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Calcium-Independent Activation of the Secretory Apparatus by Ruthenium Red in Hippocampal Neurons: a New Tool to Assess Modulation of Presynaptic Function

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Calcium-Independent Activation of the Secretory Apparatus by Ruthenium Red in Hippocampal Neurons: a New Tool to Assess Modulation of Presynaptic Function The Journal of Neuroscience, January 1, 1996, 16(1):46-54 Calcium-Independent Activation of the Secretory Apparatus by Ruthenium Red in Hippocampal Neurons: A New Tool to Assess Modulation of Presynaptic Function Louis-Eric Trudeau, Robert T. Doyle, Dennis G. Emery, and Philip G. Haydon Laboratory of Cellular Signaling, Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011 The functional plasticity of the nervous system may result in terminal membrane. This binding can be displaced by heparin part from the direct modulation of the effectiveness of the and is not associated with ultrastructural damage to the syn- release machinery of synaptic terminals. To date, direct mod- aptic terminals. The use of RR-evoked release as a tool has ulation of secretion in neurons has proven difficult to study allowed us to detect a direct modulation of the secretory ap- because of the lack of a suitable tool to probe the release paratus after activation of A, adenosine receptors on hip- machinery independently of calcium influx. We report that the pocampal neurons. polyvalent cation ruthenium red (RR) directly evokes rapid and reversible calcium-independent quanta1 secretion in hippocam- Key words: miniature synaptic currents; calcium; ruthenium pal neurons by binding to external sites on the presynaptic red; secretion; hippocampus; synapse A characteristicproperty of the nervoussystem is its plasticity, or tool to induce neurotransmitter releaseindependently of calcium capacity for reorganization, as a function of past experience or influx through calcium channels.Using invertebrate neurons,we after injury. An important site of plasticity in the nervous system and others have shown previously that one method that may be is found at chemicalsynapses. The effectivenessof synaptic trans- effective is to provide calcium to the releasemachinery directly by missioncan be modified for short or long periodsof time through flash photolysis of caged calcium (Zucker and Haydon, 1988; various possiblemechanisms. One of these is a change in the Man-Son-Hing et al., 1989; Delaney and Zucker, 1990; Mulkey amount of neurotransmitter released at presynaptic terminals and Zucker, 1993). However, the technical difficulties associated after action potential-dependentcalcium influx. This changemay with this method limit its routine use, especially in mammalian be accomplishedin a number of ways. First, there may be a neurons.Other tools that have previously been found to be useful modification in the shapeof the action potential. For example, a in probing the releasemachinery of neuronsinclude a-latrotoxin decreasein potassiumconductance could allow broadeningof the as well as hyperosmotic challenges.The usefulnessof the first action potential and a prolonged depolarization of the terminals approach is limited by its nonreversibility as well as by its mixed (Klein, 1994). A prolonged opening of calcium channels and calcium-dependentand calcium-independent modes of action influx of this ion into the terminal would result in an increasein (Rosenthalet al., 1991).The useof hyperosmoticsaline to induce neurotransmitter release. A change in transmitter releasemay neurosecretionin the context of investigationsof modulation of also result more directly from a modulation of the calcium chan- release also is limited for the moment, owing to uncertainties nels (Scholz and Miller, 1991). A third possibility is a direct about the calcium dependenceof its mode of action (Landb et al., change in the effectivenessof the release machinery of nerve 1986).For example,it hasproven difficult to usecalcium-sensitive terminals, i.e., the stepsthat are downstreamfrom calcium influx dyes to monitor intracellular calcium dynamicsbecause of poten- and culminate in quanta1release of neurotransmitter (Man-Son- tial problemscaused by changesin cell volume and intracellular Hing et al., 1989). This third possibility has proven to be more ionic concentrationscaused by this stimulus(Delaney et al., 1991). difficult to addressbut may be of great importance. Indirect Yet additional agentsthat have been usedto induce increasesin evidence hasbeen presentedto suggestthat this type of plasticity the frequency of spontaneousrelease of neurotransmitter quanta may be involved in the upregulation of excitatory synaptic trans- (minis) include ethanol, chlorpromazine, and ouabain.The action mission that characterizes long-term potentiation, a candidate of ethanol seemsto be independent of calcium (Brosius et al., mechanismfor the establishmentof long-term memory (Malga- 1992),but its effects on