Olfactory modulation by dopamine in the context of...

Olfactory modulation by dopamine in the context of aversive learning

Andrew M. Dacks,1 Jeffrey A. Riffell,1,2 Joshua P. Martin,1 Stephanie L. Gage,1 and Alan J. Nighorn1

1Department of Neuroscience, The University of Arizona, Tucson, Arizona; and 2Department of Biology, University ofWashington, Seattle, Washington

Submitted 22 February 2012; accepted in final form 27 April 2012

Dacks AM, Riffell JA, Martin JP, Gage SL, Nighorn AJ.Olfactory modulation by dopamine in the context of aversive learning.J Neurophysiol 108: 539–550, 2012. First published May 2, 2012;doi:10.1152/jn.00159.2012.—The need to detect and process sensorycues varies in different behavioral contexts. Plasticity in sensorycoding can be achieved by the context-specific release of neuromodu-lators in restricted brain areas. The context of aversion triggers therelease of dopamine in the insect brain, yet the effects of dopamine onsensory coding are unknown. In this study, we characterize themorphology of dopaminergic neurons that innervate each of theantennal lobes (ALs; the first synaptic neuropils of the olfactorysystem) of the moth Manduca sexta and demonstrate with electro-physiology that dopamine enhances odor-evoked responses of themajority of AL neurons while reducing the responses of a smallminority. Because dopamine release in higher brain areas mediatesaversive learning we developed a naturalistic, ecologically inspiredaversive learning paradigm in which an innately appetitive host plantfloral odor is paired with a mimic of the aversive nectar of herbivor-ized host plants. This pairing resulted in a decrease in feedingbehavior that was blocked when dopamine receptor antagonists wereinjected directly into the ALs. These results suggest that a transientdopaminergic enhancement of sensory output from the AL contributesto the formation of aversive memories. We propose a model ofolfactory modulation in which specific contexts trigger the release ofdifferent neuromodulators in the AL to increase olfactory output todownstream areas of processing.

insect; antennal lobe; biogenic amines

WHILE SEARCHING for food and mates, animals encounter a hugediversity of sensory stimuli that the nervous system mustencode and decide whether, and in what manner, to respond.This task is made all the more difficult because most resourceshave patchy distributions and varying reward values. Thisvariability establishes different behavioral contexts in whichsensory information is encoded by the nervous system. Thenervous system must therefore adjust its activity so that be-havioral output is maximally beneficial for survival within thepresent context. This plasticity is often accomplished when agiven context triggers the release of specific neuromodulatorswithin restricted brain regions from a small population ofneurons. Modulators can modify neural processing by alteringthe efficacy with which neurons respond to stimuli or commu-nicate with each other without directly causing inhibition orexcitation (Kupfermann 1979). For instance, in the mammalianbrain neurons from the ventral tegmental area project to manyother areas and release dopamine (DA) within the context ofreward (Alcaro et al. 2007; Berridge 2007; Schultz 2007; Wise2004).

The insect olfactory system has evolved several modulatorysystems to maximize foraging efficiency for resources that arepatchy in their distribution. For instance, the flowers of Daturawrightii (a host plant of the moth Manduca sexta) open in theearly evening, making them a temporally patchy resource.Foraging moths must therefore be maximally sensitive toolfactory cues at a specific time. The levels of serotonin in theantennal lobes (ALs; the first synaptic neuropil of the olfactorysystem) increase at this time (Kloppenburg et al. 1999), andserotonin increases the sensitivity and responsiveness of ALneurons (Dacks et al. 2008; Kloppenburg et al. 1999; Klop-penburg and Hildebrand 1995), suggesting that serotonin actsas a circadian modulator of olfactory sensitivity (Kloppenburgand Mercer 2008).

Resources can also be patchy in their reward value. Forinstance, there is natural variability in the nicotine (which isrepellant) content of floral nectar in the flowers of the hostplants of Manduca (Kessler and Baldwin 2007; Kessler et al.2008). An association may then be formed between the nico-tine content of the nectar and the floral features so that thoseflowers can be avoided in the future. DA release in brain areasdownstream of the ALs has been demonstrated to act as amolecular signal for the occurrence of an aversive stimulusduring olfactory learning in insects (Aso et al. 2010; Beggs etal. 2007; Beggs and Mercer 2009; Claridge-Chang et al. 2009;Mizunami et al. 2009; Selcho et al. 2009; Unoki et al. 2005;Vergoz et al. 2007; Wright et al. 2010; Zhang et al. 2008).However, the effects of DA on odor-evoked responses in theALs are unknown, and the consequences of the effects of DAin the AL on behavior have not been examined. We thereforesought to explore the role of DA as a modulator of olfactorycoding in the AL of Manduca within the context of aversivelearning.

MATERIALS AND METHODS

Moths were reared in the Department of Neuroscience at theUniversity of Arizona under a 17:7-h light-dark cycle as describedpreviously (Christensen and Hildebrand 1987).

Immunocytochemistry. For DA immunoreactivity (DA-ir), brainswere labeled with a protocol identical to that published by Dacks andNighorn (2011), which also included preadsorption controls for theDA antibody with Manduca brain tissue. For immunochemical label-ing of Manduca tyrosine hydroxylase (TH-ir), brains were dissected ininsect saline and fixed overnight in 4% paraformaldehyde at 4°C.Brains were then washed in PBS, embedded, and sectioned as de-scribed previously (Dacks and Nighorn 2011) except for AMIRAreconstruction, for which whole mount brains were used. Sectionswere washed in PBS with 0.5% Triton X-100 (PBST), blocked for 1h in PBST with 2% IgG-free BSA, and incubated for 2 days in1:10,000 rabbit anti-Manduca TH (a generous gift from Dr. MaureenGorman at Kansas State University) in PBST with 1% Triton X-100

Address for reprint requests and other correspondence: A. M. Dacks, MountSinai Hospital, Dept. of Neuroscience, Box 1065, Annenberg 21-06, 1468Madison Ave., New York, NY 10029 (e-mail: [email protected]).

J Neurophysiol 108: 539–550, 2012.First published May 2, 2012; doi:10.1152/jn.00159.2012.

5390022-3077/12 Copyright © 2012 the American Physiological Societywww.jn.org

and 50 mM sodium azide (PBSAT). Sections were then washed inPBST, blocked, and incubated overnight in 1:1,000 goat anti-rabbitCy3 (Jackson Immunoresearch) in PBSAT. Tissue was then washed inPBST, cleared, and mounted as described previously (Dacks andNighorn 2011). The full characterization of the anti-Manduca THantibody is described by Gorman et al. (2007). In Western blots asecond smaller, weaker band was observed (Gorman et al. 2007). Weobserved some very weak labeling in addition to the strong labelingthat matched the observed DA-ir. We therefore only traced TH-irprocesses in AMIRA (see below) that also matched the DA-ir pro-cesses innervating the AL. Anterograde fills of olfactory receptorneurons (ORCs) projecting to the macroglomerular complex (MGC)were performed by cutting the tips of the sex pheromone-sensitivesensillae of males and placing droplets of dextran-Texas red dissolvedin insect saline over the stumps. The entire antenna was then coatedin petroleum jelly to avoid desiccation (Molecular Probes), and themoths were allowed to rest overnight so that the dye could be taken upand transported throughout the ORCs. Brain were then dissected andprocessed as described above.

