The Lizard Log

The Langkilde Lab in Action


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Looking at brains and hormone receptors

Hi everyone!

Last time I talked about looking at how maternal stress effects egg nutrients and we have found that maternal stress increases yolk protein and decreases yolk lipid concentrations! With that out of the way, today I am going to talk about the next project I am working on which involves brains. We know that maternal stress can have many effects on animals, such as changing their hormone concentrations and behavior, however less is known about how maternal stress influences hormone receptors in the brain, so I wanted to explore this. First you have to preserve the brain and then slice it thin so you can eventually look at the cells.

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The white circles are brain slices, important to not let them dry out!

You also want to make some sort of barrier around the slices so fluid stays on the slides as they incubate in the different fluids. I used a barrier pen, but you can also use clear nail polish!

Next, through a series of washes and solutions, you want to punch holes in the cell membranes so your antibodies can get in and bind with the receptors.

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Carefully adding and removing solution can be done with pipettes

Because some of the solutions have to incubate for a long time, its best to have them in an enclosed container with a wet paper town so the fluids don’t evaporate. After the solutions are on, you want to let them incubate on a gently rotating surface so the solution moves and the antibodies can bind to the appropriate receptors. You can use different antibodies to bind to different receptors, so we used ones that bind to stress hormone receptors.

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Sometimes they incubate at room temperature….

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…and sometimes they incubate in a refrigerated room.

After you get the different antibodies attached to the receptors, you need to add something to differentiate those cells. For that we use a diaminobenzidine , or DAB for short, solution. However, you have to be really careful with this, as not only is DAB a hazardous material as a solid, to get it to dissolve into a fluid you need to use 10M hydrochloric acid, which has a pH of somewhere around -1.0 (measuring pH gets very weird when you get strong acids and are below 0)!

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Not only hazardous, but will also melt through organic things!

However, if we add this to our brain tissue without correcting the pH, that would be very bad! So by using a base (NaOH) and moving quickly (don’t want the DAB to come out of solution) we can correct the pH using a pH meter.

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pH meter along with solutions and protocol.

Once we bring the pH back to around neutral, we can add it to our brain slices so the dab will cause a color change in the cells with receptors for stress hormones.

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The thicker slices (50um) have more receptors so they are darker then the 15um slices.

Finally, after removing all fluids and sealing the slides with a cover slip, we can look at the slices to see what cells have receptors!

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The darker cells have stress hormone receptors compared to the tissue at the top.

The question I want to address with this technique is if maternal stress effects the amount of stress hormone receptors in the brains, so stay tuned!
Thanks,
David

 

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Testing the environmental matching hypothesis – return to Alabama!

Another summer field season has now come and gone! This summer I returned to Solon Dixon Forestry Education Center in Alabama (one of my favourite spots on earth!) to continue my research on how stress during gestation influences the offspring of eastern fence lizards (Sceloporus undulatus).

Last year, we investigated how physiological stress during gestation (at the level of a non-lethal predator encounter – for example, when a lizard encounters a couple of toxic fire ants, but isn’t killed by the ants) affects survival of mothers, and how many of their eggs successfully hatch. You can read more about this experiment, and the fieldwork that went into it, here (and stay posted for the published results soon!).

This year I wanted to build on these results and ideas to test how maternal stress influences the offspring that do hatch and make it out into the world. Do they themselves then cope better with a stressful environment, having been “primed” for it by their mothers (the “environmental matching” hypothesis)? Or are offspring born to stressed mothers poorer in quality, and less likely to survive in the wild, regardless of how stressful their environment is? In order to test these ideas, we first made the long trip south to collect gravid females from south Alabama early in the summer, and to build experimental enclosures in which to eventually release their offspring. I then repeated the maternal stress treatment from last year and once again became a lizard mama as I followed the females from laying their eggs, to incubating the eggs, and eventually seeing these bite-sized babies hatch out!

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Freshly hatched fence lizards – <1g!

Once they hatched, the offspring went into the enclosures that we’d built. These enclosures were designed to test whether maternal stress programs offspring to be able to better deal with a stressful environment. The enclosures either contained a key stressor (invasive fire ants), or were fire ant-free. Each day I conducted a mini-census, walking through enclosures to look for each lizard – as you can see in the video below, babies were marked so I could tell exactly who was present each day (and so, which lizards survived, and which didn’t). I also observed their behaviour, and how they used the habitat available to them (for example, did offspring from stressed/non-stressed mothers differ in whether they liked to be out in the open, like the lizards you see in the video – or did they hide more?).

After a great summer (if measuring 200+ baby lizards isn’t a metric of a great summer, I don’t know what is), I’m now back at Penn State with a box of data to work through. I’m excited to report back on what I found in the coming months – so stay tuned!

 

 

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Hanging out with an adult female Sceloporus at Solon Dixon

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Beautiful Solon Dixon