Pharmacology of GABA receptors in Ascaris suum muscle: an electrophysiological study

The aim of this work was to describe the pharmacological profile of the Ascaris muscle GABA receptor, and to compare its properties to those of GABA receptors in vertebrates. The interest of such study is to show the existence of pharmacological differences between Ascaris and vertebrate GABA receptors, which may be exploited in antiparasitic drug research. A short review will be presented first on the anatomy, physiology and pharmacology of the neuro-muscular system of Ascaris, and on the pharmacological characteristics of GABA receptors in vertebrates and invertebrates. It will be followed by a simplified presentation of the receptor theory and the subsequent development of drug-receptor interaction theories.The effects of GABA and various GABA-agonists on the membrane input conductance of Ascaris muscle cells were studied using a current-clamp technique. Dose-response curves were obtained and described by a modified Hill equation for each agonist tested. The relative potencies of GABA-agonists in Ascaris are compared to those obtained at vertebrate GABA-A receptors. Despite some differences: inactivity of sulphonic acid derivatives and the loss of relative potency of rigid analogues of GABA, such as muscimol, THIP and isoguvacine, the agonist profile of Ascaris muscle GABA receptor appears correlated with the agonist profile of vertebrate GABA-A receptors.In contrast, the antagonist profile of Ascaris GABA receptors differs greatly from the antagonist profile of the vertebrate GABA-A receptor. The two classical GABA-A antagonists, picrotoxin and bicuculline, were found very weak or inactive at antagonizing GABA responses in Ascaris. The steroid derivative, RU5135, a very potent competitive antagonist in vertebrate (IC50=5nM, rat cuneate nucleus, Simmonds and Turner, 1985), was found to hold back some activity in Ascaris , but in a non competitive manner and with an IC50 of 117μM.The effects of the arylaminopyridazine GABA derivatives SR95103 and SR95531 on GABA responses were investigated on GABA-induced conductance changes using a current-clamp technique. Arylaminopyridazine GABA derivatives are specific and competitive antagonists at the vertebrate GABA-A receptor, SR95531 being the most potent analogue. In Ascaris SR95103 was found more potent than SR95531 at antagonizing GABA responses. This potency order contrasts with that at vertebrate GABA-A receptors. The antagonism of SR95103 was associated with a parallel shift to the right in the GABA dose-response relationship. A modified Schild plot was used to describe the action of SR95103, the data is consistent with a competitive mechanism involving two molecules of GABA but one molecule of antagonist interacting with the receptor. The estimated KB for SR95103 is 64±13μM (mean±SE, n=14) and the pA₂=4.The actions of SR95103 were further examined on GABA-activated single-channel currents using an outside-out patch-clamp technique. The presence of SR95103 (30- 100μM) did not alter the GABA-activated channel main conductance, but reduced the open-probability of the channels. No effect on the mean burst duration, corrected mean open-time and the distribution of burst-durations, was observed with 30μM SR95103. However, when a higher concentration was used, 100μM, mean open-time and mean burst duration were reduced, and the proportion of short bursts (mainly represented by single openings) was increased. The mode of action of SR95103 is discussed, and it is suggested that most of the antagonism is competitive and produced by a single molecule of SR95103 combining with the receptor-channel complex, as suggested by the current-clamp data. However, an additional non-competitive component, possibly a channel block, is detected at high concentrations. This non-competitive component accounts only for a small proportion of the antagonism, since no reduction in the maximal response was detected by the current-clamp technique, even when high antagonist concentrations were used (ImM).Other arylaminopyridazine derivatives were tested on GABA response in Ascaris using a current-clamp technique. The order of potency of these compounds on Ascaris muscle GABA receptor, was found different from the order of potency at the vertebrate GABA-A receptor. One analogue, SR95132, virtually inactive in vertebrate preparations was found equipotent to SR95103 in Ascaris. When tested on GABA dose-response curves, SR95132 (KB=65μM) and other potent analogues, displaced GABA dose-response curves to the right without decrease in the maximal response. The modified Schild plots for these compounds, were also consistent with a competitive mechanism involving two molecules of GABA and only one molecule of antagonist interacting with the receptor. Structure-activity relationships for this series of compounds are examined in Ascaris and compared to the vertebrates. Substitution on the pyridazine ring in 4-position, while detrimental for the antagonist potency at the vertebrate GABA-A receptor, appears to be a prerequisite for antagonistic activity on Ascaris muscle GABA receptor. Newly synthesized arylaminopyridazine derivatives confirmed that the presence of a substituent in the 4-position is very important for the antagonistic activity at the Ascaris GABA receptor. The ethyl-4-substituted derivative, NCS247-90 with Kb=55μM, was found to be more potent than SR95103 and SR95132. Benzyl- (NCS251-90) and the isopropyl-(NCS252-90) 4-substituted derivatives also appeared to be potent antagonists. Besides the role of the 4-position, the length of the side chain and the acidic function were also investigated. A longer side-chain (5C), as in NCS194-83, decreaseithe potency but does not produce a complete loss of potency. Replacement of the carboxylic acid group by a sulphonic acid group produceja total inactivation of the compound, as seen with NCS249-90 and NCS250-90. This is in agreement with the lack of potency of sulphonic acid derivatives as agonists. These results are discussed in terms of the accessory binding sites theory of Ariens, and suggest the existence of different accessory sites responsible for the binding of antagonists on Ascaris GABA receptor.In conclusion, the Ascaris muscle GABA receptor differs from vertebrate GABA-A receptors in terms of antagonist properties, whereas its agonist properties are very similar to those of vertebrate GABA-A receptors. Among GABA antagonists, the exploration of the arylaminopyridazine GABA derivatives family may lead to the discovery of a new generation of anthelminthics. Already, one analogue, SR95132, shows an antagonistic activity in Ascaris but not in vertebrates

