Cecilia Gotti

Subunit Composition of Functional Nicotinic Receptors in Dopaminergic Neurons Investigated with Knock-Out Mice

Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinsons disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (α4β2*, α6β2*, and α4α6β2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromα4, α6, α4α6, and β2 knock-out mice. Our results establish that α6β2* nAChRs are functional and sensitive to α-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonα6)α4β2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of α6β2* and α4β2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonα6)α4β2* nAChRs most likely contribute to nicotine reinforcement.

Human neuronal nicotinic receptors

Nicotine is a very widely used drug of abuse, which exerts a number of neurovegetative, behavioural and psychological effects by interacting with neuronal nicotinic acetylcholine receptors (NAChRs). These receptors are distributed widely in human brain and ganglia, and form a family of ACh-gated ion channels of different subtypes, each of which has a specific pharmacology and physiology. As human NAChRs have been implicated in a number of human central nervous system disorders (including the neurodegenerative Alzheimers disease, schizophrenia and epilepsy), they are suitable potential targets for rational drug therapy. Much of our current knowledge about the structure and function of NAChRs comes from studies carried out in other species, such as rodents and chicks, and information concerning human nicotinic receptors is still incomplete and scattered in the literature. Nevertheless, it is already evident that there are a number of differences in the anatomical distribution, physiology, pharmacology, and expression regulation of certain subtypes between the nicotinic systems of humans and other species. This review will attempt to survey the major achievements reached in the study of the structure and function of NAChRs by examining the molecular basis of their functional diversity viewed mainly from pharmacological and biochemical perspectives. It will also summarize our current knowledge concerning the structure and function of the NAChRs expressed by other species, and the newly discovered drugs used to classify their numerous subtypes. Finally, the role of NAChRs in behaviour and pathology will be considered.

Structural and functional diversity of native brain neuronal nicotinic receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels present in the central and peripheral nervous systems, that are permeable to mono- and divalent cations. They share a common basic structure but their pharmacological and functional properties arise from the wide range of different subunit combinations making up distinctive subtypes. nAChRs are involved in many physiological functions in the central and peripheral nervous systems, and are the targets of the widely used drug of abuse nicotine. In addition to tobacco dependence, changes in their number and/or function are associated with neuropsychiatric disorders, ranging from epilepsy to dementia. Although some of the neural circuits involved in the acute and chronic effects of nicotine have been identified, much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions. We briefly review some recent findings concerning the structure and function of native nAChRs, focusing on the subtypes identified in the mesostriatal and habenulo-interpeduncular pathways, two systems involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the effect of chronic nicotine on the expression of native subtypes.

Diversity of vertebrate nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric neurotransmitter receptors. They are members of the Cys-loop family of ligand-gated ion channels which also include ionotropic receptors for 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) and glycine. Nicotinic receptors are expressed in both the nervous system and at the neuromuscular junction and have been implicated in several neurological and neuromuscular disorders. In vertebrates, seventeen nAChR subunits have been identified (alpha1-alpha10, beta1-beta4, gamma, delta and epsilon) which can co-assemble to generate a diverse family of nAChR subtypes. This review will focus on vertebrate nAChRs and will provide an overview of the extent of nAChR diversity based on studies of both native and recombinant nAChRs.

α4 but Not α3 and α7 Nicotinic Acetylcholine Receptor Subunits Are Lost from the Temporal Cortex in Alzheimer's Disease

Abstract : Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimers disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age‐matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for α3, α4, and α7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to α3, α4, and α7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and α4‐like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the α4 subunit) were reduced in AD cases compared with control subjects (p <0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p <0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic α4‐like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p <0.05). There were no significant changes in α3‐ or α7‐like immunoreactivity associated with AD or tobacco use. The selective involvement of α4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.

Nicotinic Acetylcholine Receptors in the Mesolimbic Pathway: Primary Role of Ventral Tegmental Area α6β2* Receptors in Mediating Systemic Nicotine Effects on Dopamine Release, Locomotion, and Reinforcement

α6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of α6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of α6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of α6* nAChR in the mesostriatal system is heterogeneous, with (non-α4)α6β2* being predominant in the mesolimbic pathway and α4α6β2* in the nigrostriatal pathway. We verified whether α6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists α-conotoxin MII (CntxMII) (α3/α6β2* selective) or α-conotoxin PIA (CntxPIA) (α6β2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the α6β2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that α6β2*-selective compounds capable of crossing the blood–brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.

