Solutions.

The medium used for slice incubation and superfusion, BBS, contained (in millimolar) NaCl 126, KCl 2.5, NaH₂PO₄ 1.25, NaHCO₃ 26, CaCl₂ 2, MgCl₂ 1, and glucose 25, bubbled with 95% O₂–5% CO₂. The slicing medium, GCS, contained (in millimolar) KGluconate 130, KCl 15, EGTA 2, Hepes 20, glucose 25, and D-AP5 0.05 (to block NMDA receptors), pH-adjusted to 7.4 with NaOH. This high-potassium/EGTA (intracellular-mimicking) medium was used solely during slicing at 4 °C (42, 44) to enable better protection despite unavoidable mechanical membrane damage produced by slicing. Immediately following slicing, slices were immerged in a recovery medium (MCS) for a transition from the high-K⁺, low-Ca²⁺ GCS to the low-K⁺, high-Ca²⁺ BBS. MCS contained (in millimolar) d-mannitol 225, KCl 2.5, NaH₂PO₄ 1.25, NaHCO₃ 25, and glucose 25. All superfusion solution contained 10 µM GABAzine. For patch-clamp experiments, normal intracellular (pipette) solution contained (in millimolar) KMeSO₄ 145, NaCl 6, Hepes 10, K_2.5EGTA 0.5, MgCl₂ 1, MgATP 4, and Na₂GTP 0.5, pH-adjusted to 7.35 with KOH. This medium was stored at −80 °C. Alexa Fluor 594 hydrazide was added at 15 µM just before patching.