Stimulus mediation, specificity and impact of menthol in rats trained to discriminate puffs of nicotine e-cigarette aerosol from nicotine-free aerosol.

RATIONALE: It is unclear if e-cigarettes have reduced abuse liability relative to traditional cigarettes, especially when considering advanced devices which deliver nicotine more efficiently. Translatable and predictive animal models are needed to addresses this question. OBJECTIVES: Our goal was to explore the subjective stimulus effects of e-cigarettes by training rats to discriminate puffs of nicotine aerosol from vehicle aerosol using an aerosol delivery system designed to model e-cigarette use patterns in humans. METHODS: Rats were trained to discriminate between ten, 10 s puffs of aerosol generated from 3 mg/ml nicotine e-liquid and nicotine-free e-liquid using a food-reinforced operant procedure. Following acquisition, tests were conducted to determine the specificity of the nicotine aerosol stimulus as well as the impact to the stimulus effects of nicotine resulting from the addition of menthol to e-liquid. RESULTS: Rats learned the nicotine aerosol puff vs vehicle puff discrimination in a mean of 25 training sessions. Injected nicotine fully substituted for the stimulus effects of nicotine aerosol. The stimulus effects of nicotine aerosol were blocked by the nicotinic receptor antagonist mecamylamine. The nicotinic receptor partial agonist, varenicline as well as the stimulant d-amphetamine substituted more robustly for nicotine aerosol puffs than did the NMDA antagonist, ketamine. Menthol enhanced the stimulus effects of nicotine aerosol without altering nicotine blood plasma levels. CONCLUSIONS: Nicotine aerosol puffs can function as a training stimulus in rats. The stimulus effects were CNS-mediated and receptor specific. Menthol appears to enhance the stimulus effects of nicotine aerosol through a pharmacodynamic rather than pharmacokinetic mechanism.

Surgical incision pain induced an increase in alcohol consumption in mice.

INTRODUCTION: Large population-based studies have suggested a link between increased alcohol use and reduced pain. In addition, these studies suggest that higher levels of pain intensity are associated with an increase in alcohol consumption and rates of hazardous drinking which potentiates the risk of developing alcohol use disorders (AUD). The mechanisms and determinants of the alcohol-pain interaction can be studied in preclinical studies. METHODS: The overall goal of this study is to use animal models to explore the impact of acute postoperative pain on alcohol intake. To achieve this, we characterized the timeline and levels of alcohol intake and preference in mice after laparotomy in the 2-bottle choice paradigm. RESULTS: Our results show that laparotomy surgery increased alcohol intake and preference in male mice but not females in the 2-bottle choice and 3-bottle choice assays. In addition, ketoprofen administration blocked the increase in alcohol consumption in male mice after laparotomy. We also found that changes in alcohol initial sensitivity and acute functional tolerance, using loss of righting reflex (LORR) response, occur after surgery in mice. CONCLUSION: Taken together, these findings suggests that sex, pain and alcohol sensitivity-related factors may modulate the relationship between alcohol consumption and pain.

Differential roles of diacylglycerol lipase (DAGL) enzymes in nicotine withdrawal.

Nicotine and tobacco-related deaths remains a leading cause of preventable death and disease in the United States. Several studies indicate that modulation of the endocannabinoid system, primarily of the endocannabinoid 2-Arachidonoylglycerol (2-AG), alters nicotinic dependence behaviors in rodents. This study, using transgenic knock-out (KO) mice, evaluated the role of the two 2-AG biosynthesis enzymes, (Diacylglycerol lipase-α) DAGL-α and DAGL-ß in spontaneous nicotine withdrawal. DAGL-α deletion prevents somatic and affective signs of nicotine withdrawal, while DAGL-ß deletion plays a role in hyperalgesia due to nicotine withdrawal. These results suggest a differential role of these enzymes in the various signs of nicotine withdrawal. Our behavioral findings relate to the distribution of these enzymes with DAGL-ß being highly expressed in macrophages and DAGL-α in neurons. This study offers new potential targets for smoking cessation therapies.

