Mifepristone decreases nicotine intake in dependent and non-dependent adult rats.

BACKGROUND: Addiction to tobacco and nicotine products has adverse health effects and afflicts more than a billion people worldwide. Therefore, there is an urgent need for new treatments to reduce tobacco and nicotine use. Glucocorticoid receptor blockade shows promise as a novel treatment for drug abuse and stress-related disorders. AIM: These studies aim to investigate whether glucocorticoid receptor blockade with mifepristone diminishes the reinforcing properties of nicotine in rats with intermittent or daily long access to nicotine. METHODS: The rats self-administered 0.06 mg/kg/inf of nicotine for 6 h per day, with either intermittent or daily access for 4 weeks before treatment with mifepristone. Daily nicotine self-administration models regular smoking, while intermittent nicotine self-administration models occasional smoking. To determine whether the rats were dependent, they were treated with the nicotinic acetylcholine receptor antagonist mecamylamine, and somatic signs were recorded. RESULTS: The rats with intermittent access to nicotine had a higher level of nicotine intake per session than those with daily access but only the rats with daily access to nicotine showed signs of physical dependence. Furthermore, mecamylamine increased nicotine intake during the first hour of access in rats with daily access but not in those with intermittent access. Mifepristone decreased total nicotine intake in rats with intermittent and daily access to nicotine. Moreover, mifepristone decreased the distance traveled and rearing in the open field test and operant responding for food pellets. CONCLUSION: These findings indicate that mifepristone decreases nicotine intake but this effect may be partially attributed to the sedative effects of mifepristone.

VNS improves VSMC metabolism and arteriogenesis in infarcted hearts through m/n-AChR-Akt-SDF-1α in adult male rats.

Vagal nerve stimulation (VNS) provides a novel therapeutic strategy for injured hearts by activating cholinergic anti-inflammatory pathways. However, little information is available on the metabolic pattern and arteriogenesis of VSMCs after MI. VNS has been shown to stimulate the expression of CPT1α, CPT1ß, Glut1, Glut4 and SDF-1α in coronary VSMCs, decreasing the number of CD68-positive macrophages while increasing CD206-positive macrophages in the infarcted hearts, leading to a decrease in TNF-α and IL-1ß accompanied by a reduced ratio of CD68- and CD206-positive cells, which were dramatically abolished by atropine and mecamylamine in vivo. Knockdown of SDF-1α substantially abrogated the effect of VNS on macrophagecell alteration and inflammatory factors in infarcted hearts. Mechanistically, ACh induced SDF-1α expression in VSMCs in a dose-dependent manner. Conversely, atropine, mecamylamine, and a PI3K/Akt inhibitor completely eliminated the effect of ACh on SDF-1α expression. Functionally, VNS promoted arteriogenesis and improved left ventricular performance, which could be abolished by Ad-shSDF-1α. Thus, VNS altered the VSMC metabolism pattern and arteriogenesis to repair the infarcted heart by inducing SDF-1α expression, which was associated with the m/nAChR-Akt signaling pathway.

Behavioral characterization of early nicotine withdrawal in the mouse: a potential model of acute dependence.

BACKGROUND: Clinical and preclinical research have demonstrated that short-term exposure to nicotine during the initial experimentation stage can lead to early manifestation of withdrawal-like signs, indicating the state of "acute dependence". As drug withdrawal is a major factor driving the progression toward regular drug intake, characterizing and understanding the features of early nicotine withdrawal may be important for the prevention and treatment of drug addiction. In this study, we corroborate the previous studies by showing that withdrawal-like signs can be precipitated after short-term nicotine exposure in mice, providing a potential animal model of acute dependence on nicotine. RESULTS: To model nicotine exposure from light tobacco use during the initial experimentation stage, mice were treated with 0.5 mg/kg (-)-nicotine ditartrate once daily for 3 days. On the following day, the behavioral tests were conducted after implementing spontaneous or mecamylamine-precipitated withdrawal. In the open field test, precipitated nicotine withdrawal reduced locomotor activity and time spent in the center zone. In the elevated plus maze test, the mecamylamine challenge increased the time spent in the closed arm and reduced the number of entries irrespective of nicotine experience. In the examination of the somatic aspect, precipitated nicotine withdrawal enhanced the number of somatic signs. Finally, nicotine withdrawal did not affect cognitive functioning or social behavior in the passive avoidance, spatial object recognition, or social interaction test. CONCLUSIONS: Collectively, our data demonstrate that early nicotine withdrawal-like signs could be precipitated by the nicotinic antagonist mecamylamine in mice, and that early withdrawal from nicotine primarily causes physical symptoms.

