Hiroki Akiyama

Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro

The effects of uniform steady state (DC) extracellular electric fields on neuronal excitability were characterized in rat hippocampal slices using field, intracellular and voltage‐sensitive dye recordings. Small electric fields (<|40| mV mm−1), applied parallel to the somato‐dendritic axis, induced polarization of CA1 pyramidal cells; the relationship between applied field and induced polarization was linear (0.12 ± 0.05 mV per mV mm−1 average sensitivity at the soma). The peak amplitude and time constant (15–70 ms) of membrane polarization varied along the axis of neurons with the maximal polarization observed at the tips of basal and apical dendrites. The polarization was biphasic in the mid‐apical dendrites; there was a time‐dependent shift in the polarity reversal site. DC fields altered the thresholds of action potentials evoked by orthodromic stimulation, and shifted their initiation site along the apical dendrites. Large electric fields could trigger neuronal firing and epileptiform activity, and induce long‐term (>1 s) changes in neuronal excitability. Electric fields perpendicular to the apical–dendritic axis did not induce somatic polarization, but did modulate orthodromic responses, indicating an effect on afferents. These results demonstrate that DC fields can modulate neuronal excitability in a time‐dependent manner, with no clear threshold, as a result of interactions between neuronal compartments, the non‐linear properties of the cell membrane, and effects on afferents.

Attractive axon guidance involves asymmetric membrane transport and exocytosis in the growth cone

Asymmetric elevation of the Ca2+ concentration in the growth cone can mediate both attractive and repulsive axon guidance. Ca2+ signals that are accompanied by Ca2+-induced Ca2+ release (CICR) trigger attraction, whereas Ca2+ signals that are not accompanied by CICR trigger repulsion. The molecular machinery downstream of Ca2+ signals, however, remains largely unknown. Here we report that asymmetric membrane trafficking mediates growth cone attraction. Local photolysis of caged Ca2+, together with CICR, on one side of the growth cone of a chick dorsal root ganglion neuron facilitated the microtubule-dependent centrifugal transport of vesicles towards the leading edge and their subsequent vesicle-associated membrane-protein 2 (VAMP2)–mediated exocytosis on the side with an elevated Ca2+ concentration. In contrast, Ca2+ signals without CICR had no effect on the vesicle transport. Furthermore, pharmacological inhibition of VAMP2-mediated exocytosis prevented growth cone attraction, but not repulsion. These results strongly suggest that growth cone attraction and repulsion are driven by distinct mechanisms, rather than using the same molecular machinery with opposing polarities.

Control of Neuronal Growth Cone Navigation by Asymmetric Inositol 1,4,5-Trisphosphate Signals

Measurements of its spatial profile reveal the crucial role of asymmetric IP3 signals in growth cone navigation. IP3 Points the Way During nervous system development, neurites navigate to their targets by responding to cues in the environment. Attractive or repulsive turning depends on the generation of asymmetric changes in Ca2+ within the growth cone of the developing neurite. The source of these intracellular Ca2+ signals is critical to determining whether the response is attractive (with the growth cone turning toward the side with increased Ca2+ concentration) or repulsive (with the growth cone turning away from the side with increased Ca2+ concentration). Here, Akiyama et al. show that attractive turning to nerve growth factor (NGF) depends on phospholipase C (PLC)–dependent generation of asymmetric changes in inositol 1,4,5-trisphosphate (IP3) and the ensuing IP3-induced Ca2+ release from the endoplasmic reticulum. Dissection of the pathway showed that the turning response required basal cAMP signaling upstream of asymmetric IP3-induced Ca2+ release and required basal phosphatidylinositol 3-kinase (PI3K)–dependent signaling downstream. Inositol 1,4,5-trisphosphate (IP3) is generally viewed as a global messenger that increases cytosolic calcium ion (Ca2+) concentration. However, the spatiotemporal dynamics of IP3 and the functional significance of localized IP3 production in cell polarity remain largely unknown. Here, we demonstrate the critical role of spatially restricted IP3 signals in axon guidance. We found that IP3 and ensuing Ca2+ signals were produced asymmetrically across growth cones exposed to an extracellular gradient of nerve growth factor (NGF) and mediated growth cone turning responses to NGF. Moreover, photolysis-induced production of IP3 on one side of a growth cone was sufficient to initiate growth cone turning toward the side with the higher concentration of IP3. Thus, locally produced IP3 encodes spatial information that polarizes the growth cone for guided migration.

