Making hippocampal cultures
The Seung lab started making hippocampal
cultures in early 2002. Because we like to study the action at single
synapses, we like low-density cultures. Our fantastic technician
Jung "the Healer" Choi
has now worked out a recipe that yields consistently good cultures
(after patiently trying out many many permutations for a year and
a half). It is loosely based on the recipe
of Yuki Goda
Here's the rough outline:
- Plate about 5,000 cells per 10mm coverslip. This is a
very low density, and no neurons should survive.
In case some neurons do survive, you can kill them by
putting the coverslips in the refrigerator overnight. Add
Ara-C one day after plating, to prevent too much proliferation
of glia. (Ara-C inhibits cell
division, and is also used in chemotherapy for certain types
- What is left after about two weeks are glia, and because of
the low density, they form "islands" (see figures below).
- 3-4 weeks later, plate about 10,000 cells per coverslip on
top of these glia. Add Ara-C one day after plating, so that
there is some new glial growth but not too much. Of the
neurons that get plated, only the neurons that start growing
on top of the glial islands survive, and consequently there
are only a few hundred neurons left after a week.
- Use only glia-conditioned medium for these cultures.
The exact recipe that Jung uses to make hippocampal cultures
(both postnatal and embryonic), and the solutions etc, are all
We made several attempts at making embryonic cultures (as in the recipes page),
but never got anything consistent. This is strange, because everyone else
seems to have much better luck with embryonic cultures, eg. Bi and Poo
Our P1 cultures look (and record) the best on days 8-11. These
are "island cultures", which have plenty of glial islands with a few neurons.
We like to record from islands of 2 neurons for our experiments; that rules
out any interactive effects. Shown below are examples of one, two and three
neurons on an island. You can click on the images to see bigger versions:
And below on the left is another example of two cells on an island.
image shows the same two cells under a 40X magnification (the previous
images were all under 10X) and the third image is of the same two
cells being recorded. The electrodes are of about 2.5 Mohm
resistance, and this was a perforated patch recording that lasted
about 40 minutes. (The depth-of-field on my microscope is very shallow,
so the the cells in the image on the right appear out of focus in
order to make the electrodes [somewhat] clear).
Whole-cell patch clamp recording notes:
- Ruptured patch recording
- For the intracellular solution, I swear by the solution used by
the great Ottavio "the King" Arancio et al.
The recipe is (mM): K Gluconate (130), KCL (10), MgCl2(5),
EGTA (0.6), HEPES (5), CaCl2 (0.06), Mg-ATP (2), GTP (0.2),
leupeptine (0.2), phosphocreatine (20), creatine phosphokinase
(50 U/ml), pH 7.2. This solution has plenty of nutrients for the
cell, so that the cell doesn't die too quickly from all the nutrients
In my experience, using this solution, I get stable synaptic transmission
baselines (along with stable leak currents and access resistances) for at
least half an hour, often 40 minutes or so. In the Arancio et al.
paper, they show stable baselines of about 25 minutes.
- Perforated patch recording
- Of course, this should be the preferred way of doing whole cell
recordings, as long as one can figure out the idiosyncrasies of
amphotericin. Amphotericin does not go into solution
very easily, and one needs to dissolve it in di-methyl sulfoxide
(DMSO), a nasty soapy substance. And as it happens, DMSO in the
intracellular solution prevents the formation of good seals.
After much experimentation, Neville Sanjana has discovered that
the trick seems to be to only use freshly dissolved amphotericin. So
our current recipe is to weigh out small amounts (a few mgs) of
amphotericin in Eppendorf tubes, label the tubes with the weight, and
dessicate them. When we are ready to do an experiment, we dissolve it
in DMSO to get it to 30 mg/ml, and then add 10 µl of this to
1 ml of intracellular solution to get the final concentration to
be 300 µg/ml of amphotericin. The recipe for the intracellular
solution is from Bi and Poo
The amounts are (mM): K gluconate (136.5), KCl (17.5), NaCl (9),
MgCl2 (1), HEPES (10), EGTA (0.2), pH 7.20. Of course,
we front-fill the pitpette with pure intracellular solution (without
Using matlab for data acquisition
We use GNU/linux and Matlab
for our data acquisition. Yes, we have really big cojones.
We use Axon
amplifiers, and Axon provides a barebones program (called Multiclamp
Commander) to control the amplifier. We connect the BNC connectors on the
front of the amplifier to a National Instruments
BNC-2110 or the
from which a
connector connects to the
DAQ board, which plugs in to the PCI bus on the acquisition computer.
The 6052E board has 16 analog inputs
and 2 analog outputs. It also has 8 digital I/O lines, but we've never been
able to get them to work under GNU/linux.
The comedi device interface library is
used to do the actual low-level acquisition and output. Installing
comedi is not a task for the faint-hearted, and using it requires a lot
of patience and a healthy cardiovascular system. Consult your doctor.
Our in-house computer guru Alan Chen has written a C program called aoi (for
Analog Output and Input) that serves as an interface between comedi and
Armed with aoi, you can write matlab subroutines in order to do actual
Below are a set of matlab routines that can be used for acquiring data.
But perhaps more importantly, they can be used to see how data acquisition
can be done and you can write your own routines based on these.
- This program does the low-level I/O to hook up matlab with comedi.
You will need this program to use any of the matlab programs
below. Compilation instructions are in the file itself. It creates a
file called "aoi.mexglx" which matlab can understand and execute.
Requires comedi to be installed and
- This matlab routine doesn't actually do anything,
but provides the documentation about how to use aoi from the
matlab command prompt. With aoi.m in your matlab execution path,
you can say "help aoi" on the matlab prompt in order to see how
to use aoi.
