Chem. Senses 27: 231-244,
2002
© Oxford University Press 2002
Spatial Representation of Odours in the Antennal Lobe of the Moth Spodoptera littoralis (Lepidoptera: Noctuidae)
Department of Crop Science, Chemical Ecology, Swedish University of Agricultural Sciences, PO Box 44, SE-230 53 Alnarp, Sweden 1 Institut für Neurobiologie, Freie Universität, Königin Luise Strasse 28-30, D-14195, Berlin, Germany
Correspondence to be sent to: Bill S. Hansson, Department of Crop Science, Chemical Ecology, Swedish University of Agricultural Sciences, PO Box 44, SE-230 53 Alnarp, Sweden. e-mail: bill.hansson{at}vv.slu.se
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Glomeruli within the antennal lobe (AL) of moths are convergence sites for a large number of olfactory receptor neurons (ORNs). The ORNs target single glomeruli. In the male-specific cluster of glomeruli, the macroglomerular complex (MGC), the input is chemotypic in that each glomerulus of the MGC receives information about a specific component of the conspecific female sex pheromone. Little is known about how neurons that detect other odorants arborize in and amongst glomeruli. The present study focuses on how sex pheromones and biologically relevant semiochemicals are represented in the ALs of both sexes of the moth Spodoptera littoralis. To assess this, we optically measured odour-evoked changes of calcium concentration in the ALs. Foci of calcium increase corresponded in size and shape with anatomical glomeruli. More than one glomerulus was normally activated by a specific non-pheromonal odorant and the same glomerulus was activated by several odorants. All odorants and pheromone components tested evoked unique patterns of glomerular activity that were highly reproducible at repeated stimulations within an individual. Odour-evoked patterns were similar between individuals for a given odorant, implicating a spatial olfactory code. In addition, we demonstrated that activity patterns evoked by host-plant related volatiles are similar between males and females.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The antennal lobe (AL) is the primary integration centre for olfactory information in insects, analogous to the olfactory bulb in vertebrates. The AL consists of a species-fixed number of spheroidal structures called glomeruli, where olfactory receptor neurons (ORNs) synapse with higher-order neurons. Olfactory glomeruli are found in both vertebrates and invertebrates (Hildebrand and Shepherd, 1997
; Strausfeld and
Hildebrand, 1999). The functional role of glomeruli is not fully
understood. However, evidence from an accumulating number of studies, using
activity-dependent staining techniques, converges on the hypothesis that
glomeruli are functional units representing different odorants or molecular
features (Sharp et al.,
1975; Kauer, 1988;
Rodrigues, 1988;
Cinelli et al., 1995;
Friedrich and Korsching, 1997,
1998;
Joerges et al., 1997;
Distler et al., 1998;
Galizia et al., 1999;
Rubin and Katz, 1999;
Uchida et al., 2000;
Meister and Bonhoeffer, 2001).
Functionally characterized ORNs have been found to project in a chemotypic
manner to identified glomeruli in moths (Hansson et al.,
1992,
1995;
Ochieng' et al.,
1995; Todd et al.,
1995; Berg et al.,
1998). Individual ORNs likely house a single olfactory receptor
type and axons expressing a given receptor gene converge onto the same
morphologically identified glomerulus (or in one case either of two glomeruli)
in Drosophila melanogaster (Gao
et al., 2000;
Vosshall et al.,
2000). Furthermore, the number of receptor gene types showing
expression in olfactory organs in D. melanogaster equals the number
of glomeruli (Vosshall, 2001).
Thus, it is highly likely that each glomerulus represents a certain olfactory
receptor.
Apart from highly specific sex-pheromone-detecting neurons, many insect
ORNs are more broadly tuned to odorants
(Todd and Baker, 1999).
Generally, the molecular receptive range of an ORN comprises closely related
compounds and a certain receptor is probably narrowly tuned to a molecular
determinant that is common to many odorants
(Araneda et al.,
2000). The same odorant often activates ORNs with differing
response spectra and a certain receptor neuron can detect different molecules.
Thus, to resolve the quality of an odour, an across-fibre comparison may be
required. If odours are detected by ORNs in an across-fibre fashion, this
would also be reflected in the AL by a multiglomerular activity pattern. In
some insect species, however, ORNs highly selective to plant-related volatiles
have been observed. In scarab beetles, for example, receptor neurons were
found that responded only to a single plant odorant and not to closely related
compounds (Hansson et al.,
1999; Larsson et al.,
2001; Stensmyr et
al., 2001).
The sensory neurons target one of 
60 glomeruli in moths
(Rospars, 1983;
Rospars and Hildebrand, 1992).
The number of `ordinary' glomeruli is equal in males and females.
Additionally, in males, a cluster of enlarged glomeruli, called the
macroglomerular complex (MGC), is targeted by axons of ORNs selectively tuned
to conspecific sexpheromone components or to interspecific behavioural
antagonists (Hansson, 1997).
Anterograde stainings of physiologically identified ORNs tuned to
sex-pheromone components, have demonstrated that each glomerulus within the
MGC receives input from a specific component (Hansson et al.,
1992,
1995;
Ochieng' et al.,
1995; Todd et al.,
1995; Berg et al.,
1998). This chemotypic organization has also been displayed in
output signals by tracing glomerular dendritic arborizations of projection
neurons (PNs) (Hansson et al.,
1991,
1994;
Berg et al., 1998;
Vickers et al.,
1998). However, the pheromone sensitive PNs (or ORNs) do not
always branch in a glomerulus as predicted by their physiological
characteristics. In the cabbage looper moth, Trichoplusia ni, a
mismatching of neuronal input and output of the MGC glomeruli was evident
(Anton and Hansson, 1999). In
the female sphinx moth, Manduca sexta, two sexually dimorphic
glomeruli, `the large female glomeruli' (LFG), have similar positions to the
MGC in males. A recent study (King et
al., 2000) showed that all PNs arborizing in the lateral LFG
were excited by the plant odorant linalool.
