Developmental changes of GABAergic synapses formed between primary cultured cortical neurons.

The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.

[1]  R. Grantyn,et al.  Development of GABAergic synaptic connections in vivo and in cultures from the rat superior colliculus. , 1990, Brain research. Developmental brain research.

[2]  C R Houser,et al.  Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[3]  T. Kosaka,et al.  GABAergic axon terminals at perisomatic and dendritic inhibitory sites show different immunoreactivities against two GAD isoforms, GAD67 and GAD65, in the mouse hippocampus: A digitized quantitative analysis , 1998 .

[4]  J. Barker,et al.  GABA receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex. , 2000, Cerebral cortex.

[5]  K. Kuriyama,et al.  GABAA receptor stimulation enhances NMDA-induced Ca2+ influx in mouse cerebral cortical neurons in primary culture. , 1994, Brain research. Molecular brain research.

[6]  S. Schuchmann,et al.  Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures. , 2002, Journal of neurophysiology.

[7]  X. Leinekugel,et al.  Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. , 1995, The Journal of physiology.

[8]  S. Schuchmann,et al.  Ca2+ signalling and changes of mitochondrial function during low‐Mg2+‐induced epileptiform activity in organotypic hippocampal slice cultures , 2001, The European journal of neuroscience.

[9]  Juha Voipio,et al.  Cation–chloride co-transporters in neuronal communication, development and trauma , 2003, Trends in Neurosciences.

[10]  Jang-Yen Wu,et al.  The fine structural localization of glutamate decarboxylase in developing axonal processes and presynaptic terminals of rodent cerebellum , 1975, Brain Research.

[11]  Y. Ben-Ari,et al.  Early sequential formation of functional GABAA and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus , 2002, The European journal of neuroscience.

[12]  N. Akaike,et al.  Regulation of Intracellular Chloride by Cotransporters in Developing Lateral Superior Olive Neurons , 1999, The Journal of Neuroscience.

[13]  Xavier Leinekugel,et al.  Ca2+ Oscillations Mediated by the Synergistic Excitatory Actions of GABAA and NMDA Receptors in the Neonatal Hippocampus , 1997, Neuron.

[14]  C. Houser,et al.  Prominent Expression of Two Forms of Glutamate Decarboxylase in the Embryonic and Early Postnatal Rat Hippocampal Formation , 1996, The Journal of Neuroscience.

[15]  J. A. Payne,et al.  The K+/Cl− co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation , 1999, Nature.

[16]  H. Thoenen,et al.  GABAergic Stimulation Regulates the Phenotype of Hippocampal Interneurons through the Regulation of Brain-Derived Neurotrophic Factor , 1996, Neuron.

[17]  W. Maxwell Cowan,et al.  Rat hippocampal neurons in dispersed cell culture , 1977, Brain Research.

[18]  Kazuo Kobayashi,et al.  Functional synapse formation between cultured neurons of rat cerebral cortex. Block by a protein kinase inhibitor which does not permeate the cell membrane.:Block by a Protein Kinase Inhibitor which does not Permeate the Cell Membrane , 1988 .

[19]  Y. Ben-Ari.  Excitatory actions of gaba during development: the nature of the nurture , 2002, Nature Reviews Neuroscience.

[20]  S. Schuchmann,et al.  Coupling of Electrical and Metabolic Activity During Epileptiform Discharges , 2002, Epilepsia.

[21]  A Kawana,et al.  Periodic synchronized bursting and intracellular calcium transients elicited by low magnesium in cultured cortical neurons. , 1993, Journal of neurophysiology.

[22]  I Khalilov,et al.  GABA is the principal fast-acting excitatory transmitter in the neonatal brain. , 1999, Advances in neurology.

[23]  J. Wolff,et al.  Plasticity in dendrites shown by continuous GABA administration in superior cervical ganglion of adult rat , 1978, Nature.

[24]  Arnold R. Kriegstein,et al.  Is there more to gaba than synaptic inhibition? , 2002, Nature Reviews Neuroscience.

[25]  A. Kriegstein,et al.  An excitatory GABAergic plexus in developing neocortical layer 1. , 2000, Journal of neurophysiology.

[26]  J. Barker,et al.  GABA Expression Dominates Neuronal Lineage Progression in the Embryonic Rat Neocortex and Facilitates Neurite Outgrowth via GABAA Autoreceptor/Cl− Channels , 2001, The Journal of Neuroscience.

[27]  J. Barker,et al.  GABA(B) receptors mediate motility signals for migrating embryonic cortical cells. , 2001, Cerebral cortex.

[28]  J. Dubinsky.  Development of inhibitory synapses among striatal neurons in vitro , 1989, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[29]  K. Muramoto,et al.  Frequency of synchronous oscillations of neuronal activity increases during development and is correlated to the number of synapses in cultured cortical neuron networks , 1993, Neuroscience Letters.

[30]  A. Ogura,et al.  Block of synapse formation between cerebral cortical neurons by a protein kinase inhibitor , 1992, Neuroscience Letters.

[31]  Masahiro Kawahara,et al.  Formation and maturation of synapses in primary cultures of rat cerebral cortical cells: an electron microscopic study , 1993, Neuroscience Research.

[32]  Y. Ben-Ari,et al.  Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons , 1993, Neuroscience Letters.

[33]  O. Garaschuk,et al.  Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus , 1998, The Journal of physiology.

[34]  J. Barker,et al.  GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[35]  M. Poo,et al.  GABA Itself Promotes the Developmental Switch of Neuronal GABAergic Responses from Excitation to Inhibition , 2001, Cell.

[36]  A. Kriegstein,et al.  GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis , 1995, Neuron.

[37]  K. Muramoto,et al.  Bicuculline induces synapse formation on primary cultured accessory olfactory bulb neurons , 2003, The European journal of neuroscience.

[38]  J. Voipio,et al.  BDNF-induced TrkB activation down-regulates the K+–Cl− cotransporter KCC2 and impairs neuronal Cl− extrusion , 2002, The Journal of cell biology.

[39]  O. Garaschuk,et al.  Large-scale oscillatory calcium waves in the immature cortex , 2000, Nature Neuroscience.

[40]  J. Yamada,et al.  Differential development of cation-chloride cotransporters and Cl− homeostasis contributes to differential GABAergic actions between developing rat visual cortex and dorsal lateral geniculate nucleus , 2003, Brain Research.

[41]  Y. Ben-Ari,et al.  Giant synaptic potentials in immature rat CA3 hippocampal neurones. , 1989, The Journal of physiology.

[42]  M. Miller,et al.  Development of Projection and Local Circuit Neurons in Neocortex , 1988 .

[43]  A. Kriegstein,et al.  Excitatory GABA Responses in Embryonic and Neonatal Cortical Slices Demonstrated by Gramicidin Perforated-Patch Recordings and Calcium Imaging , 1996, The Journal of Neuroscience.