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A Bouron
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ABSTRACT: Presynaptic action potentials trigger the exocytosis of neurotransmitters. However, even in the absence of depolarisation-dependent Ca2+ entry nearby release sites, spontaneous vesicular release still occurs. Even though this happens at low rate, such spontaneous release may play a trophic role in maintaining the shape of dendritic structures. Like evoked responses, action potential-independent release is subject to modulation. This review describes some of the regulatory factors that rapidly and presynaptically regulate the ongoing Ca2+-independent release of neurotransmitters in the hippocampus. For instance, the electrical activity of the nerve ending, neurotransmitters, hypertonic solutions, neurotoxins, polycations, neurotrophic factors, immunoglobulins, cyclothiazide and psychotropic drugs can all modify the rate of spontaneous release. This can be achieved through various mechanisms that can be Ca2+-dependent or Ca2+-independent, protein kinase-dependent or independent. Since action potential-independent release contributes to the maintenance of dendritic structures, neuromodulators are likely to influence the density and/or length of dendritic spines, which in turn may modulate information processing in the central nervous system (CNS).
Progress in Neurobiology 05/2001; 63(6):613-35. · 8.87 Impact Factor
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A Bouron
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ABSTRACT: Intracellular Ca(2+) ([Ca(2+)](i)) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca(2+), the cholinergic agonist carbachol (200 microM) and the sarcoendoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (0.4 microM) both transiently elevated [Ca(2+)](i). A subsequent addition of Ca(2+) into the bathing medium caused a second [Ca(2+)](i) change which was blocked by lanthanum (50 microM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca(2+) entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca(2+) entry in excitable cells.
FEBS Letters 04/2000; 470(3):269-72. · 3.54 Impact Factor
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ABSTRACT: Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.
Journal of Neuroscience 04/2000; 20(6):2175-82. · 7.11 Impact Factor
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A Bouron
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ABSTRACT: Cultured hippocampal neurons from neonatal rats were used to investigate the effect of adenosine on the release of glutamate. Spontaneous tetrodotoxin-resistant miniature excitatory postsynaptic currents (mEPSCs) through AMPA receptor channels were recorded by means of the whole-cell patch-clamp technique. Adenosine (50 microM) reversibly reduced the frequency of mEPSCs by approximately 50-60%, but did not change their amplitudes. The protein kinase A inhibitor Rp-cyclic adenosine monophosphate (100-150 microM) did not block the adenosine-dependent reduction of the mEPSC frequency, showing that adenosine is not depressing synaptic transmission via a protein kinase A (PKA)-dependent mechanism. The D1 dopamine agonist SKF-38393 (250 microM), forskolin (5 microM) and 8Br-cAMP (2 mM), known to activate the cAMP/PKA-dependent signalling pathway, all enhanced the mEPSC frequency. A subsequent application of adenosine (50 microM) strongly reduced the potentiation produced by any one of these three drugs. It also reversed protein kinase C (PKC)-dependent stimulation of glutamate release induced by phorbol myristate acetate (100 nM). Taken together, adenosine not only inhibits the spontaneous release of glutamate independently of protein kinases A and C but also reverses the enhancement of exocytosis produced by protein kinases A and C activators.
European Journal of Neuroscience 01/2000; 11(12):4446-50. · 3.63 Impact Factor
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ABSTRACT: Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca2+ and Na+ channels were analyzed by electrophysiological techniques (using Ba2+ as charge carrier through Ca2+ channels) and by binding studies with specific ligands. With NGF, Na+ current (I(Na)) density increased in parallel with neurite extension. Ba2+ current (I(Ba)) density and Ca2+ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I(Na) density. With forskolin, neurite outgrowth was linked to an apparent increase in I(Ba) density similar to the one induced by NGF, while no change in I(Na) was observed. Dihydropyridine-sensitive (L-type) as well as omega-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I(Ba), binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca2+ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca2+ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics.
