**3. Olfactory extracellular recording methods**

A powerful tool for studying olfactory ORCs lies in physiologically recording from individual sensilla using a recording technique termed single-unit or single-sensillum recording (Figs. 3, 4). This technique monitors the electrical events elicited by ORCs when stimulated by different odor stimuli. Despite the fact that multiple GRCs are present within a single sensillum, the distinct electrophysiological responses of each ORC can be distinguished using specialized computer-aided software for spike sorting by differing spike amplitudes. Knowledge of the number of ORCs present within a particular sensillum, as visualized with transmission electron microscopy, can greatly contribute to a better interpretation and verification of electrophysiological results.

To record from olfactory ORCs to test the effect of volatile compounds, female *M. sexta* moths reared on artificial diet (17h light: 7h dark; ca., 60% relative humidity), 1-2 days posteclosion were used for these studies (Bell and Joachim, 1976; Sanes and Hildebrand, 1976). Recordings were carried out at ambient temperatures (24-26ºC). To record extracellulary from ORCs, the cut-tip recording technique was used (Van der Pers and Den Otter, 1978; Kaissling, 1995) (Fig. 4B). This technique involved restraining each moth in a plastic tube (5.2 x 1.2 cm i.d.) so that the head protruded from one end (Fig. 3). One of the moth's antennae was stabilized with a minimal amount of low melting point paraffin wax to allow for easier manipulation of the sensilla. Alternatively, double-sided tape can be used. The tip of a trichoid type-A sensillum, positioned on the distal or proximal margins of the dorsal, ventral, and leading surfaces of an annulus, was cut between two sharpened glass knives (2-

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 131

Fig. 4. Extracellular recording technique. (A) After the sensillum is cut, shown here using sharpened forceps, the recording electrode (B) is placed over the tip of the sensillum. The stimulating electrode, containing the odorant, is used to deliver the odorant to the sensillum. Odor stimuli are presented randomly to the sensillum. Action potentials are amplified, recorded, and relayed to a computer for further analyses. Fig. (A) was taken from Kaissling, 1995.

Randomly selected, fifth instar, 12-24 hours post-molt, *Lymantria dispar* larvae, reared on artificial diet (12h light: 12h dark; ca., 60% relative humidity) (Shields et al., 2006) were used for all experiments. The larvae were food deprived at least 12 hours prior to the experiments and were naive to the test compounds. Recordings were carried out at ambient temperatures (24-26ºC). Each larva was transected just behind the head and a blunt-tipped saline-filled glass electrode was inserted into the head with sufficient force to cause the eversion of the lateral and medial styloconic sensilla. This pipette was filled with an electrically conductive solution (typically KCl or NaCl) and served as the ground or indifferent electrode (Figs. 5, 6). The cut end of the head was sealed with a minimal amount of melted bee's wax (Fig. 5A). The stimulating or recording electrode was filled with a test solution and was positioned over the tip of a single styloconic sensillum (Figs. 5A, 6) with the aid of a micromanipulator (Leitz) under visual control on a vibration-free air table (Fig.

**4. Gustatory extracellular recording methods** 

5B). The preparation lasted, on average, one to two hours.

mm diameter, Corning Inc., Horseheads, NY) positioned at 90 degrees with respect to each other (micro knife-beveling device; type EG-03, Syntech, Hilversum, The Netherlands) (Van der Pers and Den Otter, 1978; Kaissling, 1995). This method is typically feasible for sensilla less than 50 μm in length. For longer sensilla, an alternative method can be used, whereby the sensillum is cut by pinching off the tip using sharpened forceps (Fig. 4A), thus preventing the opening and subsequent damage of the ORC dendrites due their depolarization. The flat side of the forceps is coated with a thin film of Vaseline to prevent desiccation of the sensillum (i.e., loss of the sensillum lymph surrounding the dendrites) (Kaissling, 1995).

