Visualization of modulatory effects of serotonin in the silkmoth antennal lobe
Institute of Biological Sciences, University of Tsukuba, Ibaraki 305-8572, Japan
* Author for correspondence (e-mail:kanzaki{at}biol.tsukuba.ac.jp)
Accepted 14 October 2002
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Summary |
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Key words: insect, Bombyx mori, moth, olfaction, optical recording, voltage-sensitive dye, brain
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Introduction |
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However, olfactory information is embedded in the responses of neuronal
populations. Gaining an understanding of how populations of neurons respond to
olfactory stimuli is clearly vital to deciphering mechanisms of olfactory
processing. Novel techniques such as multi-unit extracellular recording arrays
and optical imaging allow for the simultaneous monitoring of neuronal
responses in spatially distinct regions of the AL. These techniques will lead
to a greater understanding of olfactory coding mechanisms in the insect brain.
For example, neural ensemble recordings in M. sexta revealed that
there is strong heterogeneity among the response profiles of closely spaced
individual neurons in the AL (Christensen
et al., 2000). Furthermore, optical imaging with a
voltage-sensitive dye in the bumblebee (Bombus terrestris) AL has
shown that odor-induced oscillations are localized to distinct glomeruli
(Okada and Kanzaki, 2001
).
Imaging of population responses in the AL will also be extremely useful in
understanding how neuromodulators can affect the dynamics of olfactory
processing.
Serotonin, a biogenic amine, may play a vital role in olfactory coding
processes in the insect AL. A pair of unique serotonin-immunoreactive (SI)
neurons that innervate both ALs has been identified in many insects
(Schürmann and Klemm,
1984; Kent et al.,
1987
; Rehder et al.,
1987
; Homberg and Hildebrand,
1989
; Breidbach,
1990
; Salecker and Distler,
1990
; Sun et al.,
1993
; Hill et al.,
2002
). The B. mori SI neuron innervates every glomerulus
in the contralateral AL, fires long-duration spontaneous action potentials and
responds to mechanosensory stimuli to the antennae
(Hill et al., 2002
). Electron
microscopic examination of the processes of the M. sexta SI neuron
has shown a predominance of output synapses in the contralateral AL
(Sun et al., 1993
), suggesting
that the SI neuron may serve a centrifugal role. Numerous studies have
revealed that serotonin modulates the responses of AL neurons. For example, in
M. sexta, serotonin enhances the responses of individual AL neurons
to both olfactory and electrical stimulation,
(Kloppenburg and Hildebrand,
1995
; Kloppenburg et al.,
1999
; Kloppenburg and
Heinbockel, 2000
), as well as enhancing the amplitude and duration
of pheromone-evoked local field potentials in the MGC
(Kloppenburg and Heinbockel,
2000
). In vitro, serotonin inhibits two types of
K+ currents, as well as a voltage-activated Ca2+
current, in M. sexta AL neurons
(Mercer et al., 1995
). It has
been proposed that the effects of serotonin on the K+ currents may
underlie the serotonin-induced increases of AL neuron excitability
(Kloppenburg et al.,
1999
).
Are the modulatory effects of serotonin homogenously distributed throughout the AL or are there area-specific differences? Does serotonin affect neural processing in the Gs as well as in the MGC? In order to answer these questions we used a voltage-sensitive dye and a high-speed optical imaging system to visualize possible modulatory effects of serotonin in the silkmoth AL. Here, we report that optical responses in both the MGC and Gs were significantly greater, and significantly longer lasting, following serotonin application. Furthermore, we found that serotonin had a significantly greater effect in the toroid than in the cumulus, and that the enhancing effects of serotonin were also non-homogenously distributed in the Gs.
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Materials and methods |
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Optical imaging
A suction electrode (Fig.
1A) was used to stimulate the antennal nerve (AN; 30-200 µA,
0.5 ms). The camera unit of the optical imaging system (Fuji HR Deltaron 1700,
Fuji Film Microdevice, Tokyo, Japan) has a resolution of 128
photopixelsx128 photopixels. Using the 10x objective, each pixel
represents an area of 7 µmx7 µm. Excitation and emission filters
used in this optical system passed light lengths of 535±25 nm and
>615 nm, respectively. A dichroic mirror was placed in the light path of a
metal halide lamp (KMH-250, BMH-250; Kiyohara Optical Laboratory, Tokyo,
Japan). Eight responses to electrical stimulation of the AN were averaged in
each recording session. Images were captured every 0.6 ms.
