Kenji Mizuseki

Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell–Derived Inducing Activity

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

Directed differentiation of telencephalic precursors from embryonic stem cells

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells (∼90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation (∼35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6+ cells yield up to 75% of Bf1+ cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1+ and/or Islet1/2+ cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.

Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity

We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH+) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH+ neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH+ neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (≈5 weeks). In addition, 8% of the colonies contain large patches of Pax6+-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinsons disease and retinitis pigmentosa.

Theta oscillations provide temporal windows for local circuit computation in the entorhinal-hippocampal loop.

Theta oscillations are believed to play an important role in the coordination of neuronal firing in the entorhinal (EC)-hippocampal system but the underlying mechanisms are not known. We simultaneously recorded from neurons in multiple regions of the EC-hippocampal loop and examined their temporal relationships. Theta-coordinated synchronous spiking of EC neuronal populations predicted the timing of current sinks in target layers in the hippocampus. However, the temporal delays between population activities in successive anatomical stages were longer (typically by a half theta cycle) than expected from axon conduction velocities and passive synaptic integration of feed-forward excitatory inputs. We hypothesize that the temporal windows set by the theta cycles allow for local circuit interactions and thus a considerable degree of computational independence in subdivisions of the EC-hippocampal loop.

Cross-frequency phase-phase coupling between theta and gamma oscillations in the hippocampus

Neuronal oscillations allow for temporal segmentation of neuronal spikes. Interdependent oscillators can integrate multiple layers of information. We examined phase–phase coupling of theta and gamma oscillators in the CA1 region of rat hippocampus during maze exploration and rapid eye movement sleep. Hippocampal theta waves were asymmetric, and estimation of the spatial position of the animal was improved by identifying the waveform-based phase of spiking, compared to traditional methods used for phase estimation. Using the waveform-based theta phase, three distinct gamma bands were identified: slow gammaS (gammaS; 30–50 Hz), midfrequency gammaM (gammaM; 50–90 Hz), and fast gammaF (gammaF; 90–150 Hz or epsilon band). The amplitude of each sub-band was modulated by the theta phase. In addition, we found reliable phase–phase coupling between theta and both gammaS and gammaM but not gammaF oscillators. We suggest that cross-frequency phase coupling can support multiple time-scale control of neuronal spikes within and across structures.

Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells

To understand the range of competence of embryonic stem (ES) cell-derived neural precursors, we have examined in vitro differentiation of mouse and primate ES cells into the dorsal- (neural crest) and ventralmost (floor plate) cells of the neural axis. Stromal cell-derived inducing activity (SDIA; accumulated on PA6 stromal cells) induces cocultured ES cells to differentiate into rostral CNS tissues containing both ventral and dorsal cells. Although early exposure of SDIA-treated ES cells to bone morphogenetic protein (BMP)4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the fourth day of coculture causes differentiation of neural crest cells and dorsalmost CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. Notably, high concentrations of Shh efficiently promote differentiation of HNF3β+ floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naïve precursors that have the competence of differentiating into the “full” dorsal–ventral range of neuroectodermal derivatives in response to patterning signals.

The log-dynamic brain: how skewed distributions affect network operations

We often assume that the variables of functional and structural brain parameters — such as synaptic weights, the firing rates of individual neurons, the synchronous discharge of neural populations, the number of synaptic contacts between neurons and the size of dendritic boutons — have a bell-shaped distribution. However, at many physiological and anatomical levels in the brain, the distribution of numerous parameters is in fact strongly skewed with a heavy tail, suggesting that skewed (typically lognormal) distributions are fundamental to structural and functional brain organization. This insight not only has implications for how we should collect and analyse data, it may also help us to understand how the different levels of skewed distributions — from synapses to cognition — are related to each other.

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

Hippocampal CA1 pyramidal cells form functionally distinct sublayers

Hippocampal CA1 pyramidal neurons have frequently been regarded as a homogeneous cell population in biophysical, pharmacological and modeling studies. We found robust differences between pyramidal neurons residing in the deep and superficial CA1 sublayers in rats. Compared with their superficial peers, deep pyramidal cells fired at higher rates, burst more frequently, were more likely to have place fields and were more strongly modulated by slow oscillations of sleep. Both deep and superficial pyramidal cells fired preferentially at the trough of theta oscillations during maze exploration, whereas deep pyramidal cells shifted their preferred phase of firing to the peak of theta during rapid eye movement (REM) sleep. Furthermore, although the majority of REM theta phase-shifting cells fired at the ascending phase of gamma oscillations during waking, nonshifting cells preferred the trough. Thus, CA1 pyramidal cells in adjacent sublayers can address their targets jointly or differentially, depending on brain states.

Relationships between Hippocampal Sharp Waves, Ripples, and Fast Gamma Oscillation: Influence of Dentate and Entorhinal Cortical Activity

Hippocampal sharp waves (SPWs) and associated fast (“ripple”) oscillations (SPW-Rs) in the CA1 region are among the most synchronous physiological patterns in the mammalian brain. Using two-dimensional arrays of electrodes for recording local field potentials and unit discharges in freely moving rats, we studied the emergence of ripple oscillations (140–220 Hz) and compared their origin and cellular–synaptic mechanisms with fast gamma oscillations (90–140 Hz). We show that (1) hippocampal SPW-Rs and fast gamma oscillations are quantitatively distinct patterns but involve the same networks and share similar mechanisms; (2) both the frequency and magnitude of fast oscillations are positively correlated with the magnitude of SPWs; (3) during both ripples and fast gamma oscillations the frequency of network oscillation is higher in CA1 than in CA3; and (4) the emergence of CA3 population bursts, a prerequisite for SPW-Rs, is biased by activity patterns in the dentate gyrus and entorhinal cortex, with the highest probability of ripples associated with an “optimum” level of dentate gamma power. We hypothesize that each hippocampal subnetwork possesses distinct resonant properties, tuned by the magnitude of the excitatory drive.