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Published in final edited form as: Neurobiol Learn Mem. 2023 Sep 25;205:107832. doi: 10.1016/j.nlm.2023.107832

Ventral hippocampal projections to infralimbic cortex and basolateral amygdala are differentially activated by contextual fear and extinction recall

Emma T Brockway 1, Sarah Simon 1, Michael R Drew 1,*
PMCID: PMC10919432  NIHMSID: NIHMS1964789  PMID: 37757953

Abstract

Fear and extinction learning are thought to generate distinct and competing memory representations in the hippocampus. How these memory representations modulate the expression of appropriate behavioral responses remains unclear. To investigate this question, we used cholera toxin B subunit to retrolabel ventral hippocampal (vHPC) neurons projecting to the infralimbic cortex (IL) and basolateral amygdala (BLA) and then quantified c-Fos immediate early gene activity within these populations following expression of either contextual fear recall or contextual fear extinction recall. Fear recall was associated with increased c-Fos expression in vHPC projections to the BLA, whereas extinction recall was associated with increased activity in vHPC projections to IL. A control experiment was performed to confirm that the apparent shift in projection neuron activity was associated with extinction learning rather than mere context exposure. Overall, results indicate that hippocampal contextual fear and extinction memory representations differentially activate vHPC projections to IL and BLA. These findings suggest that hippocampal memory representations orchestrate appropriate behavioral responses through selective activation of projection pathways.

Keywords: Ventral hippocampus, fear, extinction, c-Fos, infralimbic cortex, basolateral cortex

1. Introduction

Fear extinction—repeated exposure to a feared stimulus in a safe environment—is a model of exposure therapy, which is a common and effective treatment for maladaptive fear in patients with PTSD or phobias (Vervliet et al., 2013). A limitation of extinction and exposure therapy is that these procedures involve new learning about the absence of threat, not unlearning of fear, and fear relapse is common (Bouton et al., 2006). How memories of fear and extinction learning interact to modulate expression of emotion is thus a key question with important clinical implications.

Recent studies have identified the hippocampus as a region in which fear and extinction memories may compete for expression. The hippocampal dentate gyrus (DG) contains so-called “fear engram cells,” which are neurons active during acquisition of contextual fear that reactivate during recall of contextual fear (Denny et al., 2014). Optogenetic stimulation of these fear engram cells induces the expression of fear, whereas silencing inhibits fear behavior (Liu et al., 2012; Ramirez et al., 2013). Recent work demonstrates that extinction learning can suppress reactivation of DG fear engram cells and activate a distinct ensemble of cells potentially representing an extinction memory (Lacagnina et al., 2019). Whereas artificial stimulation of the fear memory ensemble increases fear, stimulation of the extinction ensemble reduces fear (Lacagnina et al., 2019). These findings are consistent with the interpretation that fear and extinction memories have distinct representations in the hippocampus, and that competition between these representations determines whether fear is suppressed or recovers after extinction. However, little is known about how these distinct ensembles of DG granule cells activate different emotional and behavioral states.

It is believed that hippocampus modulates expression of fear and extinction via projections from CA1 and subiculum in ventral hippocampus (vHPC) to the basolateral amygdala (BLA) and prefrontal cortex (PFC). vHPC projections to the BLA are believed to provide contextual control of fear in both contextual and auditory cued fear conditioning (Davis, 1992; Herry et al., 2008; Orsini et al., 2011; Jin and Maren, 2015; Xu et al., 2016). The BLA is an essential locus for associative synaptic plasticity during fear learning. Silencing vHPC projections to BLA attenuates expression of contextual fear, suggesting that this pathway provides information about the contextual conditioned stimulus (CS) to the amygdala. In contrast, the infralimbic (IL) PFC is implicated in fear extinction learning. Inactivation of IL projections to the amygdala impairs both extinction learning and recall (Milad and Quirk, 2002; Bukalo et al., 2015; Bloodgood et al., 2018). vHPC projections to IL appear to enable contextual control of extinction. For instance, in auditory fear conditioning, vHPC-IL projections are necessary for context-specificity of fear extinction. vHPC projection influence on IL activity is uncertain. Some studies have shown that vHPC excites IL and reduces fear, while others suggest that vHPC projections potentiate fear through feed forward inhibition of IL (Peters et al., 2010; Rosas-Vidal et al., 2014; Marek et al., 2018a). How extinction of context fear influences vHPC activity in these pathways is unclear.

