A novel role for the infralimbic cortex in conditioned fear responding

Abstract

Freezing during Pavlovian fear conditioning is the most commonly used indicator of learned fear in rodents. Although freezing and its neural underpinnings have been widely studied, the field has largely ignored other potential indicators of learned fear. Darting, an escape-like conditioned fear response, occurs more frequently in females and is reliably predicted by a distinct behavioral phenotype: heightened shock response and decreased freezing compared to rodents that do not engage in darting. The experiments in this study sought to expand the field’s understanding of conditioned fear by investigating the neural correlates of conditioned darting, focusing on the infralimbic cortex (IL) and longitudinal columns of the periaqueductal gray (PAG) due to their known roles in modulating defensive responses, including conditioned freezing. We find that fear conditioning elicits greater neural activity (measured by quantification of cFos expression) in the IL of females compared to males, but that IL activation corresponds to freezing only in Darters. We next used chemogenetic tools to excite or inhibit either the IL alone or IL-PAG projections prior to fear conditioning. IL manipulations in either direction reduced freezing in both males and females, while IL inhibition increased darting and shock response in females only, and the effects of circuit-specific manipulations were minimal. Together, our findings suggest a novel, role for the IL in driving defensive responses during fear conditioning that is not dependent on IL-PAG connectivity.

Introduction

The medial prefrontal cortex (mPFC) in rodents plays a key role in conditioned fear acquisition and extinction (Giustino and Maren 2015; Marek et al. 2013; Maren and Quirk 2004; Rozeske et al. 2015). Canonically, the dorsal regions, including the prelimbic cortex (PL), mediate the acquisition, consolidation, and recall of fear memory (Maren and Quirk 2004; Corcoran and Quirk 2007; Quirk and Mueller 2007), while the infralimbic cortex (IL) is primarily recruited during fear extinction to suppress conditioned freezing (Quirk and Mueller 2007; Laurent and Westbrook 2009). Two major limitations of this large body of work persist, however: 1) the predominant use of only male subjects (Beery and Zucker 2011; Kokras and Dalla 2014); and 2) a sole measure of learned fear: freezing.

In 2015, our lab identified an escape-like conditioned response called darting (Gruene et al. 2015). Darting is sex-biased (towards females) and not dependent on weight or the estrous cycle (Gruene et al. 2015; Colom-Lapetina et al. 2019; Mitchell et al. 2022). Darting is also accompanied by a behavioral phenotype: Darters—animals who dart as a conditioned response—show decreased freezing during fear conditioning compared to Non-darters (Gruene et al. 2015; Colom-Lapetina et al. 2019; Mitchell et al. 2022; Greiner et al. 2019) and a comparatively heightened velocity immediately following the shock (‘shock response’) (Gruene et al. 2015; Mitchell et al. 2022). Darters only need to exhibit one dart during fear conditioning in order to display this phenotype. Darting is only observed during fear conditioning, emerging as the fear conditioning trial progresses, and does not occur during extinction learning or extinction retention (Gruene et al. 2015; Mitchell et al. 2022). The emergence of darting throughout the trial indicates that conditioned darting is a function of learning which is unique to the presence of both the conditioned and unconditioned stimulus. Previous work has attempted to identify behavioral predictors of darting, such as increased acoustic startle, corticosterone levels, or heat sensitivity (Mitchell et al. 2024), but thus far no behavioral predictors have been identified. The neural correlates of darting are also unknown, but recent work has identified sex differences in mPFC activity following fear discrimination (Day and Stevenson 2020; Day et al. 2020), suggesting the mPFC might play a role in driving divergent conditioned fear responses in males and females.

Although the IL is not historically known to play a role in fear acquisition (Giustino and Maren 2015), activity in the IL is associated with decreased freezing during extinction recall (Maren and Quirk 2004; Milad and Quirk 2002; Quirk et al. 2000), and therefore may drive the reduced freezing we observe in Darters (Gruene et al. 2015; Colom-Lapetina et al. 2019; Mitchell et al. 2022). The IL communicates with regions involved in threat and fear responding, such as the dorsal and ventral columns of the periaqueductal gray (d and v PAG) (Bandler et al. 2000; Floyd et al. 2000; Keay and Bandler 2001; Vertes 2004; Gabbott et al. 2005). Heightened activity in the dPAG leads to an increase in escape responses to threatening stimuli (Lefler et al. 2020; Vianna et al. 2001; Vianna et al. 2003) and the vPAG is well established in its role in conditioned freezing (Vianna et al. 2001; Carrive et al. 1997; Tovote et al. 2016). The IL projects to the PAG on a rostral-caudal gradient, with the rostral IL (rIL) projecting to the vPAG and the caudal IL (cIL), projecting to the dPAG (Floyd et al. 2000). We have previously reported that the vPAG in both males and females showed increased cFos following fear conditioning when compared to non-fear conditioned controls (Mitchell et al. 2024). The IL is therefore well situated as a potential mediator of conditioned freezing vs. darting via projections targeting either vPAG or dPAG.

The purpose of this study was to investigate the role of the IL during Pavlovian fear conditioning in both females and males. We hypothesized that activity in the infralimbic cortex during Pavlovian Fear Conditioning would be a key mediator of conditioned darting via suppression of conditioned freezing, and that this modulation is driven through projections to downstream targets in the PAG. Using immunohistological staining and chemogenetics, we identified the IL as an inhibitor of conditioned freezing in females and males, while exclusively affecting conditioned darting and unconditioned responses in females.

