Cholinergic Modulation of Locomotion and Striatal Dopamine Release Is Mediated by α6α4* Nicotinic Acetylcholine Receptors

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 α6^(L9’S*) receptors. α6^(L9’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 α6 α4* pentamers, as α6^(L9’S) mice lacking α4 subunits displayed essentially normal behavior. In α6^(L9’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 α6^(L9’S) mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by α6^(L9’S) nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in α6^(L9’S), but not WT or α4KO/ α6^(L9’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 α6^(L9’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

Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro: Mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells

α6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of α6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-α6 and β2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 μM ACh in 26% (5/19) of cells with evenly expressed α6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional α6-eGFPβ2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into β2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased α6-eGFPβ2 nAChR current amplitude. α6-eGFPβ2 nAChRs were also activated by nicotine and by TC-2403. The α6-eGFPβ2 currents were desensitized by 1 μM nicotine, blocked by α-conotoxin MII, partially inhibited by dihydro-β-erythroidine, and potentiated by extracellular Ca^(2+). Single-channel recordings showed that α6-eGFPβ2 nAChRs had similar single-channel conductance to, but longer open time than, α4-eGFPβ2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of α6* nAChRs for both pharmacological study and drug screening

Receptor Number and Caveolar Co-Localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase

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 β1-adrenergic receptor (β1AR)-selective activation 3.7-fold, to β2AR-selective activation only 1.6-fold and to prostaglandin E2 (PGE2) not at all. Therefore, the rank order of efficacy in coupling to AC6 is β1AR \u3e β2AR \u3e prostaglandin E2 receptor (EP2R). β2AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing βARKct, 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, β1AR, and β2AR, but not EP2R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by βAR subtypes but lack thereof by PGE2. When cardiomyocytes were stimulated with a βAR agonist, β2AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of βARKct. Thus, agonist-induced translocation of β2AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of β2AR coupling to AC6 as compared with β1AR, 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

Differential Expression and Function of Nicotinic Acetylcholine Receptors in Subdivisions of Medial Habenula

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 α3 and β4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, β2, β3, and α4 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 μm 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

Structural differences determine the relative selectivity of nicotinic compounds for native α4β2^*-, α6β2^*-, α3β4^*- and α7-nicotine acetylcholine receptors

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 mesoaccumbens 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 compoundswere screened usingmembrane 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 α4b2^* and α6β2^*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for α6β2^*-nAChR

α6* Nicotinic Acetylcholine Receptor Expression and Function in a Visual Salience Circuit

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway

Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors

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

Photoactivatable drugs for nicotinic optopharmacology

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales

Insights into the Neurobiology of the Nicotinic Cholinergic System and Nicotine Addiction from Mice Expressing Nicotinic Receptors Harboring Gain-of-Function Mutations

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated, cation-selective ion channels expressed throughout the brain. Although these channels have been investigated for several decades, it is still challenging 1) to identify the important nAChR subunits in cholinergic transmission and nicotine dependence and 2) to develop nAChR subtype-specific ligands. To overcome these challenges, we and others have studied mice expressing mutant, gain-of-function nAChR subunits. In this review, we discuss this research approach and the results it has yielded to date. Gain-of-function mutations, including those in nAChR subunits, provide an approach that is complementary to loss-of-function studies such as gene knockouts; the former allows one to answer questions of sufficiency and the latter addresses questions of necessity. Mutant mice expressing gain-of-function nAChR subunits are commonly produced using traditional gene targeting in embryonic stem cells, but novel approaches such as bacterial artificial chromosome transgenesis have yielded important insights as well. α7 nAChRs were the first nAChRs to be targeted with a gain-of-function mutation, followed by a pair of α4 nAChR gain-of-function mutant mice. These α4 nAChR gain-of-function mice (α4 L9′S mice, followed by α4 L9′A mice) provided an important system to probe α4 nAChR function in vivo, particularly in the dopamine reward system. α6 nAChR gain-of-function mice provided the first robust system allowing specific manipulation of this receptor subtype. Other targeted mutations in various nAChR subunits have also been produced and have yielded important insights into nicotinic cholinergic biology. As nAChR research advances and more details associated with nAChR expression and function emerge, we expect that existing and new mouse lines expressing gain-of-function nAChR subunits will continue to provide new insights