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PageArticles
1 of 30 in PresS. Am J Physiol Regul Integr Comp Physiol (April 6, 2006). doi:10.1152/ajpregu.00902.2005
Sex differences in the effects of amiloride
on formalin test nociception in mice
Mona Lisa Chanda & Jeffrey S. Mogil
Dept. of Psychology and Centre for Research on Pain
McGill University
*Address for correspondence:
Dr. Jeffrey S. Mogil
Dept. of Psychology
McGill University
1205 Dr. Penfield Ave.
Montreal, QC H3A 1B1 Canada
(514) 398-6085
(514) 398-4896 (fax)
[email protected]
Copyright © 2006 by the American Physiological Society.
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Chanda, Mona L. and Jeffrey S. Mogil. Sex differences in the effects of amiloride
on formalin test nociception in mice. Am J Physiol Regul Integr Comp Physiol, 2006.—
Amiloride is a non-specific blocker of acid-sensing ion channels (ASICs), which have
been recently implicated in the mediation of mechanical and chemical/inflammatory
nociception. Preliminary data using a transgenic model were suggestive of sex
differences in the role of ASICs. We report here that systemic administration of
amiloride (10-70 mg/kg, i.p.) produces a robust, dose-dependent blockade of late/tonic
phase nociceptive behavior on the mouse formalin test (5%; 20 µl) in female but not male
mice, completely abolishing the known sex difference in formalin test responding.
Adult gonadectomy produced a “switching” of sex differences in amiloride efficacy,
with castrated males displaying an amiloride blockade and ovariectomized females
rendered less sensitive to amiloride. Gonadectomized mice could be switched back to
their intact status using chronic estrogen benzoate or testosterone propionate replacement
via osmotic minipump (6 µg/day or 250 µg/day, respectively). It is unclear whether this
striking sex difference is due to sex-specific involvement of ASICs in pain processing,
but the present data represent one of the first demonstrations of pain-related sex
differences with no obvious opioid involvement.
sex difference, formalin test, nociception, amiloride, gonadectomy, hormone replacement
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WOMEN ARE GREATLY OVERREPRESENTED amongst the sufferers of chronic
pain syndromes (6, 46). This clinical reality may have a basis in perceptual biology,
since women also exhibit greater sensitivity to experimental pain across a number of
modalities (40). To identify underlying mechanisms rodent models have been studied,
and rat and mouse sex differences in nociception are now well appreciated, even though
males continue to be overwhelmingly chosen as the sole subjects of basic science
research in pain (34). Studying rodent sex differences in acute, thermal nociception
(the most commonly used stimulus) may not be the best choice of model, both in terms of
its questionable clinical relevance and because of lingering confusion in the literature as
to which sex is in fact more sensitive (see ref. 35). By contrast, in the formalin test of
chemical/inflammatory pain (16), female rats and mice have consistently been found, by
a number of different investigators, to display greater sensitivity than males (1, 21, 25,
39, see ref. 12) when differences were observed. Females also appear to be more
sensitive to the hypersensitivity produced by inflammatory injury (3, 8, 14). A number of
mechanisms have been proposed to explain rodent sex differences in nociception and its
inhibition by opioids, ranging from body size and blood pressure to neurosteroids,
receptors and signal transduction molecules (see ref. 32). Gonadal hormones obviously
play a primary role in mediating these sex differences, although debate continues as to
the relative involvement of estrogen, progesterone and testosterone, as well as the relative
importance of organizational versus activational effects (see ref. 32). Thus far, however,
the existing evidence suggests that the sexes modulate pain differently, perhaps
employing distinct, sex-specific neural circuitry in the midbrain, brain stem and spinal
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cord (28, 36, 45). In the present study, we find evidence suggesting that sex differences
in pain processing may also exist at the very first stages of sensory transduction.
Acid-sensing ion channels (ASICs) are members of the ENaC/DEG superfamily of
amiloride-sensitive epithelial sodium channels (see ref. 26). ASICs are distinct amongst
the ENaC/DEGs in that they are proton-gated sodium channels proposed as transducers
of acid-evoked pain, mechanical pain and innocuous touch. Furthermore, ASICs
contribute to pain processing and central sensitization during pathological states such as
inflammation and ischemia, both of which are accompanied by moderate-to-severe tissue
acidosis (29, 47). Previously we examined the pain phenotype of mice bearing a
dominant-negative mutation of the ASIC3 gene, which renders ASIC1, ASIC2 and
ASIC3 channel subunits inactive through oligomerization, thereby abolishing all
ASIC-related currents in sensory neurons (33). We found that mutant mice exhibited
increased sensitivity to acute mechanical and chemical/inflammatory pain, as well as
increased mechanical hypersensitivity following chronic inflammation and muscle
acidification (33). However, upon further analysis, we discovered that the ASIC3
dominant negative mutation exerted a sex-specific impact on nociception: only male,
but not female, mutants had heightened nociceptive sensitivity relative to their wild-type
counterparts (9, manuscript in preparation).