Much less is known about the function of the sexually isomorphic glomeruli
in moths. These glomeruli receive input from ORNs tuned to compounds other
than those involved in long-distance mate attraction. The only study using
anterograde staining of such ORNs was performed in female T. ni
(Todd and Baker, 1996).
However, this study did not reveal any consistent spatial separation of
functionally identified neurons into single glomeruli. Neither could dendritic
arborizations of physiologically characterized PNs be shown to originate in
glomeruli at consistent positions (Anton and Hansson,
1994,
1995) (M. Sadek, personal
communication).
In the cotton leaf worm, Spodoptera littoralis, axons of ORNs
tuned to two different components of the sexual pheromone and a behavioural
antagonist have been traced to separate glomeruli within the MGC
(Ochieng' et al.,
1995). The glomerulus of the MGC located closest to the AN is
called the cumulus or glomerulus `a' and is targeted by ORNs tuned to the
major pheromone component (Z,E)-9,11-tetradecadienyl acetate
(Z9, E11-14:OAc). Two satellite glomeruli, termed `b' and
`c', receive axons from ORNs tuned to a behavioural antagonist,
(Z)-9-tetradecanol (Z9-14:OH) and to the minor component
(Z,E)-9,12-tetradecadienyl acetate (Z9,
E12-14:OAc), respectively. Figure
1 shows the position of the glomeruli within the MGC and the
physiological types of ORN innervation they receive.
|
ORNs in S. littoralis responding to other biologically relevant
odours, such as plant-associated compounds, odorants induced by larval
host-plant feeding or odorants emited from larval frass show different degrees
of specificity (Anderson et al.,
1993,
1995;
Jönsson and Anderson,
1999). Some neurons appear to be highly specific, responding only
to a single or a few compounds, whereas others are more broadly tuned.
Intracellular recordings from AL neurons have also revealed specificity
ranging from neurons responding to a single compound to neurons responding to
all compounds tested (Anton and Hansson,
1994,
1995).
In the present study, we examined spatial representation of odorants in the AL of male and female S. littoralis upon stimulation with biologically relevant stimuli. We optically measured changes in intracellular calcium concentration of in vivo preparations. The odorants used in the study evoked reproducible patterns of glomerular activity within an individual, unique to each odorant. Odour representations were generally consistent between individuals, implicating a spatial olfactory code. In addition, we have for the first time demonstrated that both similarities and differences in glomerular responses exist between males and females.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Male and female, 1-5 days post-emergence moths were used in the study. The
animals had been reared for several generations on a potato-based diet
(Hinks and Byers, 1976). The
pupae were separated according to sex and kept in plastic boxes at 70%
relative humidity, 23°C and a 16 h:8 h light/dark cycle. Adult moths were
given excess of water until the start of the experiment.
Morphology
Moth brains were dissected out and immunostained with synapsin
(Klagges et al.,
1996). The preparations were optically sectioned using a confocal
microscope (Leica). Stacks of images were further processed on a Silicon
Graphics workstation with Imaris 2.7 software (Bitplane AG, Switzerland) to
obtain surface projections of the ALs. The final images were sharpened and
contrast-enhanced in Adobe Photoshop.
Optical imaging preparation and staining
The staining procedure was similar to that previously described (Galizia
et al., 1997,
1998). The animals were
secured in plastic tubes with their head protruding and fixed with dental wax.
The head capsule was cut open between the compound eyes, and muscles, glands
and trachea were removed to expose the ALs. Great care was taken not to
stretch or damage the antennal nerves. To minimize movement, the antennal
muscles were cut off and the oesophagus was first stretched with a pair of
forceps and then cut off. The neurolemma was left intact.
Subsequently, the animal was placed in a custom-made Plexiglass recording
holder. A drop of dissolved calcium-sensitive dye was applied directly to the
brain and a cover glass was fixed with wax over the cut window in the head.
The preparation was then left in a dark and cooled (10-12°C) chamber for 1
h. After rinsing the brain, the animal was placed under the microscope
(Olympus) where the brain was constantly perfused with fresh moth Ringer
solution (Christensen and Hildebrand,
1987).
We used the calcium-sensitive probe Calcium-green-2-AM (Molecular Probes,
Eugene, OR). The dye was dissolved in 20% Pluronic F-127 in dimethyl sulfoxide
(Molecular Probes, Eugene, OR) and diluted in moth Ringer solution to a final
concentration of 30 µM.
Odour stimulation
A continuous charcoal-filtered and moistened air stream (30 ml/s) was
ventilating the antenna ipsilateral to the recorded AL through a glass tube (7
mm internal diameter). The glass tube ended 10 mm from the antenna. Through a
small hole in the glass tube, an empty Pasteur pipette was inserted blowing an
air stream of 15 ml/s. Odorants were applied to filter papers (5 x
15 mm) and inserted into Pasteur pipettes. All odorants were diluted in
cyclohexane apart from the green leaf volatiles and
(Z)-3-hexenylacetate, which were diluted in paraffin oil. Odorants
were used in concentrations proved to elicit responses in extracellular
recordings from ORNs (Anderson et al.,
1993,
1995;
Ljungberg et al.,
1993; Jönsson and
Anderson, 1999). Filter papers were loaded with a dose of 100
µg of an odorant. The compounds used are listed in
Table 1. A puff of air (15
ml/s) was blown through the odour-laden pipette for 1 s by a manually
triggered puffer device (Syntech) and into the continuous stream of air.
During stimulation, the airstream was switched from the empty to the
odour-laden pipette, thereby minimizing mechanical stimulation. In some of the
early recordings, odorant stimulations were made without switching from clean
air to stimulus, i.e. odorant stimulation also led to an increased airflow.
