Takumi Komatsu

Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks.

The aim of this study was to clarify the saturation processes of excitatory and inhibitory synapse densities during the long-term development of cultured neuronal networks. For this purpose, we performed a long-term culture of rat cortical cells for 35 days in vitro (DIV). During this culture period, we labeled glutamatergic and GABAergic synapses separately using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of γ-aminobutyric acid (VGAT). The densities and distributions of both types of synaptic terminals were measured simultaneously. Observations and subsequent measurements of immunofluorescence demonstrated that the densities of both types of antibody-labeled terminals increased gradually from 7 to 21-28 DIV. The densities did not show a further increase at 35 DIV and tended to become saturated. Triple staining with VGluT1, VGAT, and microtubule-associated protein 2 (MAP2) enabled analysis of the distribution of both types of synapses, and revealed that the densities of the two types of synaptic terminals on somata were not significantly different, but that glutamatergic synapses predominated on the dendrites during long-term culture. However, some neurons did not fall within this distribution, suggesting differences in synapse distribution on target neurons. The electrical activity also showed an initial increase and subsequent saturation of the firing rate and synchronized burst rate during long-term culture, and the number of days of culture to saturation from the initial increase followed the same pattern under this culture condition.

Contrary effects of octopamine receptor ligands on behavioral and neuronal changes in locomotion of Lymnaea.

The pond snail Lymnaea stagnalis moves along the sides and bottom of an aquarium, but it can also glide upside down on its back below the waters surface. We have termed these two forms of locomotion “standard locomotion” and “upside-down gliding,” respectively. Previous studies showed that standard locomotion is produced by both cilia activity on the foot and peristaltic contraction of the foot muscles, whereas upside-down gliding is mainly caused by cilia activity. The pedal A neurons are thought to receive excitatory octopaminergic input, which ultimately results in increased cilia beating. However, the relationship between locomotory speed and the responses of these neurons to octopamine is not known. We thus examined the effects of both an agonist and an antagonist of octopamine receptors on locomotory speed and the firing rate of the pedal A neurons. We also examined, at the electron and light-microscopic levels, whether structural changes occur in cilia following the application of either an agonist or an antagonist of octopamine receptors to the central nervous system (CNS). We found that the application of an octopamine antagonist to the CNS increased the speed of both forms of locomotion, whereas application of octopamine increased only the firing rate of the pedal A neurons. Microscopic examination of the cilia proved that there were no changes in their morphology after application of octopamine ligands. These data suggest that there is an unidentified octopaminergic neuronal network in the CNS whose activation reduces cilia movement and thus locomotory speed.

Network confluence in a rat neuronal culture

P2-f14 Involvement of Olig2 in the neural circuit formation in the fetal mouse forebrain Katsuhiko Ono 1,5 , Carlos M. Parras 2, Hirohide Takebayashi 3, Kenji Shimamura 4, Hitoshi Gotoh 1,5, Kazuhiro Ikenaka 5 1 Dept. of Biol., Kyoto Pref. Univ. Medicine 2 Institute of the Brain and Spinal Cord (ICM), Inserm-UPMC, Paris, France 3 Dept. of Morphological Neural Science, Grad. Sch. of Medical Sciences, Kumamoto Univ., Kumamoto, Japan 4 Dept. of Brain Morphogenesis, Inst. Mol. Embryol. Genetics, Kumamoto Univ., Kumamoto, Japan 5 Div. of Mol. Neurobiol. Bioinfo., NIPS, Okazaki, Japan