strychnine and picrotoxinin

The α2-glycine receptors (GlyRs) play important roles during early central nervous system development. However, these receptors' possible involvement in neurodevelopmental events occurring in the adult brain remains to be explored. Adult hippocampal neurogenesis (AHN) is the process by which new granule cell neurons are added to the dentate gyrus (DG) throughout adulthood. In this study, we observed that hippocampal adult neural stem cells (ANSCs) express α2-containing GlyRs. Pharmacological inhibition of GlyRs by strychnine or picrotoxin decreased the proliferation of ANSCs, both in vivo and in vitro. Mice knockout for glra2, the gene coding for the GlyR α2 subunit, were determined to display impaired AHN, and this phenomenon was accompanied by deficits in spatial memory. These results, which reveal neurodevelopmental roles for α2-GlyRs in the adult brain, may be clinically relevant, given that a mutation in GLAR2, as well as AHN impairments, have been reported in autism spectrum disorder. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1430-1441, 2017.

Topics: Animals; Cell Proliferation; Cells, Cultured; Exploratory Behavior; Female; GABA-A Receptor Antagonists; Glycine Agents; Hippocampus; Male; Maze Learning; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nestin; Neurogenesis; Neurons; Picrotoxin; Rats; Rats, Inbred F344; Receptors, Glycine; Sesterterpenes; Spatial Memory; Strychnine

Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, the retinal circuitry that is activated by epiretinal stimulation is not well characterized. Whole-cell patch clamp recordings were obtained from ganglion cells in normal and rd mice using flat-mount and retinal slice preparations. A stimulating electrode was positioned along the ganglion cell side of the preparation at different distances from the stimulated tissue. Pulses of cathodic current evoked action potentials in ganglion cells and less frequently evoked sustained inward currents that appeared synaptic in origin. Sustained currents reversed around E(Cl) and were inhibited by blockade of α-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionate (AMPA)-type glutamate receptors with 2,3-dihydroxy-6-nitro-sulfamoyl-benzo(f)-quinoxaline-2,3-dione (NBQX), γ aminobutyric acid a/c (GABA(a/c)) receptors with picrotoxinin, or glycine receptors with strychnine. This suggests that epiretinal stimulation activates glutamate release from bipolar cell terminals, which in turn evokes release of GABA and glycine from amacrine cells. Synaptic current thresholds were lower in ON ganglion cells than OFF cells, but the modest difference did not attain statistical significance. Synaptic currents were rarely observed in rd mice lacking photoreceptors compared to normal retina. In addition, confocal calcium imaging experiments in normal mice retina slices revealed that epiretinal stimulation evoked calcium increases in the outer plexiform layer. These results imply a contribution from photoreceptor inputs to the synaptic currents observed in ganglion cells. The paucity of synaptic responses in rd mice retina slices suggests that it is better to target retinal ganglion cells directly rather than to attempt to engage the inner retinal circuitry.

Topics: Animals; Biophysics; Calcium; Disease Models, Animal; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA-A Receptor Antagonists; Glycine Agents; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Confocal; Patch-Clamp Techniques; Picrotoxin; Quinoxalines; Retina; Retinal Degeneration; Retinal Ganglion Cells; Sesterterpenes; Strychnine; Visual Pathways

1. PMBA is a novel antagonist of strychnine-sensitive glycine receptors in the rat spinal cord, however, its mode of action is unknown. The actions of PMBA on rat glycine receptor alpha1 and alpha2 homomers in Xenopus oocytes were studied under two-electrode voltage-clamp. 2. Co-application of PMBA and glycine to both alpha1 and alpha2 homomers yielded inward currents which decayed to a steady-state. Responses rose slowly to the same steady-state amplitude following a 2 min pre-incubation in PMBA. Strychnine, but not picrotoxinin, showed similar antagonism to PMBA. The potency of PMBA was independent of membrane potential between -100 and 0 mV. 3. When tested against EC50 concentrations of glycine, PMBA was almost equally potent on alpha1 (IC50, 406+/-41 nM: Hill coefficient, 1.5+/-0.2) and alpha2 (IC50, 539+/-56 nM; Hill coefficient, 1.4+/-0.2) homomers. 4. PMBA (1-I0 microM) and strychnine (200 nM) reduced the potency of glycine and the amplitude of the maximal agonist response of alpha1 and alpha2 homomers. In 10 microM PMBA, two distinct classes of glycine response were observed on alpha2, only a single class of responses were observed on alpha1. 5. There are similarities in PMBA and strychnine antagonism, although these compounds are structurally distinct. The possibility that PMBA interacts at two binding sites which differ in alpha1 and alpha2 subunits is discussed. PMBA may provide a lead structure for novel antagonists with which to investigate structural differences in glycine receptor at alpha1 and alpha2 subunits.