Thus, in an initial attempt to further investigate sex differences in the contribution of
ASICs to nociception, we administered the non-specific ASIC-blocker (and K+-sparing
diuretic) amiloride, to outbred mice of both sexes. Administration of amiloride has
been shown to inhibit pain in laboratory animals across a variety of nociceptive assays
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(15, 18, 41), as has a novel ASIC blocker, A-317567 (15). Although the mechanisms of
its analgesic action are not fully understood, amiloride has been shown to effectively
block acid-evoked ASIC-like currents in sensory neurons (see ref. 15). In the present
study, we characterized sex differences in amiloride inhibition of responding in the
formalin test of chemical/inflammatory nociception, and then investigated the hormonal
mechanisms underlying this difference through gonadectomy and hormone replacement
experiments.
MATERIALS AND METHODS
Animals. Naïve, adult male and female CD-1® (Crl:ICR) mice were obtained from
Charles River Laboratories (Boucherville, QC). Upon our request, some 6-week-old
mice were gonadectomized or sham gonadectomized by personnel at Charles River at
least one week prior to shipment. Mice were housed in same-sex groups of four and
acclimatized to our vivarium at McGill University for at least one week prior to any
experiments. The vivarium was temperature-controlled and maintained on a 12:12 h
light-dark cycle (lights on at 07:00 h). Access to food (Harlan Teklad 8604) and water
was ad libitum. All procedures were in accordance with the guidelines specified by the
Canadian Council on Animal Care.
Drug administration. Amiloride hydrochloride hydrate, 2-hydroxypropyl- cyclodextrin (cyclodextrin), estrogen benzoate, polyethylene glycol (PEG), and
testosterone propionate were all obtained from Sigma (St. Louis, MO). Amiloride or
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PEG vehicle were administered in every experiment via intraperitoneal (i.p.) injection.
In hormone replacement experiments, estrogen benzoate, testosterone propionate or
cyclodextrin vehicle were administered via subcutaneously implanted osmotic
minipumps (ALZET Model 2002), at a rate of 0.5 µL/h over 7 (estrogen) or
14 (testosterone) days. Mice were anesthetized with isoflurane/oxygen, and osmotic
minipumps were implanted via a small (~1 cm) incision into the upper back.
The incision was closed with sterile 9-mm stainless steel wound clips, and animals were
placed in a recovery chamber for 30 min before being returned to their home cages.
Formalin Test. The formalin test was employed using procedures described in
considerable detail previously (37). Mice were habituated singly for 30 min to
Plexiglas observation chambers (15 cm diameter; 22.5 cm high) atop a glass floor, and
then treated with amiloride (0-70 mg/kg, i.p., dissolved in 30% PEG in physiological
saline) or vehicle. The 30 mg/kg dose of amiloride administered was previously reported
to induce half-maximal antinociception on the formalin test in mice (18). Thirty min
after drug administration, all mice received 20 µl of 5% formalin injected into the plantar
surface of the right hind paw. Behavioral responses to formalin were captured by video
cameras located underneath the glass floor, and scored later using ObserverTM software
(Noldus Inc.). Mice were sampled for 5 s at the start of every minute for 1 h for the
occurrence of licking/biting of the affected paw (10). The percentage of total number
of samples with licking/biting behavior in the acute, “early” phase (0-10 min) and the
tonic, “late” phase (10-60 min) were recorded. Immediately after behavioral testing,
mice were sacrificed, and hind paws were severed and weighed to quantify inflammation.
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Edema was defined by the difference in hindpaw weights divided by the weight of the
contralateral paw, expressed as a percentage. Data obtained from mice with less than
20% inflammation (n = 13) were discarded before analysis.
