Research reportFunctional synapses in synchronized bursting of neocortical neurons in culture
Introduction
Highly correlated, spontaneous neuronal bursting activities have been reported in many regions of developing mammalian brain, such as the visual system 22, 37, 42, hippocampus 5, 35, 36, 38, locus coeruleus [16], inferior olive [3]and neocortex 12, 13, suggesting important roles for them in the signal processing of central nervous systems. Furthermore, high-frequency network oscillations have been observed both in slice preparations [7]and in living animals such as rats and monkeys 6, 27, 28, indicating that synchronized electrical activities in neurons are fundamental for integrated brain functions such as memory, learning, and recognition. However, the mechanism reported for oscillatory activities is complex because it is different in different systems and in different development stages.
There have also been reports of synchronous intracellular Ca2+ oscillations in hippocampal [31]and neocortical culture systems 18, 25, 26, 29. Furthermore, simultaneous measurement of intracellular Ca2+ and electrical activity revealed that neuronal bursts are generated periodically and accompanied by slower Ca2+ transients [34], indicating a coupling of neuronal synchronized bursting and an intracellular signal transduction.
Neuronal bursting and intracellular Ca2+ oscillation in culture are believed to be synchronized via a neural network of synapses, since these phenomena are attenuated by the NMDA receptor antagonist APV 12, 34. Using electron microscopy, Ichikawa et al. [15]found a synapse formation in cultured neocortical neurons showing a synchronized intracellular Ca2+ oscillation. However, it has not been demonstrated whether the functional synapses in these neurons are actually working.
To demonstrate that synchronized bursting neurons have synaptic connections with each other, we recorded electrical activities simultaneously in a pair of neurons with a patch clamp method. The time difference between the onset of bursting in each neuron (burst latency) was analyzed and compared with a synaptic delay estimated from evoked synaptic response. Our results suggest that synchronized bursting can be attributed to connections by way of several synapses rather than by way of gap junctions and/or diffusible factors.
Section snippets
Cell culture
Neocortical cells were prepared from the cerebral cortex of embryonic day 16–18 Wistar rats and cultured as described in the work of Nakanishi et al. [29]. Briefly, the cortices were digested with 0.02% papain and mechanically dissociated by trituration. The cells were plated on coverslips, which were coated with a 2-week old monolayer of astrocytes. The cell density was approximately 3×104 cells/cm2. The cells were maintained with Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10%
Bursting in cultured neocortical neurons is synchronized
Spontaneous electrical activities of two randomly chosen neurons in culture (12–25 DIV; mean±S.E.M.=16.1±0.31 DIV) were simultaneously recorded using two patch-clamp amplifiers in current clamp mode. The distance between two neurons ranged from 37.5 μm to 600 μm (mean±S.E.M.=159.3±16.8 μm). The mean resting potential was −61.4±0.7 mV (n=118), and the mean size of somata was 18.1±0.3 μm (n=98).
The neuronal pairs showed synchronously a periodic bursting accompanying a membrane depolarization.
Synchronized bursting neurons have functional synapses
We have presented evidence of functional synapses in pairs of spontaneously synchronized bursting neurons in culture. Furthermore, we found direct (mono and/or poly) synaptic responses in about 39% of pairs, although we observed synchronized bursting in all pairs. This means that direct synaptic connections in all neurons are not necessary for synchronization of bursting, and that direct synaptic connections in one-third of neurons are sufficient for synchronization.
The synaptic delay in our
Acknowledgements
We are grateful to Dr. Muneyuki Ito of the Department of Physiology, Institute for Developmental Research, Aichi Human Service Center, for helpful discussions. We thank Dr. Taiji Kato of the Department of Bioregulation Research, Nagoya City Univ. Med. Sch. and Dr. Shunichi Yamagishi of NIPS for their kind support of this work. We also thank the members of the Lab. of Membrane Biology at NIPS for their technical support. This research was supported in part by the Aichi Cancer Research Foundation.
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2015, Journal of Neuroscience MethodsCitation Excerpt :The pattern of coordinated activity followed a similar trend; the activity of young neurons was uncoordinated but became highly synchronized by DIV 14 and settled to a plateau by DIV 18 (Fig. 5C and D). This evolution of network activity is consistent with the (simplistic) model that neurons in culture undergo a period of rapid synaptic growth and strengthening, followed by synaptic pruning, reaching the completion of developmental synaptic modification at ∼18 DIV (Nakanishi and Kukita, 1998; Nakayama et al., 2005; Passafaro et al., 2003; Tetzlaff et al., 2010; Voigt et al., 2005). Emergence of synchrony and spontaneous activity is a complicated phenomenon that also depends on maturation of cell surface receptors, changes in ionic currents, and the relative balance between excitation and inhibition, to name a few (Klueva et al., 2008; McCabe et al., 2006; Misonou et al., 2004; Opitz et al., 2002; Turrigiano et al., 1994).
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