The energetic cost of vision in eyed and eyeless Mexican tetra, Astyanax mexicanus

Vision is very important for animals that live in places with light. If there is even a hint of light present then it seems vision is an important sense to have. Many nocturnal and deep sea animals have eyes, and these are usually quite large to capture as much light as possible. Relative to body size, small fish also have large eyes (by small let's say the size of your finger and smaller), and along with it a large brain to process visual information. We know that powering photoreceptor cells and neurons is expensive (about 20-25% of our resting metabolism goes to powering our big human brains), so how much energy does a small eyed animal like a fish spend on vision?

The Mexican tetra Astyanax mexicanus can help answer this question. Within the same species there are multiple variants with eyes and brains of different sizes. This species has diversified from surface rivers into limestone caves and has repeatedly evolved a cave variant with varying degrees of eye loss.

These are all the same species of fish, the Mexican tetra (Astyanax mexicanus), but note how different the eye size is.

These are all the same species of fish, the Mexican tetra (Astyanax mexicanus), but note how different the eye size is.

My colleague Eric Warrant and I thought that this fish species would help answer the question of how much vision can cost in a small vertebrate (this question had already been investigated in insects). There was also a fair amount of discussion in the scientific literature about the mechanism of eye loss in this species. There were 3 hypotheses as to why surface fish lost their eyes over many generations as they adapted to life underground:

  1. the genes and developmental plan for eyes degraded because there was no consequence to not having eyes (the neutral mutation with relaxed selection theory)
  2. the genes and developmental plan that built eyes were re-purposed to build other features that helped with live underground (called pleiotropy)
  3. the eyes were somehow costly or detrimental to life underground and selection favoured individuals with smaller eyes

Researchers had gathered data to evaluate the first two hypotheses, but there were no studies addressing the third hypothesis, so that was another good reason to construct an experiment that could measure the energetic cost of vision in this species. I was fortunate to be awarded a Marie Curie Fellowship from the European Research Council that helped fund the project. Rowan Softley from Surrey University joined us on an Erasmus Internship and we got to work figuring out how to measure the energetic cost of vision in a small fish.

The first thing we did was measured the energy expenditure of the whole fish. We did this using a swimming chamber that could measure the oxygen consumption rate (see the video below). We accidentally tripped across an interesting feature of this species - the cave variant appears to have virtually eliminated their circadian rhythm in metabolism. Check out the webpage describing this research if you want to know more.

So we knew how much energy it cost to run the whole body of this fish. Now, how much of that cost was down to the visual system? One approach to measuring the energy use of isolated organs would be to measure blood flow and oxygen level in blood vessels entering and exiting the brain and eye in sedated fish. Unfortunately the blood vessels were too small for the equipment that is used to measure these sorts of things. A more feasible approach was to remove the eyes and brains from euthanized fish and try and keep the organs 'alive' in a kind of heart-lung machine (similar to what is done during organ transports), and then measure the oxygen demand of these organs in little glass vials. 

To do this we needed to know more about how to keep neural tissue in a viable state outside of the body. I took a trip down to the Ophthalmology Department at the Biomedical Centre at Lund University and started working in the lab of Fredrik Ghosh, a practicing ophthalmology researcher who works on in vitro culture of retina as models of eye disease. Together with his PhD student Linnéa Taylor we started playing around with ways of improving the condition and length of time we could culture large pieces of pig retina in vitro. We tripped across an interesting observation that if we stretched the pieces we could massively improve the state of these isolated retina (see the picture below, and the resulting publication here). Having the correct mechanical support for our fish eyes would be important then.

We found that providing mechanical stretch to pig retina significantly improved the viability of the tissue for in vitro culture (published here).  This knowledge helped us figure out methods to keep cavefish eyes in a good state for metabolic rate measurements.

We found that providing mechanical stretch to pig retina significantly improved the viability of the tissue for in vitro culture (published here).  This knowledge helped us figure out methods to keep cavefish eyes in a good state for metabolic rate measurements.

