Alpha7 nAChR Research Articles

New Alpha9 nAChR Ligands Based on a 5-(Quinuclidin-3-ylmethyl)-1,2,4-oxadiazole Scaffold.

Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N'-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release.

Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry.

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia-derived progenitor cells that arise after treatment with PNU-282987. Key features • Neurogenesis in the adult mammalian retina. • Flow cytometry of retinal cells. • PNU-282987-induced mitotic activity in the retina. • Dissociation of the retina for flow cytometry analysis. Graphical overview Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3-6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.

Target identification and validation of the alpha7 nicotinic acetylcholine receptor as a potential therapeutic target in retinal disease.

The role of acetylcholine (ACh) in visual processing in the mammalian retina has been the focus of research for many decades. Pioneering work on the localization of ACh discovered that the neurotransmitter is synthesized and stored in a distinct subpopulation of amacrine (starburst) cells. It has been shown that ACh release is regulated to a low resting "tonic" level, much like what is observed at the neuromuscular junction (NMJ). If there were a dysfunction in the tonic release of ACh, might post-synaptic changes render the targets of ACh [i.e., retinal ganglion cells (RGCs)] vulnerable to disease? During my time at Pharmacia & Upjohn (PNU), selective nicotinic ACh receptor (nAChR) agonists (e.g., PNU-282987) were developed as a possible therapy for central nervous system (CNS) diseases. As RGCs are the main targets of neurodegeneration in glaucoma, could the activation of this target provide neuroprotection? In response to this question, experiments to identify alpha7 nAChRs in the retina (i.e., target ID studies) followed by "proof-of-concept" experiments were conducted. Target ID studies included binding studies with retinal homogenates, [125I]-alpha-bungarotoxin (α-BTX) autoradiography, and fluorescently tagged α-BTX binding in retinal slices. Imaging studies of intracellular calcium dynamics in the retinal slice were conducted. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with alpha7 nAChR knockout mice using the "laser-capture microdissection" technique, in situ hybridization studies, and RT-PCR analysis of the human retina were conducted. Collectively, these experiments confirmed the presence of alpha7 nAChRs on specific cells in the retina. "Proof-of-concept" neuroprotection studies demonstrated that PNU-282987 provided significant protection for RGCs. This protection was dose dependent and was blocked with selective antagonists. More recently, evidence for the generation of new RGCs has been reported with PNU-282987 in rodents. Interestingly, the appearance of new RGCs is more pronounced with eye-drop application than with intravitreal injection. One could postulate that this reflects the neurogenic activation of alpha7 receptors on the retinal pigment epithelium (RPE) (eye drops) vs. a neuroprotective effect on RGCs (injections). In conclusion, there does appear to be a cholinergic retinal "tone" associated with RGCs that could be utilized as a neuroprotective therapy. However, a distinct cholinergic neurogenic mechanism also appears to exist in the outer retina that could possibly be exploited to generate new RGCs lost through various disease processes.

Localization of Acetylcholine, Alpha 7-NAChR and the Antimicrobial Peptide Piscidin 1 in the Macrophages of Fish Gut: Evidence for a Cholinergic System, Diverse Macrophage Populations and Polarization of Immune Responses

The recognition and elimination of invading pathogens are vital for host survival. Macrophages play a central role in host protection and cells functionally reminiscent of vertebrate macrophages are present in all multicellular organisms. A pattern responsible for bacterial recognition found on the surface of macrophages is CD14. These cells possess a repertoire of antimicrobial molecules stored in their granules and lysosomes. Polarization states observed in mammalian macrophages termed M1 and M2 also likely exist in fish macrophages. Markers for macrophage subtypes are slowly but definitively emerging in fish species. In the present study cell markers such as CD14, acetylcholine, alpha 7 acetylcholine nicotinic receptor (nAChR) subtype, the inducible nitric oxidase synthase (iNOS), and the antimicrobial peptide piscidin 1 are reported for the first time in the intestinal macrophages of both catfish Heteropneustes fossilis (Bloch, 1794) and the African bonytongue Heterotis niloticus (Cuvier, 1829) along the anterior and the posterior axis and the concentric muscle layers. Many antimicrobial effector responses of vertebrate macrophages including respiratory burst and NO induction are similar across the diverse animal taxa. Antibodies against calbindin coupled with ones to VAChT and tubulin revealed the localization of myenteric and submucosal plexuses, which are made up of enteric neurons, glial cells, and nerves near macrophages. Current studies allow for the elucidation of multiple roles of macrophages in disease models providing an insight into their in vivo function in fish.

