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The Journal of Neuroscience, May 15, 1998, 18(10):3650-3658
A Novel Octopamine Receptor with Preferential Expression in
Drosophila Mushroom Bodies
Kyung-An
Han1,
Neil S.
Millar3, and
Ronald L.
Davis1, 2
Departments of 1 Cell Biology and
2 Neurology, Baylor College of Medicine, Houston, Texas
77030, and 3 Wellcome Laboratory for Molecular
Pharmacology, Department of Pharmacology, University College London,
London WC1E 6BT, United Kingdom
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ABSTRACT |
Octopamine is a neuromodulator that mediates diverse physiological
processes in invertebrates. In some insects, such as honeybees and
fruit flies, octopamine has been shown to be a major stimulator of
adenylyl cyclase and to function in associative learning. To identify
an octopamine receptor mediating this function in
Drosophila, putative biogenic amine receptors were
cloned by a novel procedure using PCR and single-strand conformation
polymorphism. One new receptor, octopamine receptor in mushroom bodies
(OAMB), was identified as an octopamine receptor because human and
Drosophila cell lines expressing OAMB showed increased
cAMP and intracellular Ca2+ levels after octopamine
application. Immunohistochemical analysis using an antibody made to the
receptor revealed highly enriched expression in the mushroom body
neuropil and the ellipsoid body of central complex, brain areas known
to be crucial for olfactory learning and motor control, respectively.
The preferential expression of OAMB in mushroom bodies and its capacity
to produce cAMP accumulation suggest an important role in synaptic
modulation underlying behavioral plasticity.
Key words:
adenylyl cyclase; single-strand conformation
polymorphism; G-protein-coupled receptor; associative learning; octopamine receptor; mushroom bodies; neuromodulation
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INTRODUCTION |
Neuromodulatory systems play crucial
roles for animal behavior by modifying the synaptic output of relevant
neurons. Octopamine is a major neuromodulator with neurotransmitter and
neurohormone functions that mediates diverse physiological processes in
the peripheral nervous system and CNS of invertebrates. It acts as a
neurotransmitter for light production in the firefly lantern, has an
excitatory modulatory function in the somatic and visceral muscles of
locust, and regulates carbohydrate and fatty acid metabolism in locust
(David and Coulon, 1985 ). Female fruit flies that are devoid of
octopamine because of a biosynthetic defect display an egg retention
phenotype, suggesting a modulatory role of octopamine in oviductal
muscular contraction (Monastirioti et al., 1996). Octopamine is also
involved in the display of submissive postures in lobsters (Livingstone
et al., 1980), escape behavior of crayfish (Glanzman and Krasne, 1983),
and feeding behaviors of blowflies (Long et al., 1986) and honeybees
(Braun and Bicker, 1992). In Drosophila, inactive mutants
containing 15% wild-type levels of octopamine display hypoactivity
(O'Dell, 1993). Conversely, octopamine application to decapitated
flies induces strong stimulation of locomotion and grooming behavior
(Yellman et al., 1997). Moreover, crucial roles of octopamine are
implicated in complex behaviors, including conditioned courtship and
olfactory learning in Drosophila (Dudai et al., 1987;
O'Dell, 1994). Together, these observations illustrate the diverse
functions of octopamine in motor control and behavior. These effects
are presumably produced by octopaminergic neurons that are dispersed in
the CNS (Monastirioti et al., 1995) acting on receptors at their
targets. Although biochemical studies have revealed high-affinity
octopamine binding sites (Dudai and Zvi, 1984) and a strong potency of
octopamine to stimulate adenylyl cyclase (AC; Uzzan and Dudai, 1982),
an octopamine receptor with this activity has not been identified.