L-theanine attenuates nicotine reward and withdrawal signs in mice.

BACKGROUND: L-theanine, 2-amino-4-(ethylcarbamoyl) butyric acid, an amino acid detected in green tea leaves, is used as a dietary supplement to attenuate stress and enhance mood and cognition. Furthermore, L-theanine induces anxiolytic effects in humans. Recently, L-theanine was reported to reduce morphine physical dependence in primates, suggesting the potential usefulness of L-theanine for drug dependence intervention. OBJECTIVE: The aim of this study is to determine whether L-theanine attenuates nicotine-withdrawal (somatic and affective signs) and nicotine reward in mice. We also investigated the effects of L-theanine on nicotinic receptors binding and function. METHODS: ICR male mice rendered dependent to nicotine through implanted subcutaneous osmotic minipumps for 14 days undertook precipitated nicotine withdrawal by mecamylamine on day 15. Anxiety-like behaviors using LDB, somatic signs observation and hot plate latency were assessed consecutively after treatment with L-theanine. Furthermore, we examined the effect of L-theanine on acute nicotine responses and nicotine conditioned reward in mice and on expressed nicotinic receptors in oocytes. KEY FINDINGS: L-theanine reduced in a dose-dependent manner anxiety-like behavior, hyperalgesia and somatic signs during nicotine withdrawal. Also, L-theanine decreased the nicotine CPP, but it did not affect the acute responses of nicotine. Finally, L-theanine did not alter the binding or the function of expressed α4ß2 and α7 nAChRs. CONCLUSION: Our results support the potential of L-theanine as a promising candidate for treating nicotine dependence.

Assessment of Abuse-Related Discriminative Stimulus Effects of Nicotine Aerosol in Rodents.

The rapid increase in e-cigarette use highlights the importance of developing relevant, predictive animal models exploring their potential health implications. The goal of the present study was to examine the abuse-related effects of brief, repeated e-cigarette aerosol exposures in rodents modeling human e-cigarette user behavior. We evaluated the discriminative stimulus effects of brief, repeated puffs of inhaled nicotine in rats that had been trained to discriminate injected nicotine from saline. Locomotor activity measurement following exposure to injected and aerosolized nicotine was also assessed as an additional behavioral outcome. We hypothesized that the stimulus effects of nicotine aerosol were central nervous system (CNS)-mediated and comparable to that produced by an injected nicotine training stimulus. We further hypothesized that number of aerosol puffs and the e-liquid nicotine concentration which was aerosolized would impact the substitution of nicotine aerosol for injected nicotine. Both nicotine injections and exposures to nicotine aerosol produced a dose-dependent effect on locomotor activity. Nicotine aerosol under our puffing conditions produced e-liquid nicotine concentration-dependent and puff-number-dependent complete substitution for the injected nicotine training condition. The nicotinic antagonist, mecamylamine, completely blocked nicotine-appropriate responding produce by the training dose of 0.3 mg/kg injected nicotine as well as that resulting from exposure to aerosol puffs generated by e-liquid containing 3 mg/ml nicotine, demonstrating that the stimulus of inhaled nicotine was most likely CNS-mediated and not due to olfactory stimulus properties. Overall, the results support the hypothesis that an aerosol exposure drug discrimination model in rodents has applicability to studying the abuse-related effects of e-cigarettes. SIGNIFICANCE STATEMENT: Animal models of nicotine aerosol exposure using testing conditions resembling human e-cigarette use are lacking. In this study, we test a novel preclinical model of nicotine vaping in rodents which allows for the exploration of the abuse-related effects of e-cigarettes. This model has the potential to contribute both to our understanding of the abuse-related pharmacological effects of e-cigarettes as well as aid in the development of rationale, evidence-based e-cigarette regulatory policies.

Comparison of Pain-Like behaviors in two surgical incision animal models in C57BL/6J mice.