Functional Changes in the Adult Mouse Retina using an Alpha7 Nicotinic Acetylcholine Receptor Agonist after Blast Exposure

Currently, there is a lack of treatments for retinal neurotrauma. To address this issue, this study uses an alpha7 nAChR agonist, PNU-282987, to determine it effects on functional activity in the retina shortly after a traumatic blast exposure. The objectives of this research include: (1) examination of the cellular and functional damage associated with ocular blast exposure, and (2) evaluation of structural and functional changes that occur post PNU-282987 treatment. Significant ocular blast damage was induced in adult mice after exposure to a single blast of 35 psi to the left eye. Blast-exposed transgenic mice expressing tdTomato Müller glia were treated daily with eyedrops containing PNU-282987 for 4 weeks following the blast exposure. Antibody staining studies in these transgenic mice was conducted to examine lineage tracing and electroretinograms (ERGs) were obtained to examine functional changes. Blast exposure caused a significant loss of cells in all retinal layers after 4 weeks. Immunohistochemical analysis demonstrated tdTomato-positive labeled photoreceptors and retinal ganglion cells in blast-exposed mice treated with PNU-282987. ERG recordings were taken from control animals, from blast-damaged animals and from animals exposed to blast followed by 4 weeks of PNU-282987 treatment. Scotopic ERG recordings from blast-exposed mice had significantly decreased amplitudes of a-wave, b-wave, oscillatory potentials and flicker frequencies, which were prevented after PNU-282987 treatment. In photopic experiments, the PhNR response was reduced significantly after blast exposure but the decrease was prevented after treatment with PNU-282987. These are the first experiments that demonstrate preservation of retinal function after blast exposure using an alpha7 nAChR agonist.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Effect of Varenicline on Detrusor Overactivity in Rat Model of Parkinson’s Disease Induced by Intranigral 6-Hydroxydopamine

Background: Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by both motor and non-motor symptoms. Bladder dysfunction is the common non-motor symptom of PD, most often presenting with detrusor overactivity (DO). Treatment of DO is currently limited, poorly tolerated and sometimes ineffective. Bladder responses are not only mediated by muscarinic cholinergic receptors (mAChR) but also by nicotinic cholinergic receptors (nAChR). However, nicotinic receptor subtypes and functions in the bladder are not clearly identified. Purpose: This study aimed at investigating the effect of varenicline, an alpha7 full agonist and alpha4beta2/alpha3 partial agonist, on detrusor strips in rat PD model induced by substantia nigra injection of 6-hydroxydopamine. Method: The detrusor activity was studied in an isolated organ bath system. Results: In PD group, the detrusor activity was increased, whereas varenicline decreased the DO. Conclusion: Alpha7 nAChR agonists may have therapeutic potential in treatment of bladder overactivity in PD.

Activation of α7 Nicotinic Acetylcholine Receptor by its Selective Agonist Improved Learning and Memory of Amyloid Precursor Protein/Presenilin 1 (APP/PS1) Mice via the Nrf2/HO-1 Pathway.

BackgroundTo reveal the mechanism underlying the effect of α7 nicotinic acetylcholine receptor (nAChR) on neurodegeneration in Alzheimer disease (AD), the influence of the receptor on recognition in APP/PS1 mice was evaluated by using its selective agonist (PNU-282987).Material/MethodsAPP/PS1 and wild-type (WT) mice were treated with PNU or saline, respectively, for 7 days at the ages of 6 and 10 months.ResultsMorris water maze analysis showed that both at 6 and 10 months of age, PNU treatment enhanced the learning and memory of APP/PS1 mice. However, PNU treatment did not alter the number of senile plaques. Furthermore, a higher protein expression of Nrf2/HO-1, ADAM10, SYP, and SNAP-25, and a lower level of oxidative stress, were observed in the hippocampus of APP/PS1 mice treated with PNU compared with the control group.ConclusionsThe results indicated that the activation of α7 nAChR by PNU improved the learning and memory of mice carrying the APP/PS1 mutation, regulated the levels of enzymes that mediate APP metabolization to reduce β-amyloid peptide damage, and decreased the level of oxidative stress and maintained synaptic plasticity, in which the mechanism might be enhancement of the Nrf2/HO-1 pathway.