Methods

Subjects

Adult (8–10 weeks) male (325-350 g) and female (225-250 g) Sprague Dawley rats (Charles River) were used in all experiments. Animals were same-sex, pair-housed at Northeastern University in a 12:12 light:dark cycle and had access to food and water ad libitum. Animals acclimated to the facility, undisturbed, for one week prior to surgery or testing. Testing was conducted during the light phase between the hours of 10 AM and 3 PM. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Northeastern University Institutional Animals Care and Use Committee.

Fear conditioning paradigm

Fear conditioning (FC) setup and protocol are described in previous work from our lab (Gruene et al. 2015; Colom-Lapetina et al. 2019; Mitchell et al. 2022; Mitchell et al. 2024; Laine et al. 2022; Vincelette et al. 2026). For Experiment 1, the conditioned stimulus (CS) was a 30-second tone co-terminating with a 0.5 second foot shock (unconditioned stimulus [US]) (1 mA, 0.3 mA, or no shock for a tone-only control condition). 1 mA, 0.3 mA and tone only groups were used in Experiment 1 to determine if IL activity differentiates between shock intensities and if this differs between Darters, Non-darters and males, and the tone only group was a control for the experience of receiving a shock. For experiments 2 and 3, the US was a 0.5 mA foot shock. We selected 0.5 mA because this was a middle range that we have previously shown to be effective in eliciting darting (Mitchell et al. 2022; Mitchell et al. 2024; Laine et al. 2022; Vincelette et al. 2026), and all other parameters were the same as Experiment 1. All experiments used 7 CS-US pairings with a mean intertrial interval of 3 minutes and total test duration of 30 minutes. Each session was restricted to a single sex. Experimenters identifying as male and female conducted these experiments.

Behavior tracking and behavioral data processing

Behavior tracking and processing was done as previously described, using our custom Python program, ScaredyRat (Mitchell et al. 2022; Mitchell et al. 2024; Laine et al. 2022). Animals were defined as Darters if they exhibited at least one dart in response to CS 3–7 and their velocity during the dart reached at least 20 cm/s, as identified by Ethovision and validated in previous work (Gruene et al. 2015; Mitchell et al. 2022).

Euthanasia & tissue preparation

For experiments requiring immunohistochemical staining for cFos, euthanasia and tissue storage were performed within ninety minutes of the termination of fear conditioning, as previously described (Mitchell et al. 2024). For experiments not requiring time-specific staining, animals were perfused within a week of completion of the behavioral paradigm.

For experiments 2 and 3, successful viral placement was verified using a 2× APO lens on a Keyence BZX710. If expression was not observed in the IL or was predominantly outside of the IL, the animal was excluded from analysis. Final N’s are stated in the Results for each experiment.

Fluorescent immunohistochemistry

For experiment 1 and validation in experiment 3, 50 μm mPFC sections were collected using a vibrating microtome (Leica VT 1000S). Three PFC sections (A/P + 3.72, +3, +2.52) from each animal were isolated. Immunohistochemistry procedures were identical to previous work from our lab (Mitchell et al. 2024), with the exception of secondary antibody use. Experiment 1 used a donkey anti-mouse secondary antibody 1:1000 (Alexa 647, Jackson Immuno product #715–605-150) and validation in experiment 3 used an Alexa 488 secondary antibody (Jackson Immuno product #715–545-151), so cFos expression would be co-labeled with mCherry+ neurons. Mounted slices were imaged on a Keyence BZX-710 at 10× magnification and 3.5 s exposure and stitched together to reconstruct the mPFC.

cFos quantification

For experiment 1, images were imported to ImageJ software and cFos+ cells were quantified using a customized cell counting macro (Mitchell et al. 2024). Counts were averaged across both hemispheres and across all three bregma regions. For validation in experiment 3, images were opened in Image J and color channels were adjusted to isolate specific wavelengths (red, green, and blue). Brightness and contrast were modified to enhance the visibility of positively labeled neurons and minimize background noise. Each channel was analyzed independently to count the neurons of interest (cFos+ neurons in GFP and mCherry+ neurons in TRITC). Double-labeled neurons were calculated by selecting red and green channels. Percentage of double labeled cells were calculated by dividing the number of double-labeled cells by the total number of virus+ cells. Experimenters for all steps were blind to animal group.

Stereotaxic surgery

All animals were experimentally naive and anesthetized with isoflurane before aseptic stereotaxic surgery began. Viruses were delivered at a rate of 50 nl/min and had a post-injection waiting time of at least 5 minutes before removing the needle. Injection volumes ranged from 200-400 nl, based on strength of viral expression as observed during pilot experiments. Stereotaxic coordinates differed between males and females because during pilot surgeries these coordinates were found to be most accurate in targeting similar regions across the sexes. Animals received an injection of 1.5 mg/kg of Meloxicam on the day of and 24 hours following surgery. Animals recovered for at least 4 weeks before behavior to allow for full viral expression.

For experiment 2, a total of 60 male and 64 female rats underwent surgery. A locally expressed mCherry (pAAV-hSyn-mCherry AAV8, Addgene), Gq (pAAV-hSyn-hM3D AAV8, Addgene), or Gi (pAAV-hSyn-hM4D AAV8, Addgene) virus was injected bilaterally into the IL (AP +3.0, ML +/− 0.3, DV -5.25 (female); AP +3.0, ML +/− 0.4, DV -5.35 (male)).

