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Recombinant small GTPases were produced in E. coli, purified with glutathione-Sepharose 4B (Amersham Biosciences), and prepared by thrombin digestion according to the recommendations of the manufacturer. Rho, Rac1, and Cdc42 clones were a generous gift from Dr. Alan Hall (London, UK). H-Ras, Rap1a, and RalC clones were kindly donated by Dr. Robert Cool (Max Plank Institute, Dortmund, Germany). In vitro glucosylation of small GTPases was performed in 50 mM triethanolamine, pH 7.5, containing 2 µl of UDP-14C-glucose (DuPont NEN; 286.2 mCi/mmol; final concentration 7 µM), 2 mM MgCl2, 1 mM DTT, 0.3 mM GDP, 1 µg of recombinant GTPase, and 5 µg/ml LT82, LT82 recombinant fragment, or LT9048. The reaction was performed for 1 hr at 37°C and stopped by adding 10 µl of sample buffer (three times) followed by boiling for 3 min. Then samples were electrophoresed on a 15% SDS-PAGE and autoradiographed. When necessary, UDP-mannose, ADP-glucose, and TDP-glucose (Sigma) were used to prevent glucosylation.
The Journal of Neuroscience, September 15, 2002, 22(18):7968-7981
Rac GTPase Plays an Essential Role in Exocytosis by Controlling the Fusion Competence of Release Sites
Yann Humeau1, Michel R. Popoff2, Hiroshi Kojima1, Frédéric Doussau1, and Bernard Poulain1 1 Neurotransmission et Sécrétion Neuroendocrine, UPR2356 du Centre National de la Recherche Scientifique, IFR-37 des Neurosciences, F-67084 Strasbourg Cedex, France, and 2 Toxines Microbiennes, Institut Pasteur, F-75724 Paris Cedex 15, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The role of small GTPases of the Rho family in synaptic functions has been addressed by analyzing the effects of lethal toxin (LT) from Clostridium sordellii strain IP82 (LT82) on neurotransmitter release at evoked identified synapses in the buccal ganglion of Aplysia. LT82 is a large monoglucosyltranferase that uses UDP-glucose as cofactor and glucosylates Rac (a small GTPase related to Rho), and Ras, Ral, and Rap (three GTPases of the Ras family). Intraneuronal application of LT (50 nM) rapidly inhibits evoked acetylcholine (ACh) release as monitored electrophysiologically. Injection of the catalytic domain of the toxin similarly blocked ACh release, but not when key amino acids needed for glucosylation were mutated. Intraneuronal application of competitive nucleotide sugars that differentially prevent glucosylation of Rac- and Ras-related GTPases, and the use of a toxin variant that affects a different spectrum of small GTPases, established that glucosylation of Rac is responsible for the reduction in ACh release. To determine the quantal release parameters affected by Rac glucosylation, we developed a nonstationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action. The results indicate that neither the quantal size nor the average probability for release were affected by lethal toxin action. ACh release blockage by LT82 was only caused by a reduction in the number of functional release sites. This reveals that after docking of synaptic vesicles, vesicular Rac stimulates a membrane effector (or effectors) essential for the fusion competence of the exocytotic sites.
Key words: Aplysia; synapse; exocytosis; quantal parameters; synaptic vesicle fusion; Rho-GTPases; clostridial toxins; fluctuation analysis
INTRODUCTION
Rho proteins (Rho, Rac, Cdc42) are widely expressed monomeric GTPases. They cycle between a soluble, GDP-bound inactive state and a membrane-associated GTP-bound state that stimulates downstream effectors. The effectors include protein kinase N, Rho-kinase, the myosin-binding subunit of myosin phosphatase, several phosphatidylinositol protein kinases (PI3-kinase, PI4-kinase, PI5-kinase), and phospholipase C and phospholipase D (PLD). Despite the ubiquitous distribution of cytosolic Rho-GTPases, their translocation to specific membrane domains allows them to intervene in distinct biological functions such as regulation of actin cytoskeletal dynamics, cell cycle progression, gene transcription, and membrane transport (Hall, 1998
; Bishop and Hall, 2000
The cellular function of endogenous Rho and Ras proteins can be acutely manipulated by using bacterial protein toxins that modify and inactivate them in a highly specific manner (Busch and Aktories, 2000
We have found previously that intraneuronal injection of LT or toxins that glycosylate or ADP-ribosylate Rho-related GTPases blocks acetylcholine (ACh) release at Aplysia synapses (Doussau et al., 2000
To identify the key GTPase(s) whose inactivation is responsible for the LT-blocking action on ACh release, we have investigated the causal relationship between the glycosylating activity of two LT variants that each affect a distinct set of GTPases and inhibition of neurotransmitter release at identified Aplysia synapses. We found that ACh release blockage is caused by glucosylation of Rac GTPase. To determine the release step(s) altered after LT application, we analyzed the fluctuations in postsynaptic response amplitudes before and during LT-induced blocking action. Our results show a decrease in the number of active release sites, but quantal size and release probability are unaltered. This reveals that the Rac-mediated pathway is implicated in the activation of release sites.
