Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations.

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10-200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.

Schisandrin B, a dual positive allosteric modulator of GABAA and glycine receptors, alleviates seizures in multiple mouse models.

Epilepsy is a prevalent and severe neurological disorder and approximately 30% of patients are resistant to existing medications. It is of utmost importance to develop alternative therapies to treat epilepsy. Schisandrin B (SchB) is a major bioactive constituent of Schisandra chinensis (Turcz.) Baill and has multiple neuroprotective effects, sedative and hypnotic activities. In this study, we investigated the antiseizure effect of SchB in various mouse models of seizure and explored the underlying mechanisms. Pentylenetetrazole (PTZ), strychnine (STR), and pilocarpine-induced mouse seizure models were established. We showed that injection of SchB (10, 30, 60 mg/kg, i.p.) dose-dependently delayed the onset of generalized tonic-clonic seizures (GTCS), reduced the incidence of GTCS and mortality in PTZ and STR models. Meanwhile, injection of SchB (30 mg/kg, i.p.) exhibited therapeutic potential in pilocarpine-induced status epilepticus model, which was considered as a drug-resistant model. In whole-cell recording from CHO/HEK-239 cells stably expressing recombinant human GABAA receptors (GABAARs) and glycine receptors (GlyRs) and cultured hippocampal neurons, co-application of SchB dose-dependently enhanced GABA or glycine-induced current with EC50 values at around 5 µM, and application of SchB (10 µM) alone did not activate the channels in the absence of GABA or glycine. Furthermore, SchB (10 µM) eliminated both PTZ-induced inhibition on GABA-induced current (IGABA) and strychnine (STR)-induced inhibition on glycine-induced current (Iglycine). Moreover, SchB (10 µM) efficiently rescued the impaired GABAARs associated with genetic epilepsies. In addition, the homologous mutants in both GlyRs-α1(S267Q) and GABAARs-α1(S297Q)ß2(N289S)γ2L receptors by site-directed mutagenesis tests abolished SchB-induced potentiation of IGABA and Iglycine. In conclusion, we have identified SchB as a natural positive allosteric modulator of GABAARs and GlyRs, supporting its potential as alternative therapies for epilepsy.

[Strychnine, in The Mysterious Affair at Styles: Blocking Glycinergic Synaptic Transmission].

Strychnine is a poison that often appears in classical mysteries and has been used for medicine and various purposes. Clearly, its point of action is glycine receptors, and it inhibits glycinergic synaptic transmission. Because of its powerful stimulant effect on the nervous system, if taken orally, characteristic symptoms that are intense and agonizing, such as tonic convulsions, opisthotonus or posterior arch tension, and convulsive laughter, appear. These symptoms are linked to the pathological basis of tetanus, and the drug is an important topic ranging from neuroscience to medical care.

Regulation of ethanol-mediated dopamine elevation by glycine receptors located on cholinergic interneurons in the nucleus accumbens.

Alcohol use disorder is one of the major psychiatric disorders worldwide, and there are many factors and effects contributing to the disorder, for example, the experience of ethanol reward. The rewarding and reinforcing properties of ethanol have been linked to activation of the mesolimbic dopamine system, an effect that appears to involve glycine receptors (GlyRs) in the nucleus accumbens. On which neuronal subtypes these receptors are located is, however, not known. The aim of this study was to explore the role of GlyRs on cholinergic interneurons (CIN) in sustaining extracellular dopamine levels and in ethanol-induced dopamine release. To this end, CIN were ablated by anti-choline acetyltransferase-saporin administered locally in the nucleus accumbens of male Wistar rats. Changes in dopamine levels induced by ablation, ethanol and/or a GlyR antagonist were monitored using in vivo microdialysis. The GlyRs antagonist strychnine depressed extracellular dopamine in a similar manner independent on local ablation, suggesting that GlyRs on CIN are not important for sustaining the extracellular dopamine tone. However, a low concentration of strychnine hampered ethanol-induced dopamine release in sham-treated animals, whilst no reduction was seen in ablated animals, suggesting that GlyRs located on CIN are involved in ethanol-induced dopamine release. Further, in ablated rats, ethanol-induced increases of the extracellular levels of the GlyR agonists glycine and taurine were attenuated. In conclusion, this study suggests that CIN are not important for GlyR-mediated regulation of basal dopamine output, but that CIN ablation blunts the ethanol-induced dopamine release, putatively by reducing the release of GlyR agonists.