Serotonin application and wash
Following acquisition of the control optical responses, serotonin
(10-4-10-5moll-1) was bath applied for 12
min. During this 12-min incubation period, the saline solution containing
serotonin was changed 3-4 times. Following bath application of serotonin,
optical responses in the AL to electrical stimulation of the AN were acquired
(the same amount of current was used as in the control). Serotonin was washed
off with normal saline solution for 15 min, changing the solution in the
recording chamber every 2 or 3 min. After washing, the optical response in the
AL was acquired, again using the same amount of current as the control.
Data analysis
Following the collection of the raw optical data, we used programs to
calculate the change in fluorescence divided by the background fluorescence
level (F/F) and to compensate for photobleaching. We also
processed the raw data with a finite impulse response (FIR) filter, which cut
off signals above 90 Hz.
In order to directly visualize and to understand the time course of the effects of serotonin, we used programs to rotate and shift optical data files to match the control position (in most cases, the brain was in a slightly different position following serotonin application and wash). The rotation and shifting of the images resulted in a good correlation between the images (in all cases r>0.9). The average optical response in 100 pixels of the AN (70 µmx70 µm) was used to normalize the optical responses in the AL. The serotonin-effect optical files were calculated by subtracting the control responses from the serotonin responses and then dividing by the maximum control values for each pixel. This was necessary because the control optical responses were not of uniform strength throughout the AL, therefore the strength of the serotonin effect depended upon the strength of the control optical response in each pixel.
The duration of the optical response was calculated by measuring the time
from the point where the rising phase of the control response reached 50% of
its peak amplitude to the point where the falling phase fell to this value. As
the rising phases were virtually identical in the control and serotonin
optical responses, and as previous imaging studies have shown that the rising
phase of the optical response is purely due to the activity of sensory axons
(Ai et al., 1998), we measured
the duration of the control and serotonin optical responses using 50% of the
control peak amplitude as the reference points. This was applicable to the
control and serotonin optical data but not to the wash response. The wash
response consistently had a slightly different rising phase than either the
control or serotonin responses; therefore, this method for measuring the
duration of the response could not be applied to the wash responses.
To determine statistical significance we used a paired t-test. An asterisk indicates a significant difference (P<0.05), and two asterisks indicate a highly significant difference (P<0.005).
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Results |
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Following bath application of serotonin, in seven out of seven preparations, the normalized optical responses in the AL were greater, longer lasting and distributed in different areas of the AL compared with controls. The optical response following the wash with saline resembled that of the control (data not shown). Typical normalized control and serotonin optical responses are shown in Fig. 3 (images are 1.2 ms apart). In the control (Fig. 3A), electrical stimulation of the AN results in a wave of depolarization that traveled throughout the AL. Strong depolarizing responses were first observed in the MGC and, in later frames, throughout the entire AL. After bath application of serotonin for 12 min (Fig. 3B), the optical responses in the AL are stronger, localized to larger regions of the AL and the optical response is longer lasting. The serotonin effect (Fig. 3C) is localized mainly in the MGC and in various regions of the Gs. In the later frames, the serotonin effect can be observed to be strongest in regions of the Gs that may represent individual glomeruli (arrows in Fig. 3C).
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Bath application of serotonin caused significant increases in the optical responses in both the MGC (48.7±8.7%; P<0.001, N=7) and in the Gs (40.0±8.0%; P<0.005, N=5) compared with the controls (Fig. 4). These effects reversed significantly with washing in both the MGC and Gs (P<0.05 in both cases).
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In order to determine if serotonin significantly affected the duration of the optical responses in the MGC and Gs, we measured the time from the point where the optical response reached 50% of the peak value of the control response (see Materials and methods for details) to where it dropped to 50% of this value. Serotonin significantly enhanced the duration of the optical response in both the MGC, from 6.6±0.2 ms to 8.3±0.4 ms (P<0.005, N=7), and in the Gs, from 8.3±0.2 ms to 10.1±0.2 ms (P<0.005, N=5) (Fig. 5).