MATERIALS AND METHODS
Preparation of toxins and determination of their glucosylating activity
The toxins were purified from C. sordellii IP82 (LT82) and VPI9048 (LT9048) strains as described previously (Popoff, 1987-D-galactopyranoside. Purification was performed with a cobalt column (Novagen) according to the recommendations of the manufacturer. An inactive mutant of the LT82 N-terminal part (Ala-286-Asp, Ala-288-Asp) was prepared as described previously (Busch et al., 1998
Detection and glucosylation of small GTPases in Aplysia
Aplysia californica (70-120 gm body weight; Marinus Inc., Long Beach, CA) were anesthetized by injection of 50-75 ml of solution containing 400 mM MgCl2, and the nervous system was removed. Freshly dissected nerve ganglia (buccal, cerebral, pleural, pedal, and abominal) were homogenized in Tris buffer containing 10 mM Tris, pH 7.5, 2 mM MgCl2, 1 mM DTT, 10 µg/ml leupeptin, 1 µM pepstatin, and 0.1 mM PMSF. Complete lysis was achieved by three cycles of freeze thawing.
Detection of Aplysia GTPases. Total proteins were extracted in Tris buffer containing 1% Triton X-100 for 20 min at room temperature. For Western blotting, samples containing 20 µg Aplysia proteins were subjected to SDS-PAGE on 15% gels, transferred to nitrocellulose, and incubated with a 1:50 dilution of antibodies against small GTPases in PBS-milk at room temperature for 16 hr. Polyclonal rabbit antibodies against Rho, Ras, Rap, and Ral were from Santa Cruz Biotechnology; anti-Rac1 was from Upstate Biotechnology. Polyclonal rabbit antibodies directed against Cdc42 were kindly provided by Dr. Philippe Chavrier (Institut Curie, Paris, France). Immunoblots were processed with peroxidase conjugate and chemiluminescence kit (ECL, Amersham) and analyzed by autoradiography.
Glucosylation of Aplysia GTPases. Lysates were centrifuged (15,000 rpm for 15 min) to separate a pellet containing membrane fractions [denoted as insoluble (I) fraction] and a fraction containing cytosolic and crude vesicle fractions [denoted as soluble (S) fraction]. The pellet was washed four times with distilled water and extracted with the buffer described above, but containing 1% Triton X-100, for 20 min at room temperature. Total proteins were assayed using a manufactured protein assay (Bio-Rad Life Science, Marne la Coquette, France). Glucosylation was performed on samples (100 µg protein) of the soluble and insoluble fractions as described above.Acetylcholine release and electrical recordings at Aplysia synapses
Electrophysiological experiments were performed at identified, chloride-dependent, inhibitory cholinergic synapses in dissected buccal ganglia of Aplysia as described previously (Poulain et al., 1986). To initiate action potentials and evoke ACh release, the presynaptic neurons were depolarized by a square pulse of 50 msec duration and appropriate intensity. To avoid overlap of the postsynaptic responses originating from the two B4 and B5 presynaptic neurons, the stimulus protocols were alternated, and each presynaptic neuron was stimulated every 40 sec (0.025 Hz). Evoked ACh release was monitored by measuring the amplitude of the evoked IPSCs using the conventional two-electrodes voltage-clamp technique (AxoClamp2B, Axon Instruments). The postsynaptic currents are purely chloride dependent (Gardner and Stevens, 1980
, the IPSC amplitude was converted as an apparent membrane conductance (G) by taking into account the reversal potential Vrev (at these synapses, Vrev = ECl
Vrev). However, although the postsynaptic response amplitude has the dimension of a membrane conductance, we refer to it in this paper as the IPSC amplitude, I (nanosiemens).
