Electrophysiological Recordings of NAc Synaptic Inputs

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).