Neural circuits and nicotinic acetylcholine receptors mediate the cholinergic regulation of midbrain dopaminergic neurons and nicotine dependence

Midbrain dopaminergic(DA)neurons are governed by an endogenous cholinergic system,originated in the mesopontine nuclei.Nicotine hijacks nicotinic acetylcholine receptors(nAChRs)and interferes with physiological function of the cholinergic system.In this review,we describe the anatomical organization of the cholinergic system and the key nAChR subtypes mediating cholinergic regulation of DA transmission and nicotine reward and dependence,in an effort to identify potential targets for smoking intervention.Cholinergic modulation of midbrain DA systems relies on topographic organization of mesopontine cholinergic projections,and activation of nAChRs in midbrain DA neurons.Previous studies have revealed thatα4,α6,andβ2 subunit-containing nAChRs expressed in midbrain DA neurons and their terminals in the striatum regulatefirings of midbrain DA neurons and activity-dependent dopamine release in the striatum.These nAChRs undergo modification upon chronic nicotine exposure.Clinical investigation has demonstrated that partial agonists of these receptors elevate the success rate of smoking cessation relative to placebo.However,further investigations are required to refine the drug targets to mitigate unpleasant side-effects.     

Mysterious α6-containing nAChRs： function,pharmacology, and pathophysiology

Neuronal nicotinic acetylcholine receptors （nAChRs） are the superfamily of ligand-gated ion channels and widely expressed throughout the central and peripheral nervous systems, nAChRs play crucial roles in modulating a wide range of higher cognitive functions by mediating presynaptic, postsynaptic, and extrasynaptic signaling. Thus far, nine alpha （α2-α10） and three beta （β2,β3, and β4） subunits have been identified in the CNS, and these subunits assemble to form a diversity of functional nAChRs. Although α4β2- and α7-nAChRs are the two major functional nAChR types in the CNS, α6＊-nAChRs are abundantly expressed in the midbrain dopaminergic （DAergic） system, including mesocorficolimbic and nigrostriatal pathways, and particularly present in presynaptic nerve terminals. Recently, functional and pharmacological profiles of α6＊-nAChRs have been assessed with the use of α6 subunit blockers such as α-conotoxin MII and PIA, and also by using α6 subunit knockout mice. By modulating DA release in the nucleus accumbens （NAc） and modulating GABA release onto DAergic neurons in the ventral tegmental area （VTA）, α6＊-nAChRs may play important roles in the mediation of nicotine reward and addiction. Furthermore, α6＊-nAChRs in the nigrostriatal DAergic system may be promising targets for selective preventative treatment of Parkinson＇s disease （PD）. Thus, α6＊-nAChRs may hold promise for future clinical treatment of human disorders, such as nicotine addiction and PD. In this review, we mainly focus on the recent advances in the understanding of α6＊-nAChR function, pharmacology and pathophysiology.     

Nicotine modulates GABAergic transmission to dopaminergic neurons in substantia nigra pars compacta

Aim： Dopaminergic neurons in the substantia nigra pars compacta （SNc） play important roles in motor control and drug addiction. As the major afferent, GABAergic innervation controls the activity of SNc dopaminergic neurons. Although it is clear that nicotine modulates SNc dopaminergic neurons by activating subtypes of somatodendritic nicotinic acetylcholine receptors （nAChRs）, the detailed mechanisms of this activation remain to be addressed. Methods： In the current study, we recorded GABAA receptor-mediated spontaneous inhibitory postsynaptic currents （sIP- SCs） from dissociated SNc dopaminergic neurons that were obtained using an enzyme-free procedure. These neurons preserved some functional terminals after isolation, including those that release GABA. Results： We found that both extra- and intra-cellular calcium modulates sIPSCs in these neurons. Furthermore, both nicotine and endogenous acetylcholine enhance the frequency of sIPSCs. Moreover, endogenous acetylcholine tonically facilitates sIPSC frequency, primarily by activating the α4β2 nAChRs on the GABAergic terminals. Conclusion： Nicotine facilitates GABA release onto SNc dopaminergic neurons mainly via the activation of presynaptic α4β2 nAChRs.     

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

Cysteine-rich peptides from the venom of cone snails （Conus） target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors （nAChRs） found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson＇s disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.     