Images were collected with a Zeiss 510 Meta laser scanningconfocal microscope equipped with argon and green HeNe lasers andappropriate filters. The Zeiss LSM Image Browser was used to createimage stacks and to adjust contrast and brightness. CorelDRAW X4(Corel, Ottawa, ON, Canada) was used to organize all images andfigures. AMIRA reconstructions were created in Amira 4.1.2 fromconfocal scans of a whole mount brain labeled with the Manduca THantibody with a custom AMIRA plug-in for reconstruction of three-dimensional branching patterns (Evers et al. 2005) generously pro-vided by Dr. Felix Evers (University of Cambridge). The processeswithin the AL were not reconstructed, to avoid obscuring the branch-ing patterns of these neurons within the rest of the brain.

Cloning of Manduca dopamine receptors. Degenerate PCR andRT-PCR were performed as described by Dacks et al. (2006). Antennallobe cDNA was isolated by cutting out the ALs only (which is relativelyeasy to do because of the large size of the ALs in Manduca) for mRNAextraction. Degenerate PCR primers were designed based on sequencesfrom Bombyx mori, Drosophila melanogaster, Papilio xuthus, and Apismellifera with Primer Premiere 4.1 (Premiere Biosoft International, PaloAlto, CA). Degenerate PCR primer sequences used to clone each of theMsDA receptors were 5=-CGTGATCTCCCTGGACMGNTAY-TGGGC-3= and 5=-GAACACCATCACGAACAGAGGNARRTA-RAA-3= for the MsINDR, 5=-ACCGCYWSNATCTTCAACYTSTG-3=and 5=-CCAGCANAVNADGAAGACGCCCAT-3= for the MsDop1,and 5=-TGYTGGBTNCCNTTYTT-3= and 5=-GTRTADATNAY-NGGRTT-3= for the MsDop2 receptors. Brain and AL cDNA weregenerated with the Omniscript RT kit (Qiagen, Valencia, CA), andAccuPrime Pfx Supermix (Invitrogen) was used to generate initial frag-ments for all three DA receptors. Rapid amplification of cDNA ends(RACE) was used to generate the full-length sequence for the MsINDRand MsDop1 receptors with the use of the SMARTer PCR Synthesis Kit(Clontech, Mountain View, CA) to generate the cDNA with a universaltag sequence and Advantage 2 Polymerase Mix (Clontech) to generate 5=and 3= fragments with touchdown PCR. The RACE primer sequencesused were as follows: 5=-ACGGTGGTGAGTGAGAACAG-3= for the 5=MSINDR fragment and 5=-TTCATAGTCTGCTGGCTGCCATTC-3=for the 3= MsINDR fragment and 5=-GCATAGCAGTAGAGCCTG-CAATAT-3= for the 5= MsDop1 fragment and 5=-GACTTTTGCAG-GAGTCAACGACTTGC-3= for the 3= MsDop1 fragment. Despite manyattempts with RACE, only a 144-nucleotide fragment of the MsDop2receptor was obtained.

Sequence alignments in Fig. 3 were constructed with the programClustalW (Combet et al. 2000) (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page�/NPSA/npsa_clustalw.html). The GenBankaccession numbers for the sequences used for the sequence alignments inFig. 3 were MsINDR (JN_117928), MsDop1 (JN_117929), MsDop2 (notapplicable), DmDD2R (NP_001014758), AmDop2 (NP_001011567), Am-Dop1 (NP_001011595), AmDop3 (NP_001014983), BmDopR2

(NP_001108338), BmDopR1 (NP_001108459), TcDop1 (not published,XP_971542), and TcINDR (not published, XM_967686).

Multichannel extracellular recordings. Multichannel extracellularrecordings were performed as described previously (Dacks et al.2008). Briefly, 2- to 5-day-old moths were secured in plastic tubeswith dental wax, and the cuticle and muscles lying above the brainwere removed. The perineural sheath was removed from the AL, andphysiological saline with 8.55 g/l sucrose and 200 �M ascorbic acid(to reduce the oxidation of DA) was immediately superfused over thebrain. Pilot experiments found no effect of 200 �M ascorbic acid onAL responses. Sixteen-channel extracellular electrode arrays (NeuroNexus Technologies, Ann Arbor, MI; catalog no. 434-3mm 50-177)were inserted into the AL in parallel with the antennal nerve. Extra-cellular activity was acquired with a RX5 Pentusa base station (Tuck-er-Davis Technologies, Alachua, FL) and a RP2.1 real-time processor(Tucker-Davis Technologies), and spike data were extracted from therecorded signals and digitized at 25 kHz with the Tucker-DavisTechnologies data-acquisition software. Threshold and gain settingswere adjusted independently for each channel, and spikes were cap-tured in the tetrode recording configuration: any waveform that passedthreshold on one channel triggered the capture of waveforms recordedon the other three channels on the same shank. Offline Sorter v.3(Plexon Neurotechnology Research Systems, Dallas, TX) was used tosort extracellular waveforms, and spikes were assigned timestamps tocreate raster plots and calculate perievent histograms in Neuroex-plorer v.3 (Plexon Neurotechnology Research Systems).

The same experimental protocol was used for all concentrations ofDA applied and the saline-only controls in which saline with 200 �Mascorbic acid was applied from different superfusion containers. ALneurons were stimulated with ten 200-ms pulses of a five-componentD. wrightii blend (Riffell et al. 2009a) separated by 10 s at a 1:1,000and then a 1:10 dilution in mineral oil. After the two sets of stimuli,2 min of spontaneous activity was recorded. The ALs were thensuperfused with dopamine at 5 � 10�5 M (n � 23), 5 � 10�6 M(n � 6), or 5 � 10� 7 M (n � 6) or with physiological saline (n � 6)(a total of 106 neurons from 41 moths) with sucrose and 200 �Mascorbic acid for 5 min. The 200 �M ascorbic acid was added to thesaline to prevent the breakdown of DA and was included for allconditions. The stimulation protocol was then repeated and another 2min of spontaneous activity recorded. The ALs were then superfusedwith physiological saline for 15 min, the stimulation protocol wasagain repeated, and another 2 min of spontaneous activity wasrecorded.