Interstimulus time was at least 1 min and all odorants were used once before a
second series of stimulations with the same odorants.
|
Recordings
Recordings of series of 40 frames were made by an air-cooled CCD camera (Till Photonics) at 4 frames/s (200 ms exposure time) at 475 or 488 nm excitation. Filter settings were dichroic: 500 nm; emission LP 515 nm. Sequences were recorded through a 10x (NA 0.30; Olympus) or 20x (NA 0.50; Olympus) air objective.
Stimulation onset was at frame 12 and lasted 1 s. Images were binned 2x on chip (to 320 x 240 pixels) to increase signal-to-noise ratio. Execution of protocols and initial analyses of data were made using the software Till-vision (Till Photonics).
Background fluorescence (F) was defined as an average of frames 2-11, i.e. before onset of stimulation. F was subtracted from all frames to yield a dF and signals were expressed as dF/F, i.e. a relative change in fluorescence over background fluorescence. A sequence with pure air stimulation was first expressed as relative change in fluorescence (dF/F) and then subtracted from a sequence with odour stimulation in order to correct for bleaching. Air stimulus substraction served an additional purpose, namely to subtract a possible mechanical component of the signal during pipette switching.
For image presentation, an average of frames 14-18 (peak of activity) of a bleaching-corrected sequence was calculated and an average of frames 2-11 (prestimulation) was subtracted. The resulting image was subsequently filtered with a spatial average low-pass filter (13 x 13 pixels) and false-colour coded to its entire intensity range.
Data analyses
We measured the diameter of the normally circular or oval-shaped regions of increased activity horizontally and vertically through the centres of gravity at 50% of maximal activity. The mean diameters of the activity foci were then compared with the mean diameter of glomeruli in histological preparations. These foci will hereafter be called glomeruli.
Measurements of calcium activity were made from 11-14 glomeruli in each
animal. The intensity kinetics for the mean pixel value of the raw data of
each glomerulus was calculated for all odorants. We defined the response of a
glomerulus as the mean net response (dF/F odour stimulation —
dF/F air control) during frames 14-18 (peak of activity) minus the
mean net response during frames 6-10 (prestimulation). The response to an
odorant was expressed as the responses of all selected glomeruli. The
responses were normalized so that the most strongly activated glomerulus for
each odorant was given the value 1. The mean pixel value in a glomerulus
represents one dimension. Thus, each response to a stimulus was described as a
single point in a multidimensional space of 11-14 dimensions. The response
profiles for each odorant were then compared to all other odorants using
Pearson correlation coefficients (Systat 5.2.1). A perfect match between two
stimuli would yield the value 1 and perfectly complementary responses would
yield the value -1. As concentrations of the odorants were not corrected for
differing volatility, comparisons of absolute intensities were irrelevant. For
example, two alcohols, 1-hexanol and 1-octanol, were both used at the same
concentration. However, the vapour pressure of 1-hexanol is 10x
higher than of 1-octanol (Hass and Newton,
1975).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Confocal surface reconstructions of the month ALs revealed that 20-25 glomeruli are visible from a frontal view (Figure 2). This means that 30-40% of the population of glomeruli would be accessible for recording. The diameter of the normally circular or oval-shaped regions of increased activity were measured horizontally and vertically through the centres of gravity at 50% of maximal activity. Their size (56.5 ± 9.7 µm, mean ± SD; 46 measurements in five animals) correspond with the size of actual glomeruli measured from histological preparations (54.1 ± 4.19 µm, mean ± SD; M. Sadek, personal communication). In 15 males and 10 females, recordings were stable and most stimuli were tested at least once. Recordings from additional animals, in which only a few odorants were tested or too much movement made recordings unreliable, were not further analysed. Generally, only one lobe was recorded from each preparation. Images (Figure 5) from recordings of the right lobe were mirrored for easier comparison. The relative change in fluorescence, dF/F, of the most activated glomeruli was in the range 1-3%. Time courses, i.e. dF/F plotted as functions of time, from three glomeruli are shown in Figure 3. Glomerulus 1 showed stronger increase in activity than glomeruli 2 and 3 when the animal was stimulated with the flower odour, geraniol. Signals reached the peak
1 s after onset of stimulation and
declined to background level after another 2-3 s.
|
|
|
Odorant-specific activity pattern
Stimulations led to odour-specific activity patterns in the ALs. Figure 4 shows responses to all 14 non-sex pheromonal odorants in a single male animal. The responses to some odorants were confined to a few glomeruli, whereas other odorants elicited more distributed activity patterns. Furthermore, activity maps were often overlapping in that the same glomerulus was activated by several different odorants.
|
Figure 7 shows the overall
glomerular pattern, expressed as absolute dF/F values, for all 14
non-sex pheromones in a male individual (same as 4). In this animal,
measurements were made from 14 glomeruli. Noise levels were defined as the
mean standard deviations of frames 2-11
(Sachse et al.,
1999). Many odorants showed overlapping patterns, but each
combination of activated glomeruli is unique to a certain odorant. For
example, phenylacetaldehyde (PAA) evoked the strongest response in glomerulus
2, (+/-)-linalool in glomerulus 1, geraniol in glomerulus 9 and 1-hexanol in
glomerulus 11. The two structurally similar sesquiterpenes, 
-humulene
and ß-caryophyllene, evoked very similar patterns.
|
Normally, two or more stimulations were made with each odorant in each animal. Repeated stimulations with the same odorant showed highly reproducible activity patterns. Comparisons of overall glomerular activity patterns between two stimulations with the same odorant yielded a correlation index of 0.79 ± 0.13 (mean ± SD, 45 odorant pairs in four animals).
To compare the similarity of glomerular responses to different odorants, we
calculated the correlation indices for all possible pairs of odorants
(Table 2). We quantified the
overall patterns of glomerular activity as the relative response in all
glomeruli measured. For each odorant stimulation, the dF/F was
normalized so that the strongest activated glomerulus was set to 1.
