Neuropharmacology and analgesia

Effects of the sazetidine-a family of compounds on the bodytemperature in wildtype, nicotinic receptor β2�/� and α7�/� mice$

Edward D. Levin a,n, Hannah G. Sexton a, Karen Gordon a, Christopher J. Gordon b,Yingxian Xiao c, Kenneth J. Kellar c, Venkata Mahidhar Yenugonda d, Yong Liu d,Michael P. White d, Mikell Paige d, Milton L. Brown d, Amir H. Rezvani a

a Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, USAb US Environmental Protection Agency, USAc Department of Pharmacology and Physiology, Georgetown University School of Medicine, USAd Center of Drug Discovery, Georgetown University School of Medicine, USA

a r t i c l e i n f o

Article history:Received 22 March 2013Received in revised form21 August 2013Accepted 30 August 2013Available online 12 September 2013

Keywords:NicotinicTemperature regulationSazetidine-ADesensitizationKnockout

a b s t r a c t

Nicotine elicits hypothermic responses in rodents. This effect appears to be related to nicotinic receptordesensitization because sazetidine-A, an α4β2 nicotinic receptor desensitizing agent, produces markedhypothermia and potentiates nicotine-induced hypothermia in mice. To determine the specificity ofsazetidine-A induced hypothermia to β2 subunit-containing nicotinic receptors, we tested its efficacy inβ2 knockout (β2�/�) mice. These effects were compared with wildtype (WT) and α7 knockout (α7�/�)mice. Confirming our earlier results, sazetidine-A elicited a pronounced and long-lasting hypothermia inWT mice. In comparison, sazetidine-A induced a much attenuated and shorter hypothermic response inβ2�/� mice. This indicates that the greater proportion of sazetidine-A induced hypothermia is mediatedvia actions on β2-containing nicotinic receptors, while a smaller component of hypothermia induced bysazetidine-A is mediated by non-β2 receptors. Similar to WT mice, α7�/� mice showed the full extent ofthe sazetidine-A effect, suggesting that the hypothermia produced by sazetidine-A did not depend onactions on α7 nicotinic receptor subtype. Three other novel nicotinic receptor desensitizing agentsderived from sazetidine-A, triazetidine-O, VMY-2-95 and YL-1-127 also produced hypothermia in WTand α7�/� mice. Furthermore, unlike sazetidine-A, triazetidine-O and YL-1-127 did not show any hint ofa hypothermic effect in β2�/� mice. VMY-2-95 like sazetidine-A did show a residual hypothermic effectin the β2�/� mice. These studies show that the hypothermic effects of sazetidine-A and the relatedcompound VMY-2-95 are mainly mediated by nicotinic receptors containing β2 subunit, but that a smallcomponent of the effect is apparently mediated by non-β2 containing receptors.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Neuronal nicotinic receptors have been implicated in theregulation of body temperature in a variety of species includingpigeons (Komarova, 2009), rodents (Gordon et al., 2002; Markset al., 1984; Overstreet et al., 1998; Ruskin et al., 2008; Sack et al.,2005; Salminen and Ahtee, 2000; Zarrindast et al., 1995) andprimates (Hall and Myers, 1972). Peripheral administration ofnicotine induces hypothermia (Zarrindast et al., 2001; Zarrindast

et al., 1995) while administration of mecamylamine, a non-specificnicotinic receptor antagonist, blocks or decreases nicotine-inducedhypothermia in mice (Nordberg and Sundwall, 1983; Zarrindastet al., 2001). Studies in inbred mouse strains have demonstratedthat the high-affinity nicotine-binding sites probably mediate thehypothermic effects of nicotine (Marks et al., 1989). Moreover,although not confirmed, it appears that the hypothermic effectof nicotine may be related to its nicotinic receptor desensitizingeffects inasmuch as the selective α4β2 nicotinic receptor desensi-tizing agent sazetidine-A also produces hypothermia and poten-tiates nicotine-induced hypothermia in rodents (Rezvani et al.,2012). To better understand the involvement of nicotinic receptorsubtypes in regulation of body temperature, we compared theeffects of four selective α4β2 desensitizing compounds with differ-ent pharmacologic properties on body temperature in wildtype,β2�/� and α7 KO mice.

