2. Principle:
- Study of electrical properties of cells and tissues.
- Measure voltage change or current changes across single ion
channels to whole organs.
- Neuroscience context: Measuring activity of neurons, especially
the action potential firing.
- Large scale brain recordings from Electroencephalogram are
also considered to be electrophysiological recordings.
3. The classical technique:
- Inserting electrodes into various preparations of biological tissue.
- Electrodes include: solid conductors, tracings on printed circuit boards, hollow
glass pipettes filled with electrolytes.
- The principle preparations include : living organisms, excised tissue (acute or
cultured), dissociated cells from excised tissue, artificially grown cells, hybrids
of the above.
- In case of Neuroscience, the electrophysiology involves electrical recordings
from brain slices, cultured neuronal tissue and sometimes the whole brain.
5. Intracellular Recordings:
(Measure voltage or current across the membrane of the cell)
- Voltage Clamp Technique
- Current Clamp Technique
- Patch Clamp Technique
- Sharp Electrode Technique
6. Intracellular Recordings: Historical background
1939: Cole and Curtis placed an extracellular electrode on squid axon and
showed that the membrane resistance dropped during action potential.
“Cole, K. S. and Curtis, H. J. (1939). Electric impedance of the squid giant axon during
activity. J. Gen. Physiol ., 22 (5), 649–670.”
1939: Hodgkin Huxley inserted a glass electrode into a giant squid axon and
recorded the first action potential.
“Hodgkin, A. and Huxley, A. (1939). Action potentials recorded from inside a nerve
fibre.Nature, 144 (3651), 710”
7. Historical Background of Intracellular Recordings (contd):
1949: Hodgkin and Katz showed that the membrane potential increases
considerably during the action potential and inferred a selective increase in
sodium permeability from these observations.
“Hodgkin, A. L. and Katz, B. (1949). The effect of sodium ions on the electrical activity of
the giant axon of the squid.J. of Physiol ., 108 (1), 37.”
1952: Intracellular recordings from vertebrate cells was performed by Brock et al.
“Brock, L., Coombs, J. and Eccles, J. (1952). The recording of potentials from
motoneurones with an intracellular electrode.J. Physiol., 117 (4), 431–460.”
8. Applications:
- Measuring membrane potential distribution in vivo.
- Studying membrane potential correlation between neurons.
- Changes in effective membrane time constant using network activity.
- Excitatory and inhibitory synaptic conductances in response to visual
stimulation.
- Current voltage relationship during spike activity.
- Reproducibility of neuronal responses.
- Dendritic computational mechanisms.
9. 1.Voltage Clamp Technique :-
- Technique developed by Marmont and Cole in 1940s.
- It is an electronic and negative feedback system.
- Principle: Clamps the membrane potential at a fixed value along the axon.
- Also measures the feedback current.
- Hodgkin and Huxley used this technique to measure action potential.
- Pulled glass or sharp electrodes are used to penetrate the membrane with little
damage.
10. Voltage Clamp : Technique
- Membrane potential held at constant ( Vclamp
).
- Measurable entity is electrode current.
- The voltage clamp operates by negative
feedback. The membrane potential amplifier
measures membrane voltage and sends
output to the feedback amplifier; this
subtracts the membrane voltage from the
command voltage, which it receives from the
signal generator. This signal is amplified and
output is sent into the axon via the current
electrode.
Fig: Circuit diagram of Single electrode
voltage clamp (Source: Wikipedia)
11. Variations in Voltage Clamp Technique :
- Double Electrode Voltage Clamp :-
Two electrodes are used where one electrode used to measure the membrane
potential and one injects the feedback current.
- Single Electrode Voltage Clamp :-
Single and same electrode is used for both the purpose. So measurement of
membrane potential is contaminated.
13. Difficulties in Voltage Clamp :
-Space Clamp Problem
-Compensation Errors
-Imperfect Clamping ( Vm is not equal to Vclamp)
14. 2. Current Clamp Technique:
- Current injected into cell with an electrode of negligible resistance and
change in potential difference across the membrane is measured.
