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R Gardette

Publications and source records attributed to R Gardette.

At least 19 recordsLinked to original sources
PubMed1995

Modulation by somatostatin of glutamate sensitivity during development of mouse hypothalamic neurons in vitro.

Glutamate sensitivity development and interactions of somatostatin (SRIF) with AMPA/Kainate receptor-mediated glutamate responses were studied in dissociated hypothalamic neurons from 16-day-old mouse embryos grown in vitro. Only 18% of functionally innervated cells could be found at 6-9 DIV whereas the percentage of innervated neurons progressively increased thereafter to reach 100% at 19-22 DIV. The glutamate sensitivity, estimated from glutamate-induced peak inward current, was very low at 6-9 DIV, sharply increased at 11-14 DIV and developed at a low increase rate thereafter. SRIF either unaffected glutamate peak current (27% of the cells), or significantly decreased (50%) or increased it (23%). Pertussis Toxin pretreatment abolished the SRIF-induced decrease of the glutamate response without affecting the excitatory effect. The number of glutamate responsive neurons inhibited by SRIF increased with time in culture whereas that of neurons responding to SRIF by an increased glutamate response was not statistically modified by functional innervation. The present data suggest that increased glutamate sensitivity coincides with the onset of functional synaptogenesis in mouse hypothalamic neurons in culture. SRIF can modulate glutamate sensitivity of hypothalamic neurons with either synergistic or antagonistic effects. Since glutamate has been shown to stimulate SRIF synthesis and secretion from hypothalamic neurons, the reverse capacity of SRIF to modulate the glutamate response suggests that both transmitters exhibit complex reciprocal interactions.

Animals↗
PubMed1995

Molecular pharmacology of somatostatin receptors.

Somatostatin was discovered for its ability to inhibit growth hormone (GH) secretion. Later, it was found to be widely distributed in other brain regions, in which it fulfills a neuromodulatory role, and in several organs of the gastrointestinal tract where it can act as a paracrine factor or as a true circulating factor. In mammals, two molecules of 14 (somatostatin 14) and 28 (somatostatin 28) amino acids are the only biologically active members of the family. They originate from a single gene which gives rise to a single propeptide alternately cleaved in different tissues. In 1992, a major breakthrough in our understanding of somatostatin functions was made with the cloning of five different receptor genes (sstr1 to sstr5) which belong to the seven transmembrane domain receptor family. Their closer relatives are opioid receptors. In first approximation, the tissular expression of the sstrs matches quite well with the distribution of somatostatin binding sites in the "classical" targets of the peptide ie brain, pituitary pancreatic islets and adrenals. The pharmacology of GH inhibition is very close to sstr2 binding but other actions of somatostatins have not yet been attributed clearly to a single receptor subtype. All clinically relevant agonists tested so far (octreotide, lanreotide and vapreotide) are selective of sstr2 being less potent on sstr3 and inactive for sstr1 and sstr4. Surprisingly, rat sstr5 displays nanomolar affinities for octreotide and vapreotide while these agonists are only active at much higher concentrations on human sstr5. All five receptors can be more or less efficiently coupled to inhibition of adenylate cyclase activity in transfected cell systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Epidermal growth factor treatment induces D2 dopamine receptors functionally coupled to delayed outward potassium current (IK) in GH4C1 clonal anterior pituitary cells.

GH4C1 cells, a clonal cell line from a rat pituitary tumor, have been widely used as a model to study the regulation of prolactin secretion. These cells, however, do not express dopamine D2 receptors and are therefore not suitable for exploring mechanisms involved in dopamine inhibition of prolactin secretion. The recent demonstration that epidermal growth factor (EGF) is able to induce functional expression of D2 receptors in GH3 cells, a parental clonal cell line, overcomes this difficulty. We have thus undertaken an electrophysiological study in order to check whether coupling of D2 receptors to K+ channels could be restored in that model. Effects of dopamine on the non-inactivating voltage-dependent outward K+ current (IK) were investigated both in control and in EGF-treated GH4C1 cells. The K+ current was not modified by EGF treatment alone. In control cells, IK measured before and during dopamine application was unchanged. In contrast, dopamine application markedly enhanced the K+ current in cells that had previously been exposed to EGF. The effect was mimicked by the specific D2 receptor agonist bromocriptine and blocked by sulpiride, a D2 receptor antagonist, thus indicating that the effect of dopamine was effectively due to the activation of D2 receptors. These results bring further evidence that EGF-induced D2 receptors in clonal strains from rat pituitary tumors are functional and are coupled to the delayed outward K+ current IK.

