Gelsemium elegans extracts have been used to treat various common diseases, including neuralgia, sciatica, rheumatoid arthritis, chronic pain, and anxiety (Rujjanawate et al., 2003; Dutt et al., 2010; Jin et al., 2014; Lin et al., 2021). Nevertheless, Gelsemium elegans preparations have been also classified as strong poisons and its clinical uses are limited by their intrinsic toxicity, which may include body tremors, dyspnea, convulsions, tremors, respiratory arrest, and even death (Zhou et al., 2017; Lin et al., 2021; Qu et al., 2021; Yang et al., 2022). Interestingly, both the beneficial and toxic effects of Gelsemium elegans extracts in humans have been reproduced in experimental models (Dutt et al., 2010; Jin et al., 2014; Lin et al., 2021). Purified alkaloids from Gelsemium plants have replicated several of the actions shown by extracts. Gelsemine, one of the most abundant alkaloids in Gelsemium elegans, has shown analgesic and anxiolytic effects on murine behavioral models (Liu et al., 2013; Meyer et al., 2013; Zhang et al., 2013; Zhang and Wang, 2015) and improved sleep instabilities in mice with neuropathic pain (Wu et al., 2015).

The molecular targets underlying the therapeutic and toxic effects of gelsemine remain unclear. Several studies have reported that some of the beneficial effects of gelsemine are dependent on mechanisms involving the modulation of the enzyme 3α-hydroxysteroid oxide-reductase (3α-HSOR) (Venard et al., 2008; Shoaib et al., 2019) and of glycine receptors (GlyRs) (Lara et al., 2016; Zeilhofer et al., 2021). The proposed mechanism to explain analgesic and anxiolytic actions of gelsemine involves the GlyR-triggered stimulation of the spinal neurosteroid production through 3α-HSOR, which in turn potentiates the activity of spinal GABA_A receptors (GABA_ARs), generating anxiolysis and analgesia (Shoaib et al., 2019). On the other hand, the gelsemine induced toxicity has been related with the functional inhibition of GlyRs (Lara et al., 2016). Despite its potential relevance as a gelsemine target, potential direct actions of the alkaloid on GABA_ARs and on GABAergic synapses have been minimally studied. Since GABA_ARs are the main biological targets of benzodiazepines (BDZs), the mainstay group of drugs used to treat anxiety and sleep disorders, and of other clinically relevant drugs, a potentiation of GABA_ARs by gelsemine has been considered as a potential mechanism of action (Ghit et al., 2021; Knoflach and Bertrand, 2021). In addition, the analgesic effects of classical BDZs (Ralvenius et al., 2015) suggest a contribution of gelsemine to pain control through GABA_ARs. BDZ sensitive GABA_ARs are composed of γ2 and α1, α2, α3 or α5 subunits, mostly together with β2 or β3 subunits. Classical BDZs bind to the interface between γ2 and the mentioned α subunits at the extracellular domain of the receptor to potentiate the GABA-activated currents. Conversely, a negative modulation of GABA_ARs by the alkaloid may contribute to its toxicity. In line with this notion, a recent study showed that the toxicity induced by gelsenicine, another Gelsemium alkaloid, was significantly reverted by the systemic application of diazepam, a classical BDZ, to rats (Li et al., 2022). Therefore, using electrophysiological recordings of recombinant and native receptors, here we intend to define whether a direct modulation of GABA_ARs by gelsemine exists, Due to their relevance for the effects of BDZs and, in some cases, due to their wide expression at GABAergic synapses, we focus on recombinant γ2-containing GABA_ARs and on the impact of the alkaloid on native GABA_ARs and on GABAergic synapses.

The GABAergic neurotransmission is the main inhibitory control of the central nervous system. The activation of GABA_ARs regulates critical neurophysiological processes, such as anxiety, muscle tone, memory, pain, among others (Fritschy, 2015; Ghit et al., 2021; Knoflach and Bertrand, 2021). In line with its relevance, the GABA_ARs pharmacology has been widely developed (Ghit et al., 2021; Knoflach and Bertrand, 2021; Cerne et al., 2022). GABAergic ligands include a large variety of agonists, antagonists, and allosteric modulators coming from diverse origins, including compounds purified from fungi and plants, as well as endogenous molecules and molecules of synthetic design. Therefore, different GABA_AR subtypes possesses many acceptor sites for these modulators, which can be located in both intra-subunit and inter-subunit sites within the transmembrane or the extracellular domains of the ion channel complex (Olsen et al., 2019; Kim and Hibbs, 2021). Here, we characterize the modulation of recombinant and native GABA_ARs by gelsemine, a natural Gelsemium alkaloid. Our results indicate that gelsemine is a negative modulator, but not an agonist, of BDZ-sensitive GABA_ARs. Native GABA_ARs expressed in cultured cortical neurons were also inhibited by gelsemine. The frequency of spontaneous miniature GABAergic and glutamatergic synaptic currents was significantly diminished by the alkaloid.

