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Tadpoles and using them to understand brains

Why use tadpoles to study the brain?

A strategy to understand brains

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To understand how the brains of humans and other animals control their behaviour we need to understand their whole nervous system. This is a daunting task, especially in humans where the brain is so complex with billions of nerve cells or neurons. This is clearly not the place to start. What we need is a simple animal with simple behaviour like a very young tadpole. Even the nervous systems of animals like worms and flies have many thousands of neurons making complex connections.


Salamander tadpoles: a simple model for study

Our research on young frog tadpoles was inspired by the work starting in the 1890s by the a photo of CoghillAmerican psychologist George Ellet Coghill. He decided that the function of the brain must be studied as a whole if we are to understand how it generates behaviour. After 30 years of research he published a book in 1929 called “Anatomy and the problem of Behaviour” Cambridge University Press, Cambridge.

Coghill’s idea was to choose an easily available simple animal and to correlate the development of its behaviour with the development of the neurons in its nervous system. The animal he chose was the tadpole of the Tiger salamander (Ambystoma tigrinum: Photo thanks to Brian Gratwicke on flickr).Tiger-Salamander-hatchlingf

Coghill took motion pictures to define the development of the tadpole’s swimming behaviour in response to touch. It bends into a coil (1 to 4) and then swims off (5 to 10).

Fig5-Coghill-1929

His diagram (at the right) shows the nervous system and how the muscle segments contract during swimming. The grey shaded muscle segments are contracting

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Salamander tadpoles: circuits controlling swimming

Coghill then spent many years trying to reveal the anatomy of the neurons in the brain and spinal cord responsible for the swimming response. His book has strikingly simple circuit diagrams of the possible neuronal circuits underlying swimming.Coghill-circuit-colour

Coghill’s explanation for this circuit was that touching the skin on the left side excites sensory nerve endings among the skin cells. This leads to activity (action potentials) in sensory neurons (red). These sensory neurons excite each other so action potentials reach the brain. In the brain the excitation leads to activity in connecting neurons (black) which pass excitation on to floor plate neurons (x orange). The floor plate neurons carry activity across to the other side where they excite the motor neurons (green). Action potentials then spread from motor neuron to motor neuron towards the tail in this motor path. This excites the muscles to contract in a head to tail sequence. The muscle contractions alternate on each side to produce swimming.

Coghill’s circuits have very few different types of neuron and have inspired later detailed study on how they develop and work.  This more recent research started in the 1970s. Unfortunately, it has not confirmed many of Coghill’s pioneering conclusions about the neuronal circuits and the way they work. For example, nobody has been able to find the “Floor plate cells” in his circuit. The anatomical methods available for him to use to define neuron types were just not good enough.


Advantages of amphibian tadpoles for study of nervous systems

Frog eggs, embryos and tadpoles have been used extensively by developmental biologists to study how an egg develops into a behaving animal with a functioning brain and nervous system. The eggs are large (~ 1mm across) and, as they divide to make an embryo with many cells, the yolk divides too, so each cell has its own supply. This means that parts of the embryo can be removed and will survive and grow on their own. Small pieces can also be transplanted to new positions to see if they influence the development of other tissues (like the lens in the eye). The tissue is remarkably tough and resists bacterial attack.

During the first half of the 20th century, European and American frogs in the genus Rana were used in most circle3studies of development. But, there was a real problem in getting eggs all year round as these frogs only breed in spring. By the 1960s many researchers were using the African Clawed Toad  Xenopus laevis (Photo at right) because this breeds in the wild during the rainy season which can occur in different locations in Africa at any time of the year. Breeding could be induced around the year by injecting the hormone Chorionic gonadotrophin. This hormone is produced by pregnant women and excreted in their urine. In the mid-20th century Xenopus colonies were kept in hospitals to use for pregnancy testing. If urine from a woman was injected into a female Xenopus and she laid eggs, then the urine contained chorionic gonadotrophin and the woman was pregnant.

Developmental biologists can therefore use hormone injections to induce Xenopus laevis to breed and provide eggs, embryos and tadpoles at any time of year. Furthermore, Xenopus laevis are entirely aquatic and are  easy to keep and look after in aquaria. There is a large community of scientists working on a huge range of questions using different species of Xenopus. (This links to Xenbase.)


Using Xenopus tadpoles to study the brain

Xenopus embryos and tadpoles are used in many studies on the development and operation of the nervous system and brain: how does the nervous system form in the embryo; how do nerve cells or neurons develop from ordinary cells; how do nerve axons make the right connections with other neurons; how do axons find their way from the eye to the correct part of the brain?

The research in our Neurobiology laboratory in the Bristol University was inspired by Coghill’s ideas. We use very young Xenopus laevis tadpoles as a model organism to investigate how the nervous system is constructed and works. When the tadpole hatches, it has behaviour which will allow it to survive. This provides us with what must be one of the simplest vertebrate nervous systems. Other neuroscientists study very young zebrafish for the same reason.