A total of 24 male and 20 female rats in the validation of experiment 3 and 115 male and 121 female rats in the cIL-dPAG and rIL-vPAG portion of experiment 3 underwent surgery. Using an intersectional DREADDs approach, a cre-dependent mCherry (pAAV-hSyn-DIO-mCherry, Addgene), Gq (pAAV-hSyn-DIO-hM3D(Gq)-mCherry, Addgene), or Gi (pAAV-hSyn-DIO-hM4D(Gi)-mCherry, Addgene) was injected into the cIL (AP +2.7, ML +/− 0.4, AP -5.2 (female), −5.3 (male)) or rIL (AP +3.4 (female), +3.6 (male), ML +/− 0.4 (female), 0.5 (male), DV -5.0 (female), −5.1 (male)). A retrograde transporting AAV carrying the gene for cre-recombinase (pAAV-EF1a-Cre, Addgene) was injected into either the dPAG (AP -6.8, ML +1.3, DV -5.0 (female), 5.1 (male), 16 degrees) or vPAG (experiment 4 only) (AP -7.5 (female), −7.4 (male), ML +/− 0.7, DV -6.0 (female), −6.2 (male)). This retrograde-cre virus does not carry a fluorophore.

Drug preparation

For all DREADDs experiments, Clozapine-N-Oxide (HelloBio HB149, CNO) was prepared 30–45 minutes prior to injection. Animals were weighed in the morning before behavior began. CNO was mixed with double distilled water at a concentration of 1 mg CNO:1 ml H₂O. Injections were made intraperitoneally (I.P.) 1 hour before the start of fear conditioning (30 minutes prior to the start of habituation). All animals received 3 mg/kg CNO except for Gq-expressing animals in Experiment 2, which received 0.5 mg/kg due to observation of seizure activity in this group at higher doses. A second manuscript regarding this observation is in preparation.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism software (version 10.2.3). Outliers were removed from a group if they were +/− two standard deviations away from a group’s mean and were calculated for each experimental group. A two-by-three between-subjects ANOVA with the variables of shock intensity (CS only, 0.3 mA and 1 mA) and sex (Female/Male), and a two-by-two between subjects ANOVA with the variable of shock intensity (0.3 mA, 1 mA) and Darter identity (Darter/Non-darter) were conducted on IL cFos levels. The CS only group was not included in the Darter/Non-darter analysis because there are no Darters in response to the tone alone. Bivariate Spearman’s correlations were conducted on average conditioned freezing to tones 5–7 (when darting emerges) and cFos expression in the infralimbic cortex. For experiments 2 and 3, differences in Darter percentages in groups were analyzed using a chi-squared test. Two-way mixed effect analysis of variances (ANOVAs) were conducted on the between-subjects factors of experimental group (mCherry, Gq, and Gi) and tone (1 through 7) and sphericity was not assumed. Number of darts, percent freezing to tones 5–7, and shock response were analyzed using a one-way ANOVA with the between-subject factor of experimental group (mCherry, Gq, and Gi). Because of the unequal sample sizes in Experiment 2, Brown-Forsythe ANOVA was used, so as not to assume equal variances among groups. For validation in experiment 3, percent double labeling in the IL was analyzed using Brown-Forsyth one-way ANOVA with the between subject factors of sex or Darter identity and shock intensity. Tukey’s, Dunnett’s, or Sidak’s tests of multiple comparisons were used when comparing means with every other mean (such as when comparing across manipulation groups), with a single control group, or for specific selections of means, respectively.

Results

Experiment 1

Infralimbic cortex activity varies according to sex and conditioned responding

Behavioral results from this experiment were published previously: Darters froze less than Non-darters at both shock intensities, and males and Non-darters froze more following a 1 mA shock than a 0.3 mA shock, while Darters did not (Mitchell et al. 2024). One male and one female were excluded from cFos analyses based on outlier parameters, for a total of 40 females (14 1 mA, 17 0.3 mA, 9 CS-only) and 36 males (13 1 mA, 13 0.3 mA, 10 CS-only) included in final analyses. Figure 1A shows experimental timeline and a representative image of IL cFos staining. Although cFos+ cells were averaged across bregma points, representative image is from A/P + 3.00. There was a significant main effect of shock intensity (F(2,68) = 13.79, P < .0001) and a significant sex x shock intensity interaction (F(2,68) = 3.86, P = .026) (Fig. 1B). Tukey’s multiple comparisons revealed that females in the 0.3 mA and 1 mA groups showed greater cFos than the CS-only group (P < .0001 and P = .0007, respectively). Males in the 1 mA group had significantly less cFos than the 0.3 mA group (P = .048), no difference from the CS only group (P = .99), and the 0.3 mA group showed a trend for higher cFos than the CS only group (P = .057). We next split the females by Darter identity, finding a significant group x shock intensity interaction (F(1,27) = 6.71, P = .015) (Fig. 1C). Sidak’s multiple comparisons test revealed that Non-darters had significantly less cFos expression in the IL after 1 mA vs. 0.3 mA shocks (P = .028), and Darters did not (P = .50), suggesting that the IL may be selectively involved in discriminating shock intensity in female Non-darters. Bivariate Spearman’s correlation showed a significant negative correlation in Darters, regardless of shock intensity (r(15) = −0.647, P = .009) (Fig. 1E), and no correlations between conditioned freezing to tones 5–7 and IL cFos in males (P = .49) and Non-darters (P = .71) (Fig. 1D and F), suggesting that heightened IL activity is related to the suppression of freezing in Darters.

Figure 1.

