European Journal of Neuroscience, Vol. 11, pp. 389–397, 1999 © European Neuroscience Association Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons in culture Alberto Bacci, Claudia Verderio, Elena Pravettoni and Michela Matteoli CNR-Cellular and Molecular Pharmacology and ‘B. Ceccarelli’ Centers, Dept of Medical Pharmacology, University of Milano, via Vanvitelli 32, 20129 Milano, Italy Keywords: calcium channels, oscillations, rat hippocampal neurons, synaptic transmission Abstract We have detected spontaneous, synchronous calcium oscillations, associated with variations in membrane potential, in hippocampal neurons maintained in primary culture. The oscillatory activity is synaptically driven, as it is blocked by tetrodotoxin, by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and by toxins inhibiting neurotransmitter release from presynaptic nerve endings. Neuronal oscillations do not require for their expression the presence of a polyneuronal network and are not primarily influenced by the γ-aminobutyric acid (GABA A ) receptor antagonist picrotoxin, suggesting that they entirely rely on glutamatergic neurotransmission. Synaptic and intrinsic conductances shape the synchronized oscillations in hippocampal neurons. The concomitant activation of N-methyl-D-aspartate (NMDA) receptors and voltage-activated L-type calcium channels allows calcium entering from the extracellular medium and sustaining the long depolarization, which shapes every single calcium wave. Introduction Clusters of synchronized depolarizations, detectable in the form of burst firing or as intracellular calcium oscillations, represent a typical example of interactions in neural circuits and characterize neurons during physiological or pathophysiological events. Bursts of syn- chronous neuronal firing can be considered as units of neural information, playing a primary role in brain function, as well as in synaptic plasticity and information processing (Lisman, 1997). During the nervous system development, neuronal oscillations have been shown to regulate patterning of connections (Shatz, 1990), to direct neuronal differentiation (Spitzer, 1994; Gu & Spitzer, 1995), and to regulate the rate of neuronal cell migration (Komuro & Rakic, 1996). Bursts of firing have also been related to phenomena occurring during epileptic seizures (Miles & Wong, 1983; Patel et al., 1988; Lodge & Palmer, 1994). Synchronous neuronal activity has also been observed in cultured neurons by optical recordings of the cytosolic-free calcium concentra- tions ([Ca 2+ ] i ), a non-invasive method which makes it possible to simultaneously monitor activity in groups of neurons. Results from these studies have indicated that multiple mechanisms may be responsible for the generation of calcium oscillations, which may either originate from intracellular rapidly exchanging stores (Friel & Tsien, 1992), or reflect calcium entry across the plasma membrane (Murphy et al., 1992; Lawrie et al., 1993; Nunez et al., 1996). We have investigated spontaneous, synchronous calcium oscilla- tions, in primary cultures of embryonic hippocampal neurons. This experimental model which has been widely characterized in terms of synaptic development and functionality (for reviews see Craig & Banker, 1994; Matteoli et al., 1995), provided a convenient, accessible system for the investigation of synchronous neuronal activity within Correspondence: M. Matteoli, as above. E-mail: MichelaM@Farma.csfic.mi.cnr.it Received 23 April 1998, revised 17 July 1998, accepted 21 August 1998 a defined neuronal cell population. We demonstrate that calcium and membrane potential oscillations are dependent on the establishment of functional synaptic contacts, but do not require for their expression the presence of a polyneuronal network. We also demonstrate that synaptic and intrinsic conductances shape the synchronized oscilla- tions in cultured hippocampal neurons, with calcium entering from the extracellular medium through N-methyl-D-aspartate (NMDA) receptors and voltage-activated channels. Materials and methods Hippocampal cell culture Primary neuronal cultures were prepared from the hippocampi of 18- day-old foetal rats, as described by Banker & Cowan (1977) and Bartlett & Banker (1984). Briefly, hippocampi were dissociated by treatment with trypsin (0.25% for 15 min at 37 °C), followed by trituration with a fire-polished Pasteur pipette. Dissociated cells were plated onto glass coverslips coated with poly-L-lysine in MEM with 10% horse serum at densities ranging from 250 000–400 000 cells/ cm 2 . After a few hours, coverslips were transferred to dishes containing a monolayer of cortical glial cells (Booher & Sensenbrenner, 1972), so that they were suspended over the glial cells but not in contact with them (Bartlett & Banker, 1984). Cells were maintained in MEM (Gibco, Milan, Italy) without sera, supplemented with N2 (Gibco), 2mM glutamine and 1 mg/mL BSA (neuronal medium). A modifica- tion of the method of Furshpan and collaborators (Furshpan et al., 1976) was used to grow single neurons on a small island of substrate, consisting of a fine mist of poly-L-lysine sprayed on glass coverslips. Fura-2 videomicroscopy and electrophysiology Neurons were loaded for 30 min at 37 °C with 2–4 μM fura-2 pentacetoxy-methylester in Krebs–Ringer solution buffered with Hepes (in mM: NaCl, 150; KCl, 5; MgSO 4 , 1.2; CaCl 2 , 2; glucose,