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So I work at a neurobiology lab where we're studying intracellular signaling pathways and how the neurons control those pathways. I've been working on culturing cells and imaging them so we can do some work with fluorescence to see how the cells and their various parts interact with each other. To do this we dissect 1-day old mice brains and remove the sections we're studying (the striatum) and use various chemicals to digest all the connections between the densely packed neurons so they will all fall away from each other easily. We then take them and put them on glass slides and let them grow in nutrient-rich solutions so they'll make connections and so we can study them better. Today I took a few of the cultures I've been growing and decided to image them using a simple fluorescence technique called immunocytochemistry. This is where we use fluorescent-labeled antibodies which bind to parts of the neurons to show us what the neurons look like, so here's the results so far:

Info: The neurons are "medium spiny neurons" that have been growing for 2 days. They started as small balls without the long slender processes (dendrites). Each cell body is about 20um long (20 micrometers, or 20 millionths of a meter). The magnification is 400x, but that gets distorted with the resolution of the images. The lvlshot images are the size that they appear through the microscope at 400x.

[lvlshot]http://homepage.mac.com/tkessler/.Pictures/Cells/ID15-1.jpg[/lvlshot][lvlshot]http://homepage.mac.com/tkessler/.Pictures/Cells/ID22.jpg[/lvlshot]
[lvlshot]http://homepage.mac.com/tkessler/.Pictures/Cells/ID25.jpg[/lvlshot][lvlshot]http://homepage.mac.com/tkessler/.Pictures/Cells/ID30.jpg[/lvlshot]
[lvlshot]http://homepage.mac.com/tkessler/.Pictures/Cells/ID37.jpg[/lvlshot][lvlshot]http://homepage.mac.com/tkessler/.Pictures/Cells/ID44.jpg[/lvlshot]

I couldnt get the pretty color pictures to come out, since I saved all tthe images as 16-bit tiff grayscale images. The fluorescence makes the images look brilliant green through a microscope.

that's nice man....to which antigens does the fluoresceine bind to? And do you add growth factors to the nutrient-rich solution?

The antigen is GAD65/67 (Glutamate decarboxylase) which converts glutamate to GABA. The medium spiny neurons are all GABAergic. We used a rabbit anti-GAD primary antibody and a Goat anti-rabbit Alexa 488 secondary antibody. The growth media is all manufactured neuronal growth media and vitamin supplements that we mix together to get various percentages of each in the final mixture, and we add penicillin, streptomycin, and gentamycin to control bacteria.

very cool stuff.

Are you studying the mechanisms that underlie the way in which the neurons connect?

I understand there are natural neurotrophic factors at play that chemically attract axons over distance, and that there is competition involved.

Are you studying these particular mechanisms? Would this fall under "intracellular signaling pathway research"?

cool, are you exploring the modulation of movement pathways or the cognitive processes involving executive functions?

edit: n.m. you're studying the connections, not the striatum function itself

Canis wrote:The antigen is GAD65/67 (Glutamate decarboxylase) which converts glutamate to GABA. The medium spiny neurons are all GABAergic. We used a rabbit anti-GAD primary antibody and a Goat anti-rabbit Alexa 488 secondary antibody. The growth media is all manufactured neuronal growth media and vitamin supplements that we mix together to get various percentages of each in the final mixture, and we add penicillin, streptomycin, and gentamycin to control bacteria.

I was about to ask the same question. Sounds pretty cool. What's the procedure for making single-cell suspensions of neurons... or is that too much info to give out right now?
Also, do you use either HEPES or Nutridoma in the growth media? And what is the base for the media -- Hank's BSS or RPMI or something like that, or is it something more specialized?. I'm just curious about how easy it is to culture neurons compared to other cells...

How can you be sure that the regrown connections are the same as the natural ones? Aren't you altering the process you're trying to study by dissolving them in the first place?

Cool stuff Canis.

I remember cell culture. I worked for a bit in a lab that looked at the mechanisms behind the internalization of Campylobacter jejuni into intestinal epithelial cells. God...I hated cell culturing when it wasn't working well.

[xeno]Julios wrote:very cool stuff.

Are you studying the mechanisms that underlie the way in which the neurons connect?

I understand there are natural neurotrophic factors at play that chemically attract axons over distance, and that there is competition involved.

Are you studying these particular mechanisms? Would this fall under "intracellular signaling pathway research"?

