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P Drapeau

Publications and source records attributed to P Drapeau.

At least 55 records · Page 3Linked to original sources
PubMed1990

Loss of channel modulation by transmitter and protein kinase C during innervation of an identified leech neuron.

When serotonergic Retzius (R) neurons of the leech contact pressure-sensitive (P) neurons in culture, P cells selectively lose a protein kinase C-dependent cationic response to serotonin and the R cell reforms the inhibitory, chloride-dependent synapse seen in vivo. In P cells not contacted by R cells, cell-attached patches contained single cation channels sensitive to serotonin and phorbol ester with characteristic properties and high incidence (present in about one-half of the patches). P cells paired with R cells had a cation channel with similar biophysical properties and incidence, but channel activity was not stimulated by serotonin and phorbol ester. These results suggest that the early clearing of the non-synaptic (excitatory) response to serotonin is due to the loss of activation by protein kinase C (and not the number) of cation channels as a prelude to inhibitory synapse formation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron.

In an attempt to define the mechanism of synaptic specificity, we have been studying pairs of identified leech neurons isolated in tissue culture. The cultured neurons reform specific synapses when paired with appropriate partners in the absence of other cell types. In recent studies, we have examined in detail the reformation of a serotoninergic synapse between the Retzius cell and one of its targets, the pressure sensitive (P) cell. The P cell in vivo and its soma in vitro have two types of responses to serotonin (5-HT). From voltage clamp analysis of cultured P cells, we demonstrated the parallel activation of chloride (gCls) and monovalent cation (gCations) channels coupled to distinct receptor subtypes and gated by separate second messengers. Only gCls was activated by 5-HT released from the presynaptic Retzius cell both in vivo and in vitro. This demonstrates the remarkable specificity of the reformation of this synapse in culture since only the correct 5-HT receptor subtype is activated. An 80% reduction of gCations was observed in P cells that had failed to be innervated by Retzius cells in culture, suggesting that gCations may be lost prior to synapse formation. Retzius cells depleted of 5-HT also reduced gCations in the paired P cells and incubating single P cells in 5-HT did not reduce gCations. In addition, aldehyde-fixed Retzius cells were able to selectively reduce gCations when paired with P cells. We conclude that the loss of gCations was due to contact between the neurons. The early clearing of counter-effective receptor subtypes may be a prelude to synapse formation.

Animals↗
PubMed1989

Contact-mediated loss of the nonsynaptic response to transmitter during reinnervation of an identified leech neuron in culture.

We have examined the modification of responses to transmitter during the reformation of a specific synapse by identified leech neurons in culture. Single pressure-sensitive mechanosensory (P) neurons in culture and the soma of this cell in vivo have 2 conductances that are activated by application of 5-HT: a Cl conductance and a monovalent cation conductance (gCations); synaptic release of 5-HT by a serotoninergic Retzius cell in vivo and in culture activates only the Cl conductance and not gCations in the P cell. We have characterized the loss of gCations in P cells by manipulating the culture conditions. When 5-HT was applied from a pipette, innervated P cells and P cells paired with Retzius cells that had not formed synapses had a gCations that was markedly reduced compared with gCations in the single P cell. When 5-HT was included in the culture medium in which single P cells were grown, gCations was not reduced. When Retzius-P cell pairs were treated with reserpine, which was shown to deplete the presynaptic neuron of 5-HT, gCations in the P cell was as low as in untreated pairs. Pairing aldehyde-fixed Retzius cells with untreated P cells also resulted in the loss of gCations. The Retzius cell had both types of receptors, but synapse formation did not affect gCations in the presynaptic neuron. The results demonstrate that the loss of the nonsynaptic response of the P cell to transmitter is due to contact between the neurons rather than to the release of transmitter or trophic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Effects of kainic acid lesions on calcium uptake and dopamine release in nerve endings isolated from rat striata.

Rats were injected intrastriatally with kainic acid and the viability of dopaminergic terminals two days following the injection was determined by comparing voltage dependent calcium uptake and dopamine release in isolated nerve endings. Evoked dopamine release remained normal following the lesion, but the initial rate of potassium stimulated calcium entry decreased by approximately 1/3. These results suggest that the excitotoxic lesion caused by acute intrastriatal injection of kainic acid results in retention of functional dopaminergic terminals.

Animals↗
PubMed1988

Long-term storage of functional, isolated nerve endings by slow freezing and rapid thawing.

