Alpha1 Subunit Research Articles

N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells

N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements (ChATBACEGFP) were cultured as tissue explants for 5 days and cocultured with transfected CHO cells for an additional 2 days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing α3 and β4 nicotinic acetylcholine receptor (nAChR) subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs) and miniature EPSPs (mEPSPs). EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin (TTX). These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.

Visual Function in CaV1.4-Deficient Mice

CACNA1F encodes the alpha1 subunit of retina-specific Cav1.4 voltage-gated L type calcium channels. Mutations in this gene cause congenital stationary night blindness type 2A (CSNB2), Aland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). The clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. The reasons for this variability are not known. To gain more insights into the pathophysiology caused by loss of Cav1.4 function we analyzed the visual phenotype of Cav1.4-deficient mice. To this end, we combined immunohistochemistry, electroretinography (ERG) and vision-dependent behavioral testing. Immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in Cav1.4-deficient mice. Heterozygous Cav1.4 mice showed mosaic synaptic defects most probably caused by random X-chromosomal inactivation of the healthy allele. Electroretinography revealed a loss of scotopic and photopic photoreceptor function. This loss of retinal network function resulted in impaired perfomance of Cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. In conclusion, loss of Cav1.4 channels strongly impairs rod and cone retinal function and vision in mice.

Gem Stabilizes Voltage-Gated Calcium Channels in the Inactivated State: Implications for Human Disease

RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that often colocalize with and strongly inhibit high-voltage activated (HVA) calcium channels (L-, N-, P/Q- and R-type). The mechanism of inhibition is largely unclear. The pore-forming alpha-1 subunit of HVA calcium channels requires the beta subunit for cell surface trafficking and gating. We previously showed direct binding between alpha-1 and Gem and obtained a Gem-insensitive chimeric P/Q alpha-1containing the IIS1-S3 region of a Gem-resistant, low-voltage activated T-type channel (Fan et al., PNAS, 2010). In this study, we investigated the cause of this insensitivity. Since the IIS1-S3 region of P/Q alpha-1 has too few cytosolic amino acids to be a Gem binding site, and since we had identified a different alpha-1 region that binds Gem, we focused on altered biophysical properties of the chimera. We observed that the chimeric channel inactivated much slower than WT P/Q channel did; we therefore examined whether slow inactivation was responsible for Gem insensitivity. Mutating residues known to increase channel inactivation enhanced Gem inhibition, whereas mutating residues known to slow channel inactivation weakened or abolished Gem inhibition. Replacing beta3 with beta2a, an auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition. Furthermore, recovery from inactivation of WT P/Q channels in the presence of Gem was markedly slower than without Gem. Thus, Gem appeared to stabilize the channel in the inactivated state. These results shed light on a new factor - altered regulation by RGK proteins - that may contribute to HVA calcium channel-related channelopathies. Indeed, we found that familial hemiplegic migraine mutations that speed P/Q channel inactivation strengthened Gem inhibition, whereas Timothy syndrome mutations that slow channel inactivation greatly weakened Gem inhibition.

Changes in beta-2 adrenergic receptor and AMP-activated protein kinase alpha-2 subunit in the rat vestibular nerve after labyrinthectomy

In the present study, to elucidate the role of vestibular ganglion (VG) after the unilateral labyrinthine damage, we examined quantitative changes in mRNA expression of beta-adrenergic receptors (bARs) and AMP-activated protein kinase alpha catalytic subunits (aAMPKs) in VG after unilateral labyrinthectomy (UL) in rats. Using the real-time PCR method, beta2 AR mRNA expression in bilateral VG and AMPK alpha2 mRNA expression in the ipsilateral VG were significantly up-regulated with the maximum increase at the postoperative 7 day and 1 day, respectively. The up-regulation of beta2 AR in bilateral VG was long-lasting until 28 days after UL and that of AMPK alpha2 in the ipsilateral VG was just transient within 7 days after UL. These mRNA changes were supported by immunohistochemical data. According to previous reports, both of bARs and aAMPKs could regulate mitochondrial uncoupling protein (UCP) mRNA expression in several kinds of tissues and therefore might have thermogenic neurotransmission and antioxidant neuroprotective roles in neuronal tissues. UL requires not only long-lasting response of VG for central vestibular neuro-plasticity around 2–4 weeks but rapid response of VG against apoptosis of peripheral vestibular epithelia-neuronal synapses. The present findings suggest that beta2 AR in bilateral VG and AMPK alpha2 in the ipsilateral VG might play important signaling roles after the unilateral labyrinthine damage.

