Dorsal peduncular cortex activity modulates affective behavior and fear extinction in mice

Abstract

The medial prefrontal cortex (mPFC) is critical to cognitive and emotional function and underlies many neuropsychiatric disorders, including mood, fear and anxiety disorders. In rodents, disruption of mPFC activity affects anxiety- and depression-like behavior, with specialized contributions from its subdivisions. The rodent mPFC is divided into the dorsomedial prefrontal cortex (dmPFC), spanning the anterior cingulate cortex (ACC) and dorsal prelimbic cortex (PL), and the ventromedial prefrontal cortex (vmPFC), which includes the ventral PL, infralimbic cortex (IL), and in some studies the dorsal peduncular cortex (DP) and dorsal tenia tecta (DTT). The DP/DTT have recently been implicated in the regulation of stress-induced sympathetic responses via projections to the hypothalamus. While many studies implicate the PL and IL in anxiety-, depression-like and fear behavior, the contribution of the DP/DTT to affective and emotional behavior remains unknown. Here, we used chemogenetics and optogenetics to bidirectionally modulate DP/DTT activity and examine its effects on affective behaviors, fear and stress responses in C57BL/6J mice. Acute chemogenetic activation of DP/DTT significantly increased anxiety-like behavior in the open field and elevated plus maze tests, as well as passive coping in the tail suspension test. DP/DTT activation also led to an increase in serum corticosterone levels and facilitated auditory fear extinction learning and retrieval. Activation of DP/DTT projections to the dorsomedial hypothalamus (DMH) acutely decreased freezing at baseline and during extinction learning, but did not alter affective behavior. These findings point to the DP/DTT as a new regulator of affective behavior and fear extinction in mice.

Introduction

The medial prefrontal cortex (mPFC) is a key brain region involved in high-order functions such as attention [1, 2], decision making [3, 4], working memory [5, 6], social behavior [7–10], mood [11, 12] and anxiety [7, 13–30]. The mPFC integrates inputs from cortical sensory and motor systems, as well as subcortical brain structures [31–36] toward top-down control of output structures such as the amygdala, thalamus, hypothalamus and bed nucleus of the stria terminalis (BNST) [31, 34, 36–38]. Despite a lack of unified consensus [39], many studies consider the rodent mPFC to be anatomically subdivided into the anterior cingulate cortex (ACC), prelimbic cortex (PL), and the infralimbic cortex (IL) [40], with some dividing the mPFC into two main regions with differences in their connectivity pattern and functional properties [37, 40–42]: the dorsal mPFC (dmPFC), which includes the ACC and dorsal PL, and the ventral mPFC (vmPFC), which comprises the ventral PL, IL and the dorsal peduncular cortex (DP) and dorsal tenia tecta (DTT). Altered mPFC activity underlies many neuropsychiatric disorders, including mood, fear and anxiety disorders [38, 41, 43–50]. In rodents, disruption of mPFC activity through lesions, pharmacology, optogenetics or chemogenetics affects anxiety- [21, 51–53] and depression-like [54–57] behavior, with evidence supporting specialized contributions from PL and IL subdivisions [15–19, 30], as well as their subprojections [56, 58]. Similarly, the PL and IL mPFC subdivisions exert distinct roles in auditory fear processing [59–64]. Although there is a considerable body of work implicating the mPFC as a key regulator of affective behavior and emotional learning, most studies focus on its main subdivisions, ACC, PL and IL, leaving subregions such as the DP/DTT understudied.

The DP/DTT is located at the ventral limit of the vmPFC [65], and plays a crucial role in driving thermoregulatory and cardiovascular sympathetic responses during stress [66–68]. The DP/DTT receives inputs from stress and emotion-related brain regions like the thalamus, amygdala, insular cortex and piriform cortex [66–68], and sends glutamatergic excitatory input to the dorsomedial hypothalamus (DMH), a central hub driving sympathetic responses to various stimuli, including psychosocial stress [66, 68]. Interestingly, while dmPFC activation abrogates stress-related autonomic and neuroendocrine responses [69–72], DP/DTT activation mimics sympathetic responses to psychosocial stress [66], suggesting a distinct role for the DP/DTT in driving sympathetic stress responses. Given the high prevalence of sympathetic system impairments in anxiety disorders [73–76] and the well-established role of the mPFC in modulating anxiety-, depression-like and cognitive behavior in rodents, we investigated the contribution of the DP/DTT to affective behavior and emotional learning in mice. Our results show that chemogenetic activation of DP/DTT with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in C57BL/6J mice increases anxiety-like behaviors in the open field (OFT) and elevated plus maze (EPM) tests, passive coping in the tail suspension test (TST) and facilitates fear extinction learning and extinction retrieval. DP/DTT activation was accompanied by an increase in serum corticosterone levels. Activation of DP/DTT projections to the dorsomedial hypothalamus (DMH) acutely suppressed freezing. These findings uncover a novel role for the DP/DTT in promoting anxiety-like and fear extinction behaviors in mice.

Materials and methods

For detailed methods, please see supplemental materials

Animals

Adult male and female C57BL/6J mice and Fos^2A-iCreER (TRAP2) mice (2–4 months old; Jackson laboratory) were maintained on a 12 h light-dark cycle (lights on at 7:00 A.M.), with experimental procedures performed during the light phase. Mice were bred in-house and weaned on postnatal day 21 with same-sex siblings (2–4 per cage). Food and water were available ad libitum. All experimental procedures were approved by the Animal Care Committee at the University of Toronto.

Tamoxifen injections

4-hydroxytamoxifen (4-OHT; Sigma Aldrich) was injected i.p. at a dose of 25 mg/kg immediately following OFT in TRAP2 mice previously infused with AAV5-Ef1α-DIO-EYFP in the DP/DTT. Animals were perfused 10 days later and their brains processed for immunohistochemistry.

Stereotaxic surgery and viral injections

Mice (8–12 weeks) underwent stereotaxic surgery as previously described [77, 78]. Mice were injected with 40–100nL of AAV5-hSyn-mCherry, AAV5-hSyn-hM4D(Gi)-mCherry, AAV5-hSyn-hM3D(Gq)-mCherry AAV1-CaMKIIa-hChR2(H134R)-EYFP, AAV1-CAG-GFP (Addgene) or AAV5-Ef1α-DIO-EYFP (UNC vector core) virus into the DP/DTT (AP + 1.85, ML ± 0.38, DV −3.52) or IL (AP + 1.9, ML ± 0.26, DV −2.9) at a rate of 20nL per minute. For optogenetic experiments, an optic fiber was implanted over the DMH (AP −1.62, ML ± 0.30, DV −5.00 mm).