Nicotinic receptor subtypes in human brain ageing, Alzheimer and Lewy body diseases

Human brain ageing is associated with reductions in a variety of nicotinic receptors subtypes, whereas changes in age-related disorders including Alzheimers disease or Parkinsons disease are more selective. In Alzheimers disease, in the cortex there is a selective loss of the alpha4 (but not alpha3 or 7) subunit immunoreactivity and of nicotine or epibatidine binding but not alpha-bungarotoxin binding. Epibatidine binding is inversely correlated with clinical dementia ratings and with the level of Abeta1-42, but not related to plaque or tangle densities. In contrast, alpha-bungarotoxin binding is positively correlated with plaque densities in the entorhinal cortex. In human temporal cortex loss of acetylcholinesterase catalytic activity is positively correlated with decreased epibatidine binding and in a transgenic mouse model over expressing acetylcholinesterase, epibatidine binding is elevated. In Parkinsons disease, loss of striatal nicotine binding appears to occur early but is not associated with a loss of alpha4 subunit immunoreactivity. Tobacco use in normal elderly individuals is associated with increased alpha4 immunoreactivity in the cortex and lower densities of amyloid-beta plaques, and with greater numbers of dopaminergic neurons in the substantia nigra pars compacta. These findings indicate an early involvement of the alpha4 subunit in beta-amyloidosis but not in nigro-striatal dopaminergic degeneration.

Rodent Habenulo–Interpeduncular Pathway Expresses a Large Variety of Uncommon nAChR Subtypes, But Only the α3β4 and α3β3β4 Subtypes Mediate Acetylcholine Release

Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo–interpeduncular (Hb–IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits, thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat and wild-type (+/+) and β2 knock-out (−/−) mice to establish that the Hb and IPn contain significant β2* and β4* populations of nAChR receptors (each of which is heterogeneous). The β4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (α2β2*, α4β3β2*, α3β3β4*, α6β3β4*). Our studies on IPn synaptosomes obtained from +/+ and α2, α4, α5, α6, α7, β2, β3, and β4−/− mice show that only the α3β4 and α3β3β4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation, and Western blotting studies in β3−/− mice showed that, in the IPn of these mice, there is a concomitant reduction of ACh release and α3β4* receptors, whereas the receptor number remains the same in the Hb. We suggest that, in habenular cholinergic neurons, the β3 subunit may be important for transporting the α3β4* subtype from the medial habenula to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb–IPn system and identify α3β4 and α3β3β4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.

Brain neuronal nicotinic receptors as new targets for drug discovery.

Neuronal nicotinic receptors (nAChRs) are a heterogeneous family of ion channels differently expressed in the nervous system where, by responding to the endogenous neurotransmitter acetylcholine, they contribute to a wide range of brain activities and influence a number of physiological functions. Over recent years, the application of newly developed molecular and cellular biological techniques has made it possible to correlate the subunit composition of nAChRs with specific nicotine-elicited behaviours, and refine some of the in vivo physiological functions of nAChR subtypes. The major new findings are the widespread expression of nAChRs, outside the nervous system, their specific and complex organisation, and their relevance to normal brain function. Moreover, the combination of clinical and basic research has better defined the involvement of nAChRs in a growing number of nervous pathologies other than degenerative diseases. However, there are still only a limited number of nicotinic-specific drugs and, although some nicotinic agonists have an interesting pharmacology, their clinical use is limited by undesirable side effects. Some selective nicotinic ligands have recently been developed and used to explore the complexity of nAChR subtype structure and function in the expectation that they will become rational therapeutic alternatives in a number of neurodegenerative, neuropsychiatric and neurological disorders. In this review, we will discuss the molecular basis of brain nAChR structural and functional diversity mainly in pharmacological and biochemical terms, and summarise current knowledge concerning the newly discovered drugs used to classify the numerous receptor subtypes and treat the brain diseases in which nAChRs are involved.

Reciprocal regulation of dopamine D1 and D3 receptor function and trafficking by heterodimerization.

Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET2), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET2 signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [3H]R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves β-arrestin binding because it was blocked by mutant β-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R- and D3R-related dysfunctions.