M. Imad Damaj

Reduced antinociception in mice lacking neuronal nicotinic receptor subunits.

Nicotine exerts antinociceptive effects by interacting with one or more of the subtypes of nicotinic acetylcholine receptors (nAChRs) that are present throughout the neuronal pathways that respond to pain. To identify the particular subunits involved in this process, we generated mice lacking the α4 subunit of the neuronal nAChR by homologous recombination techniques and studied these together with previously generated mutant mice lacking the β2 nAChR subunit. Here we show that the homozygous α4−/− mice no longer express high-affinity [3H]nicotine and [3H]epibatidine binding sites throughout the brain. In addition, both types of mutant mice display a reduced antinociceptive effect of nicotine on the hot-plate test and diminished sensitivity to nicotine in the tail-flick test. Patch-clamp recordings further reveal that raphe magnus and thalamic neurons no longer respond to nicotine. The α4 nAChR subunit, possibly associated with the β2 nAChR subunit, is therefore crucial for nicotine-elicited antinociception.

Guidelines on nicotine dose selection for in vivo research

RationaleThis review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.ObjectivesThis review capitalizes on the authors’ collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.ResultsSeven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.ConclusionsThe selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose–response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Nicotine place preference in the mouse: influences of prior handling, dose and strain and attenuation by nicotinic receptor antagonists

RationaleAlthough conditioned place preferences (CPPs) are seen with most abused drugs, nicotine does not always produce a preference in this design.ObjectivesThe goals of the present experiment were to (1) examine various factors that could contribute to these inconsistent results and (2) begin to evaluate the specific nicotinic receptors involved in the nicotine CPP.MethodsThe influences of prior handling, environmental habituation, and injection habituation on a nicotine CPP were first evaluated in ICR mice. Subsequently, various nicotine doses were assessed for their abilities to produce a CPP, and the effectiveness of nicotinic receptor antagonists in attenuating this preference was examined. Finally, nicotine CPPs were assessed in C57BL/6J and DBA/2J mice to examine the influence of strain in this design.ResultsNicotine CPPs were seen in handled/environmentally habituated, but not in unhandled, ICR mice. Habituation to the injection techniques failed to strengthen the preference. In ICR mice, a CPP was seen with one intermediate dose of nicotine. This CPP was attenuated by mecamylamine and dihydro-β-erythroidine (DHβE). A nicotine CPP was also seen in C57BL/6J, but not in DBA/2J, mice.ConclusionEarlier handling experience and strain are important factors when evaluating a nicotine CPP in the mouse. In addition, certain nicotinic receptors underlie the nicotine CPP, indicating that this model can elucidate underlying mediators of nicotine reward.

The Endogenous Cannabinoid System Modulates Nicotine Reward and Dependence

A growing body of evidence suggests that the endogenous cannabinoid system modulates the addictive properties of nicotine, the main component of tobacco that produces rewarding effects. In our study, complementary transgenic and pharmacological approaches were used to test the hypothesis that the endocannabinoid system modulates nicotine reward and dependence. An acute injection of nicotine elicited normal analgesic and hypothermic effects in cannabinoid receptor (CB)1 knockout (KO) mice and mice treated with the CB1 antagonist rimonabant. However, disruption of CB1 receptor signaling blocked nicotine reward, as assessed in the conditioned place preference (CPP) paradigm. In contrast, genetic deletion, or pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for catabolism of the endocannabinoid anandamide, enhanced the expression of nicotine CPP. Although the expression of spontaneous nicotine withdrawal (14 days, 24 mg/kg/day nicotine) was unaffected in CB1 KO mice, acute administration of rimonabant (3 mg/kg) ameliorated somatic withdrawal signs in wild-type mice. Increasing endogenous levels of anandamide through genetic or pharmacological approaches exacerbated the physical somatic signs of spontaneous nicotine withdrawal in a milder withdrawal model (7 days, 24 mg/kg/day nicotine). Moreover, FAAH-compromised mice displayed increased conditioned place aversion in a mecamylamine-precipitated model of nicotine withdrawal. These findings indicate that endocannabinoids play a role in the rewarding properties of nicotine as well as nicotine dependence liability. Specifically, increasing endogenous cannabinoid levels magnifies, although disrupting CB1 receptor signaling, attenuates nicotine reward and withdrawal. Taken together, these results support the hypothesis that cannabinoid receptor antagonists may offer therapeutic advantages to treat tobacco dependence.

