**9.5 Olfactory stimulation**

142 Zoology

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

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

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.,

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).

the responses of identified PNs innervating different glomeruli in the MGC.

25 mM sucrose to balance osmolarity with that of the extracellular fluid

Longmont, Colorado) or Autospike (Syntech, Kirchzarten, Germany).

**9.3 Electrophysiological recordings and data analysis** 

**9.2 Experimental preparation** 

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 from a cartridge at 5 pulses sec-1.

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 each) were also tested.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 145

We applied several criteria to assess the ability of PNs to register each odor pulse of a series of pulses with a discrete burst of action potentials. In most cases, a stimulus pulse was scored as "tracked" if the evoked burst comprised at least five action potentials and was separated from the next burst by a period of inactivity of at least 50 ms, which corresponded to the interstimulus interval. Alternatively, in cases where PNs showed a higher level of spontaneous activity, the instantaneous spike frequency (ISF) was calculated for each spike train. A given stimulus pulse was scored as "tracked" if (a) the ISF showed a two-fold increase in response to the odor pulse and (b) there was a clear decrease in ISF before the next odor pulse (<50% of the maximum ISF observed during the previous pulse). The "tracking index" thus ranged from 1 to 5 and was defined as the number of pulses tracked in response to the 5-pulse stimulus train.

Changes in stimulus intensity can modulate the patterns of glomerular activity and output from the olfactory bulb and AL as has been shown for olfactory systems of vertebrates and invertebrates alike (Anton et al., 1997; Hildebrand and Shepherd, 1997; Christensen and White, 2000; Keller and Vosshall, 2003; Leon and Johnson, 2003; Sachse and Galizia, 2003; Stopfer et al., 2003). Until recently, little was known about how the responses of uniglomerular PNs can be influenced by specific stimulus blends (Wu et al., 1996). Below, we present direct evidence for PNs in the AL of *M. sexta* that the temporal responses of some PNs are optimized for a particular ratio of stimulus compounds in a blend and that altering this ratio dramatically changes the central representation of the blend at the first stage of

When we changed the intensity of the pheromone stimulus blend, we observed pronounced effects on the strength of the responses (IPSPs, EPSPs, number of impulses) in two major types of MGC-PNs: the BAL-selective (*BAL+ PNs*) and C15-selective cells (*C15+ PNs*) (Fig. 12). However, not all of these neurons exhibited classical concentration-dependent responses in the range of concentrations used in this study. A subset of *BAL+ PNs* innervating the T1 glomerulus and *C15+ PNs* innervating the cumulus were extremely sensitive at all concentrations tested (Fig. 12, left panel), while others with similar morphology failed to respond except at the highest stimulus concentration (Fig. 12, right panel). Such functional diversity is not an exclusive property of the male MGC as shown in a study in female *M. sexta*  (Reisenman et al., 2004). Similar diversity exists in female *M. sexta* among PNs that innervate a single glomerulus and respond selectively to one enantiomer of linalool, a common plant volatile. The results present us with evidence from both male and female moths to clearly demonstrate functional heterogeneity with respect to threshold sensitivity and concentration-

The observed diversity in the PN population of a single glomerulus could reflect distinct functional roles for different PNs under diverse environmental conditions, as previously proposed (Christensen et al., 2000). In this scenario, PNs that are little affected by a change in pheromone concentration might signal the presence of the pheromone without regard to its intensity, whereas the PNs with higher response thresholds, recruited into the coding

**9.7.1 MGC-PNs have different dynamic ranges and response thresholds** 

response characteristics among PNs innervating a single glomerulus.

**9.6 Calculating stimulus "tracking" in MGC-PNs** 

Essentially identical results were obtained with both methods.

**9.7 Olfactory processing** 

processing in the brain.

Fig. 11. Laser-scanning confocal micrographs of antennal lobe projection neurons in the moth antennal lobe of male *Manduca sexta*. Left panel: Image of a C15-specialist MGC-PN with arborizations confined to the cumulus. The inset illustrates the organization of the antennal lobe with the macroglomerular complex (MGC) and other glomeruli (G). Right panel: Two specialist MGC-PNs, one neuron, stained with Lucifer Yellow (colored red here), had arborizations confined to the cumulus (C), and the other neuron, stained with biocytin (colored green here) had arborizations confined to the toroid (T1). Areas of apparent overlap between the 2 neurons are shown in yellow and are possible sites of synaptic contact. Bottom panel: Morphological diversity in cumulus neurons. Image of two C15-specialist MGC-PNs with arborizations confined to the cumulus. While the branches of the two neurons apparently overlapped in certain parts of the cumulus (indicated in yellow), other parts were innervated by just one of the two neurons. The green neuron was stained with Lucifer Yellow and the red neuron with biocytin. C – cumulus, T1 – toroid 1, T2 – toroid 2; do, dorsal; la, lateral. Scale bar = 100 µm. Modified from Heinbockel and Hildebrand, 1998 (left panel); Heinbockel et al., 1999 (right and bottom panels).
