Microscopy techniques and the study of synapses Emma Perez-Costas *+ , Miguel Melendez-Ferro + , and Rosalinda C. Roberts Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Sparks Center, 1720 7 th Avenue South, Birmingham, Alabama, USA Microscope techniques have been largely underestimated as powerful tools for the study of cell function and with some exceptions mostly restricted to descriptive studies. The development of new techniques and methods as well as the development of more powerful image analysis software in the last decade finally has provided the conditions to use microscopy in much more extensive and powerful ways. The development of new imaging software has also resuscitated the interest in “old” techniques such as Golgi staining methods that have suddenly come back to the spot light due to the possibility to be combined with image analysis tools. Microscopy is no longer the way to only get a “pretty picture” to illustrate our papers, but also a very powerful tool to study brain function. We propose here different strategies and methodologies that can be applied to the study of synaptic development and function using fluorescence, brightfield and electron microscopy. The use of these techniques allows the study of parameters such as the expression of synaptic proteins, number and morphology of dendritic spines, and number, type and structure of synapses. Keywords: ultrastructure; immunofluorescence; brightfield microscopy; Golgi staining; stereology; spines; tree shrew, human postmortem. 1. Introduction Normal synaptic connections, number and efficacy are crucial for proper brain function. The development of microscopy techniques or strategies for the quantification of synapses, or of dendritic spines (which receive the majority of synapses) is important for the study of brain function in normal development, adulthood and in diseases of the brain. In the present chapter we will describe methods and strategies for the study of synaptic connections that can be used in a variety of species. The methods that will be presented in the following pages can be used not only in well perfused animal brain samples, but can also be successfully applied to well preserved post-mortem human brain tissue. In the particular case of post-mortem human samples, a crucial factor is the post-mortem interval before preservation that especially can affect the quality of the tissue for the performance of ultrastructural studies. Regardless of the species, the study of synapses can be challenging because of their intrinsic complexity and plasticity which requires multiple strategies for the study of different aspects of the dynamics and functionality of these structures. When studying synaptic development or synaptic pathology several questions need always to be addressed: Which proteins are expressed in the synapse? Are there changes in the expression of these proteins? Are there changes in synaptic connections, morphology or number? Many of these questions can be answered using a combination of microscopic techniques as we will present in the following paragraphs. To illustrate the usefulness of the methodology proposed we have selected a series of examples based on studies performed in our laboratory. We have intentionally chosen examples based on studies in different species to demonstrate the versatility of the methods described here. * Corresponding author: email: epcostas@uab.edu. Phone: +1 205 9967574 + Both authors contributed equally to the manuscript. + Both authors contributed equally to the manuscript. ©FORMATEX 2007 Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ 164