Background: Management of pain post-surgery is crucial for tissue healing in both veterinary and human medicine. Overuse of some analgesics such as opioids may lead to addictions and worsen pain syndromes (opioid-induced hyperalgesia), while underuse of it may affect the welfare of the patient. Therefore, the importance of using surgery models in laboratory animals is increasing, with the goal of improving our understanding of pain neurobiology and developing safer analgesics. Methods: We compared the widely used plantar incision model with the laparotomy surgery model and measured pain-related behaviors using both spontaneous and evoked responses in female and male C57BL/6J mice. Additionally, we assessed conditioned place preference (CPP) and sucrose preference tests to measure pain-induced motivation for the analgesic ketoprofen and anhedonia-like behavior. Results: Laparotomized mice showed increased abdominal sensitivity while paw-incised mice showed increased paw thermal and mechanical sensitivity up to seven days post-surgery. Laparotomy surgery reduced all spontaneous behaviors in our study however this effect dissipated by 24 h post-laparotomy. On the other hand, paw incision only reduced the percentage of cage hanging in a sex-dependent manner at 6 h post-incision. We also showed that both surgery models increased conditioned place preference for ketoprofen while preference for sucrose was only reduced at 24 h post-laparotomy. Laporatomy, but not paw incision, induced a decrease in body weight at 24 h post-surgery. Neither surgery model affected fluid intake. Conclusion: Our results indicate that post-surgery hypersensitivity and behavioral deficits may differ by the incision site. Furthermore, factors associated with the surgery including length of the incision, duration of the anesthesia, and the layers that received stitches may affect subsequent spontaneous behaviors. These findings may help to improve drug development or the choice of the effective analgesic, depending on the surgery type.

Pharmacological characterization of 5-iodo-A-85380, a ß2-selective nicotinic receptor agonist, in mice.

BACKGROUND: Because of their implications in several pathological conditions, α4ß2* 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 (5IA), has helped in delineating the function of ß2-containing subtypes in vitro; however, much is still unknown about its behavioral effects. Furthermore, its effectiveness on α6-containing subtypes is limited. AIMS: To investigate the effects of 5IA 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 ß2 and α6 nAChR subunits in these effects. Lastly, its selectivity for expressed low sensitivity (LS) and high sensitivity (HS) α4ß2 receptors is investigated. RESULTS: 5IA 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 ß2 but not α6 nicotinic subunits. Finally, we show that 5-iodo-A-85380 potently activates both stoichiometries of α4ß2 nAChRs with differential efficacies, being a full agonist on HS α4(2)ß2(3) nAChRs, and a partial agonist on LS α4(3)ß2(2) nAChRs and α6-containing subtypes as well.

Potentiation of (α4)2(ß2)3, but not (α4)3(ß2)2, nicotinic acetylcholine receptors reduces nicotine self-administration and withdrawal symptoms.

The low sensitivity (α4)3(ß2)2 (LS) and high sensitivity (α4)2(ß2)3 (HS) nAChR isoforms may contribute to a variety of brain functions, pathophysiological processes, and pharmacological effects associated with nicotine use. In this study, we examined the contributions of the LS and HS α4ß2 nAChR isoforms in nicotine self-administration, withdrawal symptoms, antinociceptive and hypothermic effects. We utilized two nAChR positive allosteric modulators (PAMs): desformylflustrabromine (dFBr), a PAM of both the LS and HS α4ß2 nAChRs, and CMPI, a PAM selective for the LS nAChR. We found that dFBr, but not CMPI, decreased intravenous nicotine self-administration in male mice in a dose-dependent manner. Unlike dFBr, which fully reverses somatic and affective symptoms of nicotine withdrawal, CMPI at doses up to 15 mg/kg in male mice only partially reduced nicotine withdrawal-induced somatic signs, anxiety-like behavior and sucrose preference, but had no effects on nicotine withdrawal-induced hyperalgesia. These results indicate that potentiation of HS α4ß2 nAChRs is necessary to modulate nicotine's reinforcing properties that underlie nicotine intake and to reverse nicotine withdrawal symptoms that influence nicotine abstinence. In contrast, both dFBr and CMPI enhanced nicotine's hypothermic effect and reduced nicotine's antinociceptive effects in male mice. Therefore, these results indicate a more prevalent role of HS α4ß2 nAChR isoforms in mediating various behavioral effects associated with nicotine, whereas the LS α4ß2 nAChR isoform has a limited role in mediating body temperature and nociceptive responses. These findings will facilitate the development of more selective, efficacious, and safe nAChR-based therapeutics for nicotine addiction treatment.