IL cFos expression following experiment 1. A. Experimental timeline, illustrations of coronal sections used for IHC and representative micrograph showing cFos staining in the IL. Scale bars are 500 and 100 μm for 4× and 20× image, respectively. B. IL cFos expression in females and males following fear conditioning. cFos expression increased in both females following 0.3 mA and 1 mA shock exposure when compared to tone-only control groups. In males, cFos expression decreased following 1 mA shock exposure compared to 0.3 mA exposure. C. IL cFos expression in female non-darters and darters following fear conditioning. In non-darters, cFos expression decreased following 1 mA shock. This decrease did not occur in darters. D-F. Correlation of freezing to the later tones and cFos expression in non-darters, darters, and males. In darters only, more cFos expression correlated with less conditioned freezing. (^*) P < .05, (^**) P < .01, (^***) P < .001, (^****) P < .0001. Error bars for all graphs represent standard error of the mean (SEM).

Experiment 2

Chemogenetic manipulation of the IL alters fear conditioning behavior

Based on our results from Experiment 1, we decided to target the infralimbic cortex using chemogenetics (DREADDs) to determine if manipulation of this region affected conditioned and unconditioned fear responses during a one-day Pavlovian fear conditioning paradigm. We hypothesized that increased IL activity would lead to a suppression of freezing and this suppression would allow for darting to emerge. Fifteen females and 16 males were excluded due to either seizure activity during fear conditioning (12 per sex, all Gq) or inaccurate viral placement (3 females, 4 males). A total of 49 female and 43 male rats were included in the analyses. Experimental design and representative micrograph are shown in Fig. 2 A,B. In females, the proportion of conditioned Darters increased significantly with inhibition of the IL compared to controls (χ²(1) = 6.88, P = .009), while there was no change in the number of Darters between excitation and control groups (χ²(1) = 1.309, P = .25) (Fig. 2C and D). To see if there was a within-Darter change in number of darts, we analyzed the total number of darts between groups (including only the Darters), and there was no difference in the total number of darts across groups (F(2,7) = 0.31, P = .74) (Fig. 2E). In females, there was a significant main effect of experimental group on conditioned freezing (F (2,46) = 3.99, P = .03). (Figure 2F), where both Gi and Gq animals froze less than those in the mCherry group. To the latter tones females in both the Gq (P = .0497) and Gi (P = .01) groups froze less than mCherry controls (F(2,34) = 4.28, P = .02) (Fig. 2G). In females, there was a significant main effect of experimental group on shock response (F (2,43) = 4.18, P = .02), though Dunnett’s T3 test of multiple comparisons only revealed a trend towards increased shock response in Gi females compared to controls (P = .11) (Fig. 2H). In males, there was no significant effect of either Gq or Gi on the proportion of Darters (χ²(2) = 2.66, P = .26) (Fig. 2 I,J), or total number of darts (F(2,7) = 1.03, P = .40) (Fig. 2K). There was a significant main effect of experimental group on conditioned freezing (F(2,40) = 5.71, P = .007). (Figure 2L), where both Gq and Gi animals froze less than those in the mCherry group. To the latter tones, males in both the Gq and Gi (P = .02) groups froze less than mCherry controls (F(2,36) = 5.54, P = .008) (Fig. 2M). There was no significant effect on shock response (F(2,36) = 1.37, P = .19) (Fig. 2N).

Figure 2.

DREADDs manipulation of the IL. A. Experimental timeline. B. Representative targeting of the IL. Brightness and contrast have been enhanced for easier viewing. Scale bar is 500 μm. C. Proportion of females darting in each experimental group. Inhibition increased the number of darters compared to controls. D. Percentage of females darting to each tone. In all groups, number of darts increased as the trail went on. E. Total number of darts throughout the fear conditioning trial in females. Groups did not differ in the total number of darts. F. Females’ percentage of time spent freezing at baseline (BL) and during tones for fear conditioning. Both inhibition and excitation led to a decrease in conditioned freezing. G. Females’ percent of time spent freezing to tones 5–7, averaged across tones. Both inhibition and excitation led to a decrease in conditioned freezing. H. Females’ shock response during fear conditioning averaged across all tones. Though not significant, inhibition increased shock response. I. Proportion of males darting in each experimental group. There was no difference between groups. J. Percentage of males darting to each tone. There was no difference between groups. K. Total number of darts throughout the fear conditioning trial in males. There was no difference between groups. L. Males’ percentage of time spent freezing at BL and during tones for fear conditioning. Both inhibition and excitation led to a decrease in conditioned freezing. M. Males’ percent of time spent freezing to tone 5–7, averaged across tones. Both inhibition and excitation led to a decrease in conditioned freezing. N. Males’ shock response during fear conditioning averaged across all tones. There was no difference across groups. (^*) P < .05, (^**) P < .01. Error bars for all graphs represent SEM.

Experiment 3

Because conditioned freezing was negatively correlated with IL activity in Darters, and manipulation of IL activity prior to fear conditioning altered conditioned responses, we next chose to explore a potential downstream target involved in conditioned fear responses, the PAG. Based on the roles of the dorsal and ventral PAGs in active threat responses and conditioned freezing, respectively (Lefler et al. 2020; Vianna et al. 2001; Watson et al. 2016; Deng et al. 2016), we hypothesized that heightened caudal IL-dorsal PAG activity would lead to an increase in conditioned darting and a decrease in conditioned freezing, and that heightened rostral IL – ventral PAG activity would lead to a decrease in conditioned darting and an increase in conditioned freezing.