AMP-activated protein kinase deficiency reduces ozone-induced lung injury and oxidative stress in mice

BackgroundAcute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5'-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.MethodsControl and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr172 phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.ResultsIn control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr172 to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.ConclusionsOur results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.

Treatment-responsive pandysautonomia in an adolescent with ganglionic α3-AChR antibodies

Autoimmune autonomic ganglionopathy (AAG) is a rare disorder that presents with pandysautonomia typically in middle age and elderly patients. AAG is typically associated with serum autoantibodies that bind to the alpha-3 subunit of the ganglionic acetylcholine receptor (α3-AChR Ab). We report a 13 year old girl who presented with gut pseudo-obstruction, bladder dysfunction and dilated pupils unresponsive to pilocarpine. She had positive α3-AChR Ab plus other autoantibodies suggesting an autoimmune diathesis. Our patient was initially resistant to steroid therapy but responded to the addition of azathioprine resulting in a near complete clinical remission. We conclude that pandysautonomia associated with α3-AChR Ab can occur in children and has multi-organ involvement.

Association of CHRNA4 polymorphism with depression and loneliness in elderly males

The cholinergic receptor, nicotinic, alpha 4 (CHRNA4) gene encodes the neuronal nicotinic acetylcholine receptor alpha-4 subunit. Recent research has shown that a variation in CHRNA4 (rs1044396) affects attention and negative emotionality in normal adults. To determine the link between CHRNA4 variation and cognitive function/depressed mood, this study conducted a genotype-phenotype correlation analysis between the common CHRNA4:rs1044396 variant and several baseline parameters of cognition and depressed mood in 192 elderly male subjects without major psychiatric disorders or dementia. Study findings identified a significant link between the CHRNA4:rs1044396 polymorphism and depression and loneliness in the aged. Compared to carriers of at least one T-allele, carriers of the homozygous C/C genotype described themselves as more depressed and lonely. This is the first evidence which may implicate CHRNA4 in depressed emotions in the elderly.

Upregulation of Hepatocellular NTBI Clearance Following Iron Loading Is Associated with Modulation of Membrane T- and L-Type Calcium Channels

