Propranolol p0884

The cholinergic, noradrenergic and glutamatergic Mecamylamine M9020, Yohimbine Y3125, Propranolol P0884, Prazosin P7791, and atropine were purchased from Sigma. Drugs were dissolved in warm cortex buffer and filtered and then injected into cortex using a glass micropipette using a picospritzer. During the initial surgery and AAV injection, a hole was drilled through each coverslip which was ultimately used to form the cranial window. This hole was plugged using kwik-sil and covered with acrylic to prevent desiccation and infection. During imaging, 2 weeks after AAV injection, the kwik-sil was removed, GCaMP6-expressing neurons were imaged for 15 minutes, and then the mouse was removed from the microscope and placed under a dissection microscope. The glass micropipette containing the filtered drugs was then inserted into the cortex via the previously-drilled hole. This permitted the maintenance of the cranial window intact. The mouse was then placed back under the 2-photon microscope, the same neurons were again found, and their activities imaged for another 15 minutes.

Eyes were carefully enucleated and placed in ice-cold PBS. Eyes were dissected in EGM2-MV supplemented (hydrocortisone was omitted) medium (CC3202; Lonza, Walkersville, MD, USA) into posterior eye cups containing sclera, RPE, and choroid complex. The peripheral choroid was separated from the central choroid, cut into 0.5 mm × 0.5 mm pieces, and placed into growth factor reduced Matrigel (#356231; Corning, Bedford, MA, USA) in a 48-well plate on ice. The Matrigel was solidified by incubating at 37°C for 10 minutes. EGM2-MV medium was changed every 2 days. Sunitinib (PZ0012) and propranolol (P0884) were purchased from Sigma (St. Louis, MO, USA). Sunitinib was added on Day 0, and propranolol was added on either Day 0 or Day 2. Pictures were taken on a Nikon Ti2 Widefield microscope (Buffalo Grove, IL, USA) using a 4× objective and Nikon NIS Elements software. Images were analyzed with the Nikon Elements General Analysis. Images were preprocessed with edge detection and segmented with thresholding. The largest area was measured for each image. The central choroidal tissue area was subtracted from the total angiogenesis area.