The effects of intersectional DREADDs in females and males

Before beginning our intersectional experiments, we wanted to verify that our intersectional DREADDs manipulations led to the expected activation patterns (increased activity for Gq and decreased for Gi). We chose to verify DREADDs efficacy in the cIL-dPAG circuit by quantifying c-fos expression in AAV-infected neurons. Figure 3A shows the experimental timeline for this experiment. We used an intersectional DREADDs approach to manipulate the cIL-dPAG pathway prior to fear conditioning and then stained mPFC sections for cFos. Seven animals were excluded based on lack of viral expression (4 female mCherry, 1 female Gq, 1 female Gi, and one male mCherry), and a final N of 14 females and 17 males were included. Because a majority of mCherry females were excluded due to lack of viral expression, the overall N’s were too low for statistical comparisons to be split by sex, and females and males were combined for cFos analysis.

Figure 3.

Manipulation of the cIL-dPAG circuit with intersectional DREADDs. A. Experimental timeline for DREADDs experiment 3. B. Representative micrograph of mCherry/GFP (cFos) dPAG projecting cIL cells. Double-labeled cells are indicated with white arrows. Image is from a male animal in the Gq group. Scale bars are 500 and 100 μm for 4× and 20× image, respectively. Image exposure, brightness and contrast have been adjusted to make visible to the naked eye. C. Proportion of female darters. There were no differences between groups. D. Proportion of male darters. There were no differences between groups. E. Percent double-labeling in the IL. Males are represented by white symbols, female non-darters by gray symbols and female darters by black symbols. Gq increased double labeling compared to mCherry, and Gi decreased double labeling compared to Gq, but not mCherry. (^**) P < .01, (^***) P < .001. Error bars for all graphs represent SEM.

Figure 3B shows a representative micrograph of double-labeling for virus and cFos in the IL. Percentage of Darters per group are displayed in Fig. 3C (females) and 3D (males). Percent time spent freezing did not differ between groups in females (F(2,11) = 0.20, P = .82) or males (F(2,15) = 0.70, P = .51) (Supplementary Fig. 1). There was a significant main effect of experimental group on number of co-labelled cells (F(2,27) = 13.77, P < .0001) (Fig. 3E). Dunnett’s T3 multiple comparisons revealed that Gq animals had significantly more double labeling than mCherry (P = .002) and Gi (P = .0006) animals. Gi animals did not have less expression than mCherry (P = .97), but this appears to be driven predominantly by heightened activity in female Darters (Fig. 3E, right-most bar, black circles).

Chemogenetic manipulation of caudal IL-dorsal PAG circuits did not affect behavior during fear conditioning

We next expanded upon our verification data and manipulated the cIL-dPAG circuit prior to fear conditioning to probe if activity in this circuit during fear conditioning is a driver of conditioned darting. Experimental design and representative dPAG targeting and cIL viral expression for these experiments are shown in Fig. 4A–C. In the cIL-dPAG circuit, 27 females and 11 males were excluded due to lack of or inaccurate viral expression in the IL and one female was excluded because no shock was delivered during the trial. A total of 41 females and 45 males were included in final analyses. In females, there was no effect of cIL-dPAG circuit manipulation on conditioned darting (χ²(2) = 1.090, P = .58) (Fig. 4D and E). There was no difference in total number of darts across groups (F(2,14) = 0.31, P = .74) (Fig. 4F). There was no difference in conditioned freezing to all tones (F(2,38) = 0.16, P = .86), to the latter tones (F(2,38) = 0.17, P = .85) or shock response (F(2,38) = 0.39, P = .68) across groups (Fig. 4G-I). In males, there was no effect of circuit manipulation on conditioned darting (χ²(2) = 4.393, P = .11) or number of darts (t(3) = 2.45, P = .09) (Fig. 4J-L). There was no effect of circuit manipulation on conditioned freezing to all tones (F(2,42) = 1.55, P = .22), to the latter tones (F(2,42) = 0.21, P = .81) or on shock response (F(2,42) = 1.693, P = .20) (Fig. 4M-O). Despite the dPAG’s involvement in threat responding (Lefler et al. 2020), manipulation of the cIL-dPAG circuit did not change behavior during FC.

Figure 4.

Conditioned and unconditioned responses following manipulation of the cIL-dPAG circuit before fear conditioning. A. Experimental timeline for cIL-dPAG experiment. B. Representative targeting of the dPAG. Dashed white outline indicates needle placement. Brightness and contrast have been enhanced for easier viewing. Scale bars are 500 μm. C. Representative image of dPAG projecting IL neurons. Scale bars are 500 and 100 μm for 4× and 20× image, respectively. Brightness and contrast have been enhanced for easier viewing. D. Proportion of females darting in each experimental group. There were no differences between groups. E. Percentage of females darting to each tone. All groups had more darts to the later tones. F. Total number of darts throughout the fear conditioning trial in females. There were no differences between groups. G. Females’ percentage of time spent freezing at BL and during tones for fear conditioning. There were no differences between groups. H. Females’ percent of time spent freezing to tone 5–7, averaged across tones. There were no differences between groups. I. Females’ shock response during fear conditioning averaged across all tones. There were no differences between groups. J. Proportion of males darting in each experimental group. There were no differences between groups. K. Percentage of males darting to each tone. Animals darted more to the later tones. L. Total number of darts throughout the fear conditioning trial in males. There were no differences between groups. M. Males’ percentage of time spent freezing at BL and during tones for fear conditioning. There were no differences between groups. N. Males’ percent of time spent freezing to tone 5–7, averaged across tones. There were no differences between groups. O. Males’ shock response during fear conditioning averaged across all tones. There were no differences between groups. Error bars for all graphs represent SEM.

