**9.1 Olfactory neurobiology**

140 Zoology

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

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

ORCs in the antenna send olfactory information as trains of action potentials to the ipsilateral ALs of the insect brain where the axon terminals of ORCs synapse onto central neurons in neural structures known as olfactory glomeruli. Central neurons in the ALs, such as projection neurons and local interneurons, can be characterized by intracellular recordings with sharp electrodes. These glass electrodes are filled with a physiological solution that mimics the intracellular fluid of the recorded neuron. In addition, the electrodes can contain intracellular markers such as fluorescent dyes. The development of new intracellular markers provides the basis for rapid and complete reconstruction of individual neurons with little or no toxicity to the neuron. For examples, central neurons are injected iontophoretically with Lucifer yellow, neurobiotin, or biocytin. Brains are then dissected and fixed overnight in formaldehyde with sucrose in phosphate buffer. To visualize biocytin-injected neurons, brains are incubated with, e.g., Cy3-conjugated streptavidin. After subsequent histological processing, neurons are further investigated by laser-scanning confocal microscopy. This approach allows the study of both the physiological as well as morphological properties of the recorded neuron in a relatively

contact artifact was quantified in 100 ms increments.

alkaloid concentration.

**9. Intracellular recording methods** 

undisturbed *in vivo* preparation as described below.

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

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; Heinbockel et al., 2004).

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,

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 143

Neurons were injected iontophoretically with either Neurobiotin (Vector Laboratories, Burlingame, California; 3-5% in 2 M KCl with 0.05 M Tris buffer, pH 7.4) or biocytin (Sigma, St. Louis, Missouri; 3-5% in 2M KCl with 0.05 M Tris buffer, pH 7.4). Alternating hyperpolarizing and depolarizing current pulses (30 sec, 1 nA) for about 10 min were used to inject either tracer. Brains were then dissected and fixed overnight in 2.5% formaldehyde with 3% sucrose in 0.1 M sodium phosphate buffer. To visualize injected neurons, brains were incubated with Cy3-conjugated streptavidin (Jackson Immuno Research Laboratories, West Grove, Pennsylvania; diluted 1:100 with 0.2 M sodium phosphate buffer containing 0.3% Triton X-100) for 3 days on a shaker at 4°C. The brains were then dehydrated with increasing concentrations of ethanol and cleared in methyl salicylate. Neurons were further investigated by laser-scanning confocal microscopy (Fig. 11) using a BioRad MRC 600 (Bio-Rad, Cambridge, Massachusetts) with a Nikon Optiphot-2 microscope, a Krypton/Argon (15 mW) laser light source, and appropriate dichromatic filter cubes (Sun et al., 1993). Serial 2-μm optical sections were imaged through whole mounts and saved as series of images on

We used synthetic pheromone compounds kindly provided by Dr. J.H. Tumlinson (USDA, Gainesville, FL). The following olfactory stimuli were applied: (1) *E*,*Z*-10,12-hexadecadienal (bombykal or BAL); (2) *E*,*Z*-11,13-pentadecadienal (C15), a synthetic mimic of the second pheromone component, *E,E,Z*-10,12,14-hexadecatrienal, and hereinafter referred to as the second component; and (3) mixtures of BAL and C15 with various blend ratios. These compounds were dissolved in *n*-hexane and applied to a piece of filter paper (1 x 2 cm), which was inserted into a glass cartridge (acid-cleaned glass syringe barrel), as described previously (Christensen and Hildebrand, 1987). The stimulus load on the filter paper was reported as the number of ng of compound applied in hexane solution. A pulse of charcoalfiltered, humidified air (1,000 ml/min) moving through the cartridge carried the stimulus, roughly proportional to the loading on the filter paper, to the proximal half of the antenna ipsilateral to the impaled AL. The antenna typically was stimulated with five 50-ms pulses

The two key pheromone components used for sexual communication in *M. sexta* are found in a 2:1 ratio in solvent rinses of intact pheromone glands of calling virgin females (Tumlinson et al., 1989). Because the mimic (C15) of the second key component is less potent than the natural component itself, a 1:1 BAL-to-C15 ratio was used to elicit physiological responses comparable to those recorded in response to the pheromone-gland extract, as found in earlier electrophysiological studies (Christensen and Hildebrand, 1987; Kaissling et al., 1989). In our experiments, different blend ratios above and below 1:1 were tested in sequence at intervals of 1 min. For one series of blend ratios, the BAL stimulus load was held constant (1 ng) while the C15 stimulus load was varied (0.01, 0.1, 1.0, and 10 ng). For the second series of blend ratios, the C15 stimulus load was held constant while the BAL stimulus load was varied in a similar fashion. In addition to these stimulus series, an elevated stimulus level (10 ng) of either BAL or C15 and a blend of BAL and C15 (10 ng of

**9.4 Intracellular staining and confocal microscopy** 

disks.