Correlation analyses were made in four males and three females. PAA, for
example, showed a low correlation with all other odorants and was generally
most related in response to another aromatic compound with an attached
aldehyde group, benzaldehyde, in both males and females. In both sexes there
was also a high response similarity between 1-hexanol and
(Z)-3-hexenylacetate. In males there was a high correlation between responses
to -humulene and ß-caryophyllene (0.78-0.93, four animals). In
contrast, the similarity in response to these two compounds was much lower in
females (0.12-0.51, three animals).
|
Consistency between individuals
Great care was taken to use equal views, angles of the preparations and
focal plane between preparations, but direct comparisons between animals are
difficult, mostly due to individual morphological differences. However,
comparisons of the relative positions of foci of activity can be made.
Figure 5 shows recordings from
three males and two females and responses to geraniol, (+/-)-linalool and PAA
(the same relative positions of the activity foci for these odorants were
observed in all 25 animals). PAA activated a glomerulus in the dorsomedial
area in all animals recorded. Both geraniol and (+/-)-linalool activated the
central (frontal) area of the lobe, but the (+/-)-linalool-activated
glomerulus was located more ventrally. Similar patterns were observed in males
and females. Thus, the glomerular activity patterns for these odorants are not
only preserved between individuals of the same sex, but also between sexes.
However, the two sesquiterpenes, -humulene and ß-caryophyllene,
did, as described above, elicit similar patterns only in male individuals. For
a few odorants in the experiment, consistent and spatially stereotyped
responses between individuals, regardless of sex, were less obvious.
1-hexanol, for example, elicited the strongest response in the same glomerulus
as PAA in some animals, whereas in other animals the peak of activity was
observed in a more centrally located glomerulus. The latter glomerulus was
always also strongly activated by another primary alcohol, 1-octanol.
Pheromone-evoked activity
Pheromone components activated glomeruli in the region close to the entrance of the antennal nerve (Figure 6). This region was not or only weakly activated by non-sex pheromones. The major pheromone component Z9,E11-14:OAc activated a glomerulus close to the antennal nerve in all preparations that corresponds in position to the large identified MGC glomerulus called the cumulus or `a' glomerulus (see Figure 1). Also, the position of the activity focus elicited by the minor pheromone component Z9,E12-14:OAc, i.e. ventrolateral to the former, corresponds with the position of the `c' glomerulus. The behavioural antagonist Z9-14:OH activated a glomerulus medial to the former and which corresponds to the position of the glomerulus termed `b'. The putative pheromone component Z7-12:OAc showed the strongest activity in an additional glomerulus, which was also activated by non-sex pheromones. Figure 8 shows the relative change in activity in four glomeruli in two different males. The major pheromone component Z9,E11-14:OAc elicited the strongest response in glomerulus `a' and the minor component Z9,E12-14:OAc in glomerulus `c'. The behavioural antagonist Z9-14:OH elicited the strongest response in glomerulus `b', whereas Z7-12:OAc elicited high activity in both glomeruli `b' and `d'.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We have studied activity in neural populations in the antennal lobes of the cotton leaf worm S. littoralis by means of calcium imaging. The results from this study contribute to the contemporary view that odorants are represented spatially in central olfactory neuropils. We have clearly showed that different odorants evoke distinct and reproducible activity patterns in the AL. These patterns are generally consistent between individuals. Furthermore, non-sex pheromones elicit similar activity patterns in males and females. Conspecific sex-pheromone components and an interspecific signal evoked responses that supported an earlier model of ORN projections in the MGC, thus facilitating the interpretation of responses evoked by other odorants.
The Ca2+ signals have been shown to originate in glomeruli in
the honeybee (Galizia et al.,
1999). By superimposing morphological images on the physiological
activity maps, the signals were shown to be confined within the borders of
glomeruli. There is no reason to believe that the signals would originate from
a different source in moths. The average size of the activity foci is close to
the actual size of glomeruli measured in histological preparations.
Furthermore, the normally circular or oval shape and the existence of a
gravity centre in the activity spots indicate a spheroidal structure. As there
are no other spheroidal structures in the ALs than glomeruli, we assume that
the signals we measure are of glomerular origin.
Activity patterns elicited by plant-related odorants in males and females
The plant-related odorants used in this study have previously been shown to
elicit responses at the peripheral level (Anderson et al.,
1993,
1995;
Jönsson and Anderson,
1999). Consequently, we also found that the odorants used elicited
patterns of elevated activity in the ALs. Several glomeruli were often
activated when the animal was stimulated with non-pheromonal odorants.
Furthermore, the same glomeruli responded to several different compounds.
However, response amplitude was unevenly distributed in that one or a few
glomeruli were more strongly activated than the rest. We can not exclude the
possibility that more glomeruli, which were inaccessible to recording, may be
activated in other parts of the AL.
All odorants tested evoked unique patterns of glomerular activity. A
correlation analysis of response profiles of repeated stimulations of the same
odorant revealed a high reproducibility. Activity patterns for some odorants
were more similar than others. Two floral odorants, PAA and geraniol, elicited
response patterns almost without overlap and showed low correlation in all
animals (-0.32 ± 0.16, mean ± SD, seven animals). These two
odorants can easily be discriminated by female S. littoralis, as has
been shown in a differential conditioning task
(Fan and Hansson, 2001).