Cellular trafficking of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors （nAChRs） play critical roles throughout the body. Precise regulation of the cellular loca- tion and availability of nAChRs on neurons and target cells is critical to their proper function. Dynamic, post-translational regulation of nAChRs, particularly control of their movements among the different compartments of cells, is an important aspect of that regulation. A combination of new information and new techniques has the study of nAChR trafficking poised for new breakthroughs.     

Mechanism-based medication development for the treatment of nicotine dependence

Tobacco use is a global problem with serious health consequences. Though some treatment options exist, there remains a great need for new effective pharmacotherapies to aid smokers in maintaining long-term abstinence. In the present article, we first discuss the neural mechanisms underlying nicotine reward, and then review various mechanism-based pharmacological agents for the treatment of nicotine dependence. An oversimplified hypothesis of addiction to tobacco is that nicotine is the major addictive component of tobacco. Nicotine binds to α4β2 and α7 nicotinic acetylcholine receptors （nAChRs） located on dopaminergic, glutamatergic and GABAergic neurons in the mesolimbic dopamine （DA） system, which causes an increase in extracelluiar DA in the nucleus accumbens （NAc）. That increase in DA reinforces tobacco use, particularly during the acquisition phase. Enhanced glutamate transmission to DA neurons in the ventral tegrnental area appears to play an important role in this process, In addition, chronic nicotine treatment increases endocannabinoid levels in the mesolimbic DA system, which indirectly modulates NAc DA release and nicotine reward. Accordingly, pharmacological agents that target brain acetylcholine, DA, glutamate, GABA, or endocannabonoid signaling systems have been proposed to interrupt nicotine action. Furthermore, pharmacokinetic strategies that alter plasma nicotine availability, metabolism and clearance also significantly alter nicotine＇s action in the brain. Progress using these pharmacodynamic and pharmacokinetic agents is reviewed. For drugs in each category, we discuss the mechanistic rationale for their potential anti-nicotine efficacy, major findings in preclinical and clinical studies, and future research directions.     

Nicotinic mechanisms influencing synaptic plasticity in the hippocampus

Nicotinic acetylcholine receptors （nAChRs） are expressed throughout the hippocampus, and nicotinic signaling plays an important role in neuronal function. In the context of learning and memory related behaviors associated with hippocampal function, a potentially significant feature of nAChR activity is the impact it has on synaptic plasticity. Synaptic plasticity in hippocampal neurons has long been considered a contributing cellular mechanism of learning and memory. These same kinds ofcellular mechanisms are a factor in the development of nicotine addiction. Nicotinic signaling has been demonstrated by in vitro studies to affect synaptic plasticity in hippocampal neurons via multiple steps, and the signaling has also been shown to evoke synaptic plasticity in vivo. This review focuses on the nAChRs subtypes that contribute to hippocampal synaptic plasticity at the cellular and circuit level. It also considers nicotinic influences over long-term changes in the hippocampus that may contribute to addiction.     

Kinetics of desensitization and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

Aim： Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human α4β2- nicotinic acetylcholine receptors （nAChR） heterologously expressed in the SH-EP1 human epithelial cell line. Methods： Whole-cell patch clamp recordings were performed to evaluate α4β2-nAChR currents. Results： Application of 0.1μmol/L nicotine or 1 mmol/L acetylcholine （ACh） for I s or longer induced two phases, with time constants of -70 and -700 ms, for the onset of α4β2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of α4β2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human α4β2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. Conclusion： These findings indicate the existence of α4β2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.     

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit： pharmacological properties of ligand binding and function

Aim： To compare pharmacological properties of heterologously expressed homomeric α7 nicotinic acetylcholine receptors （α7 nAChRs） with those of native nAChRs containing α7 subunit （α7＊ nAChRs） in rat hippocampus and cerebral cortex. Methods： We established a stably transfected HEK-293 cell line that expresses homomeric rat α7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat α7＊ nAChRs in rat hippocampus and cerebral cortex. We used [125Ⅰ]-α-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the α7 nAChRs expressed in this cell line were studied using whole-cell current recording. Results： The newly established cell line, KXα7R1, expresses homomeric α7 nAChRs that bind [125Ⅰ]-α-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kd values of native rat α7＊nAChRs from hippocampus （Kd=0.28±0.03 nmol/L） and cerebral cortex （Kd=0.33±0.05 nmol/L）. Using whole-cell current recording, the homomeric α7 nAChRs expressed in the cells were activated by acetylcholine and （-）-nicotine with EC50 values of 280±19μmol/L and 180±40μmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of α7 nAChRs, a-bungarotoxin （IC50=19±2 nmol/L） and methyllycaconitine （IC50=100±10 pmol/L）. A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric α7 nAChRs and native α7＊receptors in rat brain, but it also revealed several notable differences. Conclusion： This newly established stable cell line should be very useful for studying the properties of homomeric α7 nAChRs and comparing these properties to native α7＊ nAChRs.     

