**2. Insect chemosensory organs**

Insects are ideal models for both olfactory and gustatory studies. They bear numerous sensory organs or sensilla, which allow them to constantly monitor and respond to changes in their internal and external environments so as to maintain themselves under the most

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 125

very similar to the main olfactory pathway in vertebrates. The other system is narrowly specialized to detect and respond to information regarding the sex pheromone emitted by conspecific females. Many researchers have classified ORCs based on their individual response profiles and have used terms such as "specialist" and "generalist" to describe those ORCs. Specialist ORCs have been described as those responding with a high sensitivity and selectivity to a single type of odor molecule, such as a pheromone component or a narrow range of related odorants, whereas generalist ORCs have been classified as those responding with relatively lower sensitivity to a broad range of odorants, such as plant-associated volatiles (Schneider et al., 1964). Interestingly, several researchers have found, that some insect ORCs respond to plant-associated volatiles and exhibit relatively high selectivity and sensitivity for effective stimulus molecules (e.g., Priesner, 1979; Dickens, 1990; Anderson et al., 1995; Heinbockel and Kaissling, 1996; Pophof, 1997;

The antennae of both sexes of *M. sexta* comprise three segments, two small, basal segments (scape and pedicel) and a long distal segment (flagellum) (Fig. 1A, B). In adult *M. sexta*, the antennal flagellum is about 2 cm long, comprises approximately 80 subsegments called annuli (or flagellomeres) (Fig. 1C), and is sexually dimorphic (Sanes and Hildebrand, 1976). Each male and female antenna has approximately 4 x 105 ORCs (Sanes and Hildebrand, 1976) and 3.0 x 105 - 3.4 x 105 ORCs (Oland and Tolbert, 1988), respectively. The antenna of each sex is associated with about 105 sensilla (Sanes and Hildebrand, 1976; Keil, 1989; Lee and Strausfeld, 1990; Shields and Hildebrand, 1999a; 1999b) and each annulus may bear approximately 2100-2200 sensilla (Fig. 1C, D) (Lee and Strausfeld, 1990; Shields and Hildebrand, 1999b). Male flagella possess long and hair-like (trichoid) male-specific sensilla that house ORCs specialized to detect components of the conspecific female's sex pheromone (Sanes and Hildebrand, 1976; Kaissling et al., 1989). Females also possess trichoid sensilla (i.e., trichoid type-A) (Fig. 1C, D). They are much shorter than in males and respond to plant volatiles. In addition, both male and female antennae carry several other types of olfactory sensilla, some of which resemble short pegs, thought to contribute to the detection of plant-associated odorants. In total, there are five types present in males (Sanes and Hildebrand, 1976; Lee and Strausfeld, 1990) and six types present in females (Shields and Hildebrand, 1999a; 1999b). In the case of trichoid type-A sensilla, circumferential cuticular ridges are present which form a helical pattern (Fig. 1E). The cuticular shaft of all olfactory sensilla is pierced by a multitude of pores (Fig. 1E). These pores extend through the entire thickness of the shaft. It is generally believed that olfactory molecules gain entry through these pores and, in order to reach the dendritic receptor sites of an ORC, the small volatile lipid-soluble odor molecules must traverse an aqueous phase, perhaps with the aid of odorant-binding proteins. The binding of these odorant ligands to odor-specific receptors coupled to G-proteins initiates a cascade of intracellular second messengers (e.g. cyclic AMP, IP3) that ultimately activate cyclic nucleotide-gated cation-permeable channels (e.g.

, K+) (reviewed by e.g. Firestein, 1992; Shepherd, 1994). These events result in the

generation of action potentials in temporal patterns (in each ORC axon) and spatial patterns (across the array of ORC axons) that represent features of the stimulus and travel along the ORC axons to the primary olfactory center in the brain (i.e., the antennal lobe, AL, of insects or the olfactory bulb of vertebrates). The axons of antennal ORCs project to and terminate in

Hansson et al., 1999; Shields and Hildebrand, 2001a).

**2.2 Antennal morphology** 

Ca2+, Cl-

favorable conditions for survival. Sensilla are used in the sensory perception for smell, taste, sound, touch, vision, proprioception, and geo-, thermo-, and hygroreception. These specialized cuticular structures vary in size and shape and act as their first level of environmental perception. Receptor cells that innervate these sensilla are designed to detect environmental status and change and transmit information regarding the nature of the change to the central nervous system. In insects, it is specifically taste (gustatory) and smell (olfactory) stimuli in the environment that control the behavior of these animals.

Gustatory and olfactory sensilla are typically innervated by more than one bipolar sensory neuron. These neurons bear dendrites which are wrapped by accessory or sheath cells. Sensory information is transduced by receptor cells into an electrical signal, resulting in the generation of nerve signals comprised of action potentials. The absolute frequency and temporal distribution of action potentials in a spike train contain information about the stimulus. This information is transmitted by axons of sensory neurons to modality-specific brain centers. Unraveling the sensory code can be achieved by stimulating specific sensilla and electrophysiologically quantifying the trains of action potentials (input), as well as quantifying the behavior (output).
