imid-nocicep

OR IG INAL

ART ICLE

Participation of central imidazoline binding

sites in antinociceptive effect of ethanol and

nicotine in rats

Manish Manohar Aglawe, Brijesh Gulabrao Taksande,

Sharvari Shambabu Kuldhariya, Chandrabhan Tukaram Chopde,

Milind Janrao Umekar, Nandkishor Ramdas Kotagale*2Division of Neuroscience, Department of Pharmacology, Smt. Kishoritai Bhoyar College of Pharmacy, New Kamptee,

Nagpur 441002, Maharashtra, India

Keywords

antinociception,

ethanol,

imidazoline Receptors,

nicotine4

Received 6 July 2012;

revised 6 February 2013;

accepted 28 March 2013

*Correspondence and reprints:

[email protected]

ABSTRACT

Despite synergistic morbidity and mortality, concomitant consumption of alcohol

and tobacco is increasing, and their antinociceptive effect has been linked with

co-abuse. Present study was designed to investigate the role of imidazoline binding

sites in the antinociceptive effect of nicotine, ethanol, and their combination. Sepa-

rate group of male Sprague–Dawley rats (200–250 g) were treated with different

doses of alcohol (0.50–2 g/kg, i.p.) or nicotine (0.25–1 mg/kg, i.p.), and their

combination evaluated in tail flick test. Influence of endogenous imidazoline bind-

ing site ligands, agonist, and antagonists were determined by their prior treatment

with effective or subeffective doses of either ethanol or nicotine. Ethanol, nicotine,

or their subeffective dose combination exhibited significant antinociceptive effects

in dose-dependent manner. Antinociceptive3 effect of ethanol and nicotine was sig-

nificantly augmented by intracerebroventricular (i.c.v.) administration of endoge-

nous imidazoline receptor ligands, harmane (25 lg/rat, i.c.v.) and agmatine

(10 lg/rat, i.c.v.), as well as imidazoline I1/a2 adrenergic receptor agonist, cloni-

dine (2 lg/rat, i.c.v.), I1 agonist moxonidine (25 lg/rat, i.c.v.), and imidazoline I2agonist, 2-BFI (10 lg/rat, i.c.v.). Conversely, antinociception elicited by ethanol or

nicotine or their subeffective dose combination was antagonized by pretreatment

with imidazoline I1 antagonist, efaroxan (10 lg/rat, i.c.v.), and I2 antagonist,

idazoxan (4 lg/rat, i.c.v.), at their per se ineffective doses. These findings project

imidazoline binding ligands as important therapeutic molecules for central antino-

ciceptive activity as well as may reduce the co-abuse potential of alcohol and

nicotine.

INTRODUCT ION

Concomitant consumption of alcohol and tobacco

remains high despite the observed synergistic morbidity

in society. Repeatedly, scientific studies have cautioned

about the synergistic risks for various cancers with

concurrent drinking and smoking [1–3]. Several factors

including common genetic mechanisms, pharmacoki-

netic and pharmacodynamic interactions predisposing

to increased rewarding effects, and/or counteracting

toxic effects of alcohol by nicotine have been proposed

for co-abuse [4–16]. In addition, a number of clinical

[17,18] and animal studies [19] have provided evi-

dences for common genetic predisposition for their

co-abuse.

Pharmacokinetic data revealed that nicotine dose

dependently reduces blood alcohol levels in rats [14].

Interestingly, alcohol-induced ataxia as well as cognitive

impairments was also protected by nicotine [20–23].

Moreover, combination of alcohol and nicotine found to

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ª 2013 The Authors Fundamental and Clinical Pharmacology © 2013 Soci�et�e Franc�aise de Pharmacologie et de Th�erapeutique

Fundamental & Clinical Pharmacology 1

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exhibit increased rewarding effects associated with exag-

gerated dopamine release in the nucleus accumbens

shell of rats [24,25].

Alcohol consumption in humans [26,27] and admin-

istration to animals [28] result in analgesia or antino-

ciception. Similarly, nicotine or nicotinic agonists

produce analgesic effect in humans [29,30] and in

animals [31–35]. Available neurological and

pharmacological evidences suggest the existence of

close relationship between reinforcement and analgesia.

Moreover, it is proposed that additive or synergistic

antinociceptive effects induced by alcohol and nicotine

may contribute to their co-abuse [36,37].

Imidazoline binding sites have currently attracted

attention in nociception as well as drug addiction [38–

40]. Moreover, the brain structures that are involved

in the drug abuse and pain perception including hypo-

thalamus, hippocampus, amygdala, etc., are rich in

imidazoline binding sites and its endogenous ligands

[41].

