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The Journal of Neuroscience, March 15, 2000, 20(6):2391-2399
Response Characteristics of an Identified, Sexually Dimorphic
Olfactory Glomerulus
Jane Roche
King,
Thomas A.
Christensen, and
John G.
Hildebrand
Arizona Research Laboratories, Division of Neurobiology, University
of Arizona, Tucson, Arizona 85721-0077
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ABSTRACT |
Partitioning of synaptic neuropil into glomeruli is a common
feature of primary olfactory centers in most animal species. The
functional significance of glomeruli, however, is not yet well
understood. The present study is part of our effort to test the
hypothesis that each glomerulus is a functional unit dedicated to
processing information about a particular odorant or attribute of odor
molecules and that the glomerular array constitutes a map of "odor
space." We investigated the physiological and morphological features
of uniglomerular projection neurons (PNs) associated with an identified
glomerulus in each antennal lobe of the female sphinx moth,
Manduca sexta. This "lateral large female
glomerulus" (latLFG) is sexually dimorphic and therefore may play a
female-specific role, such as processing of information about one or
more odorants important for orientation of a female to host plants for
oviposition. Together with the medial LFG (medLFG), the latLFG resides
outside the array of spheroidal ordinary glomeruli, near the entrance of the antennal (olfactory) nerve. Each LFG is innervated by four to
five PNs. Using intracellular recording and staining, we examined the
responses of latLFG-PNs to odorants that represent major classes of
volatiles released by host plants of M. sexta. All
latLFG-PNs were excited when the ipsilateral antenna was stimulated
with low concentrations of the monoterpenoid linalool. Dose-response analysis showed that neither other monoterpenoids nor
representatives of other classes of host plant volatiles were similarly
stimulatory to latLFG-PNs. These findings are consistent with the idea
that each glomerulus has a characteristic, limited molecular receptive range.
Key words:
olfaction; antennal lobe; glomeruli; sexual dimorphism of
the CNS; insect; Manduca sexta
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INTRODUCTION |
Unlike the visual, auditory, and
somatosensory systems, the olfactory system lacks a precise spatial map
of the periphery. How central neural space is used to map olfactory
information is only beginning to be understood. Primary olfactory
centers typically exhibit condensed neuropil structures called
glomeruli. According to a long-established hypothesis, each glomerulus
is a functional unit dedicated to processing information about a particular odorant or attribute of odor molecules, and the glomerular array constitutes a chemotopic map of "odor space" (for
review, see Hildebrand, 1995 ; Buck, 1996; Hildebrand and
Shepherd, 1997; Christensen and White, 2000).
Activity-dependent labeling with 2-deoxyglucose or voltage- or
Ca2+-sensitive dyes has revealed that
odors elicit reproducible patterns of activity across the glomerular
array in the vertebrate olfactory bulb and insect antennal lobe (AL)
(Teicher et al., 1980; Rodrigues and Buchner, 1984; Rodrigues, 1988;
Kauer and Cinelli, 1993; Friedrich and Korsching, 1997, 1998; Joerges
et al., 1997; Distler et al., 1998). In addition, single-unit
recordings have shown that neighboring output neurons, presumably with
arborizations in the same glomerulus, are more likely to respond
similarly to individual odorants than are more widely separated output
neurons associated with different glomeruli (Buonviso and Chaput, 1990;
Chaput, 1990). Such studies have supported the idea that glomeruli are
functionally significant modules but have not tested their specificity
by examining the anatomical and physiological properties of output
neurons from identified glomeruli.
To understand glomerular function, it is important to study glomeruli
that are identifiable across individuals, a prerequisite that has been
met by several insect systems (Chambille et al., 1980; Vickers et al.,
1998; Rospars and Hildebrand, 1992, 2000). In the sphinx moth,
Manduca sexta, for example, the male-specific macroglomerular complex (MGC) is a cluster of identified glomeruli dedicated to processing information about individual components of the
conspecific female's sex pheromone (Hildebrand, 1996). Little is
known, however, about the olfactory tuning of other glomeruli in the ALs.
Female M. sexta use olfactory information to locate host
plants for oviposition (Willis and Arbas, 1991). Recently, a pair of
sexually dimorphic glomeruli has been described in the female AL
(Rössler et al., 1998; Rospars and Hildebrand, 2000). These LFGs
are particularly attractive for studies of glomerular function because
they are identified, characteristically situated, and therefore
favorable for physiological recording.