Recording and ground electrodes (tip diameters: 2.5-3.5 μm and 4-5 μm, respectively) were made from borosilicate glass tubing (1.5 mm o.d., 1.10 mm i.d., Sutter Instrument Co., Novato, CA) on a Flaming-Brown micropipette puller (model P-97, Sutter, Novato, CA). The recording electrode was filled with sensillum-lymph saline solution (Kaissling and Thorson, 1980) and positioned over the tip of a trichoid type-A sensillum with the aid of a micromanipulator (Leitz) and a Wild M3Z Kombistereo microscope (Leica, Germany; total magnification, 640X) (Figs. 3, 4). The ground electrode was filled with hemolymph saline solution (Kaissling and Thorson, 1980) and inserted into the moth's eye or body. Both ground and recording electrodes were held in place by mounting them in electrode holders (1.5 mm i.d., Syntech) containing 0.25 mm silver wire (>99.99 %, Aldrich Chemical Co., Milwaukee, WI) (Fig. 3). Difficulties in cutting the tip of olfactory sensilla can arise if they are very short and small. In this case, a modification of the tip-cutting technique can be used called sidewall recording. In this method, a sharpened tungsten electrode is plunged into the base of the sensillum to establish contact with the underlying olfactory ORCs. In either case, these electrophysiological recording techniques record the contribution of one or all of the ORCs present within a sensillum and preparations can remain stable for several hours. The electrophysiological components of the set-up were placed on a vibration-free air table (Figs. 3).

Fig. 3. Typical olfactory extracellular recording set-up.

mm diameter, Corning Inc., Horseheads, NY) positioned at 90 degrees with respect to each other (micro knife-beveling device; type EG-03, Syntech, Hilversum, The Netherlands) (Van der Pers and Den Otter, 1978; Kaissling, 1995). This method is typically feasible for sensilla less than 50 μm in length. For longer sensilla, an alternative method can be used, whereby the sensillum is cut by pinching off the tip using sharpened forceps (Fig. 4A), thus preventing the opening and subsequent damage of the ORC dendrites due their depolarization. The flat side of the forceps is coated with a thin film of Vaseline to prevent desiccation of the sensillum (i.e., loss of the sensillum lymph surrounding the dendrites)

Recording and ground electrodes (tip diameters: 2.5-3.5 μm and 4-5 μm, respectively) were made from borosilicate glass tubing (1.5 mm o.d., 1.10 mm i.d., Sutter Instrument Co., Novato, CA) on a Flaming-Brown micropipette puller (model P-97, Sutter, Novato, CA). The recording electrode was filled with sensillum-lymph saline solution (Kaissling and Thorson, 1980) and positioned over the tip of a trichoid type-A sensillum with the aid of a micromanipulator (Leitz) and a Wild M3Z Kombistereo microscope (Leica, Germany; total magnification, 640X) (Figs. 3, 4). The ground electrode was filled with hemolymph saline solution (Kaissling and Thorson, 1980) and inserted into the moth's eye or body. Both ground and recording electrodes were held in place by mounting them in electrode holders (1.5 mm i.d., Syntech) containing 0.25 mm silver wire (>99.99 %, Aldrich Chemical Co., Milwaukee, WI) (Fig. 3). Difficulties in cutting the tip of olfactory sensilla can arise if they are very short and small. In this case, a modification of the tip-cutting technique can be used called sidewall recording. In this method, a sharpened tungsten electrode is plunged into the base of the sensillum to establish contact with the underlying olfactory ORCs. In either case, these electrophysiological recording techniques record the contribution of one or all of the ORCs present within a sensillum and preparations can remain stable for several hours. The electrophysiological

components of the set-up were placed on a vibration-free air table (Figs. 3).

Fig. 3. Typical olfactory extracellular recording set-up.

(Kaissling, 1995).

Fig. 4. Extracellular recording technique. (A) After the sensillum is cut, shown here using sharpened forceps, the recording electrode (B) is placed over the tip of the sensillum. The stimulating electrode, containing the odorant, is used to deliver the odorant to the sensillum. Odor stimuli are presented randomly to the sensillum. Action potentials are amplified, recorded, and relayed to a computer for further analyses. Fig. (A) was taken from Kaissling, 1995.