Imidazoline binding sites are a family of unique non-

adrenergic high-affinity binding sites that exist in three

major subclasses (I1, I2, and I3) based upon their ligand

selectivity, subcellular distribution, and physiological

functions [42–45]. In human brain, I1 receptors are

distributed in regional manner with highest density in

striatum, pallidum, and gyrus dentatus of hippocam-

pus, amygdala, and substantia nigra [46]. The I2 bind-

ing sites (I2A and I2B) are allosteric and are located on

monoamine oxidases [45,47,48]. The interaction of

imidazoline binding sites and ligands on ethanol and

nicotine intake, their dependence, and withdrawal state

is well documented [38,49,50]. Furthermore, the

involvement of imidazoline I1/I2 endogenous ligands

like agmatine, harmane, and b-carboline in nociception

as well as addiction is now fairly well established. How-

ever the influence of imidazoline binding site modula-

tion on antinociceptive effect of ethanol and nicotine

remains unexplored.

In view of this background, this study was under-

taken to investigate the effect of imidazoline binding

sites ligands on ethanol- and nicotine-induced antinoci-

ceptive effect in rats using tail flick assay method

reflecting spinal nociception in rats.

MATER IALS AND METHODS

Animals

Adult healthy Sprague–Dawley rats (200–250 g) were

used. The rats were kept four per cage (640 9 410 9

250 mm height) or individually after intracerebroven-

tricular (i.c.v.) cannulation, in a room with controlled

temperature (25 � 2 °C) and maintained on a 12:12 h

light/dark cycle (on/off at 07:00 am/07:00 pm) with

free access to food and water. All experimental proce-

dures were approved by the Institutional Animal Ethi-

cal Committee and executed in strict accordance with

the guidelines for the care and use of laboratory

animals (CPCSEA, India 5).

Drugs

Nicotine hydrogen tartrate, agmatine sulfate, clonidine

hydrochloride, moxonidine hydrochloride, harmane,

efaroxan hydrochloride, and idazoxan hydrochloride

were purchased from Sigma-Aldrich Co., USA 6, while

2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI)

was purchased from Tocris Biosciences, UK 7.

Ethanol (99%) was purchased from Merck chemicals,

Mumbai, India. Ethanol was diluted or nicotine was

dissolved in physiologic saline (0.9%) and administered

by intraperitoneal (i.p.) route in a volume of 1 mL/kg

of body weight. Agmatine, moxonidine, 2-BFI, efaro-

xan, and idazoxan were injected by intracerebroven-

tricular (i.c.v., 2 lL/rat) route. For i.c.v. administration

of drugs, dilutions were made with artificial cerebrospi-

nal fluid (aCSF) of following composition: 0.2 M NaCl,

0.02 M NaH2CO3, 2 mM KCl, 0.5 mM KH2PO4, 1.2 mM

CaCl2, 1.8 mM MgCl2, 0.5 mM Na2SO4, and 5.8 mM

D-glucose.

Intracerebroventricular cannula implantation and

drug administration

For the study involving i.c.v. administration of drugs, rats

were anesthetized with thiopental sodium (60 mg/kg,

i.p.; Abbott Pharmaceuticals Ltd., Mumbai, India) and

stereotaxically (David Kopf Instruments, CA, USA 8)

implanted [49,50] 24-gauge stainless steel guide cannula

(C313G/Spc, plastic UK 9). Stereotaxic coordinates used

were �0.8 mm posterior, +1.3 mm lateral to midline,

and �3.5 mm ventral to bregma according to Paxinos

and Watson [51]. The guide cannulae were then fixed to

skull with dental cement (DPI-RR cold cure, acrylic pow-

der; Dental Product of India, Mumbai, India) and secured

in two stainless steel screws. A 28-gauge stainless steel

dummy cannula was used to occlude the guide cannula

when not in use. Following surgery, the animals were

placed individually in cages and each subject was allowed

to recover at least for 7 days before being tested for tail

flick latency. Rats were then randomly assigned to

different groups (n = 6 per group) and habituated to the

ª 2013 The Authors Fundamental and Clinical Pharmacology ª 2013 Soci�et�e Franc�aise de Pharmacologie et de Th�erapeutique

Fundamental & Clinical Pharmacology

2 M.M. Aglawe et al.

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testing environment by transferring to experimental

room and twice daily handling for 1 week. Drugs were

injected (2 lL/rat) bilaterally over a 1-min period with a

microliter syringe (Hamilton, Reno, NV, USA) connected

by PE-10 polyethylene tubing to a 33-gauge internal

cannula (C313 I/Spc, plastic one, internal diameter

0.18 mm, outer diameter 0.20 mm) that extended

0.5 mm beyond the guide cannula. The internal cannula

was held in position for another 1 min before being

slowly withdrawn to prevent backflow and promote diffu-

sion of drugs.

