Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors.

Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABAA receptors (GABAARs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC50 of 31.5 ± 1.7 and 40.6 ± 8.2 µM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABAARs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS.

Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.

Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.

Roles of the Unsaturated Fatty Acid Docosahexaenoic Acid in the Central Nervous System: Molecular and Cellular Insights.

Fatty acids (FAs) are essential components of the central nervous system (CNS), where they exert multiple roles in health and disease. Among the FAs, docosahexaenoic acid (DHA) has been widely recognized as a key molecule for neuronal function and cell signaling. Despite its relevance, the molecular pathways underlying the beneficial effects of DHA on the cells of the CNS are still unclear. Here, we summarize and discuss the molecular mechanisms underlying the actions of DHA in neural cells with a special focus on processes of survival, morphological development, and synaptic maturation. In addition, we examine the evidence supporting a potential therapeutic role of DHA against CNS tumor diseases and tumorigenesis. The current results suggest that DHA exerts its actions on neural cells mainly through the modulation of signaling cascades involving the activation of diverse types of receptors. In addition, we found evidence connecting brain DHA and ω-3 PUFA levels with CNS diseases, such as depression, autism spectrum disorders, obesity, and neurodegenerative diseases. In the context of cancer, the existing data have shown that DHA exerts positive actions as a coadjuvant in antitumoral therapy. Although many questions in the field remain only partially resolved, we hope that future research may soon define specific pathways and receptor systems involved in the beneficial effects of DHA in cells of the CNS, opening new avenues for innovative therapeutic strategies for CNS diseases.

Fast synaptic inhibition in spinal sensory processing and pain control.

The two amino acids GABA and glycine mediate fast inhibitory neurotransmission in different CNS areas and serve pivotal roles in the spinal sensory processing. Under healthy conditions, they limit the excitability of spinal terminals of primary sensory nerve fibers and of intrinsic dorsal horn neurons through pre- and postsynaptic mechanisms, and thereby facilitate the spatial and temporal discrimination of sensory stimuli. Removal of fast inhibition not only reduces the fidelity of normal sensory processing but also provokes symptoms very much reminiscent of pathological and chronic pain syndromes. This review summarizes our knowledge of the molecular bases of spinal inhibitory neurotransmission and its organization in dorsal horn sensory circuits. Particular emphasis is placed on the role and mechanisms of spinal inhibitory malfunction in inflammatory and neuropathic chronic pain syndromes.

Glycine receptors and glycine transporters: targets for novel analgesics?

Glycinergic neurotransmission has long been known for its role in spinal motor control. During the last two decades, additional functions have become increasingly recognized-among them is a critical contribution to spinal pain processing. Studies in rodent pain models provide proof-of-concept evidence that enhancing inhibitory glycinergic neurotransmission reduces chronic pain symptoms. Apparent strategies for pharmacological intervention include positive allosteric modulators of glycine receptors and modulators or inhibitors of the glial and neuronal glycine transporters GlyT1 and GlyT2. These prospects have led to drug discovery efforts in academia and in industry aiming at compounds that target glycinergic neurotransmission with high specificity. Available data show promising analgesic efficacy. Less is currently known about potential unwanted effects but the presence of glycinergic innervation in CNS areas outside the nociceptive system prompts for a careful evaluation not only of motor function, but also of potential respiratory impairment and addictive properties.

Cytotoxicity and in vivo plasma kinetic behavior of surface-functionalized PAMAM dendrimers.

Understanding the molecular features responsible for the plasma kinetics of surface-modified polyamido amine (PAMAM) dendrimers is critical to explore novel biomedical applications for these nanomaterials. In this report, polyethylene glycol (PEG) and folic acid (FA) were employed to obtain partially-substituted PAMAM dendrimers as model biocompatible nanomaterials with different size, charge and surface functionality. Cytotoxicity assays on HEK cells at 1-500 µM concentration confirmed that PEG and FA incorporation increased the cell viability of PAMAM-based nanomaterials. Measurements of plasma kinetics in vivo revealed that PEG-PAMAM has an extended circulation time in mice blood (71.7 min) over native PAMAM (53.3 min) and FA-PAMAM (41.8 min). Molecular dynamics simulations revealed a direct relationship between circulation time and dendrimer size, thus providing valuable evidence to increase understanding about the modulation of functional properties of PAMAM-based systems through surface modification, and to guide future efforts on the rational design of novel biomedical nanomaterials.

