Molecular Neurochemistry Laboratory

Focusing on Glu and GABA transporters and receptors, our lab is involved in the design and screening of potential drug candidates in a fruitful collaboration with other laboratories of the Department as well as with chemists within the Center. We also make strong efforts in understanding better the mechanisms underlying neurodegenerative diseases like epilepsy and ischemia and in searching for new targets for drug development.

Equipment

Binding and uptake assays are carried out in brain preparations or in transfected cells. To count radioactivity either a Wallac WinSpectral 1414 counter or a Perkin Elmer Microbeta 1450 LSC and luminescence plate reader is used. Release experiments are performed in a custom-made superfusion microvolume chamber. Fast kinetic studies are implemented on pressure-driven double mixing systems like the continuous/pulsed mode quenched flow machine using filter assay technique with radioactive detection or the HiTech Scientific SF-61 DX2 stopped-flow machine with UV/fluorescence detection.

Capabilities

Investigations on multiple aspects of ligand-transporter and ligand-receptor interactions give us the opportunity to comprehend the nature of these processes beyond pharmacology. As an illustration, fast kinetic studies on the potential antiepileptic agent, Q5 reveal that it effectively blocks (red squares) the fast component of Ca²⁺ ion influx evoked by AMPA in the presence of receptor desensitization blocker cyclothiazide (blue circles).

Coherent application of uptake and release approaches can be used to disclose complex transport mechanisms. In the figure below, Glu uptake coupled GABA release is monitored in uptake (left panel) and release (middle panel) assays and by microdialysis in vivo (right panel).

Electrophysiological Laboratory

Our main research interest is in the pathophysiology and pharmacology of epileptiform activity, investigated in slice cultures and acute brain slices of rat hippocampal formation and entorhinal cortex. Below, the most frequently used preparation in our laboratory, the 400 µm thick acute entorhinal-hippocampal slice is depicted.

Equipment

The facilities of the lab include three electrophysiological setups combined with a range of optical detection devices mounted on upright microscopes (see below among the facilities of Imaging Laboratory). Setups are equipped with amplifiers ranging from a bridge-balance amplifier for sharp electrode recording to a patch-clamp amplifier with cooled headstage for whole cell and cell-attached recordings of minor currents. Each setup has two or three acquisition channels.

Capabilities

With a combination of extracellular and intracellular electrophysiological recordings, fluorescence microscopy (with CCD camera or photodiode array detection) and confocal imaging, several important physiological variables, such as membrane potential fluctuation of single cells or cell populations, excitatory and inhibitory synaptic input to a particular cell and intracellular Ca²⁺ or Na⁺ concentration dynamics in specific intracellular compartments can be monitored simultaneously. Such a combined recording paradigm is exemplified by the figure below. A CA3 pyramidal cell held in whole cell voltage clamp (pipette coming from the left), and an additional extracellular electrode (coming from above) inserted into the slice can be observed on the left panel. While recording, the cell was filled with AlexaFluor 488, and subsequently partially reconstructed with confocal sectioning (planar projection image, middle panel). A simultaneous recording of field potential signal (population activity), whole cell voltage clamp current (synaptic input to a single pyramidal cell) and [Ca²⁺]-dependent (somatic) fura-2 fluorescence is depicted in the right panel.

Imaging Laboratory

The Imaging Laboratory takes part in most ongoing projects of the Department of Neurochemistry. Thus it is involved in the work on the elucidation of possible patomechanisms of epileptic (epileptiform) activity in hippocampal slices, modes of action of GHB in the nucleus accumbens or elucidation of neurometabolic signalling pathways.

Equipment

Two setups of Electrophysiological Laboratory are combined with state of the art imaging capabilities.

The Olympus FluoView 300 system with 3 Melles Griot lasers and with a suite of amplifiers and A/D converters from Axon (MultiClamp 700A, Digidata 1322) and 2 Burleigh micromanipulators for simultaneous electrophysiological experiments or cell-specific fluorescent probe loading through a patch pipette. The strength of this setup is in its high spatial resolution enabling us to visualize subcellular processes.

