Studying Synapse Formation with Confocal Microscopy and Vital Labels Noam E. Ziv, Hagit Vardinon-Friedman, Tal Bresler, and Yaron Ramati Rappaport Institute and the Dept. of Anatomy and Cell Biology, Bruce Rappaport faculty of medicine, Technion, Haifa, Israel The mammalian brain is an extremely complex network of computational units - neurons - interconnected by specialized intercellular communication devices - synapses. Most synapses are formed between axons, the major carriers of neuronal output, and dendrites, shorter fibers that constitute the major targets for neuronal input. Most synaptic connections are established during development. It has been suggested, however, that synapse formation (and elimination) continue into adulthood, where they may underlie phenomena related to learning and memory. Accordingly, the cellular, biophysical and molecular processes that underlie and regulate synapse formation, stabilization and elimination are of great interest. The genesis of an individual axodendritic synaptic connection is a concerted process that involves structural and functional rearrangements on both sides of the nascent synaptic junction. The process begins with the formation of a physical contact between potential synaptic partners, continues with the differentiation the axonal and dendritic compartments into pre- and postsynaptic compartments respectively, and may be considered to be completed when the nascent junctions acquire a capacity for synaptic transmission. Historically, attempts aimed at understanding how the brain is “wired” were based primarily on the analysis of fixed and stained tissue by means of light and electron microscopy. Although these studies provided the infrastructure of our understanding of CNS synaptogenesis, the techniques applied were inherently limited in their ability to provide information concerning the dynamics of these processes or the cellular and molecular processes involved. The introduction of numerous contemporary techniques during the last decade has resulted in much progress, yet most of this progress relates to the identification of synaptic molecules and their interactions, not so much to the processes involved. Thus, until quite recently, surprisingly simple questions have remained unanswered: What is the time frame for the formation of individual synaptic connections? What is the temporal order of assembly of pre and post synaptic compartments? How are key synaptic molecules recruited to nascent synapses? We have begun to address these questions by studying the process of synapse formation in live neurons obtained from postnatal rat hippocampi and grown in culture. Most of this work is based on the use of automated time-lapse, multi-site laser scanning confocal microscopy and vital fluorescent markers of synaptic structure and function, complemented with immunohistochemical and biochemical approaches. In order to determine the time course of presynaptic terminal formation, we used green fluorescent protein (GFP) to visualize the entire architecture of individual neurons and time-lapse confocal microscopy to monitor encounters between labeled axons and dendrites. FM 4-64, a vital fluorescent label was then used to detect the formation of new functional presynaptic terminals at axodendritic contact sites. These experiments revealed that new, apparently functional presynaptic sites can form within <30 minutes of axodendritic contact . To determine if such presumably new presynaptic sites were associated with postsynaptic molecules and, in particular, receptors for the neurotransmitter glutamate (the major excitatory neurotransmitter in the brain), we fixated the preparations at the end of the experiments and used retrospective immunohistochemistry to determine the molecular composition of such presumably new synaptic sites. This analysis revealed that within less than 90 minutes of their initial appearance, most new presynaptic sites were associated with all 4 postsynaptic molecules examined (the postsynaptic scaffolding molecules PSD-95, ProSAP1, and glutamate receptor subunits GluR1 and NR1) . Taken together, these findings suggest that new glutamatergic synapses can form within ~2 hours or less. Interestingly, two presynaptic structural molecules Bassoon and Piccolo were present in practically all new presynapytic boutons, regardless of their age [1,2]. Both molecules were found to be carried on a previously unknown 80 nm dense core vesicle that was shown to specifically contain additional presynaptic membrane components, including Syntaxin, SNAP-25 and N-Cadherin. These findings were interpreted to suggest that these vesicles constitute “prefabricated” precursor vesicles whose fusion with the presynaptic plasma membrane would result in the rapid formation of new neurotransmitter release sites . To determine if postsynaptic differentiation also occurs by the insertion of “prefabricated” complexes, we expressed a postsynaptic molecule (PSD-95) tagged with GFP in individual neurons. Time-lapse confocal microscopy revealed that this key molecule seems to be recruited to nascent synaptic sites in a gradual manner, not by the insertion of preassembled complexes. Thus, this observation together with other findings in the literature suggest that postsynaptic assembly is realized by the sequential recruitment of postsynaptic molecules to nascent synaptic sites. To summarize, several important aspects of synaptogenesis cellular physiology were studied here using confocal microscopy and fluorescent labels, illustrating how such methods can be used elucidate key cellular and molecular processes. References 1. H. Vardinon-Friedman, T. Bresler, C.C. Garner and N.E. Ziv, “Assembly of new individual excitatory synapses- Time course and temporal order of synaptic molecule recruitment”, Neuron, 27:57-79, 2000. 2. R. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E.D. Gundelfinger, N.E. Ziv, C.C. Garner, “Assembling the presynaptic active zone: Characterization of an active zone precursor vesicle”, Neuron, 29:131-143, 2001.
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