The Journal of Neuroscience, November 1994, 14(11): 6402-6411 Hippocampal Synaptogenesis in Cell Culture: Developmental Time Course of Synapse Formation, Calcium Influx, and Synaptic Protein Distribution Trent A. Basarsky, Vladimir Parpura, and Philip G. Haydon Signal Transduction Training Group and Department of Zoology and Genetics, Iowa State University, Ames, Iowa 5001 l-3223 The formation of chemical synapses between hippocampal ulation as N-type channelsthat do supply calcium to stimulate neurons in primary cell culture was studied using electro- synaptic transmission(Takahashi and Momiyama, 1993)?Little physiology, calcium imaging, and immunocytochemical ap- information is available concerning the regulation of these pre- proaches. Inhibitory and excitatory synapses formed within synaptic developmental events. 12 d in cell culture (DE) that were sensitive to the N-type Studies of nerve-muscle synapseformation have shown that calcium channel blocker w-conotoxin GVIA (w-CgTx). At 4 the synaptic target supplies retrograde signals that control the DIC, immature connections were present in which sponta- appropriate development of presynaptic machinery. Muscle cells neous, but rarely evoked, synaptic currents were detected. manipulated into contact with growth conesof neurons derived At both 4 and 12 DIC, the synaptic proteins rab3a, synapsin from the Xenopus neural tube causea local increasein resting I, and synaptotagmin were present in hippocampal neurons, calcium level (Dai and Peng, 1993), a rapid induction of secre- but the subcellular distribution changed from one in which tion (Xie and Poo, 1986), and a local reorganization of the immunoreactivity was initially distributed within soma and growth cone such that the quanta1 content of evoked synaptic neurites to a punctate varicose appearance. Correlated with transmission is augmented (Sun and Poo, 1987). In Helisoma, the transformation from immature to mature synaptic states muscle fibers activate presynaptic protein kinaseA, which caus- was the onset of w-CgTx-sensitive calcium influx. Taken es an elevation of the resting calcium level of neuronal growth together, these data suggest that the expression of func- cones, and an enhancement of the presynaptic calcium influx tional w-CgTx-sensitive calcium influx is temporally coinci- during action potentials (Funte and Haydon, 1993; Zoran et al., dent with synapse formation, and that during the maturation 1993). After many hours of contact, the calcium sensitivity of of the synapse there is a redistribution of synaptic proteins. the secretory apparatus is then increased(Zoran et al., 1991). [Key words: synaptic proteins, synaptotagmin, synapsin, These regulatory events lead to the effective coupling of pre- rab3a, synaptogenesis, calcium channel, conotoxin, w-CgTx] synaptic action potentials to secretion. In contrast to studies of neuromuscular synapseformation, Much of our knowledge about synaptogenesishas been gained there is a paucity of studies of the regulation of synaptogenesis frdm studies of neuromuscular development where agrin and between central neurons. Central neurons can contain many aria, synthesized in motoneurons, regulate the expression and types of calcium channels,yet only a restricted subsetstimulates aggregation of postsynaptic ACh receptors (Falls et al., 1990; transmitter release(Takahashi and Momiyama, 1993). A priori, McMahan, 1990). For the development of fast synaptic trans- one would expect a differential regulation of these different sub- mission, it is also necessarythat presynaptic macromolecules sets of calcium channels during synaptogenesis.Furthermore, are synthesized, transported to the presynaptic terminal, and it is unclear when specific calcium channelsdevelop in relation arranged with the appropriate mutual spatial relation. For ex- to the onset of synapse formation, when and where synaptic ample, it is necessaryfor the calcium channel to become local- associatedproteins are expressedand when excitation becomes ized in closeproximity to the secretory vesicle to supply calcium coupled to secretion. Using dissociated hippocampal cell cul- in a sufficiently high concentration to stimulate secretion(Simon tures, the goal of this study is to elucidate the temporal relation and Llinas, 1985; Zucker and Fogelson, 1986). It is also unclear between the appearanceof functional synaptic transmission,L- whether different calcium channels are subject to the samereg- and N-type calcium channels, and synaptic protein distribution. ulation during synaptogenesis.For example, are L-type channels This study demonstrates that while many components of the that do not stimulate transmitter releasesubject to similar reg- presynaptic neuron are expressedearly in cell culture the de- velopment of w-CgTx-sensitive N-type, but not nifedipine-sen- Received Nov. 8, 1993; revised Mar. 4, 1994; accepted Apr. 13, 1994. sitive L-type, calcium influx is temporally correlated with the T.A.B. and V.P. contributed equally. We thank Kerry J. Morris and Robert