Extracellular media
Dissected buccal ganglia were maintained at 22°C using a Peltier-plate system and superfused continuously (10 ml/hr) with a physiological medium containing (in mM): NaCl 460, KCl 10, CaCl2 43.8, MgCl2 76.2, MgSO4 28, Tris Buffer 10, pH 7.5. This high concentration in divalent cations was used to minimize spontaneous neuron firing activity as described (Humeau et al., 2001aIntraneuronal injection procedure
Injection electrodes were pulled from glass tubing without a capillary and contained a silver wire to allow the electrophysiological monitoring of the impalement. Samples to be injected were mixed with a vital dye (fast green FCF, 10% v/v; Sigma) as detailed elsewhere (Poulain et al., 1986Determination of IPSC amplitude and rise and decay times
IPSC amplitude (I) was determined as the peak current of recorded IPSC. The time to rise from 20 to 80% of the maximal IPSC amplitude was determined and denoted as "IPSC rise time." IPSC rise times were determined from at least 10 IPSCs recorded under the same experimental condition and averaged. The IPSC decay time constant was determined by fitting the IPSC with multi-exponential regression I(t) = Ipeak*[w1*exp(1) + w2*exp(
1 and
1 and w2
Estimation of the quantal parameters: theoretical aspects
In brief, consider the synaptic inputs attributable to the multiple contacts between a presynaptic neuron and its postsynaptic target and consider the three parameters: q, the amplitude of elementary postsynaptic response, n, the mean number of independent release sites, and p, the mean release probability at these sites. First, we assume that at each release site p is the product of the output probability po (i.e., the probability for a release-ready SV to fuse in response to presynaptic stimulus) and the probability pA that the site is eligible for release (i.e., that a primed SV is available for release) (Brown et al., 1976
and the fluctuations of the responses around the mean have a variance:
(1)
Similar to the ion channel analysis developed by Sigworth (1980)
![<UP>Var</UP>=n*(p<SUB>o</SUB>*p<SUB>A</SUB>)*[1−(p<SUB>o</SUB>*p<SUB>A</SUB>)]*q<SUP>2</SUP>.](/content/vol22/issue18/fulltext/7968/img002.gif)
(2)
which can be fitted by a simple parabola of equation:
(3)
This allows determination of two parameters: A, the initial slope of the parabola, and B, the extent factor of the parabola. Parameter A refers to q. However quantal variability (denoted as CVq, CV being the variation coefficient) also contributes to Var. Therefore, A provides an overestimate of q (Silver et al., 1998
(4)
Parameter B refers to 1/n. However, probability parameters po and pA are heterogeneous between the release sites at vertebrate synapses (Rosenmund et al., 1993
(5)
in which, CVp is the average variation of po*pA and
![1/B=n/[(1+CV<SUB>p</SUB><SUP>2</SUP>)*(1+&bgr;CV<SUB>q inter</SUB><SUP>2</SUP>)],](/content/vol22/issue18/fulltext/7968/img006.gif)
(6) Determination of Imean and Var: practical aspects
Stationary conditions. When under a given experimental condition the amplitude of the IPSCs was stable with time (i.e., during control recordings or when ACh release was stable after a change in extracellular [Ca2+/Mg2+] or stimulation frequency), Imean and Var were calculated directly from the IPSC amplitude values determined in recording epochs of at least 25 IPSCs. Because background noise can contribute to Var, its variance, Varnoise, was calculated from the baseline fluctuations in IPSC recordings, and subtracted from Var. Then the Var = f(Imean) plots were constructed. Nonstationary conditions. When IPSC amplitude does not stabilize, determination of Imean and Var can be performed by nonstationary analysis, as reported previously for analysis of channel fluctuations during the course of evoked postsynaptic responses (Robinson et al., 1991I in Figure 1C. The variance, Var, of these fluctuations was then calculated in a window of W data that was moving, with a step of one, along the whole data range to be analyzed. Var was corrected for background noise variance (Varnoise) (see above). Then, the Var = f(Imean) plots were constructed (Fig. 1D).
For each experiment analyzed, the width of the windows used for linear local fitting (w) and the variance calculation (W) needed to be adapted to the kinetics of the IPSC amplitude changes. Indeed, except in the case of a linear change in IPSC amplitude, when the window (w) used for local linear fitting is too wide, the local fit deviates from the actual Imean, and this results in an overestimation of Var (i.e., the problem of fitting a curve with a straight line); in contrast, when w is too narrow, the local fit adapts to local fluctuations but leads to an underestimation of Var. The window width used for variance calculation, W, depended also on the kinetics of IPSC amplitude changes. Here, when the window is too wide, Var is calculated from the fluctuations in amplitude of IPSCs corresponding to very different mean amplitude, and the Var = f(Imean) plot is significantly distorted. To determine the W and w to be used, experiments in which either product of po*pA, n or q was changed were simulated. They were submitted to the nonstationary analysis described above. The most appropriate w and W were determined as those minimizing the deviation of estimated quantal parameters from the parameters used for simulation. For the faster changes in IPSC amplitude (such as those caused by a rapid fall in extracellular [Ca2+/Mg2+]), a good compromise was to use w = 9 or 11 data and W = 15-17 (Fig. 1E). For slower IPSC changes, w and W were increased. Even in these conditions, there was a remaining deviation of overall estimate of Imean (as determined by local moving linear fitting) with the true Imean (as calculated by the equation that we used to simulate IPSC amplitude change). This is illustrated by the thick dotted or hatched line in Figure 1C. A consequence of this deviation was that the Var = f(Imean) plots were fitted by parabolas (Fig. 1D, solid line) that were not exactly superimposable onto the theoretical calculated parabolas (Fig. 1D, dashed line). The uncertainty in the determination of parabola parameters A and B in the faster experiments was ~10%, whereas in slower experiments this error remained marginal.
