Institute of Physiology - Our Lab

Institute of Physiology, University of Maribor

Pedagogical work

For the most part, our members are involved in the preclinical education of physiology for medical students in the form of lectures, seminars, as well as practical courses. The department is also in charge of the physiology courses for students of biology, psychology and health sciences outside the main facility. In addition, some members of our institute perform teaching in various study programs and at different levels, such as courses in biophysics, mathematical methods in physiology, complex systems, etc.. Each year, our team participates in education of around 300 students.  

We also participate in physiological courses abroad (Medical Unviversity of Vienna, Austria, University of Szeged, Hungary).


Research

Our institute has long-term experience in complex analysis of endocrine systems. Our laboratory covers a wide range of recent and classical techniques for molecular, cellular and multicellular studies. Main expertise are confocal imaging of calcium dynamics in endocrine (alpha, beta, delta) and exocrine (acinar, ductal) pancreas cells, smooth muscle cells of detrusor, and pituitary cells.

In recent years we devoted a lot of interest to the application of innovative approaches to study intercellular networks within the intact tissue, in order to gain a comprehensive understanding of the causes and mechanisms underlying health and disease. We developed approaches to study calcium oscillations in depth (frequency, duration) on several temporal (slow, fast oscillations) and spatial (waves of calcium) scales. 

The main part of our lab focuses on the physiology and pathophysiology of the pancreas tissue, covering both endocrine and exocrine cels, as well as developing a platform for testing detrusor function in situ. In parallel, our group also gives emphasis to the development of biophysical and computational approaches to unveil the functional principles of various biomedical systems. We regularly support the in situ measurements with in vivo testing of intraperitoneal glucose and insulin tolerance tests (ipGTT, ipITT). Lately, we broadened our expertise to in vivo behavioral testing of anxious and depression behavior in mice. 


Lab equipment

  • Upright confocal microscope (Leica Microsystems TCS SP5 II)

Top end confocal microscope to acquire prolonged time series of highly spatial and temporal resolved changes in intracellular calcium concentration, membrane potential, and exocytosis in human and animal model tissues using fresh tissue slices.


Figure 1: Upright confocal microscope and electrophysiology system
(multiphoton microscope Leica TCS SP5-II)

  • Inverted confocal microscope (Leica Microsystems TCS SP5 II)

Top end confocal microscope to acquire prolonged time series of highly spatial and temporal resolved changes in intracellular calcium concentration, membrane potential, and exocytosis in human and animal model tissues using fresh tissue slices and cell cultures. In addition, confocal analysis of material surfaces and classical analysis of (patho)histological slides.


Figure 2: Inverted confocal microscope
(single-photon inverted microscope Leica TCS SP5-II)

  • System for single cell electrophysiology

A patch-clamp setup to study spontaneous and stimulated electrical activity of excitable cells, ion channels and capacitance based regulated exocytosis using depolarization protocols and slow caged photolysis. 

  • Core facility for laboratory animals

The equipment to house and maintain laboratory mice and rats, both inbred and outbred strains of control, spontaneously mutated as well as transgenic strains to study physiology and pathophysiology of most organ systems.


Figure 3: Core facility for laboratory animals



Description of laboratory activities, references and possible lines of collaboration

In the last decade we have developed and optimized the fresh pancreas tissue slice method (1) and use it for classical single cell electrophysiology and slow caged compound photolysis (2-6), acquisition of intracellular calcium concentration (7-12), and membrane potential (11) in pancreatic beta cells. Our work continues to yield key knowledge to understand normal physiology of beta cells, the dysfunction of which plays a critical role in pathogenesis of diabetes mellitus. Our methods are applicable also to pancreatic acinar cells and other endocrine tissues (chromaffin cells, pituitary cells) and neurons (13, 14). The most compatible clinical departments are Pathology, Diabetology, and Endocrinology, as well as Abdominal surgery, Ophthalmology and Orthopedics. 


References

1.      Speier S, Rupnik M. A novel approach to in situ characterization of pancreatic ß-cells. Pflügers Archiv European Journal of Physiology. 2003;446(5):553-8.

2. Speier S, Yang SB, Sroka K, Rose T, Rupnik M. KATP-channels in beta-cells in tissue slices are directly modulated by millimolar ATP. Molecular and Cellular Endocrinology. 2005;230(1–2):51-8.

3. Skelin M, Rupnik M. cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A in mouse pancreatic beta cells. Cell Calcium. 2011;49(2):89-99.

4. Mandic SA, Skelin M, Johansson JU, Rupnik MS, Berggren P-O, Bark C. Munc18-1 and Munc18-2 Proteins Modulate β-Cell Ca2+ Sensitivity and Kinetics of Insulin Exocytosis Differently. Journal of Biological Chemistry. 2011;286(32):28026-40.

5. Dolensek J, Skelin M, Rupnik MS. Calcium Dependencies of Regulated Exocytosis in Different Endocrine Cells. Physiological Research. 2011;60:S29-S38.

6. Paulmann N, Grohmann M, Voigt J-P, Bert B, Vowinckel J, Bader M, et al. Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation. PLoS Biol. 2009;7(10):e1000229.

7. Skelin Klemen M, Dolenšek J, Stožer A, Rupnik M. Measuring Exocytosis in Endocrine Tissue Slices. In: Thorn P, editor. Exocytosis Methods: Humana Press; 2014. p. 127-46.

8. Stožer A, Gosak M, Dolenšek J, Perc M, Marhl M, Rupnik MS, et al. Functional Connectivity in Islets of Langerhans from Mouse Pancreas Tissue Slices. PLoS Comput Biol. 2013;9(2):e1002923.

9. Stožer A, Dolenšek J, Skelin Klemen M, Slak Rupnik M. Cell physiology in tissue slices. Studying beta cells in the islets of Langerhans. Acta medico-biotechnica. 2013;6(1):20-32.

10. Stožer A, Dolenšek J, Rupnik MS. Glucose-Stimulated Calcium Dynamics in Islets of Langerhans in Acute Mouse Pancreas Tissue Slices. PLoS ONE. 2013;8(1):e54638.

11. Dolenšek J, Stožer A, Skelin Klemen M, Miller EW, Slak Rupnik M. The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices. PLoS ONE. 2013;8(12):e82374.

12. Marquard J, Otter S, Welters A, Stirban A, Fischer A, Eglinger J, et al. Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment. Nat Med. 2015;21(4):363-72.

13. Sedej S, Klemen MS, Schlüter OM, Rupnik MS. Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca2+-Affinity Pool by Preventing Constitutive Exocytosis. PLoS ONE. 2013;8(10):e78883.

14. Marciniak A, Cohrs CM, Tsata V, Chouinard JA, Selck C, Stertmann J, et al. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology. Nat Protoc. 2014;9(12):2809-22. Epub 2014/11/14.




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