Makoto Osanai

Transretinal Electrical Stimulation with a Suprachoroidal Multichannel Electrode in Rabbit Eyes

PurposeSeveral approaches for placing an electrode device for visual prosthesis have been previously proposed. In this study, we investigated if transretinal stimulation from the suprachoroidal space can elicit an electrical evoked potential (EEP) in albino rabbits.MethodsA flat electrode array (polyimide plate, platinum electrode) was developed and used for this study. After performing a scleral incision at 2–2.5 mm from the limbus and placing an anchoring suture, the array was inserted into the suprachoroidal space in the posterior portion of the eye by direct observation under a microscope. A platinum wire was implanted into the vitreous space as a reference electrode. For electrical stimulation, a biphasic pulse was used. When the electrode was stimulated, the EEP was recorded.ResultsWhen the electrical stimulation from the suprachoroidal space was applied, the EEP could be recorded with an epidural electrode, and the threshold was 66.0 ± 32.1μA (42.0μC/cm2). Histological examination indicated the absence of major damage to the retina and choroid from the insertion and placement of the array and the electrical stimulation.ConclusionsTransretinal electrical stimulation from the suprachoroidal space could elicit EEP, suggesting that this approach may be useful for a retinal prosthesis system.

Cav2.3 (α1E) Ca2+ channel participates in the control of sperm function

To know the function of the Ca2+ channel containing α12.3 (α1E) subunit (Cav2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Cav2.3 channel. The averaged rising rates of Ca2+ transients induced by α‐D‐mannose–bovine serum albumin in the head region of Cav2.3−/− sperm were significantly lower than those of Cav2.3+/+ sperm. A computer‐assisted sperm motility assay revealed that straight‐line velocity and linearity were greater in Cav2.3−/− sperm than those in Cav2.3+/+ sperm. These results suggest that the Cav2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.

Photon starvation artifacts of X-ray CT: their true cause and a solution

When too few photons reach detector elements, strong streaks appear through paths of high X-ray attenuation and an image becomes completely useless. This photon starvation artifact phenomenon occurs frequently when a pelvis or shoulder is scanned with thin slices. The common understanding regarding photon starvation streaks is that they are a manifestation of irregularities caused by noise in the raw data profile. Therefore, the common countermeasure is local raw-data filtering, which detects and smoothes out the highly noisy part of the raw data. However, the photon starvation artifact can be solved only partly with such a method and a more effective solution is necessary. Here, we examined the mean level shift of raw data attributable to the nonlinear nature of logarithmic conversion, which is the process required for generating raw data from detected X-ray data. We judge that the real culprit of the photon starvation artifact is this mean level shift. When the noise level is very high or the photon level is very low, this mean level shift can become prominent and can become manifest as thick streaks against which the conventional local raw data filtering has no power. To solve this problem, we propose a new scheme of local raw data filtering, which consists of reverting log-converted raw data to a form that is equivalent to pre-log detector data. With this method, not only fine streaks, but also thick streaks are removed effectively. A better image quality with lower X-ray doses is possible with this method.

Electrical stimulation with a needle-type electrode inserted into the optic nerve in rabbit eyes.

PurposeTo investigate whether electrical stimulation of the optic nerve can elicit an electrical evoked potential (EEP) in rabbits and to determine whether such stimulation is a useful approach for the placement of a visual prosthesis.MethodsTwo needle-type electrodes were inserted into the optic nerve using a transvitreal approach. For electrical stimulation, monophasic and biphasic pulses were used. By stimulating the optic nerve, the EEP was elicited. After evaluation of the EEP, a histological study was carried out.ResultsWhen electrical stimulation was applied, the EEP could be recorded. The threshold with monophasic and biphasic stimulation was 10 ± 0 µA and 20 ± 8.2 µA, respectively. Histological examination revealed no major complications, such as bleeding or degeneration, which might have resulted from the insertion of electrodes or the electrical stimulation.ConclusionsElectrical stimulation of the optic nerve can elicit an EEP, suggesting that this approach may be useful for a visual prosthesis system. Jpn J Ophthalmol 2004;48:552–557

Development of a micro-imaging probe for functional brain imaging.

Multicellular neuronal activities should be investigated to reveal the dynamics of the neuronal circuit. Optical recording from neuronal populations is suitable for recording multicellular activities. We fabricated the prototype of the micro-imaging probe in combination with a gradient index lens and image fiber. This probe has a smaller diameter than traditional probes. We found an optimal optical configuration for maximizing the efficiency of the imaging probe. Using this optical configuration with the prototype of the imaging probe, the fluorescence images were captured from neurons expressing green fluorescent protein in a cerebellar block preparation, and the calcium-dependent images were sampled in a mouse brain slice preparation. Our optical system would facilitate the in vivo imaging studies with less invasive manners using thinner optic fiber than previously made.