Four separate experiments were conducted. The first compared formalin test
sensitivity of male and female mice given various doses of amiloride (n = 7-18/
dose/drug/sex). The second experiment investigated the effects of gonadectomy
(i.e., castration and ovariectomy) on amiloride’s actions in male and female mice,
respectively (n = 20-29/sex/surgery/drug). The third experiment focused on the
possible role of estrogen in mediating the sex differences in amiloride efficacy,
and attempted to reverse the effect of ovariectomy via hormone replacement with
estrogen benzoate (n = 16/hormone/drug). The final experiment focused on the possible
role of testosterone in mediating the sex difference in amiloride efficacy, and attempted
to reverse the effect of castration via hormone replacement with testosterone propionate
(n = 15-22/hormone/drug).
Estrogen Replacement. Ovariectomized female mice were treated with estrogen
benzoate (6 µg/day, dissolved in 40% cyclodextrin in physiological saline) or vehicle via
osmotic minipump. On day 7 following the beginning of this treatment, mice were
administered either amiloride (30 mg/kg, i.p.) or vehicle, and tested on the formalin test
as described above. Immediately following testing and sacrifice, the uteri of all mice in
this experiment were removed and weighed in order to confirm the efficacy of the
estrogen replacement.
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Testosterone Replacement. Castrated male mice were treated with testosterone
propionate (250 µg/day, dissolved in 40% cyclodextrin in physiological saline) or vehicle
via osmotic minipump. On day 14 following the beginning of this treatment, mice were
administered either amiloride (30 mg/kg, i.p.) or vehicle, and tested on the formalin test
as described above. Immediately following testing and sacrifice, the seminal vesicles of
all mice in this experiment were removed and weighed in order to confirm the efficacy of
the testosterone replacement.
Statistical Analysis. Dose-response data were analyzed by the method of Tallarida
and Murray (44), as implemented by the FlashCalc 21.5® program (M. Ossipov,
University of Arizona). Data were analyzed by analysis of variance (ANOVA),
with sex, surgery, hormone, and/or drug as between-subjects factors as appropriate
(see Results). ANOVAs were followed by two-tailed Student’s t-tests where appropriate.
A significance level of
= 0.05 was adopted.
RESULTS
In all experiments, the expected biphasic pattern of formalin recuperative behaviors
(in mice; mainly licking behavior; see ref. 43) was observed (see Figs. 1-4), and most
mice were displaying minimal levels of licking behavior by 60 min post-injection.
Besides decreasing formalin licking in some conditions, amiloride did not appear to
affect either the qualitative or timing aspects of formalin responding.
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Amiloride Dose-Response Experiment. As can be seen in Figure 1, amiloride
affected formalin licking behavior in the late (10-60 min) but not early (0-10 min)
phase. In female mice, the half-maximal antinociceptive dose (AD50) of amiloride
against late-phase licking was calculated as 34 mg/kg (95% confidence interval:
24-49 mg/kg). By extrapolation, the AD50 in males was estimated as 136 mg/kg, but this
estimate should be treated with great caution due to the fact that only the highest dose
(70 mg/kg, i.p.) produced any evidence of an effect. We were unable to try higher doses,
because we observed a number of lethalities in a separate group of mice given 70 mg/kg
and observed for several hours. Inspection of Figure 1B suggested that 30 mg/kg
amiloride was the most appropriate single dose for further experimentation, since it
produced a half-maximal inhibition of responding in female mice, produced absolutely no
effect whatsoever in male mice, and was fully able to abolish the preexisting sex
difference in formalin behavior. Comparing vehicle and 30 mg/kg amiloride, two-way
ANOVAs (sex x dose) revealed no main effects or interactions in the early phase, and a
significant main effect of drug (F1,67 = 9.1, p<0.01) and a significant sex x drug
interaction (F1,67 = 4.7, p<0.05) in the late phase. Figures 1C,D illustrate the robust
blockade of formalin-induced licking behavior throughout the late phase by 30 mg/kg
amiloride in female but not male CD-1 mice. This was confirmed statistically by the fact
that a two-between, one-within repeated measures ANOVA revealed a significant
repeated measures effect (F1,759= 22.9, p<0.001), but no interaction of repeated measures
with either sex, drug or sex x drug conditions.
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In gonadally intact mice, there was no effect of sex or amiloride on the inflammation
produced by formalin injection assessed immediately postmortem at 60 min
post-injection (data not shown).
Effects of Gonadectomy. Figure 2 shows the results of the gonadectomy experiment.
In the early phase, we observed a significant drug x surgery interaction effect on early
phase formalin responding (F1,173 = 6.5, p<0.05). Inspection of Figure 2A-D reveals that
this interaction was driven largely by a decrease in formalin licking produced by
amiloride in castrated males. However, this effect was not reliable, as it was not
replicated in a subsequent experiment (see Fig. 4A).