spontaneousrelease are obtained only at roli and Tsien, 1992; Malgaroli et al., 199.5). high concentrations, which also cause significant postsynaptic A major limitation in the assessmentof a direct modulation of alterations and changeminiamplitude (Gage, 1965;Quastel et al., the releasemachinery in neurons has been the lack of a suitable 1971). The action of both chlorpromazine and ouabain on mini- frequency apparently is independentof external calcium, but has Received July 18, 1995; revised Sept. 6, 1995; accepted Sept. 8, 1995. not been shownto be independentof internal calcium (Argov and This work was funded in part by NIH Grants NS233450 and NS26650 to P.G.H., Yaari, 197.5;Baker and Crawford, 1975). These two agentsalso by the McKnight Foundation, and by a Long-Term Fellowship from the Human Frontier Science Program to L.-E.T. We thank Drs. M. McCloskey and S. Shen as accelerateminifrequency only after a prolonged delay and, in the well as T. A. Basarsky for fruitful discussions and help in reviewing this manuscript. caseof ouabain,can be associatedwith ultrastructural damageto Correspondence should be addressed to Dr. L.-E. Trudeau, Laboratory of Cellular Signaling, Department of Zoology and Genetics, Iowa State University, 339 Science synaptic terminals (Haimann et al., 1985). Finally, previous work II building, Ames, IA 50011. has shown that minifrequency could be changedafter the appli- Copyright 0 1995 Society for Neuroscience 0270-6474/95/160046-09$05.00/O cation of neuromodulators,thereby suggestinga direct presynap- Trudeau et al. Calcium-IndeDendent Induction of Secretion J. Neurosci., January 1, 1996, 16(1):46-54 47 tic modulation (Scanziani et al., 1992; Scholz and Miller, 1992). and digitized at 5 or 10 kHz. The data were acquired using pClamp This later type of analysis certainly is useful but is not necessarily software from Axon Instruments (v. 6.0). Miniature synaptic currents were analyzed further with the ACSPLOUF minianalysis software (ob- a very sensitive assay of the state of the secretory apparatus. tained from Dr. Pierre Vincent, University of California at San Diego). Spontaneous minifrequency also could be influenced by changes Group data presented in the text are expressed as mean 2 SEM, unless in the resting levels of calcium in synaptic terminals. Considering stated otherwise. these limitations, we sought to develop a new tool to directly Calcium imaging. Methods for ratiometric determination of intracellu- probe the release apparatus of neurons. lar calcium were as described in Basarsky et al. (1994). Briefly, cultured neurons were loaded at 37°C with 2 pM Fura- AM (Molecular Probes, Within this context we found it intriguing that polyvalent cat- Eugene, OR) for 45 min in the presence of 0.025% Pluronic F-127. The ions such as lanthanum and ruthenium red (RR) reliably increase culture was then washed, and de-esterification was allowed to proceed for the rate of miniature end-plate potentials and synaptic currents 45 min at 37°C before starting experiments. Images were acquired using (Heuser and Miledi, 1971; Alnaes and Rahamimoff, 1975; a CH-250-cooled CCD camera (Photometrics, Tucson, AZ) and Metafluor software (Universal Imaging, West Chester, PA). Raastad et al., 1990; Grubb et al., 1991). The mechanism of action Electron microscopy. Cultures were fixed for 90 min in a mixture of 1% of this induction of release has not been characterized in detail, p-formaldehyde and 2% glutaraldehyde and 0.25% tannic acid in 0.1 M although it generally has been thought to involve changes in cacodylate buffer and postfixed for 15 min in 0.5% osmium tetroxide plus intraterminal calcium levels. Morphological observations of lan- 0.8% potassium ferricyanide in the same buffer. After en-block staining with magnesium-uranyl acetate (l%, aqueous, 2 hr), cultures were em- thanum-treated neuromuscular junctions have shown previously bedded in Epon resin. Thin sections were stained with Reynolds lead that prolonged treatments could induce depletion of clear vesi- citrate before viewing. cles, whereas tetanus-toxin pretreated junctions exposed to lan- Drugs. Tetrodoto&, RR, picrotoxin, heparin (sodium salt, MW thanum for short durations displayed an enhanced number of ~6000‘1. ME-ATP. and Tris-GTP were from Sigma (St. Louis. MO). vesicles in close apposition to the presynaptic membrane (Mel- fl-cycjopeniyladenosine, CNQX, and SR-95531u were from R&ear&h Biochemicals (Natick, MA). lanby et al., 1988). This suggests that such treatments may facili- tate interactions between vesicles and the presynaptic membrane. Lanthanum also has been reported to promote catecholamine RESULTS release from chromaffin cells (Powis et al., 1994). Here we have Rapid and reversible induction of secretion investigated in more detail the mode of action of RR at synapses We first determinedwhether the action of RR on secretionwas a formed between hippocampal neurons in primary culture and find general
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