Neuronal Signals - NBDS 5161 Session 4: Analyzing Synaptic...
Transcript of Neuronal Signals - NBDS 5161 Session 4: Analyzing Synaptic...
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Neuronal Signals - NBDS 5161
Session 4: Analyzing Synaptic Activity,
Membrane and Synaptic Currents
Lectures can be downloaded from
http://hayar.net/NBDS5161
Abdallah HAYAR
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Updated Tentative Schedule for Neuronal Signals (NBDS 5161)
One Credit–Hour, Summer 2010
Location: Biomedical Research Building II, 6th floor, conference room,
Time: 9:00 -10:20 am
Session Day Date Topic Instructor
1 Tue 6/1 Design of an electrophysiology setup Hayar
2 Thu 6/3 Neural population recordings Hayar
3 Thu 6/10 Single cell recordings Hayar
4 Fri 6/11 Analyzing synaptic activity Hayar
5 Mon 6/14 Data acquisition and analysis Hayar
6 Wed 6/16 Analyzing and plotting data using OriginLab Hayar
7 Fri 6/18 Detecting electrophysiological events Hayar
8 Mon 6/21 Writing algorithms in OriginLab® Hayar
9 Wed 6/23 Imaging neuronal activity Hayar
10
Fri
6/25
Laboratory demonstration of an electrophysiology and imaging experiment
Hayar
11 Fri 7/9 Article presentation I: Electrophysiology Hayar
12 Mon 7/12 Article presentation II: Imaging Hayar
13 Wed 7/14 Exam and students’ survey about the course Hayar
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Student List
Name E-mail Regular/Auditor Department Position
1 Simon, Christen [email protected] Regular
(form signed)
Neurobiology &
Developmental Sciences
Graduate Neurobiology –
Mentor: Dr. Garcia-Rill
2 Kezunovic, Nebojsa [email protected] Regular
(form signed)
Neurobiology &
Developmental Sciences
Graduate Neurobiology –
Mentor: Dr. Garcia-Rill
3 Hyde, James R [email protected] Regular (form signed)
Neurobiology & Developmental Sciences
Graduate Neurobiology – Mentor: Dr. Garcia-Rill
4 Yadlapalli, Krishnapraveen
[email protected] Regular (form signed)
Pediatrics Research Technologist – Mentor: Dr. Alchaer
5 Pathan, Asif [email protected] Regular
(form signed)
Pharmacology & Toxicology Graduate Pharmacology –
Mentor: Dr. Rusch
6 Kharade, Sujay [email protected] Regular
(form signed)
Pharmacology & Toxicology Graduate Pharmacology –
4th year - Mentor: Dr. Rusch
7 Howell, Matthew [email protected] Regular (form signed)
Pharmacology & Toxicology Graduate Interdisciplinary Toxicology - 3
rd year -
Mentor: Dr. Gottschall
8 Beck, Paige B [email protected] Regular (form signed)
College of Medicine Medical Student – 2nd
Year - Mentor: Dr. Garcia-Rill
9 Atcherson, Samuel R [email protected] Auditor (form signed)
Audiology & Speech Pathology
Assistant Professor
10 Detweiler, Neil D [email protected] Auditor (form not signed)
Pharmacology & Toxicology Graduate Pharmacology –1
st year
11 Thakali, Keshari M [email protected] Unofficial auditor Pharmacology & Toxicology Postdoctoral Fellow –
Mentor: Dr. Rusch
12 Boursoulian, Feras [email protected] Unofficial auditor Neurobiology & Developmental Sciences
Postdoctoral Fellow – Mentor: Dr. Hayar
13 Steele, James S [email protected] Unofficial auditor College of Medicine Medical Student – 1st Year –
Mentor: Dr. Hayar
14 Smith, Kristen M [email protected] Unofficial auditor Neurobiology &
Developmental Sciences
Research Technologist –
Mentor: Dr. Garcia-Rill
15 Gruenwald, Konstantin [email protected] Unofficial auditor Neurobiology &
Developmental Sciences
High school Student –
Mentor: Dr. Hayar
Yang, Dong [email protected] Unable to attend Pediatrics Pulmonary Research Assistant –
Accepted in Neuroscience
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Why study voltage clamping?•Historical: This is the method invented by Hodgkin and Huxley to discover the voltage-dependent behavior of sodium and potassium currents.
