Michele Zoli

Acetylcholine receptors containing the β2 subunit are involved inthe reinforcing properties of nicotine

Release of the neurotransmitter dopamine in the mesolimbic system of the brain mediates the reinforcing properties of several drugs of abuse, including nicotine. Here we investigate the contribution of the high-affinity neuronal nicotinic acetylcholine receptor to the effects of nicotine on the mesolimbic dopamine system in mice lacking the β2 subunit of this receptor. We found that nicotine stimulates dopamine release in the ventral striatum of wild-type mice but not in the ventral striatum of β2-mutant mice. Using patch-clamp recording, we show that mesencephalic dopaminergic neurons from mice without the β2 subunit no longer respond to nicotine, and that self-administration of nicotine is attenuated in these mutant mice. Our results strongly support the idea that the β2-containing neuronal nicotinic acetylcholine receptor is involved in mediating the reinforcing properties of nicotine.

Neuronal Nicotinic Receptor a6 Subunit mRNA is Selectively Concentrated in Catecholaminergic Nuclei of the Rat Brain

Although the neuronal nicotinic receptor a6 subunit was cloned several years ago, its functional significance remains to be investigated. Here we describe an in situ hybridization study of the mRNA for this subunit in the adult rat central nervous system using oligonucleotide probes. Specific a6 mRNA labelling was restricted to a few nuclei throughout the brain; it was particularly high in several catecholaminergic nuclei [the locus coeruleus (A6), the ventral tegmental area (A10) and the substantia nigra (A9)] at levels significantly higher than those found for any other known nicotinic receptor subunit mRNA. Labelling for a6 mRNA was also detected at lower levels in the reticular thalamic nucleus, the supramammillary nucleus and the mesencephalic V nucleus. Some cells of the medial habenula (medioventral part) and of the interpeduncular nucleus (central and lateral parts) were also labelled. The distribution of a6 mRNA was compared with the distribution of the other known nicotinic acetylcholine receptor subunit mRNAs. In several nuclei, the expression of a6 was complementary to those of other a subunits. Moreover, some of the cell groups (such as the substantia nigra, the ventral tegmental area and the locus coeruleus) previously thought to contain mainly a3 mRNA in fact were found to contain high levels of α6 mRNA. Finally, we found extensive colocalization of α6 and p3, indicating the possible existence of nicotinic receptor hetero‐oligomers containing both subunits. The present results show that a6 is the major nicotinic acetylcholine receptor a subunit expressed in dopaminergic cell groups of the mesencephalon and noradrenergic cells of the locus coeruleus. This suggests the involvement of the a6 subunit in some of the major functions of central nicotinic circuits, including the modulation of locomotor behaviour and reward.

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.

Integrated events in central dopamine transmission as analyzed at multiple levels. Evidence for intramembrane adenosine A2A/dopamine D2 and adenosine A1/dopamine D1 receptor interactions in the basal ganglia

An analysis at the network and membrane level has provided evidence that antagonistic interactions between adenosine A2A/dopamine D2 and adenosine A1/dopamine D1 receptors in the ventral and dorsal striatum are at least in part responsible for the motor stimulant effects of adenosine receptor antagonists like caffeine and for the motor depressant actions of adenosine receptor agonists. The results obtained in stably cotransfected cells also underline the hypothesis that the intramembrane A2A/D2 and A1/D1 receptor interactions represent functionally important mechanisms that may be the major mechanism for the demonstrated antagonistic A2A/D2 and A1/D1 receptor interactions found in vivo in behavioural studies and in studies on in vivo microdialysis of the striopallidal and strioentopeduncular GABAergic pathways. A major mechanism for the direct intramembrane A2A/D2 and A1/D1 receptor interactions may involve formation of A2A/D2 and A1/D1 heterodimers leading to allosteric changes that will alter the affinity as well as the G protein coupling and thus the efficacy to control the target proteins in the membranes. This is the first molecular network to cellular integration in the nerve cell membrane and may be well suited for a number of integrated tasks and can be performed in a short-time scale, in comparison with the very long-time scale observed when receptor heteroregulation involves phosphorylation or receptor resynthesis. Multiple receptor-receptor interactions within the membranes through formation of receptor clusters may lead to the storage of information within the membranes. Such molecular circuits can represent hidden layers within the membranes that substantially increase the computational potential of neuronal networks. These molecular circuits are biased and may therefore represent part of the molecular mechanism for the storage of memory traces (engrams) in the membranes.

Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors.

The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the α4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the α4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I α‐conotoxin MII. These receptors are comprised of the α4, β2 and α6 nAChR subunits and non‐α4, β2, and α6 nAChR subunits. Non‐α4 subunit‐containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild‐type mice. Despite the fact that both wild‐type and α4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine‐elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild‐type and mutant mice in basal activity in both habituated and non‐habituated environments. Interestingly, mutant mice sustain an increase in cocaine‐elicited locomotor activity longer than wild‐type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that α4‐containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.

