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. Author manuscript; available in PMC: 2023 Nov 2.
Published in final edited form as: J Psychopharmacol. 2022 Nov 2;36(11):1280–1293. doi: 10.1177/02698811221132214

Pharmacological characterization of 5-iodo-A-85380, a β2-selective nicotinic receptor agonist, in mice

Lois S Akinola 1, Deniz Bagdas 2,3, Yasmin Alkhlaif 1, Asti Jackson 2,3, Cenk O Gurdap 4, Elnaz Rahimpour 3, F Ivy Carroll 5, Roger L Papke 6, M Imad Damaj 1,7
PMCID: PMC9817006  NIHMSID: NIHMS1851381  PMID: 36321267

Abstract

Background:

Because of their implications in several pathological conditions, α4β2* nicotinic acetylcholine receptors (nAChRs) are potential targets for the treatment of nicotine dependence, pain, and many psychiatric and neurodegenerative diseases. However, they exist in various subtypes, and finding selective tools to investigate them has proved challenging. The nicotinic receptor agonist, 5-iodo-A-85380, has helped in delineating the function of β2-containing subtypes in vitro; however, much is still unknown about its behavioral effects. Furthermore, its effectiveness on α6-containing subtypes are limited.

Aims:

To investigate the effects of 5-iodo-A-85380 on nociception (formalin, hot-plate, and tail-flick tests), locomotion, hypothermia, and conditioned reward after acute and repeated administration, and to examine the potential role of β2 and α6 nAChR subunits in these effects. Lastly, we describe its selectivity for expressed low-sensitivity and high-sensitivity α4β2 receptors.

Results:

5-iodo-A-85380 dose-dependently induced hypothermia, locomotion suppression, conditioned place preference, and antinociception (only in the formalin test but not in the hot plate or tail-flick tests). Furthermore, these effects were mediated by β2 and not α6 nicotinic subunits. Finally, we show that 5-iodo-A-85380 potently activates both stoichiometries of α4β2 nAChRs with different efficacies, being a full agonist on high-sensitivity α4(2)β2(3) nAChRs, and a partial agonist on low-sensitivity α4(3)β2(2) nAChRs and α6-containing subtypes as well.

Funding:

This research was supported by the National Institutes of Health grants GM57481 to RLP and DA005274 and DA032246 to MID. Lois Akinola was supported in part by the T32 DA007027/DA/NIDA and the NIH IMSD training grant R25GM090084. The authors declare no conflicts of interest.

Keywords: Nicotinic acetylcholine receptors, 5-iodo-A-85380, α4β2 nAChRs, pharmacology, behavior

Introduction

Nicotinic acetylcholine receptors (nAChRs) are a family of excitatory ligand-gated cation channels that mediate fast synaptic transmission of nerve impulses. They are ubiquitously expressed in the central (CNS) and peripheral nervous systems (PNS) as well as in neuromuscular junctions and adrenal glands (Mukhin et al., 2000). These channels are pentameric in nature, composed of five protein subunits that assemble to form a NA+, K+, and Ca2+ permeable pore (Holladay et al., 1997; Mukhin et al., 2000). To date, several nAChRs subunits have been identified, including α2 - α10 and β2 - β4; some of which assemble to form homomeric pentamers (α7, α8, α9), while others assemble to form heteromeric pentamers with various combinations of α and β subunits (Moroni et al., 2006; Zwart et al., 2006). Subunit diversity yields different nAChR subtypes, each having unique biophysical properties, including permeability to calcium and ligand sensitivity (Hernández-Vivanco et al., 2014; Jones et al., 1999; Papke, 1993). Studies show that the most prevalent nAChR subtypes in the CNS contain β2 and α4 subunits, which can heterodimerize to form various subtypes, including α4β2, α4α6β2, and α4(2)α5β2(2) nAChRs (*denotes that these nAChRs can contain other α and β subunits) (Flores et al., 1992; Gotti et al., 2009; Hamouda et al., 2021; Liu et al., 2003; Moroni et al., 2006). α4β2* nAChRs have been implicated in a variety of diseases as possible targets for the treatment of nicotine dependence, pain, as well as many cognitive and neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and Tourette’s syndrome (McEvoy & Allen, 2002; Okada et al., 2013; Quik & Wonnacott, 2011); however, the heterogeneity displayed by this receptor system has made it challenging to investigate specific subunits in isolation, and finding selective tools to investigate these subtypes has proved difficult (Alkondon & Albuquerque, 2004; Fowler et al., 2008; Improgo et al., 2010; Levin, 2002; Pimlott et al., 2004). α4β2 nAChRs exist in two distinct functional receptor isoforms in the brain; a low sensitivity (LS) α4(3)β2(2) and a high sensitivity (HS) form α4(2)β2(3). Current investigative tools have exhibited shortcomings, including high rates of nonspecific binding, poor subtype selectivity, and intrinsic toxicity (Mukhin et al., 2000; Nybäck et al., 1994).

5-Iodo-A-85380 (5IA) was initially reported to be a β2-selective agonist and has proven to be of great value in delineating the functions of neuronal β2-containing nAChR subtypes (Koren et al., 1998; Vaupel et al., 1998). 5IA is a radio-iodinated analog of the 3-pyridyl ether A-85380 compound; and unlike other ligands, it has been demonstrated to possess high potency, selectivity, and specificity for α4β2* nAChRs (Kulak et al., 2002; Mamede et al., 2004; Ogawa et al., 2009; Rueter et al., 2006; Ueda et al., 2011). Although previously thought to only be selective for α4β2* subtypes, evidence now demonstrates that 5IA also binds to α-conotoxin MII-sensitive nicotinic receptors, suggesting that it binds to non-α4β2* receptors as well, including putative α6* nAChRs (Kulak et al., 2002). However, the contributions of β2 and α6 subunits to the pharmacological and behavioral effects of 5IA are not well known. In addition, while earlier reports suggest that 5IA may differentiate between the two α4β2 nicotinic acetylcholine receptor stoichiometries in a manner similar to acetylcholine (Zwart et al., 2006), the functional selectivity of 5IA to low and high sensitivity α4β2 isoforms are not reported. The present study thus aimed to characterize the behavioral effects of 5IA and investigate the specific role of β2 and α6 nAChR subunits in its behavioral effects. For that, we assessed the impact of 5IA on nociception, locomotion, hypothermia, and conditioned reward using β2 and α6 wild-type (WT) and knock-out (KO) mice. Then, we further described 5IA’s selectivity for expressed LS and HS α4β2 and α6-containing subtypes in vitro by expressing these receptors in Xenopus oocytes.