No. Although in culturing the neurons I'm getting really interested in cell growth and formation, we're currently studying the regulation of the intracellular signaling. This is the chemical process that goes on within the cell after the cell receives some external stimulus, which causes the cell to function in certain ways. There are multiple types of cell signaling pathways, and they all serve to biochemically amplify external stimuli to the cell. For instance, in rods and cones there are the rhodopsin and opsin (respectively) molecules that are the first part of a chemical pathway which transducees light signals into electrochemical signals which the cell uses to pass the interpreted light information to the optic nerve. This pathway has several chemical components (proteins and such) that are altered sequentially to send the signal from rhodopsin to other parts of the cell that relay the information to neighboring cells.

In rods and cones, this is the general process:

Light particle --> one rhodopsin molecule --> many secondary molecules activated (a G-protein called transducin) --> each 2nd molecule activates a 3rd (called Phosphodiesterase) on the chemical pathway --> each 3rd activates a 4th (converts cGMP to an inactive form). Rods and cones, like other cells, have ion channels (little proteins that set up an electrochemical gradient in the cell membrane which governs neuronal "firing" of action potentials). In rods and cones these are all opened with cGMP, so the effective decrease in cGMP caused by the signaling pathway closes the channels, reducing their conductivity, and changes the rate at which the cells fire action potentials to other cells.

This process is a feed-forward process so in the end one photon of light can deactivate millions of cGMP molecules. This is why our eyes can detect one photon of light, even though our brains filter out such small stimuli. There are multiple regulating steps (proteins and such) that inhibit each step of the signaling reaction. Once the pathway is activated (by light in retinal cells as described above), all the parts in turn need to be shut off, however not immediately or the pathway would not work properly. There are regulatory proteins that turn off each component of the pathway, and we're interested in the time delays associated with the deactivation of each step.

In the neurons I'm culturing, we're going to be stimulating them with neurotransmitters instead of light, and will be using fluorescence to see how the various parts of the signaling cascade interact with each other.

werldhed wrote:

Canis wrote:The antigen is GAD65/67 (Glutamate decarboxylase) which converts glutamate to GABA. The medium spiny neurons are all GABAergic. We used a rabbit anti-GAD primary antibody and a Goat anti-rabbit Alexa 488 secondary antibody. The growth media is all manufactured neuronal growth media and vitamin supplements that we mix together to get various percentages of each in the final mixture, and we add penicillin, streptomycin, and gentamycin to control bacteria.

I was about to ask the same question. Sounds pretty cool. What's the procedure for making single-cell suspensions of neurons... or is that too much info to give out right now?
Also, do you use either HEPES or Nutridoma in the growth media? And what is the base for the media -- Hank's BSS or RPMI or something like that, or is it something more specialized?. I'm just curious about how easy it is to culture neurons compared to other cells...

We dissect the striatum out of the brains of 1-day old mouse pups. We then put the striatum chunks in a Trypsin-like solution called TrypLE express. This enzymatically digests the connections between the cells and allows them to fall apart from each other. We then gently centrifuge the cells and get rid of the supernatant liquid. We use a pipette tip thats roughly .3-.5mm in diameter to gently suck the cell mass back and forth (a process called trituration) in order to dislodge the cells from each other. Once this occurs we spin them again, getting rid of the supernatant and replacing it with a nutrient rich growth media. We pour this on glass slides that have been pre-treated with a chemical that the neurons will stick to, and allow the neurons to settle and start growing. We then gently wash and rinse the cells every few hours to ensure they're health. Then it's a matter of waiting...

Nightshade wrote:How can you be sure that the regrown connections are the same as the natural ones? Aren't you altering the process you're trying to study by dissolving them in the first place?

Although the connections are very cool, we're not concerned about them right now. However, neurons naturally are making such connections all the time. In the brain they're all packed together instead of in a dish, but the connections should be made quite similarly, regardless of where the neurons are. I'm not sure on this though, as it's not the focus of our research (yet).

tnf wrote:Cool stuff Canis.

I remember cell culture. I worked for a bit in a lab that looked at the mechanisms behind the internalization of Campylobacter jejuni into intestinal epithelial cells. God...I hated cell culturing when it wasn't working well.

Hehe. Yeah, it can be a royal pain in the ass and a heartbreak to see all your cells die after a week or so of going strong.

Canis wrote:We dissect the striatum out of the brains of 1-day old mouse pups. We then put the striatum chunks in a Trypsin-like solution called TrypLE express. This enzymatically digests the connections between the cells and allows them to fall apart from each other. We then gently centrifuge the cells and get rid of the supernatant liquid. We use a pipette tip thats roughly .3-.5mm in diameter to gently suck the cell mass back and forth (a process called trituration) in order to dislodge the cells from each other. Once this occurs we spin them again, getting rid of the supernatant and replacing it with a nutrient rich growth media. We pour this on glass slides that have been pre-treated with a chemical that the neurons will stick to, and allow the neurons to settle and start growing. We then gently wash and rinse the cells every few hours to ensure they're health. Then it's a matter of waiting...

