Baseline movement and tactile sensitivity of Nav1.7 KOs are not different from WT/HET.

<p>Overall locomotion was evaluated in Nav1.7 WT/HET and KO animals by scoring both total rearing behavior and basic movement using beam breaks in an automated open-field box. <b>A.</b> There was no statistically significant difference in rearing behavior between WT/HET (11187±492, n = 20) and KO (11036±781, n = 17) littermates (pairwise t-test, p = 0.2438). <b>B.</b> There was no statistically significant difference in basic movement between WT/HET (935±85, n = 20) and KO (835±75, n = 17) littermates (mean ± S.E.M.; pairwise t-test, p = 0.528). <b>C.</b> Tactile sensitivity, as assayed by measuring threshold of paw withdrawal to von Frey fibers of increasing force, was not significantly different between WT/HET (1.225 g ±0.05 g, n = 11) and KO littermates (1.18 g ±0.08 g, n = 7) (mean ± S.E.M.; Wilcoxon Two-Sample Exact Test, p = 0.6434). Dashed line represents the level at which the animal’s paw was physically lifted by the von Frey monofilament and is included to show that the measured responses are due to true behavioral tactile response.</p

Genotyping and phenotyping of global Nav1.7 knockout animals.

<p><b>A.</b> Example of endogenous (top - 267 bp) and targeted (bottom - 389 bp) PCR products obtained from DNA isolated from Nav1.7 WT (lane 1), KO (lane 2) and HET (lane 3) animals, while the last lane serves as a negative control (no DNA). All PCRs were run using a 1 kb PLUS DNA ladder. <b>B.</b> Photos of postnatal day 2 littermates showing their capability in feeding naturally by the presence of milk spots in control (see arrow left) and KO (see arrow right) and slight size difference in young animals. <b>C.</b> Body weight (grams) comparison between six female littermates at nine weeks of age that represented all three genotype classes illustrating the absence of size difference in adult animals.</p

Normal anatomy of Nav1.7 KOs.

<p>Postnatal day 4 Nav1.7 WT (A to E) and KO (F to J) neonates stained with hematoxylin and eosin (H&E). <b>Wild type:</b><b>A.</b> Sagittal section of the entire pup. <b>B</b> through <b>E</b> show magnifications of various regions of the central and peripheral nervous systems: <b>Knock Out:</b><b>F.</b> Sagittal section of the entire pup. <b>G</b> through <b>J</b> show magnifications of various regions of the central and peripheral nervous systems. (<b>B, G</b>) cortex; (<b>C, H</b>) hippocampus; (<b>D, I</b>) olfactory bulb; (<b>E, J</b>) dorsal root ganglia. (Scale bars: <b>A, F</b> = 5 mm; <b>B, G</b> = 100 µm; <b>C, H</b> and <b>D, I</b> = 400 µm; <b>E, J</b> = 300 µm).</p

Nav1.7 KOs show minimal pain behaviors upon injection of veratridine or grayanotoxin III.

