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Peter K Hepler - One of the best experts on this subject based on the ideXlab platform.
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pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient effect of BAPTA type buffers and hypertonic media
1994Co-Authors: Elisabeth S Pierson, Deborah D Miller, Dale A Callaham, Alan M Shipley, Bruce A Rivers, M Cresti, Peter K HeplerAbstract:Lily pollen tubes possess a steep, tip-bcused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 pM at the apex to -0.2 FM within 20 pm from the tip, while application of the Ca2+-specific vibrating electmde indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-l. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. lnjection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-tocused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'dibromo-BAPTA (dissociation constant [Kd] is 1.5 pM) and 4,4r-difluoro-BAPTA (Kd of 1.7 pM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 pM; BAPTA, Kd of 0.21 fl; 5,5'-difluoroBAPTA, Kd of 0.25 pM) or lower affinity (gmethyl, 5'-nitro-BAPTA, Kd of 22 pM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.
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pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient effect of BAPTA type buffers and hypertonic media
1994Co-Authors: Elisabeth S Pierson, Deborah D Miller, Dale A Callaham, Alan M Shipley, Bruce A Rivers, M Cresti, Peter K HeplerAbstract:Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.
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BAPTA calcium buffers modulate cell plate formation in stamen hairs of tradescantia evidence for calcium gradients
1994Co-Authors: M Jurgens, L H Hepler, B A Rivers, Peter K HeplerAbstract:Five BAPTA buffers with differential affinities for Ca2+ have been examined for their effects on cell plate formation in stamen hair cells ofTradescantia. The five include 5,5′-dimethyl BAPTA (Kd=0.15 μM), BAPTA (Kd=0.22 μM), 5,5′-dibromo BAPTA (Kd=1.5 μM), 5-methyl,5′-nitro BAPTA (Kd=22 μM), and 5-nitro BAPTA (Kd=40 μM). At a concentration of 5 mM and 25 mM in the pipette, the buffers were iontophoretically microinjected into dividing stamen hair cells (2 nA for 1 min) prior to or at the onset of cell plate formation. At the lowest concentration (5 mM), only one buffer, 5,5′-dibromo BAPTA, inhibits cell plate formation, and is most effective if delivered at the moment of cell plate vesicle aggregation. The inhibitory effects appear as a slowing of cell plate expansion, the formation of distorted plates, or the complete dissolution of plates that might have initiated normally. When the pipette tip concentration is elevated to 25 mM, the effects of 5,5′-dibromo BAPTA become more profound. At these levels 5,5′-dimethyl BAPTA, BAPTA, and 5-nitro BAPTA also modulate cell plate formation, producing effects similar to that of 5,5′-dibromo BAPTA at the lower concentration. Independent studies using fura-2 as a fluorescent analogue of the BAPTA buffers, indicate that the apparent effective concentration for 5,5′-dibromo BAPTA is between 1.0–1.4 mM; its threshold concentration is not known but expected to be somewhat lower. For the other buffers the threshold concentration is between 1.5–2.2 mM. The concentration dependence supports the idea that the buffers facilitate diffusion of Ca2+ away from regions of elevated concentration. The results thus provide evidence that local Ca2+ gradients may be present in the vicinity of the cell plate and that they participate in the cytokinetic process.
Elisabeth S Pierson - One of the best experts on this subject based on the ideXlab platform.
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pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient effect of BAPTA type buffers and hypertonic media
1994Co-Authors: Elisabeth S Pierson, Deborah D Miller, Dale A Callaham, Alan M Shipley, Bruce A Rivers, M Cresti, Peter K HeplerAbstract:Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.
