Biomedical subjects

G Moonen

Publications and source records attributed to G Moonen.

At least 55 records · Page 3Linked to original sources

PubMed1998

Developmental regulation of neuroligand-induced responses in cultured oligodendroglia.

Using whole-cell patch-clamp techniques, we show that oligosphere-derived oligodendrocyte progenitor cells (OP) display GABA-, glutamate-, 5-HT-, glycine- and acetylcholine-gated inward currents. When OP differentiate into oligodendrocytes (ODC), the amplitude of peak currents elicited by saturating concentrations of these transmitters decreases except for 5-HT. Intracellular Ca2+ concentration changes induced by microperfusion of glutamate, 5-HT, TRH, met-enkephalin and substance P were monitored using a fluo-3-based calcium imaging system. When OP cells differentiate into ODC, a global decrease of the proportion of responding cells is observed. During type-2 astrocytes commitment, this proportion decreases for 5-HT, TRH- and metenkephalin stimulations whereas it remains constant for substance P and glutamate. These data demonstrate a development regulation of neurotransmitter- and neuropeptide-induced responses within the oligodendroglial lineage.

Acetylcholine↗

PubMed1998

Effects of macrophage transplantation in the injured adult rat spinal cord: a combined immunocytochemical and biochemical study.

Early and robust invasion by macrophages may be one of the reasons why axonal regeneration is more effective in the PNS than in the CNS. Therefore, we have grafted autologous peritoneal macrophages labeled with fluorescent latex microspheres into spinal cord compression lesions. At various survival times, we have studied their effect on the expression of neuronal (neurofilaments [NF], calcitonin gene-related peptide [CGRP], 5-hydroxytryptamine [5-HT]) and nonneuronal markers (myelin-associated glycoprotein [MAG], glial fibrillary acidic protein [GFAP], laminin) by using semiquantitative Western blot and immunohistochemical techniques. After 1 month, we observed a significant decrease of the expression of MAG as well as an important invasion of the lesion site by neurites, chiefly peptidergic axons of presumed dorsal root origin, in macrophage-grafted animals compared with controls. In addition, angiogenesis and Schwann cell infiltration were more pronounced after macrophage grafts, providing an increase in laminin, a favorable substrate for axonal regrowth. By using reverse transcription-polymerase chain reaction (RT-PCR), mRNAs for tumor necrosis factor-alpha (TNF-alpha) were detected in the transplanted cells, whereas results were negative for nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), or acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Thus, macrophage grafts may represent an interesting strategy to promote axonal regeneration in the CNS. Our study suggests that they may exert their beneficial effects by degrading myelin products, which inhibit axonal regrowth, and by promoting a permissive extracellular matrix containing notably laminin. No evidence for a direct synthesis of neurotrophic factors by the transplanted macrophages was found in this study, but resident glial cells could secrete such factors as a result of stimulation by macrophage-released cytokines.

Animals↗

PubMed1998

Expression of growth factors and their receptors in the postnatal rat cochlea.

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.

Animals↗

PubMed1998

Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform.

Using the whole-cell patch-clamp technique, we demonstrate glycine-induced currents in oligosphere-derived oligodendrocyte progenitors cultured from newborn rats. Similar inward currents are also triggered by beta-alanine and taurine, two established glycine receptor agonists. In our recording conditions, glycine-gated currents in oligodendrocyte progenitors reverse about 0 mV and are reversibly inhibited by the glycine competitive antagonist strychnine, the Cl- channel blocker picrotoxinin and the non-competitive antagonist cyanotriphenylborate. The oligodendrocyte progenitors glycine receptor (GlyR) differs from the corresponding neuronal receptor: [3H]strychnine binding data and the strychnine inhibition curve of glycine-induced currents in oligodendrocyte progenitor cultures suggest the existence of two strychnine binding sites on the oligodendroglial GlyR. Using total RNA isolated from oligodendrocyte progenitors cultures, reverse transcription-polymerase chain reaction analysis of glycine receptor subunit expression shows the presence of alpha2 and beta subunits and immunocytochemical stainings confirm that this GlyR contains an alpha subunit which is not alpha1. The molecular structure of the oligodendroglial GlyR could be either homopentameric alpha2 or heteromeric alpha2beta but in both cases, the sequence of the alpha2 or beta subunits have to be different from the known neuronal sequences in order to explain, respectively, the cyanotriphenylborate (alpha2) and picrotoxinin (beta) sensitivities. This work thus demonstrates that GlyR are expressed by oligodendrocytes obtained not only from spinal cord but also from supraspinal structures. The pharmacological properties and presumably the molecular structure of oligodendroglial GlyR are original. The physiological meaning of the presence of such receptors on developing and mature oligodendrocytes remains unknown.

