NS200: In vitro electrophysiology section September 11th, 2013

Transcription

1 NS200: In vitro electrophysiology section September 11th, 2013 Quynh Anh Nguyen, 4 th Year Nicoll Lab quynhanh.nguyen@ucsf.edu N276 Genentech Hall, Mission Bay

2 Outline Part I: Theory Review of circuit properties of a neuron Types of Recordings Patch clamp techniques Part II: Application LTP/LTD Experimental Preparation The Rig Experimental Demo References

3 Part I: Theory The Neuron as an RC Circuit

4 Current (I) Unit of current is the ampere, or amp (A) Any net flow of charge is called a current

5 Current flow in a neuron In a cell, current is generated by the flow of ions Na +, K +, Cl, Ca 2+ Ions flow through channels Concentration gradients and electrical gradients drive ions Inward currents are negative, outward currents are positive +100 Amps Time

6 Electrical Potential (V) Unit of voltage is the volt (V) Voltage = Difference between two Electrical Potentials

7 Electrical Properties as applied to the Neuron V m, membrane potential, arises from the separation of charges across the cell membrane Nernst Equation

8 Voltage properties of the neuron Voltage fluctuations (eg an action potential) are represented as below in recordings

9 Resistance (R) Unit of resistance is the Ohm ( ), which is volts/ampere Conductance (g), measured in siemens (S) is the reciprocal of resistance g=1/r

10 Resistance in the neuron In a cell, resistance is created by a lot of different cellular components Channels Cytoplasmic molecules Size of cell Access to the cell (when making a patch clamp recording)

11 Capacitance (C) Unit of capacitance is the farad (F)

12 Capacitance in the Neuron The cell membrane acts as a capacitor Mostly a function of area

13 RC Circuit Revisited

14 Ohm s Law: Everything you need to know to be an electrophysiologist V=IR I=gV IV Curve I Slope = 1/R = g = conductance V

15 Voltage dependence

16 Electrophysiology: Types of recordings

17 Electrophysiology: Types of recordings

18 Electrophysiology: Extracellular Recordings Measures population or single cell voltage fluctuations, e.g. field EPSPs, action potentials, theta rhythms RIG

19 Electrophysiology: Types of patch clamp recordings

20 Types of Patch clamp recordings: Advantages and Disadvantages

21 Types of whole cell recordings: Current clamp Measured V = IR Constant Current clamp allows the measurement of changes in intracellular voltage in response to current injection or extracellular stimuli.

22 Types of whole cell recordings: Current clamp Firing rate of cell Action potential amplitude and waveform Resting potential Response to electrical stimulus or agonist

23 Types of whole cell recordings: Current clamp Firing rate of cell Action potential amplitude and waveform Resting potential Response to electrical stimulus or agonist

24 Types of whole cell recordings: Current clamp Firing rate of cell Action potential amplitude and waveform Resting potential Response to electrical stimulus or agonist

25 Types of whole cell recordings: Current clamp Firing rate of cell Action potential amplitude and waveform Resting potential Response to electrical stimulus or agonist

26 Types of whole cell recordings: Voltage clamp Measured Held V = IR Voltage clamp allows the measurement of current flow into or out of the cell in response to voltage steps or extracellular stimuli.

27 Types of whole cell recordings: Voltage clamp AMPAR response Channel properties Current/voltage relationship Postsynaptic properties Presynaptic properties

28 Types of whole cell recordings: Voltage clamp RIG Channel properties Postsynaptic properties Excitatory postsynaptic currents Miniature excitatory postsynaptic currents Presynaptic properties

29 Types of whole cell recordings: Voltage clamp RIG Channel properties Postsynaptic properties Excitatory postsynaptic currents Miniature excitatory postsynaptic currents Presynaptic properties

30 Types of whole cell recordings: Voltage clamp Channel properties Postsynaptic properties Presynaptic properties Probability of release Short term facilitation/depression

31 Types of whole cell recordings: Voltage clamp Channel properties Postsynaptic properties Presynaptic properties Probability of release Short term facilitation/depression

32 What we ve covered so far Circuit properties of a neuron Types of recordings Advantages/disadvantages Whole cell recordings Current clamp Voltage clamp

33 Part 2: Application Real world example: LTP and LTD Experimental Prep Introduction to the rig Experimental Demo

34 Glutamate Receptors and Synaptic Plasticity AMPA NMDA LTP LTD

35

36 Experimental Preparation I. Tissue collection II. Pulling pipettes for electrodes III.Internal IV.External

37 Tissue collection The prep used depends on what kinds of experiments you want to perform Dissociated culture Organotypic slice culture Acute slice

38 Pulling pipettes for electrodes Small size of the tip of the micropipette creates a resistance Higher resistance = smaller pipette tip

39 Internal solution Chloride reversal ATP Pharmacology Osmolarity

40 External solution Calcium/Magnesium Pharmacology 95% O2, 5% C02 Osmolarity

41 7 The Rig Perfusion pump 2. Air table 3. Faraday cage 4. Patch micromanipulator 5. Stimulation micromanipulator 6. Microscope 7. Video camera 8. Motorized stage controls 9. Micromanipulator control unit 10. Fluorescent lamp 11. Mercury lamp

42 The Rig Stimulation controls 2. Video monitor 3. Micromanipulator rotors 4. Master 8 5. NIDAQ board 6. Amplifier 4 5 6

43 Experimental Demo 4 groups of 5 Meet in N272

44 Experimental Demo Group assignment: A: Jon, Kate, Bruce, QA B: QA, Jon, Kate, Bruce, C: Bruce, QA, Jon, Kate D: Kate, Bruce, QA, Jon

45 Steps in whole cell recordings I. Positive pressure through bath II. Zeroing to ground III.Sealing on to cell IV.Canceling out pipette capacitance V. Breaking in to cell VI.Calculating series resistance VII. Assessing steady state current VIII.Recording current or voltage

46 Experimental Demo Kate s rig: Current clamp: current steps, action potentials Voltage clamp: EPSCs, IPSCs, reversal potential

47 Experimental Demo Jon s Rig: All whole cell voltage clamp AMPA/NMDA EPSCs Paired Pulse Ratio Trains I/V

48 Experimental Demo Bruce s Rig: LTP Structural plasticity

49 Helpful References Basic Texts Carter and Shieh., Guide to Research Techniques in Neuroscience, Electrophysiology Chapter Kandel et al., Principles of Neural Science, Chapter on membrane potential Johnston and Wu, Foundations of cellular neurophysiology Specific Texts Hille, Ion Channels of Excitable Membranes Cowan et al., Synapses Sakmann, Single Channel Recording (intimidating but awesome) Molleman, Patch clamping: an introductory guide to patch clamp electrophysiology (great how to)