Archiv für Experimentelle Pathologie und Pharmakologie 06/1999; 359(5):370-7. · 2.65 Impact Factor
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ABSTRACT: Tricyclic antidepressants (e.g., imipramine, desipramine) are currently used in the treatment of mood disorders such as depression. At the cellular level they inhibit the re-uptake of the exocytosed monoamines serotonin and noradrenaline. However, they also stimulate phospholipase C activity and the production of the second messenger inositol 1,4,5-trisphosphate. Since phospholipase C activation can also lead to the production of the protein kinase C activator diacylglycerol, we have undertaken experiments to see whether acutely applied desipramine could change the synaptic strength of neurons in a protein kinase C-dependent manner. Experiments performed with cultured hippocampal neurons dissociated from neonatal rats revealed that desipramine rapidly enhanced the spontaneous vesicular release of glutamate. This was observed by measuring the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. Analysis of amplitude distribution histograms indicated a presynaptic site of action. The protein kinase inhibitor staurosporine and down-regulation of protein kinase C activity greatly reduced the desipramine-dependent enhancement of the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. This presynaptic modulation requires SNARE proteins because cleavage of SNAP-25 with the botulinum neurotoxin A strongly reduced the desipramine-induced glutamate release. Thus, acute applications of desipramine stimulated the ongoing neurotransmitter release pathway, probably by activating protein kinase C. Our data indicate that tricyclic antidepressant drugs not only act on serotoninergic and/or noradrenergic cells but can also modify the activity of glutamatergic neurons.
Neuroscience 04/1999; 90(3):729-36. · 3.38 Impact Factor
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ABSTRACT: The present study was undertaken to better assess the role of dopamine on exocytosis. Since direct activation of adenylate cyclase (e.g., with forskolin) enhances neurotransmitter release it was of interest to see whether the activation of D1-type dopamine receptors, which are positively coupled to adenylate cyclase, could also modulate the molecular machinery underlying the fusion of synaptic vesicles and the release of neurotransmitter. To answer this question we have looked at the effect of the D1-type dopamine receptor agonist SKF-38393 on the spontaneous release of glutamate from cultured rat hippocampal neurons. SKF-38393 enhanced the frequency but not the amplitude of tetrodotoxin-resistant excitatory postsynaptic currents which argues for a presynaptic locus of D1 action. This effect was blocked by the D1-dopaminergic receptor antagonist SCH-23390 and the protein kinase A inhibitors H-7 and Rp-cAMP whereas pertussis toxin failed to affect the dopaminergic response. In addition, carbachol and Ruthenium Red also stimulated exocytosis but did not occlude the SKF-38393-induced modulation. These results indicate that SKF-38393 presynaptically enhances the release of glutamate via a pertussis toxin-insensitive and protein kinase A-dependent mechanism, which most likely involves D1-type dopamine receptors. Our results underline the importance of protein kinase A as potent modulator of synaptic transmission and suggest that high concentrations of dopamine can greatly enhance the release of glutamate in the hippocampus.
Neuroscience 02/1999; 94(4):1063-70. · 3.38 Impact Factor
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ABSTRACT: L-type Ca2+ channels are important targets for drugs, such as dihydropyridines (DHPs), in the treatment of cardiovascular diseases. Channel expression is regulated by alternative splicing. It has been suggested that in the cardiovascular system tissue-specific expression of different L-type Ca2+ channel splice variants may underlie the observed differences in sensitivities to channel block by DHPs. We investigated the sensitivity of Ca2+ channel splice variants derived from the human alpha1C gene to the DHP isradipine. Among seven alpha1C channels we observed up to 10-fold differences in IC50 values for isradipine, as well as changes in the voltage dependence of DHP action.
FEBS Letters 06/1998; 427(2):220-4. · 3.54 Impact Factor
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ABSTRACT: We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1-43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca2+ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca2+ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4beta-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.
Proceedings of the National Academy of Sciences 11/1997; 94(22):12224-9. · 9.68 Impact Factor
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A Bouron
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ABSTRACT: Spontaneous excitatory postsynaptic currents (sEPSC) through AMPA-type channels were recorded on cultured hippocampal pyramidal neurons by means of the whole-cell patch-clamp technique. The protein kinase C (PKC) agonist 4beta-phorbol 12-myristate 13-acetate (4beta-PMA) produced a long-lasting increase in sEPSC frequency not mimicked by the inactive analogue 4alpha-PM and blocked by the protein kinase inhibitor staurosporine. The 4beta-PMA-induced change in sEPSC frequency occurred without detectable change in [Ca2+]i. After treatment with the microtubule-disrupting agent colchicine, 4beta-PMA caused a small and transient increase in sEPSC frequency. It is concluded that colchicine affects the PKC-induced functional plasticity of nerve cells most likely by disturbing the axonal transport.