While it is clear that vHPC projections to IL and BLA can modulate expression of fear and extinction memory, it remains unclear how extinction training affects their activity. Evidence discussed above regarding competing fear and extinction engrams in DG raises the question whether extinction training orchestrates a corresponding shift in ensemble activity among vHPC projection populations. One possibility is that expression of contextual fear and extinction is mediated by the levels of activity in vHPC projections to BLA and IL, with vHPC-BLA projections favoring expression of contextual fear, and activity of vHPC-IL projections favoring suppression of fear after extinction. Alternatively, relative activity in these projections could be invariant across fear and extinction, perhaps because these forms of learning are mediated by changes in synaptic strength in the target regions.

In this study we investigated whether these vHPC projections are differentially activated during recall of context fear and extinction memories. We used retrograde tracing to label vHPC neurons projecting to the IL and BLA and then assessed c-Fos as a marker of activity within these populations of cells. We show that fear and extinction recall activate vHPC projections in opposing manners, with vHPC-BLA projections more active during fear expression and IL projections more active during expression of extinction. These results suggest a circuit mechanism through which hippocampal memory representations signal valence and orchestrate appropriate behavioral responses.

2. Methods

2.1. Animals:

Adult male and female C57BL6/J mice (n=55; Experiment 1: fear (4F, 6M), extinction (5F, 5M), control (3F, 4M); Experiment 2: fear (5F, 5M), extinction (3F, 4M), control (4F, 5M)) approximately 2 months of age were used for all experiments. Mice were housed with littermates in groups of 3–4 in plastic cages with woodchip and paper crinkle bedding and maintained on a 12hr light/dark cycle (7:00–19:00 light on) in a temperature- and humidity-controlled vivarium. Food and water were provided ad libitum. Experiments were conducted during the light phase. Mice were randomly assigned to groups by cage before the start of each experiment. As sex-by-genotype statistical interactions were not present, male and female data were aggregated. All procedures were approved by the University of Texas at Austin Institutional Animal Care and Use Committee.

2.2. Surgery:

2% isoflurane (1.0 L/min) vaporized in pure oxygen was used to induce anesthesia, followed by 1.5% isoflurane (0.75 L/min) to maintain stable anesthesia after mice were placed in a stereotaxic frame. Cholera Toxin Subunit b (Alexa Fluor 555 and 647 conjugates, Invitrogen) was infused bilaterally (69nL/injection site) using a NanoJect II microinjector (Drummond) targeting the IL (M/L 0.3, A/P +1.8, D/V −2.9) and BLA (M/L 3.45, A/P +−1.65, D/V −4.9), counterbalanced among. Mice were injected subcutaneously with carprofen (10mg/kg), buprenorphine (0.1mg/kg) and buprenorphine XR (3.25mg/kg) in sterile saline (0.9%) to provide analgesia. Subjects were given ~7 days to recover and allow for CTb expression.

2.3. Context fear conditioning and extinction:

Mice were handled for 2 min per day for 4–5 days prior to behavioral testing. Mice were transported from the vivarium to a holding room adjacent to the test room at least one hour before experimentation. Mice were transported individually to and from the conditioning room in an opaque container. The transport containers were cleaned with a 70% EtOH solution between uses.

Behavioral testing occurred in 30.5 × 24 × 21 cm conditioning chambers (Med Associates), with two aluminum side walls, a Plexiglas door and ceiling, and a white vinyl back wall. Chambers were contained within a larger, sound-attenuating chamber. An overhead white light illuminated the chamber continuously throughout the procedures. The conditioning chamber contained a straight stainless-steel rod floor (36 rods, spaced 8mm from center to center), was cleaned with a 70% EtOH solution between uses and was scented with 1% acetic acid solution in the waste tray below the floor. Contextual fear conditioning consisted of three 2-sec 0.75mA scrambled foot shocks delivered through the rod floor 160, 240, and 310 seconds after mice were placed in the chamber. Mice were removed 30s after the final foot shock and returned to their home cage.