Central cholinergic transmission affects the compulsive-like behavior of mice in marble-burying test.

The presence of the cholinergic system in the brain areas implicated in the precipitation of obsessive-compulsive behavior (OCB) has been reported but the exact role of the central cholinergic system therein is still unexplored. Therefore, the current study assessed the effect of cholinergic analogs on central administration on the marble-burying behavior (MBB) of mice, a behavior correlated with OCB. The result reveals that the enhancement of central cholinergic transmission in mice achieved by intracerebroventricular (i.c.v.) injection of acetylcholine (0.01 µg) (Subeffective: 0.1 and 0.5 µg), cholinesterase inhibitor, neostigmine (0.1, 0.3, 0.5 µg/mouse) and neuronal nicotinic acetylcholine receptor agonist, nicotine (0.1, 2 µg/mouse) significantly attenuated the number of marbles buried by mice in MBB test without affecting basal locomotor activity. Similarly, central injection of mAChR antagonist, atropine (0.1, 0.5, 5 µg/mouse), nAChR antagonist, mecamylamine (0.1, 0.5, 3 µg/mouse) per se also reduced the MBB in mice, indicative of anti-OCB like effect of all the tested cholinergic mAChR or nAChR agonist and antagonist. Surprisingly, i.c.v. injection of acetylcholine (0.01 µg), and neostigmine (0.1 µg) failed to elicit an anti-OCB-like effect in mice pre-treated (i.c.v.) with atropine (0.1 µg), or mecamylamine (0.1 µg). Thus, the findings of the present investigationdelineate the role of central cholinergic transmission in the compulsive-like behavior of mice probably via mAChR or nAChR stimulation.

Vagus nerve stimulation-induced stromal cell-derived factor-l alpha participates in angiogenesis and repair of infarcted hearts.

AIMS: We aim to explore the role and mechanism of vagus nerve stimulation (VNS) in coronary endothelial cells and angiogenesis in infarcted hearts. METHODS AND RESULTS: Seven days after rat myocardial infarction (MI) was prepared by ligation of the left anterior descending coronary artery, the left cervical vagus nerve was treated with electrical stimulation 1 h after intraperitoneal administration of the α7-nicotinic acetylcholine inhibitor mecamylamine or the mAChR inhibitor atropine or 3 days after local injection of Ad-shSDF-1α into the infarcted heart. Cardiac tissue acetylcholine (ACh) and serum ACh, tumour necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) levels were detected by ELISA to determine whether VNS was successful. An inflammatory injury model in human coronary artery endothelial cells (HCAECs) was established by lipopolysaccharide and identified by evaluating TNF-α, IL-1ß and IL-6 levels and tube formation. Immunohistochemistry staining was performed to evaluate CD31-positive vessel density and stromal cell-derived factor-l alpha (SDF-1α) expression in the MI heart in vivo and the expression and distribution of SDF-1α, C-X-C motif chemokine receptor 4 and CXCR7 in HCAECs in vitro. Western blotting was used to detect the levels of SDF-1α, V-akt murine thymoma viral oncogene homolog (AKT), phosphorylated AKT (pAKT), specificity protein 1 (Sp1) and phosphorylation of Sp1 in HCAECs. Left ventricular performance, including left ventricular systolic pressure, left ventricular end-diastolic pressure and rate of the rise and fall of ventricular pressure, should be evaluated 28 days after VNS treatment. VNS was successfully established for MI therapy with decreases in serum TNF-α, IL-1ß and IL-6 levels and increases in cardiac tissue and serum ACh levels, leading to increased SDF-1α expression in coronary endothelial cells of MI hearts, triggering angiogenesis of MI hearts with increased CD31-positive vessel density, which was abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF-1α by shRNA. ACh promoted SDF-1α expression and its distribution along with the branch of the formed tube in HCAECs, resulting in an increase in the number of tubes formed in HCAECs. ACh increased the levels of pAKT and phosphorylation of Sp1 in HCAECs, resulting in inducing SDF-1α expression, and the specific effects could be abolished by mecamylamine, atropine, the PI3K/AKT blocker wortmannin or the Sp1 blocker mithramycin. Functionally, VNS improved left ventricular performance, which could be abolished by Ad-shSDF-1α. CONCLUSIONS: VNS promoted angiogenesis to repair the infarcted heart by inducing SDF-1α expression and redistribution along new branches during angiogenesis, which was associated with the m/nAChR-AKT-Sp1 signalling pathway.

