Learning-Dependent and -Independent Enhancement of Mitral/Tufted Cell Glomerular Odor Responses Following Olfactory Fear Conditioning in Awake Mice

Abstract

Associative fear learning produces fear toward the conditioned stimulus (CS) and often generalization, the expansion of fear from the CS to similar, unlearned stimuli. However, how fear learning affects early sensory processing of learned and unlearned stimuli in relation to behavioral fear responses to these stimuli remains unclear. We subjected male and female mice expressing the fluorescent calcium indicator GCaMP3 in olfactory bulb mitral and tufted cells to a classical olfactory fear conditioning paradigm. We then used awake, in vivo calcium imaging to quantify learning-induced changes in glomerular odor responses, which constitute the first site of olfactory processing in the brain. The results demonstrate that odor-shock pairing nonspecifically enhances glomerular odor representations in a learning-dependent manner and increases representational similarity between the CS and nonconditioned odors, potentially priming the system toward generalization of learned fear. Additionally, CS-specific glomerular enhancements remain even when associative learning is blocked, suggesting two separate mechanisms lead to enhanced glomerular responses following odor-shock pairings.SIGNIFICANCE STATEMENT In the olfactory bulb (OB), odors are uniquely coded in a spatial map that represents odor identity, making the OB a unique model system for investigating how learned fear alters sensory processing. Classical fear conditioning causes fear of the conditioned stimulus (CS) and of neutral stimuli, known as generalization. Combining fear conditioning with fluorescent calcium imaging of OB glomeruli, we found enhanced glomerular responses of the CS as well as neutral stimuli in awake mice, which mirrors fear generalization. We report that CS and neutral stimuli enhancements are, respectively, learning-independent and learning-dependent. Together, these results reveal distinct mechanisms leading to enhanced OB processing of fear-inducing stimuli and provide important implications for altered sensory processing in fear generalization.

Keywords: associative learning; calcium imaging; fear conditioning; generalization; olfactory bulb; sensory processing.

Figure 1.

Olfactory aversive conditioning results in robust olfactory fear and generalization. A, Schematic detailing time course of experiments, the odors used (top), and paradigms for both imaging (above dotted line) and behavioral (below dotted line) experiments for each group. B–D, Twenty-four hours after training, mice were exposed to each of the five odors (E5, E4, E6, BZ, and 2H) and freezing was measured. Odor-only mice (B) and Shock-only mice (C) did not learn to fear E5, as freezing was not significantly different from baseline (BL), and did not generalize freezing from E5 to other odors. Paired mice (D) froze significantly more to E5 than baseline, indicating acquired fear to the CS. Mice generalized fear across all other tested odors (freezing to other odors not significantly different from freezing to E5). Mice freeze specifically to odor cues (E). Data presented mean ± SEM. *p < 0.001.

Figure 2.

Olfactory aversive conditioning enhances glomerular responses. A–C, Resting light-intensity frames and pseudocolored averaged Pre2 and Post glomerular response maps from representative Odor-only (A), Shock-only (B), and Paired (C) mice where the pseudocolor scale is based on the day with the maximum observed responses (Pre2 for Odor only and Shock only and Post for Paired) to avoid oversaturation of pseudocolored maps. The approximate value of the maximum observed responses (ΔF/F) used for pseudocolor scale is listed to the right of each odor. While scaling color in this manner makes Pre2 responses appear significantly weaker in the Paired group, amplitude of Pre2 responses are similar across mice for each odor. The maximum observed responses are also statistically similar across groups (see Materials and Methods).

Figure 3.

Post-training enhancements are independent of glomerular response amplitude. A, Example traces from representative glomerulus indicating Pre1 trial-to-trial variability in amplitude of response and extracted respiratory signal. Gray box illustrates five frames around the initial peak response used for analysis. B–D, Histograms of normalized Pre1 responses illustrating range and frequency of glomerular responses for Odor-only (B), Shock-only (C), and Paired (D) mice. Responses on Pre1 exhibit unimodal distributions with an average normalized response of ∼0.8 for all groups. E, Scatterplot showing raw Pre1 (x-axis) corresponding raw Pre2 (y-axis) values for each recorded glomerulus from all Odor-only (blue), Shock-only (purple), and Paired (red) mice. Raw normalized responses display similar amplitudes and experience-dependent decreases across groups. Solid black line represents theoretical “no change” line. F, Scatterplot showing raw Pre2 (x-axis) and corresponding raw Post (y-axis) values for each recorded glomerulus from all Odor-only (blue), Shock-only (purple), and Paired (red) mice. Scatterplots of normalized responses demonstrate similar changes to those of raw responses but allow for pooling across subjects. Normalized glomerular responses generally decrease from Pre1 to Pre2 for all groups, as evidenced by the majority of points falling below the no change line (E′, E″, E‴). Normalized glomerular responses also generally decrease from Pre2 to Post for Odor-only (F′) and Shock-only (F″) mice, whereas almost all glomerular responses increase from Pre2 to Post for Paired mice (F‴), as evidenced by points falling above no change line. Importantly, almost all raw glomerular responses for Paired mice after training also fall above the no change line (F, red). The post-training increase of raw glomerular responses appears linear, given the fit line is parallel to the no change line, indicating glomerular response enhancement is independent of glomerular response amplitude.