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pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient effect of BAPTA type buffers and hypertonic media
1994Co-Authors: Elisabeth S Pierson, Deborah D Miller, Dale A Callaham, Alan M Shipley, Bruce A Rivers, M Cresti, Peter K HeplerAbstract:Lily pollen tubes possess a steep, tip-bcused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 pM at the apex to -0.2 FM within 20 pm from the tip, while application of the Ca2+-specific vibrating electmde indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-l. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. lnjection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-tocused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'dibromo-BAPTA (dissociation constant [Kd] is 1.5 pM) and 4,4r-difluoro-BAPTA (Kd of 1.7 pM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 pM; BAPTA, Kd of 0.21 fl; 5,5'-difluoroBAPTA, Kd of 0.25 pM) or lower affinity (gmethyl, 5'-nitro-BAPTA, Kd of 22 pM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.
Roger C. Hardie - One of the best experts on this subject based on the ideXlab platform.
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inhibition of phospholipase c activity in drosophila photoreceptors by 1 2 bis 2 aminophenoxy ethane n n n n tetraacetic acid BAPTA and di bromo BAPTA
2005Co-Authors: Roger C. HardieAbstract:Abstract In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at “100 nM”, ∼10-fold inhibition at 100 nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 ∼ 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 ∼ 1 mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (
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Excitation of Drosophila photo-receptors by BAPTA and ionomycin: evidence for capacitative Ca2+ entry?
1996Co-Authors: Roger C. HardieAbstract:It has been suggested that excitation in Drosophila photoreceptors may be mediated by the depletion of intracellular Ca2+ stores (capacitative Ca2+ entry). To investigate this hypothesis, simultaneous whole-cell recordings and Indo-1 Ca2+ measurements were made from dissociated Drosophila photoreceptors, whilst testing the effects of Ca2+ releasing agents. In Ca2+ free Ringer's solution, thapsigargin raised cytosolic Ca2+ by approximately 80 nM; subsequent application of ionomycin released further Ca2+ (approximately 100 nM). A possible third compartment was indicated by the ability of monensin to mobilize further Ca2+ after saturating doses of ionomycin. Under most conditions, none of these agents activated an inward conductance, and their effects on the light response were consistent with their effects on cytosolic Ca2+. However, in the absence of both external Ca2+ and Mg2+ (to relieve a Mg2+ block of the light-sensitive channels), and after loading cells with BAPTA buffering cytosolic free Ca2+ at approximately 10 nM, ionomycin (but not thapsigargin) activated inward currents of approximately 800 pA. The response to ionomycin was enhanced (10 nA) by buffering cytosolic Ca2+ at 250 nM. A similar current also developed after approximately 3 min in cells loaded with Ca-BAPTA without any ionomycin application. The current-voltage relationships of currents activated by Ca-BAPTA or ionomycin were indistinguishable from that of the light-activated conductance and were similarly affected by a null mutation of the transient receptor potential (trp) gene which is believed to encode a subunit of the light-sensitive channels. These experiments provide some evidence for the suggestion that the light-activated and trp-dependent conductance in Drosophila photoreceptors can be activated by depletion of internal stores. However, activation by Ca-BAPTA and ionomycin had an absolute requirement for cytosolic Ca2+ as no currents could be activated by ionomycin in cells loaded with BAPTA and no Ca2+.
Bradley E Britigan - One of the best experts on this subject based on the ideXlab platform.