The cumulative studies of Drosophila learning mutants have
revealed two overriding parameters for olfactory learning. First, the
cAMP signaling pathway is critical for normal olfactory conditioning. Animals defective in cAMP phosphodiesterase (dnc), AC
(rut), and protein kinase A (DCO) are impaired in
olfactory learning (Davis, 1996). Second, the mushroom bodies are a
major neuroanatomical site for associative learning. Neuroanatomical
and physiological studies have shown that mushroom bodies receive
diverse sensory inputs (Schürmann, 1987), and flies with
malformed or missing mushroom bodies have defective olfactory learning
(Heisenberg et al., 1985; de Belle and Heisenberg, 1994). Furthermore,
genes critical for learning and memory, dnc, rut,
DCO, leo, and Volado, are
predominantly expressed in the mushroom body neuropil (Nighorn et al.,
1991; Han et al., 1992; Skoulakis et al., 1993; Skoulakis and Davis,
1996; Grotewiel et al., 1997). In addition, the  subunit of
Gs-proteins (Gs) is expressed at a higher
level in the mushroom body neuropil (Forte et al., 1993), and the
targeted expression of the constitutively active
Gs-protein abolishes olfactory learning (Connolly et
al., 1996). These observations indicate that mushroom bodies are
principal neuroanatomical substrates for olfactory learning and memory
and that they use a cAMP-mediated signaling pathway mediated by
G-protein-coupled receptors (GPRs) for their physiological modulation.
However, the identity of the modulatory neurotransmitter that triggers
this biochemical cascade in mushroom bodies remains unidentified.
We report here the isolation of a novel octopamine receptor, octopamine
receptor in mushroom bodies (OAMB). This receptor is highly enriched in
the mushroom bodies and stimulates cAMP and intracellular
Ca2+
([Ca2+]i) accumulation on
octopamine application. This is the first octopamine receptor cloned
from insects with potency to activate cAMP signaling cascades. The key
roles for octopamine in behavioral plasticity, the prominent expression
of OAMB in the mushroom body neuropil, and the ability of OAMB to
activate AC make this receptor an attractive candidate for mediating
the signal transduction cascades underlying learning and memory
processes in Drosophila.
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MATERIALS AND METHODS |
Nucleic acids. Primers used for PCR were
5'-TTCGTCATCTGCTGGCTGCCCTTCTTC-3' and 5'-TGGCTGGGCTACATCAACTCG-3',
corresponding to sequences in transmembrane domains (TMs)
VI and VII of the Drosophila tyramine receptor
(TYR) (Arakawa et al., 1990; Saudou et al., 1990). The cDNA clones
corresponding to TYR (Saudou et al., 1990), DRO1 (Witz et al., 1990),
DRO2A, and DRO2B (Saudou et al., 1992) were kindly provided by Dr. R. Hen (Columbia University, School of Medicine, New York, NY). cDNAs were
synthesized using avian myeloblastosis virus reverse transcriptase
(Promega, Madison, WI) and random hexamers (Han and Kulesz-Martin,
1992). PCRs were performed with 100 ng of genomic DNA or cDNA in the
presence of 5 µCi of [32P]dCTP (Han and
Kulesz-Martin, 1992). For single-strand conformation polymorphism
(SSCP) analysis, the PCR products were diluted 10-fold in 0.1% SDS and
10 mM EDTA, denatured at 95° for 5 min, and resolved on
4% nondenaturing acrylamide gels (Orita et al., 1989). Isolation of
nucleic acids, blotting, cloning, screening, and sequencing were
performed using standard procedures.
Pharmacology. A 2461 bp fragment [nucleotides (nt)
919-3380] of OAMB cDNA containing the open reading frame was
subcloned into the Drosophila expression vector pRmHa3 and
transfected into the Drosophila S2 cell line (Millar et al.,
1994). The same OAMB cDNA fragment was also subcloned into the
mammalian expression vector pcDNA1/Amp (Invitrogen, Torrance, CA) and
transfected into human HEK-293 cells. Stably transfected S2-OAMB cells
and transiently transfected HEK-OAMB cells were assayed for
agonist-induced changes in cAMP levels using a
[3H]cAMP assay system (Amersham, Arlington
Heights, IL) as described by Han et al. (1996). Cells were incubated
with agonists in the presence of 0.1 mM
3-isobutyl-1-methylxanthine (an inhibitor of phoshodiesterase) for 15 min at room temperature. In both transfected and untransfected cells,
the level of cAMP detected in assay samples was 0.9 ± 0.3 pmol/106 cells in the absence of agonist. As in
previous studies (Han et al., 1996), we have presented the cAMP assay
data as a percentage of the maximal response. The changes in
[Ca2+]i were measured in the HEK-OAMB
cells loaded with 4 µM fura-2 AM (Molecular Probes,
Eugene, OR) after ligand treatment using a Perkin-Elmer (Emeryville,
CA) LS50B fluorescence spectrometer (Cooper and Millar, 1997).