FEBS Letters 04/1997; 404(2-3):221-6. · 3.54 Impact Factor
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ABSTRACT: The pore-forming alpha1C subunit is the principal component of the voltage-sensitive L-type Ca2+ channel. It has a long cytoplasmic carboxyl-terminal tail playing a critical role in channel gating. The expression of alpha1C subunits is characterized by alternative splicing, which generates its multiple isoforms. cDNA cloning points to a diversity of human hippocampus alpha1C transcripts in the region of exons 40-43 that encode a part of the 662-amino acid carboxyl terminus. We compared electrophysiological properties of the well defined 2138-amino acid alpha1C,77 channel isoform with two splice variants, alpha1C,72 and alpha1C,86. They contain alterations in the carboxyl terminus due to alternative splicing of exons 40-42. The 2157-amino acid alpha1C,72 isoform contains an insertion of 19 amino acids at position 1575. The 2139-amino acid alpha1C,86 has 80 amino acids replaced in positions 1572-1651 of alpha1C,77 by a non-identical sequence of 81 amino acids. When expressed in Xenopus oocytes, all three splice variants retained high sensitivity toward dihydropyridine blockers but showed large differences in gating properties. Unlike alpha1C,77 and alpha1C,72, Ba2+ currents (IBa) through alpha1C,86 inactivated 8-10 times faster at +20 mV, and its inactivation rate was strongly voltage-dependent. Compared to alpha1C,77, the inactivation curves of IBa through alpha1C,86 and alpha1C,72 channels were shifted toward more negative voltages by 11 and 6 mV, respectively. Unlike alpha1C,77 and alpha1C,72, the alpha1C,86 channel lacks a Ca2+-dependent component of inactivation. Thus the segment 1572-1651 of the cytoplasmic tail of alpha1C is critical for the kinetics as well as for the Ca2+ and voltage dependence of L-type Ca2+ channel gating.
Journal of Biological Chemistry 03/1997; 272(6):3560-6. · 4.77 Impact Factor
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ABSTRACT: Propagation of action potentials in axons and dendrites increases intracellular Na+ ([Na+]i) and Ca2+ concentrations ([Ca2+]i). While the importance of [Ca2+]i in synaptic transmission is well established, a possible functional role of [Na+]i is unclear. In cultured hippocampal cells, [Na+]i was increased by veratridine. We have then measured spontaneous excitatory postsynaptic currents (sEPSCs) and, by means of fluorescent dyes, changes in [Na+]i, in [Ca2+]i, and in the turnover of the vesicular pool of individual boutons. An elevation of [Na+]i and a concomitant rise in [Ca2+]i, led to a large increase in sEPSC frequency and in the turnover of the presynaptic vesicular pool. Extracellular Ca2+ was essential for these effects of elevated [Na+]i on synaptic transmission. They probably occur via Na+/Ca2+ exchange.
Neuron 12/1996; 17(5):969-78. · 14.74 Impact Factor
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ABSTRACT: We have investigated in Xenopus oocytes the effects of phorbol ester-induced protein kinase C (PKC) stimulation on dihydropyridine (DHP)-insensitive and -sensitive Ca2+ channels. DHP-insensitive Ba2+ currents (IBa) were recorded from endogenous channels in non-injected oocytes and in oocytes injected with cRNAs encoding the auxiliary rabbit alpha 2/delta and beta 1 Ca2+ channel subunits. A human alpha 1C cRNA, injected alone or in combination with cRNAs of the auxiliary subunits, was used for studying DHP-sensitive IBa. We found that DHP-insensitive IBa was increased by 4 beta-phorbol 12-myristate 13-acetate (PMA), while DHP-sensitive IBa was decreased. In both cases, the effects depended only on the co-expression of the beta 1 subunit.