That sounds like a pretty straightforward technique, but I'm surprised you don't add a lysis buffer or something of the sort to hinder growth of unwanted cells. Aren't you worried about blood cells in your culture?

Nightshade wrote:How can you be sure that the regrown connections are the same as the natural ones? Aren't you altering the process you're trying to study by dissolving them in the first place?

I would say that's a worry with any in vitro test, but you have to start somewhere. Even if it's not exactly the same, you do your best to mimic the in vivo environment. If you get positive results in test tubes, then you can think about moving up to small animals, then larger animals, primates, and humans.

Plus, it's cheaper, faster, and usually easier than working with live animals.

werldhed wrote:

Canis wrote:We dissect the striatum out of the brains of 1-day old mouse pups. We then put the striatum chunks in a Trypsin-like solution called TrypLE express. This enzymatically digests the connections between the cells and allows them to fall apart from each other. We then gently centrifuge the cells and get rid of the supernatant liquid. We use a pipette tip thats roughly .3-.5mm in diameter to gently suck the cell mass back and forth (a process called trituration) in order to dislodge the cells from each other. Once this occurs we spin them again, getting rid of the supernatant and replacing it with a nutrient rich growth media. We pour this on glass slides that have been pre-treated with a chemical that the neurons will stick to, and allow the neurons to settle and start growing. We then gently wash and rinse the cells every few hours to ensure they're health. Then it's a matter of waiting...

That sounds like a pretty straightforward technique, but I'm surprised you don't add a lysis buffer or something of the sort to hinder growth of unwanted cells. Aren't you worried about blood cells in your culture?

We were using Cytosine Arabinoside (ARA-C) which is an antimetabolite to abolish glial cell growth, however we found that it was killing off our neurons as well. We stopped using it and our neurons began growing very well. I figure I'll add it back if I see glial proliferation, but so far we're not getting any glia in the cultures. The only thing we're worried about is glia, bacteria, and fungi, and so far we're not getting uncontrollable growth of any.

werldhed wrote:

Nightshade wrote:How can you be sure that the regrown connections are the same as the natural ones? Aren't you altering the process you're trying to study by dissolving them in the first place?

I would say that's a worry with any in vitro test, but you have to start somewhere. Even if it's not exactly the same, you do your best to mimic the in vivo environment. If you get positive results in test tubes, then you can think about moving up to small animals, then larger animals, primates, and humans.

Plus, it's cheaper, faster, and usually easier than working with live animals.

The in vivo technique we'll be using is brain slice recording, as the neural structure is as it would be in a live mouse, so it's actually cheaper and faster than cultures because we dont have to pay for the media and wait for the neurons to grow. The benefit of the cultures is to have a more controllable environment for studying single cell function. Once we develop a working model we'll move to brain slice recording to test the model in a more representative environment.

werldhed wrote: Also, do you use either HEPES or Nutridoma in the growth media? And what is the base for the media -- Hank's BSS or RPMI or something like that, or is it something more specialized?. I'm just curious about how easy it is to culture neurons compared to other cells...

We're using a 70% DMEM/F12 (Dulbecco's Modified Eagle Media) and 30% NeuroBasal media along with one unit of B27 vitamine/nutrient supplement. We then add about 5mM glutamic acid. The Media does not contain HEPES.

Thanks for the answers. And sorry for all the questions. I just find it interesting to see what other methods people like to use.
I asked about the lysis buffer because I do splenocyte and bone marrow isolation which ends up with a lot of RBCs in suspension. I just assumed you'd have some if you harvested tissue from mice, but I guess not.
And the DMEM makes sense; I've used it a couple of times, but obviously not for neurons. :icon25:

Anyway, it sounds cool. Good luck with it. Let us know if any publications result...

are Canis and Canidae two different people?

sliver wrote:are Canis and Canidae two different people?

Yes

So Canis - are you a graduate student, undergrad, or a research assistant or what?

I wish I could still talk about shit in that much detail. My poor brain.

I'm a Research assistant and I'm also in grad school, but for another topic which I've lost interest in. The more I do the neuro stuff the more I love it. This project I'm doing is on the level of post-doctral work, and I'm going to use it in my application to grad school. Right now I'm not in the "grad school" mindset, and love the flexibility and such that being a research assistant offers.