Download 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.
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
1
Department of Neuroscience, The University of Arizona, Tucson, Arizona; and 2Department of Biology, University of
Washington, Seattle, Washington
Submitted 22 February 2012; accepted in final form 27 April 2012
insect; antennal lobe; biogenic amines
for food and mates, animals encounter a huge
diversity of sensory stimuli that the nervous system must
encode and decide whether, and in what manner, to respond.
This task is made all the more difficult because most resources
have patchy distributions and varying reward values. This
variability establishes different behavioral contexts in which
sensory information is encoded by the nervous system. The
nervous system must therefore adjust its activity so that behavioral output is maximally beneficial for survival within the
present context. This plasticity is often accomplished when a
given context triggers the release of specific neuromodulators
within restricted brain regions from a small population of
neurons. Modulators can modify neural processing by altering
the efficacy with which neurons respond to stimuli or communicate with each other without directly causing inhibition or
excitation (Kupfermann 1979). For instance, in the mammalian
brain neurons from the ventral tegmental area project to many
other areas and release dopamine (DA) within the context of
reward (Alcaro et al. 2007; Berridge 2007; Schultz 2007; Wise
2004).
WHILE SEARCHING
Address for reprint requests and other correspondence: A. M. Dacks, Mount
Sinai Hospital, Dept. of Neuroscience, Box 1065, Annenberg 21-06, 1468
Madison Ave., New York, NY 10029 (e-mail: [email protected]).
www.jn.org
The insect olfactory system has evolved several modulatory
systems to maximize foraging efficiency for resources that are
patchy in their distribution. For instance, the flowers of Datura
wrightii (a host plant of the moth Manduca sexta) open in the
early evening, making them a temporally patchy resource.
Foraging moths must therefore be maximally sensitive to
olfactory cues at a specific time. The levels of serotonin in the
antennal lobes (ALs; the first synaptic neuropil of the olfactory
system) increase at this time (Kloppenburg et al. 1999), and
serotonin increases the sensitivity and responsiveness of AL
neurons (Dacks et al. 2008; Kloppenburg et al. 1999; Kloppenburg and Hildebrand 1995), suggesting that serotonin acts
as a circadian modulator of olfactory sensitivity (Kloppenburg
and Mercer 2008).
Resources can also be patchy in their reward value. For
instance, there is natural variability in the nicotine (which is
repellant) content of floral nectar in the flowers of the host
plants of Manduca (Kessler and Baldwin 2007; Kessler et al.
2008). An association may then be formed between the nicotine content of the nectar and the floral features so that those
flowers can be avoided in the future. DA release in brain areas
downstream of the ALs has been demonstrated to act as a
molecular signal for the occurrence of an aversive stimulus
during olfactory learning in insects (Aso et al. 2010; Beggs et
al. 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 the
ALs are unknown, and the consequences of the effects of DA
in the AL on behavior have not been examined. We therefore
sought to explore the role of DA as a modulator of olfactory
coding in the AL of Manduca within the context of aversive
learning.
MATERIALS AND METHODS
Moths were reared in the Department of Neuroscience at the
University of Arizona under a 17:7-h light-dark cycle as described
previously (Christensen and Hildebrand 1987).
Immunocytochemistry. For DA immunoreactivity (DA-ir), brains
were labeled with a protocol identical to that published by Dacks and
Nighorn (2011), which also included preadsorption controls for the
DA antibody with Manduca brain tissue. For immunochemical labeling of Manduca tyrosine hydroxylase (TH-ir), brains were dissected in
insect saline and fixed overnight in 4% paraformaldehyde at 4°C.
Brains were then washed in PBS, embedded, and sectioned as described previously (Dacks and Nighorn 2011) except for AMIRA
reconstruction, for which whole mount brains were used. Sections
were washed in PBS with 0.5% Triton X-100 (PBST), blocked for 1
h in PBST with 2% IgG-free BSA, and incubated for 2 days in
1:10,000 rabbit anti-Manduca TH (a generous gift from Dr. Maureen
Gorman at Kansas State University) in PBST with 1% Triton X-100
0022-3077/12 Copyright © 2012 the American Physiological Society
539
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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 sensory
cues varies in different behavioral contexts. Plasticity in sensory
coding can be achieved by the context-specific release of neuromodulators in restricted brain areas. The context of aversion triggers the
release of dopamine in the insect brain, yet the effects of dopamine on
sensory coding are unknown. In this study, we characterize the
morphology of dopaminergic neurons that innervate each of the
antennal lobes (ALs; the first synaptic neuropils of the olfactory
system) of the moth Manduca sexta and demonstrate with electrophysiology that dopamine enhances odor-evoked responses of the
majority of AL neurons while reducing the responses of a small
minority. Because dopamine release in higher brain areas mediates
aversive learning we developed a naturalistic, ecologically inspired
aversive learning paradigm in which an innately appetitive host plant
floral odor is paired with a mimic of the aversive nectar of herbivorized host plants. This pairing resulted in a decrease in feeding
behavior that was blocked when dopamine receptor antagonists were
injected directly into the ALs. These results suggest that a transient
dopaminergic enhancement of sensory output from the AL contributes
to the formation of aversive memories. We propose a model of
olfactory modulation in which specific contexts trigger the release of
different neuromodulators in the AL to increase olfactory output to
downstream areas of processing.
540
DOPAMINERGIC MODULATION OF OLFACTION
(NP_001108338), BmDopR1 (NP_001108459), TcDop1 (not published,
XP_971542), and TcINDR (not published, XM_967686).
Multichannel extracellular recordings. Multichannel extracellular
recordings were performed as described previously (Dacks et al.
2008). Briefly, 2- to 5-day-old moths were secured in plastic tubes
with dental wax, and the cuticle and muscles lying above the brain
were removed. The perineural sheath was removed from the AL, and
physiological saline with 8.55 g/l sucrose and 200 ␮M ascorbic acid
(to reduce the oxidation of DA) was immediately superfused over the
brain. Pilot experiments found no effect of 200 ␮M ascorbic acid on
AL responses. Sixteen-channel extracellular electrode arrays (Neuro
Nexus Technologies, Ann Arbor, MI; catalog no. 434-3mm 50-177)
were inserted into the AL in parallel with the antennal nerve. Extracellular activity was acquired with a RX5 Pentusa base station (Tucker-Davis Technologies, Alachua, FL) and a RP2.1 real-time processor
(Tucker-Davis Technologies), and spike data were extracted from the
recorded signals and digitized at 25 kHz with the Tucker-Davis
Technologies data-acquisition software. Threshold and gain settings
were adjusted independently 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 on the same shank. Offline Sorter v.3
(Plexon Neurotechnology Research Systems, Dallas, TX) was used to
sort extracellular waveforms, and spikes were assigned timestamps to
create raster plots and calculate perievent histograms in Neuroexplorer v.3 (Plexon Neurotechnology Research Systems).