Potentiation of cognitive enhancer effects of Alzheimer’s disease medication memantine by alpha7 nicotinic acetylcholine receptor agonist PHA-543613 in the Morris water maze task

RationaleThere are controversial pieces of evidence whether combination therapies using memantine and cholinesterase inhibitors are beneficial over their monotreatments. However, results of preclinical studies are promising when memantine is combined with agonists and allosteric modulators of the alpha7 nicotinic acetylcholine receptor (nAChR).ObjectivesHere, we tested the hypothesis that cognitive enhancer effects of memantine can be potentiated through modulating alpha7 nAChRs in a scopolamine-induced amnesia model.MethodsMonotreatments, as well as co-administrations of selective alpha7 nicotinic acetylcholine receptor agonist PHA-543613 and memantine were tested in the Morris water maze task in rats. The efficacy of the co-administration treatment was observed on different domains of spatial episodic memory.ResultsLow dose of memantine (0.1 mg/kg) and PHA-543613 (0.3 mg/kg) successfully reversed scopolamine-induced short-term memory deficits both in monotreatments and in co-administration. When recall of information from long-term memory was tested, pharmacological effects caused by co-administration of subeffective doses of memantine and PHA-543613 exceeded that of their monotreatments.ConclusionOur results further support the evidence of beneficial interactions between memantine and alpha7 nAChR ligands and suggest a prominent role of alpha7 nAChRs in the procognitive effects of memantine.

Hippocampal asymmetry and regional dispersal of nAChRs alpha4 and alpha7 subtypes in the adult rat

To better comprehend the relationship between left/right (L/R) differences and hippocampus functions is necessary knowledge of lateral asymmetry and regional distribution. This research was design to examine hippocampal L/R asymmetry and regional distribution profile of the alpha7 and alpha4 subtypes of nicotinic acetylcholine receptors (nAChRs) in the adult rat.10–12-week-old twenty-four male wistar rats were randomly selected. After removing the brains, immunohistochemistry, real-time PCR, and western blot methods were applied to distinguish the presence of the receptors in the hippocampus. Outcomes stated that the mentioned receptors expression profile was spatial-dependent. As, the hippocampal dispersal of alpha7 and alpha4 subtypes in the left hippocampus (LH) was remarkably maximum compare with the right hippocampus (RH) (p = 0.001, p = 0.005 respectively). Furthermore, the alpha7 optical density (OD) was not significantly different in the diverse regions in hippocampus of adult rat (p = 0.057), while the maximum OD of the alpha4 was detected in the hippocampal dentate gyrus and CA3 regions of LH (p = 0.007, p = 0.009 respectively) and the minimum OD was in the CA1 of the RH (p = 0.019). In real time PCR evaluation, there is a significantly higher expression of alpha7 and alpha4 in LH compared to RH (p = 0.043, p = 0.049 respectively), also, for western blot (p = 0.042, p = 0.030 respectively). According to present data, the alpha7 and alpha4 nAChR subtypes expression profile demonstrated lateral asymmetry, the uniform regional dispersal for alpha7 and different regional dispersal for alpha4 in the adult rat hippocampus.