Analysis of multichannel data. After spikes were assigned time-stamps, the number of spikes elicited by each odor stimulus wasdetermined by counting the number of spikes that occurred in a 1-swindow after the presentation of the stimulus. A neuron was consid-ered to respond if the firing rate after stimulation rose above athreshold of 1.96 times the standard deviation of background firingrate. If the firing rate remained above the threshold for �1 s (whichoccurred infrequently), then only those spikes elicited in that 1-speriod were counted. To calculate the duration of inhibitory re-sponses, the number of 1-ms bins in which the firing rate dropped tozero within 500 ms after stimulus presentation were counted. Theduration of the postexcitatory inhibitory phase (or “I2”) was calcu-

Table 1. Tropane alkaloid concentrations in Datura wrightii andDatura discolor flower nectars

Species Undamaged Damaged

Datura wrightii 0.16 (0.01) 1.44 (0.10)Datura discolor 1.33 (0.21) 23.82 (7.56)

Values (in �M) are mean (SE) alkaloid concentrations for n � 4–11 plantsper treatment. Plants were either undamaged or Manduca sexta larva damagedfor 72 h prior to flowering.

540 DOPAMINERGIC MODULATION OF OLFACTION

J Neurophysiol • doi:10.1152/jn.00159.2012 • www.jn.org

http://page=/NPSA/npsa_clustalw.html

lated as the amount of time in which there were no spikes followingan odor-evoked excitatory burst. All measures of background ac-tivity [mean interspike interval, coefficient of variation (CV), CV2,peak interspike interval, and mean firing rate] were calculated inMATLAB, and CV2 was calculated as described previously (Holt etal. 1996).

Nectar collection and analysis. To provide a naturalistic context forthe neural basis of aversive learning, we used the nectar and floralodors from the Manduca host plants D. wrightii and D. discolor.Many plants increase their toxic alkaloid content in leaf tissue andnectar when experiencing herbivory (Adler et al. 2006; Kessler et al.2008), which subsequently can lead to decreased visitation by thefloral visitors (Gegear et al. 2007). The decreased visitation has beenshown to be mediated by aversive learning by the pollinators (Gegearet al. 2007; Wright et al. 2010). To examine the effects of herbivoryon nectar chemical composition, we placed screen cages on D.wrightii and D. discolor branches that did not have flower buds.Manduca larvae were placed inside the screen cages and allowed tofeed ad libitum on the vegetative tissue for 3 days. After 3 days, nectarstanding crops were collected from flowers on the other branches ofthe plant at dusk (2000 PST) from at least 10 newly opened flowers ofeach species with 1-ml syringes. Control experiments were conductedin parallel with plants that had the screen cages but did not have larvaefeeding on the vegetative tissue.

Individual nectar samples were partitioned into water-soluble andnon-water-soluble fractions by adding 500 �l of methylene chloride.The methylene chloride fraction was extracted and concentrated bygently blowing nitrogen gas over the sample until 20 �l remained.Each sample was stored in a 2-ml borosilicate glass vial with aTeflon-lined cap at �80°C until analysis. Samples (1 �l) wereanalyzed with a gas chromatography-mass spectrometric detectionsystem (GC-MS) consisting of an HP 7890A GC and a 5975CNetwork Mass Selective Detector (Agilent Technologies, Palo Alto,CA). A DB1 GC column (J&W Scientific, Folsom, CA; 30 m, 0.25mm, 0.25 �m) was used, and helium was used as carrier gas atconstant flow of 1 ml/min. The initial oven temperature was 50°C for5 min, followed by a heating gradient of 6°C/min to 250°C, which washeld isothermally for 6 min. Chromatogram peaks were identifiedtentatively with the aid of the NIST mass spectral library (�120,000spectra) and verified by chromatography with authentic standards(when available). Peak areas for each compound were integrated withChemStation software (Agilent Technologies) and are presented interms of nanograms per microliter of nectar.

Pharmacology and focal microinjection-conditioning experiments.To evaluate the influence of DA within the AL on olfactory learning,DA receptor antagonists were focal-microinjected into the AL of4-day-old male moths. As described previously (Lei et al. 2009),moths were restrained in a plastic tube 30 min prior to scotophase andkept at room temperature in the light awaiting surgery and injection.The head capsule was descaled and then opened, and the ALs wereexposed for microinjection. Injection was accomplished via quartzpipettes (OD 1.0 mm, ID 0.70 mm, Sutter Instruments, San Diego,CA) pulled with a model P-2000 laser puller (Sutter Instruments) andclipped to allow solution passage. Pipettes were filled with thesolution to be injected and connected with an output line of adual-channel Picospritzer (Picospritzer II, General Valve, East Ha-nover, NJ). Pipettes were inserted into the center of each AL, and twodrops (mean diameter � SD: 82 � 12.1 �m) were administered inquick succession. The diameter was determined before the experimentby placing the tip of the pipette into a beaker of mineral oil andmeasuring the droplet size with an ocular micrometer. After injectionthe cuticle window was repositioned and sealed with myristic acid(Sigma), and the moths were allowed to recuperate for 30 min beforetesting. We previously found that drugs delivered in this manner donot diffuse into other regions of the brain, and that the saline- andvehicle-injected moths do not behave significantly differently fromnoninjected moths (Lei et al. 2009).

For DA receptor antagonist experiments (see Fig. 8, B and D),moths were injected with either a mixture of 10�8 M SCH39166 (a D1receptor antagonist) and 10�8 M L-741,626 (a D2 receptor antagonist)(both from Tocris Bioscience) or vehicle (moth saline) or were notinjected. Receptor antagonists were selected for their extreme selec-tivity for DA receptors (Bowery et al. 1996; McQuade et al. 1991). Inaddition, we performed experiments in which the ALs of moths wereinjected with SCH23390 (see Fig. 8C) at 10�6 M and tested the abilityof moths to form an aversive association between the Datura odor andthe simulated toxic nectar (see below). SCH23390 (Tocris) wasselected for its effectiveness in blocking the Dop1- and INDR-typereceptors in the silk moth Bombyx mori (Ohta et al. 2009). Because ofthe high degree of sequence similarity for the DA receptors betweenManduca and Bombyx, SCH23390 is likely an effective Dop1- andINDR-type receptor antagonist in Manduca. However, it should benoted that SCH23390 has not been directly tested on serotonin oroctopamine receptors in moths, although SCH23390 is �25 timesmore effective at blocking the effects of DA compared with octo-pamine in crude membrane preparations from cockroach brain (Orr etal. 1987). All solutions included 0.1 �M DMSO. For the dopamineinjection experiments (see Fig. 8E), the ALs were injected withdopamine-HCl at 5 � 10�5 M diluted in moth saline with 200 �Mascorbic acid (to prevent the breakdown of DA), moth saline with 200�M ascorbic acid (vehicle), or saline alone.