We have begun to examine the functions of the LFGs and to test the
hypothesis of chemotopy by studying anatomical features and molecular
receptive ranges (Mori and Shepherd, 1994) of output neurons with
arborizations confined to the LFGs. For olfactory stimulation, we used
single compounds prominent in headspace-volatile mixtures emitted by
host plants. Here we report that the lateral LFG responds best to only
one of a selected set of odorants representing three chemical classes
and thus may have a narrow molecular receptive range. Our findings
suggest that like the glomeruli of the chemotopically organized male's
MGC, the LFGs process olfactory information about a limited range of
odor chemistry.
A preliminary report of some of this work has appeared previously (King
et al., 1998).
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MATERIALS AND METHODS |
Preparation. Manduca sexta (L.)
(Lepidoptera: Sphingidae) were reared in the laboratory on an
artificial diet under a long-day photoperiod regimen, as described
previously (Sanes and Hildebrand, 1976). Adult female moths were used
1-4 d after emergence and prepared for experiments as described
previously (Christensen and Hildebrand, 1987). For electrophysiological
recording, a moth was restrained in a plastic tube with its head
exposed at one end. The labial palps, proboscis, and cibarial
musculature were then removed to allow access to brain. To eliminate
movement, the head of the moth was isolated and pinned to a
Sylgard-coated glass Petri dish with the ALs facing upward. Tracheae
and a small part of the sheath overlying one AL were removed with fine
forceps. The preparation was continuously superfused for the duration
of the experiment with physiological saline solution containing (in mM): 150 NaCl, 3 CaCl2, 3 KCl, 10 TES buffer, and 25 sucrose, pH 6.9.
Stimulation. After the head of the moth had been pinned to
the Sylgard-coated dish, the tip of one antenna was removed, and the
cut end of the antenna was inserted into a thin-walled borosilicate capillary tube filled with physiological saline solution. This capillary served both as a holder to suspend the antenna and as an
electrode for electroantennogram recording to monitor responses of the
antenna to stimuli. Once the antenna was suspended, a glass tube
carrying a stream of humidified, charcoal-filtered air was positioned
~10 mm in front of the antenna. Odorants were injected into this
continuous air stream by means of a motor-driven syringe olfactometer
(Selchow, 1998). The olfactometer was activated by a
computer-controlled command pulse using customized ASYST software (Keithly Instruments, Rochester, NY). For most trials, a single, 200 msec odor pulse was delivered to the antenna, but in some cases, five
sequential 50 msec pulses were presented.
Because an intracellular impalement could be held for no more than
40-50 min at best, there was sufficient time to test several concentrations of only a selected few odor stimuli in this effort to
characterize the neurons of the latLFG. The odorants used for characterization of neuronal response profiles represent three chemical
classes: the monoterpenoids linalool
([±]3,7-dimethyl-1,6-octadien-3-ol, catalog no. L-5255; Sigma, St.
Louis, MO), nerol (cis-3,7-dimethyl-2,6-octadien-1-ol, catalog no. 26,890-9; Aldrich, Milwaukee, WI),  -ocimene
(3,7-dimethyl-1,3,6-octatriene, catalog no. 74730; Fluka, Buchs,
Switzerland), and -myrcene (3-methylene-7-methylocta-1,6-octadiene, catalog no. M 0382; Sigma); an aliphatic aldehyde,
trans-2-hexenal [(t-2-h) catalog no. H 6765, Sigma]; and
an aromatic ester, methyl salicylate [(ms) methyl-2-hydroxybenzoate,
catalog no. M 6752, Sigma]. These compounds (Fig.
1) are produced by the vegetative and/or
floral parts of host plants of M. sexta (Buttery et al., 1987; Andersen et al., 1988; Loughrin et al., 1990; Knudsen et al., 1993; Raguso and Willis, 1997; W. Mechaber and J. G. Hildebrand, unpublished observations). In addition to these single
compounds, the blend of headspace volatiles released in a syringe by a
single fresh leaf from a tomato plant (a preferred host plant of
M. sexta) was used as a stimulus. Single odor compounds were
diluted to various concentrations in odorless light mineral oil
(Sigma). A total of 50 µl of the final dilution was applied to a 20 mm disk of Whatman number-1 filter paper. The odor-bearing filter paper
was immediately inserted into a capped 20 ml syringe, which was allowed
to equilibrate for at least 4 hr before use to ensure quantitatively
reproducible odor delivery (Selchow, 1998). Dilutions resulted in a
final application of 0.005 µl (1:104) to
5 µl (1:10) of pure compound on the filter paper. Based on differences in the specific gravities and purities of the compounds, the final gravimetric loads at the 1:103
dilution ranged from 35.56 µg for -myrcene to 58.41 µg for
methyl salicylate per 50 µl (~1 µg/µl for all compounds).
