An amygdala located in the anterior hypothalamic circuit controls sensitivity to stress

Animals

All mice were bred on a C57BL/6J genetic background and were approximately 2 to 6 months old at the start of testing. Male C57BL/6J mice were obtained from Jackson Laboratories for whole-brain immediate early genetic imaging as well as pan-neuronal and axonal terminal inactivation (Jackson Laboratories, catalog no. 000664). Studies with homozygous GAD2-IRES-Cre mice (Jackson Laboratories, catalog no. 028867) used an equal mixture of female and male mice raised in-house. Importantly, we observed no gender differences in the behaviors studied, which is consistent with our previous work.¹¹and we observed no gender differences in our neural recordings. After initially observing no effect of sex, sex was dropped as a variable in the final reported analyses. The number of male and female mice per experiment is shown in Supplementary Table 3. Animals were housed in a temperature- and humidity-controlled vivarium on a 12- to 12-h light-dark cycle (lights on at 7 a.m.), and all handling and behavioral tests took place during the light phase. All experimental procedures were approved by the Icahn School of Medicine at Mount Sinai IACUC. Sample sizes were determined based on the laboratory’s previous research using the stress sensitization procedure described.¹¹ and the laboratory’s published work on calcium imaging^36.55. Where possible, the experimenters were blind to the mice’s group membership. During the experiment, experimenters were blind to viral condition, but could not be blinded to stress group membership or laser condition when these manipulations were in progress. All analyzes were automated and performed blinded to the experimental group, including calcium imaging analysis. All mice were randomly assigned to groups.

Surgery

For all surgical procedures, anesthesia was induced with 5% isoflurane and then maintained at 1–2%. Body temperature was maintained during surgery and recovery with a heating pad under the animal, and eye ointment was applied to lubricate the eyes. All surgical procedures followed aseptic surgical technique. After the operation, the animals received 20 mg kg⁻¹ ampicillin and 5 mg kg⁻¹ carprofen (subcutaneously) daily for 7 days, as well as body weight and general condition were monitored daily. Animals that underwent GRIN lens implantation were also treated with 0.2 mg kg⁻¹ dexamethasone (subcutaneously) for 7 days after surgery. All viruses were perfused through a glass micropipette at 2 nl s⁻¹ using a Nanoject III (Drummond Scientific). After viral infusion, an additional 5–10 min was allowed to elapse before retracting the micropipette. The micropipettes were slowly retracted at 0.1 mm min⁻¹ for 3 to 5 minutes before being retracted at a faster rate. All contact details given below relate to Bregma.

Calcium imaging

For AHN imaging^GAD neurons, 300 nl of AAV1-syn-flex-GCaMP6f-WPRE (1.9 × 10¹³ genome copies (GC) ml⁻¹) was perfused into the AHN of GAD2-IRES-CRE mice (anteroposterior (AP), -0.5; mediolateral (ML), 0.5; dorsoventral (DV), -5.2). For BLA imaging^AHN neurons, the same GCaMP6f virus was injected into the BLA of wild-type mice (AP, -1.6; ML, 3.1; DV, -5.25) and a cocktail of AAVrg-ef1α-Cre (7.3 × 10¹² CG ml⁻¹) and AAV5-hSyn-DIO-mCherry (7.3 × 10¹² CG ml⁻¹) was infused into the AHN. mCherry was used to localize placement in the AHN. A 0.6 mm diameter needle was then slowly lowered to 0.2 mm above the injection site and then retracted to create space for the GRIN lens (needle lowered three times). Finally, a GRIN lens (0.6 × 7.3 mm, Inscopix, catalog no. 1050-004597) was lowered to the same coordinates and fixed to the skull using cyanoacrylate and dental cement. The lens was then coated with Kwik-Sil (World Precision Instruments) followed by a thin layer of dental cement to protect the lens before base placement. Four to six weeks later, while the animals were under anesthesia, the lens was uncovered and a baseplate connected to a UCLA miniscope (v.4.4, OpenEphys) was lowered toward the lens until the optimal field of view was identified. The baseplate was then fixed to the skull with cyanoacrylate and dental cement. The miniscope was then detached and a dust cover was connected to the base plate to protect the lens when not taking pictures. For BLA^AHN animals, a head bar was also attached to the base plate using superglue.

Optogenetics

For optogenetic silencing of AHN neurons, 150 nl of AAV5-hSyn-SIO-stGtACR1-FusionRed (2.1 × 10¹³ CG ml⁻¹), or AAV5-hSyn-DIO-mCherry (1.1 × 10¹² CG ml⁻¹), was perfused at the following coordinates at an angle of 20 degrees: AP, −0.7; ML, 0.5; VM, −5.5. For pan-neuronal silencing, 150 nl of AAV9-hSyn-Cre-hGH (1 × 10¹² CG ml⁻¹) was co-infused. Subsequently, the optical fibers (diameter 200 µm, numerical aperture 0.5, RWD Life Science) were lowered to 0.6 mm above the injection site, also at an angle of 20 degrees. Optical fibers were fixed to the skull with cyanoacrylate and dental cement. For optogenetic deactivation of the BLA^AHN terminals, 125 nl of AAV5-CaMKII (0.4)-eOPN3-mScarlet-WPRE (1.05 × 10¹³ CG ml⁻¹) or AAV5-hSyn-EGFP (1.1 × 10¹² CG ml⁻¹), was infused into the amygdala (AP, −1.4; ML, 3; DV, −5.3). Optical fibers were implanted above the AHN as described above.