The same experimental protocol was used for all concentrations of
DA applied and the saline-only controls in which saline with 200 ␮M
ascorbic acid was applied from different superfusion containers. AL
neurons were stimulated with ten 200-ms pulses of a five-component
D. wrightii blend (Riffell et al. 2009a) separated by 10 s at a 1:1,000
and then a 1:10 dilution in mineral oil. After the two sets of stimuli,
2 min of spontaneous activity was recorded. The ALs were then
superfused 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 ␮M
ascorbic acid for 5 min. The 200 ␮M ascorbic acid was added to the
saline to prevent the breakdown of DA and was included for all
conditions. The stimulation protocol was then repeated and another 2
min of spontaneous activity recorded. The ALs were then superfused
with physiological saline for 15 min, the stimulation protocol was
again repeated, and another 2 min of spontaneous activity was
recorded.
Analysis of multichannel data. After spikes were assigned timestamps, the number of spikes elicited by each odor stimulus was
determined by counting the number of spikes that occurred in a 1-s
window after the presentation of the stimulus. A neuron was considered to respond if the firing rate after stimulation rose above a
threshold of 1.96 times the standard deviation of background firing
rate. If the firing rate remained above the threshold for ⬎1 s (which
occurred infrequently), then only those spikes elicited in that 1-s
period were counted. To calculate the duration of inhibitory responses, the number of 1-ms bins in which the firing rate dropped to
zero within 500 ms after stimulus presentation were counted. The
duration of the postexcitatory inhibitory phase (or “I2”) was calcu-
Table 1. Tropane alkaloid concentrations in Datura wrightii and
Datura discolor flower nectars
Species
Undamaged
Damaged
Datura wrightii
Datura discolor
0.16 (0.01)
1.33 (0.21)
1.44 (0.10)
23.82 (7.56)
Values (in ␮M) are mean (SE) alkaloid concentrations for n ⫽ 4 –11 plants
per treatment. Plants were either undamaged or Manduca sexta larva damaged
for 72 h prior to flowering.
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and 50 mM sodium azide (PBSAT). Sections were then washed in
PBST, blocked, and incubated overnight in 1:1,000 goat anti-rabbit
Cy3 (Jackson Immunoresearch) in PBSAT. Tissue was then washed in
PBST, cleared, and mounted as described previously (Dacks and
Nighorn 2011). The full characterization of the anti-Manduca TH
antibody is described by Gorman et al. (2007). In Western blots a
second smaller, weaker band was observed (Gorman et al. 2007). We
observed some very weak labeling in addition to the strong labeling
that matched the observed DA-ir. We therefore only traced TH-ir
processes in AMIRA (see below) that also matched the DA-ir processes innervating the AL. Anterograde fills of olfactory receptor
neurons (ORCs) projecting to the macroglomerular complex (MGC)
were performed by cutting the tips of the sex pheromone-sensitive
sensillae of males and placing droplets of dextran-Texas red dissolved
in insect saline over the stumps. The entire antenna was then coated
in petroleum jelly to avoid desiccation (Molecular Probes), and the
moths were allowed to rest overnight so that the dye could be taken up
and transported throughout the ORCs. Brain were then dissected and
processed as described above.
Images were collected with a Zeiss 510 Meta laser scanning
confocal microscope equipped with argon and green HeNe lasers and
appropriate filters. The Zeiss LSM Image Browser was used to create
image stacks and to adjust contrast and brightness. CorelDRAW X4
(Corel, Ottawa, ON, Canada) was used to organize all images and
figures. AMIRA reconstructions were created in Amira 4.1.2 from
confocal scans of a whole mount brain labeled with the Manduca TH
antibody with a custom AMIRA plug-in for reconstruction of threedimensional branching patterns (Evers et al. 2005) generously provided by Dr. Felix Evers (University of Cambridge). The processes
within the AL were not reconstructed, to avoid obscuring the branching patterns of these neurons within the rest of the brain.
Cloning of Manduca dopamine receptors. Degenerate PCR and
RT-PCR were performed as described by Dacks et al. (2006). Antennal
lobe cDNA was isolated by cutting out the ALs only (which is relatively
easy to do because of the large size of the ALs in Manduca) for mRNA
extraction. Degenerate PCR primers were designed based on sequences
from Bombyx mori, Drosophila melanogaster, Papilio xuthus, and Apis
mellifera with Primer Premiere 4.1 (Premiere Biosoft International, Palo
Alto, CA). Degenerate PCR primer sequences used to clone each of the
MsDA receptors were 5=-CGTGATCTCCCTGGACMGNTAYTGGGC-3= and 5=-GAACACCATCACGAACAGAGGNARRTARAA-3= for the MsINDR, 5=-ACCGCYWSNATCTTCAACYTSTG-3=
and 5=-CCAGCANAVNADGAAGACGCCCAT-3= for the MsDop1,
and 5=-TGYTGGBTNCCNTTYTT-3= and 5=-GTRTADATNAYNGGRTT-3= for the MsDop2 receptors. Brain and AL cDNA were
generated with the Omniscript RT kit (Qiagen, Valencia, CA), and
AccuPrime Pfx Supermix (Invitrogen) was used to generate initial fragments for all three DA receptors. Rapid amplification of cDNA ends
(RACE) was used to generate the full-length sequence for the MsINDR
and MsDop1 receptors with the use of the SMARTer PCR Synthesis Kit
(Clontech, Mountain View, CA) to generate the cDNA with a universal
tag sequence and Advantage 2 Polymerase Mix (Clontech) to generate 5=
and 3= fragments with touchdown PCR. The RACE primer sequences
used were as follows: 5=-ACGGTGGTGAGTGAGAACAG-3= for the 5=
MSINDR fragment and 5=-TTCATAGTCTGCTGGCTGCCATTC-3=
for the 3= MsINDR fragment and 5=-GCATAGCAGTAGAGCCTGCAATAT-3= for the 5= MsDop1 fragment and 5=-GACTTTTGCAGGAGTCAACGACTTGC-3= for the 3= MsDop1 fragment. Despite many
attempts with RACE, only a 144-nucleotide fragment of the MsDop2
receptor was obtained.
