Nicotinic receptors mediate stress-nicotine detrimental interplay via dopamine cells' activity.

Epidemiological studies report strong association between mood disorders and tobacco addiction. This high comorbidity requires adequate treatment but the underlying mechanisms are unknown. We demonstrate that nicotine exposure, independent of drug withdrawal effects, increases stress sensitivity, a major risk factor in mood disorders. Nicotine and stress concur to induce long-lasting cellular adaptations within the dopamine (DA) system. This interplay is underpinned by marked remodeling of nicotinic systems, causing increased ventral tegmental area (VTA) DA neurons' activity and stress-related behaviors, such as social aversion. Blocking ß2 or α7 nicotinic acetylcholine receptors (nAChRs) prevents, respectively, the development and the expression of social stress-induced neuroadaptations; conversely, facilitating α7 nAChRs activation specifically in the VTA promotes stress-induced cellular and behavioral maladaptations. Our work unravels a complex nicotine-stress bidirectional interplay and identifies α7 nAChRs as a promising therapeutic target for stress-related psychiatric disorders.

GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons.

Human mutations of the GRID1 gene encoding the orphan delta1 glutamate receptor-channel (GluD1) are associated with schizophrenia but the explicit role of GluD1 in brain circuits is unknown. Based on the known function of its paralog GluD2 in cerebellum, we searched for a role of GluD1 in slow glutamatergic transmission mediated by metabotropic receptor mGlu1 in midbrain dopamine neurons, whose dysfunction is a hallmark of schizophrenia. We found that an mGlu1 agonist elicits a slow depolarizing current in HEK cells co-expressing mGlu1 and GluD1, but not in cells expressing mGlu1 or GluD1 alone. This current is abolished by additional co-expression of a dominant-negative GluD1 dead pore mutant. We then characterized mGlu1-dependent currents in dopamine neurons from midbrain slices. Both the agonist-evoked and the slow postsynaptic currents are abolished by expression of the dominant-negative GluD1 mutant, pointing to the involvement of native GluD1 channels in these currents. Likewise, both mGlu1-dependent currents are suppressed in GRID1 knockout mice, which reportedly display endophenotypes relevant for schizophrenia. It is known that mGlu1 activation triggers the transition from tonic to burst firing of dopamine neurons, which signals salient stimuli and encodes reward prediction. In vivo recordings of dopamine neurons showed that their spontaneous burst firing is abolished in GRID1 knockout mice or upon targeted expression of the dominant-negative GluD1 mutant in wild-type mice. Our results de-orphanize GluD1, unravel its key role in slow glutamatergic transmission and provide insights into how GRID1 gene alterations can lead to dopaminergic dysfunctions in schizophrenia.

RACK1 cooperates with NRASQ61K to promote melanoma in vivo.

Melanoma is the deadliest skin cancer. RACK1 (Receptor for activated protein kinase C) protein was proposed as a biological marker of melanoma in human and domestic animal species harboring spontaneous melanomas. As a scaffold protein, RACK1 is able to coordinate the interaction of key signaling molecules implicated in both physiological cellular functions and tumorigenesis. A role for RACK1 in rewiring ERK and JNK signaling pathways in melanoma cell lines had been proposed. Here, we used a genetic approach to test this hypothesis in vivo in the mouse. We show that Rack1 knock-down in the mouse melanoma cell line B16 reduces invasiveness and induces cell differentiation. We have developed the first mouse model for RACK1 gain of function, Tyr::Rack1-HA transgenic mice, targeting RACK1 to melanocytes in vivo. RACK1 overexpression was not sufficient to initiate melanomas despite activated ERK and AKT. However, in a context of melanoma predisposition, RACK1 overexpression reduced latency and increased incidence and metastatic rate. In primary melanoma cells from Tyr::Rack1-HA, Tyr::NRasQ61K mice, activated JNK (c-Jun N-terminal kinase) and activated STAT3 (signal transducer and activator of transcription 3) acted as RACK1 oncogenic partners in tumoral progression. A sequential and coordinated activation of ERK, JNK and STAT3 with RACK1 is shown to accelerate aggressive melanoma development in vivo.

The α3ß4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies.

BACKGROUND AND PURPOSE: Recent data have indicated that α3ß4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES: To assess the role of α3ß4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS: BXD recombinant mouse lines demonstrated an increased expression of α3, ß4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and ß4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3ß4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4ß2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS: Overall, our findings suggest an important role for the α3ß4* nACh receptor subtype in morphine physical dependence.

Nicotine consumption is regulated by a human polymorphism in dopamine neurons.

