NE203 laboratory manual
JWL
General instructions for Labs 1-4:
The lab reports in these labs will be in the form of Powerpoint
slides. You will be instructed to incorporate screenshots or plots
into Powerpoints slides. At the end of each lab session, you should
leave a copy of your "unfinished" powerpoint file in the lab computer.
This file will be your "raw data" notebook for the record. You should
also copy the same file in a USB drive or your laptops. You need to
finish figure legends or answers to questions in the manuals, in the
"notes" section under the slides. Two out of the four members of your
group will do a 5 minute presentation using your "finished" powerpoint
file at the beginning of the next week's lab. You should also hand in
your "finished" power point files. Grades will be determined by the
quality of your presentation (50%) and your writing in the "finished"
powerpoint file (50%). All members of the same group receive the same
grade for both presentations and powerpoint files.
To prepare for the presentation, you should ask yourselves: (1)
are the contents scientifically correct? (2) Are your
statements/sentences clear and informative? (3) Are you facing the
audience and talking to them?
Note that while we would like to see that you all provide correct
answers in your presentation, mistake in answering questions are not
fatal. However, we, Lab Instructors or Learning Assistants, would like
to talk to you and clear up the mistakes you have made. The talk or
discussion will be informal, a few minutes during or after the lab
should be enough. The idea is not to leave issues or problems
unaddressed.
These four labs will account for 25% of the total lab grades. The
lab specific distributions will be: Labs 1 and 2: 5% each, Labs 3 and
4: 7.5% each.
1
NE203 laboratory
l
y manual
JWL
Lab. 1
The purpos
se of this
s lab is to introd
duce you t
to the ba
asic
propert
ties of ac
ction pot
tentials. We will f
first fam
miliarize ourselves
with th
he user in
nterface of the si
imulator. We will then perf
form current
clamp experiment
e
ts, to le
earn how action
a
pot
tentials (APs) beh
have under
differe
ent experi
imental conditions
c
s. The exp
perimenta
al conditi
ions we will
explore
e include changes in: Na+ concentrat
c
tion, g_K and g_Na
a.
1. Fami
iliarize yourself
y
with HHsi
im and act
tion pote
ential thr
reshold.
HHsim is
i a simpl
le simula
ation prog
gram that allows y
you to exp
plore
membran
ne excitab
bility. The
T
first task is t
to learn the basic
cs of this
program
m in a few
w simple steps. We
e will sta
art by le
earning ho
ow to
stimula
ate a neur
ron.
HHsim should
s
alr
ready be open on your
y
compu
uter. The
e first th
hing you
need to
o do is to
o change two displ
lay settin
ngs. Near
r the bott
tom of the
simulat
tor window
w, there is a yell
low window
w with a menu butt
ton to its
right. Select g_
_Na (μS). In the green
g
wind
dow to th
he right, select g_K
(μS)and
d select "blank"
"
in
i the cya
an window.
.
Click on
o the sti
imuli tab
b at the upper
u
left
t corner. The "St
timulus
Pattern
ns" window
w should appear (F
Fig.1). Th
he defaul
lt setting
g is to
inject a 10nA cu
urrent st
tep (a) wi
ith 1 ms d
duration (b) at th
he beginni
ing
of a cu
urrent tra
ace (c).
You can
n change these
t
variabl
les—amplit
tude,
duratio
on and tim
me of
onset—b
by typing the
require
ed numbers
s in boxe
es
next sl
liders, or
r by simp
ply
using the
t
slider
rs (d). The
T
blue li
ine above all the
boxes and
a
slider
rs is the
e
stimula
ation disp
play in
time sc
cale. Have
e a good
look at
t its X an
nd Y-axes
s
and mak
ke sure yo
ou see th
he
step on
n the blue
e line an
nd
numbers
s in the boxes
b
match.
To test
t the effe
ect of th
his
current
t step, go
o back to
o
the sim
mulator wi
indow, an
nd
click the
t
"clear
r" button
n
at the bottom of
f the
window.
. Click it
t twice, so
that tr
races in both
b
top
and bot
ttom panel
ls are
cleared
d and reap
ppear.
2
NE203 laboratory manual
JWL
To test the current injection, click "Stim1", the blue button in the
lower left corner. You should see an action potential (AP) in red in
the top panel, while your current injection will be indicated by the
blue trace below the red. In the lower panel, sodium and potassium
conductance ,g_Na (yellow) and g_K (green), will be displayed.
Project 1: What is the current threshold for AP?
Go back to the "Stimulus Patterns" window and decrease the current
amplitude in 1nA steps and write down the highest current that fails
to fire AP. Starting with this largest subthreshold current, increase
the value for current in 0.1nA steps until you find the lowest current
that can fire AP. However, finish reading this page first.
Write down your current threshold in the note window of your
powerpoint slide 1. (Don't just write the number, include unit as
well, also make clear that the number is for Project 1 etc.) Paste the
screenshot with AP initiated by your threshold current in the graph
window of slide 1. (The best way to insert screen shot is to, in
Powerpoint application, use InsertScreenshot to port your HHsim
window into powerpoint.) Save your file with the filename in the
following format: Answers_XXXX_date where XXXX should be the initials,
first and last, of every member in your group.