Abstract 1038 Background and Rationale.Under conditions of iron overload, the iron binding capacity of transferrin is exceeded and non-transferrin-bound iron (NTBI) species appear in the circulation, at concentrations typically within the range of 0.4–10 μM. NTBI species are rapidly cleared, mainly by the liver but also by the heart and endocrine tissues, causing iron overload of these organs. It is known that pre-loading of hepatocytes or cardiomyocytes with iron causes a paradoxical, dose and time-dependent, increase in NTBI clearance by these cells in vitro through mechanisms that are largely undetermined. In an iron overloaded mouse model, the uptake of ferrous iron by myocardium was related to the expression of L-type voltage–gated calcium channels and blockers of these channels reduced iron accumulation in the heart. However, it is unknown whether a similar mechanism underlies NTBI uptake by the liver. Previous work in our laboratory, using the human HuH7 hepatoma cell line, showed that increments in NTBI uptake that occurred following iron pre-loading of cells, were inhibited by the T-calcium channel inhibitor mibefradil. Methods.Flow cytometry and quantitative confocal microscopy were used to assess for changes in the abundance and subcellular distribution of L- and T-type calcium channels by iron-loading (ferric ammonium citrate [FAC]) of HuH7 hepatocytes. These cells were pre-incubated for 24 hours with increasing concentrations of FAC and then fixed (by 4% paraformaldehyde) or fixed and permeabilised (by 0.1% Triton X-100/PBS). Fixed HuH7 cells in suspension were immuno-stained with rabbit polyclonal antibodies against the alpha-1 subunits D (L-type) or H (T-type) and any changes in L- and T-type calcium channels on the cell surface measured by flow cytometry. Confocal microscopy was also performed on fixed and subsequently permeabilised adherent HuH7 cells, which were then immuno-stained by the same antibodies and subjected to fluorescence analysis using the Volocity program (Perkins Elmer) in order to assess intracellular distribution of these channels. Results.At clinically relevant pre-loading concentrations of 2 μM FAC, flow cytometry of fixed cells showed a 38% increase in T-type calcium channels on the outer membrane but no significant change in L-type channels. However, at higher FAC concentrations (20μM and 200 μM), there was a dose dependent rise in the membrane expression of L-type channels but a fall in T-type channels. With 200 μM FAC, the expression of the L-type channel was almost 3x that of the controls (p<0.0001) while the T-type channels were reduced to 65% of the untreated cells (p=0.03).Confocal microscopy of fixed and permeabilised cells showed similar effects on calcium channels following iron pre-loading. At. the clinically relevant concentrations of 2 μM iron, cellular images and line profiles exhibited greater over-all and outer membrane T-type fluorescence than controls, while L-type channels remained unaffected. By contrast, at higher pre-loading concentrations of FAC (20μM and 200μM), dose dependent increases in L-type fluorescence were observed, particularly on the cell membrane, whereas T-type fluorescence was less than that observed at 2 μM iron and similar to that of untreated cells. Conclusions.These findings show that previously demonstrated up-regulation of transferrin independent iron uptake into HuH7 hepatocytes, following cell pre-loading, are associated with an increase in membrane expression of T-type calcium channels at 2μM iron and of L-type channels at concentrations >20μM iron. Previous findings in our laboratory showing up-regulation of NTBI clearance under these conditions, but with no change in mRNA for L or T type channels, and with inhibition of this enhanced uptake by T-type channel blockers, suggest that these effects occur at a post-transcriptional level. These findings also indicate that use of selective calcium channel blockade could be useful in the treatment of iron overload conditions. Disclosures:No relevant conflicts of interest to declare.

Pharmacological Validation of Candidate Causal Sleep Genes Identified in an N2 Cross

: Despite the substantial impact of sleep disturbances on human health and the many years of study dedicated to understanding sleep pathologies, the underlying genetic mechanisms that govern sleep and wake largely remain unknown. Recently, the authors completed large-scale genetic and gene expression analyses in a segregating inbred mouse cross and identified candidate causal genes that regulate the mammalian sleep-wake cycle, across multiple traits including total sleep time, amounts of rapid eye movement (REM), non-REM, sleep bout duration, and sleep fragmentation. Here the authors describe a novel approach toward validating candidate causal genes, while also identifying potential targets for sleep-related indications. Select small-molecule antagonists and agonists were used to interrogate candidate causal gene function in rodent sleep polysomnography assays to determine impact on overall sleep architecture and to evaluate alignment with associated sleep-wake traits. Significant effects on sleep architecture were observed in validation studies using compounds targeting the muscarinic acetylcholine receptor M3 subunit (Chrm3) (wake promotion), nicotinic acetylcholine receptor alpha4 subunit (Chrna4) (wake promotion), dopamine receptor D5 subunit (Drd5) (sleep induction), serotonin 1D receptor (Htr1d) (altered REM fragmentation), glucagon-like peptide-1 receptor (Glp1r) (light sleep promotion and reduction of deep sleep), and calcium channel, voltage-dependent, T type, alpha 1I subunit (Cacna1i) (increased bout duration of slow wave sleep). Taken together, these results show the complexity of genetic components that regulate sleep-wake traits and highlight the importance of evaluating this complex behavior at a systems level. Pharmacological validation of genetically identified putative targets provides a rapid alternative to generating knock out or transgenic animal models, and may ultimately lead towards new therapeutic opportunities.

Abstract C64: Antitumor activity of GBR 500, a monoclonal VLA-2 antibody.