Animals↗
PubMed1991

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.

Acetylcholine↗
PubMed1990

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. I. Development of reciprocal graft-host interactions.

In this paper, we have morphologically studied the developmental events underlying the neuronal replacement, 3-21 days after grafting. Despite their abnormal environment, Purkinje cell progenitors proceed with their proliferation in the grafted neuroepithelium, with a time window similar to that characterizing proliferation of this neuronal class in control mouse embryos. Only postmitotic Purkinje cells leave the grafts and migrate to the host molecular layer following stereotyped pathways. These neurons invade the host molecular layer, either through a tangential migration under the pial basal lamina from the graft/host interface or breaking locally the latter, and passing directly from the lateral swellings of the graft lying on the surface of the host folia. Whatever the pathway for host invasion, the migrating Purkinje cells penetrate radially and/or obliquely into the host molecular layer until their inward-oriented processes attain the molecular/granular layer interface, which occurs about 7 days after grafting. At the end of their migration, the grafted Purkinje cells with bipolar shapes and long and smooth processes begin to build up their ultimate dendritic trees. This dendritogenesis proceeds with constructive and regressive processes, passing through the same three developmental phases described by Ramón y Cajal (Trab. Lab. Invest. Biol. Univ. Madrid 24:215-251, 1926) for control Purkinje cells (phase of the fusiform cell, phase of the stellate cell with disoriented dendrons, and phase of orientation and flattening of the dendrites). In the grafted cerebella, the duration of the second and third phases is somewhat shorter than during normal cerebellar ontogenesis. Synaptogenesis between adult host axons and grafted Purkinje cells starts when the latter attain their second phase of dendritic development. Somatic filopodia emerging from grafted Purkinje cells begin, 10-11 days after grafting, to be synaptically contacted by axonal sprouts of the host climbing fibers resulting, 2 days later, in the formation of pericellular nests. Synaptogenesis between slender dendritic spines and host parallel fibers, together with that of axon terminals from host molecular layer interneurons and the smooth surface of the grafted Purkinje cell somata, begin earlier than in control mouse development, being almost simultaneous with climbing fiber/Purkinje cell synaptogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. II. Development of synaptic responses: an in vitro study.

Solid pieces of cerebellar primordia from 12-day-old C57Bl embryos were implanted in the cerebellar vermis of 3-4-month-old "Purkinje cell degeneration" mutant mice. Ten to 22 days after grafting, mutant mice were sacrificed, and synaptic responses of grafted Purkinje cells were studied by intracellular recordings performed in 400 microns thick sagittal slices in vitro. As early as 10 days after transplantation, grafted Purkinje cells have already completed their migration from the implant into the host molecular layer. Accordingly, inhibitory as well as excitatory responses were already elicited in these cells by electrical stimulation of the host subcortical white matter. Furthermore, a transient stage of multiple innervation of Purkinje cells by climbing fibers exists between 10 and 15 days after grafting, as revealed by the stepwise variation in amplitude of the climbing fiber-mediated excitatory postsynaptic potentials recorded before 15 days after grafting. Thirteen days after transplantation, typical all-or-none climbing fiber-mediated responses, parallel fiber-mediated excitatory postsynaptic potentials, and inhibitory postsynaptic potentials were also already present. Finally, normal adult-type synaptic responses were observed in all tested cells 15 to 17 days after grafting. Together with the companion paper (Sotelo et al., 1990), these results demonstrate that grafted Purkinje cells are able to impose on host afferents a pattern of synaptogenesis which closely follows that occurring during normal development, in particular, the transient stage of multiple innervation of Purkinje cells by climbing fibers.

Action Potentials↗
PubMed1990

Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry.

