English SUMMARY

Heterogeneity and regulation of presynaptic strength

Over the course of evolution, the brain developed from a simple relay system to an intricate network that is capable of executing many cognitive functions, including the formation of memory, development of communicative skills, problem solving and expression and interpretation of emotions. The correct execution of these tasks requires intensive communication between nerve cells. The fastest and most extensively used form of communication in the brain takes place at specialized structures called synapses. A nerve cell contains hundreds to thousands of these synapses, which enables it to communicate with large a number of cells. With these synapses, nerve cells can transfer information to each other at a time scale of a few milliseconds. In addition, synapses can modify and store information. By doing so they form the foundations for learning and memory, and acts small 'computers' in the brain.
At a synapse, one can distinguish between a part that sends information (the presynapse) and a part that receives information (the postsynapse). The presynapse contains a large number of vesicles that are filled with signal molecules. When a presynapse is electrically stimulated with a so-called action potential, one of these vesicles fuses with the membrane of the presynapse, to release the signal molecules. These molecules are then detected by the postsynapse, which translates it back into an electrical signal. This dissertation is focused on the function of the presynapse.
There are different types of synapses in the brain. Some synapses are very reliable: at every electrical stimulation they will release a vesicle, meaning that they will always transfer their information to the neighboring cell. These kinds of synapses are mostly used to send information from sensory organs. Other types of synapses are very unreliable: only occasionally they will release a vesicle upon stimulation. The change that a vesicle is release (also called the strength of the synapse) is on average 20%. These types of synapses are mostly used in brain areas involved in cognitive function, like the prefrontal cortex and the hippocampus. At first sight, this seems counterintuitive: synapses involved in important brain tasks are unreliable?! However, this feature is of the upmost importance for their function. These synapses are capable of increasing or decreasing their strength, to alter the information they transfer. For instance, by increasing its strength from 20% to 90%, the presynapse can 'store' information. This can be induced by a brief increase in electrical activity of the nerve cell, or by stimulation with signal molecules from neighboring cells. It is widely assumed that the regulation of synaptic strength is essential for correct brain function.
Although every synapse has the same function (releasing vesicles), there is a wide heterogeneity among synapses in the hippocampus. For instance, there is a large variation in the volume of the presynapse, where the vesicles are stored. Some synapses contain only 40 vesicles, while others have over a 1000 vesicles. Also synaptic strength is highly variable. As stated before, the average strength is around 20%, but for some synapses this is only 5%, whereas other may reach 100%. Even presynapses formed by the same nerve cell display such variation. In is not completely understood what the nature behind this richness is, and how this is regulated in the brain.
This dissertation is aimed at gaining more insight in the variation in presynaptic strength, and to describe the underlying cellular and molecular processes. This research has been divided into 4 chapters (2 to 5), each dealing with a separate scientific question.
In chapter 2, a new method is described to measure presynaptic strength. Until recently, it was not possible to measure the strength of large numbers of synapses within a single experiment; about 20 synapses was the limit. This new method makes use of a protein called 'SypHy'. This protein is stored within the vesicles of the presynapse, and only lights up when a vesicle is released. Therefore it becomes possible with a fluorescence microscope to measure the number of released vesicles - and thus the synaptic strength. In this chapter it is demonstrated that SypHy can be used to measure synaptic strength of hundreds of synapses simultaneously at high temporal resolution. This makes it possible to determine the relationships within a large population of synapses. We demonstrate that synapses situated next to each other have the same strength, and that this relationship is independent of the electrical activity of the nerve cell.
Chapter 3 describes how the strength of a presynapse is regulated based on its location. Previous research proved that the kind of postsynaptic cell is very important. A nerve cell receives information on large, highly branch structures called the dendritic tree. We know that the strength of the postsynapse is highly dependent on the location of the synapse on the dendritic tree. The further the synapse is located from the cell body, the stronger it becomes. This is called 'distance-dependent scaling'. In this chapter we tested if a similar rule exists for the presynapse, using the techniques described in chapter 2. In other words, does a presynapse formed at the base of the dendritic tree have a different strength then synapses formed at the tip of the tree? Surprisingly, we discovered that presynapses formed close to the cell body of the receiving cell were much stronger that presynapses at the tip of the tree. Thus, the distance-dependent scaling of the presynapse is exactly opposite from the scaling of the postsynapse. We furthermore demonstrate that this rule holds for other presynaptic properties as well. The number of vesicles per presynapse is highly dependent on synapse location, and the concentration of proteins responsible for the fusion of vesicles is highly dependent on distance as well. In total we are able to explain around 30% of the variation among synapses. Finally, we demonstrate that this effect is dependent on the identity of the receiving cells. Some cells have very strong distance-dependent scaling, while others have almost no scaling at all. We suspect that this kind of scaling is very important for information processing by nerve cells, but this requires further investigation.
Chapter 4 we investigate a molecular mechanism that regulates the strength of the presynapse. Fusion of vesicles occurs in several steps. A vesicle must be mature before fusion can occur. This is mediated by a few specific proteins, with Munc13 and Munc18 being the most important. If the synapse is electrically stimulated, calcium ions will flow into the synapse. As soon as calcium binds to the protein synaptotagmin, a vesicle can fuse with the membrane. This process is highly regulated by the synapse, and can be accelerated or slowed down at any moment. One of the most effective ways to accelerate fusion is by producing the lipid diacylglycerol (DAG). DAG can increase the strength of the synapse within seconds. DAG can bind directly to Munc13, which makes it work more efficiently. In addition, DAG binds to protein kinase C (PKC), which in turn activates Munc18 and synaptotagmin. Previous research showed that activation of both Munc13 and Munc18 is essential for the effect of DAG. The role of synaptotagmin activation was only investigated in kidney cells, where it did not have any role in vesicle fusion. Because we suspected that synaptotagmin activation might be of importance in synapses, we tested this in neurons. To do so we made use of a variant of synaptotagmin that cannot be activated by PKC. It turned out that this variant completely blocks the effect of DAG in resting cells. Under these conditions, activation of synaptotagmin is essential. Previous research showed that the activation of Munc13 and Munc18 is also of great importance during strong electrical stimulation of the synapse. However, under these conditions there was no difference between normal and non-activatable synaptotagmin. This strongly suggests that activation of synaptotagmin is only of importance during low electric activity. We suspect that the high calcium concentration in the synapse during high electric activity renders the activation of synaptotagmin by PKC dispensable.
Chapter 5 deals with a part of the genetic program responsible for the formation of new synapses. In order to make synapses, the transcription of hundreds of genes must occur. This process is regulated by specialized proteins, called transcription factors. Previous research in snails demonstrated that the transcription factor Menin plays a role in the creation of new synapses. Menin is described in detail for its role in tumor formation in endocrine tissue, but its role in the brain is completely unknown. In this chapter we made use of nerve cells from which the gene encoding for Menin can be removed. In contrast to expectations, deletion of Menin had no effect on synapse formation or other morphological parameters. In addition, there was no effect on the pre- and postsynaptic strength of these cells. This suggests that Menin plays no role in the formation of synapses, at least not in cultured mouse nerve cells.