Nicotine use disorder and Neuregulin 3: Opportunities for precision medicine.

Nicotine use disorder remains a major public health emergency despite years of trumpeting the consequences of smoking. This is likely due to the complex interplay of genetics and nicotine exposure across the lifespan of these individuals. Genetics influence all aspects of life, including complex disorders such as nicotine use disorder. This review first highlights the critical neurocircuitry underlying nicotine dependence and withdrawal, and then describes the cellular signaling mechanisms involved. Finally, current genetic, genomic, and transcriptomic evidence for new drug development of smoking cessation aids is discussed, with a focus on the Neuregulin 3 Signaling Pathway.

The Role of Microglia in Sex- and Region-Specific Blood-Brain Barrier Integrity During Nicotine Withdrawal.

Background: Smoking is the largest preventable cause of death and disease in the United States, with <5% of quit attempts being successful. Microglia activation and proinflammatory neuroimmune signaling in reward neurocircuitry are implicated in nicotine withdrawal symptomology. Microglia are integral regulators of blood-brain barrier (BBB) functionality as well; however, whether the effects of nicotine withdrawal on microglia function impact BBB integrity is unknown. Methods: Mice were treated with chronic nicotine (12 mg/kg/day) and subjected to 48 hours nicotine withdrawal. Regional BBB permeability, together with messenger RNA and protein expression of tight junction proteins, were assessed. PLX5622 chow was used to deplete microglia to evaluate the role of microglia in regulating BBB integrity and nicotine withdrawal symptomology. Results: Female mice had higher baseline BBB permeability in the prefrontal cortex and hippocampus than males. Nicotine withdrawal further exacerbated the BBB permeability selectively in the prefrontal cortex of females. These effects were concurrent with prefrontal cortex alterations in a subset of tight junction proteins with increased proinflammatory responses following nicotine withdrawal in females. Depletion of microglia via PLX5622 treatment prevented all these molecular effects and attenuated withdrawal-induced anxiety-like behavior in female mice. Conclusions: These results are the first to show sex differences in regional BBB permeability during nicotine withdrawal. This represents a possible link to both the reduced smoking cessation success seen in women and women's increased risk for smoking-related neurovascular disorders. Furthermore, these findings open an avenue for sex-specific therapeutics that target microglia and BBB dysfunction during nicotine withdrawal in women.

Dynamic effects of ventral hippocampal NRG3/ERBB4 signaling on nicotine withdrawal-induced responses.

Tobacco smoking remains a leading cause of preventable death in the United States, with approximately a 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH) of male and female mice. We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

Dynamic Effects of Ventral Hippocampal NRG3/ERBB4 Signaling on Nicotine Withdrawal-Induced Responses.

Tobacco smoking remains a leading cause of preventable death in the United States, with a less than 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH). We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

Nicotine and opioid co-dependence: Findings from bench research to clinical trials.

Concomitant use of tobacco and opioids represents a growing public health concern. In fact, the mortality rate due to smoking-related illness approaches 50% among SUD patients. Cumulative evidence demonstrates that the vulnerability to drugs of abuse is influenced by behavioral, environmental, and genetic factors. This review explores the contribution of genetics and neural mechanisms influencing nicotine and opioid reward, respiration, and antinociception, emphasizing the interaction of cholinergic and opioid receptor systems. Despite the substantial evidence demonstrating nicotine-opioid interactions within the brain and on behavior, the currently available pharmacotherapies targeting these systems have shown limited efficacy for smoking cessation on opioid-maintained smokers. Thus, further studies designed to identify novel targets modulating both nicotinic and opioid receptor systems may lead to more efficacious approaches for co-morbid nicotine dependence and opioid use disorder.

Translational Research in Nicotine Addiction.

While commendable strides have been made in reducing smoking initiation and improving smoking cessation rates, current available smoking cessation treatment options are still only mildly efficacious and show substantial interindividual variability in their therapeutic responses. Therefore, the primary goal of preclinical research has been to further the understanding of the neural substrates and genetic influences involved in nicotine's effects and reassess potential drug targets. Pronounced advances have been made by investing in new translational approaches and placing more emphasis on bridging the gap between human and rodent models of dependence. Functional neuroimaging studies have identified key brain structures involved with nicotine-dependence phenotypes such as craving, impulsivity, withdrawal symptoms, and smoking cessation outcomes. Following up with these findings, rodent-modeling techniques have made it possible to dissect the neural circuits involved in these motivated behaviors and ascertain mechanisms underlying nicotine's interactive effects on brain structure and function. Likewise, translational studies investigating single-nucleotide polymorphisms (SNPs) within the cholinergic, dopaminergic, and opioid systems have found high levels of involvement of these neurotransmitter systems in regulating the reinforcing aspects of nicotine in both humans and mouse models. These findings and coordinated efforts between human and rodent studies pave the way for future work determining gene by drug interactions and tailoring treatment options to each individual smoker.

