Transcript Bioelectrical Signal Recording

Bioelectrical Signal Recording
INTRODUCTION TO BIOMEDICAL
ENGINEERING
AUTHORS:
ANA PORTEIRA Nº 67305
GUILHERME MOURA Nº 67323
SANDRO NUNES Nº67945
SUPERVISED BY:
PROF. ANA SEBASTIÃO
Master in Biomedical Engineering
1st Semester, 2009
Structure of the Presentation
Introduction
Cell to Cell
Communication
Electrophysiology
Experimental
Procedure
Patch Clamp Technique
Recording Tecnhniques
Overview
Patch Clamp
Applications
Future Prospectives
Discussion/Conclusion
Introduction to the Subject
 Bioelectrical signals are transient
pulsations propagated throughout the
membrane of living cells such as
muscular cells and neurons;
 As the propagation of these signals
is a key part on how communication
occurs inside our body, it is absolutely
necessary to find a way of studying it;
Introduction to the Subject
Recording of Bioelectrical Signals
Electrophysiology
Biomedical Engineering
Study of the electrical
properties of biological
cells and tissues, through
measurements of voltage
changes
or
electrical
current flows on a wide
variety of scales.
Biomedical
Engineering
employs resources from
various scientific fields to
create and develop new
materials, instruments and
recording techniques.
Introduction to the Subject
Recongnize how bioelectrical signals are
transmited and which are the structures involved;
Learn what are the fundamental recording
techniques and how they function;
Main
Objectives:
Elaborate on Patch-Clamp Technique, which is
the most advanced method today;
Observe, in loco, one of its applications;
Discuss the the advantages and disadvantages and
refer to the future prospectives;
Present our analysis on the subject;
Cell to Cell Communication
Gap
Junctions
Juxtacrine
Signaling
Synaptic
Transmission
Paracrine
Signaling
Endocrine
Signaling
Cell to Cell Communication
Communications
established through
channels constituted
by Connexine
Proteins;
Connexine Proteines
pair in groups of 6,
forming a central
pore and linking
both cell’s
cytoplasm;
Gap
Junctions
Ions and small
mollecules are
allowed to freely
pass from one cell to
another in a very
short period of time;
Electric Synapses
make use of Gap
Junctions.
Cell to Cell Communication
A protein on one
cell binds to its
receptor on the
adjacent cell;
Juxtacrine
Signaling
A receptor on one
cell binds to its
ligand on the
extracellular
matrix secreted by
another cell;
Type of signaling which
involves the interaction
of a protein between an
inducing and a receptor
cell without diffusion.
The signal is
transmitted
directly from the
cytoplasm of one
cell through small
conduits into the
cytoplasm of an
adjacent cell.
Cell to Cell Communication
Paracrine Signalling
permits simmultaneous
communication between
the inducting cell and
other cells in its vicinity;
It makes use of chemical
messengers, which are a
particular type of
signaling molecules called
paracrine factors;
Paracrine
Signaling
These molecules are
diffused in the
extracellular matrix and
only affect cell in a close
range;
Diffusion of
neurotransmissors is na
example of paracrine
signalling.
Cell to Cell Communication
Endocrine Signaling
• In opposition to the rest, Endocrine
Signaling is mediated by a specialized
system of organs;
• Inducing cell deploys chemical
substances called hormonesinto the
bloodstream;
• Receptors in the cell membranes bind
to these molecules;
• Signal takes more time to arrive,
however it is more stable and capable
of acting over long periods.
Synaptic Transmission
 Synaptic Transmission depends on a process called
Action Potential.
Rapid alteration of the transmembrane
voltage generated by the activity of
voltage-gated ion channels embedded
in the cell membrane.
Synaptic Transmission
Action Potential
Resting Potential
Threshold
Rising Phase
Falliing Phase
Recovering Phase
+ channels open, permitting Na+ ions to enter
A
few Na
Many
of the
voltage-gated sodium channels start to
the
neuron;
close
and potassium
begin to
open; the resting
The
membrane
repolarizes
beyond
The
increase
in
positive
ions
inside
the
depolarizes
This
causes
the membrane
potential
to cell
begin
to go
membrane
voltage
because
more
potassium
channels
Small
amount
of
potassium
channels
are
open;
the
membrane
potential;
back
thetoresting
membrane
potential.
+to
are
than
during
the the
membrane’s
state;
Kopened
ions
enter
and
exit
cell additional
basedresting
on electric
and
Threshold
potential
is
reached
and
voltageVoltage-gated
potassium
channels
are
maximally
Potassium
channels
thatopened;
opened during the action
concentration
gradients;
gated
sodium
channels
activated
and
open.
potential
now potential
close;
Membrane
is maintained
at about and
-60mV.
Voltage
across
the membrane
rapidly reverses
The
membrane
backvalue.
to the resting potential.
reaches
its mostgoes
positive
Synaptic Transmission
 How are the bioelectrical signals propagated between
cells?
Communication between
neurons and communication
between neurons and muscle
occurs at specialized
junctions called synapses;
One example is the
chemical synapse mediated
by the neurotransmitter
Acetylcholine (ACh).
Neuromuscular Junction
Synaptic Transmission
 Converting a Chemical Signal to an Electrical Signal
Synaptic transmission begins
when the action potential reaches
the axon terminal.
The resulting depolarization,
due to opening of voltage-gated
sodium channels, initiates a
sequence of events leading to the
release of the transmitter.
Synaptic Transmission
 Converting a Chemical Signal to an Electrical Signal
The Ca2+ ions trigger the release
of neurotransmitter by causing the
synaptic vesicles closest to the
active zone of the synapse to fuse
with the presynaptic membrane.