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.

N-methyl-d-aspartate receptors and glycinergic transmission, respectively, mediate muscle relaxation and immobility of pentobarbital in mice.

Pentobarbital-induced anesthesia is believed to be mediated by enhancement of the inhibitory action of γ-aminobutyric acid (GABA)ergic neurons in the central nervous system. However, it is unclear whether all components of anesthesia induced by pentobarbital, such as muscle relaxation, unconsciousness, and immobility in response to noxious stimuli, are mediated only through GABAergic neurons. Thus, we examined whether the indirect GABA and glycine receptor agonists gabaculine and sarcosine, respectively, the neuronal nicotinic acetylcholine receptor antagonist mecamylamine, or the N-methyl-d-aspartate receptor channel blocker MK-801 could enhance pentobarbital-induced components of anesthesia. Muscle relaxation, unconsciousness, and immobility were evaluated by grip strength, the righting reflex, and loss of movement in response to nociceptive tail clamping, respectively, in mice. Pentobarbital reduced grip strength, impaired the righting reflex, and induced immobility in a dose-dependent manner. The change in each behavior induced by pentobarbital was roughly consistent with that in electroencephalographic power. A low dose of gabaculine, which significantly increased endogenous GABA levels in the central nervous system but had no effect on behaviors alone, potentiated muscle relaxation, unconsciousness, and immobility induced by low pentobarbital doses. A low dose of MK-801 augmented only the masked muscle-relaxing effects of pentobarbital among these components. Sarcosine enhanced only pentobarbital-induced immobility. Conversely, mecamylamine had no effect on any behavior. These findings suggest that each component of anesthesia induced by pentobarbital is mediated through GABAergic neurons and that pentobarbital-induced muscle relaxation and immobility may partially be associated with N-methyl-d-aspartate receptor antagonism and glycinergic neuron activation, respectively.

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.

Nicotinic and muscarinic acetylcholine receptor antagonism dose-dependently decreases sign- but not goal-tracking behavior in male rats.

RATIONALE: Acetylcholinergic antagonists have shown some promise in reducing addiction-related behaviors in both preclinical and clinical studies. However, the psychological mechanisms by which these drugs are able to affect addictive behavior remain unclear. A particular key process for the development of addiction is the attribution of incentive salience to reward-related cues, which can be specifically measured in animals using a Pavlovian conditioned approach procedure. When confronted with a lever that predicts food delivery, some rats engage with the lever directly (i.e., they sign track), indicating attribution of incentive-motivational properties to the lever itself. In contrast, others treat the lever as a predictive cue and approach the location of impending food delivery (i.e., they goal track), without treating the lever itself as a reward. OBJECTIVES: We tested whether systemic antagonism of the either nicotinic or muscarinic acetylcholine receptors would selectively affect sign- or goal-tracking behavior, indicating a selective effect on incentive salience attribution. METHODS: A total of 98 male Sprague Dawley rats were either given the muscarinic antagonist scopolamine (100, 50, or 10 µg/kg i.p.) or the nicotinic antagonist mecamylamine (0.3, 1.0, or 3 mg/kg i.p.) before being trained on a Pavlovian conditioned approach procedure. RESULTS: Scopolamine dose-dependently decreased sign tracking behavior and increased goal-tracking behavior. Mecamylamine reduced sign-tracking but did not affect goal-tracking behavior. CONCLUSIONS: Antagonism of either muscarinic or nicotinic acetylcholine receptors can reduce incentive sign-tracking behavior in male rats. This effect appears to be specifically due to a reduction in incentive salience attribution since goal-tracking either increased or was not affected by these manipulations.