Expression of acetylcholine, its contribution to regulation of immune function and O2 sensing and phylogenetic interpretations of the African butterfly fish Pantodon buchholzi (Osteoglossiformes, Pantodontidae)

Acetylcholine (Ach) is the main neurotransmitter in the neuronal cholinergic system and also works as a signaling molecule in non-neuronal cells and tissues. The diversity of signaling pathways mediated by Ach provides a basis for understanding the biology of the cholinergic epithelial cells and immune cells in the gill of the species studied.NECs in the gill were not found surprisingly, but specialized cells showing the morphological, histochemical and ultrastructural characteristics of eosinophils were located in the gill filaments and respiratory lamellae.Much remains unknown about the interaction between the nerves and eosinophils that modulate both the release of acetylcholine and its nicotinic and muscarinic receptors including the role of acetylcholine in the mechanisms of O2 chemosensing. In this study we report for the first time the expression of Ach in the pavement cells of the gill lamellae in fish, the mast cells associated with eosinophils and nerve interaction for both immune cell types, in the gill of the extant butterfly fish Pantodon buchholzi. Multiple roles have been hypothesized for Ach and alpha nAChR in the gills. Among these there are the possible involvement of the pavement cells of the gill lamellae as O2 chemosensitive cells, the interaction of Ach positive mast cells with eosinophils and interaction of eosinophils with nerve terminals. This could be related to the use of the vesicular acetylcholine transporter (VAChT) and the alpha 2 subunit of the acetylcholine nicotinic receptor (alpha 2 nAChR). These data demonstrate the presence of Ach multiple sites of neuronal and non-neuronal release and reception within the gill and its ancestral signaling that arose during the evolutionary history of this conservative fish species.

Dissociation of nicotinic α7 and α4/β2 sub-receptor agonists for enhancing learning and attentional filtering in nonhuman primates.

Nicotinic acetylcholine receptors (nAChRs) modulate attention, memory, and higher executive functioning, but it is unclear how nACh sub-receptors mediate different mechanisms supporting these functions. We investigated whether selective agonists for the alpha-7 nAChR versus the alpha-4/beta-2 nAChR have unique functional contributions for value learning and attentional filtering of distractors in the nonhuman primate. Two adult rhesus macaque monkeys performed reversal learning following systemic administration of either the alpha-7 nAChR agonist PHA-543613 or the alpha-4/beta-2 nAChR agonist ABT-089 or a vehicle control. Behavioral analysis quantified performance accuracy, speed of processing, reversal learning speed, the control of distractor interference, perseveration tendencies, and motivation. We found that the alpha-7 nAChR agonist PHA-543613 enhanced the learning speed of feature values but did not modulate how salient distracting information was filtered from ongoing choice processes. In contrast, the selective alpha-4/beta-2 nAChR agonist ABT-089 did not affect learning speed but reduced distractibility. This dissociation was dose-dependent and evident in the absence of systematic changes in overall performance, reward intake, motivation to perform the task, perseveration tendencies, or reaction times. These results suggest nicotinic sub-receptor specific mechanisms consistent with (1) alpha-4/beta-2 nAChR specific amplification of cholinergic transients in prefrontal cortex linked to enhanced cue detection in light of interferences, and (2) alpha-7 nAChR specific activation prolonging cholinergic transients, which could facilitate subjects to follow-through with newly established attentional strategies when outcome contingencies change. These insights will be critical for developing function-specific drugs alleviating attention and learning deficits in neuro-psychiatric diseases.

Facilitation and inhibition of firing activity and N-methyl-D-aspartate-evoked responses of CA1 hippocampal pyramidal cells by alpha7 nicotinic acetylcholine receptor selective compounds in vivo

Alpha7 nicotinic acetylcholine receptors (nAChRs) are promising novel targets for the treatment of neurocognitive disorders. Although the cognitive enhancer potential of alpha7 nAChR agonists and positive allosteric modulators (PAMs) has been confirmed in several preclinical animal models, there are only sparse in vivo electrophysiological data on their effects on the firing activity and excitability of neurons. The present study investigated and compared local effects of alpha7 nAChR agonist PHA-543613 and PAMs PNU-120596 and NS-1738 on the spontaneous and N-methyl-D-aspartate-evoked (NMDA-evoked) firing rate of rat CA1 hippocampal pyramidal cells, in vivo. Furthermore, effects of alpha7 nAChR antagonist methyllycaconitine (MLA) and GABA were also tested. Results showed substantially different effects of the alpha7 nAChR agonist and PAMs. While PNU-120596 and NS-1738 predominantly and significantly increased both spontaneous and NMDA-evoked firing rate of the neurons, application of PHA-543613 resulted in almost equal distribution of facilitatory and inhibitory effects. The increase of the NMDA-evoked firing rate exerted by NS-1738 was superadditive over the sum of the single effects of NMDA and NS-1738. The simultaneous application of alpha7 nAChR agonist PHA-543613 and PAM NS-1738 resulted in additive increase of both spontaneous and NMDA-evoked firing rate. However, NS-1738 counteracted inhibitory effects of PHA-543613 in 5 out of 6 neurons, resulting in a synergistic potentiation of their firing responses to NMDA. Our results suggest that alpha7 nAChR PAMs increase neuronal excitability more potently than agonists, while the remarkable occurrence of inhibitory effects of PHA-543613 (possibly originating from receptor desensitization) implies that agonists may exert neuroprotective effects.

