Biochemical Pharmacology 97 (2015) 590–600
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.e lsev ier .com/ locate /b iochempharmReviewThe role of alpha5 nicotinic acetylcholine receptors in mouse models
of chronic inflammatory and neuropathic pain
Deniz Bagdas a,b,1, Shakir D. AlSharari a,c,1, Kelen Freitas a, Matthew Tracy a,
M. Imad Damaj a,*
aDepartment of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613,
United States
b Experimental Animals Breeding and Research Center, Faculty of Medicine, Uludag University, Bursa 16059, Turkey
cDepartment of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Saudi ArabiaA R T I C L E I N F O A B S T R A C T
Article history: The aim of the present study was to determine the impact ofa5 nicotinic acetylcholine receptor (nAChR)
Received 2 March 2015 subunit deletion in the mouse on the development and intensity of nociceptive behavior in various
Accepted 20 April 2015 chronic pain models.
Available online 28 April 2015
The role ofa5-containing nAChRswas explored inmousemodels of chronic pain, including peripheral
neuropathy (chronic constriction nerve injury, CCI), tonic inflammatory pain (the formalin test) and
Keywords: short and long-term inflammatory pain (complete Freund’s adjuvant, CFA and carrageenan tests) in a5
alpha5
knock-out (KO) and wild-type (WT) mice.
Nicotinic acetylcholine receptors
The results showed that paw-licking time was decreased in the formalin test, and the hyperalgesic
Neuropathic pain
Inflammatory pain and allodynic responses to carrageenan and CFA injections were also reduced. In addition, paw edema in
formalin-, carrageenan- or CFA-treated mice were attenuated in a5-KO mice significantly. Furthermore,
tumor necrosis factor-alpha (TNF-a) levels of carrageenan-treated paws were lower in a5-KO mice. The
antinociceptive effects of nicotine and sazetidine-A but not varenicline were a5-dependent in the
formalin test. Both hyperalgesia and allodynia observed in the CCI test were reduced in a5-KO mice.
Nicotine reversal of mechanical allodynia in the CCI test was mediated through a5-nAChRs at spinal and
peripheral sites.
In summary, our results highlight the involvement of the a5 nAChR subunit in the development of
hyperalgesia, allodynia and inflammation associated with chronic neuropathic and inflammatory pain
models. They also suggest the importance of a5-nAChRs as a target for the treatment of chronic pain.
 2015 Elsevier Inc. All rights reserved.Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
2.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
2.2. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3. Behavioral pain tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.1. Formalin test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592Abbreviations: nAChRs, nicotinic acetylcholine receptors; CCI, chronic constriction nerve injury; PWL, pawwithdrawal latency; CFA, complete Freund’s adjuvant; KO, knock-
out; WT, wild-type; TNF-a, tumor necrosis factor-alpha; AUC, area under the curve.
* Corresponding author. Tel.: +1 804 8282956; fax: +1 804 8282117.
E-mail address: mdamaj@vcu.edu (M.I. Damaj).
1 These authors have equally contributed to this work.
http://dx.doi.org/10.1016/j.bcp.2015.04.013
0006-2952/ 2015 Elsevier Inc. All rights reserved.
D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600 5912.3.2. Carrageenan-induced inflammatory pain model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.3. Complete Freund’s adjuvant (CFA)-induced inflammatory pain model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.4. Measurement of paw edema. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.5. Chronic constrictive nerve injury (CCI)-induced neuropathic pain model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.6. Paw withdrawal test (evaluation of thermal hyperalgesia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.7. von Frey test (evaluation of mechanical allodynia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.8. Acetic acid-induced conditioned place aversion (CPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2.3.9. Locomotor activity test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
2.3.10. Motor coordination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
2.4. Tumor necrosis factor-alpha (TNF-a) levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
2.5. Intracerebroventricular and intrathecal injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
2.6. Statistical analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
3.1. Pain behavior and paw edema in a5 knockout mice in the formalin test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
3.2. Effect of nicotine, varenicline and sazetidine-A in a5 knockout mice in the formalin test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
3.3. Hyperalgesia, allodynia and inflammation in a5 knockout mice in the carrageenan test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
3.4. Hyperalgesia, allodynia and inflammation in a5 knockout mice in the CFA test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
3.5. Hyperalgesia and allodynia in a5 knockout mice in the CCI-induced neuropathic pain model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
3.6. Relevance of a5 to anti-allodynic effects of nicotine in the CCI test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
3.7. Impact of a5 gene deletion on acetic acid-induced conditioned place aversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
3.8. Impact of a5 gene deletion on motor activity and coordination of mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5991. Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are
pentameric ligand-gated ion channels that exist as homomeric
or heteromeric complexes ofa andb subunits. To date, 12neuronal
subunits (a2–a10 and b2–b4) have been identified in mammals
[1]. The a5 nAChR subunits are widely expressed in the
mammalian central nervous system [2], including the spinal cord
[3], rat dorsal root ganglia [4], as well as peripherally in
sympathetic and parasympathetic ganglia [5].
The a5 subunit cannot form a functional homomeric receptor,
or assemble in nAChRs as the sole a subunit expressed with either
b2 or b4. Therefore, the a5 subunit is incorporated into a4b2*,
a3b2*, and a3b4* nAChRs (where * denotes the possible inclusion
of additional nAChR subunits) where it greatly influences
nicotine’s modulation of receptor function and pharmacological
properties of these receptor subtypes in response to the drug in
heterologous expression systems [6–8]. Furthermore, recent data
support an important role for a5 in nicotine’s behavioral effects.
Mice null for the a5 nAChR subunit have reduced sensitivity to
nicotine-induced seizures and hypolocomotion [9,10]. Additional-
ly, these a5 KO mice are less sensitive to nicotine-induced
antinociception and hypothermia compared to WT littermates
after acute administration of the drug inmice [11,12]. Interestingly,
a5 KOmice showed an enhancement of nicotine reward and intake
[11,13].
