Chem. Senses 28: 113-130,
2003
© Oxford University Press 2003
RESEARCH PAPERS |
Projections to Higher Olfactory Centers from Subdivisions of the Antennal Lobe Macroglomerular Complex of the Male Silkmoth
Institute of Biological Sciences, University of Tsukuba, 1-1-1 Tsukuba, Ibaraki 305-8572, Japan
Correspondence to be sent to: Ryohei Kanzaki, Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. e-mail: kanzaki{at}biol.tsukuba.ac.jp
Abstract
The macroglomerular complex (MGC) is the first-order center for synaptic processing of olfactory information about the female sex pheromone in the male moth brain. We have investigated the MGC subdivisions of the male silkmoth Bombyx mori by use of three-dimensional reconstruction of the MGC from sequential series of confocal slice images. The B. mori MGC consists of three subdivisions similar to those of Manduca sexta: the cumulus, toroid and horseshoe. Intracellular recording and staining revealed that responses of MGC projection neurons to pheromonal stimulation correlate with their dendritic arborizations in the subdivisions of the MGC (the cumulus, toroid and horseshoe) and each subdivision specific projection neuron transmits information to different regions in the calyces of the mushroom body and the inferior lateral protocerebrum. We revealed that major pheromone component information is transferred to the medial part of the inferior lateral protocerebrum through three different antennocerebral pathways. Although it is generally accepted that the calyces of the mushroom body and the inferior lateral protocerebrum are the target sites for pheromone information from the MGC in moths, our results suggest that the medial part of the inferior lateral protocerebrum may be a more important processing site for major pheromonal information in B. mori.
Key words: brain, insect, mushroom body, pheromone, three-dimensional reconstruction
Introduction
Due to the anatomical similarity of the primary olfactory centers of
diverse animals, such as the antennal lobes (ALs) of insects and the olfactory
bulbs of vertebrates, the insect ALs have been popular models for the study of
olfactory processing (Hildebrand and
Shepherd, 1997
; Christensen and
White, 2000).
Male silkmoths (Bombyx mori) exhibit a characteristic zigzagging
pattern as they walk upwind toward pheromones released by conspecific females
(Kanzaki, 1998). The female
sex-pheromone blend contains two components. The major component
(E,Z)-10,12-hexadecadienol (bombykol) is sufficient to trigger the
complete mating behavior. Conversely, the minor component
(E,Z)-10,12-hexadecadienal (bombykal) is known to have an inhibitory
effect on the mating behavior (Kaissling
and Kasang, 1978). It has been reported that silkmoth female
glands contain bombykol and bombykal in a ratio of 9:1
(Kaissling and Kasang, 1978).
Morphologically the antennae of male B. mori have slightly longer
flagellar branches and a greater number of sensory hairs (sensilla trichodea)
than the female (i.e. they are sexually dimorphic). The antennae of the male
B. mori have 
17 000 trichodial hairs, each of which contains two
types of olfactory receptor neurons (ORNs), each sensitive to one of the two
components (Kaissling and Priesner,
1970; Kaissling and Kasang,
1978).
These ORNs send axons into the first order olfactory center, the ALs where
they make synapses with postsynaptic neurons in conspicuously arranged rounded
modules known as glomeruli (Kanzaki and
Shibuya, 1986; Koontz and
Schneider, 1987). The male B. mori AL comprises 57
ordinary glomeruli (Gs) and sexually dimorphic glomeruli called the
macroglomerular complex (MGC) (Kanzaki and Shibuya,
1983,
1986;
Soo and Kanzaki, 2000). The
MGC is the first-order center for synaptic processing of olfactory information
about the female sex pheromone in the male moth brain
(Boeckh and Boeckh, 1979;
Matsumoto and Hildebrand,
1981; Kanzaki and Shibuya,
1986; Christensen and
Hildebrand, 1987). Koontz and Schneider
(Koontz and Schneider, 1987)
reported that the B. mori MGC is subdivided into four compartments,
each compartment consisting of one or more glomeruli surrounding a central
core of neuropil (MGC 1, 2, 3, 4). In the hawkmoth (Manduca sexta),
the largest MGC glomerulus is situated closest to the entrance of the antennal
nerve and is named the `cumulus', a second, doughnut-shaped MGC glomerulus is
called the `toroid' and a third subunit is named the `horseshoe'
(Strausfeld, 1989;
Hansson et al., 1991;
Heinbockel et al.,
1998).