Postsynaptic scaffolds for nicotinic receptors on neurons

Complex postsynaptic scaffolds determine the structure and signaling capabilities of glutamatergic synapses. Recent studies indicate that some of the same scaffold components contribute to the formation and function of nicotinic synapses on neurons. PDZ-containing proteins comprising the PSD-95 family co-localize with nicotinic acetylcholine receptors （nAChRs） and mediate downstream signaling in the neurons. The PDZ-proteins also promote functional nicotinic innervation of the neurons, as does the scaffold protein APC and transmembrane proteins such as neuroligin and the EphB2 receptor. In addition, specific chaperones have been shown to facilitate nAChR assembly and transport to the cell surface. This review summarizes recent results in these areas and raises questions for the future about the mechanism and synaptic role of nAChR trafficking.     

Cross-regulation between colocalized nicotinic acetylcholine and 5-HT3 serotonin receptors on presynaptic nerve terminals

Aim： Substantial colocalization of functionally independent a4 nicotinic acetylcholine receptors and 5-HT3 serotonin receptors on presynaptic terminals has been observed in brain. The present study was aimed at addressing whether nicotinic acetylcholine receptors and 5-HT3 serotonin receptors interact on the same presynaptic terminal, suggesting a convergence of cholinergic and serotonergic regulation. Methods： Ca^2＋ responses in individual, isolated nerve endings purified from rat striatum were measured using confocal imaging. Results： Application of 500 nmol/L nicotine following sustained stimulation with the highly selective 5-HT3 receptor agonist m-chlorophenylbiguanide at 100 nmol/L resulted in markedly reduced Ca^2＋ responses （28% of control） in only those striatal nerve endings that originally responded to m-chlorophenylbiguanide. The cross-regulation developed over several minutes. Presynaptic nerve endings that had not responded to m-chlorophenylbiguanide, indicating that 5-HT3 receptors were not present, displayed typical responses to nicotine. Application of m-chlorophenylbiguanide folIowing sustained stimulation with nicotine resulted in partially attenuated Ca^2＋ responses （49% of control）. Application of m-chlorophenylbiguanide following sustained stimulation with m-chlorophenylbiguanide also resulted in a strong attenuation of Ca^2＋ responses （12% of control）, whereas nicotine-induced Ca^2＋ responses following sustained stimulation with nicotine were not significantly different from control. Conclusion： These results indicate that the presynaptic Ca^2＋ increases evoked by either 5-HT3 receptor or nicotinic acetylcholine receptor activation regulate subsequent responses to 5-HT3 receptor activation, but that only 5-HT3 receptors cross-regulate subsequent nicotinic acetylcholine receptor-mediated responses. The findings suggest a specific interaction between the two receptor systems in the same striatal nerve terminal, likely involving Ca^2＋-dependent intracellular p     

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim： Several nicotinic acetylcholine receptor （nAChR） subunits have been engineered as fluorescent protein （FP） fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors, Methods： A gene construct was generated to introduce yellow fluorescent protein （YFP）, in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit （α7Y）. SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits （α7） or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin （I-Bgt） binding, laser scanning confocal microscopy, and total internal reflectance fluorescence （TIRF） microscopy. Results： Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion： nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs, α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.     