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binding of iron and inhibition of iron dependent oxidative cell injury by the calcium chelator 1 2 bis 2 aminophenoxy ethane n n n n tetraacetic acid BAPTA
1998Co-Authors: Bradley E Britigan, George T Rasmussen, Charles D CoxAbstract:Abstract Abstract. A role for increases in intracellular calcium (Ca 2+ ) has been suggested in the pathophysiology of various forms of oxidant-mediated cell injury. In recent studies, we found that iron bound to the Pseudomonas aeruginosa siderophore, pyochelin, augments oxidant-mediated endothelial cell injury by catalyzing the formation of hydroxyl radical (HO • ). To investigate the role of Ca 2+ in this process, the effects of two Ca 2+ chelating agents, Fura-2 and 1,2-bis(2-aminophenoxy)ethane N , N , N ′, N ′-tetraacetic acid (BAPTA), were assessed. BAPTA, but not Fura-2, was protective against H 2 O 2 /ferripyochelin-mediated injury. Subsequent data suggested that chelation of iron rather than Ca 2+ by BAPTA was most likely responsible. Spectrophotometry demonstrated that both ferrous (Fe 2+ ) and ferric (Fe 3+ ) iron formed a complex with BAPTA. The affinity of BAPTA for the metals was Fe 3+ > Ca 2+ > Fe 2+ . BAPTA was found to decrease markedly iron-catalyzed production of HO • and/or ferryl species when analyzed by spin trapping. Although our results do not definitively prove that BAPTA protects endothelial cells from ferripyochelin-associated damage by chelating iron, these data indicate that caution must be exercised in utilizing protective effects of intracellular “Ca 2+ chelating agents” as evidence for a role of alterations in cellular Ca 2+ levels in experimental conditions in which iron-mediated oxidant production is also occurring.
Bert Sakmann - One of the best experts on this subject based on the ideXlab platform.
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transmitter release modulation by intracellular ca2 buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2 3 of the rat neocortex indicates a target cell specific difference in presynaptic calcium dynamics
2001Co-Authors: Andrej Rozov, Bert Sakmann, Nail Burnashev, Erwin NeherAbstract:1. In connections formed by nerve terminals of layer 2/3 pyramidal cells onto bitufted interneurones in young (postnatal day (P)14-15) rat somatosensory cortex, the efficacy and reliability of synaptic transmission were low. At these connections release was facilitated by paired-pulse stimulation (at 10 Hz). In connections formed by terminals of layer 2/3 pyramids with multipolar interneurones efficacy and reliability were high and release was depressed by paired-pulse stimulation. In both types of terminal, however, the voltage-dependent Ca2+ channels that controlled transmitter release were predominantly of the P/Q- and N-subtypes. 2. The relationship between unitary EPSP amplitude and extracellular calcium concentration ([Ca2+]o) was steeper for facilitating than for depressing terminals. Fits to a Hill equation with nH = 4 indicated that the apparent KD of the Ca2+ sensor for vesicle release was two- to threefold lower in depressing terminals than in facilitating ones. 3. Intracellular loading of pyramidal neurones with the fast and slowly acting Ca2+ buffers BAPTA and EGTA differentially reduced transmitter release in these two types of terminal. Unitary EPSPs evoked by pyramidal cell stimulation in bitufted cells were reduced by presynaptic BAPTA and EGTA with half-effective concentrations of approximately 0.1 and approximately 1 mM, respectively. Unitary EPSPs evoked in multipolar cells were reduced to one-half of control at higher concentrations of presynaptic BAPTA and EGTA (approximately 0.5 and approximately 7 mM, respectively). 4. Frequency-dependent facilitation of EPSPs in bitufted cells was abolished by EGTA at concentrations of > or = 0.2 mM, suggesting that accumulation of free Ca2+ is essential for facilitation in the terminals contacting bitufted cells. In contrast, facilitation was unaffected or even slightly increased in the terminals loaded with BAPTA in the concentration range 0.02-0.5 mM. This is attributed to partial saturation of exogenously added BAPTA. However, BAPTA at concentrations > or = 1 mM also abolished facilitation. 5. Frequency-dependent depression of EPSPs in multipolar cells was not significantly reduced by EGTA. With BAPTA, the depression decreased at concentrations > 0.5 mM, concomitant with a reduction in amplitude of the first EPSP in a train. 6. An analysis is presented that interprets the effects of EGTA and BAPTA on synaptic efficacy and its short-term modification during paired-pulse stimulation in terms of changes in [Ca2+] at the release site ([Ca2+]RS) and that infers the affinity of the Ca2+ sensor from the dependence of unitary EPSPs on [Ca2+]o. 7. The results suggest that the target cell-specific difference in release from the terminals on bitufted or multipolar cells can be explained by a longer diffusional distance between Ca2+ channels and release sites and/or lower Ca2+ channels density in the terminals that contact bitufted cells. This would lead to a lower [Ca2+] at release sites and would also explain the higher apparent K(D) of the Ca2+ sensor in facilitating terminals.