•Factual: To understand the voltage and time dependence of sodium and potassium currents underlying the action potential.
•Methodological:•The same method, in principle, is still used to study many other types of membrane currents (calcium currents, chloride currents, pump currents, etc.)•The same method is used to study the currents that go through single ion channels.
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Fig. 5. The voltage-clamp technique keeps the voltage across the membrane constant
so that the amplitude and time course of ionic currents can be measured. In the two-
electrode voltage-clamp technique, one electrode measures the voltage across the
membrane while the other injects current into the cell to keep the voltage constant. The
experimenter sets a voltage to which the axon or neuron is to be stepped (the command
potential). Current is then injected into the cell in proportion to the difference between
the present membrane potential and the command potential. This feedback cycle occurs
continuously, thereby clamping the membrane potential to the command potential. By
measuring the amount of current injected, the experimenter can determine the amplitude
and time course of the ionic currents flowing across the membrane.
Voltage Clamp
2-electrode
voltage clamp
1-electrode
voltage clamp
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Voltage clamping: 3 principles(1) Injecting positive current into the cell depolarizes the cell (injecting negative current hyperpolarizes it).
V
I
Depolarizing response
V
I
Hyperpolarizing response
(2) When current is injected into the cell, it takes some time to hyperpolarize/depolarize the cell because the cell’s capacitance must be charged/discharged.
(3) When there is no net flow of ions into the cell, the membrane potential doesn’t change.
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Voltage Clamp
• If VC
> VM, current is positive
– Membrane potential increases
– VC
- VM
decreases
• If VC
< VM, current is negative
– Membrane potential decreases
– VC
- VM
decreases
• Inward current
– Negative current injected to maintain membrane potential.
– Negative current is compensating inward flow of positive ions
– Transient current
• Outward current
– Positive current injected to compensate for outward flow of positive ions
– Persistent current
• Measure VM
= Inside -Outside
• Choose Clamp potential (VC)
• Calculate VC
- VM
• Inject current = g (VC
- VM)
The voltage clamp is used by electrophysiologists to measure the ion currents across a neuronal membrane while
holding the membrane voltage at a set level. Neuronal membranes contain many different kinds of ion channels,
some of which are voltage gated. The voltage clamp allows the membrane voltage to be manipulated independently
of the ionic currents, allowing the current-voltage relationships of membrane channels to be studied
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Comparison of Na+ and K+ Currents
following a Depolarization
repolarization closes K+ channels
depolarizationopens K+ channels
opens Na+ channels
K+ efflux
Na+ influx
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Voltage clamp: what is the behavior of voltage dependent sodium current?
The pharmacological method: Block the potassium current with a drug: tetraethylammonium. The voltage-dependent current that remains is the voltage-dependent sodium current.
-10 mV
-65 mVV
I
1 msec
In the presence of tetraethylammonium (TEA)
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The pharmacological method: Block the potassium current with a drug: tetraethylammonium. The voltage-dependent current that remains is the voltage-dependent sodium current.Note: even with a constant voltage, the sodium current first increases, and then automatically, while the depolarization is maintained, the current decreases (inactivation)
-10 mV
-65 mVV
Voltage clamp: what is the behavior of voltage dependent sodium current?
Na current
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Pharmacological method: Block the voltage-dependent sodium current with tetrodotoxin. The current that remains is the voltage-dependent potassium current.
Note: (1) the potassium current is slower to activate than the sodium current. Therefore, sometimes called “delayed current”
(2) the potassium current is maintained for as long as the depolarization is maintained. (only closes after repolarization)
Voltage clamp: what is the behavior of voltage dependent potassium current?
