Combining Laser Scanning Research Articles

Synaptic organization of the uniglomerular projection neurons of the antennal lobe of the moth Manduca sexta: a laser scanning confocal and electron microscopic study.

The detailed branching pattern and synaptic organization of the uniglomerular projection neurons of the antennal lobe, the first processing center of the olfactory pathway, of the moth Manduca sexta were studied with laser scanning confocal microscopy and a technique combining laser scanning confocal microscopy and electron microscopy. Uniglomerular projection neurons, identified electrophysiologically or morphologically, were stained intracellularly with neurobiotin or biocytin. Brains containing the injected neurons were treated with streptavidin-immunogold to label the injected material for electron microscopy and with Cy3-streptavidin to label the neurons with fluorescence for laser scanning confocal microscopy, and then embedded in Epon. Labeled neurons were imaged and reconstructed with laser scanning confocal microscopy (based on the retained fluorescence of the labeled neuron in the Epon block), and thin sections were cut at selected optical levels for correlation of light microscopic data and electron microscopic detail. Each neuron had a cell body in one of the three cell-body clusters of the antennal lobe, a primary neurite that extended across the coarse neuropil at the center of the antennal lobe and then formed a dense tuft of processes within a single glomerulus, and an axon that emanated from the primary neurite and projected from the antennal lobe via the antenno-cerebral tract to the lateral horn of the ipsilateral protocerebrum and, collaterally, to the calyces of the mushroom body. In the electron microscope, the fine dendritic branches in the apical zones of the glomeruli, where sensory axons terminate, were found to receive many input synapses. In deeper layers across the glomeruli, the processes participated in both input and output synapses, and the bases of the glomeruli, the most proximal, thickest branches formed output synapses. In both of the protocerebral areas in which axonal branches terminated, those branches formed exclusively output synapses. Our findings indicate that, in addition to conveying olfactory information to the protocerebrum, uniglomerular projection neurons in the antennal lobes of M. sexta participate in local intraglomerular synaptic circuitry.

Combining laser scanning confocal microscopy and electron microscopy in studies of the insect nervous system

Experimentally determining the synaptic interconnections between neurons in the nervous system is laborious and difficult in any animal species, but especially so in many invertebrates, including insects, where neurons generally have large, finely branching neuritic trees that form both pre- and postsynaptic specializations in dense neuropils with other neuritic trees. Electron microscopy is needed to identify synapses, but correlation of synapse type and location with the overall branching patterns of neurons, which are visible readily only in the light microscope or through extensive reconstruction of serial electron-microscope sections, is very difficult. In this paper, we present a simple method that we have developed (Sun et al. (1995) J. Histochem. Cytochem., 43: 329–335) that combines laser scanning confocal microscopy and electron microscopy for the study of synaptic relationships of neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Briefly, neurons are labeled by intracellular injection with neurobiotin or biocytin, and then processed with a gold-particle tag for electron microscopic study and a fluorescent tag for confocal microscopy, and embedded in plastic. The fluorescence of the labeled neuron in the plastic blocks is imaged in three dimensions with laser scanning confocal microscopy and then the neuron is thin-sectioned at precisely chosen depths for electron microscopic study. The fluorescence pattern can be monitored repeatedly between episodes of thin-sectioning, and subtraction of a fluorescence image from the previous fluorescence image reveals which fluorescent processes have been sectioned. In this way, electron microscopic detail can be mapped onto a three-dimensional light microscopic image of the neuron.

Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.