In anesthetic-free, chronic experiments (n = 5), for the surgery the animals were anesthetized Nutlin-3a in vitro with a mixture (4 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 mg/ml). Silicon probes were implanted above the thalamus attached to a custom-manufactured microdrive. After 1 week of recovery, the probes were moved
gradually, and recordings were made at several depth locations. Tungsten wires (50 μm) were implanted to both primary somatosensory of motor cortices, also in hippocampus in three cases. Three of the five chronic animals yielded narrow spike units of clusterable quality. For juxtacellular recording and labeling, glass micropipettes (20–70 MΩ) filled with 1.5% Neurobiotin (Vector Laboratories) were used. After perfusion, 60-μm-thick coronal brain sections were cut on a Vibratome
and incubated with avidin-biotin-peroxidase complex (Vector Laboratories). The labeled cells were visualized using nickel-intensified diaminobenzidine (DAB) reaction. Labeled neurons and axonal trees were reconstructed using Camera Lucida. In case of dual nRT-VB recording experiments first a silicon probe was lowered into VB, and the receptive field of the multiunit was determined. Next, nRT units with a matching receptive field were then sought with several penetrations of a juxtacellular recording pipette. Lesion experiments were performed by recording a baseline session from VB, followed by iotophoresis of 1% kainic acid (−2 μA, 7 s on/off cycle) Volasertib cost for 20 min without moving the electrodes. Several (3–4) hours later postlesion session was recorded from the same electrode. Parvalbumin-channelrhodopsin and vesicular GABA-transporter-channelrhodopsin mouse strains were generated by crossing PV-cre or vGAT-cre (The Jackson Laboratory) mice with -129S-Gt(ROSA)26Sortm32(CAG-COP4∗H134R/EYFP-Hze (The Jackson Laboratory) reporter strains. For optical stimulation a 473 nm DPSS laser (LaserGlow) was used via a fiberport (Thorlabs) and a patch cord (Thorlabs) to the brain-implanted optic fiber. The optic fiber was either attached to the silicon probe in
also close proximity (<200 μm) of the recording site (axonal stimulation), or inserted directly into the nRT (soma-dendritic stimulation). Light intensity was modulated through the DPSS power supply, with a MATLAB-controlled DAQ-board (National Instruments). Stimulus strength was adjusted to span a range from near-threshold (∼0.1 mW) to maximal effect (∼10 mW). Extracellular signals were high-passed filtered (0.3 Hz), amplified (2,000 times) by a 64-channel amplifier, and digitized at 20 kHz with two National Instruments PCI-6259 cards. After detection, units were grouped by the semiautomatic “cluster cutting” algorithm (“KlustaKwik”; available at http://github.com/klusta-team) followed by manual clustering (Csicsvari et al., 2003). Auto- and cross-correlograms were inspected to verify the clustering procedure.