Neuro-glial plasticity of neuroendocrine networks

Neuro-glial plasticity of neuroendocrine networks is a major mechanism involved in key events of physiological functions such as parturition and lactation (oxytocinergic system) and preovulatory surge (GnRH system). This type of plasticity is classically described as rearrangements between glial cells and neuroendocrine neurones. Neuro-glial plasticity can occur within several hours. Cellular and molecular mechanisms involved are complex and imply an active regulation of neuroendocrine networks activity. In the present study we show that GnRH pulsatile secretion studied in vitro is regulated by gap junction communication between glial cells. Glial cells forming the microenvironment of GnRH neuronal network could represent a new system for integrating environmental cues and for regulating GnRH secretionLa plasticité neuro-gliale des réseaux neuroendocrines est un élément majeur de la régulation d’évènements clés de grandes fonctions, comme la plasticité du système ocytocinergique lors de la parturition et de la lactation et celle du système GnRH dans le déclenchement du pic pré-ovulatoire de LH. Cette plasticité est décrite par des réarrangements neuroanatomiques des cellules gliales associées aux neurones neuroendocrines. Elle peut se mettre en place en quelques heures. Les mécanismes cellulaires et moléculaires sont complexes et mettent en jeu une régulation active de l’activité des neurones par les cellules gliales. Dans l’étude présentée ici, nous montrons que la pulsatilité de sécrétion des neurones à GnRH étudiés in vitro est régulée par la communication des cellules gliales via des jonctions gap. Les cellules gliales du microenvironnement des neurones à GnRH pourraient ainsi représenter un nouveau système d’intégration des signaux environnementaux et de régulation de la sécrétion de GnR

Développement précoce des neurones à GnRH-1

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Stochastic modelling of GnRH neurones network

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L'hypothalamus est-il sexué ? Sécrétion pulsatile de GnRH et synchronisation neuronale

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Glial gap-jonction mediated regulation of GnRH neurons network activity