Behavioral testing

For experiments spanning Figs. 1, 2 and 3A–E, following a 2-week recovery period after surgery, cohorts of mice underwent the following behavioral tests interspersed by a 3-day washout period: 1) open field test (OFT), 2) elevated plus maze (EPM), 3) tail suspension test (TST), 4) forced swim test (FST) and 5) auditory fear conditioning. Behavioral task order was defined by degree of stress, starting from the least to greater stress-inducing tasks to minimize carryover effects across tasks [79]. For experiments in Fig. 3F–L, animals underwent fear conditioning following recovery from surgery without prior behavior. In the experiments featured in Fig. 5, all cohorts underwent the following behavioral tests interspersed by a 3-day washout period: 1) OFT, 2) EPM, 3) auditory fear training, followed the next day by either (a) fear extinction training and extinction retrieval or (b) baseline freezing testing. For chemogenetic experiments, the DREADD agonist compound 21 (C21; 2 mg/kg, 0.5 mg/mL, i.p.) was injected 1 h before the start of behavioral testing. For optogenetic experiments, light stimulation (473 nm, 20 Hz, 5 ms pulses) was applied following a baseline recording period in each task. Behavior was automatically scored using ANY-maze software (Stoelting) (OFT, EPM), DBscorer [80] (TST, FST) or VideoFreeze [77, 81] (medassociates) (fear). Detailed behavioral procedures are described in the Supplemental Methods.

Fig. 1. Chemogenetic activation of the DP/DTT increases anxiety-like behavior in the open field and elevated plus maze tests.

A, B Experimental timeline for the OFT. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT were injected with the DREADD agonist C21 one hour prior to being placed in an open field. The time spent in the center zone (red) and the outer zone (white) was measured. C The time spent in the center zone of the open field was significantly reduced in hM3D mice compared to mCherry and hM4D mice (no difference between mCherry and hM4D, p = 0.965). D The amount of time spent in the outer zone of the open field arena was significantly greater in the hM3D mice compared to mCherry and hM4D mice (mCherry versus hM4D, p = 0.938). E The total distance traveled during the test did not differ between groups (one-way ANOVA, F_2,38 = 2.292, p = 0.115). F Representative track and heat maps. G, H Experimental timeline for the EPM test. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT were injected with the DREADD agonist C21 one hour prior to being placed in an elevated plus maze. The time spent in the open arms vs closed arms was measured. I The time spent in the open arms of the elevated plus maze was significantly reduced in hM3D mice compared to mCherry and hM4D mice (no difference between mCherry and hM4D mice, p = 0.993). J The number of open arm entries was significantly reduced in hM3D mice compared to hM4D mice (no difference between hM3D and mCherry, p = 0.136; or hM4D and mCherry, p = 0.430). K The total distance traveled during the test did not differ between groups (one-way ANOVA, F_2,38 = 0.022, p = 0.98). While we found that hM4D males traveled less than hM4D females (see results), separation of the data by sex did not reveal group differences (One-way ANOVA, females F_2,16 = 0.3571, p = 0.7051; males F_2,19 = 0.093, p = 0.9117). L Representative track and heat maps. M Schematic of IL viral targeting. Mice previously injected with hM3D or control mCherry constructs into the IL were injected with the DREADD agonist C21 one hour prior to undergoing the OFT (N, O) or EPM test (P–R). N IL-hM3D mice spent the same amount of time in the center zone of the OFT as IL-mCherry mice (t₁₄ = 0.2502, p = 0.8061). O OFT distance traveled did not differ between groups (t₁₄ = 1.689, p = 0.1134). P IL-hM3D mice spent the same amount of time in the open arms of the EPM as IL-mCherry mice (t₁₄ = 1.916, p = 0.076). Q IL-hM3D and IL-mCherry mice displayed the same number of open arm entries in the EPM (t₁₄ = 0.8721, p = 0.3979). R EPM distance traveled did not differ between groups (t₁₄ = 1.999, p = 0.0654). *p < 0.05, **p < 0.01. Male (square) and female (circle) individual datapoints are displayed for transparency (see text for details). Some figure diagrams were created with the assistance of BioRender.com.

Fig. 2. Chemogenetic activation of the DP/DTT increases immobility in the tail suspension test, but has no effect on forced swim behaviors.

A, B TST experimental timeline. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT were injected with the DREADD agonist C21 one hour prior to undergoing the TST. C Representative raster plots for each mouse indicating periods of immobility (black) and mobility (white) over the tail suspension test duration. D The average percent of time spent immobile during the TST was significantly greater in hM3D mice compared to mCherry and hM4D mice (but not different between mCherry and hM4D, p = 0.838). E, F Effects of IL DREADD soma manipulation on the TST. Mice previously injected with hM3D or control mCherry constructs in the IL were injected with the DREADD agonist C21 one hour prior to undergoing the TST. E Representative raster plots for each mouse indicating periods of immobility (black) and mobility (white) over the TST duration. F IL-hM3D mice spent the same amount of time immobile in the TST as IL-mCherry mice (t₁₄ = 1.102, p = 0.2892). G, H FST experimental timeline. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT were injected with the DREADD agonist C21 one hour prior to undergoing the forced swim test. I Representative raster plots for each mouse indicating periods of immobility (black) and mobility (white) over the FST duration. J There was no effect of treatment on percent immobility in the forced swim test (one-way ANOVA, F_2,38 = 1.722, p = 0.192). While we found that hM3D females had a lower percent time spent immobile compared to males (see results), separation of the data by sex did not reveal group differences (One-way ANOVA, females F_2,16 = 2.816, p = 0.0896; males F_2,19 = 1.042, p = 0.3722). *p < 0.05. Male (square) and female (circle) individual datapoints are displayed for transparency (see text for details). Some figure diagrams were created with the assistance of BioRender.com.

Fig. 3. Chemogenetic inhibition of the DP/DTT has a modest effect on auditory fear acquisition, but activation of the DP/DTT facilitates within-session extinction, extinction retrieval and increases serum corticosterone levels.