Nerve endings (synaptosomes) were isolated from homogenized rat brain corpora striata following centrifugation on discontinuous sucrose gradients. The synaptosomes (in 0.8 M sucrose) were (i) slowly frozen by placing the tube containing the suspension in a freezer at -10 degrees C for 1 h followed by (ii) swirling in a mixture of acetone and dry ice for 15 min and (iii) were stored in liquid nitrogen for up to 6 weeks. Freshly isolated synaptosomes and synaptosomes from the same preparation that were frozen for 2, 4, or 6 weeks and rapidly thawed in a water bath at 37 degrees C were re-equilibrated with a physiological salt solution and assayed for their ability to accumulate Ca and to release transmitter (dopamine) upon depolarization in high K medium. K-dependent Ca uptake gradually declined to approximately 1/3 the value observed with freshly isolated synaptosomes after 6 weeks of storage. K-stimulated dopamine release (only from intact synaptosomes) was normal over the entire period of storage. It is concluded that synaptosomes retain their physiological properties when stored frozen for a few weeks and that cold storage may be a useful technique for experiments requiring lengthy or repeated assays or accumulation of material.

Animals↗
PubMed1988

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.

Amiloride↗
PubMed1988

Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.

We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.

Animals↗
PubMed1988

Selection of postsynaptic serotonin receptors during reinnervation of an identified leech neuron in culture.

Serotoninergic Retzius neurons reform an inhibitory synapse onto pressure-sensitive mechanosensory (P) neurons when the cells are removed from the nervous system of the leech and are juxtaposed in tissue culture. The somas of P cells in situ and single (uninnervated) P cells in culture have both a depolarizing and Cl-dependent hyperpolarizing response to application of the transmitter serotonin (5-HT). Synaptic release of 5-HT by a Retzius cell in situ and in culture evokes a Cl-dependent postsynaptic response but does not appear to activate the depolarizing receptors. We have characterized the ionic currents induced by synaptically released and applied 5-HT in voltage-clamped P cells in culture in order to determine the responses to transmitter and their modifications following innervation. When 5-HT was applied to single P cells, 2 types of channels were activated by 5-HT and could be distinguished by changing the ionic composition of the superfusion solution. In an impermeant cation (TrisCl) solution, a 5-HT-dependent Cl current was activated. When single P cells were superfused with a Cl-free solution (Cl replaced by impermeant SO4), 5-HT activated a monovalent cation current. Following innervation of a P cell by a cocultured Retzius cell, the reversal potential of the peak postsynaptic current depended on the Cl gradient and the synaptic response was blocked by the Cl channel blocker 9-anthracenecarboxylic acid. Thus, synaptic release of 5-HT activated solely the Cl channels and not the cationic channels. Pipette application of 5-HT onto innervated P cells activated a Cl conductance comparable in magnitude to the synaptic response. In contrast, the cationic conductance was reduced roughly 5-fold. It is concluded that innervation of a P cell by a Retzius cell resulted in clustering of the synaptic 5-HT receptors, which activate Cl channels and reduction of the nonsynaptic, cationic response. The result is a selection of receptors in the cultured P cell that mimics the pattern observed in vivo.

Animals↗
PubMed1987

Transmitter localization and vesicle turnover at a serotoninergic synapse between identified leech neurons in culture.

An electron microscopic study has been made of chemical synapses that develop between identified nerve cells isolated from the CNS of the leech and maintained in culture. Structures resembling synapses were observed in pairs of Retzius cells and P sensory cells at which chemical transmission had been demonstrated by recording with microelectrodes. Vesicle recycling was shown by following the uptake of extracellular markers after stimulation. The membrane separation between the presynaptic Retzius cell (which is known to liberate serotonin) and the postsynaptic P cell was wider in synaptic than in extrasynaptic regions. The Retzius cell contained clusters of clear vesicles apposed to thickenings of the presynaptic membrane. These clear vesicle clusters were capped by a layer of dense core vesicles that did not contact the presynaptic membrane thickenings. Subsynaptic cisternae were found in the postsynaptic cell opposite the presynaptic membrane thickenings. Occasional slight postsynaptic membrane thickenings were seen. Extracellular material was observed within the synaptic cleft. Similar synaptic structures developed between pairs of Retzius cells in culture; even a single Retzius cell was able to form autapses upon itself. Structures resembling transmitter release sites were found in Retzius cells at a distance from any postsynaptic membranes. These are presumed to be locations for the diffuse release of transmitter. Presynaptic structures resembling release sites were never observed in P cells apposed to Retzius cells. Antibody to serotonin (5-HT) labelled with colloidal gold showed serotonin to be localized in the dense core vesicles in Retzius cells. Stimulation of pairs of Retzius and P cells by raised concentrations of K+ resulted in uptake of extracellular markers. Only Retzius cells became labelled. Ferritin was found in cisternae, in dense core vesicles, and in clear vesicles. HRP was found in cisternae and in clear vesicles. Colloidal gold was taken up by coated vesicles and was occasionally found in both clear and dense core vesicles. The uptake of extracellular markers following stimulation was blocked by high Mg++. These results show that structures develop between pairs of cells at which chemical transmission develops and that transmitter release leads to turnover of dense core and clear vesicles.