We used a forward-paired conditioning paradigm to examine theeffects of the pharmacological manipulations while the animal learnedto associate an odor with an aversive stimulus. In the forward-pairedcondition, the artificial mimic of the D. wrightii floral scent wasdelivered in a 3-s pulse. One second after odor onset, the uncondi-tioned stimulus, which was either 1 �l of 0.1 M quinine or the “toxicnectar” (30 �M scopolamine in 20% sucrose), was applied to theproboscis for �2 s. A 10-min intertrial interval separated each trainingtrial, and moths were trained over seven or eight trials. After theconditioning trials were completed, a test trial was performed duringwhich only the D. wrightii mixture was presented to assess thebehavioral responses as a result of the conditioning treatment. Theability of moths to form aversive association in backward- or random-pairing training paradigms was not tested because of the inability ofmoths to form appetitive associations with these training procedures(Daly and Smith 2000). The toxic nectar used in the forward-pairedparadigms is based on the scopolamine levels shown in Table 1 andFig. 8A and sucrose levels normally found in nectar, as herbivory didnot affect sugar concentrations. Fourteen to forty-nine moths wereused for each drug and control treatment group (n � 247 total moths).

Statistical analyses. All statistical analyses were performed withGraphPad Prism 5.01 (GraphPad Software, La Jolla, CA) or SPSS18.0 (IBM, Armonk, NY). For electrophysiological measures aD’Agostino and Pearson omnibus normality test was applied, and ifdata were normally distributed a single-factor repeated-measuresANOVA with a Tukey honestly significant difference (HSD) post hoctest was calculated. If data were not normally distributed, a Kruskal-Wallis test with a Dunn’s multiple comparison post hoc test wasperformed (P � 0.05 was assigned as a significance threshold for bothtests). For all normalizations, response measures of a given neuron(such as peak firing rate or inhibition duration) were normalized to themaximal response for that neuron to all of the odor stimuli under allof the treatment conditions. In all behavioral experiments, repeated-measures binary logistic regression modeling was used to analyzeproboscis extension response (PER) between treatments. Fisher’sexact test was used to make specific pairwise comparisons among testodors and treatments within trials.

RESULTS

Antennal lobes of Manduca are innervated by two pairs ofdopaminergic neurons. To identify dopaminergic input to theALs of Manduca we labeled the brains of adult moths for both

541DOPAMINERGIC MODULATION OF OLFACTION


DA and Manduca TH, the rate-limiting enzyme essential to theproduction of DA. The AL is comprised of spherical neuropilcalled “glomeruli” that receive input from ORCs on the anten-nae. Olfactory information is transmitted to downstream brainareas via the projection neurons, and glomeruli are intercon-nected by a diverse population of local interneurons. DA-ir andTH-ir were observed in every glomerulus of the AL (Fig. 1, A

and D, respectively), and the DA-ir and TH-ir fibers innervatedthe entire glomerular volume (Fig. 1, B and D, respectively)with the exception of the distal area of the pheromone-sensitiveMGC of male moths (Fig. 1, C, E, and F). The glomeruli thatcomprise the MGC are distinct from the other glomeruli in theAL in that they receive input from ORCs that are selectivelytuned to single components of the female sex pheromone.Anterograde dye fills of the pheromone-sensitive ORCs on theantennae demonstrate a lack of overlap between the TH-irprocesses and the receptor neurons innervating the MGC (Fig.1F). The widespread nature of the projections of the DA-ir/TH-ir neurons within the AL suggests that the activation ofthese neurons likely affects odor-evoked responses in all glom-eruli, rather than a few specialized glomeruli. Both DA-ir andTH-ir revealed four neurites innervating the ALs (Fig. 1, C andD). These four processes were traced from the ALs in wholemount TH-ir preparations to two pairs of cell bodies (Fig. 2,A–C) in the dorsal protocerebrum, a central brain region ininsects. Each pair of cells projects from the dorso-posteriorprotocerebrum to both ALs as well as other higher areas ofprocessing, including the lateral horn, forming an archlikestructure that bridges both ALs (Fig. 2, D–F). Because of thearching morphology of these neurons, we refer to them as thedopaminergic arching (DAAr) neurons.

Although the AL is uniformly innervated by DA-ir/TH-irfibers, the effects of DA on AL neurons could be heteroge-neous depending on the DA receptors expressed in the AL. Wecloned homologs of the DA 1-type receptor (Dop1), DA 2-typereceptor (Dop2), and invertebrate DA receptors (INDRs) fromManduca, all of which share high levels of sequence identitywith other insect DA receptors (Fig. 3; MsINDR; 92% sequenceidentity with B. mori, MsDop1; 93% sequence identity with B.mori, MsDop2 fragment; 100% sequence identity with D. mela-nogaster). RT-PCR of AL cDNA revealed that all three ManducaDA receptor homologs are expressed in the AL (Fig. 4) and thusat least these three receptors are potential targets for anymodulatory effects of DA in the AL. Thus the ALs of Manducaexpress several DA receptors and receive dopaminergic inputfrom four centrifugal neurons.

Responses of the majority of antennal lobe neurons areenhanced by dopamine. In Drosophila (Yu et al. 2004) andManduca (Daly et al. 2004) the responses of AL neurons aretransiently enhanced after the formation of aversive or appet-itive associations, respectively. This is striking as it suggeststhat despite the opposing natures of appetitive and aversivecontexts, there is an increase in the responsiveness of the ALassociated with the formation of either type of olfactory asso-ciation. Similarly, both octopamine (Barrozo et al. 2010) andserotonin (Dacks et al. 2008; Kloppenburg et al. 1999) enhanceodor-evoked responses of AL neurons in moths, despite beingassociated with different behavioral contexts. We thereforesought to determine the effects of DA on odor-evoked re-sponses of AL neurons. We performed extracellular multichan-nel recordings of the responses of AL neurons to two concen-trations of the innately attractive (Raguso and Willis 2002,2005; Riffell et al. 2008, 2009a) odor of D. wrighti flowers,from which adult Manduca feed (Raguso et al. 2003; Riffell etal. 2008). DA increased the number of odor-evoked spikes(Fig. 5A) in 58% of responsive AL neurons (n � 30 of 52neurons that responded to the Datura odor), and this effect wasDA dose dependent (Fig. 5B). Of those neurons enhanced by

Fig. 1. The antennal lobe (AL) of Manduca sexta is innervated by dopami-nergic centrifugal neurons. A: dopamine-immunoreactive (DA-ir) processesinnervate every glomerulus of the AL. B: DA-ir processes innervate the entirevolume of the ordinary glomeruli (dashed border). C: the AL of M. sexta isinnervated by 4 DA-ir processes. Inset: higher-magnification view of the 4DA-ir branches (asterisks) as they enter the AL. D: tyrosine hydroxylaseimmunoreactivity (TH-ir) reveals 4 branches that innervate the AL similarly tothe 4 DA-ir branches in C and weakly labels other neurons (arrow andarrowhead). Only the 4 strongly labeled branches similar to the DA-ir pro-cesses were traced for 3-dimensional (3D) reconstructions. E: while the DA-irprocesses in the AL innervate the entire volume of the ordinary glomeruli, theyinnervate only the proximal region (bracket and “P”) of the macroglomerularcomplex (MGC) and not the distal region (bracket and “D”). F: mass fills ofolfactory receptor neurons (magenta) in the antennal nerve and TH-ir (green)reveal that the processes of the DA-ir/TH-ir neurons do not overlap with theaxons of the olfactory receptor neurons. All images are presented from afrontal perspective, and all scale bars � 100 �m except for the inset in C, inwhich the scale bar � 20 �m.