Sequence alignments in Fig. 3 were constructed with the program
ClustalW (Combet et al. 2000) (http://npsa-pbil.ibcp.fr/cgi-bin/
npsa_automat.pl?pageⴝ/NPSA/npsa_clustalw.html). The GenBank
accession numbers for the sequences used for the sequence alignments in
Fig. 3 were MsINDR (JN_117928), MsDop1 (JN_117929), MsDop2 (not
applicable), DmDD2R (NP_001014758), AmDop2 (NP_001011567), AmDop1 (NP_001011595), AmDop3 (NP_001014983), BmDopR2
DOPAMINERGIC MODULATION OF OLFACTION
For DA receptor antagonist experiments (see Fig. 8, B and D),
moths were injected with either a mixture of 10⫺8 M SCH39166 (a D1
receptor antagonist) and 10⫺8 M L-741,626 (a D2 receptor antagonist)
(both from Tocris Bioscience) or vehicle (moth saline) or were not
injected. Receptor antagonists were selected for their extreme selectivity for DA receptors (Bowery et al. 1996; McQuade et al. 1991). In
addition, we performed experiments in which the ALs of moths were
injected with SCH23390 (see Fig. 8C) at 10⫺6 M and tested the ability
of moths to form an aversive association between the Datura odor and
the simulated toxic nectar (see below). SCH23390 (Tocris) was
selected for its effectiveness in blocking the Dop1- and INDR-type
receptors in the silk moth Bombyx mori (Ohta et al. 2009). Because of
the high degree of sequence similarity for the DA receptors between
Manduca and Bombyx, SCH23390 is likely an effective Dop1- and
INDR-type receptor antagonist in Manduca. However, it should be
noted that SCH23390 has not been directly tested on serotonin or
octopamine receptors in moths, although SCH23390 is ⬃25 times
more effective at blocking the effects of DA compared with octopamine in crude membrane preparations from cockroach brain (Orr et
al. 1987). All solutions included 0.1 ␮M DMSO. For the dopamine
injection experiments (see Fig. 8E), the ALs were injected with
dopamine-HCl at 5 ⫻ 10⫺5 M diluted in moth saline with 200 ␮M
ascorbic 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 the
effects of the pharmacological manipulations while the animal learned
to associate an odor with an aversive stimulus. In the forward-paired
condition, the artificial mimic of the D. wrightii floral scent was
delivered in a 3-s pulse. One second after odor onset, the unconditioned stimulus, which was either 1 ␮l of 0.1 M quinine or the “toxic
nectar” (30 ␮M scopolamine in 20% sucrose), was applied to the
proboscis for ⬃2 s. A 10-min intertrial interval separated each training
trial, and moths were trained over seven or eight trials. After the
conditioning trials were completed, a test trial was performed during
which only the D. wrightii mixture was presented to assess the
behavioral responses as a result of the conditioning treatment. The
ability of moths to form aversive association in backward- or randompairing training paradigms was not tested because of the inability of
moths to form appetitive associations with these training procedures
(Daly and Smith 2000). The toxic nectar used in the forward-paired
paradigms is based on the scopolamine levels shown in Table 1 and
Fig. 8A and sucrose levels normally found in nectar, as herbivory did
not affect sugar concentrations. Fourteen to forty-nine moths were
used for each drug and control treatment group (n ⫽ 247 total moths).
Statistical analyses. All statistical analyses were performed with
GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA) or SPSS
18.0 (IBM, Armonk, NY). For electrophysiological measures a
D’Agostino and Pearson omnibus normality test was applied, and if
data were normally distributed a single-factor repeated-measures
ANOVA with a Tukey honestly significant difference (HSD) post hoc
test was calculated. If data were not normally distributed, a KruskalWallis test with a Dunn’s multiple comparison post hoc test was
performed (P ⬍ 0.05 was assigned as a significance threshold for both
tests). For all normalizations, response measures of a given neuron
(such as peak firing rate or inhibition duration) were normalized to the
maximal response for that neuron to all of the odor stimuli under all
of the treatment conditions. In all behavioral experiments, repeatedmeasures binary logistic regression modeling was used to analyze
proboscis extension response (PER) between treatments. Fisher’s
exact test was used to make specific pairwise comparisons among test
odors and treatments within trials.
RESULTS
Antennal lobes of Manduca are innervated by two pairs of
dopaminergic neurons. To identify dopaminergic input to the
ALs of Manduca we labeled the brains of adult moths for both
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lated as the amount of time in which there were no spikes following
an odor-evoked excitatory burst. All measures of background activity [mean interspike interval, coefficient of variation (CV), CV2,
peak interspike interval, and mean firing rate] were calculated in
MATLAB, and CV2 was calculated as described previously (Holt et
al. 1996).
Nectar collection and analysis. To provide a naturalistic context for
the neural basis of aversive learning, we used the nectar and floral
odors from the Manduca host plants D. wrightii and D. discolor.
Many plants increase their toxic alkaloid content in leaf tissue and
nectar when experiencing herbivory (Adler et al. 2006; Kessler et al.
2008), which subsequently can lead to decreased visitation by the
floral visitors (Gegear et al. 2007). The decreased visitation has been
shown to be mediated by aversive learning by the pollinators (Gegear
et al. 2007; Wright et al. 2010). To examine the effects of herbivory
on 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 to
feed ad libitum on the vegetative tissue for 3 days. After 3 days, nectar
standing crops were collected from flowers on the other branches of
the plant at dusk (2000 PST) from at least 10 newly opened flowers of
each species with 1-ml syringes. Control experiments were conducted
in parallel with plants that had the screen cages but did not have larvae
feeding on the vegetative tissue.
Individual nectar samples were partitioned into water-soluble and
non-water-soluble fractions by adding 500 ␮l of methylene chloride.
The methylene chloride fraction was extracted and concentrated by
gently blowing nitrogen gas over the sample until 20 ␮l remained.