All behavioral sessions were video recorded at 30 frames/s using a near-infrared camera mounted to the interior door of the chamber. Freezing was defined as the absence of movement, except for those related to breathing. Videos were automatically scored using a linear pixel change algorithm (VideoFreeze; Med Associates). For the fear conditioning session, the percent of time spent freezing was calculated for the 160-sec pre-shock baseline and the 30 sec following each shock. For the extinction and retrieval tests, percent freezing was averaged across the entire 5-min session.

Extinction sessions consisted of 5-min exposures to the original fear conditioning context once per day for 9 days. Subjects in the fear retrieval group stayed in home cages in a holding room during this time. Both extinction and fear retrieval groups were returned to the conditioning chamber for a 5-min retrieval session on the final test day. The control group of mice stayed in their home cage in the holding room during this time.

For context pre-exposure mice were placed in the conditioning chamber for 5-minute exposure sessions once per day for 9 days, so that mice received the same amount of context exposure as mice receiving extinction training. Mice in the pre-exposure group were fear conditioned the day following the final pre-exposure and received a retrieval test a day later.

2.4. Immunohistochemistry:

Ninety minutes after the retrieval test, mice were deeply anesthetized with ketamine/xylazine (150/15 mg/kg) and transcardially perfused with 0.01M phosphate buffered saline (1x PBS), followed by 4% paraformaldehyde (PFA) in 1× PBS. Brains were extracted and post-fixed overnight at 4°C in 4% PFA and then transferred to a 30% sucrose in 1× PBS at 4°C for two days. 35 μm coronal sections were collected on a cryostat. For immunohistochemistry, sections were washed in 1× PBS and blocked at room temperature (RT) for 1.5 h in 5% normal donkey serum (NDS) in 1× PBS with 0.5% Triton-X (PBS-T). Sections were incubated with primary antibodies (1:2,000 rabbit anti-c-Fos polyclonal antibody, Synaptic Systems #226–003) diluted in 5% NDS in PBS-T overnight at room temperature. Sections were rinsed in 1× PBS-T and incubated in secondary antibodies (1:500 Alexa Fluor 488-conjugated AffiniPure Donkey Anti-rabbit IgG, Jackson ImmunoResearch Laboratories) in 1× PBS-T for 2 h at RT. Sections were washed in 1× PBS, mounted onto slides, and stained with 1:1000 DAPI for 5 minutes before being coverslipped with Fluoromount-G (SouthernBiotech).

2.5. Imaging and quantification:

4–6 z-stacks per animal were taken of the ventral hippocampus on a Zeiss Axio Imager.M2 microscope with Apotome.2 and StereoInvestigator 64 software using an ECPlan-Neofluor 20x objective. Images were taken within −2.9 to −3.8 mm bregma, spanning a range of ventral CA1 up to the distal CA1/subiculum border. Fluorescent channels were separated in ImageJ to manually count CTb-labeled neurons, using the multi-point tool to mark and save pixel coordinates as region of interest (ROI) sets to compare between channels. A cell was considered positive for CTb labeling if a clear halo of fluorescent labeling could be identified surrounding the soma location as defined by DAPI staining. A cell was considered positive for c-Fos labeling if the fluorescence level in the soma was 2x background levels of fluorescence. Fluorescence was measured in ImageJ as the mean fluorescent intensity within a selected region of interest drawn around the soma, and the background was considered the mean fluorescent intensity of a region of surrounding tissue of uniform intensity and no visibly labeled cells. Cell counts were averaged for each subject and are displayed as means ±SEM.

2.6. Statistical analysis:

Data were analyzed using two-sided t-tests or ANOVA, using repeated measures when appropriate. Significant ANOVAs were followed by post hoc Holm-Sidak’s test for multiple comparisons. Data analysis was performed on Prism (GraphPad Software) or JMP (SAS Institute). The α value was set at 0.05 for all analyses. All data are presented as mean ±SEM.