Figure 4.

Mean instantaneous frequency (MIF) is stable. Extracted MIF from fluorescent trials (such as Fig. 3A) falls in line with previously published data for awake, head-fixed mice and ranges from ∼2–6 Hz. Pre2 MIF is similar before and after odor onset for all Odor-only (A), Shock-only (D), and Paired (G) trials. Post-MIF is stable before and after odor onset for all Odor-only (B), Shock-only (E), and Paired (H) trials. Thick black lines represent calculated average MIF, whereas colored lines represent every trial colored by odor. Odor-evoked MIF (after odor onset) is comparable on Pre2 and Post for Odor-only (C), Shock-only (F), and Paired (I) mice. Importantly, MIF is not significantly higher or lower for any particular odor.

Figure 5.

Enhanced responses are global, not odor- or glomerulus-specific. A–C, Normalized glomerular responses over time for each imaged odor. Responses for all odors continually decrease over time for Odor-only (A) and Shock-only mice (B). Glomerular responses to all odors of Paired mice (C) decrease before learning (from Pre1 to Pre2) followed by robust reinstatement of responses after learning (Post). The same learning-induced enhancement in Paired mice occurs in E5 Responsive (D) and Non-E5 Responsive (E) glomeruli, indicating glomerular overlap does not play a significant role in learning-induced alterations (F). G, H, Scatterplots showing the average correlation of spatial glomerular activation patterns between E5 and each of the neutral odors before (Pre2, x-axis) and after (Post, y-axis) for all Odor-only (G), Shock-only (H), and Paired (I) mice. Solid black line represents theoretical “no change” line. Activation patterns of E5 and neutral odors decorrelate after training in both Odor-only (G) and Shock-only (H) mice but become more correlated after training in Paired mice (I). Data presented mean ± SEM. *p < 0.001 from Pre1, ∧p < 0.001 from Pre2.

Figure 6.

Olfactory fear learning induces long-lasting behavioral fear and glomerular enhancements. A, Schematic detailing time course of experiments, the odors used (top), and paradigms for both imaging (above dotted line) and behavioral (below dotted line) experiments. B, Seventy-two hours after training, mice were exposed to each of the five odors and freezing was measured. Mice froze significantly more to E5 than baseline, indicating acquired fear to the CS. Mice generalized fear across all other tested odors (freezing to other odors not significantly different from freezing to E5). *p < 0.05. C, Glomerular responses to all odors significantly decrease from Pre1 to Pre2; however, even 72 h after training, responses are significantly greater after learning (Post) than before (Pre2). Data presented mean ± SEM. *p < 0.001 from Pre1, ∧p < 0.001 from Pre2.

Figure 7.

Learning-induced glomerular changes are variable in anesthetized mice. A, Schematic detailing time course of experiments, the odors used (top), and paradigms for both imaging (above dotted line) and behavioral (below dotted line) experiments. In this experiment mice are anesthetized (ANES) for all imaging sessions but awake during behavioral assays. B, Twenty-four hours after training, mice froze significantly more to E5 than baseline, indicating acquired fear and generalized fear across all other tested odors (freezing to other odors not significantly different from freezing to E5). *p < 0.05. C, Glomerular responses of anesthetized mice decreased from Pre1 to Pre2. After training (Post), only averaged responses of E5, E4, and 2H were significantly enhanced (relative to Pre2), whereas responses to E6 and BZ were not significantly different. D, E, Scatterplots showing normalized Pre1 (x-axis)/Pre2 (y-axis) responses (D) or Pre2 (x-axis)/Post (y-axis) responses (E) for each recorded glomerulus from anesthetized mice. Solid black line represents theoretical “no change” line. Glomerular responses generally decrease from Pre1 to Pre2, as evidenced by the majority of the points falling below the no change line (D). On average, responses slightly increase from Pre2 to Post (E); however, of the 292 glomeruli analyzed in the anesthetized mice, only 59.9% were enhanced after training, whereas 40.1% were suppressed. Data presented mean ± SEM. *p < 0.001 from Pre1, ∧p < 0.001 from Pre2.