Data is also emerging on the possible role of a5-containing
nAChRs in the regulation of important functions in the central
nervous system as well as the peripheral nervous system. For
example, these receptorswere reported to influence the autonomic
control of several organ systems [14]. Furthermore, Vincler and
Eisenach [26] observed an increased expression of the a5 nAChR
subunit in the outer laminae of the dorsal horn following spinal
nerve ligation in rats [3]. More recently, the same group reported
that intrathecal injection of a5 antisense oligonucleotides to rats
with spinal nerve ligation alleviated the mechanical allodynia in
the animals [15]. In addition, treatment of these rats with a5-
antisense was accompanied with a significant reduction in pCREB
immunoreactivity in the outer laminae of the dorsal horn of the
ligated rats. We therefore hypothesized that disruption of a5nAChR subunit will modulate pain behaviors in chronic inflam-
matory and neuropathic pain.
The present study seeks to determine the impact of a5 nAChR
subunit deletion in themouse on the development and intensity of
nociceptive behavior in various chronic pain models. Using a5
knock-out (KO) mice, the role of a5-containing nAChRs was
explored inwell-established rodentmodels of chronic pain, such as
peripheral neuropathy (chronic constriction nerve injury, CCI),
tonic inflammatory pain (the formalin test) and short- and long-
term inflammatory pain (carrageenan and complete Freund’s
adjuvant – CFA tests). We also determined to what extent is the
antinociceptive effects of nicotine, in some of these models, are
mediated by a5-containing nAChRs. Data obtained from these
studies will further the understanding of the a5 nAChR subunit in
pain regulation andmay lead to the development of a5-containing
nAChR agonists for the treatment of chronic pain.
2. Materials and methods
2.1. Animals
Male C57BL/6J mice were purchased from Jackson Laboratories
(Bar Harbor,ME) andwere used only for backcrossing.Mice null for
the a5 subunit and their wild-type littermates were bred in an
animal care facility at Virginia Commonwealth University (Rich-
mond, VA) and are maintained on a C57Bl/6J background. They
have been backcrossed to at least N12. For all experiments,
mutants and wild type controls are obtained from crossing
heterozygote mice. This breeding scheme allows us to rigorously
control for any anomalies that may occur with crossing solely
mutant animals. a5 KO mice were originally described by Salas
et al. [10]. Male animals were 8–10 weeks of age at the start of the
experiments and were group-housed in a 21 8C humidity-
controlled Association for Assessment and Accreditation of
Laboratory Animal Care-approved animal care facility with ad
libitum access to food and water. Experiments were performed
during the light cycle and were approved by the Institutional
Animal Care and Use Committee of Virginia Commonwealth
University and conducted according to the guide for the Care and
592 D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600Use of Laboratory Animals as adopted and promulgated by the
United States National Institutes of Health.
2.2. Drugs
()-Nicotine hydrogen tartrate salt was purchased from Sigma–
Aldrich Inc. (St. Louis, MO, USA). All drugs were dissolved in
physiological saline (0.9% sodium chloride) and injected subcuta-
neously (s.c.) at a total volume of 1 ml/100 g body weight unless
noted otherwise. Varenicline (7,8,9,10-tetrahydro-6,10-methano-
6H-pyrazino(2,3-h)(3)benzazepine) and sazetidine-A (6-[5-[(2S)-
2-azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol) were supplied
by the National Institute of Drug Abuse (NIDA Drug Supply
Program, Bethesda, MD). All doses are expressed as the free base of
the drug.
2.3. Behavioral pain tests
2.3.1. Formalin test
The formalin test was carried out in an open Plexiglas cage,
with a mirror placed at a 45 degree angle behind the cage to
allow an unobstructed view of the paws. Mice were allowed to
acclimate for 15 min in the test cage prior to injection. Each
animal received an intraplantar injection of 20 ml of (2.5%)
formalin to the right hindpaw. Each mouse was then immedi-
ately placed in a Plexiglas box. Up to two mice at one time were
observed from 0 to 5 min (phase 1) and 20 to 45 min (phase 2)
post-Formalin injection. The period between the two phases of
nociceptive responding is generally considered to be a phase of
weak activity. The amount of time spent licking the injected paw
was recorded with a digital stopwatch. Paw diameter (see
Section 2.3.4) was also measured before and 1 h after formalin
injection.
In a separate experiment, nicotinic analogs [nicotine (1.5 mg/
kg), varenicline (3 mg/kg) and sazetidine-A (1.5 mg/kg)] or the
control solution was injected s.c. 15 min before the formalin
injection in a5 WT and KO mice. These active doses were selected
based on our recent report in the same test [16]. All behavioral
testing on WT and KO animals in this study was performed in a
blinded manner.
2.3.2. Carrageenan-induced inflammatory pain model
a5 WT and KO mice were injected with 20 ml of (0.5%) of
lambda-carrageenan in the intraplantar region of the right
hindpaw. Paw diameter (see Section 2.3.4), thermal hyperalgesia
and mechanical allodynia (see Sections 2.3.6 and 2.3.7) were
measured before and at several hourly time-points after lambda-
carrageenan injection (3, 6 and 24 h).
2.3.3. Complete Freund’s adjuvant (CFA)-induced inflammatory pain
model
a5 WT and KO mice were injected with 20 ml of CFA (50% for
allodynia measures and 75% for hyperalgesia) in the intraplantar
region of the right hindpaw. Paw diameter (see Section 2.3.4),
thermal hyperalgesia and mechanical allodynia (see Sections
2.3.6 and 2.3.7) were measured before and after CFA injection (day
3 and 14).