Alpha7 nAChR Expression Is Correlated with Arthritis Development and Inhibited by Sinomenine in Adjuvant-Induced Arthritic Rats.

Sinomenine (SIN) is the active ingredient of the Chinese herb Sinomenium acutum that has been used to treat rheumatoid arthritis (RA) for about 30 years in China. Marked expression of the alpha7 nicotinic acetylcholine receptor (α7nAChR) in the joint synovium of RA patients suggested a relationship between α7nAChR and RA. This study investigated the relationship between α7nAChR and RA development and the effects of SIN on α7nAChR expression in vivo and in vitro. Sprague-Dawley rats were injected with complete Freund's adjuvant to induce arthritis and then treated with SIN or methotrexate (MTX) from day 0 to day 30. Four clinical parameters—paw volume, arthritic index (AI), serum TNF-α concentration, and erythrocyte sedimentation rate (ESR)—were measured. Splenic lymphocytes were isolated for Bacille Calmette Guerin (BCG) stimulation. α7nAChR expression in tissues and cells was examined by RT-PCR, western blot, immunofluorescence, flow cytometry, and immunohistochemistry. Cell proliferation was evaluated by the CCK-8 assay. The relationship between α7nAChR expression and the four clinical parameters was analyzed by single-factor correlation analysis. Our results showed that the paw volume, AI, TNF-α concentration, and ESR in adjuvant-induced arthritic (AIA) rats were reduced by SIN or MTX treatment. SIN decreased α7nAChR expression in tissues and cells compared to the model group, while MTX had no significant effect on α7nAChR expression. Moreover, there was a positive relationship between α7nAChR expression and paw swelling, AI, and TNF-α concentration. Splenic lymphocyte activation was accompanied by increased α7nAChR expression, while SIN treatment inhibited cell activation and downregulated α7nAChR expression. α7nAChR expression showed a positive correlation with the progression of RA in AIA rats that may involve lymphocyte activation. Different from MTX, the inhibition of SIN on α7nAChR expression might contribute to its antiarthritic effect, suggesting that SIN could be an important supplement to the treatment strategy for RA.

Cognitive Enhancer Effects of Low Memantine Doses Are Facilitated by an Alpha7 Nicotinic Acetylcholine Receptor Agonist in Scopolamine-Induced Amnesia in Rats.

Alpha7 nicotinic acetylcholine receptors (nAChRs) play an important role in learning and memory and are promising targets for pharmacological cognitive enhancement. Memantine, an approved substance for Alzheimer’s disease treatment, is an antagonist of the N-Methyl-D-aspartate receptor (NMDAR) and also acts as an alpha7 nAChR antagonist. Here, we tested the interaction between an alpha7 nAChR agonist (PHA-543613) and memantine. Efficacy of memantine, PHA-543613, and their co-administration were investigated on the spatial working memory of rats using the spontaneous alternation paradigm in T-maze. Scopolamine-induced transient amnesia was used to model cognitive impairment. First, the dose-response relationship was assessed for memantine, and its lowest effective dose was found to be 0.1 mg/kg. Then, co-administration treatments with subeffective doses of the alpha7 nAChR agonist PHA-543613 and different doses of memantine were tested. The co-administration of subeffective drug doses significantly improved memory performance of the rats and reversed scopolamine-induced deficits. Interestingly, a higher than effective (0.3 mg/kg) dose of memantine did not increase performance in monotreatment, only in co-administration with PHA-543613. However, the co-administration of PHA-543613 did not further increase the efficacy of the previously effective monotreatment doses of memantine. Thus, the efficacy of memantine monotreatment and its co-administration with PHA-543613 converged to create a common ceiling effect, with an additive interaction found in the behavioral effects. These results suggest that memantine and PHA-543613 may exert their cognitive enhancer effects on the same target, possibly on the alpha7 nAChRs. Results also suggest possible benefits of a combination therapy with memantine and alpha7 nAChR agonists.