In the late phase, we observed a significant main effect of drug (F1,178 = 20.2,
p<0.001) but also a significant three-way (sex x surgery x drug) interaction (F1,178 = 4.5,
p<0.05). Subsequent t-tests revealed a higher level of formalin responding in Sham +
Vehicle females versus Sham + Vehicle males (as was observed in the previous
experiment in intact vehicle-treated females and males) that only just failed to reach
significance (t42 = 2.1, p=0.051). More importantly, t-tests revealed highly significant
effects of amiloride in female (t40 = 3.3, p<0.005) but not male (t44 = 1.0, p=0.32) mice,
precisely replicating the results of the first experiment. Neither castration or ovariectomy
significantly altered formalin responding per se, although there was a trend towards
lower sensitivity in ovariectomized females relative to sham females (t39= 1.53, p=0.13).
However, in castrated males, amiloride reduced formalin responding compared to vehicle
(t53 = 3.2, p <0.001), producing an antinociceptive effect comparable to that of sham
females (see Fig. 2E). Female ovariectomy also produced a “switch” of amiloride
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efficacy. That is, in ovariectomized females amiloride no longer significantly reduced
late phase formalin responding (t41 = 1.5, p=0.14) (Fig. 2E).
Effects of Estrogen Replacement to Ovariectomized Females. A two-way ANOVA
performed on early phase data revealed a main effect of amiloride (F1,59 = 5.4, p<0.05).
The modest inhibition of early-phase responding by amiloride in this experiment
(see Fig. 3A,B) stands in contrast, however, to the results of the gonadectomy
experiment, in which no such inhibition was observed in ovariectomized female mice
(see Fig. 2C).
A two-way ANOVA performed on late-phase data revealed a significant effect of
drug (F1,59 = 16.9, p<0.001), and a trend towards a hormone x drug interaction (p=0.20).
As can be seen in Figure 3B, estrogen was clearly successful in restoring the efficacy of
amiloride, but in this experiment amiloride also trended strongly towards producing a
significant inhibition of formalin licking in ovariectomized females given vehicle
replacement (p=0.06).
Estrogen treatment to ovariectomized females significantly increased uterine weight
equally across both drug conditions (main effect of hormone: F1,58 = 226.0, p<0.001).
Effects of Testosterone Replacement to Castrated Males. A two-way ANOVA
performed on early-phase data revealed a main effect of hormone (F1,69 = 9.0, p<0.005),
with testosterone-replaced castrated males displaying greater early-phase formalin
responding relative to their vehicle-treated counterparts (t71=3.0, p<0.05) (see Fig. 4A,B).
A two-way ANOVA performed on late-phase data revealed a significant effect of
drug (F1,69 = 5.5, p<0.05) but also a significant hormone x drug interaction (F1,69 = 4.1,
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p<0.05). There was a trend towards lower formalin sensitivity in testosterone-replaced
castrated mice receiving vehicle (t35= 1.4, p=0.17). As can be seen in Figure 4,
chronic testosterone treatment completely reversed the effect of gonadectomy on
amiloride efficacy, such that amiloride reduced formalin responding during the late phase
in vehicle-treated castrated males (precisely replicating the findings of the gonadectomy
experiment), but not in testosterone-treated males.
Testosterone treatment to castrated males significantly increased seminal vesicle
weight equally across both drug conditions (main effect of hormone: F1,69 = 344.9,
p<0.001).
DISCUSSION
The main finding of this study is a robust sex difference in the ability of amiloride
to inhibit licking behavior in the late-phase formalin test in female but not male
CD-1 mice. AD50s were estimated as 34 mg/kg and 136 mg/kg for the two sexes,
respectively, a potency ratio of 4.0. However, we were unable to demonstrate amiloride
antinociception in males at any dose below 70 mg/kg, a dose found to be toxic,
suggesting that the sex difference observed may be qualitative as opposed to quantitative.
We have some reason to believe as well that the sex difference is not specific to the
formalin test, or even to chemical/inflammatory nociception, since 30 mg/kg amiloride
also increased von Frey fiber withdrawal thresholds in female but not male mice (data not
shown).