We have studied the development of synaptic transmission by innervating sympathetic neurons in vitro and monitoring synaptic activity with both physiological recording and cytochrome oxidase histochemistry. The onset of synaptic transmission was reflected in increased cytochrome oxidase reaction product within individual neurons. Within 24 hours of co-culture, relatively low frequency suprathreshold potentials were recorded in approximately 20% of the innervated neurons. At this stage the cytochrome oxidase activity of innervated neurons, as assessed by optical density of the histochemical reaction product, was increased twofold compared with uninnervated neurons. Over the next 2-4 days of innervation, changes in the pattern and extent of synaptic activity and superthreshold events were accompanied by a net fourfold increase in cytochrome oxidase activity levels compared with noninnervated neurons. The increase in density of cytochrome oxidase reaction product observed after innervation was reversed completely by blockade of synaptic transmission. Differences in the efficacy of synaptic input provided to the sympathetic neurons by appropriate versus inappropriate presynaptic sources was determined by co-culturing sympathetic neurons with explants that contained either preganglionic neurons or somatic motor neurons. Although sympathetic neurons innervated by motor neuron explants had increased levels of cytochrome oxidase activity compared with noninnervated controls, the density of cytochrome oxidase reaction product was even greater in sympathetic neurons innervated by preganglionic explants. We conclude that both the onset of innervation of sympathetic neurons as well as the subsequent maturation of synaptic function is directly reflected in graded increases in cytochrome oxidase reaction product.

Adrenergic Fibers↗
PubMed1988

Electrophysiological demonstration of a synaptic integration of transplanted Purkinje cells into the cerebellum of the adult Purkinje cell degeneration mutant mouse.

After implantation of solid pieces of cerebellar primordia from 12-day-old C57BL embryos into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice, Purkinje cells from the donor leave the implant and differentiate while migrating into the host molecular layer. Electrophysiological studies were performed using in vitro cerebellar slice preparations from "Purkinje cell degeneration" mutants 1-2 months after grafting, when grafted Purkinje cells have reached their final location in the host molecular layer and have completed their morphological differentiation. Intracellular recordings obtained from 45 Purkinje cells in mutant mice demonstrated that such grafted neurons have normal bioelectrical properties including sodium and calcium conductances and inward rectification. Moreover, all grafted Purkinje cells responded to electrical white matter stimulation by a typical all-or-none climbing fiber response. Responses mediated through the activation of mossy and parallel fibers, as well as inhibitory postsynaptic potentials, were also recorded in a significant number of grafted Purkinje cells. On the whole, all these excitatory and inhibitory responses in grafted "Purkinje cell degeneration" mutant mice have characteristics comparable to those in control mice. After electrophysiological studies, Purkinje cells were further characterized by their positive staining by calbindin antibody. Neurons of this class were dispersed throughout the molecular layer of the host folia in which the electrophysiological recordings had been performed. The ectopic location of their perikarya, the presence of dendritic trees spanning most of the molecular layer (without entering the granular layer), and the occasional presence of axons emerging from the ectopic neurons and forming loose bundles at the white matter axis of the folia, corroborate the grafted nature of the Purkinje cells studied. Therefore, these experiments demonstrate that embryonic Purkinje cells from the graft can complete differentiation in the adult host cerebellum, and establish specific synaptic contacts with the presynaptic elements previously impinging on the missing neurons of "Purkinje cell degeneration" mutants. This process leads to a qualitative functional synaptic restoration of the cortical cerebellar network.

Animals↗
PubMed1987

Postnatal development of the chemosensitivity of rat cerebellar Purkinje cells to excitatory amino acids. An in vitro study.

In vitro sagittal slices of immature rat cerebellum were used to study the development of the sensitivity of Purkinje cells (PCs) to L-aspartate (L-Asp), L-glutamate (L-Glu) and related derivatives. As early as postnatal day 0 all PCs already displayed clear excitatory responses to short iontophoretic applications of L-Asp, L-Glu and quisqualate while in the same conditions no effect of N-methyl-D,L-aspartate (NMDLA) was detected. By postnatal day 5, i.e. after the onset of the synaptogenesis, the sensitivity of PCs to L-Asp, L-Glu and quisqualate significantly increased up to values similar to those recorded in adult rat cerebellum and surprisingly nearly all (87%) the recorded cells now also displayed excitatory responses to NMDLA. Although this sensitivity of PCs to NMDLA was significantly lower than that observed with the other drugs, it persisted until the end of the first postnatal month when the adult type of connectivity is already well established but at this stage only 30 per cent of the tested cells were still sensitive to the agonist. During this period, excitatory responses elicited by NMDLA were selectively antagonized by 2-amino-5-phosphonovalerate (2-APV), suggesting that during postnatal development, NMDA receptor types are transiently expressed on PCs membranes since in the adult, NMDLA no longer had an excitatory effect. Instead, this drug now exerted a preferential antagonistic action on the excitatory response elicited by L-Asp. Also in the adult, no major changes occurred in the sensitivity of PCs to L-Asp, L-Glu and quisqualate when these drugs were ejected at a dendritic site whereas, when ejected at the somatic level, the sensitivity of the cell appeared 2-3 times lower.