Post-gastrulation transition from whole-body to tissue-specific intercellular calcium signaling in the appendicularian tunicate Oikopleuradioica.

We recently described calcium signaling in the appendicularian tunicate Oikopleura dioica during pre-gastrulation stages, and showed that regularly occurring calcium waves progress throughout the embryo in a characteristic spatiotemporal pattern from an initiation site in muscle lineage blastomeres. Here, we have extended our observations to the period spanning from gastrulation to post-hatching stages. We find that repetitive Ca2+ waves persist throughout this developmental window, albeit with a gradual increase in frequency. The initiation site of the waves shifts from muscle cells at gastrulation and early tailbud stages, to the central nervous system at late tailbud and post-hatching stages, indicating a transition from muscle-driven to neurally driven events as tail movements emerge. At these later stages, both the voltage gated Na+ â€‹channel blocker tetrodotoxin (TTX) and the T-type Ca2+ channel blocker and nAChR antagonist mecamylamine eliminate tail movements. At late post-hatching stages, mecamylamine blocks Ca2+ signals in the muscles but not the central nervous system. Post-gastrulation Ca2+ signals also arise in epithelial cells, first in a haphazard pattern in scattered cells during tailbud stages, evolving after hatching into repetitive rostrocaudal waves with a different frequency than the nervous system-to-muscle waves, and insensitive to mecamylamine. The desynchronization of Ca2+ waves arising in different parts of the body indicates a shift from whole-body to tissue/organ-specific Ca2+ signaling dynamics as organogenesis occurs, with neurally driven Ca2+ signaling dominating at the later stages when behavior emerges.

Nicotinic stimulation of splenic T cells is protective in endoscopic retrograde cholangiopancreatography-induced acute pancreatitis in mice.

It has previously been shown that current smoking is protective against endoscopic retrograde cholangiopancreatography (ERCP)-induced acute pancreatitis, but the mechanism of this effect was not identified. We tested the hypothesis that nicotine is the active factor in this protection in a mouse model of ERCP. Pretreatment with nicotine dose dependently inhibited acute pancreatitis caused by infusion of ERCP contrast solution into the main pancreatic duct in mice. 3-2,4-Dimethoxybenzylidene anabaseine (GTS-21), a specific partial agonist of the α7 nicotinic cholinergic receptor (α7nAChR), also protected the pancreas against ERCP-induced acute pancreatitis. The effects of GTS-21 were abolished by pretreatment with the nicotinic receptor antagonist mecamylamine. Surgical splenectomy performed 7 days before ERCP-induced pancreatitis blocked the protective effects of GTS-21. Intravenous injection of a crude preparation of total splenocytes prepared from mice pretreated with GTS-21 inhibited ERCP-induced pancreatitis; splenocytes from mice treated with vehicle had no effect. When T cells were removed from the crude GTS-21-treated splenocyte preparation by immunomagnetic separation, the remaining non-T-cell splenocytes did not protect against ERCP-induced acute pancreatitis. We conclude that nicotine protects against ERCP-induced acute pancreatitis and that splenic T cells are required for this effect. Stimulation of α7 nicotinic cholinergic receptors may protect against ERCP-induced acute pancreatitis and may also be a novel approach to therapeutic reversal of ongoing acute pancreatitis.NEW & NOTEWORTHY Epidemiological evidence indicated that acute smoking reduced the risk of endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis, but the mechanism has remained elusive. The current findings indicate the nicotine reduces the severity of ERCP-induced pancreatitis by stimulating a population of splenic T cells that exert a protective effect on the pancreas. These findings raise the possibility that nicotinic agonists might be useful in treating pancreatitis.