New Binding Mode of SLURP Protein to a7 Nicotinic Acetylcholine Receptor Revealed by Computer Simulations

SLURP-1 is a member of three-finger toxin-like proteins. Their characteristic feature is a set of three beta strands extruding from hydrophobic core stabilized by disulfide bonds. Each beta-strand carries a flexible loop, which is responsible for recognition. SLURP-1 was recently shown to act as an endogenous growth regulator of keratinocytes and tumor suppressor by reducing cell migration and invasion by antagonizing the pro-malignant effects of nicotine. This effect is achieved through allosteric interaction with alpha7 nicotinic acetylcholine receptors (alpha-7 nAChRs) in an antagonist-like manner. Moreover, this interaction is unaffected by several well-known agents specifically alpha-bungarotoxin. In this work, we carry out the conformational analysis of the SLURP-1 by a microsecond-long full-atom explicit solvent molecular dynamics simulations followed by clustering, to identify representative states. To achieve this timescale we employed a GPU-accelerated version of GROMACS modeling package. To avoid human bias in clustering we used a non-parametric clustering algorithm Affinity Propagation adapted for biomolecules and HPC environments. Then, we applied protein-protein molecular docking of the ten most massive clusters to alpha7-nAChRs in order to test if structural variability can affect binding. Docking simulations revealed the unusual binding mode of one of the minor SLURP-1 conformations.

Deletion of nicotinic acetylcholine receptor alpha9 in mice resulted in altered bone structure.

Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO).Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by μCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen lαl and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences.In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.

The jury remains divided on electronic cigarettes and their effects on the wider population