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Figure 1. Determination of quantal parameters by nonstationary analysis as tested on a simulated experiment. A, IPSC amplitude = f(t) values ( ) were calculated as the product binomial (n, pt)*q in which n = 600 sites and q = 2 nS. A sigmoid decrease of release probability p = po*pA with time, t, was simulated using equation pt = 0.8/[1 + exp(
and
, respectively. For these two conditions, optimal w values are indicated by arrows.
Parabola adjustment and determination of quantal parameters at Aplysia synapses
Var = f(Imean) plots were fitted by Equation 4 constrained to pass the origin using a built-in procedure of SigmaPlot5 software. The coefficient of determination r2 is reported in the Figures or corresponding text to indicate how good the fit is. CVq was not determined in this study because of the difficulty in distinguishing miniature events issuing from B4 or B5 presynatic neurons from the inhibitory inputs caused by the many other presynaptic neurons, and CVp was not estimated. Therefore, to provide insights on possible changes in q, we determined the parabola fitting parameter A (see Eq. 5), and B provides an estimate of 1/n (see Eq. 6). According to Equation 1, po*pA = Imean/(n*q), and we calculated its approximation, P, according to the equation:
P differs from actual values of average release probability po*pA by the contribution of quantal variability and probability variability (Eq. 5, 6). If not stated otherwise, results are presented as means ± SEM. When appropriate, the significance was tested by the paired or unpaired Student's t test.
(7)
RESULTS
Effect of the intraneuronal injection of lethal toxin from C. sordellii on evoked ACh release
To restrict the action of the toxin to a single presynaptic element, LT82, which in vitro glucosylates recombinant Ras, Rac, Ral, and Rap but not Cdc42 and Rho, was pressure injected into either B4 or B5 cholinergic neurons to give a final intraneuronal concentration of ~50 nM. This induced a rapid decrease in IPSC amplitude, indicating inhibition of ACh release (Fig. 2A-C). No inhibition of ACh release was detected with the noninjected control neurons (Fig. 2A,C). In all experiments, the inhibitory action of LT manifested after an average delay of 15.2 ± 3.9 (SD, n = 53) min after the injection. This may represent the time needed for LT molecules [molecular weight (MW) ~250 kDa] to diffuse up to the nerve terminals that are distant by 300-500 µm from the presynaptic soma. This delay is comparable with that (5-10 min) observed for tetanus and botulinum neurotoxins (MW ~150 kDa) at the same synapses (Poulain et al., 1988
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Figure 2. Inhibition of evoked ACh release by LT toxins that inactivate small GTPases. A, A representative experiment is illustrated. ACh release was evoked at identified synapses in the buccal ganglion of Aplysia californica. See inset for a schematic drawing of the neuronal connections. The amplitude of the IPSCs (percentage of average IPSC in control period) is plotted against time, before and after pressure injection of lethal toxin from C. sordellii (LT) into one of the two presynaptic cholinergic neurons (
Typical recordings, illustrated in Figure 2B, exhibit no detectable alteration in the action potentials recorded in the injected neurons at a time when IPSC amplitude was strongly depressed. This suggests that the inhibitory action of LT on ACh release does not result from an alteration in presynaptic membrane excitability. We also found no change in either the IPSC rise or decay time after LT had exerted its blocking action (Fig. 2D,E). This latter finding indicated that release kinetics and synchronization of released quanta remained unchanged after LT action.
Blocking of ACh release needs an LT intact catalytic site
The impressive blocking action of LT (Fig. 2A,C) was comparable, on a molar basis, to that produced by intraneuronal application of tetanus or botulinum neurotoxins (Poulain et al., 1988
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Figure 3. Mutations in catalytic domain of LT82 abolish both GTPase-glucosylating and ACh release-blocking activities. A, Protein substrate specificity of glucosylation by LT82 (top panel), recombinant LT82 fragment (amino acids 1-546) containing an intact catalytic domain (middle panel; LT82 NH2) or a D to A exchange at positions 286 and 288 (bottom panel; LT82 NH2 mutant). Recombinant GTPases (1 µg of each) were incubated with LT or fragments (5 µg/ml) in the presence of UDP-14C-glucose for 60 min at 37°C. Labeled proteins were analyzed by SDS-PAGE and autoradiography. Position of molecular weight markers is indicated. B, The effect of recombinant fragments on ACh release. Same presentation of the data as in Figure 2C. ** denotes significant difference (p < 0.001).
LT glucosylates small GTPases in Aplysia
Figure 4A shows a Western blot analysis of the small GTPases present in Aplysia nerve tissue. These immunoreactivities were detected using specific antibodies raised against members of the Rho and Ras families and correspond to proteins migrating with an apparent molecular weight of 20-30 kDa (data not shown). They cannot be strictly assigned as being Rho, Rac, Cdc42, Ras, Ral, or Rap. Indeed, because of possible divergent evolution of Rho and Ras GTPases, cross-reactivities of the mammalian antibodies with several GTPases cannot be excluded.