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Régulation des neurones à Gonadotropin Releasing Hormone (GnRH) par les cellules gliales

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Développement de la signalisation calcique dans les neurones à GnRH

La gonadotropin releasing hormone est essentielle à la reproduction. Sécrétée de manière pulsatile, elle contrôle la sécrétion des hormones gonadotropiques hypophysaires. Les neurones à GnRH, peu nombreux, dispersés dans l'aire préoptique, ont une origine extracérébrale. Une phase migratoire au cours de la vie embryonnaire précède la mise en place de la sécrétion. Les mécanismes de la migration et de la sécrétion, en particulier la pulsatilité, restent méconnus. Avec des cultures d'explants olfactifs, nous avons étudié les conductances calciques au cours du développement et les facteurs impliqués dans la synchronisation. Nos résultats montrent une maturation des conductances calciques et leur contribution relative dans les fluctuations calciques intracellulaires au cours du développement. Quant à la synchronisation, nos résultats montrent l'existence d'une rythmicité dans l'activité électrique des neurones et l'implication du GABA et de la GnRH sans écarter d'autres neurotransmetteurs.The gonadotropin releasing hormone is essential to the reproduction. Secreted in a pulsatile manner, it controls the gonadotropic hormones secretion. GnRH neurons are in small number and are spread in the hypothalamic preoptic area. A migratory phase during the embryonic life, precedes the set up of the secretion. Mechanisms involved in the migratory state, as well as those involved in secretion, in particularly pulsatility, remain unknown. Using two models of nasal explants cultures, we studied modifications in calcium conductances during the in vitro development and factors potentially involved in neuronal synchronization. Our results have underlined a maturation in calcium conductances and their relative contribution in intracellular calcium signaling occurring during the in vitro development. They have also shown the existence of rhythmicity in electrical activity, and confirmed the role for GABA and GnRH, even if the involvement of others neurotransmitters cannot be eliminated.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants.

International audienceThe aim of this study was to investigate the development of pulsatile GnRH secretion by GnRH neurones in primary cultures of olfactory placodes from ovine embryos. Culture medium was collected every 10 min for 8 h to detect pulsatile secretion. In the first experiment, pulsatile secretion was studied in two different sets of cultures after 17 and 24 days in vitro. In the second experiment, a set of cultures was tested after 10, 17 and 24 days in vitro to investigate the development of pulsatile GnRH secretion in each individual culture. This study demonstrated that (i) primary cultures of GnRH neurones from olfactory explants secreted GnRH in a pulsatile manner and that the frequency and mean interpulse duration were similar to those reported in castrated ewes, and (ii) pulsatile secretion was not present at the beginning of the culture but was observed between 17 and 24 days in vitro, indicating the maturation of individual neurones and the development of their synchronization

Primary culture of neural precursors from the ovine central nervous system (CNS)

The present study demonstrates that bipotential neural precursors isolated from an early developmental stage of the sheep embryo nervous system can be maintained in vitro in an undifferentiated state for a long period. These precursors multiplied under the action of epidermal growth factor and basic fibroblast growth factor and formed free-floating aggregates of nestin-immunoreactive cells, called neurospheres. These precursors can undergo predominantly neural or glial differentiation according to the culture conditions. Medium supplemented with foetal calf serum mainly favoured cell differentiation predominantly into astrocytes, whereas the defined SATO medium favoured neuronal differentiation. Using various immunomarkers of neurones and astroglial cells, we described the course of differentiation of neuronal and astroglial cells in different culture conditions. The ability to grow neural precursors from common laboratory animals has been useful for studying the cellular and molecular mechanisms underlying the development of the central nervous system. Furthermore, neural progenitors are already being used for in vivo cell therapy in various neurodegenerative disorders. The ovine species is a well-known model for prion diseases, since scrapie is endemic in most countries and has been studied for a long time. In this respect, the availability of ovine neural precursors will add a new perspective to the study of the pathogenicity of prion diseases