Cheetah data acquisition software

Recordings were collected using four tetrodes. Each tetrode consisted of four polyimide-coated nichrome wires (12.5 µm diameter; Sandvik) gold plated to 0.2–0.4 MΩ impedance. Electrical signals were amplified and recorded using the Digital Lynx S multichannel acquisition system in conjunction with Cheetah data acquisition software (Neuralynx). Tetrode depths, estimated by calculating the rotation of the screw affixed to the shuttle holding the tetrode (one rotation = ~250 µm), were adjusted ~75 µm between recording sessions to sample independent populations of neurons across sessions. Offline spike sorting and cluster quality analysis was performed using MCLUST software (MClust-4.0, A.D. Redish et al.) in MATLAB. Briefly, single units were isolated by manual clustering based on features of the sampled waveforms (amplitude, energy, and the first principal component normalized by energy). Clusters with L-ratio <0.75 and isolation distance >12 were deemed single units (Schmitzer-Torbert et al., 2005 (link)), which resulted in excluding 30% of clusters. Although units were clustered blind to inter-spike interval (ISI), clusters with ISIs <1 ms were excluded.

To characterize effect of HIFU on brain surface activities, we used µECoG arrays (Neuronexus) with 16 small recording electrodes (surface area ~0.03 mm²) arranged in a 4 × 4 grid, allowing us to sample small populations of neurons from the superficial layers of cortex at high resolution. Epidural neural signals were acquired with a NeuraLynx Digital Data Acquisition System, including a DL 4SX-M 32ch Base, a headstage pre-amplifier HS-18-CNR-MDR50, a HS-18-N2T-16 adaptor and Cheetah data acquisition software, at a sampling frequency of 16 kHz from freely moving animals in their home cages. High pass filtered (HPF; 900 to 5 kHz) and ECoG (0.1 and 300 Hz) signals were saved with Cheetah software (NeuraLynx). The animals’ behavioral state was simultaneously recorded and classified into Move or Resting status with HomeCageScan software (CleverSys) during electrophysiological recordings.
Ipsilateral and contralateral ECoG data were collected from different cohorts of animals. Contralateral animals were allowed to recover from implantation surgery for a week, then four-week baseline data were collected (Fig. 1A, n = 10 in both HIFU and sham groups). In ipsilateral cohort, animals were subjected to HIFU/sham treatment immediately before ECoG array implantation surgery, therefore, no baseline data were collected (Fig. 1A in purple, n = 4 in both HIFU and sham groups).

All the animals were subjected to the recording of the local field potentials (LFPs) during tone habituation session, fear recall and extinction session and extinction recall session. Auditory-evoked potentials (AEPs) were recorded by connecting the microelectrodes to a unit gain buffer head stage (HS-36-Flex; Neuralynx, Bozeman, Montana, USA) and a data acquisition system Digilynx (Neuralynx, Bozeman, Montana, USA). Neural data were amplified (1000 times) and acquired at a sampling rate of 1 kHz followed by a band-pass filter (1–500 Hz) using Cheetah data acquisition software (Neuralynx, Bozeman, Montana, USA).