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Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, Arizona
Submitted 19 December 2007; accepted in final form 27 February 2008
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ABSTRACT |
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INTRODUCTION |
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; Kupfermann 1979). The efficacy of a neural circuit therefore can be modified to produce subtle adjustments of response features by employment of different modulators or receptor subtypes. This allows for reversible adjustments of neural circuits enabling a nervous system to adapt to changes in the external and internal environments. 5HT acts as a neuromodulator in many neural circuits including the moth antennal lobes (AL) (Kloppenburg and Mercer 2008).
Each AL (the primary olfactory processing center in the insect brain) of the hawkmoth Manduca sexta is innervated by a single 5HT-immunoreactive (5HT-ir) neuron (Kent et al. 1987), here referred to as the contralaterally projecting, serotonin-immunoreactive deutocerebral (CSD) neuron which is morphologically unique and widely distributed phylogenetically (Dacks et al. 2006). The CSD neuron is activated by wind stimulation to the antennae (Hill et al. 2002) and innervates all of the glomeruli with both synaptic and nonsynaptic release sites (Sun et al. 1993). This neuron receives synaptic input within the AL from local interneurons (LNs) and projection neurons (PNs) (Sun et al. 1993) and may receive input from both lateral protocerebra (Hill et al. 2002). The levels of 5HT in the ALs of Manduca vary throughout the day, peaking when moths are most active (Kloppenburg et al. 1999); this suggests that there is a circadian rhythm to the influence of the CSD neuron. 5HT increases the excitability of both LNs and PNs by increasing their input resistance via reduction of two K+ channel conductances (Kloppenburg et al. 1999; Mercer et al. 1995, 1996), resulting in increased depolarization and number of action potentials elicited by stimulation in vivo (Kloppenburg and Hildebrand 1995; Kloppenburg et al. 1999) and in vitro (Mercer et al. 1996). Furthermore in an imaging study, Hill et al. (2003) found that 5HT enhanced responses of the ALs in the silkmoth Bombyx mori evoked by sex pheromone. The 5HT-induced increase in excitability of individual AL neurons has led to the hypothesis that 5HT increases the sensitivity and responsiveness of the AL neurons in Manduca (Kloppenburg et al. 1999).
Most studies to date have focused on the effects of 5HT on individual neurons and employed limited stimulus sets (either antennal nerve shock or a single concentration of sex-pheromone blend). Although those investigations have yielded abundant information about the effects of 5HT on the activity of individual neurons, an understanding of the global modulatory effects of 5HT is lacking. In this study, the effects of 5HT on the dose responses and sensitivity of the AL were examined using multichannel extracellular electrodes to record the responses of AL neural ensembles to olfactory stimuli that varied in concentration and chemical structure. In addition, the effects of 5HT on the efficacy and resolution of the odor-evoked global patterns of activation within the AL were tested.
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METHODS |
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M. sexta were raised and maintained in the rearing facility of the Arizona Research Laboratories Division of Neurobiology as previously described (Christensen and Hildebrand 1987). Multichannel recordings were performed as described in Lei et al. (2004). Briefly, moths were inserted into a plastic tube and their heads fixed in place with dental wax. The cuticle and muscles overlying the AL were removed as were the tracheae and perineural sheath surrounding the AL. Brains were perfused with physiological saline (Christensen and Hildebrand 1987), and 16-channel silicon microelectrode arrays (generously provided by the Center for Neural Communication Technology at the University of Michigan, http://www.engin.umich.edu/facility/cnct/backind.html) were inserted in parallel with the antennal nerve into the exposed AL.
Two separate experiments were performed using different sets of olfactory stimuli on different groups of moths. The first experiment examined the effect of 5HT on dose-response relationships in the AL. Two floral blends, hibiscus oil (Select Oils, Tulsa, OK) and ylang-ylang oil (Now Foods, Bloomingdale, IL), were applied at four serial dilutions in mineral oil, from 10–5 to 10–2 (for stimulus design protocol, see Fig. 1 A). Floral blends were chosen to increase the likelihood that recorded units would be activated by the odor stimulus.
