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Pharmacological characterization of dopamine receptors in the
corpus allatum of Manduca sexta larvae
Noelle A. Granger
a,*, Richard Ebersohl
a, Thomas C. Sparks
baDepartment of Cell Biology and Anatomy, Campus Box 7090, Taylor Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
bDowAgro Sciences, 3221 Zionsville Road, Indianapolis, IN, USA
Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
Dopamine receptors previously identified in corpora allata (CA) of Manduca sexta last instars on the basis of dopamine effects on JH (juvenile hormone)/JH acid biosynthesis and cyclic AMP (cAMP) accumulation, were characterized pharmacologically. For this study, a broad spectrum of agonists or antagonists of D1, D2, D3 or D4 dopamine receptors, together with the dopamine metabolite N-acetyl-dopamine, other neurotransmitters and their agonists/antagonists, were tested for their effects on gland activity and cAMP production. The lack of effect of other neurotransmitters supports the specificity of the effect of dopamine and the dopamine specificity of the receptors. Only the D2 receptor antagonist spiperone had a potent effect on JH biosynthesis and cAMP formation by CA taken on day 0 of the last stadium, when dopamine stimulates both activities and thus appears to be acting via a D1-like receptor. Several other D2 receptor antagonists, and D1, D2/D1 and D4,3/D2 receptor antagonists were less effective. Thus, the D1-like receptor of the Manduca CA appears to be distinct pharmacologically from vertebrate D1 receptors. By contrast, a number of D2 agonists/antagonists had a significant effect on JH acid biosynthesis and cAMP production by the CA from day 6 of the last stadium, when dopamine inhibits both activities and thus appears to be acting via a D2-like receptor. Certain D1-specific agonists/antagonists were equally effective. The Manduca D2-like receptor therefore bears some pharmacological resemblance to vertebrate D2 receptors. acetyl dopamine acted as a dopamine agonist with day 6 CA, the first identified function for an N-acetylated biogenic amine in insects. Dopamine was found to have the same differential affect on the formation of cAMP in homogenates of day 0 and day 6 brains as it did with CA, and in the same concentration range. Dopamine receptor agonists/antagonists affecting cAMP formation by day 0 and day 6 CA homogenates had similar effects with brain homogenates. By contrast, dopamine only stimulated cAMP formation by homogenates of day 0 and day 6 abdominal or ventral nerve cord. These results suggest that D1- and D2-like dopamine receptors of Manduca are regionally as well as temporally localized.2000 Elsevier Science Ltd. All rights reserved.
Keywords: Juvenile hormone biosynthesis; Cylic AMP (cAMP); Neurotransmitter; Biogenic amines; Receptor agonist; Receptor antagonist
1. Introduction
There has been considerable interest in the molecular mechanisms by which juvenile hormone (JH) synthesis/release is controlled, since interruption of these processes would affect the many individual roles JH plays in regulating development and reproduction. Emphasis has been placed largely on understanding the
* Corresponding author. Tel.:+1-919-966-3288; fax:+ 1-919-966-1856.
E-mail address: [email protected] (N.A. Granger).
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 4 7 - 3
756 N.A. Granger et al. / Insect Biochemistry and Molecular Biology 30 (2000) 755–766
by CA on days 3–6 (Granger et al., 1996). According to the traditional classification of dopamine receptors (Creese et al., 1983; Gingrich and Caron, 1993), binding of ligand to a D1 receptor increases adenylyl cyclase activity, while binding to a D2 receptor either has no effect or inhibits the activity of this enzyme. Thus these results suggested the existence of D1- and D2-like dopa-mine receptors in the CA. A preliminary investigation of the effects of a small number of vertebrate D1 and D2 receptor agonists/antagonists on JH biosynthesis and cAMP formation indicated that the D1-like receptor was pharmacologically distinct from vertebrate D1 receptors, while the D2-like receptor was more similar pharmaco-logically to vertebrate D2 receptors.
