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.