Reversal of Ethanol-induced Intoxication by a Novel Modulator of Gßγ Protein Potentiation of the Glycine Receptor.

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of Gßγ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of Gßγ. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication.

Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors.

Glycine receptors (GlyR) are inhibitory Cys-loop ion channels that contribute to the control of excitability along the central nervous system (CNS). GlyR are found in the spinal cord and brain stem, and more recently they were reported in higher regions of the CNS such as the hippocampus and nucleus accumbens. GlyR are involved in motor coordination, respiratory rhythms, pain transmission, and sensory processing, and they are targets for relevant physiologic and pharmacologic modulators. Several studies with protein crystallography and cryoelectron microscopy have shed light on the residues and mechanisms associated with the activation, blockade, and regulation of pentameric Cys-loop ion channels at the atomic level. Initial studies conducted on the extracellular domain of acetylcholine receptors, ion channels from prokaryote homologs-Erwinia chrysanthemi ligand-gated ion channel (ELIC), Gloeobacter violaceus ligand-gated ion channel (GLIC)-and crystallized eukaryotic receptors made it possible to define the overall structure and topology of the Cys-loop receptors. For example, the determination of pentameric GlyR structures bound to glycine and strychnine have contributed to visualizing the structural changes implicated in the transition between the open and closed states of the Cys-loop receptors. In this review, we summarize how the new information obtained in functional, mutagenesis, and structural studies have contributed to a better understanding of the function and regulation of GlyR.

Ischemia-like oxygen and glucose deprivation mediates down-regulation of cell surface γ-aminobutyric acidB receptors via the endoplasmic reticulum (ER) stress-induced transcription factor CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP).

Cerebral ischemia frequently leads to long-term disability and death. Excitotoxicity is believed to be the main cause for ischemia-induced neuronal death. Although a role of glutamate receptors in this process has been firmly established, the contribution of metabotropic GABAB receptors, which control excitatory neurotransmission, is less clear. A prominent characteristic of ischemic insults is endoplasmic reticulum (ER) stress associated with the up-regulation of the transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP). After inducing ER stress in cultured cortical neurons by sustained Ca(2+) release from intracellular stores or by a brief episode of oxygen and glucose deprivation (in vitro model of cerebral ischemia), we observed an increased expression of CHOP accompanied by a strong reduction of cell surface GABAB receptors. Our results indicate that down-regulation of cell surface GABAB receptors is caused by the interaction of the receptors with CHOP in the ER. Binding of CHOP prevented heterodimerization of the receptor subunits GABAB1 and GABAB2 and subsequent forward trafficking of the receptors to the cell surface. The reduced level of cell surface receptors diminished GABAB receptor signaling and, thus, neuronal inhibition. These findings indicate that ischemia-mediated up-regulation of CHOP down-regulates cell surface GABAB receptors by preventing their trafficking from the ER to the plasma membrane. This mechanism leads to diminished neuronal inhibition and may contribute to excitotoxicity in cerebral ischemia.

Chronic pain states: pharmacological strategies to restore diminished inhibitory spinal pain control.

Potentially noxious stimuli are sensed by specialized nerve cells named nociceptors, which convey nociceptive signals from peripheral tissues to the central nervous system. The spinal dorsal horn and the trigeminal nucleus serve as first relay stations for incoming nociceptive signals. At these sites, nociceptor terminals contact a local neuronal network consisting of excitatory and inhibitory interneurons as well as of projection neurons. Blockade of neuronal inhibition in this network causes an increased sensitivity to noxious stimuli (hyperalgesia), painful sensations occurring after activation of non-nociceptive fibers (allodynia), and spontaneous pain felt in the absence of any sensory stimulation. It thus mimics the major characteristics of chronic pain states. Diminished inhibitory pain control in the spinal dorsal horn occurs naturally, e.g., through changes in the function of inhibitory neurotransmitter receptors or through altered chloride homeo-stasis in the course of inflammation or nerve damage. This review summarizes our current knowledge about endogenous mechanisms leading to diminished spinal pain control and discusses possible ways that could restore proper inhibition through facilitation of fast inhibitory neurotransmission.

Control of ethanol sensitivity of the glycine receptor α3 subunit by transmembrane 2, the intracellular splice cassette and C-terminal domains.