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We observed significantly greater formalin responses during the late phase in
gonadally intact females relative to males, which is consistent with previous findings in
the rodent literature from a number of different groups (1, 21, 25, 39). This quantitative
sex difference has been attributed to the actions of gonadal steroid hormones, in which
testosterone exerts a blunting effect on formalin pain, whereas estrogen heightens
sensitivity through a reduction of pain inhibition mechanisms (2, 21, 22). The present
study revealed a strong trend towards lower pain responding during the late phase in
ovariectomized females relative to their sham-operated counterparts, consistent with the
notion that estrogen is responsible for the sex difference in sensitivity to formalin.
Castration has been shown to produce an increase in formalin responding in male rats
(21, 22), but we did not observe any significant difference in sensitivity between
castrated and sham-operated CD-1 males. Testosterone treatment to castrated males
produced a significant increase in responding in the early phase, but a strong trend
towards decreased responding in the late phase.
To our knowledge, only one study has ever tested amiloride against formalin test
nociception (18). That study reported dose-dependent inhibition of formalin test licking
behavior by amiloride injected systemically, supraspinally or spinally in both the early
and late phases, with full efficacy against late phase licking. Subjects were male Swiss
mice of unreported origin. The only other studies to test amiloride in a behavioral
nociceptive assay were performed by Sluka and colleagues (41), who reported
dose-dependent amiloride inhibition of acid-induced mechanical hypersensitivity in male
C57BL/6 mice, and by Dube and colleagues (15), who demonstrated dose-dependent
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amiloride reversal of both thermal and mechanical hypersensitivity produced by
inflammation or skin incision, respectively, in male Sprague Dawley rats.
Ours is therefore the first study to report a sex difference in the antinociceptive effects
of amiloride, in which drug treatment markedly and dose-dependently attenuated late
phase responding in female, but not male CD-1 mice, except at a toxic dose. Our data are
obviously in contradiction with those of Ferreira and colleagues (18). Possible reasons
for the discrepancy may include the more concentrated formalin solution used presently
(5% versus 2.5%), which may have rendered the amiloride dose less efficacious, and/or
genotypic differences between the two different outbred populations used (see ref. 11).
We have shown on a number of prior occasions that analgesic potency and efficacy are
dependent on genetic factors (5, 38, 48, 49), and also that sex differences interact
importantly with genotype, being observed in some strains but not others (24, 35,
see ref. 31).
Gonadectomy reversed the sex difference in the present study, producing an
unmasking of late-phase amiloride antinociception in males and attenuating amiloride’s
efficacy in females. In interpreting these effects in Figures 2-4, it is important to
remember that the efficacy of amiloride to reduce formalin responding is properly
compared only to the analogous vehicle-treated group. This is because the “baseline”
shifts from condition to condition, due to the often non-significant but nonetheless
statistically influential effects of sex, gonadectomy and hormone treatments.
Estrogen treatment reversed the effects of gonadectomy in ovariectomized females,
such that the not-quite-significant (i.e., p=0.14 and p=0.06 by t-test in Figure 2E and 3C)
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effect of 30 mg/kg amiloride after ovariectomy reverted to a strongly significant
amiloride blockade (characteristic of intact females) after hormone replacement.
These findings implicate estrogen in the modulation of amiloride’s antinociceptive
effects. The possible involvement of progesterone—shown by us previously to play an
important role in the “switching” of N-methyl-D-aspartate (NMDA)-dependence of swim
stress-induced and -opioid antinociception in male and female CD-1 mice (42)—has yet
to be evaluated.
Testosterone treatment reversed the effects of gonadectomy in castrated males,
such that hormone-replaced males reverted to the amiloride-insensitive status
characteristic of intact males. These findings therefore implicate testosterone as well in
the mediation of amiloride’s actions. A point that remains to be clarified is whether the
robust effects of testosterone are due to conversion to dihydrotestosterone, aromatization
to estrogen, or direct actions at androgen receptors.
In addition to the hormonal mediators involved, the site of the sex-specific effects
of amiloride warrant further investigation. Both amiloride and the newly reported
drug, A-317567, block ASIC-like currents in dorsal root ganglia. The common
pharmacological actions of these two drugs reported by Dube et al. (15) implicate ASICs
in the antinociceptive effects of amiloride. This hypothesis is perhaps contradicted by
our findings using ASIC3 dominant negative mutants, in which blockade of all ASIC-like
transient currents produced increases in sensitivity on the formalin test (among other
assays) in males but not females (9, manuscript in preparation). The difference between
the direction of the sex difference in these two cases does not appear to be due to genetic
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background issues, since amiloride given to FVB/J mice (the background strain of the
ASIC mutants) also produces antinociception in females but not males (data not shown).
It is possible that the transgenic data actually reflect compensatory mechanisms, and not
the direct impact of the absence of ASIC-like currents in these animals.