Intracellular recording has been invaluable in deciphering the coding
mechanisms used by AL local interneurons (LNs) and projection neurons (PNs, AL
output neurons) (Matsumoto and Hildebrand,
1981; Kanzaki and Shibuya,
1986; Christensen and Hildebrand,
1987,
1996;
Kanzaki et al., 1989;
Hildebrand, 1996;
Hansson and Christensen,
1999). M. sexta PNs that innervate the cumulus respond
selectively to the minor pheromone component, while those innervating the
toroid respond selectively to the major component
(Hansson et al.,
1991). Numerical comparison of cells in the ALs of M.
sexta males and females using anatomical techniques has demonstrated that
the males have about 30 MGC-PNs (Homberg
et al., 1988).
In some species of moths, the number of MGC compartments in the male AL
corresponds to the number of pheromone components to which the species'
olfactory receptor neurons respond, while in other moth species the number of
the MGC compartments is smaller (Vickers
et al., 1998; Hansson
and Christensen, 1999). The functional subdivisions in the MGC
have been well studied neurophysiologically in several species of moths
(Hansson et al.,
1991; Hansson and Christensen,
1999). However, the projection sites in higher olfactory centers
in the protocerebrum (PC) from each subdivision are still unclear.
In the present study we investigated the MGC subdivisions of the male B. mori by use of three-dimensional (3-D) reconstruction of the MGC from sequential series of confocal slice images. Morphological and physiological properties of PNs which have arborizations in each subdivision of the MGC were investigated. We found that the B. mori MGC consists of three subdivisions similar to those of M. sexta: the cumulus, toroid and horseshoe. Here we demonstrate that subdivision specific projection neurons transmit information about functionally different pheromone components through three different antennocerebral pathways to different regions in higher olfactory centers, the calyces of the mushroom body (Ca) and the inferior lateral protocerebrum (ILPC). Response patterns of MGC-PNs to pheromonal stimuli correlate with their dendritic arborizations in the subdivisions of the MGC. Also, we demonstrate that the ILPC may be a more important processing site than the Ca for major pheromonal information in B. mori.
Materials and methods
Physiology
Bombyx mori (Lepidoptera: Bombycidae) were reared on an artificial
diet at 26°C and 60% relative humidity. Methods employed in this study
have been described elsewhere (Kanzaki and
Shibuya, 1986; Kanzaki et
al., 1989; Mishima and
Kanzaki, 1999). Adult male moths were used 3–6 days
following eclosion. After cooling (4°C, 30 min) to achieve
anesthesia, the intact moth with its ventral side down was waxed to a plastic
chamber. The head was immobilized with a notched plastic yoke slipped between
the head and thorax. Wings and legs were also immobilized in the chamber. The
brain was exposed by opening the head capsule and removing large tracheae;
intracranial muscles were removed to eliminate brain movements. The antennal
lobe was surgically desheathed to facilitate insertion of microelectrodes.
Electrodes were filled with 4% Lucifer Yellow CH (LY) solution (Sigma, St
Louis, MO) in distilled water. The resistance of the electrodes was 100
M
. A silver ground electrode was placed in the body. The brain was
superfused with saline solution (mM): 140 NaCl; 5 KCl; 7 CaCl2; 1
MgCl2; 4 NaHCO3; 5 trehalose; 5 N-tris
(hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES); and 100 sucrose (pH
7.3). Electrical responses of AL neurons were monitored with an oscilloscope
and recorded on a DAT recorder (RD-125T; TEAC, Tokyo, Japan) at 24 kHz. The
acquired signals were stored on a computer through an A/D converter (Quik Vu
II; TEAC, Tokyo, Japan). The voltage level of the baseline was set above the
noise voltage level. The number of impulses above the baseline was measured
and processed by the computer and shown as impulse-frequency histograms (0.2 s
bins).
Olfactory stimulation
Three olfactory stimuli were used in these experiments: (i) bombykol
(E10,Z12–16:AL; primary pheromone component)
(Butenandt et al.,
1949); (ii) bombykal (E10, Z12–16:OH;
minor pheromone component) (Kaissling and
Kasang, 1978); and (iii) 1-hexanol (E6:AL; a leaf odor). Air-puff
stimuli similar to those described previously
(Kanzaki et al.,
1994) were used. Odorants were applied to a piece of filter paper
(1 x 1 cm) as a 5 µl solution and then inserted into a glass
stimulant cartridge (Pasteur pipette, 1 mm tip diameter). Odor concentration
is expressed as the amount of stimulant applied to the filter paper. In the
present study 10 ng bombykol, 10 ng bombykal and 5 µl hexanol were used.
