**9.7.3 Phasis vs. tonic patterning of postsynaptic response**

Both key pheromone components (represented in this study by BAL and C15) must be present to elicit upwind olfaction-modulated flight in a wind tunnel in *M. sexta* (Tumlinson et al., 1989). Individual MGC-PNs gave multiphasic responses (inhibitionexcitation-afterhyperpolarization) (Figs. 12-15), which are typical of PNs in the AL of *M. sexta* (Christensen and Hildebrand, 1987; Christensen et al., 1998). However, *BAL+ PNs*

These MGC-PNs integrated convergent excitatory and inhibitory afferent input and better resolved intermittent olfactory signals. This processing function was optimized at particular

suited to convey the temporal structure of the stimulus to higher brain centers, whereas responses of *BAL+-* and *C15-specialist PNs* often lacked the temporal precision to represent rapid changes in pheromone concentration. These results show that information about individual components and stimulus timing is transmitted from the MGC in different

We also observed blend effects in *BAL+* / *C15+ PNs* and the *C15+* / *BAL+ PNs* (Fig. 15). These blend effects were clearly ratio-dependent. These MGC-PNs are excited by stimulation with either of the two key pheromone components and, therefore, they are considered to be pheromone "generalists" (Christensen and Hildebrand, 1987; 1990;

Principally, the responses of these MGC-PNs to stimulation with either pheromone component were primarily excitatory. However, the cells often received different amounts of excitatory and inhibitory input when stimulated with blends of the two components. Several PNs responded with a strong, long-lasting excitatory response to stimulation with one component and a less-intense and brief response to stimulation with the other component. Stimulation with the blend evoked a "mixed" response of intermediate character depending on the concentrations of the components. The excitatory phase of this blend response was not simply the sum of the excitatory responses elicited by each of the two components alone but the response was typically weaker than expected from adding the two responses. MGC-PNs with temporally distinct blend responses could serve specific functions in olfactory information processing. They could function as the neural substrate in the AL for rapid behavioral changes in response to the appropriate component ratio of a

Both key pheromone components (represented in this study by BAL and C15) must be present to elicit upwind olfaction-modulated flight in a wind tunnel in *M. sexta* (Tumlinson et al., 1989). Individual MGC-PNs gave multiphasic responses (inhibitionexcitation-afterhyperpolarization) (Figs. 12-15), which are typical of PNs in the AL of *M. sexta* (Christensen and Hildebrand, 1987; Christensen et al., 1998). However, *BAL+ PNs*

 *PNs* were best

amounts of C15. Repetitive stimulus pulses were delivered to the ipsilateral antenna at a frequency of 5 pulses sec-1 (stimulus markers are shown beneath the records). Note the modulations of PN firing with each stimulus pulse. C15 at 10 ng led to a hyperpolarization and suppression of firing in the PN (*dashed line*). (C, F) Responses (intracellular records and ISF plots) of the same neuron to blends containing 1 ng C15 plus increasing amounts of BAL. The tracking index of this PN improved at a ratio of 10 ng BAL to 1 ng C15, but was optimized at a 1:1 ratio with 10 ng of each component. Note the distinct periods of inactivity (*arrows*) between spike bursts evoked by consecutive stimulus pulses. The concentrationdependency of this neuron was also indicated by a marked reduction in response latency as

the amount of BAL in the blend was increased. From Heinbockel et al., 2004.

blend ratios for different PNs. *C15+ / BAL- PNs* and, particularly, *BAL+ / C15-*

output channels.

Christensen et al., 1996).

pheromone blend (Heinbockel et al., 2004).

**9.7.3 Phasis vs. tonic patterning of postsynaptic response** 

had larger IPSPs, smaller EPSPs, weaker responses during 200 ms after stimulus onset, and stronger responses over 1000 ms after stimulus onset than did *C15+ PNs*. This suggests that BAL evokes sustained responses and C15 evokes shorter, more phasic responses. Corresponding response patterns were observed among ORCs in the antenna of *M. sexta* (Kaissling et al., 1989) such that responses of BAL-specific ORCs to antennal stimulation with BAL are typically more phasic-tonic whereas the phasic part of the response is more pronounced in C15-specific receptor cells. Different ORCs with selective responses to the same stimulus compound could nevertheless exhibit different response dynamics as has been shown for pheromone-specific ORCs of moths (Almaas et al., 1991) and ORCs in *D. melanogaster* (de Bruyne et al., 2000). It is tempting to speculate that MGC-PNs with phasic olfactory responses could be synaptic targets of phasic ORCs, whereas PNs giving tonic responses could receive input largely from ORCs that exhibit tonic firing patterns. In this scenario, at least some aspects of temporal coding and coding of stimulus concentration and mixture ratio are regulated through the activation of different populations of ORCs.

If MGC-PNs respond to pheromonal stimulation with sustained firing outlasting the stimulus duration they are likely to report the onset of stimulation rather than its termination and could serve as a substrate for more temporally complex spike codes (Heinbockel and Kloppenburg, 1999; Friedrich and Stopfer, 2001; Laurent et al., 2001; Christensen and Hildebrand, 2002). If MGC-PNs respond with a more phasic spike pattern and show better pulse-following, they could encode both the beginning and the end of each stimulus pulse. These MGC-PNs are able to convey information about rapidly changing signals and by doing so transmit information about the physical dynamics of the stimulus to higher olfactory centers in addition to cues about stimulus identity (Heinbockel et al., 1999; Vickers et al., 2001).