- A program that provides a testpulse (2mV in amplitude, 20msec wide)
that can be used for establishing a patch-clamp seal on the cell.
Monitors the output current of the cell, and plots access
resistance over time. Also provides estimates of the leak current,
membrane capacitance, and other patching parameters. Requires
- A low-level program that sends out (multiple) testpulses to the
amplifier, and reports back with the output and the calculations
of the various patch parameters based on the waveforms. Requires
- A low-level program that sends out the analog output to aoi
and reports back what it finds. It does the appropriate scaling so
that everyting is in the right units.
- A program to acquire a synaptic transmission baseline from a pair
of neurons. The program stimulates cell 1 and reports what it finds
on cell 2, and vice versa. It does this 2 times a minute, for as
long as the "Stop" button is not pressed.
It also allows the user to select a pair of X values on the PSC
plot and then it plots the PSC value at those points over time.
Requires vstimulate.m and
- A low-level program that sends out the a pulse to one channel
(using aoi) and reports back what it finds on both channels.
It does the appropriate scaling so that everyting is in the right units.
Recording from hippocampal (etc) slices
Here is what I've learned, both from talking to people and
my own personal experience:
- There must be glucose in the bath ACSF. The solution I was using
for the first couple of months was from the recipe in
the Stevens and Wang article, which didn't have
any glucose in it, and I kept wondering why my slices weren't
very healthy. There were hardly any healthy cells in the top
50-80µm, and the ones deeper were not well visualized.
They must have forgotten to mention glucose, because I don't
see how they got anything done without it.
The recipe I use now for the bath ACSF
comes from my friend Matt Nolan, who records from dendrites and
knows more about making healthy slices than anyone else I know.
The recipes for the bath ACSF is (mM):
NaCl (124), NaH2PO4 (1.2), KCl (2.5), NaHCO3 (25), Glucose (20),
CaCl2 (2), MgCl2 (1). And the recipe for the dissection ACSF
is: NaCl (86), NaH2PO4 (1.2), KCl (2.5), NaHCO3 (25), Glucose (25),
CaCl2 (0.5), MgCl2 (7), sucrose (75). Note that only half of the
NaCl in the dissection solution has been replaced by sucrose
(more commonly people replace all of the NaCl with sucrose).
Matt tells me that this leads to better slices, and I have to
- Another thing I learned from Matt Nolan: the vibration of the microtome
should be restricted to the x (side-to-side) dimensions, and should be minimal
in the z (up and down) dimension. Minimizing the extraneous z dimension
movement (called z-deflection) of the microtome is an important factor
in cutting healty slices.
We have a
VT1000 S mictrotome, and when I looked in the specs in the manual,
the z-deflection is not even mentioned. When I called up Leica
customer support, they had no idea what the z-deflection was
either; they said they'd call up the factory and call me back.
Then they called me back and said that the
z-deflection of our instrument is less than
3µm. That is comparable to the specs from
Vibratome. Vibratome also
claim to have something called "zero-z technology" which
supposedly dramatically reduces the z-deflections. The
microtomes from Dosaka
supposedly have even lower z-deflections, around
2µm or less.
- The angle of the blade is important. Ideally the angle should
be as shallow as possible. I find that 3-5° works fine
An important consequence of the angle
issue is that the fancy sapphire blade that Leica
sells along with the microtome (for a low, low price of $900)
is useless. The angle of the wedge of that blade is 28°,
which means that the least angle you can cut at without smushing
the tissue underneath is 14°. Not good for healthy slices.
I use simple Schick safety razor blades
and they work fine. I use each blade only once. The
blades sold by Ted Pella are also reportedly good.
- The pricing of the Leica microtomes is not fixed. The price
of our VT1000 S was about $10,500, and my friend
Tamily Weissman tells me
that when they bought one for
her lab it cost them something close to $18,000. YMMV.
- Other things to note about using the Leica VT1000 S: use a high
frequency (I use 80 Hz) and a high vibration amplitude (the Leica
technical support person recommended 0.8mm instead of the 0.6mm
default). The speed I generally use is 3, which according to
my manual corresponds to 0.15mm/s.
- The Leica VT1000 S has a serious design flaw: the panel with
all the buttons is on the side of the sectioning chamber, where
the buttons and knobs are susceptible to ice and water spills.
The knobs they use are supposedly sealed, and are not affected
by spills, but that's only in theory. In practicality, my
friend Tamily tells
me that they've had to replace one of the knobs on their Leica
vibratome twice. The Leica people denied that it had anything
to do wth spillage, and said that it must have been mishandled.
However, they've now put a little plexiglass barrier between
the panel and the sectioning chamber, and the knob is fine.
When I spoke to the Leica technical
person about this, he acknowledged that this was a common problem,
and that it would be resolved in the next version of the microtome,
coming out in a few years. In the meantime, he advised me to put
some Saran wrap on the control panel. Why
won't they write this in the manual?
- Given what I've learned about microtomes, if I had to do it all
over again, I wouldn't get the Leica at all; I'd go with the
Virbatome 3000, which costs about the same, and has a lot fewer
- For electrodes, I started out using borosilicate glass capillary
tubing (OD=1.2mm, ID=0.69mm, from
Warner). This works
fine for recording from cultured neurons. However, when I tried
recording in slices with this glass, the seals would form very very
slowly (over minutes) and would not last. Then my friend Vivek
Unni, who knows a thing or two about recording from slices,
recommended the KG-33 glass from
So I bought some, with OD=1.5mm and ID=1.0mm.
To pull these using the Sutter P-97
puller, the ramp test came to 290. So the settings I use are:
air pressure (500), heat (305), velocity (45), time (200), and
this results in the elctrodes with resistances around 5 megaohms.
Now let's see how it goes.