Also, in the note window, describe/compare g_Na and g_K in terms of
their width, heights and onset. (By onset I mean which one takes off
first?)
***This powerpoint file will be your raw data file and should not be
changed further after you finish the lab today. You should take a copy
of this file home, to polished it for the presentation next week. You
will also need to complete figure legends and answers to some of the
questions in this manual.
***Make sure that you click on the "Clear" button to clear the traces
in the window every time you test a new current amplitude. The 1st
click clears the top panel and the 2nd clears the bottom panel. It is a
good idea to repeat this "double click" a few times (3 times-6 clicks)
to be sure that the program goes back to the same numerical ground
state before a new test is carried out.
3
JWL
NE203 laboratory
l
y manual
***It should
s
be noted th
hat we are
e
definin
ng current
t thresho
old for
firing an AP. In
n your te
extbook,
when th
he thresho
old of AP
P is
mention
ned, it is
s often referring
r
to volt
tage thres
shold, na
amely the
voltage
e at which
h AP take
es off
with a sharp up-turn. In
n fact,
the pre
ecise poin
nt when "AP
"
takes
off wit
th a sharp
p up-turn
n" is not
easy to
o define unambiguo
u
ously. The
e
area wh
here AP st
tarts to take off
is enla
arged in Fig.2.
F
Th
he arrow
indicat
tes roughl
ly where the
thresho
old is.
Project
t 2: What would be
e the
current
t threshol
ld of a 2nd current
t
step if
f it were delivere
ed 5ms
after the
t
first one?
We now need to add
a
a 2nd current
step. First,
F
def
fine the interval
between
n the two steps by
y setting the numbe
er in the
e box labe
eled in
Fig.1e to 5ms. Set
S
the current
c
am
mplitude o
of both t
the 1st and
d 2nd step
ps
to 10nA
A (a&f). Click
C
"St
tim1". Wha
at happens
s?
You now
w need to find the
e current threshold
d for the
e 2nd step.
. Start b
by
nd
increas
sing the current
c
of
o the 2 step, in
n 10nA ste
eps, until
l you see
e
the sec
cond AP. Once
O
you find the current t
that fire
es AP, dec
crease it in
1nA ste
eps until you find
d the thre
eshold. (D
Don’t cha
ange the a
amplitude of
the fir
rst curren
nt step, only the second on
ne). What
t is the c
current
thresho
old of the
e second step? Wri
ite down y
your answ
wer in the
e note
window of slide 2. Paste
e the scre
eenshot wi
ith the 2nd AP init
tiated by
y
thresho
old curren
nt in sli
ide 2.
Compare th
he current
t threshold of the
e 2nd AP with that of the 1sst
AP—the one you determine
d
ed from Pr
roject 1.
The exp
e
periment you
y
have just perf
formed is an examp
ple of the
e relative
refract
tory perio
od. The relative
r
refractory
r
y period defines a time
interva
al after an
a AP, du
uring whic
ch a stron
nger stim
mulation i
is needed to
fire an
n AP. Can you expl
lain the mechanism
m
behind t
this obser
rvation?
Hint: The
T
reason
n why a stronger
s
stimulus
s
i
is requir
red for th
he 2nd AP has
to do with
w
g_K and
a
g_Na. Inspect the g_K t
traces in
n the lowe
er window.
Has g_K
K returned
d to base
eline when
n the 2nd stimulus was deliv
vered? (g
g_Na
ire
definit
tely has.)
) How wou
uld this "residual"
"
" g_K mak
kes it har
rder to fi
the 2nd AP? You don’t nee
ed to answ
wer this question immediate
ely.
However
r, you sho
ould have
e a 3-5 li
ine answer
r in the powerpoin
nt file yo
ou
hand in
n next wee
ek. The powerpoint
p
t file you
u used fo
or your pr
resentation.
(If you
u are not sure abo
out the an
nswers: ta
alk among
g yourselv
ves, ask
Learnin
ng Assista
ants or Lab
L
Instru
uctor. Kee
ep track of time t
though.
4
NE203 laboratory manual
JWL
Perhaps it is better to save the question/discussion after you have
finished all projects of this lab.)
The role of g_Na is not obvious from the g_Na trace. Why is the g_Na
associated with the 2nd AP smaller? Come back and answer this question
after you finish Project 7.
Project 3: Is AP amplitude dependent on extracellular Na+
concentration?
We are now going to recreate a historically important experiment. This
experiment provided the first conclusive evidence for the hypothesis
that Na+ influx was responsible for action potentials.
We will use
the same "Stim1" you played with in Project 1.
(1) Go to the "Stimulus Patterns" window. Click the "reset" button at
the lower left corner.
(2) Click on the "Membrane" tab in the upper left corner of the
simulator window, and you should see a "Membrane" window. Here you
have the concentrations and Nernst equilibrium potentials for Na+, K+
and Cl-. Have a good look and make sure that you find the
concentrations and Eion.