The widely characterized property of nicotinic receptor desen-sitization involves the inability of the receptor channel to openwhen bound by either the endogeneous ligand acetylcholine or

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European Journal of Pharmacology

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ejphar.2013.08.037

☆This project was supported by NIH/NIDA grants U19DA027990 and R03DA025947.The novel compounds in the publication are covered by issued patent and patentapplications. The following authors are listed as inventors: for sazetidine-A, KJK andYX; for triazetidine-O, MLB, PM, YX and KJK; for VMY-2-95, MLB, MP, YX, KJK, VMY,EDL and AHR; and for YL-1-127, YX, KJK, MLB, MP, YL, EDL and AHR. GeorgetownUniversity is the holder for issued patents.

n Correspondence to: Department of Psychiatry and Behavioral Sciences, Box104790, Duke University Medical Center, Durham, NC 27710, USA.Tel.: þ1 919 681 6273; fax: þ1 919 681 3416.

E-mail address: edlevin@duke.edu (E.D. Levin).

European Journal of Pharmacology 718 (2013) 167–172

an exogenous drug. This is an inherent property of nicotinicreceptors, as well as several other ligand-gated channels. In thecase of nicotinic receptors, the agonist activity of nicotine andother nicotinic agonists is relatively short-lived (seconds to min-utes), whereas the desensitization effects last much longer (min-utes to hours). The actions of a nicotinic agonist to both activateand desensitize nicotinic receptors have been known for morethan 50 years (Katz and Thesleff, 1957). The important contribu-tions of desensitization to effects of nicotinic ligands in vivo andin vitro have been widely characterized (Buccafusco et al., 2009;Hulihan-Giblin et al., 1990; Picciotto et al., 2008; Quick and Lester,2002; Sharp and Beyer, 1986; Xiao et al., 2006). It has been shownthat a single injection of nicotine desensitizes the response forseveral hours (Hulihan-Giblin et al., 1990; Sharp and Beyer, 1986).We reported previously that the selective α4β2 nicotinic receptorantagonist DHβE did not reduce the hypothermia effect of sazeti-dine-A; instead, it significantly prolonged the induced hypother-mia (Rezvani et al., 2012). It is clear that the desensitizationplays an important role in hypothermia reduced by nicotinicagents.

Sazetidine-A has a very high binding affinity for rat α4β2 nicotinicreceptors and, more importantly, it is 410,000-fold and 3500-foldselective for α4β2 receptors compared to its affinity at rat α3β4 andα7 nicotinic receptors, respectively (Xiao et al., 2006; Xiao et al.,2008). Consistent with its high selectivity for α4β2 receptors inbinding assays, sazetidine-A is a much weaker desensitizer of α3β4or α7 nicotinic receptor subtypes (Liu et al., 2010; Xiao et al.,2006,2010). Thus, sazetidine-A is a highly selective and potent α4β2nicotinic receptor desensitizer (Xiao et al., 2006).

Recently, three analogs of sazetidine-A have been synthesized.These analogs are triazetidine-O (Xiao et al., 2009), VMY-2-95(Yenugonda et al., in press) and YL-1-127 (Liu et al., 2013). Similarto sazetidine-A, in binding assays these three compounds havemore than 10,000 fold selectivity for rat α4β2 receptors over ratα3β4 or α7 receptors. In functional studies in cell models, thesethree sazetidine-A analogs potently and selectively desensitizeα4β2 receptors. Although similar to sazetidine-A, these com-pounds have important medicinal and pharmacological propertiesthat are different from those of sazetidine-A.

To determine the roles of specific nicotinic receptor subtypes inthe hypothermia induced by sazetidine-A and its novel analogs,we studied effects of these compounds on body temperature inwildtype (WT) mice, β2 knockout (β2�/�) mice and α7 knockout(α7� /�) mice. Based on the current literature regarding the role ofneuronal nicotinic receptors on regulation of body temperatureand our previous findings (Rezvani et al., 2012), it was hypothe-sized that the desensitization of nicotinic receptors containingβ2 subunit would significantly reduce body temperature and thatthe induced hypothermia would be absent in β2� /� mice.

2. Methods

2.1. Animals

Adult male WT, β2� /� and α7� /� mice weighing 2572 (S.E.M.)g were used in these studies. The nicotinic α7 and β2 receptorknockout mouse breeding pairs and wildtype controls (C57/BL6)were generously provided by Dr. John Dani and colleagues atBaylor University. Animals were bred in our laboratory. They werehoused in groups of three for one week before surgery and weresingle housed after the surgery. Wood chip bedding was usedthroughout. Animals had free access to food and water in anambient temperature of 2271 1C and 12 L:12D photoperiod(lights on at 1800 h). Injections were given s.c. in a volume of10 ml/kg during the dark phase when animals were active.

