Correlating physiologic measures with three-dimensional (3D) imaging at the light and electron microscopic levels is a powerful combination of methods for studying the structure and function of biological systems. Neurobiology is an ideal field for the application of these methods because neurons and glia have complex and extensive 3D structure, and their physiology is under intense study. Neurons, such as those studied here from Aplysia, can be more than 100 μm in diameter, and glia undergo large scale 3D shape change as a function of a number of physiologic parameters. The ability to accurately quantitate the 3D structure, volume and surface area of live neurons and glia is important to our understanding of the complex function of these cells.Neurons were isolated from the major ganglia of juvenile Aplysia Californica and glia were obtained from long term cultures of LRM 55 cells or as primary isolates from rats. Cultures were exposed to Dil dissolved in DMSO with or without 20% Pluronic F-127 and added to the culture media. The imaging instrument was an Olympus IMT-2 and a Bio-Rad MRC-600.

Neurons are cells with extensive dendritic and axonal arborizations extending from the cell body hundreds of micrometers or more in all three-dimensions. These structures have specializations, such as dendritic spines, that are at or just below the level of resolution of the light microscope (LM), and others, such as synapses, that can be resolved only in the electron microscope. Thus, it can be essential to correlate light and electron microscopic images from the same specimen. Due to its discrimination along the z-dimension (optic axis), the confocal light microscope is ideal for investigating neurons and correlating their structure and function. At the ultrastructural level, we use the high-voltage electron microscope (HVEM) to collect three-dimensional data, because it images thick objects. We are studying neurons in culture, and in thick acute and long term cultured brain slices. LM observations are made either after fixation or live by LM, and these images are correlated with HVEM ultrastructural observations.

Cell volume changes and regulation are thought to be important in the physiology and pathology of neurons. Osmotic challenges alter a number of physiologic parameters including electrical properties, chemosensitivity, and Na pump activity. Thus, a good method for correlating images of the living cell's surface with electrophysiologic measures is needed, and we are exploring the use of confocal light microscopy for imaging neurons in culture.Isolated neurons from ganglia of Aplvsia California were plated on polylysine coated coverslips in L15 media with 20% hemolymph at room temperature. Cells stained with “Dil” were imaged with a Bio—Rad MRC—600 and an Olympus BH—2. Images were displayed using Bio—Rad's maximum projection procedure, or Vital Images’ Voxel View software run on an IBM RISC 6000 Powerstation 320. Electrophysiologic properties were assessed after imaging.

The correlation of physiology and ultrastructure is critical to many areas of neurobiology, but is usually possible only in “simple” systems, (neuromuscular junction, isolated invertebrate or lower vertebrate preparation or in culture). Such correlation is difficult in the central nervous system (CNS) due to its complex microanatomy, large number of synapses on each neuron and lack of techniques for finding specific sites, (e.g. presynaptic terminals buried in a mass of tissue). We are developing specimen labeling and preparation methods to mark the site at which a physiologic parameter has been measured or manipulated by a microelectrode. The marker must be either electron dense or fluorescent - the former is preferred. It must bind to the cell surface without excessive diffusion into surrounding tissue, and it must be visible during block trimming and sectioning, to form a traceable link from the macroscopic to the ultrastructure.