-10 mV
-65 mVV
I
In the presence oftetrodotoxin (TTX)
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Voltage clamp: what is the behavior of voltage dependent potassium current?
-10 mV
-65 mVV
I
In the presence oftetrodotoxin (TTX)
Pharmacological method: Block the voltage-dependent sodium current with tetrodotoxin. The current that remains is the voltage-dependent potassium current.
Note: (1) the potassium current is slower to activate than the sodium current. Therefore, sometimes called “delayed current”
(2) the potassium current is maintained for as long as the depolarization is maintained. (only closes after repolarization)
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Bursting persists after blockade of Ih current
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Voltage Dependent Channels
• Diversity of firing patterns produced by myriad voltage dependent
channels
• Channels differ by
– Ion selectivity (e.g. K, Na, Ca)
– Distribution (Dendrites, soma, axon)
– Sensitivity to drugs
– Activation and Inactivation properties:
– Activation: Turning on of current with depolarization
– De-activation: Turning off of current with repolarization
– Inactivation: Turning off of current with sustained depolarization
– De-inactivation: Removal of inactivation (block) by
repolarization
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Sodium Currents
• Transient, INaF
– Responsible for Action Potential
• Persistent, INaP
– Threshold near resting potential
– Origin:
• Window current, or
• Different gating mode of INaF, or
• Separate channel protein
Function of Persistent current-Enhancement of subthreshold synaptic potentials-Depolarization activates INaP, => more depolarization-Hyperpolarization de-activates INaP, which produces
more hyperpolarization
Plateau potential-Prolonged potential that remains after synaptic inputs
or current injection is removed-Contributes to persistent firing
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Persistent sodium current is activated by a slow depolarizing voltage ramp
(> -60 mV) and is blocked by Tetrodotoxin (TTX)
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Bursting is mediated by sodium channel activation and is blocked by
intracellular application of a sodium channel blocker QX-314
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FIGURE 12 Simulation of the effects of the addition of various ionic currents to the pattern of activity generated by
neurons in the mammalian CNS. (A) The repetitive impulse response of the classical Hodgkin–Huxley model (voltage
recordings above, current traces below). With only INa and IK, the neuron generates a train of five action potentials in
response to depolarization. Addition of IC (B) enhances action potential repolarization. Addition of IA (C) delays the onset
of action potential generation. Addition of IM (D) decreases the ability of the cell to generate a train of action potentials.
Addition of IAHP (E) slows the firing rate and generates a slow afterhyperpolarization. Finally, addition of the transient
Ca2+ current IT results in two states of action potential firing: (F) burst firing at -85 mV and (G) tonic firing at -60 mV.
From Huguenard and McCormick (1994).
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Sequence
of events
involved in
transmission
at a typical
chemical
synapse
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There are three main types of ionotropic glutamate
receptors
AMPA
Kainate
NMDA
AMPA and Kainate receptors are collectively also known as non-NMDA receptors. All three receptors are
ligand-gated ion channels. AMPA and kainate receptors are permeable to Na+ and K+ ions, whereas NMDA
receptors also have a high permeability to Ca2+ ions.
Each receptor type is a multimeric protein complex comprised of either 4 or 5 subunits.
Each subunit contains 3 transmembrane domains and a re-entrant loop.
NMDA receptors are unusual in that they also require a co-agonist in addition to glutamate for them to
function properly. In addition they are blocked in a voltage dependent manner by Mg2+ ions. NMDA
receptors are also modulated/blocked by a variety of endogenous and exogenous ligands.
AMPA and NMDA receptors are co-localised at glutamatergic synapses where they mediate ‘fast’ chemical
synaptic transmission.
The precise role of kainate receptors is still a matter of much research.