Animals↗

PubMed1998

Mammalian auditory hair cell regeneration/repair and protection: a review and future directions.

Regeneration/repair and protection of auditory hair cells and auditory neurons is an exciting, rapidly evolving field. Simultaneous developments in the fields of otobiology and surgical otology have led to new and exciting possibilities in inner ear medicine and surgery; specifically, the treatment or prevention of a variety of types of hearing losses in the foreseeable future. Sensorineural hearing loss in humans is commonly associated with a loss of auditory hair cells. It has been generally accepted that hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury. An organ of Corti explant study has shown that it is possible to initiate the regeneration/repair of mammalian hair cells. In this study, ototoxin-damaged organ of Corti explants from juvenile rats were treated with a combination of retinoic acid (10-8M) and fetal calf serum (10%). TGF-alpha has been identified as a growth factor capable of evoking auditory hair cell regeneration/repair in ototoxin-damaged organ of Corti explants. Preliminary in vitro experiments with juvenile rat organ of Corti explants and in vivo studies in the cochleae of adult guinea pigs indicate that pretreatment followed by continuous treatment of the inner ear with a combination of retinoic acid and TGF-alpha can protect the auditory hair cells from the ototoxic effects of aminoglycosides. Because the integrity of spiral ganglion neurons is also essential for normal auditory function, there is a parallel series of in vitro and in vivo studies of the effects of neurotrophic factors on the survival of auditory neurons and the regeneration of injured neuronal processes. Clinical studies have demonstrated that it is now possible to perform surgeries on the inner ear, i.e., partial or total labyrinthectomies, and maintain hearing. The field of cochlear implantation has also provided insights into both the short- and long-term effects of cochlear fenestration on inner ear function. Administration of growth factors to the inner ears of animals is now possible with the use of implanted catheters and miniature infusion pumps. These advances suggest that localized application of drugs to the human inner ear may be feasible. The aim of this paper has been to provide an overview of advances in the study of the biology of auditory hair cells and auditory neurons, as well as recent relevant surgical advances. Taken together, these advances in otobiology and surgery will, in the future, be combined to devise new and innovative treatments for inner ear disorders.

Adult↗

PubMed1997

Diazepam-insensitive GABAA receptors on postnatal spiral ganglion neurones in culture.

Using dissociated spiral ganglion cell cultures obtained from 3-day-old rat cochlea, we investigated the response of auditory neurones to gamma-aminobutyric acid (GABA) using patch-clamp techniques. In our recording conditions, GABA elicited inward currents in > 95% of the neurones which reversed around 0 mV. Similar inward currents were measured using isoguvacin, a specific agonist of GABAA receptors. GABA-gated currents were reversibly inhibited by the channel blocker picrotoxin and the GABA competitive antagonist bicuculline. These functional GABAA receptors are characterized by an insensitivity to benzodiazepines and a relatively high sensitivity to beta-carbolines and barbiturates. These results show that the GABAA receptor pharmacological properties of spiral ganglion neurones are close to those of cerebellar granule cells.

Animals↗

PubMed1997

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: I. Effects of antisense oligodeoxynucleotides using explants including or excluding the preoptic area.

Using antisense oligodeoxynucleotides we aimed to study the role of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin-releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time-related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the beta subunit of the GABAA receptor and the NR2A subunit of the NMDA receptor, respectively. After 6-7 h, binding studies of tritiated ligands show a decrease in GABA- and NMDA-receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABAA and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol- and NMDA-evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABAA and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABAA and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABAA and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist-evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.

