1. INTRODUCTION TO BIOMEDICAL ENGINEERING AUTHORS: ANA PORTEIRA Nº 67305 GUILHERME MOURA Nº 67323 SANDRO NUNES Nº67945 SUPERVISED BY: PROF. ANA SEBASTIÃO Bioelectrical Signal Recording Master in Biomedical Engineering 1st Semester, 2009

2. Structure of the Presentation Introduction Cell to Cell Communication Electrophysiology Recording Tecnhniques Overview Patch Clamp Technique Experimental Procedure Patch Clamp Applications Future ProspectivesDiscussion/Conclusion

3. Introduction to the Subject  As the propagation of these signals is a key part on how communication occurs inside our body, it is absolutely necessary to find a way of studying it;  Bioelectrical signals are transient pulsations propagated throughout the membrane of living cells such as muscular cells and neurons;

4. Introduction to the Subject Recording of Bioelectrical Signals Electrophysiology Study of the electrical properties of biological cells and tissues, through measurements of voltage changes or electrical current flows on a wide variety of scales. Biomedical Engineering employs resources from various scientific fields to create and develop new materials, instruments and recording techniques.

5. Introduction to the Subject Main Objectives: Learn what are the fundamental recording techniques and how they function; Recongnize how bioelectrical signals are transmited and which are the structures involved; Elaborate on Patch-Clamp Technique, which is the most advanced method today; Observe, in loco, one of its applications; Discuss the the advantages and disadvantages and refer to the future prospectives; Present our analysis on the subject;

6. Cell to Cell Communication Gap Junctions Juxtacrine Signaling Paracrine Signaling Endocrine Signaling Synaptic Transmission

7. Cell to Cell Communication Communications established through channels constituted by Connexine Proteins; Connexine Proteines pair in groups of 6, forming a central pore and linking both cell’s cytoplasm; Ions and small mollecules are allowed to freely pass from one cell to another in a very short period of time; Electric Synapses make use of Gap Junctions. Gap Junctions

8. Type of signaling which involves the interaction of a protein between an inducing and a receptor cell without diffusion. Juxtacrine Signaling A protein on one cell binds to its receptor on the adjacent cell; The signal is transmitted directly from the cytoplasm of one cell through small conduits into the cytoplasm of an adjacent cell. A receptor on one cell binds to its ligand on the extracellular matrix secreted by another cell; Cell to Cell Communication

9. Paracrine Signalling permits simmultaneous communication between the inducting cell and other cells in its vicinity; It makes use of chemical messengers, which are a particular type of signaling molecules called paracrine factors; These molecules are diffused in the extracellular matrix and only affect cell in a close range; Diffusion of neurotransmissors is na example of paracrine signalling. Paracrine Signaling

10. Cell to Cell Communication Endocrine Signaling In opposition to the rest, Endocrine Signaling is mediated by a specialized system of organs; Inducing cell deploys chemical substances called hormonesinto the bloodstream; Receptors in the cell membranes bind to these molecules; Signal takes more time to arrive, however it is more stable and capable of acting over long periods.

11. Synaptic Transmission Synaptic Transmission depends on a process called Action Potential. Rapid alteration of the transmembrane voltage generated by the activity of voltage-gated ion channels embedded in the cell membrane.

12. Synaptic Transmission Resting Potential Threshold Rising Phase Falliing Phase Recovering Phase Action Potential Small amount of potassium channels are open; K + ions to enter and exit the cell based on electric and concentration gradients; Membrane potential is maintained at about -60mV. A few Na + channels open, permitting Na + ions to enter the neuron; The increase in positive ions inside the cell depolarizes the membrane potential; Threshold potential is reached and additional voltage- gated sodium channels opened; Voltage across the membrane rapidly reverses and reaches its most positive value. Many of the voltage-gated sodium channels start to close and potassium begin to open; This causes the membrane potential to begin to go back to the resting membrane potential. Voltage-gated potassium channels are maximally activated and open. The membrane repolarizes beyond the resting membrane voltage because more potassium channels are opened than during the membrane’s resting state; Potassium channels that opened during the action potential now close; The membrane goes back to the resting potential.

13. Synaptic Transmission How are the bioelectrical signals propagated between cells?  Communication between neurons and communication between neurons and muscle occurs at specialized junctions called synapses;  One example is the chemical synapse mediated by the neurotransmitter Acetylcholine (ACh). Neuromuscular Junction

14. Synaptic Transmission Converting a Chemical Signal to an Electrical Signal  Synaptic transmission begins when the action potential reaches the axon terminal.  The resulting depolarization, due to opening of voltage-gated sodium channels, initiates a sequence of events leading to the release of the transmitter.

15. Synaptic Transmission Converting a Chemical Signal to an Electrical Signal  The Ca 2+ ions trigger the release of neurotransmitter by causing the synaptic vesicles closest to the active zone of the synapse to fuse with the presynaptic membrane.  This fusion process is regulated by the interaction between protein complexes expressed on the vesicle and presynaptic membranes.

16. Synaptic Transmission Converting a Chemical Signal to an Electrical Signal  When the vesicles fuse with the presynaptic membrane, they empty their content of neurotransmitter into the synaptic cleft.

17. Synaptic Transmission Converting a Chemical Signal to an Electrical Signal  The neurotransmitter moves across the synaptic cleft and binds to receptors on the postsynaptic membrane;  The channel opens, sodium ions enter the postsynaptic cell, and the depolarization signal is thus propagated to the postsynaptic cell.

