Targeting α7 Nicotinic Acetylcholine Receptors in Post-Traumatic Stress Disorder: Mechanisms and Clinical Implications

Abstract

Post-traumatic stress disorder (PTSD) is a severe disorder that is characterized by intrusive thoughts, impaired mood and cognition, hyperarousal, and avoidance in the months and years following a traumatic event. PTSD is comorbid with depression, anxiety, substance abuse, and is challenging for the treatment with psycho- and pharmacotherapy. In light of novel neurobiological findings on the pathogenesis of the disease, innovative approaches to its treatment are investigated. Recently, targeting cholinergic signaling with an antagonist of α7 nicotinic acetylcholine receptors (nAChR), BNC-210, showed a significant promise in clinical trials. This opened up a novel therapeutic strategy that, considering its translational success, calls for a mechanistic understanding. In this review, we dissect the neurobiological role of α7 nAChR in regard of PTSD, and related depression and anxiety states. We discuss the synaptic role of the receptor in fear circuits, immune and endocrine role via the anti-inflammatory cholinergic pathway, and current evidence for the utility of pharmacological modulation of α7 nAChR.

1. Introduction

Post-traumatic stress disorder (PTSD) is a psychiatric disorder that develops in months and years after an exposure to severe traumatic experience. Its characteristic symptoms include re-experiencing of trauma memories, avoidance of cues that remind the individual of the trauma, negative alterations in cognition and mood, intrusive thoughts, and hyperarousal [1,2,3]. The estimated prevalence of PTSD varies; however, the risk groups include refugees and displaced populations [4], combat veterans (with up to 30% occurrence [5]), and victims of assault (up to 20% occurrence [6]).

PTSD is a multifactorial disease, and its development depends on genes [2,7], type of trauma [8], and the sex of the individual [9]. Although it nosologically overlaps with anxiety disorders and depression, The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) and the International Classification of Diseases, 11th Revision (ICD-11; World Health Organization) does not classify PTSD as an anxiety disorder [10]; instead, PTSD belongs to the category of trauma- and stressor-related disorders. Given the distinct pathological profile of PTSD, personalized and innovative therapeutic approaches are needed.

First-line treatment for PTSD involves psychotherapy, such as cognitive behavior therapy, while pharmacotherapy is often used in concert [1]. Pharmacotherapy for PTSD is usually aimed at anxiety, arousal, and depression. Currently, the only FDA-approved treatments for PTSD are the serotonin reuptake inhibitors sertraline and paroxetine. A significant proportion of patients receiving psychotherapy and standard drug therapy drop out of or are nonresponsive to therapy [11,12]. Serotonin reuptake inhibitors can be supplemented with other therapeutics for boosting efficacy or targeting comorbid symptoms, e.g., antipsychotics for intrusive thoughts, sedatives for sleep disturbance, or drugs against mood disorders and substance abuse [1,2,13]. Novel pharmacological approaches for PTSD management are searched and investigated. For example, psychedelics, such as MDMA [14,15], α-adrenergic antagonist prazosin [16], and other drugs [17] have been evaluated in clinical studies. Mechanistically, PTSD is linked to endocrine factors, neurotransmitter balance, and distinct fear-related brain circuits [3], thereby various alternative pathways and mechanisms, other than serotoninergic, may be targets for potential drug development [17].

Recently, targeting cholinergic pathway has emerged as a novel, prospective option for the treatment of fear-related pathologies [18]. The cholinergic system in the brain comprises neurons that utilize the neurotransmitter acetylcholine (ACh) for neurotransmission. ACh binds to the two types of membrane receptors: ionotropic nicotinic acetylcholine receptors (nAChR) and metabotropic muscarinic acetylcholine receptors (mAChR). nAChRs are ligand-gated cation channels that mediate fast synaptic transmission and plasticity whereas mAChRs are slow G-protein coupled receptors that modulate overall excitability of neurons. Cholinergic system also includes enzyme and transporter machinery responsible for the synthesis, transport, inactivation, and recycling of ACh [19,20,21,22,23]. This system is largely involved in cognition, learning, and memory with a pronounced role of α7 nAChR subtype [18,24,25,26]. In this review, we discuss the potential of targeting α7 nAChRs for the therapy of PTSD, the neuroimmune, endocrine, and pharmacological prerequisites for this strategy, and its current translational status.

2. Synaptic Transmission and Regulation

2.1. α7 nAChRs as Ionotropic Receptors in Neurons

nAChRs are pentameric channels that display a great receptor heterogeneity resulting from numerous subunit types that can assemble in different stoichiometries [27,28,29,30]. In humans, there are eleven neuronal (α2-7, α9, α10, β2-4) and five muscular nAChR subunits (α1, β1, γ, δ, ε) [31]. α7 subunits mostly form functional homomeric receptors with fast activation and inactivation kinetics. α7-containing heteromers have also been reported [32]. Homomeric α7 receptors demonstrate a high permeability to calcium (P_Ca/P_Na ≈ 6–20) that is comparable to NMDA receptors [33,34,35,36]. Calcium influx in neurons is generally required for neurotransmitter release and synaptic plasticity. However, excessive calcium influx can induce damaging events known as excitotoxicity, which is studied best for ionotropic glutamate receptors and is proposed to underlie several neurodegenerative diseases [37]. In a simplified form, nAChRs can adopt three functional states: closed resting, open, and closed desensitized. The desensitized state arises upon a prolonged exposure to ACh or other activators, including in their presence at subthreshold concentrations, and is unresponsive to further stimulations. The desensitized state is thus protective for the cells against receptor overstimulation and toxic calcium overload [38]. All the three functional states are considered in regard of possible therapeutic significance.

In neurons, α7 receptors can locate at the three key subcellular zones, implementing different mechanisms of regulation [39,40]. The activation of presynaptic α7 nAChRs leads to calcium influx that triggers immediate vesicle exocytosis [41,42,43]. Additionally, they can trigger voltage-gated calcium channels or calcium-induced calcium release from endoplasmic reticulum stores [44,45], which modulate the probability of neurotransmitter release [41,46,47]. Alternatively, α7 receptors can be present at the perisynaptic (synaptic cleft borders at near distance from postsynaptic density) and extrasynaptic zones (outside the synaptic cleft). There, they mediate volume transmission, responding to tonic ACh levels or ACh spillover at high-frequency stimulations [48,49]. Finally, α7 nAChRs located postsynaptically (within postsynaptic density) mediate fast depolarizing currents, activate signaling cascades, and modulate synaptic plasticity [48,50,51]. Localization in astrocytes (non-synaptic) can also contribute to the metabolic support of neurons [52,53,54,55,56].

2.2. α7 nAChRs in Metabotropic Signal Transduction Pathways

Acute physiological or psychological stress and glucocorticoids affect glutamate transmission, including effects on glutamate release, glutamate receptors, glutamate clearance and metabolism. Overstimulation of glutamate receptors (AMPAR and NMDA receptors) triggers excitotoxicity, causing cell death [37]. The α7 nAChR counteracts excitotoxicity through several distinct biological pathways, including lowering neuronal hyperactivation, blocking inflammatory cascades, and neutralizing oxidative cell death [57,58,59].

