**7. Staining and mapping of ORC axonal projections**

136 Zoology

Fig. 8. Representative extracellular olfactory electrophysiological recordings from five different olfactory receptor cells (ORCs) from female *Manduca sexta* trichoid type-A sensilla showing excitatory responses (A-N, P, Q) and inhibitory (O) responses. Note the increasing excitatory activity of the ORC to increasing concentrations of trans-nerolidol (C-F) and *cis*-3 hexenylbenzoate (J-M). Note the two different types of excitatory responses to geraniol (B and H). The stimulus bar is represented by a filled rectangle and represents 200 ms and represents the onset of odor delivery. This figure from modified from Shields and

Hildebrand, 2001a.

The axons of ORCs project to and terminate in compartments of condensed synaptic neuropil (i.e., glomeruli) in the primary olfactory centers in the CNS of invertebrates and vertebrates (Hildebrand and Shepherd, 1997). Each olfactory glomerulus is a discrete anatomical and functional unit and represents a specific anatomical "address" dedicated to collecting and processing specific molecular features about the olfactory environment, conveyed to it by ORC axons expressing specific olfactory receptor proteins (Buck, 1996; Buonviso and Chaput, 1990; Christensen et al., 1996; Hildebrand and Shepherd, 1997; Mombaerts, 1996). Over approximately the past two decades, there has been mounting evidence that the arrays of glomeruli in the ALs of insects and the olfactory bulbs of vertebrates are organized chemotopically (e.g., Sharp et al., 1975; Rodrigues and Buchner, 1984; Hansson et al., 1992; Mombaerts, 1996; Friedrich and Korsching, 1997; 1998; Galizia et al., 1999), analogous to visuotopy, in visual systems, and tonotopy, of auditory systems.

The axons of antennal ORCs project via the antennal nerve to the ipsilateral AL in *M. sexta*. In the ALs, they form synapses with processes of a subset of the approximately 1,200 central neurons (Homberg et al., 1988; Christensen et al., 1995; Rössler et al., 1998; 1999). In both sexes of *M. sexta*, each AL bears 60 ordinary, sexually isomorphic glomeruli and three sexually dimorphic glomeruli (Rospars and Hildebrand, 2000). Male *M. sexta* moths bear three prominent glomeruli (cumulus, toroid 1, toroid 2), which constitutes the male-specific macroglomerular complex (MGC). This complex processes information about the conspecific female's sex pheromone (Hansson et al., 1991; Heinbockel et al., 1999; Rospars and Hildebrand, 2000) (discussed in more detail, below). Interestingly, females also bear two homologous, sexually dimorphic glomeruli (i.e., large female glomeruli or LFG, lateral and medial) (Rössler et al., 1998; 1999; Rospars and Hildebrand, 2000). Central neurons with arborizations leading to the lateral LFG were found to display a preferential response to linalool and certain other monoterpenoids (King et al., 2000). LFGs have been implicated in being involved in olfactory information attributed to the interactions of females with hostplants or with courting males.

To determine where the axons of ORCs of trichoid type-A sensilla project in the AL of female *M. sexta*, we performed anterograde labeling using dextrantetramethylrhodamine of ORCs in groups of 5-10 sensilla on various surfaces of a single annulus in the middle of an antennal flagellum (Fig. 1C, D). This was carried out by restraining an adult female moth in a plastic tube (Fig. 4) and cutting the tips of 5-10 trichoid type-A sensilla (Fig 4); (see Shields and Hildebrand, 1999a; Shields and Hildebrand, 2001) using the method outlined, above. In order to stain the ORCs associated with these cut sensilla, we created a small well from melted paraffin wax around the selected annulus and filled it with a small amount of 1% solution of dextran-tetramethylrhodamine (3000 MW, anionic, lysine-fixable, D-3308, Molecular Probes, Eugene, OR) until the annulus became completely submerged. The area was then covered with a small amount of petroleum jelly. We transferred the preparation to a humid chamber and kept it in darkness for 2-3 days. Following this period, we excised the brain and fixed it for 24-48 h in 2.5% formaldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) containing 3% sucrose. Following this, the tissue was dehydrated in a graded ethanol series, cleared in methyl salicylate, and viewed as a temporary whole mount in a laser-scanning confocal microscope.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 139

For all gustatory experiments, stimulus compounds were dissolved in 30 mM potassium chloride (KCl) (control) (Fisher Scientific, Fair Lawn, New Jersey) in distilled water to enhance the electrical conduction of the recording electrode and to improve the signal-tonoise ratio. This inorganic salt was chosen since the hemolymph of plant-feeding (i.e., phytophagous) feeders typically shows high K+ and low Na+ concentrations (Kaissling, 1995). KCl was also used to fill the indifferent electrode (Figs. 5, 6). Selected carbohydrates (i.e., sucrose and inositol), as well as alkaloids (Fig. 10) were tested in this study to observe

**8. Gustatory stimuli and responses from the medial styloconic sensilla** 

Fig. 10. Representative extracellular gustatory electrophysiological recordings from the medial styloconic sensillum of *Lymantria dispar*. The deterrent-sensitive cell responds to (A) potassium chloride (control) and to the alkaloids, (B-D) caffeine, (E) strychnine, and (F) atropine. The recordings in G, H, and I are higher magnifications of the action potentials

from the deterrent-sensitive cell to (D) caffeine, (E) strychnine, and (F), atropine, respectively. The deterrent-sensitive cell displays a typical phasic-tonic response (i.e., a transient pattern of firing changing to that of a more sustained pattern of firing) to these alkaloids. This feature is more apparent at higher concentrations (compare (C) and (D) with (B)) and is shown for caffeine. A weaker concentration of caffeine (B) elicits fewer spikes from the deterrent-sensitive cell. This figure was modified from Shields and Martin, 2010.