Increased neurodegeneration during ageing in mice lacking high-affinity nicotine receptors

We have examined neuroanatomical, biochemical and endocrine parameters and spatial learning in mice lacking the β2 subunit of the nicotinic acetylcholine receptor (nAChR) during ageing. Aged β2−/− mutant mice showed region‐specific alterations in cortical regions, including neocortical hypotrophy, loss of hippocampal pyramidal neurons, astro‐ and microgliosis and elevation of serum corticosterone levels. Whereas adult mutant and control animals performed well in the Morris maze, 22‐ to 24‐month‐old β2−/− mice were significantly impaired in spatial learning. These data show that β2 subunit‐containing nAChRs can contribute to both neuronal survival and maintenance of cognitive performance during ageing. β2−/− mice may thus serve as one possible animal model for some of the cognitive deficits and degenerative processes which take place during physiological ageing and in Alzheimers disease, particularly those associated with dysfunction of the cholinergic system.

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.

D2R striatopallidal neurons inhibit both locomotor and drug reward processes.

The specific functions of dopamine D2 receptor–positive (D2R) striatopallidal neurons remain poorly understood. Using a genetic mouse model, we found that ablation of D2R neurons in the entire striatum induced hyperlocomotion, whereas ablation in the ventral striatum increased amphetamine conditioned place preference. Thus D2R striatopallidal neurons limit both locomotion and, unexpectedly, drug reinforcement.

Neuroprotection via nAChRs: the role of nAChRs in neurodegenerative disorders such as Alzheimer's and Parkinson's disease.

Epidemiological studies have identified a negative correlation between smoking and the development of neurodegenerative disorders such as Parkinsons disease, and in some studies, Alzheimers disease. These findings have been attributed to the ability of nicotine to act as a neuroprotective agent. A large number of studies demonstrate that nicotine can protect against neuronal death in vitro and in vivo, and the mechanisms underlying the ability of nicotine to protect against excitotoxicity and amyloid-? toxicity are beginning to be elucidated. Despite the compelling evidence that nicotine is neuroprotective, it is clear that nicotine can be toxic under some circumstances. The balance between nicotine neuroprotection and toxicity depends on dose, developmental stage and regimen of administration. Therefore, a full understanding of the molecular and cellular effects of nicotine on signaling pathways relevant to neuronal survival is critical for informed drug discovery of nicotinic compounds to combat human neurodegeneration. This review summarizes recent studies related to the mechanisms underlying nicotine-mediated neuroprotection, and addresses issues that are relevant to use of nicotine as a neuroprotective agent in vivo.

Mapping and computer assisted morphometry and microdensitometry of glucocorticoid receptor immunoreactive neurons and glial cells in the rat central nervous system.

By means of a monoclonal mouse immunoglobulin G2a antibody against the rat liver glucocorticoid receptor and the indirect immunoperoxidase technique, the distribution of glucocorticoid receptors in neuronal and glial cell populations was mapped in the central nervous system of the male rat. The mapping was complemented by computer-assisted morphometric and microdensitometric evaluation of glucocorticoid receptor immunoreactivity in many brain regions. The quantitative analysis allowed us to achieve for the first time an objective characterization of glucocorticoid receptor distribution in the CNS, thus avoiding the ambiguities of previous mapping studies based on subjective evaluations. In addition, a taxonomic analysis of central nervous system regions containing glucocorticoid receptor immunoreactivity was carried out utilizing the quantitative parameters obtained in the morphometric evaluation. Nuclei of neuronal and glial cells containing glucocorticoid receptor immunoreactivity were detected in a widespread, but still highly heterogeneous, fashion in the central nervous system, underlining the view that glucocorticoids can control a large number of central nervous system target cells via effects on gene expression. Many nerve cell populations have been shown to contain substantial amounts of nuclear glucocorticoid receptor immunoreactivity, whereas only a low density of glial cells, in both gray and white matter, show nuclear glucocorticoid receptor immunoreactivity. Thus, in most brain areas, the major target for glucocorticoids appears to be the nerve cells. Interestingly, an inverse correlation was found in the regional density of glucocorticoid receptor-immunoreactive nerve and glial cells, suggesting that glucocorticoids may influence a brain area either via glial cells or, more frequently, via nerve cells. The results on mapping highlight the impact of glucocorticoids in areas both traditionally and not traditionally involved in stress responses. The distribution of glucocorticoid receptor immunoreactivity also emphasizes a role of glucocorticoids in the regulation of the afferent regions of the basal ganglia and the cerebellar cortex, and of both afferent and efferent layers of the cerebral cortex. Glucocorticoid receptor immunoreactivity is widely distributed over the thalamus, probably leading to modulation of activity in the various thalamocortical pathways transmitting inter alia specific sensory information to the cerebral cortex. Many unspecific afferents to the cerebral cortex are potentially regulated by glucocorticoid receptors such as the noradrenaline and 5-hydroxytryptamine afferents, since their nerve cells of origin contain strong glucocorticoid receptor immunoreactivity. Eight brain regions involving sensory, motor and limbic areas were shown to have a similarity with regard to glucocorticoid receptor-immunoreactive parameters at the level of 95%. The density of glucocorticoid receptor-immunoreactive nerve cells appeared to be the main factor in determining such a very high level of similarity. Overall, our results emphasize that glucocorticoids may appropriately tune networks of different areas to obtain optimal integration and in this way improve survival of the animal under challenging conditions.