2.2. Surgery and telemetry system

To measure core body temperature in mice, an automatedtelemetry system was used. Animals were anesthetized witha dose of 60 mg/kg ketamine (Fort Dodge Animal Health, FortDodge, IA, USA) mixed with 15 mg/kg domitor (Pfizer AnimalHealth, New York, NY, USA) injected IP. The fur over the ventralabdominal cavity was shaved and the area was cleaned with betaiodine first and a solution of 70% (v/v) alcohol. After the alcoholdried, a 2-cm longitudinal incision was made along the midline ofthe abdomen about 1 cm below the sternum, and a transmitterweighing about 3.5 g (Model TA-F20) was inserted into theabdominal cavity. The incision was sutured and the outer skinwas closed with wound staples. After two weeks of recovery, eachcage was positioned on a receiver board, which decoded thetemperature of the transmitter within the body cavity. The bodytemperature was collected automatically every 5 min and stored ina computer using the Data Quest IV software (Data Sciences, Inc.,St. Paul, MN, USA). The data for analysis were collected for threehours after drug administration.

2.3. Experimental protocol

Dose effect functions of four novel α4β2 nicotinic desensitizingcompounds with slightly different properties were tested on bodytemperature of WT, β2�/� and α7�/� mice using a repeated measurescounterbalanced design for each study. In experiment one, mice wereinjected subcutaneously with 0.3, 1 or 3 mg/kg of sazetidine-A or anequal volume of saline (N¼7 WT, N¼7 β2� /� and N¼8 α7� /�). Inanother set of mice, a dose-response function for triazetidine-O wasestablished. Mice were injected s.c. with 1.5, 3, 6 or 18 mg/kgtriazetidine-O or saline (N¼11 WT, N¼11 β2� /�and N¼8 α7�/�).Three weeks after the last triazetidine-O administration, a dose-response function was established for VMY-2-95 in the same mice.Mice were injected s.c. with 0.3, 1, or 3 mg/kg VMY-2–95 or vehicle(N¼11 WT, N¼11 β2� /� and N¼8 α7 KO�/�). In another group ofmice the acute effects of YL-1-127 on body temperature wereassessed. Mice were injected with 0.3, 1, 3, 6, 12 and 18mg/kg ofYL-1-127 or the vehicle (N¼11 WT, N¼9 β2�/�and N¼7 α7�/�). Allanimals in each group received all treatments and the intervalsbetween injections were at least 3 days.

2.4. Drug preparation

Sazetidine-A HCl was synthesized at RTI, (Research Triangle, NC,USA) and supplied by the National Institute on Drug Abuse; triaze-tidine-O, VMY-2-95 and YL-1-127 were synthesized as the free baseby methods reported previously (Brown et al., 2009; Liu et al., 2013;Xiao et al., 2006; Yenugonda et al., 2013). The compounds were498.5% pure. Sazetidine-A and triazetidine-O were dissolved insaline solution, while VMY-2-95 and YL-1-127 were dissolved in asolution of 100 mM HCl. All of the drugs were diluted in saline toneeded concentrations. The pH of the drug solutions were adjusted to6.5–7.0. All drugs doses are referred to as the salt. Drug solutions wereprepared weekly and kept refrigerated between experiments. Thedrug was injected (s.c.) in volume of 10 ml/kg body weight.

2.5. Statistical analysis

The temperature data were assessed with analysis of variancefor repeated measures. The between subjects factor was genotypeand the within subjects factors were drug treatment dose and five-min block over 3-h. The studies were run in repeated cohorts, eachcontaining all of the genotypes and cohort was included as acontrol factor. The threshold for significance for the main effectsand interactions was po0.05. Comparisons of the treated groups

E.D. Levin et al. / European Journal of Pharmacology 718 (2013) 167–172168

to control were done using the Bonferroni correction. With threecomparisons of each dose vs. control we have tightened topo0.01 as the threshold for significance for the comparisonsbetween treatment conditions.