DA, there was an average 58.5% increase in elicited spikes(Fig. 5C; single-factor repeated-measures ANOVA: P �0.0001). Thus DA increased the magnitude of odor-evokedresponses of most AL neurons. DA did not affect the responsesof 31% of AL neurons and significantly reduced the responsesof 11% of AL neurons (Fig. 5D). DA also decreased thethreshold for activation of a few AL neurons (Fig. 5E), sug-gesting that DA may increase the sensitivity of AL neurons,resulting in more cells participating in the encoding of olfac-tory stimuli.

DA also affected the slow temporal dynamics of AL re-sponses (i.e., firing patterns that evolve over hundreds ofmilliseconds), which can provide information about the iden-tity of an odor (Laurent et al. 1998) and the physical structureof an odor plume (Vickers et al. 2001). The excitatory re-sponses of Manduca PNs are often preceded by a rapidGABAA-dependent inhibition (referred to as “I1”) and fol-lowed by a period of spike suppression (“I2”) lasting anywherebetween 10 ms and 1.5 s (Christensen et al. 1996). The I2 phaseshortens the duration of PN responses after stimulus offset,allowing tracking of odor intermittency (Lei et al. 2009;Tripathy et al. 2010) that is both inherent in the odor plumestructure (Vickers et al. 2001) and produced by the beating ofthe moth’s wings (Sane and Jacobson 2006). DA caused a24.5% decrease in the duration of I2 (Fig. 6, A and B; Kruskal-Wallis test: P � 0.0001) in 73.5% (25 of 34 units) of units thatdisplayed the I2 phase. This effect was not due to the enhance-ment of the magnitude of the excitatory phase overwhelmingthe I2 phase, as higher odor concentrations (which increaseresponse magnitudes) did not affect I2 duration (Fig. 6C). DAdid not, however, affect the duration of purely inhibitoryresponses (Fig. 6D), suggesting that odor-evoked inhibitionand the I2 phase are mediated by different cellular or networkmechanisms. Furthermore, DA had no apparent effects on I1phase. In addition to odor-evoked activity, 2 min of back-ground activity was recorded before, during, and after DAapplication to determine whether DA affected the backgroundactivity of AL neurons. However, there was no effect of DA on

several measures of background activity of AL neurons (meaninterspike interval, CV, CV2, peak interspike interval, andmean firing rate; Fig. 6, E–I, respectively; Kruskal-Wallis test).

The global release of a neuromodulator should result inwidespread effects on the representation of a stimulus by anensemble of neurons within a network. However, DA did nothomogeneously affect the responses of individual neurons andmay therefore alter the population response to an odor in oneof several ways. If DA sharpened the overall AL response, wewould expect an enhancement of the strongest responses and adecrement of the weakest responses (Sachse and Galizia 2002),increasing the contrast or signal-to-noise ratio. Conversely, ifDA broadened the AL responses, then DA would either de-crease or have little effect on the strongest responses andincrease the weaker responses (Olsen et al. 2007; Silbering etal. 2008), thus recruiting more cells to participate in the odorrepresentation. In contrast to these two options, we observedthat the relative strength of a neuron’s response does notpredict the modulatory effects of DA (Fig. 7A). Thus theensemble response is not homogeneously sharpened or broad-ened but reorganized by the enhancement of most neuronsacross the spectrum of response strength, the decrement ofsome responses, and the recruitment of additional neurons.Comparing pre-DA responses to post-DA responses across thepopulation (Fig. 7B), the best-fit line is positively shifted froma 1:1 pre- to post-DA response ratio, indicating that responsesare increased across the population. However, the slope of thisline is close to 1 (slope � 0.9756), indicating that the overallpopulation response was increased and that neither significantsharpening (slope � 1) nor broadening (slope � 1) of thepopulation response occurred.

Injection of dopamine receptor antagonists into the ALprevents formation of an aversive association. Given the cleareffects of DA on the AL responses of Manduca, and the factthat the release of DA within the mushroom bodies of Dro-sophila is necessary (Schwaerzel et al. 2003) and sufficient(Claridge-Chang et al. 2009) for aversive learning, we inves-tigated the role of DA within the AL in aversive learning.

Fig. 2. Morphology of the dopaminergicarching neurons. A–C: consecutive horizon-tal sections through a TH-ir-labeled M. sextabrain following the projections of the proto-cerebral neurons from the ALs to their cellbodies. Arrowheads indicate the origin of theprotocerebral neurons from the AL in A orthe previous section in B and C. Asterisksindicate the termination point of the dopa-mine arching (DAAr) neurons within thegiven section including 2 large TH-ir-posi-tive cell bodies in C. All scale bars � 100�m. D–F: 3D reconstructions of 1 pair ofDA/TH-ir AL neurons viewed from frontal,horizontal, and sagittal perspectives, respec-tively, of a whole mount preparation. Theprocesses of these neurons within the ALs(which are indicated by brackets) have beenomitted to avoid obscuring the branchingpatterns of these neurons outside the AL.The DAAr cell bodies are indicated by as-terisks in D–F.



Fig. 3. ClustalW sequence alignments for the 3 M. sexta DA receptor homologs. Asterisks indicate conserved amino acid sequence. A: alignment of the MsDop1receptor with dopamine 1-like receptor homologs from Tribolium casteneum (TcDop1), Bombyx mori (BmDopR1), and Apis mellifera (AmDop1). B: alignmentof the MsINDR receptor with invertebrate dopamine (INDR) receptor homologs from T. casteneum (TcINDR), B. mori (BmDopR2), and A. mellifera (AmDop2).C: alignment of the MsDop2 receptor with dopamine 2-like receptor homologs from A. mellifera (AmDop3) and D. melanogaster (DmDD2R).



Aversive learning assays in insects have predominantly reliedon electric shock as an aversive stimulus. Here we take advan-tage of the natural variation in the amounts of aversive com-pounds in the floral nectar of the host plants of Manduca(Kessler et al. 2008). Plant herbivory has cascading effects onplant physiology that, in turn, influence diverse processes.Herbivory alters the volatile chemicals emitted by host plantsof Manduca (Kessler et al. 2008) as well as the time of day atwhich these plants flower (Kessler et al. 2010). We thereforetested whether herbivory could also alter the chemical contentof host plant nectar. Manduca caterpillars were allowed to feedon the vegetative (but not floral) tissue for 3 days, and then thenectar content of the flowers was collected and analyzed witha GC-MS system. Herbivory increased the amount of thetropamine alkaloids (e.g., scopolamine) in the floral nectar ofboth D. wrightii and D. discolor (Fig. 8A and Table 1). This, incombination with natural variability in the nectar content ofnicotine, which is repellant to Manduca (Kessler et al. 2008)and honeybees (Kohler et al. 2012), suggests that there areinstances in the field in which pollinators encounter flowerswith aversive nectar. We therefore tested whether moths couldform an association between an appetitive olfactory stimulus(the Datura floral blend) (Riffell et al. 2009b) and an aversivetastant (a synthetic blend of the nectar of herbivorized Daturaplants), and whether the formation of such an association couldbe blocked by DA receptor antagonists.