Since the female gland is known to contain bombykol and bombykal in a ratio of
9:1, 9 ng bombykol + 1 ng bombykal were used as blend stimulation
(Kaissling and Kasang, 1978).
Olfactory stimulation was applied to the antenna ipsilateral to the AL. Each
stimulus was a 500 ms puff at a velocity of 17.5 cm/s and the interval
between puffs was at least 30 s.
3-D reconstruction of a single neuron
We stained each neuron by iontophoretic injection of LY with constant hyperpolarizing current (–1 nA) for 5–10 min. The brain was fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for 1 h at room temperature and dehydrated with an ethanol series and cleared in methyl salicylate. Each stained neuron was imaged frontally using a confocal imaging system (LSM-510; Carl Zeiss, Jena, Germany) with plan apochromat x20 (n.a. = 0.75) and x40 (n.a. = 1.0) objectives. The LY-stained neurons were examined with 458 nm excitation and a long-pass emission filter (>475 nm) in whole mount. In cases where the subdivisions of the MGC were unclear, the brain was immersed in Alexa Fluor 568 (Molecular Probes Europe BV) for 1 h for background staining. The background was examined with 543 nm excitation and a long-pass emission filter (>560 nm) in whole mount. Serial optical sections were acquired at 0.7 µm intervals throughout the entire depth of a neuron. 3-D reconstructions of the labeled neurons were made from these sections.
3-D reconstruction of the MGC
The dissected brains were prefixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for 2 min and immersed in 0.1% LY solution for 4 h at room temperature. The brains were then fixed in 4% formaldehyde for 1 h at room temperature and dehydrated with an ethanol series and cleared in methyl salicylate. Each stained brain was imaged frontally using a confocal imaging system with a plan apochromat x40 (n.a. = 1.0) objective. Serial optical slices were acquired at 0.7 µm intervals. These images were read into an image processing software (Amira; TGS, Berlin, Germany) and analyzed. MGCs which were traced in each optical slice were three-dimensionally reconstructed.
Results
Subdivisions of the B. mori MGC
As with males of many other species of moths, the AL of the male B.
mori consists of coarse neuropil and a specific number of spheroidal
neuropil compartments called glomeruli. According to the shape, position and
size, two types of glomeruli were characterized
(Figure 1A). A male specific
macroglomerular complex (MGC; diameter 150–200 µm) exists at the
entrance of the AL and 57 small-sized glomeruli (ordinary glomeruli,
diameter 30–50 µm) exist ventro-medial to the MGC and are arranged
surrounding the coarse neuropil (Soo and
Kanzaki, 2000).
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As shown in Figure 1A, the
MGC consists of three subdivisions. To know the detailed structure of these
subdivisions 3-D reconstructions of the MGC were made from series confocal
slice images (Figure
1B–E). Three distinct subdivisions were easily observed.
These are: a globular region situated at the entrance of the AL; a
doughnut-shaped region of neuropil adjacent to the globular region; and a
small neuropil ventral to the doughnut-shaped region. These subdivisions
correspond to the `cumulus', `toroid' and `horseshoe' MGC subdivisions
described in M. sexta
(Strausfeld, 1989;
Hansson et al.,
1991). Koontz and Schneider
(Koontz and Schneider, 1987)
reported that the MGC of the male B. mori is subdivided into
compartments consisting of a central glomerulus (MGC 1) and three ring-shaped
glomeruli (MGC 2, 3, 4). The MGC 2, 3 and 4 correspond to the cumulus, toroid
and horseshoe, respectively. 3-D reconstruction of the MGC revealed that the
MGC 1 is a protuberant part of the toroid (MGC 3) rising dorsally into the
cumulus (MGC 2) from a posterior part of the toroid (Figures
1C,
8).
