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Calcium-Dependent Glutamate Release During Neuronal Development and Synaptogenesis: Different Involvement of &O-Agatoxin IVA- and O-Conotoxin GVIA-Sensitive Channels

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Calcium-Dependent Glutamate Release During Neuronal Development and Synaptogenesis: Different Involvement of &O-Agatoxin IVA- and O-Conotoxin GVIA-Sensitive Channels Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6449-6453, July 1995 Neurobiology Calcium-dependent glutamate release during neuronal development and synaptogenesis: Different involvement of &o-agatoxin IVA- and o-conotoxin GVIA-sensitive channels CLAUDIA VERDERIO*, SILVIA Coco*, GUIDO FUMAGALLIt, AND MICHELA MATTEOLI*t *Consiglio Nazionale delle Ricerche Center of Cytopharmacology and Bruno Ceccarelli Center, Department of Pharmacology, University of Milan, Milan, Italy; and tDepartment of Pharmacology, University of Verona, Verona, Italy Communicated by Vittorio Erspamer, University of Rome La Sapienza, Rome, Italy, March 15, 1995 ABSTRACT Hippocampal neurons maintained in primary MATERIALS AND METHODS culture recycle synaptic vesicles and express functional gluta- mate receptors since early stages of neuronal development. By Hippocampal Cell Culture. Primary neuronal cultures were analyzing glutamate-induced cytosolic calcium changes to sense prepared from the hippocampi of 18-day fetal rats as described presynaptically released neurotransmitter, we demonstrate that (8, 9). Briefly, hippocampi were dissociated by treatment with the ability of neurons to release glutamate in the extracellular trypsin (0.25% for 15 min at 37°C), followed by trituration with space is temporally coincident with the property of synaptic a fire-polished Pasteur pipette. Dissociated cells were plated vesicles to undergo exocytotic-endocytotic recycling. Neuronal on a poly(L-lysine)-treated glass coverslips in minimum essen- differentiation and maturation ofsynaptic contacts coincide with tial medium (MEM) with 10% horse serum at densities ranging a change in the subtype of calcium channels primarily involved from 10,000 cells per cm2 to 20,000 cells per cm2. After a few in controlling neurosecretion. Whereas o-agatoxin IVA- hours, coverslips were transferred to dishes containing a sensitive channels play a role in controlling neurotransmitter monolayer of cortical glial cells (10), so that they were sus- secretion at all stages of neuronal differentiation, ao-conotoxin pended over the glial cells but not in contact with them (9). GVIA-sensitive channels are primarily involved in mediating Cells were maintained in MEM (GIBCO) without serum, glutamate release at early developmental stages only. supplemented with 1% HL1 (Ventrex Laboratories, Portland, ME), 2 mM glutamine, and 1 mg of bovine serum albumin per Synaptic transmission is the process by which signals are ml (neuronal medium). transferred from a neuron to a target cell. At the nerve [Ca2+], Measurements with Fura-2. Neurons were loaded terminal synaptic vesicles undergo exocytotic-endocytotic re- for 30 min at 37°C with 2-4 ,M fura-2 pentakisacetoxymethyl cycling and at each cycle they release neurotransmitter in the ester in Krebs-Ringer-Henseleit solution (KRH) buffered extracellular space (1, 2). Released neurotransmitter binds to with Hepes (150 mM NaCl/5 mM KCl/1 mM MgSO4/2 mM postsynaptic receptors that are strategically localized at CaCl2/10 mM glucose/10 mM Hepes-NaOH, pH 7.4), washed postsynaptic sites and that in turn translate the external signals in the same solution to allow deesterification of the dye, and into a postsynaptic response (3). The anatomical relation transferred to the heated stage of the microscope, where between pre- and postsynaptic compartments is the basis on temperature was maintained at 35°C throughout the experi- which the point-to-point signaling typical of synaptic transmis- ment. Cells under different experimental conditions (see sion relies. below) were observed with an inverted Zeiss IM35 microscope However, several lines of evidence indicate that the func- equipped with a calcium-imaging unit (11). Fluorescence tionality of pre- and postsynaptic components is not dependent images were obtained by alternately illuminating the cells at on their spatial organization at synaptic sites. It has been 345 nm and 380 nm and emission filtered with a 418-nm demonstrated recently that synaptic vesicles present in devel- long-pass filter; images (345 nm or 380 nm) consisted of 0.125 oping processes of cultured hippocampal neurons are able to megabyte (MB) each and were acquired rhythmically at 2 Hz undergo exocytotic-endocytotic recycling (4, 5), with mecha- frequency. Fluorescence images were collected with an inten- nisms that appear to be already calcium-dependent (6). More- sified charge-coupled device camera (Hamamatsu Photonics, over, functional glutamate receptors are expressed already at Middlesex, NJ) and the camera output was analyzed by a digital stages preceding synapse formation (7). image processor (Argus 100; Hamamatsu Photonics) where We now investigate whether synaptic vesicle recycling before video frames were digitized and integrated