In situ hybridization and immunohistochemistry. A clone
containing the 5' half of the OAMB cDNA (nt 1-1614) served to make RNA
probes using digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN).
Ten-micrometer frontal sections of Canton-S flies fixed in 4%
paraformaldehyde were hybridized with riboprobes and processed for the
immunological detection of hybridized transcripts as described by
Han et al. (1996).
A 542 bp fragment (nt 1916-2458) of the OAMB cDNA was subcloned in
pGEX-KT (Pharmacia, Piscataway, NJ) to generate a fusion protein with
the glutathione S-transferase. After the third injection of
the fusion protein into specific pathogen free rabbit, the antiserum
was collected and affinity-purified for immunostaining (Han et al.,
1996). Canton-S flies were fixed in 2% paraformaldehyde for 3 hr and
soaked in 25% sucrose solution overnight at 4°C. Ten-micrometer
cryosections were incubated with the affinity-purified anti-OAMB
antibody or the preimmune serum and processed as described by Han et
al. (1996).
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RESULTS |
Isolation of novel biogenic amine receptors
To identify new biogenic amine receptors expressed in
Drosophila, we performed PCR with fly head cDNA and the
primers made from conserved amino acids in TMs VI and VII of a
Drosophila tyramine-octopamine receptor (Arakawa et al.,
1990; Saudou et al., 1990). Known biogenic amine receptors are
relatively uniform in length between TMs VI and VII, which minimizes
the differential amplification of receptor subsets attributable to
heterogeneity in length between the PCR primers. Because of this,
however, distinguishing novel PCR products from known products on the
basis of size becomes problematic. To circumvent this, we used SSCP
(Orita et al., 1989) to screen the PCR products. The PCR products were
denatured to generate single strands and then subjected to
nondenaturing gel electrophoresis. Denatured single-stranded DNA forms
some secondary structure based on primary sequence, allowing for
resolution of DNA fragments by length and conformation.
The resolution and sensitivity of SSCP were first tested using PCR
products made from head and body cDNA as well as genomic DNA (Fig.
1). Independent PCRs using the same
templates were loaded on the gel to eliminate PCR artifacts and to
align the bands in the various lanes. Approximately a dozen strongly
labeled bands were observed using head cDNA as a template (Fig. 1). The
spectrum of bands observed with body cDNA, however, was quite distinct. This suggested that the predominant receptor types found in the body
are generally nonoverlapping with the predominant receptors found in
the head. The PCR products from genomic DNA would be expected to be the
sum of those produced from head and body cDNA. Although this was
observed, the bands representing body receptor products from genomic
DNA were very weak, and most major products were identical to those
using head cDNA as a template. The primers used for PCR were not
degenerate but represented the sequences of the TYR. Thus, receptor
RNAs with the highest sequence identity would be amplified
preferentially. It seems possible that head cDNA contains receptor
sequences more similar to the primers used than does the body cDNA.
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Figure 1.
PCR and SSCP. PCR products were generated from fly
head cDNA (H), body cDNA
(B), or genomic DNA (G) and
resolved on a 4% nondenaturing gel. The cDNAs for the tyramine
receptor (TYR) and three different serotonin receptors
(DRO1, DRO2A, and DRO2B)
were used for counterscreening. The arrowheads mark some
of the unique PCR products identified with head cDNA.
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The cDNA clones representing four known biogenic amine receptors
identified from Drosophila were also used in the analysis. These included one TYR (Saudou et al., 1990) and three serotonin receptors (DRO1, DRO2A, and DRO2B) (Witz et al., 1990; Saudou et al.,
1992). These known receptors produced PCR products that co-migrated
with approximately two-thirds of the major PCR products made from head
cDNA (Fig. 1). This indicated that several unidentified receptor RNAs
exist in the head RNA population with high sequence similarity to the
primers. To identify these RNAs and others that might not be resolved
in the gel, the complete population of PCR products amplified from head
cDNA was cloned. Approximately 100 independent clones were screened by
SSCP for unique mobilities against the four known receptors, yielding
four clones representing novel receptor sequences, as revealed after
isolation of the corresponding cDNAs and sequencing. One of these is a
novel D1 dopamine receptor, DAMB (Han et al., 1996); a second, OAMB, is
described here.
