Research School of Biological Sciences, Australian National University, Canberra 2601, Australia
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ABSTRACT |
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Oleskevich, Sharon.
Cholinergic Synaptic Transmission in Insect Mushroom Bodies
In Vitro.
J. Neurophysiol. 82: 1091-1096, 1999.
The mushroom body of the bee brain is an important site for
learning and memory. Here we investigate synaptic transmission in the
mushroom body using extracellular recording techniques in a whole bee
brain in vitro preparation. The postsynaptic response showed
attenuation by cadmium and paired-pulse facilitation, similar to in
vivo findings. This confirms the viability of the in vitro preparation
and supports the isolated whole bee brain as a useful model of the in
vivo preparation. Bath application of the acetylcholine receptor
antagonists, D-tubocurarine and -bungarotoxin attenuated the postsynaptic response by 61 and 62% of control, respectively. The
glutamate receptor antagonists, (+)-2-amino-5-phosphonopentanoic acid
and 6-cyano-7-nitroquinoxaline-2,3-dione, had no effect. The
invertebrate monoamine and neuromodulator, octopamine, transiently increased the postsynaptic response by 130% of control. These results
suggest that synaptic transmission of the olfactory input pathway in
the mushroom body is 1) mediated primarily by
acetylcholine and 2) modulated by octopamine.
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INTRODUCTION |
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The honeybee, Apis mellifera, has a
highly developed olfactory learning capacity and relatively simple CNS
and therefore is well suited for electrophysiological investigation of
learning and memory. The honeybee olfactory pathway has been described in detail (Mobbs 1982). Higher order processing of
olfactory information occurs in the mushroom body (MB) of the
protocerebrum. The MB structure is found in all insects and occupies a
large portion of the insect brain (Strausfeld et al.
1995
). The MB has been likened to the vertebrate hippocampus
and is implicated in learning and memory by anatomic, behavioral, and
molecular studies (Davis 1993
; Hammer and Menzel
1995
). The MB receives multiple sensory inputs (Mobbs
1982
), and partial or complete ablation of this structure
results in defective olfactory learning (Heisenberg et al.
1985
). Localized cooling of the MB induces retrograde amnesia of olfactory learning (Erber et al. 1980
), and several
genes that are essential for associative learning show a preferential
expression in the MB (Han et al. 1992
; Nighorn et
al. 1991
; Skoulakis et al. 1993
).
The MB is a bilateral structure consisting of 340,000 Kenyon cells.
The Kenyon cells are organized such that the dendrites and terminals
are segregated into the calyx (input region), and the and
lobes
(output region), respectively (Mobbs 1982
). The
relatively small size of the Kenyon cells (4-7 µm diam) has precluded in vivo intracellular recording except in the locust MB
(Laurent and Naraghi 1994
). In the honeybee MB,
extracellular field potentials have been recorded following
presentation of external stimuli such as light and scent (Kaulen
et al. 1984
; Mercer and Erber 1983
). More
recently, an extracellular field potential consisting of pre- and
postsynaptic events was recorded in vivo in the honeybee MB following
electrical stimulation of the olfactory input pathway
(Oleskevich et al. 1997
).
Despite extensive anatomic and physiological investigations of the
honeybee MB, the primary neurotransmitter mediating synaptic transmission in this region has not yet been identified. Histochemical and immunocytochemical labeling studies show that the neurotransmitter acetylcholine (ACh) and receptors for acetylcholine (AChR) are present
in the MB (Kreissl and Bicker 1989; Scheidler et
al. 1990
). Calcium imaging studies demonstrate that cultured MB
cells respond to ACh via nicotinic AChR (Bicker and Kreissl
1994
; Cayre et al. 1999
; Goldberg et al.
1999
). Glutamate immunoreactivity and excitatory responses to
glutamate have been reported in the MB of different insects
(Bicker et al. 1988
; Cayre et al. 1999
).
Biogenic amines such as dopamine, serotonin, norepinephrine, and
octopamine (OA) are also present in the MB (Cayre et al.
1999
; Mercer et al. 1983
). OA, an invertebrate
monoamine and neuromodulator, has been shown to facilitate olfactory
learning in the honeybee (Hammer 1993
). Activation of an
OA receptor specifically localized in the MB results in an increase in
intracellular calcium and cyclic 3',5'-AMP (cAMP) (Han et al.
1998
). The cAMP pathway has also been implicated in olfactory
learning in Drosophila, because mutants with a defective cAMP signaling system show impaired learning (for review see
Davis 1993
).