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While slight variations in the form of the subdivisions of the MGC were
observed in different individuals (n = 11), subdivisions into three
compartments appeared to be a constant feature. Here we use the nomenclature
of these subdivisions according to those of M. sexta
(Strausfeld, 1989;
Hansson et al.,
1991)
MGC-projection neurons
Thirty-seven MGC-projection neurons (PNs) were three-dimensionally characterized by intracellular staining with LY (Table 1). Olfactory responses were recorded from 14 out of 37 MGC-PNs. Thirty-one out of 37 MGC-PNs had their cell bodies in the medial cell group (MC) in the AL. Twenty-nine out of 31 had axons running in the inner antennocerebral tract (IACT) leading to the protocerebrum, including the calyces of the mushroom body (Ca) and the inferior lateral protocerebrum (ILPC). Two out of 31 had axons in the IACT leading to the ILPC, but not to the Ca. Six MGC-PNs had their cell bodies in the lateral cell group (LC) in the AL. The axons were in the medial antennocerebral tract (MACT, n = 3) or the outer antennocerebral tract (OACT, n = 3) and sent terminal branches exclusively to the ILPC. None of these PNs projected to the Ca (Table 1). According to the branching areas of dendritic arborizations in the subdivisions of the MGC, MGC-PNs characterized in this study were classified into the following four groups (Table 1).
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Cumulus-PNs
Morphology
Nine PNs were classified into this group (Table 1). In all of these PNs, dendritic arborizations were restricted to the cumulus. Dendritic arborizations of most of the cumulus-PNs appeared to be uniformly distributed throughout the cumulus with numerous fine spines (Figures 2 and 3). A few cumulus-PNs had arborizations separated into some distinct tufts in partitions similar to those of the toroid-PNs (data not shown).
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The cumulus-PN cell bodies (diameter 10–15 µm) resided in the MC of the AL and the axons passed through the IACT to the Ca and ILPC (Figures 2 and 4 and Table 1). The axons of the PNs sent some blebby axonal branches into the whole calyces and extended blebby terminals into the ILPC (Figures 2 and 4). Small-field branches were restricted to the lateral part of the ILPC (Figure 4).
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Physiology
Olfactory responses were recorded from five out of nine PNs in this group.
One example is shown in Figure
5. The morphology of this cell is shown in
Figure 3E. This PN showed
strong excitatory responses to bombykal and pheromone blend applied to the
ipsilateral antenna, whereas no responses were elicited by bombykol, hexanol
or clean air stimulus (control). The background activity was <2 Hz. The
peak firing frequency evoked by bombykal was 85 Hz. The initial period of
phasic firing with a membrane depolarization was followed by a reduction in
spike frequency. The membrane potential was hyperpolarized and firing was
suppressed for 2.5s before restoration of the normal background firing
rate. Another two sets of olfactory stimulation consistently elicited similar
response patterns.
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Spike frequency histograms of four cumulus-PNs are shown in Figure 6. Cell 1 (top row) of the histograms are of the PN shown in Figure 5. All of these PNs showed excitatory responses to bombykal and the pheromone blend. No response was elicited by bombykol, hexanol or control in the cumulus-PNs tested. Thus, the cumulus-PNs tested in this study responded exclusively to the minor pheromone component, bombykal.
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Toroid-PNs
Morphology
Seventeen PNs were classified into this group
(Table 1). Fifteen out of 17
toroid-PNs had dendritic arborizations separated into some distinct tufts in
the toroid subdivisions (Figures
7A and
8A–D). A variety of
distinct tufts in partitions were observed. In one toroid-PN a few thin
processes with fine blebs were observed projecting into the cumulus (arrows in
Figure 8A). The toroid-PNs had
branchings with tufts in the medio-dorsal part of the MGC
(Figure 8A–8D, thick
arrows). This region is a protuberant structure rising dorsally into the
cumulus from a posterior part of the toroid
(Figure 1C). Two out of 17
toroid-PNs had dendritic arborizations with numerous fine spines, similar to
those of the cumulus-PNs (Figures
7B and
8E,F). In case of the PN shown
in Figure 8E, arborizations
were restricted to a distinct partition, the lateral posterior part of the
toroid.
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All the toroid-PN cell bodies (diameter 10–15 µm) resided in the MC of the AL and the axons passed through the IACT (Table 1). Fifteen out of 17 toroid-PNs sent only a single or a few short blebby branches into the Ca (Figures 7A and arrows in Figure 9A). In contrast, wide-field branches with thick blebs were consistently observed in the medial part of the ILPC (Figure 9). Two toroid-PNs which had dendritic arborizations with numerous fine spines sent wide-field thick blebby branches extending into the medial part of the ILPC bypassing the Ca (Figure 7B).
