**6. Olfactory responses from trichoid type-a sensilla**

Alternating current (AC) signals were recorded for 6 s, starting 1 s before stimulation (olfactory experiments) and for 10 s, beginning 0 s before stimulation. In either case, these signals were pre-amplified 10X using a Syntech Probe run through a 16-bit analog-to-digital interface (IDAC-02, olfactory, or IDAC-4, gustatory) (Syntech), and then analyzed off-line with Autospike software (Syntech). This software allowed the user to more easily interpret how many cells were responding to specific compounds, as determined by their size, shape, and firing frequency of the biphasic action potentials. The net number of spikes within the first 500 ms after stimulus delivery was considered to be a measure of the strength of excitation and was calculated as the mean number of spikes within 500 ms after the termination of stimulus delivery minus the mean number of spikes within 500 ms before stimulus onset. The responses of ORCs were classified as excitatory (Figs. 7A-F, 7H-M, 8A-N, 8P, 8Q) or inhibitory (Figs. 7G, 8O) if there was cessation in spiking activity for more than 500 ms after stimulation. Doseresponse experiments were conducted after the general screening of the odorants was completed and only if the response of an ORC remained constant throughout the experimental session. In order to increase the accuracy of the data analyses, vapor pressure of a particular odorant was taken into consideration when analyzing the sensitivities of selected ORCs.

For testing individual compounds, each odorant was dissolved in odorless mineral oil (light white oil, Sigma, St. Louis, MO) at a dilution of 1:10 (v:v). A 30-µl aliquot was pipetted onto a 1.5-cm2 piece of grade-1 filter paper (Whatman, Kent, ME) and inserted into a disposable 20 ml syringe. Control syringes, loaded with 30 µl of mineral oil, were also prepared. All syringes were capped and allowed to equilibrate, similar to that described above. After each experimental session, the capped odorant syringes were stored at 4°C. Some odorants eliciting the strongest responses were tested in dose-response experiments. For these experiments, dilutions were prepared in decadic steps (v:v) over at least four log units from stock solutions. Stimulus loads ranged from 3 x 10-4 to 3 μl of the test compound (approximately 0.3 μg to 3 mg). Odorants were tested in order of increasing concentration to prevent adaptation of the ORCs. Also included among the odorants, were two key components of the female moth's sex pheromone, E10,E12-hexadecadienal (bombykal) and E11,Z13-pentadecadienal ("C15"), a relatively stable mimic of E10,E12,Z14-hexadecatrienal (see below) (Kaissling et al., 1989). Each compound was dissolved in *n*-hexane and applied

The odor was delivered to the female antenna using a stimulating device (type CS-01, Syntech) (Shields and Hildebrand, 2001a). The tip of the syringe containing the odorant was introduced into a 20 cm long, L-shaped glass tube (1 cm i.d.) whose open end was positioned 5-7 mm from the test antenna (Fig. 3). The end of the tube was flattened to 0.5 cm to correspond with the length of the antenna. The antenna was continuously flushed with a 5 cms-1 stream of charcoal-filtered, humidified air. To deliver a stimulus, 2 ml of odor-laden air was injected at a constant rate over 200 ms from the syringe into the airflow using a motor-driven syringe olfactometer, resulting in increased airflow of 20 cm s-1 (measured by a thermo-anemometer) (Fig. 3). A glass funnel (3.5 cm i.d.) attached to an air evacuation line was positioned near the preparation to draw away any odor-bearing air following stimulus delivery. Odor stimuli were presented randomly to the sensilla to be tested. Blank controls were tested repeatedly, but never elicited a response from the ORCs. An interstimulus interval of at least 60 s, or longer, if necessary for spontaneous activity of the ORC to return

Alternating current (AC) signals were recorded for 6 s, starting 1 s before stimulation (olfactory experiments) and for 10 s, beginning 0 s before stimulation. In either case, these signals were pre-amplified 10X using a Syntech Probe run through a 16-bit analog-to-digital interface (IDAC-02, olfactory, or IDAC-4, gustatory) (Syntech), and then analyzed off-line with Autospike software (Syntech). This software allowed the user to more easily interpret how many cells were responding to specific compounds, as determined by their size, shape, and firing frequency of the biphasic action potentials. The net number of spikes within the first 500 ms after stimulus delivery was considered to be a measure of the strength of excitation and was calculated as the mean number of spikes within 500 ms after the termination of stimulus delivery minus the mean number of spikes within 500 ms before stimulus onset. The responses of ORCs were classified as excitatory (Figs. 7A-F, 7H-M, 8A-N, 8P, 8Q) or inhibitory (Figs. 7G, 8O) if there was cessation in spiking activity for more than 500 ms after stimulation. Doseresponse experiments were conducted after the general screening of the odorants was completed and only if the response of an ORC remained constant throughout the experimental session. In order to increase the accuracy of the data analyses, vapor pressure of a particular odorant was taken into consideration when analyzing the sensitivities of selected ORCs.

directly (without mineral oil) at 1 ngμl-1 and 10 ngμl-1 to the filter paper.

to their initial value, was allowed between stimulations.

**6. Olfactory responses from trichoid type-a sensilla** 

Fig. 7. Representative extracellular olfactory electrophysiological recordings of from three different olfactory receptor cells (ORCs) from female *Manduca sexta* trichoid type-A sensilla showing excitatory (A-F, H-M) and inhibitory (G) responses. The responses in (F) and (G) are from a different ORC than those in (A-E) and (H-M). 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, 2001b.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 137

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

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

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

hostplants or with courting males.

laser-scanning confocal microscope.

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.