Naive Manduca extend their proboscis in response to theDatura floral blend in both a tethered feeding assay (seeMATERIALS AND METHODS) and flight assays (Raguso and Willis2002, 2005; Riffell et al. 2009a, 2009b), indicating that theDatura odor is an innately attractive stimulus. This feedingresponse was eliminated when a synthetic nectar mimickingthe chemical composition of the nectar from larva-damagedplants was repeatedly paired with the Datura odor (Fig. 8B;Fisher’s exact test; P � 0.005) in a forward-paired learningparadigm. Trained moths did not respond to the Datura blend2 h after training (Fig. 8B; Fisher’s exact test; P � 0.005), thusrepresenting a learned aversion for the Datura blend. To testthe necessity of DA in the AL for aversive memory formation,DA receptor antagonists were injected directly into the ALs ofadult moths. Moths injected with either a mixture of D1 and D2receptor antagonists (Fig. 8B; SCH39166 and L-741,626, re-spectively) or a D1 receptor antagonist (Fig. 8C; SCH23390)responded in the same proportion at the end of the trainingprotocol as at the start, and significantly more than controlanimals (Fig. 8B; logistic regression: �1

2 � 7.15, P � 0.01). In

particular, animals injected with the mixture of D1 and D2receptor antagonists produced significantly more PER re-sponses than control animals at the end of the training protocol(Fisher’s exact test; P � 0.001) and had elevated responsescompared with animals injected with the D1 receptor antago-nist SCH23390 (Fig. 8C; logistic regression: �1

2 � 6.84, P �0.009). In control experiments in which moths injected withsaline only were repeatedly exposed to either the Datura blendor a mineral oil blank without exposure to an aversive stimulus,the proportion of moths responding to either olfactory stimulusremained unchanged (Fig. 8D). This indicated that the decreasein the proportion of moths responding to the Datura odor whenit is paired with the aversive stimulus is due to associativeprocesses. Moths injected with the DA receptor antagonistscontinued to produce feeding responses at the same proportion

Fig. 4. Intrinsic neurons of the AL express the Dop1-, Dop2-, and INDR-typedopamine receptors. RT-PCR for the Manduca homologs of the dopamine1-like (MsDop1), dopamine 2-like (MsDop2), and invertebrate dopaminereceptor (MsINDR) using cDNA from the AL demonstrates that all 3 receptorsare expressed in the AL. RT� and RT� indicate lanes in which reversetranscriptase was included or omitted, respectively. White lines between lanesindicate that RT-PCR products were run on separate gels.

Fig. 5. DA enhances the responses of AL neurons to the Datura floral blend.A: peristimulus time histogram demonstrating that the responses of an ALneuron to the Datura floral blend (10�1 dilution) are enhanced by DA. Squarewave pulses indicate the presentation of the 200-ms odor stimulus. Responsesduring the control, DA, and washout phases are shown in black, white, andgray, respectively. B: effects of DA on all AL neurons at several concentrationsof DA. The peak evoked firing rate when all cells were grouped was enhancedby DA at 5 � 10�5 M (P � 0.016) and 5 � 10�6 M (P � 0.047) but not byDA at 5 � 10�5 M (P � 0.14) or saline (P � 0.86). C: the responses of unitsthat were significantly enhanced by DA had a 58.5% increase in the number ofodor-evoked spikes from control levels (P � 0.0001). Values are means � SE.D: peristimulus time histogram demonstrating that for some cells DA causeda decrease in magnitude of the odor-evoked responses. E: for some cells DAapplication resulted in a shift in the threshold of activation such that responseswere evoked by odor concentrations that were subthreshold during the controland wash phases.



2 h after training (Fig. 8, B, C, and E). The DA receptorantagonists could not have prevented the detection of theDatura odor, as injected moths still produced feeding re-sponses when the odor was presented. Furthermore, the injec-tion of the DA receptor antagonists did not cause a nonasso-ciative increase in the PER that masked the decrease in re-sponses, as there was no difference between treatments in theproportion of naive moths responding at the start of the ex-periment (trial 0; Fig. 8B, C, and E). The injection of themixture of DA receptor antagonists also prevented the forma-tion of an aversive association between the Datura floral blendand a high-concentration bitter tastant, 0.1 M quinine (Fig. 8E),both immediately after training and 2 h later (Fisher’s exacttest; P � 0.001). When DA was injected directly into the ALs,very few moths exhibited feeding responses to the Datura odorshortly after the injections (Fisher’s exact test; P � 0.01),whereas the majority of moths injected with either saline orvehicle extended their proboscis in response to the Datura odor(Fig. 8F). However, 2 h after injection moths in all treatmentgroups responded at the same proportion (Fig. 8F).

DISCUSSION

The nervous system must adjust its activity to best suit thebehavioral state of the individual animal, which, in turn, isoften established by the external and internal environments. Inthis study, we explored the effect of an aversive food stimuluson the encoding of an associated odor in the primary olfactorysystem. The nectar resources that Manduca encounters whileforaging in the field vary in their reward value, with someexpressing aversive compounds (like nicotine and scopol-amine). The nervous system of Manduca must be able toaccentuate sensory cues associated with aversive stimuli. Wedemonstrate that, in addition to the established role of medi-ating the formation of aversive olfactory associations in higherbrain areas, DA plays a more transient role of enhancing theresponse to olfactory stimuli in the primary sensory neuropil.

DA acts as a molecular mediator of aversive learning inhigher brain areas in a variety of insect species (Aso et al.2010; Beggs et al. 2007; Beggs and Mercer 2009; Claridge-Chang et al. 2009; Keene and Waddell 2005; Mizunami et al.2009; Schwaerzel et al. 2003; Selcho et al. 2009; Unoki et al.2005, 2006; Vergoz et al. 2007; Wright et al. 2010; Zhang etal. 2008) and has also been implicated in appetitive learning ininsects (Kim et al. 2007; Krashes et al. 2009; Selcho et al.2009). Despite the extensive study DA has received in olfac-tory learning, the effects of DA release in the primary olfactoryneuropil were unknown. We found that two pairs of DA-ir/TH-ir neurons (the DAAr neurons) innervated widely through-out each AL, innervating all of the glomeruli, although only theproximal portion (with relation to the center of the AL) of thesex pheromone-sensitive MGC glomerulus, which is occupiedby the ORCs (Fig. 1, E and F). Anterograde fills of the ORCsrevealed a lack of overlap between the TH-ir processes and theORCs, reminiscent of the innervation pattern of GABA (Re-isenman et al. 2011) and serotonin (Sun et al. 1993) in theMGC of Manduca. This suggests that the MGC may be lesssubject to presynaptic modulation relative to the other glom-eruli. In fruit flies and bees the ALs are innervated by dopa-minergic neurons that are local to the ALs (Chou et al. 2010and Kirchhof et al. 1999, respectively) and are thus morpho-logically quite distinct from the DAAr neurons. Neurons sim-