3. Results

3.1. Sazetidine-A

With sazetidine-A there were significant main effects ofsazetidine-A treatment (F(3,57)¼36.37, Po0.0005), genotype (F(2,19)¼6.21, Po0.01) and time (F(35, 665)¼12.79, Po0.0005).There were significant interactions of sazetidine-A� genotype (F(6,57)¼7.72, Po0.0005), sazetidine-A x time (F(105, 1995)¼5.41,Po0.0005) and sazetidine-A� genotype� time (F(210, 1995)¼6.94, Po0.0005). In the wildtype mice, the sazetidine-A main effectwas significant (F(3,18)¼37.38, Po0.0005), with an mean decreasefrom 37.070.03 averaged over the three hours after injection ofsaline to 28.970.2 after injection of 3 mg/kg of sazetidine-A. In theβ2� /� mice the hypothermic effect of sazetidine-A was muchdiminished, but the sazetidine-A main effect was still significant(F(3,18)¼13.18, Po0.0005) with the β2� /� mice showing a declinefrom 36.670.1 after saline injections to 35.270.1 after 3 mg/kg ofsazetidine-A averaged over the three hours after administration.The α7�/� mice showed a relatively undiminished hypothermiceffect of sazetidine-A with a significant main sazetidine-A effect(F(3,21)¼7.82, Po0.0005) with temperature declining from36.770.04 after saline to 31.970.2 after 3 mg/kg of sazetidine-Aaveraged over the three hours after administration. Within thethree hours after administration, the maximum drop in tempera-ture with the high dose of sazetidine-A (3 mg/kg) in wildtype micewas approximately 10 1C, while this value for the β2�/� mice andthe α7� /� mice was approximately 4 and 7 1C, respectively (Fig. 1).In the β2� /� mice the late phase of the sazetidine-A inducedhypothermia was eliminated, but the early phase was left intact.

3.2. Triazetidine-O

Similar to sazetidine-A, triazetidine-O also caused significanthypothermia (F5, 120)¼14.57, Po0.0005). There was a significantmain effect of genotype (F2,24)¼9.05, Po0.005) and a significantgenotype� triazetidine-O interaction (F(10,120)¼5.26, Po0.0005).In the WT mice there was a significant main effect of triazetidine-O(F(5,45)¼12.02, Po0.0005), but the extent of hypothermia was

much less than seen with sazetidine-A (�10 1C with 3 mg/kgsazetidine-A vs. �4 1C with 18 mg/kg triazetidine-O). Furthermore,in contrast to sazetidine-A the β2�/� mice showed no indication ofhypothermia with triazetidine-O. The α7�/� mice showed a signifi-cant (F(5,30)¼3.73, Po0.01) hypothermic effect of triazetidine-O(Fig. 2), indicating that as with sazetidine-A, the α7 receptor subtypeis not essential for the hypothermic response.

3.3. VMY-2–95

With VMY-2–95 there was a significant main effect of VMY-2-95treatment (F(3,66)¼18.46, Po0.0005) and genotype (F(2,22)¼3.85,Po0.05) and time (F(35, 770)¼1.88, Po0.005). Significant interac-tions were seen for time� genotype (F(70,770)¼1.57, Po0.005),VMY-2-95� time (F(105,2310)¼4.52, Po0.0005) and VMY-2-95 xgenotype x time (F(210,2310)¼1.41, Po0.0005). Follow-up tests ofthe simple main effects were made for the highest order significantinteraction VMY-2–95� genotype� time. Fig. 3 shows the effects ofVMY-2-95 on body temperature over the course of 3 h afterinjection in WT, β2� /� and α7� /� mice. The WT mice showed asignificant effect of VMY-2-95 (F(3,27)¼5.19, Po0.01) with the3 mg/kg VMY-2-95 dose causing an overall significant hypothermia(F(1,27)¼12.55, Po0.005). With the WT mice there was a signifi-cant interaction of VMY-2-95� time (F(105,945)¼1.28, Po0.05).Tests of the dose-effect for the simple main effects at each timepoint showed for the wildtype mice that the 3 mg/kg dose causedsignificant (F(1945), Po0.0005) hypothermia for each time pointstarting 10 minutes post injection and continuing throughout therest of the 3-h period after injection. The 1 mg/kg doses caused amore modest but still significant (F(1945), Po0.01–0.0005)hypothermia from 25 min through the 3-h period after injection.The 0.3 mg/kg dose had no discernable effect on body temperature.