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Glutamatergic synaptic currents have a fast and
slow component
Control
10 pA25 ms
In the presence of AP5, an
NMDA receptor antagonist,
only the fast component remains
‘Fast’ component mediated
by AMPA receptors
‘Slow’ component mediated
by NMDA receptors
Figure from: Clark, Farrant & Cull-Candy (1997) Journal of Neuroscience 17, 107-116.
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-- NMDA receptors (Glutamate),
are ligand-gated ion channels that
are also voltage dependent.
-- For the channel to be open, the
receptor must bind glutamate and
the membrane must be
depolarized. This behavior is due
to a magnesium-dependent block
of the receptor at normal
membrane resting potentials.
-- Second, the receptor permits a significant
influx of Ca and increases in intracellular Ca
activate a variety of processes that alter the
properties of the neuron.
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NMDA-receptors – physiological and
pathophysiological roles
NMDA receptor-channels have been implicated in many CNS ‘processes’ ranging
from synaptogenesis to excitotoxicity.
Blocking NMDA receptors prevents ‘normal’ synaptic connections to be made during
development e.g. in barrel or visual cortex.
During stroke, overactivation of NMDA receptors results in cell death.
Models of learning and memory implicate a fundamental role of the NMDA receptor
as a ‘co-incidence’ detector.
Drugs that block NMDA receptors may be of use in the treatment of
stroke and Parkinson’s disease, while those that potentiate receptor function may be
of benefit in the treatment of Alzheimer’s disease.
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Binding sites for:
Benzodiazepines (BZ)
(sedatives hypnotics)
Steroids
(New General
Anaesthetics)
Barbiturates
(Old Sedatives hypnotics)
Ethanol
(sedation)
Anticonvulsants
(Spasticity motor
disorders)
GABAA Receptor : Structure & Activation
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The bursts of EPSP/Cs are action-potential dependent
Cell#001201c8
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Baclofen reduces the frequency of action potential-independent EPSCs (mEPSCs)
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µ-opioid receptor
2-adrenoceptor
Glutamate GABA
RVL Bulbospinal neuron
Heart
Blood vessels
Spinal cord
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500 ms
50 pA
500 ms
50 pA
mEPSCS (in presence of TTX + Gabazine)
mIPSCS (in presence of TTX + CNQX)
Excitatory and inhibitory miniature postsynaptic currents
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0 2 4 6 8 10 12 14 16 180
4
8
12
16
Bin = 10 sec
Met-Enkephalin (3 µM) In presence of TTX (1 µM)
+ Gabazine (3 µM) + Strychnine (10 µM)
Fre
qu
en
cy o
f m
EP
SC
s (
Hz)
Time (min)
20 pA
ME decreases the frequency of miniEPSCs
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0 2 4 6 8 10 12 14 160.0
0.1
0.2
0.3
0.4
0.5
Fre
qu
en
cy o
f m
IPS
Cs (
Hz)
Time (min)
50 pA
ME (3 µM) in presence of TTX + CNQX
ME decreases the frequency of miniIPSCs
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0 5 10 15 20 25 30
0.0
0.2
0.4
0.6
0.8
1.0
Control
ME
Re
lative
fre
qu
en
cy
Amplitude of mEPSCs (pA)
0 2 4 6 80.2
0.4
0.6
0.8
1.0
Inter-event interval (ms)
ME reduces the frequency but not the amplitude of miniEPSCs
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200 400 600 800
Bin = 30 sec
Endomorphin (3 µM)
In presence of TTX (1 µM) +Stry (10 µM) +Gabazine (3 µM)
Barium (1 mM)
0 200 400 600 8000.0
0.2
0.4
0.6
0.8
1.0
Fre
qu
en
cy o
f m
EP
SC
s (
Hz)
Time (sec)
-60
-40
-20
0
20
40Control
Endomorphin (3 µM)
Mem
bra
ne c
urr
en
t (p
A)
The decrease in miniEPSC frequency by EM persisted in barium
