Brain slice preparation and electrophysiology

Brain slices were prepared as described previously [27]. After cardiac blood collection, the mice were transcardially perfused with cooled cutting solution (in mM: choline chloride 120, KCl 3, NaHCO₃ 28, NaH₂PO₄ 1.25, glucose 22, and MgCl₂ 8) from the left ventricle, and their brains were rapidly excised and soaked in ice-cold cutting solution. Brains were placed on a vibrating-blade microtome (VT1000S; Leica, Wetzlar, Germany). Two or three 450 μm thick coronal brain slices (450 μm) containing the ACC were prepared from the brain submerged in ice-cold cutting solution saturated with 95% O₂ and 5% CO₂. The prepared slices were trimmed on both sides, placed on a film sheet (Nucleopore®, CORNING, Corning, NY), and transferred to a chamber filled with O₂/CO₂-saturated artificial cerebrospinal fluid (ACSF, in mM: NaCl 120, KCl 3, CaCl₂ 2.5, MgCl₂ 1.3, NaHCO₃ 26, NaH₂PO₄ 1.25, glucose 15). The brain slices were incubated in the chamber for at least 1 h before electrophysiology experiments.

Thereafter, the brain slice attached to the film sheet was transferred to a submerged recording chamber, and the slice was glued with agar at the four corners of the film to the bottom of the chamber. The slices were then perfused with O₂/CO₂-saturated ACSF at a rate of 6mL/min. The solution was maintained at 26–28°C by a controller (TC-324B, WARNER Instrument, Holliston, MA).

Extracellular recordings were performed to obtain field potentials from the Cg1 region of the right ACC. Glass electrodes were made using a micropipette puller (P-97, SUTTER, Novato, CA, USA) and filled with NaCl (0.5 M). Electrodes were placed at the recording points: the superficial layer (layer II/III) of the dorsal-midline corner in the Cg1 region of the ACC and at approximately 150 μm inside the brain surface, as estimated under a microscope (Fig 2A).

(A) Microscopic image of the ACC. Extracellular field potentials were recorded from the superficial layer of the ACC slices. The black line indicates the position of the recording electrode. The dotted line indicates Cg1 region of ACC. (B) Timeline of drug perfusion protocol for extracellular recordings. Extracellular field potentials were recorded under the absence (top) or presence (bottom) of 10 μM histamine. Network oscillation was induced by KA perfusion. KA was perfused with increasing concentrations (0.3, 1.0, and 3.0 μM for 5 min each). Abbreviations: ACC, anterior cingulate cortex; KA, kainic acid.

To induce network oscillation in vitro, KA (K0250, Sigma-Aldrich, St. Louis, MO) was perfused for 5 min at 0.3, 1.0, and 3.0 μM concentrations in series (control group: n = 8, LPS-treated group: n = 9). To investigate the effect of histamine on KA-induced network oscillation, 10 μM histamine (H-7250, Sigma-Aldrich St. Louis, MO) was first perfused for 10 min, and then KA was added in the presence of histamine at the abovementioned concentrations (control group: n = 7, LPS-treated group: n = 10, Fig 2B). The signal was amplified by a differential amplifier (DAM80, WPI, Sarasota, FL) and acquired at a sampling rate of 10 kHz via an interface (Digidata 1440A, Molecular Devices, San Jose, CA) with a band-pass filter between 0.1 Hz and 1 kHz and recorded on a PC.

The 5-minute time window was adopted for the analysis of network oscillations, from the time when the corresponding concentration of KA perfusion reached the slice (1 min after the start of KA perfusion). The power spectrum density (PSD) was obtained by fast Fourier transform using Clampfit 11.1 software (Molecular Devices, San Jose, CA, USA). The oscillation power component within each frequency range (theta: 3–8 Hz, alpha: 8–12 Hz, beta: 12–30 Hz, low gamma: 30–50 Hz, high gamma: 50–80 Hz, total: 3–80 Hz) was calculated from the area under the PSD curve. In this study, the delta (0.5–3 Hz) range was excluded from the analysis because of its low power.