(3) Go back to the main window and click on the "Stim1" button. You
will get your AP as before.
(4) Along the top edge of the main window, there is a cluster of three
buttons: Cursor, Zoom and Pan. Make sure that "Cursor" is highlighted
in red.
You now need to measure the AP amplitude, by taking the difference
between voltage at the peak of AP and at resting membrane potential
(Vm). Click on the part of the red line where you think resting Vm
should be. (Hint: It should be at the part of the red trace before the
current step starts.) You should see an oval cursor on the red line,
and the numerical readout indicated in the pink window at the lower
right.
Click on the peak of the AP, and you will again find the voltage level
of the AP peak in the pink readout window. The difference between AP
peak and resting Vm should give you AP amplitude, or height. You will
also need to enter Na+ equilibrium potential (ENa), found in the
"Membrane" window.
You can now measure AP amplitude for the Na+ concentrations (Na_cn_out)
listed in the table below. You change Na+ concentration by going to
"Membrane" window and entering numbers in the box below Cout (mM). By
the end of this project, you should have all the boxes filled, except
perhaps the last row. Don’t forget the double clicking "Clear" button
routine.
Resting Vm
(mV)
AP peak (mV)
AP amp (mV)
5
Na_cn_out
(mM)
ENa (mV)
NE203 laboratory manual
JWL
55
75
100
200
400
600
700
It should be obvious why we are not going to record the reading for
700 mM Na+. This is not a software glitch in HHsim. When Na+ influx is
very strong as a result of high extracellular Na+, there is enough
residual Na+ current after each AP to push Vm beyond threshold and fire
AP again. No need to go into detail here, but you should be aware that
many neurons in our brain are capable of doing something like this,
namely firing continuously in an autonomous way.
We now can use these measurements to make a nice graph, as good
looking as that of the historical experiment. (The effect of sodium
ions on the electrical activity of giant axon of the squid.HODGKIN AL,
KATZ B.J Physiol. 1949 Mar 1;108(1):37-77)
Use "Alt_Tab" to find the "Lab 1" folder, and open the "Project 3
AP_Na_cn plot.pxt". You should see a table with cells filled with 0,
and a blank graph. Enter the relevant numbers from your measurements
into the cells. As the numbers are entered, you should see the graph
gradually taking shape. Look through the labels on the axes. The keys
for the data, namely the red dots and grey line, are also indicated
and the colors of the axes are matched with those of the data. The key
point is that the AP_amp vs Na+ concentration plot has a slope similar
to that of the Nernst equation, suggesting that Na+ permeability plays
a key role in AP generation. For the IGOR file, use "Save As" to save
your graph, with the file name in the format of "Project 3 AP_Na_cn
XXXX", where XXXX should be the initials of everyone in your team.
Highlight the graph by clicking on it. Go to "EDIT" menuExport
Graphics. There will be an "Export Graphics" dialogue box. Click "OK".
You can then paste the graph in the slide 3. Here, you need to write
a figure legend. Figure legend should have an informative title: what
is this figure meant to show. In the legend, you need to describe the
graph. Although axes are labeled, you still need to say what have you
manipulated and what have you measured. Although normally not donein
scientific papers, we would like you to finish the legend with a 1-2
sentence conclusion about the meaning/significance of this graph.
(Statements about the significance of a figure belong to text section
of a paper, not figure legends. Also see Appendix for an example of
figure legend.) As the answers associated with other slides, you don’t
need to have the legend in its finished form right now. However, it
should be finished/polished when you hand in your powerpoint file next
week.
Project 4: What happens to AP when you change g_K?
6
JWL
NE203 laboratory
l
y manual
The pur
rpose of this
t
project is to
o gain som
me insigh
hts to the
e importan
nce
There are
of K+ channels.
c
e about 100
1
genes for K+ ch
hannels. T
This larg
ge
number gives ris
se to gre
eat variet
ty of K+ c
channels. K+ channe
els are
often the
t
key to
o determi
ining the shape, am
mplitude, firing f
frequency
and fir
ring patte
ern of AP
P. We will
l look at only one
e specific
c aspect of
K+ chan
nnel funct
tion here,
, namely how g_K v
values sha
ape AP am
mplitude a
and
duratio
on.
(1) Go to the "M
Membrane"
" and "Sti
imulus Pat
tterns" w
windows an
nd click on
the res
set button
ns.
(2) Mak
ke sure th
hat the current
c
step in
n Stim1 is
s 10nA.
(3) Cli
ick on the
e "Channe
els" tab
near th
he top lef
ft corner
r, and
the cha
annels win
ndow shou
uld
appear.
.
