Potentiation of synaptic transmission in Rat anterior cingulate cortex by chronic itch

Young adult (3–5 weeks old) Sprague-Dawley rats were raised in the Animal Center of Key Laboratory of Brain Genomics at East China Normal University. They were housed in a vivarium (temperature: 22–24 °C; humidity: 50–60 %) with a 12-h light/dark cycle and a sufficient food and water supply. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee at Shanghai Jiaotong University.

Chronic itch was simulated in accordance with the method described by Sun et al. [13]. Diphenylcyclopropenone (DCP) was dissolved in acetone for reserve. Before DCP application, the skin on the rat’s buttock (~4 cm²) was exposed by shaving the hair. DCP (4 %, 0.5 ml) was applied to the exposed skin for sensitization on Day 1 (D1). Seven days after the first application, rats were challenged by applying 2 % DCP (0.5 ml) at the same site once daily until D15. Five minutes after applying DCP, the number of scratching behaviors toward the application site was counted over a 30-min period (Fig. 1a).

On each day of testing, rats were placed in a small transparent plastic chamber (21.5 × 21.5 × 12.5 cm) and were allowed 30 min to acclimate prior to DCP application. One bout of scratching was defined as lifting of a forelimb toward the application site and then returning the forelimb back to the floor, regardless of the number of scratching strokes that were performed between these two movements. Scratching behavior was observed by an investigator blinded to the treatment groups.

Coronal brain slices of the ACC were prepared following the procedures reported in our previous work [14]. Rats were anesthetized by sodium pentobarbital (80 mg/kg, i.p.) and decapitated on D16-D22, after the behavioral tests were completed. The brain was rapidly excised from the skull and placed in ice-cold artificial cerebrospinal fluid (ACSF) solution (in mM: 119 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.3 MgSO₄, 11.0 D-Glucose, 1.0 NaH₂PO₄, 26.2 NaHCO₃) bubbled with carbogen (95 % O₂ + 5 % CO₂). One or two minutes later, coronal slices (350 μm) of the ACC were cut from the brain in ice-cold ACSF using a Vibroslicer (VT1000 S, Leica Microsystems, USA). Then, the slices were transferred to the ACSF, which was oxygenated with carbogen at room temperature for incubation. At least 1 h later, the slices were examined in a recording chamber, which was bubbled with carbogen, and the temperature in the chamber was maintained at 28–30 °C. Neurons were visualized using a microscope (DX50WI, Olympus, Japan) via differential interference contrast optics and infrared video microscopy.

Standard whole-cell voltage-clamp recordings from the ACC were performed with a MultiClamp 700B amplifier (Axon Instruments, USA) and digitized at 5 kHz by a data acquisition card (DigiData 1440, Axon Instruments, USA) in all electrophysiological experiments. The series resistance (15–30 MΩ) was monitored during the entire recording process. Data were discarded if the series resistance changed by more than 20 % during a single recording. The micropipettes (3.5–6.5 MΩ) were pulled from borosilicate glass (1.5 mm outer diameter and 1.1 mm inner diameter; Sutter Instruments, USA) using a P-97 (Sutter Instruments, USA) and were filled with an internal solution containing (in mM) 125.0 K-gluconate, 10.0 Na₂-phosphocreatine, 2.0 KCl, 0.5 EGTA, 10.0 HEPES, 4.0 Mg-ATP, 0.3 Na₃GTP (adjusted to pH of 7.2 with KOH). Miniature excitatory postsynaptic currents (mEPSCs) were recorded from ACC neurons located in cortical layers II/III, and the neurons were voltage clamped at -70 mV in the presence of tetrodotoxin (TTX, 0.5 μM). The extracellular solution routinely contained picrotoxin (0.1 mM) to blockγ-aminobutyric acid A receptor(GABA_AR) mediated currents except in the recording of miniature inhibitory postsynaptic currents (mIPSCs). For investigating an obvious GABAergic transmission at resting condition (-70 mV), we used the internal solution with a high Cl^- concentration, which contained (in mM) 11 EGTA, 2 MgCl₂, 1 CaCl₂, 10 HEPES, 115 K-Glu, 2 K₂ATP, 25 KCl (adjusted to pH of 7.2 with KOH). It has been proved that high intracellular concentrations of Cl^- would not be altering resting cell properties [15]. The perfusion solution contained TTX (0.5 μM), 2-amino-5-phosphonopentanoate (AP5, 50 μM) and 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX, 20 μM).

