Sachiko Yoshida

GABA imaging in brain slices using immobilized enzyme-linked photoanalysis

GABA plays an important role in inhibitory neurotransmission. In the developing brain, GABA also acts as a paracrine chemical mediator. To evaluate the ambient GABA gradients in the brain, an enzyme-linked imaging system that consisted of GABase and NADP(+) was developed. In rat cerebellar slices, GABA release was observed in the layers containing GABAergic neurons. In telencephalic slices from embryonic GAD67-GFP knock-in mice, ambient GABA levels were high in the ganglionic eminence, where GABA cells are generated, but missing in homozygotes. This study indicates that this method will be useful to study the topography and dynamics of ambient GABA concentrations.

Accumulation of GABAergic Neurons, Causing a Focal Ambient GABA Gradient, and Downregulation of KCC2 Are Induced During Microgyrus Formation in a Mouse Model of Polymicrogyria

Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase–green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl– transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl– concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca2+ oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca2+ oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development.

Numerical analysis of ultrasound propagation and reflection intensity for biological acoustic impedance microscope

This paper proposes a new method for microscopic acoustic imaging that utilizes the cross sectional acoustic impedance of biological soft tissues. In the system, a focused acoustic beam with a wide band frequency of 30-100 MHz is transmitted across a plastic substrate on the rear side of which a soft tissue object is placed. By scanning the focal point along the surface, a 2-D reflection intensity profile is obtained. In the paper, interpretation of the signal intensity into a characteristic acoustic impedance is discussed. Because the acoustic beam is strongly focused, interpretation assuming vertical incidence may lead to significant error. To determine an accurate calibration curve, a numerical sound field analysis was performed. In these calculations, the reflection intensity from a target with an assumed acoustic impedance was compared with that from water, which was used as a reference material. The calibration curve was determined by changing the assumed acoustic impedance of the target material. The calibration curve was verified experimentally using saline solution, of which the acoustic impedance was known, as the target material. Finally, the cerebellar tissue of a rat was observed to create an acoustic impedance micro profile. In the paper, details of the numerical analysis and verification of the observation results will be described.

Properties of Wave Propagation in the Oscillatory Neural Network in Limax marginatus

Abstract The olfactory center (procerebral lobe; PC lobe) of the terrestrial slug, Limax marginatus, shows oscillatory behavior with a frequency of about 1 Hz and an activity wave that propagates from the apical to distal regions of the PC surface. To study the dynamic properties of this oscillatory network, serotonin, glutamate and acetylcholine were applied to the PC lobe. Serotonin and acetylcholine increased the frequency of PC oscillation and decreased the velocity of wave propagation. The effect of serotonin on the frequency was long-lasting and there was a delay before it caused a decrease in the wave propagation velocity. In contrast, the effect of acetylcholine on the frequency was phasic, and no delay was observed. Glutamate first decreased, then increased, the frequency. However, specific changes in the wave propagation velocity were not observed. From these experimental results, it is suggested that the oscillatory neural network of PC lobe has a potential to represent odor information as a series of spatially and temporally distributed ensembles of coherent firing neurons.

P1H-8 Development of Cerebella Tissue of Rat Characterized by Acoustic Impedance Microscope

Acoustic microscopy is expected to be a powerful tool for observing biological matters without chemical staining. We have proposed a new method for two-dimensional acoustic impedance imaging for biological tissue that can perform micro-scale observation without preparing a sliced specimen. A tissue was attached on a 0.5 mm-thick plastic substrate. An acoustic pulse was transmitted from the rear side of the substrate. The reflection intensity was interpreted into local acoustic impedance of the target tissue. In the previous report, we demonstrated the outline of the system and the result of preliminary observation, showing its feasibility. This report deals with the optimization of the observation method, and characterization of the tissue of developing cerebellum. The result shows that change in acoustic impedance of each cerebellar layer depending on postnatal day was correspond to change of structure with growth

Measurement of the signal from a cultured cell using a high-Tc SQUID

Stem cells in developing tissues give rise to the multiple specialized cell types that make up the heart, lung, skin, and other tissues. For scientists, it is important to know the stages of stem cell development. We propose a new method to obtain knowledge of the cell development stages by using a high-Tc SQUID magnetometer. In the first step of the research, we used Wister rat myocardial cells incubated for 12–14 days in a culture dish as a sample, which were not derived from stem cells. With the cells we tried to measure the signal using a SQUID magnetometer. We were able to measure the magnetic signals, which may be generated by ion transport through the cell membrane. A beat signal with a period of 0.6 s was observed. The peak-to-peak value was about 400 pT. This showed good agreement with the result of microscopic observation.