OAMB sequence predicts a biogenic amine receptor
A 114 bp clone obtained by PCR was used to screen a genomic DNA
library. A fragment from one positive phage was, in turn, used to
screen a head cDNA library. The subsequently identified cDNA clone
(OAMB) of 3387 bp contained a methionine followed by a long open
reading frame predicting a protein of 637 amino acids (Fig.
2A).
Hydropathy profiles (data not shown) revealed seven hydrophobic domains
with striking similarities to the TMs of GPRs (Vernier et al., 1993).
An Asp residue was found in TM III, and two Ser residues were found in
TM V (Fig. 2A). These residues constitute part of the
binding site for biogenic amines of other receptors (Vernier et al.,
1993), implying that OAMB belongs to the biogenic amine receptor
superfamily. This putative receptor clone also contained two consensus
sites for N-linked glycosylation (N-X-S/T) in the putative
extracellular N terminus and the second extracellular loop. Ten
consensus phosphorylation sequences for protein kinase C
[(R/K) ... S/T-X-R/K], two for protein kinase A (K/R-R-X-T/S), and five for calcium-calmodulin-dependent
protein kinase II (R-X-Y-S/T) were found in the cytoplasmic
domains (Fig. 2A). These sites might be involved in
dynamic regulation of receptor activity. A unique feature of OAMB is
its long extracellular loop (130 amino acids) between TM IV and V. Most
biogenic amine receptors contain a relatively short loop ranging from
10 to 30 amino acids. Although no recognizable motif was found in this
extended sequence, it is attempting to speculate its function for
interaction with cell adhesion molecules or extracellular matrix for
regulation of receptor activity.
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Figure 2.
Deduced amino acid sequence of OAMB and alignment
of OAMB with other biogenic amine receptors. A, The
seven putative TMs are indicated by overlining and
roman numerals. Circles,
asterisk, triangles, and
squares mark putative N-linked glycosylation sites,
protein kinase A, protein kinase C, and calcium-calmodulin-dependent
protein kinase II phosphorylation sites, respectively. The serine
(S) marked as a diamond is a
putative phosphorylation site by all kinases indicated above. The Asp
residue (D) in TM III and the two Ser residues
(S) in TM V thought to interact with octopamine
are indicated in bold. OAMB has been mapped to the
cytological location 92F. B, The deduced amino acid
sequence of OAMB is aligned with the barnacle GPR
(GPR-BAR), the Drosophila tyramine
receptor (TYRR-DRO), the human 1 adrenergic receptor
(A1AB-HUM), the 2 receptor
(A2AA-HUM), the  1 receptor
(B1AR-HUM), and the Drosophila
dopamine DAMB receptor (DAMB-DRO). Predicted TMs I-VII
are overlined. Numbers in
parentheses correspond to the number of amino acids at
the N and C termini and in the second and the third cytoplasmic loops
that are not represented in the figure. The amino acids that are
conserved in all compared receptors are shaded.
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Comparison of the predicted amino acid sequence of OAMB with protein
data banks revealed the highest sequence identity (39%) with a
barnacle GPR with unknown ligand specificity (Isoai et al., 1996).
Other biogenic amine receptors displayed similar overall identity
(25-30%) with OAMB, confined primarily to the seven TMs. When the TMs
were compared (Fig. 2B), the degree of sequence
identity increased to 72% for the barnacle GPR; 52-55% for human
1 adrenergic receptors (Ramarao et al., 1992), the
Drosophila DAMB (Han et al., 1996), and the TYR (Saudou et
al., 1990); and 45-50% for human 2 and adrenergic receptors
(Frielle et al., 1987; Fraser et al., 1989). The failure to find a high
identity to any one receptor subfamily suggests that OAMB represents
the prototypic member of a new receptor subfamily that may include the
barnacle GPR.
Octopamine-induced cAMP and
[Ca2+]i increase through OAMB
To investigate the functional properties of OAMB, a stable cell
line was established by transfection of Drosophila S2 cells with the OAMB cDNA and assayed for intracellular cAMP accumulation in
the presence of various neuromodulators (Han et al., 1996). No
significant changes in cAMP levels were detected in either transfected
(S2-OAMB) or untransfected S2 cells treated with serotonin, dopamine,
or histamine (up to 10 µM; data not shown). In contrast, octopamine at 10 µM induced a marked increase in cAMP
levels ~10-fold in transfected but not in untransfected cells.
