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

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

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.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 141

Female moths release sex pheromones that attract conspecific males over long distances (Kaissling, 1987; Hildebrand, 1996). An outstanding challenge in olfactory neurobiology is to understand how a male moth is able to locate a mate, namely, a conspecific female releasing sex-pheromone. The olfactory brain of a male moth must integrate information about qualitative, quantitative, and spatiotemporal features of an attractive blend of volatile compounds, the sex pheromone, released by a conspecific female (Hildebrand, 1995; 1996; Christensen et al., 1996). Central processing of this information occurs in a sexually dimorphic cluster of olfactory glomeruli, the MGC, in the male moth's AL. Since information about the sex-pheromone is primarily processed in the MGC, it serves as a model for studies of the functional architecture of glomeruli, as well as the physiological relationships between glomeruli in the olfactory system (Hansson and Christensen, 1999; Christensen and White,

Several species of moths use the same chemical compounds in their sex-pheromone blend, but the attractant signal produced by each species is nevertheless unique because each blend has characteristic proportions of the components (Arn et al., 1992; Kaissling, 1996). Chemical studies revealed that in the sphinx moth *M. sexta*, the sex-pheromone blend comprises eight components. Behavioral data shows that two of the eight components are required to evoke olfaction-modulated flight in males (Tumlinson et al., 1989). The antennae of male *M. sexta* are covered by different types of sensilla (see above). The long trichoid sensilla on the male's antennae house highly selective and sensitive ORCs that detect one or the other of the two key pheromone components (Kaissling et al., 1989). Each of these ORC populations sends their axons to a different glomerulus in the MGC (Christensen et al., 1995). Projection neurons (PNs) that arborize in one of these glomeruli, the toroid 1 [T1] (Strausfeld, 1989; Homberg et al., 1995) respond to antennal stimulation with *E*,*Z*-10,12-hexadecadienal (bombykal or BAL, one of the two key components of the female's sex pheromone). PNs that arborize in a neighboring glomerulus (the cumulus) respond selectively to *E*,*E*,*Z*-10,12,14 hexadecatrienal (the second key component) or its more stable mimic, *E*,*Z*-11,13 pentadecadienal (C15; Kaissling et al., 1989; Hansson et al., 1991). An MGC-PN with arborizations in both glomeruli is activated by both BAL and C15 (Hansson et al., 1991;

For a number of moth species it has been demonstrated in behavioral studies in the field and in laboratory wind tunnels that pheromone-modulated upwind flight is regulated in a dosedependent manner (Baker, 1989). Similarly, electrophysiological studies of AL neurons in male *M. sexta* have shown how the responses of MGC-PNs to pheromone components and extracts of the female's pheromone gland are influenced by stimulus intensity (Christensen and Hildebrand, 1990). In a technological advance, neural-ensemble recordings have revealed concentration-dependent patterns of synchronous firing between MGC-PNs responding to the two key pheromone components (Christensen et al., 2000). A growing body of evidence suggests that glomeruli can receive converging excitatory and inhibitory input. Subpopulations of MGC-PNs receive excitatory input driven by BAL and inhibitory input driven by C15 (or vice versa) (Christensen and Hildebrand, 1987; Hansson et al., 1991; Heinbockel et al., 1999; Lei et al., 2002). Input from both excitatory and inhibitory pathways enhances the ability of MGC-PNs to resolve multiple pulses of pheromone such as intermittent odor stimuli found in natural stimulus situations (Christensen and Hildebrand,

2000; Christensen and Hildebrand, 2002; Reisenman et al., 2008; Lei et al., 2010).

**9.1 Olfactory neurobiology** 

Heinbockel et al., 2004).

the responses of the deterrent-sensitive, inositol-sensitive, sucrose-sensitive, and saltsensitive cells (see also Shields and Martin, 2010; 2012). The alkaloids were diluted in 10% ethanol solution, which was added to the electrolyte solution. Ethanol, at this concentration, was found to have no discernible effect on the activity of cell(s) (referred to as KCl-sensitive cell(s)) (see below) responding to the control (i.e., KCl) alone. Each stimulus to be tested was applied to the tip of the styloconic sensillum by way of the stimulating electrode (Figs. 5, 6).

Electrical activity from individual styloconic sensilla was amplified and conditioned (bandpass filter set at 100-1200 Hz) prior to being digitized by a 16-bit analog-to-digital interface (IDAC-4, Syntech). For each electrophysiological recording, we stimulated a sensillum for total of 10 sec. Action potentials generated 50 ms after contact with the sensillum were analyzed off-line using a computer equipped with Autospike (Syntech).A pause of at least 2 minutes was allowed between successive stimulations to minimize any carry-over effects. For dose-response experiments, only those phytochemicals that elicited a robust response were tested. Test stimuli were presented to the sensillum from lowest to highest concentration. To ensure a reproducible response to a particular concentration, 1-2 replications of each were made. The number of action potentials generated 0.05 s after the contact artifact was quantified in 100 ms increments.

We found two cells (i.e., one large spike amplitude and one small spike amplitude) in the medial styloconic sensillum that responded to KCl (Fig. 10A). Another cell in the same sensillum responded robustly to various alkaloids (i.e., deterrent-sensitive cell) (Fig. 10B-F). This cell displayed a typical phasic-tonic response (i.e., the initial high firing frequency of the cell changed rapidly to a more sustained pattern of firing). The ability of the deterrentsensitive cell to respond to more than one alkaloid may be due to either the presence of multiple receptor sites, each with their own structure-function specificity, or a broadly tuned single receptor site (Blaney et al., 1988). These results correlated well with feeding behavioral studies using gypsy moth larvae (Shields et al., 2006), where it was shown that an increase in feeding deterrency (decrease in consumption) occurred with increasing alkaloid concentration.