British Journal of Healthcare ManagementVol. 24, No. 11 CommentThe jury remains divided on electronic cigarettes and their effects on the wider populationGraham F CopeGraham F CopeSearch for more papers by this authorGraham F CopePublished Online:14 Nov 2018https://doi.org/10.12968/bjhc.2018.24.11.530AboutSectionsView articleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareShare onFacebookTwitterLinked InEmail View article References Action on Smoking and Health. 2015. Smoking still kills. Protecting children, reducing in equalities. http://www.ash.org.uk/files/documents/ASH_962.pdf (accessed 2 November 2018) Google ScholarAction on Smoking and Health. 2018. New data shows smokers are getting the message on e-cigarettes. http://ash.org.uk/media-and-news/press-releases-media-and-news/ash-news-release-new-data-shows-smokers-are-getting-the-message-on-e-cigarettes/ (accessed 2 November 2018) Google ScholarBinns C, Lee MK, Low WY. Children and e-cigarettes: a new threat to health. Asia-Pacific J Pub Health. 2018;30(4):315–320. https://doi.org/10.1177/1010539518783808 Crossref, Google ScholarBritton J, Arnott D, McNeill A et al.. Nicotine without smoke – putting electronic cigarettes in context. BMJ. 2016;353:i1745. https://doi.org/10.1136/bmj.i1745 Crossref, Google ScholarCope GF. The effects of smoking on wound healing. Wounds UK. 2014;10(2):10–18. Google ScholarCope GF. Smoking – what all healthcare professionals need to know. Kewick: M&K Publishing; 2016. Google ScholarCope GF. 2017. Written evidence submitted to the Parliamentary enquiry on e-cigarettes. http://data.parliament.uk/WrittenEvidence/CommitteeEvidence.svc/EvidenceDocument/Science%20and%20Technology/ECigarettes/written/74062.html (accessed 2 November 2018) Google ScholarEngland L, Aagaard K, Bloch M et al.. Developmental toxicity of nicotine: A transdisciplinary synthesis and implications for emerging tobacco products. Neurosci Biobehavl Rev. 2017;72: 176–189. https://doi.org/10.1016/j.neubiorev.2016.11.013 Crossref, Google ScholarFeldman C, Anderson R. Cigarette smoking and mechanisms of susceptibility to infections of the respiratory tract and other organ systems. J Infection. 2013;67(3):169–184. https://doi.org/10.1016/j.jinf.2013.05.004 Crossref, Google ScholarFujii M, Mashimo M, Moriwaki Y et al.. Expression and function of the cholinergic system in immune cells. Frontiers Immunol 2017;6;8:1085. https://doi.org/10.3389/fimmu.2017.01085 Crossref, Google ScholarHahn HL, Kleinschrot D, Hansen D. Nicotine increases ciliary beat frequency by a direct effect on respiratory cilia. Clin Investigator 1992;70(3–4):244–251. Google ScholarJohnson JM, Naeher LP, Yu X et al.. Air monitoring at large public electronic cigarette events. Int J Hyg Envir Health 2018;221(3);541–547. https://doi.org/10.1016/j.ijheh.2018.02.003 Crossref, Google ScholarKamerow D. Start-up e-cigarette brand aims to “improve smokers' lives. BMJ. 2018;362:k2930. https://doi.org/10.1136/bmj.k2930 Crossref, Google ScholarMorley K. 2018. Exclusive: Dubious sales tactics of Philip Morris salesmen revealed. https://www.telegraph.co.uk/news/2018/10/15/tobacco-sales-reps-push-free-drinks-sell-wares/?playlist=structure%3Anews%2Fuk-news (accessed 30 October 2018) Google ScholarOnoda N, Nehmi A, Weiner D et al.. Nicotine affects the signaling of the death pathway, reducing the response of head and neck cancer cell lines to DNA damaging agents. Head and Neck. 2001;23(1):860–870 Crossref, Google ScholarHouse of Commons Science and Technology Committee. 2018. E-cigarettes. https://publications.parliament.uk/pa/cm201719/cmselect/cmsctech/505/505.pdf (accessed 5 November 2018) Google ScholarPublic Health England. 2017. Cost of smoking to the NHS in England: 2015. https://www.gov.uk/government/publications/cost-of-smoking-to-the-nhs-in-england-2015/cost-of-smoking-to-the-nhs-in-england-2015 (accessed 5 November 2018) Google ScholarPoirier G, Huang W, Tam K, DiFranza JR, King JA. 2017. Evidence of altered brain responses to nicotine in an animal model of attention deficit/hyperactivity disorder. Nic Tob Res. 2017;19(9):1016–1023. https://doi.org/10.1093/ntr/ntx088 Crossref, Google ScholarZhang Y, Jia Y, Li P et al.. Reciprocal activation of alpha 5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation. J Genet Genomics. 2017;44(7):355–362. https://doi.org/10.1016/j.jgg.2017.03.003 Crossref, Google Scholar FiguresReferencesRelatedDetails 2 November 2018Volume 24Issue 11ISSN (print): 1358-0574ISSN (online): 1759-7382 Metrics History Published online 14 November 2018 Published in print 2 November 2018 Information© MA Healthcare LimitedPDF download

Nicotine improves probabilistic reward learning in wildtype but not alpha7 nAChR null mutants, yet alpha7 nAChR agonists do not improve probabilistic learning