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Figure 1.
Chemical structure of host plant-related volatiles
used to test the responses of uniglomerular PNs associated with the
latLFG. Three classes of odorants were used: monoterpenoids (top
panel), an aliphatic aldehyde (middle
panel), and an aromatic ester (bottom
panel). Note the structural relatedness of the
monoterpenoids.
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Intracellular recording and staining. Intracellular
recording was performed as described previously (Christensen and
Hildebrand, 1988). Borosilicate glass microelectrodes filled with a 4%
solution of Lucifer Yellow CH (LY) (Sigma) in 0.2 M
LiCl, and having resistances of 100-400 M , were used for
intracellular recording and staining. Microelectrodes were manipulated
to penetrate the glomerular region of the AL above the known location
of the LFGs, so that LFG-PNs could be targeted in these experiments.
The responses of PNs to stimulation of the ipsilateral antenna with
odorants were monitored on an oscilloscope and directly digitized with
a computer using Axoscope software (Version 1.1, Axon Instruments,
Foster City, CA). After physiological characterization, cells were
injected with LY by passing hyperpolarizing current (up to 1 nA) for
5-15 min. Longer injections usually resulted in more dense and
complete staining of neurons.
Immediately after the completion of an experiment, the brain was
excised and immersed in formaldehyde fixative solution (2.5% formaldehyde in 0.1 M phosphate buffer with 3% sucrose
added) in a glass vial. Brains were fixed for at least 1 hr, after
which they were dehydrated by passage through a graded series of
ethanol solutions and finally cleared with methyl salicylate. Brains
used for initial glomerular identification were post-fixed in 2%
glutaraldehyde in 0.1 M phosphate buffer to enhance
contrast. Cleared brains were viewed as whole mounts with a
laser-scanning confocal microscope [Bio-Rad MRC-600 (Bio-Rad,
Cambridge, MA) equipped with a Nikon Optiphot-2 microscope and both 15 mW krypton/argon and 100 mW argon laser light sources]. Preparations
found to contain stained neurons were returned to 100% ethanol and
embedded in Spurr's resin (Electron Microscopy Sciences, Ft.
Washington, PA) in preparation for serial sectioning. Sections (48 µm) were then cut on a sliding microtome, and images were collected
from each section at 2 µm optical steps with the confocal microscope.
The stained neuron was reconstructed from these images so that the
glomerulus housing its arborization could be determined.
Data analysis. Frequency analysis of spike trains was
performed with the aid of a DOS-based program written in Turbo Pascal by Dr. J. Douglass (ARL Division of Neurobiology, University of Arizona). Electrophysiological records saved as text files were imported into the program, where they were converted to instantaneous spike frequency (ISF) values. These values were then plotted against time of occurrence of each spike to yield histograms plotting instantaneous frequency versus time. For dose-response curves, the
peak value of ISF obtained during each 6 sec recording period was used
to calculate the mean (± SEM) for each odor compound at each concentration.
Three-dimensional reconstructions. Methods described by
Vickers et al. (1998) were used for three-dimensional (3-D)
reconstruction of ALs containing LY-injected neurons. Briefly, 2 µm
optical sections from glutaraldehyde-fixed whole mounts and from 48 µm Spurr's sections were collected with the laser-scanning confocal
microscope. Using Confocal Assistant software (version 4.02, Todd
Brelje, Bio-Rad), five individual optical sections were stacked to
produce an image representing an optical slab of tissue ~10 µm
thick. These images were then printed with a 600 dpi laser printer. The borders of the AL and individual glomeruli were traced and digitized using a digitizing tablet (model XGT, Kurta, Phoenix, AZ) to
yield a bitmap file representing each section. These bitmap files were then rendered in 3-D, using software custom-designed by Nirav Merchant
and Amit Pandy (ARL Division of Neurobiology, University of Arizona),
on a Silicon Graphics (Mountain View, CA) Indigo computer. With Cosmo
Worlds software (Silicon Graphics), 3-D images were rotated in any
plane to allow visualization of glomeruli from various aspects.