Currently, it has been extensively shown that α7 nAcRs can mediate metabotropic, or mixed signaling, in addition to their well-recognized ionotropic function [60]. Specifically, the α7 nAChR is linked to PI3K/Akt pathway, which promotes cell survival and neuroprotection, via Src tyrosine kinases. Src/Fyn tyrosine kinase activates PI3K (Phosphoinositide 3-kinase) that restores the surface level of NR2A receptors after tyrosine dephosphorylation [61]. Activation of α7 nAChRs recruits the adaptor protein β-arrestin [62] that modulates Src tyrosine kinase via the exposed SH3 domain for PI3K binding [63,64]. Activated PI3K converts PIP2 into PIP3. The latter recruits AKT to the cell membrane, while PDK1 and mTORC2 phosphorylates it into the active form. AKT next inhibits GSK-3β by phosphorylation, resulting in cell growth and survival, protein synthesis, angiogenesis, and glucose homeostasis [65]. The direct target of GSK-3β is β-catenin, a transcriptional co-activator of the WNT signaling pathway. WNT signaling is linked to the pathogenesis of various diseases such as autoimmune, cardiovascular, myocardial, neurodegenerative, metabolic disorders, and cancer. Phosphorylation of β-catenin by GSK-3β leads to the inhibition of WNT signaling and, consequently, to the enhancing of the NF-κB (nuclear factor kappa-B) pathway, which induces inflammation responses [66,67]. In sum, stimulation of α7 nAChRs triggers a chain of activation events with following inhibition of GSK-3β, that maintains WNT activity and prevents the development of inflammation (α7 nAChRs → Src → PI3K → PIP3 → AKT ⊣ GSK-3β → NF-κB pathway).

The α7 nAChRs are the main players of cholinergic anti-inflammatory pathway that links the brain and the immune system. Endotoxins/LPS, cytokines or injury generate signals traveling via vagus nerve fibers to the celiac-mesenteric ganglion which stimulates splenic nerve, resulting in activation of choline acetyltransferase (ChAT)-expressing T cells and acetylcholine release [68]. In immune cells, activation of α7 nAChRs leads to stimulation of JAK2 (Janus Kinase-2) that phosphorylates STAT3 (signal-transducer and activator of transcription-3). Activated STAT3 dimerizes and translocates to the nucleus in which it prevents the transcription of genes for inflammatory cytokines by interfering with the binding of NF-κB to promotor regions [69]. However, GSK-3β can also activate the transcription factors STAT3 and STAT5 that drive the release of pro-inflammatory IL-6 and lead to pro-inflammation response [66]. Sustained peripheral inflammation breaches the blood–brain barrier and activates resident microglia; thus, systemic immune response translates into central neuroinflammation and excitotoxicity.

As shown, PTSD arises from a disruption of the normal mechanism of fear generation and regulation within the prefrontal cortex, the hippocampus, and the amygdala [70,71]. Acute traumatic experience causes cortisol spikes; however, PTSD patients show paradoxically low baseline cortisol levels and enhanced glucocorticoid receptor (GR) sensitivity. This dysregulation of the hypothalamic–pituitary–adrenal axis, combined with intense stress-induced glutamate release, results in a decrease in BDNF levels in the cortex and hippocampus [72,73,74]. BDNF promotes brain plasticity, adaptation to new experiences, learning, and regeneration [75,76,77,78,79,80,81]. BDNF directly binds to TrkB receptors, activating the MAPK pathway, phosphorylating GRs, and, consequently, promoting healthy synaptic plasticity by enhancing long-term potentiation (LTP). Deficit in BDNF level causes morphological weakening of neurons and severe synaptic depression characterized by impaired LTP. Additionally, BDNF induces TrkB-PI3K-AKT signaling and non-canonical JAK/STAT pathway in neurons, which controls neurogenesis, neuroinflammation, and synaptic plasticity [82,83]. Contrastingly, in amygdala, the level of BDNF increases during the trauma experience, which causes its remodeling and hyperactivation, increasing fear and anxiety [72].

In the prefrontal cortex, α7 nAChRs are present on glutamatergic neurons and GABAergic interneurons [84,85,86]; thus, the receptor activation exerts a bidirectional effect on synaptic plasticity. Selective activation of α7 nAChRs on glutamatergic terminals by endogenous ACh (in the presence of a positive allosteric modulator) leads to glutamate release and spontaneous excitatory postsynaptic currents (EPSCs), thereby inducing synaptic plasticity and normal or facilitated long-term potentiation (LTP) [87]. In contrast, global activation of α7 receptors on GABAergic cell bodies by a combination of a positive allosteric modulator (PNU-120596) and an agonist (PNU-282987) triggers GABA release and spontaneous inhibitory postsynaptic currents (IPSCs), impairing plasticity and reducing LTP [87]. In the hippocampus, α7 nAChRs also regulate excitation and inhibition as in the prefrontal cortex, due to their presence on glutamatergic neurons and GABAergic interneurons. The receptors mediate cholinergic signaling at presynaptic terminals contributing to modulation of glutamate release and synaptic vesicle organization [88]. Thus, the α7 nAChR modulation can restore stress-disrupted signaling from cortex and hippocampus to amygdala [89] and potentially alleviate the behavioral and physiological symptoms of PTSD.

2.3. α7 nAChRs and Fear Circuits of the Brain

The brain regions most consistently associated with PTSD are amygdala complex, hippocampus, and medial prefrontal complex that work in concert for the acquisition and later expression of fear memory [2,90,91,92]. The complementary roles of thalamus and other structures also emerge in recent studies [2]. Conditioned fear stimuli are received by the lateral and basolateral nuclei of the amygdala leading to the fear memory consolidation, which is associated with increased synaptic efficacy and neural remodeling [93,94,95,96,97]. Axons from the centromedial subdivision of the amygdala project to the network of efferent structures that trigger fear and panic responses to traumatic cues [2]. The medial prefrontal cortex provides inhibitory control over amygdala activity, reducing subjective distress [98,99,100], whereas the hippocampus has a role both in the coding of fear memories as well as in the regulation of the amygdala [101,102]. Multiple studies demonstrated a smaller hippocampus volume in PTSD patients compared to healthy individuals, thus establishing hippocampal dysfunction as a pathological marker of PTSD [103,104].

The localization of α7 nAChR shows certain correlation with the neuroanatomical structures involved in PTSD. Extensive data on the nAChR subunit distribution within the mammalian brain is summarized by the “Human protein atlas” project that collects data on protein and RNA profiles over tissue and cell types [105]. According to the single cell RNA-seq data available on that platform, nAChR subunit abundances have distinct mapping patterns within the brain cell clusters (Figure 1).

Figure 1. Transcriptomic profile of nAChR subunits in the human brain based on single nuclei RNA sequencing (snRNA-seq). Both dot size and color intensity correspond to the mean expression level (nCPM). The plot was generated using R package ggplot2 from the publicly available data (http://www.proteinatlas.org, [105,106]).

CHRNA7 (gene encoding α7 nAChRs) demonstrates a unique expression pattern that is principally different from the other nAChR genes. High (mammillary body cluster) to moderate (CA1-3, 4, dentate gyrus) expression is observed in hippocampal structures (Figure 1), which supports the older report on the α7 transcript distribution within the human hippocampus based on in situ hybridization [107]. This, on the one hand, is consistent with the α7 role in calcium-dependent synaptic plasticity required for fear extinction. Additionally, the dominant expression in the mammillary body cluster might correlate with the receptor involvement in the spatiotemporal context of memory, the impairment of which in PTSD manifests as generalized pathological fear and flashbacks [108,109]. High expression peaks for CHRNA7 are observed in GABAergic inhibitory cell clusters (CGE, midbrain-derived inhibitory interneurons). They do not form anatomically distinct structures but rather designate cell clusters of the similar transcriptome, ontogenesis, and functional tasks. The preference to these clusters suggests that α7 receptors coordinate gamma- and theta-oscillations required for neural synchronization and prevent excessive excitation of fear circuits [110,111,112,113,114]. The prominent presence of CHRNA7 RNA in the amygdala cell cluster integrates into the neural model of pathological fear. CHRNA7 preference to inhibitory cell populations supports the electrophysiological data, indicating the α7 regulatory role in GABAergic inhibition in basolateral amygdala (BLA) [41]. In sum, simultaneous CHRNA7 expression in hippocampal structures and amygdala interneurons provides evidence that the receptor modulates the hippocampus–amygdala axis.

