Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.

Neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (ChR2). Here, we quantitatively investigated the rapidness of the light-gated current of ChR2 expressed in PC12 cells using blue light-emitting diode (LED) light. The light-gated current consists of two components, inactivating and non-inactivating. The magnitude of inactivating component was almost linearly related to the light intensity. The non-inactivating component showed a tendency to saturate at high illumination. Both the activation and inactivation rates of the light-gated current were linearly dependent on the light intensity. However, the activation rate (turning-on rate) is about 10-fold faster than the inactivation rate. Although the turning-off time constant was little dependent on the light intensity, that at the end of 1s light pulse was about two-fold larger than that at 20 ms. Neurons are also made photosensitive by the expression of ChR2 in the living animal. Since both the turning-on and turning-off time constants of light-gated current was smaller than the membrane time constant of neurons, the LED light illumination of the photosensitive neurons was enough to evoke action potentials in a pulse-to-pulse manner in an acute slice of hippocampus.

Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.

Organotypic slice culture preserves the morphological and physiological features of the hippocampus of live animals for a certain time. The hippocampus is one of exceptional regions where neurons are generated intrinsically and spontaneously throughout postnatal life. We investigated the possibility that neurons are generated continuously at the dentate granule cell layer (GCL) in slice culture of the rat hippocampus. Using 5-bromodeoxyuridine (BrdU) labelling and retrovirus vector transduction methods, the phenotypes of the newly generated cells were identified immunohistochemically. At 4 weeks after BrdU exposure, BrdU-labelled cells were found in the GCL and were immunoreactive with a neuronal marker, anti-NeuN. There were fibrils immunoreactive with anti-glial fibrillary acidic protein (GFAP), an astrocyte marker, in the layer covering the GCL and occasionally encapsulated BrdU-labelled nuclei. When the newly divided cells were marked with the enhanced green fluorescent protein (EGFP) using a retrovirus vector, these cells had proliferative abilities throughout the following 4-week cultivation period. Four weeks after the inoculation, the EGFP-expressing cells consisted of various phenotypes of both early and late stages of differentiation; some were NeuN-positive cells with appearances of neurons in the GCL and some were immunoreactive with anti-Tuj1, a marker of immature neurons. Some EGFP-expressing cells were immunoreactive with anti-GFAP or anti-nestin, a marker of neural progenitors. The present study suggests that slice cultures intrinsically retain spontaneous neurogenic abilities for their cultivation period. The combination of slice culture and retrovirus transduction methods enable the newly divided cells to be followed up for a long period.