Mice were anesthetized with 1.5 to 2.0% isoflurane for surgical procedures and placed into a stereotactic frame (David Kopf Instruments, Tujunga, CA). Lidocaine (2%; Akorn, Lake Forest, IL) was applied to the sterilized incision site as an analgesic, while subcutaneous saline injections were administered throughout each surgical procedure to prevent dehydration. In addition, carprofen (5mg/kg) and dexamethasone (0.2mg/kg) were administered both during surgery and for 7 days post-surgery with amoxicillin.
For calcium imaging experiments, mice underwent two separate surgical procedures. First, mice were unilaterally microinjected with 500 nanoliters of AAV1.Syn.GCaMP6f.WPRE.SV40 virus at 50nl/min into the dorsal CA1 using the stereotactic coordinates: −2.1 mm posterior to bregma, 2.0 mm lateral to midline and −1.65 mm ventral to skull surface. Two weeks later, the microendoscope (a gradient refractive index lens) was implanted above the previous injection site. For the procedure, a 2.0mm diameter circular craniotomy was centered 0.5mm medial to the virus injection site. Artificial cerebrospinal fluid (ACSF) was repeatedly applied to the exposed tissue to prevent drying. The cortex directly below the craniotomy was aspirated with a 27-gauge blunt syringe needle attached to a vacuum pump. The microendoscope (0.25 pitch, 0.50 NA, 2.0mm in diameter and 4.79 in length, Grintech Gmbh) was slowly lowered with a stereotaxic arm above CA1 to a depth of 1.35mm ventral to the surface of the skull at the most posterior point of the craniotomy. Next, a skull screw was used to anchor the microendoscope to the skull. Both the microendoscope and skull screw were fixed with cyanoacrylate and dental cement. Kwik-Sil (World Precision Instruments) covered the microendoscope. Two weeks later, a small plastic baseplate was cemented onto the animal’s head atop the previously formed dental cement. Debris was removed from the exposed lens with ddH2O, lens paper and forceps. The microscope was placed on top of the baseplate and locked in a position in which the field of focus was in view, so that cells and visible landmarks, such as blood vessels, appeared sharp and in focus. Finally, a plastic cover was fit into the baseplate and secured by magnets.
For aged DREADD experiments, mice were bilaterally microinjected with 700 nanoliters of Lentivirus CaMK2.hM3Dq.T2A.EGFP/dTomato virus at 100nl/min into the dorsal CA1 using the stereotactic coordinates: −1.80 mm posterior to bregma, +/−1.50 mm lateral to midline, −1.60 mm ventral to skull surface; −2.50 mm posterior to bregma, +/−2.00 mm lateral to midline, −1.70 mm ventral to skull surface.
For calcium imaging experiments, mice underwent two separate surgical procedures. First, mice were unilaterally microinjected with 500 nanoliters of AAV1.Syn.GCaMP6f.WPRE.SV40 virus at 50nl/min into the dorsal CA1 using the stereotactic coordinates: −2.1 mm posterior to bregma, 2.0 mm lateral to midline and −1.65 mm ventral to skull surface. Two weeks later, the microendoscope (a gradient refractive index lens) was implanted above the previous injection site. For the procedure, a 2.0mm diameter circular craniotomy was centered 0.5mm medial to the virus injection site. Artificial cerebrospinal fluid (ACSF) was repeatedly applied to the exposed tissue to prevent drying. The cortex directly below the craniotomy was aspirated with a 27-gauge blunt syringe needle attached to a vacuum pump. The microendoscope (0.25 pitch, 0.50 NA, 2.0mm in diameter and 4.79 in length, Grintech Gmbh) was slowly lowered with a stereotaxic arm above CA1 to a depth of 1.35mm ventral to the surface of the skull at the most posterior point of the craniotomy. Next, a skull screw was used to anchor the microendoscope to the skull. Both the microendoscope and skull screw were fixed with cyanoacrylate and dental cement. Kwik-Sil (World Precision Instruments) covered the microendoscope. Two weeks later, a small plastic baseplate was cemented onto the animal’s head atop the previously formed dental cement. Debris was removed from the exposed lens with ddH2O, lens paper and forceps. The microscope was placed on top of the baseplate and locked in a position in which the field of focus was in view, so that cells and visible landmarks, such as blood vessels, appeared sharp and in focus. Finally, a plastic cover was fit into the baseplate and secured by magnets.
For aged DREADD experiments, mice were bilaterally microinjected with 700 nanoliters of Lentivirus CaMK2.hM3Dq.T2A.EGFP/dTomato virus at 100nl/min into the dorsal CA1 using the stereotactic coordinates: −1.80 mm posterior to bregma, +/−1.50 mm lateral to midline, −1.60 mm ventral to skull surface; −2.50 mm posterior to bregma, +/−2.00 mm lateral to midline, −1.70 mm ventral to skull surface.