Abstract VLA-2 is the alpha2beta1 integrin which mediates interactions of cells with collagens, laminin and other extracellular matrix components. In tumor cells, VLA-2 has been described to be involved in neovascularization, tumor-stroma interactions and metastasis. The objective of this study was to assess the potential of VLA-2 as a target in a range of tumor models. We used GBR 500, a monoclonal IgG4 antibody directed against the alpha2 subunit of the alpha2beta1 integrin heterodimer. The antibody binds to human but not to mouse VLA-2. GBR 500 is currently undergoing clinical phase I studies. We first characterized the expression of VLA-2 in a set of 96 commonly used human cancer cell-lines from different tissues/organs (including colorectal, skin, breast, prostate, pancreas, lung, cervix, kidney, ovary, CNS, bone, liver, thyroid, and blood) and screened for growth inhibition in solid tumor subcutaneous xenografts. In an orthotopic mouse xenograft model using a DU145-luc cell line we explored the importance of VLA-2 for tumor growth in a physiological setting. We finally assessed the effect of VLA-2 antagonism on metastasis in a PC-3 luc metastasis model. Results: VLA-2 inhibition leads to modest growth reduction in subcutaneous xenografts. In the orthotopic xenograft stronger tumor growth inhibition was achived. The strongest tumor growth inhibition effects were achieved in the mestastasis model. Conclusion: According to our finding growth inhibition of tumors by VLA-2 inhibition increases with increased stroma exposition Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C64.

Morphological aspects of neuromuscular junctions and gene expression of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle of rats with heart failure

HF is syndrome initiated by a reduction in cardiac function and it is characterized by the activation of compensatory mechanisms. Muscular fatigue and dyspnoea are the more common symptoms in HF; these may be due in part to specific skeletal muscle myopathy characterized by reduced oxidative capacity, a shift from slow fatigue resistant type I to fast less fatigue resistant type II fibers and downregulation of myogenic regulatory factors (MRFs) gene expression that can regulate gene expression of nicotinic acetylcholine receptors (nAChRs). In chronic heart failure, skeletal muscle phenotypic changes could influence the maintenance of the neuromuscular junction morphology and nAChRs gene expression during this syndrome. Two groups of rats were studied: control (CT) and Heart Failure (HF), induced by a single intraperitoneal injection of monocrotaline (MCT). At the end of the experiment, HF was evaluated by clinical signs and animals were sacrificed. Soleus (SOL) muscles were removed and processed for morphological, morphometric and molecular NMJ analyses. Our major finding was an up-regulation in the gene expression of the alpha1 and epsilon subunits of nAChR and a spot pattern of nAChR in SOL skeletal muscle in this acute monocrotaline induced HF. Our results suggest a remodeling of nAChR alpha1 and epsilon subunit during heart failure and may provide valuable information for understanding the skeletal muscle myopathy that occurs during this syndrome.

GABAA-positive modulator selective discriminative stimulus effects of 1,1,1-trichloroethane vapor

BackgroundThe abuse-related behavioral effects of inhalant vapors are poorly understood but probably involve multiple neurotransmitter receptor mechanisms. The present study examined the receptor systems responsible for transducing the discriminative stimulus of the abused chlorinated hydrocarbon 1,1,1-trichloroethane (TCE) in mice. MethodsThirty mice were trained to discriminate 10min of 12000ppm TCE vapor exposure from air using an operant procedure. Substitution tests were then conduced with positive GABAA receptor modulators and/or NMDA receptor antagonists. ResultsThe nonselective benzodiazepines midazolam and diazepam produced 62% and 61% and the barbiturate pentobarbital produced 68% TCE-lever selection. Zaleplon, an alpha1 subunit-preferring positive GABAA receptor benzodiazepine-site positive modulator resulted in 29% TCE-lever selection. The direct extrasynaptic GABAA agonist gaboxodol (THIP) and the GABA reuptake inhibitor tiagabine failed to substitute for TCE. No substitution was elicited by a competitive (CGS-19755), noncompetitive (dizocilpine) or glycine-site (L701,324) NMDA antagonist. The mixed benzodiazepine/noncompetitive NMDA antagonist anesthetic Telazol and the anticonvulsant valproic acid exhibited low levels of partial substitution for TCE (38% and 39%, respectively). Ethanol and nitrous oxide failed to substitute for TCE. ConclusionsThe results suggest that the discriminative stimulus effects of TCE are fairly selectively mediated by positive modulation of GABAA receptors. The failure of gaboxadol to substitute and the poor substitution by zaleplon suggests that extrasynaptic GABAA receptors as well as GABAA receptors containing alpha1 subunits and are not involved in transducing the discriminative stimulus of TCE. Studies with additional GABAA benzodiazepine-site positive modulators will be necessary to confirm and extend these findings.