Glial cells as therapeutic targets for smoking cessation.

Smoking remains the leading cause of morbidity and mortality in the United States, with less than 5% of smokers attempting to quit succeeding. This low smoking cessation success rate is thought to be due to the long-term adaptations and alterations in synaptic plasticity that occur following chronic nicotine exposure and withdrawal. Glial cells have recently emerged as active players in the development of dependence phenotypes due to their roles in modulating neuronal functions and synaptic plasticity. Fundamental studies have demonstrated that microglia and astrocytes are crucial for synapse formation and elimination in the developing brain, likely contributing to why glial dysfunction is implicated in numerous neurological and psychiatric disorders. Recently, there is increasing evidence for the involvement of glial cells in drug dependence and its associated behavioral manifestations. This review summarizes the newly evaluated role of microglia and astrocytes as molecular drivers of nicotine dependence and withdrawal phenotypes. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology.

Molecular Mechanisms Associated with Nicotine Pharmacology and Dependence.

Tobacco dependence is a leading cause of preventable disease and death worldwide. Nicotine, the main psychoactive component in tobacco cigarettes, has also been garnering increased popularity in its vaporized form, as derived from e-cigarette devices. Thus, an understanding of the molecular mechanisms underlying nicotine pharmacology and dependence is required to ascertain novel approaches to treat drug dependence. In this chapter, we review the field's current understanding of nicotine's actions in the brain, the neurocircuitry underlying drug dependence, factors that modulate the function of nicotinic acetylcholine receptors, and the role of specific genes in mitigating the vulnerability to develop nicotine dependence. In addition to nicotine's direct actions in the brain, other constituents in nicotine and tobacco products have also been found to alter drug use, and thus, evidence is provided to highlight this issue. Finally, currently available pharmacotherapeutic strategies are discussed, along with an outlook for future therapeutic directions to achieve to the goal of long-term nicotine cessation.

Inhibition of the Dead Box RNA Helicase 3 Prevents HIV-1 Tat and Cocaine-Induced Neurotoxicity by Targeting Microglia Activation.

HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 µM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 µM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1ß. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.

Microglia morphology and proinflammatory signaling in the nucleus accumbens during nicotine withdrawal.

Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.

Neuregulin 3 Signaling Mediates Nicotine-Dependent Synaptic Plasticity in the Orbitofrontal Cortex and Cognition.

Neuregulin 3 (NRG3) and ErbB4 have been linked to nicotine addiction; however, the neuronal mechanisms and behavioral consequences of NRG3-ErbB4 sensitivity to nicotine remain elusive. Recent literature suggests that relapse to smoking is due to a lack of impulsive control, which is thought to be due to altered functioning within the orbitofrontal cortex (OFC). Therefore, we examined circuitry changes within this structure following nicotine application. We report that nicotine controls synaptic plasticity in the OFC through NRG3/ErbB4-dependent regulation of GABAergic inhibition. We observed that both nicotine and NRG3 facilitated the conversion of long-term potentiation into long-term depression at cortical layer 3/5 synapses. Induction of long-term depression by nicotine relied on nicotinic receptor activation and key regulators of NRG3 signaling: (1) release of intracellular calcium, (2) activation of the BACE1 beta-secretase, and (3) ErbB4 receptor activation. Nicotine-induced synaptic plasticity was also associated with accumulation of intracellular GABA and was completely blocked by GABAA/GABAB antagonists. To test whether these mechanisms underlie OFC-dependent behavior, we evaluated the effects of nicotine in the go/no-go task. Nicotine-impaired stimulus discrimination in this task was rescued by pharmacologic disruption of the NRG3 receptor, ErbB4. Altogether, our data indicate that nicotine-induced synaptic plasticity in the OFC and cognitive changes depend on NRG3-ErbB4 signaling. We propose that nicotine activation of this pathway may contribute to nicotine addiction, particularly in individuals with genetic variation in NRG3.

Distinct Roles of CREB Within the Ventral and Dorsal Hippocampus in Mediating Nicotine Withdrawal Phenotypes.

