Summary α6-containing (α6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function α6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating α6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. α6L9’S mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of α6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at α6* provides fine-tuning of DA release and locomotor responses. α6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms, provide a new method for manipulating DA transmission in Parkinson’s disease, nicotine dependence, or attention deficit hyperactivity disorder.

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to ␤ 1 -adrenergic receptor (␤ 1 AR)-selective activation 3.7-fold, to ␤ 2 AR-selective activation only 1.6-fold and to prostaglandin E 2 (PGE 2 ) not at all. Therefore, the rank order of efficacy in coupling to AC6 is ␤ 1 AR > ␤ 2 AR > prostaglandin E 2 receptor (EP 2 R). ␤ 2 AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing ␤ARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, ␤ 1 AR, and ␤ 2 AR, but not EP 2 R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by ␤AR subtypes but lack thereof by PGE 2 . When cardiomyocytes were stimulated with a ␤AR agonist, ␤ 2 AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of ␤ARKct. Thus, agonist-induced translocation of ␤ 2 AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of ␤ 2 AR coupling to AC6 as compared with ␤ 1 AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.

The RGS7 (R7) family of RGS proteins bound to the divergent Gβ subunit Gβ5 is a crucial regulator of G protein–coupled receptor (GPCR) signaling in the visual and nervous systems. Here, we identify R7BP, a novel neuronally expressed protein that binds R7–Gβ5 complexes and shuttles them between the plasma membrane and nucleus. Regional expression of R7BP, Gβ5, and R7 isoforms in brain is highly coincident. R7BP is palmitoylated near its COOH terminus, which targets the protein to the plasma membrane. Depalmitoylation of R7BP translocates R7BP–R7–Gβ5 complexes from the plasma membrane to the nucleus. Compared with nonpalmitoylated R7BP, palmitoylated R7BP greatly augments the ability of RGS7 to attenuate GPCR-mediated G protein–regulated inward rectifying potassium channel activation. Thus, by controlling plasma membrane nuclear–shuttling of R7BP–R7–Gβ5 complexes, reversible palmitoylation of R7BP provides a novel mechanism that regulates GPCR signaling and potentially transduces signals directly from the plasma membrane to the nucleus.

Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express α6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive α6L9′S* receptors. α6L9′S mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by α6α4* pentamers, as α6L9′S mice lacking α4 subunits displayed essentially normal behavior. In α6L9′S mice, receptor numbers are normal, but loss of α4 subunits leads to fewer and less sensitive α6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires α4 subunits, implicating α6α4β2* nAChRs in α6L9′S mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by α6L9′S nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in α6L9′S, but not WT or α4KO/α6L9′S, mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from α6L9′S presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate α6α4β2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we ®nd that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to signi®cant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3±Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-speci®c deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3D and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability. Keywords: histone deacetylase/nucleolus/rDNA/RNA polymerase I/target of rapamycin IntroductionControl of cell growth and proliferation requires that cells rapidly change their protein biosynthetic capacity in response to nutrients and mitogens as well as stressful conditions. Modulation of protein synthesis involves coordinated changes in both the rate of translational initiation and the abundance of ribosomes. Ribosome concentration¯uctuates in response to growth conditions (Venema and Tollervey, 1999;Warner, 1999;Leary and Huang, 2001;Fatica and Tollervey, 2002;Moss and Stefanovsky, 2002;Peculis, 2002;Grummt, 2003). When cells are growing under favorable conditions, a high ribosome concentration is needed to meet the demand for protein synthesis. However, ribosome biogenesis is a high energy-and nutrient-consuming process. In yeast, for instance, several hundred genes participate in the production of ribosomes, involving all three RNA polymerases and accounting for a major portion of the total cellular biosynthetic output (Venema and Tollervey, 1999;Warner, 1999;Leary and Huang, 2001;Fatica and Tollervey, 2002;Moss and Stefanovsky, 2002;Peculis, 2002;Grummt, 2003). Ribosomal DNA (rDNA) transcription is an important initial step for ribosome biogenesis, producing rRNA products that represent 60% of total yeast transcripts during normal growth. To conserve resources, cells must limit the production of new ribosomes during nutrient starvation. On the other hand, deregulation of ribosome biogenesis has been implicated in uncontrolled growth and tumorigenesis in mammalian cells (Ruggero and Pandol®, 2003).The nucleolus is the site of rDNA transcription by RNA polymerase I (Pol I), rRNA maturation and assembly of ribosomes (Scheer and Hock, 1999). There are~150 tandem copies of rDNA genes in yeast. In addition to the components of Pol I and rDNAs, the nucleolus contains many other nucleolar proteins and small nucleolar RNAs...

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that ␣3 and ␤4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, ␣6, ␤2, ␤3, and ␣4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 M nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson’s disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6β2β3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the β3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4β2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6β2β3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, α4β2* and α6β2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The α4β2*-nAChRs are widely distributed, while α6β2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the meso-accumbens pathway thought to be essential for reward and addiction. α4β2*-nAChR also modulate GABA release in these areas.Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing hα7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, α4β2*, α6β2*, α7 and α3β4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at α4β2* and α6β2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for α6β2*-nAChR.