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Tadpoles and using them to understand brains

Tadpole Laboratory

What goes on in the Tadpole laboratory

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This is where we will give a simple account of how we study tadpoles. More details can be found on the science pages. The main reason to study tadpoles is because,  when they are just hatched,  their brains and nervous system are remarkably simple. We have a chance to understand how they allow the tadpole to swim when touched with a hair and to stop when they bump into the side of the dish:entiregoodcold-crop

More about why we study tadpoles.


Producing Tadpoles

We have a breeding colony of South African Clawed Toads (latin name: Xenopus laevis). Each week we give a pair of adult toads  a hormone injection to induce mating and egg laying. This is what the adults look like. The females are in the top row and the males in the bottom:

S GROUPf

Adult Xenopus for breeding

We bring the eggs back from the animal facility and after one day in the lab they look like this:Eggs-in-dish-P1000990f

When you look more closely you can see that some are beginning to get longer and look like tadpoles:Eggs-in-dish-closer-P1000991f

At room temperature they reach the hatching stage  (white arrows) after two days. The photos show how they develop in their first three days:

Xenopus stage chart

Xenopus tadpole development stages


Tadpole behaviour and responses

The newly hatched Xenopus tadpole looks like this and spends most of its time doing nothing.tadpole-37-38f

But, if you touch it anywhere on the body it will swim until it bumps into something like the side of the dish. The tadpole is only 5 mm long and moves fast so we have to take slow motion video to see what it does.

MORE details on Science page


Tadpole brain and nervous system

When it hatches, the tadpole brain and nervous system contains some 3,000 nerve cells (neurons). The front part of the brain is only just starting to develop. Most of the neurons are in the hindbrain and spinal cord (between the red arrows in the photo)tadpole-37-38-CNS-arrowsf

If we look at the part between the red arrows, the spinal cord is very small: about the same thickness as a human hair (~ 0.1 mm across)! The hindbrain (between the ears and above the gills) is bigger and has a fluid filled space or ventricle in the middle. The little yellow dots near the ventricle are neurons (~ 0.01 mm across).Tadpole-CNS-oblique-labelledf

To expose neurons we open the hindbrain and spinal cord along the top (the colours are artificial). This immediately reveals sensory neuron cell bodies. These neurons have nerve fibres in the skin which are excited by touch.CNS-opened

By removing small areas of the wall of the ventricle (dull pink) other types of neuron can be seen under the microscope. This allows us to record their activity.


 

How we record the activity of nerve cells (neurons)

When the spinal cord is opened, the tadpole is held on a small platform in a dish of salty solution to imitate frog’s blood. We  put the dish on a powerful 500x microscope to see the cell bodies of neurons exposed at the opened top edge of the spinal cord. The long red arrow shows the position of the dish:

recording-microscopef

We use electronically controlled manipulators (short red arrow) to place a glass recording electrode onto a single neuron which we can see down the microscope. In the picture below the electrode is on a sensory neuron. These are the largest neurons in the spinal cord and are excited when the skin is touched. You can see another neuron to the left.: Recording-electrode-on sensory neuron

The recording below was made using similar methods. The neuron sits at its resting voltage but when the skin is touched (blue arrow at time 0) a nerve impulse or action potential occurs. The voltage suddenly goes positive for about 3 milliseconds (3/1000 second):Recording2

This action potential is the electrical signal which travels along nerve fibres or axons so one nerve cell can excite or inhibit another at connections called synapses.


Recording connections between neurons

To look for synaptic connection or synapses, we record from two neurons at the same time. We inject current through the electrode into neuron one (black) to make it fire an action potential. If the neurons are connected, we will see an obvious response in neuron two (red). The response in neuron two (red arrow) starts shortly after the action potential in neuron one. The delay is ~ 2ms:synapsef

The delay means that electric current is not flowing directly from  neuron to neuron. What actually happens is that the action potential in neuron one initiates the release of a chemical messenger which spreads across the synapse gap to neuron two. The “transmitter” chemical then binds onto special receptor sites on neuron two. This results in channels opening so electrical current moves into neuron two. A positive increase in voltage appears called a “synaptic potential” (red arrow above). If the synaptic potential is big enough, it will initiate an action potential in neuron two. This happens here.

To see the anatomy of the neurons after the recording, we let a chemical marker spread into them from the recording electrode. We process this so it is visible under the microscope and take photos. This photograph from one side of the spinal cord shows the sensory neuron (neuron one black) and neuron two (coloured red) in the recording above. White arrows show possible synaptic connections. The black arrow shows the axon or nerve fibre of neuron one.sensory and pathway neuronsf


The methods described here can be used to study many types of neuron in the tadpoles brain and spinal cord. For more details go to the Science pages.

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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

frog

         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:

synapse_labelled_blank

 

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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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