HRP-filling of neurons and axonal arbors in fixed brain slabs

Visually-guided introduction of HRP into perfusion-fixed hamster brain slabs was carried out by three procedures: pressure or extracellular iontophoretic injections, introduction of a small HRP-crystal on the closed tip of a micropipette, and by dropping a very small drop of HRP solution on the ventricular surface. The prefixation was made with a solution of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer in time periods ranging from 4 to 26 days. The introduction of HRP-crystals into brain slabs did not produce HRP-filling of cells and processes, but only a granular background of HRP diffusion. Injections into adjacent brain slabs of an HRP-containing solution did produce, in every case, some degree of labelling of neuronal somata, dendrites, individual axons, and fibre bundles. Several cells showed dense HRP-filling of the soma and processes, including dendritic spines and axonal varicosities. Dense HRP-filled axonal terminal arbors were seen in the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) after iontophoretic injections in the optic tract. The good ultrastructural preservation achieved by perfusion of the fixative solution was not affected by the subsequent injection procedures, and the pre- and post-synaptic specializations of HRP-filled axons were recognizable. This approach may prove to be useful in correlative light and electron microscope studies of the synaptic relationships between HRP-filled neurons and axons.