Physiology
Olfactory responses were recorded from 8 out of 17 toroid-PNs. One example
is shown in Figure 10. The
morphology of this cell is shown in Figure
7B. This toroid-PN showed strong excitatory responses with
membrane depolarization to bombykol and the pheromone blend applied to the
ipsilateral antenna, whereas no responses were elicited by bombykal, hexanol
or clean air. Membrane depolarization gave rise to a train of spikes and the
firing was suppressed for 1s before restoration to the normal background
firing rate. The background activity was <5 Hz. The peak firing frequency
evoked by bombykol was 80 Hz. Another two sets of olfactory stimulation
showed similar response patterns.
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Another example is shown in Figure
11. The morphology of this cell is shown in
Figure 7A. This toroid-PN also
showed strong excitatory responses to bombykol and the pheromone blend.
Moreover, inhibitory responses with membrane hyperpolarizations were elicited
by bombykal. No responses were elicited by hexanol or control. The background
activity was <10 Hz. The peak firing frequency evoked by bombykol was
75 Hz. Another two sets of olfactory stimulation showed similar response
patterns.
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Olfactory responses of seven toroid-PNs are shown as spike-frequency histograms in Figure 12; cells 2 and 3 are of the PNs shown in Figures 10 and 11, respectively. All of these toroid-PNs showed excitatory responses selectively to bombykol. In the present study six out of eight toroid-PNs showed excitatory responses to bombykol and no response to bombykal, while two out of eight toroid-PNs showed excitatory responses to bombykol and inhibitory responses to bombykal. None of the eight toroid-PNs tested responded to hexanol or control. Thus, the toroid-PNs tested in this study showed excitatory responses selectively to the major component of the pheromone, bombykol.
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Cumulus and toroid-PNs (c + t-PNs)
Morphology
Six PNs were classified into this group
(Table 1). All these c + t-PNs
had a major neurite extending into two or three subdivisions of the MGC. Each
of the neurites gave rise to numerous filamentous branches with fine spines
throughout the whole MGC including the cumulus and toroid
(Figure 13). One c + t-PN had
fine branches in the horseshoe as well (arrows in
Figure 13C). The cell bodies
(diameter 10–15 µm) of these PNs resided in the LC of the AL. Only
the PNs classified into this group had their cell bodies in the LC
(Table 1). The axons passed
through the medial antennocerebral tract (MACT, n = 3) or the outer
antennocerebral tract (OACT, n = 3) to the ILPC. The axons sent
wide-field blebby branches into the medial part of the ILPC. No projections
were observed in the Ca (Figure
13A,D and Table 1).
Although the paths to the PC are different, i.e. MACT or OACT, it seems that
the projection areas of these two types of c + t-PNs overlap in the ILPC
(Figure 13D).
Physiology
Olfactory responses were recorded from one out of six c + t-PNs. Olfactory
responses of this cell are shown in Figure
14. The morphology of the cell is not shown because of weak
staining of the cell. However, the morphological characteristics were similar
to those of the PN shown in Figure
13A. This c + t-PN showed strong excitatory responses with
membrane depolarization to bombykol, bombykal and the pheromone blend. Weak
excitation was also recorded in response to control and hexanol, suggesting a
weak mechanosensory response. The background activity was <2 Hz. The peak
firing frequencies evoked by bombykol and bombykal were 75 and 65
Hz, respectively. Another two sets of olfactory stimulation showed similar
response patterns.
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Horseshoe-PN
Morphology
Five PNs were classified into this group
(Table 1). Four out of five PNs
had dendritic arborizations with numerous fine spines restricted to the
horseshoe (Figure 15B). One
out of five horseshoe-PNs had arborizations mainly in the horseshoe and partly
in the toroid (data not shown). All horseshoe-PNs had cell bodies (diameter 10
µm) in the MC of the AL. The axons of the PNs passed through the IACT to
the Ca and ILPC (Table 1). The
axons sent some blebby branches into the medio-ventral part of the Ca and into
the lateral part of the ILPC with small-field blebby branchings
(Figure 15A).
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Physiology
Olfactory responses were recorded from one out of five horseshoe-PNs.