Tyramine and norepinephrine at 10 µM also produced
smaller but significant elevations in cAMP levels only in transfected
cells. Dose-response curves for octopamine, tyramine, and
norepinephrine (Fig. 3A)
indicated that the octopamine-induced cAMP increase was
concentration-dependent and saturable, with an EC50 of
1.9 ± 0.5 × 10 7 M.
Tyramine and norepinephrine were ~100-fold less potent than octopamine and generated ~70-80% of the maximal response seen with
octopamine (Fig. 3A).
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Figure 3.
Agonist modulation of cAMP and
[Ca2+]i levels in
Drosophila S2 cells and human HEK cells.
A, Dose-response curves for agonist-induced elevation
in cAMP levels in the S2-OAMB cells. All data points are the means of
three independent determinations and have been normalized to the
maximum response to octopamine. The curves were fitted by least squares
method. The mean calculated EC50 value for octopamine is
0.19 ± 0.05 µM. B, Agonist-induced
elevation of [Ca2+]i in transiently
transfected HEK-OAMB or HEK-DAMB cells
loaded with fura2-AM. Agonists were applied at 10 µM.
Increase in [Ca2+]i is represented by
the measured fluorescence ratio (340:380 nm).
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We also examined the functional properties of OAMB in transiently
transfected human embryonic kidney (HEK-OAMB) cells. As shown for the
S2-OAMB cells, octopamine and, to a lesser extent, tyramine induced a
marked elevation in cAMP in transfected cells but had no effect in
untransfected HEK cells (data not shown). The EC50 for both
octopamine and tyramine determined in HEK-OAMB cells was essentially
indistinguishable from the values determined in S2-OAMB cells.
Together, these results indicate that OAMB represents a functional
octopamine receptor that can couple positively with AC to stimulate
cAMP production in both Drosophila and human cells.
The ability of OAMB to alter [Ca2+]i
was also examined in HEK cells expressing OAMB. After loading cells
with the Ca2+-sensitive dye fura-2 AM, a clear
increase in [Ca2+]i was observed in
response to octopamine at 105 M but
not to dopamine (Fig. 3B). No response was detected in nontransfected cells (data not shown) or in cells expressing the DAMB
receptor. The latter cells did show, however, an increase in
[Ca2+]i in response to dopamine but
not to octopamine. These data indicate that the increase in
[Ca2+]i by octopamine in
OAMB-expressing cells is mediated by OAMB. Thus, activation of OAMB
stimulated intracellular accumulations of cAMP and
Ca2+ in transfected HEK cells.
The OAMB receptor is preferentially expressed in
mushroom bodies
To examine the tissue distribution of OAMB, RNA blots of head and
body fractions were probed with an OAMB cDNA clone. Two major mRNA
species of 4.2 and 3.5 kb were detected in the head fraction but not in
the body fraction (Fig. 4), indicating
that the OAMB transcripts were specific to fly heads. In
situ hybridization was performed to determine cell types that
express OAMB. A series of frontal head sections was hybridized with
digoxigenin-labeled riboprobes representing the 5' half of the OAMB
cDNA (Fig. 5). The OAMB transcripts,
detected with antisense (Figs. 5A) but not sense (Fig.
5D) RNA probes, were present preferentially in perikarya of
mushroom bodies situated in the dorsal and posterior brain cortex.
However, OAMB expression was not uniform among all mushroom body
neurons; some mushroom body neurons exhibited intense signals, whereas
others stained at relatively lower levels (Fig. 5C).
Similarly, two clusters of cells located in the anterior brain cortex
near the mushroom body lobes stained intensely for OAMB transcripts (Fig. 5B). A relatively low signal was observed in cells
scattered in the central brain (Fig. 5A) and medulla of the
optic lobes (data not shown). No significant signal was detectable in
other tissues, including muscle (Fig. 5A) and fat cells
(data not shown).
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Figure 4.
RNA blotting of OAMB. Ten micrograms of
poly(A+) RNA from heads
(H) or bodies (B)
were resolved by gel electrophoresis, transferred to a nylon membrane,
and hybridized with a 32P-labeled OAMB cDNA clone
(top) or a ribosomal protein rp49 cDNA clone
(bottom) (O'Connell and Rosbash, 1984) as a loading
control. Molecular size markers (in kilobases) are indicated.
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Figure 5.
Expression of OAMB in mushroom bodies.
A-D, In situ hybridization.