FEBS Letters 01/1996; 377(2):159-62. · 3.54 Impact Factor
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ABSTRACT: Whole-cell patch-clamp measurements were made in nerve-growth-factor (NGF)-treated PC12 cells. External application of bradykinin (BK) activated an outward and an inward current which could be separated by using KCl- or CsCl-containing pipette solutions. The slowly activating inward current could be induced by BK independently of the filling of intracellular Ca2+ stores. By using GDP-beta-S in the pipette medium, we showed that BK-induced outward and inward currents were differentially regulated through G-protein-sensitive and -insensitive mechanisms, respectively. While the outward current was inhibited by GDP-beta-S, the inward current was not affected. Our results show that occupancy of BK receptors activates different signaling pathways for the induction of outward and inward currents.
Neuroscience Letters 08/1995; 195(1):37-40. · 2.11 Impact Factor
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ABSTRACT: Voltage-dependent inhibition by 1,4-dihydropyridines is a characteristic property of L-type Ca2+ channels. Six out of 50 exons of the channel alpha 1C subunit gene are subjected to alternative splicing, thus generating channel isoform diversity. Using Xenopus oocytes as an expression system, we have found that transmembrane segment IIIS2 of human alpha 1C subunit is involved in the control of voltage dependence of dihydropyridine action. This segment is genetically regulated through alternative splicing of exons 21/22. Site-directed mutagenesis points to two amino acids in IIIS2, which determine the difference of the splice variants in their sensitivities to dihydropyridines. This finding provides new insight into molecular mechanisms of Ca2+ channel inhibition by this important class of drugs.
Journal of Biological Chemistry 06/1995; 270(18):10540-3. · 4.77 Impact Factor
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ABSTRACT: Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3-4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca2+ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca2+ level and are most likely due to the inhibition of PP-1 class phosphatases.
Neuroscience Letters 02/1995; 183(3):198-201. · 2.11 Impact Factor
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ABSTRACT: We have studied the effects of protein kinase C (PKC) activators 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca2+ and K+ channels in nerve-growth-factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba2+ and K+ currents were recorded at room temperature with the patch-clamp technique. By using omega-conotoxin (CgTX) and isradipine, two specific Ca2+ channel blockers, we found three types of Ba2+ currents (IBa): (1) a omega-CgTX-sensitive IBa; (2) an isradipine-sensitive IBa; and (3) a omega-CgTX plus isradipine-resistant IBa. The external application of 4 beta-PMA or OAG down-modulated the isradipine-sensitive IBa whereas the two other IBa were not affected. 4 beta-PMA-induced inhibition of IBa was prevented by staurosporine (a protein kinase inhibitor) and PKC (19-31) (a specific PKC inhibitor). The delayed rectifier K+ current (IK) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the IBa in different manners. The presence of okadaic acid decreased the isradipine-sensitive IBa more than the omega-CgTX-sensitive IBa. The omega-CgTX plus isradipine-resistant IBa was not affected. Calyculin A down-modulated all three components of IBa to a similar degree. Our results suggest a differential modulation of voltage-activated Ca2+ and K+ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.
Pflügers Archiv - European Journal of Physiology 08/1994; 427(5-6):510-6. · 4.46 Impact Factor
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ABSTRACT: We have studied morphological differentiation and ion channel expression in PC12 cells under different culture conditions. Differentiation mediated by nerve growth factor (NGF) was compared with that induced by depletion and inhibition of protein kinases (phorbol ester beta-PMA plus staurosporine). Morphological differentiation was similar under both conditions. However, ion channel densities, studied by means of the patch-clamp technique, were enhanced by NGF and reduced by beta-PMA+staurosporine. Similar changes were also observed for omega-conotoxin-sensitive Ca2+ channels by measuring radioligand binding. The decrease in Ca2+ channel density, after treatment of the cells with beta-PMA+staurosporine, resulted in a reduced increase in the intracellular Ca2+ concentration during K+ depolarization. We conclude that morphological differentiation, but not ion channel expression, can occur during depression of protein kinase activities in PC12 cells.
Proceedings of the Royal Society B: Biological Sciences 09/1992; 249(1325):211-6. · 5.41 Impact Factor
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Renal physiology and biochemistry 17(3-4):132-3.