Confirmation of cannula placement

At the end of all experiments, dilute India ink

(5 lL/rat, i.c.v.) was injected and animals were killed

by an overdose of thiopental sodium. The brain of each

animal was dissected out and sliced in coronal plane to

verify the placement of the guide cannula and distribu-

tion of ink in the ventricles. In 13% of animals, guide

cannulae were found incorrectly placed. Data of only

those animals that showed uniform distribution of ink

in the ventricles were considered for statistical analysis.

Assessment of nociception

Nociception was assessed with tail flick apparatus

(INCO, India10 ). It consisted of an electrically heated

nichrome wire (1/8 mm) as a radiant heat source.

A desired intensity was adjusted by supplying constant

current (6 �A) to wire so that a sudden flick of the tail

occurred in about 5–6 s. The basal reaction time (pre-

drug reaction time) to radiant heat was determined by

placing the tip (dorsal 2–3 cm) of the tail on hot wire.

A cutoff period of 15 s was observed to avoid damage

to the tail [52,53]. Animals failing to withdraw tail in

5–6 s were rejected from study. At least three basal

reaction times for each rat with the interval of 1 min

were recorded. Animals were then randomly divided

into different groups each containing six animals for

further studies as described below. All the measure-

ments were taken by the skilled observer blind to the

treatment given.

Effect of ethanol and nicotine on nociception

Animals were randomly divided into different groups

(n = 6), and each group was assigned to one of the fol-

lowing treatment. Firstly, we determined the acute

antinociceptive effect of various doses of ethanol (8%

w/v in saline, 0.50–2 g/kg, i.p.) or nicotine (0.25–

1 mg/kg, i.p.) or their combination or saline (1 mL/kg,

i.p.) in tail flick assay. For combination studies,

submaximal or ineffective doses of both the drugs were

used. Ethanol was always administered 30 min before

and nicotine 20 min before the tests. Individual rat

was subjected to tail flick test, and reaction latency

was determined.

Effect of endogenous ligands of imidazoline

binding sites on ethanol- or nicotine-induced

antinociception

In these experiments, rats were treated with endogenous

ligands of imidazoline binding sites viz. agmatine

(10 lg/rat, i.c.v.) or harmane (25 lg/rat, i.c.v.) or aCSF

(2 lL/rat, i.c.v.) 10 min before i.p. injection of vehicle or

subeffective dose of ethanol (8% w/v; 0.5 g/kg, i.p.) or

nicotine (0.25 mg/kg, i.p.) or saline (1 mL/kg, i.p.).

Effect of imidazoline receptor agonists on

ethanol- and nicotine-induced antinociception

Separate group of rats (n = 4–16) were administered

either with aCSF (2 lL/rat, i.c.v.) or agmatine (10–

40 lg/rat, i.c.v.) or harmane (25–100 lg/rat, i.c.v.) or

clonidine (2–8 lg/rat, i.c.v.) or moxonidine (25–

100 lg/rat, i.c.v.) or 2-BFI (10–40 lg/rat, i.c.v.) or

efaroxan (10–40 lg/rat, i.c.v.) or idazoxan (4–

16 lg/rat, i.c.v.) 10 min before being subjected to tail

flick test to determine the reaction latency of individual

animal.

Separate groups of rats were treated with imidazoline

I1/a2 receptor agonist moxonidine (25 lg/rat, i.c.v.) or

clonidine (2 lg/rat, i.c.v.) or imidazoline I2 receptor

agonist 2-BFI (10 lg/rat, i.c.v.) or aCSF (2 lL/rat,

i.c.v.) 10 min before i.p. injection of vehicle or subeffec-

tive dose of ethanol (8% w/v; 0.5 g/kg, i.p.) or nicotine

(0.25 mg/kg, i.p.) or saline (1 mL/kg, i.p.). Individual

rat was subjected to tail flick test, and reaction latency

was determined as mentioned earlier.

Effect of imidazoline receptor antagonists on

ethanol- and nicotine-induced antinociception

For the antagonism study, rats were treated with imi-

dazoline I1 receptor antagonist, efaroxan (10 lg/rat,

i.c.v.), or imidazoline I2 receptor antagonist, idazoxan

(4 lg/rat, i.c.v.), or aCSF (2 lL/rat, i.c.v.) 10 min prior

to antinociceptive dose of ethanol (8% w/v; 1 g/kg,

i.p.) or nicotine (0.5 mg/kg, i.p.) or combination of

subeffective dose of ethanol (0.5 g/kg, i.p.) and nicotine

(0.25 mg/kg, i.p.) or saline (1 mL/kg, i.p.).

In combination studies, the same chronological

sequence as described above was applied, that is,

imidazoline receptor antagonist administration was



Ethanol/nicotine antinociception modulated by imidazoline receptor 13

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followed by alcohol and 10 min later by nicotine, and

the tests were conducted 20 min after nicotine admin-

istration. Individual rat was subjected to tail flick test,

and reaction latency was determined.