3. Results

3.1. Fear and Extinction retrieval activate different ventral hippocampal projections

To label hippocampal projection neurons, male and female C57BL6/J mice were injected with the retrograde tracer Cholera Toxin subunit B (conjugated to Alexa Fluor 566 and 648), with injections targeted to IL and BLA, counterbalanced by fluorophore across mice (Fig. 1A–B). A week later, mice were divided into three groups: fear, extinction, and home cage control. Mice in all groups were context fear conditioned, with no differences in freezing between groups (Fig. 1C; two-way RM ANOVA; no interaction effect, F(6,72) = 0.099, p = 0.996; effect of time, F(3,72) = 40.510, p <0.001; no effect of group, F(2,24) = 0.258, p = 0.775). Mice in the Extinction condition then received 9 days of extinction training, followed by a test for recall of extinction on the final day. Mice in the Fear group did not receive extinction and were tested for recall of fear on the final test day. Mice in the home cage control group did not receive any further testing. Freezing to the conditioning context decreased across days for the extinction group (One-way RM ANOVA, F(8,72) = 4.341, p < 0.001), and freezing was significantly higher during retrieval testing for the fear group than the extinction group (t(19) = 2.942, p = 0.008). Mice were perfused 90 min after the final session (or taken from their home cage for the control), and immunohistochemistry against c-Fos was used to assess activity within vHPC neurons. We predicted that mice in the Fear group would display more c-Fos activity in BLA-projecting vHPC neurons than in IL-projecting neurons, whereas Extinction mice would exhibit the opposite pattern.

Figure 1: Labeling of ventral hippocampal projections in CFC and Extinction.

Figure 1:

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(A) Experimental design and timeline. (B) Representative images of CTb injections in IL and BLA, with cell bodies labeled in vHPC. (C) Freezing behavior during CFC, Extinction, and Retrieval sessions. The left panel depicts freezing during the pre-shock period and post-shock intervals of the conditioning session. The middle and right panels display mean freezing during each 5-min extinction or retrieval session. During extinction, freezing decreased across sessions. In the retrieval test, the fear group exhibited higher freezing than the extinction group. (D) Representative images of c-Fos-labeled vHPC neurons and overlap with CTB as imaged for quantification.

The overall density of c-Fos-positive cells did not differ among the extinction, fear, and home cage groups, although the home cage level was numerically lower and the ANOVA bordered on significance (Fig. 2A; ANOVA, F(2,24) = 3.248, p = 0.056). Activity within specific projection populations was measured as the percentage of CTb-positive neurons that co-expressed c-Fos (Fig. 1D). A repeated-measures ANOVA revealed a significant Projection X Behavior Group interaction (Fig. 2B; F(2,24) = 12.33, p < 0.001). Extinction mice displayed more c-Fos expression in IL-projecting neurons compared to BLA-projecting neurons, whereas Fear mice had more c-Fos expression in BLA projections compared to IL projections (Holm-Sidak’s test, Ext: p = 0.004; Fear: p = 0.007). We additionally computed a within-subjects measure comparing the relative levels of IL and BLA projection activity, which was the percentage of IL-projecting cells that are positive for c-Fos divided by the overall percentage of IL- and BLA-projecting cells that were c-Fos positive (Fig. 2C). This metric confirmed that fear and extinction recall activate these vHPC projection pathways with different strengths: there was higher c-Fos expression in BLA projections compared to IL projections during fear retrieval, whereas this balance was shifted towards more c-Fos expression in IL-projecting neurons during extinction retrieval (t(18) = 3.815, p = 0.001).

Figure 2: Fear and Extinction retrieval activate different ventral hippocampal projections.

Figure 2:

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(A) Fear and Extinction retrieval groups exhibited similar overall c-Fos density in the ventral hippocampus, and c-Fos density trended lower in the home cage control group. (B) Percentage of IL-projecting and BLA-projecting CTb-labeled neurons that were positive for c-Fos. (C) A projection activity ratio was calculated for each subject (each bar represents one mouse). The ratio was calculated as the percentage of IL-CTb labeled cells positive for c-Fos as a function of the percentage of all CTb labeled cells positive for c-Fos.