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Figure 4. LT induces glucosylation of GTPases in Aplysia. A, Immunochemical detection of small GTPases present in Aplysia nerve tissue. Total protein homogenates (20 µg) were subjected to gel electrophoresis and transferred to nitrocellulose sheets, and the GTPases were detected using anti-Rho, anti-Rac1, anti-Cdc42, anti-Ras, anti-Ral, and anti-Rap antibodies. B, Samples of Aplysia nerve tissue fractions (S, soluble; I, insoluble) containing 100 µg of neuronal proteins were incubated with LT82 or LT9048 (5 µg/ml each) and UDP-14C-glucose alone (denoted as
The intraneuronal targets affected by LT were examined by determining incorporation of 14C-glucose into Aplysia proteins. Because UDP-glucose, the LT cosubstrate, cannot cross the plasma membrane, these experiments were performed on Aplysia neuronal tissue fractions. Fractions containing either soluble (i.e., mostly cytosolic) or insoluble (i.e., membrane associated) protein material were treated with LT82 or LT9048 (5 µg/ml for 1 hr) in the presence of 7 µM UDP-14C-glucose, and then proteins were separated by SDS-PAGE. Figure 4B, (first, second, fifth, and sixth lanes from left) shows that 14C-glucose incorporation was detectable only in proteins of 18-30 kDa MW and may correspond to the glucosylation of the Aplysia monomeric GTPases revealed by Western blotting (Fig. 4A).
Blocking activity of LT on ACh release is causally linked to glucosylation of Rac
Substitution of glucose in the nucleotide-sugar by other sugar moieties inhibits the glucosylation reaction (Hofmann et al., 1998
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Figure 5. Glucosylation of Rac and inhibition of ACh release by LT. A, The substrate specificity of LT82 and LT9048 was determined as described in Figure 3A except that in vitro glucosylation was performed in the absence or presence of glucosylation competitors: 1 mM unlabeled UDP-mannose (UDP-man.) or 10 mM unlabeled TDP-glucose (TDP-gluc.). The position of molecular weight markers is indicated. B, The ACh release inhibition determined 90 min after injection of LT82 alone (50 nM, final intrasomatic) or together with UDP-mannose (1 mM final) or TDP-glucose (10 mM final), or LT9048 (50 nM final). Data are expressed as a percentage of the average IPSC amplitude determined before injection. The horizontal dashed line means no inhibition. The number of experiments in each conditions is indicated. ** denotes significant difference (p < 0.001) as compared with the other conditions. All other comparisons are nonsignificant. C, Same kind of experiment as in Figure 2A, except that both presynaptic neurons were injected with LT82 (50 nM, final, white arrow), but one of them (
Interestingly, we found that the glucosylation reaction could be assigned more selectively to Rac by using ADP- or TDP-glucose as competitive inhibitors for UDP-glucose. Indeed, when the glucosylation reaction was performed, in vitro, in the presence of 10 mM ADP-glucose (data not shown) or TDP-glucose (Fig. 5A), 14C-glucose incorporation into Ras-related proteins (Ras, Rap, and, at a lesser extent, Ral) was markedly reduced, whereas glucosylation of Rac was mostly unaffected. Therefore, intraneuronal application of TDP-glucose (10 mM, final) with LT82 (50 nM, final) was used to minimize intraneuronal glucosylation of Ras, Rap, and Ral. In this experimental condition, we observed that the release of ACh was blocked to a similar extent as that produced by LT82 alone (Fig. 5B; compare first and fourth bar from left).
To determine whether the residual glucosylation of Ral participates in the LT82-induced blockage of ACh release, we used LT9048, which has hardly any effect on Ral but efficiently glucosylates Rac, Ras, and Rap as LT82, but also Cdc42 (Fig. 5A, fourth lane from left). The blocking action of LT9048 (50 nM, final) was comparable to that induced by injection of LT82 (Fig. 5B, fourth bar from left). This indicated that glucosylation of Ral by LT82 does not contribute to the LT-induced blockage of ACh release. To summarize, these combinations of treatments and toxins indicate that the glucosylation of Rac is sufficient to explain the inhibitory action of LT on ACh release.