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0.5 sec
20 pA
b
a
ba
4 sec
20 pA
Stimulation paradigm
SpinalCord
Glu, monosynapticGlu,
disynaptic
Stim Rec
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0 1 2 3 4 5 6 7 80
10
20
30
40
20 pA
Num
ber o
f eve
nts
Time (sec)
EPSCs afterdischarge
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0 5 10 15 20 250
4
8
12
16
Fre
q. o
f sp
on
t. E
PS
Cs (
Hz)
Time (min)
NE (30 µM)NE (30 µM) 2-MOI (1 µM)
50 pA
Effect of NE on EPSCs afterdischarge
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Pharmacological characterization of evoked PSCs in the SubCD
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ON stimulation evokes constant latency single EPSCs in ET cells:latency = 2.1 0.1 ms, SD = 87 9 µs, n = 8
Cell#001201c3 Cell#001211c2
Cell#001201c8Cell#001117c4
Cell#001129c3
Cell#000629c4Cell#000629c3
Cell#001204c2
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ON stimulation evokes bursts of EPSCs in SA and PG cellslatency = 6.1 0.4 ms, SD = 1.6 0.4 ms, n = 12
Cell#001204c4
Cell#001211c4
Cell#001116c9
Cell#000629c2
Cell#001204c3 Cell#001117c7
Cell#001201c4
Cell#001116c3
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Effect of increasing intensity of stimulation on evoked bursts of EPSCs
Cell#001204c4
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Wash +
SKF 86466
Control + Gabazine
+ Strychnine
+ Barium
(100 nM)
+ Moxonidine
(1 µM)
150 ms
20 pA
(10 µM) (30 µM)
Effect of moxonidine on evoked EPSCs in barium
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0
40
80
120
160
200
240
Moxonidine
Am
pli
tud
e o
f ev
ok
ed
EP
SC
s (
pA
)
100 nM 300 nM 1 µM 3 µM 10 µM wash+2-MOI 3 µM
0 4 8 12 16 20 24 28 32 36
Time (min)
0.1 1 100
20
40
60
80
100
EC50
~ 1 µM
BA
% I
nh
ibit
ion
[Moxonidine] (µM)
Concentration-dependent inhibition of the evoked EPSCs by moxonidine
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0 5 10 15 20 250
40
80
120
160
Moxonidine (10 µM)
Time (min)
Am
pli
tud
e o
f evok
ed
EP
SC
(p
A) Moxonidine (10 µM) SKF 86466 (10 µM)
a
50 ms
50 pA
d ecb
dc eba
Inhibition of the evoked EPSCs by moxonidine
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B
A
CNQX GabazineWash+ NE+ Barium
Tau = 11 ms
Control
Tau = 21 ms
CNQX
Tau = 21 ms
50 pA
50 ms
50 pA
50 ms
+ NE
NE inhibits the evoked IPSCs in barium
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10 ms
50 pA
Control NE (30 µM) Wash
2 ms
50 pA
Effect of NE on mono- and disynaptic evoked EPSCs
SpinalCord
Glu, monosynapticGlu,
disynaptic
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GluGlu
5-HT
5-HT1-R
2-AR
Tyramine
Fluoxetine
Presynaptic monoaminergic modulation in RVL
RVL bulbospinal
neuron
2-AR
Glu
5-HT
DA
D2
2-AR1-AR
5-HT3
5-HT2
RVL
neuron
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Tyramine inhibits evoked and spontaneous EPSCs
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The effect of tyramine is mimicked by other monoamine releasing agents
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Time
Voltage
Response types at single CNS
synapses with different #s of release sites.
failure
1 vesicle2 ves.
1 ves.
fail
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If transmission is robust on the first
stimulus most readily releasable vesicles
will be gone and depression results.
Squire Fundamental Neurosci. 2002
Short term plasticity, history dependent changes in responsiveness
Stim.
Residual Ca can facilitate
transmission if not all quanta are
released on the first stimulus.
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ET cells are entrained by ON input at physiological theta frequencies
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Paired-Pulse Depression Depends on Neuronal Type