The human brain is an organ of staggering complexity, with hundreds of billions of neurons and glial cells forming something like a quadrillion connections, or synapses. Generating this connectivity is a mammoth task, and the growing brain uses a number of strategies to ensure that the process runs smoothly – it produces far more cells than it needs, and these in turn form many more synapses than are required. Redundant connections are then removed in a process called pruning.
We now know that pruning occurs well into adulthood, but exactly how the brain disposes of its unwanted connections was unclear. A team of Italian researchers now reveals the surprising mechanism by which pruning occurs – it is carried out by cells called microglia, which patrol the developing brain and engulf unwanted synapses as if they were invading microbes.
Microglia are non-neuronal cells that have numerous branches and are highly abundant in the brain. They are related to immune system cells called macrophages, and carry out a similar function, forming the first line of defence against invaders, as well as performing various housekeeping functions. They crawl, amoeba-like, through the spaces between neurons, using their protrusions to detect viruses and microbes that have infiltrated the brain and quickly engulf those they find. They also actively migrate towards parts of the brain that have been damaged by stroke or disease, eating up dead neurons and clearing away any cellular debris left behind.
Even in the absence of injury or invaders, microglial cells are constantly moving, extending and retracting their protrusions as they migrate through the brain tissue. Exactly why they do so is unclear, but the recent finding that they come into frequent, brief contact with synapses suggests that they are actively surveying their micro-environment and monitoring the state of the connections.
To investigate, Rosa Paolicelli of the European Molecular Biology Laboratory in Monterotondo, Italy, and her colleagues analysed the brains of two week-old mice, a stage of postnatal development during which synapse pruning is at its peak. First, they determined the distribution of a protein called PSD-95 – a key component of the dense protein network found in excitatory synapses – and compared it to the location of microglia.
These experiments, performed in slices of brain tissue taken from the hippocampus, revealed that PSD-95 and microglia overlapped in a small number of locations. In most cases, the protein appeared to be surrounded by the microglia, suggesting that it was actually inside the cells. The researchers used an electron microscope to examine the samples more closely and, sure enough, found fragments of neuronal material containing PSD-95 in the microglia. The fragments were observed within membrane-bound vesicles, which are involved in a process called phagocytosis.
Phagocytosis means “cell-eating” and is the process by which microglia and other cells take up solid materials. First, the material is pulled towards the cell membrane, which then begins to invaginate, or fold in on itself, to envelop the material. As the in-folding continues, the outer edges of the membrane are drawn together until they eventually meet, producing a globule (the vesicle), which then buds off and moves into the cell. The contents of the vesicle are then processed appropriately – microbes are destroyed and membrane proteins and other cellular components recycled.
Next, the researchers created a strain of mutant mice lacking the gene encoding the fractalkine receptor. Fractalkine is a small signalling molecule which is known to play many roles in the immune system. Neurons in the brain ramp up fractalkine production when they are forming synapses; the fractalkine receptor is synthesized exclusively by microglia and is essential for their survival and migration.
The mutants had significantly greater numbers of synapses at two and three weeks of age than their normal litter-mates, leading to an increase in the frequency of spontaneous electrical impulses. Consequently, long-term depression, a form of synaptic plasticity in which synaptic connections are weakened, was enhanced, but this effect was reversible and had disappeared by the time the mice were 40 days old. The three-week-old mice were also less susceptible to drug-induced epileptic seizures but this, too, was not seen in the adults.
This suggests that synapse pruning in the mutants’ brains was diminished, causing the connections to mature more slowly than they normally would, leading to a delay in the development of brain circuitry.
Thus, the developing brain treats unwanted synapses as if they were unwanted invaders. It dispatches microglial cells to survey the state of synapses in their surroundings and to dispose of the ones that are wired incorrectly or superfluous. Abnormal neural connectivity has been implicated in neurodevelopmental disorders such as autism, so deficiencies in microglial surveillance may contribute to such conditions.
“We are very interested to see if there are long-lasting behavioural changes due to the deficient pruning in the mutant mice,” says senior author Cornelius Gross. “In particular, we are interested to see if autism-like behavioural phenotypes are revealed in these mutants. The second question is: What is the local ‘eat me’ signal from synapses that indicate they are ready to be pruned? We think it might be fractalkine [and] we also suspect that the complement cascade proteins are involved.”
The making and breaking of synapses also occurs throughout life, and is essential for learning and memory. It is, therefore, intriguing to speculate that microglia could also be involved in these processes. “It is certainly possible,” Gross says, “but so far, there is no evidence that microglia are required for the loss of synapses under these circumstances. Synapse loss and gain can be seen in cultured cells, so microglia are not absolutely essential.”
Reference: Paolicelli, RC et al (2011) Synaptic Pruning by Microglia Is Necessary for Normal Brain Development. Science DOI: 10.1126/science.1202529
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