A–E DP/DTT manipulation during auditory fear acquisition. A Experimental timeline. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT were injected with the DREADD agonist C21 one hour prior to undergoing auditory fear conditioning. During acquisition, mice were placed in a sound-attenuated fear conditioning chamber and received 6 tone-shock pairings (0.5 mA). Twenty-four hours later, auditory fear memory was evaluated in a different context in the absence of shocks. B During fear acquisition, DP/DTT manipulations did not affect average tone freezing (one-way ANOVA, F_2,38 = 2.828, p = 0.072). C hM4D mice displayed significantly more freezing than hM3D mice at tone presentations 4 and 6 during acquisition. Two-way rmANOVA found no main effect of treatment F_2,38 = 2.828, p = 0.072, but a significant main effect of tone (F_5,190 = 100.6, p < 0.001, and no treatment by tone interaction: F_10,190 = 1.633, p = 0.998). During fear retrieval, DP/DTT manipulations did not affect freezing during average tone (D; one-way ANOVA, F_2,38 = 0.507, p = 0.607). Individual tone analysis (E) showed no main effect of treatment (Two-way rmANOVA, F_2,38 = 0.507, p = 0.607), but a significant main effect of tone (F_5,190 = 4.00, p = 0.002, no treatment by tone interaction: F_10,190 = 1.213, p = 0.285), likely driven by within-session extinction. indicates a significant difference between hM3D and hM4D groups (p < 0.05). F–L DP/DTT manipulation during fear extinction. F Experimental timeline. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT (no prior behavior) underwent fear acquisition (as previously; 6 tone-shock pairings). The following day, mice were injected with the DREADD agonist C21 one hour prior to undergoing extinction training. During extinction training, mice were placed in an alternate context (context B) and received 12 tone presentations. The following day, fear memory was evaluated during extinction retrieval, in the absence of C21. During extinction retrieval, mice were placed in context B and received 5 tone presentations. During auditory fear acquisition, mice from all groups displayed similar average tone freezing (G; one-way ANOVA F_2,31 = 0.5886, p = 0.5612) but decreased freezing in tone 6 in the hM3D group (H; Two-way rmANOVA significant effect of tone F_5,155 = 74.69, p < 0.0001 and interaction F_10,155 = 1.984, p = 0.0385; but no main effect of treatment F_2,31 = 0.8356, p = 0.4431; tone 6 mCherry vs hM3D p = 0.0023 and hM3D vs hM4D p = 0.0109). During extinction training, 1 h after C21 treatment, hM3D mice showed decreased average tone freezing (I; Two-way rmANOVA main effect of treatment and tone but no interaction F_22,341 = 1.072, p = 0.3758) and tone-by-tone freezing (J) compared to hM4D and mCherry control mice. During extinction retrieval, hM3D mice froze less compared to hM4D and mCherry control mice in the average tone (K) and tone-by-tone (L; Two-way rmANOVA significant main effect of treatment and tone but no interaction F_8,124 = 0.6037, p = 0.7733) analyses. Minimal sex differences were found in this dataset (see Supplementary Materials). M Experimental timeline for serum corticosterone assay. Mice previously injected with hM3D, hM4D or control mCherry constructs in the DP/DTT were injected with the DREADD agonist C21 and euthanized 90 minutes later. Blood was extracted at the time of perfusion for corticosterone measurements. N Serum corticosterone was significantly higher in hM3D mice compared to mCherry mice (but no change between mCherry and hM4D, p = 0.440, hM4D vs hM3D, p = 0.103). indicates a significant group difference between mCherry and hM3D conditions; indicates a significant group difference between hM3D and hM4D conditions; indicates a significant difference between hM3D and all other groups. *p < 0.05. Male (square) and female (circle) individual datapoints are displayed for transparency. Some figure diagrams were created with the assistance of BioRender.com.

Fig. 5. Optogenetic activation of the DP/DTT-DMH pathway acutely suppresses freezing but has no effect on affective behavior.

A Experimental timeline. Mice were injected with ChR2 or control GFP AAV constructs in the DP/DTT and implanted with an optic fiber in the DMH. Ten days later, animals underwent behavior, starting with the OFT. Three days later, animals went through the EPM and, following another 3 days, one of two auditory fear conditioning protocols (see below). B–D Behavior in the OFT. B Light activation did not affect the time spent in the center of the open field between groups (Two-way rmANOVA significant main effect of stimulation F_1,18 = 5.657, p = 0.0287; but no main effect of treatment F_{1, 18} = 0.1118, p = 0.7420 or interaction F_1,18 = 0.02443, p = 0.8775; no significant posthoc tests, p > 0.17). C Light activation did not affect the time spent in the outer zone of the open field between groups (Two-way rmANOVA significant main effect of stimulation F_1,18 = 6.549, p = 0.0197; but no main effect of treatment F_{1, 18} = 0.11240, p = 0.7288 or interaction F_1,18 = 0.1744, p = 0.6811; no significant posthoc tests, p > 0.097). D The total distance traveled during the test was not affected by light activation (Two-way rmANOVA no main effect of treatment F_{1, 18} = 2.724, p = 0.1162; stimulation F_1,18 = 3.054, p = 0.0976; or interaction F_1,18 = 2.728, p = 0.1160). E–G Behavior in the EPM. Light activation did not affect the time spent in the open arms of the EPM (E; Two-way rmANOVA no main effect of treatment F_1,14 = 0.9766, p = 0.3398; stimulation F_1,14 = 2.631, p = 0.1271; or interaction F_1,14 = 0.0100, p = 0.9217), the number of entries to the open arms of the EPM (F; we note a non-significant trend toward a decrease in open arm entries in the EPM for the ChR2 animals: Two-way rmANOVA significant effect of stimulation F_1,14 = 10.53, p = 0.0059; but no main effect of treatment F_1,14 = 3309, p = 0.5743 or interaction F_1,14 = 0.1645, p = 0.6912; Šídák’s multiple comparisons test, ChR2 p = 0.057, GFP p = 0.097) or the total distance traveled (G; Two-way rmANOVA no main effect of treatment F_1,14 = 1.207, p = 0.2904; stimulation F_1,14 = 0.0676, p = 0.7986; or interaction F_1,14 = 0.00511, p = 0.0940). H–L Effect of DP/DTT-DMH activation on fear extinction and extinction retrieval. H Experimental timeline for the fear extinction cohort. Three days after the EPM, mice were trained in auditory fear conditioning as described previously (6 tone-shock pairings). We note a significant effect of tone on fear training in this cohort (data not shown; Two-way rmANOVA significant effect of tone F_{3.537, 35.37} = 50.62, p < 0.0001; but no main effect of treatment F_1,10 = 1.792, p = 0.2103 or interaction F_5,50 = 0.5963, p = 0.7029). The following day, mice underwent extinction training in context B (12 tones, as previously) with light activation for the duration of each tone. On the next day, they underwent extinction retrieval in context B (5 tones, same protocol as before) in the absence of light. I During extinction training, DP/DTT-DMH optogenetic activation reduced average freezing (I) and freezing across tones (J). K, L During extinction retrieval in the absence of light, average tone freezing (K; t₁₀ = 0.1392, p = 0.8921) and freezing across tones (L; Two-way rmANOVA no significant main effect of treatment F_1,10 = 0.0193, p = 0.8921; tone F_2.707,27.07 = 2.575, p = 0.0798 or interaction F_4,40 = 1.592, p = 0.1953) were similar between groups. M–O Effect of DP/DTT-DMH activation on baseline fear after conditioning. M Experimental timeline for the fear baseline cohort. Three days after the EPM, a separate cohort of mice was trained in auditory fear conditioning as previously (6 tone-shock pairings). The following day, mice were placed in context B in the absence of tones, and after 2 min of baseline exploration the light was turned on for 2 min. N DP/DTT-DMH optogenetic activation decreased freezing in the absence of tones. Two-way rmANOVA significant main effect of stimulation and interaction, but not virus F_1,11 = 0.006, p = 0.9396; Šídák’s multiple comparisons test, light OFF minus Light ON ChR2 p = 0.0011, GFP p = 0.7607. O DP/DTT-DMH optogenetic activation increased rearing instances. Two-way rmANOVA significant main effect of stimulation and interaction, but not virus F_1,11 = 0.8904, p = 0.3656; Šídák’s multiple comparisons test, light OFF minus Light ON ChR2 p = 0.0008 GFP p = 0.8609. *p < 0.05. Male (square) and female (circle) individual datapoints are displayed for transparency. Some figure diagrams were created with the assistance of BioRender.com.

