Bibliometric analysis of nicotinic acetylcholine receptors channel research (2000-2020)

ABSTRACT To explore the research status, hotspots, and trends in research on nicotinic acetylcholine receptor (nAChR) channel. The Web of Science core collection database from 2000 to 2020 was used as the data source. The visual analysis software VOSviewer1.6.16 and Citespace5.7 R3 were used to visualize the studies of the nAChR channel. The national/institutional distribution, journal distribution, authors, and related research were discussed. A total of 5,794 articles were obtained. The USA and the Utah System of Higher Education were the most productive country and institution for nAChR channel research. Journal of Biological Chemistry was the most productive journal (212) and the most productive researcher was McIntosh, J. Michael. The first highly co-cited article was “Refined structure of the nicotinic acetylcholine receptor at 4A resolution.” The most researched area was neurosciences neurology. The hot spots of nAChR channel research were “subunit and structure of nAChR,” “activation/agonist of nAChR channel,” and “Changes in nAChRs With Alzheimer’s Disease.” The top three research frontiers of nAChR channel research were “neuropathic pain,” “neuroinflammation,” and “α7 nACHR.” The study provides a perspective to visualize and analyze hotspots and emerging trends in the nAChR channel.

There are two types of acetylcholine receptors (AChR): nicotinic AChR (nAChR) and muscarinic AChR. nAChRs are fast cationic channels, initially discovered in muscles and autonomic ganglia function, while muscarinic AChRs are class I heptahelical G-proteincoupled receptors with a slower signaling pace [1,2].
nAChRs are members of the Cys-loop ligand-gated ion channel superfamily, which also comprises the γaminobutyric acid type A (GABAA), glycine (Gly), and serotonin type 3 (5-HT3) receptors. nAChR is a pentamer with five homomeric or heteromeric subunits. The diversity of nAChR subunit combinations determines their ligand-binding sites, which regulate various physiological processes. For example, nAChRs can affect the cation permeability, from monovalent Na +and K+ ions to divalent Ca2+ ions. In turn, Ca2+ ions influence signal transduction, which may affect their modulation by external Ca2+ and Zn2+ cations [3][4][5].
Bibliometric analysis has been widely used to calculate the productivity of countries, institutions, authors, and the frequency of keywords to explore research hotspots/frontiers in specific fields [12][13][14]. Although nAChR channels have been a hotspot of multidisciplinary research for decades, no bibliometric studies regarding the trends in nAChR channels research activity have been published. Here, we collected scientific publications on nAChR channels research in the past 21 years, then used CiteSpace and VOSviewer for data analysis and visualization to provide researchers with some direction regarding nAChR channels research [15,16].

Data collection
The data search was conducted on 1 December 2020. The search keywords entered into the database were as follows: TS = (nicotinic acetylcholine receptor channel * CONTACT Yuanhui Hu huiyuhui55@sohu.com **Co-first authors OR nicotinic ACh receptor channel* OR nAChR *), language: (English) and year range: . The data were extracted from the Science Citation Index Expanded (SCI-expanded) of Web of Science Core Collection (WoSCC) bibliographic database, and the data were collected within 1 day to avoid any potential deviation due to the daily updating of the database. In this study, the data were downloaded directly from the database as secondary data without further animal experiments. Therefore, no ethical approval was required. Six thousand seventeen publications were obtained, and the following documents were excluded: proceedings paper (176), early access (27), book chapter (19), and retracted publication (1). In total, 5,794 articles were analyzed. The retrieval strategy of the experiments is shown in Figure 1. The VOSviewer 1.6.16 was used to identify top countries, institutions, authors, and journals. The CiteSpace 5.7 R3 was used to analyze keywords, co-cited references, and trends.

General information and annual publication output
Five thousand seven hundred and ninety-four articles were obtained. To explore the trends in nAChR channel research, we showed the number of articles per year in the form of a histogram. As shown in Figure 2, the number of publications on nAChR channel research increased gradually since 2004, reached a peak in 2013, and then began to decline, but it was still above 250. The average annual number of publications was 275.9.

Active countries and institutions
The co-occurrence map provides valuable information and helps researchers to identify the cooperative relationship [17,18]. Table 1 lists the top 10 countries and institutions that contributed to publications on the nAChR channel. Countries and institutions co-occurrence maps are shown in Figure 3 (A, B).
The 5,794 articles on nAChR channel research were published by more than 2,931 research institutions in 87 countries/territories. The USA, Peoples R China, England, Germany, and Japan were the top five productive countries ( Table 1). The United States published the most papers (2,905 articles), followed by China (672 articles), and they were the two critical countries in nAChR channel research. Figure 3(a) shows that the United States attached great importance to cooperation and had close collaborations with China, Japan, Germany, England France Australia, and Italy. Table 1 shows that American institutions published most of the publications. The Utah System of Higher Education (University of Utah) produced the highest number of publications on nAChR channels (417), followed by the University of California System (306). The co-occurrence map of institutions showed that scientific cooperation among institutions was greatly affected by the geographical location, and there are more cooperations among institutions in the same region ( Figure 3).