A, Frontal section of the posterior brain. Central brain
(CB) and optic lobe (OL) areas are
indicated. K, Kenyon (mushroom body) cells.
B, Frontal section of the anterior brain.
C, D, Frontal section at the level of the
calyces. A-C were hybridized with an antisense OAMB
probe, and D was hybridized with a sense OAMB probe.
E-H, Immunohistochemistry. E, Frontal
section at the level of calyces. F, Frontal section at
the level of lobes. , lobes; , lobes;  , lobes.
G, Horizontal section. e, Ellipsoid body;
p, pedunculus. H, Sagittal section.
c, Calyx. The head sections were incubated with
anti-OAMB antibody. For all frontal sections, dorsal is
top. Anterior is top in G
and to the right in H. Magnification:
D, F, 200×; A-C, E, G, H, 400×.
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To determine the distribution of the OAMB receptor within the mushroom
bodies and other brain structures, head sections were stained with a
polyclonal antibody generated against the third cytoplasmic loop of the
receptor. Strong immunoreactivity was observed in the neuropil that
house the mushroom body dendrites (calyces; Fig. 5E), axons
(pedunculi; Fig. 5G,H), and axon
terminals (, , and lobes; Figs.
5F-H). Distinct immunoreactivity was also
detected in ellipsoid body of central complex (Fig. 5G). This observation suggests that some cells in the anterior brain cortex
that hybridized to antisense OAMB (Fig. 5B) may represent neurons projecting to the ellipsoid body. No specific immunoreactivity was observed in other regions of heads or bodies. We conclude that the
novel octopamine receptor OAMB is highly enriched in the mushroom body
and ellipsoid body neuropils.
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DISCUSSION |
In the present study, we have identified a novel octopamine
receptor that stimulates both cAMP and
[Ca2+]i accumulation. The PCR-SSCP
approach used to isolate the receptor is unique and advantageous. SSCP
analysis relies on the electrophoretic mobility of single-stranded DNA
conferred primarily by secondary structure, detecting even
single-nucleotide alterations. Therefore, a parallel comparison of PCR
products from various tissue samples along with known sequences allows
for a rapid way of identifying novel sequences of low abundance among
closely related genes. We obtained four novel GPR clones from fly heads
using this approach (K.-A. Han and R. L. Davis, unpublished
observations), and further analysis identified one of them as a novel
dopamine receptor DAMB and another as an octopamine receptor OAMB.
Although we have restricted our analysis by screening a small fraction
of the recombinant colonies (<0.01%), it should be possible to
perform a near-saturation screen for a family of genes by designing
various combinations of primer sets and screening recombinant colonies
on a larger scale by colony hybridization and sequencing.
Although octopamine is a principal neuromediator in insects, its
receptors have proven difficult to clone. Pharmacological profiles of
octopamine receptors are well characterized in locust. One neuronal and
three non-neuronal receptor subtypes with distinct affinity for ligands
and different effectors have been found (Evans and Robb, 1993; Roeder,
1995). One octopamine-sensitive receptor has been cloned from
Drosophila heads, but tyramine is two orders of magnitude
more potent than octopamine in inhibiting AC activity through this
receptor. However, both ligands show similar potencies for increasing
[Ca2+]i. Thus, it has been classified
as a tyramine-octopamine receptor (Saudou et al., 1990; Robb et al.,
1994). Octopamine binds with high affinity at sites distinct from
tyramine in membranes prepared from fly heads and is a potent activator
of AC (Uzzan and Dudai, 1982; Dudai and Zvi, 1984). The OAMB receptor
identified here satisfies such observations, because OAMB stimulated AC
with higher efficacy to octopamine over tyramine in both mammalian and
Drosophila cell lines. In addition, OAMB mediated a
[Ca2+]i increase in response to
octopamine. It is possible that the [Ca2+]i increase results from the
activation of enzymes such as phospholipase C. A recently cloned
octopamine receptor from the pond snail Lymnaea is coupled
to both AC and phospholipase C (Gerhardt et al., 1997).