Our knowledge of neurotransmitter receptors in insects is limited, but growing (for reviews see Roeder, 1994; Osborne, 1996). D1 and D2 dopamine receptors belong to a large superfamily of G protein-coupled receptors, and the genes or cDNAs of more than 60 are known (O’Dowd, 1993), including octopamine, octopamine/tyramine, and serotonin receptors for
Droso-phila (Arakawa et al., 1990; Witz et al., 1990; Saudou
et al., 1992; Robb et al., 1994; Vanden Broeck et al., 1995; Von Nickisch-Rosenegk et al., 1996). Molecular cloning has revealed five pharmacologically distinct ver-tebrate dopamine receptor subtypes. Two of these cloned receptor subtypes (D1A and D1/D5B) exhibit the func-tional and pharmacological properties of the classical D1 receptor sub-family, while the other three (D2S, D2L, D3, and D4) are D2-like (Gingrich and Caron, 1993; Strader et al., 1995). Several dopamine receptors have been sequenced in Drosophila (Gotzes et al., 1994; Sugamori et al., 1995; Feng et al., 1996; Han et al., 1996). While these receptors demonstrate relatively little sequence homology to cloned vertebrate D1-like recep-tors, they are nevertheless linked to increases in adenylyl cyclase activity when expressed in cell lines or Xenopus oocytes. The DopR99B receptor (Feng et al., 1996; Reale et al., 1997) is also coupled directly to the gener-ation of an intracellular Ca2+signal.
Both cloned (Drosophila: Gotzes et al., 1994; Suga-mori et al., 1995; Feng et al., 1996; Reale et al., 1997) and native (Periplaneta americana: Orr et al., 1987; Downer, 1990; Apis mellifera: Kokay and Mercer, 1996, 1997) have been examined for their ability to bind com-pounds which act as agonists and antagonists of dopam-ine receptors in vertebrates. The results of these studies strongly suggest that the D1-like and D2-like sub-famil-ies of dopamine receptors in insects are substantially dis-tinct with different and variable subtype groups, parti-cularly between different orders of insects.
The objective of the present study was to characterize pharmacologically the dopamine receptors in the CA of
Manduca last instars, by examining the effects of a broad
range of vertebrate D1 and D2 receptor agonists/antagonists on JH/JH acid biosynthesis and
cAMP formation. This has enabled a comparison of these receptors to those of vertebrates, as well as to known insect dopamine receptors. Also examined were the effects of other neurotransmitters, to confirm the specificity of the dopamine effects, and the effects of dopamine and certain dopamine receptor agonists/antagonists on cAMP formation in the Manduca nervous system, specifically the brain and ventral nerve cord.
2. Materials and methods
2.1. Animals
Larvae of M. sexta were reared on artificial medium (Bell and Joachim, 1976), as previously detailed (Granger et al., 1996), at 27°C, high humidity (60–70%), and a non-diapausing photoperiod (L/D 16:8). The gate II larvae used for this study were taken on days 0 and 6 of the fifth stadium. Under these rearing conditions, the majority of gate II larvae ecdysed to fifth instars between 2 and 6 pm, and CA from day 0 larvae (V0) were routinely taken for assay (JH biosynthesis) or frozen (adenylyl cyclase assay) the following morning. In this colony, gate II larvae undergo dorsal vessel exposure and display wandering behavior early on day 5 of the fifth stadium, and thus CA were taken from day 6 larvae (V6) the morning after wandering.
2.2. Chemicals
For incubation of CA in vitro, bovine serum albumin (BSA) was purchased from Miles Inc. (Kankakee, IL) and Grace’s medium from GIBCO (Grand Island, NY). For the adenylyl cyclase assay, adenosine deaminase, creatine kinase, creatine phosphate, and dithiothreitol (DTT) were all obtained from Boehringer Mannheim (Indianapolis, IN); rabbit muscle myokinase from ICN Biochemicals (Cleveland, OH); and alumina, adenosine triphosphate, cyclic adenosine monophosphate (cAMP), Dowex-50W, ethylenediamine tetraacetic acid (EDTA), HEPES buffer, leupeptin, and phenylmethylsulfonyl fluoride (PMSF) from Sigma (St Louis, MO). New England Nuclear Corp. (Boston, MA) supplied a-[32
P]-ATP (s.a. 3000 Ci mmol21).
For the JH RIAs, JH I was purchased from SciTech (Prague, Czech Republic), JH III was obtained from Calbiochem/Behringer Diagnostics (San Diego, CA) and [3H]–JH I and [3H]–JH III (11.6 Ci mmol21) came from
New England Nuclear.