A previous study reported that native GABA_ARs expressed in cultured spinal neurons were inhibited by gelsemine (Lara et al., 2016). Our results using recombinant receptors of defined composition showed that gelsemine inhibited the GABA-activated chloride currents through GABA_ARs configurations that are widely expressed in the CNS and that have been associated with the actions of BDZs (Ghit et al., 2021; Knoflach and Bertrand, 2021; Cerne et al., 2022). The inhibitory actions exerted by gelsemine on these GABA_ARs were similar, implying the absence of subunit-selective effects. In line with these observations, native GABA_ARs expressed in cultured cortical neurons, which are also BDZ-sensitive, were inhibited by gelsemine. Despite the heterogeneity of GABA_AR configurations expressed on these neurons, the IC₅₀ and the maximal current inhibition were comparable with the results from recombinant GABA_ARs. In a wider context, the results reported here allow us to generate a more complete profile of the gelsemine actions on the two inhibitory neurotransmitter-gated ion channels, GABA_ARs and GlyRs. Previous findings reported an average IC₅₀ values of ≈40 ± 4 μM and a maximal percentage of inhibition ≈ −87 ± 5% (applying 300 μM gelsemine) for recombinant GlyRs (Lara et al., 2016). Thus, a comparison of the experimental IC₅₀ values and the maximal inhibition of both chloride-permeable channels establish that GlyRs are more sensitive to the gelsemine inhibition than GABA_ARs. Although we cannot discard other mechanisms, we think that these evidences contribute to provide, at least in part, the neurophysiological bases of the toxicity of gelsemine and of Gelsemium elegans extracts (Lin et al., 2021). The direct inhibitory actions of gelsemine on GABAergic and glycinergic currents correlate well with a general depression of the inhibitory control of the central nervous system and with the symptomatology of these alkaloids in mammals. In agreement with this concept, a recent study in rats found that diazepam reverted the toxicity induced by gelsenicine, another Gelsemium alkaloid (Li et al., 2022). Further studies on inhibitory receptors may generate a rational framework to design targeted pharmacological approaches to treat acute Gelsemium alkaloid toxicity through, for example, positive allosteric modulators of GlyRs or of GABA_ARs (Lara et al., 2020; Cerne et al., 2022).

The molecular mechanisms underlying the gelsemine inhibition of GABA_ARs are unknown. Previous studies have shown that gelsemine increased the EC₅₀ for glycine of recombinant GlyRs (Lara et al., 2016). Additional reports showed that gelsemine displaced the binding of ³H-strychnine on spinal cord tissue (Shoaib et al., 2019). Likewise, our results showed that the gelsemine significantly decreased the apparent agonist sensitivity of native GABA_ARs without significant changes on the maximal currents or on the desensitization rates. Although these data collectively suggest that gelsemine may act as competitive antagonist on both ion channels, molecular or structural corroborations of the gelsemine binding to the orthosteric site of GABA_ARs or GlyRs has been not reported yet. Our results however have shown that the negative gelsemine effects on GABA_ARs require only α and β subunits, discarding the participation of γ2 subunits and hinting at a potential binding to the orthosteric site. Nevertheless, GABA_ARs composed of α and β subunits still have modulatory sites for other ligands (e.g., propofol, neurosteroids, among others) (Olsen et al., 2019; Kim and Hibbs, 2021) and, therefore, the actions of gelsemine through these sites cannot be discarded.

Our analysis of the regulation of GABAergic synapses by gelsemine revealed additional biological actions. Our findings showed that gelsemine significantly decreased the frequency of GABAergic mIPSCs and glutamatergic mEPSCs without changes on the average amplitude nor the kinetics of the synaptic currents, suggesting the participation of a presynaptic target. Based on previous studies (Lara et al., 2016) and the data presented here, one can argue that gelsemine exerted the synaptic modulation through presynaptic inhibitory receptors. The relevance of presynaptic chloride-permeable ion channels has been demonstrated in previous reports (Jang et al., 2006; Schicker et al., 2008; Choi et al., 2013; McCracken et al., 2017). Nevertheless, a role of presynaptic GABA_ARs or GlyRs on the gelsemine actions is not supported by several lines of evidence. First, our electrophysiological analyses were performed under the presence of strychnine. Second, the isolation of glutamatergic events was done using bicuculline and strychnine. Third, the gelsemine concentration used to assess the synaptic effects (i.e., 50–100 μM) will not be able to generate a strong modulation of post-synaptic GABA_ARs. Altogether, these observations suggest that gelsemine reduces excitatory and inhibitory transmission by a mechanism not related with inhibitory ion channels. In this context, it is feasible that the function of voltage-gated calcium channels, presynaptic GPCRs, or other proteins involved in the presynaptic calcium regulation could be directly affected by the alkaloid (Zhu and Pan, 2005; Pernía-Andrade et al., 2009; Booker et al., 2020). Further experiments of intracellular calcium dynamics may help to define the molecular nature of additional gelsemine-sensitive synaptic proteins in future projects.