- The membrane potential (voltage difference between the inside and the
outside of the cell) is measured by comparing the potential at the amplifier
end of the intracellular electrode with the potential of a reference electrode
(outside of the cell).
- Considering resistance of intracellular electrode to be 0 and neglecting
junction potentials, measured Vr equals membrane potential Vm.
15. Current Clamp Technique: Non-ideal case
- Measuring spontaneous activity:
- Development of LJP (Liquid Junction Potential)
- Damage induced by the sharp electrodes
- Electrode filtering
- Measuring response to an injected current:
- Bridge balance
- Discontinuous current clamp
-Active electrode compensation
16. Conductance Measurement: Current Clamp
- Simplistic Model with only one non-zero conductance
- Complex model with varying linear conductances
17. 3. Patch Clamp Technique:
- Technique was developed by Erwin Neher and Bert Sakmann in 1970s.
- Used to record the currents of single / multiple ion channel molecules to
understand their role in action potential and nerve activity.
- patch clamp microelectrode is a micropipette of large tip diameter.
- Automated patch clamp, is a recent technique, which collects large amount of
data inexpensively in a short period of time. It uses microfluidic device to capture
cells and integrated electrode.
18. Fig. 2: A phase contrast image of
a patch pipette attached to the
membrane of a cultured murine
hippocampal neuron. Courtesy of
Dr. Ainhara Aguado, Ruhr
University Bochum, Germany
21. Patch Clamp : Technique
- In excised patch techniques, the patch is removed from the main body of cell.it is
used to study role of single ion channel in the section of membrane.
- In cell attached patch, gentle suction is applied through the microelectrode to
draw a piece of membrane into the tip to form a seal. Used to study ion channel
activity in the patch.
- In whole cell patch, more amount of suction is applied to displace the patch in
the tip leaving the electrode sealed to the rest of the cell. Used to take stable
intracellular recordings.
22. Patch Clamp : Technique
- In perforated patch, small pores are made on the patch with pore forming
agent so that large molecules can stay inside and ion can pass freely. Used
to analyse membrane properties of the patch pharmacologically.
- In loose patch, a loose seal is formed by moving the pipette slowly to the cell
increasing the resistance and pipette does not get close to cell. Loose seal
creates small gap ensuring the movement of ion channel outside the cell and
the membrane remains intact.
24. 4. Sharp Electrode Technique :-
- This technique is used to record membrane potential with minimal effect of ionic
constitution of intracellular fluid.
- The micropipette used is same as patch clamp micropipette but the pore size is
small to reduce the ion exchange between intracellular fluid and electrolyte.
- Tip of the electrode is filled with various dyes like Lucifer yellow for the study of
morphology of the cell under microscope.
- Dyes are injected by applying DC or pulsed voltage to the electrode.
26. 1. Amperometry 2. Field Potential
- Carbon electrode to record changes in the
chemical composition of oxidised
components in a biological solution.
- Oxidation is achieved by potential
changes at the membrane by scanning.
- Used to study exocytosis in nervous and
endocrine systems.
- Oxidisable chemicals are Nor-epinephrine,
Epinephrine and other monoamine
receptors.
- Types: Single Potential Amperometry and
Pulsed Amperometry
- Local Current sinks or sources generated
by activity of multiple cells at a time.
- Hippocampal Synaptic field potentials:
CC BY-SA 3.0,
https://en.wikipedia.org/w/index.php?curid=23818
60
C
27. 3.Single Unit Recording 4.Multi Unit Recording
- High impedance microelectrode having a tip
size of 1 micrometer detecting at most one
neuron.
- Action potential recorded here are same as
intracellular AP recording but signal are smaller.
- Provide brain processes information and show
how a single neuron responds to different
stimulus in different area.
- This technique is used in human cognition and
cortical mapping.
- Used for anesthetized or conscious animals.
