Amane Koizumi

Spatiotemporal Recapitulation of Central Nervous System Development by Murine Embryonic Stem Cell-Derived Neural Stem/Progenitor Cells

Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here we demonstrate that we can recapitulate the spatiotemporal regulation of central nervous system (CNS) development in vitro by using a neurosphere‐based culture system of embryonic stem (ES) cell‐derived NS/PCs. This ES cell‐derived neurosphere system enables the efficient derivation of highly neurogenic fibroblast growth factor‐responsive NS/PCs with early temporal identities and high cell‐fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming epidermal growth factor‐responsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the glial fibrillary acidic protein promoter, similar to that observed in the developing brain. Moreover, the rostrocaudal and dorsoventral spatial identities of the NS/PCs can be successfully regulated by sequential administration of several morphogens. These NS/PCs can differentiate into early‐born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild‐type and disease model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders.

Potential functional neural repair with grafted neural stem cells of early embryonic neuroepithelial origin

The fate of grafted neuroepithelial stem cells in the normal mature brain environment was assessed both morphologically and electrophysiologically to confirm their feasibility in the functional repair of damaged neural circuitry. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of transgenic green fluorescence protein-carrying rat embryos, and implanted into the normal adult rat striatum. The short- and long-term differentiation pattern of donor-derived cells was precisely monitored immunohistochemically. The functional abilities of the donor-derived cells and communication between them and the host were investigated using host-rat brain slices incorporating the graft with whole-cell patch-clamp recording. Vigorous differentiation of the neuroepithelial stem cells into mostly neurons was noted in the short-term with positive staining for tyrosine hydroxylase, suggesting that the donor-derived cells were exclusively following their genetically programmed fate, together with gamma-aminobutyric acid (GABA) and glutamate expression. In the long-term, the large number of donor-derived neurons was sustained, but the staining pattern showed expression of dopamine- and adenosine 3:5-monophosphate-regulated phosphoprotein 32, suggesting that some neurons were following environmental cues, together with the appearance of some cholinergic neurons. Some donor-derived astrocytes were also seen in the graft. Many action potentials indicating the presence of both dopaminergic and non-dopaminergic patterns could be elicited and recorded in the donor-derived neurons in addition to spontaneous glutamatergic and GABAergic post-synaptic currents which were strongly shown to be of host origin. Neuroepithelial stem cells are therefore an attractive candidate as a source of donor material for intracerebral grafting in functional repair.

NMDA receptor-mediated depolarizing after-potentials in the basal dendrites of CA1 pyramidal neurons

It was shown recently that the basal dendrites of cortical pyramidal neurons generate NMDA-spikes. In the present study, we made whole-cell recordings from hippocampal CA1 pyramidal neurons and examined whether NMDA receptor activation was involved in synaptic responses. At low input stimulus intensity, EPSPs with a fast decay time were induced. As the intensity of stimulation was increased in the presence of GABA receptor antagonists, a depolarizing after-potential (DAP) was generated in addition to a fast decaying potential. A DAP was never observed when the input was applied to the apical dendrites. The DAP was suppressed by hyperpolarization or by NMDA receptor antagonists, but not by Na+, K+, or Ca2+ channel blockers. One possible mechanism is that the morphology of the basal dendrites favors DAP generation. A compartmental model simulation showed that synaptic inputs to thinner shorter dendrites generated a potential that resembled a DAP. Our study shows that a synaptic input to the basal dendrites of a hippocampal pyramidal neuron can generate a NMDA receptor-mediated potential in the presence of GABA receptor blockade.

Tetrodotoxin-sensitive persistent current boosts the depolarization of retinal amacrine cells in goldfish

To light illumination retinal amacrine cells respond with graded depolarization accompanied by a spike discharge. It has been assumed that the graded depolarization is produced solely by the excitatory synaptic inputs from bipolar cells. Here we demonstrate that a tetrodotoxin (TTX)-sensitive persistent current also contributes to the graded depolarization. This current was isolated in amacrine cells of the goldfish retinal slice preparations by the whole-cell patch clamp technique. The activation threshold of the persistent current was about -50 mV, approximately 10 mV more positive than the membrane potential in the dark. Therefore, it is highly likely that the TTX-sensitive persistent current is a booster of the excitatory postsynaptic potential in amacrine cells.