Phosphatidylinositol 3-kinase facilitates microtubule-dependent membrane transport for neuronal growth cone guidance

The activity of PI3K is necessary for polarized cell motility. To guide extending axons, environmental cues polarize the growth cone via asymmetric generation of Ca2+ signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, it remains unclear how PI3K is involved in such events for axon guidance. Here, we demonstrate that PI3K plays a permissive role in growth cone turning by facilitating microtubule (MT)-dependent membrane transport. Using embryonic chick dorsal root ganglion neurons in culture, attractive axon turning was induced by Ca2+ elevations on one side of the growth cone by photolyzing caged Ca2+ or caged inositol 1,4,5-trisphosphate. We show that PI3K activity was required downstream of Ca2+ signals for growth cone turning. Attractive Ca2+ signals, generated with caged Ca2+ or caged inositol 1,4,5-trisphosphate, triggered asymmetric transport of membrane vesicles from the center to the periphery of growth cones in a MT-dependent manner. This centrifugal vesicle transport was abolished by PI3K inhibitors, suggesting that PI3K is involved in growth cone attraction at the level of membrane trafficking. Consistent with this observation, immunocytochemistry showed that PI3K inhibitors reduced MTs in the growth cone peripheral domain. Time-lapse imaging of EB1 on the plus-end of MTs revealed that MT advance into the growth cone peripheral domain was dependent on PI3K activity: inhibition of the PI3K signaling pathway attenuated MT advance, whereas exogenous phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K-catalyzed reactions, promoted MT advance. This study demonstrates the importance of PI3K-dependent membrane trafficking in chemotactic cell migration.

βIV-spectrin forms a diffusion barrier against L1CAM at the axon initial segment

Axonal and somatodendritic plasma membranes of polarized neurons express distinct sets of functional molecules. It is known that the neuronal polarity can be maintained by a barrier that impedes diffusional mixing of membrane components between the two domains. Using betaIV-spectrin knockout mice, we demonstrate the involvement of this cytoskeletal protein in the formation of a barrier that selectively blocks lateral mobility of L1 cell adhesion molecule (L1CAM) at the axon initial segment of hippocampal neurons. We also show that the betaIV-spectrin-based barrier is required for the axon-specific distribution of L1CAM both in vitro and in vivo. The barrier activity against L1CAM may depend on direct interactions of L1CAM with ankyrinG, a protein binding to betaIV-spectrin, rather than on steric hindrance by other transmembrane proteins clustered at the axon initial segment. Our results highlight the role of betaIV-spectrin and ankyrinG as critical components of a selective barrier against L1CAM.

Optical detection of dendritic spike initiation in hippocampal CA1 pyramidal neurons.

Previous studies have shown that spikes can be generated in the dendrites of CA1 pyramidal neurons. Some have suggested that, in response to synaptic inputs, spikes are initiated near the soma and propagate back into the dendrites, but some recent studies have shown that intense synaptic inputs initiate spikes in the dendrite. Here, we report the optical detection of spike propagation along the apical dendrites of hippocampal pyramidal neurons. Rat hippocampal slices were stained with the fluorescent voltage-sensitive dye, JPW1114, and optical signals monitored using a 16 x 16 photodiode array system at a frame rate of 4 kHz. A stimulating electrode was placed at the boundary between the stratum (str.) lacnosum-moleculare and the str. radiatum to stimulate the Schaffer collateral, and fast and slow signal components were detected in the dendritic and somatic regions. By comparing the optical signals with whole-cell recordings, we confirmed that the fast component was due to a population of dendritic spikes in pyramidal neurons. The fast component appeared in dendritic locations near the input sites in response to synaptic activation, and signal onset at the soma was delayed by a few milliseconds compared with that at the input sites. Local perfusion of a Na(+) channel blocker near the soma eliminated the fast component at the soma, but had no effect on the fast component at the input sites. Our results indicate that dendritic spikes can be initiated in dendrites near the input site and propagate orthodromically toward the proximal dendrites and the soma.

