**9.7.2 Effects of blend ratio on temporal response dynamics in PNs**

In subpopulations of *BAL+ PNs* and *C15+ PNs*, a change in the ratio of BAL and C15 clearly affected pulse tracking of these PNs (Figs. 13, 14).

Fig. 13. Responses and anatomy of a *C15+ PN* from male *Manduca sexta* that gave depolarizing responses to C15 and inhibitory responses to BAL (*C15+ / BAL- PN*). (A) Responses from left to right are to blends containing 1 ng C15 plus increasing amounts of BAL. The ipsilateral antenna received five 50-ms stimulus pulses at 5 pulses sec-1 (stimulus markers are shown beneath the records). This neuron had a low threshold for excitation and gave strong responses to stimuli containing only 0.1 ng of C15. The horizontal lines in the records indicate the membrane potential observed prior to stimulation. Notice that BAL alone hyperpolarized the PN and suppressed spiking activity (*C15+* / *BAL- PN*). (B) Responses of the same PN to different pulse rates of antennal stimulation (BAL, C15, 10 ng each) ranging from 1 to 5 sec-1. At higher frequencies, the PN was unable to repolarize sufficiently to track each of the five stimulus pulses. The tracking index improved dramatically at lower pulse frequencies. (C) The neuron in (A) branched in the cumulus and not in the toroid 1 of the MGC (frontal view). *C*, cumulus; *do*, dorsal; *la*, lateral; *me*, medial; *T1*, toroid; *T2*, toroid 2. (D) More anterior view of the ramifications of the axon in the protocerebrum. *ILP*, inferior lateral protocerebrum. (E) More posterior aspect of protocerebral ramifications of the axon. *LH*, lateral horn. Scale bar = 100 μm. From Heinbockel et al., 2004.

In subpopulations of *BAL+ PNs* and *C15+ PNs*, a change in the ratio of BAL and C15 clearly

Fig. 13. Responses and anatomy of a *C15+ PN* from male *Manduca sexta* that gave depolarizing responses to C15 and inhibitory responses to BAL (*C15+ / BAL- PN*). (A) Responses from left to right are to blends containing 1 ng C15 plus increasing amounts of BAL. The ipsilateral antenna received five 50-ms stimulus pulses at 5 pulses sec-1 (stimulus markers are shown beneath the records). This neuron had a low threshold for excitation and gave strong

responses to stimuli containing only 0.1 ng of C15. The horizontal lines in the records indicate the membrane potential observed prior to stimulation. Notice that BAL alone hyperpolarized the PN and suppressed spiking activity (*C15+* / *BAL- PN*). (B) Responses of the same PN to different pulse rates of antennal stimulation (BAL, C15, 10 ng each) ranging from 1 to 5 sec-1. At higher frequencies, the PN was unable to repolarize sufficiently to track each of the five stimulus pulses. The tracking index improved dramatically at lower pulse frequencies. (C) The neuron in (A) branched in the cumulus and not in the toroid 1 of the MGC (frontal view). *C*, cumulus; *do*, dorsal; *la*, lateral; *me*, medial; *T1*, toroid; *T2*, toroid 2. (D) More anterior view of the ramifications of the axon in the protocerebrum. *ILP*, inferior lateral protocerebrum. (E) More posterior aspect of protocerebral ramifications of the axon. *LH*, lateral horn. Scale bar =

**9.7.2 Effects of blend ratio on temporal response dynamics in PNs** 

affected pulse tracking of these PNs (Figs. 13, 14).

100 μm. From Heinbockel et al., 2004.

Fig. 14. Intracellular recordings and ISF plots from one *BAL+ PN* from male *Manduca sexta* that gave depolarizing responses to BAL and an inhibitory response to C15 (*BAL+* / *C15- PN*). Responses to blanks are shown in (A) and (D). (B), (E) Intracellularly recorded responses and corresponding ISF plots to blends containing 1 ng BAL plus increasing

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 151

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

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;

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

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.

populations of ORCs.

Vickers et al., 2001).

different ambient conditions.

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.

These MGC-PNs integrated convergent excitatory and inhibitory afferent input and better resolved intermittent olfactory signals. This processing function was optimized at particular blend ratios for different PNs. *C15+ / BAL- PNs* and, particularly, *BAL+ / C15- PNs* were best 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 output channels.

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; Christensen et al., 1996).

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 pheromone blend (Heinbockel et al., 2004).