Each sample was stored in a 2-ml borosilicate glass vial with a
Teflon-lined cap at ⫺80°C until analysis. Samples (1 ␮l) were
analyzed with a gas chromatography-mass spectrometric detection
system (GC-MS) consisting of an HP 7890A GC and a 5975C
Network Mass Selective Detector (Agilent Technologies, Palo Alto,
CA). A DB1 GC column (J&W Scientific, Folsom, CA; 30 m, 0.25
mm, 0.25 ␮m) was used, and helium was used as carrier gas at
constant flow of 1 ml/min. The initial oven temperature was 50°C for
5 min, followed by a heating gradient of 6°C/min to 250°C, which was
held isothermally for 6 min. Chromatogram peaks were identified
tentatively with the aid of the NIST mass spectral library (⬃120,000
spectra) and verified by chromatography with authentic standards
(when available). Peak areas for each compound were integrated with
ChemStation software (Agilent Technologies) and are presented in
terms 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 of
4-day-old male moths. As described previously (Lei et al. 2009),
moths were restrained in a plastic tube 30 min prior to scotophase and
kept at room temperature in the light awaiting surgery and injection.
The head capsule was descaled and then opened, and the ALs were
exposed for microinjection. Injection was accomplished via quartz
pipettes (OD 1.0 mm, ID 0.70 mm, Sutter Instruments, San Diego,
CA) pulled with a model P-2000 laser puller (Sutter Instruments) and
clipped to allow solution passage. Pipettes were filled with the
solution to be injected and connected with an output line of a
dual-channel Picospritzer (Picospritzer II, General Valve, East Hanover, NJ). Pipettes were inserted into the center of each AL, and two
drops (mean diameter ⫾ SD: 82 ⫾ 12.1 ␮m) were administered in
quick succession. The diameter was determined before the experiment
by placing the tip of the pipette into a beaker of mineral oil and
measuring the droplet size with an ocular micrometer. After injection
the cuticle window was repositioned and sealed with myristic acid
(Sigma), and the moths were allowed to recuperate for 30 min before
testing. We previously found that drugs delivered in this manner do
not diffuse into other regions of the brain, and that the saline- and
vehicle-injected moths do not behave significantly differently from
noninjected moths (Lei et al. 2009).
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DOPAMINERGIC MODULATION OF OLFACTION
DA and Manduca TH, the rate-limiting enzyme essential to the
production of DA. The AL is comprised of spherical neuropil
called “glomeruli” that receive input from ORCs on the antennae. Olfactory information is transmitted to downstream brain
areas via the projection neurons, and glomeruli are interconnected by a diverse population of local interneurons. DA-ir and
TH-ir were observed in every glomerulus of the AL (Fig. 1, A
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Fig. 1. The antennal lobe (AL) of Manduca sexta is innervated by dopaminergic centrifugal neurons. A: dopamine-immunoreactive (DA-ir) processes
innervate every glomerulus of the AL. B: DA-ir processes innervate the entire
volume of the ordinary glomeruli (dashed border). C: the AL of M. sexta is
innervated by 4 DA-ir processes. Inset: higher-magnification view of the 4
DA-ir branches (asterisks) as they enter the AL. D: tyrosine hydroxylase
immunoreactivity (TH-ir) reveals 4 branches that innervate the AL similarly to
the 4 DA-ir branches in C and weakly labels other neurons (arrow and
arrowhead). Only the 4 strongly labeled branches similar to the DA-ir processes were traced for 3-dimensional (3D) reconstructions. E: while the DA-ir
processes in the AL innervate the entire volume of the ordinary glomeruli, they
innervate only the proximal region (bracket and “P”) of the macroglomerular
complex (MGC) and not the distal region (bracket and “D”). F: mass fills of
olfactory 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 the
axons of the olfactory receptor neurons. All images are presented from a
frontal perspective, and all scale bars ⫽ 100 ␮m except for the inset in C, in
which the scale bar ⫽ 20 ␮m.
and D, respectively), and the DA-ir and TH-ir fibers innervated
the entire glomerular volume (Fig. 1, B and D, respectively)
with the exception of the distal area of the pheromone-sensitive
MGC of male moths (Fig. 1, C, E, and F). The glomeruli that
comprise the MGC are distinct from the other glomeruli in the
AL in that they receive input from ORCs that are selectively
tuned to single components of the female sex pheromone.
Anterograde dye fills of the pheromone-sensitive ORCs on the
antennae demonstrate a lack of overlap between the TH-ir
processes 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 of
these neurons likely affects odor-evoked responses in all glomeruli, rather than a few specialized glomeruli. Both DA-ir and
TH-ir revealed four neurites innervating the ALs (Fig. 1, C and
D). These four processes were traced from the ALs in whole
mount TH-ir preparations to two pairs of cell bodies (Fig. 2,
A–C) in the dorsal protocerebrum, a central brain region in
insects. Each pair of cells projects from the dorso-posterior
protocerebrum to both ALs as well as other higher areas of
processing, including the lateral horn, forming an archlike
structure that bridges both ALs (Fig. 2, D–F). Because of the
arching morphology of these neurons, we refer to them as the
dopaminergic arching (DAAr) neurons.
Although the AL is uniformly innervated by DA-ir/TH-ir
fibers, the effects of DA on AL neurons could be heterogeneous depending on the DA receptors expressed in the AL. We
cloned homologs of the DA 1-type receptor (Dop1), DA 2-type
receptor (Dop2), and invertebrate DA receptors (INDRs) from
Manduca, all of which share high levels of sequence identity
with other insect DA receptors (Fig. 3; MsINDR; 92% sequence
identity with B. mori, MsDop1; 93% sequence identity with B.
mori, MsDop2 fragment; 100% sequence identity with D. melanogaster). RT-PCR of AL cDNA revealed that all three Manduca
DA receptor homologs are expressed in the AL (Fig. 4) and thus
at least these three receptors are potential targets for any
modulatory effects of DA in the AL. Thus the ALs of Manduca
express several DA receptors and receive dopaminergic input
from four centrifugal neurons.
