NS6740, an α7 nicotinic acetylcholine receptor silent agonist, disrupts hippocampal synaptic plasticity

doi:10.1016/j.neulet.2018.04.025

Neuroscience Letters

Volume 677, 11 June 2018, Pages 6-13

https://doi.org/10.1016/j.neulet.2018.04.025 Get rights and content

Highlights

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NS6740, a potent desensitizer of α7 nAChR, antagonizes the effects of α7 agonists that improve cognition in animals.
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We show that NS6740 disrupts synaptic function and reduces LTP in the rat hippocampus.
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The effects of NS6740 are specific for α7 nAChR over other nAChR, as well as ionotropic glutamate and GABA receptors.
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The specific effect of NS6740 indicate that α7 nAChR activity is essential for normal synaptic function in the hippocampus.

Abstract

Long-term potentiation (LTP) in the dentate gyrus was previously shown to be enhanced by nicotine, an effect dependent on both homomeric α7 and heteromeric α2β2 nicotinic acetylcholine receptors (nAChR). In our experiments, bath-applied nicotine produced no significant enhancement of LTP. The α7 nAChR silent agonist NS6740, a weak activator of α7 nAChR ion channels but an effective modulator of the cholinergic anti-inflammatory pathway, decreased LTP and, additionally, produced a substantial reduction in the baseline synaptic function prior to the high frequency stimulation used to induce LTP. The effects of NS6740 on the various ligand-gated ion channels associated with the generation and modulation of dentate LTP were evaluated with receptors expressed in Xenopus oocytes. A 60 s pre-application of 5 μM NS6740 to α7 receptors blocked the response to subsequent applications of acetylcholine (ACh). In contrast, the responses of α2β2 nAChR to control applications of ACh were not significantly affected by NS6740. Likewise, responses of cells expressing GluR1 + GluR2 AMPA-type glutamate receptor subunits or GABA_A α1, β2, and γ2L subunits to control agonist applications (100 μM kainic acid or 10 μM GABA, respectively), were unaffected by NS6740. The effects of NS6740 on α7 were inconsistent with simple antagonism since, while unresponsive to ACh, the receptors exposed to NS6740 were effectively activated by the positive allosteric modulator PNU-120596. The results support the hypothesis that NS6740 switches the mode of α7 signaling in a channel-independent manner that can reduce synaptic function.

Introduction

The α7 subtype of nicotinic acetylcholine receptor (nAChR) has long been considered a potential therapeutic target for treating cognitive disorders, and α7-selective drugs have been shown to be effective at improving baseline or compromised behavior in animal models of learning and memory [5], [13], [27], [45], [51], [53], [55]. Drugs targeting α7 have also been identified as effective at reducing inflammation and associated pain [43], [47]. While α7 channel activation appears to be important for cognitive effects [8], it does not seem to be important for the modulation of inflammation [4], [26], [49], [54]. In fact, NS6740, which is a compound with good activity in models of inflammation and pain, is an extremely weak partial agonist of α7 and is otherwise a strong desensitizer of α7 channel activity [32]. NS6740 is, however, able to activate large α7 ion channel currents in the presence of a positive allosteric modulator (PAM), identifying it as a “silent agonist”. The concept that there are two distinct types of α7-mediated signaling is supported by reports that NS6740 is not only ineffective in cognitive tests, it is able to block the effects of cognition-enhancing α7 channel activators, for example, the effects of the α7 agonist BMS-902483 on improving novel object recognition [44]. On its own, BMS-902483 has been also shown to increase hippocampal long-term potentiation (LTP), an effect hypothesized to be related to its positive cognitive effects.

LTP is arguably the most widely accepted neurophysiological correlate of learning and memory, involving long-lasting change in synaptic efficacy, which can be experimentally induced by high-frequency stimulation (HFS) of presynaptic axons. Mechanisms that have been shown to be important for LTP have also been demonstrated essential for spatial learning [28], [41], fear conditioning [48], and passive avoidance learning [60]. LTP in the dentate gyrus of the hippocampus has been reported to be enhanced by acute application of nicotine [59]. This effect was reportedly blocked by methyllycaconitine (MLA) and absent in α7 nAChR knockout mice. However, other nAChR, including heteromeric α2β2 receptors, have also been implicated in the induction and modulation of hippocampal LTP [30], [31].

Here we report the effects of NS6740 on synaptic function and plasticity in the dentate gyrus of the rat hippocampus. Using heterologously expressed receptors, we investigated the effects of NS6740 on several ion channel receptors associated with synaptic function in the hippocampus, including important subtypes of AMPA-sensitive glutamate receptors, GABA_A receptors, and α2β2 subtypes of nAChR.

Section snippets

Chemicals

NS6740 was supplied by Dr. Ganesh Thakur (Northeastern Univ., Boston MA). PNU-120596 was supplied by Professor Nicole Horenstein (University of Florida, Gainesville FL). All other chemicals and reagents were purchased from Sigma (St. Louis MO).