Synthesis and biological evaluation of bupropion analogues as potential pharmacotherapies for smoking cessation.

Bupropion (2a) analogues were synthesized and tested for their ability to inhibit monoamine uptake and to antagonize the effects of human alpha3beta4*, alpha4beta2, alpha4beta4, and alpha1* nAChRs. The analogues were evaluated for their ability to block nicotine-induced effects in four tests in mice. Nine analogues showed increased monoamine uptake inhibition. Similar to 2a, all but one analogue show inhibition of nAChR function selective for human alpha3beta4*-nAChR. Nine analogues have higher affinity at alpha3beta4*-nAChRs than 2a. Four analogues also had higher affinity for alpha4beta2 nAChR. Analogues 2r, 2m, and 2n with AD(50) values of 0.014, 0.015, and 0.028 mg/kg were 87, 81, and 43 times more potent than 2a in blocking nicotine-induced antinociception in the tail-flick test. Analogue 2x with IC(50) values of 31 and 180 nM for DA and NE, respectively, and with IC(50) of 0.62 and 9.8 microm for antagonism of alpha3beta4 and alpha4beta2 nAChRs had the best overall in vitro profile relative to 2a.

Synthesis and Biological Evaluation of Bivalent Ligands for the Cannabinoid 1 Receptor

Dimerization or oligomerization of many G-protein-coupled receptors (GPCRs), including the cannabinoid 1 (CB1) receptor, is now widely accepted and may have significant implications for medications development targeting these receptor complexes. A library of bivalent ligands composed of two identical CB1 antagonist pharmacophores derived from SR141716 linked by spacers of various lengths were developed. The affinities of these bivalent ligands at CB1 and CB2 receptors were determined using radiolabeled binding assays. Their functional activities were measured using GTP-γ-S accumulation and intracellular calcium mobilization assays. The results suggest that the nature of the linker and its length are crucial factors for optimum interactions of these ligands at CB1 receptor binding sites. Finally, selected bivalent ligands (5d and 7b) were able to attenuate the antinociceptive effects of the cannabinoid agonist CP55,940 (21) in a rodent tail-flick assay. These novel compounds may serve as probes that will enable further characterization of CB1 receptor dimerization and oligomerization and its functional significance and may prove useful in the development of new therapeutic approaches to G-protein-coupled receptor mediated disorders.

The CB2 cannabinoid receptor-selective agonist O-3223 reduces pain and inflammation without apparent cannabinoid behavioral effects

Although Δ(9)-tetrahydrocannabinol (THC) and other mixed CB(1)/CB(2) receptor agonists are well established to elicit antinociceptive effects, their psychomimetic actions and potential for abuse have dampened enthusiasm for their therapeutic development. Conversely, CB(2) receptor-selective agonists have been shown to reduce pain and inflammation, without eliciting apparent cannabinoid behavioral effects. In the present study, we developed a novel ethyl sulfonamide THC analog, O-3223, and compared its pharmacological effects to those of the potent, mixed CB(1)/CB(2) receptor agonist, CP55,940, in a battery of preclinical pain models. Competitive cannabinoid receptor binding experiments revealed that O-3223 was approximately 80-fold more selective for CB(2) than CB(1) receptors. Additionally, O-3223 behaved as a full CB(2) receptor agonist in [(35)S]GTPγS binding. O-3223 reduced nociceptive behavior in both phases of the formalin test, reduced thermal hyperalgesia in the chronic constriction injury of the sciatic nerve (CCI) model, and reduced edema and thermal hyperalgesia elicited by intraplantar injection of LPS. These effects were blocked by pretreatment with the CB(2) receptor-selective antagonist SR144528, but not by the CB(1) receptor antagonist, rimonabant. Unlike CP55,940, O-3223 did not elicit acute antinociceptive effects in the hot-plate test, hypothermia, or motor disturbances, as assessed in the rotarod test. These data indicate that the CB(2) receptor-selective agonist, O-3223, reduces inflammatory and neuropathic nociception, without affecting basal nociception or eliciting overt behavioral effects. Moreover, this compound can serve as a template to develop new CB(2) receptor agonists with increased receptor selectivity and increased potency in treating inflammatory and neuropathic pain.