Olfactory responses of this cell are shown in
Figure 16. Morphological
characteristics of the cell were similar to those of the horseshoe-PN shown in
Figure 15. This horseshoe-PN
showed excitatory responses with membrane depolarization to bombykal applied
to the ipsilateral antenna, whereas no responses were elicited by bombykol,
mixture, hexanol or clean air. Although bombykal (10 ng) elicited excitatory
responses, the mixture which contained 1 ng bombykal did not excite the PN.
This might be caused by low concentration (sub-threshold) of bombykal
stimulation or perhaps mixture suppression. The background activity was <5
Hz. The peak firing frequency evoked by bombykal was 50 Hz. Another two
sets of olfactory stimulation showed similar response patterns.
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Discussion
Subdivisions of the MGC
Initial sex-pheromone processing in the insect brain takes place in the
male-specific MGC of the AL (Boeckh and
Tolbert, 1993; Christensen and
Hildebrand, 1996; Hildebrand,
1996; Christensen and White,
2000). It has also been reported that the MGC consists of several
functional subdivisions in M. sexta and Heliothis virescens
(Hansson et al.,
1991; Vickers et al.,
1998). Koontz and Schneider
(Koontz and Schneider, 1987)
reported that the MGC of the male B. mori is subdivided into four
compartments consisting of a central glomerulus (MGC 1) and three ring-shaped
glomeruli (MGC 2, 3, 4). In the present study 3-D reconstruction of the B.
mori MGC has led to some important advances in understanding the
structure of the MGC. Our results revealed that MGC 2, 3 and 4 correspond to
the cumulus, toroid and horseshoe, respectively. The structure of the B.
mori MGC is fundamentally similar to that of M. sexta
(Hansson et al.,
1991); however, some different structures were observed. That is,
Koontz and Schneider (Koontz and
Schneider, 1987) reported that MGC 1 is a central glomerulus
independent from the other ring-shaped glomeruli. However, we found that MGC 1
is a part of the toroid (MGC 3). It is a protuberant structure rising dorsally
into the cumulus (MGC 2) from a posterior part of the toroid—MGC 3
(Figures 1C and
8). This is supported by the
morphological structure of the toroid-PNs characterized in this study (e.g.
Figures 7 and
8). Most of the toroid-PNs,
constituent neurons of the toroid, had branchings with tufts in the
medio-dorsal part of the MGC (Figure
8A–D, thick arrows), which corresponds to a protuberant
structure rising dorsally into the cumulus from a posterior part of the toroid
(Figure 1C). Moreover,
projection patterns of some types of antennal sensory fibers restricted to MGC
1 and 3 were observed by cobalt staining from cut hairs on the antenna
(Koontz and Schneider, 1987).
Judging from our physiological results, that toroid-PNs respond exclusively to
bombykol, these sensory fibers may carry bombykol information to the toroid
including the protuberant structure.
MGC-projection neurons
In M. sexta MGC-PNs, the pheromone blend or a component of the
pheromone evokes a characteristic triphasic response consisting of a brief,
hyperpolarizing inhibitory potential followed by depolarization with firing of
action potentials and then a delayed period of hyperpolarization
(Christensen and Hildebrand,
1987; Hansson and Christensen,
1999). In contrast, B. mori MGC-PNs showed a biphasic
response to pheromonal stimulation; typically the firing of action potentials
followed by a period of hyperpolarization. Initial hyperpolarizing inhibition
was not observed (Figures 5,
10,
11,
14 and
16). Similar response
characteristics to B. mori were reported in MGC-PNs of the noctuids
H. virescens and H. zea
(Vickers et al.,
1998; Hansson and Christensen,
1999).
It has been reported that M. sexta males have 30 MGC-PNs,
based on numerical comparison of cells in the ALs of male and female moths
using anatomical techniques (Homberg
et al., 1988). Since the total number of cell bodies
contained in the MC is almost the same in M. sexta and B.
mori—200 (Homberg et
al., 1988; Seki and
Kanzaki, 2002)—B. mori males are also thought to
have similar number of MGC-PNs. In the present study we have characterized
morphologically and/or physiologically 37 MGC-PNs in B. mori males.
These MGC-PNs were classified into four groups according to the branching
areas of dendritic arborizations in the subdivisions of the MGC: cumulus-PNs;
toroid-PNs; cumulus and toroid-PNs (c + t-PNs); and horseshoe-PNs
(Table 1).
