****p<0.0001. See also Fig. S10, S11.