Dorsal Unpaired Median Neurons of Locusta migratoria Express Ivermectin- and Fipronil-Sensitive Glutamate-Gated Chloride Channels

2007 ◽  
Vol 97 (4) ◽  
pp. 2642-2650 ◽  
Author(s):  
Daniel Janssen ◽  
Christian Derst ◽  
Roeland Buckinx ◽  
Jimmy Van den Eynden ◽  
Jean-Michel Rigo ◽  
...  
Keyword(s):  
Chloride Channels ◽  
Locusta Migratoria ◽  
Functional Expression ◽  
Equilibrium Potential ◽  
Current Response ◽  
Metathoracic Ganglion ◽  
Continuous Application ◽  
Dum Neurons ◽  
Induced Changes ◽  
Dorsal Unpaired Median

Together with type A GABA and strychnine-sensitive glycine receptors, glutamate-gated chloride channels (GluCl) are members of the Cys-loop family of ionotropic receptors, which mediate fast inhibitory neurotransmission. To date, GluCls are found in invertebrates only and therefore represent potential specific targets for insecticides, such as ivermectin and fipronil. In this study, we identified the functional expression of GluCls in dorsal unpaired median (DUM) neurons of the metathoracic ganglion of Locusta migratoria using electrophysiological and molecular biological techniques. In whole cell patch-clamped DUM neurons, glutamate-induced changes in both their membrane potentials (current-clamp) and currents (voltage-clamp) were dependent on the chloride equilibrium potential. On continuous application of glutamate, the glutamate-elicited current response became rapidly and completely desensitized. Application of glutamate in the presence of 10 μM fipronil or 100 μM picrotoxin reversibly decreased GluCl-mediated currents by 87 and 39%, respectively. Furthermore, 1 μM ivermectin induced a persistent chloride current, suggesting the expression of ivermectin-sensitive GluCl α subunits. A degenerate PCR/RACE strategy was used to clone the full-length L. migratoria LmGlClα subunit. Finally, RT-PCR experiments demonstrated the presence of LmGluClα transcripts in locust DUM neurons. Our results provide the first direct evidence of a functional ivermectin-sensitive GluCl channel on the cell surface of DUM neurons of L. migratoria.

scholarly journals Cys-Loop Ligand-Gated Chloride Channels in Dorsal Unpaired Median Neurons of Locusta migratoria

2010 ◽  
Vol 103 (5) ◽  
pp. 2587-2598 ◽  
Author(s):  
Daniel Janssen ◽  
Christian Derst ◽  
Jean-Michel Rigo ◽  
Emmy Van Kerkhove
Keyword(s):  
Gaba Receptors ◽  
Chloride Channels ◽  
Locusta Migratoria ◽  
Ibotenic Acid ◽  
Ionic Currents ◽  
Sequencing Analysis ◽  
Anion Channels ◽  
Continuous Application ◽  
Dum Neurons ◽  
Dorsal Unpaired Median

In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABAA nor GABAC receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter.

Coupling of Efferent Neuromodulatory Neurons to Rhythmical Leg Motor Activity in the Locust

1998 ◽  
Vol 79 (1) ◽  
pp. 361-370 ◽  
Author(s):  
Sylvie Baudoux ◽  
Carsten Duch ◽  
Oliver T. Morris
Keyword(s):  
Motor Activity ◽  
Motor Neurons ◽  
Spike Activity ◽  
Motor Pattern ◽  
Rhythmic Activity ◽  
Motor Output ◽  
Variable Activity ◽  
Metathoracic Ganglion ◽  
Dum Neurons ◽  
Dorsal Unpaired Median

Baudoux, Sylvie, Carsten Duch, and Oliver T. Morris. Coupling of efferent neuromodulatory neurons to rhythmical leg motor activity in the locust. J. Neurophysiol. 79: 361–370, 1998. The spike activity of neuromodulatory dorsal unpaired median (DUM) neurons was analyzed during a pilocarpine-induced motor pattern in the locust. Paired intracellular recordings were made from these octopaminergic neurons during rhythmic activity in hindleg motor neurons evoked by applying pilocarpine to an isolated metathoracic ganglion. This motor pattern is characterized by two alternating phases: a levator phase, during which levator, flexor, and common inhibitor motor neurons spike, and a depressor phase, during which depressor and extensor motor neurons spike. Three different subpopulations of efferent DUM neurons could be distinguished during this rhythmical motor pattern according to their characteristic spike output. DUM 1 neurons, which in the intact animal do not innervate muscles involved in leg movements, showed no change apart from a general increase in spike frequency. DUM 3 and DUM 3,4 neurons produced the most variable activity but received frequent and sometimes pronounced hyperpolarizations that were often common to both recorded neurons. DUM 5 and DUM 3,4,5 neurons innervate muscles of the hindleg and showed rhythmical excitation leading to bursts of spikes during rhythmic activity of the motor neurons, which innervate these same muscles. Sometimes the motor output was coordinated across both sides of the ganglion so that there was alternating activity between levators of both sides. In these cases, the spikes of DUM 5 and DUM 3,4,5 neurons and the hyperpolarization of DUM 3 and DUM 3,4 neurons occurred at particular phases in the motor pattern. Our data demonstrate a central coupling of specific types of DUM neurons to a rhythmical motor pattern. Changes in the spike output of these particular efferent DUM neurons parallel changes in the motor output. The spike activity of DUM neurons thus may be controlled by the same circuits that determine the action of the motor neurons. Functional implications for real walking are discussed.