Materials and methods

Animals

Adult (8–10 weeks of age at the beginning of experiments) male and female C57BL/6J (The Jackson Laboratory; JAX - Bar Harbor, ME USA), as well as adult male and female ICR mice (8 weeks old upon arrival; Harlan Laboratories, Indianapolis, IN) were used in the study. Mice were randomly chosen and assigned to treatment groups and groups were blinded to the experimenters. Initially, ICR mice were used in the acute thermal pain tests (hot plate & tail flick tests), then later studies were conducted using C57BL/6J mice due to the background of the KO mice. Male and female mice null (KO) for the β2 and α6 nicotinic subunits (Institut Pasteur, Paris, France) and their WT littermates were bred on a C57BL/6J background in an animal care facility at Virginia Commonwealth University. Mutant and wild-type animals were obtained by backcrossing heterozygous mice for at least 12 to 15 generations. This breeding scheme controlled for any irregularities that might occur with crossing solely mutant animals. Male and female animals were used in each experiment and groups were equally balanced such that each experiment contained mixed-sex cohorts. Mice were housed in a temperature- (21°C) and humidity-controlled Association for Assessment and Accreditation of Laboratory Animal Care-approved animal care facility at Virginia Commonwealth University on a 12-hour light/dark cycle (lights on at 7:00 AM). Mice were housed in groups of five with Teklad corn cob bedding (#7097, Envigo Teklad, Madison, WI, USA), and received free access to food (#7012, Teklad LM-485 mouse sterilized diet, Envigo, Madison, WI) and water. They also received enrichment consisting of shreddable 5 × 5cm nesting materials (Nestlets™, Avantor Inc., Radnor Township, PA) per cage made of sterilized pulped virgin cotton fiber, along with a red Safe Harbor Mouse Retreat™ hut (Bio-Serv, Flemington, NJ). All experiments were performed during the light cycle, and the study was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. All studies were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Drugs

5-Iodo-A-85380 2HCl was synthesized at Research Triangle Institute and generously provided by Dr. Ivy Carroll (RTI International, Research Triangle Park, North Carolina). 5IA was dissolved in physiologic saline (0.9% sodium chloride) and freshly prepared solutions were injected subcutaneously (s.c.) at a total volume of 1mL/100g body weight. 5IA doses are expressed as the salt forms. The range and pretreatment time of 5IA doses were chosen based on published literature (Anderson et al., 2015; Caldarone et al., 2011).

Behavioral measures.

1. Antinociceptive measures

a. Tail-flick test.

Antinociception was assessed by the tail-flick method of D’amour and Smith (1941) as modified by Dewey et al. (1970). A control response (2–4 sec) was determined for each mouse before treatment, then, a test latency was determined after drug administration. To minimize tissue damage, a maximum latency of 10 sec was imposed. Antinociceptive response was calculated as percent maximum possible effect (% MPE), where %MPE = ((test-control)/(10-control)) x 100. Six ICR mice per group were injected subcutaneously with 5IA (0.001, 0.01, 0.1, and 0.5 mg/kg) or vehicle (saline) and tested at different time points (5, 10,15, 30, 60, and 120 min) after injection. In a separate group, C57BL/6J mice (n=6/group) were injected subcutaneously with vehicle (saline) or 0.5 mg/kg 5IA and tested at different time points (5, 10,15, 30, 60, and 120 min) after injection.

b. Hot-plate test.

Antinociception was also assessed using the tail-flick method. This method is a modification of that described by Eddy and Leimbach (1953) and Atwell & Jacobson (1978). Mice were placed into a 10 cm wide glass cylinder on a hot plate (Thermojust Apparatus) maintained at 55.0 °C. Two control latencies at least ten min apart were determined for each mouse. The normal latency (reaction time) was six to ten seconds. Antinociceptive response was calculated as percent maximum possible effect (% MPE), where %MPE = ((test-control)/(40-control) x 100). The reaction time was scored when the animal jumped or licked its front or hind paws. Six ICR mice per group were injected subcutaneously with 5IA (0.001, 0.01, 0.1, and 0.5 mg/kg) or vehicle (saline) and tested at different times (5, 10,15, 30, 60, and 120 min) after injection. In a separate group, C57BL/6J mice (n=6/group) were injected subcutaneously with vehicle (saline) or 0.5 mg/kg 5IA and tested at different time points (5, 10,15, 30, 60, and 120 min) after injection.

c. Formalin test.

The formalin test was conducted in an open Plexiglas cage (29 × 19 × 13 cm each). Male and female C57BL/6J mice (8/group) were allowed to acclimate in the test cage for 15 min before injection. Then, each animal was injected with either 20 μL of 2.5% formalin or vehicle (saline) in the right hind paw intraplantarly (i.pl.). Mice were then observed from 0 to 5 min (phase I) and 20 to 45 min (phase II) post-injection. The amount of time spent licking the injected paw was then recorded with a digital stopwatch. In a separate cohort (8/group), a time course of the effects of 5IA was established in the formalin assay after injection of saline or 5IA (0.5 mg/kg, s.c.) and tested at different time points (5, 15, 30, and 60 min). For the dose-response, vehicle (saline), or 5IA (0.01, 0.1, and 0.5 mg/kg) was injected subcutaneously 15 min before formalin injection. In a different group (8/group), the effects of 5-IA (0.5 mg/kg, s.c.) on paw licking behavior in the formalin test were measured in β2 and α6 WT and KO mice. In all antinociceptive measures, mice were used in one experiment only and euthanized at the end of the experiment.

