Alexa 568

In this study, we used several different charged (negative, positive, and neutral) molecules of fluorescent dyes. Alexa-488-biocytin, Alexa-568, and sulforhodamine-101 were purchased from Molecular Probes. Lucifer Yellow, sulforhodamine-B, 1,1-Diethyl-2,2-cyanine iodide (decynium22, D22) 2-NBD-glucose, and 4-(4-(dimethylamino)-styryl)-N-methylpyridinium, (ASP+) were obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). For testing the compounds, one of these negatively or positively charged, or polar molecules, was added to the ICS at the following concentrations: 2 mg/mL (~3.6 µM) 2-NBD-Glucose [59 (link)]; 2 µM ASP+ [72 ]; 1 µM D-22 [72 ,73 (link)]; 1 µM sulforhodamine-101 [74 (link),75 (link),76 (link)]; 100 µM Alexa 488-biocytin [65 ]; 1 mM Lucifer Yellow [24 (link),77 (link)]; 200 µM Alexa 568 [65 ]; and 2 mM sulforhodamine-B [57 (link)].
Carbenoxolone (200 µM CBX), a gap junction uncoupler, used in this study to block fluorescent dye propagation was purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA).

Calcium imaging was performed as previously described (Müller and Davis, 2012 (link); Müller et al., 2015 (link)). Final stocks of OGB-1 488 (1 mM, Sigma) and Alexa-568 (5 mM, Sigma) were prepared in HL3 (0 mM Ca²⁺). Third instar Drosophila larvae was dissected and incubated on ice for 10 min in HL3 with zero calcium (1 mM OGB-1; 5 mM Alexa 488, Invitrogen). Indicators were removed and larvae were washed for 10 min with HL3, then placed into the recording chamber for imaging. A scanning confocal microscope (Ultima, Prairie Technologies) with a 60× objective (1.0 NA, Olympus) was used for imaging. 488 nm excitation wavelength from a krypton-argon laser used for excitation and emitted photons were collected through a pinhole at a photocathode photomultiplier tube (Hamamatsu). All line scans were performed at type 1b boutons of muscle 6/7, segments A2-A3. Loading efficiency of the dye was assessed by the intensity of co-loaded Alexa 568. Single stimuli (1 ms) and stimulus trains (5 pulse, 1 ms duration, 50 Hz) were used. Changes in the fluorescence were quantified as previously described (Müller and Davis, 2012 (link); Müller et al., 2015 (link)).

Measurements were performed at room temperature in a recording chamber that was perfused with oxygenated aCSF. Pyramidal neurons from the CA3 area were visualized by means of a BW51WI microscope (Olympus, Tokyo, Japan) equipped with an oblique illumination condenser. Visually guided patch-clamp recordings of CA3 pyramidal neurons were performed in whole-cell configuration with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) in voltage-clamp mode. The pipette solution contained 122 mM K-gluconate, 2.5 mM MgCl₂, 5.6 mM Mg-gluconate, 5 mM K-HEPES, 5 mM H-HEPES, 5 mM Na₂ATP, 1 mM EGTA, and 2.5 mM biocytine (pH adjusted to 7.4 with KOH) and the fluorescent dye Alexa 488 or Alexa 568 (10 μM; Sigma-Aldrich). The electrodes had an input resistance of 4–8 MΩ. Fast synaptic transmission that is mediated by NMDA and γ-aminobutyric acid (GABA) receptors was blocked by adding 50 μM DL-AP5 and 10 μM SR 95531 hydrobromide (gabazine). Recordings were sampled at 20 kHz with an analog-to-digital converter (DIGIDATA 1440A, Axon Instruments, Union City, CA, USA) and displayed by means of Clampex software.