In this study, we investigate the primary neurotransmitter that
mediates synaptic transmission of the olfactory input pathway in the
MB. A newly developed whole bee brain in vitro preparation allowed a
pharmacological identification of the neurotransmitter. The viability
and stability of the new preparation was confirmed by comparison of the
synaptic response to a previously reported in vivo synaptic response in
the MB (Oleskevich et al. 1997). The effect of
cholinergic antagonists and the neuromodulator OA was investigated to
determine their role in modulation of synaptic transmission in the
insect olfactory pathway.
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METHODS |
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Tissue preparation
Whole bee brains (n = 46) were prepared from
adult foraging honeybees, Apis mellifera. The bees were
deeply anesthetized by cooling to minimize animal suffering. A new
dissection method allowed isolation and removal of the whole bee brain
from the head capsule (Fig.
1A). The whole brain (~600
µm thick) was submerged in a recording chamber (0.5 ml), anchored
with a nylon net, and continuously perfused (1.5 ml/min) with
physiological saline containing (in mM) 140 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 1.2 NaH2PO4, 14 glucose, 4 Na-HCO3, and 6 HEPES (pH 7.4; osmolarity, 322 mosM) at room temperature (24°C; Fig. 1B). After
dissection, bee brain activity was allowed to recover for 20-40 min
before the start of the experiment (Kirov et al. 1999).
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Electrophysiology
Extracellular recordings (n = 69) were made as
previously described (Oleskevich et al. 1997). Glass
recording electrodes (0.5-1 M
) were filled with physiological
saline and placed in the center of the median calyx of the MB at a
depth of 50-150 µm (Fig. 1C). The recording electrode was
placed in the center of the calyx, close to the Kenyon cell somata.
Focal stimulation of the antennal lobe (0.1 ms; 3-9 V; 0.2 Hz) was
delivered via a parylene-coated tungsten bipolar electrode (1-2 µm
tip, 2 M
). The exposed tips of the stimulating electrode were
positioned in the dorsomedial antennal lobe (~750 µm from the
recording electrode) where the antenno-glomerular tracts originate
(Mobbs 1982
). Stimulation of the antennal lobe evoked a
characteristic extracellular field response in the median calyx of the
MB, presumably via activation of the antenno-glomerular tract. The
characteristic response helped ensure accurate and reproducible
placement of the stimulating and recording electrode. Additional
experiments confirmed focal stimulation and allowed separate
experiments to be performed on each side of the brain
(Oleskevich et al. 1997
). Paired-pulse facilitation
(PPF) was evoked by two consecutive stimuli separated by 24 ms. The
amplitude of the population spike or afferent volley was measured from
a horizontal line joining the maximum peaks that appear before and
after the response (see Fig. 1C). If the afferent volley
amplitude changed by >15% during the course of the experiment, the
results were not used. Data acquisition and analysis was performed with
pClamp 6.0 and AxoGraph 4.0 (Axon Instruments). Extracellular responses
were low-pass filtered on-line at 3 kHz. All changes in response
amplitudes are expressed as a percent change from the control value.
Results are expressed as means ± SE, and statistical tests of
significance were determined with parametric (paired or unpaired
t-tests) or nonparametric (paired sign) tests, as appropriate.
Materials
Cadmium (50-100 µM), D-tubocurarine (30-60 µM;
RBI), -bungarotoxin (0.2-0.5 µM; RBI),
(+)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 µM;
Tocris), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 µM; Tocris),
and OA (100 µM; RBI) were added to the physiological saline and
applied by bath perfusion.
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RESULTS |
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Synaptic response in the isolated bee brain
Whole bee brains were isolated and maintained in vitro for up to
2.5 h. Extracellular field potentials were recorded in the MB
calyx, following focal stimulation of the antennal lobe (Fig. 1C). As elucidated below, the field potential consisted of a
presynaptic afferent fiber volley and a postsynaptic population spike
superimposed on the rising phase of the field excitatory postsynaptic
potential (Fig. 2A). The mean
amplitudes of the afferent volley and population spike were 0.42 ± 0.04 (SE) mV and 0.65 ± 0.10 mV, respectively (n = 21). The latency from the onset of stimulation was
2.7 ± 0.1 ms for the afferent volley and 6.8 ± 0.2 ms for
the population spike (n = 14). A comparison of the
field potential to that previously reported in vivo (Oleskevich
et al. 1997) shows that the latency from the onset of
stimulation to the population spike was significantly greater in vitro
(P < 0.005). The amplitude of the afferent volley and
the population spike were 62 and 50% smaller, respectively, in vitro
(P < 0.05; Fig. 2A).