Responses to the two structurally similar sesquiterpenes, -humulene and
ß-caryophyllene, were very similar in all male individuals. In both males
and females a type of ORN tuned to -humulene and ß-caryophyllene
showed equal responses to both compounds
(Anderson et al.,
1995; Jönsson and
Anderson, 1999; Carlsson and Hansson, unpublished observation). In
contrast to males, the glomerular activity patterns elicited by the two
compounds in females showed low similarity. A previous study
(Anderson et al.,
1995) found a single ORN in a female that responded solely to
-humulene, thereby offering the animal a possible mechanism to
discriminate between the compounds. Furthermore, intracellular recordings of
AL neurons revealed that several interneurons responded solely to either of
the compounds in females (M. Sadek, personal communication). The far weaker
correlation between responses in females may indicate that only females
possess an ability to discriminate between the compounds, or at least have a
higher ability than males. The two sesquiterpenes are emitted from leaves of
host plants, for example cotton, and may be important olfactory cues to guide
a mated female to a suitable site for oviposition. It is not unlikely that the
ratio between closely related compounds is important for recognition of a host
plant, as has been demonstrated, for example in the Colorado potato beetle
Leptinotarsa decemlineata (Visser
and Avé, 1978). Intersexual differences are very
interesting as they may reflect adaptations to sex-specific requirements.
Pheromone evoked activity patterns in males
As with the moth Heliothis virescens
(Galizia et al.,
2000), pheromone-evoked calcium responses in the male-specific MGC
in S. littoralis corroborated previous studies of AL projections of
physiologically characterized single ORNs
(Ochieng' et al.,
1995; Berg et al.,
1998). In contrast to H. virescens, there does not seem
to be a consistent correspondence between input and output of the MGC in
S. littoralis (Anton and Hansson,
1995; Vickers et al.,
1998). The major component, Z9,E11-14:OAc,
activated an area close to the entrance of the AN, which is not activated by
non-pheromones. This glomerulus is most likely identical to the `cumulus' or
`a' glomerulus, which has been shown to be the convergence site for ORNs tuned
to the major component. Also, the minor component
Z9,E12-14:OAc and the behavioural antagonist
Z9-14:OH activated areas that corresponded in location to glomeruli
`c' and `b', respectively. The putative pheromone component Z7-12:
OAc activated the `b' glomerulus and an additional glomerulus located further
dorsally. It is unclear if this second glomerulus responding to
Z7-12:OAc belongs to the cluster of glomeruli comprising the MGC.
Projection neurons responding exclusively to Z7-12:OAc have been
found to arborize in ordinary glomeruli
(Anton and Hansson, 1995). The
glomeruli within the MGC were not (or only weakly) activated by odorants other
than sex pheromones.
Interindividual comparisons
Direct comparisons between animals would be facilitated if we could see the
outlines of glomeruli and if we could compare this anatomy with a glomerular
atlas of the AL. However, visual inspection of the relative positions of the
most strongly activated glomeruli in S. littoralis clearly showed
that the patterns for a number of odorants were conserved between individuals
of both sexes. For instance, PAA always elicited high activity in the
dorsomedial part of both lobes. Another floral odour, geraniol, activated
glomeruli closer to the entrance of the antennal nerve in all animals tested
and a third odour, (+/-)-linalool, evoked the highest activity ventral to the
glomeruli activated by geraniol. Most of the odorants tested elicited activity
patterns that were similar in all animals, which may indicate a spatial
olfactory code as has been suggested for the honeybee
(Galizia et al.,
1999). However, similar patterns do not constitute evidence for
involvement of homologous glomeruli. A glomerular map of the AL in S.
littoralis is currently under construction (M. Sadek, in preparation) and
future experiments may prove that a homology does indeed exist. For a few
odorants, however, we did not observe patterns that were spatially stereotyped
between all animals. For example, the response to 1-hexanol varied between
individuals in that the most strongly activated glomerulus in a number of
animals was the same, as was that most strongly activated by 1-octanol,
whereas in some animals the peak of activity was seen in the same glomerulus
that was always strongly activated by PAA. Thus, there may be some individual
differences in response patterns. It has been reported
(Galizia et al.,
1999) that intraspecific variability does indeed exist and is not
due to variations in glomerular positions between animals.
Glomerular activity patterns and receptor specificity
Pheromone components are detected by extremely specific ORNs, whereas most
non-pheromonal odorants are detected by less specific receptor types with
different but overlapping response spectra. These less specific ORNs may
indeed be narrowly tuned with respect to a certain molecular feature, but the
number of odorants sharing this determinant can potentially be large. An
olfactory system relying simply on a system in which each input channel is
devoted to a specific odorant would leave the animal with severe restrictions.
Only a limited number of odorants may be detected and the animal would have
difficulties coping with novel olfactory-related situations. On the other
hand, such a one compound/receptor type system would be advantageous for
processing highly predictable signals such as those involved in intraspecific
communication. In vertebrates, most odours are detected by a combination of
ORNs (Buck, 1996). ORNs in the
olfactory epithelium are broadly tuned (Duchamp-Viret et al.,
1999,
2000) and the best
characterized receptor, 17 from the rat, responds to an entire family of
molecules, but with a molecular determinant in common
(Araneda et al.,
2000). This fact is also reflected in the main olfactory bulb,
where all odorants are represented by different combinations of activated
glomeruli (Rubin and Katz,
1999; Uchida et al.,
2000; Meister and Bonhoeffer,
2001). However, it was recently demonstrated
(Leinders-Zufall et al.,
2000) that sensory neurons in the vomeronasal organ in mice are
highly selectively tuned to pheromones. Similar mechanisms thus seem to exist
in insects and vertebrates.
If receptors have broad and overlapping response profiles, an odorant would
be detected by several different receptor types and each receptor type would
detect a number of different odorants. Consequently, if all receptor neurons
representing a certain receptor type terminate in the same glomerulus and the
stimulating odorant is detected by several types of receptors, an array of
glomeruli would be activated. The number of activated glomeruli depends on the
specificity of the ORNs. The glomeruli of the moth MGC receive highly
specialized afferents and it is very likely that each of the MGC glomeruli
represents a specific receptor type. It is still not known if all ORNs housing
a certain receptor type terminate in the same ordinary glomerulus (or
glomeruli) in moths, as has recently been demonstrated in D.
melanogaster (Vosshall et
al., 2000) and earlier in vertebrates
(Mombaerts, 1996;
Mombaerts et al.,
1996). It can not be excluded that several types of ORNs innervate
the same glomerulus and that one type of ORN innervates several glomeruli.
