Department of Molecular Neurophysiology

Department of Molecular Neurophysiology

Department of Molecular Neurophysiology

 

 

Head: Govindan Dayanithi,  MSc, Ph.D.

E-mail: gdaya@biomed.cas.cz, gdaya@univ-montp2.fr

Tel.: +420 241 062 725

 

The Department of Molecular Neurophysiology studies the role of vasopressin and oxytocin in the central and peripheral nervous system and their therapeutic implications for a number of human diseases. The division uses three models of transgenic rats, which allow the visualization of fluorescent vasopressin and oxytocin. These models are used to study calcium signalling and calcium homeostasis in magnocellular neurons and nerve terminals to illustrate the signalling mechanisms of vasopressin and oxytocin in DRG neurones and glial cells. Recently, the department is also focusing on the fundamental aspects of Ca2+ signalling mechanisms in stem cells from different species (humans, murine and non-human primate animal models for Alzheimer’s disease) and of different origin obtained under different experimental conditions. This approach will lead to the development of better tools (e.g. for accurate modelling of the disease, for drug discovery or for toxicity screening) and novel approaches for cell-based therapies by improving both the differentiating potential and survival of all types of stem cells after transplantation.

 
 
Research Scientists:
 
Govindan Dayanithi, , MSc, Ph.D.
Research Director in CNRS
 
 
PhD Students:
 
Oksana Forostyak, MD
Štěpán Kortus, MSc
 
Technican:
 
Dominika Dušková

Important result in 2015





1. Physiology of vasopressin and oxytocin and Ca2+ signalling in the supraoptic nucleus neuronesk
The magnocellular vasopressin (AVP) and oxytocin (OT) neurones of the rat supraoptic nucleus (SON) exhibit specific electrical behaviour, synthesize AVP and OT peptides, and secrete them into the neurohypophysial system in response to various physiological stimulants. The electrical activities of these neurons are regulated by the release of AVP and OT, either somato-dendritically or when applied to supraoptic neurones or slice preparations in vitro. In these neurones, both AVP and OT bind to specific autoreceptors, which induce distinct Ca2+ signals and regulate cellular events (ref: # 1, 2, 3, 5, 7, 9, 15). We have recently demonstrated that freshly isolated single SON neurones from adult rats exhibited distinct spontaneous [Ca2+]i oscillations. The oscillations could be observed simultaneously in both soma and dendrites (see video-1). But the high 50 mM K+-induced [Ca2+]i response could be observed only in the soma (see video-2). The computational estimations of Ca2+ fluxes have also been proposed (Ref # 16). In addition, we have also identified the major mechanisms that underlie these oscillations. To achieve this, we used Ca2+ imaging techniques (fast fluorescence photometry and video imaging) to measure [Ca2+]i oscillations from individual neurones. These neurones were treated with various drugs targeting individual mechanisms of cellular Ca2+ homeostasis such as voltage-dependent-calcium-channels (VDCC; L-N-P/Q-R and T-type), endoplasmic reticulum (ER), plasma membrane Ca2+ pumps (PMCA) or intracellular Ca2+ signalling pathway.

 

  
The schematic diagram shows Ca2+ transport mechanisms with highlights (red) of those that were identified as essential for [Ca2+]i oscillations in the magnocellular neurons.

 

 

 

 

2. Physiology of vasopressin and oxytocin and Ca2+ signalling in the peripheral nervous system DRG neurones and non-neuronal cells

Besides their important role in the central nervous system it has recently been suggested that both AVP and OT also play a role in the peripheral sensory system, in particular that AVP may play a regulatory role in nociception and OT has been shown to have strong spinal anti-nociceptive action and analgesic properties (ref: # 6). In our department we aimed to study the role of OT and AVP in the dorsal root ganglia neurons isolated from transgenic rats tagged by visible fluorescent (AVP-eGFP ,OT-mRFP and double transgenic AVP-eGFP/OT-mRFP) proteins. We showed that in these rats, AVP and OT are expressed and can be visualized directly in the freshly isolated dorsal root ganglia and in the cultured neurons and that this expression increases significantly during pregnancy and lactation. The [Ca2+]i measurements results revealed that capsaicin-sensitive DRG neurons also expressed functional receptors for AVP and OT, indicating the novel physiological effect of these neurohormones in pain (pregnancy-related) and possible physiological consequence of OT in DRGs during lactation.

