Fn e600

Entire procedures were approved by Institutional Animal Care and Use Committee in Anhui, China. The cortical slices (400 μm) were made from FVB-Tg(Gad GFP)4570Swn/J mice (Jackson Lab, Bar Harbor, ME 04609, USA) in postnatal day 22–25. These mice were anesthetized by inhaling isoflurane and decapitated by guillotine. The cortical slices in coronal direction were cut with Vibratome in the oxygenated (95% O₂/5% CO₂) artificial cerebrospinal fluid (ACSF) in concentrations (mM) of 124 NaCl, 3 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 0.5 CaCl₂, 4 MgSO₄, 20 dextrose, and 5 HEPES (pH 7.35 at 4°C). These slices were held in the oxygenized ACSF (124 NaCl, 3 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 2.4 CaCl₂, 1.3 MgSO₄, 10 dextrose and 5 HEPES with pH 7.35) at 25°C for 2 hours. Each slice was then transferred to a submersion chamber (Warner RC-26G) that was perfused by the oxygenated ACSF at 31°C for whole-cell recording [47 (link),48 (link)]. Chemical reagents were purchased from Sigma.
GABAergic neurons for whole-cell recordings were selected in layer II~III of the sensory cortices based on GFP-labeled neurons under the DIC-fluorescent microscope (Nikon, FN-E600, Japan), in which an excitation wavelength was 488 nm. The neurons demonstrated fast spiking and less adaptation in spike amplitudes and frequencies, the typical properties for interneurons [49 (link)–52 (link)].

The cortical slices (400 μm) were made from CR-formation F1 mice that were anesthetized by inhaling isoflurane and decapitated by a guillotine. The slices were sectioned by a Vibratome in the oxygenated (95%O₂/5%CO₂) artificial cerebrospinal fluid (ACSF), in which the chemical concentrations (mM) were 124 NaCl, 3 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 0.5 CaCl₂, 4 MgSO₄, 10 dextrose, and 5 HEPES, pH 7.35 at 4 °C. The slices were held in the oxygenated ACSF (124 NaCl, 3 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 2.4 CaCl₂, 1.3 MgSO₄, 10 dextrose, and 5 HEPES, pH 7.35) at 25°C for 2 hours, and then were transferred to a submersion chamber (Warner RC-26G) perfused with the oxygenated ACSF at 31°C for whole-cell recording [54 (link)].
Electrophysiological recordings on barrel cortical neurons in layers II-III were done under DIC-fluorescent microscope (Nikon FN-E600, Japan), in which the wavelength at 488 nm excited GFP and the wavelength at 575 nm excited YFP. GABAergic neurons appeared basket shape and fast spiking with less adaptation in spike amplitudes and frequency [55 (link), 56 (link)]. Glutamatergic neurons showed pyramidal shape and regular spikes with adaptations of spike amplitude and frequency [51 ]. The cerebral slices were coronal sections including the barrels correspondent to the projection from long whiskers that were stimulated in paired-WS/OS training.

Cultured hTM/pTM cells were loaded with 5 μM Fura-2-AM for 15–30 minutes and were perfused with isotonic saline (pH 7.4) containing (in mM): 98.5 NaCl, 5 KCl, 3 MgCl₂, 2 CaCl₂, 10 HEPES, 10 D-glucose, 93 mannitol. Epifluorescence imaging was performed as described22 (link)23 (link) using inverted Nikon Ti or upright Nikon E600 FN microscopes with 20x (0.75 N.A. oil) and 40x (1.3 N.A. oil & 0.8 N.A. water) objectives and Nikon Elements software. In a subset of experiments, [Ca²⁺]_pTM levels were calibrated using the standard equation (K_d at RT = 224 nM). Results represent averages across cells (3–6 slides, each containing 20–40 cells) from at least three separate experiments.