However, it is highly unlikely that the glomeruli receive identical input. As
a major contributor to the calcium signals is presynaptic influx of
Ca2+ in the ORNs (Galizia
et al., 1998), the odour-evoked patterns in the AL would
reflect the array of receptors activated at the antennal level. The fact that
the activity patterns evoked by sex-pheromone components in the males
correspond with single ORN projections further indicates that a substantial
part of the signal is of afferent origin, as prediction of the dendritic
arborizations of interneurons based on their physiology often fails
(Anton and Hansson, 1995). It
is, however, important to note that application of the
Ca2+-sensitive dye directly on the brain tissue results in a
non-selective dye uptake. Therefore, [Ca2+] changes in the AL
interneurons may also contribute to a composite signal. Interneurons confined
to the AL generally synapse within most glomeruli and may explain why we often
observed some weak activity in most parts of the lobe.
Combinations of responding glomeruli in the ALs may constitute a spatial
olfactory code that underlies final odour perceptions as results from
recordings in the honeybee AL suggest
(Galizia et al.,
1999). However, both slow temporal patterns in individual PNs and
synchrony of PN ensembles have been shown to be odorant-specific
(Laurent and Davidowitz, 1994;
Laurent et al.,
1996). Thus, spatial as well as temporal features of an olfactory
response carry information about the odour identity. We can not exclude the
possibility that these mechanisms work in parallel and are required for fine
odour discrimination.
Calcium recordings have an obvious drawback; namely, they tell us little about how odour information is computed and integrated in the ALs. However, these recordings provide us with information about many other interesting aspects of olfaction—for example about ORN specificity and the topographic organization of glomeruli—and allow us to make interindividual and intersexual comparisons. To solve questions regarding olfactory information processing in the lobes, we intend to integrate calcium recordings with single-cell recordings of AL interneurons in future experiments.
This study builds on previous behavioural and physiological work in S. littoralis. Our results confirm the model of functional organization in the MGC in this species. Biologically relevant host-plant-related odorants are represented in an across-glomerular fashion. These patterns of glomerular activity are roughly conserved between individuals. In addition, we show for the first time that representations of non-pheromonal odorants are generally similar between males and females, despite obvious differences in olfactory-related ecological requirements.
|
Acknowledgments |
|---|
We would like to thank Mr M. Stensmyr and Mrs K. Johnsson for help with AL reconstruction and immunostaining. This research was supported by grants from the Swedish Natural Science Research Council (NFR) to B.S.H.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Anderson, P., Hilker, M., Hansson, B.S., Bombosch, S., Klein, B. and Schildknecht, H. (1993) Oviposition deterring components in larval frass of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae): a behavioural and electrophysiological evaluation. J. Insect Physiol., 39,129 -137.
Anderson, P., Hansson, B. S. and Löfqvist, J. (1995) Plant-odour-specific receptor neurons on the antennae of the female and male Spodoptera littoralis. Physiol. Entomol., 20,189 -198.
Anton, S. and Hansson, B.S. (1994) Central processing of sex pheromone, host odour, and oviposition deterrent information by interneurons in the antennal lobe of female Spodoptera littoralis (Lepidoptera: Noctuidae). J. Comp. Neurol.,350 , 199-214.[ISI][Medline]
Anton, S. and Hansson, B.S. (1995) Sex pheromone and plant-associated odour processing in antennal lobe interneurons of male Spodoptera littoralis (Lepidoptera: Noctuidae). J. Comp. Physiol. A,176 , 773-789.
Anton, S. and Hansson, B.S. (1999) Physiological mismatching between neurons innervating olfactory glomeruli in a moth. Proc. R. Soc. Lond. B,266 , 1813-1820.
Araneda, R.C., Kini, A.D. and Firestein, S. (2000) The molecular receptive range of an odorant receptor. Nat. Neurosci., 3,1248 -1255.[ISI][Medline]
Berg, B.G., Almaas, T.J., Bjaalie, J.G. and Mustaparta, H. (1998) The macroglomerular complex of the antennal lobe in the tobacco budworm moth Heliothis virescens: specified subdivision in four compartments according to information about biologically significant compounds. J. Comp. Physiol. A,183 , 669-682.
Buck, L.B. (1996) Information coding in the vertebrate olfactory system. Ann. Rev. Neurosci.,19 , 517-544.[ISI][Medline]
Campion, D.G., Hunter-Jones, P., McVeigh, L.J., Hall, D.R., Lester, R. and Nesbitt, B.F. (1980) Modification of the attractiveness of the primary pheromone component of the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), by secondary pheromone components and related chemicals.Bull. Ent. Res. , 70,417 -434.[ISI]
Christensen, T.A. and Hildebrand, J.G. (1987) Male-specific, sex pheromone-selective projection neurons in the antennal lobes of the moth Manduca sexta. J. Comp. Physiol. A, 160,553 -569.[Medline]
Cinelli, A.R., Hamilton, K.A. and Kauer, J.S.
(1995) Salamander olfactory bulb neuronal activity observed
by video rate, voltage-sensitive dye imaging. III. Spatial and temporal
properties of responses evoked by odorant stimulation. J.
Neurophysiol., 73,2053
-2071.
Distler, P.G., Bausenwein, B. and Boeckh, J. (1998) Localization of odor-induced neuronal activity in the antennal lobes of the blowfly Calliphora vicina: a[3H] 2-deoxyglucose labeling study. Brain Res., 805,263 -266.[ISI][Medline]
Duchamp-Viret, P., Chaput, M.A. and Duchamp, A.
(1999) Odor response properties of rat olfactory receptor
neurons. Science, 284,2171
-2174.
Duchamp-Viret, P., Duchamp, A. and Chaput, M.A.
(2000) Peripheral odor coding in the rat and frog: quality
and intensity specification. J. Neurosci.,20
, 2383-2390.