<p><b>A.</b> hNav1.7 currents recorded from HEK 293 cells in control (left) and after addition of 30 µM veratridine (right). Currents were evoked by a family (traces overlaid) of depolarizing voltage pulses incremented by +10 mV from a holding voltage of −100 mV, with repolarization to −80 mV followed by return to the holding voltage of −100 mV. Inward currents were evoked by depolarizations to −50 mV and more positive. Note the prolonged opening following repolarization in veratridine. Scale bars, 500 pA and 10 ms. <b>B.</b> Nav1.7 currents recorded with 300 µM grayanotoxin III in the internal (pipette) solution. Currents shown at left are in response to a family (traces overlaid) of step depolarizations in +5 mV increments from −120 mV to −50 mV, followed by repolarization to −120 mV. Depolarizations to −95 mV and more positive evoked inward currents, and these currents showed little or no inactivation during the test pulse. Dotted line marks the zero current level (I = 0). Currents shown at right are records from the same cell, after switching the holding voltage to −80 mV. Note that the holding current increased despite the decreased driving force (compare current at −80 mV to the dashed line marking the holding current at −120 mV), presumably reflecting steady influx of sodium ions through grayanotoxin-modified Nav1.7. Step depolarizations from −80 mV to −40 mV at +10 mV increments evoked additional currents with slow inactivation and deactivation kinetics. Scale bars, 500 pA and 20 ms. <b>C.</b> Licking and lifting in male CD-1 mice in response to increasing intraplantar doses of veratridine. At doses of 1 µg and 10 µg, veratridine caused a statistically significant increase in paw licking and lifting behaviors compared to saline or vehicle (1% ethanol/99% PBS) controls. The licking and lifting caused by 1 µg veratridine was completely prevented by pre-dosing the animals with mexiletine (MEX; 30 mg/kg, i.p. or p.o.). A separate animal cohort was used for each dose. <b>D.</b> Licking and lifting in male CD-1 mice in response to increasing intraplantar doses of grayanotoxin III. At doses of 0.1 µg and 1 µg, grayanotoxin III caused a statistically significant increase in paw licking and lifting behaviors compared to saline or vehicle (1% ethanol/99% PBS) controls. A separate animal cohort was used for each dose. <b>E.</b> In a separate experiment, the licking and lifting induced by grayanotoxin III was prevented by pre-dosing the animals with mexiletine (MEX; 30 mg/kg, i.p. or p.o.). <b>F.</b> Total paw licking and lifting behavior time in Nav1.7 KO (n = 5) and WT/HET littermates (n = 7) mice in the 20 minutes following i.pl. injection of 1 µg veratridine. Responses from KO (44.4 sec ±26.6 sec) were smaller than from WT/HET (292.1 sec ±34.7 sec) (p = 0.0073) (mean ± S.E.M., homogeneous ANOVA model with Tukey-Kramer adjusted t-test). <b>G.</b> Total painful paw lifting and licking behavior time in Nav1.7 KO (n = 3) and WT/HET littermates (n = 7) in the 15 minutes following i.pl. injection of 0.1 µg grayanotoxin III. Responses from KO (7.33 sec ±3.5 sec) were smaller than from WT/HET (170.9 sec ±23.1 sec) (p = 0.0003) (mean ± S.E.M., heterogeneous ANOVA model with Welch’s test).</p

Nav1.7 KOs display inability to smell and insensitivity to intra-dermal histamine.

<p><b>A.</b> Total time spent to find a scented food pellet buried in cage bedding for Nav1.7 KO (n = 19) and WT/HET littermates (n = 21). Only one KO found the pellet before the test was cut off at 15 minutes; for statistical purposes the other 18 KO animals were each assigned a time of 15 minutes (900 sec) for an average time of 838.4 sec ±40.2 sec. All WT/HET animals found the pellet, in average time 214.9 sec ±39.3 sec (p = 2.209 E-09) (mean ± S.E.M., Wilcoxon two-sample exact test). <b>B.</b> Total number of scratching bouts following i.d. injection of histamine for Nav1.7 KO (n = 8) and WT/HET littermates (n = 17). Nav1.7 KO animals showed a 98% reduction in scratching bouts (average 1.25±0.6) compared to WT/HET (60.9±13.7) (mean ± S.E.M., heterogeneous ANOVA analysis, p<0.0001).</p

TTX-sensitive currents and mechanically-evoked spiking of C-fibers are reduced in KO mice.

<p><b>A</b>. Representative current traces showing the effect of 200 nM TTX on a KO DRG neuron. Overlaid sweeps show the time course of selective TTX blockade of fast-inactivating currents. Sweeps shown are the three before and the three after TTX addition (inter-sweep interval 10 s). Scale bars, 200 pA and 2 ms. <b>B</b>. Average current densities from acutely dissociated Nav1.7 KO and age-matched WT adult DRG neurons. TTX-S currents: 10.1±3.06 pA/pF, n = 12 neurons from 2 KO animals; and WT/HET, 27.2±4.53 pA/pF, n = 16 neurons from 4 control animals (2 WT and 2 HET) (p = 0.0074, unpaired t-test, mean ± S.E.M.). TTX-R currents: 16.9±4.44 pA/pF from KO, 11.9±3.26 from WT/HET (p = 0.3638, unpaired t-test, mean ± S.E.M.). Cell capacitance averaged 50.2 pF ±5.7 pF from WT/HET and 65.4 pF ±8.6 pF from KO (p = 0.069, unpaired t-test, mean ± S.E.M.). <b>C</b>. Representative mechanically-evoked action potentials in saphenous nerve. Upper trace shows the stimulation protocol (mN); middle and lower traces show representative action potentials from a single C-fiber from a Nav1.7 HET and a Nav1.7 KO preparation respectively. <b>D.</b> Frequency of mechanically-evoked action potentials from C fibers was significantly reduced in Nav1.7 KO mice (filled squares, n = 9 fibers) at forces ≥150 mN when compared to those recorded in C fibers from Nav1.7 control animals (open circles, n = 13 fibers) (150 mN: 8.71±0.86 Hz in control; 3.20±0.76 Hz in KO (mean ± S.E.M., two way ANOVA; p<0.01); 200 mN: 8.1±1.11 in control, 4.14±1.14 Hz in KO (mean ± S.E.M., two way ANOVA, p<0.05)).</p

Compound 52 reduced the ongoing spontaneous activity of C-fiber nociceptors but not Aδ-fiber nociceptors.