Active journals
The 5,794 articles were published in 1,003 journals.

Active authors
Author co-occurrence map can provide information on influential research groups and potential collaborators. It can help researchers to find potential collaborators [17,18]. Approximately 17,830 authors contributed 5,794 articles related to nAChR channel research. The networks shown in Figure 4 indicate the cooperation among authors, and the top 10 active authors are listed in Table 3. McIntosh, J. Michael ranked first in nAChR channel publication with 160 articles, who mainly focused on alpha9 nAChRs and their role in chronic pain [19,20]. Papke, Roger L. was the second highly published author (82 articles). His research focused on the mechanisms of nAChR ligands and signaling and contributed to addiction, pain, inflammation, and other medically important issues [21][22][23].

Co-cited references
Five thousand seven hundred and ninety-four articles were visualized and analyzed using CiteSpace with a period time from 2000 to 2020, and a time slice of 1 was chosen for the analysis of the co-cited references. The network of co-cited references on nAChR channels consists of references with higher centrality and citation counts which is presented in Figure 5. The highly cited references were analyzed to determine the key knowledge base in the field. The top 10 highest co-cited references are summarized in Table 4.  The 10 co-cited references on nAChR channel are mainly basic research from structure to function. The first highly co-cited article was "Refined structure of the nicotinic acetylcholine receptor at 4A resolution."(460 citation rate), in which Unwin N confirmed that two ligand-binding subunits had a different extended conformation from three other subunits in the closed channel, identified several interactions on both pairs of subunit interfaces and within the subunits, and finally proposed a refined structure of the nicotinic acetylcholine Receptor at 4 A°Resolution. There were five co-cited references published in Nature: In 2001, Brejc K studied the crystal structure of the molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of a nAChR a-subunit, which assisted in the development of anti-Alzheimer's disease and nicotine addiction drugs [24]. In 2003, Miyazawa A mainly focused on the structure and gating mechanism of the acetylcholine receptor pore, present an atomic model of the closed pore to shield the inner ring from the lipids [25]. In 2008, Hilf RJC reveals the first structure of pentameric ligand-gated ion channels (pLGICs) at 3.3 A°resolution and provides an important model system for the investigation of the general mechanisms of ion permeation and gating within the family [26]. One year later, Bocquet N presented the X-ray structure at 2.9 A°resolution of the bacterial Gloeobacter violaceus pLGICs at pH 4.6 in an apparently open conformation [27]. In 2011, Hibbs RE presented the first threedimensional structure at 3.3 A°resolution to explain the principles of activation and permeation in an anion-selective Cys-loop receptor [28]. These articles laid the foundation for studying the structure and mechanism of nAChR channels. Albuquerque EX and Karlin A systematically reviewed nAChR channels, including nAChR subunit structure, nAChR expression, nAChR function, and relationship with disease [29,30]. And these reviews provided a theoretical basis for the study of nAChR channels.   Bertrand D 59 7 Adams DJ 57 8 Gotti C 55 9 Marks MJ 54 10 Changeux JP 52 Research area analysis Figure 6 shows the top 15 research areas that appeared in publications related to nAChR channel research from 2000 to 2020. NEUROSCIENCES NEUROLOGY, PHARMACOLOGY PHARMACY, and BIOCHEMISTRY MOLECULAR BIOLOGY are the top three areas where nAChR channels are more studied.

Subunit and structure of nAChR
The AChR is an integral membrane protein that responds to the binding of acetylcholine. nAChR is a pentamer with five homomeric or heteromeric subunits, consisting of 10 α subunits (α1-10), four β subunits (β1-4), and γ, δ, and ε subunits. Although these subunits combine to form various nAChR subtypes, only two subtypes are highly expressed in the central nervous system. One is α4β2 heteromeric subunits, which have a high affinity for nicotine. Another is five α7 homomeric subunits, referred to as  α7nAChR, mainly contribute to α-bungarotoxin [32,33]. The structure of nAChR subunits is composed of a large extracellular N-terminal domain (NTD; the location of the Cys-loop), three hydrophobic transmembrane regions (M1-M3), a variable intracellular loop, a fourth transmembrane region (M4), and a short extracellular C-terminus. The M1, M3, andM4 segments separate the pore-lining region from the hydrophobic membrane [2,33,34].