 

 

  
Both AVP and OT are expressed and can be visualized in freshly isolated dorsal root ganglia (DRG) and neurohypophysis (NH) from transgenic AVP-eGFP (left panel), Double AVP-eGFP/OT-mRFP transgenic (middle panel) and OT-mRFP (right panel) rats. The NH (upper row) and the DRG (lower row) were isolated from the same animal and analyzed with a confocal microscope. Scale bars 50 µm.

 

 

 

 

3. Calcium signalling in stem cells: molecular physiology and multiple roles.
Stem cells (SCs) of different origins have brought hope as a potential tool for use in cell replacement therapies. Ca2+ signalling plays a key role in SC differentiation and proliferation, and dysregulation of Ca2+ homeostasis may instigate pathological scenarios. Currently, the role of ion channels and receptors in SCs is not fully understood. In recent years, we found that (i) the pre-differentiation of human embryonic SCs (hESCs) led to the activation of Ca2+ signalling cascades and enhanced the functional activities of these cells ; (ii) the Ca2+ homeostasis and the physiological properties of hESC-derived neural pre-cursors (NPs) changed during long term propagation in vitro ; (iii) differentiation of NPs derived from human induced pluripotent SCs affects the expression of ion channels and receptors; (iv) these neuronal precursors exhibited spontaneous activity, indicating that their electrophysiological and Ca2+ handling properties are similar to those of mature neurones , and (v) in mesenchymal SCs isolated from the adipose tissue and bone marrow of rats the expression profile of ion channels and receptors depends not only on the differentiation conditions but also on the source from which the cells were isolated, indicating that the fate and functional properties of the differentiated cells are driven by intrinsic mechanisms. Together, identification and assignment of a unique ion channel and a Ca2+ handling footprint for each cell type would be necessary to qualify them as physiologically suitable for medical research, drug screening, and cell therapy.

 

  
Schematic drawing showing the change in functional expression of Ca2+ channels and receptors linked to Ca2+ signalling in ADSCs and BMSCs during pre-differentiation

 

 

 

Important result in 2014

 


1. The peripheral chimerism of bone marrow–derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice
In our study we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP+) transplanted cells into non-haematopoietic tissues. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and the entry of transplanted cells into the small intestine and liver. Grafted bone marrow cells or GFP+lin-Sca-1+ cells are not transient in the GI tract and thus could be used for the long-term treatment of various pathologies.

 

 

 

Histological analysis of cell transplant in the intestine. Scale: (A-D) 100 μm, (E) 25 μm, (F) 20 μm.

 

  

Collaboration: Prof. S. Filip, Faculty of Medicine and Teaching Hospital, Charles University in Prague, Hradec Králové, Czech Republic

Publication:

Filip S, Mokrý J, Vávrová J, Sinkorová Z, Mičuda S, Sponer P, Filipová A, Hrebíková H, Dayanithi G,J (2014): The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice, Cell Mol Med. May;18(5):832-43. IF 3.698


2. Full-length transient receptor potential vanilloid 1 channels mediate calcium signals and possibly contribute to osmoreception in vasopressin neurones in the rat supraoptic nukleus
The purpose of the present study was to uncover the structure and function of molecules related to the central osmoreceptorin the supraoptic nucleus (SON) of rats. For this purpose, we performed RT-PCR and immunohistochemistry for TRPV1-related molecules, and patch-clamp and imaging of the cytosolic Ca2+ concentration ([Ca2+]i) to measure responses to osmolality changes and TRPV-related drugs. Arginine vasopressin (AVP), synthesized in the SON is a key factor in systemic osmolality regulation. We found that AVP neurones in the SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

 

 

  

 

Schematic drawing showing the mechanisms of TRPV1-mediated Ca2+ responses in vasopressin neurons of rat supraoptic nucleus.Highlights: Full-length TRPV1 and a new splice variant, TRPV1_SON were expressed in the rat SON. Immunoreactivity of the N-terminal portion of TRPV1 was colocalized with that of AVP. Mannitol-induced [Ca2+]i responses were observed in AVP but not in OT neurones. Mannitol-induced [Ca2+]i responses were blocked by the TRPV1 antagonist CPZ and BCTC. At 36 °C, the TRPV1 agonist capsaicin evoked [Ca2+]i and ionic current responses.

