DEPARTMENT OF CELLULAR NEUROPHYSIOLOGY

Laboratory of Neurobiology

Laboratory of Molecular Neurophysiology

Identification of cellular and molecular mechanisms of integration in neural networks, through characterisation of intercellular signalling pathways within neuronal-glial circuits and intracellular signalling mechanisms in neurones and glia in physiological and pathological conditions.

By employing the complex of electrophysiological, video-imaging and molecular biological techniques we shall identify main receptors responsible for these signalling and localise intracellular signalling cascades. Further we shall develop a complex understanding of information processing in neuronal-glial circuits thus contributing to a more inclusive theory of brain function, which emphasises a continuous interplay of discreet neuronal networks with reticular and internally continuous astroglial web.

Research at the Laboratory of Cellular Neurophysiology focuses on these main topics:

• role of glutamatergic and purinergic pathways in neuronal-glial signalling in cortex, hippocampus and spinal cord with a specific emphasis on the glial NMDA and P2X receptors;
• purinoreceptor-mediated signalling in neurones and glia in the context of their role in sensory transduction and in acute and chronic pain;
• glial representation of TRP channels and their role in glial signalling;
• ion channels and Ca2+ signalling cascades in various types of neural stem cells at different stages of differentiation;
• in vivo imaging on neuronal-glial circuits in physiological and pathophysiological conditions;
• calcium signalling cascades in neurodegeneration and in Alzheimer disease in particular;
• morphology and physiology of glia in normal brain ageing.

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(A) Green fluorescent protein (GFP)-labeled primary embryonic stem cells (D6/GFP) express the typical neuronal markers βIII-tubulin,  MAP-2 and DCX (doublecortin), six days after in vitro differentiation. (B) A  typical current pattern of D6/GFP-derived neuron-like cells. Using the patch clamp technique in the whole-cell configuration,  large outwardly rectifying K+  current amplitudes2 together with  tetrodotoxin-sensitive Na+ currents, were detected in these cells (right, see the voltage protocol in the inset).

Fig. 2: Neuronal loss and astrogliosis in the hippocampus after global cerebral ischemia

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Coronal sections of the rat hippocampus 7 days after ischemia/ reperfusion. The slices were stained with an antibody against glial fibrilary acidic protein (GFAP)  and NeuN or nestin. Note the decreased NeuN immunoreactivity and increased GFAP/nestin immunoreactivity (reactive gliosis) in the CA1 region. The enclosed region is shown in detail below.

Fig. 3: Membrane properties of astrocytes and NG2-glia after global cerebral  ischemia

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(A) Immunohistochemical identification and electrophysiological characterization of NG2-glia and astrocytes in the rat hippocampus (CA1 region) after bilateral carotid occlusion. During patch clamp recording2 the cells were labeled with Alexa Fluor 488-hydrazide and were stained post-recording with antibodies against glial fibrilary acidic protein (GFAP) and NG2-proteoglycan. (B) Typical current patterns of NG2-glia and astrocytes in the rat hippocampus(CA1 region) NG2- glia express a typical complex current pattern of outwardly and inwardly rectifying K+ currents; astrocytes display predominantly  time- and voltage-independent passive K+ currents (right, see  the voltage protocol in the inset).

Fig.4. Synaptic currents mediated by NMDA receptors in astrocytes.

Astrocytes in layer II of the cortical slice (obtained from the transgenic mice with green fluorescent protein selectively expressed in astroglia), were identified by their fluorescence and voltage-clamped; electrical stimulation of synaptic inputs was applied to layer IV. Synaptically evoked currents are predominantly sensitive to NMDA antagonist MK801 (10 µM), the residual current is partially blocked by DL-TBOA (100 µM). Each point on the time graphs represents the mean ± SEM for five EPSCs; illustrative EPSCs are shown below.

Fig.5. Miniature spontaneous excitatory currents in cortical astrocytes mediated by NMDA and AMPA glutamate receptors.

All recordings were made at holding potential -80 mV in the presence of TTX (1 µM), picrotoxin (100 µM) and DL-TBOA (100 µM). A. Representative whole-cell recordings in control and after application of NBQX (30 µM). Right panels represent generalized waveform of spontaneous currents (average of 50 events). Lower graph shows probability density function of spontaneous currents in control and in the presence of NBQX. B. Representative whole-cell recordings from astrocytes in cortical slice in control and after application of D-AP5 (30 µM). Right panels show generalized waveform of spontaneous currents (average of 50 events). Lower graph shows probability density function of spontaneous currents in control and in the presence of D-AP5.

List of current grants:

Name of the grant	Grant agency	Number of the grant	Principal investigator (coinvestigator)	Duration of the grant
Research project: "Molecular, cellular and systems mechanisms of serious diseases of the human organism, their diagnosis, therapy and pharmacotherapy "	AVČR	AV0Z50390512	Syková (Chvátal)	2005-10
Research centre: “Centre for cell therapy and tissue repair”.	MŠMT ČR	1M0021620803	Syková (Chvátal)	2005-09
Research center: "Centre of neuroscience"	MŠMT ČR	LC554	Syka (Chvátal, Verkhratsky)	2005-09
Role of glial cells during ischemia/reperfusion and the subsequent regeneration of the nervous tissue	GAČR	305/06/1316	Anděrová	2006-08
Study of the membrane and morphological properties of astrocytes, oligodendrocytes and neurons in the CNS during pathological states using fluorescent proteins	GAČR	305/06/1464	Chvátal	2006-08
Pre-clinical evaluation of stem cell therapy in stroke	EU-STREP	LSHB-CT-2006-037328	Syková (Chvátal, Anděrová)	2006-09
Electrophysiological and morphological properties of neuronal progenitor cells after transplantation into the site of photochemical lesion	GAUK	62/2006/C/2LF	Prajerová	2006-07