Targeting Peroxisome Proliferator-Activated Receptor-α (PPAR- α) to reduce paclitaxel-induced peripheral neuropathy.

BACKGROUND AND PURPOSE: Paclitaxel, a widely used anti-cancer drug, is frequently associated with prolonged and severe peripheral neuropathies (PIPN), associated with neuroinflammation. Currently, PIPN effective treatments are lacking. Peroxisome Proliferator-Activated Receptor-α (PPAR-⍺) can modulate inflammatory responses. Thus, the use of PPAR-⍺ agonists, such as fibrates (fenofibrate and choline-fenofibrate), currently used in dyslipidemia treatment, could represent an interesting therapeutic approach in PIPN. EXPERIMENTAL APPROACH: Our studies tested the efficacy of fenofibrate (150 mg/kg, daily, i.p.) and choline fenofibrate (60 mg/kg daily, p.o.) in reversing and preventing the development of PIPN (paclitaxel: 8 mg/kg, i.p., every other day for 4 days) in male and female C57BL/6J mice. Mechanical and cold hypersensitivity, conditioned place preference, sensory nerve action potential (SNAP), as well as the expression of PPAR-⍺, TNF-⍺, IL-1ß and IL-6 mRNA were evaluated. KEY RESULTS: While fenofibrate treatment partially reversed and prevented the development of mechanical hypersensitivity, this was completely reversed and prevented by choline-fenofibrate. Both fibrates were able to completely reverse and prevent cold hypersensitivity induced by paclitaxel. The reduction of SNAP amplitude induced by paclitaxel was also reversed by both fenofibrate and choline-fenofibrate. Our results indicate that suppression of paclitaxel-induced hypersensitivity by fibrates involves the regulation of PPAR-⍺ expression and decrease neuroinflammation in DRG. Finally, the co-treatment of Paclitaxel and fenofibric acid (fibrates active metabolite) was tested on different cancer cell lines, no decrease in the antitumoral effect of paclitaxel was observed. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show for the first time the therapeutic potential (prevention and reversal) of fibrates in PIPN and opens to a potential pharmacological repurposing of these drugs.

Assessing nicotine dependence using an oral nicotine free-choice paradigm in mice.

Models to assess the addictive-like properties of nicotine in mice are limited. Therefore, we aimed to characterize and validate an addiction index by using an oral nicotine free-choice paradigm in mice. Adult C57BL/6J, DBA/2J, or genetically modified mice carrying deletions for nicotinic acetylcholine receptor (nAChR) subunits, (n = 8-10/sex/group) were given a choice of water or nicotine (10-960 µg/ml) solution using a two-bottle free-choice (2BC) paradigm. In general, oral nicotine intake and preference were higher in female mice compared to males. Absence of nicotine led to withdrawal, and intermittent access resulted in an escalation in consumption and greater nicotine withdrawal than continuous exposure. Additionally, oral nicotine consumption increased nucleus accumbens tyrosine hydroxylase levels. While ß2 and α6 KO mice showed a significant decrease in nicotine intake, deletion of α5 nAChRs increased nicotine consumption at high concentrations. Deletion of the α7 subunit altered the observed sex difference in nicotine consumption, with females consuming less than males. The α4ß2 partial agonist varenicline decreased oral nicotine consumption. Although addition of quinine to the nicotine solution lowered nicotine intake, mice primed with nicotine did not lower their intake after quinine addition. Nicotine deprivation followed by re-exposure showed increased nicotine consumption, and DBA/2J mice consumed less nicotine compared to C57BL/6J. We validated the mouse 2BC paradigm to study nicotine's addictive-like properties including nicotine intake, preference, withdrawal, and escalation of nicotine consumption during binge drinking or after reinstatement of a deprivation period.