(4) Cli
ick on the
e "Detail
ls"
button next to "Delayed
"
Rectifi
ier". (Del
layed rec
ctifier
is the main K+ channel
c
in
n the
squid giant
g
axon
n. Naming
g a K+
current
t Delayed Rectifie
er, a
term or
riginated from ele
ectric
enginee
ering, is a bit
unfortu
unate. Hod
dgkin and
d Huxley
built their
t
own voltage clamp
amplifi
iers, they
y must ha
ave been
familia
ar with al
ll concep
pts in
electro
onics and electric
c
enginee
ering. Thi
is term is
i still
commonl
ly used to
oday, to intimidat
te student
ts with b
bio and ps
sy
backgro
ound.) You
u should see the "Delayed
"
R
Rectifier
r" window (Fig.3). It
is a co
omplicated
d window but we wi
ill change
e only on
ne paramet
ter, gmax,
which is
i indicat
ted in Fi
ig.3 by th
he arrow. gmax repre
esents the
e maximal
l
conduct
tance for the dela
ayed recti
ifier and its valu
ue is prop
portional to
the num
mber of de
elayed re
ectifier K+ channel
ls availab
ble to th
his neuron
n—
namely the large
er the nu
umber, the
e more K+ channels.
. You wil
ll test th
he
effect of changi
ing gmax, according
g to the v
values in
n the tabl
le below, on
AP amp and durat
tion. (Th
he default
t gmax is 3
36μS, res
set button
n doesn't
change this numb
ber.)
You know how
h
to mea
asure AP amp from "Project 3". What about
measuri
ing AP dur
ration? It
I is some
ewhat arbi
itrary. W
We will us
se the AP
width at
a -40 mV as its duration.
d
To be abl
le to pla
ace the cu
ursor
precise
ely, you will
w
need
d to zoom in a bit.
. Click o
on the Zoo
om tab at
the top
p of the simulator
s
r window. The curso
or will t
turn into a little
magnify
ying glass
s. Zoom in
i on the AP you wa
ant to me
easure at the level
l
of -40 mV, then go back to the to
op of the window a
and click on the
cursor tab in or
rder to measure
m
th
he times a
at which the red, AP lines
7
NE203 laboratory manual
JWL
cross -40 mV line. (Zooming in by 2 clicks should be enough. If you
zoom in the wrong place, just right click, zoom out and try again.)
You will now be able to measure the times at which the rising and
falling phases of the AP cross the -40 mV line, by clicking on the red
trace and placing the oval indicator there. The time corresponding to
the pink oval is displayed in the black window to the right of the
pink window you used to read voltage. (At this level of zoom, the oval
cursor may not fall exactly on the point at which AP crosses the -40mV
line. Don't get stressed, just get the cursor as close to the -40mV
line as possible.) Write down the times at which the AP line crosses
the -40 mV line. The difference between the two time points is the
duration. Write down your duration in the table.
gmax ( μS)
AP peak (mV)
AP amp (mV)
AP duration (ms)
33
36
40
50
60
70
Zoom out and measure AP amplitude. (Don't forget to take the
difference between baseline and the peak to obtain AP amplitude.)
Bring up the "Lab 1" folder and open the "Project_4 g_K variation
plot.pxt" file. You can now enter the numbers you have just measured.
Save the IGOR file and paste the graph in powerpoint slide 4 and write
a figure legend for it.
Pay attention to the trend in AP duration and amplitude as the g_K
value is increased. In general, higher g_K level results in lower AP
amplitude and narrower AP duration. Explain why this is so in your
presentation next week. Don’t forget to add your explanation in the
powerpoint file.
8
NE203 laboratory
l
y manual
JWL
Project
t 5: What happens to AP whe
en you cha
ange g_Na
a?
There is
i another
r way to change
voltage
e gated co
onductanc
ce, namely
y
using channel
c
bl
lockers.
(1) Res
set the "M
Membrane"
",
"Channe
els" and "Stimulus
"
s
Pattern
ns" window
ws.
(2) Cli
ick on the
e "Drug" tab in
the sim
mulator wi
indow. Yo
ou should
see a window
w
lab
beled "Dr
rugs"
(Fig.4)
). We are going to
o ignore
the cya
an writing
g that in
nvites you
u
to clic
ck on the graph. (Click on
Reset at
a the low
wer left corner if
f
you hav
ve already
y clicked
d on the
graph.)
) For the purpose of
graphin
ng, it is more con
nvenient
to defi
ine the le
evel of blocking
b
numeric
cally. We will ent
ter the
percent
tage of Na
a+ channel
l block
by hand
d (Fig.4 arrow).
a
Let's
L
start by
b increas
sing the level of
block in
i 5% incr
rements. Measure
AP amp as before
e, you wi
ill also
need to
o measure g_Na by placing the
t
cursor
r on g_Na
a trace, l
lower pane
el.
% inhib
bition
0
5
10
25
30
40
50
AP amp (m
mV)
g_
_Na (μS)
u the "La
ab 1" fol
lder and open
o
the "
"Project 5 g_Na bl
lock
Bring up
plot.px
xt" file. You can now enter
r the numb
bers you have just
t measured
d.
e a
Don’t forget
f
to save you
ur plot an
nd paste t
the graph
h in slide
e 5, write
figure legend. Explain,
E
in your own
o
words,
, why the
e reductio
on in g_Na
results
s in reduc
ced AP am
mplitude. Explain i
in your p
presentati
ion next
week an
nd in the note win
ndow of sl
lide 5.