Chemogenetics

For chemogenetic activation of AHN neurons, 100 nl of AAV5-hsyn-DIO-HM3Dq-mCherry (2.5 × 10¹³ CG ml⁻¹) or AAV5-hSyn-DIO-mCherry (4.5 × 10¹² CG ml⁻¹), was perfused at the following coordinates at an angle of 20 degrees: AP, −0.5; ML, 0.5; VM, −5.5. For chemogenetic silencing of AHN inputs, a mixture of 100 nl of rgAAV-efla-Cre (7.3 × 10¹² CG ml⁻¹) and AAV8-hSyn-EGFP (1.5 × 10¹² GC;ml⁻¹) was infused at the following coordinates: AP, −0.7; ML, 0.5; VM, −5.5. Separately, 150 nl of AAV5-hSyn-DIO-hM4Di (2.4 × 10¹³ CG ml⁻¹) or AAV5-hSyn-DIO-mCherry (7.3 × 10¹² CG ml⁻¹) were infused into the amygdala (AP, -1.4; ML, 3.1; DV, -5.2) or vHC (AP, -3; ML, 3.2; DV, -4.5).

Retrograde tracing

rgAAV-hSyn-EGFP (75 nl; titer, 1.36 × 10¹³ CG ml⁻¹) was infused unilaterally at the following coordinates: AP, −0.5; ML, 0.5; VM, −5.5.

Behavioral tests

Individual housing and habituation

All animals were housed alone for 1–2 weeks before beginning behavioral testing. During this time, they were handled in the vivarium for 1 minute per day for 3 to 5 days. Additionally, the animals were accustomed to being transported to the laboratory for 3–5 days, where they were also handled. Animals participating in the chemogenetic experiments were accustomed to being restrained three times during this period. Animals in the Miniscope and optogenetic experiments were habituated to the connection to the Miniscope and/or patch cords approximately five times. During each of these habituation sessions, animals were connected to the miniscope and/or patch cords and returned to their home cage with the lid removed while remaining connected for 3 to 5 minutes. For experiments involving head fixation, in addition to the habituation procedures described above, animals were habituated to the head restraint for 5–6 days, starting with 5 min and continuing up to 20 min.

Experimental contexts

Stressor 1 (footshock stress) and Stressor 2 (auditory stressor), as well as their respective booster sessions, took place in unique experimental contexts, consisting of very distinct visual, olfactory, auditory, and spatial cues. For Stressor 1, animals were transported from the vivarium in their cages on a trolley to the experimental testing room, which was well lit and had an air filter producing ambient sound. Animals were then placed in a well-lit experimental testing chamber with a grid floor (Med Associates) scented with 5% Simple Green solution. For Stressor 2, with the exception of the iDISCO experiment (see details in the Stressor 2 description below), animals were transported from the vivarium in P1000 pipette tip boxes and transported in a dark cardboard box to the experimental testing room, which was dark except for a faint red light. Animals were then placed in a dark testing chamber (Med Associates) with a flat Plexiglas floor and a curved back wall. The chamber was scented with 1% acetic acid solution.

Stress factor 1

After a 5 min baseline exploration period, animals received ten 1 s, 1 mA scrambled foot shocks, with an intershock interval of 30 s. The animals were removed from the test chamber 30 seconds after the last shock. For the iDISCO experiment, animals received the same number of shocks, pseudo-randomly distributed over a 60-min session. When optogenetic inhibition of cell bodies was applied during stressor 1, blue laser light was administered continuously, starting 30 seconds before the first shock and continuing until the end of the session. See “Chemogenetic and Optogenetic Actuation” for laser parameters.

Stress factor 2

For most experiments, after a 3 min baseline period, animals were exposed to a single loud auditory stimulus (3 s, 125–130 dB white noise, 0 ms rise time) delivered through a loudspeaker attached to the chamber wall. Animals were removed 10 seconds later and returned to the vivarium. For the eOPN3 experiment, mice were removed from the chamber after 180 seconds, given the potentially slow nature of metabotropic signaling. For the iDISCO experiment, Stressor 2 was administered in the home cage to avoid transport-evoked c-Fos. For calcium imaging, a series of five auditory stimuli were presented, allowing us to define whether cells respond reliably to the auditory stimulus. Here, the baseline period was 2 minutes and the interstimulus interval was 2 minutes. For optogenetic inhibition of AHN neurons during Stressor 2, laser light was administered continuously throughout the session. For optogenetic inhibition of the amygdala at AHN terminals, light illumination began 15 s before stimulus onset and…