2-Amino-5-phosphonovalerate↗
PubMed1987

Differential effects of serotonin on the spontaneous discharge and on the excitatory amino acid-induced responses of deep cerebellar nuclei neurons in rat cerebellar slices.

The effects of steady iontophoretic applications of serotonin on the spontaneous discharge and on the excitatory responses induced in deep cerebellar nuclei neurons by iontophoretic pulse applications of L-glutamate, L-aspartate, N-methyl-D,L-aspartate and quisqualate were studied in rat cerebellar slices maintained in vitro. Serotonin increased the spontaneous firing rate of deep cerebellar nuclei neurons in 91% of the tested cells by 109% on the average and had no effect on the remaining recorded neurons. Conversely, the monoamine induced a depression of the excitatory responses induced by four agonists tested and the depressant potency of serotonin was in the order quisqualate, glutamate, aspartate, N-methyl-D,L-aspartate. These effects persisted in low calcium high magnesium solution, suggesting that the serotonin receptors involved in these phenomena were, at least partially, postsynaptically located. The serotonin-induced increase in the cell firing rate appeared to be methysergide-resistant whereas the serotonin-induced decrease in the responses elicited by excitatory amino acids was depressed by this antagonist, which could indicate that these differential effects of serotonin are mediated via different mechanisms and/or serotonin receptor subtypes.

Action Potentials↗
PubMed1986

Chemoresponsiveness of intracellular nuclei neurones to L-aspartate, L-glutamate and related derivatives in rat cerebellar slices maintained in vitro.

The sensitivity of intracerebellar nuclei neurones to pulse applications of L-aspartate, L-glutamate, N-methyl-D,L-aspartate and quisqualate was tested in rat cerebellar slices maintained in vitro. The responses of the nuclear neurones to the four agonists consisted of a transient and dose-dependent increase in their firing of simple spikes. When suprathreshold currents were used, quisqualate induced the highest increase in the spike discharge frequency of the cells. Quisqualate mediated responses were unaffected by steady applications of 2-amino-5-phosphonovalerate, whereas the sensitivity of the responses induced by the three other agonists was in the order N-methyl-D,L-aspartate, L-aspartate, L-glutamate. When the superfusing solution was devoid of Mg2+ ions, N-methyl-D,L-aspartate and L-aspartate mediated responses were much potentiated, while quisqualate induced responses were not enhanced. In such a medium, L-glutamate elicited responses were more or less potentiated depending on cells. These results suggest that rat intracerebellar nuclei neurones bear both N-methyl-D-aspartate and non-N-methyl-D-aspartate, probably quisqualate, receptors, and that L-aspartate and L-glutamate have a mixed action upon both types. L-Aspartate preferentially activates N-methyl-D-aspartate receptors, whereas L-glutamate predominantly acts via non-N-methyl-D-aspartate receptors. Furthermore, the potency of L-glutamate in activating N-methyl-D-aspartate receptors appears to vary as a function of the cells.

Animals↗
PubMed1985

Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. II. Membrane conductances.

The development of membrane conductances of intracerebellar nuclei neurons was studied in the rat since birth up to the weaning period by the use of thick sagittal cerebellar slices maintained in vitro. Mature nuclear neurons express fast sodium and slowly inactivating sodium conductances, as well as calcium conductances. As early as birth, fast sodium and calcium conductances appear well developed whereas slowly inactivating sodium conductances mature within the first postnatal week.

Animals↗
PubMed1985

Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. I. Postsynaptic potentials.

The development of the synaptic responses of intracerebellar nuclei neurons was studied in the rat by the use of thick sagittal cerebellar slices maintained in vitro. It has been shown that functional excitatory synapses are present on these neurons from birth, probably due to climbing and/or mossy fiber collaterals; functional inhibitory synapses, due to monosynaptic projections of Purkinje cell axons onto intracerebellar nuclei, are present as early as postnatal day 2; and a more complex pattern of synaptic responses, including short latency excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs), longer latency IPSPs, and late depolarizing responses, can be elicited in nuclear neurons as early as postnatal day 3, indicating an early development of some complete functional cerebellar circuits involving the intracerebellar nuclei.