The mRNA of the other nAChR genes (Figure 1), particularly CHRNA4 (encoding α4 subunit) and CHRNB2 (encoding β2 subunit), is less abundant, although this contradicts the reports that showed the major prevalence of α4β2 nAChR receptors in the brain using radioligand- and immunolabeling [115,116,117,118]. Such a discrepancy highlights the importance of complementing transcriptomic data with reliable protein-level evidence. Nevertheless, the patterns of CHRNA4 and CHRNB2 are highly similar, hinting co-expression, which supports the fact that α4 and β2 co-assemble [27,119,120]. Other subunits show highly region-restricted expression. The α3 pattern marks brainstem/midbrain inhibitory pathways. It was reported that α3-containing receptors participate in the pathways underlying nicotine-induced aversion, the mechanism that prevents nicotine intoxication [121,122,123]. The tandem peaks of CHRNA6 (α6) and CHRNB3 (β3) are consistent with the reports that β3 incorporates into α6-containing receptors (α6β2β3) [124,125]. As shown before, their function is prominent in the dopaminergic system [126,127].

2.4. Genetic Variants of α7 nAChRs as Risk Factors

Genetic variants of nAChRs are robustly linked to several diseases. To date, nine human nAChR genes have been associated with Mendelian disorders [31]. The human CHRNA7 gene is located on chromosome 15q13.3, a highly unstable region of the genome prone to DNA rearrangements. Heterozygous deletion of 15q13.3 is associated with a highly variable phenotype: from no to moderate cognitive deficits and epilepsy incidences [128], while patients with homozygous deletions in this region have severe neurodevelopmental problems, with epileptic encephalopathy, hypotonia, and seizures [129]. In contrast, the role and significance of CHRNA7 duplications in the etiology of neuropsychiatric disease is less certain [31]. Duplications spanning the CHRNA7 gene were associated with attention deficit hyperactivity disorder [130]. CHRNA7 duplications were found to be enriched in adolescents with major depressive and anxiety disorders [131]. The identified copy number variants may represent risk factors that interact with other DNA risk variants or environmental factors. CHRNA7 copy increase can affect neuronal development and function. Stem cell-derived neurons obtained from a boy who had autism and CHRNA7 duplications (the affected proband) showed a distinct phenotype, developmental disruptions, and dysregulated gene expression patterns in comparison to the neurons obtained from his unaffected mother with CHRNA7 duplications or unrelated control individuals lacking the duplication. The affected proband and the unaffected mother-derived neurons both had increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity. However, the mother’s neurons exhibited upregulated gene expression in developmental pathways suggesting that molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in this model [132]. This simultaneously illustrates CHRNA7-associated cell anomalies and phenotypic variance in individuals with 15q13.3 duplications.

A CHRFAM7A gene that was found specifically in humans is a chimeric gene that consists of a partial duplication of CHRNA7 (exons 5–10), forming a hybrid with exons from ULK4 gene (A-C from the original gene and D of unknown provenance) [133]. Copy number of CHRFAM7A is highly variable. It is usually co-expressed with CHRNA7 in several tissues including leukocytes, where its product dup-α7-nAChR is presumed to form hetero-pentamers with α7 nAChR, acting as a dominant negative regulator of the receptor [133,134]. Given that CHRFAM7A is human-restricted and is not accounted for in rodent studies, it might contribute to the translational gap between preclinical and clinical trials [135].

Cognitive deficits in patients with schizophrenia are marked by diminished sensory gating, i.e., ability to filter out unimportant stimuli [136]. Sensory gating can be evaluated using P50 auditory evoked potential, which is a positive electroencephalographic waveform that occurs 50 milliseconds after presentation of an auditory stimulus. In testing conditions, subjects are presented with two consecutive auditory stimuli with a 0.5 s interval. While normal subjects show a reduced P50 to the second stimulus, patients with schizophrenia do not demonstrate the suppression of it [136]. Polymorphisms in the 5′ core promoter region of the CHRNA7 gene were found to be associated with diminished RNA expression and P50 suppression deficits [137]. A major source of P50 wave is hippocampal interneurons. Furthermore, diminished hippocampal function captured by neuroimaging techniques and decreased volume of hippocampal grey matter associated with schizophrenia is interpreted as a deficit of hippocampal inhibitory control [138]. Given that combined studies of smoking behaviors, genetics, receptor distribution and function, animal models and nicotinic effects on schizophrenia symptoms showed significant promise for targeting α7 nAChRs, no such drugs succeeded in larger phase 3 clinical trials [25]. Multiple studies showed impaired sensory gating in individuals with PTSD [139,140,141], as well as deficits in attention, processing speed, executive function, and memory [142]. Taking into account a major α7 presence in hippocampal circuits, CHRNA7 genetic variants may be a pre-existing factor in developing PTSD; however, further studies are required.

3. Cholinergic Receptors in the Neuroimmune and Endocrine Regulation

3.1. Systemic and Central Inflammation

Numerous studies have suggested that inflammation plays a significant role in the pathogenesis of diseases of central nervous system (CNS) (Figure 2) [143,144,145,146]. Infections, tissue injury, or other external stimuli activate components of the innate immune system (macrophages and dendritic cells) that produce pro-inflammatory cytokines (TNF, IL-1, IL-6, and IL-18), leading to the activation of T cells and B cells and thereby amplifying inflammation [147]. Inflammatory cytokines can enter the CNS via cellular, humoral, and neural pathways that bypass the blood–brain barrier (BBB) [147,148]. Consequently, activation of the peripheral immune system can significantly increase pro-inflammatory cytokine levels in the CNS [143,147]. Activation of endothelial cells by inflammatory cytokines (e.g., TNF-α and IL-1β) leads to the prostaglandin and nitric oxide (NO) production [149], activating microglia and astrocytes and initiating neuroinflammation [148]. Microglia, the resident immune cells of the CNS, can generally be divided into basal state, inflammatory state, and anti-inflammatory state microglia [150,151]. Microglia survey the microenvironment in real time for potential signals, including cytokines, chemokines, purines, hormones, and neurotransmitters [151]. Activated microglia and astrocytes produce numerous inflammatory mediators, thereby, promoting neuroinflammation, which can initiate or maintain psychological or neurodegenerative pathology by affecting neurotransmitter synthesis and release, the hypothalamic–pituitary–adrenal (HPA) axis, and the balance of neurotrophic factors in the body [152,153,154]. Removal of microglia or reduction of their activation in neurodegenerative diseases restores synaptic integrity and CNS function [155].

Figure 2. Schematic representation of the impact of systemic inflammation on the pathogenesis of CNS diseases.

The disruption in the circuits responsible for fear learning and extinction is a result, at least in part, from the interaction of brain structures with systemic inflammation and neuroinflammation [142,156]. PTSD is characterized by significant inflammatory activation and immune dysregulation [157,158]. It includes elevated pro-inflammatory cytokines and markers (TNF-α, IL-1β, IL-2, IL-6, IL17, IFN-γ, C-reactive protein) in both peripheral blood and the central nervous system [156,159], excessive activation of immune cells and microglia [160,161], and disrupted neuroimmune interactions [159,162].