Fig. 6. DA decreases the postexcitation inhibitory phase of odor-evokedresponses. A: DA decreased the duration of the postexcitatory inhibitory phase(I2, arrow) of an AL neuron response. The preexcitatory inhibition phase (I1),which was not affected by DA, is also indicated with an arrow. B: a decreaseof 24.5% was observed for I2 duration of units significantly affected by DA(P � 0.005). Values are means � SE. C: responses of the cell in A to a higherconcentration of the Datura wrightii blend. Note that the postexcitatoryinhibition (I2) phase of the responses is still very prominent during the controland wash phases yet is significantly reduced during the DA superfusion similarto what was observed for the lower odor blend concentration. This suggeststhat the reduction of the I2 phase is not due to an enhancement of excitabilityof the AL neuron, as a higher odor concentration would therefore be expectedto produce a shortened I2 phase. D: DA superfusion had no effect on theduration of odor-evoked inhibition, suggesting that the mechanisms underlyingthe I2 phase and odor-evoked inhibitory responses are not the same. DAapplication had no effect on several measures of background activity includingmean interspike interval (ISI; E), coefficient of variation (CV; F), mean CV2

(G), peak ISI (H), and mean firing rate (I).

Fig. 7. DA increases AL responses without sharpening or broadeningensemble responses. A: mean frequency (spikes/s) elicited by 10�1 dilutionof Datura blend from all of the recorded AL neurons. The identity of eachcell was maintained between population response curves for before DAapplication (black line) and during DA application (gray line). The effectsof DA on responses were independent of a cell’s response strength beforeDA. B: scatterplot of odor-evoked responses (plotted as peak firing rate)before and during DA. The best-fit line (solid line; slope � 0.9756) ofresponses before and during DA is positively shifted from a theoretical1-to-1 ratio (dashed line).



ilar in morphology to the DAArs have been partially filled fromthe AL of Manduca (Homberg et al. 1988), and the location ofthe cell bodies of the DAAr neurons is similar to the PPL1neurons in Drosophila, although the morphologies of theDAAr and the PPL1 neurons are not similar (Mao and Davis2009).

Despite the widespread projections of the DAAr neurons inthe AL, DA did not affect all AL neurons in the same manner,suggesting that the MsDA receptors may not be homoge-neously expressed in AL neurons. In insects, activation ofdifferent DA receptors has opposing effect on cAMP levels(reviewed in Mustard et al. 2005), lending support to a modelin which homogeneous DA release results in heterogeneouseffects on neural responses. For instance, in Drosophila, se-lective dopamine 1-like receptor activation has been attributedto the modulation of cholinergic postsynaptic excitatory poten-tials (Yuan and Lee 2007). Thus the DA-induced decrease inresponse magnitude observed in some cells (Fig. 5D) may beattributable to a direct effect of the activation of specific DAreceptors, although it could also be due to the enhancement oflocal interneurons that provide inhibitory input. Furthermore,different MsDA receptors could mediate distinct effects of DAon AL neuron responses such as the observed enhanced exci-tation (Fig. 5) or shortened postexcitation inhibition (Fig. 6).For instance, the Dop1-type receptor of Drosophila expressionin the mushroom bodies is required for both appetitive andaversive conditioning (Kim et al. 2007), and rescuing theexpression of this receptor in the gamma lobes of the mush-room bodies can fully restore the ability of flies to makeaversive associations (Qin et al. 2012). In addition, the Dro-sophila INDR (DAMB) is highly expressed in the mushroombodies in a manner highly overlapping with rutabaga adenylatecyclase (Han et al. 1996), which is thought to act as a molec-ular coincidence detector during classical conditioning. Theframework of this model is similar to vertebrate systems in thata small number of neuromodulatory neurons act on diverse andheterogeneously expressed receptors in a sensory area. Forinstance, there is a diverse array of serotonin receptors ex-pressed by different cell types within the olfactory bulb(McLean et al. 1995; Morilak et al. 1993; Pazos and Palacios1985), which is innervated by centrifugal neurons from theraphe nuclei (McLean and Shipley 1987). Not only doesserotonin have diverse effects on the response properties of thedifferent cell types in the olfactory bulb (Hardy et al. 2005; Liuet al. 2012; Petzold et al. 2009), but it also facilitates, but is notnecessary for, olfactory learning (Langdon et al. 1997). Theheterogeneity of the effects of biogenic amines within anyneural circuit underlines the importance of future studies inwhich the expression patterns and the effects of selectiveactivation of the receptors are examined.

Different behavioral contexts can establish similar levels ofarousal. Although DA, serotonin, and octopamine differ in thebehavioral context in which they are released, they eachproduce a general increase in the magnitude of the majority ofodor-evoked responses in the ALs of moths, although there aresubtle differences in the effects of these modulators on odor-evoked responses such as the effects of DA on I2 duration (Fig.6). It should also be reiterated that not all AL neuron responseswere enhanced by either DA or serotonin. Serotonin increasedthe odor-evoked responses of 50% of AL neurons in Manduca,while causing a reduction in 6% of AL neurons (Dacks et al.2008). Furthermore, in some instances the decreases in odor-evoked responses caused by serotonin were odor dependent,suggesting that serotonin modulated the inhibitory input to theAL neurons. There are multiple serotonin (Dacks et al. 2006)and DA receptors expressed in the AL, further complicatingthe consequences of modulator release, which could therefore

Fig. 8. DA is necessary for the formation of aversive olfactory associations.A: ion chromatogram of undamaged (hatched line) and larva-damaged (solidline) flower nectar from D. discolor flowers. Herbivory induces higher alkaloidcontent in the floral nectar [peaks a and b; corresponding to scopolamineanalogs (m/z � 65, 81, 94, 108, 138, 154, 273 and m/z � 77, 94, 108, 138, 154,285, respectively)]. B: moths with ALs injected with vehicle (n � 35) anduninjected moths (n � 19) stopped extending their proboscis [proboscisextension response (PER)] in response to the appetitive Datura floral odorwhen it was paired with a synthetic blend of the high-alkaloid nectar. Mothsin which the ALs were injected with a mixture of DA receptor antagonists(“Mix” � SCH39166 and L-741,626 each at 10�8 M; n � 49) respon-ded significantly higher than saline-injected and uninjected moths (*P � 0.05,**P � 0.005, Fisher’s exact test). C: moths in which the ALs were injectedwith the DA receptor antagonist SCH23390 (n � 25) did not form an aversiveassociation between the synthetic toxic nectar and the Datura odor, whilevehicle-injected control moths (n � 19) did decrease their feeding responsesafter repeated pairings of the toxic nectar to the Datura odor. D: moths inwhich the ALs were injected with saline continued to respond at the sameproportions after repeated exposure to either the Datura blend (n � 19) or amineral oil control (n � 20). E: after repeated pairings with 0.1 M quinine,vehicle-injected control moths (n � 18) stopped extending their proboscis(***P � 0.001, Fisher’s exact test) to the odor while moths injected with themixture of DA receptor antagonists (n � 15) continued to respond at the samelevels throughout training. F: injection of DA (n � 19) directly into the ALssignificantly decreased the feeding behavior (proboscis extension) response tothe Datura odor (P � 0.01, Fisher’s exact test) immediately after the injectionbut did not affect the probability of response 2 h after injection. Injection ofsaline (n � 10) or vehicle (saline with 200 �M ascorbic acid; n � 28) did notaffect the immediate or 2 h postinjection responses of moths to the Daturaodor.