2. Evaluation of locomotor activity.

ICR mice (12/group) were placed into individual Omnitech photocell activity cages (28 × 16.5 cm) 15 min after acute s.c. administration of either 0.9% vehicle (saline) or 5IA (0.01, 0.1, 0.5 and 1 mg/kg). Interruptions of the photocell beams (two banks of eight cells each) were then recorded for the next 30 min. Data were expressed as the number of photocell interruptions. In a separate group (8/group), the effects of 5IA (0.5 mg/kg, s.c.) on locomotor activity were measured in β2 WT and KO mice. Lastly, we tested for possible locomotor sensitization responses to 5IA in a separate cohort of mice (12/group), by injecting mice with saline or 5IA (0.01, 0.1, 0.5, and 1 mg/kg, sc.) for five days followed by a two-day abstinence (no injection). Then, on day 8, mice received their respective challenge dose of 5IA or saline.

3. Evaluation of body temperature.

Rectal temperature was measured using a thermistor probe (inserted 24 mm) and digital thermometer (Yellow Springs Instrument Co., Yellow Springs, OH). Readings were taken just before and at different times after the s.c. injection of either vehicle (saline) or 5IA in ICR mice (8/group). The difference in rectal temperature before and after treatment was calculated for each mouse. In a separate group (8/group), the effects of 5IA (0.5 mg/kg, s.c.) on body temperature were measured in β2 WT and KO mice. The ambient temperature of the laboratory varied from 21–24°C from day to day.

4. Evaluation of acute reward.

As previously described, an unbiased CPP paradigm was performed (Kota et al., 2007). The CPP apparatus (Med Associates, St. Albans, VT, ENV3013) consisted of three chambers in a linear arrangement; a white, black, and neutral chamber, which differed in overall color and floor texture (wire mesh, grid rod, and smooth PVC) respectively. The white and black chambers (16.76 × 12.7 × 12.7 cm) are separated by a narrow gray (neutral) chamber (9.78 × 12.7 × 12.7 cm). Partitions could be removed to allow access from the gray chamber to the black and white chambers. On day 1, male and female C57BL/6J mice (10/group) were confined to the middle chamber for a 5-min habituation and then allowed to move freely between all three chambers for 15 min. Time spent in each chamber was recorded, and these data were used to populate groups of approximately equal bias in baseline chamber preference. Mice that displayed more than a 60% preference for any one of the chambers at baseline (before conditioning) were excluded from the study. In our studies, no animals displayed a significant preference (>60%) for any of the chambers at baseline. Twenty-minute conditioning sessions occurred twice a day (days 2 – 4). During conditioning sessions, mice were confined to one of the larger chambers. The vehicle groups received saline in one large chamber in the morning and saline in the other large chamber in the afternoon. The 5IA group received the drug (0.01, 0.05, 0.1, 0.5, or 1 mg/kg, s.c., n=10/treatment group) in one large chamber and saline in the other large chamber. Treatments were counterbalanced equally to ensure that some mice received the unconditioned stimulus in the morning while others received it in the afternoon and then alternated so that animals who received the unconditioned stimulus in the morning on one day would receive it in the afternoon the next day and vice versa. The 5IA-paired chamber was randomized among all groups. Sessions were 4 hours apart and were conducted by the same investigator. On test day (day 5), mice were allowed access to all chambers for 15 min in a drug-free state. The preference score was calculated by determining the difference between time spent in the drug-paired side during test day versus the time spent in the drug-paired side during the baseline day. In a separate cohort (8–10/group), 5IA-induced CPP (0.05 mg/kg, s.c.) was measured in β2 and α6 WT and KO mice.

5. Oocytes studies.

a. Expression in Xenopus oocytes.

The human nAChR clones and concatemers were obtained from Dr. J. Lindstrom (University of Pennsylvania, Philadelphia, PA). Subsequent to linearization and purification of the plasmid DNAs, RNAs were prepared using the mMessage mMachine™ in vitro RNA transcription kit (Ambion, Austin, TX). High sensitivity forms of α4β2 receptor with the subunit stoichiometry α4(2)β2(3) were formed by co-expressing the β2–6-α4 concatemer (Zhou et al., 2003) with monomeric β2. Low sensitivity forms of α4β2 receptor with the subunit stoichiometry α4(3)β2(2) were formed by co-expressing the β2–6-α4 concatemer (Zhou et al., 2003) with monomeric α4. Receptors containing α6 and β3 receptors in combination with α4 and β2 were formed by expressing the five-subunit β3α4β2α6β2 concatemer (Kuryatov & Lindstrom, 2011). Oocytes were surgically removed from mature female Xenopus laevis frogs (Nasco, Ft. Atkinson, WI) as previously described (Pismataro et al., 2020). Frogs were maintained in the Animal Care Service facility of the University of Florida, and all procedures were approved by the University of Florida Institutional Animal Care and Use Committee.

b. Two-electrode voltage clamp electrophysiology.

Experiments were conducted using OpusXpress 6000A (Molecular Devices, Union City, CA) (Papke & Stokes, 2010). Both the voltage and current electrodes were filled with 3 M KCl. Oocytes were voltage-clamped at −60 mV at room temperature (24°C). The oocytes were bath-perfused with Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, and 1 μM atropine, pH 7.2) at 2 ml/min. To evaluate the experimental compounds, responses were compared to control ACh-evoked responses, defined as the average of two initial applications of 60 μM ACh made to the same cells before test applications. Multi-cell averages were calculated for comparisons of complex responses. Means of the normalized data were calculated for each of the 10,322 points in each of the 206.44 s traces (acquired at 50 Hz), as well as the standard errors for those averages. Solutions were applied from 96-well plates via disposable tips. Following a 30 s period to define baseline holding currents, drug applications were 12 s in duration and followed by 181 s washout periods. The responses were calculated as both peak current amplitudes and net charge, as previously described (Papke & Papke, 2002). Data were collected at 50 Hz, filtered at 20 Hz, and analyzed by Clampfit (Molecular Devices) and Excel (Microsoft, Redmond, WA). Data were expressed as means ± SEM from at least four oocytes for each experiment and plotted with Kaleidagraph 4.5.2 (Abelbeck Software, Reading, PA). In order to show actual raw data traces, means of the normalized data were calculated for each of the 10,322 points in each of the 206.44 s traces (acquired at 50 Hz), as well as the standard errors for those averages.

Statistical analysis.