3.2. Hypothalamic–Pituitary–Adrenal (HPA) Axis

The HPA axis is responsible for a slow and prolonged hormonal response to stress (Figure 3) [163]. Sensory input (noxious temperature, injury, inflammation) and psychological stress (fear, anxiety, emotional trauma) activate the HPA axis. The hypothalamus secretes corticotropin-releasing hormone (CRH), activating CRH receptors in the brain, resulting in release of adrenocorticotropic hormone (ACTH) from the pituitary gland [163]. ACTH then triggers the adrenal cortex to produce the stress hormone cortisol. Cortisol binds to glucocorticoid receptors (GRs) exerting widespread immune effects, acting as a negative feedback loop whereby elevated cortisol levels inhibit the hypothalamus and pituitary gland [163,164]. Generally, cortisol is a powerful anti-inflammatory hormone that inhibits the immune system and blocks inflammatory pathways in the body. Short-term elevation of cortisol levels effectively controls acute inflammation. However, long-term high cortisol levels can lead to glucocorticoid resistance of immune cells, enhancing systemic inflammation [165].

Figure 3. Schematic representation of hypothalamic–pituitary–adrenal axis and cholinergic anti-inflammatory pathway.

Significant alterations in HPA axis function have been found in patients with major depressive disorder (MDD) and post-traumatic stress disorder (PTSD). Approximately 25–30% of individuals suffering from MDD display higher cortisol levels [166,167,168], whereas previously depressed subjects showed no difference in cortisol levels compared with never-depressed individuals [168]. Different alterations in HPA axis function have been observed in PTSD patients, namely, reduced cortisol levels compared with healthy subjects [169]. The hypoactivity of the HPA axis found in PTSD patients is related to enhanced responsiveness and expression of GRs, which in turn facilitates the negative feedback mechanism [170,171]. In addition, when directly exposed to sudden acute stress or traumatic triggers, the HPA axis of PTSD patients can display either an exaggerated, sharp spike in cortisol [172,173,174] or a reduced release of cortisol [175,176], depending on the stress test used in the experiment.

3.3. Cholinergic Anti-Inflammatory Pathway

The cholinergic system plays an important role in regulation of the immune system both in peripheral tissues and in the CNS (Figure 3) [177,178]. ACh signals affect certain immune cells, barrier structures, autonomic efferent pathways, and systemic inflammatory feedback loops [179] with a great involvement of α7 nAChRs [180]. The receptor activation on macrophages, microglia, astrocytes, and other cell types decreases production of pro-inflammatory cytokines and provides anti-inflammatory effects [179]. mAChRs and other nAChR subtypes also contribute to cholinergic neuroimmune communication, but to a lesser extent [181,182]. The cholinergic anti-inflammatory pathway (CAP) is a reflex response of the nervous system to inflammation that limits peripheral immune system activation [183,184]. The brain modulates systemic inflammatory responses through the HPA axis and the cholinergic anti-inflammatory pathway via the parasympathetic nervous system [185]. The afferent fibers of the vagus nerve monitor the presence of inflammatory markers in the body. When the brain detects systemic inflammation, it can activate the HPA axis, prompting the adrenal glands to secrete glucocorticoids into the bloodstream; additionally, the efferent fibers of the vagus nerve release acetylcholine. Acetylcholine can significantly attenuate the release of pro-inflammatory cytokines (tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, and IL-18) from human macrophages [184]. The vagus nerve classically does not innervate lymphoid organs, and it is generally unclear whether ACh from efferent fibers of the vagus nerve has a significant effect on immune cells. However, vagus nerve fibers can activate the celiac ganglion, which is the starting point for innervation of the spleen by the splenic nerve [186]. Nerve fibers in the spleen utilize norepinephrine as the primary neurotransmitter [187], which is able to activate acetylcholine-producing memory T cells [188]. Activated specific populations of T cells provide sufficient ACh production, which interacts with the α7 nAChR expressed on macrophages in the spleen [188,189], leading to a significant decrease in pro-inflammatory cytokine release and attenuation of systemic inflammatory responses [184,188].

Expression of different components of the cholinergic system in non-neuronal cells of the CNS and in immune system cells underscores the complexity of cholinergic anti-inflammatory pathway regulation. α7 nAChRs are recognized as key players while α9α10 nAChRs are also shown to participate in stress-related responses via cholinergic anti-inflammatory pathway [190,191,192]. The cholinergic system, as well, includes the enzymes responsible for ACh synthesis (choline acetyltransferase, ChAT), transporters (vesicular acetylcholine transporter, VAChT), metabotropic receptors (mAChR), and the enzymes required for ACh degradation (acetylcholinesterase, AChE; butyrylcholinesterase, BChE) [179]. The Protein Atlas database [106] and numerous studies have revealed that expression of ChAT in non-neuronal cells is restricted to certain populations of mononuclear and T cells, whereas AChE and BChE are intensively produced in a variety of cells and organs, providing high levels of ACh-degrading enzymes in the serum [179,193]. The α7 nAChR, important for decreasing inflammation, is present on macrophages, dendritic cells, neutrophils, basophils, NK cells, T and B cells, microglia, and astrocytes. Mononuclear and T cells do not use classical VAChT-driven exocytosis to release ACh to affect the spleen, lymph nodes, or sites of inflammation [193,194]. Most probably, direct cell-to-cell interaction of ACh-producing cells and α7 nAChR-expressing cells is essential to transmit the signal, because of the high activity of AChE and BChE in the serum.

4. Pharmacological Modulation of α7 nAChRs

4.1. Agonists

Substantial overlap exists among PTSD, anxiety disorders, and depression in terms of symptoms, neurobiological mechanisms, current pharmacotherapy, and potential therapeutic targets. Accordingly, in this review we consider pharmacological studies of α7 nAChRs across anxiety disorders, depression, and PTSD, with particular attention to their potential relevance to fear learning and extinction. The data on animal models are summarized in Table 1 and clinical studies on humans are summarized in Table 2.

Table 1. In vivo effects of α7 nAChR modulators in animal models relevant to anxiety, depression, stress, and fear extinction.