Systemic injection of nicotinic acetylcholine receptor antagonist mecamylamine affects licking, eyelid size, and locomotor and autonomic activities but not temporal prediction in male mice.

Nicotinic acetylcholine receptors are thought to be associated with a wide range of phenomena, such as movement, learning, memory, attention, and addiction. However, the causal relationship between nicotinic receptor activity and behavior remains unclear. Contrary to the studies that examined the functions of muscarinic acetylcholine receptors, the role of the nicotinic acetylcholine receptors on behavior has not been examined as extensively. Here, we examined the effects of intraperitoneal injection of mecamylamine, a nicotinic acetylcholine receptor antagonist, on the performance of male mice in a head-fixed temporal conditioning task and a free-moving open-field task. The head-fixed experimental setup allowed us to record and precisely quantify the licking response while the mice performed the behavioral task with no external cues. In addition, by combining the utility of the head-fixed experimental design with computer vision analysis based on deep learning algorithms, we succeeded in quantifying the eyelid size of awake mice. In the temporal conditioning task, we delivered a 10% sucrose solution every 10 s using a blunt-tipped needle placed within the licking distance of the mice. After the training, the mice showed increased anticipatory licking toward the timing of sucrose delivery, suggesting that the mice could predict the timing of the reward. Systemic injection of mecamylamine decreased licking behavior and caused eye closure but had no effect on learned conditioned predictive behavior in the head-fixed temporal conditioning task. In addition, the injection of mecamylamine decreased spontaneous locomotor activity in a dose-dependent manner in the free-moving open-field task. The results in the open-field experiments further revealed that the effect of mecamylamine on fecal output and urination, suggesting the effects on autonomic activities. Our achievement of successful eyelid size recording has potential as a useful approach in initial screening for drug discovery. Our study paves a way forward to understanding the role of nicotinic acetylcholine receptors on learning and behavior.

Nicotine promotes the utility of short-term memory during visual search in macaque monkeys.

RATIONALE: The central cholinergic system is a major therapeutic target for restoring cognitive functions. Although manipulation of cholinergic signaling is known to alter working memory (WM), the underlying mechanism remains unclear. It is widely accepted that WM consists of multiple functional modules, one storing short-term memory and the other manipulating and utilizing it. A recently developed visual search task and a relevant model can be used to assess multiple components of WM during administration of acetylcholine receptor (AChR)-related substances. OBJECTIVES: The effects of systemic administration of AChR-related agents on WM and eye movements were examined during the oculomotor foraging task. METHODS: Three monkeys performing the task received an intramuscular injection of saline or the following AChR-related agents: nicotine (24 or 56 µg/kg), mecamylamine (nicotinic AChR antagonist, 1.0 mg/kg), oxotremorine (muscarinic AChR agonist, 3.0 µg/kg), and scopolamine (muscarinic AChR antagonist, 20 µg/kg). The task was to find a target among 15 identical objects by making eye movements within 6 s. The data were analyzed according to the foraging model that incorporated three parameters. RESULTS: Nicotine and mecamylamine significantly increased the utility but not the capacity of short-term memory, while muscarinic AChR-related agents did not alter any WM parameters. Further regression analyses with a mixed-effect model showed that the beneficial effect of nicotine on memory utility remained after considering eye movement variability, but the beneficial effect of mecamylamine disappeared. CONCLUSIONS: Nicotine improves visual search, mainly by increasing the utility of short-term memory, with minimal changes in oculomotor parameters.

TLR2 and TLR4 Modulate Mouse Ileal Motility by the Interaction with Muscarinic and Nicotinic Receptors.

