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fter finishing postdoctoral training at the University of California at Berkeley, Dr. Peter Scheiffele, 32, arrived at Columbia Health Sciences in June to set up his laboratory. He is the first of four recruits in a new developmental neurobiology initiative of the departments of physiology & cellular biophysics and pathology.

Dr. Scheiffele, assistant professor of physiology & cellular biophysics, was recruited because of his seminal research, published in the June 9, 2000, issue of Cell, which showed for the first time how a single protein, called neuroligin, could trigger the formation of a synapse. At Columbia, Dr. Scheiffele and his laboratory are continuing to study the role of neuroligin as well as other proteins involved in creating new synapses.

Nerve cells communicate with each other through synapses. During synaptic transmission, neurotransmitters released from the presynaptic neuron into the synaptic cleft, a small gap between the two nerve cells, bind to and activate receptors in the postsynaptic neuron. How new synaptic connections are formed is of great interest to scientists because changes in the number and function of synapses affect learning, memory, and behavior.

Most neuroscience researchers believed synapse formation would require significant cross-talk between what ultimately would become pre- and postsynaptic cells. But Dr. Scheiffele showed neuroligin alone was able to stimulate the formation of presynaptic specializations.

Dr. Scheiffele discovered the unique role of neuroligin in generating synapses by using an in vitro co-culture system. This system assays how individual proteins introduced into a non-neuronal cell affect the distribution of synaptic components in a presynaptic neuron. In the Cell study, he transfected the cDNAs of six neuronal adhesion proteins (which had been previously identified) into non-neuronal cells to create six cell lines that expressed these factors. He then put each cell line in culture with pontine cells, neurons that deliver information from the brainstem to the cerebellum, and monitored changes in the presynaptic neurons.

Of the six adhesion proteins, only neuroligin could induce presynaptic-like structures in the pontine neurons. (See illustration.) In response to neuroligin, the end of the pontine axon flattened to form a large, intimate contact area with the transfected non-neuronal cell and gathered clusters of synaptic vesicles, which contain neurotransmitters. When the researchers depolarized the pontine neurons, the cells released these vesicles by regulated exocytosis, as they would during synaptic transmission between two neurons.

A candidate binding partner for neuroligin in the presynaptic neuron is a protein named neurexin, Dr. Scheiffele says. While scientists have not been able to directly demonstrate the presence of neurexin in the presynaptic membrane, indirect evidence suggests it might be there. When Dr. Scheiffele added soluble neurexin to his co-culture system, essentially blocking neuroligin from binding to the pontine cells, he detected a reduction in the number of presynaptic structures by 80 percent.

To see if neuroligin worked in a more realistic situation, he blocked neuroligin’s activity by adding neurexin to a co-culture of pontine axons and their natural postsynaptic target, granule cells from the cerebellum. The result: The number of synapses developing under these conditions was reduced by half. Such findings suggest neuroligin binds to neurexin to trigger synapse formation in vivo and is part of the general synapse-building machinery of many or all other central nervous system synapses.

Finding more direct evidence that neuroligin acts through neurexin as a presynaptic receptor and identifying the signaling downstream of this receptor should keep Dr. Scheiffele’s lab busy. The functional cell culture assays also allow lab members to find other components that respond to neuroligin to build a synapse. Dr. Francisco Gomez-Scholl is purifying proteins that attach to the neuroligin-neurexin complex, especially those that assemble and cluster vesicles. Dr. Marta Benedetti is using the assays to investigate the potential role of voltage-dependent calcium channels in synaptogenesis. The lab also has identified novel candidate proteins involved in synapse formation and function by using microarray studies, and an important focus of the lab will be to unravel the role these molecules play during the development of the central nervous system.

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