Perfusions and sectioning

For the c-Fos analysis, mice were euthanized 90 minutes after C21 treatment. Mice were transcardially perfused with 0.1 M phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde fixative (PFA). The brains were extracted and stored in PFA overnight at 4 °C. Brains were sectioned at 50 µm in the coronal plane with a vibratome (model VT1000, Leica).

Serum corticosterone

Behaviorally-naive mice were injected with C21 and, 90 min later, prepared for blood extraction for serum corticosterone assessment using an immunoassay kit (Enzo Life Sciences).

Immunofluorescence

Immunofluorescence staining was performed using the primary antibodies rabbit anti-mCherry (RRID: AB_2571870), rabbit anti c-Fos (RRID: AB_2891278), rat anti-mCherry (RRID: AB_2536611), polyclonal chicken anti-GFP (RRID: AB_371416), polyclonal rabbit anti-GluR2/3 (RRID: AB_90710), or monoclonal mouse anti-GAD67 (RRID: AB_2278725) and secondary goat anti-rabbit Alexa Fluor 568 (RRID: AB_143157), goat anti-rat Alexa Fluor 568 (RRID: AB_2534121), goat anti-mouse Alexa Fluor 633 (SAB4600138, Millipore) goat anti-chicken Alexa Fluor 488 (RRID: AB_2534096) or anti-rabbit HRP (RRID: AB_2340590) followed by tyramide signal amplification.

Image acquisition & quantification

Images were acquired on a Nikon Eclipse Ni-U epifluorescence microscope or a Leica Stellaris 5 laser scanning confocal microscope. ImageJ Software (version 1.53e) [77] was used for c-Fos quantification. For the DP/DTT TRAP neurochemical analysis, confocal 1um Z-stack images of the DP/DTT were obtained and manually counted using LASX Office (version 1.4.5 27713) software (Leica).

Slice electrophysiology

Acute brain slices containing the mPFC or the DMH were prepared from adult male and female C57BL/6J mice at least two weeks after AAV injection. Brains were placed in ice-cold sucrose based cutting solution containing the following (in mM): 180 sucrose, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 1 CaCl₂, 2 MgCl₂, 2 Na⁺ pyruvate and 0.4 L-Ascorbic acid with 95% O₂ and 5% CO₂. Acute brain slices (coronal, 300 µm) were obtained using a vibratome (VT1000S, Leica). Brain slices were allowed to recover for 30 min in a holding chamber containing a recovery solution at 30 °C, made up of 50% sucrose-based cutting solution and 50% artificial cerebrospinal fluid (ACSF) (in mM): 120 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 11 glucose, 2 CaCl₂, 1 MgCl₂, 2 Na⁺ pyruvate and 0.4 L-Ascorbic acid with 95% O₂ and 5% CO₂. Slices then underwent an additional 30 min recovery in regular ACSF at room temperature before patch-clamp recordings.

For DREADD experiments, current-clamp recordings were obtained from hM4D- or hM3D-mCherry+ pyramidal neurons in layers 2/3 or 5 of the DP/DTT with borosilicate pipettes (3–5 MΩ) that were filled with intracellular solution containing the following (in mM): 126 K D-Gluconate, 5 KCl, 10 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na-phosphocreatine. C21 (1 µM, HelloBio) [82] was bath applied for at least 10 min, with the last 5 min being compared to the baseline period.

For optogenetic experiments, voltage clamp recordings were obtained from DMH neurons with borosilicate pipettes (3–5 MΩ) filled with intracellular solution containing the following (in mM): 130 Cs-methanosulfonate, 10 HEPES, 0.5 EGTA, 8 NaCl, 4 Mg-ATP, 0.4 Na-GTP, 10 Na-phosphocreatine, 1 N-ethyl lidocaine (QX-314). DP/DTT axon terminals were stimulated using a TTL-pulsed microscope objective-coupled light-emitting diodes (460 nm, ∼1.36 mW/mm2, 5 ms; Prizmatix).

Electrophysiological data was acquired using a MultiClamp 700B amplifier (Molecular Devices). Signals were low-pass filtered at 2 kHz and digitized at 20 kHz using Digidata 1550 A and pClamp10 software (Molecular devices).

Statistical analysis

Statistical analysis was performed using Prism 9.1 (GraphPad) and presented as the mean ± standard error of the mean (SEM) with significance defined at p < 0.05. Non-statistically significant results are reported in the figure legends due to space limits. All data were analyzed for sex differences, with non-statistically significant results reported in the supplemental methods. To facilitate interpretation of the data, and in the absence of major differences within and across tasks, we pooled male and female mice, with individual data points presented as squares (males) and circles (females) for transparency.

For within subject comparisons, a paired t-test was used. Comparisons of independent groups were made using a one-way ANOVA, followed by Tukey’s Post Hoc test when appropriate. Parametric data with multiple comparisons were analyzed using a two-way repeated measures ANOVA, followed by Tukey’s or Šídák’s Post Hoc test with corrections for multiple comparisons. Normality of parametric datasets were confirmed by the D’Agostino and Pearson normality test (Prism 9.1). For detailed statistical analysis of sex differences please see Supplemental Materials.

Results

To explore the contribution of the DP/DTT to anxiety-, depression-like and fear behavior in mice, we first bidirectionally modulated DP/DTT activity across a series of behavioral tasks with excitatory and inhibitory DREADDs. AAV vectors encoding excitatory (hM3D), inhibitory (hM4D) DREADDs or the control fluorophore mCherry were injected into the DP/DTT of C57BL/6J mice. Viral expression was strongly localized to the DP/DTT across each treatment condition (Supplementary Fig. S1).