Inhibition of the rostral IL-ventral PAG trended towards behavioral changes in females only

We next manipulated the rIL-vPAG circuit because of the vPAG’s known role in conditioned freezing (Vianna et al. 2001; Deng et al. 2016; Brandão et al. 2008; De Oca et al. 1998) and to probe if activity in this circuit is determinative of conditioned response. Experimental design and representative vPAG targeting and rIL viral expression for these experiments are shown in Fig. 5A–C. A total of 14 females and 22 males were excluded from analysis due to lack of or inaccurate viral expression in the IL and one male was excluded because no shock was delivered during the trial, resulting in 40 females and 37 males in the final analyses. Surprisingly, there were very few Darters in our female cohort (Figs. 5D and E), although the proportion of Darters was not different between groups (χ²(2) = 1.153, P = .56). N’s were too low to run statistics on total numbers of darts (Fig. 5F). In females, there was no significant effect of DREADDs on conditioned freezing (F(2,36) = 2.71, P = .08; Fig. 5G), though the effect was trending where Gi animals froze less than Gq and mCherry. When the latter tones were compared, the was a significant difference in percent time spent freezing (F(2,36) = 4.24, P = .02), with Gi females freezing less than Gq (P = .032) and mCherry (P = .048) animals (Fig. 5H). There was also a trending effect of DREADDs on shock response during FC (F(2,37) = 2.51, P = .0950) (Fig. 5I). Gi animals trended towards higher shock responses than other groups. In males, we observed no DREADDs effect on proportion of Darters (χ²(2) = 2.242, P = .33) (Fig. 5J and K). N’s were too low run statistics on total numbers of darts (Fig. 5L). There was no effect of DREADD manipulation on conditioned freezing to all tones (F(2,34) = 0.57, P = .57) (Fig. 5M), conditioned freezing to latter tones (F(2,34) = 0.90, P = .42) (Fig. 5N) or shock response (F(2,34) = 0.681, P = .51) (Fig. 5O).

Figure 5.

Conditioned and unconditioned responses following manipulation of the rIL-vPAG circuit before fear conditioning. A. Experimental timeline for rIL-vPAG experiment. B. Representative targeting of the vPAG. Dashed black circles indicate needle placement. Brightness and contrast have been enhanced for easier viewing. Scale bars are 500 μm. C. Representative image of vPAG projecting IL neurons. Scale bars are 500 and 100 μm for 4× and 20× image, respectively. Brightness and contrast have been enhanced for easier viewing. D. Proportion of females darting in each experimental group. There were no differences between groups. E. Percentage of females darting to each tone. Animals darted more to the later tones. F. Total number of darts throughout the fear conditioning trial in females. There were no differences between groups. G. Females’ percentage of time spent freezing at BL and during tones for fear conditioning. There were no differences between groups. H. Females’ percent of time spent freezing to tone 5–7, averaged across tones. Inhibition decreased conditioned freezing compared to mCherry and Gq. I. Females’ shock response during fear conditioning averaged across all tones. Though not significant, inhibition increased shock response. J. Proportion of males darting in each experimental group. There were no differences between groups. K. Percentage of males darting to each tone. Animals darted more to the later tones. L. Total number of darts throughout the fear conditioning trial in males. There were no differences between groups. M. Males’ percentage of time spent freezing at BL and during tones for fear conditioning. There were no differences between groups. N. Males’ percent of time spent freezing to tone 5–7, averaged across tones. There were no differences between groups. O. Males’ shock response during fear conditioning averaged across all tones. There were no differences between groups. (^*) P < .05. Error bars for all graphs represent SEM.

Discussion

These studies investigated the role of the infralimbic cortex and infralimbic cortex-periaqueductal gray circuits in Pavlovian Fear Conditioning using a combination of chemogenetic and immunohistological approaches. In Non-darter females and males, we found that activity in the IL during FC is dependent on the intensity of the shock. Non-darters exhibited the highest levels of cFos expression following a 0.3 mA shock, and a decrease in activation following a 1 mA shock. This pattern was the same for males—an increase in cFos after 0.3 mA shock and then a decrease after 1 mA—but the changes between the two shock groups did not reach significance. A higher shock could lead to an increase in stress, which could lessen activity in the prefrontal cortex and allow other brain regions to control stress responding (Arnsten 2009; Salehi et al. 2010). Importantly, female Darters did not follow this pattern: cFos expression in the IL following FC remained elevated regardless of shock intensity. Female Darters also showed a negative correlation between cFos expression in the IL and conditioned freezing. This correlation did not exist in males or female Non-darters. Observing elevated IL cFos expression in Darters is perhaps unsurprising because of the IL’s known role in the suppression of freezing during extinction (Ng and Sangha 2023), and suggests that the IL might also play a role during fear conditioning for Darters only.

