**2.2 Antennal morphology**

124 Zoology

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

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

Olfactory stimuli play an important role in the orientation of many animals in their environment. Moths detect odor cues with their main olfactory organs, paired antennae. The antennae detect diverse mixtures of volatiles by means of ORCs residing in various types of sensilla. These sensilla are the crucial interface between the outer world and the central nervous system of the moth. The olfactory systems of both invertebrates and vertebrates share many similarities (see Hildebrand and Shepherd, 1997) and are capable of detecting and discriminating among a large number of odorants that differ in size, shape, and complexity. The olfactory organs of invertebrates (e.g., paired antennae of insects) and vertebrates (e.g., nose in mammals) are adapted to detect a vast array of odorants by means of receptors that are located on ORCs. These ORCs are associated with various types of sensilla in invertebrates (e.g., insects) or the olfactory epithelium lining a portion of the nasal cavity of vertebrates (e.g., mammals). Clyne et al. (1999) and Vosshall et al. (1999) identified a novel family of seven-transmembrane-domain proteins, which are encoded by 100-200 genes and are likely to function as *Drosophila melanogaster* olfactory receptors. An individual ORC in the antenna of *D. melanogaster* is thought to express one or a few of the candidate olfactory receptor genes and therefore, each ORC is functionally distinct (Vosshall, 2001).

Because of the fundamental morphological and physiological similarities between invertebrate and vertebrate olfactory systems and since current evidence indicates that basic olfactory processing is similar across all phyla (Hildebrand and Shepherd, 1997), it is feasible to use insects, such as the sphinx moth *Manduca sexta*, as a model system and thereby gain insights into the neural mechanisms of odor recognition and discrimination common to insects and to other animals (Hildebrand, 1995; 1996; Hildebrand and Shepherd, 1997). Insects are ideal experimental models because they possess readily accessible olfactory sensilla, have relatively simple peripheral and central nervous systems, possess individually identifiable neural structures, and have a relatively small number of sensory cells mediating olfactory mechanisms. In moths, the olfactory system comprises two parallel subsystems. One system processes information about plant-associated volatiles odors and is

(olfactory) stimuli in the environment that control the behavior of these animals.

quantifying the behavior (output).

**2.1 Peripheral olfactory processing** 

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. Ca2+, Cl- , 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

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 127

Insect larvae depend largely on their sense of taste and smell to find food. These larvae possess elaborate sensory organs (i.e., sensilla) located on the antennae and mouthparts that serve to gather olfactory and gustatory information on the chemical composition of the food plant. The feeding response in lepidopterous larvae is controlled by input from gustatory sensilla located on the mouthparts (Schoonhoven and Dethier, 1966; Shields, 1994). Food plant recognition is thought to be primarily mediated by the input from a bilateral pair of styloconic sensilla (Schoonhoven and Dethier, 1966; de Boer et al., 1977; de Boer and Hanson, 1987) located on the mouthparts. Each sensillum houses four GRCs (Figs. 2, 6) that are thought to play a primary role in hostplant discrimination. They have been referred to as the salt- sugar-, inositol-, and deterrent-sensitive cells (Schoonhoven, 1972; Schoonhoven et al., 1992), since they typically respond to salt, sweet, inositol, and bitter compounds, respectively (e.g. Schoonhoven, 1972; Frazier, 1986; Shields and Mitchell, 1995; Bernays et al., 1998; Glendinning et al., 1999). During feeding, the sensilla are in continuous contact with the sap liberated from the plant leaf and are capable of detecting different chemicals (i.e., phytochemicals) present in the plant. This gustatory sensory input is encoded as patterns of nerve impulses by GRCs and this information is then transferred to taste centers in the brain of the insect. Therefore, GRCs form the first layer of the decision-making process that ultimately determines whether food is acceptable or should be rejected. Thus, the insect faces the task to decipher individual tastants in a complex multimolecular mixture

Fig. 2. Scanning electron micrographs showing the (A) whole head of a gypsy moth, *Lymantria dispar*, larva. The arrow denotes the location of the styloconic sensilla. (B) Higher magnification view of the lateral (L) and medial (M) styloconic sensilla located on the galea (G). (C) Higher magnification view of a lateral styloconic sensillum. The sensillum is

comprised of a cone (C) or peg inserted into a style (S) or column. A terminal pore (arrow) is

Insect GRCs transduce the quality and quantity of the complex plant chemistry into a neural code of action potentials. Complex stimuli resulting from e.g. plant saps often evoke spike trains in several receptor cells innervating one or more sensilla. The frequency of action potentials and the temporal distribution of action potentials in a spike train contain information about the stimulus. The axons of GRCs travel to, and converge in, the first relay station, the subesophageal ganglion (SOG), without intermittent synapses. Unraveling the sensory code occurs by analyzing "input-output" relationships (Schoonhoven and van Loon, 2002) and can be achieved by stimulating specific sensilla and quantifying

**2.3 Peripheral gustatory processing** 

and to make appropriate feeding choices.

visible at the apex of the cone.

compartments of condensed synaptic neuropil (glomeruli) in the AL (Hildebrand and Shepherd, 1997). Mounting evidence indicates that glomeruli are discrete anatomical and functional units, each dedicated to collecting and processing olfactory information about a subset of odor molecules (see below). Information about the odor stimulus is conveyed to a particular glomerulus by the axons of ORCs that express a particular olfactory receptor protein (Buck, 1996; Hildebrand and Shepherd, 1997; Mombaerts, 1996).

Fig. 1. Female *Manduca sexta* antenna. (A) Light micrographs showing a female *M. sexta* moth and its antenna. The antenna comprises three segments, two small, basal segments (scape and pedicel) and a long distal segment (flagellum). The antennal flagellum is about 2 cm long and comprises approximately 80 subsegments called annuli (or flagellomeres). (B) Higher magnification view of (A) showing the multitude of annuli. (C) Scanning electron micrograph of a portion of an adult female antennal flagellum showing a higher magnification of three annuli. (D) Higher magnification view of a single annulus. Long, hair-like sensory organs (sensilla) called trichoid sensilla are abundant on the surface of an annulus. (E) Higher magnification view of a single trichoid type-A sensillum showing the cuticular shaft and pores that extend through its entire thickness. The shaft bears circumferential cuticular ridges which form a helical pattern. Odorant molecules diffuse through these pores and interact with the underlying dendrites.