Previous studies have shown that the effect of ethanol onglycine receptors (GlyRs) containing the a1 subunit is affected by interaction with heterotrimeric G proteins (Gßγ). GlyRs containing the α3 subunit are involved in inflammatory pain sensitization and rhythmic breathing and have received much recent attention. For example, it is unknown whether ethanol affects the function of this important GlyR subtype. Electrophysiologic experiments showed that GlyR α3 subunits were not potentiated by pharmacologic concentrations of ethanol or by Gßγ. Thus, we studied GlyR α1­α3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in a1 in the α3(A254G) ­α1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 ± 5%, 100 mM) and Gßγ activation (80 ± 17%). Interestingly,insertion of the intracellular α3L splice cassette into GlyR α1 abolished the enhancement of the glycine-activated current by ethanol (5 ± 6%) and activation by Gßγ (21 6 7%). In corporation of the GlyR α1 C terminus into the ethanol-resistant α3S(A254G) mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 ± 6%)together with a current enhancement after G protein activation (68 ± 25%). Taken together, these data demonstrate that GlyRα3 subunits are not modulated by ethanol. Residue A254 in TM2, the α3L splice cassette, and the C-terminal domain of α3GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.

Endoplasmic reticulum-associated degradation controls cell surface expression of γ-aminobutyric acid, type B receptors.

Metabotropic GABAB receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABAB receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABAB receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABAB receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABAB receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys(48)-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABAB2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABAB receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABAB receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABAB receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.

Extracellular signal-regulated kinase and glycogen synthase kinase 3ß regulate gephyrin postsynaptic aggregation and GABAergic synaptic function in a calpain-dependent mechanism.

Molecular mechanisms of plasticity at GABAergic synapses are currently poorly understood. To identify signaling cascades that converge onto GABAergic postsynaptic density proteins, we performed MS analysis using gephyrin isolated from rat brain and identified multiple novel phosphorylation and acetylation residues on gephyrin. Here, we report the characterization of one of these phosphoresidues, Ser-268, which when dephosphorylated leads to the formation of larger postsynaptic scaffolds. Using a combination of mutagenesis, pharmacological treatment, and biochemical assays, we identify ERK as the kinase phosphorylating Ser-268 and describe a functional interaction between residues Ser-268 and Ser-270. We further demonstrate that alterations in gephyrin clustering via ERK modulation are reflected by amplitude and frequency changes in miniature GABAergic postsynaptic currents. We unravel novel mechanisms for activity- and ERK-dependent calpain action on gephyrin, which are likely relevant in the context of cellular signaling affecting GABAergic transmission and homeostatic synaptic plasticity in pathology.

Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin.

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3ß (GSK3ß) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3ß inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca(2+)-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

TG2 promotes amyloid beta aggregates: Impact on ER-mitochondria crosstalk, calcium homeostasis and synaptic function in Alzheimer's disease.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly affects the elderly. AD's main features have been related to cellular and molecular events, including the aberrant aggregation of the amyloid beta peptide (Aß), Ca2+ dyshomeostasis, and increased mitochondria-associated membranes (MAMs). Transglutaminase type 2 (TG2) is a ubiquitous enzyme whose primary role is the Ca2+-dependent proteins transamidation, including the Aß peptide. TG2 activity has been closely related to cellular damage and death. We detected increased TG2 levels in neuronal cells treated with Aß oligomers (AßOs) and hippocampal slices from J20 mice using cellular and molecular approaches. In this work, we characterized the capacity of TG2 to interact and promote Aß toxic aggregates (AßTG2). AßTG2 induced an acute increase in intracellular Ca2+, miniature currents, and hiperexcitability, consistent with an increased mitochondrial Ca2+ overload, IP3R-VDAC tethering, and mitochondria-endoplasmic reticulum contacts (MERCs). AßTG2 also decreased neuronal viability and excitatory postsynaptic currents, reinforcing the idea of synaptic failure associated with MAMs dysregulation mediated by TG2. Z-DON treatment, TG2 inhibitor, reduced calcium overload, mitochondrial membrane potential loss, and synaptic failure, indicating an involvement of TG2 in a toxic cycle which increases Aß aggregation, Ca2+ overload, and MAMs upregulation. These data provide novel information regarding the role TG2 plays in synaptic function and contribute additional evidence to support the further development of TG2 inhibitors as a disease-modifying strategy for AD.