Animals

Procedures involving animal subjects have been reviewed and approved by the Institutional Animal Care and Use Committee and were in accordance with guidelines established by the U.S. Public Health Service Policy on Humane Care and Use of Laboratory

Nicotine effects on LTP

We hypothesized that the nicotinic modulation of LTP in the dentate gyrus would be most effectively facilitated by α7 nAChR-selective ligands that are efficacious for ion channel activation. We first established the control LTP data in standard ACSF containing 100 μM picrotoxin to suppress the GABA_A-mediated inhibition in the dentate gyrus. The HFS-induced control LTP attained a peak amplitude of 180 ± 4% over baseline immediately following the stimulation and declined to stable potentiation of

Discussion

Our experimental design was based the methods described by Welsby et al. [59], who previously reported that 5 μM nicotine produced a robust enhancement of dentate LTP, with an early potentiation of 250% compared to a control of 200%, and increase at 60 min after HFS of 182% compared to 143% in the control slices. Although we saw significant LTP under all conditions (P < 0.05), we observed lower levels of LTP under control conditions and no significant effects of 5 or 10 μM nicotine compared to

Acknowledgements

This work was supported by National Institutes of Health Grants GM57481 and AG052258, and the Evelyn F. McKnight Brain Research Foundation. We thank Dr. Michael King for technical assistance in the LTP experiments. We thank Dr. Ganesh Thakur (Northeastern University) for gift of the NS6740.

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NS6740 is an α₇ nicotinic acetylcholine receptor-selective partial agonist with low efficacy for channel activation, capable of promoting the stable conversion of the receptors to nonconducting (desensitized) states that can be reactivated with the application of positive allosteric modulators (PAMs). In spite of its low efficacy for channel activation, NS6740 is an effective activator of the cholinergic anti-inflammatory pathway. We observed that the concentration-response relationships for channel activation, both when applied alone and when co-applied with the PAM PNU-120596 are inverted-U shaped with inhibitory/desensitizing activities dominant at high concentrations. We evaluated the potential importance of recently identified binding sites for allosteric activators and tested the hypotheses that the stable desensitization produced by NS6740 may be due to binding to these sites. Our experiments were guided by molecular modeling of NS6740 binding to both the allosteric and orthosteric activation sites on the receptor. Our results indicate that with α₇C190A mutants, which have compromised orthosteric activation sites, NS6740 may work at the allosteric activation sites to promote transient PAM-dependent currents but not the stable desensitization seen with wild-type α₇ receptors. Modeling NS6740 in the orthosteric binding sites identified S36 as an important residue for NS6740 binding and predicted that an S36V mutation would limit NS6740 activity. The efficacy of NS6740 for α₇S36V receptors was reduced to zero, and applications of the compound to α₇S36V receptors failed to induce the desensitization observed with wild-type receptors. The results indicate that the unique properties of NS6740 are due primarily to binding at the sites for orthosteric agonists.
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¹: Present address: Department of Pharmacology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, United States.

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Research articleNS6740, an α7 nicotinic acetylcholine receptor silent agonist, disrupts hippocampal synaptic plasticity

Highlights

Abstract

Introduction

Section snippets

Chemicals

Animals

Nicotine effects on LTP

Discussion

Acknowledgements

Brain Res.

Pharm. Biochem. Behav.

Bioorg. Med. Chem. Lett.

Exp. Neurol.

Neuropharmacology

J. Biol. Chem.

Behav. Brain Res.

Biochem. Pharmacol.

NeuroPharm

J. Biol. Chem.

Methods

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Neuropharmacology

Neuropharmacology

Trends Pharmacol. Sci.

J. Neuroimmunol.

Neuron

Gastroenterology

The alpha7 nicotinic acetylcholine receptor positive allosteric modulator attenuates lipopolysaccharide-induced activation of hippocampal IkappaB and CD11b gene expression in mice

Drug Discov. Ther.

Metabolism and disposition of GTS-21, a novel drug for Alzheimer's disease

Xenobiotica

New insights on neuronal nicotinic acetylcholine receptors as targets for pain and inflammation: a focus on alpha7 nAChRs

Curr. Neuropharmacol.

The novel alpha7 nicotinic acetylcholine receptor agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2- carboxamide improves working and recognition memory in rodents

J. Pharmacol. Exp. Ther.

Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin

Nature

Role of channel activation in cognitive enhancement mediated by alpha7 nicotinic acetylcholine receptors

Br. J. Pharmacol.

Whole blood cytokine attenuation by cholinergic agonists ex vivo and relationship to vagus nerve activity in rheumatoid arthritis

J. Intern. Med.

Mechanism of action of benzodiazepines on GABAA receptors

Br. J. Pharmacol.

Cholinergic agonists regulate JAK2/STAT3 signaling to suppress endothelial cell activation

Am. J. Physiol. Cell Physiol.

Targeting the alpha 7 nicotinic acetylcholine receptor to reduce amyloid accumulation in Alzheimer's disease pyramidal neurons

Curr. Pharm. Des.

The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation

Br. J. Pharmacol.

Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus

J. Neurosci.

Research article
NS6740, an α7 nicotinic acetylcholine receptor silent agonist, disrupts hippocampal synaptic plasticity