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; Raguso et al. 2003), were used at a single concentration of 5 µg/µl mineral oil (for stimulus protocol design, see Fig. 1B). These odorants represented three different odor classes; monoterpenoids (LIN and GER), aromatics (MES and PAA), and aliphatic aldehydes (ZHP). Odors belonging to similar and dissimilar odor classes were used because the spatiotemporal responses of the AL of Manduca are more similar for odorants from the same odor class compared with odorants from different odor classes (Hansson et al. 2003; Lei et al. 2004).
For both experimental designs, a total of 10 µl of odor compound(s) was applied to a piece of filter paper in a 5-ml syringe. Odor stimuli were delivered by injecting air from the syringe into an air flow that was directed onto the antenna ipsilateral to the AL in which the probes were inserted. Each stimulus consisted of 10 200-ms pulses of odor with a 5-s interpulse interval. To remain consistent with previous studies, 5HT was applied at a concentration of 10–4 M (Kloppenburg and Hildebrand 1995; Kloppenburg et al. 1999; Mercer et al. 1995, 1996) to examine network level consequences of observations made in previous studies on 5HT in the AL of Manduca. Thus the entire experiment consisted of presenting all the odors in a stimulus set (control phase), a 20-min bath application of 10–4 M 5HT followed by a repeated presentation of the odor set (5HT phase), and finally a 20-min washout phase followed by a third presentation of the odor set (washout phase; Fig. 1, A and B). Odor cartridges loaded were used only once per experiment.
Data analysis
Ensemble activity from the 16 recording sites was acquired simultaneously using a Pentusa amplifier (Tucker-Davis Technologies, Alachua, FL), and spike data were extracted from the recorded signals and digitized at 25 kHz using the Tucker-Davis Technologies data-acquisition software. Threshold and gain settings were adjusted for each channel, and spikes were captured in the tetrode recording configuration: any waveform that passed threshold on one channel triggered the capture of waveforms recorded on the other three channels in a given shank. Spikes were sorted by a clustering algorithm based on a combination of integrated waveform information extracted from principal-components analysis (PCA) and specific waveform parameters for each individual channel within a tetrode such as peak-valley ratio, linear and nonlinear energy, and half-peak spike width (Off-Line Sorter; Plexon, Dallas, TX). Only those units that were statistically separable (multivariate ANOVA; P < 0.05) in three-dimensional space defined by a combination of any three parameters were used for further analysis. Spikes were assigned timestamps to create raster plots and calculate perievent histograms in Neuroexplorer (Nex Technologies, Winston-Salem, NC). The odor-evoked responses of 135 units were obtained from 21 male animals (10 moths for experiment 1 and 11 moths for experiment 2). Although there are 
1,200 neurons in the AL of Manduca (Homberg et al. 1988), we feel that a data set of 135 units provides an adequate sampling of the activity within the AL.
Response metrics
The response of a unit was defined as "excitatory" if the firing rate during the first second after odor onset exceeded 1.96 SD (corresponding to 
= 0.05) above the average background firing rate (measured during the 500 ms before odor onset) for that unit during the specific treatment (i.e., the control, 5HT, or washout phase). The average firing rate over time across the odor pulses was calculated as the raw probability of a spike occurring within a 10-ms time bin. For presentation purposes only, responses were depicted as firing rate (Hz) convolved with a 3 bin Gaussian curve. Response magnitude or "response integral" was defined as the area of the response curve above threshold for the first second after odor onset. In addition, the duration of the response and the maximum firing rate above threshold were calculated. For these measures, the values were normalized to the maximal evoked response for a given unit across all odors. After normalization, comparisons were made between units. To determine if 5HT affects odor evoked responses to the same relative degree at each odor concentration, the values of responses were normalized to the highest response magnitude for each concentration of an odor. This was performed for the analysis depicted in Fig. 3C. The latency to response was calculated as the time between odor stimulus onset and the first point in time during which the firing rate of a given unit remains above the 1.96 SD threshold described in the preceding text. To determine if 5HT affected the regularity of spiking within odor-evoked responses, we calculated the coefficient of variation (CV) for each odor-evoked response for each odor pulse and compared the average CV for each unit before, during, and after 5HT treatment.
