Naturally expressed nicotinic acetylcholine receptors composed of ␣4 and ␤2 subunits (␣4␤2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human ␣4␤2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human ␣4 and ␤2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed ␣4␤2-nAChR engage in high-affinity, specific binding of Rbϩ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-␤-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human ␣4␤2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human ␣4␤2-nAChR.

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified. We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.

Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR ␣6 subunit message is abundant in the VTA, but ␣6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for ␣6␤2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. ␣-Conotoxin (␣-Ctx) MII labeling coupled with immunocytochemical staining localizes putative ␣6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA A receptors. These increases are abolished by ␣6*-nAChR-selective ␣-Ctx MII or ␣-Ctx PIA (1 nM) but not by ␣7 (10 nM methyllycaconitine) or ␣4* (1 M dihydro-␤-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR ␤2 knock-out mice. Moreover, ACh induces an ␣-Ctx PIA-sensitive elevation in intraterminal Ca 2ϩ in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nM nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that ␣6␤2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic ␣6␤2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.

Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.

Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10 mM) enhanced GABA receptor (GABA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100 mM) had no effect on optically evoked GABA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse.

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing ␣6 subunits (␣6* nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human ␣6␤2, ␣6␤4, ␣6␤2␤3, ␣6␤4␤3, or ␣6␤4␤3␣5 nAChR. Cells expressing subunits needed to form ␣6␤4␤3␣5 nAChR exhibited saturable [ 3 H]epibatidine binding (K d ϭ 95.9 Ϯ 8.3 pM and B max ϭ 84.5 Ϯ 1.6 fmol/mg of protein). The rank order of binding competition potency (K i ) for prototypical nicotinic compounds was ␣-conotoxin MII (6 nM) Ͼ nicotine (156 nM) ϳ methyllycaconitine (200 nM) Ͼ ␣-bungarotoxin (Ͼ10 M), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that ␤3 and ␣5 subunits are likely partners of the ␣6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that ␣6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human ␣5 subunit makes a critical contribution to ␣6␤4␤3␣5 nAChR assembly into a ligand-binding form with native ␣6*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.Distinct nicotinic acetylcholine receptor (nAChR) subtypes expressed in mesostriatal dopaminergic neurons are involved in modulation of striatal dopamine (DA) release (Wonnacott, 1997;Champtiaux et al., 2002;Luetje, 2004); nAChR containing ␣4 and ␤2 subunits (␣4␤2* nAChR) or containing ␣6 subunits (␣6* nAChR) participate directly, and ␣7* nAChR participate indirectly. Whereas the biochemistry of ␣4␤2* and ␣7* nAChR is well characterized, the subunit composition and functional properties of ␣6* nAChR remain unclear. Suggested roles for ␣6* nAChR in modulation of DA transmission imply their potential importance in locomotion, reward, schizophrenia, and Parkinson's disease (le Novère et al

Unlike many other sexually dimorphic systems, gonadal secretions do not explain sex differences in the morphology of the telencephalic song control nuclei of zebra finches. It is important to understand whether a novel mechanism for controlling structure is restricted to the forebrain regions specialized for song, and whether other areas respond more typically to gonadal steroids. Therefore, the effects of sex and adult androgen manipulation on the neuromuscular end of the song control system (tracheosyringeal portion of the hypoglossal nucleus, nXIIts, and the syrinx, or vocal organ) were investigated. Because lesion and axotomy experiments indicate a right-side bias in the functional control of song, asymmetry in the motor nucleus and in the ventralis and dorsalis syrinx muscles was also tested. Male-biased dimorphisms existed in the volume of nXIIts, and in syrinx mass and size of muscle fibers, but not in motoneuron number or size. Asymmetries favoring the right side were detected in nXIIts volume and motoneuron number in males, as well as in ventralis and dorsalis fiber size in both sexes. Hormone manipulations had no effect on nXIIts size, neuron size, or number. Testosterone treatment of adult females increased all of the syringeal measures, but the only effect of flutamide in males was to decrease syrinx weight. Thus, male-biased sexual dimorphisms and right side dominance in both nXIIts and the syrinx may facilitate singing behavior. Adult androgen exposure can induce partial masculinization of the syrinx, but other factors must be important in mediating the sex differences in both that structure and the volume of nXIIts.

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