2.3.4. Measurement of paw edema
The thickness of the formalin, carrageenan, CFA treated and
control pawsweremeasured both before and after injections at the
time points indicated above, using a digital caliper (Traceable
Calipers, Friendswood, TX). Data were recorded to the nearest
0.01 mm and expressed as change in paw thickness (DPD = differ-
ence in the ipsilateral paw diameter before and after injection paw
thickness).2.3.5. Chronic constrictive nerve injury (CCI)-induced neuropathic
pain model
Mice were anesthetized with pentobarbital (45 mg/kg, i.p.). An
incision was made just below the hip bone, parallel to the sciatic
nerve. The right common sciatic nerve was exposed at the level
proximal to the sciatic trifurcation and a nerve segment 3–5 mm
long was separated from surrounding connective tissue. Two loose
ligatures with 5–0 silk suture were made around the nerve with a
1.0–1.5 mm interval between each of them. Muscles were closed
with suture thread and the wound with wound clips. This
procedure resulted in chronic constrictive injury (CCI) of the
ligated nerve. Thermal hyperalgesia andmechanical allodynia (see
Sections 2.3.6 and 2.3.7) were measured at several weekly time-
points after operation (1, 3, and 7 weeks).
In a separate experimental group, nicotine (0.9 mg/kg) or
vehicle were injected intraperitoneally (i.p.) toa5WT and KOmice
7 weeks after CCI surgery and tested for mechanical allodynia.
Mechanical stimuli thresholds were determined for each animal 5,
10, 15, 30, and 60 min after injection of nicotine.
We also tested anti-allodynic effects of nicotine using different
administration routes to understand its sites of action. Hence, we
administered nicotine intracerebroventricularly (25 mg/5ml, i.c.v.)
for central; intrathecally (20mg/5ml, i.t.) for spinal/supraspinal
and intraplantarly (45mg/5ml, i.pl.) for peripheral sites of action in
a5 WT and KOmice 2 weeks after CCI surgery. We chose the doses
according to our previous results and dose–response curves
determinations (data not shown).
2.3.6. Paw withdrawal test (evaluation of thermal hyperalgesia)
Thermal hyperalgesia was measured via the Hargreaves test.
Mice were placed in clear plastic chambers (7  9  10 cm) on an
elevated surface and allowed to acclimate to their environment
before testing. The radiant heat source was directed to the plantar
surface of each hind paw in the area immediately proximal to the
toes. The paw withdrawal latency (PWL) was defined as the time
from the onset of radiant heat to withdrawal of the animal’s hind
paw. A 20-s cut-off time was used. Three measures of PWL were
taken and averaged for each hind-paw using the Hargreaves test.
Withdrawal latencies were measured in each hind paw. Results
were expressed either as withdrawal latency for each paw or as
DPWL (s) = contralateral latency  ipsilateral latency.
2.3.7. von Frey test (evaluation of mechanical allodynia)
Mechanical allodynia thresholds were determined according to
the method of Chaplan et al. [17]. Mice were placed in a Plexiglas
cage withmeshmetal flooring and allowed to acclimate for 30 min
before testing. A series of calibrated von Frey filaments (Stoelting,
Wood Dale, IL) with logarithmically incremental stiffness ranging
from2.83 to 5.88 expressed as dsLog 10 of [10 £ force in (mg)]were
applied to the paw with a modified up–down method [18]. In the
absence of a paw withdrawal response to the initially selected
filament, a thicker filament corresponding to a stronger stimulus
was presented. In the event of paw withdrawal, the next weaker
stimulus was chosen. Each hair was presented perpendicularly
against the paw, with sufficient force to cause slight bending, and
held 2–3 s. The stimulation of the same intensity was applied
5 times to the hind paw at intervals of a few seconds. The
mechanical threshold was expressed as Log 10 of [10 £ force in
(mg)], indicating the force of the von Frey hair to which the animal
reacted (paw withdrawn, licking or shaking).
2.3.8. Acetic acid-induced conditioned place aversion (CPA)
To evaluate the negative affective component of pain, the CPA
test was performed. In brief, groups ofa5WT and KOmice (n = 6–8
per group) were handled for 3 days prior to initiation of CPA
testing. The CPA apparatus consisted of a three-chambered box
D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600 593with awhite compartment, a black compartment, and a center gray
compartment. The black and white compartments had different
floor textures to help the mice further differentiate between the
two environments. On day 1, mice were placed in the gray center
compartment for a 5 min habituation period, followed by a 15 min
testing period where mice were free to explore all compartments
to determine their baseline responses. A baseline score was
recorded and used to randomly pair each mouse with either the
black or white compartment. Drug-paired sides were randomized
so that an even number of mice received drug on the black and
white side. On day 2 (conditioning session), conditioning was
performed as follows: themicewere given an i.p. injection of saline
(10 ml/kg) as a control non-noxious stimulus or 1.2% acetic acid
(AA) (10 ml/kg) as a noxious stimulus and then immediately
confined in the drug-paired compartment for 40 min. On test day
(day 3), mice were allowed to freely explore all the compartments,
and day 1 procedure was repeated. Data were expressed as time
spent on the drug-paired side post-conditioning minus time spent
on the drug-paired side pre-conditioning. A positive number
indicated a preference for the drug-paired side, whereas a negative
number indicated an aversion to the drug-paired side. A number at
or near zero indicated no preference for either side.
2.3.9. Locomotor activity test
Mice were placed into individual Omnitech (Columbus, OH)
photocell activity cages (28  16.5 cm). Interruptions of the
photocell beams (two banks of eight cells each), which assess
walking and rearing, were then recorded for the next 30 min. Data
were expressed as the number of photocell interruptions.
2.3.10. Motor coordination
In order to measure motor coordination, we used the rotarod
test (IITC Inc. Life Science). The animals were placed on textured
drums (1¼ inch diameter) to avoid slipping. When an animal falls
onto the individual sensing platforms, test results were recorded.
Five mice were tested at a time using a rate of 4 rpm. Naive mice
were trained until they remained on the rotarod for 5 min. If a
mouse fell from the rotarod during this time period, it was scored
as motor impaired. Percent impairment was calculated as follows:
% impairment = [(180  test time)/180  100].