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) generated by Neuroexplorer (Nex Technologies). Cross-correlation is calculated as the raw average correlation of spike times during stimulation between unit pairs. Because correlation is highly sensitive to changes in the firing rate of individual neurons, the cross-covariance of the spike times between unit pairs was also calculated as a measure of synchrony that is insensitive to firing rate (Aertsen et al. 1989). First, a "shift predictor" was generated for each pair of units by calculating the average correlation for all combinations of time-shifted odor pulses (for example, between the spike train generated by unit A for the 1st odor pulse and unit B for the 2nd odor pulse). For each bin, cross-covariance was calculated by subtracting the shift predictor from the raw correlation and dividing by the SD of the shift predictor. A threshold of 1.96 SD of the mean spontaneous (500 ms before stimulus onset) cross-correlation and -covariance was established for every odor that stimulated both units of a given pair, and the sum of these measures in a 500-ms period after stimulus onset was used to quantify stimulus-driven synchrony.
PCA was used to measure the similarity of ensemble responses to different stimuli to determine if 5HT could affect odor-evoked responses at an ensemble level. Many researchers have used PCA to examine the similarity of AL ensemble responses to different odors or concentrations over time with the use of "response trajectories" in a multidimensional principal component (PC) space based on the covariance between units responses (Bhandawat et al. 2007; Daly et al. 2004; Stopfer et al. 2003). Similarity between response trajectories is calculated as the Euclidean distance between different trajectories over time. The distances between ensemble response trajectories to different odors (Daly et al. 2004) or to different odorant concentrations (Stopfer et al. 2003) is used as a measure of the similarity between ensemble responses to different stimuli with greater distance indicating less similar responses. For instance, Fig. 1C depicts the average response trajectories across all of the ensembles in experiment 2 for GER, LIN, and MES. Furthermore, Daly et al. (2004) demonstrated that measures of similarity obtained by PCA predicted the ability of Manduca moths to discriminate odors in a learning task (Daly et al. 2001). This analysis was performed to obtain measures of distance between the response trajectories for each ensemble to all odor stimuli tested before, during, and after treatment with 5HT.
The PCA and the calculations of trajectory distance were performed in Matlab (The MathWorks, Natick, MA) for each odor comparison in a manner similar to Stopfer et al. (2003) except that the PCA was calculated for every odor comparison (recorded from a given ensemble) and the distances were averaged across preparations. Thus all Euclidean distance values in the Results represent averages across preparations. For demonstrative purposes, the response trajectories depicted in Fig. 1C were calculated using an ensemble in which all of the recorded units across all of the ensembles were combined into a single large ensemble. Only those PCs with eigenvalues of 
1 were used. To obtain single measures of distance, a threshold of 1.96 SD of the spontaneous (500 ms before stimulus onset) distance between two response trajectories was established for every odor comparison. The sum of the distance values above this threshold during the 500 ms after stimulus onset was used as a measure of response similarity for all odor comparisons. These values were then normalized to the highest distance between response trajectories for that ensemble. For comparisons using different odors at single concentrations, LIN with GER and PAA with MES were considered to be comparisons of odors from the same odor class and all other comparisons were considered to be comparisons of odors from different odor classes (see preceding text). All ANOVAs (both 1-way and repeated-measures ANOVAs) performed for all of the comparisons in this study used a Tukey honestly significantly different (HSD) post hoc test and were performed using Statistica (StatSoft, Tulsa, OK).