Chemogenetic activation of DP/DTT increases anxiety-like behavior

We first examined the impact of manipulating DP/DTT activity on anxiety-like behavior in freely behaving mice in the OFT (Fig. 1A–F; Supplementary Fig. S2A, B). Mice expressing hM3D, hM4D or mCherry constructs in the DP/DTT region were injected with C21 one hour prior to OF testing (Fig. 1A, B). Chemogenetic activation of DP/DTT neurons (hM3D mice) decreased the time spent in the center zone of the OF arena compared to mCherry and hM4D mice (Fig. 1C; One-way ANOVA, F_2,38 = 7.795, p = 0.002, Tukey’s post hoc tests, hM3D versus mCherry p = 0.002; hM3D versus hM4D p = 0.011; Supplementary Fig. S2B). In contrast, hM3D mice spent significantly more time in the outer zone of the open field apparatus compared to both mCherry control and hM4D mice (Fig. 1D, one way ANOVA, F_2,38 = 7.283, p = 0.002; Tukey’s post hoc tests, hM3D versus mCherry, p = 0.007 and hM3D versus hM4D, p = 0.006). Furthermore, the total distance traveled was similar across groups (Fig. 1E, one way ANOVA, F_2,38 = 2.292, p = 0.115), suggesting that the effect of C21-mediated activation of DP/DTT neurons on anxiety-like behavior was not driven by changes in locomotor activity levels. These data show that chemogenetic activation of the DP/DTT induces anxiety-like behavior in the OFT (Fig. 1F).

Next, we examined the effects of manipulating DP/DTT activity on anxiety-like behavior using the EPM test (Fig. 1G–L; Supplementary Fig. S2C–F). Consistent with the anxiogenic effect of DP/DTT activation observed in the OFT, hM3D mice spent less time in the open arms of the EPM apparatus compared to mCherry control and hM4D mice (Fig. 1I; one-way ANOVA, F_2,38 = 5.87, p = 0.006, Tukey’s post hoc tests, hM3D versus mCherry, p = 0.010, hM3D versus hM4D, p = 0.025; Supplementary Fig. S2D). Additionally, hM3D mice displayed fewer entries to the open arms of the EPM compared to hM4D mice (Fig. 1J, one-way ANOVA, F_2,38 = 4.86, p = 0.013, Tukey’s post hoc test, hM3D versus hM4D, p < 0.011). Moreover, hM3D mice spent significantly more time in the closed arms of the EPM compared to mCherry control and hM4D mice (Supplementary Fig. S2E). We saw no group differences in the number of entries to the closed arm (Supplementary Fig. S2F), middle platform (F_2,38 = 2.130, p = 0.133; data not shown), or in the time spent in the middle platform (F_2,38 = 1.749, p = 0.187; data not shown). All three experimental groups showed equivalent total distance traveled in the EPM apparatus (Fig. 1K). We found a significant effect of sex on the distance traveled in the EPM, with hM4D males traveling significantly less distance compared to hM4D females (F_1,35 = 8.23, p = 0.007; Sidak’s multiple comparisons test p > 0.109; no main effect of treatment or interaction. For more detailed sex differences analysis, see supplemental materials). Importantly, chemogenetic activation of the IL did not affect performance in the OFT or in the EPM (Fig. 1M–R). Taken together, our data suggest that acute activation of DP/DTT, but not IL neurons results in increased anxiety-like responses in the OFT and EPM.

Chemogenetic activation of DP/DTT increases passive coping behavior

To examine whether chemogenetic modulation of DP/DTT activity affects passive coping behavior, we next subjected mice to the TST (Fig. 2A, B). The hM3D mice spent significantly more time immobile compared to mCherry and hM4D groups following C21 injection (Fig. 2C, D, one-way ANOVA, F_2,38 = 5.07, p = 0.11; Tukey’s post hoc tests, hM3D vs mCherry, p = 0.041, hM3D vs hM4D, p = 0.017; Supplementary Fig. S2G, H), consistent with increased passive coping in the hM3D group. All three groups showed equivalent length of the longest bout of immobility, and number of transitions from mobility to immobility states (Supplementary Fig. S2I, J). Chemogenetic activation of IL did not affect immobility in the TST (Fig. 2E, F).

To expand our findings of increased passive coping behavior in the TST following DP/DTT activation, we conducted the FST (Fig. 2G, H). C21 treatment did not affect time spent immobile (Fig. 2I, J), longest bout of immobility or the number of transitions between mobility and immobility states (Supplementary Fig. 2K–N) in any of the experimental groups in the FST. When we analyzed sex differences, we found that hM3D females spent significantly less time immobile than hM3D males and hM4D females (Two-way ANOVA, F_1,35 = 6.561, p = 0.015; Sidak’s multiple comparisons test, hM3D females versus hM3D males p = 0.0093; hM3D females versus hM4D females p = 0.0482). Taken together, our results demonstrate that activation of DP/DTT neurons promotes passive coping behavior in the TST, but did not alter behavior in the FST.

Chemogenetic activation of DP/DTT activity facilitates auditory fear extinction

We next sought to explore whether manipulating DP/DTT activity might affect fear memory. To test this, we first inhibited or activated the DP/DTT one hour prior to training mice to associate an auditory tone with an electric foot shock (Fig. 3A). During fear acquisition, chemogenetic manipulation of DP/DTT activity did not affect baseline freezing (one-way ANOVA, F_2,38 = 2.367, p = 0.107; data not shown), or the mean percent freezing across all tones (Fig. 3B). However, we found that hM4D mice displayed a right shift in their fear learning curve, with significantly higher freezing during tone 4 and 6 of the training protocol compared to hM3D mice (Fig. 3C, two-way rmANOVA, significant effect of tone F_5,190 = 100.6, p < 0.001; Tukey’s post hoc tests, minute 4 hM3D versus hM4d, p = 0.012, and minute 6 hM3D versus hM4D, p = 0.012) suggesting a slight facilitation of auditory fear acquisition upon DP/DTT inhibition. We found a significant main effect of sex on average freezing to tone (Two-way ANOVA, F_1,35 = 7.786, p = 0.009, see supplemental materials).

Auditory fear memory retrieval was evaluated 24 h later in a different context (Fig. 3A). All three experimental groups showed similar baseline freezing (one-way ANOVA, F_2,38 = 0.153, p = 0.858; data not shown), and equivalent freezing to the tones (Fig. 3D, E). Overall, these data suggest that manipulating DP/DTT activity has no major impact on auditory fear acquisition, except for a marginal facilitation of fear acquisition following DP/DTT inhibition.