Irritable bowel syndrome (IBS) is a chronic functional bowel disorder characterized by intestinal dysmotility. Changes in intestinal microbiota (dysbiosis) can lead to alterations in neuro-muscular functions in the gut. Toll-like receptors (TLRs) 2 and 4 recognize intestinal bacteria and are involved in the motor response induced by gastrointestinal (GI) neurotransmitters. Acetylcholine (ACh) is a well-known neurotransmitter involved in the regulation of GI motility. This study aimed to evaluate the role of TLR2 and TLR4 in the intestinal motor-response induced by ACh in the mouse ileum, as well as the expression and function of the muscarinic and nicotinic ACh receptors. Muscle contractility studies showed that the contractions induced by ACh were significantly lower in TLR2-/- and TLR4-/- with respect to WT mice. In WT mice, the contractions induced by ACh were reduced in the presence of AF-DX AF-DX 116 (a muscarinic ACh receptor (mAChR) M2 antagonist), 4-DAMP (a mAChR M3 antagonist), mecamylamine (a nicotinic AChR receptor (nAChR) α3ß4 antagonist) and α-bungarotoxin (a nAChR α7 antagonist). In TLR2-/- mice, the contractions induced by ACh were increased by AF-DX 116 and mecamylamine. In TLR4-/- mice, the contractions induced by ACh were reduced by α-bungarotoxin and 4-DAMP. The mRNA and protein expressions of M3 and α3 receptors were diminished in the ileum from TLR2-/- and TLR4-/- with respect to WT mice. However, the levels of mRNA and protein of ß4 were diminished only in TLR4-/- but not in TLR2-/- mice. In conclusion, our results show that TLR2 and TLR4 modulates the motor responses to ACh in the mouse ileum. TLR2 acts on muscarinic M2 and M3 and nicotinic α3ß4 ACh receptors, while TLR4 acts on muscarinic M3 and nicotinic α3ß4 and α7 ACh receptors.

Prenatal Hypoxia Affects Nicotine Consumption and Withdrawal in Adult Rats via Impairment of the Glutamate System in the Brain.

The role of damaging factors in the prenatal period as a basis for drug addiction in offspring is of great interest. In this study, we aim at deciphering the effects and possible mechanisms of prenatal severe hypoxia (PSH) on predisposition to nicotine addiction in adult rats. In PSH rats, we found an increasing tendency to nicotine consumption in the two-bottle choice test. After 2 weeks of chronic treatment with nicotine via osmotic minipump (9 mg/kg per day), we assessed the symptoms of withdrawal in the conditioned place aversion test after mecamylamine (an antagonist of nicotinic acetylcholine receptors, nAChR) treatment. We showed that the mecamylamine-precipitated withdrawal aversion was stronger in the PSH group than in the control group. This suggests that PSH acts as a predisposing factor for developing nicotine addiction in adulthood. PSH rats also demonstrated an increased level of phosphorylated DARPP-32 protein (known as the relay for dopamine and glutamate signaling) at 34 threonine (pThr34DARPP-32) in relation to its total amount in the nucleus accumbens of the striatum (NAc). Meanwhile, no changes in both the content of dopamine in the mesolimbic pathway and the first type of dopamine receptors (DAR1) in NAc were found. The increased rate of DARPP-32 phosphorylation in adult PSH rats might result from excessive glutamatergic stimulation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) caused by activation of presynaptic nAChR by nicotine. This hypothesis is supported by the observed increase in VGluT2-positive terminals to Nurr1-positive neuronal bodies in VTA in PSH animals. Thus, the altered glutamate signaling phenotype might play a significant role in the development of PSH-related nicotine addiction.

Co-administration of morphine and levamisole increases death risk, produces neutropenia and modifies antinociception in mice.