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RESULTS |
Spatial organization of the large female glomeruli
With the aid of 3-D reconstructions, we have identified
morphologically a number of glomeruli in the AL of M. sexta
that are favorable, by virtue of their size and location, for
physiological studies. Among these are two prominent glomeruli in the
female AL, previously designated "lateral female glomeruli"
(Rössler et al., 1998). In view of their locations within the AL
and their sizes, these two identified, sexually dimorphic glomeruli
have been renamed the large female glomeruli (Rospars and
Hildebrand, 2000). Based on 3-D reconstructions of eight ALs from eight
different female moths, we have found that like the MGC in each AL of
male moths (Matsumoto and Hildebrand, 1981; Hansson et al., 1991;
Vickers et al., 1998), the LFGs reside near the entrance of the
antennal nerve into the AL, outside the array of ordinary glomeruli
(Fig. 2A,B). The LFGs
directly abut each other, with one, designated the latLFG, at the
lateral edge of the AL. This glomerulus lies slightly anterior to the
more posteromedially situated LFG, designated the medLFG (Fig.
2C). The sizes of the LFGs and their positions, relative to
landmarks such as the entrance of the antennal nerve and the lateral
group of neuronal cell bodies in each AL, are relatively constant among
individuals, so that the LFGs are readily identifiable in confocal
stacks. Each LFG is innervated by the axon terminals of numerous
olfactory receptor cells (ORCs), at least four to five uniglomerular
projection (output) neurons (PNs) (Fig.
3), a number of multiglomerular local
interneurons, and possibly multiglomerular projection neurons (Homberg
et al., 1988). In this study, we examined olfactory responses of the
uniglomerular PNs.
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Figure 2.
Sexually dimorphic glomeruli in M. sexta.
A, B, Confocal images of ALs from a male
(A) and a female (B)
M. sexta. In the male AL, the macroglomerular complex
(MGC) resides at the entrance of the antennal nerve
(AN), outside of the shell-like array of
spheroidal "ordinary" glomeruli (G).
Occupying a similar position in the female AL are the two LFGs. The
lateral group of neuronal cell bodies (LC) appears in
each section. C, 3-D reconstruction of a female AL
showing the positions of the medLFG and latLFG from three different
views. The top panel shows a frontal view, the
middle panel shows a back or posterior view, and the
bottom panel shows a view of the LFGs along the axis of
the antennal nerve. The medLFG is shown in light gray,
and the latLFG is shown in black. D,
Dorsal; M, medial; P, posterior. Scale
bar, 50 µm.
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Figure 3.
Multiple uniglomerular projection neurons with
arborizations confined to an LFG. A, A confocal stack
showing four or five PNs with dendritic arborizations restricted to the
latLFG. The medLFG, directly adjacent to the latLFG, contains no
neurites of these PNs. B, A confocal stack showing four
PNs with dendritic arborizations restricted to the medLFG. In this
case, the latLFG contains no processes of these neurons. Each of these
PNs has a soma located in the medial group of neuronal cell
bodies (MC). AN, Antennal nerve;
D, dorsal; L, lateral. Scale bars, 100 µm.
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Physiological response properties of uniglomerular projection
neurons innervating the lateral large female glomerulus
Because glomerular assignment was crucial to this study of AL PNs,
analysis and conclusions are based only on neurons that were
physiologically characterized and stained intracellularly with Lucifer
Yellow. We report on 10 physiologically characterized and stained PNs,
in 10 different female moths, which had dendritic arborizations
restricted to the latLFG (Figs. 3A, 4). Each of these PNs
had its soma located in the medial group of AL neuronal somata (Homberg
et al., 1988). In five preparations in which the PN axons were
completely stained, it could be ascertained that these PNs were PIa
neurons, based on the nomenclature of Homberg et al. (1988). The axons
of these PNs projected via the inner antenno-cerebral tract to the
calyces of the ipsilateral mushroom body and the lateral horn of the
protocerebrum (Fig.
4E,F,G).
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Figure 4.
Morphology of uniglomerular PNs arborizing within
the latLFG. A, Confocal stack collected from a
whole-mount preparation, showing PNs with somata in the medial group of
neuronal cell bodies (MC), dendritic arborizations
restricted to the latLFG, and axons (Ax) projecting from
the antennal lobe (AL). B,
C, Confocal images collected from the same preparation
shown in A after sectioning, illustrating more clearly
the close anatomical relationship between the medLFG and latLFG. The
dendritic arborizations of these PNs were restricted to the latLFG.
Somata are not visible in either section. D, High
magnification (60×) confocal stack of the latLFG shown in
B. The fine dendritic arborizations of these PNs were
highly branched and extended throughout the entire glomerulus.