3.2. Effect of context exposure on projection activity

Although our results are consistent with the idea that fear and extinction differentially recruit hippocampal output pathways, there is the alternative possibility that ventral hippocampal activity is shaped by the amount of context exposure rather than by extinction per se. In the prior experiment, the extinction group received 9 more days of exposure to the context than the fear recall group, raising the possibility that context exposure, rather than extinction, shifted the balance of vHPC projection activity. To control for the amount of context exposure between groups, we conducted a second behavioral experiment in which the fear and extinction groups received the same total amount of context exposure. As before, CTb was used to label vHPC projections to IL and BLA. One group of mice (pre-exposure group, Fig. 3) then received 9 sessions of pre-exposure to the context prior to context fear conditioning, followed by a fear retrieval session. The extinction group was treated as before: mice were context fear conditioned (without pre-exposure), then given 9 days of extinction and an extinction retrieval test. Home cage control mice were context fear conditioned and later taken straight from their home cage for perfusion. In this design, mice in the fear and extinction recall groups had the same amount of exposure to the conditioning context.

Figure 3: Fear conditioning and extinction in groups with equivalent context exposure.

Figure 3:

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(A) Experimental design and timeline. (B) Freezing during context preexposure, conditioning, extinction and recall. The fear recall group exhibited significantly higher freezing than the extinction group during retrieval.

The pre-exposed group of mice exhibited low levels of freezing during pre-exposure sessions. All mice showed evidence of fear acquisition during conditioning, with freezing increasing across the 3 footshock presentations and no differences between groups (Fig. 3b; two-way RM ANOVA; no interaction effect, F(6,69) = 0.478, p = 0.823; effect of time, F(3,69) = 44.63, p <0.001; no effect of group, F(2,23) = 1.350, p = 0.279). Freezing decreased across trials during extinction for the extinction group of mice (One-way RM ANOVA, F(8,48) = 8.613, p < 0.001). In the retrieval test, the fear group of mice froze significantly more than the mice that received extinction training (t(15) = 4.422, p < 0.001). Mice were perfused 90 min after the retrieval session (or taken from their home cage for the control) and immunohistochemistry against c-Fos was used to assess activity within vHPC neurons as before.

The extinction and pre-exposure groups showed similar densities of c-Fos labeling in the vHPC; c-Fos density showed comparatively lower activity in the home cage control group (Fig. 4a; F(2,23)=10.710, p < 0.001; Holm-Sidak’s, Ext vs Fear p = 0.358, Ext vs HC p = 0.001, Fear vs HC p = 0.003). Like the previous experiment, the extinction group displayed more c-Fos expression in IL-projecting neurons compared to BLA-projecting neurons (Fig. 4b). In contrast, the pre-exposed group exhibited similar levels of c-Fos expression in IL and BLA projections during fear retrieval. A two-way repeated measures ANOVA revealed a significant Projection X Behavior Group interaction (F(2,23) = 4.699, p = 0.019). A Holm-Sidak post hoc test confirmed that the extinction group had higher activity in the IL-projecting than BLA-projecting neurons, whereas in the pre-exposure group activity was not significantly different between the two populations (Ext: p = 0.014, Pre-exp: p = 0.610). This pattern was also observed in the activity ratio (Fig. 4c), where the extinction group ratios skew higher, closer to the IL end of the scale, whereas the ratios for the pre-exposure group are centered on 0.5, indicating comparable percentages of c-Fos activation within vHPC-IL and vHPC-BLA projections (Effect of Behavior Group: t(15) = 2.576, p = 0.021). These results replicate our previous finding that extinction recall is associated with proportionally more activation of vHPC cells projecting to IL than in those projecting to BLA. Differing from the prior experiment, the fear recall group that was pre-exposed to the context had comparable amounts of c-Fos activation in both projection populations.