Fan, R.-J. and Hansson, B.S. (2001) Olfactory conditioning in the moth Spodoptera littoralis.Physiol. Behav. , 72,159 -165.[Medline]
Friedrich, R.W. and Korsching, S.I. (1997) Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging.Neuron , 18,737 -752.[ISI][Medline]
Friedrich, R.W. and Korsching, S.I.
(1998) Chemotopic, combinatorial, and non-combinatorial
odorant representations in the olfactory bulb revealed using a
voltage-sensitive axon tracer. J. Neurosci.,18
, 9977-9988.
Galizia, G.C., Joerges, J., Küttner, A., Faber, T. and Menzel, R. (1997) A semi-in-vivo preparation for optical recording of the insect brain. J. Neurosci. Meth.,76 , 61-69.[ISI][Medline]
Galizia, C.G., Nägler, K., Hölldobler, B. and Menzel, R. (1998) Odour coding is bilaterally symmerical in the antennal lobes of honeybees (Apis mellifera).Eur. J. Neurosci. , 10,2964 -2974.[ISI][Medline]
Galizia, G.G., Sachse, S., Rappert, A. and Menzel, R. (1999) The glomerular code for odor representation is species specific in the honeybee Apis mellifera. Nat. Neurosci.,2 , 473-478.[ISI][Medline]
Galizia, G.G., Sachse, S. and Mustaparta, H. (2000) Calcium responses to pheromones and plant odours in the antennal lobe of the male and female moth Heliothis virescens.J. Comp. Physiol. A , 186,1049 -1063.[Medline]
Gao, Q., Yuan, B. and Chess, A. (2000) Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat. Neurosci.,3 , 780-785.[ISI][Medline]
Hansson, B.S. (1997) Antennal lobe projection patterns of pheromone-specific olfactory receptor neurons in moths. In Cardé, R.T. and Minks, A.K.(eds), Insect Pheromone Research: New Directions. Chapman & Hall, New York, pp.164 -183.
Hansson, B.S., Christensen, T.A. and Hildebrand, J.G. (1991) Functionally distinct subdivisions of the macroglomerular complex in the antennal lobe of the male sphinx moth Manduca sexta. J. Comp. Neurol., 312,264 -278.[ISI][Medline]
Hansson, B.S., Ljungberg, H., Hallberg, E. and
Löfstedt, C. (1992) Functional specialization of
olfactory glomeruli in a moth. Science,256
, 1313-1315.
Hansson, B.S., Anton, S. and Christensen, T.A. (1994) Structure and function of antennal lobe neurons in the male turnip moth Agrotis segetum. J. Comp. Physiol. A,175 , 547-562.
Hansson, B.S., Almaas, T.J. and Anton, S. (1995) Chemical communication in heliothine moths. V. Antennal lobe projection patterns of pheromone-detecting olfactory receptor neurons in the male Heliothis virescens (Lepidoptera: Noctuidae). J. Comp. Physiol. A,177 , 535-543.
Hansson, B.S., Larsson, M.C. and Leal, W.S. (1999) Green leaf volatile-detecting olfactory receptor neurones display very high sensitivity and specificity in a scarab beetle. Physiol. Entomol., 24,121 -126.
Hass, H.B. and Newton, R.F. (1975) Correction of boiling points to standard pressure. In Weast, R.D. (ed.), Handbook of Chemistry and Physics. CRC Press, Cleveland, OH, pp. 176-177.
Hildebrand, J.G. and Shepherd, G.M. (1997) Mechanisms of olfactory discrimination: converging evidence for common principles across phyla. Ann. Rev. Neurosci., 20,595 -631.[ISI][Medline]
Hinks, C.F. and Byers, J.R. (1976) Biosystematics of the genus Euxoa (Lepidoptera: Noctuidae). V. Rearing procedures and life cycles of 36 species. Can. Entomol., 108,1345 -1357.
Joerges, J., Küttner, A., Galiza, C.G. and Menzel, R. (1997) Representations of odours and odour mixtures visualized in the honeybee brain. Nature,387 , 285-288.
Jönsson, M. and Anderson, P. (1999) Electrophysiological response to herbivore-induced host plant volatiles in the moth Spodoptera littoralis. Physiol. Entomol.,24 , 377-385.
Kauer, J.S. (1988) Real-time imaging of evoked activity in local circuits of the salamander olfactory bulb.Nature , 331,166 -168.[Medline]
Kehat, M., Greenberg, S. and Tamaki, Y. (1976) Field evaluation of the synthetic sex pheromone, as an attractant for males of the cotton leafworm, Spodoptera littoralis (Boisd.). Isr. Appl. Entomol. Zool.,11 , 45-52.
King, J.R., Christensen, T.A. and Hildebrand, J.G.
(2000) Response characteristics of an identified, sexually
dimorphic olfactory glomerulus. J. Neurosci.,20
, 2391-2399.
Klagges, B.R.E., Heimbeck. G., Godenschwege, T.A, Hofbauer, A.,
Pflugfelder, G.O., Reifegerste, R., Reisch, D., Schaupp, M., Buchner, S.
and Buchner, E. (1996) Invertebrate synapsins: a
single gene codes for several isoforms in Drosophila. J.
Neurosci., 16,3154
-3165.
Larsson, M.C., Leal, W.S. and Hansson, B.S. (2001) Olfactory receptor neurons detecting plant odours and male volatiles in Anomala cuprea beetles (Coleoptera: Scarabidae). J. Insect Physiol.,47 , 1065-1076.[ISI][Medline]
Laurent, G. and Davidowitz, H. (1994)
Encoding of olfactory information with oscillating neuronal
assemblies. Science, 265,1872
-1875.
Laurent, G., Wehr, M. and Davidowitz, H.
(1996) Temporal representations of odors in an olfactory
network. J. Neurosci., 16,3837
-3847.