E, F, Axonal projection patterns of these
latLFG-PNs. Axons (arrows in E) projected
through the inner antennocerebral tract to the calyces of the
ipsilateral mushroom body (CMB) and the lateral horn of
the protocerebrum. Axon terminals are visible in the CMB indicated in
F by arrows. G, Schematic
illustration (horizontal view) showing the general morphology of
latLFG-PNs. All latLFG-PNs observed to date shared the same basic
morphology and are classified as type PIa neurons (Homberg et al.,
1988). LH, Lateral horn of the protocerebrum;
OL, optic lobe; D, dorsal;
L, lateral. Dotted lines indicate the
outlines of the ALs and the mushroom bodies. Scale bars:
A-C, E, F,
100 µm; D, 50 µm. All images were inverted using
Corel Photo-Paint.
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Intracellular recordings from latLFG-PNs showed that linalool was
strongly excitatory even at the lowest concentration tested (Figs.
5, 6).
Increasing the concentration of linalool led to increases in ISF during
the initial excitatory phase of the responses (Fig. 5,
insets) (at 1:10,000, peak ISF was 140.6 sec 1; at
1:1000, peak ISF was 196.08 sec1). A
higher concentration of linalool also resulted in a decrease in the
latency of the excitatory response (Fig. 5) (at 1:10,000, latency from
the beginning of the stimulus pulse to the first spike was 302.3 msec;
at 1:1000, latency was 220.6 msec). The excitatory phase of the
response to linalool was followed by membrane hyperpolarization,
resulting in cessation of spiking activity (Figs. 5, 6). The duration
of both the excitatory and inhibitory phases increased with increasing
concentration. A summary of these physiological findings is presented
in Table 1.
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Figure 5.
Physiological records collected from one of the
stained latLFG-PNs of Figure 4. This neuron was tested with various
concentrations of three single odorants [linalool,
trans-2-hexenal (t-2-h), methyl
salicylate (ms)], along with the volatile blend emitted
by a whole tomato leaf and a mineral oil blank. Stimulus onset is
indicated by the vertical dotted line. Stimulus duration
was 200 msec, marked by the solid line under each
record. This neuron was strongly excited when the ipsilateral antenna
was stimulated with linalool even at the lowest concentration tested
(1:10,000). Insets in the first two records show a 300 msec segment on an expanded time scale. These insets
illustrate the increase in instantaneous spike frequency with an
increase in the concentration of linalool. The other odor compounds
tested did not evoke an excitatory response from this cell, even at
dilutions up to 1:100. Volatiles from a whole tomato leaf and a mineral
oil blank also failed to elicit an excitatory response from this
PN.
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Figure 6.
Morphology and physiological properties of another
latLFG-PN. The panel in the top left
corner is a confocal stack through a section showing the two
LFGs. The dendritic arborization of this single, uniglomerular PN was
restricted to the latLFG. Scale bar, 50 µm. Image was inverted in
Corel Photo-Paint. Representative physiological records demonstrate
that this latLFG-PN was strongly excited by linalool, whereas the other
single compounds (t-2-h and ms), as well
as the blend of volatiles from a tomato leaf and the mineral oil blank,
failed to cause any change in background spiking activity. The response
to linalool was biphasic, consisting of an excitatory phase followed by
a period of hyperpolarization. The response profile obtained from this
latLFG-PN is almost identical to the pattern of responses illustrated
in Figure 5. Stimulus onset is indicated by the vertical dotted
line. Stimulus duration was 200 msec, as indicated by the
solid line under each record.
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The two other single odorants (t-2-h and ms, even at 10-fold higher
concentration), the blend of tomato-leaf volatiles, and the mineral oil
blank all failed to evoke an excitatory response from these PNs (Figs.
5, 6). Higher concentrations of the single odorants sometimes elicited
an inhibitory response resulting in a cessation of background spiking
activity (Fig. 5).
These observations were consistent across all 10 PNs that had
arborizations exclusively in the latLFG (Fig.
7). Each latLFG-PN was strongly and
selectively excited in response to antennal stimulation with linalool.
Although their response profiles were similar, these PNs varied in
their overall sensitivity to linalool as indicated by the variation in
the ISF on stimulation with a concentration of 1:1000 (Fig. 7,
top row). In addition, two of the PNs (1 and 3) were excited in response to the blend of tomato-leaf
volatiles. That stimulus was likely to be variable from one experiment
to another, owing to differences among leaf samples used in the
stimulus syringe, which probably accounts for the excitation observed
in these PNs. It is also possible that these two neurons may represent a different class of latLFG-PNs that respond to other compounds in
addition to linalool.
