Grin lens

A Gradient-Refractive Index (GRIN) lens and metal headcap were implanted following previously described procedures (Namboodiri et al., 2019 (link)) with the following modifications. In most mice, once the dura was removed from the craniotomy, we performed two injections of 0.5 $\mu L$ of virus (1 $\mu L$ total) containing the GCaMP gene construct (AAVDJ-CamKIIa-GCaMP6s, $5.3*{10}^{12}$ viral particles/mL from UNC Vector core lot AV6364) using a glass pipette microinjector (Nanoject II) at Bregma +1.94 mm AP, 0.3, and 1.2 mm ML, –2 mm DV. Ten minutes elapsed before the microinjector withdrawal to allow the virus to diffuse away from each infusion site. Then, mice were implanted with a 1 × 4 mm GRIN lens (Inscopix) aimed at +1.94 mm AP, 0.6 mm ML, and –1.8 mm DV. A subset of mice did not receive viral injections; instead, a lens with the imaging face coated 1 $\mu L$ of the GCaMP6s virus mixed with 5% aqueous silk fibroin solution (Jackman et al., 2018 (link)) was implanted at the same coordinate. GCaMP expression and transients were similar in both preparations. Mice were allowed to recover for at least 5 weeks before experiments began.

Integrated microendoscopic GRIN lens (0.5 mm in diameter × 6 mm in length, Inscopix, #1050-002211) were implanted into the target areas of the mice for monitoring of calcium signals. For data analysis, fluorescence videos were processed offline with Inscopix data processing software (version 1.1.6).

Crh-IRES-Cre;Ai148 mice were maintained under isoflurane anesthesia in the stereotaxic apparatus. For fiber photometry, a 400 µm diameter mono fiber optic cannula (Doric Lenses, MFC_400/430/0.48_5mm_MF2.5_FLT) was implanted dorsal to the PVN (AP, −0.7 mm; L, −0.2 mm from the bregma; DV, −4.5 mm from the dura. The GRIN lens (7.3 mm length; Inscopix) was lowered dorsal to the PVN at a 100 µm/min speed using a motorized stereotaxic apparatus. The implantations were targeted to the PVN and were affixed to the skull with METABOND® and dental cement. At least one month after lens implantation a baseplate was installed on the head. Experiments started after an additional two weeks of recovery and handling.

Eight-week-old mice were anaesthetized with 1.5–3.0% isoflurane and placed in a stereotaxic apparatus (Kopf Instruments). AAV9.Syn.GCaMP6s.WPRE.SV40 were packaged and supplied by UPenn Vector Core at titers 7.5 × 10¹³ viral particles per ml and viral aliquots were diluted prior to use with artificial cortex buffer to a final titer of 5 × 10¹² viral particles per ml. After performing a craniotomy, 100 nl of virus was injected into the dPAG (coordinates in mm, from skull surface): −4.20 anteromedial, −0.85 lateral, −2.3 depth, 15-degree angle. Five days after virus injection, the animals underwent a second surgery in which two skull screws were inserted and a microendoscope was implanted above the injection site. A 0.5 mm diameter, ~4-mm-long gradient refractive index (GRIN) lens (Inscopix, Palo Alto, CA) was implanted above the dPAG (−2.0 mm ventral to the skull surface) (Resendez et al., 2016 (link)). The lens was fixed to the skull with cyanoacrylate glue and adhesive cement (Metabond; Parkell, Edgewood, NY, USA). The exposed end of the GRIN lens was protected with transparent Kwik-seal glue and animals were returned to a clean cage. Two weeks later, a small aluminum base plate was cemented onto the animal’s head on top of the previously formed dental cement. Animals were provided with analgesic and anti-inflammatory (carprofen).