Activation/agonist of nAChR channel
The cyclic C tyrosines and the cyclic B tryptophan behave similarly at all of the sites; the cyclic A tyrosine and γW55 are larger energy sources at αγ; αY190 provides most of the free energy from the affinity change in adult AChRs, but in Fetal αγ site and γW55 rank first together [35]. And in terms of agonist energy, the effects of the two agonist sites are almost independent in adult and fetal AChR because the sum of the energy measured from one site is approximately equal to the total energy of the two sites AChRs [36,37]. However, Mukhtasimova N. once proposed that there were two states before the opening of nAChR: the first state elicits brief openings, whereas the second elicits long-lived openings in 2009. And he found whether with or without agonists, long-lived openings and related activation states could be detected and showed the same dynamic characteristics [38].

Changes in nAChRs with alzheimer's disease
George AA demonstrated that oligomeric forms of Aβ1-42 (oAβ42) interact with α7β2-containing nAChR, and altered the intrinsic excitability of specific populations of basal forebrain cholinergic neurons (BFCNs). And he found α7β2-nAChR signaling could weaken spatial reference memory deficits in the APP/ PS1 mouse model of AD [39].
Cholinesterase inhibitors, such as galantamine, are first approved to treat mild to moderately severe Alzheimer's disease. However, the clinical potency of these drugs does not correlate well with their activity as cholinesterase inhibitors nor is their action as shortlived as purely symptomatic treatment. A subgroup of cholinesterase inhibitors can directly interact with nAChR, sensitize nicotinic receptors by increasing channel opening probability and by slowing down receptor desensitization [40].In 2008, Kowal NM evaluated galantamine actions at α4β2 and α7 nAChR in Xenopus laevis oocytes and subjected them to twoelectrode voltage-clamp electrophysiological experiments. Finally, he concluded that galantamine is not a positive allosteric modulator of α7 or α4β2 receptors [41].
A new study found SK family, calcium-sensitive potassium channels, mediated inhibition exert powerful negative feedback on nicotinic excitation, dampening attention-relevant signaling in the TgCRND8 brain. These findings may contribute to a novel therapeutic target for early attention deficits in AD [42].

Neuropathic pain
Neuropathic pain is usually chronic, caused by a lesion or disorders of the peripheral or the central nervous system. And it influences 7-10% of the general population [43]. The most common conditions of neuropathic pain include trigeminal neuralgia, peripheral nerve injury, painful polyneuropathy, postherpetic neuralgia, and painful radiculopathy [44]. Studies show that neuropathic pain seriously impairs quality of life [45,46] and cause enormous economic losses to the country and society [47].

Neuroinflammation
Neuroinflammation is common in the majority of neurological conditions. It can be both an inducement and a secondary reaction to nervous system insult [48]. The Neuroinflammation process is marked by the production of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-18 and tumor necrosis factor (TNF), chemokines, small-molecule messengers, and reactive oxygen (The format of Table 6 is variable, and the picture format is as follows:) species by innate immune cells in the CNS, and microglia and astrocytes mainly involved in this process [49][50][51]

α7 nACHR
The α7 nAChR is a ligand-gated ion channel and is highly expressed in the brain regions (e.g., the cerebral cortex and hippocampus) responsible for cognitive functions. The α7 nAChR is distributed both presynaptically and postsynaptically, to activate intracellular signaling cascades [32]. Burn injury (BI) pain consists of inflammatory and neuropathic components. BI significantly increased spinal cord microgliosis, microglia activation, and pain-transducer (protein and/or messenger RNA) expression. Zhou Yinhui found that GTS-21, a selective α7AChR agonist, mitigated paintransducer changes, while the α7AChR antagonist could nullify the beneficial effects of GTS-21 [51].
Chronic cerebral hypoperfusion (CCH) induces an inflammatory response and contributes to cognitive impairment. It is demonstrated that activating of α7nAChR and its downstream JAK2-STAT3 pathway could promote cognitive function and improve neuroprotective effects against inflammation in CCH rats [52].

Conclusions
Based on the WOSCC database, bibliometric and Visual analysis was used to study the characteristics of nAChR channel research results from 2000 to 2020. Since 2004, the number of publications on nAChR channels has maintained approximately 300 per year. The three hot spots of nAChR channel research were "subunit and structure of nAChR," "activation/agonist of nAChR channel," and "Changes in nAChRs With Alzheimer's Disease." The top three research frontiers were "neuropathic pain," "neuroinflammation," and "α7 nAChR." Bibliometric analysis of the literature on the nAChR channels contributes researchers to identify cooperations, find research hotspots, and predict the frontiers of nAChR channel research.