Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

PKC and CaMKII dependent synaptic potentiation in cultured cerebral neurons.

We have reported that the long-lasting potentiation of spontaneous excitatory postsynaptic currents (SEPSCs) was induced by a Mg(2+)-free treatment in cultured chick cerebral neurons and a factor(s) extracellularly released during the treatment could induce the potentiation by itself. In this paper, protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase type II (CaMKII) but not protein kinase A (PKA) were reported to contribute to the potentiation mechanism during a step between the activation of the N-methyl-D-aspartate receptors by the Mg(2+)-free treatment and the secretion of the protein factor(s).

Synaptic potentiation induced by a protein factor in cultured cerebral neurons.

1. We reported in a previous paper that long-lasting enhancement of spontaneous excitatory post synaptic currents (SEPSCs) in cultured chick cerebral neurons was induced by exposure to a conditioned medium (CM) prepared by Mg(2+)-free treatment of neurons. This suggested that the CM contained a diffusible factor(s) for the potentiation. 2. In this paper, the factor(s) was shown to be a protein(s) by heat and trypsin treatment of the CM. 3. The factor induced the potentiation within 5 min, but it was not required for maintenance of increased SEPSCs. 4. The factors in CM induced the potentiation without protein synthesis. 5. Protein synthesis at least in postsynaptic neurons, was indispensable to induce the potentiation by the Mg(2+)-free condition.

Long-lasting enhancement of synaptic activity in dissociated cerebral neurons induced by brief exposure to Mg2+-free conditions.

The long-lasting enhancement of periodic clusters of spontaneous excitatory postsynaptic currents (SEPSCs) was examined in dissociated chick cerebral neurons that had been transiently exposed to Mg2+-free solution for 15 min. Since the enhancement was diminished by blockade of synaptic transmission, it clearly depended on synaptic activities. A specific antagonist of N-methyl-D-aspartate receptors (NMDARs) also inhibited the potentiations. Furthermore, the presence of inhibitors of protein and RNA synthesis in the Mg2+-free solution blocked the potentiation. In the potentiated neurons, the frequency of miniature excitatory postsynaptic currents (mEPSPs) increased. In addition, a diffusible molecule(s) that promoted the potentiation appeared to be involved in this phenomenon, since the conditioned medium of Mg2+-free treated neurons enhanced synaptic activity in other dish.