Kwik cast

To continuously apply muscimol in vivo, we used EVAFLEX (EV40W, DuPont-Mitsui Polychemicals, Japan), the commercial counterpart of ELVAX. Pieces of EVAFLEX (1 mm × 1 mm × 0.2 mm) were prepared [22 (link)]; each piece contained the vehicle or 100 mM of muscimol. To implant the pieces of EVAFLEX, mice were anesthetized using isoflurane; a craniotomy (1.5 mm×1.5 mm) was performed above the primary somatosensory cortex at P12 or P21 in WT and mGluR1-KO mice. After removal of dura using a needle, a piece of EVAFLEX was placed onto the surface of the brain. The craniotomy was sealed with Kwik-Cast (World Precision Instruments, USA), covered with dental cement, and sutured.

Unilateral retrograde tracing from the superior colliculus was performed as described by Nadal-Nicolás et al. [40 (link)]. Mice were anesthetized by an intraperitoneal injection of a mixture of 75 mg/kg body weight ketamine (Anesketin; Eurovet, Bladel, The Netherlands) and 1 mg/kg medetomidine (Domitor; Pfizer, New York City, NY, USA). The mouse head was fixed in a stereotaxic frame and a 2 × 2 mm² cranial window was made above the superior colliculus. The visual cortex was aspirated, and a gelatin sponge soaked in 0.9% saline containing 4% hydroxystilbamidine metanesulfonate (OHSt) and 10% dimethylsulfoxide (Life Technologies, Carlsbad, CA, USA) was applied on the left superior colliculus. The craniotomy was sealed with elastomer (Kwik-cast, World Precision Instruments), and the skin was sutured. After the procedure, mice were given meloxicam (5 mg/kg, Metacam, Boehringer-Ingelheim, Ingelheim am Rhein, Germany) for post-operative analgesia. Anesthesia was reversed with an intraperitoneal injection of 1 mg/kg atipamezole hydrochloride (Antisedan, Pfizer). A lubricant (Vidisic, Bausch + Lomb, Bridgewater, NJ, USA) was used to prevent dry eyes. Mice were sacrificed six days after tracing. Given the unilateral tracing, we used retinas of different, vehicle-injected mice as controls.

To test the possibility of patch cable vibration effects on fluorescent signals captured with TCSPC we devised a benchtop testing system (Supplementary Fig. 3). The tip of patch cable connect to our in vivo photometry system was mounted on an optical table, and a sample tube containing a fluorescent polymer (World Precision Instruments, cat # KWIK-CAST) was placed at the tip of patch cable. Single-photon counting measures of fluorescence using the same laser and detector used for in vivo photometry were carried out for 5 min under conditions in which the patch cable was stable, and when the cable was vibrated by movements simulating those that occur was mice traverse the behavioral apparatuses used in our experiments.

The care and experimental manipulation of animals were performed in accordance with institutional and United Kingdom Home Office guidelines. Homozygous Pvalb-IRES-Cre mice (JAX stock #008069) of both genders were used in this study. At 6 weeks, auditory cortex was injected unilaterally with AAV-EF1a-DIO-hChR2(H134R)-EYFP virus to express selectively channelrhodopsin (ChR2) and EYFP proteins in parvalbumin-positive (PV⁺) interneurons. Mice were anesthetized with 1%–2% isoflurane under aseptic conditions and held using ear bars on a stereotaxic mount (Angle 2, Leica). A small burr hole was made with a dental drill ∼1 m lateral from midline and ∼2.7 mm caudal to bregma. A small glass pipette holding the virus was advanced to reside in auditory cortex; 0.5 μl was then slowly injected into cortex over a period of 15 min. The pipette was then removed and the burr hole sealed with Kwik-Cast (World Precision Instruments), once dry acrylic dental cement was layered over the top forming a hard seal over the site. The area was then cleaned (iodine) and the tissue sealed (Histoacryl, Braun). Analgesia was administered during the procedure via intraperitoneal injection (Carprofen; 5 mg/kg). The animal was then recovered and the virus left to express for 2 weeks, during which time buprenorphine (0.8 mg/kg) jelly was used for postoperative analgesia.