Genetically modified mouse lines were purchased from Jackson Laboratories and bred in-house. The PV-Cre mouse line (B6;129P2-Pvalbtm1(cre)Arbr/J; JAX stock #008069) was used to target PV-expressing interneurons. The PV-Cre mouse line was bred with Ai9 mice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J; JAX stock #007909) to identify PV neurons. PV interneurons of the PV:Cre/Ai9 line express robust tdTomato fluorescence following Cre-mediated recombination. To selectively evoke APs in PV neurons, PV-cre mice were bred with Ai27D mice (B6.Cg-Gt[ROSA]26Sortm27.1[CAG-COP4*H134R/tdTomato]Hze/J; JAX stock #012567). PV neurons of the PV-Cre/Ai27D line express the improved channelrhodopsin-2/tdTomato fusion protein. The PV-Cre, Ai27D, and Ai9 transgenic lines were bred as homozygotes. Mice were housed under a 12-h light-dark cycle with ad libitum access to food and water. Only male mice were used to avoid the potential effect of the estrus cycle. All care procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Korea Brain Research Institute (M2-IACUC-19-00040).
Viral vectors and stereotactic surgeries
Animals were anesthetized with ketamine (100 mg/kg) supplemented with dexmedetomidine hydrochloride (0.4 mg/kg) by intraperitoneal injection and positioned in a stereotaxic injection frame (Kopf instruments). Ketoprofen (5 mg/kg) was subcutaneously injected for its anti-inflammatory effects. During the surgery, responses to pedal withdrawal reflex stimuli were absent. Virus injection was conducted followed by a stereotaxic surgical procedure.
An EF1a-driven, Cre-dependent, humanized channel rhodopsin (hChR2) H134R mutant fused to enhanced yellow fluorescent protein (eYFP) for optogenetic activation (Addgene # 20298-AAV1) and hSyn-Cre (Addgene # 105553-AAV1) was transduced by adeno-associated virus serotype 1 (AAV1). Between postnatal days 40−50, the mixture (50:50) of AAV1-double floxed-H134R and AAV1-Cre was unilaterally injected into the MD of the PV:Cre/Ai9 mouse. To derive PV-induced inhibitory postsynaptic currents (IPSCs) in the PFC, AAV1-double floxed-H134R was bilaterally injected into the PV-Cre mouse PFC. Approximately 70 nL of the virus solution (1012 viral particles/mL) was delivered with a glass micropipette (Drummond Scientific) through a small skull window (1−2 mm2). To avoid leakage into surrounding brain areas, we left the injection pipettes in the brain for 6 min after the injection before slowly withdrawing them. The injections were performed using following stereotaxic coordinates. The MD coordinates from the bregma were as follows: anterior-posterior, -1.58 mm; medial-lateral, ±0.30 mm; dorsal-ventral, -3.10 mm. The PFC coordinates from the bregma were as follows: anterior-posterior, 1.75 mm; medial-lateral, ±0.30 mm; dorsal-ventral, -1.00 mm. During all surgical procedures, the animals were kept on a heating pad in an isolated cage and were brought back to their home cages when they regained movement. For optimal viral expression, all mice were euthanized at least 3 weeks after the surgery.
Electrophysiology and optogenetics
Mice aged 9−10 postnatal weeks were euthanized by exposure to CO2 followed by decapitation. The brains were quickly and carefully removed in ice-cold dissection solution: 25 mM sodium bicarbonate (NaHCO3), 1.25 mM sodium monophosphate (NaH2PO4), 25 mM D-glucose, 2.5 mM KCl, 7 mM MgSO4·6H2O, 0.5 mM CaCl2, 110 mM choline chloride, 11.61 mM (+) sodium-L-ascorbate, and 3 mM sodium pyruvate. The measured osmotic concentration was between 320−330 mOsm. Acute 300-µm thick brain slices were prepared via coronal sections with a vibratome (Leica VT1200S) in ice-cold dissection solution. The composition of artificial cerebrospinal fluid (aCSF) was as follows: 119 mM NaCl, 2.5 mM KCl, 1 mM MgSO4·7H2O, 26 mM sodium bicarbonate (NaHCO3), 1.25 mM sodium monophosphate (NaH2PO4), 20 mM D-glucose, 0.4 mM L-ascorbic acid, 2 mM CaCl2, and 2 mM pyruvic acid. The measured osmotic concentration was between 305−310 mOsm. After 30 min of recovery time in warmed aCSF (32°C), slices were transferred to room temperature. For each mouse, PFC slices were prepared first, and then slices around the MD were prepared to ensure that slices of the injection sites were obtained. Mice were excluded from data analysis whenever expression of the virus was observed outside of the MD. The dACC L2/3 pyramidal neurons and PV interneurons were recorded either by voltage clamping or current clamping for the following procedures.