be heterogeneous because of the different effects of the acti-vation of each receptor type or the changes in the lateral inputreceived by different neurons as has been shown for serotoninin insects and vertebrates (Dacks et al. 2008, 2009; Hardy et al.2005; Liu et al. 2012; Petzold et al. 2009). In this study, theenhancement by DA selectively increases the activity of par-ticular AL neurons. However, the responses of this subpopu-lation are uniformly elevated, increasing the proportion ofneurons participating in the response without altering (e.g.,broadening or sharpening) the signal-to-noise ratio of thepopulation activity in the AL. Theoretically, this could result inan increase in the overall sensitivity of the olfactory system ina manner similar to lateral excitation evoked at low odorconcentrations (Yaksi and Wilson 2010). In addition to en-hancing odor-evoked responses, DA also affected the slowtemporal patterning of olfactory-driven responses by decreas-ing the postexcitatory inhibition phase (I2). Although the ef-fects of DA on the conductances of Manduca AL neurons areunknown, DA decreases a Ca2�-dependent K� conductance inin vitro honeybee AL neurons (Perk and Mercer 2006), whichcould in theory result in a decreased postexcitatory inhibitionduration. Whatever the biophysical properties modulated byDA, the decreased I2 phase could allow for a faster recoveryfrom excitation, while still preserving the mechanism by whichAL neurons can track the temporal dynamics of an odorstimulus (Lei et al. 2009; Tripathy et al. 2010). It should alsobe noted that it is not known whether the AL neurons affectedby one amine are also affected by other amines. However, thelarge proportion of neurons in the AL affected by biogenicamines makes it likely that there must be some overlap.

The injection of DA receptor antagonists prevented theformation of an aversive olfactory association, suggesting thatDA increases the likelihood that signals from the ALs areassociated with input encoding the aversive stimulus in higher-order associative centers. However, DA injection into the ALalone was not sufficient to induce an aversion to a specificodor. There are two possible explanations for this result. Thismay have been due to a lack of temporal pairing between theDA injection and the presentation of the Datura odor. Weattempted to perform repeated forward pairings of DA injec-tion into the AL with presentation of the Datura odor; how-ever, this had adverse consequences for the health of the moths,which began to extend their proboscis in response to theinjections alone after a few trials. Although it is possible that atight temporal correlation between the DA application and theodor stimulus is required for the formation of an aversion to theDatura odor, it is also possible that the effects of DA on ALneurons are not sufficient to induce the formation of an aver-sive memory. DA release within the AL may serve as asensitizing modulator within the context of aversive learning,increasing AL output and occurring in combination with therelease of DA in the mushroom bodies observed in Drosophila(Aso et al. 2010; Claridge-Chang et al. 2009; Schwaerzel et al.2003). The MB-MP1 dopaminergic neurons in the mushroombodies of Drosophila are sufficient (Aso et al. 2010) but notnecessary for the formation of aversive olfactory associations(Krashes et al. 2009), so it is conceivable that the release of DAin the ALs of Manduca could be necessary but not sufficientfor aversive learning. Furthermore, rescue of the rutabagaadenylate cyclase exclusively in the mushroom bodies ofDrosophila is sufficient to restore aversive learning (Mao et al.

2004; Zars et al. 2000), further suggesting that the ALs them-selves are not the site of memory formation in the brain,despite the observations that olfactory representations in theAL change during learning. However, DA injection did causean immediate decrease in feeding responses to the normallyappetitive Datura floral odor, similar to an aversive response.Thus there may be some information encoded in DA-modu-lated AL output provided in the short term about the aversivecontext in which an odor is encountered, perhaps encoded inthe identity of neurons that are selectively enhanced by DA.

We therefore propose that the release of modulatory aminesin the AL from a small number of neurons that reside outsidethe AL serves to enhance olfactory responses in a particularbehavioral context. This enhancement occurs in tandem withthe processing of relevant contextual cues in other brain re-gions, leading to appropriate behavioral responses. Becauseresources are patchy in their temporal and spatial distributionas well as their reward value, there are several modulatorysystems that alter olfactory processing within these differentcontexts. In addition to the proposed role of serotonin as acircadian modulator of olfactory sensitivity in moths (Klop-penburg and Mercer 2008), serotonin has been proposed inhoneybees to signal the malaise induced after consumption oftoxic foods (Wright et al. 2010) and octopamine is thought tosignal rewarding stimuli during the formation of appetitiveolfactory associations (Hammer and Menzel 1998; Schwaerzelet al. 2003). We found that DA enhances the odor-evokedresponses of AL neurons and that DA receptor antagonistsinjected directly into the AL prevent the formation of aversiveolfactory associations. Our data suggest that, similar to octo-pamine and serotonin, DA enhances olfactory responses in theALs, but within a different context, that of aversion.

ACKNOWLEDGMENTS

We thank K. Teter and O. Zaninovich for cloning the MsDARs, L. Oland,N. Gibson, and P. Jansma for technical assistance with immunocytochemistryand confocal microscopy, M. Gorman for supplying the rabbit anti-MsTHantibody, F. Evers, C. Duch, and M. Landis for assistance with AMIRA, E.Constantopolis, B. Medina, and E. Lutz for help with behavioral experiments,and A. Paulk, and P. Dacks for constructive comments on the manuscript.

GRANTS

This work was supported by National Institutes of Health Grant DC-42092to A. J. Nighorn and NIH Training Grant 1 K12 Gm00708 to A. M. Dacks.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.M.D., J.A.R., and A.J.N. conception and design ofresearch; A.M.D., J.A.R., and A.J.N. performed experiments; A.M.D., J.A.R.,J.P.M., S.L.G., and A.J.N. analyzed data; A.M.D., J.A.R., J.P.M., and A.J.N.interpreted results of experiments; A.M.D., J.P.M., S.L.G., and A.J.N. preparedfigures; A.M.D. drafted manuscript; A.M.D., J.A.R., J.P.M., S.L.G., and A.J.N.edited and revised manuscript; A.M.D., J.A.R., J.P.M., S.L.G., and A.J.N.approved final version of manuscript.

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