Elephant Black Garlic's Beneficial Properties for Hippocampal Neuronal Network, Chemical Characterization and Biological Evaluation.

Garlic has been used for decades as an important food and additionally for its beneficial properties in terms of nutrition and ancestral therapeutics. In this work, we compare the properties of fresh (WG) and aged (BG) extract obtained from elephant garlic, harvested on Chiloe Island, Chile. BG was prepared from WG with a 20-day aging process under controlled temperature and humidity conditions. We observed that in BG, compounds such as diallyl disulfide decrease, and compounds of interest such as 5-hydroxymethylfurfural (69%), diallyl sulfide (17%), 3H-1,2-Dithiole (22%) and 4-Methyl-1,2,3-trithiolane (16%) were shown to be increased. Using 2,2-diphenyl-1-picrylhydrazyl (DPPH, BG: 51 ± 5.7%, WG: 12 ± 2.6%) and 2,20-azino-bis-(3-ethylbenzothiazoline-6 sulfonate) diammonium salt (ABTS, BG: 69.4 ± 2.3%, WG: 21 ± 3.9%) assays, we observed that BG possesses significantly higher antioxidant activity than WG and increased cell viability in hippocampal slices (41 ± 9%). The effects of WG and BG were shown to improve the neuronal function through an increased in intracellular calcium transients (189 ± 4%). In parallel, BG induced an increase in synaptic vesicle protein 2 (SV-2; 75 ± 12%) and brain-derived neurotrophic factor (BDNF; 32 ± 12%) levels. Thus, our study provides the initial scientific bases to foster the use of BG from Chiloe Island as a functional food containing a mixture of bioactive compounds that may contribute to brain health and well-being.

The basic property of Lys385 is important for potentiation of the human α1 glycine receptor by ethanol.

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the α1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gßγ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 ± 1 versus 41 ± 0.5 µM) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human α1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gßγ, is critical for ethanol potentiation of the receptor.

Glycine Receptor Subtypes and Their Roles in Nociception and Chronic Pain.

Disruption of the inhibitory control provided by the glycinergic system is one of the major mechanisms underlying chronic pain. In line with this concept, recent studies have provided robust proof that pharmacological intervention of glycine receptors (GlyRs) restores the inhibitory function and exerts anti-nociceptive effects on preclinical models of chronic pain. A targeted regulation of the glycinergic system requires the identification of the GlyR subtypes involved in chronic pain states. Nevertheless, the roles of individual GlyR subunits in nociception and in chronic pain are yet not well defined. This review aims to provide a systematic outline on the contribution of GlyR subtypes in chronic pain mechanisms, with a particular focus on molecular pathways of spinal glycinergic dis-inhibition mediated by post-translational modifications at the receptor level. The current experimental evidence has shown that phosphorylation of synaptic α1ß and α3ß GlyRs are involved in processes of spinal glycinergic dis-inhibition triggered by chronic inflammatory pain. On the other hand, the participation of α2-containing GlyRs and of ß subunits in pain signaling have been less studied and remain undefined. Although many questions in the field are still unresolved, future progress in GlyR research may soon open new exciting avenues into understanding and controlling chronic pain.

Antiseizure medication in early nervous system development. Ion channels and synaptic proteins as principal targets.

The main strategy for the treatment of epilepsy is the use of pharmacological agents known as antiseizure medication (ASM). These drugs control the seizure onset and improves the life expectancy and quality of life of patients. Several ASMs are contraindicated during pregnancy, due to a potential teratogen risk. For this reason, the pharmacological treatments of the pregnant Women with Epilepsy (WWE) need comprehensive analyses to reduce fetal risk during the first trimester of pregnancy. The mechanisms by which ASM are teratogens are still under study and scientists in the field, propose different hypotheses. One of them, which will be addressed in this review, corresponds to the potential alteration of ASM on ion channels and proteins involved in relevant signaling and cellular responses (i.e., migration, differentiation) during embryonic development. The actual information related to the action of ASM and its possible targets it is poorly understood. In this review, we will focus on describing the eventual presence of some ion channels and synaptic proteins of the neurotransmitter signaling pathways present during early neural development, which could potentially interacting as targets of ASM. This information leads to elucidate whether these drugs would have the ability to affect critical signaling during periods of neural development that in turn could explain the fetal malformations observed by the use of ASM during pregnancy.

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine.

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system.