 

 

Collaboration: Prof. Izumi Shibuya, Laboratory of Veterinary Physiology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan

Publication: 

Moriya T, Shibasaki R, Kayano T, Takebuchi N, Ichimura M, Kitamura N, Asano A, Hosaka YZ, Forostyak O, Verkhratsky A, Dayanithi G, Shibuya I, (2015): Full-length transient receptor potential vanilloid 1 channels mediate calcium signals and possibly contribute to osmoreception in vasopressin neurones in the rat supraoptic nucleus. Cell Calcium 57(1):25-37. IF 4.210

 

 

Important result in 2013

 



1. Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.
2. Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons.
Neural precursors (NPs) derived from human embryonic or fetal stem cells are considered to be a promising tool for cell replacement therapy in CNS injuries and neurodegenerative diseases. The characterization and analysis of NP populations derived from hESCs remain open. Our aim was to study the role and physiology of Ca2+ and its signaling mechanisms to understand the functional properties of NPs during during differentiation. Our results show that these cells respond to different physiological stimuli by an increase in [Ca2+]i that varies during the course of hESC differentiation. The NPs express functional glutamate receptors, purinoreceptors, voltage-dependent Ca2+ channels and show spontaneous Ca2+ oscillations as typically observed in neuronal cells. Understanding the functional properties of stem cells may allow us to better control their regenerative potential and help to improve strategies for their use in transplantation and the treatment.

Collaboration:
The James Black Centre, Department of Neuroscience, King’s College London, UK
University of Manchester, School of Biological Sciences, Manchester, UK
Department of Physiology, School of Medicine, Birmingham University, Birmingham, UK
Department of Veterinary Physiology, Faculty of Agriculture, Tottori University, Tottori, Japan
Institut National de la Santé et de la Recherche Médicale, Unité de recherche U710, Université Montpellier 2, Montpellier; and Ecole Pratique des Hautes Etudes, Sorbonne, France

 

 

Intracellular Ca2+ stores (Ryanodine) and spontaneous [Ca2+]i transients in passage 7 of human embryonic stem cell-derived neural precursors.

 

 

Running Grants

 

January 2017 – December 2022
Source: European Union/ Czech Republic Structural Fund
Grant ID# CZ.02.1.01/0.0/15_003/0000419
Role: Member of the team
Název: Center of Reconstructive Neuroscience

 

January 2017 – December 2019
Source: Czech Grant Agency;
Grant ID# GA CR P303/17-21146S
Role: Principal Investigator
Title: Induction of human mesenchymal stromal cells to secrete neural growth factors for the purpose of spinal cord regeneration after the injury

 

January 2015 – December 2017
Source: Czech Grant Agency;
Grant ID# GA CR P304/15/09161S
Role: Project participant
Title: Muscle remodelling on the basis of extracellular matrix seeded with functionally characterized stem cells.

2016

Dayanithi, G., Verkhratsky, A.: (2016) Calcium signalling in stem cells: Molecular physiology and multiple roles. Cell Calcium., 59(2-3):55-6.
Forostyak, O., Forostyak, S., Kortus, Š., Syková, E., Verkhratsky, A., Dayanithi, G.: (2016) Physiology of Ca(2+) signalling in stem cells of different origins and differentiation stages. Cell Calcium, 59(2-3):57-66.

Forostyak, O., Butenko, O., Anděrová, M., Forostyak, S., Syková, E., Verkhratsky, A., Dayanithi, G.: (2016) Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells. Stem Cell Res., 16(3):622-634.

Kortus, S., Srinivasan, C., Forostyak, O., Ueta, Y., Syková, E., Chvátal, A., Zápotocký, M., Verkhratský, A., Dayanithi, G.: (2016) Physiology of spontaneous [Ca2+]i oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus. Cell Calcium, 59(6):280-8.

Kortus, Š., Srinivasan, C., Forostyak, O., Zápotocký, M., Ueta, Y., Syková, E., Chvátal, A., Verkhratský, A., Dayanithi, G.: (2016) Sodium-calcium exchanger and R-type Ca2+ channels mediate spontaneous [Ca2+]i oscillations in magnocellular neurones of the rat supraoptic nucleus. Cell Calcium, 59(6):289-98.

Skoloudik, L., Chrobok, V., Kalfert, D., Koci, Z., Sykova, E., Chumak, T., Popelar, J., Syka, J., Laco, J., Dedková, J., Dayanithi, G., Filip, S.: (2016) Human Multipotent Mesenchymal Stromal Cells in the Treatment of Postoperative Temporal Bone Defect: An Animal Model. Cell Transplant., 25(7):1405-14.