The doses of endogenous imidazoline binding site

ligands, imidazoline receptor agonists, and antagonists

used here are selected on the basis of preliminary

experiments conducted at our laboratory and available

literature.

Data analysis

Antinociception was calculated as the percentage of

maximum possible effect by the (% MPE) formula: %

MPE = 100 9 (drug time control time/cutoff time con-

trol time). Percentage of MPE values were calculated

for each animal and were statistically analyzed with

one-way analysis of variance (ANOVA). Means were com-

pared with post hoc Dunnett’s or Newman–Keuls test.

P-values < 0.05 were considered significant.

RESULTS

Antinociceptive effect of ethanol and nicotine

As shown in Figure 1a, ethanol (1–2 g/kg, i.p.) dose

dependently increased tail flick latency as compared to

saline-treated group [F(5, 35) = 18.56, P < 0.001]

demonstrating its analgesic potential. Post hoc Dunnett’s

test revealed that ethanol 1, 1.5, and 2 g/kg, i.p.

increased baseline latency by 114% (P < 0.001), 150%

(P < 0.001), and 243% (P < 0.001), respectively. How-

ever, lower doses of ethanol (0.5 g/kg, i.p.) failed to

produce any analgesic effect.

Similarly, administration of nicotine (0.5, 0.75, and

1 mg/kg, i.p.) dose dependently increased baseline tail

flick latency by 141% (P < 0.05), 198% (P < 0.001),

and 222% (P < 0.001), respectively [F(4, 29) = 15.87,

P < 0.001] (Figure 1b). Nicotine at the dose of

0.25 mg/kg, i.p. could not produce significant antinoci-

ception in rats.

Figure 1c depicts the effects executed by combination

of submaximal doses of alcohol (0.5 g/kg, i.p.) and nico-

tine (0.25 mg/kg, i.p.) in the tail flick assay. Subeffective

dose combination of alcohol and nicotine produced sig-

nificant increases in latency time as compared to saline-

treated group [F(3, 21) = 15.76, P < 0.001]. Post hoc

Dunnett’s comparisons indicated the significant potenti-

ation of the antinociceptive effect of per se ineffective

doses of nicotine and ethanol. The doses of nicotine or

ethanol used here did not influenced latency time in tail

flick assay as compared to saline-treated rats.

(a)

(b)

(c)

Figure 1 Antinociceptive effect of ethanol (a), nicotine (b), and

ineffective dose combination of ethanol and nicotine (c) in tail

flick test. Separate group of rats (n = 6) were treated either with

saline (1 ml/kg, i.p.) or ethanol (0.1–2 g/kg), 30 min before or

nicotine (0.1–2 mg/kg, i.p.) 20 min before or ineffective dose

combination of ethanol (0.5 g/kg, i.p.) and nicotine (0.25 mg/kg,

i.p.) at appropriate time before the test and reaction latency was

measured. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective

control group (one-way ANOVA post hoc Dunnett’s test).

$P < 0.001 vs. ethanol treatment, #P < 0.001 vs. nicotine

treatment (one-way ANOVA post hoc Newman–Keuls test).




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Endogenous imidazoline ligands potentiated

antinociceptive effect of ethanol and nicotine

As shown in Figure 2, administration of agmatine [20

(P < 0.05) and 40 (P < 0.01) lg/rat, i.c.v. but not

10 lg/rat] [F(3, 35) = 5.75, P < 0.01], harmane [50

(P < 0.05) and 100 (P < 0.001) lg/rat, i.c.v. but not

25 lg/rat] [F(3, 32) = 14.56, P < 0.001], clonidine

[4 (P < 0.05) and 8 (P < 0.001) lg/rat, i.c.v. but not

2 lg/rat] [F(3, 31) = 13.78, P < 0.001], moxonidine

[50 (P < 0.05) and 100 (P < 0.001) lg/rat, i.c.v. but

not 25 lg/rat] [F(3, 31) = 15.33, P < 0.001], and

2-BFI [20 (P < 0.01)–40 (P < 0.001) lg/rat, i.c.v. but

not 10 lg/rat] [F(3, 30) = 8.20, P < 0.001] signifi-

cantly increased baseline tail flick latency. However,

efaroxan (10–40 lg/rat, i.c.v.) as well as idazoxan (4–

16 lg/rat, i.c.v.) in the doses administered here failed

to influence the basal tail flick latency.

As depicted in Figure 3, administration of ineffective

dose of ethanol (0.5 g/kg, i.p.) to animals pretreated

with the endogenous ligands of imidazoline binding sites

significantly potentiated the antinociceptive effect of eth-

anol [FDrug 9 Pretreatment (2, 28) = 18.84, P < 0.001;

FDrug (1, 28) = 67.44, P < 0.001; FPretreatment

(2, 28) = 20.40, P < 0.001] (two-way ANOVA). Post hoc

Bonferroni mean comparisons indicated the significant

augmentation of analgesic effect of ethanol by agmatine

(10 lg/rat, i.c.v.; P < 0.001) and harmane (25 lg/rat,

i.c.v.; P < 0.001).