We next manipulated IL activity during FC to determine whether it could alter conditioned response types. Interestingly, both inhibition and excitation of the IL reduced conditioned freezing across sex. One possible explanation for this bidirectional effect is that freezing behavior follows an inverted-U relationship with IL activity, such that both hypoactivity and hyperactivity suppress conditioned freezing, consistent with other inverted-U models of prefrontal cortical function (Arnsten 2009; Arnsten 2015). Contrary to our hypothesis, excitation of the IL did not significantly increase the proportion of Darters relative to controls, whereas inhibition of the IL did selectively in females. It is possible that the heightened IL cFos seen in Darters in Experiment 1 was disproportionately representative of inhibitory interneurons, such as parvalbumin (Ho et al. 2025), though we did not stain for cell-type specificity. If Darters have a greater amount of excitation in interneurons, an overall inhibition of the IL via DREADDs could disproportionately excite interneurons, increasing overall activity in the IL and leading to a suppression of conditioned freezing and an increase in conditioned darting. The DREADDs viruses used in Experiment 2 were not cell type-specific and therefore could have infected both excitatory and inhibitory neurons within the IL, leading to the observed increase in Darters following inhibition. Future work will investigate how cell type-specific activity shapes darting, freezing, and other related behaviors. One caveat to this experiment is the low number of Gq males compared to mCherry and Gi males (8 vs. 18). This was an unfortunate consequence of having to exclude animals from analysis that experienced seizures. We acknowledge that comparing different sized groups risks skewed results due to unequal variances, and so to mitigate this risk we performed Brown-Forsythe one-way ANOVAs (on total number of darts, time spent freezing to tones 5–7, and shock response), which does not assume that all groups have equal variances, and we did not assume sphericity when performing two-way ANOVAs (percent time spent freezing across tones).

We next investigated a downstream target of the IL known for its involvement in fear and threat responding, the PAG. Based on the dPAG’s role in escape-like defensive responses (Lefler et al. 2020; Vianna et al. 2003) and the vPAG’s role in conditioned freezing (Vianna et al. 2001; Tovote et al. 2016; De Oca et al. 1998; Lowery-Gionta et al. 2018), we hypothesized that excitation of the cIL-dPAG circuit would lead to an increase in Darters and their associated phenotype (less freezing, higher shock response (Gruene et al. 2015; Mitchell et al. 2022)), while excitation of the rIL-vPAG would do the opposite. Surprisingly, manipulation of the cIL-dPAG circuit, regardless of excitation or inhibition, produced no effects in females or males. Although the dPAG plays a well-established role in escape-like responses to innate threats (Lefler et al. 2020; Reis et al. 2021; Reis et al. 2023), and even in conditioned freezing (Kim et al. 2013; Liu et al. 2022), it is possible that its role does not extend to learned, escape-like responses. In addition, the columns of the periaqueductal gray can have contrasting functions depending on the rostral/caudal location examined (Depaulis et al. 1992; Keay and Bandler 2015). It is possible that cIL projections to the dPAG target a different locus along this axis than the primary hub for conditioned darting or freezing and instead mediate another form of fear-related behavioral or cognitive outcome not examined here. It is intriguing to note that in our DREADD efficacy cohort, the female Darters exhibited heightened cIL-dPAG activity regardless of administration with excitatory or inhibitory DREADD. In experiment 3, we observed a decrease in freezing following both excitation and inhibition, which we hypothesize is due to a difference in cell-type activation in the IL between Darters and Non-darters. The heightened cFos expression of cIL-dPAG neurons following excitation and inhibition in female Darters could be an extension of this: Darters having a disproportionate number in interneurons infected and activated. This would appear as an increase in activation in the individual cells, even after infection with inhibitory DREADD. This heightened activity could be a crucial component of darting behavior, but the behavioral outcome might be regulated by a different brain region with connections to the IL and PAG, such as the amygdala (Vertes 2004; Price 2003). In addition, our N’s in the DREADDs verification portion of this study are too low to make meaningful statistical comparisons by sex and Darter identity and future studies will expand on these results and investigate the differences in DREADD infection and activation in Darters, Non-darters and males.

Inhibition of the rIL-vPAG circuit led to a significant decrease in freezing to the later tones in females, while there were no changes following excitation or following any manipulation in males. This decrease in freezing following inhibition was accompanied by a trending increase in shock response, suggesting that inhibition of the rIL-vPAG circuit reproduces two key aspects of the Darter phenotype (decreased freezing, increased shock response), but does not lead to an increase in conditioned darting as a behavior. In Experiment 2, we observed that broad inhibition of the IL decreased freezing and increased the number of Darters. The IL neurons which project to the vPAG could highlight a subset of IL neurons whose activity is dampened in order for darting to emerge, but that do not contribute to the increase in conditioned darting itself. In Darters, heightened shock response, decreased freezing, and conditioned darting might be the outcome of coordinated activation and inhibition of region-specific microcircuits and interconnected circuits throughout the brain. Brain regions known for their role in conditioned fear behavior, such as the lateral, central and basolateral amygdala (Ciocchi et al. 2010; Davis 1992; Gore et al. 2015; Janak and Tye 2015; Pitkänen et al. 1997) and the prelimbic cortex (Burgos-Robles et al. 2017; Salzman and Fusi 2010), should be investigated, because of their reciprocal projections with the IL (Marek et al. 2013; Rozeske et al. 2015; Vertes 2004; Janak and Tye 2015; Burgos-Robles et al. 2017) and projections to the PAG (Depaulis et al. 1992; Keay and Bandler 2015; Butler et al. 2011; Zhang et al. 2024).

Although our lack of behavioral change following activation of either circuit is not consistent with work investigating the role of these regions individually, it is consistent with work investigating IL-PAG circuits in other escape-like behaviors, such as active avoidance (Li et al. 2026). Previous studies have shown that stimulation of the dPAG provokes escape and running behavior (Lefler et al. 2020), vPAG activation leads to an increase in freezing (Vianna et al. 2001; Tovote et al. 2016; De Oca et al. 1998; Lowery-Gionta et al. 2018), and results from Experiment 1 and 2 in this study show that heightened IL activity leads to a decrease in freezing. But previous work examining the role of IL-PAG circuitry in defensive behaviors found that although animals exhibiting active avoidance show an increase in IL activity, they do not show an increase in activity in IL-PAG circuitry. In addition, the likelihood of showing an active avoidance strategy was not altered by optogenetic manipulation of the circuit (Li et al. 2026). The lack of behavioral change following excitation of IL-PAG circuits could be due to a disruption of disinhibitory microcircuits within each or either region. This could negate any behavioral changes observed when targeting each area individually, such as the decrease in freezing seen in females and males following IL excitation in experiment 2. Similar to experiments 1 and 2, we do not know the cell types that the IL projects to or targets in the PAG. Local circuit and cell-type specific manipulations in these regions would offer further insight into the role of the IL, PAG, and IL-PAG circuits in learned fear.

