Instrumentation for Electrophysiological Studies of Excitable Tissues

Transcription

1 BME462/ELE562/NEU503 Joint Lecture Instrumentation for Electrophysiological Studies of Excitable Tissues Ying Sun, Ph.D. Professor Dept. of Electrical, Computer and Biomedical Engineering University of Rhode Island

2 Allen L. Hodgkin Andrew Huxley

3 A. L. H o d g k i n a n d A. F. H u x l e y B r a n c h i a l n e r v e s N e u r o n a l c u r r e n t - v o l t a g e r e l a t i o n s d e t e r m i n e d b y v o l t a g e c l a m p e x p e r i m e n t s i n t h e s q u i d g i a n t a x o n, T e n t a c l e s A r m s C e r e b r a l g a n g l i a O p t i c g a n g l i a V i s c e r a l g a n g l i a V i s c e r a l n e r v e M a n t l e n e r v e H e a d E y e S i p h o n M a n t l e M a n t l e S t e l l a t e g a n g l i a F i n n e r v e G i a n t a x o n L a t e r a l f i n

4 V o l t a g e C l a m p E x p e r i m e n t a l S e t u p Microscope To voltage clamp amplifier Microelectrode holder Height adjustment

5 Neuroscience Voltage Clamp Invented by G. Marmont & K. Cole (independently) in Used by A. L. Hodgkin & A. F. Huxley in 1952 to identify the ionic currents responsible for the action potentials in the squid giant axon (Nobel Prize 1963).

6 Hodgkin-Huxley Equations

7

8

9

10 Electrophysiology Microelectrode experiments with Lymnaea stagnalis (pond snail)

11 Universal Clamp A novel instrument for controlling neuronal signals via feedback control provided by a digital signal processor chip (US and international patents 2009), funded by the National Institutes of Health

12 Intermittent Voltage Clamp

13 Possible Connectivity for a Universal Clamp with 2 Headstages V: voltage measurement VC: voltage clamp IC: current clamp DC: dynamic clamp V/VC: voltage controlled VC R: right L: left

14 Universal Clamp α prototype ± 1 2 V D C S u p p l y ± V D C - D C U C l a m p C i r c u i t D S P B o a r d

15 Universal Clamp β prototype Top view

16 Analog Devices Blackfin BF548 EZ-Lite Kit Bottom view Universal Clamp β prototype

17 Universal Clamp β prototype

18 Neurotransmitters are released from synaptic vesicles of the presynaptic neuron and bind to receptors on the postsynaptic neuron, thereby triggering an impulse through the second neuron.

19 Can we monitor exocytosis/endocytosis activities? Specific cell membrane capacitance = 0.9 μf/cm 2 (fairly constant) Whole cell capacitance = 3 12 pf (pico: p = ) Capacitance signal from a single vesicle = 1 15 ff (femto: f = ) A vesicle event changes the cell capacitance by ~1/1000. A delicate and fast instrument can detect the resulting phase shift in a sinusoidal wave.

20 High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis. Rituper et al., Nature Protocols 8:169 83, 2013

21 Lock-in Amplifier

22

23 Cellular Capacitance Measurement with the Universal Clamp

24 Instrumentation for cell capacitance measurements: switching sinusoidal excitations for studying cell membrane transport. 40th Northeast Bioengineering Conference, Boston, MA, April 25-27, 2014.

25

26

27

28 Signal flow diagram for continuous monitoring of cell capacitance

29 Summary 1. Time-domain formulation for a linear least-squares estimator has the advantage of providing fast, accurate, and continuous measurements of the elctrical properties of a cell. 2. The concept of the lock-in amplifier improving signal-tonoise ratio with sinusoidal modulations was used to simplify the formulation and integrated into the Universal Clamp based a high-speed signal processor. 3. For future work, very small phase shifts related to vesicle activities remain to be a challenge and may require a phaselock-loop type of algorithm for accurate measurements.

30 Acknowledgements Major Contributors for this Project: Ying Sun, Ph.D. Professor, Biomedical Engineering, URI Jiang Wu, Ph.D., Analog Devices, Inc. John DiCecco, Ph.D., Naval Undersea Warfare Center Leon Collis, Ph.D., Pfizer Robert B. Hill, Ph.D., Professor, Biological Sciences, URI Kiyoaki Kuwasawa, Ph.D., Professor, Okayama University, Japan This project was supported by NIH SBIR Phase I and Phase II grant (2R44NS A1) and is currently supported by another Phase I grant (1R43NS A1).

31 In Memory of Prof. Robert B. Hill

32 Exam Question for NEU 503 Describe the four different configurations of the patch clamp in terms of the instrumentation technique and the experimental preparation.