Rc 21bdw

For D1ER measurements in Figure 1H and Figure S3, fluorescence images were acquired from transfected cultured neurons (4–5 DIV) using a Zeiss Axiovert 200M inverted microscope. The bath solution was identical to that used for patch-clamp recording, but omitted TTX. Neurons on glass coverslips were mounted in a sealed-top, diamond-shaped chamber (RC-21BDW, Warner Instruments). Gravity-driven continuous laminar flow (~1 ml/min) through the chamber allowed rapid exchange between control, glutamate- and blocker-containing solutions.

Fast confocal imaging was performed using an EC Plan-Neofluar 40x/1.30 NA oil objective on an Axio-Observer Z1 microscope (Carl Zeiss) coupled to a CSU-X1 spinning disk (Yokogawa), an Evolve 16-bit EMCCD camera (Photometrics), and Slidebook 5.0–5.5 software. DIV 12–15 neurons were grown on glass coverslips and transfected 24–48 hours prior to imaging. Neurons were transfected with pSilencer (empty vector control) or AKAP150 shRNAi; YFP, AKAP79 WT or ΔPKA-YFP/mCh; and RGECO-1 (Addgene 32444) or GCaMP6F (Addgene 40755) as indicated using Lipofectamine 2000 (Invitrogen). Following pretreatment with vehicle or nimodipine, coverslips were mounted in an imaging chamber (Warner Instruments, RC-21BDW) and perfused in normal Tyrode’s saline containing TTX and vehicle or nimodipine for the duration of the experiment. Neurons were maintained at 34°C while single xy-plane were acquired at 0.5 Hz for 1–2 min encompassing a 15 or 30 s 90 mM K⁺ perfusion. Changes in RGECO-1/GCaMP6F fluorescence were measured for regions of interest at the base of a primary dendrite.