Action of locust neuromodulatory neurons is coupled to specific motor patterns

1995 ◽  
Vol 74 (1) ◽  
pp. 347-357 ◽  
Author(s):  
M. Burrows ◽  
H. J. Pfluger
Keyword(s):  
Motor Neurons ◽  
Motor Pattern ◽  
Intracellular Recordings ◽  
Flexor Muscle ◽  
Motor Patterns ◽  
Specific Subset ◽  
Metathoracic Ganglion ◽  
Flexor Muscles ◽  
Dum Neurons ◽  
Dorsal Unpaired Median

1. Many muscles of the locust are supplied by dorsal unpaired median neurons (DUM neurons) that release octopamine and alter the contractions caused by spikes in motor neurons. To determine when these neuromodulatory neurons are normally activated during behaviour, intracellular recordings were made simultaneously from them and from identified motor neurons during the specific motor pattern that underlies kicking. A kick consists of a rapid and powerful extension of the tibia of one or both hind legs that is produced by a defined motor pattern. Only 3 identified DUM neurons of the 20 in the metathoracic ganglion spike during a kick, and they supply muscles involved in generating the kick. Their spikes occur in a distinctive and repeatable pattern that is closely linked to the pattern of spikes in the flexor and extensor tibiae motor neurons. When the extensor and flexor muscles cocontract, these three DUM neurons produce a burst of spikes at frequencies that can rise to 25 Hz, and with the number of spikes (3-15) related to the duration of this phase of the motor pattern. The spikes stop when the flexor muscle is inhibited and therefore before the tibia is extended rapidly. The other DUM neurons which supply muscles that are not directly involved in kicking are either inhibited or spike only sporadically. 2. The activation of a specific subset of DUM neurons during kicking may thus be timed to influence the action of the muscles that participate in this movement and appear to be controlled by the same circuits that determine the actions of the participating motor neurons. These modulatory neurons thus have specific individual actions in the control of movement.

scholarly journals Ion Transport and the Development of Hydrogen Ion Secretion in the Stomach of the Metamorphosing Bullfrog Tadpole

1969 ◽  
Vol 54 (1) ◽  
pp. 76-95 ◽  
Author(s):  
John G. Forte ◽  
Liangchai Limlomwongse ◽  
Dinkar K. Kasbekar
Keyword(s):  
Ion Transport ◽  
Active Transport ◽  
Hydrogen Ion ◽  
Equilibrium Potential ◽  
Flux Analysis ◽  
Cell Interior ◽  
Ion Secretion ◽  
Hcl Secretion ◽  
Bullfrog Tadpole ◽  
Induced Changes

Isolated bullfrog tadpole stomachs secrete H+ by stage XXIV of metamorphosis, when tail reabsorption is nearly complete. At this stage the PD shows characteristic responses identical to those of the adult. The appearance of HCl secretion correlates well with other studies showing the morphogenesis of oxyntic cells. Prior to the development of H+ secretion tadpole stomachs maintain a PD similar in polarity and magnitude to that of the adult; i.e., secretory (S) side negative with respect to the nutrient (N) side. The interdependence with aerobic metabolism appeared to increase progressively through metamorphosis; however, glycolytic inhibitors always abolished the PD. Isotopic flux analysis showed that the transepithelial movement of Na+ was consistent with passive diffusion, whereas an active transport of Cl- from N to S was clearly indicated. Variations in [Na+], [K+], and [Cl-] in the bathing solutions induced changes consistent with the following functional description of the pre-H+-secreting tadpole stomach. (a) The S side is relatively permeable to Cl-, but not to Na+ or K+. (b) An equilibrium potential for K+ and Cl- exists at the N interface. (c) Ouabain abolishes the selective K+ permeablity at the N interface and reduces the total PD. (d) Effects of Na+ replacement by choline in the N solution become manifest only below 10–20 mM. It is concluded that prior to development of H+ secretion, the tadpole gastric PD is generated by a Cl- pump from N to S and a Na+ pump operating from the cell interior toward the N side.