Pharmacological Class	Compound	Dose and Route of Administration	Model/Animal Strain	Effect	Ref.
Agonists	PNU-282987	3–30 mg/kg, s.c.	mFST; female NMRI mice; combined with a sub-active dose of citalopram	Marked antidepressant-like effect	[195]
		1 mg/kg, i.p., single administration	4-week unpredictable chronic mild stress (CMS); male Sprague–Dawley rats	Reversed CMS-induced increased immobility in FST, reduced number of spontaneously active VTA dopamine neurons, and increased BLA projection-neuron firing	[196]
		10 mg/kg, i.p.	Open field and novelty-suppressed feeding tests; Sprague–Dawley rats	Increased anxiety-like behavior and latency to feed; effect on latency was reduced by WAY-100135 but not reversed by MLA	[197]
		0.5 mg/kg, i.p., daily for 14 days	TST and FST; CaMKIV-null mice	Rescued depressive-like behavior	[198]
	NS9775 (α7 agonist + triple monoamine reuptake inhibitor)	0.3–3 mg/kg, p.o.	Modified Y-maze and 5-trial inhibitory avoidance; male NMRI and C57BL/6 mice	Reversed scopolamine-induced cognitive deficits and MK-801-induced learning deficits	[26]
		10–30 mg/kg, p.o.	FST, marble-burying, four-plate test; male NMRI and C57BL/6 mice	Increased swim distance; reduced digging; increased punished crossings, consistent with antidepressant- and anxiolytic-like effects
	DMXBA	4 mg/kg, i.p.	Chronic restraint stress; Kunming mice	Reduced immobility in FST and TST and attenuated neuroinflammation and neuronal damage; effects abolished by α-bungarotoxin	[199]
	Nicotine	0.003–0.3 mg/kg, s.c., daily for 14 days	TST and FST; CaMKIV-null mice	Rescued depressive-like behavior	[198]
		0.3 mg/kg, s.c.	FST; kynurenine 3-monooxygenase knockout mice	Increased locomotor activity and prolonged immobility; effects antagonized by MLA	[200]
		0.3 mg/kg/day, s.c., once daily for 7 days	Swim stress/swim test, social interaction, conditioned place preference; male C57BL/6J mice	Attenuated stress-induced social behavioral impairments; effects blocked by MLA; restored Akt, CaMKII, and ERK phosphorylation and reduced hippocampal morphological abnormalities	[201]
	GTS-21	1 mg/kg, i.p., daily for 14 days	TST and FST; CaMKIV-null mice	Rescued depressive-like behavior	[198]
	Galantamine	1 mg/kg, p.o.	FST; kynurenine 3-monooxygenase knockout mice	Increased locomotor activity and prolonged immobility; effects antagonized by MLA	[200]
	PHA568487	0.3 mg/kg/day, i.p., once daily for 7 days	Swim stress/swim test, social interaction, conditioned place preference; male C57BL/6J mice	Attenuated stress-induced social behavioral impairments; effects blocked by MLA	[201]
	JN403	10 and 30 mg/kg, p.o.	Social exploration model; Sprague–Dawley resident rats and young Lister Hooded intruder rats	Increased duration of social contacts; effect persisted after 6 h pretreatment and subchronic administration	[202]
	PHA-543613	6 or 12 μg/20 μL, i.t.	Single prolonged stress (SPS); adult Sprague–Dawley rats	Dose-dependently attenuated SPS-induced mechanical allodynia and suppressed spinal glial activation and pro-inflammatory cytokines; effects abolished by MLA	[203]
PAMs	NS-1738 (type I PAM)	1.0 and 10.0 mg/kg; i.p.	FST and TST; male C57BL/6J mice	Antidepressant-like activity after subchronic treatment; effects inhibited by MLA	[204]
	PAM-2 (type II PAM)	0.5 and 1.0 mg/kg; i.p.	FST and TST; male C57BL/6J mice	Antidepressant-like activity after subchronic treatment; also active after chronic treatment; effects inhibited by MLA; synergism with bupropion (10 mg/kg)
		1.0 mg/kg, i.p., daily for 3 weeks	FST; male and female C57BL/6J mice	Antidepressant-like effects after 1 week of subchronic and 2 weeks of chronic treatment; no effect on locomotor activity	[205]
		0.1 and 0.5 mg/kg, i.p.	Elevated plus maze; naïve male Swiss mice	Acute anxiolytic-like effect; blocked by MLA (3 mg/kg, i.p.); reversed nicotine (0.1 mg/kg)-induced anxiogenic effects	[206]
		1 mg/kg, i.p.	Novelty-suppressed feeding test; naïve male Swiss mice	Acute anxiolytic-like effect
	PNU-120596 (type II PAM)	1 or 4 mg/kg, i.p.	LPS-induced depressive-like behavior; male C57BL/6J mice	Prevented LPS-induced changes in FST, TST, and sucrose preference; reduced Iba-1 expression in hippocampus and PFC; effects blocked by MLA (3 mg/kg, i.p.)	[207]
		1.0 and 10.0 mg/kg; i.p.	FST and TST; male C57BL/6J mice	Antidepressant-like activity after subchronic treatment; effects inhibited by MLA	[204]
		1 or 4 mg/kg, i.p.	LPS-induced disorder; male C57BL/6J mice	Anxiolytic-, pro-cognitive-, and antidepressant-like effects in EPM, Y-maze, and FST, respectively	[208]
		1 or 3 mg/kg, i.p., daily for 6 days	Cortical contusion TBI model; adult C57BL/6:129SV mice	Improved neurological motor performance and motor coordination in neuroscore, beam-walk, rotarod, and burrowing tests	[209]
	Cotinine	1 or 5 mg/kg, via gavage, daily for 8 days	Contextual fear conditioning, EPM, open field; two-month-old male C57BL/6J mice	Reduced anxiety- and depressive-like behavior and enhanced fear extinction	[210]
		0.24 mg/mL, i.n., daily for 3 weeks	Restraint, novel object recognition, FST; C57BL/6 male mice	Reduced stress- and depressive-like behavioral abnormalities and improved cognitive performance	[211]
		6 or 18 mM; intrahippocampal infusion	Fear-extinction paradigm; male Wistar rats	Enhanced fear extinction; effect prevented by hippocampal MLA	[212]
NAMs/Antagonist	Mecamylamine	1.0–10.0 mg/kg, s.c.	TST and mFST; female NMRI mice	Antidepressant-like effects; no confounding motor stimulation	[213]
		1.0 mg/kg, i.p., 30 min before testing	TST and FST; C57BL/6J mice and α7-knockout mice	Reduced immobility without altering baseline locomotor activity; effect in FST absent in α7-knockout mice	[214]
		2 μg/mL, intra-amygdala	Light–dark exploration box, TST, FST, social defeat paradigm; male C57BL/6J mice	Produced robust anxiolytic- and antidepressant-like effects	[215]
	MLA	1.0–10.0 mg/kg, s.c.	TST and mFST; female NMRI mice	Antidepressant-like effects; no confounding motor stimulation	[213]
		5 mg/kg, i.p.	TST and FST; male and female C57BL/6J mice	Antidepressant-like effects in males but not females	[216]
	BNC210	0.1–50 mg/kg, p.o.	EPM under basal and stress-induced conditions; male CD-1 and C57BL/6 mice and Wistar rats	Reduced anxiety-like behavior	[217]
		1–10 mg/kg, p.o.	Marble-burying test; male CD-1 and C57BL/6 mice and Wistar rats	Reduced stress-/anxiety-related behavior
		10–100 mg/kg, p.o.	Conditioned fear extinction; male CD-1 and C57BL/6 mice and Wistar rats	Facilitated conditioned fear extinction
		10–100 mg/kg, p.o.	FST; male CD-1 and C57BL/6 mice and Wistar rats	Reduced stress/depressive-like behavior
		0.1–50 mg/kg, p.o.	Light–dark box; male CD-1 and C57BL/6 mice and Wistar rats	Reduced anxiety-like behavior

BLA, basolateral amygdala; CMS, chronic mild stress; EPM, elevated plus maze; FST, forced swim test; LPS, lipopolysaccharide; mFST, mouse forced swim test; MLA, methyllycaconitine; PFC, prefrontal cortex; SPS, single prolonged stress; TBI, traumatic brain injury; TST, tail suspension test; VTA, ventral tegmental area.

Table 2. Clinical studies of α7 nAChR modulators in anxiety- and trauma-related disorders.

Pharmacological Class	Compound	Dose and Route of Administration	Model/Participants	Effect	Ref.
Agonist	VQW-765 (AQW051)	10 mg, single oral dose	Randomized, double-blind, placebo-controlled TSST; 230 adults with public-speaking anxiety (116 VQW-765; 114 placebo; 70% women, 30% men; mean age ≈ 40 years)	Trend toward reduced anxiety intensity during the performance phase measured by SUDS (p = 0.1443); statistically significant response in women (p = 0.034)	[218]
		—	Phase III clinical trial in social phobia, USA	Ongoing clinical development	NCT07221578
NAM	BNC210	2000 mg, oral	Cholecystokinin tetrapeptide-induced panic model; 59 healthy participants	Significantly reduced the number and overall intensity of panic symptoms measured by the Panic Symptom Scale	[18]
		300 mg or 2000 mg, oral suspension	Phase 2 study; 24 participants with generalized anxiety disorder (21 women)	300 mg, but not 2000 mg, reduced amygdala responses to fearful faces and task-related anterior cingulate functional connectivity	[219]
		300 mg or 2000 mg, oral suspension	Same Phase 2 GAD cohort; 24 participants (21 women)	Low-dose BNC210 reduced Spielberger state anxiety and flight intensity in the female subgroup and altered defense-related behavior in the joystick-operated runway task	[220]
		225 or 675 mg, single oral dose	Phase 2 social anxiety disorder study (NCT05193409); 151 adults, mean age 36 years, 37% men and 63% women; simulated public-speaking challenge	Primary endpoint based on mean SUDS change was not met; post hoc analysis showed reduced stress AUC with 225 mg vs. placebo (p = 0.044); good tolerability	[221]
		900 mg twice daily, oral dispersible tablet, 12 weeks	Phase 2b ATTUNE trial (NCT04951076); modified ITT population of 182 adults with PTSD, mean age ≈42 years, majority women	Statistically significant and clinically meaningful reduction in PTSD symptom severity on CAPS-5 at week 12; marked reduction in depressive symptoms on MADRS	[222]
		—	Phase 2 PTSD study (NCT02933606)	Clinical development in PTSD	[18]
		—	Phase III AFFIRM study (NCT06510504)	Ongoing development for stressor-related disorders/PTSD	[221]

CAPS-5, Clinician-Administered PTSD Scale for DSM-5; GAD, Generalized anxiety disorder; MADRS, Montgomery–Åsberg Depression Rating Scale; SUDS, Subjective Units of Distress Scale; TSST, Trier Social Stress Test.