Addiction to nicotine and the inability to quit smoking are influenced by genetic factors, emphasizing the importance of understanding how genes and drugs of abuse mechanistically impact each other. One well-characterized protein responsible for regulating both response to drugs and gene expression is the transcription factor CREB (cAMP-responsive element binding protein). Previous work indicates that hippocampal-specific alterations in CREB signaling and synaptic plasticity may underlie certain nicotine withdrawal phenotypes. However, the structure of the hippocampus possesses dorsal and ventral subregions, each differing in behavioral, anatomic and gene expression characteristics. This study examines the effects of CREB deletion specifically in the ventral or dorsal hippocampus of animals chronically treated with saline, nicotine, or undergoing 24 h withdrawal. After region-specific viral injections of AAV-GFP or AAV-CRE in CREBloxP/loxP animals, behavioral testing measured anxiety levels, using the Novelty-Induced Hypophagia test, and cognition, using a contextual fear conditioning paradigm. Deletion of CREB in the ventral, but not dorsal, hippocampus resulted in amelioration of nicotine withdrawal-induced anxiety-like behavior in the Novelty-Induced Hypophagia test. In contrast, CREB deletion in the dorsal hippocampus resulted in learning and memory deficits in fear conditioning, whereas CREB deletion in the ventral hippocampus showed an enhancement in learning. Gene expression analysis showed differential treatment- and region-dependent alterations of several CREB target genes that are well-known markers of neuroplasticity within the hippocampus. Collectively, these data provide persuasive evidence towards the distinct roles of CREB within the dorsal and ventral hippocampus separately in mediating select nicotine withdrawal phenotypes.

Role of the Neuregulin Signaling Pathway in Nicotine Dependence and Co-morbid Disorders.

Smoking is currently the leading cause of preventable death in the United States and is responsible for over four million deaths annually worldwide. Therefore, there is a vast clinical unmet need with regards to therapeutics targeting smoking cessation. This is even more apparent when examining smokers co-morbid with psychiatric illness, as rates of smoking in this population are ~4× higher than in the general population. Examining common genetic and molecular signaling pathways impinging upon both smoking behavior and psychiatric illness will lead to a better understanding of co-morbid disorders and potential development of novel therapeutics. Studies have implicated the Neuregulin Signaling Pathway in the pathophysiology of a number of psychiatric illnesses. Additionally, recent studies have also shown an association between the Neuregulin Signaling Pathway and smoking behaviors. This review outlines basic mechanisms of the Neuregulin Signaling Pathway and how it may be exploited for precision medicine approaches in treating nicotine dependence and mental illness.

Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine.

BACKGROUND: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine. METHODS: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35 mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity. RESULTS: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and phosphorylated cAMP-response element-binding protein in the medial prefrontal cortex of impoverished condition but not enriched condition rats. CONCLUSION: These findings suggest that environmental enrichment, via upregulation of prefrontal microRNA-221 expression, suppresses the nicotine-induced activation of extracellular signal-regulated kinase and cAMP-response element-binding protein, which provides a potential mechanism underlying enhanced locomotor sensitivity to nicotine.

Activation of α4ß2*/α6ß2* nicotinic receptors alleviates anxiety during nicotine withdrawal without upregulating nicotinic receptors.

Although nicotine mediates its effects through several nicotinic acetylcholine receptor (nAChR) subtypes, it remains to be determined which nAChR subtypes directly mediate heightened anxiety during withdrawal. Relative success in abstinence has been found with the nAChR partial agonist varenicline (Chantix; Pfizer, Groton, CT); however, treatment with this drug fails to alleviate anxiety in individuals during nicotine withdrawal. Therefore, it is hypothesized that success can be found by the repurposing of other nAChR partial agonists for cessation therapies that target anxiety. It is noteworthy that the selective partial agonists for α4ß2, ABT-089 [2-methyl-3-[2(S)-pyrrolidinylmethoxy]pyridine], and α7, ABT-107 [5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole] (AbbVie, North Chicago, IL), have not been evaluated as possible therapeutics for nicotine cessation. Therefore, we examined the effect of ABT-089 and ABT-107 on anxiety during withdrawal from nicotine in the novelty-induced hypophagia (NIH) paradigm. We found that short-term administration of ABT-089 and ABT-107 alleviate anxiety-like behavior during withdrawal from nicotine while long-term administration of ABT-089 but not ABT-107 reduces anxiety-like behavior during withdrawal. After behavioral testing, brains were harvested and ß2-containing nAChRs were measured using [(3)H]epibaditine. ABT-089 and ABT-107 do not upregulate nAChRs, which is in contrast to the upregulation of nAChRs observed after nicotine. Furthermore, ABT-089 is anxiogenic in nicotine naive animals, suggesting that the effects on anxiety are specifically related to the nicotine-dependent state. Together, these studies identify additional nAChR partial agonists that may aid in the rational development of smoking cessation aids.

Chronic sazetidine-A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain.