in real time on four synaptogenesis is associated with neurotransmitter release. To 0.5-MB memory boards. The digital data were then transferred this aim, we used a biological assay that allows us to perform at high rate via a connecting board placed on the VME bus of an on-line analysis of glutamate released by hippocampal Argus 100 into a Motorola 68020-based host computer and neurons by analyzing the cytosolic calcium ([Ca2+]j) changes stored in two 300-MB hard disks. Background and calibration induced by N-methyl-D-aspartate (NMDA) receptor activa- images were similarly acquired at the two wavelengths; the tion. We show that hippocampal neurons are able to release calculation of Ca2+ concentration was carried out pixel by pixel glutamate by a calcium-dependent mechanism even before on pairs of corresponding 345-nm and 380-nm images according synapse formation. Moreover, we demonstrate that c-agatoxin to Grynkiewicz et at (12). The time resolution of the measure- IVA (w-Aga-IVA) and w-conotoxin GVIA (co-CTx-GVIA) have different effects on blocking glutamate release at various Abbreviations: [Ca2+]i, cytosolic calcium concentration; NMDA, N- developmental stages, indicating that formation and matura- methyl-D-aspartate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; tion of synaptic contacts are associated with changes in the APV, D-2-amino-5-phosphonopentanoic acid; w-Aga-IVA, w-aga- subtypes of voltage-activated calcium channels controlling toxin IVA; co-CTx-GVIA, w-conotoxin GVIA; BoNT/F, toxin from presynaptic glutamate release. Clostridium botulinum of F serotype; PDC, L-trans-pyrrolidine-2,4- dicarboxylic acid. 4To whom reprint requests should be addressed at: Consiglio Nazio- The publication costs of this article were defrayed in part by page charge nale delle Ricerche Center of Cytopharmacology and B. Ceccarelli payment. This article must therefore be hereby marked "advertisement" in Center, Department of Medical Pharmacology, University of Milan, accordance with 18 U.S.C. §1734 solely to indicate this fact. via Vanvitelli 32, 20129 Milan, Italy. 6449 Downloaded by guest on September 30, 2021 6450 Neurobiology: Verderio et aL Proc. Natl. Acad. Sci. USA 92 (1995) ments was 1 s. All temporal plots were obtained from neuronal A cell bodies. APV CNQX Experimental Treatments. Incubations were carried out either in 2 ml of KRH or in 2 ml of its Mg2+-free version, which contained no MgCl2 and was supplemented with 1 ,uM glycine. - Application of NMDA (25 ,tM) or glutamate (20 j,M) was performed in a medium free of Mg2> ions. Incubations with Ca2+ channel blockers (w-Aga-IVA and cw-CTx-GVIA) were performed at 35°C for 20-25 min in KRH before the Mg2+- 'Us- BoNT/F IGlutamate free/glycine treatment. Drugs were maintained in the incuba- tion medium for the entire length of the experiment. Incuba- + 400- tions with toxin from Clostridium botulinum of F serotype (BoNT/F) were performed at 30°C for 2 hr. For the experi- 100 p J f - Mg2- free/Gly ments aimed at accumulating glutamate in the extracellular medium, coverslips were maintained in a fixed amount of KRH -C Cd2+ NMDA NMDA (1 or 2 ml) in the presence of the glutamate uptake inhibitor 700- Cd2^ L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (20 ,tM). The collected medium was divided in two aliquots, one of which 400- was supplemented with the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 100- -_____J_ Mg2+ free/Gly j.tM). The same neuronal culture was then exposed subse- min quently to the two aliquots of medium. In other experiments, the same coverslip was first incubated for 1 hr with 1 ml of FIG. 1. Activation of NMDA receptors by presynaptic release of KRH and then incubated with a new 1 ml of KRH containing glutamate in neurons rich in synaptic contacts. (A) Temporal plot of the calcium channel blocker Cd2+ (100 ,tM). Before challeng- [Ca]2+i changes induced by the Mg2+-free/glycine medium in the cell ing a new coverslip with the two aliquots of conditioned body of a 14-day-old hippocampal neuron. [Ca]2+ responses are medium in sequence, Cd2> was added to the Cd2+-free aliquot inhibited by 100 ,uM D-2-amino-5-phosphonopentanoic acid (APV) (final concentration, 100 ,uM). but not by 10 ,uM CNQX. (B) [Ca2+]i responses to the Mg2+-free/ Materials. Most of the chemicals used were purchased from glycine medium in the soma of a 10-day-old neuron before and after Tocris Neuramin (Bristol, U.K.). Fura-2 was from Calbiochem. incubation (2 hr) with 7 nM BoNT/F. Note that [Ca2+], changes are International and totally prevented by toxin treatment. The presence of BoNT/F does c-Aga-IVA was purchased from Peptides not impair the neuronal ability to respond to 20 t,M glutamate. (C) w-CTx-GVIA was from Bachem. [Ca2+]i responses to Mg2+-free/glycine medium in the cell body of a 12-day-old neuron in the presence or in the absence of 200 AM Cd2+ RESULTS Cd2+ strongly inhibits the response to the Mg2+-free/glycine medium and reduces only partially the postsynaptic response to 25 ,tM NMDA. Mg2+-Free/Glycine Protocol as a Tool To Detect Glutamate Note that the kinetics of [Ca2+]i responses to the Mg2+-free/glycine Release from Hippocampal Neurons.
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