All behavioral data were analyzed using the GraphPad Prism software, version 9.3.1 (GraphPad Software, Inc., La Jolla, CA) and expressed as the mean ± S.E.M. The data were first assessed for normality of residuals and equal variance. Normality was assessed using the Shapiro-Wilk test and variances were assessed using either the F-test or the Brown–Forsythe test and Bartlett’s test. All data passed the normality test. For data that did not pass the sphericity test, a Geisser-Greenhouse correction was used. Statistical analysis was thus conducted using either a repeated measure (RM) or non-RM parametric analysis of variance (ANOVA) test followed by a Dunnett’s or Sidak post hoc test where appropriate. A value of p <0.05 was used to constitute a minimum level of significance. All time course data were analyzed using a RM two-way ANOVA except the formalin data, which was assessed using a non-RM two-way ANOVA, all dose-response data were assessed using a one-way ANOVA, except the formalin data which as assessed using a non-RM two-way ANOVA, and all KO studies used a non-RM two-way ANOVA. Comparison of results of oocyte studies were made using a one-way ANOVA or using t-tests between the pairs of experimental measurements. In cases where multiple comparisons were made, a Bonferroni correction for multiple comparisons (Aickin & Gensler, 1996) was applied to correct for possible false positives. A value of p <0.05 was used to constitute a minimum level of significance. The statistics were calculated using an Excel template provided in Microsoft Office or ANOVA protocols in Kaleidagraph (4.5.2 Abelbeck Software, Reading, PA).

Results

Antinociceptive effects of 5IA in acute and tonic nociceptive tests

Tail-flick and hot plate tests

Initially, the effects of acute 5IA administration on acute thermal nociception in ICR mice was tested. Six animals per group were injected s.c. with vehicle or 5IA (0.001, 0.01, 0.1 and 0.5 mg/kg) and tested at different time points (5, 10,15, 30, 60 and 120 min) in each test. Results are presented in Table 1. A RM two-way ANOVA revealed no significant effect of 5IA on acute thermal antinociception in the hot plate test but revealed a main effect of dose in the tail-flick test (Fdose (4,25) = 3.102, p=0.0334). However, there were no significant effects of dose at any of the observed time points according to a Dunnett’s post hoc test, and no overall interaction of time and dose factors were observed. We then evaluated the same effects in C57BL/6J mice with the highest dose used on ICR mice, 0.5 mg/kg 5IA. Six animals per group were injected s.c. with either vehicle or 5IA (0.5 mg/kg) and tested at different time points (5, 10,15, 30, 60, and 120 min) in each test. A RM two-way ANOVA revealed no significant effect of 5IA on acute thermal antinociception in the hot plate or tail-flick test and no interaction of time and dose factors were observed in either test (Table 1). Since there were no observed differences in 5IA’s effects in both thermal pain tests in ICR mice compared to C57BL/6J mice, we decided to test 5IA’s effects only in C57BL/6J mice moving forward.

Table 1.

Summary of 5IA’s effects in ICR mice in the tail-flick and hot plate tests after s.c. administration at various doses and multiple time points. Antinociceptive response was calculated as the percent maximum possible effect (% MPE). Data are expressed as mean ± SEM of % MPE of six ICR mice per group.

Tail-Flick Test
Time (min) 5 10 15 30 60 120
Vehicle 1 ± 1 1 ± 1 1 ± 1 0 ± 0 2 ± 2 2 ± 1
0.001 2 ± 2 3 ± 1 3 ± 2 0 ± 0 1 ± 1 4 ± 2
0.01 1 ± 1 2 ± 1 2 ± 1 2 ± 2 1 ± 1 1 ± 1
0.1 0 ± 0 3 ± 1 3 ± 1 6 ± 4 3 ± 2 5 ± 2
0.5 10 ± 4 4 ± 3 11 ± 4 7 ± 4 6 ± 3 7 ± 3
Hot-Plate Test
Time 5 10 15 30 60 120
Vehicle 10 ± 3 12 ± 4 12 ± 4 15 ± 4 14 ± 5 17 ± 6
0.001 8 ± 3 10 ± 3 10 ± 5 4 ± 3 8 ± 5 11 ± 5
0.01 9 ± 3 7 ± 3 7 ± 3 5 ± 3 13 ± 6 5 ± 3
0.1 7 ± 2 6 ± 3 3 ± 2 0 ± 0 8 ± 4 14 ± 6
0.5 17 ± 5 11 ± 8 18 ± 9 10 ± 6 11 ± 7 11 ± 4
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Formalin Test

In the formalin test, we evaluated the antinociceptive effects of 5IA after intraplantar injection of 2.5% formalin in C57BL/6J mice (8/group). We first determined a time-course of its effects. Mice were administered either vehicle (saline) or 5IA (0.5 mg/kg, s.c.) at various pretreatment time points in a between subject manner and evaluated for paw licking responses after intraplantar injection of formalin. An ordinary two-way ANOVA revealed significant effects of 5IA treatment and pretreatment time in both phase I (Ftreatment (1,56) = 201.6, p<0.0001; Fpretreatment (3,56) = 40.52, p<0.0001; Finteraction (3,56) = 6.616, p=0.0007) and phase II (Ftreatment (1,56) = 24.87, p<0.0001; Fpretreatment (3,56) = 5.931, p=0.0014; Finteraction (3,56) = 7.738, p=0.0002) (Figure 1(a) and (b)). Sidak post hoc analyses show significant effects of 5IA treatment at 5, 15, 30, and 60 min in phase I, while the effects were less pronounced in phase II with significant differences observed at only 5 and 15 minutes.

Figure 1. Antinociceptive effects of 5IA in the formalin test.

Figure 1.

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Paw licking responses after intraplantar injection of vehicle (saline) or 2.5% formalin into the right hind paw of C57BL/6J mice were measured. Mice were observed from 0 to 5 min (phase I) and 20 to 45 min (phase II) post-injection. (a) Shows the dose-responsive antinociceptive effects of 5IA (0.01, 0.1 and 0.5 mg/kg) in the formalin test. (b), (c) Time course of 5IA (0.5 mg/kg s.c.) as a function of pretreatment time in phase I or phase II. Data are expressed as the amount of time spent licking the injected paw and presented as the mean ± SEM of 8 mixed sex cohorts of C57BL/6J mice per group. *p < 0.05 vs vehicle control within groups. 5IA = 5-I-A-8538.