2.4. Tumor necrosis factor-alpha (TNF-a) levels
The injected hind paw samples treated with either saline or
carrageenan (0.5%) in a5 KO and WT mice were collected 6 h after
carrageenan injection and homogenized in 1 ml of Tris–HCl buffer
containing protease inhibitors (Sigma–Aldrich Inc., St. Louis,
MO, USA; P8340). Samples were centrifuged at 3000 rpm at a([FTiIg$.)_D1FG]
Fig. 1. Pain behavior and paw edema in a5 KO andWTmice in the formalin test. The paw
the right paw of botha5 KO and theirWT littermate mice. Changes in paw edema (B), as m
(DPD), ina5WT and KOmice 1 h after intraplantar injection of formalin. Data were given
WT group.temperature of 4 8C for 30 min, and the supernatant was frozen at
80 8Cuntil theassay.Theproteinconcentrationwasdeterminedby
the Bradford assay [19]. TNF-a level (pg/mg protein) was deter-
mined using an enzyme-linked immunosorbent assay commercial
kit (Quantikine M murine; R&D Systems, Minneapolis, MN).
2.5. Intracerebroventricular and intrathecal injections
I.c.v. injections were performed according to the method of
Pedigo et al. [20]. Mice were lightly anesthetizedwith ether and an
incision was made in the scalp such that the bregma was exposed.
Injectionswere performed using a 26 gauge needle with a sleeve of
PE 20 tubing to control the depth of the injection. An injection
volume of 5ml was administered at a site 2 mm rostral and 2 mm
caudal to the bregma at a depth of 2 mm. All experiments were
conducted with the investigator blind to genotype and treatment.
At the end of the study, animals were injected i.c.v. with 5ml of
cresyl violet dye, and were perfused to confirm drug diffusion into
both the lateral ventricles.
I.t. injections were performed free-hand between the fifth and
sixth lumbar vertebra in unanesthetized male mice according to
the method of Hylden and Wilcox [21].
2.6. Statistical analysis
Pain behavioral data are presented as mean  SEM. Allodynia
over multiple minutes after nicotine injection in CCI mice were
calculated as area under the curve (AUC) using the trapezoidal rule.
For simple comparisons of two groups, a two-tailed Student t test was
used. Otherwise, statistical analysis was performed with mixed-
factor ANOVA. Two-way ANOVAs were used to evaluate pain
sensitivity at the different time points in different genotypes.
Significant overall ANOVA are followed by Bonferroni’s or Student
Newman Keuls post hoc test when appropriate. All differences were
considered significant at p < 0.05. The GraphPad Prism program was
used for data calculations, graphical representations, and statistical
analysis (GraphPad Software Inc., San Diego, CA).
3. Results
3.1. Pain behavior and paw edema in a5 knockout mice in the
formalin test
First, we evaluated the paw licking response of a5 KO and WT
mice (n = 8/each group) in the formalin test at 2.5% concentration.
No significant difference was found in phase I behaviors between
WT and KO animals (Fig. 1A). However, in the phase II response,
there were significant reductions of pain behavior in a5 KO micelicking response after intraplantar injection of (A) 2.5% formalin concentration into
easured by the difference in the ipsilateral paw diameter before and after injection
as themean  S.E.M. of 8 animals for each group. *p < 0.05 significantly different from
594 D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600compared to WT mice [F(1,14) = 32.278 p < 0.001] (Fig. 1A).
Additionally, the changes of paw edema were measured in a5 WT
and KO mice 1 h after intraplantar injection of 2.5% formalin. The
degree of paw edema in a5 KO mice was significantly lower than
WT littermate controls [t = 5.842, df = 14; p < 0.001] (Fig. 1B).
3.2. Effect of nicotine, varenicline and sazetidine-A in a5 knockout
mice in the formalin test
We tested the effects of nicotine, varenicline and sazetidine-A
in formalin test (2.5%) ina5 KO andWTmice (n = 8/each group). All
three drugs reduced significantly the paw licking time of phase I
response [F(1,14) = 18.92, F(1,14) = 18.181 and F(1,14) = 24.81;
p < 0.001, respectively] (Fig. 2A) and phase II responses
[F(1,14) = 320.69, F(1,14) = 61.792 and F(1,14) = 335.338;
p < 0.001, respectively] (Fig. 2B) in a5 WT mice. However, nicotine
and sazetidine-A failed to show antinociception in phase I
[F(1,14) = 0.169 and F(1,14) = 1.303, p > 0.05, respectively] and
phase II [F(1,14) = 0.00618, p > 0.05] responses in a5 KO mice
(Fig. 2A and B). In contrast to nicotine and sazetidine-A, varenicline
showed antinociceptive effect in both phases in KO mice
[F(1,14) = 37.399 (phase I) and F(1,14) = 48.351 (phase II)p< 0.001].
3.3. Hyperalgesia, allodynia and inflammation ina5 knockout mice in
the carrageenan test
Mice (n = 6/each group) were given an intraplantar injection of
carrageenan (0.5%) and then tested for hyperalgesia and allodynia
at 3, 6 and 24 h later. In addition, paw edema was measured 6 h
after injection of carrageenan. Prior to carrageenan injection,a5 KO
and WT mice did not differ in heat and mechanical baselines
(Fig. 3A and B). However, our results indicated that there is
significant difference betweenWT and KO animals in development
of carrageenan-induced inflammation as seen in the degree of paw
edema [t = 8.685, df = 10; p < 0.001] and hyperalgesic responses
[F(1,10) = 10.088; p < 0.01 after 3 h, F(1,10) = 8.282; p < 0.05 after
6 h and F(1,10) = 5.478; p < 0.05 after 24 h] (Fig. 3A and C). On the
other hand, a significant reduction in mechanical allodynia
response in the KO mice compared to WT animals was only
observed at the time point of 24 h post carrageenan injection
[F(1,10) = 4.878; p = 0.05] (Fig. 3B).