Cannabinoid receptors involved in descending inhibition on spinal seizure-like activity in the phrenic output.

Seizure-like burst activities are induced by blockade of GABAA and/or glycine receptors in various spinal ventral roots of brainstem-spinal cord preparation from neonatal rodents. We found that this is not applicable to the phrenic nerve and that a new inhibitory descending pathway may suppress seizure-like activity in the phrenic nerve. Experiments were performed in brainstem-spinal cord preparation from newborn rats (age: 0-1 day). Left phrenic nerve and right C4 activities were recorded simultaneously. When GABAA and glycine receptors were blocked by 10 µM bicuculline and 10 µM strychnine (Bic+Str), seizure-like burst activities appeared in the fourth cervical ventral root (C4) but not the phrenic nerve. After making a transverse section at C1, the inspiratory burst activity disappeared from both C4 and the phrenic nerve, whereas seizure-like activity appeared in both nerves. We hypothesized that inhibitory descending pathways other than those via GABAA and/or glycine receptors (from the medulla to the spinal cord) work to avoid disturbance of regular respiratory-related diaphragm contraction by seizure-like activity. We found that cannabinoid receptor antagonist, AM251 was effective for the induction of seizure-like activity by Bic+Str in the phrenic nerve in brainstem-spinal cord preparation. Cannabinoid receptors may be involved in this descending inhibitory system.

Conformational transitions and allosteric modulation in a heteromeric glycine receptor.

Glycine Receptors (GlyRs) provide inhibitory neuronal input in the spinal cord and brainstem, which is critical for muscle coordination and sensory perception. Synaptic GlyRs are a heteromeric assembly of α and ß subunits. Here we present cryo-EM structures of full-length zebrafish α1ßBGlyR in the presence of an antagonist (strychnine), agonist (glycine), or agonist with a positive allosteric modulator (glycine/ivermectin). Each structure shows a distinct pore conformation with varying degrees of asymmetry. Molecular dynamic simulations found the structures were in a closed (strychnine) and desensitized states (glycine and glycine/ivermectin). Ivermectin binds at all five interfaces, but in a distinct binding pose at the ß-α interface. Subunit-specific features were sufficient to solve structures without a fiduciary marker and to confirm the 4α:1ß stoichiometry recently observed. We also report features of the extracellular and intracellular domains. Together, our results show distinct compositional and conformational properties of α1ßGlyR and provide a framework for further study of this physiologically important channel.

Origin of Retinal Oscillatory Potentials in the Mouse, a Tool to Specifically Locate Retinal Damage.

To determine the origin of oscillatory potentials (OPs), binocular electroretinogram (ERG) recordings were performed under light and dark adaptation on adult healthy C57BL/6J mice. In the experimental group, 1 µL of PBS was injected into the left eye, while the right eye was injected with 1 µL of PBS containing different agents: APB, GABA, Bicuculline, TPMPA, Glutamate, DNQX, Glycine, Strychnine, or HEPES. The OP response depends on the type of photoreceptors involved, showing their maximum response amplitude in the ERG induced by mixed rod/cone stimulation. The oscillatory components of the OPs were affected by the injected agents, with some drugs inducing the complete abolition of oscillations (APB, GABA, Glutamate, or DNQX), whereas other drugs merely reduced the oscillatory amplitudes (Bicuculline, Glycine, Strychnine, or HEPES) or did not even affect the oscillations (TPMPA). Assuming that rod bipolar cells (RBC) express metabotropic Glutamate receptors, GABAA, GABAC, and Glycine receptors and that they release glutamate mainly on Glycinergic AII amacrine cells and GABAergic A17 amacrine cells, which are differently affected by the mentioned drugs, we propose that RBC-AII/A17 reciprocal synapses are responsible for the OP generation in the ERG recordings in the mice. We conclude that the reciprocal synapses between RBC and AII/A17 are the basis of the ERG OP oscillations of the light response, and this fact must be taken into consideration in any ERG test that shows a decrease in the OPs' amplitude.