A combined 5 Mhz Micromax CCD camera / NeuroPDA-III photodiode matrix imaging setup built on an Olympus BX51WI microscope with a suite of Axon amplifiers and converters (Axopatch 200B and Multiclamp 700A, Digidata 1320). The NeuroPDA-III, WuTech H-469IV photodiode array is made up of 464 individual photodiodes in a hexagonal array, each having its own data channel providing a maximal 1600 picture/sec rate of acquisition. The Princeton Instruments 782 x 582 cooled CCD camera has a readout rate of 5 MHz which affords a lower temporal but higher spatial resolution, something between the capabilities of the laser scanning confocal and the photodiode matrix system.

Capabilities

The imaging systems are generally used to measure intrinsic optical signals or fluorescence in brain (hippocampus, nucleus accumbens) slices either acutely isolated or cultured, or cells from cell cultures. Fluorescent probes for following [Ca²⁺]_i, [Na⁺]_i, pH and membrane polarization changes are used.

Visualization of cell swelling in the hippocampal formation during epileptiform activity
The intrinsic optical signal depends on the amount of light coming through the tissue. The swelling of cells is visible because as they let more water inside, they also become more permeant to light and thus is more bright. Brightness is converted in the movie into color (less bright – blue, more bright - red). The upper part of the movie shows the electrophysiological signal confirming the timing of ictal and inter-ictal events.

Visualization of the membrane potential during epileptiform activity in the hippocampus
Electrophysiological measurements offer great temporal resolution, however spatial information is lost. The two ways to circumvent this problem is either the use of multielectrode recordings or fast imaging in combination with membrane-potential sensitive fluorescent dyes. An example is shown to the left (click for the movie) where a hippocampal slice was loaded with RH-414, a voltage-sensitive dye. The upper part contains the electrophysiological recording (from a single electrode), the image shows the spatiotemporal recruitment of the hippocampal regions to the epileptiform activity of the slice.

Time-resolved fluorescent analysis with confocal microscopy

Pyramidal cell from area CA3 in the hippocampus loaded through a micropipette with Rh-123, which is a voltage-sensitive fluorescent dye, preferentially distributed to the mitochondrial membrane. The cell was optically reconstructed from a series of images taken at different focal planes. Yellow boxes in the right part of the image show magnified parts labelled with smaller boxes on the left.

When cells are loaded with Rh-123, one can distinguish between individual mitochondria and how these mitochondria change their membrane potential during ictal and interictal activity during epileptiform activity. It was possible to show, that independent fluctuations can occur in remote dendrites while some mitochondria show remarkable coincidence in neighbouring dendrites.

Molecular Modelling Laboratory

To investigate binding interactions between ligands and their targets (CNS receptors and transporters) at the molecular level, we use high resolution crystal structures of protein-ligand complexes or their homology models to gain a closer understanding of ligand binding to receptors and transporters as well as to disclose conformational changes underlying their functions. Ligand positions are searched by using a number of docking programs based on different algorithms. In silico screening results are validated experimentally.

Equipment

Molecular mechanics calculations are performed in SYBYL (Tripos Inc.). Molecular docking is most frequently performed using GOLD 3.1.1 (Genetic Optimisation for Ligand Docking, Cambridge Crystallographic Data Center Software Ltd. Cambridge, UK), FlexX (implemented in SYBYL) and Autodock. Application of a number of scoring functions (GOLD and ChemScore in GOLD; GScore, DScore, FScore, ChemScore, PMF Score and Fscore in FlexX) provide a powerful tool for investigating the docked ligand positions in the selected drug target.
Molecular dynamics processes are performed on the Research Center’s Fujitsu-Siemens cluster of 114 dual core processors.

Capabilities

Molecular docking using high resolution crystal structures is able to predict ligand positions, thereby providing structural information on the binding mode. Structural data together with binding and function experiments enhance our understanding on ligand-protein interaction at the atomic level, that may lead to further information on protein function.

Guanosine monophosphate (A) and inhibitors sildenafil (B) and zaprinast (C) docked to the type 6 phosphodiesterase binding crevice. Inhibitors, but not GMP interacted with Phe778 and Met759 (sildenafil) or Met759 (zaprinast), the key residues involved in the interaction between the catalytic binding domain and the inhibitory γ subunit of type 6 phosphodiesterase. Agreeing with predictions obtained by modelling binding, both inhibitors enhanced the amplitude of electric light responses of the isolated rat retina however the enhancement was smaller for the more efficacious inhibitor sildenafil. These paradoxical responses can be explained as a result of the enhancement of light activation of PDE6 through the competition between the catalytic site inhibitors and the γ subunit residues for catalytic domain residues Phe778 and Met759.