Smoking is the most important preventable cause of morbidity and mortality worldwide. Recent genome-wide association studies highlighted a human haplotype on chromosome 15 underlying the risk for tobacco dependence and lung cancer. Several polymorphisms in the CHRNA3-CHRNA5-CHRNB4 cluster coding for the nicotinic acetylcholine receptor (nAChR) α3, α5 and ß4 subunits were implicated. In mouse models, we define a key role in the control of sensitivity to nicotine for the α5 subunit in dopaminergic (DAergic) neurons of the ventral tegmental area (VTA). We first investigated the reinforcing effects of nicotine in drug-naive α5(-/-) mice using an acute intravenous nicotine self-administration task and ex vivo and in vivo electrophysiological recordings of nicotine-elicited DA cell activation. We designed lentiviral re-expression vectors to achieve targeted re-expression of wild-type or mutant α5 in the VTA, in general, or in DA neurons exclusively. Our results establish a crucial role for α5*-nAChRs in DAergic neurons. These receptors are key regulators that determine the minimum nicotine dose necessary for DA cell activation and thus nicotine reinforcement. Finally, we demonstrate that a single-nucleotide polymorphism, the non-synonymous α5 variant rs16969968, frequent in many human populations, exhibits a partial loss of function of the protein in vivo. This leads to increased nicotine consumption in the self-administration paradigm. We thus define a critical link between a human predisposition marker, its expression in DA neurons and nicotine intake.

Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement.

Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.

Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration.

The identification of the molecular mechanisms involved in nicotine addiction and its cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were developed to match this aim. Although the beta2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement properties of nicotine, little is known about the contribution of the different alpha subunit partners of beta2 (i.e., alpha4 and alpha6), the homo-pentameric alpha7, and the brain areas other than the ventral tegmental area (VTA) involved in nicotine reinforcement. In this study, nicotine (8.7-52.6 microg free base/kg/inf) self-administration was investigated with drug-naive mice deleted (KO) for the beta2, alpha4, alpha6 and alpha7 subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT mice, beta2-VEC mice with the beta2 subunit re-expressed exclusively in the VTA, alpha4-VEC mice with selective alpha4 re-expression in the VTA, alpha6-VEC mice with selective alpha6 re-expression in the VTA, and alpha7-KO mice promptly self-administer nicotine intravenously, whereas beta2-KO, beta2-VEC in the substantia nigra, alpha4-KO and alpha6-KO mice do not respond to nicotine. We thus define the necessary and sufficient role of alpha4beta2- and alpha6beta2-subunit containing nicotinic receptors (alpha4beta2*- and alpha6beta2*-nAChRs), but not alpha7*-nAChRs, present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drug-naive mice.

The cholinergic mesopontine tegmentum is a relatively neglected nicotinic master modulator of the dopaminergic system: relevance to drugs of abuse and pathology.

The mammalian mesopontine tegmentum (MPT) contains two cholinergic nuclei, the pedunculopontine tegmental nucleus (PPTg) and the laterodorsal tegmental nucleus (LDTg). These provide the cholinergic innervation of, among other brain areas, the dopaminergic A9 and A10 cell groups. Their axons are thus the source of endogenous acetylcholine (ACh) acting on somato-dendritic acetylcholine receptors in the substantia nigra (SN) and ventral tegmental area (VTA). The anatomy, physiology, functional and pathological implications of these interactions with the nicotinic subtype of acetylcholine receptors (nAChRs) are discussed with a view of the important role of the MPT as a master regulator of nicotinic dopaminergic signalling in the brain, including for nicotine addiction.

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.

Targeted in vivo expression of nicotinic acetylcholine receptors in mouse brain using lentiviral expression vectors.

Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiquitous distribution. Yet, except for providing a receptor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lentiviral expression vector to re-express the beta2 subunit specifically in the ventral tegmental area (VTA) of beta2-/- mice. The viral vector efficiently expresses beta2- subunit protein leading to new nAChR-binding sites. VTA neurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing beta2-/- mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and beta2-subunit re-expressing beta2-/- mice, but not in beta2-/- mice. Furthermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for beta2* nAChR, specifically expressed in the VTA, in mammalian cognitive function.

Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors.

Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the beta2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying beta2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that beta2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.

Neural cells without functional N-Methyl-D-Aspartate (NMDA) receptors contribute extensively to normal postnatal brain development in efficiently generated chimaeric NMDA R1 -/- <--> +/+ mice.