Antiviral activities of plant-derived indole and ß-carboline alkaloids against human and avian influenza viruses.

The persistent evolution of drug-resistant influenza strains represents a global concern. The innovation of new treatment approaches through drug screening strategies and investigating the antiviral potential of bioactive natural-based chemicals may address the issue. Herein, we screened the anti-influenza efficacy of some biologically active indole and ß-carboline (ßC) indole alkaloids against two different influenza A viruses (IAV) with varied host range ranges; seasonal influenza A/Egypt/NRC098/2019(H1N1) and avian influenza A/chicken/Egypt/N12640A/2016(H5N1). All compounds were first assessed for their half-maximal cytotoxic concentration (CC50) in MDCK cells and half-maximal inhibitory concentrations (IC50) against influenza A/H5N1. Intriguingly, Strychnine sulfate, Harmalol, Harmane, and Harmaline showed robust anti-H5N1 activities with IC50 values of 11.85, 0.02, 0.023, and 3.42 µg/ml, respectively, as compared to zanamivir and amantadine as control drugs (IC50 = 0.079 µg/ml and 17.59 µg/ml, respectively). The efficacy of the predefined phytochemicals was further confirmed against influenza A/H1N1 and they displayed potent anti-H1N1 activities compared to reference drugs. Based on SI values, the highly promising compounds were then evaluated for antiviral efficacy through plaque reduction assay and consistently they revealed high viral inhibition percentages at non-toxic concentrations. By studying the modes of antiviral action, Harmane and Harmalol could suppress viral infection via interfering mainly with the viral replication of the influenza A/H5N1 virus, whilst Harmaline exhibited a viricidal effect against the influenza A/H5N1 virus. Whereas, Strychnine sulfate elucidated its anti-influenza potency by interfering with viral adsorption into MDCK cells. Consistently, chemoinformatic studies showed that all studied phytochemicals illustrated HB formations with essential peptide cleft through the NH of indole moiety. Among active alkaloids, harmalol displayed the best lipophilicity metrics including ligand efficiency (LE) and ligand lipophilic efficiency (LLE) for both viruses. Compounds geometry and their ability to participate in HB formation are very crucial.

The role of medial olivocochlear activity in contralateral suppression of auditory steady-state responses.

OBJECTIVE: The auditory steady-state response (ASSR) amplitudes fall in the presence of contralateral noise. However, whether and to what extent medial olivocochlear (MOC) activity involves in contralateral suppression of ASSR remain unclear. Therefore, we assess the role of MOC activity in contralateral suppression of ASSR. METHODS: Mice were treated with strychnine to completely eliminate MOC activity and then measured ASSR amplitudes in the presence of contralateral noise. RESULTS: The contralateral noise reduces ASSR amplitudes at some stimulus intensity. After treating with the strychnine to eliminate MOC activity, ASSR amplitudes recovered again. CONCLUSIONS: MOC activity participated in contralateral suppression of ASSR.

Functional analysis of a bitter gustatory receptor highly expressed in the larval maxillary galea of Helicoverpa armigera.

Many plant secondary substances are feeding deterrents for insects and play a key role in the selection of host plants. The taste sensilla of phytophagous insects contain gustatory sensory neurons sensitive to deterrents but the molecular basis of deterrent chemoreception remains unknown. We investigated the function of Gr180, the most highly expressed bitter gustatory receptor in the maxillary galea of Helicoverpa armigera larvae. Functional analyses using the Xenopus oocyte expression system and two-electrode voltage clamp revealed that the oocytes expressing Gr180 responded to coumarin. Tip recording results showed that the medial sensilla styloconica of the maxilla of fifth instar larvae exhibited electrophysiological responses to coumarin. Two-choice feeding bioassays confirmed that coumarin inhibited larval feeding. A homozygous mutant strain of H. armigera with truncated Gr180 proteins (Gr180-/-) was established using the CRISPR-Cas9 system. The responses of the medial sensilla styloconica in Gr180-/- to coumarin were almost abolished, and the responses to sinigrin and strychnine were also significantly decreased. Knockout of Gr180 alleviated the feeding deterrent effects of coumarin, sinigrin, and strychnine. Thus, we conclude that Gr180 is a receptor responding to coumarin,and also participates in sensing sinigrin and strychnine. These results enhance our understanding of the gustatory sensing mechanisms of phytophagous insects to deterrents.

