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.

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.

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.

Sometimes they incubate at room temperature….

…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)!

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.

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.

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!

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

 

What’s in an egg?

Hello again everyone!

While most of my work has been on measuring hormones and metabolites from blood, or recording behaviors, I decided to try my hand at something new. I wanted to see if I could measure the contents of a lizard egg!

As eggs can vary widely in the volume of water they contain, the first thing I had to do was dry the egg. Because I wanted to measure proteins and lipids, I wasn’t able to heat the egg up though, so instead I used a freeze-dryer.  Once dried, I carefully removed the shell (because shells are reaaaaally hard to grind) and then homogenized the yolk sample.

Once the yolk was ground up, I needed a way to extract the proteins and lipids from the yolk. To do so, I weighed out a specific amount of the egg, added some dangerous chemicals, and then filtered that solution through an incredibly tiny filter. The size of the holes in a coffee filter are 20 microns, while the size of a bacteria is 0.6 microns. This filter had holes that were 0.2 microns!

 

After filtering the solution, I could then try to measure the amount of proteins and lipids. To do so, I added a tiny drop of the solution to a piece of quick dry paper.

Once the paper completely dried, I was able to shine a light through it and get an absorbance value.

Stay tuned for the results of what I found!

Cheers,
David

Solon Dixon!

Hey everyone!

Even though I am continuing the same project from last year (how maternal stress affects the offspring in fence lizards), there are still some striking differences. One of the biggest is that there are fellow grad students and a post doc this summer!

From left to right: Cameron (PhD), myself (PhD), Kirsty (post-doc), and Dustin (PhD).

Also, last year we made the drive in one day, however this year we broke the drive up over 2 days. This gave us an excellent opportunity to experience different parts of the USA on our drive. For the night we stopped in Knoxville, TN and had dinner at an amazing place called Calhoun’s On The River. True to its namesake, it had a beautiful view of the Tennessee River!

After all the driving, we finally made it back to Solon Dixon and started catching lizards. As usual, the lizards’ personalities were very evident.

Male fence lizard unamused with our attempts to catch him

Apparently the female lizards found that corner of the tub to be very interesting.

As I went to put this female back in her tub, she refused to let go of my fingers!

On top of finding many fence lizards, we were also about to see many other reptiles and amphibians!

A barking tree frog tightly hugging my finger.

An American alligator, at a very reasonable size to handle.

A yellow bellied slider who found a little bit of water to sit in.

A glass lizard!

As I spend more time down here, I find it rubbing off on me more and more.

Very tempted to get a cowboy hat.

After catching the females, our first trip came to an end. However, we were quickly back down to release the females and run experiments with the hatchlings. With us this time we had an undergraduate researcher, Jen!

The Bayfront Park, overlooking Mobile Bay. Located right next to one of our field sites, Blakeley State Park.

As we wait for more hatchlings to emerge, we have been focusing on removing fire ants from some of the enclosures we built. As fire ants are highest in the mounds earlier in the day, this means some early mornings. On the up side, it also means we always get to see the sunrise.

Sunrise right near the enclosures.

Most things have gone well, with only one piece of equipment starting to show signs of wear, but this just gave me an excuse to do some handywork!

Used some steel epoxy to seal a leak in the pot we boil water in for fire ants.

Things have started to pick up in terms of hatchling, so soon you should be able to hear about how things are going with them. Until then, here is a pic from right here at Solon Dixon

With the drier weather they are finally able to do prescribed burns.

Cheers,
David

 

Alabama Part II: Prep for experiments!

Hello again! After my first trip down to Alabama, I ended up catching a lot of gravid female fence lizards. I ended bringing them back to Penn State so they could lay their eggs.

Individual housing so we could figure out who laid which eggs

They took to their new homes very well, as they were furnished with a half log for basking/hiding, and sand for laying their eggs.

She wasn’t going to share her new home with anyone!

We spent most of every day with them, whether it was cleaning their housing, doing behavioral observations, checking for eggs, or feeding them.

You can imagine which part was their favorite!

It took nearly a month for all the mothers to lay their eggs, and it was not without excitement. Most of them laid their eggs in the nice, moist sand we provided, and their eggs looked wonderful. However some decided to rebel against that idea and laid their eggs directly under the heat lamp! Needless to say, those eggs needed some TLC.

Finally, when it was reaching about the time for the eggs to start hatching, we headed back down to Solon Dixon, AL to get ready for the second round of field work. I brought 2 volunteers with me again, and they have been an incredible help, not only with doing the work down here, but also giving great ideas and making the trip much more enjoyable.