Responses of the majority of antennal lobe neurons are
enhanced by dopamine. In Drosophila (Yu et al. 2004) and
Manduca (Daly et al. 2004) the responses of AL neurons are
transiently enhanced after the formation of aversive or appetitive associations, respectively. This is striking as it suggests
that despite the opposing natures of appetitive and aversive
contexts, there is an increase in the responsiveness of the AL
associated with the formation of either type of olfactory association. Similarly, both octopamine (Barrozo et al. 2010) and
serotonin (Dacks et al. 2008; Kloppenburg et al. 1999) enhance
odor-evoked responses of AL neurons in moths, despite being
associated with different behavioral contexts. We therefore
sought to determine the effects of DA on odor-evoked responses of AL neurons. We performed extracellular multichannel recordings of the responses of AL neurons to two concentrations 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 et
al. 2008). DA increased the number of odor-evoked spikes
(Fig. 5A) in 58% of responsive AL neurons (n ⫽ 30 of 52
neurons that responded to the Datura odor), and this effect was
DA dose dependent (Fig. 5B). Of those neurons enhanced by
DOPAMINERGIC MODULATION OF OLFACTION
543
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-evoked
responses of most AL neurons. DA did not affect the responses
of 31% of AL neurons and significantly reduced the responses
of 11% of AL neurons (Fig. 5D). DA also decreased the
threshold for activation of a few AL neurons (Fig. 5E), suggesting that DA may increase the sensitivity of AL neurons,
resulting in more cells participating in the encoding of olfactory stimuli.
DA also affected the slow temporal dynamics of AL responses (i.e., firing patterns that evolve over hundreds of
milliseconds), which can provide information about the identity of an odor (Laurent et al. 1998) and the physical structure
of an odor plume (Vickers et al. 2001). The excitatory responses of Manduca PNs are often preceded by a rapid
GABAA-dependent inhibition (referred to as “I1”) and followed by a period of spike suppression (“I2”) lasting anywhere
between 10 ms and 1.5 s (Christensen et al. 1996). The I2 phase
shortens 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 plume
structure (Vickers et al. 2001) and produced by the beating of
the moth’s wings (Sane and Jacobson 2006). DA caused a
24.5% decrease in the duration of I2 (Fig. 6, A and B; KruskalWallis test: P ⬍ 0.0001) in 73.5% (25 of 34 units) of units that
displayed the I2 phase. This effect was not due to the enhancement of the magnitude of the excitatory phase overwhelming
the I2 phase, as higher odor concentrations (which increase
response magnitudes) did not affect I2 duration (Fig. 6C). DA
did not, however, affect the duration of purely inhibitory
responses (Fig. 6D), suggesting that odor-evoked inhibition
and the I2 phase are mediated by different cellular or network
mechanisms. Furthermore, DA had no apparent effects on I1
phase. In addition to odor-evoked activity, 2 min of background activity was recorded before, during, and after DA
application to determine whether DA affected the background
activity of AL neurons. However, there was no effect of DA on
several measures of background activity of AL neurons (mean
interspike interval, CV, CV2, peak interspike interval, and
mean firing rate; Fig. 6, E–I, respectively; Kruskal-Wallis test).
The global release of a neuromodulator should result in
widespread effects on the representation of a stimulus by an
ensemble of neurons within a network. However, DA did not
homogeneously affect the responses of individual neurons and
may therefore alter the population response to an odor in one
of several ways. If DA sharpened the overall AL response, we
would expect an enhancement of the strongest responses and a
decrement of the weakest responses (Sachse and Galizia 2002),
increasing the contrast or signal-to-noise ratio. Conversely, if
DA broadened the AL responses, then DA would either decrease or have little effect on the strongest responses and
increase the weaker responses (Olsen et al. 2007; Silbering et
al. 2008), thus recruiting more cells to participate in the odor
representation. In contrast to these two options, we observed
that the relative strength of a neuron’s response does not
predict the modulatory effects of DA (Fig. 7A). Thus the
ensemble response is not homogeneously sharpened or broadened but reorganized by the enhancement of most neurons
across the spectrum of response strength, the decrement of
some responses, and the recruitment of additional neurons.
Comparing pre-DA responses to post-DA responses across the
population (Fig. 7B), the best-fit line is positively shifted from
a 1:1 pre- to post-DA response ratio, indicating that responses
are increased across the population. However, the slope of this
line is close to 1 (slope ⫽ 0.9756), indicating that the overall
population response was increased and that neither significant
sharpening (slope ⬎ 1) nor broadening (slope ⬍ 1) of the
population response occurred.
Injection of dopamine receptor antagonists into the AL
prevents formation of an aversive association. Given the clear
effects of DA on the AL responses of Manduca, and the fact
that the release of DA within the mushroom bodies of Drosophila is necessary (Schwaerzel et al. 2003) and sufficient
(Claridge-Chang et al. 2009) for aversive learning, we investigated the role of DA within the AL in aversive learning.
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Fig. 2. Morphology of the dopaminergic
arching neurons. A–C: consecutive horizontal sections through a TH-ir-labeled M. sexta
brain following the projections of the protocerebral neurons from the ALs to their cell
bodies. Arrowheads indicate the origin of the
protocerebral neurons from the AL in A or
the previous section in B and C. Asterisks
indicate the termination point of the dopamine arching (DAAr) neurons within the
given section including 2 large TH-ir-positive cell bodies in C. All scale bars ⫽ 100
␮m. D–F: 3D reconstructions of 1 pair of
DA/TH-ir AL neurons viewed from frontal,
horizontal, and sagittal perspectives, respectively, of a whole mount preparation. The
processes of these neurons within the ALs
(which are indicated by brackets) have been
omitted to avoid obscuring the branching
patterns of these neurons outside the AL.
The DAAr cell bodies are indicated by asterisks in D–F.
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DOPAMINERGIC MODULATION OF OLFACTION
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Fig. 3. ClustalW sequence alignments for the 3 M. sexta DA receptor homologs. Asterisks indicate conserved amino acid sequence. A: alignment of the MsDop1
receptor with dopamine 1-like receptor homologs from Tribolium casteneum (TcDop1), Bombyx mori (BmDopR1), and Apis mellifera (AmDop1). B: alignment
of 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).