烟碱对毒蕈碱受体及其效应器系统的调节(英文)

Nicotinic and muscarinic receptors are distinctively different, and belong to ion channel-gated and GTP binding protein-coupled receptor superfamily respectively. But they have the same endogenous agonist acetylcholine (ACh), and they are the 2 types of cholinergic receptors and mediate the physiologic functions of cholinergic nervous system. The innervation and regulatory effects between nicotinic and muscarinic receptors have been studied extensively in peripheral nervous system (PNS). The functions of muscarinic receptors in the tissues innervated by the parasympathetic cholinergic postfibers, can be modulated by     

Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors （nAChRs）, they are grouped into either heteromeric （comprising both a and β subunits） or homomeric （comprising only a subunits） receptors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the a-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals： （1） direct calcium influx through the nAChRs, （2） indirect calcium influx through voltage-dependent calcium channels （VDCCs） which are activated by the nAChR-mediated depolarization, and （3） calciuminduced calcium release （CICR） （triggered by the first two sources） from the endoplasmic reticulum （ER） through the ryanodine receptors and inositol （1,4,5）-triphosphate receptors （IP3Rs）. Downstream signaling events mediated by nAChR- mediated calcium responses can be grouped into instantaneous effects （such as neurotransmitter release, which can occur in milliseconds after nAChR activation）, short-term effects （such as the recovery of nAChR desensitization through cellular signaling cascades）, and long-term effects （such as neuroprotection via gene expression）. In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understanding the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.     

Pharmacological and immunochemical characterization of α2＊ nicotinic acetylcholine receptors （nAChRs） in mouse brain

Aim： α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2＊ nAChRs in these mouse brain regions. Methods： α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing （α2＊） nAChRs in these two regions. Results： [125Ⅰ]Epibatidine （200 pmol/L） autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus （by 4.8±1.7 and 92＋26 fmol·mg^-1 protein, respectively）. Pharmacological characterization using the β2-selective drug A85380 to inhibit [125Ⅰ]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2＊ nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good （〉90% of β2＊ and 〉80% of β4＊ nAChRs were routinely recovered）. Conclusion： Immunoprecipitation experiments indicated that interpeduncular nucleus α2＊ nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits.     

Stable expression of amiloid protein precursor gene in SH- EPI cells transfected with nicotinic acetylcholine receptor α7 subtype gene

The senile plaques in the brain are thought to contribute to the pathogenesis of Alzheimer＇s disease （AD） and are mainly consisted of β - amyloid peptide, which is the proteolytic product of amyloid precursor protein （APP）. Previous work suggested that nicotine could effectively reduce β - amyloid peptide aggregation in the brain of animal models and improve the cognition impairment, indicating that the nicotinic acetylcholine receptors （nAChR） might play an important part in the function of memory and cognition and might be a target in the therapy of Alzheimer＇s disease. The α7 nicotinic acetylcholine receptors are highly expressed in hippocampus and in cholinergic neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer＇s disease. To test whether the α7 nicotinic acetylcholine receptors can block the processing of amyloid precursor protein into β - amyloid peptide, we transfected human APP695 into the native nAChR - null SH - EP1 human epithelial cells which had been transfected with the gene of α7nAChR and confirmed their expression by RT - PCR and Western Blot.     

Neuronal nicotinic acetylcholine receptors are important targets for alcohol reward and dependence

Neuronal nicotinic acetylcholine receptors are important targets for alcohol reward and dependence. Alcoholism is a serious public health problem and has been identified as the third major cause of preventable mortality in the world. Worldwide, about 2 billion people consume alcohol, with 76.3 million having diagnosable alcohol use disorders. Alcohol is currently responsible for the death of 4% of adults worldwide （about 2.5 million deaths each year）, and this number will be significantly increased by 2020 unless effective action is taken. Alcohol is the most commonly abused substance by humans. Ethanol （EtOH） is the intoxicating agent in alcoholic drinks that can lead to abuse and dependence. Although it has been extensively studied, the mechanisms of alcohol reward and dependence are still poorly understood. The major reason is that, unlike other addictive drugs （eg, morphine, cocaine or nicotine） that have specific molecular targets, EtOH affects much wider neuronal functions. These functions include phospholipid membranes, various ion channels and receptors, synaptic and network functions, and intracellular signaling molecules. The major targets in the brain that mediate EtOH＇s effects remain unclear. This knowledge gap results in a therapeutic barrier in the treatment of alcoholism. Interestingly, alcohol and nicotine are often co-abused, which suggests that neuronal nicotinic acetylcholine receptors （nAChRs）, the molecular targets for nicotine, may also contribute to alcohol＇s abusive properties. Here, we briefly summarize recent lines of evidence showing how EtOH modulates nAChRs in the mesolimbic pathway, which provides a perspective that nAChRs are important targets mediating alcohol abuse.     