In general, MGC-PNs that responded exclusively to bombykol (the principal component of the pheromone blend) had arborizations confined to the toroid, including a protuberant structure rising dorsally into the cumulus from a posterior part of the toroid (Figures 7, 8 and 10, 11, 12). Whereas PNs that responded selectively to bombykal (the minor pheromone component) had arborizations within the cumulus (Figures 2, 3, 5 and 6) or the horseshoe (Figures 15and 16). In this study we present the first report of morphological and/or physiological properties of horseshoe-PNs in moths. A horseshoe-PN showed excitatory responses selectively to bombykal (Figure 16).
PNs that showed excitatory responses to both pheromone components had arborizations in both the cumulus and toroid (c + t-PNs; Figure 14). One c + t-PN had fine branches in the horseshoe as well (Figure 13C). This is interesting because we found a horseshoe-PN which showed excitatory responses to bombykal. It is a possibility that the c + t-PN may receive bombykal information not only in the cumulus but also in the horseshoe. These results indicate that information about these two pheromone components is processed initially in different regions of the MGC.
In two of the toroid-PNs bombykol and bombykal elicited excitatory and
inhibitory responses, respectively. These PNs perform a higher level of
processing than other PNs that arborize in the same subdivisions. Similar
responses have been reported in PNs of M. sexta
(Christensen and Hildebrand,
1987) and H. virescens
(Christensen et al.,
1995, Mustaparta,
1996). In the present study, c + t-PNs had cell bodies in the LC
in the AL and axons in the MACT or OACT
(Table 1 and
Figure 13). We previously
reported that the c + t-PNs showed dose–response characteristics
(Kanzaki and Shibuya, 1986).
M. sexta PNs which have cell bodies in the LC have been reported, but
which ACT their axons pass through is not known
(Heinbockel et al.,
1998).
We found toroid-PNs which have distinct tufts in a variety of confined
regions in the toroid (Figure
8). Koontz and Schneider
(Koontz and Schneider, 1987)
reported that the terminal arborizations of all olfactory receptor neurons
(ORNs) in the MGC are confined to a region 50 µm in diameter in the
MGC. Most of the PNs characterized in the study had arborizations separated
into some distinct tufts in the toroid within 50 µm
(Figure 8). The diameter of
these arborizations corresponds to that of the terminal region of ORNs. Such a
confined field size suggests the possibility that different ORN types may be
segregated into different distinct tufts of PNs. Local interneurons and
feedback neurons from the PC also have their arborizations in this field
(Hill et al., 2002;
Seki and Kanzaki, 2002). It
seems that this confined field may play a role as a local circuit for
olfactory coding. Further, it is possible that there is a functional
connection between the toroid and cumulus. In the present study, two
toroid-PNs showed excitatory responses to the major pheromone component,
bombykol, and inhibitory responses to the minor component, bombykal
(Figure 11). There may be some
connections between the cumulus and toroid, possibly mediated by some
GABAergic local interneurons (Christensen
et al., 1998). In M. sexta, indirect routes of
excitatory input to PNs via intercalated excitatory interneurons
(Sun et al., 1997) or
through disinhibitory circuits involving two inhibitory interneurons arranged
in series have been found (Distler and Boeckh,
1997a,
b;
Christensen et al.,
1998).
None of the MGC-PNs tested (n = 12) in this study responded to
1-hexanol. Although we must investigate responsiveness to many other
non-pheromonal odors, as reported in some species of moths, B. mori
MGC-PNs may also be specialized only for pheromone processing
(Hansson and Christensen,
1999).
Projections in the calyces of the mushroom body and the lateral protocerebrum
MGC-PNs transfer pheromonal information to higher olfactory centers in the
PC, including the Ca and the ILPC through the antennocerebral tracts (ACTs).
In M. sexta five different ACTs have been reported: inner (IACT),
medial (MACT), outer (OACT), dorsal (DACT) and dorsolateral ACTs (DLACT)
(Homberg et al.,
1988). Most of the physiologically characterized MGC-PNs of M.
sexta run through the IACT (Hansson
et al., 1991; Hansson
and Christensen, 1999). Besides the IACT pathways, cobalt
injection into the MGC of M. sexta revealed that some MGC-PNs have
axonal pathways in the OACT and MACT
(Homberg et al. 1988;
Kanzaki et al.,
1989). However, physiological and morphological properties of
M. sexta MGC-PNs which pass through the OACT or MACT have not yet
been well characterized. In the present study, we physiologically and/or
morphologically characterized B. mori MGC-PNs which take three
different pathways to the PC: IACT (n = 31), MACT (n = 3)
and OACT (n = 3) (Table
1).