A dose-response curve was then determined in C57BL/6J mice (8/group) using a pretreatment time of 15 min based on the results of the time course. Various doses of 5IA (0.01, 0.1 and 0.5 mg/kg) were tested in the formalin test after subcutaneous injection of 2.5% formalin. 5IA significantly induced antinociception in a dose-related fashion (Ftreatment (3, 56) = 29.39, p<0.0001) (Figure 1(c)). A Dunnett’s post hoc analysis following an ordinary two-way ANOVA shows significant attenuation of paw licking responses at 0.1 and 0.5 mg/kg compared to the vehicle group. Although the effects of 5IA treatment were congruent in both phases of the formalin test, paw licking latencies were significantly pronounced in phase II compared to phase I of the formalin test (Fphase (1, 56) = 24.90, p<0.0001). A RM two-way ANOVA showed significant differences in paw licking latency between the saline- and 0.01 mg/kg 5IA-treated groups but not in the 0.1 or 0.5 mg/kg groups in phase II compared to phase I of the formalin test. Also, a significant interaction of treatment and phase factors was observed (Finteraction (3, 56) = 4.266, p=0.0088).

5IA-induced antinociception in the formalin test is mediated by the β2 nicotinic subunit

To investigate the role of α6 and β2 nicotinic receptors in 5IA-induced antinociception in the formalin test, vehicle (saline) or 5IA (0.5 mg/kg, s.c.) was administered to male and female α6 and β2 WT and knockout (KO) mice (8/group) 15 minutes before intraplantar injection of 2.5% formalin. An ordinary two-way ANOVA revealed significant effects of 5IA treatment on paw licking responses in phase I (Ftreatment (1,28) = 131.4, p<0.0001; Figure 2(c)) and phase II (Ftreatment (1,28) = 64.48, p<0.0001; Figure 2(d)) in both α6 WT and KO animals. However, no significant effects of genotype were observed between WT and KO animals. While 5IA completely blocked the paw licking behavior in both α6 WT and KO mice in phase I of formalin test, it only partially inhibited it in phase II compared to vehicle treated mice. However, responses against 5IA in α6 WT and KO mice did not differ (Figure 2(c) and (d)). Conversely, while an ordinary two-way ANOVA revealed significant effects of 5IA treatment on paw licking responses in phase I (Ftreatment (1,28) = 8.514, p=0.0008; Figure 2(a)) and phase II (Ftreatment (1,28) = 11.95, p=0.0018; Figure 2(b)) in β2 WT animals, it revealed an effect of genotype in both phase I (Ftreatment (1,28) = 14.07, p=0.0008) and phase II (Ftreatment (1,28) = 7.527, p=0.0105), such that, β2 KO animals treated with 5IA showed similar paw licking responses compared to their KO vehicle controls. 5IA treatment significantly attenuated paw licking time in phase I (Figure 2(a)) and partially decreased paw licking time in phase II (Figure 2(b)) compared to vehicle treatment in β2 WT mice. Hence, the antinociceptive effects of 5IA are diminished in β2 KO animals suggesting an important role for this nicotinic subunit in the mediation of 5IA-induced antinociception in the formalin test.

Figure 2. 5IA-induced antinociception in the formalin test is mediated by the β2 nicotinic subunit in mice.

Figure 2.

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Paw licking responses after intraplantar injection of vehicle or 2.5% formalin into the right paw of β2 or α6 WT and KO mice were measured. Mice were observed from 0 to 5 min (phase I) and 20 to 45 min (phase II) post-injection. Effect of 5IA (0.5 mg/kg s.c.) on nociception in (a), (b) β2 KO and WT mice and (c), (d) α6 KO and WT mice in the formalin test. Data are expressed as the amount of time spent licking the injected paw and presented as the mean ± SEM of 8 mixed sex cohorts of C57BL/6J mice per group. *p < 0.05 vs vehicle control within groups. #p < 0.05 vs WT control. 5IA = 5-I-A-85380, WT = wild-type, KO = knock-out.

Impact of 5IA on locomotor activity in mice after acute and repeated injection

Locomotor activity was measured using Omnitech photocell activity cages. First, we assessed the dose-responsive effects of 5IA on locomotion in C57BL/6J mice (12/group). Mice were administered either vehicle (saline) or 5IA (0.01, 0.1, 0.5, and 1 mg/kg, s.c.) with a 15-minute pretreatment time, then, interruptions of the photocell beams were recorded 30 min following the pretreatment period. Overall, 5IA produced dose-dependent depression of spontaneous activity in mice. An ordinary one-way ANOVA revealed significant effects of dose on locomotion (F (4,55) = 22.23, p<0.0001; Figure 3(a)). A Dunnett’s post hoc test showed significant attenuation of locomotion only at 0.5 and 1 mg/kg compared to the vehicle group. Since the α6 subunit did not seem to mediate 5IA’s effects in the formalin test, we tested only the effects of β2 moving forward. In β2 WT and KO mice only, we then measured the effects of 5IA (0.5 mg/kg, s.c.) on locomotor activity (8/group). An ordinary two-way ANOVA revealed significant effects of treatment, genotype, and their interaction on locomotion (Ftreatment (1,28) = 10.68, p=0.0029; Fgenotype (1,28) = 14.25, p=0.0008; Finteraction (1,28) = 42.01, p<0.0001; Figure 3(b)). According to a Sidak post hoc analysis, 5IA produced significant attenuation of locomotion compared to the saline-treated group. However, this effect was only observed in β2 WT mice and not in β2 KO mice; thus, suggesting a role for β2 nicotinic subunit in the mediation of 5IA-induced attenuation of locomotor activity.

Figure 3. Effects of 5IA on locomotor activity after acute and repeated administration.

Figure 3.

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C57BL/6J mice were administered drug (s.c.) with a pretreatment time of 15 min; then, interruptions of the photocell beams were recorded 30 min following the pretreatment period. (a) Dose-response of the effects of 5IA (0, 0.01, 0.1, 0.5 and 1 mg/kg) on locomotion (b) β2 mediation of 5IA-induced (0.5 mg/kg s.c.) locomotion suppression (c) Locomotor sensitization to effects of 5-IA after five days repeated injections. Data are expressed as the number of photocell interruptions and are presented as the mean ± SEM of 8 (fig 3b) and 12 (figs 3a and 3c) mixed-sex cohorts of C57BL/6J mice per group. *p < 0.05 vs vehicle control within groups. #p < 0.05 vs WT control. 5IA = 5-I-A-85380, veh = vehicle, WT = wild-type, KO = knock-out.