Topics: Animals; Dose-Response Relationship, Drug; Glycine; Glycine Agents; Oocytes; Organophosphonates; Phenylalanine; Picrotoxin; Receptors, Glycine; Recombinant Proteins; Sesterterpenes; Strychnine; Xenopus laevis

Expression of the glycine receptor was investigated in membranes prepared from primary cultures of mouse cerebellar granule cells and postnatal mouse cerebellum using the antagonist [3H]strychnine for ligand binding. Scatchard analysis of the binding data obtained from P17 cerebellum showed a single population of binding sites (K(D) approximately 6 nM) and [3H]strychnine binding to membranes prepared from cultured neurons and P17 cerebellum was found to have the same sensitivity to the glycinergic agonists glycine, beta-alanine and taurine. The development of [3H]strychnine binding sites in cultured cerebellar granule cells and cerebellum showed opposing profiles. [3H]strychnine binding to primary cultures increased significantly during the culture period whereas during development in vivo the number of binding sites decreased over time and was hardly detectable in the adult cerebellum. Release of preloaded D-[3H]aspartate evoked by 40 mM K+ from granule cells cultured for seven days was inhibited by glycine by about 50%. Beginning after seven days in culture the ability of glycine to inhibit transmitter release declined to no inhibition after 17 days in culture. Experiments with the non-competitive antagonist, picrotoxinin, showed no blocking effect of 150 microM picrotoxinin on the glycine-induced inhibition of transmitter release. This contrasted with the inhibitory effect of 100 microM picrotoxinin in whole-cell patch-clamp recordings on responses to 500 microM glycine (56% block). Furthermore, it was demonstrated that the amplitude of the glycine activated peak current had the same size after six to seven days and after 16-17 days in culture. Northern blot analysis, and co-injection of messenger RNA plus antisense oligonucleotides into Xenopus oocytes revealed glycine receptor alpha2 and beta messenger RNAs in the cultured granule cells. These findings suggest that granule cells in culture express glycine receptor isoforms containing alpha2 picrotoxinin-sensitive and alpha2/beta picrotoxinin-insensitive receptors.

Topics: Animals; beta-Alanine; Binding, Competitive; Cell Membrane; Cells, Cultured; Cerebellum; Cerebral Cortex; Female; Glycine; Isonicotinic Acids; Kinetics; Mice; Mice, Inbred Strains; Neurons; Oocytes; Patch-Clamp Techniques; Picrotoxin; Radioligand Assay; Receptors, Glycine; RNA, Messenger; Sesterterpenes; Strychnine; Taurine; Time Factors; Tritium; Xenopus laevis