PV neurons were discerned visually and electrophysiologically by measurements of intrinsic properties. PV interneurons exhibited higher firing rates with minimal adaptation and a lower AP threshold. All recordings, including voltage holding at -30 mV, were performed with patch pipettes (3.5−4 MΩ) filled with an internal solution that consisted of the following components: 20 mM KCl, 125 mM K-gluconate, 10 mMHEPES, 4 mM NaCl, 0.5 mM EGTA, 4 mM ATP, 0.3 mM Tris-GTP, and 10 mM phosphocreatine with a pH adjusted to 7.20 with KOH. The measured osmotic concentration was between 307−314 mOsm. Recordings were performed at room temperature.
Optogenetic stimulation was applied with a 1-ms light pulse from a 470-nm laser source; the light was guided with an optic fiber placed within 1 mm of the recorded neurons. The total power of the laser measured at the tip of the fiber by Power Meter (Thorlabs) was ~5 mW/mm2.
We measured the resting potential of all neurons in current-clamp mode immediately after rupture of the neuronal membrane. Input resistance was determined by measuring the voltage change in response to a small hyperpolarizing current pulse (5 pA, 50 ms) at resting potential. Spike threshold was acquired by 20-pA step increments of current injection and determined as the point at which the first AP was evoked. Series resistance was observed throughout the entire experiment and was not compensated. Cells with series resistances over 20 MΩ were excluded.
All solutions were kept saturated with 95% O2 and 5% CO2. Acute slices were continuously perfused with aCSF at room temperature. All data except the experiment performed with PV-Cre/Ai27D mice were sampled at 20 kHz by the EPC-10 amplifier (HEKA Elektronik) with Patchmaster software (HEKA Elektronik) and further analyzed by MATLAB (Mathworks). Electrophysiological data for the experiment using PV-Cre/Ai27D mice were recorded using an Axopatch 700B amplifier (Molecular Devices), and command pulse generation was performed using Digidata 1550 (Molecular Devices). The data were further analyzed using Clampfit 10.4 (Axon Instruments) and IGOR Pro software (Wavemetrics).
The following drugs were perfused in aCSF: 100 µM AP5 ([2R]-amino-5-phosphonovaleric acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, Tocris; IC50 = ~50 µM), 10 µM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, an AMPA/kainate receptor antagonist, Tocris; IC50 = 1.5 µM), 2 µM bicuculline (ionotropic g-aminobutyric acid or GABAA receptor antagonist, Sigma-Aldrich; IC50 = 2 µM), 0.5 µM TTX (tetrodotoxin, a Na+ channel blocker, Abcam; IC50 < 10 nM), and 100 µM 4-AP (4-aminopyridine, a Kv1 channel blocker, Tocris; IC50 = 147 µM). All drugs were perfused throughout the experimental protocol and washed out for at least 30 min after the end of the protocol.
Data analysis was performed using MATLAB (Mathworks), and GraphPad Prism 6.0 (GraphPad Software). Data are presented as the mean ± standard deviation unless otherwise noted. Parametric or non-parametric tests were employed according to the normality tests. Statistical analyses were performed using a two-tailed Student’s t-test for the comparison of two groups. For comparisons across more than two groups, data were analyzed using one-way analysis of variance followed by Tukey’s post hoc analysis to correct for multiple comparisons. For data with a non-normal distribution, the non-parametric Mann-Whitney or Wilcoxon signed-rank tests were used. A P value < 0.05 was considered statistically significant.