Lexi (L) and Michaleia (R)

The first order of business was returning the mothers back to where we caught them. Luckily, they all posed for me to take nice picture,

Lizards going back home!

After the mothers were safely back to their natural habitats, we got started on the biggest part of the preparation phase, making the outdoor enclosures. To do this we used aluminum fencing buried 6-10 inches into the dirt, supported by metal electrical poles. To make our lives easier, we rented a trench digger to dig the trenches for us!

Imagine a giant circular saw that you pull behind you.

Using that helped speed up enclosure building a lot, especially since we had 4 to build.

The finished product.

Even using the trencher, it took many long, hot days in the field to get everything ready. Luckily there are a few cool and refreshing escapes close by!

A freshwater spring at Solon Dixon.

We returned once again to the spring at Solon Dixon, whose 60F water made the 100F days much more bearable. In addition to the cool temperature, the spring also has a wealth of different organisms. This time, in addition to all the fish and spiders, we saw a crawdad and a frog!

Also, while waiting for some aluminum flashing to come in, we were able to take a short trip to Destin, FL. The sand and water were beautiful, and the amount of fish, jellyfish, and invertebrates (with a surprise appearance from a shark!) was incredible.

O’Steen Beach

But after the relaxation time, it was back to work. Once the enclosures were up, we had to remove the fire ants from half of the enclosures to give us high and low stress enclosures.

The “Fire ant removal” gear.

To do this, you pound a piece of metal into the colony, and then flood it with hot water. Around this time, we started seeing wild fence lizard hatchlings.

A little guy trying to blend in with white rocks…    he will learn how to blend in better!

With those sightings, we knew that our hatchlings would be next. And sure enough, a few days later, we had our very first eggs hatch.

3 little guys just hatching.

Now that the hatching has started, we get to start stocking the enclosures with baby fence lizards and running behavioral trials. I am very excited to be able to finally run experiments with the hatchlings! I look forward to letting you all know how the experiments went.
Till next time,
David

Stress and Fence Lizards

Through the lens of conservation, I became interested in stress. My biggest interest is in how humans can cause stress in animals, and how that stress impacts them. To this end, I started studying the effects of stress within an individual. I looked at how stress changed different hormones and metabolites in adult male northern elephant seals, and how that response differed during different times of the year.

Adult male northern elephant seal (Mirounga angustirostris), about 1,500kg, asleep

This experience was great and fueled my excitement and curiosity about studying stress. I started talking with 2 professors at Penn State (Drs. Sheriff and Langkilde) about what they were looking into and fell in love with the project I am working on now. Instead of studying stress within an individual (stress the animal, see what happens to them), we are going to look at the effects of maternal stress (stress the mother, see what happens to their offspring). The other large difference was instead of being in California working on seals, I was going to be in Alabama working on eastern fence lizards!

A female fence lizard (L), the ones I am studying, and a male (R).

Even more striking then their back color is the color of their underside!

The dark blue chest and neck of a male eastern fence lizard.

I had never been to Alabama or worked with herps before, but I was incredibly excited. Last summer I was able to go down with Chris Thawley and he showed me where to look, how to catch the fence lizards, and many other things. That time, combined with Tracy, Gail, and his answering of my constant questions throughout the year (thank you so much!) helped alleviate some of my own stress about the field work.

So I had a plan laid out. I would get gravid female lizards from the field and have 1/2 be stressed, 1/2 not stressed. I would then take them back to the lab and have them lay their eggs, and then look at differences in the offspring. I thought that sounded pretty simple, but lizards prove to be an elusive bunch. So far we have all the females we need from Conecuh National forest, but we are being thwarted by the lizards in Geneva State Forest and Blakeley State Park. Luckily, I have 2 skilled field techs working with me, Michaleia and Miranda.

Michaleia drying a colored mark on the back of a lizard (L), and Miranda out in the field (R).

They have been a tremendous help in not only finding the lizards, but also collecting data and samples as well as catching the lizards. We have been learning so much about the area, the wildlife, and how to handle the lizards. The lack of lizards from Geneva and Blakeley has not curbed my enthusiasm about studying this amazing lizards, and I am looking forward to working with them more and getting into the meat of my research with them.

Cheers

David
P.S. Here are some other interesting things we have been seeing down here.

Fire ants eating the tail of a fence lizard

And some other herps!

A gray rat snake, Pantherophis spiloides, blending into the leaf litter.

A southern toad, Anaxyrus terrestris, hanging out.

A ground skink, Scincella lateralis, found at our field station.

A green anole, Anolis carolinensis, perched on the side of a log.