delayed detection of evoked synaptic transmission. Doyle for technical assistance, Drs. R. Jahn and A. Czemik for providing anti- bodies, and Dr. S. Shen for helpful discussion. This work was supported by the Some of these data have appeared in preliminary form (Bas- Euileosv Foundation of America, NIH Grants NS26650 and NS24233, the Alfred arsky et al., 1992). P: SIban foundation. and a Medical Research Council of Canada studentship (T.A.B.). Materials and Methods Correspondence should be addressed to P. G. Haydon, Signal Transduction Training Group and Department of Zoology and Genetics, 339 Science II, Iowa Cell culture.Hippocampi were dissected from l&d-old Sprague-Daw- State University, Ames, IA 5001 l-3223. ley rats. Tissue was incubated for 1 hr at 37°C in Ca2+/Mg2+-free Earle’s Copyright 0 1994 Society for Neuroscience 0270-6474/94/146402-10$05.00/O balanced salt solution (EBSS; pH 7.35; GIBCO) containing papain (20 The Journal of Neuroscience, November 1994, 74(11) 6403 U/ml; Sigma), HEPES (10 mM), L-cysteine (0.2 mg/ml), glucose (20 In some cases, to gain enhanced spatial resolution, a Noran Odyssey mM), penicillin (100 U/ml), and streptomycin (100 &ml). Tissue was real-time laser scanning confocal microscope equipped with IMAGE-~/ washed once with fresh EBSS and then placed in EBSS (pH 7.35) con- AT software (v. 4.0) was used. Optical sections were collected through taining HEPES (10 mM) and trypsin inhibitor (10 mg/ml, type II-O; a 60x plan-apochromatic objective that gave a single section optical Sigma) for 5 min. After being rinsed, hippocampi were mechanically thickness of 0.7 pm (full width at half-maximum, FWHM). The initial dispersed by triturating through a fire-polished glass pipette. Cells were and final optical planes were specified to ensure that all immunoreactive plated into poly+lysine (1 mg/ml, MW 100,000; Sigmaboated glass- areas were captured. bottomed dishes. Cultures were maintained at 37°C in a humidified 5% Calcium imaging. Calcium levels were estimated using fura-2/AM CO,, 95% air atmosphere. Culture medium consisted of Eagle’s mini- and ratiometric imaging techniques (Grynkiewicz et al., 1985). All mea- mum essential medium (Earle’s salts, GIBCO) supplemented with 10% surements were made from somatic regions. Cells were loaded with heat-inactivated fetal bovine serum (FBS; Sigma) and containing 40 mM fura-Z/AM (2 PM) for 40 min at 37°C. One microliter of 25% (w/w) of glucose, 2 mM L-glutamine, 1 mM pyruvate, 14 mM NaHCO,, 100 U/ml Pluronic F-127 was mixed per 1 ml of fura-2/AM loading solution (2 penicillin, and 100 &ml streptomycin (pH 7.35). The dissociation PM) to aid solubilization of the ester into aqueous medium. After wash- procedure was modified from previously described procedures (Leifer ing, fura-2/AM was deesterified for 40 min at 37°C. All experiments et al., 1984; Huettner and Baughman, 1986; Mattson and Kater, 1989). took place at 22-24°C. Normal external saline was (in mM) NaCl, 140; To suppress proliferation of non-neuronal cells, arabinosylcytosine (ARA- KCl, 5; MgCl,, 2; CaCl,, 2; and HEPES, 10 (pH 7.35). In zero-calcium C; 5 PM) was added after 2-3 d in culture. Cultures were fed once a saline, Ca2+ was replaced with Mg2+ and 2 mM EGTA was added. week by exchanging 30% of the medium with fresh medium. To test for the presence of voltage-sensitive calcium influx, neurons Electrophysiology. Conventional dual whole-cell recording techniques were incubated in TTX (1 PM) to prevent spontaneous action potentials, (Hamill et al., 1981) were employed to stimulate and record synaptic and were depolarized using elevated potassium saline that was applied currents from cultured hippocampal neurons that had been grown in by a 30 set pressure ejection pulse from a puffer pipette (-2-3 pm cell culture for 1-21 d. All experiments were performed at room tem- opening; 5-8 psi). In this saline, KC1 (50 mM) replaced NaCl. perature (22-24°C). Normal external saline contained (in mM) NaCl, Image processing and analysis was performed using either QFM ra- 140; HEPES, 10; KCl, 5; CaCl,, 2; MgCl,, 2; pH 7.35 with NaOH. tiometric software and a QX-7 processor (Quantex Corp.) or IMAGE- l/ Pipette solutions contained (in mM) K-gluconate, 140; EGTA, 10; Mg- FLUOR (v. 1.63g, Universal Imaging Corp.). Background subtracted ratio ATP, 4; GTP, 0.1; HEPES, 10; pH 7.35 with KOH. The osmolarity of images (340/380 nm or 3501380 nm) were used to calculate the [Caz+], the external saline was adjusted with sucrose to be 10 mOsm higher according to Equation 5 of Grynkiewicz et al. (1985). than the internal pipette solution. Pipettes fabricated from 1.5 mm o.d. Calibration of fura- was performed in situ (Thomas and Delaville, borosilicate glass had D.C. resistances of 30-7 Mn. Postsynaptic cells 199 1). Briefly, cells were permeabilized with the calcium ionophore were voltage clamped at a range of -80 to -30 mV to test for the 4-bromo-A23187 (10 PM; Molecular Probes) in the presence of 4 mM presence ofexcitatory and inhibitory synaptic transmission.