Studies with different α7 nAChR agonists have provided evidence for anxiolytic- and antidepressant-like effects, although the behavioral consequences of receptor activation are non-linear and highly dependent on the compound used. Some studies have revealed that α7 nAChR agonists were more effective at lower doses, while higher doses had no effect, showing an inverted U-shaped dose–response curve [223,224,225].

The α7-selective full agonist PNU-282987 [226], administered subcutaneously at 3–30 mg/kg in combination with a sub-active dose of the selective serotonin reuptake inhibitor citalopram, produced marked antidepressant-like effects in female NMRI mice in the mouse forced swim test (mFST) [195]. In another study, the mechanisms underlying the antidepressant-like effects of PNU-282987 were examined in a model of unpredictable chronic mild stress (CMS) in male Sprague–Dawley rats. Animals were exposed to CMS for four weeks and subsequently received a single systemic administration of PNU-282987 (1 mg/kg, i.p.). CMS increased immobility in the FST, decreased the number of spontaneously active dopamine neurons in the ventral tegmental area, and increased the firing rate of putative projection neurons in the basolateral amygdala. PNU-282987 reversed these stress-induced behavioral and electrophysiological alterations, suggesting that α7 nAChR activation can modulate interactions between amygdala activity and dopaminergic neurotransmission under chronic stress [196].

However, α7 activation does not invariably produce anxiolytic effects. PNU-282987 administered intraperitoneally at 10 mg/kg to Sprague–Dawley rats increased anxiety-like behavior in the open field test and significantly increased latency to feed in the novelty-suppressed feeding test. The latter effect was significantly reduced by the serotonin 5-HT1A receptor antagonist WAY-100135, whereas the α7 nAChR antagonist methyllycaconitine (MLA) failed to reverse the anxiety-like effects induced by PNU-282987 [197]. These findings emphasize that α7 agonism does not necessarily translate into a uniform anxiolytic phenotype and that downstream serotonergic mechanisms may contribute to some behavioral effects of PNU-282987.

NS9775 represents a pharmacologically distinct approach because, in addition to α7 nAChR agonism (EC₅₀ = 280 nM), it inhibits the reuptake of three monoamines. In male NMRI and C57BL/6 mice, NS9775 (0.3–3.0 mg/kg, p.o.) reversed scopolamine-induced deficits in a modified Y-maze and MK-801-induced learning deficits in the 5-trial inhibitory avoidance test. At 30 mg/kg, NS9775 increased swim distance in the forced swim test, whereas 10 and 30 mg/kg reduced digging behavior in the marble-burying paradigm and increased the number of punished crossings in the four-plate test, indicating pro-cognitive, antidepressant-like, and anxiolytic-like activity [26]. Because NS9775 combines α7 agonism with triple monoamine reuptake inhibition, its behavioral profile cannot be attributed exclusively to α7 nAChR activation.

The selective α7 nAChR agonist DMXBA also demonstrated antidepressant-like effects in a chronic restraint stress (CRS) model. DMXBA (4 mg/kg, i.p.) significantly reduced the duration of immobility in both the FST and tail suspension test (TST) in Kunming mice and attenuated CRS-associated neuroinflammation and neuronal damage. These effects were abolished by the selective α7 nAChR antagonist α-bungarotoxin (1 μg/kg, i.p.), supporting the involvement of α7 nAChRs [199].

Additional evidence comes from genetically modified models. In CaMKIV-null mice, which exhibit deficits in contextual and cued fear-conditioning memory and decreased anxiety-like behavior [227,228] 14-day administration of nicotine (0.003–0.3 mg/kg, s.c.), PNU-282987 (0.5 mg/kg, i.p.), or GTS-21 (1 mg/kg, i.p.) significantly rescued depressive-like behavior in the TST and FST [198]. These findings are particularly relevant to the relationship between α7 nAChRs and stress-related disorders because they indicate that pharmacological stimulation of the receptor can compensate, at least partially, for deficits in intracellular signaling pathways associated with synaptic plasticity.

In kynurenine 3-monooxygenase knockout mice, a model relevant to mechanisms implicated in major depressive disorder [229], nicotine (0.3 mg/kg, s.c.) and galantamine (1 mg/kg, p.o.) increased locomotor activity and prolonged immobility in the FST. These effects were antagonized by MLA (2 mg/kg, s.c.), further implicating α7 nAChRs in the behavioral response [200].

Nicotine and the selective α7 nAChR agonist PHA568487 have also been investigated in stress-related social dysfunction. In male C57BL/6J mice, nicotine (0.3 mg/kg/day, s.c.) or PHA568487 (0.3 mg/kg/day, i.p.), administered once daily for seven consecutive days (days 2–8), attenuated stress-induced impairments in the swim stress and swim tests, social interaction test, and conditioned place preference test. The beneficial effects were blocked by MLA (2 mg/kg, s.c.). At the molecular level, nicotine ameliorated stress-associated reductions in Akt, CaMKII, and ERK phosphorylation and reduced morphological abnormalities in the hippocampus without inducing conditioned place preference, suggesting that hippocampal α7 nAChR signaling contributes to social behavioral deficits after stress [201].

The partial α7 nAChR agonist JN403 [230] (10 and 30 mg/kg, p.o.) also demonstrated anxiolytic-like properties in the social exploration model. In Sprague–Dawley “resident” rats exposed to young Lister Hooded “intruder” rats, JN403 significantly increased the duration of social contacts. This effect persisted after a 6 h pretreatment period and following subchronic administration [202].

The potential relevance of α7 nAChR agonism to PTSD-related somatic symptoms has also been demonstrated in a single prolonged stress (SPS) model in adult Sprague–Dawley rats, an established model of PTSD and its pain comorbidity. Intrathecal administration of the selective α7 nAChR agonist PHA-543613 (EC₅₀ = 65 nM [231]) at 0.3 or 0.6 μg/μL attenuated SPS-induced mechanical allodynia, assessed by paw withdrawal mechanical threshold, in a dose-dependent manner. PHA-543613 also suppressed SPS-induced spinal glial activation and increase in spinal pro-inflammatory cytokines. These effects were abolished by MLA (0.5 μg/μL, i.t.), supporting the involvement of α7 nAChRs [203].

The translational potential of α7 nAChR agonism has also been explored clinically. VQW-765, formerly known as AQW051, is a selective α7 nAChR agonist with an EC₅₀ of 7.5 μM that demonstrated effects on cognitive function in animal models [232]. A randomized, double-blind, placebo-controlled trial included 230 adults (70% women and 30% men; mean age approximately 40 years) with a history of public-speaking anxiety. Participants received a single oral dose of VQW-765 (10 mg; n = 116) or placebo (n = 114) before the Trier Social Stress Test (TSST). Anxiety was assessed using the Subjective Units of Distress Scale (SUDS). Notably, only women showed a statistically significant response to the treatment [218]. VQW-765 is currently in a Phase III clinical trial for social phobia in the USA (oral administration; NCT07221578).