The second part of the study was aimed at determining the release process altered by LT (i.e., implicating Rac-mediated pathway). Previously, we showed that ACh release can recover from near total inhibition to initial values in ~1 sec when sustained high-frequency stimulations (> 10 Hz) are applied to poisoned nerve terminals (Doussau et al., 2000
Determination of quantal size and number of active release sites at the identified cholinergic synapses in the buccal ganglion of Aplysia
To estimate the quantal size, q, and number of functional release sites, n, we generated IPSC variance versus mean plots (see Materials and Methods) by submitting buccal ganglion synapses to protocols aimed at changing average release probability, (i.e., the product po*pA) and thus ACh release. Average product po*pA was altered by two different manipulations. The first one involved a change in the extracellular [Ca2+/Mg2+] ratio and was aimed at changing the effectiveness of an action potential in activating a release site (i.e., to alter mainly but not exclusively po). The second manipulation was based on changes in stimulation frequency inducing a variable degree of depression (Silver et al., 1998
In the first series of seven experiments, the release probability was changed by manipulating the extracellular [Ca2+/Mg2+] ratio. In the first part of the experiments, we analyzed (stationary analysis) the fluctuations in amplitude of IPSCs during stable epochs of ~30 min after the ganglia had been superfused with medium containing different [Ca2+/Mg2+] ratios (five distinct levels between 2.1 and 0.14) (Fig. 6A). In the second part of these experiments, nonstationary analysis was performed on the fluctuations in IPSC amplitude during the reduction in ACh release caused by a fast transition between high (2.1) and low (0.14) extracellular [Ca2+/Mg2+] ratios (Fig. 6B). As shown in Figure 6, C and D, the A and B parameters of the parabolas (Eq. 4) adjusting the Var = f(Imean) plots were the same for both protocols (compare black-filled and open bars). The duration of the second protocol (45-60 min) was short enough to be performed twice on the same neuron, before and after injection of LT (see below).
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Figure 6. Stationary and nonstationary analysis of the fluctuations in IPSC amplitude when release probability is modified. The experiment illustrated in A and B is representative of a series of seven performed under the same experimental conditions. The Aplysia ganglion was superfused with physiological medium containing modified extracellular [Ca2+/Mg2+] (as indicated in A1 and B1) to change release probability. A1, The I = f(t) plot reporting IPSC amplitude (I) measured after stabilization or evoked ACh release at the indicated extracellular [Ca2+/Mg2+]. B1, The I = f(t) plot during transition between high and low extracellular [Ca2+/Mg2+]. A2, B2, The result of subtracting Imean from the corresponding I as determined by stationary (A) or nonstationary analysis using a window for local linear fitting w = 9, and variance window width w = 16 (B). For details, see Materials and Methods and Figure 1. A3, B3, The corresponding Var = f(Imean) plots. The solid line denotes the approximation of the data by a simple parabola using Equation 4. The regression coefficients r2 are indicated to indicate how good the fits are. A and B are taken from the same experiment in which the corresponding values of A that refer to q are 2.91 nS (A3) and 2.83 nS (B3) and 1/B that refer to n are 426 sites (A3) and 389 sites (B3). C, D, Averaged parabola parameters A and 1/B. Black-filled bars, open bars, and gray-filled bars refer to A and 1/B determined by stationary and nonstationary analysis when extracellular [Ca2+/Mg2+] was changed as described in A and B and when stimulation rate was modified as described in E, respectively. These two experimental conditions are denoted as [Ca2+/Mg2+] and Stim. rate. The number of experiments is indicated, and ns denotes nonsignificant difference. E, To change release probability, presynaptic neurons were submitted to various stimulation frequencies. A representative experiment of a series of six is illustrated. IPSC amplitudes from epochs of 50 recordings at the indicated stimulation frequency were reported against the total number of IPSCs analyzed. E2, Using stationary analysis, the fluctuation of IPSC around Imean was determined. E3, The corresponding Var = f(Imean) plot. The solid line denotes the parabola adjusted to the plot using Equation 4.
In a second series of experiments, we attempted to modify product po*pA by changing the rate of stimulation. Increasing the stimulation rate resulted in synaptic depression (Fig. 6E1), which is thought to reflect synaptic vesicle depletion at release sites. After stabilization of ACh release in each stimulation condition, the amplitude of fluctuation in IPSC around Imean was determined using stationary analysis. The Var = f(Imean) plots (Fig. 6E3) were satisfactorily fitted (r2 > 0.90) by a simple parabola (Eq. 4). This graphically confirmed our assumption (see above) that when the rate of stimulation is increased, release probability is diminished. Figure 6, C and D (gray-filled bars), shows that the A and B parameters determined by this protocol were not significantly different from those determined by manipulating extracellular [Ca2+/Mg2+] ratios to change product po*pA (Fig. 6C,D, compare the gray-filled bars with open and black-filled bars).