Whether our results help to clarify the medicinal effects of gelsemine and of Gelsemium elegans extracts in mammals is complex to determine. Gelsemine concentrations within the nanomolar range have been reported to stimulate the production of spinal neurosteroids and/or to produce analgesia or anxiolysis (Shoaib et al., 2019). Since the direct effects of gelsemine on GABA_ARs occurs using concentrations >10–25 μM, a contribution of this type of fast modulation to the beneficial effects is unlikely. Thus, we believe that our findings discard that the anxiolytic (Liu et al., 2013; Meyer et al., 2013), hypnotic (Wu et al., 2015) and analgesic effects (Zhang et al., 2013; Zhang and Wang, 2015; Shoaib et al., 2019) of gelsemine are mediated by the direct potentiation of BDZ-sensitive GABA_ARs. On the other hand, the regulation of the excitatory and inhibitory synaptic activity mediated by gelsemine may contribute to explain some of its therapeutic actions. Presynaptic attenuation of synaptic activity is a common mechanism of action of clinically relevant drugs (Taylor et al., 2007; Alles et al., 2020) and a fast reduction of neurotransmission may contribute to control neural circuits implicated in anxiety and pain. In this context, it should be noted that the time scales of the beneficial behavioral effects of Gelsemium alkaloids have shown a degree of variability. For example, Venard et al. (2008)) showed that gelsemine enhanced the spinal neurosteroid production after 3 h incubation. Analgesic effects have been detected 10 min after the gelsemine intrathecal injection (Zhang et al., 2013), while anxiolytic effects have been reported after 30 min of subcutaneous administration or after 7 consecutive days of treatment with the alkaloid (Liu et al., 2013; Meyer et al., 2013). Therefore, we believe that further research on the fast effects of gelsemine at the synaptic level will complement the proposed indirect mechanisms centered on the gelsemine-induced stimulation of neurosteroids production and the subsequent modulation of GABA_ARs, contributing to generate a broader and more detailed mechanistic representation of the gelsemine actions on the CNS.

In summary, our findings establish gelsemine as a negative modulator of the GABA_ARs subtypes that contribute to the therapeutic BDZ effects. Thus, the functional pharmacological profile of gelsemine reject direct anxiolytic and analgesic actions through BDZ-sensitive GABA_ARs. However, the reduction of GABAergic currents elicited by gelsemine, together with the previously reported GlyR inhibition (Lara et al., 2016), may explain a significant part of the toxicity profile of gelsemine in mammals. On the other hand, our results showed that the alkaloid diminished the GABAergic synaptic function likely through a presynaptic mechanism. These presynaptic actions of the alkaloid may provide a complementary hypothesis to explain its beneficial effects. Future studies focused on the presynaptic compartment will help to further understand the fast actions elicited by gelsemine on the mammalian central nervous system.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AM, JG, VS, CL, AS, CB, PC, and EL-S performed the experiments and analyzed the results. JF, CM-M, LA, and GM-C contribute with reagents, critical equipment, and general resources. GM-C, JF, EL-S, and GY conceived and designed the study, analyzed the data, and wrote the article. All authors have read and approved the final manuscript.

This work was supported by ANID-FONDECYT 1211082 (to GY), ANID-FONDECYT 1211095 (GM-C), ANID-FONDECYT 1200908 (to JF) and ANID-FONDECYT 1220680 (to EL-S). This work was also supported by the Millennium Nucleus for the Study of Pain (MiNuSPain). MiNuSPain is a Millennium Nucleus supported by the Millennium Science Initiative NCN19_038 of the Ministry of Science, Technology, Knowledge and Innovation, Chile. AM, JG, VS, AS, and CL were supported by ANID-CONICYT doctoral fellowships. AS was also supported by the Graduate School Fellowship of the University of Concepción (MSc in Neurobiology).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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