Forostyak, O., Dayanithi, G., Forostyak, S.: (2016) CNS Regenerative Medicine and Stem Cells. Opera Med Physiol., 2(1):55-62.
Dayanithi, G., Kortus, S., Forostyak, O., Sykova, E., Verkhratsky, A.: (2016) Calcium Oscillations in the Isolated Vasopressin Neurons of the Rat Supraoptic Nucleus. Endocrine Reviews, 37(2), Supplement.

Forostyak, O., Dayanithi, G., Forostyak, S., Sykova, E.: (2016) Oxytocin and Vasopressin-Induced Calcium Signals in Rat Adipose and Bone Marrow Derived Stem Cells. Endocrine Reviews, 37(2), Supplement.

 

 

2015

Forostyak, O., Romanyuk, N., Verkhratsky, A., Syková, E., Dayanithi G.: (2015) Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors. Stem Cells Dev. 22(10):1506-1521.

Školoudik, L., Chrobok, V., Kalfert, D., Koči, Z., Syková, E., Chumak, T., Popelář, J., Syka. J., Laco, J., Dedková, J., Dayanithi, G., Filip, S.:(2015) Human multipotent mesenchymal stromal cells in the treatment of postoperative temporal bone defect: an animal model. Cell Transplant. 25(7):1405-1414.

Moriya, T., Shibasaki, R., Kayano, T., Takebuchi, N., Ichimura, M., Kitamura, N., Asano, A., Hosaka, YZ., Forostyak, O., Verkhratsky, A., Dayanithi, G., Shibuya, I., (2015) Full-length transient receptor potential vanilloid 1 channels mediate calcium signals and possibly contribute to osmoreception in vasopressin neurones in the rat supraoptic nucleus. Cell Calcium. 57(1): 25-37.

Filipová, A., Diaz-Garcia, D., Bezrouk, A., Čížková, D., Havelek, R., Vávrov,á J., Dayanithi, G., Řezacová, M.: (2015) Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts. Cell Biol. Int. 39(8): 943-953.
Kortus, S., Dayanithi, G., Zapotocky, M.: (2015). Computational estimation of calcium fluxes in isolated magnocellular neurons. BMC Neuroscience 16: (suppl 1) P299.

 

 

2014

Filip, S., Mokrý, J., Vávrová, J,, Sinkorová, Z., Mičuda, S., Sponer, P., Filipová, A., Hrebíková, H., Dayanithi. G,: (2014). The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice. J Cell. Mol. Med 18: 832-843.

Viero, C., Forostyak, O., Sykova, E., Dayanithi, G.: (2014). Getting it right before transplantation: example of a stem cell model with regenerative potential for the CNS. Front. Cell Dev. Biol 2:36. doi: 10.3389/fcell.2014.00036.

 

 

2013

Forostyak O, Kozubenkoa N, Verkhratsky A, Sykova E & Dayanithi G (2013). Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors. Stem Cells and Development 22: 1506-1521.

Cocks G, Romanyuk N, Amemori T, Forostyak O, Dayanithi G, Jendelova P, Jeffries A, Thuret S, Miljan E, Sinden J, Sykova E & Price J (2013). Conditionally immortalised stem cell lines from human spinal cord retain regional identity and generate V2a interneurons and motorneurons. Stem Cell Research and Therapy 4: article 69.

Filip S, Mokrý J, Vávrová J, Sinkorová Z, Mičuda S, Sponer P, Filipová A, Hrebíková H & Dayanithi G (2014). The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice. Journal of Cellular and Molecular Medicine 18: 832-843.

Calcium imaging high K induced calcium response in vasopressin neurone 

 

Calcium oscillations in GFP transgenic neuron 

 

Prof. Izumi Shibuya, Laboratory of Veterinary Physiology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan

Prof. Yoichi Ueta, Department of Physiology, University of Occupational and Environmental Health, Kitakyushu, Japan

Prof. Alexei Verkhratsky, University of Manchester, School of Biological Sciences, Manchester, UK

Prof. Jose R. Lemos, University of Massachusetts Medical School, Worcester, MA, USA.

Prof. Jean-Michel Verdier, INSERM U 1198, University of Montpellier , France;

École Pratique des Hautes Études-Sorbonne, Paris, France