Yasuhiro Nakagami

Optical recording of trisynaptic pathway in rat hippocampal slices with a voltage-sensitive dye

Changes in membrane potentials were recorded from rat hippocampal slices with a voltage-sensitive dye using a real-time optical recording system, which had high spatial resolution of 128 x 128 points with a high time resolution of 0.6 ms. Serial excitatory propagation was recorded in the dentate gyrus. CA3 and CA1 after stimulation of the perforant pathway, and the optical signals were clearly divided into two components in the dentate gyrus adjacent to the stimulus site. The slow component was suppressed in Ca(2+)-free solution, but the fast component in the molecular layer was not affected. However, the application of 1 microM tetrodotoxin fully abolished both components. These results suggest that the fast and slow components mainly reflect Na(+)-dependent action potentials and excitatory postsynaptic potentials, respectively. The excitatory response duration in the stratum radiatum of CA3 was significantly longer than that in other hippocampal areas. The long-lasting excitation in CA3 is probably related to the CA3 associational projections, because direct stimulation of CA3 pyramidal cell layer also produced similar results. The long-lasting dendritic excitation is probably important to integrate synaptic transmission and may be related to epileptogenesis. When long-term potentiation was induced by a tetanic stimulation (100 Hz for 1 s), the onset latency in the stratum radiatum of CA1 was reduced to as much as 65%, suggesting an increase of excitatory propagation. The analysis of the spatial-temporal optical signals contributes to understanding information processes in the hippocampus, related to learning and memory including long-term potentiation.

Optical recording of rat entorhino-hippocampal system in organotypic culture

It is difficult to comprehend the entorhino-hippocampal information processing using acute transverse hippocampal slice, because the dorsally inclined connections of the entorhino-hippocampal projections can be damaged easily. Therefore, we investigated the spatial-temporal propagation in organotypic cultures of the hippocampus attaching to the entorhinal cortex using a real-time optical recording system with a voltage-sensitive dye and suitability as an in vitro model. Real-time imaging demonstrated that the stimulation of the perforant pathway induced excitatory propagation in trisynaptic pathway of the hippocampus and sequentially in the layer V from the medial to the lateral entorhinal cortex. The horizontal propagation from the lateral to the medial site was also seen after the stimulation of the lateral entorhinal cortex. The analysis of the entorhino-hippocampal organotypic culture would contribute to understanding of the mechanism of learning and memory.

The regional vulnerability to blockade of action potentials in organotypic hippocampal culture.

We investigated how blockade of spontaneous action potentials influenced the synaptogenesis by measuring the field population spike using hippocampal organotypic cultures. Although respective blockade of inhibitory and excitatory neurotransmission by picrotoxin and CNQX did not significantly induce cell death in all hippocampal area, sodium channel blocking drugs (tetrodotoxin or lidocaine) caused specific and severe damage and affected the formation of functional synapse in CA1 and the entorhinal cortex but not in CA3. It is suggested that the spontaneous action potentials would play a critical role during synaptogenesis.