The influence of pretreatment of imidazoline binding

site ligands on the analgesic effect of nicotine is shown

in Figure 3. Pretreatment of agmatine (10 lg/rat, i.c.v.;

P < 0.001) or harmane (25 lg/rat, i.c.v.; P < 0.001)

prior to ineffective dose of nicotine (0.25 mg/kg, i.p.)

produced significant increase in latency time as

compared to their control group [FDrug 9 Pretreatment

(2, 29) = 15.63, P < 0.001; FDrug (1, 29) = 61.75,

P < 0.001; FPretreatment (2, 29) = 18.66, P < 0.001]

(two-way ANOVA). The doses of agmatine and

harmane administered here failed to increase the

latency time when compared with the vehicle-treated

animals.

Imidazoline receptor agonists potentiated


As demonstrated in Figure 4, administration of ineffec-

tive dose of ethanol (0.5 g/kg, i.p.) to the animals pre-

treated with the imidazoline I1 receptor agonist,

moxonidine (25 lg/rat, i.c.v.; P < 0.01), or I2 agonist,

2-BFI (10 lg/rat, i.c.v.; P < 0.001), or mixed I1/a2adrenergic receptor agonist, clonidine (2 lg/rat, i.c.v.;

P < 0.001), significantly potentiated the latency time,

respectively, as compared to ethanol-treated control

animals [FDrug 9 Pretreatment (3, 37) = 4.01, P < 0.05;

FDrug (1, 37) = 43.19, P < 0.001; FPretreatment

(3, 37) = 5.18, P < 0.001] (two-way ANOVA post hoc

Bonferroni mean comparisons).

Similarly, pretreatment of moxonidine, clonidine, or

2-BFI prior to ineffective dose of nicotine (0.25 mg/kg,

i.p.) produced significant increase in latency time as

compared to respective control group [FDrug 9 Pretreatment

Figure 2 Antinociceptive effect of imidazoline receptor agonists and antagonists in tail flick test. Separate group of rats (n = 4–16) were

treated either with artificial cerebrospinal fluid (aCSF; 2 lL/rat, i.c.v.) or agmatine (10–40 lg/rat, i.c.v.) or harmane (25–100 lg/rat,

i.c.v.) or clonidine (2–8 lg/rat, i.c.v.) or moxonidine (25–100 lg/rat, i.c.v.) or 2-BFI (10–40 lg/rat, i.c.v.) or efaroxan (10–40 lg/rat,

i.c.v.) or idazoxan (4–16 lg/rat, i.c.v.), and reaction latency was measured. *P < 0.05, **P < 0.01, ***P < 0.001 vs. aCSF-treated

control animal (one-way ANOVA post hoc Dunnett’s test).




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(3, 39) = 3.40, P < 0.05; FDrug (1, 39) = 47.82,

P < 0.001; FPretreatment (3, 39) = 5.78, P < 0.01] (two-

way ANOVA). Post hoc Bonferroni multiple mean compari-

sons with aCSF + nicotine-treated control group

exhibited significant potentiation in % MPE by moxoni-

dine (25 lg/rat, i.c.v.; P < 0.001) or 2-BFI (10 lg/rat,

i.c.v.; P < 0.01) or clonidine (2 lg/rat, i.c.v.;

P < 0.001). Moxonidine, clonidine, and 2-BFI in the

dose used here did not influence the basal latency time

in tail flick assay (Figure 4).

Imidazoline receptor antagonists attenuated


Two-way ANOVA indicated that pretreatment of rats with

imidazoline I1 antagonist, efaroxan (10 lg/rat, i.c.v.), or

I2 antagonist, idazoxan (4 lg/rat, i.c.v.), significantly

attenuated the ethanol (1 g/kg, i.p.)-induced increased

latency time in tail flick assay [FDrug 9 Pretreatment

(2, 28) = 4.06, P < 0.05; FDrug (1, 28) = 31.11,

P < 0.001; FPretreatment (2, 28) = 3.98, P < 0.05] (Fig-

ure 5). Post hoc Bonferroni multiple comparisons

between the means demonstrated the decreased latency

time in efaroxan (10 lg/rat, i.c.v.) and idazoxan (4 lg/

rat, i.c.v.) pretreated animals indicating the antagonistic

potential of these agents in the antinociceptive effect of

ethanol.

Similar treatment of imidazoline receptor antago-

nists, efaroxan (10 lg/rat, i.c.v.) and idazoxan

(4 lg/rat, i.c.v.), before nicotine (0.5 mg/kg, i.p.) also

antagonized the nicotine-induced increases in latency

[FDrug 9 Pretreatment (2, 28) = 4.77, P < 0.05; FDrug

(1, 28) = 23.99, P < 0.001; FPretreatment (2, 28) =

3.96, P < 0.05] (two-way ANOVA post hoc Bonferroni

Test; Figure 5). Efaroxan or idazoxan administered

alone or with saline did not influence the latency time

in tail flick assay.

