Oxytocin Modulation of PPG Neurons

Coronal brainstem slices (200μm) were obtained from PPG-Cre:GCaMP3 mice and used to assess the effects of bath-applied oxytocin on PPG^NTS neuron calcium dynamics using a previously-optimised protocol ^{43 (link)}. Oxytocin was dissolved in aCSF (3mM KCl, 118mM NaCl, 25mM NaHCO₃, 5mM glucose, 1mM MgCl₂, 2mM CaCl₂; pH 7.4) to give a bath concentration of 100nM, based on reports that this concentration elicits robust activation of vagal afferent neurons under ex vivo conditions ^{44 (link)}. Slices were superfused with aCSF for ≥10 minutes, with the final 5 minute period prior to oxytocin application used to determine baseline fluorescence intensity. Slices were then superfused with oxytocin solution for 3-5 minutes, washed with aCSF for ≥10 minutes, then finally superfused with 100μM glutamate for 1 minute as a positive control to confirm imaged neurons were healthy and responsive to glutamatergic input. GCaMP3 fluorescence was excited at 460 ± 25 nm using an LED light source, for 250ms every 5 seconds. Imaging was conducted using a widefield microscope (Zeiss) with 40x water immersion lens and captured at 12-bit on a CCD camera (QClick, QImaging). Data were obtained from 8 experiments (i.e. recordings from single slices) from 3 mice.