Elsevier

Brain Research

Volume 795, Issues 1–2, 8 June 1998, Pages 137-146
Brain Research

Research report
Functional synapses in synchronized bursting of neocortical neurons in culture

https://doi.org/10.1016/S0006-8993(98)00283-2Get rights and content

Abstract

Spontaneous electrical activities in pairs of neocortical neurons in culture were simultaneously recorded using a whole cell current clamp technique. Synchronous bursting activities were observed in all 59 pairs tested. In 52 pairs of neurons electrically stimulated, EPSPs were recorded in 20 pairs (39%), among which 3 pairs (6%) showed bidirectional coupling. The response latency observed was 4.05±0.61 ms (mean±S.E.M.). The synaptic delay was estimated at 1.5–1.9 ms, suggesting the response latency is derived from a polysynaptic connection. The burst latency which was defined as the time difference of the onset of bursting in each neuron was 5.87±0.47 ms (mean±S.E.M.), and was weakly correlated with the spatial distance between the neurons (37.5–600 μm apart) (Rs=0.362, tied P value=0.0065). No synchronized bursting was observed in bathing solution with a low Ca2+ concentration (0.4 mM) or in bathing solution containing 50 μM D-AP5 and 15 μM CNQX. No dye-coupling between bursting neurons was observed on injection of the small molecule dye Lucifer yellow or the neurotracer neurobiotin. Disrupting neural connections completely by cutting the cell layer, caused disappearance of synchronized bursting with each neuron bursting independently. In conclusion, these results are consistent with the hypothesis that synchronized bursting in cultured neocortical neurons is attributed to connections by way of several synapses rather than by way of gap junctions and/or diffusible factors.

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.

References (43)

  • R. Yuste et al.

    Neuronal domains in developing neocortex: mechanisms of coactivation

    Neuron

    (1995)
  • N. Akaike et al.

    Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms

    Jpn. J. Physiol.

    (1994)
  • T. Araki et al.

    Correlation of the inhibitory postsynaptic potential of motoneurones with latency and time course of inhibition of monosynaptic reflexes

    J. Physiol. (London)

    (1960)
  • T. Bal et al.

    Synchronized oscillations in the inferior olive are controlled by the hyperpolarization-activated cation current Ih

    J. Neurophysiol.

    (1997)
  • L.S. Benardo

    Recruitment of GABAergic inhibition and synchronization of inhibitory interneurons in rat neocortex

    J. Neurophysiol.

    (1997)
  • Y. Ben Ari et al.

    Giant synaptic potentials in immature rat CA3 hippocampal neurons

    J. Physiol. (London)

    (1989)
  • A. Bragin et al.

    Gamma (40–100 Hz) oscillation in the hippocampus of the behaving rat

    J. Neurosci.

    (1995)
  • G. Buzsaki et al.

    High-frequency network oscillation in the hippocampus

    Science

    (1992)
  • B.W. Connors et al.

    Electrophysiological properties of neocortical neurons in vitro

    J. Neurophysiol.

    (1982)
  • S.M. Crain et al.

    Organotypic bioelectric activity in cultured reaggregates of dissociated rodent brain cells

    Science

    (1972)
  • S.M. Crain, Neurophysiologic Studies in Tissue Culture. Raven Press, New York, 1976, pp....
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