To elicit synaptic responses from ACC neurons located in cortical layers II/III, stimulation was delivered via a bipolar tungsten electrode placed in layer V of the ACC. The amplitude of the evoked excitatory postsynaptic currents (EPSCs) was adjusted within 30–100 pA at the holding potential of -70 mV in the presence of AP5 (50 μM), except during the experiment in which the AMPA-to-NMDA (N-methyl-D-aspartate) receptor ratio was measured. In the paired-pulse ratio (PPR) experiments, the intervals were 50, 75, 100, 150 ms. For experiments in which AMPA-to-NMDA receptor ratio was measured, the intracellular solution contained (in mM) 115.0 Cs-gluconate, 8.0 NaCl, 20.0 HEPES, 5.0 TEA-Cl, 0.2 EGTA, 0.3 Na₃GTP, and 4.0 Mg-ATP (adjusted to pH of 7.2 with CsOH). AMPAR-mediated synaptic responses were measured at -70 mV, whereas NMDAR-mediated responses were measured at +40 mV and at a latency where AMPAR responses had fully decayed (60 ms) [16]. Synaptic responses were averaged over 50–100 trials. For calculating the rectification of AMPA receptor-mediated EPSCs, the internal solution consisted of the following (in mM): 140.0 cesium methanesulfonate, 5.0 NaCl, 0.5 EGTA, 10.0 HEPES, 0.1 Na₃-GTP, 2.0 Mg-ATP, 0.1 spermine, 2.0 QX314, and 10.0 phosphocreatine disodium (adjusted to pH of 7.2 with CsOH). We recorded current at the holding potentials of +35 and -65 mV and used the ratio of the peak amplitude of EPSCs at negative (-65 mV) and positive (+35 mV) holding potentials as the rectification index [17].

Failure rate experiments were performed using the minimum stimulation, as described previously [18, 19]. Cs⁺-based internal solution, which contained (in mM) 115.0 Cs-gluconate, 8.0 NaCl, 20.0 HEPES, 5.0 TEA-Cl, 0.2 EGTA, 0.3 Na₃GTP, 4.0 Mg-ATP, and 2.0 QX314 (adjusted to pH 7.2 with CsOH), was used. Stimulation with 2-sec intervals were produced using a pulse generator (Master-8, A.M.P. Instruments, Israel) and through a stimulus isolator (ISO-Flex, A.M.P. Instruments, Israel). The stimulus intensity was first identified around the threshold stimulus. The stimulus intensity was then adjusted until the percentage at which the stimulation failed to evoke a response, or the failure rate, was approximately 50 % at the holding voltage of -65 mV. We used this final stimulus intensity to evoke responses in the same cell at the holding voltage of +40 mV. For each cell, 200–300 sweeps were recorded at -65 mV and +40 mV. The failure rate was defined as the percentage of failed evoked responses over sweeps with holding potentials of -65 and +40 mV. All failure responses were visually checked and verified. The fraction of silent synapses was calculated using the equation 1-ln(F_-65)/ln(F₊₄₀) as described previously [19]. F_-65 was defined as the failure rate at -65 mV, whereas F₊₄₀ was defined as the failure rate at +40 mV.

The results are shown as the mean ± SEM. Statistical significance was assessed using Student’s t test when two groups were compared or a paired t-test when scores before and after an intervention were compared within the same group. The Mann-Whitney rank sum test was used for nonparametric tests. A two-way ANOVA was performed with group as a between-group factor and time as a repeated-measures factor. ANOVA was followed by the Bonferroni post hoc test. In all experiments, p < 0.05 was considered statistically significant.