OCTOPAMINERGIC MODULATION OF THE FOREWING STRETCH RECEPTOR IN THE LOCUST LOCUSTA MIGRATORIA

1990 ◽  
Vol 149 (1) ◽  
pp. 255-279 ◽  
Author(s):  
JAN-MARINO RAMIREZ ◽  
IAN ORCHARD
Keyword(s):  
Intact Animal ◽  
Locusta Migratoria ◽  
Beat Frequency ◽  
Motor Pattern ◽  
Stretch Receptor ◽  
Maximal Effect ◽  
Naturally Occurring ◽  
Entire Central Nervous System ◽  
Dorsal Unpaired Median ◽  
Stimulation Of

Modulatory actions of various biogenic amines and peptides on the locust forewing stretch receptor (SR) were examined. The response of the SR to sinusoidal wing movements was unaffected by physiological concentrations (5×10−8moll−1) of the peptides AKHI, AKHII, proctolin and FMRFamide. The biogenic amine octopamine, however, enhanced the SR response in a dosedependent manner when injected into the haemolymph of an almost intact animal or perfused over an isolated thorax preparation in which head, abdomen, gut and the entire central nervous system were removed (threshold at 5×10−8moll−1, maximal effect at 5×10−4moH−1 DL-octopamine). The SR was as sensitive to D-octopamine, the naturally occurring isomer of octopamine, as it was to DLoctopamine. Serotonin was equal to octopamine in effectiveness, followed in order of potency by synephrine, metanephrine and tyramine. Dopamine was ineffective. Phentolamine, but not DL-propranolol, antagonized the action of octopamine. The threshold of the modulatory effect of octopamine on the SR suggests that the increased haemolymph octopamine level which occurs during flight is sufficient to increase the SR activity. Two observations suggest that dorsal unpaired median (DUM) cells are involved in the octopaminergic modulation of the SR during flight: (1) selective stimulation of these cells modulated the SR response and this effect was blocked by phentolamine; and (2) a number of DUM cells were activated during flight. These results suggest that the SR activity is enhanced by octopamine following the onset of flight. Since the SR is involved in the control of wing beat frequency, the modulation of the SR might influence the generation of the motor pattern in flying locusts.

scholarly journals Mechanisms of spreading depolarization in vertebrate and insect central nervous systems

2016 ◽  
Vol 116 (3) ◽  
pp. 1117-1127 ◽  
Author(s):  
Kristin E. Spong ◽  
R. David Andrew ◽  
R. Meldrum Robertson
Keyword(s):  
Locusta Migratoria ◽  
Model Systems ◽  
Control Mechanisms ◽  
Cellular Mechanisms ◽  
Nervous Systems ◽  
Spreading Depolarization ◽  
Metathoracic Ganglion ◽  
Evolutionarily Conserved ◽  
Central Nervous Systems ◽  
The Central Nervous System

Spreading depolarization (SD) is generated in the central nervous systems of both vertebrates and invertebrates. SD manifests as a propagating wave of electrical depression caused by a massive redistribution of ions. Mammalian SD underlies a continuum of human pathologies from migraine to stroke damage, whereas insect SD is associated with environmental stress-induced neural shutdown. The general cellular mechanisms underlying SD seem to be evolutionarily conserved throughout the animal kingdom. In particular, SD in the central nervous system of Locusta migratoria and Drosophila melanogaster has all the hallmarks of mammalian SD. Locust SD is easily induced and monitored within the metathoracic ganglion (MTG) and can be modulated both pharmacologically and by preconditioning treatments. The finding that the fly brain supports repetitive waves of SD is relatively recent but noteworthy, since it provides a genetically tractable model system. Due to the human suffering caused by SD manifestations, elucidating control mechanisms that could ultimately attenuate brain susceptibility is essential. Here we review mechanisms of SD focusing on the similarities between mammalian and insect systems. Additionally we discuss advantages of using invertebrate model systems and propose insect SD as a valuable model for providing new insights to mammalian SD.

scholarly journals Heterogeneity of chloride channels in the apical membrane of isolated mitochondria-rich cells from toad skin.

