Fiber systems in the olfactory bulb and cortex: A study in adult and developing rats, Using the Timm method with the light and electron microscope
Authors: Friedman Beth , Price Joseph L. 
Published: May 2005

The structure of the olfactory bulb and cortex of rats is described using the Timm method, in which endogenous transition or “heavy” metals are precipitated with sulfide and then stained with silver. With the light microscope, two types of staining, a fine, “colored” reaction and a coarse, “granular” reaction, are found in the neuropil of the adult forebrain; neuronal Somata, fiber tracts, and some specific areas of the neuropil are almost unstained. With the electron microscope the Timm method consists of small silver granules, which are associated with axon terminals. These granules are located over the external surface of the presynaptic membrane, facing the synaptic cleft, and over synaptic vesicles, especially the internal surface of the vesicle membrane. This localization suggests that the reactive metals are related to a membrane component which is recycled during secretion of synaptic transmitters. The colored and granular reactions seen with the light microscope appear to be correlated with different intensities of staining of different types of axon terminals.

The full extent of the olfactory cortex can be delineated by a bilaminar staining pattern in layer I, which coincides with the pattern of termination of fiber systems to the cortex demonstrated by axonal tracing experiments. The superficial part of layer I is very palely stained; this corresponds precisely to layer la, the zone of termination of fibers from the olfactory bulb. In contrast, the deep part of the layer is stained with an intense colored reaction; this corresponds precisely to layer Ib, the zone of termination of the major association fiber system within the olfactory cortex. A prominent granular reaction, which does not correspond to any known long axon system, is found in all layers of the cortex, although it is concentrated at the superficial edge of layer Ib and in layer II. Variations in staining in the several subdivisions of the olfactory cortex are illustrated and described. In the olfactory bulb, the most prominent staining is a relatively fine, granular reaction in the glomerular formations, which apparently corresponds to the olfactory nerve terminals.

In immature rats, the Timm method stains the neuropil throughout the olfactory system, although the patterns and quality of staining differ from that found in adults. Before birth, the best–differentiated reaction is seen in glomeruli of the olfactory bulb and correlates well with the pattern of olfactory nerve ingrowth. The earliest and most prominent reaction is found in the “modified glomerular complex” in the caudodorsal part of the bulb. In the olfactory cortex the earliest staining occurs before birth in the posterior piri‐form cortex. However, cortical staining is not extensive until the first postnatal week, and the islands of Calleja in the olfactory tubercle back a distinct positive reaction until postnatal day 7. The staining patterns become adultlike in all parts of the olfactory cortex during the third postanatal week.