The average A values (Fig. 6C) as determined by these three procedures indicate a quantal size, q, of ~2.5-2.8 nS at the studied synapse. This value overestimates true q because quantal variability (CVq) contributes to IPSC fluctuations (Eq. 5). Nevertheless, the A value is comparable to the uncorrected q values determined previously (1.5-2 nS) at the same Aplysia synapse using a different approach (Simonneau et al., 1980
Quantal parameters determined by manipulating extracellular [Ca2+/Mg2+] after LT action
To determine which of the quantal parameters were modified and accounted for the decrease in IPSC amplitude that characterizes LT action, the fast change in extracellular [Ca2+/Mg2+] protocol was applied before and after LT82 had significantly inhibited ACh release. Figure 7A shows a typical experiment from a series of five. To minimize the difficulty in analyzing the combined blocking action of LT82 and the reduction in synaptic efficacy caused by [Ca2+/Mg2+] change, the protocol was applied at a time when the LT-induced block was at least 80% (on average by 84%) (Fig. 8A). During the high to low [Ca2+/Mg2+] transition protocol (40-60 min), we determined that the blockage caused by LT82 was 11.7 ± 2% (SEM) as compared with the 91.8 ± 1% decrease in IPSC amplitude observed when [Ca2+/Mg2+] was lowered from 2.1 to 0.14. The corresponding nonstationary analysis of fluctuations in IPSC amplitude and Var = f(Imean) plots is shown in Figure 7, B and C. After LT had blocked ACh release, the Var = f(Imean) relationship still appeared parabolic (Figs. 7D, 8B). The initial slope of the parabola, A, remained unmodified (Figs. 7D,E, 8C), suggesting that quantal size, q, was not changed. However, if the studied synapse is saturating, it is conceivable that small changes in the presynaptic component of q (SV content) may not have been detected.
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Figure 7. The effect of changing extracellular [Ca2+/Mg2+] before and during LT action. The experiment illustrated is representative of a series of five performed under the same experimental conditions. A, To determine the quantal parameters before and after LT82 application, extracellular [Ca2+/Mg2+] was modified between 2.1 and 0.14 as indicated in the bottom panel. Arrow denotes the time of LT injection. Solid lines denote the set of Imean determined by nonstationary analysis (with w = 9). The horizontal, long-dashed line denotes average I in the control period with [Ca2+/Mg2+] = 0.42. Dashed line (after LT) denotes the extrapolated I values (by sigmoid fitting) that would have been obtained without changing extracellular [Ca2+/Mg2+]. B, C, Top panels, Magnification of the I = f(t) plot portions boxed in A; bottom panels, the result of subtracting I values from the Imean estimated using nonstationary analysis. D, E, The corresponding Var = f(Imean) plots before (
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Figure 8. Determination of the parabola parameters affected by LT action using the fast change in extracellular [Ca2+/Mg2+] protocol. Five experiments similar to that illustrated in Figure 6B were analyzed before (open bars or open symbols) and 190 ± 20 min (mean ± SD) after LT injection (closed symbols or filled bars). A, The mean IPSC amplitude. B, Mean Var = f(Imean) plot was obtained by averaging the Var values for Imean over a 5% amplitude interval. C, The average parameter A of the adjusted parabolas, which refers to quantal size, q. D, The relationships between IPSC amplitude and extracellular [Ca2+/Mg2+]. The data were normalized to values observed at [Ca2+/Mg2+] = 0.14 to highlight possible differences. E, The average 1/B ratio, which refers to n. F, Relationships between P parameter (see Eq. 8), which refers to po*pA, and extracellular [Ca2+/Mg2+]. In A, B, and E, a significant difference (p < 0.001) was found compared with controls; C, D, and F show a nonsignificant difference (p > 0.5).
The relationship between ACh release and extracellular Ca2+ was examined by plotting the I = f([Ca2+/Mg2+]). To highlight possible changes in this relationship, the IPSC amplitude values were normalized to the amplitudes observed at the lowest extracellular [Ca2+/Mg2+] used. However, no apparent modification could be found after LT treatment compared with the control period (Fig. 8D). In all experiments, we observed that the portion of the parabola depicted in the Var = f(Imean) plots during decreasing extracellular [Ca2+/Mg2+] started at the same initial average probability as that observed before applying LT (Figs. 7E, 8B, straight lines), thereby indicating that average product po*pA was unchanged. Moreover, the relationship between P (which refers to product po*pA, Eq. 7) and extracellular [Ca2+/Mg2+] did not significantly change before and after LT (Fig. 8F). This suggests that the coupling between Ca2+ influx and exocytosis is unlikely to be altered by LT action.
After LT, the only difference that we detected was the decrease in parabola parameter B. Indeed, 1/B (Fig. 8E) was reduced to an extent similar to the reduction in mean IPSC amplitude induced by LT (Fig. 8A). This suggests that the blocking action of LT action is caused by a diminution in n, the number of active release sites.
Intracellular application of the Ca2+-buffer EGTA has been shown to interfere with LT action (Doussau et al., 2000
Quantal parameters determined by changing stimulation frequency after LT action
The "change in stimulation rate" protocol was applied before and after LT82 had inhibited ACh release (Fig. 9A). In preliminary experiments, we noticed that a significant inhibitory effect of LT developed during the long duration needed for this protocol (120-180 min to collect enough data for variance determination). To avoid this problem, the effects of LT were stopped by intraneuronal injection of UDP-mannose (1-10 mM, final) after that ACh release was inhibited by ~80% (Figs. 9A, 10A). Because of the technical difficulty in injecting the same neuron twice without trauma, only four experiments could be analyzed. The "stimulation rate protocol" was performed ~60 min after the UDP-mannose injection to verify that LT action was indeed stopped. Fluctuations in IPSC amplitude were analyzed before (control) and after LT injection by the stationary procedure (Fig. 9B,C).