- Microelectrode having a large tip recording
neuronal activity of several neuron at once.
- Record changes in activity in a discrete brain
area during normal activity.
- Used for conscious animals.
- Help to understand that how collective
computation in brain occurs.
29. 1.BERA Assay : 2.SSM Based Technique :
- Determine various chemical and biological
molecules by measuring changes in AP of cells
immobilized in a gel matrix.
- Used primarily in biosensor application.
- Also used for detection of human viruses,
veterinary disease agents, plant viruses,
pesticides and superoxide anions .
- It is rapid, reproducible and cost efficient.
- It is a core technology behind pan-European
FOODSCAN Project about pesticides and food
risk.
- A lipid monolayer is painted over the
functionalized microelectrode.
- It absorbs proteoliposomes, membrane
vesicles and membrane fragments containing
channels.
- Painted membrane supported by electrode, so
called as a solid support membrane.
- Mechanical perturbations do not destroy SSM.
- Capacitive electrode is mechanically stable
and help to investigate the electrogenic activity
of proteins of interest.
30. 3. Planar Patch Clamp
- High throughput electrophysiology technique
- Cell suspension is prepared on a chip containing microstructured aperture.
- Advantages of the planar geometry:
i) Allows integration of microfluidics, that helps compound
screening of ion channels.
ii) Accessible for optical and scanning probe techniques.
iii) Perfusion and intracellular slide can be prepared.
31. 4. Computational Electrophysiology:
- Measure of conductive properties of proteins and membranes in silico.
- Mainly Molecular dynamics simulations in which a model system is subjected
to an externally applied voltage.
- Advantage: Enormous and rigorous amount of clear and detailed data (less
noise) obtained which also has a very high resolution that can’t be obtained
from atomistic experiments.
- Disadvantage: Reproducibility of the data in vivo, validation of the data
obtained is often difficult, applicability of the model in living system is often
questionable, cost involved with the computational modelling is high.
32. Fig: The
electrophysiology
rig
Keys:
1. Micromanipulator
controller
2. DIC Microscope
3. Micromanipulator with
the motors that move
in three axis
4. perfusion manifold
5. Camera
6. electrode mounted on
a head stage amplifier
which is fixed on the
micromanipulator
7. microscope stage
where slides or
cultures are kept
34. References:
1. Intracellular recording by ROMAIN BRETTE AND ALAIN DESTEXHE
2. Hodgkin AL, Huxley AF (1952d): A quantitative description of membrane current and its
application to conduction and excitation in nerve. J. Physiol. (Lond.) 117: 500-44
3. Hodgkin AL, Huxley AF (1952a): The components of membrane conductance in the
giant axon of Loligo. J. Physiol. (Lond.) 116: 473-96.
4. Scanziani, Massimo; Häusser, Michael (2009). "Electrophysiology in the age of light".
Nature. 461 (7266): 930–9. doi:10.1038/nature08540. PMID 19829373.
5. https://en.wikipedia.org/wiki/Electrophysiology
6. Patch-Clamp Analysis ADVANCED TECHNIQUES , Second Edition , Edited by
Wolfgang Walz Department of Physiology, University of Saskatchewan, Saskatoon,
https://en.wikipedia.org/wiki/Electrophysiology
http://www.hopkinsmedicine.org/heart_vascular_institute/education/electrophysiology/
https://www.axolbio.com/page/whole-cell-patch-clamp-protocol
http://www.abcam.com/protocols/brain-slice-electrophysiology-video-protocol
http://sci-hub.cc/10.1002/0471142301.ns0601s00
Electrophysiology is the science and branch of physiology that pertains to the flow of ions (ion current) in biological tissues and, in particular, to the electrical recording techniques that enable the measurement of this flow. Classical electrophysiology techniques involve placing electrodes into various preparations of biological tissue.
Intracellular Recording: Intracellular recording is an electrophysiology technique that uses a microelectrode inserted into a single cell, usually a neuron, to measure its electrical activity.