Figure 8.

The expression of learning-induced glomerular response enhancements is independent of general fear states. A, Schematic detailing time course of experiments, the odors used (top), and paradigms for both imaging (above dotted line) and behavioral (below dotted line) experiments. B, Tone-shock conditioned mice learn to fear the conditioned tone and freeze to it significantly more than baseline. *p < 0.005. C, Odor-evoked glomerular responses of mice conditioned to fear a tone decreased from Pre1 to Pre2 and again from Pre2 to Post1. Importantly, there was no significant difference between odor-evoked glomerular responses during Pre1, when awake mice were imaged normally, and Post2, when we experimentally induced fear by preceding each odor imaging trial with a presentation of the fear-inducing tone. Data presented mean ± SEM. *p < 0.001 from Pre1, ∧p < 0.001 from Pre2.

Figure 9.

Generalized, but not CS-specific, glomerular enhancements are associative learning-dependent. A, Schematic detailing time course of experiments, the odors used (top), and paradigms for both imaging (above dotted line) and behavioral (below dotted line) experiments, including drug administration. B, C, Mice were exposed to all five odors 24 h after training and freezing was measured. In mice receiving VEH infusions before training, presentations of E5 elicited significantly more freezing than baseline, indicating they learned to fear the CS. Additionally, VEH mice generalized fear to all odors, except BZ. In contrast, mice receiving MUSC infusions before training did not freeze significantly more to presentations of E5 than baseline, indicating that they did not learn. *p < 0.001. D, E, Resting light-intensity frames and pseudocolored averaged Pre2 and Post maps from representative VEH (D) and MUSC (E) mice, where the pseudocolor scale is based on the day with the maximum observed responses (Post for Vehicle, Pre2 for Muscimol for all odors except E5) to avoid oversaturation of pseudocolored maps. Although scaling color in this manner makes Pre2 responses appear significantly weaker in the VEH group, amplitude of Pre2 responses are similar across mice for each odor. The approximate value of the maximum observed responses (ΔF/F) used for pseudocolor scale is listed to the right of each odor. F, G, Normalized glomerular responses over time for each imaged odor for all VEH (F) and MUSC (E) mice. Responses for all odors in both groups significantly decrease from Pre1 to Pre2. Responses to all odors are significantly increased after training in VEH mice. In contrast, only responses to presentations of E5 are enhanced in MUSC mice; all other odor responses are continually suppressed. In MUSC mice, the same post-training suppression of nonconditioned odors occurs regardless of whether glomeruli are E5 Responsive (H) or Non-E5 Responsive (I). *p < 0.001 from Pre1, ∧p < 0.001 from Pre2. J, Glomerular overlap does not affect the percentage change of glomerular responses (Pre2 to Post) in VEH (top; with the exception of BZ) or MUSC (bottom) mice, indicating glomerular changes are odor- rather than glomerulus-specific (J). Data presented mean ± SEM. *p < 0.05.

Figure 10.

Amygdala inactivation during expression of learning does not impact glomerular responses. A, Schematic detailing time course of experiments, the odors used (top), and paradigms for both imaging (above dotted line) and behavioral (below dotted line) experiments, including drug administration. B, Confirmation of BLA cannula placement and evaluation of possible MUSC spread. PCx, Piriform cortex; CoA, cortical amygdala; MeA, medial amygdala; ac, amygdalar capsule; ec, external capsule. C, D, Freezing to all five odors was measured 24 h after training and confirms that both groups of mice froze significantly more to the CS than baseline and generalized that fear to all odors except BZ. *p < 0.001. E, F, Averaged glomerular responses for both groups decreased significantly from Pre1 and Pre2, but were reinstated after learning (Post1). Infusions of either VEH or MUSC occurred between Post1 and Post2. Following infusions, both groups exhibited mixed-profile responses, with some odor responses increasing, others decreasing, and some not changing significantly. Additional analysis quantifying the average percentage change from Post1 to Post2 (G) indicates no significant difference between groups, indicating BLA inactivation does not alter glomerular responses in a meaningful way. *p < 0.001 from Pre1, ∧p < 0.001 from Pre2, #p < 0.001 from Post1.