The strychnine-sensitive glycine receptor, a member of a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system, forms an anion-specific transmembrane channel in response to glycine binding. A rapid-reaction technique, a cell-flow method with a 10-ms time resolution, was adapted for measurements with cultured embryonic mouse spinal cord cells containing glycine receptors. Whole-cell current responses resulting from the opening of glycine receptor channels were measured at pH 7.4, 22-24 degrees C, and transmembrane voltages of -40 and -75 mV. Two different receptor forms, A alpha and A beta, were detected. At saturating glycine concentrations, an average of 70% of the whole-cell current amplitude was associated with form A alpha and 30% with A beta. The constants pertaining to the minimum mechanisms that account for the concentration of the two open-channel receptor forms over a 100-fold range of glycine concentration were determined by cell-flow measurements of the current amplitudes and of the falling (desensitizing) rate of the current. The dissociation constant of the site controlling channel opening was 220 microM on the basis of three binding sites for A alpha and 380 microM on the basis of two binding sites for A beta. The channel-opening equilibrium constant, phi-I, was 170 for A alpha and 110 for A beta. The rate coefficients for desensitization, alpha and beta, associated with these two forms have maximum values of 0.7 and 0.1 s-1, respectively. The rates at which the receptors recovered from desensitization were also measured, using a double-flow mixing device, and were found to be 0.06 s-1 for A alpha and 0.02 s-1 for A beta. In the presence of 100 microM glycine, the apparent dissociation constant for the inhibitor picrotoxinin from receptor form A alpha was 80 microM, and that from A beta was 460 microM. This suggests that A beta contains beta-subunits (58 kD), because this subunit confers picrotoxinin insensitivity to glycine receptors (Pribilla, I., et al. (1992) EMBO J. 11, 4305). In the case of one receptor form (A alpha), the chemical mechanism and its constants led to two measurements that could be assessed by an independent method, the single-channel current-recording technique: (i) the fraction of receptor channels open at a given glycine concentration (ALn)o and (ii) the rate coefficient for desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Cell Membrane; Cells, Cultured; Electric Conductivity; Glycine; Hydrogen-Ion Concentration; Ion Channel Gating; Ion Channels; Mice; Neurons; Picrotoxin; Receptors, Glycine; Sesterterpenes; Spinal Cord; Strychnine

Control over stress protects against many of the deleterious effects of stress exposure, but the endogenous mediators responsible for these prophylactic effects have remained elusive. Using behavioral pharmacology, in vitro radioligand binding and neurochemical analyses, we demonstrate that exposure to escapable stress results in brain and behavior changes reminiscent of benzodiazepine administration. The stress control group shows significant protection against picrotoxinin-induced seizures, reductions in [35S]t-butylbicyclophosphorothionate (TBPS) binding and a 3-fold increase of benzodiazepine-like substances in brain in comparison to both yoked-inescapable shock and non-shock controls. These observations suggest that coping behavior leads to the release of endogenous benzodiazepine-like compounds in brain which protect the organism from stress pathology.

Topics: Analysis of Variance; Animals; Brain; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cerebral Cortex; Convulsants; Electroshock; Escape Reaction; Flumazenil; GABA-A Receptor Agonists; Hippocampus; Male; Picrotoxin; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Seizures; Sesterterpenes; Stress, Psychological; Strychnine; Sulfur Radioisotopes; Tritium

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel, which mediates post-synaptic inhibition in spinal cord and other brain regions. Heterologous expression of the ligand binding alpha subunits of the GlyR generates functional agonist-gated chloride channels that mimic most of the pharmacological properties of the receptor in vivo. Here, nuclear injection into Xenopus oocytes of a human alpha 1 subunit cDNA, engineered for efficient expression, was used to create GlyR channels over a wide density range, resulting in whole-cell glycine currents of 10 nA to 25 microA. Notably, the pharmacology of these channels changed at high expression levels, with the appearance of a novel receptor subpopulation of 5- to 6-fold higher apparent agonist affinity at current values > 4 microA. The low-affinity receptors were readily blocked by nM concentrations of the competitive antagonist strychnine, whereas the high-affinity receptors were more resistant to antagonism by this alkaloid. Picrotoxinin, a chloride channel blocker, inhibited both GlyR populations with equal potency. Our data suggest that receptor interactions, occurring at high receptor density, modify the agonist response of the GlyR. This phenomenon may contribute to neurotransmitter efficacy at fast synapses.

Topics: Animals; DNA, Complementary; Female; Glycine; Humans; Peptide Fragments; Picrotoxin; Receptors, Glycine; Recombinant Proteins; Sesterterpenes; Strychnine; Xenopus laevis

gamma-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian nervous system, is known to operate bicuculline-sensitive Cl- channels through GABAA receptors and bicuculline-insensitive cation channels through GABAB receptors. Recent observations indicate that the retina may contain GABA receptors with unusual pharmacological properties. Here we report that GABA gates bicuculline-insensitive Cl- channels in rod bipolar cells of the rat retina, which were not modulated by flunitrazepam, pentobarbital and alphaxalone and were only slightly blocked by picrotoxinin. Moreover, the GABAB receptor agonist baclofen, and the antagonist 2-hydroxysaclofen had no effect. The underlying single-channel conductance was 7 pS and the open time 150 ms. These values are clearly different from those obtained for GABAA receptor channels recorded in other neurons of the same preparation, and in other parts of the brain. The bicuculline- and baclofen-insensitive GABA receptors were activated selectively by the GABA analogue cis-4-aminocrotonic acid (CACA). Hence they may be similar to those receptors termed GABAC receptors.