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RESULTS |
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There were several general effects of 5HT on the responses of AL units observed across both experiments performed in this study. To characterize the general effects of 5HT in the AL, the effects of 5HT on individual responses and pair-wise co-activity of units within an ensemble were examined in both experiments (see METHODS). Serotonin caused a 14.5 ± 1.8% increase in the maximum evoked firing rate (Fig. 2, A and B) and a 16.8 ± 2.1% increase in the burst duration (A and C) of AL units. For those units that generated a phasotonic response, only the tonic phase appeared to increase in duration. These effects contributed to an overall increase of 24.3 ± 2.3% in the response integral across all the AL units recorded (Fig. 2D). The responses of only a subset (52%, n = 70 of 135 units) of the AL units recorded were affected by 5HT, and Fig. 2, E and F, depicts two units from the same ensemble, one unaffected (E) and one affected by 5HT (F). There were no effects of 5HT on the response latency (P = 0.53, 1-way ANOVA, Tukey HSD post hoc test) nor were there any obvious effects of 5HT on the variability of the first spike elicited in the odor-evoked responses. There were also no effects of 5HT on the regularity of spike timing within individual responses as measured by comparing the CV of spike times within spike trains following each odor pulse before, during, and after 5HT treatment (P = 0.38, 1-way ANOVA, Tukey HSD post hoc test).
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). There was a 26.2 ± 4.6% increase in the amount of odor-evoked cross-covariance between pairs of units (Fig. 2H), indicating that the increase in firing rate alone could not account for the increase in cross-correlation. However, 5HT did not affect the amount of spontaneous correlation between pairs of units (data not shown). 5HT significantly increased the background firing rate in only 4.7% (n = 6 of 135 units) of the units recorded but on average had no effect on the background firing rate of AL units (P = 0.893, 1-way ANOVA, Tukey HSD post hoc test). There was, however, a significant decrease in the background firing rate over time from the control to the washout treatment (P = 0.0215, 1-way ANOVA, Tukey HSD post hoc test), although there was no effect on the evoked firing rate from the control to the washout treatment (P = 0.341, 1-way ANOVA, Tukey HSD post hoc test). In some instances, 5HT caused a stimulus dependent decrease in the response magnitudes of some units (5.9%, n = 8 of 135 units). For these neurons, there were no obvious commonalities in terms of evoked firing rate or slow temporal properties (i.e., phasic vs. tonic responses) nor did these units respond to similar sets of odors. Figure 3A depicts a reduction in the response magnitude of an AL unit to ZHP during 5HT application, yet the response of the same unit to LIN is enhanced (Fig. 3B) during 5HT application. There was an obvious period of spike suppression following the initial period of excitation of this unit in response to ZHP during the 5HT treatment that was not present during the control or wash phases (Fig. 3A) nor was it apparent for responses of this unit to LIN (Fig. 3B). In addition, there was an obvious increase in the spontaneous firing rate of this unit (Fig. 3A). The 5HT-induced reduction in response magnitude also appeared to be concentration dependent. Figure 3C depicts the response of a single unit to increasing concentrations of hibiscus oil. As concentration increased, the response magnitude of this unit decreased. However, during 5HT application, the response magnitude of the unit decreased to a greater extent and was abolished at odorant concentrations that elicited responses during the control and washout phase. Unlike the neuron depicted in Fig. 3, A and B, there were no effects of 5HT on the spontaneous firing rate of the unit depicted in Fig. 3C. That these reductions in response magnitude were dependent on odorant identity and concentration suggests that this phenomenon was not due to an overall decrease in sensitivity or responsiveness of individual AL units but rather that 5HT altered the influence of other elements of the network (such as LNs) on the response of the recorded unit.
In addition to the odor- and concentration-dependent 5HT-induced decreases in responses, 5HT caused some units to respond to concentrations of odors that were previously subthreshold. Although the use of discrete odor concentrations limited the likelihood of observing a shift in the activation threshold of an AL unit (see DISCUSSION), there were four AL units (of 49 units that responded to the odors tested in this study) that responded during 5HT treatment to an odor presented at a concentration that was subthreshold during the control and washout phases (Fig. 3D). This suggests that 5HT likely increases the sensitivity of individual AL neurons.