To test whether DP/DTT manipulation might affect fear extinction and retrieval, we infused AAV vectors encoding either hM3D, hM4D or mCherry into the DP/DTT of a separate cohort of mice, and trained them in auditory fear conditioning as previously (Fig. 3F–H). The next day, we injected C21 one hour prior to extinction training. During extinction training, hM3D animals froze less than hM4D and mCherry animals (Fig. 3I: One-way ANOVA F_2,31 = 12.91, p < 0.0001, mCherry versus hM3D p = 0.0003, hM3D versus hM4D p = 0.0002; Fig. 3J: Two-way rmANOVA main effect of treatment F_2,31 = 12.91, p < 0.0001 and tone F_11,341 = 7.869, p < 0.0001 only; mCherry versus hM3D p = 0.0003, hM3D versus hM4D p = 0.0002), suggesting that DP/DTT activation impairs auditory fear retrieval and facilitates within session extinction. The next day, animals underwent extinction retrieval in the absence of C21. At extinction retrieval, hM3D animals showed less freezing than both hM4D and mCherry animals (Fig. 3K: One-way ANOVA F_2,31 = 5.853, p = 0.0070, mCherry versus hM3D p = 0.0078, hM3D versus hM4D p = 0.0245; 3L: Two-way rmANOVA significant main effect of treatment F_2,31 = 4.526, p = 0.0189 and tone F_4,124 = 3.021, p = 0.0204 only; mCherry versus hM3D p = 0.0255, hM3D versus hM4D p = 0.0385). Overall, these data show that DP/DTT activation leads to reduced fear recall and a facilitation of fear extinction learning and extinction retrieval.

Chemogenetic activation of DP/DTT increases serum corticosterone levels

Given the important role of the mPFC in regulating the hypothalamic-pituitary-adrenal (HPA) axis response to emotional stress [70, 83–85], and the regulation of stress-induced sympathetic responses by the DP/DTT [66], we next tested the effects of DP/DTT chemogenetic modulation on serum levels of the stress hormone corticosterone. We injected C21 into a subset of mCherry, hM3D, and hM4D mice that remained in their home cage, and subsequently extracted blood to evaluate serum corticosterone (Fig. 3M). Notably, these mice had not undergone any previous behavioral testing. Interestingly, hM3D mice displayed higher serum corticosterone levels compared to mCherry control mice (Fig. 3N, one-way ANOVA, F_2,12 = 6.325, p = 0.013, Tukey’s post hoc tests, hM3D versus mCherry, p = 0.011). Chemogenetic inhibition of DP/DTT activity did not affect baseline serum corticosterone levels (Fig. 3N). These data indicate that activation of DP/DTT neurons is sufficient to trigger a neuroendocrine stress response by increasing serum corticosterone levels. We validated our chemogenetic manipulations using cFos immunohistochemistry and slice electrophysiology (Supplementary Fig. S3).

Characterization and mapping of DP/DTT downstream projections

To explore potential downstream effectors mediating the effects of DP/DTT activation on affective, fear behavior and neuroendocrine responses, we first analyzed brain-wide patterns of DP/DTT axonal innervation with the help of the mCherry tag in AAV-injected mice (Fig. 4A–C). Projections from the DP/DTT were observed in the lateral septum (medial and lateral nuclei), indusium griseum (IG), BNST, dorsal endopiriform nucleus (DEn), thalamus (nucleus reuniens, RE; paraventricular nuclei, PVT; submedius nucleus, Sub), hypothalamus (DMH; lateral hypothalamus, LH; paraventricular nucleus, PVN; posterior hypothalamus, PH; and retromammillary nuclei, RM) and brainstem (periaqueductal gray, PAG) (Fig. 4A–C). Posterior brainstem sections containing the raphe nuclei also showed DP/DTT mCherry+ projections (data not shown).

Fig. 4. Mapping of DP/DTT downstream projections and characterization of activity-tagged DP/DTT neurons following the OFT.

A–C Representative projections of the DP/DTT in anterior, intermediate, and posterior sections. A In anterior sections, we observed notable viral expression in the indusium griseum (IG), septum, bed nucleus of the stria terminalis (BNST), dorsal endopiriform nucleus (DEn), nucleus reuniens (RE) and paraventricular thalamus (PVT). B In intermediate sections, we observed continued viral expression in the PVT and RE. Viral expression was also observed in the paraventricular nucleus (PVN) submedius thalamic nucleus (Sub), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH), and posterior hypothalamus (PH). C In the most posterior sections we examined, viral expression was detected in the periaqueductal gray (PAG), retromammillary nuclei (RM), PVT, LH and PH. In posterior brainstem sections, we also observed DP/DTT projections to the raphe nuclei (data not shown). D–F Characterization and downstream projection patterns of activity-tagged DP/DTT neurons following the OFT. D Experimental timeline. TRAP2 mice were infused with AAV expressing a DIO-YFP construct in the DP/DTT. Seven days later, animals underwent the OFT and were injected with tamoxifen immediately afterwards. Ten days later, animals were perfused and their brains processed for immunohistochemistry against AMPA-type glutamate receptor subunits 2/3 (GluR2/3) and glutamate decarboxylase 67 (GAD67). E Representative image of DP/DTT activity tagged neurons (green). Scale bars: 400 µm (left), 100 µm (right inset). F Left: Representative image showing DP/DTT OFT activity-tagged neurons (green) co-stained with GluR2/3 (magenta; arrows) or GAD67 (cyan; arrow heads). Scale bar: 50 µm. Right: Distribution of DP/DTT OFT activity-tagged neurons indicates that these cells predominantly co-label with GluR2/3 (67.9%), with 13.9% of neurons co-labeled with GAD67, 18.2% negative for either marker, and a minority of cells (2.5%) co-labeled for both GluR2/3 and GAD67 (n = 6).

To further characterize the DP/DTT neuronal subpopulations actively recruited during behavior, we next used viral tracing in Fos^2A-iCreER (TRAP2) transgenic mice [86, 87] to tag DP/DTT neurons active during the OFT. We infused an AAV expressing Cre-dependent eYFP (AAV-DIO-eYFP) into the DP/DTT of TRAP2 mice and, following recovery, injected animals with tamoxifen immediately after OFT testing to tag the soma of activated DP/DTT neurons through YFP expression (Fig. 4D, E). We found that 67.9% of DP/DTT neurons active during OFT testing co-labeled with the AMPA-type glutamate receptor subunits GluR2/3, 13.9% with the GABAergic marker GAD67, and 2.5% of labeled cells were positive for both markers (Fig. 4F). Overall, this points to OFT activating a heterogeneous DP/DTT population containing a minority of interneurons.