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transmitter release modulation in nerve terminals of rat neocortical pyramidal cells by intracellular calcium buffers
1998Co-Authors: Ora Ohana, Bert SakmannAbstract:Dual whole-cell voltage recordings were made from synaptically connected layer 5 (L5) pyramidal neurones in slices of the young (P14-P16) rat neocortex. The Ca2+ buffers BAPTA or EGTA were loaded into the presynaptic neurone via the pipette recording from the presynaptic neurone to examine their effect on the mean and the coefficient of variation (c.v.) of single fibre EPSP amplitudes, referred to as unitary EPSPs. The fast Ca2+ buffer BAPTA reduced unitary EPSP amplitudes in a concentration dependent way. With 0.1 mm BAPTA in the pipette, the mean EPSP amplitude was reduced by 14 ± 2.8% (mean ±s.e.m., n = 7) compared with control pipette solution, whereas with 1.5 mm BAPTA, the mean EPSP amplitude was reduced by 72 ± 1.5% (n = 5). The concentration of BAPTA that reduced mean EPSP amplitudes to one-half of control was close to 0.7 mm. Saturation of BAPTA during evoked release was tested by comparing the effect of loading the presynaptic neurone with 0.1 mm BAPTA at 2 and 1 mm[Ca2+]o. Reducing [Ca2+]o from 2 to 1 mm, thereby reducing Ca2+ influx into the terminals, decreased the mean EPSP amplitude by 60 ± 2.2% with control pipette solution and by 62 ± 1.9% after loading with 0.1 mm BAPTA (n = 7). The slow Ca2+ buffer EGTA at 1 mm reduced mean EPSP amplitudes by 15 ± 2.5% (n = 5). With 10 mm EGTA mean EPSP amplitudes were reduced by 56 ± 2.3% (n = 4). With both Ca2+ buffers, the reduction in mean EPSP amplitudes was associated with an increase in the c.v. of peak EPSP amplitudes, consistent with a reduction of the transmitter release probability as the major mechanism underlying the reduction of the EPSP amplitude. The results suggest that in nerve terminals of thick tufted L5 pyramidal cells the endogenous mobile Ca2+ buffer is equivalent to less than 0.1 mm BAPTA and that at many release sites of pyramidal cell terminals the Ca2+ channel domains overlap, a situation comparable with that at large calyx-type terminals in the brainstem. Release of transmitter in synapses is dependent on Ca2+ influx into the nerve terminals (Katz, 1969) and the binding of Ca2+ to a putative Ca2+ sensor on synaptic vesicles which in turn controls the exocytosis of a vesicle. The free cytoplasmic Ca2+ concentration ([Ca2+]i) in the vicinity of the Ca2+ sensor depends on the magnitude and time course of the Ca2+ influx into the terminal during an action potential and on the distance between the Ca2+ sensor and the site of Ca2+ entry. Fixed and mobile Ca2+ buffers affect the size and time course of the [Ca2+]i transient, depending on their concentration, association and dissociation kinetics and their mobility (Sala & Hernandez-Cruz 1990; Nowycky & Pinter, 1993; Roberts, 1994; Neher, 1995). Exogenous Ca2+ buffers, which compete for free Ca2+ with the endogenous buffers and the vesicular Ca2+ sensor, were used previously to examine the coupling between presynaptic [Ca2+]i transients and transmitter release. The lack of effect of presynaptic EGTA (80 mm), a slowly binding Ca2+ buffer, on phasic release in the squid giant synapse indicated a very fast rise of [Ca2+]i near the sensor and suggested that the distance between Ca2+ channels and the Ca2+ sensor was short (Adler et al. 1991). In contrast, at the calyx-type giant synapse of the rat medial nucleus of the trapezoid body (MNTB) relatively low concentrations of EGTA (1 mm) were sufficient to reduce phasic transmitter release (Borst & Sakmann, 1996) indicating a longer diffusional distance of Ca2+ between the Ca2+ channel