Lastly, we investigated possible locomotor sensitization in response to repeated 5IA treatment by injecting C57BL/6J mice (12/group) with vehicle (saline) or 5IA (0.01, 0.1, 0.5, and 1 mg/kg, sc.) for 5 days. Mice were then challenged with respective doses of 5IA or vehicle two days after final injection on day 8. A RM two-way ANOVA revealed significant effects of time (Ftime (3.974, 218.5) = 12.16, p<0.0001), dose (Fdose (4, 55) = 17.29, P=0.0001) and an interaction of both factors (Ftimexdose (20, 275) = 2.583, p=0.0003) (Figure 3(c)). Dunnett’s post hoc test further revealed dichotomous effects of 5IA on locomotion over time. At low doses (0.01 mg/kg), 5IA produced stimulant effects on locomotion at days 3 and 5 but produced depressive effects at higher doses (0.5 and 1 mg/kg) compared to vehicle control on days 1 and 2. Tolerance to the depressant effects of higher doses of 5IA developed progressively after day 2 of repeated injection of the drug. Furthermore, no sensitized locomotor responses were observed at challenge day 8 to 5IA at any of the doses tested.

Effects of 5IA on body temperature in mice

We then tested the effect of 5IA on the body temperature after s.c. injection in C57BL/6J mice (8/group). We determined a time-course of the effects of 5IA at a dose of 0.5 mg/kg. Mice were administered vehicle (saline) or 5IA at various pretreatment time points and then evaluated for changes in body temperature. A RM Two-way ANOVA revealed that 5IA-treated animals had significant reductions in body temperature in comparison to their vehicle controls at 15-, 30- and 60-min post drug administration (Ftreatment (1, 14) = 31.05, p<0.0001; Ftime (1.476, 20.67) = 2.222, p=0.1437; Finteraction (3, 42) = 31.05, p=0.0637; Figure 4(a)). In a separate cohort (8/group), a dose-response curve was determined 15 min after s.c. injection of various doses of 5IA. According to a one-way ANOVA, 5IA induced significant hypothermia at 0.1 and 0.5 mg/kg in comparison to the vehicle counterparts (F (3, 28) = 9.321, p=0.0002; Figure 4(b)). In addition, a two-way ANOVA followed by Sidak post hoc test showed that the hypothermic effect of 5IA was significantly abolished in β2 KO mice compared to 5IA treated β2 WT (Ftreatment (1, 28) = 9.328, P=0.0049; Fgenotype (1, 28) = 10.03, p=0.0037; Finteraction (1, 28) = 10.76, p=0.0028; Figure 4(c)). These results suggested that 5IA-induced hypothermia is mediated by β2 nicotinic subunit.

Figure 4. The effects of β2-selective nicotinic receptor agonist 5IA on body temperature.

Figure 4.

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(a) Time course of 5IA-induced hypothermia (0.5 mg/kg s.c.). 5IA-treated mice exhibited significant attenuation in body temperature in comparison to their vehicle controls at 15, 30, and 60 min after administration of the drug. (b) Dose-response curve of 5IA (0.01, 0.1, 0.5 mg/kg) determined 15 min after s.c. injection of the drug. 5IA elicited significant hypothermia at 0.1 and 0.5 mg/kg in comparison to vehicle controls. (c) The hypothermic effect of 5IA (0.5 mg/kg, s.c.) was significantly reduced in β2 KO mice compared to WT animals. *p < 0.05 vs. vehicle control groups. Each point represents the mean ± SEM of 8 mixed sex cohorts of C57BL/6J mice per group. *p < 0.05 vs vehicle control within groups. #p < 0.05 vs WT control. 5IA = 5-I-A-85380, veh = vehicle, WT = wild-type, KO = knock-out.

Development of preference to 5IA in mice

To determine if 5IA elicits intrinsic rewarding properties, CPP was conducted using a range of doses (0.01, 0.05, 0.1, 0.5 or 1 mg/kg, s.c.) in C57BL/6J mice (10/group). One-way ANOVA followed by Dunnett’s multiple comparisons test revealed significant 5IA induced-CPP at 0.05 and 0.1 mg/kg, but not at 0.01, 0.5, and 1 mg/kg (F (5,54) = 4.610, p=0.0014) (Figure 5). Furthermore, to investigate the role of β2 and α6 nicotinic subunits in the observed 5IA-induced CPP, we administered the drug in β2 and α6 KO mice (8–10/group). An ordinary two-way ANOVA accompanied by Sidak post hoc test demonstrated that β2 WT mice treated with 5IA (0.05 mg/kg) showed significant CPP in comparison to their vehicle counterparts (Ftreatment (1, 28) = 4.934, p=0.0346), but the effect of 5IA was significantly abolished in β2 KO mice (Fgenotype (1, 28) = 15.48; p=0.0005). In addition, the interaction between 5IA and genotype was significant between the same subjects (Finteraction (1, 28) = 14.47; p=0.0007; Figure 6(a)). However, there was no significant difference in 5IA-induced CPP between α6 WT and KO mice (Fgenotype (1, 36) = 0.004555; p=0.9466; Figure 6(b) although α6 mice treated with 5IA showed significant CPP in comparison to their vehicle counterparts (Ftreatment (1, 36) = 39.42, p< 0.0001). These results suggest that 5IA induces CPP on its own and that this preference is mediated via β2 nicotinic subunits and not α6.

Figure 5. Dose-response curve of the β2-selective nicotinic receptor agonist, 5IA, in the CPP test.

Figure 5.

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To evaluate the intrinsic rewarding properties of 5IA, C57BL/6J mice were conditioned with either vehicle or 5IA at different doses (0.01, 0.05, 0.1, 0.5, or 1 mg/kg, s.c.) in the CPP paradigm. 5IA significantly induces CPP on its own and in a dose-related fashion. Data are expressed as preference score (sec) calculated by determining the difference between time spent in the drug-paired side during test day versus the time spent in the drug-paired side during the baseline day. Each bar represents the mean ± SEM of 10 mixed sex cohorts of C57BL/6J mice per group. *p < 0.05 vs vehicle control group. 5IA = 5-I-A-85380.