With the β2� /� mice there was also a significant main effect ofVMY 2–95 (F(3,24)¼4.53, Po0.05), but the magnitude ofhypothermia was much less than with the wildtype mice. Therewas a significant VMY 2-95� time interaction (F(105,840)¼2.07,Po0.0005). The 3 mg/kg dose caused significant hypothermiastarting 10 min post-injection and lasting throughout the restof the 3-h period after injection (F(1840), Po0.01–0.0005). Thelower doses caused modest but significant episodes of hyperther-mia with the 1 mg/kg doses causing significant hyperthermia 140–150 min post-injection and the lower 0.3 mg/kg dose causingsignificant hyperthermia 75–80 min post-injection (F(1840),Po0.01–0.005).

Fig. 1. The hypothermic effect of sazetidine-A in wildtype mice and mice with knockouts of α7 and β2-containing nicotinic receptors (mean7S.E.M.), Wildtype N¼7, β2� /�

N¼7, α7�/� N¼8.

E.D. Levin et al. / European Journal of Pharmacology 718 (2013) 167–172 169

With the α7�/� mice, there was a significant main effect of VMY-2-95 (F(3,15)¼35.07, Po0.0005) with the 3 mg/kg VMY-2-95 dosecausing significant hypothermia (F(1,15)¼77.98, Po0.0005). Therewas a significant VMY 2-95� time interaction (F(105,525)¼8.85,Po0.0005). In the α7� /� mice the 3 mg/kg VMY-2-95 dose caused asignificant degree of hypothermia starting 10 min post injection andlasting throughout the rest of the 3-h period after injection (F(1525),Po0.0005). The lower doses caused lesser and shorter extents ofhypothermia, The 1 mg/kg dose caused modest but significanthypothermia 10–50 min post injection (F(1525), Po0.005–0.0005)and at the end of the 3-h post-injection period (F(1525), Po0.0.05).

3.4. YL-1-127

The next experiment tested the hypothermic effects of YL-1-127.The lower dose range of YL-1-127 (0.3, 1 and 3 mg/kg) did notproduce any significant hypothermia (Fig. 4). Even the higher doserange of YL-1-127 (6, 12 and 18 mg/kg) did not produce significanthypothermia (Fig. 5), although there was a trend toward ahypothermic effect in the WT and the α7�/� mice (Fig. 5).

4. Discussion

The results of this study confirm our earlier finding (Rezvaniet al., 2012) that sazetidine-A causes a robust and long lastinghypothermia in wildtype mice. The threshold for this effect is

between 0.3 and 1 mg/kg. This dramatic hypothermic effect wascertainly truncated in the β2�/� mice, however there was still asmaller and shorter hypothermic effect of sazetidine-A in thesemice during the early phase (0–90 min) despite the absence ofα4β2 receptors. Thus, sazetidine-A must have produced this effectthrough a non-α4β2 nicotinic receptor, either some other subtypesof the nicotinic receptor or a non-nicotinic receptor. The identity ofthis receptor is not currently known. Compared with sazetidine-A,triazetidine-O was considerably less potent in producinghypothermia in WT and α7� /� mice. It only produced significanthypothermia at a high dose of 18 mg/kg. This effect was not seenin the β2� /� mice even at the high dose of 18 mg/kg. VMY-2–95cause much less hypothermia than sazetidine-A. There was somehypothermic effect also seen with the β2� /� and α7� /� mice butthe temperature decrease was much less in extent than was seenwith sazetidine-A. In contrast to the other drugs, YL-1–127produced no significant hypothermia up to an acute high dose of18 mg/kg. There was a non-significant trend toward hypothermiawith the highest dose tested in the WT and α7�/� mice, whichwas not apparent in the β2� /� mice.

The differential hypothermic effects of these four α4β2 desen-sitizing drugs provide important information concerning possibleclinical use when laid upon the information concerning thepotency and efficacy of these drugs for reducing nicotine self-administration. Sazetidine-A significantly reduces nicotine self-administration at a dose of 3 mg/kg, but not lower (Levin et al.,2010). At this dose sazetidine-A causes dramatic hypothermia in

Fig. 2. The hypothermic effect of triazetidine-O in wildtype mice and mice with knockouts of α7 and β2-containing nicotinic receptors (mean7S.E.M.), Wildtype N¼11, β2� /� N¼11,α7�/� N¼8.