Glycine and GABAA receptors suppressively regulate the inspiratory-related calcium rise in the thoracic inspiratory cells of the neonatal rat.

We previously demonstrated that in an isolated brainstem-spinal cord preparation from neonatal rats, a local bath application of strychnine (a broad antagonist of glycine and GABAA receptors) to the spinal cord enhances thoracic inspiratory motor activity. Herein, to investigate the involvement of the inspiratory spinal interneurons that provide excitatory input to the motoneuron, we conducted calcium imaging using this preparation. Oregon Green 488 BAPTA-1 AM, a fluorescent calcium indicator, was injected into the ventromedial surface of the thoracic cord. In all cells that showed inspiratory-related fluorescence changes > 2% of the baseline fluorescence intensity, the inspiratory-related fluorescence change decreased when the focal depth was deepened. The application of strychnine to the spinal cord increased the inspiratory-related intracellular calcium rise in these cells. These results suggest that the enhancement of inspiratory interneuron activity could be involved in this enhancement of inspiratory motor activity.

Action of the Photochrome Glyght on GABAergic Synaptic Transmission in Mouse Brain Slices.

Glyght is a new photochromic compound described as an effective modulator of glycine receptors at heterologous expression, in brain slices and in zebrafish larvae. Glyght also caused weak inhibition of GABAA-mediated currents in a cell line expressing α1/ß2/γ2 GABAA receptors. However, the effects of Glyght on GABAergic transmission in the brain have not been analysed, which does not allow a sufficiently comprehensive assessment of the effects of the compound on the nervous system. Therefore, in this study using whole-cell patch-clamp recording, we analysed the Glyght (100 µM) action on evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in mice hippocampal slices. Two populations of cells were found: the first responded by reducing the GABAergic eIPSCs' amplitude, whereas the second showed no sensitivity to the compound. Glyght did not affect the ionic currents' amplitude induced by GABA application, suggesting the absence of action on postsynaptic GABA receptors. Additionally, Glyght had no impact on the paired-pulse modulation of GABAergic eIPSCs, indicating that Glyght does not modulate the neurotransmitter release mechanisms. In the presence of strychnine, an antagonist of glycine receptors, the Glyght effect on GABAergic synaptic transmission was absent. Our results suggest that Glyght can modulate GABAergic synaptic transmission via action on extrasynaptic glycine receptors.

Structural basis for strychnine activation of human bitter taste receptor TAS2R46.

Taste sensing is a sophisticated chemosensory process, and bitter taste perception is mediated by type 2 taste receptors (TAS2Rs), or class T G protein-coupled receptors. Understanding the detailed molecular mechanisms behind taste sensation is hindered by a lack of experimental receptor structures. Here, we report the cryo-electron microscopy structures of human TAS2R46 complexed with chimeric mini-G protein gustducin, in both strychnine-bound and apo forms. Several features of TAS2R46 are disclosed, including distinct receptor structures that compare with known GPCRs, a new "toggle switch," activation-related motifs, and precoupling with mini-G protein gustducin. Furthermore, the dynamic extracellular and more-static intracellular parts of TAS2R46 suggest possible diverse ligand-recognition and activation processes. This study provides a basis for further exploration of other bitter taste receptors and their therapeutic applications.

The ethanol inhibition of basolateral amygdala neuron spiking is mediated by a γ-aminobutyric acid type A-mediated tonic current.