Imidazoline receptor antagonists attenuated

synergistic antinociceptive effect of ethanol and

nicotine

As depicted in Figure 6, pretreatment with efaroxan

(10 lg/rat, i.c.v.) or idazoxan (4 lg/rat, i.c.v.) blocked

the potentiated antinociceptive effect elicited by

ineffective dose combination of nicotine (0.25 mg/kg,

i.p.) and ethanol (0.5 g/kg, i.p.) [FDrug 9 Pretreatment

(2, 27) = 8.28, P < 0.01; FDrug (1, 27) = 21.24,

P < 0.001; FPretreatment (2, 27) = 11.36, P < 0.001]

(two-way ANOVA post hoc Bonferroni test).

Figure 3 Potentiation of antinociceptive effect of ethanol and

nicotine by endogenous imidazoline binding site ligands in tail

flick test. Separate group of rats (n = 5–6) were treated either

with artificial cerebrospinal fluid (aCSF; 2 lL/rat, i.c.v.) or

harmane (25 lg/rat, i.c.v.) or agmatine (10 lg/rat, i.c.v.) 10 min

before saline (1 mL/kg, i.p.) or ethanol (0.5 g/kg, i.p.) or nicotine

(0.25 mg/kg, i.p.), and reaction latency was measured 30 min

after alcohol or 20 min after nicotine administration. *P < 0.001

vs. aCSF + saline control group, $P < 0.001 vs. aCSF + ethanol,

#P < 0.001 vs. aCSF + nicotine (two-way ANOVA post hoc

Bonferroni multiple comparison test).

Figure 4 Potentiation of antinociceptive effect of ethanol and

nicotine by imidazoline receptor agonist in tail flick test. Separate

group of rats (n = 4–6) were treated either with artificial

cerebrospinal fluid (aCSF; 2 lL/rat, i.c.v.) or moxonidine (25 lg/

rat, i.c.v.) or 2-BFI (10 lg/rat, i.c.v.) or clonidine (2 lg/rat, i.c.v.)

10 min before saline (1 mL/kg, i.p.), ethanol (0.5 g/kg, i.p.), or

nicotine (0.25 mg/kg, i.p.), and reaction latency was measured

20 min after alcohol or 10 min after nicotine administration.

*, $P < 0.01; **, $$P < 0.01 vs. ethanol/nicotine-treated

respective control group (two-way ANOVA post hoc Bonferroni

multiple comparison test).




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DISCUSS ION

Over the years, it has been a great scientific challenge to

produce centrally acting analgesic without abuse poten-

tial. Currently, opioidergic drugs are the only clinically

available central analgesics. Present study provides

functional evidences for the involvement of imidazoline

binding sites in antinociceptive effects of alcohol and

nicotine. It is well known that alcohol and nicotine

traditionally being used as strong pain-relieving sub-

stances. In present study, we found that ethanol and

nicotine as well as their subeffective combination pro-

duce dose-dependent increase in latency time in tail flick

assay of spinal nociception. These results are in well

agreement with earlier findings of Campbel et al.

[54,55]. Indeed, these drugs are known for their

co-abuse potential and share common pathways for

their reward as well as analgesic property [36,37]. Con-

siderable efforts are being directed toward developing

more effective therapy for treatment of alcoholism and

nicotine addiction. The challenge is compounded by

conditions of comorbid dependence on alcohol and nico-

tine [56]. The 12imidazoline receptor binding sites are of

particular relevance to the comorbid occurrence of alco-

holism and heavy smoking. It is intimately involved in

perception and modulation of pain, while it plays major

role in reward [57]. It is not surprising therefore that

the rewarding and antinociceptive effects of alcohol and

nicotine, either individually or combined, may be closely

intertwined with the endogenous imidazoline system.

Several clinical and preclinical studies have demon-

strated therapeutic potential of imidazoline binding site

ligands [58,59]. Interestingly, several behavioral effects

of ethanol and nicotine including conditioned hyperlo-

comotion, anhedonia, anxiety, withdrawal, locomotor

sensitization, intake, and development of dependence or

withdrawal syndrome are found to be associated with

imidazoline binding sites [38,40,49,50,60–66]. Immu-

nocytochemical studies suggest that imidazoline bind-

ing sites are expressed in brain regions involved in

pain perception and response to painful stimuli [41].