Topics: Animals; Animals, Newborn; Bicuculline; Cells, Cultured; Chloride Channels; Flunitrazepam; gamma-Aminobutyric Acid; Ion Channels; Membrane Potentials; Membrane Proteins; Neurons; Pentobarbital; Photoreceptor Cells; Picrotoxin; Pregnanediones; Rats; Receptors, GABA-A; Sesterterpenes; Strychnine

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cells, Cultured; Cerebellum; Cloning, Molecular; DNA; Glycine; Humans; Ion Channels; Molecular Sequence Data; Mutagenesis, Site-Directed; Picrotoxin; Rats; Receptors, Glycine; Receptors, Neurotransmitter; Sequence Homology, Amino Acid; Sesterterpenes; Spinal Cord; Strychnine

A volatile anesthetic-gated current was characterized in patch-clamped cultured postnatal rat hippocampal neurons. In this preparation, the major volatile anesthetics, isoflurane, halothane, and enflurane, open an anion-selective conductance. This volatile anesthetic-gated current exhibits anion selectivity with a chloride-to-acetate permeability ratio of 15, shows outward rectification well described by the constant field equation, and is activated in a dose-dependent fashion with half-maximal response to isoflurane at 0.8 mM (0.032 atm). The current persists in the absence of external Ca2+ and is not blocked by strychnine, a glycine antagonist. However, the gamma-aminobutyric acidA (GABAA) antagonists, bicuculline and picrotoxinin, and the nonspecific anion channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), completely block the response. These observations suggest that volatile anesthetics, like several other general anesthetics such as barbiturates, steroids, and etomidate, have a GABA-mimetic effect on vertebrate central neurons in culture. It is not clear whether this GABAA-gating property is a prerequisite for all general anesthetics. However, under normal physiological conditions of low intracellular Cl-, it is likely that drugs with both direct GABA agonist and GABA modulatory properties will produce overall depression of the central nervous system by increasing the normal inhibitory synaptic influence and by directly hyperpolarizing neurons.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anesthetics; Animals; Animals, Newborn; Bicuculline; Chlorine; Chromatography, Gas; Culture Techniques; Dose-Response Relationship, Drug; Electrophysiology; Enflurane; gamma-Aminobutyric Acid; Halothane; Hippocampus; Ion Channel Gating; Isoflurane; Neurons; Picrotoxin; Rats; Sesterterpenes; Strychnine

The anticonvulsant effect of inhibitors of GABA-T (R/S-gamma-vinyl-GABA, ethanolamine-O-sulfate, gabaculine, aminooxyacetic acid) was enhanced by 10 mmol/kg glycine in animal seizure models which are based on a functional GABA deficit. Similar to glycine in their action, although less effective, were its close structural analogues (sarcosine, N,N-dimethylglycine) and homologous omega-aminoacids (beta-alanine, taurine, gamma-aminobutyric acid, delta-aminovaleric acid). It is assumed that glycine and its structural analogues act on supraspinal glycine receptors as glycine agonists. Our observation is the first example of the synergistic interaction of two inhibitory neuronal systems resulting in the amplification of the anticonvulsant effect. Combined treatment with GABA-T inhibitors and glycine may turn out to be of practical importance in the therapy of seizure disorders and other diseases, for which treatment with GABA-T inhibitors is considered a potentially useful therapeutic approach.

Topics: 3-Mercaptopropionic Acid; 4-Aminobutyrate Transaminase; Amino Acids; Aminocaproates; Aminooxyacetic Acid; Animals; Anticonvulsants; Bicuculline; Brain Chemistry; Drug Synergism; Ethanolamines; Glycine; Male; Mice; Pentylenetetrazole; Picrotoxin; Rats; Rats, Inbred Strains; Sesterterpenes; Strychnine; Vigabatrin