The Smoking Gun: Fish Brains and Nicotine

In researching neural pathways, it helps to establish an analogous relationship between a region of the human brain and the brains of more-easily studied animal species. New work from a team led by Carnegie’s Marnie Halpern hones in on one particular region of the zebrafish brain that could help us understand the circuitry underlying nicotine addiction. It is published the week of December 9 by Proceedings of the National Academy of Sciences.

The mammalian habenular nuclei, in a little-understood and difficult-to-access part of the brain, are involved in regulating both dopamine and serotonin, two neurotransmitters involved in motor control, mood, learning, and addiction. But unlike the mammalian habenulae, the habenular nuclei of fish are located dorsally, making them easy for scientists to access and study. However, some outstanding questions remained about the properties of the zebrafish habenulae, creating a roadblock for truly linking these structures as analogous in fish and humans. In particular, it was unresolved whether zebrafish habenular neurons produce the neurotransmitter acetylcholine, which is enriched in this region of the mammalian brain and activates the same receptors to which nicotine is known to bind.

The new work by lead author Elim Hong and colleagues confirms that the pathway between the habenula and another part of the brain called the midbrain interpenduncular nucleus utilizes acetylcholine in zebrafish, as it does in humans. The work also shows that there is a left-right difference in this part of the fish brain.

The purpose of this asymmetry is unknown, but, as demonstrated by electrophysiological recordings with collaborator Jean-Marie Mangin of the University of Pierre and Marie Curie, it results in differences in neural activity between the brain hemispheres. Other research in Halpern’s lab indicates that such left-right differences could influence behavior. Hong performed these experiments through a European Molecular Biology Organization Short-Term Fellowship while hosted in the laboratory of Claire Wyart in Paris, France.

The team further showed that this acetylcholine pathway in zebrafish responds in a similar way to nicotine as does the analagous pathway in the mammalian brain. This makes the zebrafish a good model for studying the brain chemistry of nicotine addiction.

“Our work demonstrates broader uses for zebrafish in studying the function of the habenula and addresses a major weakness in the field, which was the poor characterization of neurotransmitter identity in this area,” said Hong. “Going forward, these results will help us study how brain circuitry influences nicotine addiction.”

The pig, the fish and the jellyfish: Tracing nervous disorders in humans

What do pigs, jellyfish and zebrafish have in common? It might be hard to discern the connection, but the different species are all pieces in a puzzle. A puzzle which is itself part of a larger picture of solving the riddles of diseases in humans.

The pig, the jellyfish and the zebrafish are being used by scientists at Aarhus University to, among other things, gain a greater understanding of hereditary forms of diseases affecting the nervous system. This can be disorders like Parkinson’s disease, Alzheimer’s disease, autism, epilepsy and the motor neurone disease ALS.

In a project, which has just finished, the scientists have focussed on a specific gene in pigs. The gene, SYN1, encodes the protein synapsin, which is involved in communication between nerve cells. Synapsin almost exclusively occurs in nerve cells in the brain. Parts of the gene can thus be used to control an expression of genes connected to hereditary versions of the aforementioned disorders.

The pig
The SYN1 gene can, with its specific expression in nerve cells, be used for generation of pig models of neurodegenerative diseases like Parkinson’s. The reason scientists bring a pig into the equation is that the pig is well suited as a model for investigating human diseases.

- Pigs are very like humans in their size, genetics, anatomy and physiology. There are plenty of them, so they are easily obtainable for research purposes, and it is ethically easier to use them than, for example, apes, says senior scientist Knud Larsen from Aarhus University.

Before the gene was transferred from humans to pigs, the scientists had to ensure that the SYN1 gene was only expressed in nerve cells. This was where the zebra fish entered the equation.

The zebrafish and the jellyfish
- The zebrafish is, as a model organism, the darling of researchers, because it is transparent and easy to genetically modify. We thus attached the relevant gene, SYN1, to a gene from a jellyfish (GFP), and put it into a zebrafish in order to test the specificity of the gene, explains Knud Larsen.

This is because jellyfish contain a gene that enables them to light up. This gene was transferred to the zebrafish alongside SYN1, so that the scientists could follow where in the fish activity occurred as a result of the SYN1 gene.

- We could clearly see that the transparent zebrafish shone green in its nervous system as a result of the SYN1 gene from humans initiating processes in the nervous system. We could thus conclude that SYN1 works specifically in nerve cells, says Knud Larsen.