Several lines of evidence indicate that octopamine plays essential
roles in behavioral plasticity in Drosophila. Although no
information is available on the performance of flies with reduced octopamine content (inactive) and without octopamine
(th) in classical olfactory conditioning, flies fed with
formamidines display impaired olfactory learning, although they can
sense the relevant stimuli presented during training (Dudai et al.,
1987). These drugs not only interact with octopamine binding sites but also exhibit antagonistic effects on octopamine-induced cAMP
production. This suggests that an octopamine receptor positively
coupled to AC mediates olfactory learning. In addition,
inactive mutant males display impaired experience-dependent
courtship modification toward mature males (O'Dell, 1994). The
necessity for functional octopamine binding sites and cAMP metabolism
for normal learning suggests that OAMB may serve as a receptor for
octopaminergic input to mediate synaptic plasticity. Intriguingly, OAMB
is highly enriched in the mushroom body neuropil, a principal
neuroanatomical site mediating normal olfactory conditioning and
conditioned courtship (O'Dell et al., 1995; Davis, 1996; Davis and
Han, 1996). These observations suggest that OAMB may modulate the
physiology of mushroom bodies underlying associative learning. However,
multiple biogenic amine receptors are likely to be important for
learning, because we have previously shown that the novel dopamine
receptor DAMB is localized preferentially to the mushroom body lobes
and pedunculi (Han et al., 1996).
Therefore, the anatomical and biochemical architecture may be arranged
such that conditioned stimulus (CS) and unconditioned stimulus (US)
inputs converge at the mushroom bodies through distinct receptors and
activate signal transduction cascades to modulate the synaptic output
of mushroom bodies (Fig. 6). Odor
information received during olfactory conditioning is conveyed to the
mushroom body calyces (Strausfeld, 1976), presumably through
cholinergic transmission (Restifo and White, 1990), and encoded as a
CS, perhaps in part through modulatory actions of the octopamine
receptor OAMB. This receptor may be activated by modulatory neurons
delivering US information to trigger cAMP accumulation through AC or
[Ca2+]i increase through phospholipase
C. How odors are encoded and how simultaneous US input might convert an
odor into a CS are unknown, although studies in larger insects (Laurent
and Davidowitz, 1994) implicate synchronous oscillation in the antennal
lobes or the mushroom bodies as part of the physiological basis for odor recognition. It is possible that OAMB may modify this process to
convert odor information into a CS. Because formamidine treatment significantly reduces the learning of rut flies (Dudai et
al., 1987), other ACs (Levine et al., 1992) distinct from the
rut AC may be coupled to OAMB. Alternatively, OAMB may use
[Ca2+]i to mediate the US information.
That octopamine mediates US information has been demonstrated using the
proboscis extension reflex conditioning of the honeybee, in which
octopamine injection into the calyces during training substitutes for a
sugar reward given to the proboscis (Menzel et al., 1996). Given the
diversity of neuromodulatory systems, it is also conceivable that OAMB
may process different sensory inputs. The newly identified receptors DAMB and OAMB with distinct biochemical and anatomical properties should help dissect the elaborate neuromodulatory systems underlying behavioral plasticity.
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Figure 6.
Model for the cAMP cascade triggered by OAMB
during olfactory conditioning. Olfactory information
(CS) is received at the antennae and conveyed to the
dendrites of mushroom body cells via the antennoglomerular tract. The
olfactory information presented to the mushroom body cells may be
modified from activation of OAMB by concurrent US inputs
from modulatory neurons. Increases in
[Ca2+]i or cAMP from activation of
OAMB may be responsible for modifying or processing the
CS information. Information representing the
US may also be mediated by dopaminergic
(DA) modulatory neurons that activate the dopamine
receptor (DAMB) situated on the pedunculi and lobes.
Activated DAMB stimulates AC, leading to
increased cAMP production. The elevated cAMP then modulates the
synaptic output of mushroom body neurons to motor circuits, either
directly through cyclic nucleotide-sensitive potassium channels or
indirectly through protein kinase A (PKA), which in turn
phosphorylates ion channels and other molecules to encode memory
(Davis, 1996).
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FOOTNOTES |
Received Dec. 11, 1997; revised Feb. 25, 1998; accepted March 3, 1998.
This work was supported by grants from the Human Frontiers Science
Project, National Institutes of Health, and the R. P. Doherty-Welch Chair in Science to R.L.D. Additional support was from
the Wellcome Trust to N.S.M. We thank S. Ahmed and J. Volmer for expert
technical assistance. Correspondence should be addressed to Dr. Ronald
L. Davis, Department of Cell Biology and Neurology, Baylor College of
Medicine, Houston, TX 77030.
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Top
Abstract
Introduction
