Hiroyoshi Miyakawa

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.

Reversal of long-term potentiation (depotentiation) induced by tetanus stimulation of the input to CA1 neurons of guinea pig hippocampal slices

The reduction of the long-term potentiated response induced by tetanus (depotentiation (DP) of LTP) was investigated by the delivery of a train of low-frequency afferent stimuli (depotentiating stimulation: DPS) after the tetanus (100 Hz, 100 pulses) in CA1 neurons of the guinea pigs hippocampal slice. The parameters of DPS (frequencies of 1, 2, 5 and 10 Hz; number of pulses of 200 and 1000; and the time-lag after tetanus of 20 and 100 min) were altered systematically and their effects on LTP were evaluated through the analysis of the slope of field EPSP (S-EPSP) and amplitude and peak latency of population spike (A- and L-PS). DPS of 1 Hz, 1000 pulses, given 20 min after tetanus, reduced the potentiated component of S-EPSP, A-PS and L-PS by 68.5%, 80.1% and 56.1%, respectively (mean, n = 6), whereas it reduced the control response by 4.3%, 7.1%, and 1.9%, respectively (n = 6). Significantly less effectiveness was observed for DPS at higher frequencies (2-10 Hz), with smaller numbers of pulses, featuring a longer time-lag after tetanus and under APV administration. When DPS was applied before tetanus, significantly less robust LTP was observed. However, these effects were blocked by the administration of APV during DPS.

Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels

Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CA1 pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.

Cytoplasmic calcium elevation in hippocampal granule cell induced by perforant path stimulation andl-glutamate application

Calcium-dependent fluorescence of a Ca2+ indicator (fura-2) loaded in the slice of guinea pig hippocampus was measured by a microscope/video-camera/photometry system. Tetanic stimulation of the perforant path (PP) or application of L-glutamate caused increment of the fluorescence from the dendritic and somatic layers of the granule cells in the dentate gyrus. Magnitude of the increment depended on the frequency and intensity of the PP-stimulation or on the dose of L-glutamate. 2-Aminophosphonovaleric acid, a glutamate-receptor antagonist, suppressed both PP-stimulus-induced and L-glutamate-evoked responses, while tetrodotoxin blocked the former only. Thus the fluorescence increment should represent an elevation of Ca2+ concentration in the postsynaptic cytoplasm of the granule cells.

Low-threshold potassium channels and a low-threshold calcium channel regulate Ca2+ spike firing in the dendrites of cerebellar Purkinje neurons: a modeling study

Various types of voltage-gated ion channels are distributed along the dendrites of neurons in the central nervous system. We have recently shown experimentally that the dendrites of cerebellar Purkinje neurons contain low-threshold voltage-gated Ca(2+) channels and low-threshold voltage-gated K+ channels. Although we found that these channels are involved in regulating the onset of Ca(2+)-dependent action potentials in the dendrites, we were unable to identify which of the known types of low-threshold Ca2+ channels and K+ channels were responsible, since there was no reliable method of discriminating between them. Here, we have built a detailed compartmental model of a Purkinje neuron by incorporating two types of low-threshold Ca2+ channel (T-type and class-E, or R-type) and two types of low-threshold K+ channel (A-type and D-type), in addition to another eight voltage-gated channel types, using a compartmental model neuron simulator. The model reproduces the basic features of the depolarization-induced responses of Purkinje neurons, such as fast Na+ spikes in the soma, Ca2+ spikes in the dendrites, the slow onset of Ca2+ spikes, repetitive Ca2+ spikes in the presence of TTX, the marked shortening of Ca2+ spike onset in the presence of 4-aminopydridine, and the longer Ca2+ spike onset in the presence of Ni2+. Our model shows that the D-type K+ channel and the class-E Ca2+ channel regulate the onset of depolarization-induced Ca2+ spikes in Purkinje neurons. These channels might be involved in integrating synaptic inputs in Purkinje neurons.

PACAP/PAC1 autocrine system promotes proliferation and astrogenesis in neural progenitor cells

The Pituitary adenylate cyclase‐activating peptide (PACAP) ligand/type 1 receptor (PAC1) system regulates neurogenesis and gliogenesis. It has been well established that the PACAP/PAC1 system induces differentiation of neural progenitor cells (NPCs) through the Gs‐mediated cAMP‐dependent signaling pathway. However, it is unknown whether this ligand/receptor system has a function in proliferation of NPCs. In this study, we identified that PACAP and PAC1 were highly expressed and co‐localized in NPCs of mouse cortex at embryonic day 14.5 (E14.5) and found that the PACAP/PAC1 system potentiated growth factor‐induced proliferation of mouse cortical NPCs at E14.5 via Gq‐, but not Gs‐, mediated PLC/IP3‐dependent signaling pathway in an autocrine manner. Moreover, PAC1 activation induced elongation of cellular processes and a stellate morphology in astrocytes that had the bromodeoxyuridine (BrdU)‐incorporating ability of NPCs. Consistent with this notion, we determined that the most BrdU positive NPCs differentiated to astrocytes through PAC1 signaling. These results suggest that the PACAP/PAC1 system may play a dual role in neural/glial progenitor cells not only differentiation but also proliferation in the cortical astrocyte lineage via Ca2+‐dependent signaling pathways through PAC1.

