Sulforhodamine 101

Worms were immobilized on 2% agarose pads with WormGlu (GluStitch), dissected, and patch-clamped as described in Eastwood et al. (2015) (link). Recordings were performed on the ALMR neuron due to geometric constraints of the stimulator system; ALMR is bilaterally symmetric to the previously used ALML neuron. The extracellular solution contained (in mM): NaCl (145), KCl (5), MgCl₂ (5), CaCl₂ (1), and Na-HEPES (10), adjusted to pH 7.2 with NaOH. Before use, 20 mM D-glucose was added, bringing the osmolarity to ~325mOsm. The intracellular solution contained (in mM): K-Gluconate (125), KCl (18), NaCl (4), MgCl₂ (1), CaCl₂ (0.6), K-HEPES (10), and K₂EGTA (10), adjusted to pH to 7.2 with KOH. Before use, 1 mM sulforhodamine 101 (Invitrogen) was added to help visualize successful recording of the neuron.
Membrane current and voltage were amplified and acquired with an EPC-10 USB amplifier and controlled through Patchmaster software (HEKA/Harvard Biosciences). The liquid junction potential between the extracellular and intracellular solutions was −14 mV and was accounted for by the Patchmaster software. Data were sampled at 10 kHz and filtered at 2.9 kHz.
Electrophysiology source data from Eastwood et al. (2015) (link) are available upon request.

Bicuculline, strychnine, tetrodotoxin, 6,7-dinitroquinoxaline-2,3-dione (DNQX), L-glutamic acid were obtained from Sigma (St. Louis, MO, USA). (2S,3S)-3-[3-[4-(Trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), D-2-amino-5-phosphonopentanoate (D-AP5) were obtained from Tocris Bioscience (Minneapolis, MN, USA). Sulforhodamine 101 and LPS-RS were purchased from Invitrogen (San Diego, CA). [³H] L-glutamic acid was obtained from Perkin Elmer.

Oxygenated (95% O₂, 5% CO₂) artificial cerebrospinal fluid (ACSF) contained in mM: 129 NaCl; 1.23 NaH₂PO₄; 10 glucose; 1.6 CaCl₂.H₂O; 3 KCl; 21 NaHCO₃; 1.8 mM MgSO₄. To induce epilepsy MgSO₄ was eliminated and 2 mM KCl was added (low-[Mg²⁺] ACSF). Stock solutions of cell permeant Fluo-4 AM (2.5 mM, Life Technologies) and Oregon Green BAPTA-1 AM (OGB-1, 800 μM, Life Technologies) were diluted in 20% Pluronic F-127 (Life Technologies). Final DMSO concentration was 0.16% (Fluo-4) or 0.2% (OGB-1), which did not significantly altered epileptiform activity. Astrocytic γ-aminobutyric acid (GABA) transporter inhibitor SNAP-5114 (Tocris) stock solution (100 mM) was diluted in DMSO (final DMSO concentration in these experiments: 0.1%). Stock solution (50 mM, Sigma) of carbenoxolone hemisuccinate (CBX) was diluted in distilled water. Stock solution of astroglia-specific marker dye sulforhodamine101 (SR101, 10 mM, Invitrogen) was diluted in distilled water.

One day prior to each glucose depletion assay, a single colony was inoculated in 5 mL of galactose media and grown at 30°C at 250 rpm overnight. On the day of each experiment, 1 mL of overnight culture was transferred into 50 mL of glucose medium and grown to OD₆₀₀ 0.3–0.6 (∼ 8 hr at 30°C, 250 rpm) before loading into the microfluidic chip. The setup of the microfluidic device and the time-lapse fluorescence microscopy have been described previously [15 (link), 28 (link)]. Briefly: A microfluidic chip was first primed with water. Five reservoirs were connected to the chip using 0.02in x 0.06in Tygon Micro-Bore tubing (Saint Gobain Performance Plastics) with 23-gauge Luer stub adapters (Becton Dickinson) and connection pins. Two reservoirs contained 20 mL of galactose and glucose media. Fluorescent tracer dye (Sulforhodamine 101, 1 μg/ml, Invitrogen) was added to glucose media to track the environmental concentration of glucose [15 (link)]. Two other reservoirs acted as waste outlets. The fifth reservoir contained the yeast culture. The chip was left in the growth chamber, attached to the microscope, for 30 min to allow it to reach thermal equilibrium at 30°C. Cells were loaded into the cell trapping chamber and were imaged every 5 min for 15 h in red, yellow fluorescence and transmitted light while a preprogrammed controller dynamically changed the input media (see below).