Tpmpa

Perinatal mice are hypothermically anesthetized for 2–3 minutes before surgery, and kept cold for the brief period during surgical injection. Pulled glass micropipettes (1B150F-4; World Precision Instruments) were loaded with AAV-Syn-GCaMP6f or AAV-Syn-jRCaMP1b and then 1–2uL was pressure injected into each ocular vitreous or transverse sinus at P0-P1. Mice are then placed on a temperature-controlled heat pad after surgery and returned to their mothers upon recovery from anesthesia. After retinal injections, we waited 4–5 days for virus to transfect and fill retinal ganglion cell axons. For acute eye injections, either (1) 0.5–1 µl of 10 µM (±)-epibatidine dihydrochloride hydrate (Sigma no. E1145) dissolved in saline, or (2) a cocktail of 1 mM AP5 (Tocris) and 200 µM NBQX (Tocris), or (3) a cocktail of 500 µM 1,2,5,6-Tetrahydropyridine-4-yl-methylphosphinic acid (TPMPA, Sigma), 50 µM gabazine (Tocris), and 5 uM strychnine (Sigma) was pressure injected using a glass micropette inserted into the vitreous.

To distinguish between direct stimulation of RGCs and indirect stimulation of
RGCs through the inner retina, INL to RGC transmission was blocked using excitatory and
inhibitory blockers. If responses are still present after blocking synaptic transmission,
this would indicate RGCs are being directly stimulated. If no responses remain after
blocking synaptic transmission, we can conclude that the INL is stimulated without
involving RGCs directly. We compared untreated PVA-evoked responses to those evoked after
intravitreal injection of saline (2.5μl) and to PVA-stimulation 3–5 min
after intravitreal injection of synaptic blockers (2.5μl). The synaptic blocker
cocktail consisted of two glutamate receptor antagonists (DAP-5 (50 μM) and CNQX
(10 μM), Sigma-Aldrich) and three inhibitory receptor antagonists (picrotoxin 10
μM, TPMPA 50 μM, strychnine 10 μM; Sigma-Aldrich) dissolved in
lactated Ringer’s solution. These concentrations represent the final
concentrations in the vitreous and were calculated using the estimated vitreal volume for
the rat eye (52.5 μl; Sha and Kwong 2006 ).
A complete block of synaptic signaling was characterized by a complete or nearly complete
decline in spontaneous activity within 5 minutes of synaptic blocker injection.