J Neurophysiol • doi:10.1152/jn.00159.2012 • www.jn.org
DOPAMINERGIC MODULATION OF OLFACTION
Fig. 4. Intrinsic neurons of the AL express the Dop1-, Dop2-, and INDR-type
dopamine receptors. RT-PCR for the Manduca homologs of the dopamine
1-like (MsDop1), dopamine 2-like (MsDop2), and invertebrate dopamine
receptor (MsINDR) using cDNA from the AL demonstrates that all 3 receptors
are expressed in the AL. RT⫹ and RT⫺ indicate lanes in which reverse
transcriptase was included or omitted, respectively. White lines between lanes
indicate that RT-PCR products were run on separate gels.
particular, animals injected with the mixture of D1 and D2
receptor antagonists produced significantly more PER responses than control animals at the end of the training protocol
(Fisher’s exact test; P ⬍ 0.001) and had elevated responses
compared with animals injected with the D1 receptor antagonist SCH23390 (Fig. 8C; logistic regression: ␹21 ⫽ 6.84, P ⫽
0.009). In control experiments in which moths injected with
saline only were repeatedly exposed to either the Datura blend
or a mineral oil blank without exposure to an aversive stimulus,
the proportion of moths responding to either olfactory stimulus
remained unchanged (Fig. 8D). This indicated that the decrease
in the proportion of moths responding to the Datura odor when
it is paired with the aversive stimulus is due to associative
processes. Moths injected with the DA receptor antagonists
continued to produce feeding responses at the same proportion
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Aversive learning assays in insects have predominantly relied
on electric shock as an aversive stimulus. Here we take advantage of the natural variation in the amounts of aversive compounds in the floral nectar of the host plants of Manduca
(Kessler et al. 2008). Plant herbivory has cascading effects on
plant physiology that, in turn, influence diverse processes.
Herbivory alters the volatile chemicals emitted by host plants
of Manduca (Kessler et al. 2008) as well as the time of day at
which these plants flower (Kessler et al. 2010). We therefore
tested whether herbivory could also alter the chemical content
of host plant nectar. Manduca caterpillars were allowed to feed
on the vegetative (but not floral) tissue for 3 days, and then the
nectar content of the flowers was collected and analyzed with
a GC-MS system. Herbivory increased the amount of the
tropamine alkaloids (e.g., scopolamine) in the floral nectar of
both D. wrightii and D. discolor (Fig. 8A and Table 1). This, in
combination with natural variability in the nectar content of
nicotine, which is repellant to Manduca (Kessler et al. 2008)
and honeybees (Kohler et al. 2012), suggests that there are
instances in the field in which pollinators encounter flowers
with aversive nectar. We therefore tested whether moths could
form an association between an appetitive olfactory stimulus
(the Datura floral blend) (Riffell et al. 2009b) and an aversive
tastant (a synthetic blend of the nectar of herbivorized Datura
plants), and whether the formation of such an association could
be blocked by DA receptor antagonists.
Naive Manduca extend their proboscis in response to the
Datura floral blend in both a tethered feeding assay (see
MATERIALS AND METHODS) and flight assays (Raguso and Willis
2002, 2005; Riffell et al. 2009a, 2009b), indicating that the
Datura odor is an innately attractive stimulus. This feeding
response was eliminated when a synthetic nectar mimicking
the chemical composition of the nectar from larva-damaged
plants was repeatedly paired with the Datura odor (Fig. 8B;
Fisher’s exact test; P ⬍ 0.005) in a forward-paired learning
paradigm. Trained moths did not respond to the Datura blend
2 h after training (Fig. 8B; Fisher’s exact test; P ⬍ 0.005), thus
representing a learned aversion for the Datura blend. To test
the necessity of DA in the AL for aversive memory formation,
DA receptor antagonists were injected directly into the ALs of
adult moths. Moths injected with either a mixture of D1 and D2
receptor antagonists (Fig. 8B; SCH39166 and L-741,626, respectively) or a D1 receptor antagonist (Fig. 8C; SCH23390)
responded in the same proportion at the end of the training
protocol as at the start, and significantly more than control
animals (Fig. 8B; logistic regression: ␹21 ⬎ 7.15, P ⬍ 0.01). In
545
Fig. 5. DA enhances the responses of AL neurons to the Datura floral blend.
A: peristimulus time histogram demonstrating that the responses of an AL
neuron to the Datura floral blend (10⫺1 dilution) are enhanced by DA. Square
wave pulses indicate the presentation of the 200-ms odor stimulus. Responses
during the control, DA, and washout phases are shown in black, white, and
gray, respectively. B: effects of DA on all AL neurons at several concentrations
of DA. The peak evoked firing rate when all cells were grouped was enhanced
by DA at 5 ⫻ 10⫺5 M (P ⫽ 0.016) and 5 ⫻ 10⫺6 M (P ⫽ 0.047) but not by
DA at 5 ⫻ 10⫺5 M (P ⫽ 0.14) or saline (P ⫽ 0.86). C: the responses of units
that were significantly enhanced by DA had a 58.5% increase in the number of
odor-evoked spikes from control levels (P ⬍ 0.0001). Values are means ⫾ SE.
D: peristimulus time histogram demonstrating that for some cells DA caused
a decrease in magnitude of the odor-evoked responses. E: for some cells DA
application resulted in a shift in the threshold of activation such that responses
were evoked by odor concentrations that were subthreshold during the control
and wash phases.
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546
DOPAMINERGIC MODULATION OF OLFACTION
DISCUSSION
2 h after training (Fig. 8, B, C, and E). The DA receptor
antagonists could not have prevented the detection of the
Datura odor, as injected moths still produced feeding responses when the odor was presented. Furthermore, the injection of the DA receptor antagonists did not cause a nonassociative increase in the PER that masked the decrease in responses, as there was no difference between treatments in the
proportion of naive moths responding at the start of the experiment (trial 0; Fig. 8B, C, and E). The injection of the
mixture of DA receptor antagonists also prevented the formation of an aversive association between the Datura floral blend
and a high-concentration bitter tastant, 0.1 M quinine (Fig. 8E),
both immediately after training and 2 h later (Fisher’s exact
test; P ⬍ 0.001). When DA was injected directly into the ALs,
very few moths exhibited feeding responses to the Datura odor
shortly after the injections (Fisher’s exact test; P ⬍ 0.01),
whereas the majority of moths injected with either saline or
vehicle extended their proboscis in response to the Datura odor
(Fig. 8F). However, 2 h after injection moths in all treatment
groups responded at the same proportion (Fig. 8F).
Fig. 7. DA increases AL responses without sharpening or broadening
ensemble responses. A: mean frequency (spikes/s) elicited by 10⫺1 dilution
of Datura blend from all of the recorded AL neurons. The identity of each
cell was maintained between population response curves for before DA
application (black line) and during DA application (gray line). The effects
of DA on responses were independent of a cell’s response strength before
DA. B: scatterplot of odor-evoked responses (plotted as peak firing rate)
before and during DA. The best-fit line (solid line; slope ⫽ 0.9756) of
responses before and during DA is positively shifted from a theoretical
1-to-1 ratio (dashed line).