Thus, it is perfectly conceivable that the sex-specific effects of amiloride on
nociception shown here are not related to the drug’s known effects on ASIC channel
activity, and that we have uncovered sex differences in another neurochemical system.
Amiloride is known to act on other pharmacological target sites implicated in pain
processing, such as biosynthetic pathways for proinflammatory cytokines (23), the
Na+/H+ exchanger (NHE) (30), ATP-sensitive K+ channels (7), the adenosine A1 receptor
(20) and the GABA-A receptor complex (19). None of these systems have ever been
directly implicated in the mediation of sex differences in formalin nociception, but in
many cases there is evidence of their regulation by gonadal hormones (4, 13, 17, 27).
We have begun to investigate these possibilities one by one. Preliminary experiments
using ethylisopropyl-amiloride (EIPA; 3 and 10 mg/kg, i.p.), a high-potency
NHE-blocking compound (30), do not reveal efficacy against late-phase formalin
nociception in either sex.
Whatever the mechanism of the sex differences in amiloride efficacy shown here
turns out to be, this finding represents one of the first demonstrations of sex differences in
pain processing not obviously related to opioid modulation. We believe that further study
will eventually reveal sex differences in pain mechanisms to be pervasive, and evident at
all levels of the neuraxis. This finding is also representative of a class of recent
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pain-related discoveries that are only made possible by the simultaneous testing of both
sexes, still a strategy only rarely used in the field (34).
GRANTS
This research was supported by a Louise Edwards Foundation Award in Pain
Research to J.S. Mogil. We thank Dr. Jim Pfaus for his generous contributions.
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FIGURE LEGENDS
Fig.1. Amiloride (0-70 mg/kg, i.p.) dose-dependently reduces sensitivity to
5% formalin nociception (20 µl, intraplantar) in female, but not male, CD-1 mice.
A,B) Dose-response relationships in female (open circles) and male (closed squares)
mice in the early (A; 0-10 min) and late (B; 10-60 min) phases of the formalin test.
Symbols represent mean ± S.E.M. percentage of samples in which licking/biting was
observed (of 10 samples in the early phase and 50 samples in the late phase). There is
no evidence of amiloride antinociception in the early phase. Amiloride AD50s (see text)
were calculated as 34 mg/kg and 136 mg/kg for female and male mice, respectively.
C,D) Time-course data of vehicle (“0” dose in graphs A,B) and 30 mg/kg amiloride
groups. Symbols represent mean ± S.E.M. percentage of samples in which licking/biting
was observed in each 5-min time bin post-injection of formalin.
Fig.2. Gonadectomy produces a “female-like” pattern of amiloride (30 mg/kg, i.p.)
efficacy against late-phase formalin test responding in male mice, and reduces the
efficacy of amiloride in female mice. A-D) Time-course data of the four groups:
sham-operated females (A), sham-operated males (B), ovariectomized females (C)
and castrated males (D). Symbols represent mean ± S.E.M. percentage of samples in
which licking/biting was observed in each 5-min time bin post-injection of formalin.
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E) Summed late-phase data from graphs A-D. Bars represent mean ± S.E.M percentage
of positive samples from 10-60 min post-injection. ***p<0.005 compared to
corresponding vehicle-treated group (t-test).
Fig. 3. Estrogen benzoate replacement reverses the effect of gonadectomy on amiloride
efficacy against late-phase formalin test responding in ovariectomized female mice,
restoring their robust sensitivity to amiloride. A,B) Time-course data. Symbols represent
mean ± S.E.M. percentage of samples in which licking/biting was observed in each
5-min time bin post-injection of formalin. C) Summed late-phase data from Graphs A,B.
Bars represent mean ± S.E.M percentage of positive samples from 10-60 min
post-injection. ***p<0.005 compared to corresponding vehicle-treated group (t-test).
Fig. 4. Testosterone propionate replacement reverses the effect of gonadectomy on
amiloride efficacy against late-phase formalin test responding in castrated male mice,
restoring their insensitivity to amiloride. A,B) Time-course data. Symbols represent
mean ± S.E.M. percentage of samples in which licking/biting was observed in each
5-min time bin post-injection of formalin. C) Summed late-phase data from Graphs A,B.
Bars represent mean ± S.E.M percentage of positive samples from 10-60 min
post-injection. **p<0.01 compared to corresponding vehicle-treated group (t-test).
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Figure 1
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Figure 2
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Figure 3
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Figure 4