It is somewhat
s
surprisin
s
ng that AP
P can stil
ll be gen
nerated af
fter 50% o
of
the cha
annels are
e blocked
d. This is
s not unre
ealistic. Most neu
urons in our
brain have
h
a lot
t more Na
a+ channel
ls than is
s necessar
ry to fir
re AP,
general
lly by a factor
f
of
f 2-10. In
n neurophy
ysiology terms, we
e say
neurons
s have ver
ry high safety
s
fac
ctor for g
generatin
ng AP.
9
NE203 laboratory
l
y manual
JWL
Lab.2
In Lab. 1, we explo
ored AP behavior b
by inspect
ting how AP amplit
tude
+
and dur
ration cha
anged as we manipu
ulated [Na
a ]out, g_N
Na and g_K
K.
Experim
ments this
s week ar
re designe
ed to inve
estigate mechanism
ms
underly
ying AP in
n greater
r depth. We
W will st
tart by u
using volt
tage clamp
experim
ments to examine
e
iNa and iK activated
a
by volta
age steps.
. We will
then co
onsider th
he direct
tions of current
c
fl
low by in
ncorporati
ing the co
ore
concept
t of Nerns
st equili
ibration with
w
volta
age clamp
p experime
ents. We
will fi
inally con
nsider th
he behavio
or and sig
gnificanc
ce of Na+ channel
inactiv
vation.
Project
t 6: How to
t you an
nalyze and
d present Na+ and K+ current
t recorde
ed
with vo
oltage cla
amp?
d
(1) HHs
sim allows
s us eith
her to inj
ject curre
ent into an axon a
and record
voltage
e response
es (curre
ent clamp)
) or to pe
erform vo
oltage cla
amp and
measure
e current.
. We can switch be
etween the
ese two m
modes with
h the tab at
the low
wer right corner of
o the sim
mulator wi
indow. Sw
witch to V
Voltage
Clamp now.
n
As
A a remin
nder, all projects in Lab. 1 were cu
urrent clamp
experim
ments wher
re we man
nipulated current i
injection
n into an axon and
observe
ed membran
ne potent
tial (Vm) and AP ch
hanges. In lab. 2,
, we will use
both cu
urrent cla
amp and voltage
v
cl
lamp techn
niques. V
Voltage cl
lamp is a
techniq
que where we contr
rol Vm of a neuron and measure curre
ent flow
through
h ion chan
nnels, wh
hile the voltage
v
is
s maintai
ined at a constant
level.
ou turn on
n
Once yo
Voltage
e Clamp, the
t
"Stimul
lus Patter
rns"
window will chan
nge into
"Voltag
ge Clamp
Protoco
ol", Fig.5
5. The
red lin
ne in this
s window
defines
s how we will
w
control
l membrane
e
potenti
ial: start
ts at 60mV fo
or 10ms (a
a) then
step to
o -40mV fo
or 20ms
(b), th
hen step back to
-60mV for
f
10ms (c).
(
These voltage
v
le
evels
are not
t ideal. We
W are
going to
t change the
holding
g potentia
al to 90 mV by
b changin
ng the
numbers
s in (a) and
a
(c)
to -90m
mV. For th
he
purpose
e of illus
stration, our volt
tage step will be -40mV (b)
). There i
is
no need
d to chang
ge time.
10
NE203 laboratory
l
y manual
JWL
(2) We want to keep
k
thin
ngs simple
e and anal
lyze one current a
at a time.
t
"Chann
nels" win
ndow and uncheck
u
"F
Fast sodi
ium" so th
hat we can
Go to the
n
analyze
e iK in is
solation.
n
(3) Cli
ick "Run" in the simulator
s
window. (
(A remind
der, use "
"Run" when
you sim
mulate vol
ltage cla
amp but us
se "Stim1"
" when yo
ou perform
m current
clamp, by inject
ting curr
rent and record
r
Vm responses
s.)
The cur
rrent in the
t
upper window
w
wil
ll look
a bit odd.
o
Don't
t worry
about the
t
detail
ls. We
will fo
ocus on ho
ow to
measure
e this cur
rrent.
In theo
ory, we sh
hould
use an approach similar
to that
t for the
measure
ement of AP
A
amplitu
ude, namel
ly by
taking the diffe
erence
between
n the base
eline
(Fig.6a
a) and the
e
maximal
l iK ampli
itude
(b). In
n the case
e of iK,
the max
ximal curr
rent is
always at the en
nd of
the vol
ltage step
p.
