In Vivo Single-Unit Extracellular Recordings
Local drug administration. A cannula was lowered into targeted regions at the following coordinates on the right hemisphere (AP relative to bregma, ML relative to midline, DV relative to brain surface): BLA; AP -2.8 mm, ML +4.8 mm, DV -6.8 mm / VP; AP -0.3 mm, ML +2.0 mm, DV -6.8 mm. Drug solution were prepared with Dulbecco’s PBS Buffer: TTX, 25 nmol/0.5 µL/infusion (Tocris, CAS 4368-28-9), kynurenic acid (5 µg in .5 µl ) (Sigma, CAS 492-27-3). Kynurenic acid, a glutamate receptor antagonist, is known to block glutamatergic inputs onto VP (Chang and Grace, 2014). Drugs or vehicle were infused 1 min after cannula lowering by slow manual infusion. Cannula was left in place for an additional 1 minute and then removed before VTA DA neurons recordings.
For acute withdrawal, extracellular recordings were performed 18h up to 72h after the last cocaine self-administration session. For protracted withdrawal, recordings were performed from 26 days to 45 days after the last session. Naïve rats have also been used for recording of DA activity. Since there was no difference in DA activity between naïve rats and saline WD1-3 and saline WD26-45, data have been pooled.
Single-unit extracellular recordings . Rats were anesthetized with isoflurane (5 % induction, 2.5 % maintenance) and placed in a stereotaxic frame. Body temperature was maintained at 37 °C with a temperature-controlled heating pad. The scalp was exposed and burr holes were drilled in the skull overlying the VTA, the BLA or the VP. Extracellular recording electrodes were pulled from glass micropipettes (WPI; impedance 10-20 MΩ for BLA recordings, 6-8 MΩ for VTA recording). The tip of the glass microelectrode was broken to a diameter of 2 µm, and filled with a 2 % Chicago Sky Blue dye (Sigma, CAS 2610-05-01) solution in 2 M NaCl.
For DA neurons, recording electrode was lowered using a microdrive through the right VTA in 9 sequential vertical tracks separated by 0.2mm (Supplementary figure 1). Spontaneously active cells encountered identified as DA neurons (online analysis) were recorded to determine population activity as previously described (Salin et al., 2021). Single-unit activity recorded from the VTA was amplified 10 times, filtered (low pass: 30 Hz; high pass: 16 kHz), and further amplified 50 times (Multiclamp 700b, Axon Instruments, Union City, CA). The signal was digitized at 16 kHz (CED 1401) and acquired on a computer using Spike 2 7.0 software (Cambridge Electronics Design, Cambridge, UK). Three parameters were analyzed: the number of neurons/electrode track, firing rate and the percentage of spikes in bursts.
For BLA recordings, electrode was lowered through the right BLA at the following coordinates: AP -2.8mm to -3.2mm (from bregma), ML +4.6mm to 5.2mm (from midline), DV -7.0mm to -9.5 (from brain surface). Encountered cells in the targeted area meeting BLA putative pyramidal neuron’s criteria (action potential from peak to trough ≥ 0.5ms (Bienvenu et al., 2012) and with a signal-to-noise ratio of 3:1 (or greater) were recorded for at least five minutes. Single-unit signals were amplified 10 times, filtered using a high-pass filter at 30 Hz and a low-pass filter at 10kHz, and further amplified 50 times (Multiclamp 700b, Axon Instruments, USA). The signal was digitized at 16 kHz (CED 1401) and acquired on a computer using Spike 2 7.0 software (Cambridge Electronics Design, Cambridge, UK). Bursts were detected using characteristic parameters of pyramidal neurons (Vitrac et al., 2014) (maximum interval to start a burst: 40 ms, maximum interval to end a burst: 10 ms, minimum interval between burst: 20 ms, minimum duration of a burst: 5 ms and minimum number of spikes in a burst: 2) and analyzed using NeuroExplorer® (Nex Technologies, Colorado Springs, USA).
For all recorded cells, the following electrophysiological parameters were analyzed: basal firing rate, bursting rate, and the percentage of spikes in bursts.