Since each AL is innervated by fewer than 50 MGC-PNs (Homberg et al., 1989), each MGC-PN contributes significantly to information flow from the MGC to higher order olfactory centers. Different features of the olfactory stimulus are likely to be encoded by these different outputs arising from a single MGC glomerulus, i.e., functional heterogeneity exists in the population of olfactory output channels from the MGC. In the AL of male *M. sexta*, both component-specific and blend-specific MGC-PNs exist that are affected by changes in concentration and/or blend ratio, but the performance of different PNs is optimized under different ambient conditions.

Advances in intracellular recording, staining, confocal microscopy and data analysis allowed us to characterize the responses of individual MGC-PNs and their ability to encode features of the stimulus, e.g., to follow intermittent olfactory stimuli. PNs that integrated information about the two-component pheromone blend (i.e., they received excitatory input from one component and inhibitory input from the other) were particularly well suited to track a train of stimulus pulses. Stimulus-pulse tracking was furthermore optimized at a synthetic blend ratio that mimics the physiological response to an extract of the female's pheromone gland. Our results show that optimal responsiveness of a PN to repetitive stimulus pulses depends not only on stimulus intensity but also on the relative strength of the two opposing synaptic inputs that are integrated by MGC-PNs.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 153

response to BAL followed by inhibition. Addition of increasing amounts of BAL to the blend resulted in improved stimulus-pulse tracking. Note the distinct periods of inactivity (*arrows*) between spike bursts evoked by consecutive stimulus pulses. (C) In this neuron, stimulation with C15 evoked a brief excitatory response preceded by an IPSP and followed by a strong and prolonged inhibitory response phase. Stimulation with BAL resulted in a mixed response (inhibition / excitation / inhibition) comprising a strong IPSP and a strong

This chapter details both extracellular and intracellular recording methods used with insect preparations in the fields of insect olfaction and/or gustation. Using one or both olfactory recording techniques contributes to a better understanding of (i) how and what olfactory information is processed in the insect brain, (ii) the chemical identification of important plant volatiles for insect-plant interactions, and (iii) which components of the female sex pheromone, as well as plant-associated volatiles, play important roles in male and female moth orientation, respectively. Complementing these recording techniques with the use of fluorescent markers allows axonal projections to be traced to the brain or central neurons to be labeled individually. Gustatory extracellular recording methods can ultimately (i) lead to a clearer understanding of the importance of gustatory cues involved in larval host-plant interactions, (ii) give us a better perception on how taste stimuli code for different behavioral responses, and (iii) provide ideas and strategies for crop protection from insect predation. Overall, either one or both of these methods (i.e., extracellular or intracellular) can contribute to an increased understanding of how and what chemosensory information is processed in the insect brain and shed more light on how nervous systems recognize,

This work was supported by NIH grants 1R15DC007609-01 and 3R15DC007609-01S1 to V.D.S., NIH grant DC-02751, as well as grants from Towson University, the FCSM URG and TU URG. Grants from the Whitehall Foundation, NIH-NIGMS (S06GM08016) and NIH-NINDS (U54NS039407) to T.H. also supported this work. The authors would like to gratefully acknowledge J.G. Hildebrand, T.A. Christensen, T.L. Martin, C.E. Reisenman, R.

Andersen, R.A., Hamilton-Kemp, T.R., Loughrin, J.H., Hughes, C.G., Hildebrand, D.F., &

Anderson, P., Hansson, B.S., & Löfqvist, J. (1995). Plant-odour-specific receptor neurones on

No. 1, (January 1988), pp. 295-299. ISSN 0021-8561

20, No. 3, (September 1995), pp. 189-198

Sutton, T.G. (1988). Green leaf headspace volatiles from *Nicotiana tabacum* lines of different trichome morphology. *Journal of Agricultural and Food Chemistry,* Vol. 36,

the antennae of female and male *Spodoptera littoralis*. *Physiological Entomology,* Vol.

excitatory response phase (*BAL+ / C15+ PN*). From Heinbockel et al., 2004.

analyze, and respond to complex sensory information.

**10. Conclusions** 

**11. Acknowledgments** 

**12. References** 

Bennett, J. Klupt, R. Kuta, and M. Chen.

Fig. 15. Responses of *BAL+* / *C15+ PNs* and *C15+ / BAL+ PNs* from male *Manduca sexta.* These MGC-PNs were depolarized by antennal stimulation with either BAL or C15. (A) This neuron responded with a strong response to C15 and a more sustained response to BAL (*C15+ / BAL+ PNs*). Stimulation with C15 also evoked a distinct IPSP (marked by asterisk) resulting in a mixed response (inhibition / excitation / inhibition). Varying the blend ratio changed the response character to more phasic or more tonic, depending on the load in the stimulus blend. (B) ISF response plots of a *C15+ / BAL+ PN* to various BAL and C15 stimulus loads and ratios revealed a sustained phasic-tonic response to C15 and a brief excitatory

response to BAL followed by inhibition. Addition of increasing amounts of BAL to the blend resulted in improved stimulus-pulse tracking. Note the distinct periods of inactivity (*arrows*) between spike bursts evoked by consecutive stimulus pulses. (C) In this neuron, stimulation with C15 evoked a brief excitatory response preceded by an IPSP and followed by a strong and prolonged inhibitory response phase. Stimulation with BAL resulted in a mixed response (inhibition / excitation / inhibition) comprising a strong IPSP and a strong excitatory response phase (*BAL+ / C15+ PN*). From Heinbockel et al., 2004.