Multiple spatiotemporal patterns of dendritic Ca2+ signals in goldfish retinal amacrine cells

Although it has been reported that dendritic neurotransmitter releases from amacrine cells are regulated by the intracellular Ca(2+) concentration ([Ca(2+)](i)), their spatiotemporal patterns are not well explained. Fast Ca(2+) imagings of amacrine cells in the horizontal slice preparation of goldfish retinas under whole-cell patch-clamp recordings were undertaken to better investigate the spatiotemporal patterns of dendritic [Ca(2+)](i). We found that amacrine cell dendrites showed inhomogeneous [Ca(2+)](i) increases in both Na(+) spiking cells and cells without Na(+) spikes. The spatiotemporal properties of inhomogeneous [Ca(2+)](i) increases were classified into three patterns: local, regional and global. Local [Ca(2+)](i) increases were observed in very discrete regions and appeared as discontinuous patches, presumably evoked by local excitatory postsynaptic potentials. Regional [Ca(2+)](i) increases were observed in either a single or a small number of dendrites, presumably reflecting the result of dendritic action potentials. Global [Ca(2+)](i) increases were observed in the entire dendrites of a cell and were mediated by Na(+) action potentials or multiple Na(+) action potentials riding on slow depolarization. Ca(2+)-mediated potentials also evoked global [Ca(2+)](i) increase in cells without Na(+) spikes. These spatiotemporal dynamics of dendritic Ca(2+) signals may reflect multiple modes of synaptic integration on the dendrites of amacrine cells.

Fast Calcium Imagings of Amacrine Cell Dendrites in Horizontally Sliced Goldfish Retina

Amacrine cells are interneurons that make lateral and vertical connections in the inner plexiform layer of the retina. Amacrine cells have no axon and their dendrites function as both presynaptic and postsynaptic sites. GABAergic amacrine cells constitute 80% of the amacrine cell population in goldfish retina and mediate lateral inhibition between neighboring amacrine cells. Their light-evoked responses consist of regenerative action potentials and excitatory postsynaptic potentials (EPSPs). These depolarizing voltage changes induce Ca2+ influx into dendrites, which triggers transmitter release from the storage site in the dendrite. Thus, it is crucial to know how and where Ca2+ influx is caused in amacrine cells.

Functional Roles of Action Potentials and Na Currents in Amacrine Cells

Amacrine cells are retinal interneurons that play important roles in information processing in the inner plexiform layer. It is known that the major population of amacrine cells are y-aminobutyric acid (GABA)-ergic or glycinergic. However, of over 20 morphological subtypes [1], functional roles are known in only two subtypes: glycinergic A2 amacrine cells and cholinergic starburst amacrine cells. GABAergic cells are thought to be inhibitory and their major roles are thought to send inhibitory feedback to bipolar cells, mutual inhibition to neighboring amacrine cells, and feed-forward inhibition to ganglion cells. Most amacrine cells lack an axon and their dendrites function not only as the input site but also as the output site. Therefore, the strength of inhibition is expected to depend on the magnitude of depolarization and the length of its propagation within dendritic processes. Here, we summarize our recent works on the functional role of the action potential and the sustained depolarization induced by transient and persistent Na currents in the information processing of amacrine cells.

Spatial Asymmetry and Temporal Delay of Inhibitory Amacrine Cells Produce Directional Selectivity in Retina

Directional selectivity is a unique function relating to agility that some portion of ganglion cells in the retina fire only for moving light signals with specific direction and speed [1]. Taylor et al. [2] showed that inhibitory synaptic outputs from inhibitory amacrine cells to directional selective ganglion cells are playing a critical role in directional selectivity in the rabbit retina. We previously reported that dendrites of inhibitory amacrine cells have active regenerative properties to propagate action potentials [3]. γ-Aminobutyric acid releases from the dendrites were driven by action potential propagation into the dendrites. The speed of action potential propagation in dendrites of amacrine cells was approximately 10 m/s, which is one tenth slower than that of an axon. Thus, initiation and propagation of action potentials on amacrine cell dendrites cause a temporal delay in synaptic outputs to ganglion cells. In addition, asymmetric expansion of dendrites of amacrine cells causes asymmetric synaptic outputs to ganglion cells. Here, we established a novel hypothesis that these features of inhibitory amacrine cells might play an important role in forming directional selectivity. In order to clarify the mechanism of directional selectivity, we numerically analyzed the whole retina activity using a recently developed neural-network simulation powered by NEURON, which models each cell’s electrophysiological activity. All known experimental facts reported to date are explained in a consistent manner by our hypothesis on the connection of cells that direction-selective ganglion cells are receiving inhibitory synaptic inputs from amacrine cell dendrites with a random spatial asymmetry and a temporal delay.