Prolonged zymosan-induced inflammatory pain hypersensitivity in mice lacking glycine receptor alpha2

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons under normal conditions and during persistent pain. However, the role of different glycine receptor (GlyR) subtypes in pain processing has only begun to be unraveled. Here, we analysed whether the GlyR alpha2 subunit might be involved in the processing of acute or persistent pain. Real-time RT-PCR and in situ hybridization analyses revealed that GlyR alpha2 mRNA is enriched in the dorsal horn of the mouse spinal cord. Mice lacking GlyR alpha2 (Glra2 −/− mice) demonstrated a normal nociceptive behavior in models of acute pain and after peripheral nerve injury. However, mechanical hyperalgesia induced by peripheral injection of zymosan was significantly prolonged in Glra2 −/− mice as compared to wild-type littermates. We conclude that spinal GlyRs containing the alpha2 subunit exert a previously unrecognized role in the resolution of inflammatory pain.

Effects of d-cycloserine and midazolam on the expression of the GABA-A alpha-2 receptor subunits in brain structures of fear conditioned rats

The role of the GABA-A alpha-2 receptor subunit in the basolateral amygdala (BLA), dentate gyrus of the hippocampus (DG) and prefrontal cortex (M2 area) during a fear session (performed one week after the conditioned fear test), was studied. We employed a model of high (HR) and low anxiety (LR) rats divided according to their conditioned freezing response. Pretreatment of rats with d-cycloserine immediately before the fear session attenuated fear response in HR and LR rats and increased the density of alpha-2 subunits in the BLA, M2 area and DG of HR animals. The less potent behavioural influence of midazolam (in HR group only) was linked to the increased expression of alpha-2 subunit in M2 area and DG. These results support a role of the GABA-A receptor alpha-2 subunit in processing of emotional cortico-hippocampal input to the BLA.

Fiber connections of the caudal mesopallium in the pigeon

In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44 ± 5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01 ± 3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85 ± 10.04% compared to 34.96 ± 5.11% at and caudal to area postrema (P > 0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03 ± 4.83% at and caudal to area postrema compared to 42.98 ± 3.90% in rostral areas (P = 0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency.

S17 * ALCOHOL AND GABAA RECEPTORS * S17.1 * EXTRASYNAPTIC GABAA-RS: PHARMACOLOGY AND REGULATION OF GENE EXPRESSION

S17 * ALCOHOL AND GABAA RECEPTORS * S17.1 * EXTRASYNAPTIC GABAA-RS: PHARMACOLOGY AND REGULATION OF GENE EXPRESSION

Association between CHRNA5 genetic variation at rs16969968 and brain reactivity to smoking images in nicotine dependent women

Background Tobacco smoking is the leading preventable cause of death in the developed world. Identifying risk factors for smoking may lead to more effective treatments. Genome wide association studies revealed a relationship between development of nicotine dependence and a single-nucleotide polymorphism (SNP, rs16969968) of the nicotine acetylcholine receptor (nAChR) alpha-5 subunit gene (CHRNA5). The relationship between this SNP and other factors contributing to smoking behavior such as smoking cue reactivity is unclear. Methods We assessed the role of rs16969968 on brain functional MRI (fMRI) reactivity to smoking cues by studying nicotine dependent women with the nicotine dependence ‘risk’ allele (A allele, N = 14) and without the ‘risk’ allele (G/G smokers, N = 10). Nicotine dependence severity, as assessed with the Fagerstrom test for nicotine dependence, smoking pack-years, and expired carbon monoxide levels, were equivalent in these groups. Results We observed a group difference in fMRI reactivity; women without the A allele (G/G smokers) showed greater fMRI reactivity to smoking images in brain areas related to memory and habitual behavior such as the hippocampus and dorsal striatum. Conclusions Our finding suggests that nicotine-dependent smokers lacking the rs16969968 A allele are more likely to recall smoking-related memories and engage in habitual responding to smoking cues than A allele smokers. Although more studies are necessary to determine the mechanism underlying and significance of this cue reactivity difference, these data suggest that smokers may develop and remain nicotine dependent due to different factors including genetics and cue reactivity. This finding may have implications for personalizing smoking treatment.