<p>Intravenous injection of compound 52 decreased ongoing spontaneous activity of C-fiber nociceptors sensitized by intraplantar administration of CFA (upper panel). The level of ongoing spontaneous activity of sensitized C-fiber nociceptors was significantly lower following administration of compound 52 than following administration of vehicle, from 12 minutes post-administration on. The attenuation of ongoing spontaneous activity by compound 52 continued until the end of the monitoring period (26 minutes after administration of drug). In contrast, administration of compound 52 did not decrease the level of ongoing spontaneous activity in Aδ-fiber nociceptors when compared to vehicle (lower panel). *p < 0.05; **p < 0.01.</p

Nav1.7 KOs do not develop CFA-induced thermal hyperalgesia.

<p>Latency time to withdrawal from radiant heat stimulus for Nav1.7 KO (n = 4) and WT/HET littermates (n = 9) at baseline and 1, 3, and 7 days following CFA injection. WT/HET mice developed robust hyperalgesia in the affected paw 24 hours post-CFA injection that returned to baseline at day 7. KO mice did not develop hyperalgesia. Differences between KO and WT/HET were statistically significant at baseline (WT/HET = 11.5 sec ±0.8 sec; KO = 16 sec ±0.8 sec, p = 0.0001), day 1 (WT/HET = 4.0 sec ±0.5 sec, KO = 15.7 sec ±0.9 sec, p<0.0001) and day 3 (WT/HET = 9.6 sec ±1.0 sec, KO = 17.8 sec ±0.7 sec, p = 0.0028), but not at day 7 (WT/HET = 11.3 sec ±1.1 sec, KO = 15.3 sec ±1.0 sec, p = 0.5381), at which time control animals had recovered back to baseline (all values mean ± S.E.M, ANOVA).</p

Testing of representative aminotriazine compound A in the formalin model of pain.

<p><b>A</b>, Timecourse of flinching binned every minute for each of five cohorts: vehicle, positive control morphine (2 mg/kg), and 10 mg/kg, 30 mg/kg, and 60 mg/kg compound A. All cohorts were n = 8 animals, except morphine was n = 5. <b>B</b>, Total flinches in phase 2 (10 minutes to 40 minutes) (bars and left y-axis) and terminal plasma concentrations of compound A (symbols and right y-axis). Total reduction in phase 2 flinching as percent of vehicle: morphine 68%, 10 mg/kg compound A 32%, 30 mg/kg compound A 66%, 60 mg/kg compound A 85%. Terminal plasma and brain concentrations of compound A: for the 10 mg/kg dose plasma 3.02 uM ± 0.21 μM and brain 0.28 μM ± 0.029 μM; for the 30 mg/kg dose plasma 8.07 μM ± 0.65 μM and brain 0.88 μM ± 0.14 μM; for the 60 mg/kg dose plasma 13.5 μM ± 1.60 μM and brain 1.54 μM ± 0.10 μM (for each dose cohort mean ± SEM, plasma n = 8, brain n = 4). <b>C</b>, Effects of compound A on spontaneous locomotor behaviors. Graph shows basic movement counts (bars and left y-axis) and terminal plasma concentrations (symbols and right y-axis) following indicated doses of compound A. Total percent reduction and the corresponding terminal plasma concentrations: for the 10 mg/kg dose -4.2% and 2.75 μM ± 0.26 μM; for the 30 mg/kg dose 14% and 5.53 μM ± 0.57 μM; for the 60 mg/kg dose 46% and 13.0 μM ± 1.7 μM (brains were not analyzed) (for each dose cohort mean ± SEM, n = 8). **p < 0.01, ***p < 0.001.</p

Compound 52 attenuated CFA-induced sensitization of C-fiber nociceptors to heat stimuli.

<p>Graphs plot number of action potentials (Aps) in response to thermal stimulus of indicated temperature. Administration of vehicle did not alter heat response thresholds in sensitized C-fiber nociceptors (upper panel). By contrast, heat response thresholds of sensitized C-fiber nociceptors were decreased 30 minutes following intravenous injection of compound 52 (lower panel). The two panels represent separate experiments taken from different cohorts of animals. *p < 0.05.</p