4.2. Positive Allosteric Modulation

An alternative strategy is to enhance endogenous cholinergic signaling rather than directly activate α7 nAChRs. This can be achieved with positive allosteric modulators (PAMs)—compounds that enhance ACh-induced currents but do not activate the receptors when used alone. α7 nAChR PAMs are commonly divided into two classes. Type I PAMs increase α7-mediated currents without substantially altering receptor desensitization kinetics, whereas type II PAMs both potentiate receptor-mediated currents and slow the rate of desensitization [233].

The first evidence for antidepressant activity of an α7 type II PAM was obtained with 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). C57BL/6J mice received PAM-2 (1.0 mg/kg, i.p.) daily for three weeks and were evaluated in the FST. PAM-2 produced antidepressant-like effects in both sexes after one week of subchronic treatment and after two weeks of chronic treatment without affecting locomotor activity [205].

A subsequent study compared the effects of the type I PAM NS-1738 (1.0 and 10.0 mg/kg, i.p.) with those of the type II PAMs PNU-120596 (1.0 and 10.0 mg/kg, i.p.) and PAM-2 (0.5 and 1.0 mg/kg, i.p.) in male C57BL/6J mice. All three compounds produced antidepressant-like activity in the FST and TST following subchronic treatment, whereas PAM-2 also remained active following chronic treatment. MLA inhibited these effects, and synergism was observed between PAM-2 and the antidepressant bupropion (10.0 mg/kg). Analysis of signaling pathways in the prefrontal cortex and hippocampus of mice chronically treated with PAM-2 indicated activation of both mTOR and ERK pathways. Importantly, the data suggested that the antidepressant-like effects of α7 PAMs were associated with receptor potentiation rather than with α7 desensitization or neurotransmitter transporter blockade [204].

PAM-2 has also demonstrated anxiolytic-like effects. In naïve male Swiss mice, acute i.p. administration of PAM-2 produced anxiolytic-like activity at 0.5 mg/kg in the elevated plus maze and at 1 mg/kg in the novelty-suppressed feeding test. Following chronic treatment (i.p.), the compound remained active at a lower dose of 0.1 mg/kg in the elevated plus maze. The anxiolytic-like effect of PAM-2 at 0.5 mg/kg was inhibited by MLA (3 mg/kg, i.p.), suggesting α7 nAChR involvement. Furthermore, PAM-2 (0.5 mg/kg) reversed the anxiogenic effects induced by nicotine (0.1 mg/kg) [206].

PNU-120596 has been extensively studied in models of inflammation-associated depression. In male C57BL/6J mice, systemic administration of lipopolysaccharide (LPS; 1 mg/kg, i.p.) increased immobility in the FST and TST and reduced sucrose preference. PNU-120596 (1 or 4 mg/kg, i.p.) dose-dependently prevented these LPS-induced depressive-like changes and reduced the LPS-induced increase in expression of ionized calcium-binding adaptor molecule 1 (Iba-1), a marker of microglial activation, in the hippocampus and prefrontal cortex. Pretreatment with MLA (3 mg/kg, i.p.) significantly attenuated the antidepressant-like effects of PNU-120596 (4 mg/kg, i.p.) [207]. In the same LPS-induced disorder model (LPS 1 mg/kg, i.p.), PNU-120596 (1 or 4 mg/kg) produced anxiolytic-, pro-cognitive-, and antidepressant-like effects in the elevated plus maze, Y-maze, and FST, respectively [208].

The neuroprotective potential of α7 potentiation has also been demonstrated following traumatic brain injury. In adult C57BL/6:129SV mice subjected to cortical contusion injury, PNU-120596 (1 or 3 mg/kg, i.p.), administered for six days, significantly improved neurological motor performance, assessed with a seven-item, four-point neuroscore, as well as motor coordination in the beam-walk, rotarod, and burrowing tests [209]. Although this study did not directly assess fear extinction, it supports the broader concept that α7 nAChR potentiation can influence neural recovery and plasticity following severe stress or brain injury.

Cotinine, the predominant metabolite of nicotine, represents another potentially relevant pharmacological approach. Cotinine has been proposed to act as a positive allosteric modulator of α7 nAChRs, possibly by stabilizing the active receptor conformation [234]. Cotinine reduced anxiety- and depressive-like behavior and facilitated fear extinction in contextual fear conditioning, elevated plus maze, and open field tests [210,235]. Beneficial effects were also reported in restraint, novel object recognition, and FST [211]. Particularly relevant to fear extinction, direct infusion of cotinine into the hippocampus of rats enhanced extinction, and this effect was prevented by the selective α7 nAChR antagonist MLA, providing pharmacological evidence for a role of hippocampal α7 nAChRs in this process [212]. Together, these findings make cotinine one of the more direct experimental links between α7 nAChR modulation and the extinction of conditioned fear.

4.3. Antagonists and Negative Allosteric Modulation

At first glance, the evidence that α7 nAChR agonists and PAMs can improve stress-related phenotypes appears inconsistent with reports that α7 nAChR antagonists or negative allosteric modulators (NAMs) can produce similar behavioral effects. However, these apparently opposing findings may reflect the physiological role of α7 nAChRs as regulators of cholinergic tone rather than a simple linear relationship between receptor activation and therapeutic efficacy. In conditions characterized by excessive or dysregulated cholinergic signaling, reducing α7 nAChR activity may normalize circuit excitability and alter maladaptive stress responses.

Early evidence for this concept came from studies of central nicotinic receptor blockade. Antagonism of central α7 nAChRs produced antidepressant-like effects in mice [213]. In female NMRI mice, the effects of nicotine (0.1–1.0 mg/kg), the α7-selective agonist PNU-282987 (3.0–30.0 mg/kg), the non-selective antagonist mecamylamine (1.0–10.0 mg/kg), the α7-selective antagonist MLA (1.0–10.0 mg/kg), and the non-brain-penetrant non-selective antagonist hexamethonium (3.0–10.0 mg/kg) were examined after subcutaneous administration in the TST and FST. Mecamylamine and MLA produced antidepressant-like effects without confounding motor stimulation, whereas hexamethonium was inactive, supporting the involvement of central rather than peripheral nicotinic receptors. None of the tested nicotinic agonists produced an antidepressant-like profile [213]. These findings are consistent with the cholinergic/adrenergic hypothesis of depression, which proposed that excessive cholinergic activity contributes to depressive states [236].

The role of central nicotinic signaling was further investigated using mecamylamine. In C57BL/6J mice, intraperitoneal administration of mecamylamine 30 min before behavioral testing reduced immobility in the TST at 1.0 mg/kg without affecting baseline locomotor activity. At the same dose, mecamylamine also reduced immobility in the FST. Notably, α7-knockout mice were insensitive to mecamylamine in the FST, suggesting that the antidepressant-like effects of reduced central nicotinic signaling may depend, at least in part, on α7 subunits [214].

More direct evidence for a role of α7 nAChRs in amygdala was obtained in male C57BL/6J mice. Local infusion of mecamylamine into the amygdala at 2 μg/mL, as well as viral-mediated downregulation of the α7 nAChR subunit in the amygdala, produced robust anxiolytic- and antidepressant-like effects in the light–dark exploration box, TST, FST, and social defeat paradigm [215]. These findings are particularly relevant to PTSD, given a central role of amygdala in the fear acquisition, threat appraisal, and extinction.