Experiment 1: the effect of 5HT on responses to odors varying in concentration
The effects of 5HT on responses to olfactory stimuli over a range of intensities were tested to determine how 5HT affected the sensitivity and gain of individual AL neurons. The responses of AL units to hibiscus and ylang-ylang oil at different concentrations spanning four log steps were recorded before, during, and after the ALs were superfused with saline solution containing 5HT at 10–4M (for experimental design, see Fig. 1A). As reported in the preceding text, 5HT increased the responses of a subset of AL units at each effective odor concentration, an effect that could be reversed during the washout phase (Fig. 4 A). When the response magnitudes for every stimulus tested for all of the responsive units were calculated (regardless of whether a response was above threshold), the effect of 5HT on the overall level of activation of all of the AL neurons was significant only at the highest concentration (Fig. 4B). The slope of the dose response curve for the AL units was significantly steeper during 5HT treatment compared with the control and washout phases [ANCOVA: F(2,586) = 4.28, P < 0.05], indicating that 5HT increased the gain of the average dose-response curve of the AL units. When only the suprathreshold responses were normalized to the maximal response of an individual unit at each odor concentration, 5HT significantly increased the responses by the same relative proportion for each concentration (except for 10–3 dilution, P = 0.072; Fig. 4C). These results indicated that while 5HT increased the responses of individual units at each odor concentration that evoked a response, the overall enhancing effect of 5HT was greatest at the highest concentration because of an increase in global AL activation with increased odor concentration.
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). 5HT significantly enhanced this effect at each concentration disparity tested (Fig. 4D), suggesting that 5HT may indeed increase the resolution of odor concentration. Experiment 2: the effect of 5HT on responses to odors varying in chemical structure
The effects of 5HT on responses to olfactory stimuli differing in chemical structure were tested to determine how 5HT affected the similarity of responses of AL ensembles to odorants from the same or different odor classes. The responses of AL units to GER, LIN, MES, PAA, and ZHP at 5 µg/µl were recorded while the ALs of moths were superfused with saline solution, treated with 5HT at 10–4 M, and then rinsed again with fresh saline (for experimental design, see Fig. 1B). The time courses of individual unit responses often differed for different odorants. As reported previously for this species (Hansson et al. 2003; Lei et al. 2004), odorants of the same odor class, such as GER and LIN (which are both monoterpenoids), elicited similar response patterns (Figs. 1C and 5, A and B) while odorants from different odor classes, such as LIN (a monoterpenoid) and MES (an aromatic compound) elicited dissimilar response patterns (Figs. 1C and 5, B and C). While 5HT enhanced the responses of individual AL units by increasing firing rate and response duration, 5HT did not drastically alter general response patterns (Fig. 5, A–C) with the exception of the 5.9% of units depicted in Fig. 3. When the effects of 5HT on response similarity for odors from different odor classes were examined using PCA, 5HT significantly enhanced the differences between AL ensemble responses to odorants from different odor classes but not for comparisons of responses to odorants from the same class (Fig. 5D).
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DISCUSSION |
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. The heterogeneity of the effects of 5HT suggests either that only a subset of AL neurons express 5HT receptors or that for a subset of AL neurons, there is a balance of enhanced excitability and enhanced suppression from the local inhibitory network. Previous studies on the effects of 5HT on spontaneous AL activity reported a depolarization in the resting membrane potential of some AL neurons (Kloppenburg and Hildebrand 1995; Mercer et al. 1995, 1996), which likely resulted from a 5HT-induced shift in the inactivation voltage of an IA-type K+-channel (Kloppenburg et al. 1999). Although the resting membrane potential could not be measured in this study, the background firing rate of a few units increased significantly with the application of 5HT. However, on average, 5HT did not affect background firing rate, suggesting that 5HT increases excitatory responses relative to spontaneous activity. In addition to increasing the response magnitude, 5HT significantly increased the odor-evoked cross-correlation and (to a lesser degree) cross-covariance between units in an active ensemble. The significant increase in cross-covariance suggests that the 5HT-induced increase in firing rate alone cannot explain the increase in cross-correlation. One possible mechanism underlying this effect could be an enhancement of the influence of LNs causing increased coordination of precise spike timing across AL units as observed for the pheromone system of Manduca (Lei et al. 2002). Regardless of the underlying mechanism, the 5HT-induced increase in the correlated activity of AL units could result in an increase in the probability of activation of downstream neurons in the ipsilateral mushroom body or lateral protocerebrum, potentially increasing behavioral sensitivity without relying on a shift in the response threshold of individual AL units. By enhancing the correlated firing of two neurons, 5HT might increase the dynamic range of coding of a stimulus without relying on an increase in firing rate.