19. Electrophysiology Different types of electrophysiological recordings Peri T Kurshan, Asli Oztan & Thomas L Schwarz, Nature Neuroscience

20. Electrophysiology Overview through Recording Conditions  The tissue location where we are stimulating;  What sorts of stimulation are used (chemical or electrical);  What type of synaptic potential (spontaneous or induced)  In what type of cell the signal is being recorded;  Which cell receptors are blocked, in order to know that don‘t interfere in the result; Different types of electrophysiological recordings

21. Evaluate Neuronal Communication H W? Measures ion currents across a neuronal membrane while holding the membrane potential at a set level.  Keeps the electrical current through the recording electrode;  Records the membrane potential by injecting current into a cell through a microelectrode Current Clamp Voltage Clamp

22. Electrodes in Electrophysiology Sharp Electrodes  Records the potential inside the cell membrane with minimal effect on the ionic constitution of the intracellular fluid.  There is very little ion exchange between the intracellular fluid and the electrolyte in the pipette (small hole)  Records the potential inside the cell membrane with minimal effect on the ionic constitution of the intracellular fluid.  There is very little ion exchange between the intracellular fluid and the electrolyte in the pipette (small hole) Voltage Clamp Current can be injected into the cell to change the membrane potential of the cell. Measure Recording Membrane Potential, Resistance, Synaptic potentials, Action potentials Current Clamp Electrólito líquido e.g. KCl, NaCl

23. Electrodes in Electrophysiology Patch Electrodes  The interior of the pipette is filled with a solution matching the ionic composition of the bath solution (cytoplasm for whole-cell recording.)  A chloride silver wire is placed in contact with this solution and conducts electrical current to the amplifier.  The interior of the pipette is filled with a solution matching the ionic composition of the bath solution (cytoplasm for whole-cell recording.)  A chloride silver wire is placed in contact with this solution and conducts electrical current to the amplifier. It is sealed onto the surface of the cell membrane, rather than inserted through it. High resistance seal Micropipette Cell Membrane Pressure … Suction

24. Recording Techniques Overview Extracellular EEG ECG Patch-Clamp The cell is impaled with a sharp glass electrode and the voltage or the current is recorded across the membrane Intracellular The electrode is placed on the surface (skin) and record cell’s populations. The electrode is placed on the surface (skin) and record cell’s populations.

25. Patch Clamp Technique Inside-Out/Outside- Out Advantages: To record individual channels; good pharmacology and the inside/outside solutions can be changed. Disadvantage: channel properties can be changed. Advantages: To record individual channels; good pharmacology and the inside/outside solutions can be changed. Disadvantage: channel properties can be changed. Whole-Cell Advantages: good pharmacology and high definition. Disadvantage: dialysis of cytoplasmatic contents Advantages: good pharmacology and high definition. Disadvantage: dialysis of cytoplasmatic contents

26. Reduces the dialysis Higher access resistance; Impossible to record from single channels. Higher access resistance; Impossible to record from single channels. Patch Clamp Technique Advantage Disadvantages Whole -cell Disadvantage: Dialysis of cytoplasmatic contents Perforated Patch Makes small holes on the patch with pore-forming agents instead of applying suction.

27. Experimental Procedure - Recordings from CA1 pyramidal cells After we clamp the cell What was the goal? Decrease in the frequency and amplitude due to the deterioration of the cell – for e.g. dialysis of intracellular molecules. Decrease in the frequency and amplitude due to the deterioration of the cell – for e.g. dialysis of intracellular molecules. Evaluate the changes in the frequency and amplitude of spontaneous events in the absence of any drugs 1.Apply positive pressure through the patch electrode; 2.Place the electrode near its surface and applied a negative pressure forming the giga-seal. 3.Wait for the membrane to rupture. 1.Apply positive pressure through the patch electrode; 2.Place the electrode near its surface and applied a negative pressure forming the giga-seal. 3.Wait for the membrane to rupture. Our own experiment Patch Clamp Experiment Results :

28. Patch-Clamp Applications Alcohol affects a specific type of neurotransmitter receptor, NMDA. The entire single-channel methods have the capability of being applied to human samples (e.g., slices or cultures from biopsies), both from normal tissue removed during surgery, and from diseased brains. Importance of the study of drug interactions with ionic channels, channel gating mechanisms, ion channel permeation dynamics among others

29. Limitations Careful and precise fabrication of electrodes; Skillful manipulation of the patch pipette towards a cell; Clever design of electronics and apparatus to allow low-noise recordings. High resolution measurements allowed by the Patch Clamp Tecnhique have some downsides: Highly Dependant in Nanotecnhnology

30. Future Prospectives Advances in micro-fabrication offer promising technologies for less laborious and cheaper patch clamp recordings. Listed below are the two main improvements that could result from advancements in nanotechnology: Superior designs of integrated nanoelectronic patch clamp amplifiers By improving the design of patch clamp amplifiers, it is possible to further lower background noise and increase signal bandwidth. Development of new nanomaterials The implementation of more resistive nanomaterials in the construction of the coating or the pipette may facilitate the gigaseal formation.

31. Patch clamp NEUROPSYCOPHARMAC OLOGY GENETICS AND MOLECULAR BIOLOGY MENTAL DISEASE ELECTROPHYSIOLOGICAL TECHNIQUES NEW METHODS AND ELECTODES REAL PHYSIOLOGY SIMULATION IONS AND NEUROTRANSMIT TERS STUDY Discussion/Conclusion BIOMEDICAL ENGINEERING

32. THE END Questions?