Cyclothiazide

For field EPSP (fEPSP) recordings, baseline responses were collected at 0.07 Hz with a stimulation intensity that yielded a half-maximal response. Once a stable baseline response was acquired, excitatory transmissions were evoked at a set series of increasing stimuli using an isolated pulse stimulator (A-M Systems). fEPSP slopes and fiber volleys were then interpolated via linear fits to obtain an input/output model of basal evoked excitatory transmissions. For paired-pulse facilitation, pairs of peak amplitude responses were obtained at indicated inter-pulse intervals and divided to obtain the PPF ratios for each inter-pulse interval. For release probability, a single 20 Hz, 5-s stimulation was given in the presence of 2 μM kynurenic acid (Tocris), and 0.05 μM cyclothiazide (Tocris). Signals were filtered at 2 kHz and digitized at 1 kHz under control of Multiclamp 700B Amplifier and Digidata 1550 Digitizer. The acquired data were analyzed using Clampfit 10. theta burst stimulation (TBS)-long-term potentiation (LTP) was induced by four episodes of TBS with 10 s intervals. TBS consisted of 10 stimulus trains delivered at 5 Hz; each train consisted of four pulses at 100 Hz. High-frequency stimulation (HFS)-LTP was induced by a single delivery of a 100 Hz, 1-s stimulus. Average responses (±SEM) are expressed as percentages of baseline responses.

Drugs used were D-AP5, SR 95531, NBQX, Cyclothiazide, Amiloride and QX314.HCl from Tocris Bioscience UK or Sigma-Aldrich.

Neurobasal^® medium, B-27 supplement, antibiotic-antimycotic, calcein acetoxymethyl ester (calcein-AM), JC-1 and rhodamine 123 were purchased from Invitrogen (Barcelona, Spain). N-Methyl-D-aspartic acid (NMDA), mdivi-1, HBSS, glycine, poly-L-ornithine, glutamine, thapsigargin, tunycamicin, EGTA and FCCP were obtained from Sigma (St. Louis, MO, USA). α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and cyclothiazide (CTZ) were obtained from Tocris Biosciences (Minneapolis, MN, USA). Cytotox 96^® for LDH release quantification was purchased from Promega (Madison, WI, USA). The plasmid expressing mitochondria-targeted Ca²⁺ indicator (2mtD4cpv) was kindly provided by Roger Tsien (University of California, San Diego, CA, USA). Lentiviral particles carrying a Drp1-shRNA vector were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).