Further I2 sites can be detected in spinal cord and

implicated in modulation of nociceptive processing

[67]. However, it remains unknown as to whether they

enhanced or inhibit nociception. In view of this, pres-

ent study investigated the effect of endogenous imidaz-

oline ligands on antinociceptive effect of ethanol and

nicotine in tail flick assay representing spinal

nociception. Agmatine and harmane are the potent

endogenous ligands that activate imidazoline I1 and I2

Figure 5 Influence of imidazoline receptor antagonists on the

antinociceptive effect executed by ethanol and nicotine in tail flick

test. Separate group of rats (n = 6) were treated either with

artificial cerebrospinal fluid (aCSF; 2 lL/rat, i.c.v.) or idazoxan

(4 lg/rat, i.c.v.) or efaroxan (10 lg/rat, i.c.v.) 10 min before saline

(1 mL/kg, i.p.), ethanol (1 g/kg, i.p.), or nicotine (0.5 mg/kg, i.p.),

and reaction latency was measured 20 min after alcohol or 10 min

after nicotine administration. *P < 0.001 vs. vehicle control group,

$P < 0.01 vs. alcohol-treated group, # 11vs. nicotine-treated

group (two-way ANOVA post hoc Bonferroni multiple comparison

test).

Figure 6 Influence of imidazoline receptor antagonists on the

antinociceptive effect executed by noneffective dose combination

of ethanol and nicotine in tail flick test. Separate group of rats

(n = 6) were treated either with artificial cerebrospinal fluid

(aCSF; 2 lL/rat, i.c.v.) or idazoxan (4 lg/rat, i.c.v.) or efaroxan

(10 lg/rat, i.c.v.) 10 min before saline (1 mL/kg, i.p.) or ethanol

(0.5 g/kg, i.p.) and nicotine (0.25 mg/kg, i.p.), and reaction

latency was measured 20 min after alcohol or 10 min after

nicotine administration. #P < 0.001 vs. vehicle control group,

*P < 0.001 vs. alcohol- and nicotine-treated group (two-way

ANOVA post hoc Bonferroni multiple comparison test).




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receptor. It is important to note that these agents

themselves exhibit antinociceptive profile in neuropath-

ic pain [68,69]. Interestingly, we found that pretreat-

ment of these agents potentiated the antinociceptive

effect of ethanol as well as nicotine. This suggests that

imidazoline binding sites act as common neural sub-

strate responsible for analgesic effect of alcohol and

nicotine. Moreover, evidences also suggest the involve-

ment of imidazoline binding sites in addiction liability

[38–40]. In our earlier study, we also demonstrated

the involvement of imidazoline binding sites in ethanol

withdrawal anxiety [50]. Moreover, agmatine, an

endogenous receptor ligand attenuated the develop-

ment as well as expression of nicotine, induced locomo-

tor sensitization [49]. This is interesting in view of the

therapeutic potential of imidazoline agents to counter-

act the comorbidity of alcohol and nicotine.

It is also reported that several imidazoline receptor

agonists including moxonidine, clonidine, 2-BFI, and

BU-224 possess analgesic property [67,70]. Hence to

confirm above finding, we observed the effect of imidaz-

oline receptor agonists on antinociceptive potential of

ethanol and nicotine. We found that clonidine, mixed

agonist of imidazoline I1/adrenergic a2 receptor, moxo-

nidine, imidazoline I1 receptor agonist, and 2-BFI,

imidazoline I2 receptor agonist, significantly increased

the antinociceptive effect of nicotine and ethanol. Con-

versely, antinociceptive effect of ethanol and nicotine

or their subeffective dose combination was significantly

inhibited by pretreatment with imidazoline I1 antago-

nist, efaroxan, as well as imidazoline I2/adrenergic a2receptor antagonist, idazoxan.

Thus, our result clearly suggests the involvement of

imidazoline I1 and I2 receptors in analgesic effects of

ethanol and nicotine. However, most of the agents

used in this study like clonidine, agmatine, and idazo-

xan possess moderate affinity toward a2 adrenergic

receptors. Further analgesic property of clonidine and

agmatine has been linked to a2 adrenergic receptors

modulation [68]. Thus, possible involvement of a2adrenergic receptors in antinociceptive effect of ethanol

and nicotine needs further investigation. Moreover,

agmatine and harmane also act on NMDA [71,72] and

GABAA receptors [73], respectively, and hence the con-

tribution of these targets could not be ruled out and

needs further investigation.

The antinociceptive activity of nicotine or alcohol

has been linked to their co-abuse [36,37]. Therefore, it

can be inferred from the present study that imidazoline

agents may act as important therapeutic molecules for

the treatment of alcohol and nicotine addiction as well

as their co-abuse potential. However, extensive bio-

chemical and pharmacological studies are required to

prove this hypothesis.

CONFL ICT OF INTEREST

All the authors report no conflict of interest.

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binding sites are of…’.