Cognitive impairments, e.g., reward learning, are present in various psychiatric disorders and warrant treatment. Improving reward-related learning could synergistically enhance psychosocial treatments and cognition generally. A critical first step is to understand the mechanisms underlying reward learning. The dopamine system has been implicated in such learning, but less known is how indirect activation of this system may affect reward learning. We determined the role of alpha7 nicotinic acetylcholine receptors (nAChR) on a probabilistic reversal learning task (PRLT) in mice that includes reward and punishment. Male alpha7 knockout (KO), heterozygous (HT), and wildtype (WT) littermate mice (n = 84) were treated with vehicle, 0.03, or 0.3 mg/kg nicotine. Two cohorts of C57BL/6NJ male mice were treated with various alpha7 nAChR ligands, including the full agonists PNU282877 and AR-R-17779, the positive allosteric modulator CCMI, the partial agonist SSR180711, and the antagonist methyllycaconitine. All mice were then tested in the PRLT. Nicotine (0.3 mg/kg) significantly improved initial reward learning in alpha7 WT and HT mice but did not improve learning in KO mice, suggesting an involvement of the alpha7 nAChR in the pro-learning effects of nicotine. Neither alpha7 nAChR treatments (PNU282987, AR-R-17779, CCMI, SSR180711, nor methyllycaconitine) affected mouse PRLT performance however. Nicotine improved reward learning via a mechanism that may include alpha7 nAChRs. This improvement unlikely relied solely on alpha7 nAChRs however, since no alpha7 nAChR ligand improved reward learning in normal mice. Future assessments of the effects of other nAChR subtypes on reward learning are needed.

S232. ALPHA7 NICOTINIC RECEPTOR AGONISTS REVERSE THE HYPERDOPAMINERGIC STATE IN THE MAM MODEL OF SCHIZOPHRENIA

BackgroundMost investigations into the pharmacology of schizophrenia have revolved around dopaminergic and glutamatergic neurotransmission; however, one neurotransmitter that has not received adequate attention is the cholinergic system. Indeed, several post-mortem, genetic and epidemiologic studies link specifically the alpha7 nicotinic receptor (nAChR) to schizophrenia, and the potential use of alpha7 modulators as a treatment strategy is an active field of research. Nevertheless, studies to date have been limited to normal animals rather than on a validated neurodevelopmental model of schizophrenia. Moreover, knowledge about the differential impact of orthosteric and allosteric modulators in vivo is lacking. Thus, we investigated the effects of alpha7 nAChR modulation on dopamine (DA) neuron activity in the ventral tegmental area (VTA) in the methylazoxymethanol acetate (MAM) animal model of schizophrenia.MethodsAll experimental procedures were conducted according to NIH guidelines and were approved by University of Pittsburgh Institutional Animal Care and Use Committee. Sprague-Dawley pregnant dams were treated with MAM or saline on gestational day 17. Recordings of VTA dopamine neuron activity was performed on the male offspring at adulthood. The effects of four different drugs were evaluated: PNU282987 (full agonist), SSR180711 (partial agonist) NS1738 (type I positive allosteric modulator - PAM) and PNU120596 (PAM type II).ResultsIntravenous administration of alpha7 selective ligands did not induce a major change in the firing profile of spontaneously active DA neurons when dosed during dopamine neuron recording. PNU120596 increased in the number of active DA neurons found in the VTA of normal rats, their mean firing rate and percentage of spikes in bursts. In contrast, the full agonist PNU282987 and the partial agonist SSR180711 reduced the hyperdopaminergic tone in MAM rats, with a more prominent decrease in the number of DA neurons recorded in the lateral VTA. In order to investigate the drug site of action, both PNU282987 and SSR1800711 were infused into the ventral hippocampus (vHipp) and basolateral amygdala (BLA). After vHipp infusion, the alpha7 nAChR agonists significantly decreased the number of active DA neurons in MAM rats, with no significant impact in control rats. Once more, the effects were more robust in the lateral VTA. In contrast, the same drugs when infused directly into the BLA increased the number of spontaneously active DA neurons in the VTA of normal rats, but not in the MAM model.DiscussionIn summary, our results show that alpha7 nAChR positive modulators can affect midbrain dopaminergic neuronal activity in vivo in a state-dependent manner. Interestingly, alpha7 nAChR agonists counterbalanced the hyperdopaminergic state of MAM rats and this effect is partially mediated by their action in the vHipp. This effect is consistent with the potential use of alpha7 nAChR agonists for schizophrenia treatment and fits the current search for drugs able to control dopaminergic function acting in structures upstream from the dopamine receptor. The predominant inhibition of the lateral VTA points to a lower propensity to produce unwanted side effects in comparison to current employed antipsychotic agents. Our data show that drug effects can vary according to the basal level of activity of specific brain circuits and highlights the importance of using appropriated animal models to make inferences about potential therapeutic use of new neuropsychiatric drug candidates.