2.6. Whole cell patch-clamp electrophysiology

At least three DRG neurons from the CFA-injected side (CFA) and contralateral (Cntrl) side from each animal were recorded. To try and maintain an even distribution of animals across all time points at which recordings were made, the same culture was used for 4-h, 24-h and 48-h wherever possible, however, due to the paucity of labeled neurons and variability of culture conditions, this was not possible in some cases. In total, each of the conditions contained DRG neurons from 6 to 8 mice. At each time point recordings were made for up to a maximum of 4-h. The extracellular solution contained (in mM): NaCl (140), KCl (4), MgCl₂ (1), CaCl₂ (2), glucose (4) and HEPES (10) adjusted to the required pH (>6.0) with NaOH. For solutions with pH < 6.0, MES was used instead of HEPES. Patch pipettes of 4–9 MΩ were pulled with a P-97 Flaming/Brown puller (Sutter Instruments; Novato, CA, USA) from borosilicate glass capillaries and the intracellular solution used contained (in mM): KCl (110), NaCl (10), MgCl₂ (1), EGTA (1), HEPES (10), Na₂ATP (2), Na₂GTP (0.5) adjusted to pH 7.3 with KOH. Recordings were made using a HEKA EPC-10 amplifier (Lambrecht, Germany) and the corresponding Patchmaster software. FB labeled neurons were identified by their fluorescence upon excitation with a 365 nm LED (Cairn Research; Faversham, United Kingdom). For all experiments, DRG neurons were held at −60 mV and whole cell currents were acquired at 20 kHz. Only neurons where an action potential (AP) could be evoked in response to current injections and had a resting membrane potential more negative than −40 mV were analyzed. Images of neurons were captured using a 40× objective on a Nikon Eclipse Ti—S microscope and a Zyla 5.5 sCMOS camera (Andor; Belfast, United Kingdom), followed by pixel to μm conversion to calculate their diameter using ImageJ software.

APs were generated by 80 ms current injections of 150–1050 pA in 50 pA steps (Fig. 2A). If an AP was generated at 150 pA, the neuron was retested with current injections of 0–1000 pA, in 50 pA steps. Threshold, amplitude, half peak duration (HPD), afterhyperpolarization (AHP) duration and AHP amplitude (Fig. 2A) were measured using Fitmaster software (HEKA) or IgorPro software (Wavemetrics) as described before (Bonin et al., 2013; Djouhri et al., 2001; Kim et al., 1998). AP amplitude is defined as the peak of the AP from the repolarized membrane potential; HPD is the AP width at half-amplitude, AHP was obtained by fitting the decay to a single exponential function and AHP amplitude was obtained by subtracting the peak voltage during AHP from the repolarized membrane potential (see Fig. 2A for more detail).

Action potential properties of knee neurons following acute knee inflammation. A) Action potential generation protocol (inset) and response along with a close-up of an evoked action potential from a knee neuron showing the different parameters measured. Example of a labeled knee neuron (blue arrow) and an unlabeled neuron (white arrow) shown in inset. Scale bar indicates 25 μm. B) Frequency distribution of neuronal diameter in μm of CFA (red, n = 25) and Cntrl (black, n = 27) neurons. C-F) Distribution of threshold (C), half peak duration (HPD) (D), afterhyperpolarization (AHP) (E) and AHP amplitude (F) after 4-h in culture. Error bars indicate SEM, * indicates p < 0.05, ** indicates p < 0.01, unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Solutions for determining acid sensitivity (pH 7, pH 6, pH 5) and TRP-agonist sensitivity (capsaicin, cinnamaldehyde and menthol) were applied in a random order through a gravity-driven 12 barrel perfusion system (Dittert et al., 2006) to DRG neurons in 5 s pulses with at least 30 s wash period (with pH 7.4) between stimuli. Solutions of 10 μM capsaicin (1 mM stock in 100% ethanol; Sigma-Aldrich), 100 μM cinnamaldehyde (10 mM stock in 100% ethanol; Merck) and 100 μM menthol (20 mM stock in 100% ethanol; Alfa Aesar) were made up in pH 7.4 extracellular solution from their respective stock solutions. Neurons that produced an inward current, time-locked to drug application, were counted as responders; in control experiments using the vehicle control (1% EtOH, n = 3) no such inward currents were observed. Current amplitude was measured in Fitmaster (HEKA) by subtracting the maximum peak response from the baseline (average of the first 3 s before stimulation), which was then normalized by dividing by neuron capacitance to give current density.

100 μM GABA (100 mM stock in water; Sigma-Aldrich) was applied for 5 s followed by a 20 s wash with pH 7.4, then 100 μM GABA and 250 μM bicuculline (100 mM in DMSO; Sigma-Aldrich) were applied together for 5 s. Recovery from bicuculline block was assessed after 1 min by a subsequent 100 μM GABA application for 5 s. To test for the presence of the GABA_A-δ subunit, 100 μM of the δ agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP, 100 mM stock in distilled water; Sigma-Aldrich) was applied for 5 s.