However
r, since the
t
baselin
ne (a) is very
near to
o zero, ta
aking
the rea
ading from
m (b)
d
alone, without subtracti
s
ion, shoul
ld be good
d enough. Again, y
you should
click on
o the par
rt of the
e trace yo
ou want to
o measure
e, and tak
ke the
reading
g from the
e pink bo
ox at the lower rig
ght. (Pay
y attentio
on to the
own
units in
i the cur
rrent rea
adout.) Yo
ou need to
o run the
e voltage steps sho
in the table bel
low, by going
g
back
k to the "
"Voltage Clamp Pro
otocol"
window and chang
ging the number in
n the volt
tage step
p box (Fig
g.5b).
(Don't change an
nything in
i the low
wer, time box.) Al
lso note t
that the
voltage
e you ente
er should
d be the absolute
a
v
voltage, i.e. the voltage
level where
w
the steps ju
ump to, no
ot the hei
ight of t
the voltag
ge step.
Voltage
e (mV)
iK (nA)
iNNa (nA)
-80
-60
-40
-20
0
20
40
(4) Aft
ter you fi
inish all
l the volt
tage steps
s and mea
asure the
corresp
ponding iK. Go bac
ck to the
e "Channel
ls" window
w. Check
k "Fast
11
NE203 laboratory manual
JWL
Sodium" and uncheck "Delayed Rectifier". Use the same voltage steps to
activate iNa and fill in the column of iNa in the table.
Note the signs on the current reading. Also note that iNa measurement
should be taken from the minimal point of the current.
Switch to the "Lab 2" folder and open the "Project 6 I-V plot" file.
You know the routine by now. Enter the numbers in the appropriate
cells in the table, and you will get your beautiful I-V plot. Save
your plot and export your plot to slide 1. Write a figure legend for
this slide.
Compare your plot with Fig.3.3 in your textbook. Your iK plot should be
similar to the "late" line in Fig.3.3, while your iNa line should be
similar to the "Early" line. Hopefully, this exercise will have given
you a better idea about what an I-V plot is. Construction of an I-V
plot is a way to summarize the behavior of iNa and iK as the Vm is
changed by voltage clamp. Since this is a survey course, we will not
torture you about detailed reasons why the I-V curves of iNa and iK look
so different. However, it is educational to think about parameters
that determine the direction of current flow with one small slice of
the voltage clamp experiment.
Project 7: What does the concept of Nernst equilibration have to do
with the direction of iNa flow?
In the I-V plot you constructed for Project 6, iNa was inward and
negative going while iK was outward and positive. The question we will
address here is: what factors determine the directions of Na+ or K+
flow? The answer hinges on the understanding of Nernst equilibrium.
The voltage clamp experiment in this project is designed to help us
think through this problem.
Here, we are going to use one voltage step, -60mV to 0mV, to open
Na channels. However, the voltage step will be tested in three
[Na+]out: 440, 50 and 10 mM.
+
(1) Click "Reset" of the "voltage Clamp Protocol" window then change
the voltage step from -60mV to 0mV (the upper box of Fig.5b).
(2) "Channels" window: make sure that "Fast Sodium" is checked and
"Delay Rectifier" is unchecked.
(3) "Membrane" window: click "Reset"
(4) "Simulator" window: Click "Clear" a few time. Click "Run". iNa
activated by the voltage step show appear.
(5) "Membrane" window: change Na+_Cout to 50mM. Head over to "Simulator"
window: Don’t click the clear window this time. Just click "Run". Blue
iNa and voltage traces should appear. (We want to overlay traces so we
can compare them.)
12
NE203 laboratory manual
JWL
(6) "Membrane" window: change Na+_Cout to 10mM. Head over to "Simulator"
window: Don’t clink the clear window this time. Just click "Run".
Green iNa and voltage traces should appear.
Inset the screenshot in slide 2.and write figure legend.
Of the three iNa traces, the red is negative, blue is flat and
green positive. Explain this observation in the note section of slide
2, following the figure legend.
Hint: Think about what we have changed, Na+_Cout.
What happen to ENa as Na+_Cout was changed?
What happen to Na+ channel conductance change, due the voltage step to
0mV, as Na+_Cout was changed? (Note that Na+ channel conductance is
mainly determined by depolarization and that we use the same voltage
step for all of the three Na+_Cout values.)
What kind of force, electric or diffusional, is driving Na+ when Na+
channels open and Na+ is allowed to pass through the channel pore?
Try to think through these questions for each Na+ concentration.
13
NE203 laboratory
l
y manual
JWL
Project
t 8: How does
d
Na+ channel
c
inactivati
i
ion modify
y the eas
se/difficu
ulty
in AP initiation
i
n?
By now, we
e all have
e the expectation that when
n Vm is de
epolarized,
Na+ and
d K+ chann
nels will open. How
wever, Na
a+ channel
ls have an
nother lay
yer
of comp
plexity, namely
n
in
nactivatio
on process
s. Figure
e 7A-D ill
lustrates
the fou
ur states a Na+ cha
annel can
n have. At
t a negati
ive Vm, m
most chann
nels
are in close and
d not_ina
activated state (C&
&E). The rising ph
hase of an
AP is caused
c
by inward Na
N + curren
nt after t
the channe
el pore o
open (A&E)
).