In our local manipulation study (Experiment 2), and in the rIL-vPAG circuit manipulations, there were low amounts of female Darters. This was a surprising result, given previous work by our lab and others (Gruene et al. 2015; Colom-Lapetina et al. 2019; Mitchell et al. 2022; Greiner et al. 2019; Mitchell et al. 2024; Laine et al. 2022; Borkar et al. 2020; Manzano Nieves et al. 2023). In Experiment 2, the number of female Darters in the Gi group (61%) was still greater than the average seen in other experiments (roughly 40% (Gruene et al. 2015; Colom-Lapetina et al. 2019; Mitchell et al. 2022; Mitchell et al. 2024)), lending support to the hypothesis that inhibition of the IL leads to an increase in conditioned darting. In the rIL-vPAG manipulations in experiment 3, the lack of Darters extends across groups, including the mCherry control group. We do not believe that the low number of Darters in the rIL-vPAG circuit is the result of CNO administration, because animals in the cIL-dPAG circuit cohorts all showed an expected proportion of Darters. We hypothesize that the lack of Darters in this circuit could be due to trauma occurring during surgeries, either because of the more rostral targeting of the IL (+3.4 compared to +3.0 or + 2.7), or because of the lack of an angle for the vPAG injections. The trauma of the needle through the lateral PAG (lPAG) could have affected the later emergence of conditioned darting in the current experiment. Indeed, previous work has identified the lateral PAG (directly dorsal to the vPAG) as having increased cFos expression in Darters compared to Non-darters (Mitchell et al. 2024), and activation of the lPAG around the same bregma region as used in this study (A/P – 7.5) produces escape or flight reactions in response to stress (Keay and Bandler 2001). Future studies investigating escape behaviors and the vPAG should consider the role of the lPAG, as well.

Different shock intensities were used in Experiments 1 (0.3 mA and 1 mA), and Experiments 2 and 3 (0.5 mA). We chose a large range in shock intensity for Experiment 1 in order to determine if the IL differentiates between shock intensities, and if this differs between Darters, Non-darters, and males, and because we previously found that increases in shock intensities reduces the number of Darters (Mitchell et al. 2022). The behavior data for the cFos results in Experiment 1 was published previously (Mitchell et al. 2024), and in that experiment and in an earlier experiment examining the effect of shock intensity on conditioned fear behaviors (Mitchell et al. 2022), we found comparatively similar levels of freezing following a 1 mA shock (roughly 40–60%) to our results in Experiments 2–3 following a 0.5 mA shock. The 0.3 mA shock is lower intensity and leads to less freezing (Mitchell et al. 2022; Mitchell et al. 2024), as it is less aversive. The results of Experiment 1 illustrate that the IL does not differentiate between shock intensities in Darters, whereas it does in Non-darters and males. We returned to a 0.5 mA shock for later experiments because previous work has extensively shown that 0.5 mA is effective in consistently eliciting darting and conditioned freezing (Mitchell et al. 2022; Mitchell et al. 2024; Laine et al. 2022; Vincelette et al. 2026), and because the results from Experiment 1 suggest that heightened IL activity is a hallmark of darting, regardless of shock intensity.

The current work identifies heightened IL, but not IL-PAG circuit activity, as a potential neurobiological indicator of conditioned darting. The data from these studies suggest that IL activity affects both darting and freezing as a conditioned response, with a suppression of IL activity leading to a decrease in freezing and an increase in darting, particularly in females. Future work will continue examining specifically how the suppression of IL activity during fear conditioning could allow for the emergence of alternate fear behaviors, such as darting, to occur.

Authors' contributions

Julia Ruth Mitchell (Conceptualization [equal], Data curation [lead], Formal analysis [lead], Investigation [lead], Methodology [lead], Project administration [lead], Supervision [lead], Visualization [equal], Writing - original draft [equal], Writing - review & editing [lead]), Samantha Tuberman (Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], Visualization [equal], Writing - original draft [equal], Writing - review & editing [supporting]), Rylin Lubash (Investigation [equal]), Leticia C Takasumi (Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal]), Emmett Bergeron (Investigation [supporting]), Roberto Calitri (Investigation [equal], Methodology [equal], Supervision [supporting]), Mikaela A Laine (Methodology [equal], Validation [equal]), Maryclare Pikus (Investigation [supporting]), Victoria Vance (Investigation [supporting]), Leena Ziane (Investigation [supporting]), Rebecca M Shansky (Conceptualization [lead], Formal analysis [equal], Funding acquisition [lead], Methodology [lead], Resources [lead], Supervision [lead], Visualization [equal], Writing - review & editing [equal])

Competing interests

The authors have nothing to disclose.

Study funding and APC funding

This work was funded by NIH grant R01MH123803 awarded to RMS.

Data availability

Raw data will be made available by the corresponding author upon reasonable request.

Ethics statement

All animal procedures were approved by Northeastern University Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to achieve scientific objectives.