Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures

Synapse formation is a fundamental process in neurons that occurs throughout development, maturity, and aging. Although these stages contain disparate and fluctuating numbers of mature neurons, tactics employed by neuronal networks to modulate synapse number as a function of neuronal density are not well understood. The goal of this study was to utilize an in vitro model to assess the influence of cell density and neuronal maturity on synapse number and distribution. Specifically, cerebral cortical neurons were plated in planar culture at densities ranging from 10 to 5000 neurons/mm², and synapse number and distribution were evaluated via immunocytochemistry over 21 days in vitro (DIV). High-resolution confocal microscopy revealed an elaborate three-dimensional distribution of neurites and synapses across the heights of high-density neuronal networks by 21 DIV, which were up to 18 μm thick, demonstrating the complex degree of spatial interactions even in planar high-density cultures. At 7 DIV, the mean number of synapses per neuron was less than 5, and this did not vary as a function of neuronal density. However, by 21 DIV, the number of synapses per neuron had jumped 30- to 80-fold, and the synapse-to-neuron ratio was greatest at lower neuronal densities (< 500 neurons/mm²; mean approximately 400 synapses/neuron) compared to mid and higher neuronal densities (500-4500 neurons/mm²; mean of approximately 150 synapses/neuron) (p<0.05). These results suggest a relationship between neuronal density and synapse number that may have implications in the neurobiology of developing neuronal networks as well as processes of cell death and regeneration.

[1]  T. Arendt,et al.  Transgenic activation of Ras in neurons increases synapse formation in mouse neocortex , 2005, Journal of Neural Transmission.

[2]  M. Browning,et al.  Differential distribution of the synapsins in the rat olfactory bulb , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[3]  B. Pakkenberg,et al.  Aging and the human neocortex , 2003, Experimental Gerontology.

[4]  Ari Glezer,et al.  A microperfused incubator for tissue mimetic 3D cultures , 2009, Biomedical microdevices.

[5]  P. De Camilli,et al.  Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[6]  A. N. van den Pol,et al.  Early synaptogenesis in vitro: Role of axon target distance , 1998, The Journal of comparative neurology.

[7]  Virginia M. Y. Lee,et al.  Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytes , 1999, The Journal of comparative neurology.

[8]  Early synaptogenesis in vitro: Role of axon target distance , 1998 .

[9]  Paul J. Harrison,et al.  Synaptophysin protein and mRNA expression in the human hippocampal formation from birth to old age , 2006, Hippocampus.

[10]  Michelle C LaPlaca,et al.  Neuronal response to high rate shear deformation depends on heterogeneity of the local strain field. , 2006, Journal of neurotrauma.

[11]  Thomas J. O'Shaughnessy,et al.  Functional synapse formation among rat cortical neurons grown on three-dimensional collagen gels , 2003, Neuroscience Letters.

[12]  R. Oppenheim Cell death during development of the nervous system. , 1991, Annual review of neuroscience.

[13]  R. Moore,et al.  Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin I immunoreactivity , 1989, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[14]  P. De Camilli,et al.  The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[15]  A. N. van den Pol,et al.  Excitatory actions of GABA in developing rat hypothalamic neurones. , 1996, The Journal of physiology.

[16]  B. Pakkenberg,et al.  Ageing of substantia nigra in humans: 
cell loss may be compensated by hypertrophy , 2002, Neuropathology and applied neurobiology.

[17]  Paul J. Harrison,et al.  Synaptophysin gene expression in human brain: A quantitative in situ hybridization and immunocytochemical study , 1994, Neuroscience.

[18]  J. DeFelipe,et al.  Microstructure of the neocortex: Comparative aspects , 2002, Journal of neurocytology.

[19]  Steve M. Potter,et al.  An extremely rich repertoire of bursting patterns during the development of cortical cultures , 2006, BMC Neuroscience.

[20]  A. Frankfurter,et al.  The distribution of tau in the mammalian central nervous system , 1985, The Journal of cell biology.

[21]  Michelle C LaPlaca,et al.  High rate shear strain of three-dimensional neural cell cultures: a new in vitro traumatic brain injury model. , 2005, Journal of biomechanics.

[22]  D. Bernard,et al.  Appearance of neurofilament subunit epitopes correlates with electrophysiological maturation in cortical embryonic neurons cocultured with mature astrocytes. , 1996, Brain research. Developmental brain research.

[23]  G. D. de Polavieja,et al.  Age-Independent Synaptogenesis by Phosphoinositide 3 Kinase , 2006, The Journal of Neuroscience.

[24]  L. Martin,et al.  Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. , 2002, Journal of neurobiology.

[25]  Bruce C Wheeler,et al.  Added astroglia promote greater synapse density and higher activity in neuronal networks. , 2007, Neuron glia biology.

[26]  P. Greengard,et al.  Differential expression of synapsins I and II among rat retinal synapses , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[27]  B. R. Sastry,et al.  Activity-mediated shift in reversal potential of GABA-ergic synaptic currents in immature neurons. , 2005, Brain research. Developmental brain research.

[28]  R. Khazipov,et al.  γ-Aminobutyric acid (GABA): a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life , 1994 .

[29]  Gong Chen,et al.  Sequential Postsynaptic Maturation Governs the Temporal Order of GABAergic and Glutamatergic Synaptogenesis in Rat Embryonic Cultures , 2007, The Journal of Neuroscience.

[30]  Hwai-Jong Cheng,et al.  Axon Pruning and Synaptic Development: How Are They per-Plexin? , 2006, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[31]  E. Hernández-Echeagaray,et al.  Similar Synapse Density in Layer IV Columns of the Primary Somatosensory Cortex of Transgenic Mice with Different Brain Size: Implications for Mechanisms Underlying the Differential Allocation of Cortical Space , 2004, Brain, Behavior and Evolution.

[32]  D. K. Cullen,et al.  Strain rate-dependent induction of reactive astrogliosis and cell death in three-dimensional neuronal–astrocytic co-cultures , 2007, Brain Research.

[33]  Robert H. Lee,et al.  Three-dimensional neural constructs: a novel platform for neurophysiological investigation , 2008, Journal of neural engineering.

[34]  G. Brewer Serum‐free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus , 1995, Journal of neuroscience research.

[35]  L. Lanfumey,et al.  Life-Long Hippocampal Neurogenesis: Environmental, Pharmacological and Neurochemical Modulations , 2007, Neurochemical Research.

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

[37]  R. Crowther,et al.  Molecular characterization of microtubule-associated proteins tau and map2 , 1991, Trends in Neurosciences.

[38]  G. Brewer,et al.  Optimized survival of hippocampal neurons in B27‐supplemented neurobasal™, a new serum‐free medium combination , 1993, Journal of neuroscience research.

[39]  M. Oudega,et al.  Neurotrophins Reduce Degeneration of Injured Ascending Sensory and Corticospinal Motor Axons in Adult Rat Spinal Cord , 2002, Experimental Neurology.

[40]  D. K. Cullen,et al.  Collagen-Dependent Neurite Outgrowth and Response to Dynamic Deformation in Three-Dimensional Neuronal Cultures , 2007, Annals of Biomedical Engineering.

[41]  A. Glezer,et al.  Microfluidic engineered high cell density three-dimensional neural cultures , 2007, Journal of neural engineering.

[42]  M. Poo,et al.  Excitatory GABA Action Is Essential for Morphological Maturation of Cortical Neurons In Vivo , 2007, The Journal of Neuroscience.