BACKGROUND: The basolateral nucleus of the amygdala (BLA) plays an important role in the development of fear and anxiety-related behaviors. The BLA receives inputs from all sensory stimuli. After processing those stimuli, BLA neurons signal neurons within the central amygdala and other brain regions, including the ventral and dorsal striatum and frontal cortex. Studies suggest that the BLA is involved in drug dependence and in the reinforcing actions of ethanol. For example, acute exposure to ethanol reduces anxiety, while withdrawal from chronic ethanol exposure alters BLA synaptic transmission, which increases anxiety, a common underlying cause of relapse. Exposure to and withdrawal from chronic alcohol also disrupts many brain areas that connect with the BLA. Despite these important findings, the acute actions of alcohol on the intrinsic excitability of BLA neurons have not been fully characterized. METHODS: Brain slices containing the BLA were prepared from adult C57BL/6J male mice. Whole-cell and sharp electrode electrophysiological recordings were performed to characterize the effects of acute ethanol on BLA neuronal and astrocyte function, respectively. RESULTS: Ethanol inhibited action potential (AP) firing of BLA neurons but had no effect on BLA astrocyte resting membrane potential. The ethanol-induced inhibition of firing was concentration-dependent (11 to 66 mM) and accompanied by a reduction in the input resistance and an increase in the rheobase of BLA neurons. The inhibitory effect of ethanol was suppressed by picrotoxin, which blocks both γ-aminobutyric acid type A (GABAA ) and glycine receptors, but not by the selective glycine receptor antagonist strychnine, which suggests an involvement of GABAA receptors. Ethanol did not affect spontaneous inhibitory postsynaptic currents suggesting that the inhibition of BLA neuronal excitability by ethanol was not due to an increase in GABAA -mediated synaptic transmission. However, acute ethanol enhanced the amplitude of the holding current of BLA neurons, an effect that was prevented by picrotoxin, which by itself reduced the holding current. CONCLUSIONS: These results suggest that BLA neurons express a GABA-mediated tonic current that is enhanced by acute ethanol, which leads to reduced excitability of BLA neurons.

Exploration by molecular networking of Strychnos alkaloids reveals the unexpected occurrence of strychnine in seven Strychnos species.

INTRODUCTION: Plants of the Strychnos genus, which include about 200 species, are used for multiple traditional purposes as hunting poison, for example, and have shown interesting pharmacological properties, especially curarizing and tetanizing, but also against malaria. Many monoterpene indole alkaloids have already been isolated and identified. Among them, there is strychnine, a famous alkaloid that can cause death by asphyxiation. OBJECTIVE: Investigate alkaloidic molecular diversity from Strychnos genus using molecular networking technique and study the Strychnos genus from a chemotaxonomic point of view. MATERIAL AND METHODS: Twenty-eight different species and different plant parts were ground into powder using a grinder. The methanolic extracts were carried out using a pressurized solvent extraction and the alkaloid extract was performed manually with a separating funnel. The extracts were analyzed by HPLC-ESI(+)-Q/TOF. The data were processed using MZmine 2 software and the molecular network was generated on the GNPS platform. The study of the generated molecular network allowed the detection of various alkaloids. Among these is the famous strychnine which has been detected in 7 new Strychnos species not yet described as strychnine producers. This identification was investigated using orthogonal approaches, namely TLC, NMR, HPLC-UV and UHPLC-ESI(+)-Q/TOF analyses. The LOD by HPLC-UV of strychnine was also determined. RESULTS: Further analyses allowed to confirm the presence of strychnine in S. densiflora trunk barks but also to show the presence of strychnine with high probability in the trunk barks of S. camptoneura, S. congolana, S. boonei, and S. tchibangensis, and in the leaves of S. usambarensis. About the trunk barks of S. tricalyisoides, the probability of a strychnine content remains low. CONCLUSION: This work exemplified the efficiency of molecular networking in identifying known metabolites (major and minor alkaloids) involved in the chemotaxonomic study of plants from Strychnos genus.

Strychnine and its mono- and dimeric analogues: a pharmaco-chemical perspective.

Covering: up to November 2021Since its isolation in 1818, strychnine has attracted the attention of a plethora of chemists and pharmacologists who have established its structure, developed total syntheses, and examined its complex pharmacology. While numerous reviews on structure elucidation and total synthesis of strychnine are available, reports on structure-activity relationships (SARs) of this fascinating alkaloid are rare. In this review, we present and discuss structures, synthetic approaches, metabolic transformations, and the diverse pharmacological actions of strychnine and its mono- and dimeric analogues. Particular attention is given to its SARs at glycine receptors (GlyRs) in light of recently published high-resolution structures of strychnine-GlyR complexes. Other pharmacological actions of strychnine and its derivatives, such as their antagonistic properties at nicotinic acetylcholine receptors (nAChRs), allosteric modulation of muscarinic acetylcholine receptors as well as anti-cancer and anti-plasmodial effects are also critically reviewed, and possible future developments in the field are discussed.