Although the majority of units affected by 5HT demonstrated an increase in response magnitude, some units exhibited an odorant identity and concentration-dependent decrease in response magnitude (Fig. 3). The stimulus-dependent nature of this effect suggests that, in these instances, 5HT did not decrease the overall responsiveness or sensitivity of these specific units but rather affected the lateral interactions mediated by the LNs activated by a given stimulus. Although these effects of 5HT were not commonly observed (5.9% of units exhibited a stimulus-dependent decrease in response), it occurred commonly enough to suggest that 5HT does influence the local network and therefore the lateral interactions within the AL. In addition, 5HT also shifted the response threshold of a few units to lower odor concentrations, suggesting that 5HT may also increase the sensitivity of some AL units. However, the proportion of units with lowered activation thresholds was likely underestimated due to the discrete nature of the stimuli tested. To observe a shift in the activation threshold, odor concentrations between the thresholds of activation for control conditions and 5HT exposure must be tested. If 5HT was not to shift the threshold to a previously subthreshold concentration, then there would be no apparent shift in the activation threshold. Although the resolution of the present study is limited, it is reasonable to suggest that 5HT offsets the dose-response function of AL neurons, potentially leading to increased sensitivity. Kloppenburg et al. (1999) hypothesized that the increased input resistance induced by 5HT would result in less input required from ORCs to elicit a response from AL neurons and thus in a shift in the threshold of activation. Furthermore an increase in the membrane resistance would also produce an increased time and space constant, therefore providing greater opportunity for input from multiple ORCs to temporally sum and initiate a response.
Use of graded odor stimuli revealed that 5HT modulated the gain of AL unit response functions by increasing the slope of the dose-response curve of the AL units. Application of 5HT led to similar proportionate increases in response magnitude at all odor concentrations tested. This resulted in a greater enhancement of absolute response magnitude at higher concentrations. Previous studies have demonstrated that higher odor concentrations recruit more AL neurons and evoke stronger responses than lower odor concentrations (Carlsson and Hansson 2003; Ng et al. 2002; Sachse and Galizia 2003; Wang et al. 2003; although see Stopfer et al. 2003). This would provide a greater substrate for 5HT to enhance and result in a steeper slope of the dose-response function. It is therefore possible that 5HT increases the resolution of the AL for coding odor intensity by increasing the range of response magnitudes over which the AL can respond to given range of odor concentration. To further examine if 5HT could enhance the resolution of concentration coding by AL ensembles, PCA was employed to compare the effects of 5HT on the similarity of ensemble responses to different concentrations of the same odorant. Similarity between response trajectories in PC space correlate highly with behavioral measures of discrimination, such that large distances between odor-evoked ensemble response trajectories are correlated with a high probability that an animal can discriminate two odors (Daly et al. 2001, 2004). In the ALs of locusts (Stopfer et al. 2003) and honeybee (Sachse and Galizia 2003), ensemble responses become increasingly different with increasing concentration disparity for presentations of a single odor. 5HT significantly enhanced distances at each concentration disparity and increased the gain of the dose response functions of the individual AL units, thereby providing a greater range of responses for odor concentration. These findings suggest that 5HT enhances the resolution with which the AL encodes odor concentration, although behavioral assays will be required to test this hypothesis.