The results of this investigation pave the way for the SYN1 gene being used in pig models for research into human diseases. The pig with the human gene SYN1 can presumably also be used for research into the development of the brain and nervous system in the foetus.

- I think it is interesting that the nervous system is so well preserved, from an evolutionary point of view, that you can observe a nerve-cell-specific expression of a pig gene in a zebrafish. It is impressive that something that works in a pig also works in a fish, says Knud Larsen.

Read the scientific article here.

"Brainbow" zebra fish.

Neurons are labeled in multiple colors with Brainbow fluorescence microscopy. Three fluorescent proteins (cyan, yellow and red) are randomly taken up by various neurons, offering a palette of dozens of colors to help scientists follow complex neural pathways. Shown here is a five-day-old zebra fish larva viewed from the dorsal side, captured using a 20X objective.

Image credit: Dr. Albert Pan, Harvard University, Cambridge, Mass., U.S.

(Source: scientificamerican.com)

Visualizing a memory trace

Whole brain imaging of zebrafish reveals neuronal networks involved in retrieving long-term memories during decision making

In mammals, a neural pathway called the cortico-basal ganglia circuit is thought to play an important role in the choice of behaviors. However, where and how behavioral programs are written, stored and read out as a memory within this circuit remains unclear. A research team led by Hitoshi Okamoto and Tazu Aoki of the RIKEN Brain Science Institute has for the first time visualized in zebrafish the neuronal activity associated with the retrieval of long-term memories during decision making.

The team performed experiments on genetically engineered zebrafish expressing a fluorescent protein that changes its intensity when it binds to calcium ions in neurons and thereby acts as an indicator of neuronal activity. “Neurons in the fish cortical region form a neural circuit similar to the mammalian cortico-basal ganglia circuit,” says Okamoto.

The fish were trained on an avoidance task by placing individual fish into a two-compartment tank and shining a red light for several seconds into the compartment containing the fish. If the fish did not move into the other compartment in response to the light, it was ‘punished’ with a mild electric shock. After several repetitions, the fish learned to avoid the shock by switching compartments as soon as the light came on. 

The researchers then examined the neuronal activity of the fish under the microscope in response to exposure to red light. One day after the learning task, the fish showed specific activity in a discrete region of the telencephalon, which corresponds to the cerebral cortex in mammals, when presented with the red light. However, just 30 minutes after the learning task no activity was observed in this part of the brain. The results suggest that this telencephalonic area encodes the long-term memory for the learned avoidance behavior. Confirming this, removing this part of the telencephalon abolished the long-term memory but did not affect learning or short-term storage of the memory. 

In humans, the ability to choose the correct behavioral programs in response to environmental changes is indispensable for everyday life, and the ability to do so is thought to be impaired in various psychiatric conditions such as depression and schizophrenia. 

“Combining the neural imaging technique with genetics, we will be able to investigate how neurons in the cortico-basal ganglia circuit choose the most suitable behavior in any given situation,” says Okamoto. “Our findings open the way to investigate and understand how these symptoms appear in human psychiatric disorders.”

Zebrafish study paves the way for new treatments for genetic disorder

Scientists from the University of Sheffield have paved the way for new treatments for a common genetic disorder thanks to pioneering research on zebrafish – an animal capable of mending its own heart.

Charcot Marie Tooth disease (CMT) is the most common genetic disorder affecting the nervous system. More than 20,000 people in the UK suffer from CMT, which typically causes progressive weakness and long-term pain in the feet, leading to walking difficulties. There is currently no cure for CMT.

A research project conducted at the Sheffield Institute for Translational Neuroscience (SITraN) and the MRC Centre for Developmental and Biomedical Genetics (CDBG) by Dr Andrew Grierson and his team has revealed that zebrafish could hold the key to finding new therapeutic approaches to treat the condition.

Dr Grierson said: “We have studied zebrafish with a genetic defect that causes CMT in humans. The fish develop normally, but once they reach adulthood they start to develop difficulties swimming.

"By looking at the muscles of these fish we have discovered that the problem lies with the connections between motor neurons and muscle, which are known to be essential for walking in humans and also swimming in fish."

CMT represents a group of neurodegenerative disorders typically characterised by demyelination (CMT1), a process which causes damage to the myelin sheaths that surround our neurons, or distal axon degeneration (CMT2) of motor and sensory neurons. The distal axon is the terminal where neurotransmitter packages within neurons are docked.