Embryonic stem (ES) cells have revolutionised our understanding of animal physiology. Analysis of chimaeric mice generated from these cells allows us to study the role of genes in development and function of the nervous system. The NMDA receptor, one of the two major ionotropic glutamate receptors, has been proposed to play fundamental roles in the survival, migration, differentiation, and activity-dependent maturation of neural cells. The NMDA receptor subunit 1 (NR1) gene is indispensable for receptor function, and knock-out mice die at birth, inhibiting the study of glutamate signalling in postnatal neurons. Homozygous NR1-/- ES cells were derived from matings of heterozygous mice under feeder-free conditions. Chimaeras were made by incorporating these ES cells into wild-type blastocysts and by the classical aggregation of morulae between wild-type and NR1-/- embryos. The resulting chimaeras survive and develop normally. NR1-/- neurons, identified by their lacZ label, were analysed and quantified in developing and adult brains with varying knock-out contributions in every single brain region. Specifically, postnatal ontogenesis of cerebellum and hippocampus was normal. Accordingly, in chimaeric mice, NMDA receptor-initiated signals are not required for the migration, differentiation, and survival of most types of neurons in the central nervous system, in a cell-autonomous way.

Long-term survival, migration, and differentiation of neural cells without functional NMDA receptors in vivo.

The NMDA receptor, one of the two major ionotropic glutamate receptors, has been proposed to play fundamental roles in the survival, migration, differentiation, and activity-dependent maturation of neural cells. The NR1 gene encodes the major subunit that is responsible for channel function, and NR1 -/- mice die at birth, inhibiting the study of glutamate signaling in postnatal neurons. The properties of cells lacking the NR1 subunit of NMDA receptors were studied by transplanting dissociated telencephalic, diencephalic, and mesencephalic cells of E14 mouse embryos with a targeted deletion of the NR1 gene into the ventricles of embryonic rats using intrauterine transplantation (Brüstle et al., 1995, Neuron 15, 1275-1285). The transplanted cells took part in the normal development of the host brain where they survived after migration into a large number of brain structures. Morphological and immunohistochemical analysis suggests that NR1 -/- cells can differentiate normally in these sites. The results provide evidence that NMDA-receptor-initiated signals are not required for the postnatal differentiation and survival of many types of neurons in the central nervous system, in a noncell autonomous fashion after transplantation into a wild-type environment.

Host-guided migration allows targeted introduction of neurons into the embryonic brain.

The stereotyped positions occupied by individual classes of neurons are a fundamental characteristic of CNS cytoarchitecture. To study the regulation of neuronal positioning, we injected genetically labeled neural precursors derived from dorsal and ventral mouse forebrain into the telencephalic vesicles of embryonic rats. Cells from both areas were found to participate in the generation of telencephalic, diencephalic, and mesencephalic brain regions. Donor-derived neurons populated the host brain in distinct patterns and acquired phenotypic features appropriate for their final location. These observations indicate that neuronal migration and differentiation are predominantly regulated by non-cell-autonomous signals. Exploiting this phenomenon, intrauterine transplantation allows generation of controlled chimerism in the mammalian brain.

Parallel synthesis and analysis of large numbers of related chemical compounds: applications to oligonucleotides.

This review presents methods for addressing the issue of molecular complexity in biological systems. Biomolecules immobilised on solid phases can be used to probe biological interactions. As a specific example, arrays of large numbers of oligonucleotides allow the analysis of DNA sequences and complex populations of DNA molecules. The specific embodiments of this new method are presented, research up to mid-1992 outlined and requirements for future development discussed.

A study of oligonucleotide reassociation using large arrays of oligonucleotides synthesised on a glass support.

An extensive analysis of oligonucleotide interactions was carried out by hybridising a synthetic pool of 256 10mers, A(C,T)8A, representing all oligopyrimidine octamer sequences to an array of four copies of all 256 different octapurine sequences. The resulting 256 duplexes were quantified by phosphorimaging and analysed to determine the dependence of duplex formation on base composition, sequence, and salt concentration. The results show that the base composition dependence of duplex formation can be reduced by high concentrations of tetramethylammonium chloride. This chaotropic solvent also increases duplex yield by up to fifty-fold.

Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

An efficient method was developed for making complete sets of oligonucleotides of defined length, covalently attached to the surface of a glass plate, by synthesizing them in situ. A device carrying all octapurine sequences was used to explore factors affecting molecular hybridization of the tethered oligonucleotides, to develop computer-aided methods for analyzing the data, and to test the feasibility of using the method for sequence analysis. Further development is needed before the method can be used routinely, but our work shows that it has a number of potential advantages over gel-based methods: it should be easy to automate; the quality of the sequence results can be evaluated statistically; it provides a powerful way of comparing related sequences and detecting mutation; it can be applied to both DNA and RNA; and specific motifs can be incorporated into all sequences of the array to focus analysis on sequences of biological interest.