Neuritic differentiation and synaptogenesis in serum-free neuronal cultures of the rat cerebral cortex

Abstract

To better understand the dynamics of the cellular processes involved in early neocortical development, we studied the neuritic differentiation and synaptogenesis of dispersed neurons grown in serum-free cultures under a wide variety of culture conditions. Microtubule-associated protein (MAP2), phosphorylated neurofilament (SMI 31) and synaptophysin immunocytochemistry was complemented with time-lapse studies. During the first week in vitro dissociated cortical neurons developed from roundish cells without processes to neurons with axons and differentiated dendrites, going through five distinct phases. The sequence of these phases was unaltered in a wide range of culturing methods, but the timing of the steps varied among cultures started with different cell densities. Synaptic terminals were first observed after 3-4 days in vitro, coincident with the beginning of dendritic differentiation. Synaptogenesis progressed at least until the end of the third week in vitro, despite a decline in cell density during the second week in vitro. The process of cellular differentiation of cerebral cortical neurons in vitro resembled the development of these cells in the intact tissue, suggesting that organized cell migration is not a prerequisite for the differentiation of single cortical neurons.

Odor-Induced, Activity-Dependent Transneuronal Gene InductionIn Vitro: Mediation by NMDA Receptors

“…E xperimental manipulations were only performed on OB cultures after they had been 5 d in vitro. This "pre-experimental" culture time was essential to allow cells damaged by dissociation to perish and for surviving cells to form dendrites, axons, and synapses, which does not occur in cortical neuron cultures until 3-4 d in vitro (De Lima et al, 1997), and to recover electrophysiological responses to amino acids. Both IPSPs and EPSPs are observed in OB cultures at 5 d in vitro (Trombley and Westbrook, 1990;Trombley and Shepherd, 1992), indicating that OB cells at the "age" used in the present experiments are electrophysiologically competent and respond to excitatory amino acid stimulation.…”

The microtubule-stabilizing drug Epothilone D increases axonal sprouting following transection injury in vitro

“…For our particular investigations it was of great importance to determine maturity in vitro since previous investigations have demonstrated that the neuronal response to injury is dependent on neuronal maturity (Chuckowree and Vickers, 2003;Blizzard et al, 2007). Development of primary murine cortical neurons in vitro occurs via a sequence of steps across a defined time-course, involving neurite outgrowth, polarization and elongation followed by loss of immaturity markers such as growth cone markers and localization of synaptic proteins in dendritic spines as an indication of mature synapses (Dotti et al, 1988;Dichter, 1978;Picken Bahrey and Moody, 2003;De Lima et al, 1997;King et al, 2006). Neuronal development in culture in this study was assessed by the use of morphological, synaptic and electrophysiological characteristics.…”

The Effect of Including the C2 Insert of Nonmuscle Myosin II-C on Neuritogenesis

“…In our culture system, neuritogenesis followed a well defined stereotyped program: stage 1, within a few h (0 -2 h after changing to DM) of neuritogenesis, most cells started sprouting but were devoid of neurites; stage 2, within 2-4 h of neuritogenesis, neurons developed neurites and, depending on the number of neurites, cells' morphology could be divided into groups like unipolar, bipolar, and multipolar but none with established axon; stage 3, after 4 h of neuritogenesis, neurites started to grow and form axons, and the remaining neurites could not extend during this stage, consistent with a similar observation by de Lima et al (37). In control cells treated with nonspecific siRNA, we found that stage 1 and stage 2 were interchangeable Plasmid DNA containing GFP-tagged NMHC II-C isoform was transfected into 24-h post-siRNA-treated cells.…”