All the cumulus-PNs, toroid-PNs and horseshoe-PNs characterized in this
study had their cell bodies in the MC of the AL and passed through the IACT to
the PC including the Ca and the ILPC. Interestingly, the projection patterns
and areas in the Ca and the ILPC were different between the cumulus- and
horseshoe-PNs, and the toroid-PNs (Table
1). The axons of the cumulus- and horseshoe-PNs sent blebby axonal
branches into the whole Ca and extended branchings with blebby terminals to a
small field in the lateral part of the ILPC (Figures
2,
4 and
15). However, toroid-PNs sent
only a single or a few short blebby branches into the Ca and extended
wide-field branchings with blebby terminals to the medial part of the ILPC
(Figures 7 and
9). Two toroid-PNs which had
dendritic arborizations with numerous fine spines sent wide-field thick blebby
branches extending into the medial part of the ILPC bypassing the Ca
(Figure 7B and
Table 1). Cumulus- and
horseshoe- and toroid-PNs showed excitatory responses selectively to bombykal
and bombykol, respectively. These results suggest that information about each
pheromone component is conserved at the level of the Ca and the ILPC, and it
seems that the Ca may not be a prominent processing site for the major
pheromone component, bombykol. The Ca may be instead a processing site for
minor pheromonal information and non-pheromonal information. We have
characterized some ordinary glomerular PNs, whose axons sent dense blebby
branches into the whole calyces (data not shown). In contrast, all the
characterized types of M. sexta PNs (cumulus- and toroid-PNs) which
transfer pheromone information, have prominent branchings with varicosities
clustered in certain areas of the Ca
(Homberg et al.,
1988; Hansson et al.,
1991). While, it is generally accepted that the Ca and the ILPC
are the target sites for olfactory information from the MGC in moths, our
results suggest that the ILPC, especially the medial part of the ILPC may be
the more important processing site for major pheromonal information in B.
mori.
Physiological, behavioral and genetic studies in several insect species
suggest that the MB is critical for olfactory and other forms of associative
memory (Heisenberg, 1998;
De Belle and Kanzaki, 1999). In
B. mori the major pheromone component, bombykol is sufficient to
release the complete odor modulated behavior
(Kanzaki, 1998). It is
interesting that toroid-PNs which selectively transfer bombykol information to
the PC had major projections to the ILPC, especially to the medial part of the
ILPC but not to the Ca. This suggests that the bombykol-driven behavior of
B. mori may be an independent behavior isolated from olfactory
associative memory.
In the present study we revealed the projection sites of horseshoe-PNs. They had projections into the Ca and the lateral part of the ILPC through the IACT. These morphological properties are similar to those of cumulus-PNs. Interestingly, the physiologically characterized horseshoe-PN in this study also showed similar response characteristics to those of cumulus-PNs, that is, the cell responded selectively to bombykal.
In the present study we morphologically and/or physiologically characterized c + t-PNs (n = 6). The c + t-PN tested showed excitatory responses to both bombykol and bombykal (Figure 14). These c + t-PNs are morphologically different from toroid-PNs and cumulus-PNs in the following three points (Table 1): (i) all of these c + t-PNs had their cell bodies in the LC instead of the MC; (ii) all of these c + t-PNs passed through the MACT or the OACT to ILPC instead of the IACT; and (iii) all these c + t-PNs projected only to the ILPC, bypassing the Ca (e.g. Figure 13D). Although the paths to the ILPC are different, i.e. MACT or OACT, it seems that the projection areas of these two types of c + t-PNs overlap in the ILPC. However, we still do not know whether the projection areas of c + t-PNs overlap with those of toroid-PNs, cumulus-PNs and horseshoe-PNs. Multiple staining of these PNs in the same brain is our ongoing work in order to investigate precise target areas of these PNs running through each pathway to the ILPC, which will lead to some important advances in understanding the processing of pheromone information in a higher olfactory center, the ILPC.
Acknowledgments
The authors thank Dr Evan Hill for his critical comments on the manuscript. This research was supported by a grant from the basic research activities for innovative bioscience (BRAIN) and a grant-in-aid for scientific research on priority area (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (11168207).
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Accepted November 28, 2002
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