and the Ca2+ sensor. In terminals of retinal bipolar neurones, EGTA and BAPTA differentially affected two phases of transmitter release which presumably represent two pools of vesicles (Mennerick & Matthews, 1996). Adding exogenous Ca2+ buffers to terminals was also used to estimate the concentration of endogenous mobile Ca2+ buffers. This yielded estimates of endogenous buffers being equivalent to 1.6 mm and 50 μm in inner ear cells and the calyx-type synapse, respectively (Roberts, 1993; Borst et al. 1995). In addition, exogenous Ca2+ buffers were used for elucidating the function of [Ca2+]i transients in short- and long-term changes in synaptic efficacy (Delaney et al. 1991; Bain & Quastel, 1992; Van der Kloot & Molgo 1993; Winslow et al. 1994; Kobayashi et al. 1995; Tank et al. 1995; Bao et al. 1997). We examined the effect of exogenous fast and slow Ca2+ buffers on evoked transmitter release in terminals of the axodendritic synaptic contacts between neighbouring pyramidal neurones in layer 5 (L5) of rat neocortex (Markram et al. 1997). Simultaneous pre- and postsynaptic whole-cell voltage recordings (WCR) from L5 pyramidal neurones were made and multiple, sequential WCRs from the same presynaptic neurone with different pipette solutions containing either the fast binding Ca2+ buffer BAPTA or the slow binding buffer EGTA or with control solution. The mean EPSP amplitudes were measured before, during and after buffer loading of the presynaptic neurone. The results show that both the slow and the fast binding buffers, at relatively low concentrations, reversibly reduced the evoked transmitter release comparable with the results obtained at the axosomatic synapse of the MNTB.
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pre and postsynaptic whole cell recordings in the medial nucleus of the trapezoid body of the rat
1995Co-Authors: J G G Borst, Fritjof Helmchen, Bert SakmannAbstract:1. Simultaneous whole-cell recordings in a rat brain slice preparation are described from presynaptic terminals (calyces of Held) and postsynaptic somata which form an axosomatic synapse in the medial nucleus of the trapezoid body (MNTB). 2. Presynaptic action potentials evoked suprathreshold excitatory postsynaptic potentials (EPSPs). The minimum synaptic delay was around 0.4 ms at 36 degrees C and 0.9 ms at 23-24 degrees C. The amplitude of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated component of the excitatory postsynaptic currents (EPSCs) was 2-13 nA (at -80 mV). 3. Current-voltage relations showed that presynaptic Ca2+ channels were of the high voltage-activated type. 4. A single action potential evoked a presynaptic fluorescence transient that decayed with a time constant of 0.3-0.7 s, depending on the concentration (60-200 microM) of the Ca2+ indicator Calcium Green-5N (CG-5N). The peak amplitude of the [Ca2+]i transient was severalfold larger in the terminal than in the preterminal axon. 5. EPSC peak amplitudes were stable for more than 30 min after establishing the whole-cell configuration in the presynaptic terminal when the pipette contained 50 microM BAPTA. In contrast, with 1 mM BAPTA, peak amplitudes of EPSCs were reduced to one-third. 6. Trains of presynaptic action potentials evoked EPSCs with progressively smaller amplitudes. Little change was observed in the depression when the terminals were dialysed with 50 microM BAPTA, whereas depression was reduced with 1 mM BAPTA. 7. In low (1 mM) [Ca2+]o, facilitation instead of depression of EPSCs was observed. 8. The effects of presynaptic BAPTA suggest that the endogenous mobile Ca2+ buffer capacity of giant presynaptic terminals in the MNTB is lower than in other terminals of fast transmitting synapses.