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Fig. 6. DA decreases the postexcitation inhibitory phase of odor-evoked
responses. 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 decrease
of 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 higher
concentration of the Datura wrightii blend. Note that the postexcitatory
inhibition (I2) phase of the responses is still very prominent during the control
and wash phases yet is significantly reduced during the DA superfusion similar
to what was observed for the lower odor blend concentration. This suggests
that the reduction of the I2 phase is not due to an enhancement of excitability
of the AL neuron, as a higher odor concentration would therefore be expected
to produce a shortened I2 phase. D: DA superfusion had no effect on the
duration of odor-evoked inhibition, suggesting that the mechanisms underlying
the I2 phase and odor-evoked inhibitory responses are not the same. DA
application had no effect on several measures of background activity including
mean interspike interval (ISI; E), coefficient of variation (CV; F), mean CV2
(G), peak ISI (H), and mean firing rate (I).
The nervous system must adjust its activity to best suit the
behavioral state of the individual animal, which, in turn, is
often established by the external and internal environments. In
this study, we explored the effect of an aversive food stimulus
on the encoding of an associated odor in the primary olfactory
system. The nectar resources that Manduca encounters while
foraging in the field vary in their reward value, with some
expressing aversive compounds (like nicotine and scopolamine). The nervous system of Manduca must be able to
accentuate sensory cues associated with aversive stimuli. We
demonstrate that, in addition to the established role of mediating the formation of aversive olfactory associations in higher
brain areas, DA plays a more transient role of enhancing the
response to olfactory stimuli in the primary sensory neuropil.
DA acts as a molecular mediator of aversive learning in
higher brain areas in a variety of insect species (Aso et al.
2010; Beggs et al. 2007; Beggs and Mercer 2009; ClaridgeChang 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 et
al. 2008) and has also been implicated in appetitive learning in
insects (Kim et al. 2007; Krashes et al. 2009; Selcho et al.
2009). Despite the extensive study DA has received in olfactory learning, the effects of DA release in the primary olfactory
neuropil were unknown. We found that two pairs of DA-ir/
TH-ir neurons (the DAAr neurons) innervated widely throughout each AL, innervating all of the glomeruli, although only the
proximal portion (with relation to the center of the AL) of the
sex pheromone-sensitive MGC glomerulus, which is occupied
by the ORCs (Fig. 1, E and F). Anterograde fills of the ORCs
revealed a lack of overlap between the TH-ir processes and the
ORCs, reminiscent of the innervation pattern of GABA (Reisenman et al. 2011) and serotonin (Sun et al. 1993) in the
MGC of Manduca. This suggests that the MGC may be less
subject to presynaptic modulation relative to the other glomeruli. In fruit flies and bees the ALs are innervated by dopaminergic neurons that are local to the ALs (Chou et al. 2010
and Kirchhof et al. 1999, respectively) and are thus morphologically quite distinct from the DAAr neurons. Neurons sim-
DOPAMINERGIC MODULATION OF OLFACTION
ilar in morphology to the DAArs have been partially filled from
the AL of Manduca (Homberg et al. 1988), and the location of
the cell bodies of the DAAr neurons is similar to the PPL1
neurons in Drosophila, although the morphologies of the
DAAr and the PPL1 neurons are not similar (Mao and Davis
2009).
Despite the widespread projections of the DAAr neurons in
the AL, DA did not affect all AL neurons in the same manner,
suggesting that the MsDA receptors may not be homogeneously expressed in AL neurons. In insects, activation of
different DA receptors has opposing effect on cAMP levels
(reviewed in Mustard et al. 2005), lending support to a model
in which homogeneous DA release results in heterogeneous
effects on neural responses. For instance, in Drosophila, selective dopamine 1-like receptor activation has been attributed
to the modulation of cholinergic postsynaptic excitatory potentials (Yuan and Lee 2007). Thus the DA-induced decrease in
response magnitude observed in some cells (Fig. 5D) may be
attributable to a direct effect of the activation of specific DA
receptors, although it could also be due to the enhancement of
local interneurons that provide inhibitory input. Furthermore,
different MsDA receptors could mediate distinct effects of DA
on AL neuron responses such as the observed enhanced excitation (Fig. 5) or shortened postexcitation inhibition (Fig. 6).
For instance, the Dop1-type receptor of Drosophila expression
in the mushroom bodies is required for both appetitive and
aversive conditioning (Kim et al. 2007), and rescuing the
expression of this receptor in the gamma lobes of the mushroom bodies can fully restore the ability of flies to make
aversive associations (Qin et al. 2012). In addition, the Drosophila INDR (DAMB) is highly expressed in the mushroom
bodies in a manner highly overlapping with rutabaga adenylate
cyclase (Han et al. 1996), which is thought to act as a molecular coincidence detector during classical conditioning. The
framework of this model is similar to vertebrate systems in that
a small number of neuromodulatory neurons act on diverse and
heterogeneously expressed receptors in a sensory area. For
instance, there is a diverse array of serotonin receptors expressed by different cell types within the olfactory bulb
(McLean et al. 1995; Morilak et al. 1993; Pazos and Palacios
1985), which is innervated by centrifugal neurons from the
raphe nuclei (McLean and Shipley 1987). Not only does
serotonin have diverse effects on the response properties of the
different cell types in the olfactory bulb (Hardy et al. 2005; Liu
et al. 2012; Petzold et al. 2009), but it also facilitates, but is not
necessary for, olfactory learning (Langdon et al. 1997). The
heterogeneity of the effects of biogenic amines within any
neural circuit underlines the importance of future studies in
which the expression patterns and the effects of selective
activation of the receptors are examined.
Different behavioral contexts can establish similar levels of
arousal. Although DA, serotonin, and octopamine differ in the
behavioral context in which they are released, they each
produce a general increase in the magnitude of the majority of
odor-evoked responses in the ALs of moths, although there are
subtle differences in the effects of these modulators on odorevoked responses such as the effects of DA on I2 duration (Fig.