We found that a majority of axonal projections from these ORCs terminated in the two LFG located in the dorsolateral region of the ipsilateral (AL) (Fig. 9A), more specifically, near the entrance of the antennal nerve into the AL. We also found that in addition to the LFGs, a subset of the other 60 spheroidal, ordinary, sexually isomorphic glomeruli received sparse projections of a subset of ORC afferent axons (Fig. 9). The results of these anatomical studies and our electrophysiological results that some trichoid type-A sensilla are tuned mainly to terpenoids and aromatic esters, provide a basis to hypothesize that information about odorants belonging to those chemical classes is processed in the LFGs (King et al., 2000; Shields and Hildebrand, 2001a; b). To improve visualization (i.e., resolution) of successfully labeled preparations, samples were embedded in Spurr's low-viscosity embedding medium (Electron Microscopy Sciences, Fort Washington, PA) and sectioned at 48 μm. The preparations were then viewed in a laser scanning confocal microscope (Bio-Rad MRC-600; Cambridge, MA) equipped with a Nikon Optiphot-2 microscope and both 15-mW krypton-argon and 100-mW argon laser light sources and YHS filter cube (excitation wavelength 568 nm). Serial optical sections were collected at 3-μm intervals through the whole mount or 2 μm intervals from the embedded sectioned preparations (Fig. 9). Image processing and analysis were performed using Confocal Assistant 4.02 (copyrighted by Todd Brelje, distributed by Bio Rad, Cambridge, MA), Corel Photopaint 8, and Corel Draw 8 (Corel Corporation, Ottawa, Ontario, Canada).

Fig. 9. Confocal microscopic images (A-D) showing serial optical sections of female *Manduca sexta* specimens embedded in plastic and sectioned to improve resolution taken at different depths through the antennal lobe. Images show the central projections of axons from olfactory receptor cells from trichoid type-A sensilla stained with the fluorescent dye dextrantetramethylrhodamine. Askterisks indicate the sexually dimorphic large female glomeruli located in the dorsolateral region of the antennal lobe, near the site of entry of the antennal nerve (AN). The arrows indicate ordinary sexually isomorphic glomeruli. d, dorsal; l, lateral. Scale bars = 100 µm. Figs. (A) and (B) were modified from Shields and Hildebrand, 2001a.

We found that a majority of axonal projections from these ORCs terminated in the two LFG located in the dorsolateral region of the ipsilateral (AL) (Fig. 9A), more specifically, near the entrance of the antennal nerve into the AL. We also found that in addition to the LFGs, a subset of the other 60 spheroidal, ordinary, sexually isomorphic glomeruli received sparse projections of a subset of ORC afferent axons (Fig. 9). The results of these anatomical studies and our electrophysiological results that some trichoid type-A sensilla are tuned mainly to terpenoids and aromatic esters, provide a basis to hypothesize that information about odorants belonging to those chemical classes is processed in the LFGs (King et al., 2000; Shields and Hildebrand, 2001a; b). To improve visualization (i.e., resolution) of successfully labeled preparations, samples were embedded in Spurr's low-viscosity embedding medium (Electron Microscopy Sciences, Fort Washington, PA) and sectioned at 48 μm. The preparations were then viewed in a laser scanning confocal microscope (Bio-Rad MRC-600; Cambridge, MA) equipped with a Nikon Optiphot-2 microscope and both 15-mW krypton-argon and 100-mW argon laser light sources and YHS filter cube (excitation wavelength 568 nm). Serial optical sections were collected at 3-μm intervals through the whole mount or 2 μm intervals from the embedded sectioned preparations (Fig. 9). Image processing and analysis were performed using Confocal Assistant 4.02 (copyrighted by Todd Brelje, distributed by Bio Rad, Cambridge, MA), Corel Photopaint 8, and Corel Draw 8 (Corel Corporation, Ottawa, Ontario, Canada).

Fig. 9. Confocal microscopic images (A-D) showing serial optical sections of female *Manduca sexta* specimens embedded in plastic and sectioned to improve resolution taken at different depths through the antennal lobe. Images show the central projections of axons from olfactory

receptor cells from trichoid type-A sensilla stained with the fluorescent dye dextrantetramethylrhodamine. Askterisks indicate the sexually dimorphic large female glomeruli located in the dorsolateral region of the antennal lobe, near the site of entry of the antennal nerve (AN). The arrows indicate ordinary sexually isomorphic glomeruli. d, dorsal; l, lateral. Scale bars = 100 µm. Figs. (A) and (B) were modified from Shields and Hildebrand, 2001a.