Animals↗
PubMed1986

Voltage dependence of 5-hydroxytryptamine release at a synapse between identified leech neurones in culture.

The release of 5-hydroxytryptamine (5-HT) from presynaptic terminals has been studied by the voltage-clamp technique at synapses made by isolated Retzius and pressure (P) sensory neurones dissected from the leech C.N.S. and maintained in tissue culture. At these synapses facilitation, depression and modulation of release occur with action potentials and with voltage-clamp pulses. Depolarization of Retzius cells from a constant holding potential by steps of varying amplitude (5 ms in duration) caused graded release of 5-HT. The steep transfer function for release using these short test pulses resembled that seen at the giant synapse of the squid: synaptic potentials increased markedly with presynaptic depolarizations beyond -25 mV and decreased with large depolarizing pulses beyond +40 mV. When the steady holding potential of voltage-clamped Retzius cells was suddenly displaced to a new value within the range of -40 mV to -85 mV, there followed a slow but smaller change of the post-synaptic P-cell membrane potential in the same direction. After an initial delay of about 40 ms, the post-synaptic potential reached its new level with an exponential time course and a time constant of 0.7 s. Since Retzius and P cells are not electrically coupled, these effects can be accounted for by alterations in tonic release of transmitter. Changes of presynaptic holding potential to a more depolarized level resulted in an increase in voltage noise recorded in the P cell. Conversely, hyperpolarization from a depolarized level reduced noise. Noise analysis showed that these changes could be accounted for by quantal events with a mean amplitude of about 0.15 mV. This value is similar to that for spontaneous miniature potentials and quantal fluctuations observed at synapses between Retzius and P cells. Changes in steady holding potential also had marked effects upon the transfer function observed with brief depolarizing pulses of the Retzius cell. The post-synaptic responses evoked by depolarizations to 0 mV with pulses of 5 ms duration were reduced in amplitude as the holding potential of the Retzius cell was increased from the resting value of -45 to -75 mV. For example, depolarization to 0 mV starting from -45 mV evoked synaptic potentials as much as ten times larger than those evoked by depolarizations to 0 mV starting from -75 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1984

Manganese fluxes and manganese-dependent neurotransmitter release in presynaptic nerve endings isolated from rat brain.

The uptake and efflux of 54Mn and 45Ca, and the release of dopamine (DA) were measured in pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain. The uptake of Mn and Ca was increased when forebrain or striatal synaptosomes were incubated in a depolarizing, K-rich solution. The time courses of K-stimulated Mn and Ca entry were similar: there was initially a high rate of ion accumulation, lasting 1-3 s, that gradually levelled off. The initial uptake of Mn, like that of Ca, was greatly diminished by a 10 s pre-incubation in K-rich solution prior to the addition of radiotracer. Several Ca channel blockers, including Ni (0.03 mM), Sr (2.0 mM), Co (0.04 mM), Ba (1.5 mM) and La (0.2 mM), suppressed the K-stimulated uptake of Mn and of Ca to a similar extent. The K-stimulated uptake of Mn increased as a function of the external Mn concentration, and saturated at high external concentrations of Mn. These high concentrations of Mn also blocked the K-stimulated uptake of Ca. There was a decreased efflux of Ca, but not of Mn, from the synaptosomes when the external Na concentration was reduced. The Na-dependent efflux of Ca was diminished by external Mn, but was unaffected when the synaptosomes were loaded with Mn. The rate of [3H]DA release from striatal synaptosomes was less than 0.001 s-1 in non-depolarizing, low-K solutions, in the absence or presence of Mn and Ca (1 mM). The rate of release was also unchanged in depolarizing, K-rich solutions in the absence of these divalent cations. The addition of 1 mM-Mn to a K-rich solution increased the rate of DA release by about 40%, and the time course of release was linear for at least 30 s. The addition of 1 mM-Ca increased the rate of release nearly 100-fold during the first second, and thereafter the rate of release rapidly declined. Ni (1 mM) and, to a lesser extent, Mg (10 mM) reduced the rate of K-stimulated DA release that is dependent on either Mn or Ca. The pattern of inhibition of DA release resembled the pattern of inhibition of K-stimulated uptake of Mn and Ca. The addition of Mn to K-rich solutions stimulated the release of the neurotransmitters 5-hydroxytryptamine and gamma-aminobutyric acid, but not acetylcholine, from striatal synaptosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Changes in Na+-ATPase and Na,K-pump during maturation of sheep reticulocytes.

