Science-related notes of Naveen Agnihotri

• Making hippocampal cultures
• Whole-cell patch clamp recording
• Using matlab for data acquisition
• Recording from hippocampal slices

Making hippocampal cultures

1. 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 of leukemias).

2. What is left after about two weeks are glia, and because of the low density, they form "islands" (see figures below).

3. 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.

4. 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 found here.

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 (1998).

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. The middle 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. (1995). 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 washing out.

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 (1998). 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 amphotericin).

Using matlab for data acquisition

We use Axon Multiclamp 700A 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 breakout box (eg, the BNC-2110 or the BNC-2090), from which a SH68-68-EP connector connects to the NI PCI-6052E 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 matlab.

Armed with aoi, you can write matlab subroutines in order to do actual acquisiton. 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.

aoi.c

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 functioning.

aoi.m

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.

axontp.m

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 testpulse.m.

testpulse.m

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 vstim.m.

vstim.m

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. Requires aoi.

baseline_acquire.m

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 testpulse.m.

vstimulate.m

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. Requires vstim.m.

Recording from hippocampal (etc) slices

• 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 agree.

• 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 Leica 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 for me.
       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 feather 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 hassles.

• 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 Garner Glass. 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.