PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg.   2018.09.21.. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

BASICS OF NEUROBIOLOGY - Methods Peter Pazmany Catholic University Faculty of Information Technology www.itk.ppke.hu Neurobiológia alapjai - Módszerek BASICS OF NEUROBIOLOGY - Methods By Imre Kalló 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu METHODS IN NEUROBIOLOGY VII. Electrophysiological approaches Imre Kalló Pázmány Péter Catholic University, Faculty of Information Technology Histology techniques: light microscopic studies Applications using fluorescent dyes Histology techniques: electron microscopic studies Techniques to map neuronal connections Molecular biological techniques Living experimental models Electrophysiological approaches Behavioral studies Dissection, virtual dissection, imaging techniques 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu ELECTROPHYSIOLOGICAL APPROACHES Electrophysiology is the study of electrical properties of biologycal cells and tissues. In vivo electrophysiology Anesthetised and moving animals Measuring field potentials Multiunit recordings Single cell recordings Juxtacellular recording Intracellular recording In vitro electrophysiology Patch-clamp recording Voltage clamp, Current clamp modes Loose patch, Cell-attached, Whole cell, Perforated patch modes 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu MEASURING FIELD POTENTIALS The field potential is the summation of spatial and temporal alterations of synaptic and voltage-dependent currents in a defined region of the brain. Consequently, it refers to and characterizes the activity of a certain cell or afferent population. From the surface of the skull (EEG) From the surface of the brain (ECoG) From the brain (LFP, MUA) V 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu MEASURING FIELD POTENTIALS Electroencephalography: Neural oscillations: δ oscillation: < 3.5 Hz θ oscillation: 4 – 7.5 Hz α oscillation: 8 – 13 Hz (Berger rythm) β oscillation: 14 – 30 Hz γ oscillation: > 30 Hz slow oscillations: < 1 Hz From the surface of the skull (EEG) From the surface of the rain (ECoG) From the brain (LFP, MUA) 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu MEASURING FIELD POTENTIALS From the surface of the skull (EEG) From the surface of the rain (ECoG) From the brain (LFP, MUA) Tetrodes allow to visualisation of single neurons from different positions Spike sorting, clustering Local Field Potential (generated by membrane currents) input of the cells Multi Unit Activity (spiking of local neurons) output of the cells 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu IN VIVO EXTRACELLULAR RECORDING 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu IN VIVO EXTRACELLULAR RECORDING MULTIELECTRODES 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology I. www.itk.ppke.hu IN VIVO JUXTACELLULAR RECORDING An extracellular recording technique It makes a correlated electrophysiological and morphological examination possible Electrode and marker of cell filling (neurobiotin) Fine positioning of the electrode – changes of the amplitude of action potentials Current impulse (1-10 nA, anode impulse) – modulation of neuronal activitity Duration of filling: 15-20 min 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu IN VITRO ELECTROPHYSIOLOGY VOLTAGE CLAMP The cell’s potential is clamped at a chosen value. The size of ionic current crossing the cell’s membrane at the chosen value is measured. Current meter Membrane potential amplifier Signal generator Feedback amplifier Intracellular electrode Extracellular electrode AXON Circuit diagram depicting the feed-back amplifier charging the cell membrane of the recorded cell with the digitally subtracted transient capacitive currents. Xu C et al Histamine innervation and activation of septohippocampal GABAergic neurones: involvement of local ACh release. J Physiol. 2004 Dec 15;561(Pt 3):657-70. 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology I. www.itk.ppke.hu IN VITRO ELECTROPHYSIOLOGY CURRENT CLAMP MODE The current clamp technique records the membrane potential  by injecting current into a cell through the recording electrode. The membrane potential generated by the cell „spontaneously” or in response to external stimulus is measured. Xu C et al Histamine innervation and activation of septohippocampal GABAergic neurones: involvement of local ACh release. J Physiol. 2004 Dec 15;561(Pt 3):657-70. 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology I. www.itk.ppke.hu IN VITRO ELECTROPHYSIOLOGY: PATCH CLAMP TECHNIQUE Cell-attached mode: Transfer is only through ion channels entrapped Whole cell mode: Content of the pipette redistribute with that of the cytoplasm Perforated patch mode: Ion transfer through the perforations, but large molecules stay within the cytoplasm 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006

Basics of neurobiology: Methods in Neurobiology VII. www.itk.ppke.hu IN VITRO RECORDINGS – PAIRED RECORDINGS 2018.09.21. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006