Levamisole is a veterinary anthelmintic drug and a common adulterant of misused drugs. This study analyses the lethal, antinociceptive and haematological effects produced by acute or repeated levamisole administration by itself or combined with morphine. Independent groups of male Swiss Webster mice were i.p. injected with 100 mg/kg morphine, 31.6 mg/kg levamisole (lethal doses at 10%, LD10 ) or the same doses combined. Naloxone pretreatment (10 mg/kg, i.p.) prevented morphine-induced death, as did 2.5 mg/kg, i.p. mecamylamine with levamisole. Co-administration of levamisole and morphine (Lvm + Mor) increased lethality from 10% to 80%. This augmented effect was prevented by 30 mg/kg, i.p. naloxone and reduced with 10 mg/kg naloxone plus 2.5 mg/kg, i.p. mecamylamine. In independent groups of mice, 17.7 mg/kg, i.p. levamisole antagonized the acute morphine's antinociceptive effect evaluated in the tail-flick test. Repeated 17.7 mg/kg levamisole administration (2×/day/3 weeks) did not affect tolerance development to morphine (10 mg/kg, 3×/day/1 week). Blood samples obtained from mice repeatedly treated with levamisole showed leukopenia and neutropenia. Morphine also produced neutropenia, increased erythrocyte count and other related parameters (e.g. haemoglobin). Lvm + Mor had similar effects on leukocyte and neutrophil counts to those seen with levamisole only, but no erythrocyte-related alterations were evident. Blood chemistry analysis did not indicate liver damage but suggested some degree of electrolyte balance impairment. In conclusion, Lvm + Mor increased death risk, altered morphine-induced antinociceptive effects and produced haematologic abnormalities. The importance of studying combinations of drugs of abuse lies in the fact that drug users frequently combine drugs, which are commonly adulterated.

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways.

BACKGROUND AND OBJECTIVE: Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000 ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10-5 M) alone, treated with 100 ng/mL LPS alone, or treated with different concentrations of ACh (10-9/10-8/10-7/10-6/10-5 M) before LPS stimulation. HUVECs were also pre-treated with 10-6 M ACh with or without mecamylamine (an nAChR blocker) (10 µΜ) and methyllycaconitine (a specific α7 nAChR blocker) (10 µΜ) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways. RESULTS: LPS (at 10 ng/mL, 100 ng/mL and 1,000 ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100 ng/mL and 1,000 ng/mL). ACh (10-9 M-10-5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10-5 and 10-6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10-6M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10-6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38 MAPK in HUVECs, which was blocked by mecamylamine. CONCLUSIONS: ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

Exploration of potential role of Rho GTPase in nicotine dependence-induced withdrawal syndrome in mice.

The RhoA gene showed an important genotypic association with nicotine dependence and smoking initiation. The current study aims to investigate the effect of the Rho GTPase inhibitor ML141 in the progression of nicotine dependence in a mice model of precipitated nicotine withdrawal syndrome by mecamylamine.The experimental procedure involved administration of 2.5 mg/kg nicotine dissolved in normal saline subcutaneously (s.c) four times a day consecutively for 7 days and last single dose in the morning on 8th day. ML-141 was dissolved in dimethyl sulfoxide (DMSO) and was administered daily with nicotine as corrective treatment at a dose of 1,5 and 10 mg/kg (p < 0.05). An injection of 3 mg/kg of mecamylamine intraperitoneal (ip) was given an hour later than the last nicotine dose on the day 8 to precipitate withdrawal of nicotine and withdrawal severity was assessed by measuring hyperalgesia, piloerection, jumping frequency, tremors, and withdrawal severity score (WSS). Various behavioural changes such as hyperalgesia, piloerection, jumping frequency, and tremors were monitored and WSS was calculated. ML-141 a selective Rho GTPase inhibitor was found to show dose-dependent effect on all these parameters. Inhibition of Rho GTPase was found to reduce the severity of withdrawal syndrome; therefore, it can be concluded that Rho GTPase would serve as a suitable biological target by regulating the reward system in brain and could be used as new target for drug discovery.

Sensory Effects of Nicotine and Tobacco.