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Figure 7.
Plots of instantaneous spike frequency versus time
for the 10 latLFG-PNs included in this study. Shown are representative
records from each neuron when stimulated with linalool,
trans-2-hexenal and methyl salicylate, each at a
concentration of 1:1000. Also shown are representative responses from
each neuron when it was stimulated with the volatile blend from a whole
tomato leaf and the mineral oil blank. PNs were numbered in order of
ascending peak instantaneous spike frequencies. Stimulus onset is
indicated by the vertical dashed line in each column.
The stimulus was a single, 200 msec odor pulse. All latLFG-PNs
exhibited a marked excitatory response when the ipsilateral antenna was
stimulated with linalool, with instantaneous spike frequencies reaching
up to 250 sec1 (top
row). Two of the 10 latLFG-PNs were also excited by the tomato
leaf volatiles (second row, neurons 1 and
3). None of the other compounds tested elicited an
excitatory response from these PNs. NT, Not
tested.
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Dose-response analysis further demonstrates the sensitivity and
selectivity of latLFG-PNs to linalool (Fig.
8). Some of the latLFG-PNs were tested
with a larger set of single odorants at various concentrations to
determine whether latLFG-PNs were responsive only to linalool or if
other compounds with similar chemical structures could also elicit
responses. Data from two latLFG-PNs (Fig. 8) show that linalool was the
strongest stimulus, driving the cells to a greater ISF than any of the
other odorants delivered at a similar concentration. Of the
monoterpenoids tested, only nerol could elicit excitatory responses in
these PNs, but with a potency roughly 1% that of linalool (Fig. 8).
The ISF values recorded after delivery of the other compounds,
including tomato-leaf volatiles, were comparable to background firing
and to the responses to stimulation with the mineral oil blank. Thus,
it appears that the latLFG-PNs were narrowly tuned to linalool or a
compound related to it. These dose-response curves also suggest that
latLFG-PNs may vary in sensitivity to linalool as reflected in the peak
ISF for linalool at the various concentrations tested.
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Figure 8.
Dose-response curves for two latLFG-PNs. To
explore their response characteristics, some of the latLFG-PNs were
tested with a wider selection of monoterpenoids (Fig. 1) at various
concentrations. Dose-response curves were constructed by plotting the
mean peak instantaneous spike frequency (means ± SEM) versus
concentration. The plots show that linalool was the strongest stimulus
for both neurons. Nerol could also elicit an excitatory response from
these cells, but a much higher concentration was required, and the peak
instantaneous frequency was still substantially lower than that
attained during the excitatory response to linalool. These two
latLFG-PNs differed in their background firing rates and their overall
sensitivities to linalool. The PN in the top panel (PN
6 in Fig. 7) exhibited a higher background firing rate
(~50 sec1) and greater
overall sensitivity to linalool in comparison to the PN in the
bottom panel (PN 5 in Fig. 7).
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In addition to chemical information, the moth's olfactory system also
encodes information about the temporal features of odor stimuli
(Christensen et al., 1996). Several latLFG-PNs were tested with 50 msec
pulses of odor delivered at various rates to test the ability of these
PNs to follow pulses of linalool. The latLFG-PNs could follow odor
pulses delivered at rates of three or four pulses per second (Fig.
9). Each odor pulse evoked a discrete
onset of action potentials that was abruptly terminated by a strong
membrane hyperpolarization. This inhibitory mechanism therefore ensures that the neurons will remain inactive during the interval before the
next stimulus arrives, thus increasing the "contrast" between odor
pulses. This inhibition may last for several seconds, as is evident in
the hyperpolarizations evoked by the fifth pulse in the stimulus train
of Figure 9. These findings illustrate that the ability of the
olfactory system to encode information about the spatiotemporal
properties of the stimulus is not limited to males or to pheromone
information processing (Hansson and Christensen, 1999).
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Figure 9.
Temporal resolution of linalool pulses by two
latLFG-PNs. A, Response of a latLFG-PN (PN
10 in Fig. 7) on stimulation of the ipsilateral antenna
with linalool (1:1000) delivered at a rate of four pulses per
second. B, Response of a latLFG-PN (PN
6 in Fig. 7) to linalool (1:1000) delivered at a rate of
three pulses per second. Each pulse was 50 msec in duration, as
indicated by the bars under each record.
Insets in each panel are plots of instantaneous spike
frequency (ISF) versus time for the physiological
record. In both cases, the latLFG-PN closely followed the presentation
of the pulsed stimulus.
