Metabolic-Electrophysiological Coupling in Single Neurons

AnimalsAll procedures were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China (Ethical Approval Number: USTCACUC26100124003). Male C57BL/6J mice (4-5 weeks old, Beijing Vital River Laboratory Animal Technology) were group-housed (4-6 per cage) under a 12-h light:12-h dark cycle (23 °C, ad libitum food/water) for 2-3 weeks prior to experiments.Acute Brain Slice PreparationMice were euthanized via rapid decapitation. Brains were dissected and immersed in ice-cold, oxygenated (95% O₂/5% CO₂) cutting solution containing (in mM): 30 NaCl, 26 NaHCO₃, 194 sucrose, 10 D-glucose, 4.5 KCl, 1.2 NaH₂PO₄, and 1 MgCl₂ (pH 7.4, 300–310 mOsm/kg). Coronal hypothalamic sections (270 µm) were prepared using a vibratome (VT1200S, Leica). Slices recovered for 30 min at 32 °C in oxygenated artificial cerebrospinal fluid (ACSF: 124 mM NaCl, 26 mM NaHCO₃, 10 mM D-glucose, 4.5 mM KCl, 2 mM CaCl₂, 1.2 mM NaH₂PO₄, 1 mM MgCl₂; pH 7.4, 300–310 mOsm/kg), then held at 23-25 °C until recording.Combined Electrophysiology and Single-Neuron Mass Spectrometry (SNMS) 1.Electrophysiological Recordings Slices were transferred to a recording chamber perfused with oxygenated ACSF (23-25 °C). Whole-cell patch-clamp recordings from the core region of suprachiasmatic nucleus (SCN) neurons were performed using borosilicate pipettes (3-5 MΩ) filled with 185 mM NH₄HCO₃/80 mM NH₄Cl. Signals were amplified (MultiClamp 700B), digitized (Digidata 1550B), and acquired via pCLAMP (Molecular Devices). Neurons were voltage-clamped at −70 mV. Only cells with tight seals (>1 GΩ) and uncompromised membranes were analyzed. 2.Cytoplasmic Aspiration for SNMS After 1-min recordings of postsynaptic currents (PSCs), gentle negative pressure was applied to aspirate cytoplasmic contents into the pipette. Pipettes were promptly withdrawn and coupled to an Exactive Plus mass spectrometer (Thermo Fisher Scientific).Raw MS files (.raw) were converted to .mzML format.