Tc 324b

To measure the temperature of microenvironment, we employed electrical resistance thermometry using a glass microelectrode (Palmer and Williams, 1974 (link); Shapiro et al., 2012 (link); Yao et al., 2009 (link)). To elucidate the relationship between the electrical resistance of an electrode and the temperature of the external saline solution (Xiang et al., 2010 (link)), we monitored the electrical resistances at various values of temperature of the solution. First, we heated the saline solution in a petridish up to 50°C with an inline heater (SF-28, Warner Instruments, Hamden, CT) that was regulated by a thermo-controller (TC-324B, Warner Instruments). Then, we turned off the heater and recorded the electrical resistances (R) of the electrode and the temperatures (T) of the solution simultaneously during natural cooling. The electrical resistances of glass microelectrodes were 5–10 MΩ at ambient temperature (~25°C). We measured the electrical resistances by giving square pulses (50 ms, 10 mV; 1 Hz) in the voltage clamp mode. The reciprocal of temperature (1/T) was plotted against the log of the electrical resistance (log R), so that the Arrhenius equations were estimated as a R-T transformation formula by linear regression.

Neural retinas were prepared for recordings as described in detail with minor modifications17 (link). Briefly, rodents were anesthetized with sevoflurane (Abbott Japan, Osaka, Japan) and sacrificed. The eyes were enucleated and the neural retinas were isolated from the RPE and sclera and vitreous under dim red light. After trimming 2 to 3 mm off of the margins of the retina, the retina was mounted over a MED64 probe with the ganglion cell against the electrodes. The MED64 probe consisted of 64 electrodes (50 μm × 50 μm square, interpolate distance 150 μm, MED-P5155, Alphamed Scientific Inc., Osaka, Japan). The MED probes were treated with 0.1% polyethyleneimine (Sigma-Aldrich, St Louis, MO, USA) in 25 mM borate buffer (pH 8.4) over night at room temperature.
Custom-made anchors were made of stainless steel washers (weighing 1.0 or 2.0 g), with nylon and polyurethane mesh attached with glue for every experiment. Then, the mounted retina was placed in a chamber and continuously perfused with bicarbonate-based Ames’ medium (Sigma-Aldrich, St Louis, MO, USA) gassed with 95% O₂ and 5% CO₂ maintained at 34–37 °C with a temperature controller (TC324B, Warner Instruments, Hamden, CT).

Recordings were performed in a chamber perfused with ACSF consisting of the following (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 2 MgCl₂, 2 CaCl₂, 26 NaHCO₃, and 10 glucose, held at 32–34 °C using an inline heater (TC-324B, Warner Instruments). Cells were visualized via infrared differential interference contrast under an Olympus BX51WI microscope equipped with a Dage-MTI IR-1000 camera. All recordings were made using borosilicate glass pipettes (3–6 MΩ) filled with intracellular solution containing (in mM): 133 K gluconate, 1 KCl, 2 MgCl₂, 0.16 CaCl₂, 10 HEPES, 0.5 EGTA, 2 Mg-ATP, and 0.4 Na-GTP (adjusted to 290 mOsm and pH 7.3). For NAc recordings, we used epifluorescence to target areas with strong eYFP expression in aPVT^CRF synaptic afferents. Putative CINs were differentiated from neighboring MSNs by their larger cell bodies and the presence of rebound firing following a hyperpolarizing current step^{56 (link)}. Pair of MSNs and CINs located within 50 µm and at similar depth were recorded in sequence^{57 (link)}. For PVT recordings, neurons labeled retrogradely from the NAc were targeted using epifluorescence. Excitatory postsynaptic responses were evoked using blue light pulses (1 ms) generated by an LED light source (UHP-T-450- EP, Prizmatix), and delivered through a ×60, 0.9 NA water-immersion objective (Olympus).