PROTOPLAST PATCH-CLAMPING USING AN UPRIGHT MICROSCOPE WITH A MOVABLE STAGE

Investigating membrane properties of plants is a challenging task, considering that success of experiments is highly dependent on the possibility to isolate metabolically active protoplasts that can withstand membrane current recordings. The aim of the present work is to obtain viable protoplasts derived from root cells of Pisum sativum that can be used for the whole-cell patch clamp. We designed the procedure of the pea protoplasts isolation that delivers stable protoplasts with preserved membrane integrity suitable for electrophysiological experiments. We applied a custom approach for patch-clamping protoplasts using a microscope with a movable microscope stage. We recorded prominent inward and prominent outward types of membrane current profiles of protoplasts. Obtained data indicate that optimized isolation protocol and custom system for patch clamping, can be applied to study membrane properties of root protoplasts.kategorija M3

MEMBRANE CURRENTS OF C6 CELLS IN PHARMACOLOGICAL INVESTIGATION

Living cells exhibit multiple ion channel proteins and malfunction of these channels underlies numerous diseases. Channelopathies include diseases of the nervous, cardiovascular, respiratory, endocrine, urinary, and immune system. Currently, ion channels represent the second-largest target for existing drugs. Here, using the whole-cell patch-clamp technique, we explored the epinephrine effect on membrane ionic currents in glioma C6 cells. We demonstrate that epinephrine specifically evokes an increase of C6 cells outward ionic currents that is stable within 10 min, while it does not affect inward currents. Our results thus provide fine resolution and time frame for targeting ion channel activity that is crucial in pharmacological investigations.kategorija M3

The effects of nembutal on the intracerebellar EEG activity revealed by spectral and fractal analysis

A detailed analysis of the anesthetic-induced modulation of intracerebellar electrical activity is an important step to understand the functional brain responses to anesthesia. We examined the electrical activity recorded from different cortical layers: molecular layer (ML), Purkinje cell layer (PCL), granular layer (GL) and the white matter (WM) in the vermian part of rat cerebellar lobule V during Nembutal anesthesia using spectral and fractal analysis. Spectral analysis revealed a difference in the mean relative power of delta (0.1-4.0 Hz) and theta (4.1-8.0 Hz) frequencies through the cerebellar layers. Compared to the ML, delta activity increased significantly in the GL, while theta activity decreased in the GL and the WM. Fractal analysis revealed that the mean value of Higuchi's fractal dimension (HFD) increased, starting from the ML to the WM. Theta activity exhibited a negative correlation with the HFD value in the ML. In contrast, the gamma activity showed a positive correlation with the HFD value in the ML and the GL. The combined use of spectral and fractal analyses revealed that Nembutal displays different effects on rat cerebellar electrical activity, which largely depends on the neurochemical and electrophysiological organization of the cerebellar layers