Effects of alpha 7 positive allosteric modulators in murine inflammatory and chronic neuropathic pain models

Agonists and positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChRs) are currently being considered as novel therapeutic approaches for managing cognitive deficits in schizophrenia and Alzheimers disease. Though α7 agonists were recently found to possess antinociceptive and anti-inflammatory properties in rodent models of chronic neuropathic pain and inflammation, the effects of α7 nAChRs PAMs on chronic pain and inflammation remain largely unknown. The present study investigated whether PAMs, by increasing endogenous cholinergic tone, potentiate α7 nAChRs function to attenuate inflammatory and chronic neuropathic pain in mice. We tested two types of PAMS, type I (NS1738) and type II (PNU-120596) in carrageenan-induced inflammatory pain and chronic constriction injury (CCI) neuropathic pain models. We found that both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema caused by a hind paw infusion of carrageenan. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the CCI model, PNU-120596 had long-lasting (up to 6 h), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Systemic administration of the α7 nAChR antagonist MLA reversed PNU-120596s effects, suggesting the involvement of central and peripheral α7 nAChRs. Furthermore, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results indicate that the type II α7 nAChRs PAM PNU-120596, but not the type I α7 nAChRs PAM NS1738, shows significant anti-edematous and anti-allodynic effects in inflammatory and CCI pain models in mice.

Enhanced nicotine reward in adulthood after exposure to nicotine during early adolescence in mice

Approximately one million adolescents begin smoking cigarettes every year. Studies show that adolescents may be particularly vulnerable to various aspects of nicotine dependence. Work on rodents demonstrates parallel findings showing that adolescence is a time of changed sensitivity to both rewarding and aversive effects of nicotine. However, it is unclear if these effects are long-lasting and whether they contribute to a lifetime of nicotine addiction. In this study we have characterized the effects of adolescent nicotine exposure on the rewarding properties of nicotine in adulthood using the CPP model. Specifically, we have addressed whether the phase of adolescence (early, middle, or late adolescence) plays a role in the susceptibility to the enhanced rewarding effects of nicotine. Furthermore, we have investigated the long-term effects of adolescent nicotine exposure on nicotine reward in adulthood and have correlated these behavioral adaptations with possible molecular mechanisms. We observed that early adolescence in the mouse is a unique phase for elevated sensitivity to nicotine reward using a CPP model. In addition, exposure to nicotine during this phase, but not during late adolescence or adulthood, resulted in a lasting enhancement of reward in adulthood. Finally, we have shown that early adolescent nicotine exposure significantly elevates nAChR function in adulthood. Overall, we demonstrate that early adolescence represents a period of development, distinct from middle and late adolescence, during which nicotine exposure can cause persistent changes in behavior and molecular adaptations.

Alpha-7 nicotinic acetylcholine receptor agonists selectively activate limbic regions of the rat forebrain: An effect similar to antipsychotics

It is considered that activation of nicotinic α7 receptors (α7 nAChR) is useful for the treatment of cognitive disturbances in schizophrenia and Alzheimers disease. Recently, selective α7 nAChR agonists have been discovered and are used to validate the α7 nAChR as a drug target for the treatment of cognitive disturbances in schizophrenia. One important feature shared by all known antipsychotics is their capacity to induce expression of the neuronal immediate‐early gene c‐fos in the limbic forebrain. Using two novel and selective α7 nAChR agonists, PNU‐282987 and SSR180711, we investigated their ability to induce c‐Fos expression in the limbic forebrain with particular emphasis on the same regions reported to be activated by antipsychotics. Both α7 nAChR agonists increased c‐Fos dose‐dependently in the prefrontal cortex and the shell of nucleus accumbens, while leaving the core of nucleus accumbens and the dorsolateral striatum unaffected. The accumbal and cortical effect of SSR180711 was blocked completely by pre‐administration of the α7 nAChR antagonist methyllycaconitine. Also, SSR180711 displayed no c‐Fos‐inducing effect in α7 nAChR knock‐out mice. In conclusion, these results show that selective pharmacologic stimulation of α7 nAChR function results in activation of forebrain regions similar to conventional antipsychotics.