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Figure 9. The effects of changing stimulation rate before and during LT action. A representative experiment of a series of four is illustrated. A, Top panel, To determine the quantal parameters before and after LT82 injection (white arrow), ACh release was modified by increasing the stimulation rate between 0.01 and 10 Hz to change release probability. The inhibitory action of LT was arrested by injection (black arrow) of UDP-mannose (10 mM, final). Bottom panel indicates the different stimulation intervals used during the experiment. B, C, Top panels, The IPSC amplitudes are observed at the indicated stimulation rate and correspond to the data boxed in A. Bottom panels, The result of subtracting I values from Imean (stationary analysis). D, The corresponding Var = f(Imean) plots before (
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Figure 10. Determination of the parabola parameters before and after LT action using the change in stimulation rate protocol. Same presentation as in Figure 8, except that changes in the stimulation rate were used to modify release probability. In B, Imean and Var values were averaged for similar stimulation rate conditions. Averages were made from four successful experiments. In A, B, and E, a significant difference (p < 0.001) was found compared with controls; C, D, and F show a nonsignificant difference (p > 0.5).
The Var = f(Imean) plots were well fitted (r2 > 0.90) by a simple parabola (Figs. 9D,E, 10B). As expected, the parabola parameter A was not significantly modified by LT treatment, confirming that quantal size is unchanged (Fig. 10C). Here also, the Var = f(Imean) plots depicted the same portion of parabola (Figs. 9D, 10B) before and after blockage of ACh release by LT. Neither the shape of the Imean = f (stimulation interval) plots (Fig. 10D) nor the relationships between P and the stimulation interval were modified after LT injection, as compared with controls (Fig. 10F). We could not explore the responsiveness of the synapse to very high frequency because at a stimulation rate over 10 Hz, LT-induced block is relieved (Doussau et al., 2000
Variance to mean relationship during LT-induced blockage of ACh release confirms a reduction in the number of release sites
If LT82-induced blockage of ACh exocytosis is caused by a decrease in n, this should also manifest as a linear relationship between Imean and Var in the Var = f(Imean) plots made by nonstationary analysis of IPSC amplitude fluctuations during the ACh release blockage produced by LT injection. Indeed, when IPSC amplitude changes as the result of a reduction of n, Var should decrease linearly with Imean according to equation:
A fall in IPSC amplitude caused by a change in the product po*pA should be described by Equation 3, which is a parabola (see Eq. 4), similarly as this has been illustrated above when po*pA was experimentally modified by distinct procedures. After progressive q changes, the Var = f(Imean) relationship should be described by a quadratic function of positive curvature of equation:
(8)
Data were submitted to a nonlinear regression analysis using the quadratic function Var = a*Imean + b*I2mean. Indeed, this procedure avoids any a priori on the linearity or nonlinearity of the Var = f(Imean) relationship. A linear slope should manifest with b = 0 and a > 0 (Var = a*Imean). If a = 0 and b >0, Var = b*I2mean, and the corresponding Var = f(Imean) plot would display a positive curvature pinpointing a change in q size (Eq. 9). If a > 0 and b < 0, Var = f(Imean) plot would exhibit a negative curvature indicating a change in product po*pA (Eq. 3).
![<UP>Var</UP>=[(1−p<SUB>o</SUB>*p<SUB>A</SUB>)/(n*p<SUB>o</SUB>*p<SUB>A</SUB>)]*I<SUB><UP>mean</UP></SUB><SUP>2</SUP>.](/content/vol22/issue18/fulltext/7968/img009.gif)
(9) In Figure 11A3, the parameters for nonlinear regression analysis of the Var = f(Imean) plot were a = 0.68 and b = 10
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Figure 11. Graphical determination of n as the quantal parameter modified by LT action. A, IPSC fluctuations during the course of LT82-induced blockage of ACh release were analyzed by nonstationary analysis using a window width for local linear fitting w = 33, and variance window W = 21. A1, I = f(t) plot. The set of Imean is represented by a solid white line. A2, The result of subtracting I values from Imean. A3, The corresponding Var = f(Imean) plot. B, C, The Var = f(Imean) plots from experiments in which either LT82 (n = 11) or LT9048 (n = 5) was injected. Individual plots are indicated by different symbols and are scaled to the Imean and Var values determined before LT injection. For clarity, only 1 point of 10 is plotted. A3-C, The adjustment of the data by linear (solid straight line) or nonlinear regression analysis (dashed curves) is presented. For r2 values see Results. The linear Var = f(Imean) relationships indicate that Imean diminution is caused by a decrease in n, the number of active release sites.
As an outcome of this study, one may anticipate that the graphical procedure described above [see also Humeau et al. (2001b)