The majority of CMT2 cases are caused by mutations in mitofusin 2 (MFN2), which is an essential gene encoding a protein responsible for fusion of the mitochondrial outer membrane. Mitochondria are known as the cellular power plants because they generate most of the supply of adenosine triphosphate (ATP), which is used as a source of chemical energy.

Dr Grierson said: “Previous work on this disorder using mammalian models such as mice has been problematic, because the mitofusin genes are essential for embryonic development. Using zebrafish we were able to develop a model with an adult onset, progressive phenotype with predominant symptoms of motor dysfunction similar to CMT2.

"Motor neurons are the largest cells in our bodies, and as such they are highly dependent on a cellular transport system to deliver molecules through the long nerve cell processes which connect the spinal cord to our muscles. We already know that defects in the cellular transport system occur early in the development of diseases such as Alzheimer’s disease, Motor Neuron Disease and spastic paraplegia. Using our zebrafish model we have found that similar defects in transport are also a key part of the disease process in CMT."

Dr Grierson and his team are now seeking funding to identify new treatments for CMT using the zebrafish model. Because of their size and unique biology, zebrafish are ideal to be used in drug screens for the identification of new therapies for untreatable human conditions.

(Image courtesy: University College London)

Eyes on the prey: Researchers analyse the hunting behaviour of fish larvae in virtual reality

Moving objects attract greater attention – a fact exploited by video screens in public spaces and animated advertising banners on the Internet. For most animal species, moving objects also play a major role in the processing of sensory impressions in the brain, as they often signal the presence of a welcome prey or an imminent threat. This is also true of the zebrafish larva, which has to react to the movements of its prey. Scientists at the Max Planck Institute for Medical Research in Heidelberg have investigated how the brain uses the information from the visual system for the execution of quicker movements. The animals’ visual system records the movements of the prey so that the brain can redirect the animals’ movements through targeted swim bouts in a matter of milliseconds. Two hitherto unknown types of neurons in the mid-brain are involved in the processing of movement stimuli.

In principle, the visual system of zebrafish larvae resembles that of other vertebrates. Moreover, its genome has been decoded, it is a small organism, and it has transparent skin, which is easily penetrated by light in the fluorescent microscope. Therefore, these animals are very suitable for studying visual motion perception. They also display very clear prey capture behaviour. With the help of their finely-tuned visual system, they pursue and catch small ciliates. To do this, they execute a series of swimming manoeuvres in a matter of one or two seconds, during which they repeatedly verify the direction and distance of the prey so that they can adapt their subsequent movement steps. The larva’s brain must, therefore, filter and evaluate visual information extremely rapidly so that it can select appropriate motor patterns.

Using high-speed video recordings, researchers working with Johann Bollmann at the Max Planck Institute for Medical Research began by studying the natural course of prey capture by the larvae under a variety of starting conditions. It emerged that the larvae repeatedly execute a basic motion pattern and can apply an orientation component that re-directs the hunter towards the prey with each swim bout. To do this, the larvae must process visual information in just a few hundreds of milliseconds.

Using an innovative experimental design, the scientists then modelled, in a second step, the natural swimming environment as a “virtual reality”, in which the larvae execute typical prey capture sequences without actually moving. The virtual prey consisted of computer-controlled images, which were projected onto a small screen. In this way, the role of motion parameters, for example the size and speed of the “prey”, could be studied quantitatively in relation to the processing of visual stimuli by the animals.

In the “virtual reality”, the scientists can test how the fish larvae respond to unexpected shifts in the prey after a swim bout. “When we direct our gaze at a target through movements of our eyes and head, we expect the object to appear in a central position in our field of view. In the larvae, very slight deviations from the target position or delays in the re-appearance of the virtual prey increased the reaction times. When it receives unexpected visual feedback, the larva’s brain presumably needs extra processing time to calculate the next swim bout,” explains Johann Bollmann from the Max Planck Institute in Heidelberg.

In addition, with the help of fluorescent microscopes, the researchers can examine the activity of groups of neurons in the larval brain which are likely to control the targeted prey capture movements. In a previous study, they discovered cell types that react specifically to opposing directions of movement. These previously unknown neurons in the dorsal region of the midbrain (tectum) differ in their directional sensitivity and in the structure of their finely branched projections. “It appears that different directions of motion are processed in different layers of the tectum, since the dendritic ramifications of these cell types are spatially separated from each other,” says Bollmann.