PCA was used also to examine the effects of 5HT on AL ensemble responses to different odorants. Previously Daly et al. (2001) found that the ability of adult Manduca to discriminate odorants increased with dissimilarity in chemical structure and that the response trajectories of AL ensembles were most dissimilar for structurally dissimilar odorants (Daly et al. 2004). In addition, odorants from the same odor class evoked similar spatiotemporal responses in the ALs of Manduca compared with odorants from different odor classes (Hansson et al. 2003; Lei et al. 2004). Similarly in the present study, comparisons of response trajectories of AL ensembles for pairs of odorants from different odor classes showed greater distances in PC space. In fact, 5HT only significantly increased the distances between the response trajectories for odorants from different odor classes but not for odorants from the same odor class. Although 5HT increased the magnitude of the responses of individual units, it rarely drastically changed the pattern of the response (with the exception of the 5.9% of units that exhibited a stimulus dependent decrease in response magnitude). The 5HT effect of increasing the overall magnitude of the response patterns of AL units likely does little to enhance the differences between responses to odorants from the same odor class. On the other hand, this 5HT effect of increasing response magnitude enhances the differences already present for responses to odorants from different odor classes, which could have resulted in the observed increase in distance between the AL response trajectories for dissimilar odors.
Neural circuits must operate at an appropriate level within a range of functionality and resolution corresponding to the current physiological state. In this manner, 5HT adjusts the sensitivity and responsiveness of the AL of Manduca depending on the arousal state by modulating the excitability of individual neurons. The level of 5HT in the ALs of Manduca peaks during the time of day when moths rely most heavily on their olfactory systems (Kloppenburg et al. 1999). By coordinating the sensitivity and responsiveness of the AL, 5HT likely enhances the ability of moths to detect and discriminate olfactory cues when stimuli such as female sex pheromone and host plant volatiles are present. Injection of 5HT into male cabbage looper moths results in broadening of the time span of behavioral responsiveness to pheromone (Linn and Roelofs 1986) and 5HT offsets the behavioral response of male B. mori to sex pheromone (Gatellier et al. 2004). These studies suggest that the increase in excitability of individual AL neurons could translate into increased behavioral responsiveness and sensitivity.
There are similarities between the effects of 5HT on olfactory processing in the AL of Manduca and in the rat olfactory bulb. 5HT enhances the responses of juxtaglomerular and mitral cells in the olfactory bulb of rats (Hardy et al. 2005) and plays a facilitative role in olfactory learning in rat pups (Langdon et al. 1997; McLean et al. 1993; Price et al. 1998). Furthermore, Murakami et al. (2005) demonstrated that the pedunculopontine tegmental nucleus (although not serotonergic) modulates the responsiveness and sensitivity of the anterior piriform cortex and olfactory tubercle in a sleep-state-dependent manner. It therefore appears that both vertebrate and invertebrate nervous systems may rely on centrifugal feedback to modulate olfactory processing based on arousal state.
By enhancing response magnitudes and lowering the activation threshold of units in the AL of Manduca, it appears that 5HT modulates both the gain and the sensitivity of the AL units with little effect on background activity. These effects enhance the contrast between units that are excited by an olfactory stimulus and other units that are either inhibited or unaffected, thus increasing the specificity of the overall response pattern. The effects of 5HT likely coordinate large-scale refinement of the response properties of the AL and allow the AL to remain plastic in the context of the arousal state of the individual. Although 5HT does not dramatically change the specific spatiotemporal patterns of response in the AL, it enhances AL responses, which could improve the abilities of moths to process information about the olfactory environment in which they are immersed.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. M. Dacks. Arizona Research Laboratories, Division of Neurobiology, University of Arizona, P.O. Box 210077, Tucson, AZ 85721 (E-mail: adacks{at}email.arizona.edu)
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) and ylang-ylang oil (
) were tested at logarithmically increasing concentrations depicted as increasing bar height. After the odors were tested once, 5HT was applied (- - -, onset) for 20 min, and then the same battery of olfactory stimuli was tested again. The 5HT was washed out (- - -, onset) for 20 min, and the stimuli were repeated. B: stimulus protocol for experiment 2. Five odorants, methyl salicylate (MES; 
), Z-3-hexenyl propionate (ZHP; 
), linalool (LIN; 
), geraniol (GER; 
), and MES (dark gray dashed line with diamond markers) are depicted. 
, direction of the time course of the trajectories. Distances between response trajectories are obtained by measuring the Euclidean distance between trajectories at each point in time.