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DISCUSSION |
The insect AL is an experimentally favorable model system for
studies of the function of glomeruli in olfactory information processing because (1) the glomerular array is relatively invariant across individuals of a species, and all glomeruli can be identified from individual to individual within a species (Chambille et al., 1980;
Rospars, 1983; Rospars and Hildebrand, 1992, 2000); (2) output PNs with
arborizations confined to a specific glomerulus are tractable for
intracellular and extracellular recording; and (3) many of the odorants
important for various aspects of behavior are known.
The lateral and medial LFGs are prominent glomeruli in the ALs of
female M. sexta. Although the MGC glomeruli in ALs of male M. sexta have been studied extensively, this is the first
investigation of the anatomical and physiological properties of
neurons with arborizations in the LFGs. Because they are sexually
dimorphic and assume their characteristic sizes, shapes, and positions
only in the female ALs, the LFGs may play important roles in
female-specific behavior, such as upwind orientation toward, and
recognition of, host plants and/or oviposition-related cues.
The MGC and LFGs are similarly but distinctively situated in the ALs of
males and females, respectively (Fig. 2), and are among the first
glomeruli to arise during postembryonic development (Rössler et
al., 1998). Recent findings suggest that the two LFGs are homologous to
the two main glomeruli of the MGC (Rospars and Hildebrand, 2000).
Developmental studies of gynandromorphic female moths with
trans-sexually grafted male antennae suggest that PNs that normally
arborize within the LFGs are recruited to innervate the induced MGC
(Rössler et al., 1999). In addition, some gynandromorphic females
with male antennae and induced MGCs orient and fly upwind toward a
source of sex pheromone, a stimulus to which normal females are
unresponsive. On reaching the pheromone source, gynandromorphic females
sometimes exhibit oviposition behavior, which is ordinarily elicited by
host plant stimuli (Schneiderman et al., 1986). These findings indicate
that the MGC and LFGs share common features of development and possibly
downstream connections mediating odor-modulated behavior, and also
highlight the importance of understanding the functional significance
of these identified glomeruli. In moths, glomeruli specific to the
female ALs have been described in a few different species (Rospars,
1983; Schneider and Wunderer, 1990), but the roles these glomeruli play
in processing of olfactory information or in female behavior are not known.
Female M. sexta use olfactory cues to orient toward and
locate host plants (Willis and Arbas, 1991; W. Mechaber and J. G. Hildebrand, unpublished observations). By testing latLFG-PNs for their responses to antennal stimulation with various host plant-related volatiles, we have found that the latLFG-PNs respond preferentially to
a monoterpenoid, linalool. Although technical constraints limited this
study to only six individual odorants, the fact that monoterpenoids similar in structure to linalool did not elicit a comparable excitatory response (Figs. 1, 8) suggests that the molecular receptive range of
the latLFG may be limited to linalool and perhaps other closely related
compounds. The responses to linalool recorded from latLFG-PNs were
typically biphasic, with an early excitatory phase followed by a period
of hyperpolarization. The peak instantaneous firing frequency of the
excitatory phase of the response increased, and the latency to the
excitation decreased, with greater odorant concentrations (Table 1).
The latLFG-PNs were also capable of following 50 msec pulses of
linalool presented at three to four pulses per second. Taken
together, these findings indicate that the pattern of spiking in a
particular PN depended on both the concentration and the pattern of the
odor stimulus (Fig. 9; Table 1). Our observations therefore suggest
that latLFG-PNs respond to a particular host plant odorant and encode
information about both its concentration and intermittency, as has been
documented for MGC-PNs processing sex pheromonal information in the ALs
of male moths (Christensen et al., 1998).
Although physiological characteristics of the linalool-responsive
latLFG-PNs described here are similar to those described for sex
pheromone-specific MGC-PNs in males (Christensen and Hildebrand, 1987,
1997; Hansson et al., 1991), latLFG-PNs typically were not as sensitive
to linalool as MGC-PNs are to sex pheromone components. MGC-PNs exhibit
response thresholds in the range 0.1-10 ng (Matsumoto and Hildebrand
1981; Hansson et al., 1991), whereas most of the latLFG-PNs in this
study responded with excitation at a dosage as low as 4.17 µg of
linalool (Figs. 5, 8, top panel; Table 1).