However
r, even fo
or a brie
ef event such
s
as an
n AP (~1 ms in dur
ration), a
signifi
icant frac
ction of Na+ chann
nels becom
me inactiv
vated and
d stop
carryin
ng Na+ inf
flux on the
t
fallin
ng phase (
(Fig.7B&E
E). After the AP is
over an
nd Vm retu
urn to res
sting lev
vel, the c
channel po
ore will return to
o
closed state (Fi
ig.7D&E) and then the inact
tivation gate will
l disengage
(Fig.7C
C&E). This
s inactiv
vation pro
ocess is e
extensive
ely exploi
ited by
mother nature an
nd can be
e found in
n many var
riants of
f Na+, K+ a
and Ca++
channel
ls.
More
M
inter
restingly,
, Na+ chan
nnels can also "sn
neak" into
o
inactiv
vated stat
te from closed
c
sta
ate if res
sting Vm o
of a neur
ron is
slightl
ly depolar
rized for
r a long time
t
(Fig.
.7F). For
r example,
, if the
resting
g Vm of a neuron stays at -80mV,
90%
% of its channels will be i
in
be
closed and not_i
inactivat
ted stage (Fig.7F).
. (The re
emaining 1
10% will b
%
in clos
sed and in
nactivate
ed state (Fig.7F).)
(
) Under t
this condi
ition, 90%
of the Na+ chann
nels will open and carry cu
urrent whe
en the neuron is
stimula
if the same
ated. In contrast,
c
s
neuro
on shifts
s its rest
ting Vm to
o 55 mV, only 50% of its Na
N + channel
ls will b
be availab
ble—namely in clos
sed
14
JWL
NE203 laboratory
l
y manual
and not
t_inactiva
ated stat
te—while the
t
remain
ning chan
nnels are not
availab
ble becaus
se they are
a
inacti
ivated alr
ready (Fi
ig.7F). (N
Note that
those "closed
"
an
nd inacti
ivated" ch
hannels ca
annot con
ntribute t
to AP
because
e their ch
hannels pores
p
are "plugged"
" by the inactivat
tion gate
even th
hough depo
olarizati
ion could open the pore.) T
Thus, it w
would be
more di
ifficult to
t fire AP
A when th
his neuron
n has a m
more depol
larized Vm.
In fact
t, many ne
eurons in
n our brai
in can sub
btly adju
ust their resting Vm
and shi
ift the fr
raction of
o Na+, K+ or even Ca2+ chann
nels betw
ween close
ednot_ina
activated and clos
se-inactiv
vated stat
tes. You can then imagine
that de
epending on
o the ty
ype of ina
activating
g channel
ls a neuro
on has,
depolar
rizing res
sting Vm could
c
red
duce excit
tability i
if Na+ cha
annels is
s
the mai
in channel
l type in
nactivated
d by depol
larized Vm.
(1)Swit
tch back to
t "Volta
age Record
ding" in t
the simul
lation win
ndow. Reset
paramet
ters in th
he follow
wing windo
ows and th
hen close
e them: "D
Drugs",
"Membra
ane" and "Channels
"
s". (Make sure that
t both "F
Fast Sodiu
um" and
"Delaye
ed Rectifi
ier" are checked. Reset the
e "Stimul
lus Patter
rns" windo
ow.
(2) We are going
g to chan
nge restin
ng Vm
for 50m
ms and see
e how thi
is change in
Vm alte
ers AP amp
plitude. Refer
R
to
Fig.8 to
t set up this pro
otocol. Se
et
the con
nditioning
g current
t to 1.2nA
A (a)
and the
e duration
n to 50ms
s (b). Set
t the
next ti
ime window
w to 0ms (c). Fina
ally,
set the
e 2nd curr
rent step to 6nA (d
d)
and 1ms
s (e). The
e 50ms pe
eriod befo
ore
the sti
imulus (d and e) current
c
st
tep
is call
led the co
onditioni
ing step. Use
the cur
rrent step
ps listed
d in the table
t
below to
t see how
w these conditioni
c
ing
current
t steps af
ffect res
sting Vm and
a
AP ampl
litude. (D
Don’t for
rget to do
o the
followi
ing: Near the bott
tom of the
e
simulat
tor window
w, there is a yell
low
window with a me
enu butto
on to its
right. Select g_
_Na (μS). In the green
g
window to the ri
ight, sel
lect g_K
(μS)and
d select "blank"
"
in
i the cya
an
window.
.)
Note: resting
r
Vm should be
b measur
red right before th
he 2nd, 6n
nA, curren
nt
is deli
ivered, at
t ~50ms point.
p
We want to m
measure Vm that ha
as been
modifie
ed/conditi
ioned by the 50ms condition
ning curr
rent steps
s. (Note
that th
he conditi
ioning cu
urrent her
re is used
d to modi
ify restin
ng Vm for
50ms. Although
A
only
o
50ms
s, it is already
a
en
nough to significa
antly alter
AP ampl
litude bec
cause it is long enough
e
to engage i
inactivati
ion proces
ss
e
and shi
ift some channels
c
from the closed-no
ot_inacti
ivated sta
ate to the
cloased
d_inactiva
ated stat
te (Fig.7F
F). Many n
neurotran
nsmitters in our
brain can
c
modula
ate resti
ing Vm ove
er the tim
me scale o
of minute
e to hours
s.