Animals↗

PubMed1997

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: II. Reduced NR2C- and GABAA-receptor-mediated inhibition at initiation of sexual maturation.

N-methyl-D-aspartate (NMDA) receptors and gamma-aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin-releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol-evoked release of GnRh is similarly inhibited by the GABAA receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRh interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABAA receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA-evoked release of GnRH and the GnRh interpulse interval are similarly inhibited by 100 or 500 microM of the NMDA receptor antagonist 7-chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 microM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRh interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABAA receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.

Animals↗

PubMed1997

Jean Marquet Award. Regeneration of the neurosensory structures in the mammalian inner ear.

Regeneration of the neurosensory structures in the mammalian inner ear. Motivated by the absence of treatment of neurosensory deafness apt to restore auditory function or alter the course of progressive hearing loss, we developed two different experimental strategies to approach these diseases: one is otoprotection, designed to prevent further degradation of auditory function; the other is regeneration which is defined as the replacement of hair cells in the deaf ear and their reconnection to the central nervous system through primary auditory neurons. In this paper, we summarize our data on the regeneration of auditory neurons and hair cells. Neuronal maintenance and regeneration was studied through an initial investigation of growth factors during inner development. Once the effect of various neurotrophic molecules was determined, the factors were tested on mature auditory neurons in vitro and in vivo. The hair cell regeneration was investigated on the basis of a concept derived from comparative physiology and from the study of the development of the inner ear. We showed that in young rats it is possible to induce the regeneration/repair of hair cells in the organ of Corti in cultures using retinoïc acid and transforming growth factor alpha. The clinical prospects of these findings of inner ear regeneration are discussed.

Animals↗

PubMed1996

Beta-carbolines induce apoptotic death of cerebellar granule neurones in culture.

Apart from its role in fast inhibitory transmission, only neurotrophic effects have been reported following activation of the GABAA receptor. Here, we show that n-butyl-beta-carboline-3-carboxylate and n-methyl-beta-carboline-3-carboxamide, which are negative allosteric modulators of the GABAA receptor acting at the benzodiazepine site, are neurotoxic for cerebellar granule neurones in culture. The beta-carboline-induced neuronal death is apoptotic since DNA internucleosomal fragmentation was induced and the neurotoxicity could be prevented by inhibitors of mRNA or protein synthesis. As GABA and benzodiazepine ligands (diazepam and Ro 15-1788) protect cerebellar granule cells against beta-carboline-induced toxicity, these data raise the possibility that the interaction between the beta-carbolines and the GABAA receptor is the triggering event leading to neuronal apoptosis.

Animals↗

PubMed1996

Cytokine production by human thymic epithelial cells: control by the immune recognition of the neurohypophysial self-antigen.

Oxytocin (OT) has been shown to be the dominant peptide of the neurohypophysial family expressed by thymic epithelial and nurse cells (TEC/TNC) in various species. Thymic OT is not secreted but, after translocation of a hybrid neurophysin/MHC class I protein, is integrated within the plasma membrane of TEC, thus allowing its presentation to pre-T cells. In order to further demonstrate that thymic OT behaves like a membrane antigen, we assessed the effect of mAbs to OT on cytokine productions by cultures enriched in human TEC. 75-85% pure TEC cultures were prepared from human thymic fragments. Using immunofluorescence and confocal microscopy, ir-OT, ir-interleukin-1 beta (IL-1 beta), ir-interleukin-6 (IL-6) and ir-leukemia inhibitory factor (LIF) could be detected in these TEC cultures. ir-OT was restricted to TEC, while some ir-IL-6 and ir-LIF were also seen in occasional fibroblasts. In basal conditions, ir-IL-6 and ir-LIF (but not ir-OT and ir-IL-1 beta) were detected in the supernatants of human TEC cultures. MAbs to OT induced a marked increase of ir-IL-6 and ir-LIF secretion in TEC cultures. No significant effect was observed using mAbs against vasopressin, mouse immunoglobulins, or control ascitic fluid controls. These data show that OT is fully processed and recognized by specific mAbs at the outer surface of TEC plasma membrane. They further support that thymic OT behaves as the self-antigen of the neurohypophysial family.