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The effects of serotonin varied depending on the region of the AL (Fig. 6). Optical responses were measured in six compartments of the MGC (each compartment representing an area of 81 µmx81 µm) and in five compartments of the Gs (each compartment representing an area of 144 µmx144 µm) (see inset in Fig. 6). Among preparations, there was variation in the regions that had the strongest enhancement due to serotonin. In all preparations, the enhancing effects of serotonin were non-homogenously distributed throughout the AL. Fig. 6 shows the effects of serotonin in six regions of the MGC and five regions of the Gs in one preparation. In this preparation, the modulatory effects of serotonin were stronger in the toroid than in the cumulus compartments of the MGC, and the enhancing effects were greater in some Gs regions (i.e. Gs3) than in others (i.e. Gs2 and Gs4).
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Serotonin had a stronger enhancing effect in the toroid than in the cumulus (Fig. 7A). The average peak increase in the optical response in the toroid (54.9±8.0%; N=7) was significantly greater than in the cumulus (42.6±10.2%; P<0.05, N=7). Additionally, the effects of serotonin in the central cumulus were significantly greater than in the lateral cumulus (49.3±11.4% versus 39.0±11.4%; P<0.05, N=7; Fig. 7B). There were no significant differences among the enhancing effects of serotonin in the medial, central and lateral toroid (Fig. 7B).
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In the Gs, the enhancing effects of serotonin were also non-homogenously distributed (Fig. 7C). The enhancement due to serotonin was significantly greater in Gs3 than in Gs1, Gs4 and Gs5 (60.8±8.8% versus 32.0±11.8%, 37.7±5.8% and 21.2±13.8%, respectively; P<0.05, N=5).
Application of a lower concentration of serotonin (10-5 mol l-1) did not result in a significant enhancement of the optical responses in the AL. Following application of serotonin at 10-5 mol l-1, the peak optical responses in the MGC and Gs were 3.5±14.4% weaker (N=3) and 9.2±10.9% stronger (N=3), respectively; these did not represent significant changes.
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Discussion |
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The concentrations of serotonin used in this study
(10-4-10-5 mol l-1) are similar to the
concentrations used by other studies examining the effects of serotonin on
moth AL neurons (Kloppenburg and
Hildebrand, 1995; Kloppenburg
et al., 1999
; Kloppenburg and
Heinbockel, 2000
). While it is impossible to determine the exact
concentration of serotonin that reaches the synaptic regions, it is safe to
assume that, due to the glial sheath encasing each glomerulus and the presence
of serotonin uptake mechanisms, it is a few orders of magnitude lower than the
bath applied concentration.
An ultrastructural study of the SI putative AL feedback neuron in M.
sexta demonstrated the presence of dense-cored vesicles in the
contralateral AL and also found a low number of synaptic sites
(Sun et al., 1993). Taken
together, these data suggest the possibility of non-synaptic release of
serotonin. Serotonin could potentially diffuse throughout a glomerulus and
bind to receptors in outer regions of the glomerulus where the SI neuron does
not branch (Kent et al., 1987
;
Sun et al., 1993
;
Hill et al., 2002
). Thus,
serotonin released from the SI neuron could potentially bind to receptors on
the sensory terminals (located in the outer regions of glomeruli). The
enhancement of the optical responses in the MGC and the Gs reported here could
be due, in part, to serotonin acting on the sensory terminals. For example, by
altering Ca2+ influx into the presynaptic terminal, serotonin could
lead to an increase in neurotransmitter release. Such presynaptic effects of
serotonin have been described in the crayfish (Procambarus clarkii)
neuromuscular junction, where serotonin increases the number of vesicles
released (Southard et al.,
2000
) and increases the amount of reliable vesicles
(Wang and Zucker, 1998
). A
similar effect in B. mori could lead to an increased release of
transmitter from sensory terminals, which could lead to a larger number of
action potentials in postsynaptic neurons. Such increases in postsynaptic
action potentials could underlie the increases in the amplitude and duration
of the optical responses reported here.
Serotonin could also be acting on the postsynaptic neurons, the LNs and
PNs. Studies of M. sexta AL neurons in vitro have
demonstrated that serotonin leads to an increase in input resistance, spike
broadening, a decreased response latency and an increase in the number of
spikes elicited by electrical stimulation
(Mercer et al., 1995). Mercer
et al. proposed that these effects are due, at least in part, to effects on
three types of K+ channels. The observed enhancement of the optical
responses in the MGC and Gs in the present study could result from these kinds
of effects on populations of LNs and PNs. If a population of AL neurons had
increased input resistances, increased excitability and fired broader spikes,
one would expect that stronger and longer-lasting membrane potential
depolarizations would be observed throughout the AL.
