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1. In order to understand the functional organization of the crustacean olfactory system, we are using intracellular recording and staining techniques to correlate the structure and function of single, odorant-sensitive interneurons in the brain of the crayfish Procambarus clarkii. We describe here the anatomy and physiology of interneurons that connect the brain with the medullae terminales or other eyestalk ganglia. 2. All of the interneurons in our study are at least third-order olfactory neurons (second-order olfactory interneurons) because they respond to chemostimulation of the olfactory organ (the antennules) but do not branch in the olfactory lobe (the neuropil to which primary olfactory receptor cells of the antennules project). 3. Much of the central nervous system, including the three main divisions of the brain (protocerebrum, deuterocerebrum, tritocerebrum) and the medullae terminales, are involved in integrating olfactory or multimodal (including olfactory) information, since these areas contain neurites of olfactory interneurons. Previous studies have indicated that regions involved in such processing include the olfactory lobes and accessory lobes of the deuterocerebrum, and regions I, II, IV, and VII (in some species) of the medullae terminales. Our results show that also prominent among regions involved in olfactory or multimodal (including olfactory) integration are the anterior and posterior optic neuropils of the protocerebrum, the lateral and medial antennular neuropils of the deuterocerebrum, the tegumentary neuropils and the antennal neuropils of the tritocerebrum, and neuropils III, VI, XII of the medullae terminales. 4. These olfactory interneurons were sensitive to chemostimulation (unimodal), chemo- and mechanostimulation (bimodal), or chemo-, mechano-, and photostimulation (trimodal). Responses could be excitatory or inhibitory, even for a given neuron. Morphologically complex interneurons (those having bilateral branching) were more likely to have complex response characteristics (trimodal sensitivity) than were morphologically simpler interneurons (those having unilateral branching). Olfactory interneurons with a soma in the medulla terminalis showed the most complex response profiles: they were trimodal, and were exicted by odorants but were inhibited by touch and/or light. This finding suggests that these are complex, high order interneurons. 5. Our studies reveal that olfactory and other sensory information is transmitted between the brain and the medullae terminales (and possibly other eyestalk ganglia) by a coactivated, parallel array of structurally and functionally diverse neurons.

A unique probe--biotinylated adenosine-5'-monophosphate (5'AMP-biotin)--was used in transmission electron microscopic (TEM) studies to localize 5'AMP odorant binding sites on the dendrites of olfactory receptor neurons in the aesthetasc sensilla of the spiny lobster, Panulirus argus. This probe is capable of both binding to and exciting 5'AMP-sensitive olfactory receptor neurons, as revealed through biochemical and electrophysiological assays. TEM studies showed that 5'AMP-biotin binding sites are distributed along the entire dendritic region that is exposed to odorants, including the transitional zone (between the inner and outer dendritic segments, including the ciliary segment) and all of the outer dendritic segment. The density of 5'AMP binding sites per micron2 of membrane is similar along the length of the olfactory dendrite. However, the relative number of 5'AMP-biotin binding sites per micron2 of sensillar area diminishes in the distal 30% of the aesthetasc due to a decrease in the amount of dendritic membrane in that region. The distribution of these 5'AMP binding sites is therefore much more extensive than that of enzymes that inactivate 5'AMP--5'ectonucleotidase/phosphatase--which are restricted to the transitional zone (Gleeson et al., 1991). Taken together, these results suggest that 5'AMP-biotin is labeling 5'AMP-specific olfactory receptor sites that are located along the entire outer dendritic segment and that can be coupled to olfactory transduction. This study represents the first in situ localization of specific olfactory receptor sites using a specific, functionally defined ligand.

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