Detecting cells using non-negative matrix factorization on calcium imaging data.

We propose a cell detection algorithm using non-negative matrix factorization (NMF) on Ca2+ imaging data. To apply NMF to Ca2+ imaging data, we use the bleaching line of the background fluorescence intensity as an a priori background constraint to make the NMF uniquely dissociate the background component from the image data. This constraint helps us to incorporate the effect of dye-bleaching and reduce the non-uniqueness of the solution. We demonstrate that in the case of noisy data, the NMF algorithm can detect cells more accurately than Mukamels independent component analysis algorithm, a state-of-art method. We then apply the NMF algorithm to Ca2+ imaging data recorded on the local activities of subcellular structures of multiple cells in a wide area. We show that our method can decompose rapid transient components corresponding to somas and dendrites of many neurons, and furthermore, that it can decompose slow transient components probably corresponding to glial cells.

Active properties of dendritic membrane examined by current source density analysis in hippocampal CA1 pyramidal neurons

The laminar profile of extracellular field potentials evoked by alveus or stratum radiatum stimulation was recorded at the CA1 region of guinea pig hippocampal slices. One-dimensional approximation of the current source density analysis method was applied to the data. When the alveus was stimulated, the location of inward membrane current (sink) moved from the cell body layer to the dendritic region for a distance of at least 200 micron at a velocity of 0.16 +/- 0.03 m/s (n = 6). This sink movement was not blocked by either low- Ca2+ or Ca2+-free/high-Mg2+ medium, but was blocked by tetrodotoxin application restricted to the dendritic region by a local perfusion method. When the stratum radiatum was stimulated, sink movement from the dendritic region to the cell body layer was not distinct. Retrograde sink movement from the cell body layer to the dendritic region subsequent to the cell body spike was not observed. These findings indicated that the dendrites of the CA1 pyramidal neurons can generate action potentials which propagate along the dendrite and that they might primarily be mediated by Na ion.

Voltage-gated Ca2+ channel blockers, ω-AgalVA and Ni2+, suppress the induction of θ-burst induced long-term potentiation in guinea-pig hippocampal CA1 neurons

Abstract It is widely believed that a rise in post-synaptic calcium concentration ([Ca 2+ ];) is a necessary step in the induction of long-term potentiation (LTP) (Bliss and Collingridge, Nature, 361 (1993) 31–39). In this experiment, we examine the involvement of voltagegated Ca 2+ channels (VGCC) in the induction of AP5-sensitive LTP induced by θ-burst stimulation in guinea-pig hippocampal CA1 neurons. The VGCC blockers, Ni 2+ , (25 μM, T -channel blocker) or ω-AgaIVA (60 nM, P-channel blocker), which have no effect on synaptic transmission, suppress 60% or 78% of the Θ-burst induced UP, respectively. This implies that Ca 2+ entry through VGCC is an important step in this form of LTP.

Differential roles of two types of voltage-gated Ca2+ channels in the dendrites of rat cerebellar Purkinje neurons.

The distribution and function of voltage-gated Ca2+ channels in Purkinje neurons in rat cerebellar slices were studied using simultaneous Ca2+ imaging and whole-cell patch clamp recording techniques. Voltage-gated Ca2+ channels were activated by applying depolarizing voltage steps through the pipette attached at the soma in a voltage-clamp mode in the presence of tetrodotoxin. Poor space clamp due to extensive arborization of the dendrites allowed the dendrites to fire Ca2+ spikes. Ca2+ imaging with Fura-2 injected through the pipette, showed a steady [Ca2+]i increase at the soma and transient, spike-linked [Ca2+]i jumps in the dendrites. omega-Agatoxin-IVA (200 nM) abolished the depolarization-induced Ca2+ spikes, the spike-linked [Ca2+]i increase in the dendrites, and the steady [Ca2+]i increase at the soma. omega-Conotoxin-GVIA (5 microM) and nifedipine (3 microM) had no significant effect on the depolarization-induced responses. In the presence of 4-aminopyridine(2 mM) and omega-Agatoxin-IVA, transient [Ca2+]i increases remained in the dendrites. Low concentrations of Ni2+(100 microM) reversibly suppressed this [Ca2+]i increase. The voltage for half-maximal activation and inactivation of this component were lower than -50 mV and -31 mV, respectively. In normal conditions, low concentration of Ni2+ slowed the onset of the Ca2+ spike without changing the time course of the spikes or the amplitude of the accompanying [Ca2+]i increase. These results show that omega-Agatoxin-IVA-sensitive Ca2+ channels are distributed both in the soma and the dendrites, and are responsible for dendritic Ca2+ spikes, whereas low-voltage activated, Ni2+-sensitive Ca2+ channels are distributed in the whole dendrites including both thick and fine branches, and provide boosting current for spike generation.