Electrophysiology ——patch clamp

1. Electrophysiology——patch clamp

2. Outlines • Electrophysiology fundamentals • Types of Patch clamp recordings • Patch clamp procedures • A glimpse of electrophysiological data visualization

3. Fundamentals Types Procedures Visualization What is Electrophysiology ? • The study of the electrical properties of biological cells and tissues. • Voltage change or electric current  • On a wide variety of scales, • from single  ion channels to whole organs

4. Fundamentals Types Procedures Visualization Nature of neuronal electricity Neuron: a enclosed lipid membrane envelope, Lipid layer has a high resistance. Voltage difference across the membrane. Selective  permeability to ions. Ion channels, pumps in the membrane

5. Fundamentals Types Procedures Visualization Nature of neuronal electricity Electrical equivalent circuit Rin Cm Circuits & Brain: organized complexes consisting of this electric unit.

6. Fundamentals Types Procedures Visualization Mission of Electrophysiology • Measurements of electrical activity of neurons, particularly synaptic and action potential (AP) activities.

7. Fundamentals Types Procedures Visualization Methods of Electrophysiology • Extracellular recording EEG (electroencephalogram), LFP (Local field potential ), Single-unit ( SU ), Multi-unit (MU) • Intracellular recording • Patch-clamp recording

8. 各种电生理记录的优缺点： 细胞外记录 记录群体或单细胞的电活动； 容易得到信号，机械稳定性要求低。 电极粗大，对组织损伤大。 细胞内记录 记录单个神经元，电位变化精确可控，可进行 电压钳、电流钳实验。对细胞内过程影响小。 只能记录细胞胞体，机械稳定性要求高。 全细胞记录 记录单个神经元，电位变化精确可控，可进行 电压钳、电流钳实验，对亚细胞结构进行记录。 机械稳定性要求低。 对细胞内过程影响大（除穿孔全细胞记录）。

9. Fundamentals Types Procedures Visualization Resting membrane potential Vm = Vin – Vout (Vout = Vfar∞ = 0mV) Electrical potential Vs. Concentration gradient

10. Fundamentals Types Procedures Visualization Resting membrane potential R: 气体常数; T: 绝对温度; F: 法拉第常数; z: 离子电荷数

11. Fundamentals Types Procedures Visualization Resting membrane potential

12. Fundamentals Types Procedures Visualization Resting membrane potential

13. Fundamentals Types Procedures Visualization Equivalent circuit

14. Hardware Amplifier DAC

15. Fundamentals Types Procedures Visualization Hardware • Patch-clamp system Monitor /oscillograph Stimulus controller Digital/Analog signal converter (DAC) Amplifier pipette Neuron Signal collection and procession platform (interface)

16. 减法运算器 If=(Vp-Vo)/Rf Fundamentals Types Procedures Visualization Voltage Clamp: 运算放大器 VP VP VP

17. Fundamentals Types Procedures Visualization Current Clamp: 电压跟随器 （也是运算放大器的一种） 加法运算器 17

18. Fundamentals Types Procedures Visualization Hardware : patch clamp amplifier • Axon • 􀁺 Axoclamp 2系列：Axoclamp 2B（已停产） • 􀁺 Axopatch1系列：Axopatch 1D（已停产） • 􀁺 Axopatch 200系列：Axopatch 200B • 􀁺 GeneClamp500：GeneClamp500B（已停产） • 􀁺 MultiClamp 700系列：MultiClamp 700B • two headstages • software control on PC: Commander • Voltage & current clamp • 􀁺 Axoclamp 900A • HEKA

19. Fundamentals Types Procedures Visualization Software: Commander panel

20. Fundamentals Types Procedures Visualization Methods of Electrophysiology • 4 kinds of patch clamp • cell-attached • whole-cell • outside-out • inside-out • Patch-clamp recording http://www.bem.fi/book/04/04x/0427x.htm

21. Fundamentals Types Procedures Visualization Methods of Electrophysiology

22. Fundamentals Types Procedures Visualization Glass pipette • Sharp electrode • Patch-Clamp pipette

23. Fundamentals Types Procedures Visualization Breaking target cell

24. Fundamentals Types Procedures Visualization Intracellular solution Dye loading Morphological characterization: Alexa 488594Lucifer yellow

25. Fundamentals Types Procedures Visualization Voltage clamp • Ion channel properties, Current changes • Sodium Current Activation curve 快门 Inactivation curve 慢门 Li et al. (2014). PLoS Biol

26. Fundamentals Types Procedures Visualization Voltage clamp • Ion channel properties, Current changes • Postsynaptic currents I-V curve 40 0 -40 V (mV) -80 -120 I (pA) 25 pA mV 0.1 s

27. Fundamentals Types Procedures Visualization Current clamp • Threshold analysis • PV & SST cells Li et al. (2014). PLoS Biol

28. Fundamentals Types Procedures Visualization Current clamp 10 mV RF • Voltage changes, physiological functions 0.5 s I-V curve RF 40 mV 0.4 s

29. Fundamentals Types Procedures Visualization Current clamp • Brain/ network state: Vm characteristics depend on network state • synchronous & asynchronous states Tan et al. (2014). Nature

30. Fundamentals Types Procedures Visualization Current clamp • Brain/ network state: • Vm skewness • Whole-cell Vm measurements from V1 of behaving monkeys Tan et al. (2014). Nature

31. Fundamentals Types Procedures Visualization Current clamp • Brain/ network state: Vm fluctuations • Whole-cell Vm measurements from V1 of behaving monkeys Tan et al. (2014). Nature

32. Fundamentals Types Procedures Visualization Current clamp • Brain/ network state: Vm & LFP relationship Tan et al. (2014). Nature

33. Fundamentals Types Procedures Visualization Current clamp • Analog mode of excitatory synaptic transmission disynaptic IPSPs Zhu et al. (2011). PLoS Biol

34. Fundamentals Types Procedures Visualization Current clamp • Analog mode of excitatory synaptic transmission EPSP summation window Zhu et al. (2011). PLoS Biol

35. Fundamentals Types Procedures Visualization Current clamp • Analog transmission: mediated by SST but not PV interneurons Zhu et al. (2011). PLoS Biol

36. Summary • Electrophysiology fundamentals • Types of Patch clamp recordings • Patch clamp procedures • A glimpse of electrophysiological data visualization • Versatile interpretation & presentation according to specific questions/studies

37. The brain is really a brain as complex as the universe when it is understood in a electrophysiological point of view !

38. Thanks for your attention ! Q & A