Figure 4: Fear and extinction recall activate different vHPC projections even with cumulative context exposure is equated.

Figure 4:

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(A) Overall c-Fos density in vHPC. Density of Extinction and Fear groups exceeded that of the Home Cage group. (B) Percentage of IL-projecting and BLA-projecting CTB+ neurons expressing c-Fos. The extinction group showed higher c-Fos expression in IL projections than BLA projections. In the fear retrieval and home cage groups, levels of c-Fos expression did not differ by projection. (C) Projection activity ratio for each subject.

Lastly, we looked for a relationship between freezing behavior and the relative activation of vHPC projections (Fig. 5). Combining data from both experiments (excluding home cage mice), we observed a significant inverse correlation between the projection activity ratio (as described above) and freezing behavior (r2 = 0.141, p = 0.023). Low freezing was associated with a high ratio, reflecting more activity in IL-projecting neurons, whereas high freezing was associated with relatively greater activity in BLA-projecting neurons.

Figure 5: Projection activity ratio correlates with freezing.

Figure 5:

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Linear regression of activity ratio plotted against percent of time spent freezing during the recall test for both experiments.

4. Discussion

We investigated the role of vHPC projections to BLA and IL in expression of fear and fear extinction memories. Previous research has shown that the hippocampus generates context fear and extinction memory representations that give rise to opposing behavioral responses to a shared context. The current study addresses the circuit mechanisms through which these hippocampal representations might influence behavior. Our results demonstrate that recall of context fear and extinction differentially activate ventral hippocampal projections to the amygdala and IL. Although both projections are active at both retrieval tests, the relative levels of activity shift. Fear recall was associated with more c-Fos expression in vHPC cells projecting to the BLA than in cell projecting to IL, whereas extinction recall was associated with more c-Fos activity in cells projecting to the IL than those projecting to BLA. This differential activation may provide a mechanism for the hippocampus to influence downstream regions involved in fear processing by weighting system level-activity more towards fear activation in the amygdala or extinction-related processing in the IL.

Previous studies of hippocampal memory have shown that the hippocampus encodes emotional valence in addition to contextual components of episodic memory. Learning new valence associations in a stable environment, such as fear conditioning to a previously neutral environment or extinction of context fear, causes remapping in place cells as well as population-level changes in ensemble activity. Place fields of CA1 cells are typically stable over time in the same environment; however, when the valence of an environment changes because of fear conditioning or extinction, place fields remap, perhaps forming new representations of the same context (Moita, 2004; Wang et al., 2012, 2015). Activity-dependent tagging studies support this idea. Extinction learning suppresses reactivation of DG fear ensembles and activates a distinct population of neurons potentially representing the extinction memory (Lacagnina et al., 2019). These studies show that hippocampal coding of context memory is sensitive to changing valence within a stable context.

The current study suggests a mechanism through which putative fear and extinction representations in the hippocampus might influence behavior. The hippocampus may activate context fear expression through preferential activation of projections to BLA, which is consistent with other work investigating hippocampal-amygdala interactions. The BLA is necessary for acquisition and expression of fear conditioning for both contextual and tone conditioned stimuli (Davis, 1992; Fanselow and Ledoux, 1999). Hippocampal projections to the amygdala are believed to convey context memory representations for contextual fear conditioning and contextual regulation of cued fear (Herry et al., 2008; Orsini et al., 2011; Jin and Maren, 2015; Xu et al., 2016). Disrupting activity in this projection, through optogenetic inhibition or stimulation, impairs encoding and retrieval of contextual fear (Xu et al., 2016; Jimenez et al., 2018; Graham et al., 2021). As the current study focused on activity within the vHPC, we cannot determine whether the projections we assessed target excitatory or inhibitory neurons or their overall influence on BLA processing. However, our finding that context fear recall is associated with more c-Fos expression in ventral hippocampal projections to the amygdala, rather than IL, is consistent with the idea that preferential activation of the hippocampus-to-amygdala pathway initiates recall of contextual fear responses.