We then measured paw TNF-a concentrations after intraplan-
tar injection of 0.5% carrageenan. As shown in Fig. 3D, TNF-a levels
[()FFiDgG.T_$2I]
Fig. 2. Effect of nicotine, varenicline and sazetidine-A in a5 KO and WT mice in the f
varenicline (3 mg/kg) and sazetidine-A (1.5 mg/kg) before intraplantar formalin (2.5%) in
were given as the mean  S.E.M. of 8 animals for each group. *p < 0.05 significantly differe
WT group.in the paw were significantly increased in a5-WT mice after
carrageenan injection compared with vehicle treated WT mice
[F(1,10) = 22.58; p < 0.001]. On the other hand,a5 KOmice showed
significantly lower levels of TNF-a 6 h after carrageenan injection
compared with carrageenan-treated WT mice [F(1,10) = 6.793;
p < 0.05] (Fig. 3D).
3.4. Hyperalgesia, allodynia and inflammation ina5 knockout mice in
the CFA test
Mice were given an intraplantar injection of CFA and then
tested for hyperalgesia, allodynia and edema 3 and 14 days later.
Prior to CFA injection,a5 KO andWTmice did not differ in heat and
mechanical baselines (Fig. 4A and B). However our results
indicated that there is significant difference between WT (n = 6)
and KO (n = 8) animals in development of CFA-induced inflamma-
tion as observed by changes in heat latencies and paw edema
[F(1,12) = 19.876; p < 0.001 on day 3 and t = 4.458, df = 12;
p < 0.001, respectively] (Fig. 4A and C). a5-KO mice showed
reduction in heat hypersensitivity response on day 3 only.
Additionally, there is significant attenuation of mechanical
allodynia formation in KO (n = 5) CFA-treated mice
[F(1,11) = 18.672; p < 0.01 at day 3 and F(1,11) = 18.97;
p < 0.01 at day 14] when compared with WT mice (n = 7)
(Fig. 4B) on both day 3 and 14.
3.5. Hyperalgesia and allodynia in a5 knockout mice in the CCI-
induced neuropathic pain model
Prior to any surgical procedure, a5 KO and WT mice did not
differ in heat and mechanical baselines (Fig. 5A and B). The
development of neuropathic pain was compared in a5 WT and KO
mice in the CCI model. Our results show thata5 KOmice displayed
a significant attenuation of heat hypersensitivity compared with
WT mice [F(1,18) = 14.457; p < 0.01] at 3 weeks post-surgery
(Fig. 5A; n = 10/each group). However, a significant decrease in
mechanical allodynia was observed at the 1-week time point post-
surgery [F(1,10) = 46.116; p < 0.01] (Fig. 5B; n = 6/each group).
3.6. Relevance ofa5 to anti-allodynic effects of nicotine in the CCI test
Nicotine itself exerts anti-allodynic effects after both inflam-
matory and neuropathic injuries [22]. We tested the ability of i.p.,ormalin test. The effects of subcutaneous administration of nicotine (1.5 mg/kg),
jection on paw licking time at phase I (A) and phase II (B) in the formalin test. Data
nt from corresponding vehicle and #p < 0.05 significantly different from corresponding
([FTi3g_.)DF$IG] D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600 595
Fig. 3. Carrageenan-induced hyperalgesia, allodynia, edema and increase in TNF-a paw levels in a5 KO and WT mice. Differences in paw withdrawal latencies
(DPWL = contralateral  ipsilateral hindpaw latencies) (A) andmechanical pawwithdrawal thresholds (PWT) (B) ina5 KO and theirWT littermate mice different times after
intraplantar injection of carrageenan (0.5% solution/20 ml). Degree of edema (C), as measured by the difference in the ipsilateral paw diameter before and after injection
(DPD), ina5 KO andWTmice 6 h after intraplantar injection of carrageenan. Effect of carrageenan on TNF-a paw levels (D) at 6 h after intraplantar injection of carrageenan in
a5 KO andWTmice. Data were expressed as the mean  S.E.M. of 6 animals for each group. *p < 0.05 significantly different from the value of saline treated WT group, #p < 0.05,
significantly different from the value of carrageenan treated WT group. BL: baseline.
_[D()FIi4gT.FG]$
Fig. 4. CFA-induced hyperalgesia, allodynia and edema ina5 KO andWTmice. Differences in pawwithdrawal latencies (DPWL = contralateral  ipsilateral hindpaw latencies)
(A) and mechanical paw withdrawal thresholds (PWT) (B) in a5 KO and their WT littermate mice different times after intraplantar injection of CFA (75% and 50% solution/
20ml, respectively). Degree of edema (C), as measured by the difference in the ipsilateral paw diameter before and after injection of 75% CFA (DPD), in a5 KO and WT mice
days after intraplantar injection of CFA. Data were expressed as the mean  S.E.M. of 5–8 animals for each group. *p < 0.05 significantly different from WT group. BL: baseline.
[()F_i.g5T$DFIG] 96 D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600
Fig. 5. Chrna5 (a5 nAChR) deficiency impairs thermal hyperalgesia andmechanical allodynia responses in the injured sciatic nerve CCImodel of neuropathic pain. Differences
in pawwithdrawal latencies (DPWL = contralateral  ipsilateral hindpaw latencies) (A) and pawwithdrawal thresholds (PWT) (B) were determined inWT anda5 KOmice at
weekly different time points after chronic constrictive nerve injury (CCI) operation. Data were given as themean  S.E.M. of 6–10 animals for each group. *p < 0.05 significantly
different from the value of WT group. BL: baseline.i.c.v., i.t. and i.pl. (-)-Nicotine to reverse mechanical allodynia
produced by CCI and CFA in WT and KO mice. Although potency
and efficacy varied by route of administration, nicotine was
significantly effective against allodynia in WTmice by all injection
routes (Figs. 6 and 7). The anti-allodynic effect of systemic nicotine
administration (0.9 mg/kg, i.p.) in a5 WT and KO mice was tested
7 weeks after CCI surgery. Nicotine induced significant anti-
allodynic effect in WT mice in a time dependent manner
[F(5,30) = 32.921; p < 0.001]. On the other hand, KO mice failed
to show any significant anti-allodynic effect at any of the time
points tested [F(5,30) = 1.36; p = 0.267] (Fig. 6A; n = 6/each group).
