GoKawiilMice
All procedures were approved by the New York University Langone Medical Center Institutional Animal Care and Use Committee in compliance with the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Adult mice 12–38 weeks old were used for all of the studies. Mice were housed at 18–23 °C with 40–60% humidity under a 12 h–12 h light–dark cycle (dark cycle, 22:00 to 10:00), with food and water available ad libitum. For C57 and SW mixed-background mice, we bred the F 1 mixed-background mice by crossing a male C57BL/6N (Charles River, 027), C57 Esr1-2A-cre (Jackson, 017911), Esr1-2A-Flpo (Jackson, 037009), Npy2r-ires-cre (Jackson, 029285), or Oxt-ires-cre (Jackson, 024234) with a female SW (Taconic, SW-F) mouse. For mixed-background Npy2r-ires-cre/Esr1-2A-Flpo double transgenic female mice, we bred the F 3 mixed-background mice by crossing F 2 mixed-background homozygous Npy2r-ires-cre female mice with C57 homozygous Esr1-2A-Flpo male mice. For hybrid Oxtrflox/flox female mice (Jackson, 008471), we bred the F 2 mixed-background mice by crossing F 1 SW and C57 mixed background Oxtrflox/+ mice. These mixed-background breeding strategies enabled cell-type-specific manipulations in aggressive lactating female mice. C57 Esr1-ZsGreen mice were generated and provided by the Y. Xu lab originally and then bred in-house. Stimulus animals were adult (>8 weeks old) and juvenile (15 days old in one case, and 19–28 days old in all other cases) C57BL/6N female and male mice, originally purchased from Charles River and then bred in-house. Stimulus animals were group-housed. After surgery, all test virgin animals were singly housed, and all lactating animals were co-housed with their pups except during the pup-separation procedure. All experiments were performed during the dark cycle (10:00 to 16:00) of the animals.
Viruses
AAV2-CAG-FLEX-GFP (4.00 × 1012 viral genomes (vg) per ml), AAV2-hSyn-GFP (7.30 × 1012 vg per ml), AAV2-Ef1α-DIO-ChR2-eYFP (4.00 × 1012 vg per ml), AAV5-hSyn-Chronos-GFP (2.10 × 1013 vg per ml) and AAV2-Syn-Flex-ChrimsonR-tdTomato (6.00 × 1012 vg per ml) viruses were purchased from the University of North Carolina vector core. AAV1-CMV-Hl-eGFP-cre (2.63 × 1013 vg per ml), AAV2-Ef1α-DIO-hM4Di-mCherry (5.00 × 1012 vg per ml), AAV2-Ef1α-fDIO-mCherry (8.60 × 1012 vg per ml), AAV8-Ef1α-fDIO-GCaMP6s (1.80 × 1013 vg per ml), AAVrg-Ef1α-Flpo (7 × 1012 vg per ml) and AAV8-Ef1α-Coff/Fon-mCherry48 (2.00 × 1013 vg per ml) were purchased from Addgene. AAV2-CAG-FLEX-GCaMP6f-WPRE-SV40 (2.27 × 1013 vg per ml) was custom-made by the University of Pennsylvania vector core facility. AAVDJ-Ef1α-fDIO-hM4Di-mCherry (2.65 × 1013 vg per ml) was custom-made by Vigene Biosciences, and the plasmid was provided by B. Lim at USCD. HSV-hEf1α-LS1L-Flpo (1.00 × 109 vg per ml) was purchased from the Massachusetts General Hospital Neuroscience Center vector core.
Drugs
For chemogenetic inhibition, 5 mg per kg CNO (Sigma-Aldrich, C0832) dissolved in saline was administered i.p. To block OXTRs in the PA and VMHvl, 250 nl per side of 100 µM L-371,257 hydrochloride (Tocris, 2410) in saline was bilaterally injected through the implanted cannulae.
Stereotaxic surgery
Mice (12–20 weeks old) were anaesthetized with isoflurane (1–1.5%) and secured in a stereotaxic apparatus (Kopf Instruments, Model 1900). Viruses and tracers were delivered into the targeted brain regions as previously described16,49. In brief, injections were performed using glass capillaries and a nanoinjector (World Precision Instruments, Nanoliter) at rates of 10 nl min−1 for viruses and 5 nl min−1 for tracers. Only animals with correctly verified injection sites were included in the final analysis (n = 176 out of 194).
For retrograde tracing experiments, 50 nl of Alexa-Fluor-555-conjugated CTB (Thermo Fisher Scientific, C22843) was unilaterally injected into the VMHvl (bregma coordinates: anteroposterior (AP) −1.45 mm; mediolateral (ML) ±0.680 mm; dorsoventral (DV) −5.680 mm). Brains were collected 10 days later. Only mice with confirmed targeting of the VMHvl were included in the final analysis (n = 5 out of 8).
For pharmacogenetic inhibition of PAEsr1 cells, 120 nl of AAV2-hSyn-DIO-hM4Di-mCherry was bilaterally injected into the PA of Esr1-2A-cre mice (bregma coordinates: AP, −2.30 mm; ML, ±2.40 mm; DV, −4.80 mm). Only animals with confirmed bilateral targeting of the PA were included in the final analysis (n = 7 out of 8).
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