BIOELECTRICAL SIGNALS RECORDS WHOLE CELL PATCH CLAMP Joana Tremoceiro | José Maria Moreira | Manuel Figueiral | Rita Gil Mestrado Integrado em Engenharia Biomédica | Fall SemesterIntroduction This project has two central aim: 1- Study the techniques used to record bioelectrical signals – intracellular and extracellular recordings – and some applications; 2- Develop an experiment, using Patch-clamp technique, to analyze the ion channel’s behaviors, particularly, the effect of A1 receptors (using adenosine) at GABAergic tonic and spontaneous currents, at a stratum pyramidale’s cell of a Wistar rat’s hippocampusIntroduction This project has two central aim: 1- Study the techniques used to record bioelectrical signals – intracellular and extracellular recordings – and some applications; 2- Develop an experiment, using Patch-clamp technique, to analyze the ion channel’s behaviors, particularly, the effect of A1 receptors (using adenosine) at GABAergic tonic and spontaneous currents, at a stratum pyramidale’s cell of a Wistar rat’s hippocampus Electrophysiology Electrophysiology is a branch of physiology that studies the electrical properties of biological cells and tissues, through measurement of voltage changes and electrical current. Electrophysiology Electrophysiology is a branch of physiology that studies the electrical properties of biological cells and tissues, through measurement of voltage changes and electrical current. Conclusion By the experiment we may conclude that activation of A1 receptors leads to inhibition of GABAergic tonic current. That may be observed at figure 3 where phasic currents disappear but, unlike the graphic above, the baseline does not change, which means that there was no tonic currents.Conclusion ResultsResults Future Prespective It is expected that researches of this area will focus, not only on the development of equipment like EEG’s and HVR’s, but also on studying ion channels behavior, since the majority of inherited diseases are causes by this channels mutations. An example are drugs that actuate on ion channels, like some epileptic drugs. Future Prespective It is expected that researches of this area will focus, not only on the development of equipment like EEG’s and HVR’s, but also on studying ion channels behavior, since the majority of inherited diseases are causes by this channels mutations. An example are drugs that actuate on ion channels, like some epileptic drugs. Synaptic Transmission One of the types of cells commonly studied are neurons, nervous system cells specialized at passing signals to individual targets by synapses (inducing the post synaptic cell to a variation of it’s membrane potential): Membrane Potential Resting potential = -70 mV Hyperpolarized - membrane potential decreases, due to the entrance of Cl - Depolarized - membrane potential increases, due to the entrance of Na + or exit of K - Brain’s neurotransmitters Glutamate - has excitatory effects (activates Na + channels - depolarizations) GABA - has inhibitory effects (activates Cl - channels - hyperpolarizations). Synaptic Transmission One of the types of cells commonly studied are neurons, nervous system cells specialized at passing signals to individual targets by synapses (inducing the post synaptic cell to a variation of it’s membrane potential): Membrane Potential Resting potential = -70 mV Hyperpolarized - membrane potential decreases, due to the entrance of Cl - Depolarized - membrane potential increases, due to the entrance of Na + or exit of K - Brain’s neurotransmitters Glutamate - has excitatory effects (activates Na + channels - depolarizations) GABA - has inhibitory effects (activates Cl - channels - hyperpolarizations). Chemical Synapse Electrical Synapse Literature Cited: [1] Enderle, J, Blanchard S, Bronzino J (2000). Introduction to Biomedical Medicine, Academic Press [2] Nolte J (2007) Elsevier’s Integrated Neuroscience (chap 1-3). Elsevier’s Integrated Series, Elsevier [3] [4] Acknoledgements : Professor Ana Sebastião and Dr. Diogo Rombo, Departamento de Neurociências, FMUL, Lisbon, Intracellular Recording Recordings across the membrane of the cell Patch-Clamp Techniques: Extracellular Recording Recording from outside the cell (examples: nerve fibers, brain slices, heart) Techniques:  Single Unit Recording  Multi-Unit Recording  Field Potentials  Amperometry Fig1: Electroencephalogram (EEG) Three electrodes: positive, negative, earth Presynaptic neuron induces voltage changes in the postsynaptic cell through conducting ion channels Presynaptic neuron releases a neurotransmitter that binds to the postsynaptic cell’s receptors. Activation of ion channels Changes of the ionic permeability of the cell membrane Whole-Cell Patch 1st Rupture of the membrane patch 2nd Access to the interior of the cell 3rd Study the entire membrane as one Voltage Clamp Used to measure ion currents across a neuronal membrane while holding (clamping) the membrane potential at a set level. The cell’s potential was kept on -70 mV A blocker of GABAA receptors was added Whole-Cell Patch 1st Rupture of the membrane patch 2nd Access to the interior of the cell 3rd Study the entire membrane as one Voltage Clamp Used to measure ion currents across a neuronal membrane while holding (clamping) the membrane potential at a set level. The cell’s potential was kept on -70 mV A blocker of GABAA receptors was added Fig2: Voltage Clamp proceder (negative feedback) Phasic currents (miniature post synaptic currents): spontaneous release of neurotransmitters Tonic currents: continuous presence of small amounts of neurotransmitters around the neuron. Fig3: a) squematic tonic and phasic currents; b) experiment’s results Before introduction of Gabazine After introduction of Gabazine Control Cell Presence of: phasic currents (don´t know yet about tonic current) Desapearence of: tonic current phasic current Pre-incubated cell (CPA solution) Desapearence phasic current