Chronic nicotine administration increases the density of brain α4ß2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4ß2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4ß2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.

Divergent functional effects of sazetidine-a and varenicline during nicotine withdrawal.

Smoking is the largest preventable cause of death in the United States. Furthermore, a recent study found that <10% of quit attempts resulted in continuous abstinence for 1 year. With the introduction of pharmacotherapies like Chantix (varenicline), a selective α4ß2 nicotinic partial agonist, successful quit attempts have significantly increased. Therefore, novel subtype-specific nicotinic drugs, such as sazetidine-A, present a rich area for investigation of therapeutic potential in smoking cessation. The present studies examine the anxiety-related behavioral and functional effects of the nicotinic partial agonists varenicline and sazetidine-A during withdrawal from chronic nicotine in mice. Our studies indicate that ventral hippocampal-specific infusions of sazetidine-A, but not varenicline, are efficacious in reducing nicotine withdrawal-related anxiety-like phenotypes in the novelty-induced hypophagia (NIH) paradigm. To further investigate functional differences between these partial agonists, we utilized voltage-sensitive dye imaging (VSDi) in ventral hippocampal slices to determine the effects of sazetidine-A and varenicline in animals chronically treated with saline, nicotine, or undergoing 24 h withdrawal. These studies demonstrate a functional dissociation of varenicline and sazetidine-A on hippocampal network activity, which is directly related to previous drug exposure. Furthermore, the effects of the nicotinic partial agonists in VSDi assays are significantly correlated with their behavioral effects in the NIH test. These findings highlight the importance of drug history in understanding the mechanisms through which nicotinic compounds may be aiding smoking cessation in individuals experiencing withdrawal-associated anxiety.

Translational research in nicotine dependence.

Nicotine addiction accounts for 4.9 million deaths each year. Furthermore, although smoking represents a significant health burden in the United States, at present there are only three FDA-approved pharmacotherapies currently on the market: (1) nicotine replacement therapy, (2) bupropion, and (3) varenicline. Despite this obvious gap in the market, the complexity of nicotine addiction in addition to the increasing cost of drug development makes targeted drug development prohibitive. Furthermore, using combinations of mouse and human studies, additional treatments could be developed from off-the-shelf, currently approved medication lists. This article reviews translational studies targeting manipulations of the cholinergic system as a viable therapeutic target for nicotine addiction.

Genetic background influences the effects of withdrawal from chronic nicotine on learning and high-affinity nicotinic acetylcholine receptor binding in the dorsal and ventral hippocampus.

RATIONALE: The effects of nicotine on cognitive processes may play an important role in nicotine addiction. Nicotine withdrawal impairs hippocampus-dependent learning and genetic factors influence this effect. However, the neural changes that contribute to these impairments are unknown. Chronic nicotine upregulates hippocampal nicotinic acetycholine receptors (nAChRs), which may contribute to cognitive deficits when nicotine administration ceases. If nAChR upregulation underlies withdrawal deficits in learning, then strains of mice exhibiting withdrawal deficits in hippocampus-dependent learning should also show upregulation of hippocampal nAChRs. OBJECTIVES: Here, we examined the effects of nicotine withdrawal on fear conditioning and [(3)H]epibatidine binding in the dorsal and ventral hippocampus in two inbred mouse strains and their F1 hybrids. METHODS: Male C57BL/6NTac, 129S6/SvEvTac, and B6129SF1/Tac mice were administered chronic nicotine (18 mg/kg/day) for 12 days through osmotic pumps and then were trained and tested in fear conditioning 24 h after cessation of nicotine treatment. RESULTS: Nicotine withdrawal impaired hippocampus-dependent contextual conditioning in C57BL/6NTac mice but not 129S6/SvEvTac or B6129SF1/Tac mice; no changes were observed in hippocampus-independent cued fear conditioning. Upregulated [(3)H]epibatidine binding was found in the dorsal, but not ventral, hippocampus of C57BL/6NTac mice and in the ventral hippocampus of B6129SF1/Tac mice after chronic nicotine. CONCLUSIONS: Upregulation of high-affinity binding sites in the dorsal hippocampus of C57BL/6NTac mice, the only strain that exhibited nAChR upregulation in this region and withdrawal deficits in contextual conditioning, suggests that upregulation of high-affinity binding sites in the dorsal hippocampus mediates, in part, nicotine withdrawal deficits in contextual conditioning and genetic background modulates these effects.

Chronic sazetidine-A at behaviorally active doses does not increase nicotinic cholinergic receptors in rodent brain.

Chronic nicotine administration increases α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This up-regulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at α4ß2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were ∼150 times its affinity for α4ß2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not up-regulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all α4ß2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.