1996 ◽  
Vol 108 (5) ◽  
pp. 421-433 ◽  
Author(s):  
J B Sørensen ◽  
E H Larsen
Keyword(s):  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
Single Cells ◽  
Membrane Depolarization ◽  
Equilibrium Potential ◽  
Toad Skin ◽  
Patch Clamp Technique ◽  
Cl Channels ◽  
Inside Out

The isolated epithelium of toad skin was disintegrated into single cells by treatment with collagenase and trypsine. Chloride channels of cell-attached and excised inside-out apical membrane-patches of mitochondria-rich cells were studied by the patch-clamp technique. The major population of Cl- channels constituted small 7-pS linear channels in symmetrical solutions (125 mM Cl-). In cell-attached and inside-out patches the single channel i/V-relationship could be described by electrodiffusion of Cl- with a Goldmann-Hodgkin-Katz permeability of, PCl = 1.2 x 10(-14) - 2.6 x 10(-14) cm3. s-1. The channel exhibited voltage-independent activity and could be activated by cAMP. This channel is a likely candidate for mediating the well known cAMP-induced transepithelial Cl- conductance of the amphibian skin epithelium. Another population of Cl- channels exhibited large, highly variable conductances (upper limit conductances, 150-550 pS) and could be activated by membrane depolarization. A group of intermediate-sized Cl(-)-channels included: (a) channels (mean conductance, 30 pS) with linear or slightly outwardly rectifying i/V-relationships and activity occurring in distinct "bursts," (b) channels (conductance-range, 10-27 pS) with marked depolarization-induced activity, and (c) channels with unresolvable kinetics. The variance of current fluctuations of such "noisy" patches exhibited a minimum close to the equilibrium-potential for Cl-. With channels occurring in only 38% of sealed patches and an even lower frequency of voltage-activated channels, the chloride conductance of the apical membrane of mitochondria-rich cells did not match quantitatively that previously estimated from macroscopic Ussing-chamber experiments. From a qualitative point of view, however, we have succeeded in demonstrating the existence of Cl-channels in the apical membrane with features comparable to macroscopic predictions, i.e., activation of channel gating by cAMP and, in a few patches, also by membrane depolarization.

Temporal Development of Cyclic Nucleotide-Gated and Ca2+-Activated Cl− Currents in Isolated Mouse Olfactory Sensory Neurons

2007 ◽  
Vol 98 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Anna Boccaccio ◽  
Anna Menini
Keyword(s):  
Sensory Neurons ◽  
Flash Photolysis ◽  
Olfactory Receptors ◽  
Cell Voltage ◽  
Equilibrium Potential ◽  
Olfactory Sensory Neurons ◽  
Second Phase ◽  
Current Response ◽  
Intact Mouse ◽  
Secondary Current

A Ca2+-activated Cl− current constitutes a large part of the transduction current in olfactory sensory neurons. The binding of odorants to olfactory receptors in the cilia produces an increase in cAMP concentration; Ca2+ enters into the cilia through CNG channels and activates a Cl− current. In intact mouse olfactory sensory neurons little is known about the kinetics of the Ca2+-activated Cl− current. Here, we directly activated CNG channels by flash photolysis of caged cAMP or 8-Br-cAMP and measured the current response with the whole cell voltage-clamp technique in mouse neurons. We measured multiphasic currents in the rising phase of the response at −50 mV. The current rising phase became monophasic in the absence of extracellular Ca2+, at +50 mV, or when most of the intracellular Cl− was replaced by gluconate to shift the equilibrium potential for Cl− to −50 mV. These results show that the second phase of the current in mouse intact neurons is attributed to a Cl− current activated by Ca2+, similarly to previous results on isolated frog cilia. The percentage of the total saturating current carried by Cl− was estimated in two ways: 1) by measuring the maximum secondary current and 2) by blocking the Cl− channel with niflumic acid. We estimated that in the presence of 1 mM extracellular Ca2+ and in symmetrical Cl− concentrations the Cl− component can constitute up to 90% of the total current response. These data show how to unravel the CNG and Ca2+-activated Cl− component of the current rising phase.

scholarly journals Agonist and antagonist properties of an insect GABA-gated chloride channel (RDL) are influenced by heterologous expression conditions

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254251
Author(s):  
Charles L. C. Smelt ◽  
Victoria R. Sanders ◽  
Alin M. Puinean ◽  
Stuart J. Lansdell ◽  
Jim Goodchild ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Chloride Channels ◽  
Bombus Terrestris ◽  
Functional Expression ◽  
Glycine Receptors ◽  
Competitive Antagonist ◽  
Inhibitory Neurotransmitter ◽  
Apparent Affinity ◽  
Chaperone Protein ◽  
Agonist And Antagonist

Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of ‘resistance to dieldrin’, that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3’ untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs.

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