Leinders-Zufall, T., Lane, A.P., Puche, A.C., Ma, W., Novotny, M.V., Shipley, M.T. and Zufall, F. (2000) Ultrasensitive pheromone detection by mammalian vomeronasal neurons.Nature , 405,792 -796.[Medline]
Ljungberg, H., Anderson, P. and Hansson, B.S. (1993) Physiology and morphology of pheromone-specific sensilla on the antennae of male and female Spodoptera littoralis (Lepidoptera: Noctuidae). J. Insect Physiol.,39 , 253-260.
Meister, M. and Bonhoeffer, T. (2001)
Tuning and topography in an odor map on the rat olfactory bulb.J. Neurosci.
, 21,1351
-1360.
Mombaerts, P. (1996) Targeting olfaction. Curr. Opin. Neurobiol.,6 , 481-486.[ISI][Medline]
Mombaerts, P., Wang, F., Dulac, C., Chao, S.K., Mendelsohn, M., Edmondson, J. and Axel, R. (1996) Visualizing an olfactory sensory map. Cell, 87,675 -686.[ISI][Medline]
Ochieng', S.A., Anderson, P. and Hansson, B.S. (1995) AL projection patterns of olfactory receptor neurons involved in sex pheromone detection in Spodoptera littoralis (Lepidoptera: Noctuidae). Tissue Cell,27 , 221-232.[ISI][Medline]
Paré, P.W. and Tumlinson, J.H. (1998) Cotton volatiles synthesized and released distal to the site of insect damage. Phytochemistry,47 , 521-526.[ISI]
Rodrigues, V. (1988) Spatial coding of olfactory information in the antennal lobe of Drosophila melanogaster.Brain Res. , 453,299 -307.[ISI][Medline]
Rospars, J.P. (1983) Invariance and sex-specific variations of the glomerular organization in the antennal lobes of a moth, Mamestra brassicae, and a butterfly, Pieris brassicae. J. Comp. Neurol., 220,80 -96.[ISI][Medline]
Rospars, J.P. and Hildebrand, J.G. (1992) Anatomical identification of glomeruli in the antennal lobes of the male sphinx moth Manduca sexta. Cell Tissue Res., 270,205 -227.[ISI][Medline]
Rubin, B.D. and Katz, L.C. (1999) Optical imaging of odorant representations in the mammalian olfactory bulb. Neuron, 23,499 -511.[ISI][Medline]
Sachse, S., Rappert, A. and Galizia, G.G. (1999) The spatial representation of chemical structures in the antennal lobe of honeybees: steps toward the olfactory code.Eur. J. Neurosci. , 11,3970 -3982.[ISI][Medline]
Sharp, F.R., Kauer, J.S. and Shepherd, G.M. (1975) Local sites of activity related glucose metabolism in rat olfactory bulb during olfactory stimulation. Brain Res., 98,596 -600.[ISI][Medline]
Stensmyr, M.C., Larsson, M.C., Bice, S.B. and Hansson, B.S. (2001) Detection of fruit- and flower-emitted volatiles by olfactory receptor neurons in the polyphagous fruit chafer Pachnoda marginata (Coleoptera: Cetoniinae). J. Comp. Physiol. A, 187,509 -519.[ISI][Medline]
Strausfeld, N.J. and Hildebrand, J.G. (1999) Olfactory systems: common design, uncommon origins? Curr. Opin. Neurobiol., 9,634 -639.[ISI][Medline]
Todd, J.L. and Baker, T.C. (1996) Antennal lobe partitioning of behaviorally active odors in female cabbage looper moths. Naturwissenschaften,83 , 324-326.
Todd, J.L. and Baker, T.C. (1999) Function of peripheral olfactory organs. In Hansson, B.S. (ed.),Insect Olfaction. Springer, Berlin, pp.67 --96.
Todd, J.L., Anton, S., Hansson, B.S. and Baker, T.C. (1995) Functional organization of the macroglomerular complex related to behaviorally expressed olfactory redundancy in male cabbage looper moth. Physiol. Entomol., 20,349 -361.
Uchida, N., Takahashi, Y.K., Tanifuji, M. and Mori, K. (2000) Odor maps in the mammalian olfactory bulb: domain organization and odorant structural features. Nat. Neurosci., 3,1035 -1043.[ISI][Medline]
Vickers, N.J., Christensen, T.A. and Hildebrand, J.G. (1998) Combinatorial odor discrimination in the brain: attractive and antagonist odor blends are represented in distinct combinations of uniquely identifable glomeruli. J. Comp. Neurol.,400 , 35-56.[ISI][Medline]
Visser, J.H. and Avé, D.A. (1978) General green leaf volatiles in the olfactory orientation of the colorado beetle, Leptinotarsa decemlineata. Entomol. Exp. Appl.,24 , 538-549.
Vosshall, L.B. (2001) The molecular logic of olfaction in Drosophila. Chem. Senses,26 , 207-213.
Vosshall, L.B., Wong, A.M. and Axel, R. (2000) An olfactory sensory map in the fly brain.Cell , 102,147 -159.[ISI][Medline]
Accepted November 28, 2001
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. A. Carlsson, K. Y. Chong, W. Daniels, B. S. Hansson, and T. C. Pearce Component Information Is Preserved in Glomerular Responses to Binary Odor Mixtures in the Moth Spodoptera littoralis Chem Senses, June 1, 2007; 32(5): 433 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Reisenman, T. A. Christensen, and J. G. Hildebrand Chemosensory Selectivity of Output Neurons Innervating an Identified, Sexually Isomorphic Olfactory Glomerulus J. Neurosci., August 31, 2005; 25(35): 8017 - 8026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Masante-Roca, C. Gadenne, and S. Anton Three-dimensional antennal lobe atlas of male and female moths, Lobesia botrana (Lepidoptera: Tortricidae) and glomerular representation of plant volatiles in females J. Exp. Biol., March 15, 2005; 208(6): 1147 - 1159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|