Figure 6. The role of β2 and α6 nicotinic subunits in the CPP enhancement post 5IA administration.

Figure 6.

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β2 and α6 KO and WT mice were injected with either 5IA (0.05 mg/kg s.c.) or vehicle for three conditioning days in the CPP test. (a) β2 WT mice exhibited significant CPP in comparison to their saline counterparts, however, this effect was not observed in β2 KO mice. The 5IA-treated β2 KO mice showed significant attenuation of CPP compared to their WT. (b) 5IA treated α6 WT, and KO mice displayed significant CPP compared to their vehicle controls. There was no effect of genotype in α6 mice. *p < 0.05 vs. Saline groups, #p < 0.05 vs. WT mice. Each point represents the mean ± SEM of n=8 mice per group. 5-IA = 5-I-A-85380. WT = wild-type, KO = knock-out.

Oocyte expression experiments

Concentration-response studies were conducted on oocytes expressing the two forms of α4β2 receptors and α6-containing receptors. Data were initially measured relative to the acetylcholine (ACh) controls which were 10 μM, 100 μM, or 30 μM, for α4(2)β2(3), α4(3)β2(2), and α4β2α6β2β3, respectively, and then adjusted by the ratio between the ACh control responses and the ACh evoked maximum responses determined in other experiments (Papke et al., 2013). 5-I-A-85380 was a potent drug for all the receptors tested (Table 2) evoking responses at submicromolar concentration. Interestingly, it was much more efficacious for activating the receptor subtype with high sensitivity α4(2)β2(3) for nicotine and ACh than for the low sensitivity α4(3)β2(2) form (Figure 7). The efficacy for the α6-containing receptors was intermediate between that for the HS and LS α4β2 receptors (Table 2, Figure 7). Interestingly, an additional effect of 5IA on the HS α4β2 receptors was that it prolonged the currents (Figures 7 and 8) so the Imax value for the net charge was much greater than for peak currents (Table 2).

Table 2.

Oocyte concentration-response data.

Receptor Peak Currents Net Charge
Imax EC50 Imax EC50
α4(2)β2(3) 1.3 ± 0.02 42 ± 2 nM 3.0 ± 0.18 62 ± 11 nM
α4(3)β2(2) 0.22 ± 0.1 37 ± 6.3 nM 0.22 ± 0.04 25 ± 14 nM
β3α4β2α6β2 0.78 ± 0.1 11 ± 0.01 nM 0.76 ± 0.01 19 ± 0.4 nM
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Figure 7. Responses of LS α4β2, HS α4β2 and β3α4β2α6β2 nAChR receptors to 5IA.

Figure 7.

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The effects of 5IA on LS α4β2, HS α4β2, and β3α4β2α6β2 nAChR subtypes were assessed in oocytes expressing the different nicotinic subunits. The high sensitivity (HS) α4(2)β2(3) nAChR was generated by co-expressing the β2–6-α4 concatemer with monomeric β2 subunits. The low sensitivity form α4(3)β2(2) was formed by co-expressing the β2–6-α4 concatemer with monomeric α4. Receptors containing α6 and β3 receptors in combination with α4 and β2 were formed by expressing the five-subunit β3α4β2α6β2 concatemer. (a) Averaged traces of nAChR receptor activation (± SEM in tan) by acetylcholine 300 nM 5IA compared to ACh controls from the same cells (see methods). The 300 nM 5IA responses were normalized to the ACh controls obtained prior to the 5IA applications. The ACh controls were 100 μM, 10 μM and 30 μM ACh for the LS α4β2 (n = 6), HS α4β2 (n = 4) and β3α4β2α6β2 (n = 5) receptors, respectively. (b) The averaged ACh control response (±SEM) obtained prior to (black line with tan SEM range) and after (blue line with light blue SEM) the 5IA applications.

Figure 8. Concentration-response curves obtained from oocytes expressing human neuronal nAChR LS and HS α4β2 or α6/4β2β3 subunits to 5IA.

Figure 8.

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Oocytes expressing human neuronal LS α4β2, HS α4β2 (upper plot) or the β3α4β2α6β2 concatemer were tested for their responses to varying concentrations of applied 5IA. Responses to varying concentrations of applied 5IA were measured as peak currents and net charge. Each point shows the averaged data ± SEM, n ≥ 4. Responses were initially measured relative to ACh controls obtained in the same cells and normalized to the maximum ACh responses determined in separate experiments.

Discussion

This study investigated the effects of 5-iodo-A-85380 on multiple behavioral outcomes after acute and repeated administration, including its effects on nociception, locomotion, hypothermia, and conditioned reward, and examined the potential role of β2 and α6 nAChR subunits in these effects. Then, we further described its selectivity for expressed low-sensitivity and high-sensitivity α4β2 receptors. Key findings reveal that 5IA dose-dependently induces hypothermia, locomotion suppression, conditioned place preference, and antinociception only in the formalin test but not in acute thermal pain tests like the hot plate and tail-flick tests. We also report that β2 nicotinic subunits play an important role in these effects. In contrast, α6 nicotinic subunits did not mediate its antinociceptive effects in the formalin test or in the development of conditioned place preference in the CPP test. Finally, we show that 5IA potently activates both stoichiometries of α4β2 nAChRs with different efficacies, being a full agonist on HS α4(2)β2(3) nAChRs, whereas it had very low efficacy on LS α4(3)β2(2) nAChRs and on α6-containing subtypes as well.