Whether linalool is behaviorally significant for M. sexta is
not known. Antennal ORCs that respond preferentially to linalool have been described in female M. sexta (Shields and
Hildebrand, 1998), as in the silkmoth, Bombyx mori
(Heinbockel and Kaissling, 1996). Linalool is commonly emitted by
floral and vegetative parts of many plants (Knudsen et al.,
1993), including favored host plants of M. sexta (Buttery et
al., 1987; Andersen et al., 1988; Loughrin et al., 1990; Raguso
and Willis, 1997; Raguso and Pichersky, 1999). Tomato plants are one of
the preferred oviposition sites for M. sexta (Yamamoto and
Fraenkel, 1960). If the LFGs are involved in processing olfactory
information related to oviposition, one might expect tomato-leaf
volatiles to be odor stimuli for LFG-PNs. In the present study,
although linalool evoked consistent, excitatory responses from
latLFG-PNs, the headspace volatiles emitted by an isolated tomato leaf
did so for only 2 of the 10 latLFG-PNs included in this study (Fig. 7).
Among possible explanations for this apparent discrepancy are that
either the volatiles emitted by an isolated tomato leaf in our
olfactometer probably varied significantly from experiment to
experiment or that a single leaf (as used in our experiments) might
have released a level of linalool that is below the threshold for
excitation of the latLFG-PNs.
Another possible function for the LFGs is that they might be
responsible for processing information about components of a male
pheromone. Male moths of many species possess scent organs that produce
and disseminate male pheromones (Birch et al., 1990). Given the
relatedness between the LFGs and the glomeruli of the MGC, as described
above, it is possible that the LFGs may be involved in detection of a
pheromone released by courting males. Male pheromone is usually
produced by sequestering compounds from plants eaten as larvae and
therefore is often made up of a mixture of volatiles normally
associated with plants (Birch et al., 1990). In fact, linalool is one
of the components of the male pheromone produced by a different moth
species, Trichoplusia ni (Landolt and Heath, 1990). One of
our future goals will be to determine the composition of the emissions
from a putative scent organ in the abdomen of male M. sexta.
In addition, determining the response specificity of PNs with
arborizations in the other LFG, the medLFG, may shed light on the
functional role of the LFGs and may suggest the suite of odorants that
can effect female-specific behaviors.
Reproducible and consistent tuning of individual, identified glomeruli
is to be expected if the glomeruli of primary olfactory centers are
organized chemotopically. Extending and complementing our previous
studies of the glomeruli of the male-specific MGC, our current studies
of the latLFG suggest that glomeruli in addition to those of the MGC
consistently exhibit limited molecular receptive ranges and may even
constitute labeled line representations of particular behaviorally
significant odorants. Our findings indicate that within and among
individuals, uniglomerular PNs (output neurons) with arborizations in a
particular glomerulus exhibit similar response properties when tested
with a small but varied panel of odorants. Although the latLFG appears
to have a relatively narrow molecular receptive range, reflecting the
tuning characteristics of the ORCs that converge on that glomerulus, it
remains to be seen whether other glomeruli in the AL have more or less
narrow molecular receptive ranges.
In mammals, ORCs that express the same olfactory receptor protein
project to a very small number of glomeruli, and it is possible that
each glomerulus receives input from ORCs that express a single or
closely related receptor type(s) (for review, see Buck, 1996). In
moths, ORCs that respond selectively to individual sex pheromone components project exclusively to specific glomeruli of the MGC (Hansson et al., 1992; Christensen et al., 1995; Hildebrand, 1996; Hildebrand and Shepherd, 1997). It will be of interest to determine whether this is also the case for the LFGs and other ordinary glomeruli
in the AL of M. sexta. Demonstrating that single glomeruli receive common inputs and yield similar outputs promises to provide a
clearer understanding of how olfactory information is represented in
the brain and to shed light on the function of the olfactory glomeruli
of diverse animals.
| |
FOOTNOTES |
Received Oct. 21, 1999; revised Dec. 23, 1999; accepted Dec. 28, 1999.
This work was supported by National Institutes of Health Grant DC02751.
We thank Heather Stein, Patricia Jansma, Nirav Merchant, and Amit Pandy
for expert technical assistance; John Douglass for writing the
spike-analysis software; A. A. Osman and Zenzele Mpofu for rearing
Manduca; and Reg Chapman, Ann Fraser, and Eileen Hebets
for helpful comments on this manuscript.
Correspondence should be addressed to Dr. John G. Hildebrand, Arizona
Research Laboratories, Division of Neurobiology, University of Arizona,
611 Gould-Simpson Building, 1040 E. 4th Street, Tucson, AZ 85721-0077. E-mail: jgh{at}neurobio.arizona.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
