Postnatal capsaicin treatment
All animal manipulation was approved by the Animal Care Committee of the Jikei University School of Medicine and conformed to the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan (1998) and to the guidelines of the International Association for the Study of Pain .
Neonatal Wistar rats of either sex were anesthetized with diethyl ether 12–48 hours after birth and either a capsaicin solution (capsaicin-treated group) or vehicle solution (vehicle-treated group) was subcutaneously injected. The capsaicin injection solution contained 1% or 5% capsaicin (Sigma), 10% ethanol and 10% Tween-80 in saline. The vehicle solution contained 10% ethanol and 10% Tween-80 in saline. The injection volume was adjusted in each animal according to its body weight and solution concentration so that the dose of capsaicin in this solution became 50 mg/kg in the same manner as a previous report showing maximal degeneration of unmyelinated afferent neurons . The pups were placed on a soft water bed heated at approximately 37°C until recovery from anesthesia and were returned to their mothers afterwards. They were weaned approximately at 3 weeks and maintained under standard laboratory conditions under a 12 hr light/dark cycle. They had free access to food and drinking water. There were no crucial differences in general behavior or food intake between these two groups. The body weights at the third postnatal week were 29.3 ± 0.7 g (mean ± standard error of the mean; n = 27) and 26.7 ± 1.3 g (n = 13), for the capsaicin- and vehicle-treated groups, respectively. Because we could not detect any apparent difference between the results from male and female rats, their data were pooled. All animals of both capsaicin- and vehicle-treated groups survived until the SNL and decapitation for electrophysiological recordings (see below).
Evaluation of capsaicin-induced eye-wiping
The sensitivity to capsaicin in each animal belonging to either the capsaicin- or vehicle-treated group was evaluated by observing the behavioral responses to instillation of capsaicin solution to the cornea according to a previously described method ("eye-wiping test" in [53, 54]). The 0.01% capsaicin eye instillation solution was made by diluting the stock solution containing 1% capsaicin, 10% ethanol and 10% Tween-80 with saline to 1:100. The solution of the same content but without capsaicin was called "solvent" and was used as a control for capsaicin application. On postnatal day 19–20, an approximately 10 μl drop of 0.01% capsaicin solution or of solvent was applied to the eyes of an unrestrained rat. The interval between instillations in one rat was more than 30 min. The order of application of capsaicin solution and solvent, as well as that of right and left eyes, was randomized in a blind manner by two experimenters. The number of eye-wiping behaviors in the first 2 min after the application was counted by visual inspection of one of these experimenters also in a blind manner.
Neuropathic pain model
At postnatal day 20–28, rats were separated from their dam and anesthetized initially with diethyl ether and then mounted on a surgical platform in a prone position with the limbs fixed while being kept continuously anesthetized with isoflurane inhalation (1.5-2% in 100% O2). A longitudinal incision (0.5-1 cm in length) was made at the midline of the lower lumbar using a sterile surgical blade. The left paraspinal muscles were isolated and the left L6 transverse process was exposed. Under a dissecting microscope, the left L6 transverse process covering the L4 and L5 spinal nerves was carefully removed. The left L5 spinal nerve was isolated and tightly ligated with 6–0 silk threads. After surgery, the muscles were sutured in layers, the skin was closed using silk thread (4–0), and anesthesia was discontinued. Animals fully recovered from anesthesia within 30 min and showed no signs of distress as evidenced by the monitoring of breathing patterns, grooming behavior and locomotion in the cage. The rats were housed in cages filled with soft cushion-like flooring in a temperature- (approximately 25°C) and humidity- (approximately 50%) controlled room until decapitation as described below. Water and food were available ad libitum, and there was no apparent difference in daily consumption of water and food and in body weight measured before decapitation between the groups. Behavior was monitored regularly, and no aberrant symptoms arising from excessive nociception were observed except for a mild deformity of the lesioned paw .
Von Frey filament test
The paw withdrawal threshold in response to mechanical stimuli was evaluated by well-trained experimenters, according to the previously reported method . Mechanical stimuli were applied using von Frey filaments of different rigidity (0.4 - 15.0 g). Each rat was placed on a metal mesh floor (25 cm x 25 cm) and a von Frey filament was applied manually from beneath. The 50% threshold for the paw withdrawal behavior (50%-paw withdrawal threshold, PWT) was estimated by the up-and-down method . Care was taken to reduce the number of trials to avoid unnecessary pain sensations. The tests were performed 3 times for each rat; immediately before the SNL, once within post-operational day 2–4, and once immediately before decapitation for the slice preparation.