We observed that the enhancing effects of serotonin were non-homogenously distributed in the AL. This phenomenon could, in theory, be due simply to differences in the penetration of serotonin into different parts of the AL. However, we feel that this is unlikely due to the fact that the entire AL was desheathed in each preparation. Also, as serotonin was bath applied, it should have penetrated equally throughout the AL. It seems unlikely that the glial sheaths encasing some glomeruli pose more of a diffusional barrier than others. While it is feasible that the glomeruli situated in the interior of the AL may not have been exposed to as high a concentration of serotonin as more peripherally located glomeruli, our imaging technique mainly records membrane potential changes from the surface of the brain, so these considerations are essentially moot.
The non-homogeneity of the effects of serotonin in the AL could be due to
differences in the distribution and density of serotonin receptors in
different regions of the AL. The observed differences in the enhancing effects
of serotonin in the MGC are particularly interesting because the SI neuron in
several insect species has branches in both the cumulus and the toroid
(Kent et al., 1987;
Sun et al., 1993
;
Hill et al., 2002
). In B.
mori and M. sexta, the toroid is the region of the MGC in which
PNs that respond to the major pheromone component branch, while the cumulus is
the compartment in which PNs that respond to the minor component branch (R.
Kanzaki, unpublished observations; Hansson
et al., 1991
). The behavioral implications of the present data are
that serotonin release into both the cumulus and the toroid could lead to a
greater increase in the moth's sensitivity to the major pheromone component
compared with the minor component.
This is the first report of serotonin enhancing neural responses in the Gs.
Intracellular recording from Gs PNs in B. mori and M. sexta
have revealed that they respond to general odors
(Kanzaki and Shibuya, 1987;
King et al., 2000
). Therefore,
serotonin released into the AL could potentially enhance the responses of PNs
to general odors as well as to pheromones. General odors play very important
roles in the lives of insects, in behaviors such as locating food sources and
host plants for oviposition (Willis and
Arbas, 1991
). The finding that serotonin enhances neuronal
responses in the Gs as well as the MGC could have been predicted from the
branching pattern of the B. mori SI neuron, which branches in every
glomerulus of the contralateral AL (Hill
et al., 2002
). The non-homogenous distribution of the enhancing
effects of serotonin in the Gs could also stem from differences in the
distribution and density of serotonin receptors in the Gs. Alternatively, it
is possible that serotonin enhances the activity of populations of both
inhibitory and excitatory LNs with branches in many Gs. The net serotonin
effect would therefore depend on the relative activation of inhibitory and
excitatory LNs in each region of the AL. At any rate, due to the
non-homogeneity of the serotonin effect in the Gs, release of serotonin
throughout the Gs (i.e. by the SI neuron) could result in greater increases in
sensitivity to some general odors than to others. It would be worthwhile
recording intracellularly from Gs PNs and/or LNs and examining whether or not
serotonin differentially modulates the responses to different general
odors.
The serotonin-induced enhancement of neural responses in the AL described
here and previously in M. sexta
(Kloppenburg and Hildebrand,
1995; Mercer et al.,
1995
; Kloppenburg et al.,
1999
; Kloppenburg and
Heinbockel, 2000
) may in part underlie behavioral modification in
moths caused by exogenous application of serotonin. In the moth
Trichoplusia ni, it has been reported that serotonin applied
exogenously extends the time window during which pheromone-induced behavior
can be elicited (Linn and Roelofs,
1986
). In B. mori, exogenous application of serotonin
increases the sensitivity of the moth to pheromone, while application of
serotonin receptor antagonists decreases the sensitivity (L. Gatellier,
unpublished observations). The serotonin-induced behavioral changes cited
above could stem, in part, from the effects of serotonin in the AL. Enhanced
neuronal responses in the AL would be relayed to higher olfactory centers in
the protocerebrum that are involved in the generation of descending signals
that produce pheromone-oriented behaviors (Kanzaki et al.,
1991
,
1994
).
In the future, we plan to use optical-imaging techniques to examine the effects of serotonin application on pheromone and general odor-evoked optical responses in the AL. Optical imaging is a technique well-suited to the study of olfactory systems and will continue to increase our understanding of olfactory processing mechanisms and the role(s) that neuromodulation plays in olfaction.
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Acknowledgments |
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