Localization of GABA‐A receptor alpha subunits on neurochemically distinct cell types in the rat locus coeruleus

The locus coeruleus (LC) provides the major source of noradrenaline to the central nervous system and is modulated by neurochemically diverse afferents. LC function is central to arousal, memory, cognition and the stress response, with dysfunction of the LC-noradrenergic axis implicated in debilitating psychiatric disorders. The precise targeting of neurotransmitter receptors within the LC is essential for processing the information contained in diverse afferents and thus LC output. The inhibitory modulation of LC neurons is thought to be effected mainly through GABA-A receptors (GABA(A)Rs). Diverse GABA(A)Rs are pentameric complexes assembled from a repertoire of subunits resulting in substantial diversity in their molecular, functional and pharmacological properties throughout the brain. The precise location of distinct GABA(A) R subunits in subregions of the LC, and the neurochemical identity of the cells that express them, remains to be determined. Here, we show that the GABA(A)R alpha1 subunit is expressed exclusively in neurochemically and morphologically diverse non-noradrenergic cell types within the LC, which may innervate the principal noradrenergic cells. Thus, the GABA(A)R alpha1 subunit could provide a neurochemical signature for a pool of local circuit interneurons in the LC. In contrast, non-overlapping GABA(A)R alpha2 and alpha3 subunit-immunoreactive puncta were enriched on noradrenergic dendrites and, to a lesser extent, on somata. The study reveals a cell-type- and domain-specific expression pattern of distinct GABA(A)R subunits in the LC. These data will serve as a template for understanding inhibitory modulation of this region and facilitate more directed pharmacological strategies for disorders arising from the impairment of LC function.

Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation. Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) by 1.4-fold. Treatment of human adipocytes with fatty acids and tumor necrosis factor α (TNFα) induced insulin resistance and down-regulation of mitochondrial genes, which was restored by ANP treatment. These results show that ANP regulates lipid catabolism and enhances energy dissipation through AMPK activation in human adipocytes.

Antibodies against Extracellular Domains of alpha4 and alpha7 Subunits Alter the Levels of Nicotinic Receptors in the Mouse Brain and Affect Memory: Possible Relevance to Alzheimer's Pathology

Nicotinic acetylcholine receptors (nAChRs) of α4β2 and α7 subtypes expressed in the brain neurons are involved in regulating memory and cognition. Their level is decreased upon several neurodegenerative disorders including Alzheimer's disease (AD), although the reasons for such a decrease are not completely understood. To test whether the nAChR-specific antibodies can affect the brain nAChRs and influence the behavior, we either immunized mice with recombinant extracellular domains of α4 and α7, subunits α4(1-209) and α7(1-208), or injected them with α7(1-208)-specific antibodies. A decrease of α4β2- and α7-nAChRs accompanied with an increase of α4β4-nAChRs in brain membranes of immunized mice was observed. Both α4(1-209)- and α7(1-208)-specific antibodies were detected in the brain membrane lysates of immunized mice. Antibody injection resulted in brain nAChR decrease only if mice were co-injected intraperitoneally with bacterial lipopolysaccharide. Brain sections of immunized mice were analyzed for the binding of [125I]-α-bungarotoxin and [125I]-epibatidine. A decrease in α-bungarotoxin binding in striatum (nucleus accumbens and caudate putamen) accompanied with an increase of epibatidine binding in the forebrain and caudate putamen was observed in mice immunized with either α4 or α7 nAChR domains compared to those immunized with BSA. Mice immunized with α7(1-208) demonstrated significantly worse episodic memory measured in a novel object recognition task compared to non-immunized animals but did not differ from the controls in locomotor or anxiety-related tests. These results suggest that nAChR-specific antibodies are able to penetrate the brain upon inflammation with resulting decreases of brain nAChRs and worsening episodic memory.