Early development of  Xenopus laevis

After 24 hours of development the Xenopus embryo has made the beginning of a nervous system. During the next 29 hours it grows into a tadpole and the nervous system and muscles start to function. Then it hatches from the egg and starts to swim. The stages of development have been defined and given numbers:stages-final-web

We use stage 37/38  when the tadpole usually hatches. The behaviour of the hatchling tadpole is simple and described on the tadpole behaviour page.

Tadpole swims when touched at *

The details of swimming movements which hatchling Xenopus tadpoles make in response to touch with a fine hair  have been studied by making high speed videos at 200 fps. In these examples touch on the left (*) leads to a bend to the right followed by swimming. Waves of bending travel from the head to tail (at ~ 14 cm per second) and increase in amplitude as they travel along the body. They move the tadpole in the direction shown by the arrows. Swimming speeds at ~ 20 oC range from 4 to 6 cm per second.hatchling tadpole swims when touched at *

Kahn J.A., Roberts A. & Kashin S. (1982) The neuromuscular basis of swimming movements in embryos of the amphibian Xenopus laevis. J. exp. Biol.  99, 175‑184. http://jeb.biologists.org/cgi/reprint/99/1/175

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Adult South Africal Clawed toad Xenopus laevis

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         Xenopus laevis

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What Tadpoles Look Like

Tadpoles can start swimming spontaneously or when they are stimulated but it is just as important that they can stop. This normally happens when their head and cement gland bumps into the surface of the water or some other solid tadpoles swimmingobject like a plant or the side of a dish. This kind of stimulus and the tension in the mucus strand when the tadpole is hanging attached have an inhibitory effect on the tadpole. While hanging, it never moves spontaneously and is much less responsive to stimulation. This ability to keep still may make it more difficult for predators to detect and eat tadpoles. 

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Types of Neurons

There are different types of neurons in a nervous system and they are named depending on their function.

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Interneuron from Xenopus laevis tadpole

Broadly there are 3 main types:

  1. Sensory neurons
  2. Motor neurons
  3. Interneurons 

 

 

 

flex-behaviour

Flexion behaviour of hatchling tadpole in response to skin stimulation (represented by arrow).

When the skin of Xenopus laevis hatchling is touched, sensory neurons are activated, passing on exitation to sensory pathway neurons (interneurons) in the spinal cord, which in turn excite motor neurons, causing flexion behaviour

For more info on research into flexion behaviour click here. 

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Synaptic transmission (blank diagram)

pencil22small_foka.tkPrint and fill in the blank diagram with the key steps in the process of synaptic transmission:

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Resting potential and action potential confusion!

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The terms resting potential and action potential can be confusing, as they seem to suggest that one is an active process and the other not.

 

Action potentials are actually produced by a passive process- sodium ions diffusing into the axon, causing depolarisation. 

Resting potentials are generated by an active process, which needs ATP. The sodium-potassium pump carries out active transport of ions in and out of the axon to generate a potential difference across the cell and a voltage of -60/70 mV inside the axon.

So even though the axon is said to be at “rest”, an active process involving energy in the form of ATP is actually going on. And even though the action potential sounds like it needs energy, it is actually a passive process.

Make sure you are clear on this!

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Axon, membrane or axon membrane?!

General_Revisionstar

 

Some exam boards prefer you to mention simply the “axon”, others just the “membrane”, or the “membrane of the axon”. 

 

When we are talking about a difference in charge over an area, we always refer to what area that is- for example a potential difference over the axon membrane. 

Some exam boards will prefer you say that the “membrane” or “axon membrane is depolarised”…but others will be happy with just the “axon is depolarised”

Check what is preferred by your exam board and incorporate into your notes links below:

 ⇒Edexcel

AQA

WJEC

OCR

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Exam Board Links

pencil22small_foka.tkClick on the links below to access the specifications for listed exam boards:

 

 ⇒Edexcel

AQA

WJEC

OCR

 

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Toad spawn

Toad eggs are the same size as Frog eggs but are laid in a string, often among weeds in the pond. The string can be more than 1 meter long and contain a double row of eggs. Here is a photo of a small piece from a pond in Hampshire.

A small piece of toad spawn from a pond in Hampshire.

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American Bullfrog

Below is an American Bullfrog. The Latin name of this frog is Lithobates catesbeianus. It is sometimes also called Rana catesbeianus. 

American_Bullfrog_(Rana_catesbeiana)_-_Algonquin_Provincial_Park_Ontario_By Ryan Hodnett Own work

Click here for more information on the American Bullfrog from the ARKive website.


Media credits: American Bullfrog-Ryan Hodnett

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