There were significant differences between WT and KO groups
interaction [t = 8.055, df = 10; p < 0.001] (Fig. 6B).
We then performed a head-to-head comparison of supraspinal,
spinal and peripheral nicotine-induced anti-allodynia (25mg,
i.c.v.; 20 mg, i.t.; 45mg, i.pl.) in a5 null mutant mice after
neuropathic (CCI) injury (2 weeks after surgery). All routes of
administration produced robust reversal of mechanical allodynia
in WT mice at these doses (Fig. 7; n = 6/each group). Supraspinal
nicotine anti-allodynia was preserved in a5 KO mice
[F(4,40) = 13.16; p < 0.001] (Fig. 7A). In contrast, spinal nicotine
anti-allodynia was abolished in a5 KO mice [F(4,40) = 0.5268;
p = 0.8609] (Fig. 7B). Similarly, anti-allodynia resulting from
injection of nicotine directly into the hind paw was abolished in
a5 KO mice [F(4,40) = 2.313; p = 0.0741] (Fig. 7C). These data
suggest that nicotine blocks mechanical allodynia in the periphery
and/or spinal cord in a a5-dependent manner, except supraspin-
ally, where it appears to have minimal contribution.[()FFiDgG.T_$6I]
Fig. 6. Altered anti-allodynic efficacy of nicotine in Chrna5 (a5 nAChR) mutant mice. Nico
CCI (week 7 post surgery) in the a5 WT but not in KOmice. The mechanical pawwithdraw
were expressed as the mean  S.E.M. of 6 animals for each group. *p < 0.05 significantly d
group. BL: baseline.3.7. Impact of a5 gene deletion on acetic acid-induced conditioned
place aversion
Mice were given an i.p. injection of acetic acid (AA) (1.2%, n = 8/
WT and n = 7/KO) or saline (n = 6/each group) and then tested for
place preference. In the CPA test, WT mice showed significant AA-
induced aversion [t = 3.79, p < 0.05]. On the other hand, KO mice
failed to show a significant aversive effect [t = 0.75, p = 0.469]. The
a5-lacking mice showed reduction in aversive response [t = 3.404,
p < 0.05] (Fig. 8).
3.8. Impact of a5 gene deletion on motor activity and coordination of
mice
To determine whether the effects of a5 gene deletion of mice in
behavioral pain tests are not due to disruption of locomotor
activity and coordination during testing, we evaluated motor
activity and coordination of mice. As seen in Table 1, WT (n = 10)
and KO (n = 8) mice in this study did not significantly differ in
motor activity (t = 0.03628, df = 16; p = 0.9715) or motor coordi-
nation (t = 1.398, df = 10; p = 0.1923, n = 6/each group) in any time-
point after testing.
4. Discussion
Nicotine and nicotinic receptors have been explored for the past
three decades as a strategy for pain control. These receptors are
widely expressed throughout the central and peripheral nervoustine (0.9 mg/kg, i.p.) reversed already-developed mechanical allodynia produced by
al threshold of ipsilateral paw (A) and AUC of threshold values (B) are shown. Data
ifferent from test 0. #p < 0.05 significantly different from corresponding value of WT
([F.i7g_)DT$FIG] D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600 597
Fig. 7. Anti-allodynic effects of supraspinal, spinal and/or peripheral nicotine in a5 mutant mice. A head-to head comparison of supraspinal (25 mg/5 ml, i.c.v.) (A), spinal
(20 mg/5 ml, i.t.) (B) and peripheral (45mg/20 ml, intraplantar) (C) nicotine anti-allodynia against CCI-induced neuropathic pain in a5 WT and KO mice were tested using
identical parameters at the peak of allodynia (14 days post-CCI). Data were expressed as themean  S.E.M. of 6 animals for each group. *p < 0.05 significantly different from test
0. #p < 0.05 significantly different from corresponding value of WT group. BL: baseline.system as well as immune cells. Despite encouraging results with
many a4b2* agonists in animal models of pain, human studies
showed a narrow therapeuticwindow of these drugs. However, the
lack of understanding of the composition of nicotinic receptor
subtypes mediating analgesia has hampered the clinical use of
nicotinic agonists. The present study investigates the role of a5
subunits in the development and modulation of pain behaviors in
mice. The a5 subunit is an accessory subunit as it can only form
functional receptors when co-expressed with a principal subunit
(such as a3, or a4) and one complementary subunit (b2 or b4, e.g.
as a4b2a5 or a3b4a5 receptors) [23]. Our results showed that
genetic deletion of a5 nicotinic subunits reduced pain-like[()F_i.g8T$DFIG]
Fig. 8. Conditioned place aversion test in a5 KO and WT mice. Intraperitoneal
injection of 1.2% acetic acid induced conditioned place aversion as a negative
affective component of pain. Datawere given as themean  S.E.M. of 6–8 animals for
each group. *p < 0.05, compared to the vehicle-injected mice. #p < 0.05, compared to
the acetic acid-injected WT mice.behaviors, thermal hyperalgesia and mechanical allodynia in
tonic, inflammatory and neuropathic pain mouse models. The
reduction of inflammation as measured by the degree of paw
edema in the carrageenan inflammatory test was accompanied by
a reduction of carrageenan-induced increase in TNF-a paw levels.