Fig. 3. The hypothermic effect of VMY-2-95 in wildtype mice and mice with knockouts of α7 and β2-containing nicotinic receptors (mean7S.E.M.), Wildtype N¼11, β2�/�

N¼11, α7�/� N¼8.

E.D. Levin et al. / European Journal of Pharmacology 718 (2013) 167–172170

mice, which may be a useful effect if hypothermia is somehowrelated to the mechanism mediating reduction of nicotine self-administration, but if not it could be an undesired side effect. Inrats a much smaller magnitude hypothermic effect is seen with the3 mg/kg dose but it is still stastically significant (Rezvani et al.,2012). Importantly, the hypothermia induced by sazetidine-A isreduced in β2�/� mice but it is not eliminated, indicating thatsazetidine-A might have actions on other non β2 containingreceptors involved in regulation of body temperature.

The hypothermia induced by triazetidine-O and VMY-2-95 ismuch less robust than with sazetidine-A. Triazetidine-O in ourother studies was not found to significantly reduce nicotine self-administration in rats in doses up to 18 mg/kg (unpublished data),so the lack of a hypothermic side effect at this dose range is notrelevant to possible development for smoking cessation. In con-trast, VMY-2–95 has been shown in our other studies to signifi-cantly reduce nicotine self-administration at 3 mg/kg to a similarextent as sazetidine-A (Slade et al., 2012). Sazetidine-A inducedhypothermia might be considered an unsought side effect in itsuse for smoking cessation. The finding that VMY-2–95 causesmuch less hypothermia than sazetidine-A at this dose, whicheffectively reduces nicotine self-administration points to thepossible superiority of this drug for development as a smokingcessation treatment. The finding that the modest hypothermiceffect of VMY-2-95 was greatly minimized (though not eliminated)in β2�/� mice demonstrates the greater specificity of this com-pound compared with sazetidine-A. Finally, YL-1-127 had theleast effect at producing hypothermia. Although no significant

hypothermic effects were seen at doses up to 18 mg/kg, there wasa modest trend toward hypothermia seen at 18 mg/kg in WT andα7�/� mice. No hint of an effect was seen in the β2�/� mice. YL-1-127 was previously shown in our studies (Liu et al., 2010; Xiaoet al., 2006,2010) to have a potency to desensitize α4β2 nicotinicreceptors is 24-fold lower than that of sazetidine-A. As shownin Fig. 4, YL-1-127 did not elicit significant hypothermia, at doseup to 3 mg/kg). However, as shown in Fig. 5, YL-1-127 produced atrend of hypothermia when the doses were increased. The potencyof producing hypothermia correlates well with the potency ofdesensitizing the receptor.

In summary, sazetidine-A, which significantly reduces nicotineself-administration, also produces robust hypothermic effects.Furthermore, part of the hypothermic effect of sazetidine-Aappears to derive from actions at non-β2 nicotinic receptors(either other nicotinic receptors or non-nicotinic receptors). It isinteresting to note that the hypothermic effect of nicotine wasreported to be mediated both by nicotinic receptors containingα4 sub-units and those without α4 subunits (Tapper et al., 2007).The sazetidine-A analog VMY-2-95, which also seems to have avery good profile for reducing nicotine self-administration, pro-duces only modest hypothermia. YL-1–127 is the least effectivecompound in causing hypothermia but its efficacy in reducingnicotine self-administration has not been fully explored.

In conclusion, given the hypothermic effects induced bysazetidine-A in β2�/� mice, it is apparent that sazetidine-Aprobably has some non-β2 nicotinic receptor actions, However,the long duration of sazetidine-A′s hypothermic effects seems to

Fig. 4. Body temperature and the low dose range of YL-1-127 in wildtype mice and mice with knockouts of α7 and β2-containing nicotinic receptors (mean7S.E.M.),Wildtype N¼11, β2�/� N¼9, α7�/� N¼7.

Fig. 5. Body temperature and the high dose range of YL-1-127 in wildtype mice and mice with knockouts of α7 and β2-containing nicotinic receptors (mean7S.E.M.),Wildtype N¼11, β2�/� N¼9, α7�/� N¼7.

E.D. Levin et al. / European Journal of Pharmacology 718 (2013) 167–172 171

require β2-containing nicotinic receptors since the persistenthypothermia induced by sazetidine-A was markedly truncated inβ2� /� mice.

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