Sex-dependent effects have also been reported. MLA (5 mg/kg, i.p.) produced significant antidepressant-like effects in male C57BL/6J mice in the TST and FST, whereas the same effect was not observed in female mice. Conversely, the α7 nAChR agonist GTS-21 (17 mg/kg) did not produce antidepressant-like effects in this study [216]. Such sex-dependent responses should be considered when interpreting the apparently divergent behavioral effects of α7 nAChR activation and inhibition.

Interestingly, α7 nAChR inhibition may also contribute to the pharmacological actions of conventional antidepressants. Duloxetine, a prototype dual antidepressant [237], inhibits human α7 nAChR-mediated currents with an IC₅₀ of 0.1 μM [238]. Other antidepressants, including fluoxetine and sertraline, as well as tricyclic antidepressants such as imipramine, amitriptyline, and doxepin, have also been reported to inhibit α7 nAChRs at micromolar concentrations in cellular systems and hippocampal preparations [239,240,241,242]. Thus, α7 nAChR inhibition may represent an underappreciated component of the pharmacological profile of some established antidepressants.

4.4. BNC210: Translational Evidence for α7 nAChR Negative Allosteric Modulation

Among α7 nAChR NAMs, BNC210 provides the strongest translational evidence for therapeutic development in anxiety- and trauma-related disorders. BNC210 was originally developed in a quinolone program by Bionomics Limited (Thebarton, Australia) for the treatment of anxiety, trauma, and stressor-related disorders [243]. It acts as a selective α7 nAChR negative allosteric modulator, with an IC50 of approximately 3 μM for human α7 nAChRs [217].

BNC210 has demonstrated an anxiolytic-like profile across several rodent species and behavioral paradigms. Oral administration to male CD-1 and C57BL/6 mice and Wistar rats reduced anxiety- and stress-related behaviors in the light–dark box (0.1–50 mg/kg), marble-burying test (1–10 mg/kg), conditioned fear extinction (10–100 mg/kg), elevated plus maze under basal and stress-induced conditions (0.1–50 mg/kg), and forced swim test (10–100 mg/kg) [217]. Of particular relevance to the present discussion, BNC210 was active in the conditioned fear extinction paradigm, providing direct preclinical evidence that negative allosteric modulation of α7 nAChRs can facilitate extinction of conditioned fear.

Clinical studies have subsequently provided evidence that BNC210 engages α7 nAChRs in humans. In healthy volunteers, target engagement was demonstrated using a nicotine-shift assay combined with quantitative electroencephalography. In a cholecystokinin tetrapeptide-induced panic model involving 59 participants, BNC210 (2000 mg) significantly reduced both the number and overall intensity of panic symptoms as measured by the Panic Symptom Scale [18].

Functional neuroimaging has provided further evidence for modulation of fear- and anxiety-related circuitry. In a Phase 2 study involving 24 individuals with generalized anxiety disorder (21 women), a low dose of BNC210 administered as an oral suspension (300 mg), but not the higher 2000 mg dose, reduced amygdala responses to fearful faces and decreased task-related anterior cingulate functional connectivity [219]. In the same patient group, low-dose BNC210 significantly reduced Spielberger state anxiety and flight intensity in 21 female participants. BNC210 also altered activity in brain regions involved in anxiety and produced changes in defense-related behavior assessed using the joystick-operated runway task [220].

A spray-dried dispersion tablet formulation was subsequently developed to overcome the solubility and bioavailability limitations of the aqueous suspension. This formulation was used in the Phase 2 social anxiety disorder (SAD) trial (NCT05193409) and the second trial in PTSD patients (NCT02933606) [18]. In the Phase 2 study of SAD (NCT05193409), the efficacy and tolerability of acute BNC210 administration were evaluated using a simulated public-speaking challenge [221]. A total of 151 adults with social anxiety disorder (mean age 36 years; 37% men and 63% women) received a single dose of BNC210 (225 or 675 mg) or placebo before the simulated public-speaking test. Notably, the study did not meet its primary endpoint for the mean change in SUDS scores, although post hoc analysis showed a statistically significant reduction in stress, calculated as the area under the curve (AUC), in the 225 mg group compared with placebo (p = 0.044). BNC210 was well tolerated, supporting further clinical development in the ongoing Phase 3 AFFIRM study (NCT06510504) [221].

Most importantly for PTSD, BNC210 has progressed to clinical testing as a chronic treatment for trauma- and stressor-related disorders. In the 12-week Phase 2b ATTUNE trial (NCT04951076), a new dispersible tablet formulation of BNC210 administered at 900 mg twice daily was evaluated in patients with PTSD. The modified intention-to-treat (ITT) population included 182 participants with a mean age of approximately 42 years, most of whom were women; physical or sexual assault represented the predominant causes of PTSD. At week 12, BNC210 produced a statistically significant and clinically meaningful reduction in overall PTSD symptom severity measured by the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) compared with placebo. A marked reduction in depressive symptoms was also observed at week 12 according to the Montgomery–Åsberg Depression Rating Scale (MADRS), supporting further development of BNC210 in PTSD [222].

5. Conclusions

Pharmacological modulation of fear-related disorders is generally aimed at restoring or enhancing the synaptic plasticity that underlies adaptive communication between the medial prefrontal cortex, hippocampus, and amygdala. The α7 nAChR is increasingly recognized as one of the cholinergic regulators of these processes. The α7 subtype is generally present in the associated brain regions and cell clusters. Considering its profound role in cognition, learning, and memory, α7 may represent a molecular node for the fine-tuning of fear learning and extinction.

Novel findings position PTSD as a disorder of neuroimmune dysfunction, with important contributions from systemic and central inflammation. The available data suggest that stimulating cholinergic anti-inflammatory pathway may serve as a potential therapeutic strategy for treating fear and anxiety disorders, including PTSD, in the future; however, the current evidence is scarce [156,244]. Given that various components of the cholinergic system are relevant for the neuroendocrine regulation, including, primarily, α7 and α9α10 nAChR receptors, as well as enzyme and transport machineries, targeting them with selective approaches can be investigated.

The pharmacological studies indicate that α7 nAChRs represent a complex and context-dependent target in fear, anxiety, depression, and PTSD. The agonists of α7 represent a straightforward strategy aimed at enhancing cholinergic signaling; however, they produce non-linear effects. Because α7 nAChRs rapidly desensitize following fast activation as well as convert to the desensitized state upon prolonged activation or in the presence of subthreshold activator concentrations, behavioral effects upon the exposure to direct agonist can be complex to interpret or negligible [38]. PAMs represent an interesting alternative to direct agonists, because they enhance the basal level of cholinergic signaling. Both direct agonism and positive allosteric modulation can enhance synaptic plasticity, reduce stress-related behavioral abnormalities, and, in some experimental settings, facilitate fear extinction. Cotinine provides particularly relevant evidence for hippocampal α7-dependent enhancement of extinction. Notably, preclinical animal studies on α7 nAChRs poorly translate to the success of clinical trials, possibly due to massive species-specific differences in cholinergic biology between rodents and humans and insufficient selectivity of the compounds used. Notably, to date, clinical trials of α7 nAChR agonists, partial agonists, and PAMs produced uncertain outcomes, with only a limited number demonstrating durable cognitive improvements in schizophrenia and Alzheimer’s disease patients, despite encouraging preclinical results [225].

At the same time, α7 nAChR antagonism and negative allosteric modulation can also produce anxiolytic- and antidepressant-like effects and may facilitate fear extinction, as exemplified most clearly by BNC210. Rather than indicating a contradiction, these findings might suggest that the therapeutic window of α7 nAChR modulation depends on the basal level of cholinergic signaling, receptor state, brain region, stress history, and behavioral context. The clinical development of BNC210 is especially important because it demonstrates that modulation of α7 nAChRs can possibly be translated into clinical studies of stress-related disorders and PTSD.