Effects of methyllycaconitine (MLA), an α7 nicotinic receptor antagonist, on nicotine- and cocaine-induced potentiation of brain stimulation reward

It has been shown that nicotine facilitates intracranial self-stimulation (ICSS) reward and that nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA) are of primary importance for its reinforcing and dependence-producing actions. Recently, we have shown that α₇ nicotinic receptors in the VTA contribute to both the acute effects of nicotine on the mesolimbic dopamine system, as well as to nicotine withdrawal reactions. However, it is not yet known whether the same receptor conformation is directly involved in the reinforcing actions of nicotine. Here, using the curve-shift method we studied the effects of methyllycaconitine (MLA), a selective α₇ receptor antagonist, microinjected (graded doses: 1, 3, 9 μg/μl per side) into the VTA on the rewarding efficacy of lateral hypothalamic self-stimulation and on the systemic nicotine-induced potentiation of brain stimulation reward. MLA did not affect baseline self-stimulation. Nicotine produced a significant reduction in ICSS threshold, without altering maximal rates of responding, while MLA attenuated the effect of nicotine at the two lower doses. Given the reported interaction between nicotine and cocaine at both the neuronal and the behavioral level, we also examined whether α₇ receptor antagonism within the VTA can affect the reinforcing action of cocaine, as measured with ICSS. Interestingly, MLA attenuated the reinforcing effect of cocaine in all doses tested, without altering the maximal rate of responding, i.e. the performance of the animals. These results suggest that α₇ nAChRs in the VTA are involved in mediating the reinforcing actions of drugs of abuse, such as nicotine and cocaine, and provide evidence that α₇ nAChR antagonists may be clinically useful in attenuating the rewarding effects of addictive drugs. Received: 1 September 1999 / Final version: 18 December 1999     

Real-time detection of α1A-AR movement stimulated by phenylephrine in single living cells

Aim： To investigate the movement of α1A-adrenergic receptors（α1A-AR） stimulated by agonist, phenylephrine （PE）, and the dynamics of receptor movement in real time in single living cells with millisecond resolution. Methods： We labeled α1A-AR using the monoclonal, anti-FLAG （a kind of tag） antibody and Cy3-conju- gated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. Results： The specific detection of α1A-AR on the surface of living HEK293A-α1A cells was achieved, α1A-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20min, apparent colocalization was found between α1A-AR and F-actins. After 40 rain stimulation of PE, trajectories of approximate linear motion in HEK293A-α1A cells were recorded, and their velocity was calculated. Conclusion： The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect α1A-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.     

Individual differences in responses to nicotine： tracking changes from adolescence to adulthood

Aim： The present study determined the extent to which individual differences in responses to the psychostimulating effect of nicotine during adolescence predict similar individual differences during adulthood in rats. We also examined the possible long-term effects of adolescent nicotine exposure on adult prepulse inhibition （PPI） of the acoustic startle response, a measure of sensorimotor gating ability. Methods： During the adolescent phase, rats were administered saline, 0.10, 0.40, or 0.60 mg/kg nicotine via subcutaneous injections for 8 days, and motor activity was measured daily. During the adult phase, these rats were treated with the same nicotine dose as in adolescence for 8 additional days. The adolescent saline rats （now adults） were subdivided into four groups and administered saline, 0.10, 0.40, or 0.60 mg/kg nicotine, respectively. PPI was assessed 12 days after the last nicotine treatment. Results： During both phases, nicotine increased motor activity across test days in a dose-dependent manner. Motor activity of rats treated with nicotine during adolescence was positively correlated with the activity recorded from the same rats during adulthood. In both phases, there were profound individual differences in the responses to the nicotine treatments. In addition, adolescent rats treated with nicotine did not show decreased motor response to the initial exposure to nicotine. Finally, adolescent exposure to nicotine at 0.4 mg/kg, but not adulthood exposure to the same dose of nicotine, produced a robust disruption of PPI, with individual rats showing different degrees of PPI disruption. Conclusion： These findings suggest that adolescent rats have increased sensitivity to the psychostimulating effect and decreased sensitivity to the aversive effect of nicotine. Also, nicotine exposure during adolescence may have long-term detrimental effects on sensorimotor gating ability.