The antinociceptive effects of 5IA in the tonic pain model of formalin-induced nociception were both time- and dose-dependent. This is the first study to report the analgesic-like properties of 5IA in animal models of pain after systemic administration, albeit previous studies have reported the antinociceptive effects of 5IA in rat neuropathic pain models after central administration (Ueda et al., 2010; Ueda et al., 2011). Furthermore, most of the animal pain studies reported are done with the 5IA precursor, A-85380, showing efficacy in multiple in vivo assays of antinociception, and anti-allodynic activity after systemic administration (Rueter et al., 2006). Indeed, 5IA is a derivative of A-85380 which has been iodinated at the 5-position of the pyridine ring (Vaupel et al., 1998). Surprisingly, and in contrast to A-85380 (Curzon et al., 1998), we failed to observe an antinociceptive effect of 5IA in two acute thermal pain tests (hot plate and tail flick tests) in ICR or C57BL/6J mice. While these results suggest that agonism of HS α4β2 receptors failed to induce antinociception in the acute thermal pain tests, it was sufficient to show activity in a more chronic pain test. In support, evidence has emerged that demonstrates that desensitization rather than activation of α4β2* nAChRs plays a more prominent role in the antinociceptive effects of nicotinic compounds (Zhang et al., 2012). In addition, recent evidence in animal models suggest that chronic pain leads to plasticity of nAChRs in various peripheral and central neuronal regions involved in pain transmission (Brunori et al., 2018; Jareczek et al., 2017; Vincler and Eisenach, 2004). Consequently changes in nAChRs signaling in the formalin test but not in acute thermal stimuli may explain the antinociceptive effect of 5IA observed in this model. Indeed, our results further showed that the effects of 5IA in the formalin test were mediated by the β2 and not the α6 nicotinic subunit, again, to our knowledge, this is the first study to report receptor mediation of 5IA’s effects in animal models of pain.

In addition to its antinociception properties, 5IA induced locomotion suppression, and hypothermia, and facilitated the development of place preference in the CPP test consistent with the profile of a nicotine-like response. Nicotine and several nicotine-like compounds have been known to induce significant hypothermia, induce CPP and alter locomotion (Clarke & Kumar, 1983; Domino, 2001; Grabus et al., 2006; Umezu et al., 2012). Further, in all three behavioral outcomes, we found the β2 nAChR subunit to play a modulatory role in the effects of 5IA. In the locomotion test, spontaneous activity was tested following administration of 5IA, which produced a dose-dependent depression of activity in mice. Interestingly, mice null for the β2 nicotinic subunit displayed protection against its locomotion suppressive effects compared to WT animals. When we investigated potential locomotor sensitization in response to repeated 5IA administration, at low doses, 5IA produced stimulant effects on locomotion on specific days (day 3 and 5) but produced depressive effects at higher doses on other days (day 1 and 2) compared to vehicle controls. These findings are consistent with the majority of previous studies showing a dichotomy to nicotine’s effects on locomotion in rodents in different conditions; demonstrating a reduction in activity after large doses of drug are given before brief test sessions, and increased activity which may or may not develop during prolonged test sessions (Bovet et al., 1967; Stolerman et al., 1974; Stolerman & Jarvik, 1973). When tested, repeated administration of 5IA did not elicit significant locomotor sensitization after challenge with drug on day 8. Generally, upon repeated administration of psychostimulants, animals display a progressively greater behavioral response. This is thought to be regulated by “experience-dependent neuronal plasticity” (Nestler, 2001). This has been shown in cocaine and methamphetamine administration in rodents (Kumar et al., 2013). Although nicotine is generally considered to be a psychostimulant, we have previously reported a lack of locomotor sensitization to nicotine at low and high doses in C57BL/6J mice (Akinola et al., 2019). Therefore, it would be rational to assume that nicotinic agonists would display the same behavioral profile in the locomotor test.

In the effects of 5IA on body temperature, we found that 5IA time- and dose-dependently induced hypothermia in mice. The hypothermic effects of 5IA were significantly greater at higher doses and appeared to be transient but persisted up to 120 minutes post-administration. Further, in mice null for the β2 nicotinic subunit, 5IA failed to produce significant hypothermia compared to their WT counterparts again suggesting a role of the β2 subunit in 5IA-induced hypothermia. 5IA also produced dose-responsive increases in CPP with significant effects at moderate doses (0.05 & 0.1 mg/kg) demonstrating some intrinsic rewarding properties. These properties also appear to be mediated by the β2 subunit as the development of CPP is attenuated in β2 KO mice compared to α6 KO mice. This is consistent with the relatively recent paper that showed that 5IA dose-dependently produced facilitated intracranial self-stimulation (ICSS) in rats, and this effect was blocked by the selective β2 antagonist dihydro-ß-erythroidine (DHßE) (Freitas et al., 2016).

We further described 5IA’s selectivity for expressed α4β2 (LS and HS) subtypes as well as α6-containing subtypes in vitro and demonstrate that 5IA potently activates all the receptors tested, evoking responses at submicromolar concentrations (Table 2). It was much more efficacious in activating the high sensitivity α4(2)β2(3) receptor subtype, being a full agonist, whereas it had very low efficacy on low sensitivity α4(3)β2(2) nAChRs and intermediate efficacy on α6-containing subtypes. Additionally, 5IA prolonged the currents on the HS α4β2 receptors so the Imax value for the net charge was much greater than for peak currents. This effect was not observed in the LS form or in α6-containing subtypes. Some of the functional differences have been proposed to relate to the presence of a putative low-affinity agonist binding site at the α4-α4 subunit interfaces present in LS but not HS receptors (Lucero et al., 2016). It has been shown that both HS and LS forms of α4β2 receptors exist in the brain (Grady et al., 2010), and the relative abundance of the two forms varies among brain areas (Fasoli et al., 2016). Under normal conditions, the cortex has been reported to have a relatively high basal expression of LS α4β2 receptors, while in the thalamus the expression of HS receptors is higher. Nicotine has been shown to increase the relative abundance of HS receptors in vitro (Srinivasan et al., 2011). It is well known that chronic nicotine increases the overall nAChR expression in the brain (Huang & Winzer-Serhan, 2006; Nashmi et al., 2007), and in the cortex chronic nicotine selectively increases the expression of HS α4β2 receptors (Fasoli et al., 2016). It should be noted that, while HS receptors respond more sensitively to low levels of nicotine or ACh, they also desensitize more readily than LS receptors and typically generate smaller currents (Kuryatov et al., 2008; Nelson et al., 2003). It is therefore interesting that some of these features appear to be overcome by 5IA, with the protracted HS receptor responses suggesting that 5IA is notably less desensitizing than is nicotine. While both α4β2 and α6-containing nAChR have been implicated in the rewarding effects of nicotine (Papke et al., 2020), our data with the KO mice would suggest that the enhanced HS α4β2 activity is the more important component of 5IA reward than the α6 partial agonist activity.

Acknowledgments

Oocyte recordings were conducted by Lu Wenchi Corrie.

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