Antibodies, Monoclonal↗

PubMed1996

Protein kinase- and staurosporine-dependent induction of neurite outgrowth and plasminogen activator activity in PC12 cells.

We analysed how interactions between protein kinase-dependent intracellular signalling pathways were implicated in the control of the production of tissue-type plasminogen activator (tPA) and the generation of neurite outgrowth by PC12 cells. To that aim, cells were treated with agents that interact with the trk receptor and with protein kinases A and C. Nerve growth factor induced only the formation of large neurites. The release of the protease and the production of short neurite outgrowth were found to be protein-kinase-A-dependent events that could be enhanced by simultaneous activation of protein kinase C with phorbol ester. At high concentration, staurosporine, a nonselective inhibitor of protein kinases, induced the production of short neurites and mimicked the protein-kinase-A-dependent effect on tPA release. Such a response was not observed with K-252a, an analogue of staurosporine devoid of neurite-outgrowth-promoting activity. The responses to protein kinase A stimulation and the addition of staurosporine, although similar, seemed to occur through an activation of distinct, yet interacting, signalling pathways. In conclusion, tPA release and large neurite outgrowth from PC12 cells are controlled by parallel, albeit interacting, pathways, suggesting that these two potentially antagonistic events in PC12 cell differentiation can be modulated in a concerted way or independently of each other, depending on the activity of several protein kinases.

Animals↗

PubMed1996

NT-3 has a tropic effect on process outgrowth by postnatal auditory neurones in vitro.

CONFOCAL analysis of early postnatal auditory neurones in a bicompartmental culture system was used to test for chemoattractant properties of NGF, BDNF and NT-3 on neuronal process outgrowth. NT-3 exerted a strong tropic effect on neuritic outgrowth from auditory neurones in this system. BDNF and NGF did not have any tropic activity that directed processes outgrowth from auditory neurones. However, BDNF was important for the support of neuronal survival in NGF-treated cultures and for neuritogenesis in NT-3-treated cultures. Since NT-3 has been identified as both a survival factor and a chemotropic agent for auditory neurones, it is likely that this neurotrophin will be a useful therapeutic agent in the treatment of damaged cochleae for the recovery of hearing.

Animals↗

PubMed1996

Alpha-MSH stimulates neurite outgrowth of neonatal rat corticospinal neurons in vitro.

Peptides related to melanocortin (alpha MSH) and corticotropin (ACTH), collectively termed melanocortins, exert trophic effects on the outgrowth of neurites from peripheral and central nervous system in vitro. Here we study the neurite outgrowth promoting effect of alpha-MSH on corticospinal (CS) neurons in vitro. Corticospinal neurons were identified in cell culture of neonatal rat cortex by immunostaining of cholera toxin subunit B (CTB), retrogradely transported from the cervical parts of the spinal cord. The CTB-immunoreactive neurons represent a small percentage (3-5%) of the total cell population after 72 h in vitro. The axons or dendrites of cortical and CTB-labelled layer V neurons were visualized using antibodies against axon- or dendrite-specific markers and measured using a semi-automatic quantification device. Here we report that alpha-MSH stimulates axonal as well as dendrite outgrowth from both total and CTB-labelled neurons with a bell-shape response curve. Axonal outgrowth of CTB-labelled neurons was dose-dependently stimulated with a maximal effect of 50% at 10(-10) M alpha-MSH. The maximal effect for stimulation of axon outgrowth for the total cortex population was observed at 10(-8) M alpha-MSH. In addition dendrite outgrowth of both total and CTB-labelled neurons is stimulated in a dose-dependent manner with maximal effects (varying between 46 and 48%) at 10(-8) M alpha-MSH. Explanations in the shift for the optimal alpha-MSH concentration for stimulation of axonal outgrowth of CTB-labelled layer V neurons as compared to total cortex neurons are discussed.

Animals↗

PubMed1996

Effects of Schwann cell transplantation in a contusion model of rat spinal cord injury.