Numerical analysis of acoustic impedance microscope utilizing acoustic lens transducer to examine cultured cells

A new technique is proposed for non-contact quantitative cell observation using focused ultrasonic waves. This technique interprets acoustic reflection intensity into the characteristic acoustic impedance of the biological cell. The cells are cultured on a plastic film substrate. A focused acoustic beam is transmitted through the substrate to its interface with the cell. A two-dimensional (2-D) reflection intensity profile is obtained by scanning the focal point along the interface. A reference substance is observed under the same conditions. These two reflections are compared and interpreted into the characteristic acoustic impedance of the cell based on a calibration curve that was created prior to the observation. To create the calibration curve, a numerical analysis of the sound field is performed using Fourier Transforms and is verified using several saline solutions. Because the cells are suspended by two plastic films, no contamination is introduced during the observation. In a practical observation, a sapphire lens transducer with a center frequency of 300 MHz was employed using ZnO thin film. The objects studied were co-cultured rat-derived glial (astrocyte) cells and glioma cells. The result was the clear observation of the internal structure of the cells. The acoustic impedance of the cells was spreading between 1.62 and 1.72 MNs/m(3). Cytoskeleton was indicated by high acoustic impedance. The introduction of cytochalasin-B led to a significant reduction in the acoustic impedance of the glioma cells; its effect on the glial cells was less significant. It is believed that this non-contact observation method will be useful for continuous cell inspections.

Non-contact observation of cultured cells by acoustic impedance microscope

We are proposing the acoustic microscope for imaging cross sectional acoustic impedance of biological tissues. A focused acoustic beam is transmitted to the object placed on the ldquorear surfacerdquo of a plastic substrate. By scanning the focal point on the surface, a 2-D reflection intensity profile is obtained. A reference material is observed under the same condition. The reflection is interpreted into characteristic acoustic impedance. This paper deals with a new system with a high resolution for observing cultured cells. A pulse voltage was applied to a ZnO type transducer with a sapphire rod. The frequency range of the acoustic wave employed was 200 - 400 MHz. A plastic film of 50 - 70 mum in thickness was used as the substrate. Rat-derived glial (astrocyte) cells were cultured on the film. The reflection was converted into local acoustic impedance at the focal spot. The distance between adjacent pixels was 2 mum, however, the diameter of the focal spot would be as large as 3 - 5 mum. The image reveals the morphological features of astrocyte. Calibration and compensation of acoustic impedance by considering oblique incidence is demonstrated. The acoustic impedance of the cells was spreading between 1.5 - 1.7 MN s/m3. We believe that this quantitative method is useful for observing cultured cells in vivo.

Sound field analysis for biological acoustic impedance microscope for its precise calibration

Acoustic impedance microscopy for biological soft tissues was proposed. A target is placed on a plastic substrate. Ultrasonic beam, which is focused on the target, is transmitted and the reflection is received by the same transducer. The reflection is normalized by using pure water, and interpreted into acoustic impedance. As the beam is focused, oblique incident analysis is required to acquire a precise interpretation. Sound potential is calculated at a particular plane, and decomposed into plane wave components with different wave numbers using Fourier Transform. Both pressure and shear waves are generated and taken into account, when oblique incident impinging the substrate. As a pulsed wave is propagating, pressure and shear waves can be separated in time domain. Reflection signal is calculated for each plane wave component, and the integral through the k-space represents the received signal. As the substrate has higher acoustic impedance than target, the normalized reflection intensity reduces with the increase in acoustic impedance of the target. The experimental plots were acquired by using different contents of saline solutions. They agreed with the calculation results by sound field analysis. Frequency dependence is negligible in the region of 30 - 100 MHz. By scanning the transducer, an acoustic impedance microimage was acquired and calibrated based on the above analysis.

Charactarization techniques of particular proteins in cerebellar cortex using acoustic impedance microscope

Two-dimensional acoustic impedance imaging is useful for observation of living organs without invasion. Because acoustic impedance is proportional to sonic speed and density, organelle having larger density, e.g. nucleus, showed higher impedance. We have proved that cerebellar cortical layers and Purjinje cell bodies were identified using the acoustic impedance microscopy. In order to visualize the distribution of specific functional proteins in acoustic imaging, we proposed direct or complex-including heavy metal treatment to elevate the density and acoustic impedance of a particular protein. Heavy metal binding was useful for acoustic impedance imaging; however, metal binding to a protein molecule was not always specific. To observe the distribution of wanted molecules, we investigated two types of heavy metal binding materials; one was p-cymene ruthenium (Ru)-binding calcium channel binding peptides, and another was cadmiun nanocrystal binding antibodies, QdotTM. We could observe the characterized acou...