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The identities of the components of the field response were confirmed
with cadmium application and paired-pulse stimulation. Bath application
of cadmium (50-100 µM) transiently attenuated the amplitude of the
population spike while the afferent volley amplitude was unchanged
(Fig. 2B). In all animals (n = 8), cadmium significantly depressed the amplitude of the population spike by
54 ± 7% without affecting the afferent volley (7 ± 3;
P < 0.01; Fig. 2C). Inhibition of the
population spike by cadmium resembled in vivo inhibition by cadmium
(1-10 µM; 41 ± 6%; n = 6; P < 0.05; Fig. 2C). Two consecutive stimuli induced a
significant PPF of the population spike (126 ± 33%;
n = 9; P < 0.005; Fig. 2D).
The afferent volley did not show PPF (2 ± 5%; n = 9). PPF of the population spike (72 ± 11%; P < 0.01) but not of the afferent volley (11 ± 5%) was also
observed in vivo. Together, the inhibition by cadmium and PPF of the
population spike suggest that the field potential recorded in the MB
consists of a presynaptic afferent fiber volley and a postsynaptic
population spike.
Synaptic response is mediated by ACh
Bath application of D-tubocurarine (30-60
µM), a competitive antagonist at the nicotinic AChR subtype, produced
a transient reduction of the population spike without affecting the
afferent volley (Fig. 3A).
D-Tubocurarine significantly inhibited the population spike
by a mean of 61 ± 6%, while the afferent volley was unchanged (4 ± 5%; n = 9; P < 0.005).
The irreversible nicotinic AChR antagonist,
-bungarotoxin (
-Bgt;
0.2-0.5 µM), was bath applied for 15-30 min to ensure maximal
diffusion through the preparation.
-Bgt attenuated the population
spike that showed no recovery 15-20 min after drug wash out (Fig.
3B).
-Bgt significantly attenuated the response by
62 ± 5% without significantly affecting the afferent volley
(
21 ± 8%; n = 5; P < .0001).
Antagonists at the glutamate N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
subtypes, D-AP5 (50 µM) and CNQX (5 µM), respectively,
did not affect the amplitude of the population spike (Fig.
3C). Coapplication of D-AP5 and CNQX had no
effect on the population spike (2 ± 3%; n = 7;
Fig. 3D).
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Modulation by OA
Bath application of the invertebrate monoamine, OA (100 µM),
transiently increased the population spike without affecting the
afferent volley (Fig. 3E). On average, the population spike more than doubled in the presence of OA. The mean increase of the
population spike was 129 ± 68% (n = 7;
P < 0.01) and 5 ± 4% for the afferent volley
(n = 6; Fig. 3F). In three preparations, the
enhancement of the population spike by OA was followed by a depression
of both the afferent volley (
85 ± 4%) and the population spike
(
93 ± 2%; data not shown).
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DISCUSSION |
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A newly developed whole bee brain in vitro preparation allowed an
investigation of the pharmacology and modulation of a synaptic response
in the MB of the honeybee. The suitability of the new preparation was
confirmed by comparison of the synaptic response to a response
previously recorded in vivo. Inhibition by cadmium suggested an
effective drug perfusion, whereas paired-pulse facilitation demonstrated a viable tissue preparation. The synaptic response was
inhibited by the cholinergic receptor antagonists,
D-tubocurarine and -bungarotoxin but not by the
glutamate receptor antagonists, AP5 and CNQX. OA transiently increased
the synaptic response. The results suggest that the synaptic response
is mediated primarily by ACh and can be modulated by OA.
Isolated whole bee brain preparation
The whole brain in vitro preparation provided access to the neural
circuitry responsible for olfactory learning in the honeybee. The
viability of the preparation is evident in the similarity of the
synaptic response to the response recorded in vivo with regard to time
course, PPF, and inhibition by cadmium (Oleskevich et al.
1997). This preparation has the advantage over the in vivo honeybee preparation in that the addition of pharmacological agents to
the physiological saline ensures thorough application at an effective
concentration. In the in vivo preparation, higher drug concentrations
were required to evoke similar effects (Oleskevich et al.
1997
). The in vitro preparation has an advantage over mammalian slice preparations and cultured honeybee MB cells, in that the brain is
kept whole and the neural circuitry remains intact. A "semi-slice"
preparation has been described for the blow fly brain (Brotz et
al. 1995
), and an in vitro preparation was recently employed
for characterization of honeybee antennal lobe neurons (Kloppenburg et al. 1999
). Our in vitro recordings of
honeybee MB neurons helped to identify the neurotransmitter and
receptor subtype, which may underlie higher-order olfactory information processing in the honeybee.