The phosphorylation status of eukaryotic elongation factor-2 indicates neural activity in the brain.

Assessment of neural activity in the specific brain area is critical for understanding the circuit mechanisms underlying altered brain function and behaviors. A number of immediate early genes (IEGs) that are rapidly transcribed in neuronal cells in response to synaptic activity have been used as markers for neuronal activity. However, protein detection of IEGs requires translation, and the amount of newly synthesized gene product is usually insufficient to detect using western blotting, limiting their utility in western blot analysis of brain tissues for comparison of basal activity between control and genetically modified animals. Here, we show that the phosphorylation status of eukaryotic elongation factor-2 (eEF2) rapidly changes in response to synaptic and neural activities. Intraperitoneal injections of the GABA A receptor (GABAAR) antagonist picrotoxin and the glycine receptor antagonist brucine rapidly dephosphorylated eEF2. Conversely, potentiation of GABAARs or inhibition of AMPA receptors (AMPARs) induced rapid phosphorylation of eEF2 in both the hippocampus and forebrain of mice. Chemogenetic suppression of hippocampal principal neuron activity promoted eEF2 phosphorylation. Novel context exploration and acute restraint stress rapidly modified the phosphorylation status of hippocampal eEF2. Furthermore, the hippocampal eEF2 phosphorylation levels under basal conditions were reduced in mice exhibiting epilepsy and abnormally enhanced excitability in CA3 pyramidal neurons. Collectively, the results indicated that eEF2 phosphorylation status is sensitive to neural activity and the ratio of phosphorylated eEF2 to total eEF2 could be a molecular signature for estimating neural activity in a specific brain area.

ATF3 contributes to brucine-triggered glioma cell ferroptosis via promotion of hydrogen peroxide and iron.

Ferroptotic cell death is characterized by iron-dependent lipid peroxidation that is initiated by ferrous iron and H2O2 via Fenton reaction, in which the role of activating transcription factor 3 (ATF3) remains elusive. Brucine is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica, which has shown potent antitumor activity against various tumors, including glioma. In this study, we showed that brucine inhibited glioma cell growth in vitro and in vivo, which was paralleled by nuclear translocation of ATF3, lipid peroxidation, and increases of iron and H2O2. Furthermore, brucine-induced lipid peroxidation was inhibited or exacerbated when intracellular iron was chelated by deferoxamine (500 µM) or improved by ferric ammonium citrate (500 µM). Suppression of lipid peroxidation with lipophilic antioxidants ferrostatin-1 (50 µM) or liproxstatin-1 (30 µM) rescued brucine-induced glioma cell death. Moreover, knockdown of ATF3 prevented brucine-induced accumulation of iron and H2O2 and glioma cell death. We revealed that brucine induced ATF3 upregulation and translocation into nuclei via activation of ER stress. ATF3 promoted brucine-induced H2O2 accumulation via upregulating NOX4 and SOD1 to generate H2O2 on one hand, and downregulating catalase and xCT to prevent H2O2 degradation on the other hand. H2O2 then contributed to brucine-triggered iron increase and transferrin receptor upregulation, as well as lipid peroxidation. This was further verified by treating glioma cells with exogenous H2O2 alone. Moreover, H2O2 reversely exacerbated brucine-induced ER stress. Taken together, ATF3 contributes to brucine-induced glioma cell ferroptosis via increasing H2O2 and iron.

Linear and nonlinear chromatic integration in the mouse retina.

The computations performed by a neural circuit depend on how it integrates its input signals into an output of its own. In the retina, ganglion cells integrate visual information over time, space, and chromatic channels. Unlike the former two, chromatic integration is largely unexplored. Analogous to classical studies of spatial integration, we here study chromatic integration in mouse retina by identifying chromatic stimuli for which activation from the green or UV color channel is maximally balanced by deactivation through the other color channel. This reveals nonlinear chromatic integration in subsets of On, Off, and On-Off ganglion cells. Unlike the latter two, nonlinear On cells display response suppression rather than activation under balanced chromatic stimulation. Furthermore, nonlinear chromatic integration occurs independently of nonlinear spatial integration, depends on contributions from the rod pathway and on surround inhibition, and may provide information about chromatic boundaries, such as the skyline in natural scenes.

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.