INTRODUCTION: Ingestion of nicotine by smoking, vaping, or other means elicits various effects including reward, antinociception, and aversion due to irritation, bitter taste, and unpleasant side effects such as nausea and dizziness. AIMS AND METHODS: Here we review the sensory effects of nicotine and the underlying neurobiological processes. RESULTS AND CONCLUSIONS: Nicotine elicits oral irritation and pain via the activation of neuronal nicotinic acetylcholine receptors (nAChRs) expressed by trigeminal nociceptors. These nociceptors excite neurons in the trigeminal subnucleus caudalis (Vc) and other brainstem regions in a manner that is significantly reduced by the nAChR antagonist mecamylamine. Vc neurons are excited by lingual application of nicotine and exhibit a progressive decline in firing to subsequent applications, consistent with desensitization of peripheral sensory neurons and progressively declining ratings of oral irritation in human psychophysical experiments. Nicotine also elicits a nAChR-mediated bitter taste via excitation of gustatory afferents. Nicotine solutions are avoided even when sweeteners are added. Studies employing oral self-administration have yielded mixed results: Some studies show avoidance of nicotine while others report increased nicotine intake over time, particularly in adolescents and females. Nicotine is consistently reported to increase human pain threshold and tolerance levels. In animal studies, nicotine is antinociceptive when delivered by inhalation of tobacco smoke or systemic infusion, intrathecally, and by intracranial microinjection in the pedunculopontine tegmentum, ventrolateral periaqueductal gray, and rostral ventromedial medulla. The antinociception is thought to be mediated by descending inhibition of spinal nociceptive transmission. Menthol cross-desensitizes nicotine-evoked oral irritation, reducing harshness that may account for its popularity as a flavor additive to tobacco products. IMPLICATIONS: Nicotine activates brain systems underlying reward and antinociception, but at the same time elicits aversive sensory effects including oral irritation and pain, bitter taste, and other unpleasant side effects mediated largely by nicotinic acetylcholine receptors (nAChRs). This review discusses the competing aversive and antinociceptive effects of nicotine and exposure to tobacco smoke, and the underlying neurobiology. An improved understanding of the interacting effects of nicotine will hopefully inform novel approaches to mitigate nicotine and tobacco use.

Dynamic activity of interpeduncular nucleus GABAergic neurons controls expression of nicotine withdrawal in male mice.

A critical brain area implicated in nicotine dependence is the interpeduncular nucleus (IPN) located in the ventral midbrain and consisting primarily of GABAergic neurons. Previous studies indicate that IPN GABAergic neurons contribute to expression of somatic symptoms of nicotine withdrawal; however, whether IPN neurons are dynamically regulated during withdrawal in vivo and how this may contribute to both somatic and affective withdrawal behavior is unknown. To bridge this gap in knowledge, we expressed GCaMP in IPN GABAergic neurons and used in vivo fiber photometry to record changes in fluorescence, as a proxy for neuronal activity, in male mice during nicotine withdrawal. Mecamylamine-precipitated withdrawal significantly increased activity of IPN GABAergic neurons in nicotine-dependent, but not nicotine-naive mice. Analysis of GCaMP signals time-locked with somatic symptoms including grooming and scratching revealed reduced IPN GABAergic activity during these behaviors, specifically in mice undergoing withdrawal. In the elevated plus maze, used to measure anxiety-like behavior, an affective withdrawal symptom, IPN GABAergic neuron activity was increased during open-arm versus closed-arm exploration in nicotine-withdrawn, but not non-withdrawn mice. Optogenetic silencing IPN GABAergic neurons during withdrawal significantly reduced withdrawal-induced increases in somatic behavior and increased open-arm exploration. Together, our data indicate that IPN GABAergic neurons are dynamically regulated during nicotine withdrawal, leading to increased anxiety-like symptoms and somatic behavior, which inherently decrease IPN GABAergic neuron activity as a withdrawal-coping mechanism. These results provide a neuronal basis underlying the role of the IPN in the expression of somatic and affective behaviors of nicotine withdrawal.