Furthermore, we also show that the antinociceptive effects of
nicotinic agonists vary in their dependency on a5 nicotinic
subunits in these chronic pain models. Moreover, our results with
a5 KO mice showed that nicotine induces anti-allodynic effects
through spinal and peripheral mechanisms. Finally, mice with the
a5-deletion showed reduction in aversive response to acetic acid
i.p. injection in the CPA test. Taken together, our results suggest a
direct link of nicotinic mechanisms in the development of
inflammatory and pain mechanisms, which are at least partly
dependent on the a5 nAChRs.
The a5 subunit is expressed in different pain pathways in
peripheral and central nervous systems such as primary afferentsTable 1
Impact of alpha5 gene deletion on motor activity and coordination of mice.
Group Spontaneous Rotarod activity
activity (time to fall in s)
(# interrupts/10min)
WT mice 50930 179.60.4
KO mice 49225 1811.4
Mice were placed into photocell activity cages for 30min or placed on the rotarod
for 5min. Data were presented as mean SEM as the number of photocell
interruptions and time to fall in s for each group, respectively (n =6–10).
598 D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600in the spinal cord, postsynaptic autonomic and somatic motor
neurons, in glial cells [24] and in locus coeruleus small cells
[25,26]. Moreover, individual neurons can express multiple
subtypes of nAChRs. nAChRs subtypes containing a5 (such as
a4a5b2*) are expressed on both inhibitory and excitatory
interneurons in the spinal dorsal horn [27], thalamus [11,28]
and hindbrain [11]. In vitro studies have shown that the expression
of the a5 subunit may act to modulate the amount of a3b4 nAChR
function in the habenulopeduncular tract and in other tissues,
which express a3b4a5 nAChR [29]. This distribution suggests the
possible involvement of a5* nAChRs in peripheral and/or central
pain behavior responses.
We first explored the involvement of a5* nAChRs in the mouse
formalin test, which has been used as a model for inflammatory
tonic pain. Thermal nociception and early phase response (phase I)
in the formalin test are caused predominantly by direct activation
of peripheral C-fibers, whereas the late response (phase II) in the
formalin test involves functional changes in the dorsal horn of the
spinal cord (i.e., central sensitization) [30,31]. In this study, a5 KO
and WT mice were tested in the formalin test at 2.5% concentra-
tions. Our results suggest that a5* nAChRs are minimally involved
in the acute (nociceptive) pain of the first phase but contribute to a
large extent to the inflammatory pain, spinal cord neuronal
sensitization, or both, which occur during the second phase.
Furthermore, we investigated the possible role of a5 subunits in
the antinociceptive effects of nicotine and two nicotinic partial
agonists (varenicline and sazetidine-A) in the formalin test. Our
results showed that the nicotine and sazetidine anti-nociceptive
effects were fully dependent on a5 subunits in both phases I and II
of the formalin test. These results alongwith our recent report [16],
suggest that the antinociceptive effects of nicotine and sazetidine-
A in the formalin test appears to be mediated largely by a4a5b2*-
nAChRs subtypes. In contrast, a5*-nAChRs subtypes do not
mediate the effects of varenicline in the same test.
Carrageenan produces a short-term local inflammation involv-
ing both neurogenic and non-neurogenic mechanisms, in which
several mediators are involved including cytokines and cyclooxy-
genase products [32,33]. Based on the present results, participation
of a5*-nAChRs in carrageenan-evoked mechanical allodynia is
limited. In contrast, carrageenan-evoked thermal hyperalgesia was
greatly prevented in a5-KO animals. In addition, inflammation-
induced paw edema and increase in paw TNF-a levels were also
reduced. It is plausible that the attenuation of the increase of TNF-
amay contribute to themechanism bywhicha5*-nAChRs reduces
carrageenan-induced thermal hyperalgesia. Indeed, a a5-depen-
dent mechanism regulating the synthesis or release of pro-
inflammatory chemokines such as TNF-a through a NF-kB
pathway could mediate the inflammatory response to carrageen-
an. In line with this suggestive mechanism, it is well reported that
activation of a7 nAChRs engages this anti-inflammatory signaling
[34]. The results from the present study suggest that a5*-nAChRs
are tonically involved in the modulation of subacute inflammatory
pain. Similarly, disruption of a5 nicotinic gene in the mouse
resulted in the reduction of chronic inflammation as seen in CFA-
induced mechanical allodynia and thermal hyperalgesia. Taken
together, our results showed that a5-null mice exhibited impaired
development of hyperalgesia and allodynia after inflammatory
injury, namely that elicited by carrageenan or CFA, suggesting the
importance of a5*-nAChRs as a target for the treatment of both
acute and chronic inflammatory pain. Interestingly, intraplantar
injection of CFA in themouse was recently reported to increase the
dorsal root ganglia (DRG) levels of a3 and b4 mRNAs [35]. While
changes in the levels of a5 mRNAs were not reported in this study,
it is possible that peripheral a5-containing nAChRs in DRGs
contribute to nicotine anti-inflammatory and antinociceptive
actions, and that a5-containing nAChRs (possibly a3a5b4*) inthe DRGs undergo plasticity during inflammation. Finally, our
results with CFA and carrageenan seem to be in contrast with a
study showing that a5 KO mice have significantly more severe
experimental colitis than WT controls when treated with dextran
sulphate sodium (DSS) [36]. One possible explanation for this
discrepancy could be that a5-dependent mechanisms play a
differential role in the nociceptive circuitry associated with the
chronic inflammatory and visceral pain signals. It is also possible
that changes in the genetic background of the a5 KO mice over
generations may have played a role in this discrepancy (the
number of backcrossing generations was not reported in that
study).