Preparation of transverse brain slices
Immediately after the final von Frey filament test on post-operational day 7–9, the rats were decapitated under isoflurane anesthesia (5% in 100% O2). We obtained coronal brain slices both ipsilateral (left) and contralateral (right) to the operation side (left) from the capsaicin-treated group (56 unilateral slices from 27 rats) and from the vehicle-treated group (31 unilateral slices from 13 rats) according to previously described procedures . Briefly, a transverse block of forebrain containing the amygdaloid complex was dissected out and cut at the midline. The dissected hemisphere was secured on the cutting stage of a vibrating blade slicer (DSK-1000, Dosaka EM) with the rostral end upwards. Coronal slices of 400-μm thickness containing the amygdala were cut in ice-cold cutting artificial cerebrospinal fluid (ACSF) composed of (in mM) 125 NaCl, 3 KCl, 0.1 CaCl2, 5 MgCl2, 1.25 NaH2PO4, 10 D-glucose, 0.4 L-ascorbic acid and 25 NaHCO3 (pH 7.4 bubbled with 95% O2 + 5% CO2; osmolarity, approximately 310 mOsm/kg). The slices were first incubated in a holding chamber with a constant flow of standard ACSF, of which the concentrations of CaCl2 and MgCl2 were 2 mM and 1.3 mM, respectively, at 37°C for 30 to 45 min. The slices were kept at room temperature (20-25°C) in the same chamber until the electrophysiological recording. Each slice was transferred to a recording chamber (approximately 0.4 ml volume) and fixed with nylon grids to a platinum frame. The slice was submerged in and continuously superfused at a rate of 1–2 ml/min with standard ACSF. To isolate excitatory synaptic inputs, 100 μM picrotoxin (Sigma) and 1 μM strychnine HCl (Sigma) were dissolved in ACSF and bath-applied throughout the recording.
Neurons in the CeC were visually identified under an upright microscope (BX-51WI, Olympus) with oblique illumination or infrared interference contrast (IR-DIC) optics. Images were captured using a CCD camera (IR-1000, DAGE-MTI) and stored digitally on a computer. Whole-cell transmembrane current was recorded from neurons in the left and right CeC (i.e., ipsi- and contralateral, respectively, to the SNL). Patch-clamp electrodes were made from borosilicate glass pipettes (1B120F-4; World Precision Instruments). The composition of the internal solution was (in mM) 120 potassium gluconate, 6 NaCl, 1 CaCl2, 2 MgCl2, 2 ATP Mg, 0.5 GTP Na, 12 phosphocreatine Na2, 5 EGTA, 5 QX-314 and 10 HEPES hemisodium (pH 7.2 as adjusted with KOH; osmolarity, approximately 310 mOsm/kg) was added to the internal solution. The tip resistance of the electrode was 3–6 MΩ. The evoked cEPSCs were recorded at a holding potential of −70 mV. The input resistance, resting membrane potential and whole-cell capacitance were measured immediately after the establishment of whole-cell mode by membrane rupture. Membrane currents were recorded using an Axopatch 200B amplifier (Axon Instruments), low-pass filtered at 2 kHz and digitized at 4 or 10 kHz and 16-bit resolution with a PowerLab interface (ADInstruments).
All recordings were made at room temperature (20-25°C). The order of recordings from the right and left amygdala was randomized to avoid side-dependent differences due to changes in the viability of neurons during the time from slice preparation to recording. All compounds except those noted above were purchased from WAKO (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).
Afferent pathway stimulation
To activate action potential-dependent glutamate release from afferent fibers arising from the LPB, we carefully located the stimulating electrode on the fiber tract ventromedial to the CeC under microscopic control. The following two types of stimulation protocol with different electrodes were performed. (1) Gross stimulation to evoke cEPSCs. A bipolar concentric steel electrode (interpolar distance, approximately 100 μm; Unique Medical, Tokyo, Japan) was used, and the stimulation intensity was set at 50, 100, 200, 400, 800 and 1000 μA. The pulse duration was 100 μs. The cEPSC amplitude increased in a graded manner as the stimulation intensity was elevated, suggesting that the amplitude depended on the number of afferent fibers recruited in response to a single stimulation. Double pulses with an inter-stimulus interval of 50 ms were delivered to calculate the PPR of the cEPSC amplitude by normalizing the amplitude of second cEPSC by that of first cEPSC . (2) Minimal stimulation to evoke sfEPSC . The stimulation electrodes for minimal stimulation were made from "theta" glass pipettes (TST150-6, World Precision Instruments), which were pulled with a similar procedure to that used for making patch pipettes. The theta stimulation pipette had a very small interpolar distance at the tip (approximately 1 μm) and was filled with ACSF. The stimulation intensity was first set at a moderate value and gradually decreased while the evoked EPSCs were observed. Then, the sfEPSC was identified in an "all-or-none" manner by the appearance of EPSCs with a similar waveform and latency at a consistent probability in response to a fixed stimulation intensity .
Data and statistical analysis
The recorded membrane current was analyzed off-line with Igor Pro 5 (WaveMetrics, OR, USA) using procedures written by F. K. The peak amplitude was measured based on the averaged waveform of 8 consecutive evoked cEPSCs. Values are expressed as the mean values ± standard error of the mean (SEM). Differences in the values were compared using one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test and Mann–Whitney U test. Differences with a probability (P) less than 0.05 were considered significant.