Extracellular Recording: used to monitor neuronal activity from outside of the cell. Measures patterns of action potential.
Intracellular Recordings: See manual notes
Hodgkin and Huxley got nobel prize for measuring AP by voltage clamp method in 1963.
Giant squid axon was used as it’s diameter is large.
**Mention about double electrode voltage clamp where another electrode is inserted to inject current into the cell to change the voltage
1.Ideal situation-- v.clamp=v.m , dv.memb/dt=0(stabilised system), sum of all ionic current + current flowing through electrode = 0
2.Speed of clamping is imp. How fast the memb potential follows command potential.
3.Clamp error = v.m - v.clamp. To reduce this feedback current is provided which is I= gc(v.clamp-v.m)+Ic where gc= large gain and infinite. I.c= control current
Double electrode advantage :- Vm is not distorted by IC. . VM matches Vclamp .because no current passes through the measuring electrode in the stationary regime. Both current and voltage measurements are correct in stationary regime.
DE Problem :- capacitive coupling between two electrodes, lower feedback gain, destabilised system, poorer clamp, longer settling time, distortion in measurement.
Problem sorted by inserting two electrodes at a wide angle.
Single electrode problem:- contaminate the measurement of membrane potential.
Prob. sorted by 1. Series resistance compensation
2. Active electrode compensation (AEC)
3. Discontinuous voltage clamping
Space clamp-- problem in recording currents originating from dendrites because remote dendritic locations are not clamped at a given potential.
Compensation error-- use of single electrode causes compensation error and destabilise the system .to overcome this prob. Electrode compensation technique is used.( in case of single electrode set up)
Imperfect Clamping or capacitive coupling -- large feedback gain (gc) can destabilise the system due to various capacitive currents and imperfections. So lower feedback gain is applied which causes imperfect clamping.( in case of double electrode set up.)
In the inside-out method, a patch of the membrane is attached to the patch pipette, detached from the rest of the cell, and the cytosolic surface of the membrane is exposed to the external media.the pipette is attached to the cell membrane as in the cell-attached mode, forming a gigaseal, and is then retracted to break off a patch of membrane from the rest of the cell.
Advantage :- access to the intracellular surface , can change the chemical composition of what the surface of the membrane is exposed to.
Outside-out :- After the whole-cell configuration is formed, the electrode is slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode with the original outside of the membrane facing outward from the electrode. to examine the properties of an ion channel when it is isolated from the cell and exposed successively to different solutions on the extracellular surface of the membrane.
Disadvantage of whole cell mode is that the intracellular fluid of the cell mixes with the solution inside the recording electrode and makes it diluted. Perforated patch technique is used to minimise this prob.
Spike sorting in multi unit recording. Recording tells about the no of cell around the closely placed electrodes as well as which spike come from which cell. Spike characterisation.
SSM :- This electrode consists of a glass support, a chromium layer, a gold layer, and an octadecyl mercaptane monolayer. The capacitive electrode (composed of the SSM and the absorbed vesicles) is so mechanically stable that solutions may be rapidly exchanged at its surface. This property allows the application of rapid substrate/ligand concentration jumps to investigate the electrogenic activity of the protein of interest, measured via capacitive coupling between the vesicles and the electrode.
BERA: increased stability of the electrode-cell interface, immobilization preserves the viability and physiological functions of the cells.assay analytes that can interact with the immobilized cells by changing the cell membrane potential. In this way, when a positive sample is added to the sensor, a characteristic, "signature-like" change in electrical potential occurs.
human viruses (hepatitis B and C viruses andherpes viruses),] veterinary disease agents (foot and mouth disease virus, prions, and blue tongue virus), and plant viruses (tobacco and cucumber viruses)....... environmental toxins, such as pesticides and mycotoxins in food, and 2,4,6-trichloroanisole in cork and wine.
A BERA sensor has two parts:
The consumable biorecognition elements
The electronic read-out device with embedded artificial intelligence