We then used CCI of the right sciatic nerve as a model of
neuropathic pain to characterize pain behaviors ofa5 KOmice. Not
surprisingly, a5 deficiency led to a decrease in thermal hyper-
algesia and to a lesser degree tactile allodynia. The reduction in
sensory pain responses in a5 KO mice are in line with a recent
study that has shown a role for the a5 nAChR subunit on pain
mechanisms in spinal nerve-ligated rats [15]. In this study, the
ligation of spinal nerves L5 and L6 induced an up-regulation of the
a5 nAChR subunit levels in the outer lamina of the spinal cord
dorsal horn ipsilateral to ligation. In addition, knock down of thea5
subunit in spinal nerve-ligated rats moderately alleviated me-
chanical allodynia. Furthermore, a5 antisense treatment in these
rats resulted in a decrease in pCREB immuno-reactive nuclei in the
outer laminae and enhancement ACh-evoked excitatory amino
acid release. Interestingly, peripheral nerve injury greatly upre-
gulated transcripts for the a5 and b2 nicotinic acetylcholine
receptor subunits within the spinal cord dorsal horn [37]. In
another study, spinal nerve ligation resulted in an increase of the
expression of several nAChR subunits ipsilaterally, including a5
subunit [38]. Taken together, these results suggest that a5*-
nAChRs play an important role in changes in spinal connectivity
(i.e., central sensitization) induced by nerve injury in rodents.
The discrepancies observed between a5 KO andWTmice in the
heat hyperalgesia and mechanical allodynia changes of the CFA
and CCI tests (Figs. 4 and 5) were unexpected. Several explanations
could account for these discrepancies, with one possibility being
the fact that heat and pain are transmitted through the anterior
spinothalamic tract of the spinal cord, while tactile sensation is
transmitted through the dorsal column of the spinal cord. These
two different pathways could account for different a5-nAChRs
receptor expression based on the pain stimulus used. However, the
expression of these nicotinic subunits in these different pathways
is not known.
Since nicotine itself has been shown to exert anti-allodynic
effects after neuropathic injuries [22], we tested the ability of
systemic nicotine to reversemechanical allodynia produced by CCI
in a5 WT and KO mice. Intraperitoneal nicotine was significantly
effective against allodynia in WT mice, but Chrna5 (nAChR a5
gene) KO mice displayed no significant nicotine-induced anti-
allodynia in the CCI test. The anti-allodynic activity of nicotine in
CCI-induced neuropathic pain seems to be mediated by a5
nicotinic receptors expressed in spinal and/or peripheral sites.
The changes in pain behaviors tests after a5 gene deletion of
mice are not due to disruption in locomotor activity and
coordination of the animals during testing since a5 KO mice did
not show any significant changes in these measures when
compared to WT mice.
Since the experience of pain has both sensory and emotional-
affective dimensions, we evaluated the role of a5*-nAChR in
aversion, an important affective component of pain, using a
conditioned place aversion (CPA) test [39,40]. In the present study,
acetic acid i.p. injection was paired to a compartment and induced
an avoidance response in comparison to a saline-paired compart-
ment, indicating that the acetic acid-produced aversion. Our
D. Bagdas et al. / Biochemical Pharmacology 97 (2015) 590–600 599results indicated also that there is a significant decrease in acetic
acid-induced CPA ina5 KO compared toa5WT animals. This result
suggests that a5 nAChRs may play an important role in negative
affective components of pain.
The mediation of nicotine’s anti-nociceptive effects in inflam-
matory and neuropathic pain by a5*-nAChRs extends previous
results from our lab and others on the role of this modulatory
subunit in nicotine pharmacological and behavioral effects.
Indeed, an important role for a5 nicotinic subunit in the initial
pharmacological effects of nicotine (antinociception in acute
pain models) and nicotine reward and withdrawal was reported
[10–13,41,42].
To summarize, we have provided strong evidence that a5*-
nAChRs seems to play an important role in the genesis of
inflammatory and neuropathic pain sensitization and hypersensi-
tivity. However, this conclusion does not exclude the possibility
that our results may be impacted by compensatory mechanisms
resulting from the genetic deletion of the a5 subunit. While a5 KO
mice were reported with no visible abnormality or neurological
deficits [9], normal brain anatomy, normal mRNA expression of
other nAChR subunits, and normal nicotinic binding in the brain
[10], it is possible that mutation of a5*-nAChRs in a complex
circuitry involved in chronic pain may be compensated by changes
in other elements in the circuitry.
Finally, our results may have an impact on the development of
nicotinic ligands as possible therapeutic targets for chronic pain.
Several high affinity a4b2* nAChR agonists were reported to have
potent analgesic activity in rodent models of acute and chronic
pain. However, much less is known about the exact composition of
the native a4b2* receptor mediating the analgesic effect and their
sites of action. Indeed, various additional nAChR subunits can
associatewith thea4 andb2 nicotinic subunits, includinga5,a6,a7
andb3 subunits, to formmultiple functional subtypes (for example
a4a5b2* subtypes), which have been identified in the spinal cord
and DRG tissues. Our current data demonstrate that, in both
chronic inflammatory and neuropathic pain models, nicotine
blocks mechanical allodynia in a a5*-dependent manner. It is
therefore possible that the modest efficacy of some a4b2* agonists
reported in animal models of chronic inflammatory pain [31] and
initial clinical studies [43–45]; may be related to their insufficient
binding and/or functional activity at a5* subtypes. We believe that
the refocusing of nicotinic analgesic development on a5*-containing
receptors will lead to more efficacious compounds.
Conflict of interest statement
The authors have no conflict of interest associated with this
study to declare.
Acknowledgements
This work was supported by NIDA DA-12610. Deniz Bagdas
would like to thank The Scientific and Technical Research Council
of Turkey (TUBITAK) for her postdoctoral research scholarship
(2219-2013). The authors wish to thank Tie Han, Lisa Merritt and
Cindy Evans for their technical assistance and maintenance of the
breeding colony. The authors wish to thank Asti Jackson for her
editing of the manuscript.
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