6). It should also be reiterated that not all AL neuron responses
were enhanced by either DA or serotonin. Serotonin increased
the 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 odorevoked responses caused by serotonin were odor dependent,
suggesting that serotonin modulated the inhibitory input to the
AL neurons. There are multiple serotonin (Dacks et al. 2006)
and DA receptors expressed in the AL, further complicating
the consequences of modulator release, which could therefore
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Fig. 8. DA is necessary for the formation of aversive olfactory associations.
A: ion chromatogram of undamaged (hatched line) and larva-damaged (solid
line) flower nectar from D. discolor flowers. Herbivory induces higher alkaloid
content in the floral nectar [peaks a and b; corresponding to scopolamine
analogs (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) and
uninjected moths (n ⫽ 19) stopped extending their proboscis [proboscis
extension response (PER)] in response to the appetitive Datura floral odor
when it was paired with a synthetic blend of the high-alkaloid nectar. Moths
in 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) responded 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 injected
with the DA receptor antagonist SCH23390 (n ⫽ 25) did not form an aversive
association between the synthetic toxic nectar and the Datura odor, while
vehicle-injected control moths (n ⫽ 19) did decrease their feeding responses
after repeated pairings of the toxic nectar to the Datura odor. D: moths in
which the ALs were injected with saline continued to respond at the same
proportions after repeated exposure to either the Datura blend (n ⫽ 19) or a
mineral 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 the
mixture of DA receptor antagonists (n ⫽ 15) continued to respond at the same
levels throughout training. F: injection of DA (n ⫽ 19) directly into the ALs
significantly decreased the feeding behavior (proboscis extension) response to
the Datura odor (P ⬍ 0.01, Fisher’s exact test) immediately after the injection
but did not affect the probability of response 2 h after injection. Injection of
saline (n ⫽ 10) or vehicle (saline with 200 ␮M ascorbic acid; n ⫽ 28) did not
affect the immediate or 2 h postinjection responses of moths to the Datura
odor.
547
548
DOPAMINERGIC MODULATION OF OLFACTION
2004; Zars et al. 2000), further suggesting that the ALs themselves are not the site of memory formation in the brain,
despite the observations that olfactory representations in the
AL change during learning. However, DA injection did cause
an immediate decrease in feeding responses to the normally
appetitive Datura floral odor, similar to an aversive response.
Thus there may be some information encoded in DA-modulated AL output provided in the short term about the aversive
context in which an odor is encountered, perhaps encoded in
the identity of neurons that are selectively enhanced by DA.
We therefore propose that the release of modulatory amines
in the AL from a small number of neurons that reside outside
the AL serves to enhance olfactory responses in a particular
behavioral context. This enhancement occurs in tandem with
the processing of relevant contextual cues in other brain regions, leading to appropriate behavioral responses. Because
resources are patchy in their temporal and spatial distribution
as well as their reward value, there are several modulatory
systems that alter olfactory processing within these different
contexts. In addition to the proposed role of serotonin as a
circadian modulator of olfactory sensitivity in moths (Kloppenburg and Mercer 2008), serotonin has been proposed in
honeybees to signal the malaise induced after consumption of
toxic foods (Wright et al. 2010) and octopamine is thought to
signal rewarding stimuli during the formation of appetitive
olfactory associations (Hammer and Menzel 1998; Schwaerzel
et al. 2003). We found that DA enhances the odor-evoked
responses of AL neurons and that DA receptor antagonists
injected directly into the AL prevent the formation of aversive
olfactory associations. Our data suggest that, similar to octopamine and serotonin, DA enhances olfactory responses in the
ALs, 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 immunocytochemistry
and confocal microscopy, M. Gorman for supplying the rabbit anti-MsTH
antibody, 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-42092
to 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 of
research; 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. prepared
figures; 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.
REFERENCES
Adler LS, Wink M, Distl M, Lentz AJ. Leaf herbivory and nutrients increase
nectar alkaloids. Ecol Lett 9: 960 –967, 2006.
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be heterogeneous because of the different effects of the activation of each receptor type or the changes in the lateral input
received by different neurons as has been shown for serotonin
in insects and vertebrates (Dacks et al. 2008, 2009; Hardy et al.
2005; Liu et al. 2012; Petzold et al. 2009). In this study, the
enhancement by DA selectively increases the activity of particular AL neurons. However, the responses of this subpopulation are uniformly elevated, increasing the proportion of
neurons participating in the response without altering (e.g.,
broadening or sharpening) the signal-to-noise ratio of the
population activity in the AL. Theoretically, this could result in
an increase in the overall sensitivity of the olfactory system in
a manner similar to lateral excitation evoked at low odor
concentrations (Yaksi and Wilson 2010). In addition to enhancing odor-evoked responses, DA also affected the slow
temporal patterning of olfactory-driven responses by decreasing the postexcitatory inhibition phase (I2). Although the effects of DA on the conductances of Manduca AL neurons are
unknown, DA decreases a Ca2⫹-dependent K⫹ conductance in
in vitro honeybee AL neurons (Perk and Mercer 2006), which
could in theory result in a decreased postexcitatory inhibition
duration. Whatever the biophysical properties modulated by
DA, the decreased I2 phase could allow for a faster recovery
from excitation, while still preserving the mechanism by which
AL neurons can track the temporal dynamics of an odor
stimulus (Lei et al. 2009; Tripathy et al. 2010). It should also
be noted that it is not known whether the AL neurons affected
by one amine are also affected by other amines. However, the
large proportion of neurons in the AL affected by biogenic
amines makes it likely that there must be some overlap.
The injection of DA receptor antagonists prevented the
formation of an aversive olfactory association, suggesting that
DA increases the likelihood that signals from the ALs are
associated with input encoding the aversive stimulus in higherorder associative centers. However, DA injection into the AL
alone was not sufficient to induce an aversion to a specific
odor. There are two possible explanations for this result. This
may have been due to a lack of temporal pairing between the
DA injection and the presentation of the Datura odor. We
attempted to perform repeated forward pairings of DA injection into the AL with presentation of the Datura odor; however, this had adverse consequences for the health of the moths,
which began to extend their proboscis in response to the
injections alone after a few trials. Although it is possible that a
tight temporal correlation between the DA application and the
odor stimulus is required for the formation of an aversion to the
Datura odor, it is also possible that the effects of DA on AL
neurons are not sufficient to induce the formation of an aversive memory. DA release within the AL may serve as a
sensitizing modulator within the context of aversive learning,
increasing AL output and occurring in combination with the
release of DA in the mushroom bodies observed in Drosophila
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