Optogenetic activation of the DP/DTT-DMH pathway acutely suppresses freezing but does not alter affective behavior

We next wanted to test whether one of the main downstream pathways revealed by our previous analysis, the DP/DTT-DMH pathway [66], might underlie our effects on affective and fear extinction behavior. We optogenetically targeted the DP/DTT-DMH pathway by expressing the excitatory opsin channelrhodopsin (ChR2) or control GFP into the DP/DTT and implanting an optic fiber over the DMH, and tested the animals in the OFT, EPM and auditory fear (Fig. 5A, Supplementary Fig. S4A, B). Optical activation of the DP/DTT-DMH pathway did not alter behavior in the OFT (Fig. 5B–D) or EPM tasks (Fig. 5E–G). In contrast, activation of the DP/DTT-DMH during tone exposure at extinction learning significantly suppressed freezing responses (Fig. 5H, I: t₁₀ = 7.119, p < 0.0001; Fig. 5J: Two-way rmANOVA significant main effect of stimulation F_1,10 = 50.68, p < 0.0001; tone F_3.961,39.61 = 3.026, p = 0.0291, and interaction F_11,110 = 1.883, p = 0.0491; Tone 1 p = 0.0042, Tone 2 p = 0.0461) without affecting extinction retrieval (Fig. 5K, L). To test whether this suppression of freezing was specific to the tone-shock association, we fear conditioned a separate cohort of mice and activated the DP/DTT-DMH pathway in the alternate context in the absence of tones (Fig. 5M). DP/DTT-DMH activation led to a reduction in baseline freezing behavior (Fig. 5N; Two-way rmANOVA significant main effect of stimulation F_1,11 = 15.88, p = 0.0021 and interaction F_1,11 = 9.375, p = 0.0108 only; ChR2 p = 0.0011) and an increase in rearing (Fig. 5O; Two-way rmANOVA significant main effect of stimulation F_1,11 = 16.30, p = 0.002 and interaction F_1,11 = 11.28, p = 0.006 only; ChR2 p = 0.0008). These data suggest that activation of the DP/DTT-DMH pathway acutely suppresses freezing at baseline and during extinction training without affecting extinction retrieval.

Discussion

Here we examined the effects of modulating the activity of DP/DTT neurons to anxiety-, depression-like and fear behavior in stress-naïve mice. We found that chemogenetic activation of the DP/DTT reduced the time spent in the center zone of the OFT and in the open arms of the EPM. DP/DTT activation also increased passive coping in the TST, but did not affect behavior in the FST. DP/DTT activation facilitated fear extinction training and extinction retrieval, whereas DP/DTT-DMH activation acutely suppressed freezing. Interestingly, DP/DTT activation led to an increase in serum corticosterone levels, suggesting that the DP/DTT also modulates HPA axis activity. DP/DTT projection mapping showed strong innervation of lateral septum, thalamus, hypothalamus, and brainstem, consistent with tracing studies [33, 65, 88, 89].

Our data showed that activation of the DP/DTT increased anxiety-like behavior in the OFT and EPM. A considerable body of work implicates the rodent mPFC in the regulation of anxiety-like behavior [7, 13–30], with contradictory findings supporting anxiogenic [14, 15, 27–30, 51–53] or anxiolytic [16–19] roles. Manipulations selective to the vmPFC/IL also see conflicting effects on EPM and open field performance [16, 18, 30, 58]. Recently, Chen and colleagues found that optogenetic activation of IL soma drives anxiety-like behavior in the EPM and open field [58], similar to our data with DP/DTT activation. In contrast, they found that optical stimulation of the DP increased entries to the open arms in the EPM and time spent in the center of the open field [58], contrary to our findings. Our data support an anxiogenic role for the DP/DTT, which is in line with findings from Kataoka and colleagues showing that inhibition of the DP/DTT pathway abolishes stress-induced avoidance behavior [66], and our finding of increased serum corticosterone following DP/DTT activation. Importantly, the viral spread over the DP in the study by Chen and colleagues is more dorsal (spreading to IL, but not DTT) compared to ours, and targets glutamatergic neurons under the αCaMKII promoter [58], in contrast to the pan-neuronal synapsin and EF1a promoters used here and by Kataoka and colleagues [66]. These discrepancies favor additional cell-type and subregion specialization across the dorso-ventral axis of the vmPFC in regulating anxiety-like behavior and neuroendocrine responses.

DP/DTT activation increased immobility time in the TST, but not in the FST. Given the differential implications of animal weight on water-based (FST) and suspension (TST) task demands, it is possible that the relatively high weight of our animals at the time of testing (>30 g for males) contributed to the differences in performance between these tasks. The mPFC has a well-established role in modulating depression-like behavior [90] in both stress-naïve [54–57, 91, 92] and stress conditions [91, 93–98], including IL-specific manipulations [55, 57]. Interestingly, Warden and colleagues identified mPFC neuronal population coding for FST active and inactive behavioral states, which reflected specialization within mPFC downstream projections to the dorsal raphe nucleus and lateral habenula [99]. Accordingly, somatic activation of the mPFC as a whole did not affect behavior in the FST [99], suggesting that further probing of specific DP/DTT projections could lead to modulation of FST behavior despite our negative findings.

Overall, we did not see major sex differences in baseline OFT or EPM behavior. While studies in rats show consistent sex differences [100] in the EPM [100–106] and OFT [95, 97, 98, 100, 101] (but see refs. [102, 103]) which are often replicated in mice [107–111], several mouse studies show reversals or no sex differences [100, 112–115], particularly in the OFT [100, 107, 115, 116]. Although our lack of sex differences in the OFT replicates similar findings in C57BL6 mice [107, 115, 116], we unexpectedly found sex differences only in hM4D mice in the EPM. Similarly, our sex differences in the TST were only found among DREADD injected animals. While we cannot explain the lack of sex differences in the matched controls in these experiments, factors such as age [117, 118] and estrous cycle [119, 120] are known to modulate sex differences in affective behavior, and could have contributed to our findings. Interestingly, one study reported sex-specific increased recruitment of the DP in female mice following auditory fear conditioning [121]. Due to the well documented sex and gender differences in the prevalence of anxiety and posttraumatic stress (PTSD) disorders [122–124], as well as in sympathetic and autonomic function [125–128], this raises the intriguing possibility of sex differences in the long-term consequences of stress-induced activation of DP pathways.

Brain-wide mapping of DP/DTT projections showed strong downstream connectivity with the hypothalamus, thalamus, lateral septum and brainstem. Importantly, many of the upstream brain regions projecting to the DP [66] also regulate anxiety-like behavior in rodents, including the mediodorsal (MD) [129–132] and paraventricular (PVT) [133–137] thalamus, insular cortex (IC) [137–142], nucleus reuniens [143] and amygdala [18, 23, 144–147]. We found that 13.9% of DP/DTT neurons activated during the OFT co-expressed GAD67, and 67.9% co-labeled with the AMPA receptor subunits GluR2/3. Although we cannot claim that GluR2/3-expressing cells are glutamatergic, expression of this particular combination of AMPA receptor subunits shows minimal overlap with interneurons in the hippocampus [148], dorsal horn [149], and in the mPFC of macaques (<2.9%) [150], consistent with the very low (2.5%) co-labeling with GAD67 in our tagged population. Nevertheless, a fuller characterization of cell types will be important in future experiments, ideally using in-situ hybridization approaches that can more definitively identify whether neurons are glutamatergic or GABAergic due to clear cell body staining. Presently, the precise neurochemical profile, as well as the patterns of afferent and efferent innervation of DP/DTT neurons modulating anxiety-like behavior remain unknown.