Changes in the sodium pump and sodium-activated ATP hydrolysis (Na+-ATPase) were followed during maturation of low potassium (LK) and high potassium (HK) sheep reticulocytes released into circulation following massive bleeding. In vitro maturation of LK cell reticulocytes resulted in a progressive decrease in cellular K+, ouabain-sensitive 86Rb+ uptake, and Na+-ATPase activity. Although in vitro maturation (up to 10 days) did not result in changes in kinetic behaviour from that typical of reticulocytes to that typical of mature cells, kinetic changes were clearly evident after longer term maturation in vivo. This was shown for HK cells by comparing the characteristics of Na+-ATPase (response to K+) of the lightest (youngest) and heaviest (oldest) cell fractions of blood obtained at various periods during recovery following massive bleeding. It is concluded that the kinetic difference between immature and mature cells is due to cell maturation and reflects either a selective change and (or) loss of certain type(s) of pump sites.

Anemia↗
PubMed1982

A buffering model for calcium-dependent neurotransmitter release.

A simple model is proposed, whereby a single buffering system for intracellular calcium accounts for the steep external Ca dependence of neurotransmitter release during depolarization of the presynaptic nerve terminal. Ca entry and buffering in the nerve terminal are assumed to be saturable; release is assumed to be proportional to intracellular Ca. The novel feature of this model is that it explains the apparent cooperative relationship between transmitter release and extracellular calcium, without invoking cooperative Ca binding.

Animals↗
PubMed1981

Enzymic diversification of the sodium pump in sheep red cells.

It is well-established that sheep are genetically dimorphic with respect to the K+ content of their mature red cells and that high-K+ (HK) and low-K+ (LK) red cells differ with respect to the activity and kinetic properties of the sodium pump and Na+-ATPase. In contrast, reticulocytes of both types have a high K+ content and are similar with respect to their pump and Na+-ATPase kinetic characteristics. Reticulocytes differ, however, from mature cells of either HK or LK sheep. The first part of this paper describes a comparative study of the side-specificity of Na+ and K+ interactions with Na--ATPase of inside-out membrane vesicles prepared from mature HK and LK red cells. The results indicate that the genetic difference between the two is expressed as differences in apparent affinities for both Na+ and K+ and that these differences are evident at the cytoplasmic as well as at the extracellular membrane surface. The second part of this paper describes a study of the nature of the maturation-associated changes in the sodium pump system of sheep reticulocytes. Maturation was followed both in vitro (long-term incubation at 37 degrees of reticulocytes) and in vivo (changes in density gradient fractionated HK cells followed at intervals during recovery from massive bleeding. The results support the conclusion that the dimorphism in the sodium pump of sheep red cells is a result of specific changes in Na+-ATPase and that these changes proceed differently during maturation of the two types of sheep red cells.

Anemia↗
PubMed1980

Interactions of K+ with (Na,K)-ATPase orientation of K+-phosphatase sites studied with inside-out red cell membrane vesicles.

Inside-out membrane vesicles from human red cells were used to investigate the side specificity of K+ interactions with the K+-activated phosphatase, a partial reaction of the (Na, K)-ATPase. In the absence of Na+ and ATP, K+ at moderate affinity sites at the extravesicular surface (cytoplasmic K+) stimulates activity, whereas intravesicular K+ (K+ normally at the extracellular surface) is without effect. In contrast, under conditions of phosphorylation of (Na, K)-ATPase (Na+ and ATP present), K+ ions acting at high affinity sites at both surfaces are required. It is concluded that an enzyme x K complex is involved in K+-activated phosphatase activity and that it is formed either by interaction of cytoplasmic K+ with the dephosphoenzyme, or as a consequence of extracellular K+ binding and dephosphorylation of the phosphoenzyme formed in the presence of Na+ plus ATP.

4-Nitrophenylphosphatase↗
PubMed1980

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells.

Na+-ATPase of high-K+ and low-K" sheep red cells was examined with respect to the sidedness of Na+ and K+ effects, using inside-out membrane vesicles and very low ATP concentrations (less than or equal to 2 muM). With varying amounts of Na+ in the medium, i.e., at the cytoplasmic surface, Na+cyt, the activation curves show that high-K+ Na+-ATPase has a higher affinity for Na+cyt compared to low-K+. The apparent affinity for Na+cyt is also increased by increasing the ATP concentrations in high-K+ but now low-K+. With Na+cyt present, Na+-ATPase is stimulated by intravesicular Na+, i.e., Na+ at the originally external surface, Na+cyt, to a greater extent in low-K+ than high-K+. Intravesicular K+ (K+ext) activates Na+-ATPase in high-K+ but not in low-K+ vesicles and extravesicular K+ (K+cyt) inhibits low-K+ but not high-K+ Na+-ATPase. Thus, the genetic difference between high-K+ and low-K+ is expressed as differences in apparent affinities for both Na+ and K+ and these differences are evident at both cytoplasmic and external membrane surfaces.

Adenosine Triphosphatases↗