Armed with a better understanding of what is needed to keep eyes and and brains alive, we set to work on developing a method to keep the eyes and brains of eyed and eyeless variants of Mexican tetra alive outside the body so we could measure how much oxygen they used. Here is our experimental sequence (for those who might want to repeat it):

  1. We wanted:
    • lots of measurements from single organ to maximise the statistical power of the study
    • an automated system so that we could maximise reproducibility and go home at night while the machine kept measuring
    • nice linear oxygen decreases when measuring oxygen demand, as that would indicate that we were supplying the organs with nutrients as best we could (the circulatory system was not working so we were relying solely on diffusion through the tissue to supply nutrients). These organs are very nutrient hungry (about 5-10 times that of other tissues) and will quickly drain the nutrients at the tissue - fluid interface
  2. We built a system with a peristaltic pump ('the heart') that would pump aerated artificial cerebrospinal fluid ('the lungs and blood') through the vials holding the organs to ensure an adequate supply of nutrients.
  3. Next issue: how to ensure that a boundary layer of depleted nutrients didn't build up around the tissues. We knew we had to properly mix the artificial cerebrospinal fluid around the organs, but we had quite small volumes of fluid (about 2 mL) and conical shaped vials. Most magnetic stirring systems weren't suited because they spun too fast and the stirring bars didn't fit our vials. After months of mucking around with stirring and mixing we ended up building our own magnetic rotors and stir bars. The stir bars were designed to go quite close to the tissue, yet stir at a relatively slow rate (about 40 rpm) to ensure maximum bulk fluid mixing while not spinning so fast that the bits of tissue were minced up. One line in a paper, several months of work!
  4. Yet another problem was the warming of the artificial cerebrospinal fluid due to friction with the wall of the tubing. The tubing we used (our 'circulatory system') needed to be quite small in diameter and we had relatively long tube runs. The artificial cerebrospinal fluid was warming up by 1-3°C, so we needed a large volume heat sink after the peristaltic pump that could be pressurised to maintain delivery. Solution: a 1L Schott bottle with holes drilled in the lid for the tubes.
  5. Now to the vials that housed the tissue and oxygen probe. We used microreaction vials (I think used for chemical synthesis). These could be used with a septum (handy for accessing the vial in a sealed manner) and had a conical bottom, which improved the hydrodynamics of fluid mixing. I drilled a hole in the wall of the vial near the bottom as the influent artificial cerebrospinal fluid port and had the effluent port in the septum. We found this configuration to give a good exchange of fluid. I drilled another hole in the wall at tissue level for the oxygen optode. Good luck to you if you decide to drill holes in these vials! Use a high quality diamond drill bit and distilled water while drilling. Go very slowly, especially towards the end of the hole. I had a success rate of 4/12 vials.
  6. And finally, how to hold the little eyes and brains in these vials. The key design principle was that as much of the tissue needed to be presented to the fluid as possible so that the fluid could swirl around it and replenish the nutrients at the boundary layer. The tissue also needed to be held quite firmly as there was quite some fluid movement inside the vials. The solution was a ball head pin and cyanoacrylate glue. The pointed end of the pin could be pushed through the rubber septum to hold it in place and allowed some height adjustment. The ball head was just the right size to hold an everted the eye, and the glue held the sclera taut and presented the retina to the artificial cerebrospinal fluid without it forming a floppy sheet (well, 2/20 times the retina detached from the rest of the eye and floated off). A pin was also a handy implement to handle and transfer the organs with. Again, months of trials, a single line in a paper.
The experimental set up used to measure oxygen demand in brains and eyes of Mexican tetra. Click through to Science Advances to enlarge.

The experimental set up used to measure oxygen demand in brains and eyes of Mexican tetra. Click through to Science Advances to enlarge.

And here is a picture of the eyes on a ball head pin. Please note, we did all of the organ dissections on euthanized animals.

The eyes from surface variants of Mexican tetra were glued to ball head pins for use in the oxygen consumption measurement system.