The ß3 subunit of the nicotinic acetylcholine receptor is required for nicotine withdrawal-induced affective but not physical signs or nicotine reward in mice.

Nicotinic acetylcholine receptors (nAChRs) are the primary target for nicotine, the addictive component in tobacco products. These pentameric receptors are made up of various subunits which contribute to the diverse functions of nAChR subtypes. The ß3 subunit of the nAChR has been understudied in nicotine dependence, even though it is expressed in brain regions important for drug reward. Therefore, we assessed nicotine dependence behaviors in ß3 wildtype (WT) and knockout (KO) male and female mice. We evaluated nicotine reward in the conditioned place preference (CPP) test and then measured nicotine withdrawal signs after chronic exposure to the drug. For the withdrawal studies, mice were continuously infused with 24 mg/kg/day of nicotine using surgically implanted osmotic mini-pumps for 14 days. Mini-pumps were removed at day 15, and withdrawal signs (somatic signs, hyperalgesia, anhedonia-like measure using the sucrose preference test and anxiety-like behaviors using the light dark boxes) were collected at 24 h intervals for three days following spontaneous withdrawal of nicotine. Nicotine-induced CPP did not differ between ß3 KO and WT mice. ß3 KO mice displayed similar somatic symptoms and hyperalgesia compared to WT mice but showed significant absence in affective (anhedonia and anxiety-like behaviors) withdrawal signs in nicotine-dependent mice. These observations suggest that the ß3 nicotinic subunits do not seem to influence nicotine reward but plays an important role in affective nicotine withdrawal signs. Given the health burden of tobacco use disorder and the modest effect of smoking cessation aids, it is important to understand underlying factor contributing to nicotine dependence. The results of this study will further our knowledge of the role of the ß3 nAChR subunit in nicotine reward and withdrawal behaviors in hopes of finding new molecular targets for smoking cessation aids.

New insights on the effects of varenicline on nicotine reward, withdrawal and hyperalgesia in mice.

Varenicline, a partial agonist for α4ß2* nicotinic acetylcholine receptors (nAChRs) and a full agonist for α3ß4 and α7 nAChRs, is approved for smoking cessation treatment. Although, partial agonism at α4ß2* nAChRs is believed to be the mechanism underlying the effects of varenicline on nicotine reward, the contribution of other nicotinic subtypes to varenicline's effects on nicotine reward is currently unknown. Therefore, we examined the role of α5 and α7 nAChR subunits in the effects of varenicline on nicotine reward using the conditioned place preference (CPP) test in mice. Moreover, the effects of varenicline on nicotine withdrawal-induced hyperalgesia and aversion are unknown. We also examined the reversal of nicotine withdrawal in mouse models of dependence by varenicline. Varenicline dose-dependently blocked the development and expression of nicotine reward in the CPP test. The blockade of nicotine reward by varenicline (0.1 mg/kg) was preserved in α7 knockout mice but reduced in α5 knockout mice. Administration of varenicline at high dose of 2.5 mg/kg resulted in a place aversion that was dependent on α5 nAChRs but not ß2 nAChRs. Furthermore, varenicline (0.1 and 0.5 mg/kg) reversed nicotine withdrawal signs such as hyperalgesia and somatic signs and withdrawal-induced aversion in a dose-related manner. Our results indicate that the α5 nAChR subunit plays a role in the effects of varenicline on nicotine reward in mice. Moreover, the mediation of α5 nAChRs, but not ß2 nAChRs are probably needed for aversive properties of varenicline at high dose. Varenicline was also shown to reduce several nicotine withdrawal signs.

Curcumin Acts as a Positive Allosteric Modulator of α7-Nicotinic Acetylcholine Receptors and Reverses Nociception in Mouse Models of Inflammatory Pain.

Effects of curcumin, a major ingredient of turmeric, were tested on the function of the α7-subunit of the human nicotinic acetylcholine (α7-nACh) receptor expressed in Xenopus oocytes and on nociception in mouse models of tonic and visceral pain. Curcumin caused a significant potentiation of currents induced by acetylcholine (ACh; 100 µM) with an EC50 value of 0.2 µM. The effect of curcumin was not dependent on the activation of G-proteins and protein kinases and did not involve Ca2+-dependent Cl- channels expressed endogenously in oocytes. Importantly, the extent of curcumin potentiation was enhanced significantly by decreasing ACh concentrations. Curcumin did not alter specific binding of [125I]α-bungarotoxin. In addition, curcumin attenuated nociceptive behavior in both tonic and visceral pain models without affecting motor and locomotor activity and without producing tolerance. Pharmacological and genetic approaches revealed that the antinociceptive effect of curcumin was mediated by α7-nACh receptors. Curcumin potentiated the antinociceptive effects of the α7-nACh receptor agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU282987). Collectively, our results indicate that curcumin is a positive allosteric modulator of α7-nACh receptor and reverses nociception in mouse models of tonic and visceral pain.

Assessment of nicotine withdrawal-induced changes in sucrose preference in mice.

Anhedonia, induced by nicotine withdrawal, may serve as an important affective sign that reinforces tobacco use and smoking relapse rates in humans. Animal models provide a way to investigate the underlying neurobiological factors involved in the decrease in responding for positive affective stimuli during nicotine withdrawal and may aid in drug development for nicotine dependence. Thus, we explored the use of the sucrose preference test to measure nicotine withdrawal-induced reduction in response for positive affective stimuli in mice. C57BL/6J and knockout (KO) mice were chronically exposed to different doses of nicotine through surgically implanted subcutaneous osmotic minipumps for 14days and underwent spontaneous nicotine withdrawal on day 15. A sucrose preference time course was performed and the results were compared to another well-established affective sign of nicotine withdrawal, the reduction in time spent in light side, using the Light Dark Box test. Subsequently, our results demonstrated a time-dependent and dose-related reduction in sucrose preference in nicotine withdrawn male C57BL/6J mice, indicative of a decrease in responding for positive affective stimuli. Furthermore, the sucrose preference reduction during nicotine withdrawal was consistent with decrease in time spent in the light side of the Light Dark Box test. We also found the reduction for positive affective stimuli and time spent in the light side was not present in nicotine withdrawn ß2 and α6 KO mice, suggesting that these nicotinic subunits are involved in the affective signs of nicotine withdrawal. Thus, this report highlights the potential utility of the sucrose preference test as a useful measure of the decrease in responding for positive affective stimuli during spontaneous nicotine withdrawal.

Effects of paclitaxel on the development of neuropathy and affective behaviors in the mouse.

Paclitaxel, one of the most commonly used cancer chemotherapeutic drugs, effectively extends the progression-free survival of breast, lung, and ovarian cancer patients. However, paclitaxel and other chemotherapy drugs elicit peripheral nerve fiber dysfunction or degeneration that leads to peripheral neuropathy in a large proportion of cancer patients. Patients receiving chemotherapy also often experience changes in mood, including anxiety and depression. These somatic and affective disorders represent major dose-limiting side effects of chemotherapy. Consequently, the present study was designed to develop a preclinical model of paclitaxel-induced negative affective symptoms in order to identify treatment strategies and their underlying mechanisms of action. Intraperitoneal injections of paclitaxel (8 mg/kg) resulted in the development and maintenance of mechanical and cold allodynia. Carboplatin, another cancer chemotherapeutic drug that is often used in combination with paclitaxel, sensitized mice to the nociceptive effects of paclitaxel. Paclitaxel also induced anxiety-like behavior, as assessed in the novelty suppressed feeding and light/dark box tests. In addition, paclitaxel-treated mice displayed depression-like behavior during the forced swim test and an anhedonia-like state in the sucrose preference test. In summary, paclitaxel produced altered behaviors in assays modeling affective states in C57BL/6J male mice, while increases in nociceptive responses were longer in duration. The characterization of this preclinical model of chemotherapy-induced allodynia and affective symptoms, possibly related to neuropathic pain, provides the basis for determining the mechanism(s) underlying severe side effects elicited by paclitaxel, as well as for predicting the efficacy of potential therapeutic interventions.