Role of Cav2.3 (α1E) Ca2+ channel in ischemic neuronal injury

We investigated the role of the Ca v 2.3 (α 1E ) channel in ischemic neuronal injury using Ca v 2.3 mutant mice. In focal ischemia model with a complete occlusion of the middle cerebral artery in vivo, infarct at 24 h was significantly larger in Ca v 2.3 mutant mice compared with that in wild-type controls. In vitro Ca 2+ imaging studies using hippocampal slices revealed that oxygen-glucose deprivation induced a [Ca 2+ ] i increase in the hippocampal CA1 region more vigorously in Ca v 2.3 mutant mice than in wild-type controls, and that tetrodotoxin or bicuculline application abolished the difference between the genotypes. These results suggest that the Ca v 2.3 channel plays a protective role in ischemic neuronal injury by a mechanism in which GABAergic neuronal actions are involved.

Long-lasting spontaneous calcium transients in the striatal cells.

The striatum plays an important role in linking cortical activity to basal ganglia output. We conducted the calcium (Ca2+) imaging to investigate the spontaneous activities of the striatum using acute slice preparations. Corticostriatal slices of rat brain were stained with Fura-PE3-AM. Long-lasting spontaneous intracellular Ca2+ ([Ca2+]i) transients, which lasted up to about 250 s, were observed. The amplitudes of the transients were variable even in a single cell. Most cells exhibited irregular frequencies, but some exhibited oscillatory features. These [Ca2+]i transients were not induced by action potentials because they were not inhibited by tetrodotoxin. Antagonists of the ionotropic glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione and D,L-2-amino-5-phosphonovaleric acid, did not block these transients. These results suggested that the action potentials and the excitatory synaptic inputs in these striatal network were not involved in the induction of the [Ca2+]i transients. In contrast, the number of the active cells, which exhibited the [Ca2+]i transients, was greatly reduced by the intracellular Ca2+ store depletor, thapsigargin. Therefore, the intracellular Ca2+ store is likely to contribute to the [Ca2+]i transients.

Spatiotemporal Properties of the Action Potential Propagation in the Mouse Visual Cortical Slice Analyzed by Calcium Imaging

The calcium ion (Ca2+) is an important messenger for signal transduction, and the intracellular Ca2+ concentration ([Ca2+]i) changes in response to an excitation of the cell. To reveal the spatiotemporal properties of the propagation of an excitatory signal with action potentials in the primary visual cortical circuit, we conducted a Ca2+ imaging study on slices of the mouse visual cortex. Electrical stimulation of layer 4 evoked [Ca2+]i transients around the stimulus electrode. Subsequently, the high [Ca2+]i region mainly propagated perpendicular to the cortical layer (vertical propagation), with horizontal propagation being restricted. When the excitatory synaptic transmission was blocked, only weak and concentric [Ca2+]i transients were observed. When the action potential was blocked, the [Ca2+]i transients disappeared almost completely. These results suggested that the action potential contributed to the induction of the [Ca2+]i transients, and that excitatory synaptic connections were involved in the propagation of the high [Ca2+]i region in the primary visual cortical circuit. To elucidate the involvement of inhibitory synaptic connections in signal propagation in the primary visual cortex, the GABAA receptor inhibitor bicuculline was applied. In this case, the evoked signal propagated from layer 4 to the entire field of view, and the prolonged [Ca2+]i transients were observed compared with the control condition. Our results suggest that excitatory neurons are widely connected to each other over the entire primary visual cortex with recurrent synapses, and inhibitory neurons play a fundamental role in the organization of functional sub-networks by restricting the propagation of excitation signals.

Both neurons and astrocytes exhibited tetrodotoxin-resistant metabotropic glutamate receptor-dependent spontaneous slow Ca2+ oscillations in striatum.

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinsons disease. The group I mGluRs are known to modulate the intracellular Ca2+ signaling. To characterize Ca2+ signaling in striatal cells, spontaneous cytoplasmic Ca2+ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca2+ transients (referred to as slow Ca2+ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca2+ oscillation. Depletion of the intracellular Ca2+ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca2+ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca2+ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca2+ oscillation in both putative-neurons and astrocytes. The slow Ca2+ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca2+ oscillation may involve in the neuron-glia interaction in the striatum.

Quantitative activation-induced manganese-enhanced MRI reveals severity of Parkinson’s disease in mice

We demonstrate that activation-induced manganese-enhanced magnetic resonance imaging with quantitative determination of the longitudinal relaxation time (qAIM-MRI) reveals the severity of Parkinson’s disease (PD) in mice. We first show that manganese ion-accumulation depends on neuronal activity. A highly active region was then observed by qAIM-MRI in the caudate-putamen in PD-model mice that was significantly correlated to the severity of PD, suggesting its involvement in the expression of PD symptoms.