The eyes from surface variants of Mexican tetra were glued to ball head pins for use in the oxygen consumption measurement system.

The details of how the eyes and brains were dissected and attached to the pins are well described in the Science Advances paper. Click on the mini images below to go through to the articles and see how we did this.

Detail of how eyes were glued to pin and everted. Click through to Science Advances to enlarge.

Detail of how eyes were glued to pin and everted. Click through to Science Advances to enlarge.

Detail of how brain were dissected and glued to pin. Click though to Science Advances to enlarge.

Detail of how brain were dissected and glued to pin. Click though to Science Advances to enlarge.

So with all of the wrinkles ironed out we began the data gathering process. We measured the weight of eyes and brains in the four different variants pictured at the top of the page, and made eye and brain oxygen consumption measurements on surface and Pachón cave variants. The 'heart-lung' machine pumped artificial cerebrospinal fluid through the vials housing the tissues, then it stopped pumping and measured the rate of oxygen consumption in the vial, then pumped through more artificial cerebrospinal fluid, and so on, repeating this process for a day or more. The raw data looked like that given in the picture below, with a rhythmic increase and decrease in oxygen level as the fluid was alternately flushed and kept static.

An example of the rise and fall of oxygen levels inside the vials containing the eyes and brains.

An example of the rise and fall of oxygen levels inside the vials containing the eyes and brains.

The oxygen consumption rate of the brain per gram tissue did not differ between eyed and eyeless Mexican tetra (you wouldn't expect it to, as far as we know it's not possible to make energetically cheaper brains, the only way to make them cheaper is to shrink them in size). The oxygen consumption rate of the brain was in the middle of the range of values measured for fish (and about 8 times that of the whole body rate). The oxygen consumption rate of an eye from a surface fish was in the ball park of measurements made on isolated trout retina (the only other comparable study, and about 2.5 times that of the whole body rate).

So, we had the oxygen consumption rates of eyes, brains and the whole body, and we knew the relative weight of brains and eyes (as % body weight), so now we could put this information together and make an estimate of how much energy it costs to have brains and eyes for surface and Pachón and Micos cave variants. We made this estimate for body weights ranging from 1 to 8.5 grams (the range of weights we worked on) and put together a series of graphs that plotted the various relative weights and costs of eyes and brains. Below is a mini-version of this, click through to Science Advances for an enlarged version.

Energetic cost of vision model. Click to enlarge.

Energetic cost of vision model. Click to enlarge.

One of the great things about working on this species is that there is already quite a lot of research on the brains of different variants. It is known that the main difference in brain volume between eyed and eyeless fish is the much smaller size of the optic tectum in eyeless variants (the optic tectum is the part of the brain that initially processes the electrical signals from the eye). This means that we can reasonably assume that differences in brain oxygen demand between eyed and reduced eye variants represents a reduction in the cost of the visual processing parts of the brain. If we have a measure of the cost of the eye and a measure of the cost of visual processing area in the brain, we can get a pretty good estimate of the cost of vision (eyes + optic tectum). Isn't that cool? I was super excited when I tripped across this idea! Thanks other researchers for the ground work!

So, when you have a look at panel G in the above figure you can see that the cost of vision is quite high, especially in small fish with relatively large eye and brain sizes. Imagine spending about 15% of your resting metabolism just on vision! In fact, eyed versions of these fish spend about as much energy running their brains and eyes as we humans do running our brains (20-25% of resting metabolism). Vision and brains must be important for the survival of these fish if they spend so much energy on them (note: these eyed Mexican tetra have fairly average eye sizes, so this observation is probably true for most small fish with eyes). It makes sense then that if an animal doesn't need vision because they live in a perpetually dark (and probably food poor) environment, then it helps balance the energy budget to reduce eye size and visual processing capability.

I hope this was useful.

This research has been published

Moran D, Softley, R, Warrant, EJ (2015) The energetic cost of vision and the evolution of eyeless Mexican cavefish. Science Advances 1:e1500363. (article pdf here)

...and has been summarised by these sites: