Sm 25a

We used the Multi-Linc method to effectively identify pyramidal neurons sending direct projections to specific areas by combining multi-areal optogenetic stimulation and multi-neuronal recordings. Details of this procedure were described previously^{52 (link)}. Briefly, prior to the insertion of silicon probes, the optical fibers (FT400EMT, FC, NA, 0.39; internal/external diameters, 400/425 μm; Thorlabs, Newton, NJ, USA) for stimulation were vertically inserted into the mPFC (4100 μm deep) and CA1 (2300 μm deep) using micromanipulators (SM-25A, Narishige). To evoke antidromic spikes in specific axonal projections from LEC neurons (mPFC- and CA1-projecting cells), a blue LED light pulse (intensity, 5–10 mW; duration, 0.5–2 ms, typically 1 ms) was applied through each of the two optical fibers using an ultra-high-power LED light source (UHP-Mic-LED-460, FC, Prizmatix Ltd., Givat-Shmuel, Israel) and a stimulator (SEN-8203, Nihon Kohden, Tokyo, Japan). To be classified as projecting neurons, neurons were required to meet several criteria, including constant latency, fixed frequency (frequency-following test, two pulses at 100 and 200 Hz), and collision test^{45 ,46 (link),50 –52 (link),59 (link),72 (link)}.

Detailed recording procedures are the same as those described in previous works (McAlinden et al., 2015 (link); Scharf et al., 2016 (link); Yague et al., 2017 (link)). All electrophysiological recordings were performed in a single-walled acoustic chamber lined with three inches of acoustic absorption foam (MAC-3, IAC Acoustics). Mice were head-fixed and either a 32 or 64 channel silicon probe (A1 × 32–10 mm–25 s–177-A32 or A4 × 16–10 mm-50 s-177-A64, respectively, NeuroNexus Technologies) was inserted using a manual micromanipulator (SM-25A, Narishige) for AC recordings. Probes were inserted at a 40–50° angle to be perpendicular to the cortical surface (800–1000 μm depth from the cortical surface). The location of the electrode in AC was assessed by evaluating the local field potential (LFP) and multiunit activities (MUA) in response to white noise stimulation (see below).
Broadband signals were amplified (RHD2132, Intan Technologies, LLF) relative to the ground and were digitized at 20 kHz (RHD2132 and RHD2000, Intan Technologies, LLC). The recording session was initiated >30 min after the probe was inserted to its target depth, to allow for signal stabilization. A typical recording session consisted of >15 min baseline recording of spontaneous activity, followed by an optical stimulation protocol, sound presentation, and then another baseline of spontaneous activity.

We performed online Multi-Linc experiments in the frontal motor cortices of unanesthetized rats under head-fixation. For the multi-channel recording, we used two 32-channel silicon probes, although the system can accommodate 128 channels. Approximately 1 h before each recording session, the probes were inserted to a depth of 1.0–1.5 mm from the cortical surface, typically in layer 5, where intratelencephalic (IT)-type projection neurons are distributed most abundantly, using three-axis micromanipulators (SMM-200B and SMM-100, Narishige). On the last recording day, the probe tracks were marked with the red fluorescent dye DiI (DiIC18(3), PromoKine, Heidelberg, Germany) applied to the back of each shank for histological conformation. In the optogenetic stimulation, we used micromanipulators (SM25A, Narishige) to place two to six optic fibers (FT1000EMT, diameter: 1000 μm, Thorlabs, New Jersey, USA) on the cortical surface in a symmetrical position contralaterally from the silicon probes.