Extracellular DC electric fields induce nonuniform membrane polarization in rat hippocampal CA1 pyramidal neurons

Non-synaptic interactions among neurons via extracellular electric fields may play functional roles in the CNS. Previously in a study using voltage-sensitive dye imaging, we reported characteristic membrane polarization profiles in the CA1 region of hippocampal slices during exposure to extracellular DC fields: slow monophasic polarization in somatic region and biphasic polarization (fast polarization and following slow repolarization) in mid-dendritic region. Here, using optical imaging and patch-clamp recordings, we showed that CA1 pyramidal neurons indeed show the characteristic polarization in response to DC fields, and investigated the mechanism underlying the profiles. Both the monophasic and biphasic polarization could be fitted with a double exponential function. The τs (ms) were 12.6±2.5 and 56.0±4.7 for the monophasic polarization, and 14.2±1.2 and 42.2±2.8 for the biphasic polarization. Based on our previous theoretical studies, we hypothesized that lower resistivity in the distal apical dendrites is responsible for generating the characteristic polarization profiles. We tested this hypothesis by removing the distal apical dendrites or by blocking ion channel-mediated conductance. Removal of distal dendrites caused drastic changes in the polarization profiles, e.g. biphasic polarization was damped. However, none of the blockers tested had a marked effect on the biphasic polarization. Our results demonstrate the importance of the apical dendrite for generating the characteristic polarization profiles, and suggest that voltage-activated conductance, including HCN channel-mediated conductance, had only minor contributions to these profiles. These findings provide a better understanding of how neurons in the CNS respond to extracellular electric fields.

Cyclic Nucleotide Control of Microtubule Dynamics for Axon Guidance.

Graded distribution of intracellular second messengers, such as Ca2+ and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca2+ have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca2+ signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca2+-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. SIGNIFICANCE STATEMENT Developing neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cones leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Second messenger networks for accurate growth cone guidance

Growth cones are able to navigate over long distances to find their appropriate target by following guidance cues that are often presented to them in the form of an extracellular gradient. These external cues are converted into gradients of specific signaling molecules inside growth cones, while at the same time these internal signals are amplified. The amplified instruction is then used to generate asymmetric changes in the growth cone turning machinery so that one side of the growth cone migrates at a rate faster than the other side, and thus the growth cone turns toward or away from the external cue. This review examines how signal specification and amplification can be achieved inside the growth cone by multiple second messenger signaling pathways activated downstream of guidance cues. These include the calcium ion, cyclic nucleotide, and phosphatidylinositol signaling pathways.

Enhancement of Critical Current Densities in (Ba,K)Fe2As2 by 320 MeV Au Irradiation in Single Crystals and by High-Pressure Sintering in Powder-in-Tube Wires

We demonstrate a large enhancement of critical current density (Jc) up to 1.0×107 A/cm2 at 5 K under self-field in (Ba,K)Fe2As2 single crystals by irradiating 320 MeV Au ions. With the very promising potential of this material in mind, we have fabricated a (Ba,K)Fe2As2 superconducting wire through a powder-in-tube method combined with the hot isostatic pressing technique, whose effectiveness has been proven in industrial Bi2223 tapes. The Jc in the wire at 4.2 K has reached 37 kA/cm2 under self-field and 3.0 kA/cm2 at 90 kOe. Magneto-optical imaging of the wire confirmed the large intergranular Jc in the wire core.