During this peri
iod, neur
ronal exci
itability will be altered b
because the
15
JWL
NE203 laboratory
l
y manual
balance
e between closed-n
not_inacti
ivated sta
ate and c
closed_ina
activated
state of
o channel
ls will be
b shifted
d.)
Conditi
ioning cur
rrent
(nA)
1.2
1
0
-1
-2
-5
-10
Resting
g Vm (mV)
AP amplitud
de (mV)
Don't f
forget the
e routine of
clickin
ng on the clear bu
uttons etc
c. Open “P
Project 8 Inactiva
ation.pxt”
to plot
t these me
easuremen
nts (Table
e 0: I_con
nd,AP_amp
p). Leave the
plottin
ng file op
pen, sinc
ce there is
i a 2nd p
part to th
his projec
ct.
The sec
cond part of this project is
i to obse
erve inac
ctivation with
voltage
e clamp (s
switch th
he mode fr
rom “Volta
age Recor
rding” to “Voltage
Clamp”)
). Since we
w are go
oing to lo
ook at iNaa alone, o
open the "
"Channels
s"
window,
, and uncl
lick "Del
layed Rect
tifier" to
o turn iK off.
Set the
e paramete
ers in th
he "Voltag
ge Clamp P
Protocol"
" window e
exactly as
indicat
ted in Fig
g.9. The main para
ameter we are goin
ng to chan
nge
systema
atically is
i the va
alue in "a
a". This v
value set
ts the lev
vel of
voltage
e for the 40ms (b) conditio
oning puls
se. We wi
ill then m
measure iNa
d).
amplitu
ude activa
ated by a 5ms depo
olarizing step to 0mV (Fig.
.9 c and d
Also se
et numbers
s in boxe
es e, f, g and h as
s indicat
ted in Fig
g.9.
Note: Since
S
we are
a
inter
rested in how the c
condition
ning pulse
es at
differe
ent Vm may
y affect iNa by an inactivat
tion proce
ess, we n
need to
16
NE203 laboratory
l
y manual
JWL
monitor
r iNa usin
ng a const
tant volta
age step, namely t
the 5ms st
tep to 0m
mV
defined
d by c and
d d in Fi
ig.9.
Also no
ote that we
w need to
t measure
e iNa ampl
litude by taking th
he
differe
ence betwe
een basel
line and peak
p
iNa, namely be
etween a a
and b in
Fig.10 because baseline
b
iNa may no
ot be zer
ro.
Conditi
ioning vol
ltage (mV
V)
-90
-80
-60
-50
-40
-35
iNa (n
nA)
Type in
n the valu
ues Table
e1: V_cond
d,I_Na. Ex
xport bot
th plots t
to slide 3
3.
Write a figure legend
l
fo
or each.
What is
s the sign
nificance
e of inact
tivation?
Look at th
he iNa vs condition
c
ning volta
age plot and consi
ider the f
fact
that a normal ne
een
euron has
s a restin
ng membran
ne potent
tial range
e of betwe
-50 to -70mV: su
uch a neu
uron can dial
d
its a
available
e Na+ chan
nnels down
n to
<20%, at
a -50mV, or dial upward to
o ~70% of maximal iNa at -70
0 mV. In
other words,
w
as this neu
uron shift
ts its res
sting Vm, which in
n turn
regulat
tes the pe
ercentage
e of Na+ channels
c
i
inactivate
ed, its a
ability to
o
ing
fire AP
P (called “excitab
bility”) can
c
be fin
nely cont
trolled by
y regulati
17
NE203 laboratory manual
JWL
the fraction of Na+ channels available for opening and generating AP.
(Not to belabor the point, but Na+ channels that are not inactivated
are the ones that can carry current and generate AP.)
***Don’t forget to go back to the Answers file and answer the last
part of Project 2 on the role of inactivation in relative refractory
period.
Consider and discuss these questions:
1. Are there more Na+ channels in closed-inactivated state when the
second stimulus was delivered than the moment right before the first
stimulus? Why?
2. Can you then explain the reason why the threshold for firing the
second AP is higher than that of the first AP on the basis of Na+
channel inactivation?
3. If we wait for a long time before delivering the second stimulus,
say 500 ms instead of 5 ms as we did in Project 2, there threshold for
firing the second AP will be the same as that of the first AP. Can you
explain this observation?
18
NE203 laboratory
l
y manual
JWL
Appendi
ix:
This fi
igure and legend below
b
illu
ustrate ho
ow you sh
hould writ
te figure
legend.
. You desc
cribe exp
perimental
l conditio
ons under
rlying eac
ch trace. As
a conve
ention, th
hat is al
ll you nee
ed to do, namely d
describe t
the figure.
19