Generating Transgenic Fly Lines

To generate the UAS-PV and UAS-GluRIIB transgenes, we obtained the cDNAs of PV from Addgene (#17301) and GluRIIB from the Drosophila Genomics Resource Center (DGRC #1374682). We inserted the PV and GluRIIB cDNA sequences into the pACU2 vector^{37 (link)} (#31223; Addgene). To generate SynapGCaMP8f (MHC-CD8-GCaMP8f-Sh), we obtained the SynapGCaMP6f transgenic construct^{24 (link)} and replaced the sequence encoding GCaMP6f with the GCaMP8f⁴⁰ sequence (#162379; Addgene) using Gibson assembly as described³⁹ . Transgenic stocks were generated by Bestgene, Inc (Chino Hills, CA 91709, USA) and inserted into w¹¹¹⁸ (#5905, BDSC) fly strains by P-element-mediated random insertion. To generate the UAS-GluRIIB^IIAtail transgenes, the 5′ fragments of GluRIIB (1–2511 bp) and the 3’ fragment of GluRIIA (2510–2724 bp) were cloned from the cDNAs of GluRIIB and GluRIIA^{65 (link)}. The GluRIIB^IIAtail fragment was then generated by overlap extension PCR from the GluRIIB 5′ fragment and the GluRIIA 3′ fragment. The GluRIIB^IIAtail fragment was then inserted into the pUAST vector^{66 (link)}. The transgenic stock of UAS-GluRIIB^IIAtail was generated by Eppendorf InjectMan (Hamburg, Germany) and inserted into the w¹¹¹⁸ strain.