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Restless Receptors

New Insights Into the Dynamic Organization of Synapses

Synapses are the switch-points in our brain for information transmission, learning and memory. And there is evidence to suggest that changes and malfunctions in synapses are partly responsible for a number of neurological and psychological disorders. Neuroscientists already know a lot about how signals are transmitted from neuron to neuron. Yet many synaptic processes are still not fully understood.

Dr. Daniel Choquet, Research Director at the Institute for Interdisciplinary NeuroSciences (IINS) of the CNRS and Bordeaux 2 University and Head of the Bordeaux Imaging Center (BIC) is researching signal transmission in the postsynaptic membrane. His studies and his developments of imaging techniques have provided new insights into the dynamics of glutamate receptors, which are involved in synaptic transmission in 80 % of excitatory cerebral nerve cells and play a key role in synaptic plasticity. The use of superresolution technologies is making an essential contribution to this research.


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Receptor movements connected with learning and memory

“The understanding of the role of postsynaptic receptors has changed significantly in recent years”, says Choquet.When I started working in Bordeaux Neuroscience Institute (INB) in 1997, receptors for neurotransmitters were believed to be stable and rather immobile molecules whose activity and regulation were purely based on phosphorylation and structural modification. However, my earlier experience of cell biology made me wonder why the dynamics of neuronal  receptors should be any less complex than those of other cell components.” Various studies then proved that receptors are not firmly anchored in the membrane, but move in permanent exchange processes by endo- and exocytosis.

Some years later, the team of Choquet was able to show that the receptors also move in the plane of the cell membrane by lateral diffusion and travel relatively long distances within the synapse. In the last few years the Laboratory ‘Cellular Physiology of the Synapse‘ has been in close cooperation with the physics group of Brahim Lounis at the University of Bordeaux 1 to start characterizing this mobility and  examining the way it is regulated. In doing so, they made an amazing discovery for the knowledge of the time: The movements of the receptors are regulated by the neuron activity which, in turn, is directly connected with learning and memory.

Receptor mobility controls neuronal transmission

Today, it is known that receptors move very rapidly and that this mobility plays an essential role in the signal transmission between neurons. In fact the mobility of receptors controls the reliability of neuronal transmission. “We have revealed that a minor modification of the mobility has a major impact on high frequency transmission. In addition this mobility enables the replacement of desensitized receptors by naïve receptors within a few milliseconds,” Choquet states. This reduces synaptic depression and allows the neuron to transmit information at a higher frequency. These results have radically changed the understanding of neuronal physiology. “Besides the electrophysiological techniques, the light microscopic techniques have played a decisive role,” Choquet explains. “They have literally shed new light on signal transmission functionality and synaptic plasticity.”

Fig. 1: Fluorescence image of a rat neuron labelled with three colors: a presynaptic marker (blue), a postsynaptic marker (red), and a glutamate receptor (green). The white color at the tip of the dendritic spines indicates an accumulation of receptors. © CNRS Photothèque / Magali Mondin, Daniel Choquet, Laboratory: UMR5091 – Physiologie cellulaire de la synapse (PCS) – Bordeaux.

Investigating receptor regulation processes

Currently, the team of Choquet is consolidating this knowledge by pursuing two different lines of research. “We are extending our experiments, which have concentrated up to now on cell cultures and brain sections, to include ex vivo investigations and even studies of living organisms”, explains Choquet. “Only then can we gain a better understanding of how learning and memory are actually influenced by the regulation of receptor movements. On the other hand, we intend to research receptor mobility within the synapse on a nanometer scale down to the smallest detail in order to find out, for example, how scaffold proteins are involved in the regulation of receptor mobility. Our working hypothesis is: different information speeds and regulations are directly related to learning and memory.”

Impact for research of neuronal diseases

The group’s work has quite a relevance for the research of neuronal diseases. “Today, we assume that changes or malfunctions at synapses play a definite role in neuronal and psychological disorders. That’s why not only neurodegenerative diseases, but also epilepsy or autism are also called synaptopathies. Of course, our basic molecular research of the animal model is still far from being clinically relevant, but our work is already linked to that of the colleagues in pathology.” For instance, they have begun, using animal models, to examine the defects in receptor trafficking that are observed in Alzheimer’s and Parkinson’s disease. A whole department of the INB is occupied with neurodegenerative diseases.

Developing imaging technologies

Choquet considers heading the BIC an added advantage for his research. “In Bordeaux, I had the dual function as head of a research group and Head of the Imaging Facility right from the beginning. I have taken care to make the imaging tools we develop for our own experiments available to the entire community", he says. "Since then, the facility has steadily grown – like my research group. In its present size and function as core facility, the BIC has evolved from the fusion of the light microscopy (LM), electron microscopy (EM) and the plant imaging facility."

 The team is very successful with the further development of imaging technologies. In 2002, they managed to obtain the first live images of the movements of AMPA receptors (AMPAR), a sub-group of the glutamate receptors, in the cell membrane by using a relatively crude approach of tracking by video microscopy of micrometer-sized latex beads bound via antibodies to AMPAR subunits. However, this method is not suitable for tracking receptors inside the synapse. Together with the physics group of Bordeaux 1, they then started developing single molecule detection techniques.

They were the first group in Europe to apply this technique successfully to living neurons. Another method they developed is the  photothermal imaging of nanogold particles to track receptors in live neurons for long periods. "Although the gold particles do not bleach or blink, and allowing theoretically infinite recording times, spontaneous photothermal signals from mitochondria may interfere with the tracking of gold particles", the scientist says.

Impact of superresolution

Choquet and his team are currently devoting a lot of our attention to superresolution technologies, which are extremely important for neuroscientific research. Apart from Single Molecule Detection, the group uses the Leica Microsystems STED system and a PALM system. Beyond this, they have also developed a new superresolution technique in the last few years called PAINT. This point-accumulation-forimaging-in-nanoscale-topography method allows dynamic superresolution imaging of arbitrary membrane proteins in living cells. In a recent paper they published a further development of this approach called uPAINT  (universal PAINT). This method is based on continuously and stochastically labeling membrane surface in the national Excellence Initiative.

"And that’s not all by any means – it rarely gets boring here", resumes Choquet. "In spite of all the work, it is extremely exciting to play a part in developing the achievement potential and the scientific reputation of the INB."