Our data showed minimal effects of DP/DTT soma manipulation on auditory fear acquisition, but a marked reduction in freezing when the DP/DTT was activated during fear extinction, which persisted during extinction retrieval in the absence of DP/DTT manipulation. This points to the DP/DTT as sharing a similar function to IL [59, 124–127] (but see ref. [128]) in the modulation of fear inhibition. In contrast, a recent study elegantly showed that activation and inhibition of DP neurons tagged during auditory fear conditioning increases and reduces freezing at retrieval, respectively [151]. The opposing direction of these findings in relation to ours could be driven by a differential contribution of the whole DP/DTT (our manipulations) compared to the selective activation of DP neurons responding to fear acquisition [151], whose connectivity is presently unknown. This is also underscored by a lack of activity signatures by these fear-trapped DP neurons during extinction [151], whereas we saw an effect of DP/DTT soma manipulation on extinction learning and retrieval.

We found that DP/DTT-DMH stimulation had no effect on affective behavior in the OFT or EPM, but acutely suppressed freezing. Notably, in contrast to our DP/DTT soma manipulation, DP/DTT-DMH-mediated suppression of freezing at extinction learning did not affect extinction retrieval, suggesting that the DP/DTT effects on extinction retrieval might be mediated by a distinct downstream target. Furthermore, the increased rearing seen in the DP/DTT-DMH ChR2 animals at baseline post-conditioning might also signal stress-related behavior [152], consistent with this pathway’s role in mediating sympathetic responses and social avoidance following social defeat stress in rats [66]. Alternatively, DP/DTT-DMH activation after foot shock might be triggering flight-like behavior as recently reported for the DP-central amygdala (CeA) pathway [153]. In this comprehensive study, inhibition of the DP-CeA pathway increased freezing during fear conditioning [153], similar to our trends with DP soma inhibition. It is also important to note that the small magnitude of our fear acquisition findings might have been affected by previous exposure to stressful manipulations such as the FST. Interestingly, stimulation of the DP-CeA pathway in a safe context after conditioning reduces freezing [153], similar to our results with DP/DTT and DP/DTT-DMH stimulation. Importantly, the authors reported an anxiolytic effect of inhibition of the DP-CeA pathway in the OFT and EPM, but no effect of activation [153], suggesting that our effects of DP soma manipulation on affective behavior might be independent of the CeA. This is in line with the absence of a strong projection to CeA with our viral strategy. Finally, Chen and colleagues demonstrated that IL downstream projections to the lateral septum and central amygdala (CeA) promote and inhibit anxiety-like behavior, respectively [58], suggesting a high degree of specialization within the vmPFC for the modulation of anxiety-like behavior.

We found that chemogenetic activation of the DP/DTT was sufficient to trigger an increase in serum corticosterone levels. This is consistent with the finding that optogenetic stimulation of the DP/DTT-DMH pathway in stress-naïve animals drives sympathetic responses mimicking those triggered by social defeat stress [66], suggesting that the DP/DTT might transduce stress responses from upstream corticolimbic regions toward downstream autonomic, behavioral/motor [66] and endocrine responses. Importantly, while most of the literature supports an inhibitory effect of the mPFC over HPA secretory responses following stress [69, 154–157] (but see ref. [158]), Radley and colleagues saw a dichotomy between the dorsal and ventral mPFC in modulating stress-induced HPA activation [70]. Specifically, while dmPFC lesions increased corticosterone levels following acute restraint stress, vmPFC lesions decreased corticosterone levels [70]. This suggests that mPFC inhibition of the HPA axis response is mostly driven by the dorsal mPFC, with the ventral portion of the mPFC (possibly spanning the DP/DTT) exerting the opposite effect, consistent with our findings. Importantly, Kataoka and colleagues saw no evidence of DP/DTT inhibition affecting basal autonomic homeostasis [66], consistent with our lack of effects of DP/DTT inhibition in stress-naïve animals. Similarly, inhibition of the ventral portion of the mPFC did not affect baseline corticosterone levels [70].

While we observed that the DP/DTT can modulate neuroendocrine responses, the downstream targets mediating this response are unclear. The hypothalamus is necessary for the effects of frontal cortex stimulation on HPA activity [159], and mediates the autonomic effects of DP/DTT activation [66]. It is hypothesized that (dorsal) mPFC inhibition of HPA activity occurs through stimulation of GABAergic projections from DMH to the paraventricular nucleus of the hypothalamus (PVN) [160]. Consistent with what has been described for the vmPFC [155], we saw DP/DTT projections to hypothalamic nuclei such as the PVN, DMH and lateral hypothalamus (LH). While Radley and colleagues proposed that the stimulating effects of ventral mPFC on HPA activity might be mediated through connections with BNST [70], studies suggest an inhibitory effect of the BNST over HPA activity following acute stress [161], and fail to see recruitment of mPFC-BNST projections following stress [157, 162]. Altough we see DP/DTT projections to the BNST, their contribution to the regulation of DP/DTT effects on HPA secretory activity is unknown.

Overall, our data expand an emerging literature examining the contributions of the DP/DTT to the modulation of psychosocial stress and sympathetic responses by revealing the DP/DTT as a regulator of anxiety-like, passive coping and fear extinction behaviors. As its topography allows for the integration of higher order information toward autonomic and sympathetic effectors, whose dysfunction is often a feature of anxiety disorders, the DP/DTT may be uniquely placed to mediate the effects of stress on affective function. Our findings support further specialization across the dorsoventral axis of the mPFC in the processing of anxiety- and depression-like behavior and stress responsivity, in line with rising evidence of pathway-specific modulation of anxiety-like behavior among mPFC subregions.

Author contributions

Designed Research: JJB, AK, MAC. Performed Research: JJB, AK, RA, JW, FV, HP, ACS, AEC, AP, SDZ, MAC. Analyzed/Interpreted Data: JJB, AK, JW, FV, AEC, AP, MAC. Wrote the paper: JJB, AK, MAC. All authors reviewed and approved the manuscript.

Funding

This work was supported by grants from CIHR (PJT 399790), Human Frontier Science Program Organization (CDA00009/2018 and RGY0072/2019), the SickKids Foundation and Canadian Institutes of Health Research (CIHR) – Institute of Human Development, Child and Youth Health (NI19-1132R), and Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-06344) to MAC.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41386-024-01795-5.