COMBINING LASER-SCANNING CONFOCAL MICROSCOPY AND ELECTRON-MICROSCOPY IN STUDIES OF THE INSECT NERVOUS-SYSTEM

Experimentally determining the synaptic interconnections between neuro ns in the nervous system is laborious and difficult in any animal spec ies, but especially so in many invertebrates, including insects, where neurons generally have large, finely branching neuritic trees that fo rm both pre- and postsynaptic specializations in dense neuropils with other neuritic trees. Electron microscopy is needed to identify synaps es, but correlation of synapse type and location with the overall bran ching patterns,of neurons, which are visible readily only in the light microscope or through extensive reconstruction of serial electron-mic roscope sections, is very difficult. In this paper, we present a simpl e method that we have developed (Sun et al. (1995) J. Histochem; Cytoc hem., 43: 329-335) that combines laser scanning confocal microscopy an d electron microscopy for the study of synaptic relationships of neuro ns in the antennal lobe, the first central neuropil in the olfactory p athway, of the moth Manduca sexta. Briefly, neurons are labeled by int racellular injection with neurobiotin or biocytin, and then processed with a gold-particle tag for electron microscopic study and a fluoresc ent tag for confocal microscopy, and embedded in plastic. The fluoresc ence of the labeled neuron in the plastic blocks is imaged in three di mensions with laser scanning confocal microscopy and then the neuron i s thin-sectioned at precisely chosen depths for electron microscopic s tudy. The fluorescence pattern can be monitored repeatedly between epi sodes of thin-sectioning, and subtraction of a fluorescence image from the previous fluorescence image reveals which fluorescent processes h ave been sectioned. In this way, electron microscopic detail can be ma pped onto a three-dimensional light microscopic image of the neuron.