On the other hand, extinction recall was associated with relatively more c-Fos expression in neurons projecting to the IL, a region important for extinction learning and recall (Quirk et al., 2000; Milad and Quirk, 2002; Laurent and Westbrook, 2009; Bukalo et al., 2015; Do-Monte et al., 2015; Bloodgood et al., 2018). While the IL is well known for its role in extinction, the function of vHPC projections to this region is less clear. Activity in vHPC-IL projections can both increase and decrease fear expression, perhaps because the projections can directly excite IL principal cells and elicit feed-forward inhibition through interneurons (Gabbott et al., 2002; Hoover and Vertes, 2007; Liu and Carter, 2018). Marek et al. recently demonstrated that activity of this projection evokes feed-forward inhibition of IL principal neurons through parvalbumin-expressing interneurons. In addition, they showed that pharmacogenetic activation of vHPC-IL projections promotes cued-fear recall whereas inhibition diminishes fear renewal (Marek et al., 2018b), suggesting that activity in this pathway increases fear expression. However, there is also evidence that strengthening of vHPC projections to IL supports extinction learning. NMDA receptor currents at vHPC-IL synapses are reduced after fear conditioning, and extinction was shown to reverse this effect (Soler-Cedeño et al., 2019). Furthermore, brain derived neurotrophic factor (BDNF), of which release in IL is both necessary and sufficient for extinction, is elevated in vHPC following extinction. BDNF infused into the vHPC enhances IL firing rates, and BDNF levels in vHPC inputs to IL are reduced in rats that fail to extinguish fear (Peters et al., 2010; Rosas-Vidal et al., 2014). These findings suggest that vHPC-provided BDNF is necessary for extinction-induced plasticity in IL. Taken together, the vHPC appears able to bidirectionally modulate IL excitability and thus may exert fine control over extinction memory recall. In our experiments we observed higher activity in IL-projecting neurons in the extinction recall group, which fits with a model in which vHPC-IL activity promotes extinction, potentially by exciting IL principal neurons. However, IL projection activity was not absent in our fear recall condition, and we did not assess the cellular targets in IL of these projections.

Importantly, our studies demonstrate that the increase in vHP-IL projection activity during extinction recall was not caused by mere context exposure. Our second experiment included pre-exposure to the context prior to fear conditioning in the fear recall group to control for potential effects of context exposure. Even when the amount of context exposure was equivalent between the fear recall and extinction groups, the relative activity of vHPC-IL and vHPC-BLA projections differed between groups. The extinction group displayed more activation of vHPC-IL-projections than vHPC-BLA projections, whereas in the fear recall group the two projections were equally active. It is important to note that because the total amount of context exposure was equated, the time between conditioning and the retrieval test was shorter for the pre-exposure group. It is possible that fear conditioning only a day prior to test could influence the balance of projection activity—for instance, heightening vHPC-BLA activity or dampening vHPC-IL recruitment from pre-exposure. That the pre-exposed fear recall group did not show increased vHPC-BLA projection (in contrast with the non-pre-exposed group in the first experiment), suggests that pre-exposure to the context prior to conditioning suppresses recruitment of vHPC-BLA projections during later retrieval. An intriguing possibility is that vHPC-BLA projections are recruited most strongly when the training history endows a CS unambiguous valence, whereas ambiguous cues (such as those that have been conditioned and then extinguished, or pre-exposed and then conditioned) recruit more distributed circuit activity.

Alternately, outcomes in the pre-exposure group could reflect latent inhibition, wherein prior exposure to the conditioned stimulus (CS) weakens its associability during acquisition of the CS-shock association, leading to less learned fear (LeDoux, 2014; Miller et al., 2022). Whether latent inhibition actually occurred is unclear. Context pre-exposure had no effect on context acquisition and, during the recall test, mice in the pre-exposure group froze at levels comparable to mice that did not receive pre-exposure. However, such comparisons are to be interpreted with caution because they are made across experiments and across groups with different acquisition-to-test intervals. Thus, we cannot rule out an effect of latent inhibition in this experiment. Indeed, it is possible that the relatively equal activity in BLA- and IL-projecting cells in the pre-exposed group reflect a weaker fear association. It is further possible that context pre-exposure recruits mechanisms similar to those recruited by extinction. Manipulations to ventral hippocampus, BLA, and IL all influence latent inhibition and extinction, and both latent inhibition and extinction of tone-shock associations are context dependent (Miller et al., 2022). A prominent theory of latent inhibition is that competing context-US and context-noUS memories are retrieved during testing, with the CS-noUS association dominating (Miller et al., 1986; Bouton, 1993). This theory of latent inhibition resembles our view of extinction as establishing a memory that competes with the original fear association. Perhaps under experimental conditions that promote stronger latent inhibition, patterns of activity in vHP would more closely resemble those exhibited during extinction.