This fusion process is regulated
by the interaction between protein
complexes expressed on the vesicle
and presynaptic membranes.
Synaptic Transmission
 Converting a Chemical Signal to an Electrical Signal
When the vesicles fuse with
the presynaptic membrane,
they empty their content of
neurotransmitter into the
synaptic cleft .
Synaptic Transmission
 Converting a Chemical Signal to an Electrical Signal
The neurotransmitter moves
across the synaptic cleft and binds
to receptors on the postsynaptic
membrane;
The channel opens, sodium ions
enter the postsynaptic cell, and the
depolarization signal is thus
propagated to the postsynaptic
cell.
Electrophysiology
Different types of electrophysiological recordings
Peri T Kurshan, Asli Oztan & Thomas L Schwarz, Nature Neuroscience
Electrophysiology
Different
Overview
typesthrough
of electrophysiological
Recording Conditions
recordings
 The tissue location where we are stimulating;
 What sorts of stimulation are used (chemical or
electrical);
 What type of synaptic potential (spontaneous or
induced)
 In what type of cell the signal is being recorded;
 Which cell receptors are blocked, in order to
know that
don‘t interfere in the result;
Evaluate Neuronal Communication
H
W?
Voltage Clamp
Current Clamp
Keeps the electrical current
through the recording
electrode;
Measures ion currents across a
neuronal membrane while holding the
membrane potential at a set level.
Records the membrane
potential by injecting current
into a cell through a
microelectrode
Electrodes in Electrophysiology
Sharp Electrodes
 Records the potential inside the cell
membrane with minimal effect on the ionic
constitution of the intracellular fluid.
 There is very little ion exchange between
the intracellular fluid and the electrolyte in
the pipette (small hole)
Measure Recording
Membrane
Voltage
Clamp
Potential, Resistance, Synaptic
potentials, Action potentials
Electrólito líquido
e.g. KCl, NaCl
Current can be injected into the cell to change the
membrane potential of the cell.
Current
Clamp
Electrodes in Electrophysiology
Patch Electrodes
It is sealed onto the surface of the cell
membrane, rather than inserted through it.
Micropipette
 The interior of the pipette is filled
with a solution matching the ionic
composition of the bath solution
(cytoplasm for whole-cell recording.)
 A chloride silver wire is placed in
contact with this solution and
conducts electrical current to the
amplifier.
Pressure … Suction
High resistance seal
Cell Membrane
Recording Techniques Overview
Intracellular
Extracellular
The
cellelectrode
is impaled
with aon
sharp
glass
The
is placed
the surface
electrode and(skin)
the voltage
and or the
current
is recorded
across the
record
cell’s populations.
membrane
ECG
Patch-Clamp
EEG
Patch Clamp Technique
Inside-Out/Outside- Out
Advantages: To record individual
channels; good pharmacology and
the inside/outside solutions can be
changed .
Disadvantage: channel properties
can be changed.
Whole-Cell
Advantages: good pharmacology
and high definition.
Disadvantage: dialysis of
cytoplasmatic contents
Patch Clamp Technique
Whole -cell
Disadvantage:
Dialysis of
cytoplasmatic contents
Advantage
Reduces the dialysis
Disadvantages
• Higher access resistance;
• Impossible to record
from single channels.
Perforated
Patch
Makes small holes on the
patch with pore-forming
agents instead of applying
suction.
Experimental Procedure
Our own
experiment
Patch Clamp
Experiment
1. Apply positive pressure through the
patch electrode;
2. Place the electrode near its surface
and applied a negative pressure
forming the giga-seal.
3. Wait for the membrane to rupture.
After we clamp
- Recordings
the cell
Evaluate the changes in the
frequency and amplitude of
spontaneous events in the
absence of any drugs
from CA1 pyramidal cells
Decrease in the
frequency and
amplitude due to
the deterioration
of the cell –
for e.g. dialysis of
intracellular
molecules.
Patch-Clamp Applications
N
e
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r
o
p
s
y
c
o
p
h
a
r
m
a
c
o
l
o
g
y
Alcohol affects a specific type of
neurotransmitter receptor, NMDA.
The entire single-channel methods have the capability
of being applied to human samples (e.g., slices or
cultures from biopsies), both from normal tissue
removed during surgery, and from diseased brains.
Importance of the study of drug
interactions with ionic channels, channel
gating mechanisms, ion channel
permeation dynamics among others
Limitations
High resolution measurements allowed by the Patch
Clamp Tecnhique have some downsides:
 Careful and precise fabrication of electrodes;
 Skillful manipulation of the patch pipette towards a cell;
 Clever design of electronics and apparatus to allow low-noise
recordings.
Highly Dependant in Nanotecnhnology
Future Prospectives
Advances in micro-fabrication offer promising technologies for
less laborious and cheaper patch clamp recordings. Listed below
are the two main improvements that could result from
advancements in nanotechnology:
Superior designs of
integrated nanoelectronic
patch clamp amplifiers
Development of new
nanomaterials
By improving the design of patch
clamp amplifiers, it is possible to
further lower background noise and
increase signal bandwidth.
The implementation of more resistive
nanomaterials in the construction of
the coating or the pipette may
facilitate the gigaseal formation.
Discussion/Conclusion
IONS AND
NEUROTRANSMIT
TERS STUDY
Patch clamp
NEUROPSYCOPHARMAC
OLOGY
REAL PHYSIOLOGY
SIMULATION
BIOMEDICAL
ENGINEERING
GENETICS AND
MOLECULAR
BIOLOGY
NEW METHODS
AND
ELECTODES
ELECTROPHYSIOLOGICAL
TECHNIQUES
MENTAL
DISEASE
THE END
Questions?