O n c e y o u h a v e A c r o b a t R e a d e r o p e n o n y o u r c o m p u t e r , c l i c k o n t h e C o m m e n t t a b a t t h e r i g h t o f t h e t o o l b a r :

S t r i k e s a l i n e t h r o u g h t e x t a n d o p e n s u p a t e x tb o x w h e r e r e p l a c e m e n t t e x t c a n b e e n t e r e d .‚ H i g h l i g h t a w o r d o r s e n t e n c e .‚ C l i c k o n t h e R e p l a c e ( I n s ) i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ T y p e t h e r e p l a c e m e n t t e x t i n t o t h e b l u e b o x t h a ta p p e a r s .

T h i s w i l l o p e n u p a p a n e l d o w n t h e r i g h t s i d e o f t h e d o c u m e n t . T h e m a j o r i t y o ft o o l s y o u w i l l u s e f o r a n n o t a t i n g y o u r p r o o f w i l l b e i n t h e A n n o t a t i o n s s e c t i o n ,p i c t u r e d o p p o s i t e . W e ’ v e p i c k e d o u t s o m e o f t h e s e t o o l s b e l o w :S t r i k e s a r e d l i n e t h r o u g h t e x t t h a t i s t o b ed e l e t e d .

‚ H i g h l i g h t a w o r d o r s e n t e n c e .‚ C l i c k o n t h e S t r i k e t h r o u g h ( D e l ) i c o n i n t h eA n n o t a t i o n s s e c t i o n .

H i g h l i g h t s t e x t i n y e l l o w a n d o p e n s u p a t e x tb o x w h e r e c o m m e n t s c a n b e e n t e r e d .‚ H i g h l i g h t t h e r e l e v a n t s e c t i o n o f t e x t .‚ C l i c k o n t h e A d d n o t e t o t e x t i c o n i n t h eA n n o t a t i o n s s e c t i o n .‚ T y p e i n s t r u c t i o n o n w h a t s h o u l d b e c h a n g e dr e g a r d i n g t h e t e x t i n t o t h e y e l l o w b o x t h a ta p p e a r s .

M a r k s a p o i n t i n t h e p r o o f w h e r e a c o m m e n tn e e d s t o b e h i g h l i g h t e d .‚ C l i c k o n t h e A d d s t i c k y n o t e i c o n i n t h eA n n o t a t i o n s s e c t i o n .‚ C l i c k a t t h e p o i n t i n t h e p r o o f w h e r e t h e c o m m e n ts h o u l d b e i n s e r t e d .‚ T y p e t h e c o m m e n t i n t o t h e y e l l o w b o x t h a ta p p e a r s .

I n s e r t s a n i c o n l i n k i n g t o t h e a t t a c h e d f i l e i n t h ea p p r o p r i a t e p a c e i n t h e t e x t .‚ C l i c k o n t h e A t t a c h F i l e i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ C l i c k o n t h e p r o o f t o w h e r e y o u ’ d l i k e t h e a t t a c h e df i l e t o b e l i n k e d .‚ S e l e c t t h e f i l e t o b e a t t a c h e d f r o m y o u r c o m p u t e ro r n e t w o r k .‚ S e l e c t t h e c o l o u r a n d t y p e o f i c o n t h a t w i l l a p p e a ri n t h e p r o o f . C l i c k O K .

I n s e r t s a s e l e c t e d s t a m p o n t o a n a p p r o p r i a t ep l a c e i n t h e p r o o f .‚ C l i c k o n t h e A d d s t a m p i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ S e l e c t t h e s t a m p y o u w a n t t o u s e . ( T h e A p p r o v e ds t a m p i s u s u a l l y a v a i l a b l e d i r e c t l y i n t h e m e n u t h a ta p p e a r s ) .‚ C l i c k o n t h e p r o o f w h e r e y o u ’ d l i k e t h e s t a m p t oa p p e a r . ( W h e r e a p r o o f i s t o b e a p p r o v e d a s i t i s ,t h i s w o u l d n o r m a l l y b e o n t h e f i r s t p a g e ) .

A l l o w s s h a p e s , l i n e s a n d f r e e f o r m a n n o t a t i o n s t o b e d r a w n o n p r o o f s a n d f o rc o m m e n t t o b e m a d e o n t h e s e m a r k s . .‚ C l i c k o n o n e o f t h e s h a p e s i n t h e D r a w i n gM a r k u p s s e c t i o n .‚ C l i c k o n t h e p r o o f a t t h e r e l e v a n t p o i n t a n dd r a w t h e s e l e c t e d s h a p e w i t h t h e c u r s o r .‚

T o a d d a c o m m e n t t o t h e d r a w n s h a p e ,m o v e t h e c u r s o r o v e r t h e s h a p e u n t i l a na r r o w h e a d a p p e a r s .‚

D o u b l e c l i c k o n t h e s h a p e a n d t y p e a n yt e x t i n t h e r e d b o x t h a t a p p e a r s .

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