Thus far, we have interpreted the vHP projection activity as controlling expression of fear and extinction. It is equally possible, however, that vHP activity is associated with behavioral correlates of fear and extinction, rather than fear and extinction per se. An example is exploratory behavior, which tends to correlate inversely with freezing. It is possible that vHP-IL activity promotes exploration (or vice versa). If so, then similar patterns of vHP-IL activity should be observed with other behavioral manipulations that modulate exploration. For example, exposure to a novel context, which typically evokes exploration, might increase activity in vHP-IL projections. Our data illustrate that expression of fear and extinction are associated with shifting patterns of activity within vHP-IL and vHP-BLA projections, but further experimentation will be required to understand the causal significance of these shifts with respect to behavior and mental processing.

This study focused on vHPC projections to BLA and IL because of the abundant evidence linking these two target regions to conditioned fear acquisition and expression. However, it is likely that other hippocampal projections modulate these forms of learning and behavior. For instance, vHPC projections to the lateral septum are involved in exploratory behavior and may help differentiate extinction and exploration (Trent and Menard, 2010). Prelimbic cortex (PL) activity increases during freezing behavior to a conditioned auditory tone, and PL neurons projecting to BLA have increased c-Fos activity after fear renewal compared to extinction (Burgos-Robles et al., 2009; Orsini et al., 2011). vHPC projections have a direct role in this activity, as contralateral lesions to disrupt vHPC-PL connectivity eliminate fear renewal (Orsini et al., 2011). Additionally, the IL and PL mutually inhibit each other, with vHPC inputs preferentially driving the relevant corticocortical neurons (Liu and Carter, 2018). Disparity in IL and PL firing correlates with auditory fear expression, with high freezing animals having higher PL firing rates compared to IL rates (Giustino et al., 2016). This finding leads us to speculate that the relative activity in hippocampal projections to PL and IL shifts between fear and extinction, with extinction recall potentially activating a larger percentage of vHPC-IL projections and fear recall associated with more vHPC-PL projection activity. Ventral hippocampal projections can also both excite and inhibit IL and PL, allowing for further modulation of the balance of activity in these regions. Hippocampal projections to the nucleus accumbens (NAc) are also a potential target for future studies. The NAc is known for its role in reward processing, latent inhibition, and activity in vHPC-NAc projections is necessary for conditioned place preference (LeGates et al., 2019; Miller et al., 2022). It is conceivable that the reduction of fear during extinction is associated with increased activity in the vHPC-NAc pathways, coinciding with the re-emergence of appetitive behaviors such as foraging.

In conclusion, we have shown that pyramidal neurons in the ventral hippocampus that project to the IL and BLA exhibit different patterns of c-Fos activation during fear or extinction recall. Although these vHPC projections are active during both fear recall and extinction recall, extinction recall is associated with more c-Fos expression in vHPC cells projecting to IL compared to those projecting to BLA, whereas fear recall is associated with more c-Fos expression in vHPC-BLA projections. These results support the view that hippocampal representations for a context evolve in conjunction with changes in the context valence, and this evolution may provide a mechanism for orchestrating appropriate behavioral responses through selective activation of projection pathways.

Acknowledgements

E.T.B. was supported by NIH T32 MH106454-05. Research supported by NIH R01 MH102595 and NIH R01 MH117426 to M.R.D.

Footnotes

Declaration of Interests

The Authors declare no competing interests.

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