Cultured Schwann cells were transplanted at various delays into a spinal cord contusion injury performed at low thoracic level in adult female rats. The Schwann cells were purified from the dorsal root ganglia of adult syngeneic animals. the transplants were well tolerated, and the transplanted Schwann cells invaded the injured spinal cord. As quantified using video image analysis, the survival and growth of the transplanted cells were poor when the grafting procedure was performed 3-4 days after injury and very good when performed immediately or 10 days after injury, in which cases post-traumatic micro- and macrocavitation were strongly reduced. In animals grafted immediately after injury but not in animals grafted after 10 days, post-traumatic astrogliosis was much reduced. The Schwann cells transplanted area was invaded by numerous regenerating axons, the vast majority of which were, based on the neurotransmitter (CGRP and SP) profile, originating from dorsal root ganglion. No regeneration of the corticospinal tract as assessed after anterograde tracing or of descending aminergic fibers could be demonstrated.

Animals↗

PubMed1996

Astroglia-released factor with negative allosteric modulatory properties at the GABA A receptor.

We have previously shown, using whole-cell patch-clamp techniques, that astrocytes release a negative allosteric modulator of the gamma-aminobutyric acid type A receptor (GABAA receptor) with beta-carboline-like properties, thus, likely to act at the benzodiazepine site. Here, using patch-clamp and binding techniques, we confirm that the low-molecular-weight fraction of astroglia-conditioned medium (ACM lmf) contains a factor(s) that negatively modulates GABAA-receptor function. This factor, like beta-carbolines, enhances the specific binding of [35S]t-butyl bicyclophosphorothionate (TBPS) to adult rat cortical membranes in the presence of GABA. However, it fails to interact with various ligands of the benzodiazepine (BZD) site of the GABAA receptor ([3H]flunitrazepam, [3H]Ro 15-1788 and [3H]Ro 15-4513). The question of the actual binding site of the astroglia-derived factor on the GABAA receptor, thus, remains open and can be addressed only after the purification of the active molecule(s) of ACM Imf has been completed, and a labeled form of the endogenous ligand becomes available. Taken together, however, the data suggest that type 1 astrocytes are able to modulate the effects of the main inhibitory neurotransmission in the central nervous system.

Allosteric Regulation↗

PubMed1996

NGF, BDNF and NT-3 play unique roles in the in vitro development and patterning of innervation of the mammalian inner ear.

Developing cochleovestibular ganglion (CVG) neurons depend upon interaction with the otocyst, their peripheral target tissue, for both trophic support and tropic guidance. RT-PCR of E11 through E14 otocyst-CVG RNA extracts have shown that NGF as well as BDNF and NT-3 are expressed in the developing inner ear (in situ RT-PCR on tissue sections of E12 otocysts localized all three neurotrophins to the otocyst). To evaluate the functional significance of NGF, BDNF and NT-3 expression, E10.5 otocyst-CVG explants were treated with antisense oligonucleotides and compared to sense treated and control cultures. Confocal microscopic analysis revealed that treatment with BDNF antisense resulted in extensive neuronal cell death, downregulation of NGF caused an inhibition of neuritogenesis and a decrease in the neuronal population of the CVG, whereas treatment with NT-3 antisense resulted in a loss of target directed CVG neuritic ingrowth in this in vitro model. The effect of NGF or BDNF antisense treatment could be prevented by the simultaneous addition of the respective growth factor. These findings demonstrate that each of the three neurotrophins have important roles during the onset of neuritic ingrowth of the CVG neurons to the otocyst.

Animals↗

PubMed1996

Effects of neurotrophins on early auditory neurones in cell culture.

During the first week of postnatal development, the innervation of the organ of Corti changes from an immature to an adult pattern. Dissociated cell cultures of early postnatal spiral ganglia were used to investigate the effects of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on maturing auditory neurones. BDNF was the most potent stimulator of neuritogenesis, NT-3 provided the strongest support for neuronal survival, while NGF supported limited neuritogenesis, and only at pharmacological levels. These findings suggest that both BDNF and NT-3 participate in the postnatal maturation of cochlear innervation and that NGF is most probably not involved in this process.

Animals↗