Animals↗
PubMed1984

Effect of excitatory amino acids on Purkinje cell dendrites in cerebellar slices from normal and staggerer mice.

The sensitivity of Purkinje cells to short pulse applications of L-aspartate, L-glutamate and related derivatives in their dendritic fields was tested in normal and staggerer mutant mice using cerebellar slices maintained in vitro. In normal mice, the response of Purkinje cells to L-aspartate and L-glutamate consisted of a transient and dose-dependent increase of their firing of simple spikes. The potency of L-aspartate in exciting Purkinje cells was lower than that of L-glutamate when the two drugs were released from adjacent barrels of the same iontophoretic electrode. Quisqualate was an even more potent excitant of these cells than L-aspartate and L-glutamate, whereas N-methyl-DL-aspartate had little or no effect. In staggerer mutant mice, the sensitivity of Purkinje cells to L-aspartate, L-glutamate and quisqualate was not significantly altered. On the contrary, N-methyl-DL-aspartate had a much stronger potency than normal in exciting Purkinje cells although this was still smaller than that of the other agonists tested. These results suggest that the sensitivity of Purkinje cells to L-aspartate and L-glutamate, i.e. the putative neurotransmitters of the climbing and parallel fibers respectively, remains largely normal in staggerer mice. In contrast, in the mutant, N-methyl-D-aspartate receptors are likely to be much more developed than normal.

Amino Acids↗
PubMed1984

Selective absence of calcium spikes in Purkinje cells of staggerer mutant mice in cerebellar slices maintained in vitro.

The bioelectrical properties of Purkinje cells (intrasomatic recordings) were studied in sagittal cerebellar slices of both adult staggerer and control mice. Mean input resistances of Purkinje cells were 25 +/- 4 M omega and 48 +/- 7 M omega in normal and staggerer mice respectively. In both groups, time-dependent inward rectifications were apparent in the hyperpolarizing voltage-responses. In normal mice, tetrodotoxin (TTX)-sensitive simple spikes and slower-rising multiphasic spikes, abolished when Ca was replaced by Cd in the bath, spontaneously occurred in Purkinje cells. These Na- and Ca-dependent spikes were also elicited by depolarizing current pulses. In the mutant, Ca spikes were never observed, even in strongly depolarized cells. On the contrary, TTX-sensitive simple spikes occurred spontaneously or were elicited by depolarizing current pulses. When Ca was replaced by Ba in the bath, the Ca spikes evoked in normal Purkinje cells by direct stimulation were first enhanced and then replaced by prolonged action potentials (1-6 s in duration) which were TTX-resistant and Cd-sensitive. These (Ba) action potentials were also triggered by climbing fibre activation of the cells. In staggerer mice, Ca spikes were never elicited by direct stimulation in Ba-containing medium, although in a few cells prolonged action potentials were occasionally elicited by depolarizing current pulses. However, this latter type of response was never evoked by climbing fibre activation of Purkinje cells. In the mutant, extracellular application of tetraethylammonium (TEA) generated prolonged action potentials, the plateaux of depolarization of which were less positive than those elicited by Ba in control mice. These plateaux were abolished by TTX and left unaffected by the substitution of Ca by Cd in the bath, suggesting that they were due to a non-inactivating Na conductance. On the whole, the present study strongly suggests that voltage-dependent Ca channels are missing in most staggerer Purkinje cells or at least that their characteristics and/or distribution are such that they cannot be activated. Na channels appear unaffected.

Action Potentials↗
PubMed1983

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro.

A voltage clamp analysis of the effects of L-aspartate, L-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications of L-aspartate and L-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl-D,L-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl-D,L-aspartate much more severely depressed L-aspartate than L-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due to L-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due to L-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl-D-aspartate and quisqualate receptors.

Amino Acids↗
PubMed1983

Bioelectrical properties of cerebellar Purkinje cells in reeler mutant mice.

Electroresponsive properties of intracellularly recorded Purkinje cells (PCs) in reeler mice were studied by using sagittal cerebellar slices maintained in vitro. In normally as well as in abnormally located PCs, fast spikes and slower rising multiphasic spikes were elicited by depolarizing currents, and they were abolished by tetrodotoxin and by cadmium respectively. This demonstrates that these neurons, as PCs in normal animals, do have sodium- and calcium-dependent spikes, thus indicating that these bioelectrical properties do not depend on the connectivity of PCs.

Action Potentials↗