Focal stimulation in the antennal lobe evoked an extracellular field
response in the MB. The field response was composed of a presynaptic
afferent volley and a postsynaptic population spike. The amplitude of
the afferent volley and the population spike was smaller in vitro than
in vivo. The synaptic efficacy was not compromised because both the
afferent volley and the population spike were similarly attenuated. The
efficiency of stimulation may have been reduced due to a change in
orientation of the stimulating electrodes and the antennal lobe in
vitro versus in vivo. The latency from the onset of stimulation to the
peak of the afferent volley and the population spike was similar to
that observed in vivo, further reinforcing the presence of a mono-
rather than polysynaptic response (Oleskevich et al.
1997).
The population spike showed attenuation by cadmium and PPF. PPF is
generally assumed to be a synaptic phenomenon in which presynaptic
residual calcium causes an increase in neurotransmitter release when a
second stimulation is applied. Cadmium is a calcium channel blocker and
can inhibit calcium-dependent neurotransmitter release and hence the
postsynaptic response, without affecting the sodium channel-dependent
presynaptic volley. The concentration of cadmium used did not fully
block the calcium channels. Together, these results further support
that the field potential recorded in the MB consists of a presynaptic
afferent fiber volley and a postsynaptic population spike
(Oleskevich et al. 1997).
Pharmacology and modulation of the synaptic response
The inhibition of the population spike by
D-tubocurarine and -Bgt provided the first direct
physiological evidence that ACh mediates synaptic transmission between
the antennal lobe and the MB. Specifically, the inhibition of the
synaptic response by
-Bgt suggests that ACh is acting via a
nicotinic AChR subtype containing the
7 subunit (Couturier et
al. 1990
). Nicotinic, muscarinic, and mixed
nicotinic/muscarinic ACh receptors have been observed in the nervous
tissue of different insects (Breer and Sattelle 1987
).
In the honeybee, labeling studies with acetylcholinesterase, AChR
antibodies, and iodinated
-Bgt demonstrate an abundance of ACh and
AChRs in the calyx of the MB, where the afferent input fibers from the
antennal lobe terminate on the dendrites of the Kenyon cells
(Kreissl and Bicker 1989
; Mobbs 1982
;
Scheidler et al. 1990
). These studies were unable to
exclude the possibility of presynaptic nicotinic AchRs, which modulate
rather than mediate synaptic transmission in vertebrate CNS
(Wonnacott 1997
).
There could be several explanations for the component of the population
spike, which remained following -Bgt application. An
-Bgt-insensitive subtype of nicotinic AChR has been described in
insects (Benke and Breer 1989
), and an incomplete
-Bgt mediated inhibition of an ACh response has been reported in the
honeybee MB (Bicker and Kreissl 1994
; Goldberg et
al. 1999
). However, this cannot account for the component
remaining after D-tubocurarine application, which is known
to block both the
-Bgt-sensitive and insensitive nAChRs in
vertebrates (Zhang et al. 1996
). A
D-tubocurarine- and
-Bgt-insensitive nicotinic AChR
may exist in insects, or the remaining component may result from the
co-release of a secondary transmitter. There is evidence for glutamate
immunoreactivity in the calyx of the locust MB (Bicker et al.
1988
), and glutamate excitatory responses were observed in most
cultured MB neurons in the cricket (Cayre et al. 1999
).
However, the lack of effect of AP5 and CNQX in the honeybee suggests
that the remaining component is not glutamate acting at glutamate
receptors with known mammalian synaptic receptor pharmacology.
The invertebrate monoamine, OA, increased the amplitude of the
population spike. Previous studies have shown that OA can increase the
extracellular field response to external olfactory stimuli (Mercer and Erber 1983). Activation of an OA receptor in
the MB of Drosophila elevates intracellular calcium and
stimulates cAMP (Han et al. 1998
). Perhaps the
activation of a cAMP signaling pathway provides a molecular basis for
the ability of OA to facilitate olfactory learning in the honeybee
(Hammer 1993
). The cAMP pathway has been implicated in
learning in Drosophila mutants, which are defective in cAMP
phosphodiesterase (dnc), adenylyl cyclase (rut) or protein kinase A
(DCO) and show impaired associative learning (for review see
Davis 1993
). The biphasic nature of the OA modulation that we observed in some animals has been reported elsewhere
(Cayre et al. 1999
). OA may have complex actions that
include increased excitation and decreased inhibition.
Conclusion
This study provides physiological evidence that synaptic transmission of the olfactory input pathway to the MB is mediated primarily by ACh acting at the nicotinic AChR. The Ach response was enhanced by OA, an important neuromodulator for olfactory learning in the honeybee.
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ACKNOWLEDGMENTS |
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The author is grateful to Drs. J. D. Clements and M. V. Srinivasan for useful discussions and comments on the manuscript.
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FOOTNOTES |
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Address for reprint requests: S. Oleskevich, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, A.C.T 2601, Australia.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 March 1999; accepted in final form 29 April 1999.
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REFERENCES |
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