**9.5 Olfactory stimulation** 

from a cartridge at 5 pulses sec-1.

each) were also tested.

1997; Christensen et al., 1998; Heinbockel et al., 1999). Here, we describe how this processing function is optimized at particular stimulus concentrations or ratios in different PNs (Heinbockel et al., 2004). By using intracellular recording and staining methods, we examined the effect of changing these quantitative attributes of the pheromonal stimulus on the responses of identified PNs innervating different glomeruli in the MGC.

## **9.2 Experimental preparation**

Male *M. sexta* were reared on an artificial diet (modified from that of Bell and Joachim, 1976) at 25°C and 50-60% relative humidity under a long-day photoperiod regimen (17h light : 7h dark) as described previously (Sanes and Hildebrand, 1976; Prescott et al., 1977). Adult moths (1-3 days post-eclosion) were immobilized and prepared by standard methods (Christensen and Hildebrand, 1987). With the antennae intact, the head was separated from the rest of the body and pinned in a Sylgard-coated (Dow Corning, Midland, Michigan) recording chamber (volume <0.5 ml) (Heinbockel and Hildebrand, 1998; Heinbockel et al., 1998). Isolation of the head from the rest of the thorax had no detectable adverse effect on neural responses (Christensen and Hildebrand, 1987). Part of the AL was then desheathed with fine forceps to facilitate insertion of the recording electrode. The brain was superfused constantly with physiological saline solution (modified from that of Pichon et al., 1972; ca. 2 ml/min) containing 150 mM NaCl, 3 mM KCl, 3 mM CaCl2, 10 mM TES buffer (pH 6.9), and 25 mM sucrose to balance osmolarity with that of the extracellular fluid

## **9.3 Electrophysiological recordings and data analysis**

Sharp glass microelectrodes for intracellular recording were produced from borosilicate tubing (o.d. 1.0 mm, i.d. 0.5 mm, World Precision Instruments, Sarasota, Florida) with a Flaming-Brown Puller (P-2000, Sutter Instrument Co., Novato, California). The tip of each electrode was filled with a solution of an intracellular stain (see below), and the shaft was filled with filtered (0.2 μm pore size) 2.5 M KCl. Electrode resistance ranged from 60-100 MΩ, measured in the tissue. Movements of microelectrodes were controlled with a Burleigh Inchworm (Model 6000/ULN; Burleigh Instruments Inc., Fishers, New York) attached to a Leitz micromanipulator (Leitz, Wetzlar, Germany). Recordings were made from neurites in the synaptic neuropil of the MGC. Because the site of the electrode impalement in a neuron can affect the amplitude of postsynaptic potentials, impalements targeted the same area of neuropil in all preparations. Typically, one neuron was recorded per animal, except when a second recorded neuron had different pheromone response patterns (excited by BAL vs. excited by C15). Intracellular recording and current injection were carried out in bridge mode with an Axoclamp-2A amplifier (Axon Instruments – Molecular Devices, Sunnyvale, California), and data were initially stored on magnetic tape (Hewlett Packard Instrumentation Tape Recorder 3968A, Palo Alto, California) and subsequently transferred to a computer and analyzed with Experimenter's Workbench (Datawave Technologies Co., Longmont, Colorado) or Autospike (Syntech, Kirchzarten, Germany).

The data presented below were analyzed statistically for differences using one-way analysis of variance (Kruskal-Wallis One-Way Analysis of Variance by Ranks, ANOVA) (Zar, 1984). A multiple-comparison procedure was used to isolate groups from each other (All Pairwise Multiple-Comparison Procedures, Dunn's Method, or Student-Newman-Keuls Method; P<0.05) (Sigma Stat, Jandel Scientific Software, Richmond, California, Version 1.0, Statistical Software).