(±)-SKF38393 N-allyl HBr, and (±)-6-chloro-APB HBr; D1 antagonists — R(+)-SCH23390 HCl, R(+ )-SCH23390 deschloro HCl; D2 agonists — R(2 )-2,10,11-trihydroxy-N-propyl noraporphine (TNPA) HBr, (±)-PPHT HCl; D2 antagonists — haloperidol, spiperone HCl, pimozide, fluphenazine 2HCl, thiothixine HCl, S(2)-eticlopride HCl, R(+)-eticlopride HCl, (±) sulpir-ide; D1/2 antagonist — pergolide methanesulfonate; D2/1 antagonists — (±) butaclamol HCl, (+) bulbocap-nine HCl; D3 agonists — S(+)-PD-128,970 HCl, (± )-7-hydroxy DPAT HBr; and D4/3/2 agonist — clozapine. The dopaminergic neurotoxin EEDQ was also obtained from RBI (Natick, MA).
2.3. Dissection of CA for the assay of JH/JH acid synthesis and the adenylyl cyclase assay
CA were dissected in Grace’s tissue culture medium containing 0.17.B5A, and two (V6) or four (V0) glands were used for each incubation, as previously described (Janzen et al., 1991). For the adenylyl cyclase assay, individual CA were frozen on the pestle of a Kontes glass microhomogenizer embedded in dry ice and stored on the pestle in the homogenizer at 280°C until used (Granger et al., 1995).
2.4. Radioimmunoassay
The amounts of JH/JH acid synthesized and released by the CA in vitro were determined by either JH I or JH III RIA of incubation medium, using antisera which recognize each JH and its acid equivalently. The assay protocol has been described in detail elsewhere (Granger and Goodman, 1988; Janzen et al., 1991). Synthesis is expressed in ng JH equivalents. There were no differ-ences in the effects of dopamine and dopamine receptor agonists/antagonists on the synthesis of JH/JH acid whether synthesis is measured by JH I or JH III RIA (Granger, unpublished results).
2.5. Adenylyl cyclase assay
The assay was performed as previously detailed (Granger et al., 1994, 1995). Tissues were homogenized in a Kontes Potter Elvehjem tissue grinder with a glass pestle (VWR Scientific, Atlanta, GE). For assays of adenylyl cyclase in whole glands, a maximum of 100µl of homogenization buffer (10 mM Na+/K+ PO4 buffer,
pH 7.2; 1 mM DTT; 100 µM leupeptin; 10 µg ml21
PMSF) was added to the frozen homogenizer. The glands were allowed to thaw during the homogenization process, and the resulting homogenate was used without further preparation. The standard number of CA equiva-lents used per assay tube was six.
Adenylyl cyclase activity was determined by measur-ing the conversion of [a-32
P]-ATP to [32
P]-cAMP by the
basic method of Combest et al. (1985), incorporating some of the modifications of Meller et al. (1988) to acco-modate the tissue limitations imposed by the CA system (Granger et al., 1996). Homogenization buffer was used as the control, and each assay point was run in triplicate.
2.6. Data analysis
Data were analyzed for significant differences between experimental groups by one way ANOVA and EC50 values were calculated by log probit regression
analysis.
3. Results
3.1. Effects of dopamine and N-acetyl-dopamine
Dopamine has previously been shown to be an effec-tive stimulator of JH biosynthesis by V0 CA and an effective inhibitor of JH acid biosynthesis by V6 CA, at concentrations ranging from 1026 to 1024 M (Granger
et al., 1995, 1996). Similar stage specific effects of dopa-mine on cAMP formation were also noted (Granger et al., 1996) at even lower concentrations. When the effects of dopamine were re-examined at the lower end of the concentration range (1028 and 1027 M), it was found
that statistically significant stimulation of V0 CA was obtained at 1027 M, while significant inhibition of V6
CA was achieved at 1028M (Table 1).
N-acetyl-dopam-ine is considered to be the first step in the degradative pathway for dopamine (Krueger et al., 1990) but is not detectable by electrochemical array high pressure liquid chromatography (EC-HPLC) in either the V0 or the V6 gland (lower limit of detection=1 pg; Granger et al., 1996). This compound had no effect on the synthesis of JH by V0 CA, but was as effective as dopamine itself in inhibiting JH acid synthesis by CA on day 6, at equiv-alent concentrations (Table 1).
3.2. Effects of other neurotransmitters
The effects of octopamine, serotonin and norepi-nephrine on JH/JH acid synthesis by V0 and V6 CA were examined first. Octopamine has previously been shown to have no effect on adenylyl cyclase activity in homogenates of V6 CA over a wide range of concen-trations (Granger et al., 1995). In this study, neither octo-pamine nor serotonin had an effect on the JH and JH acid biosynthetic activity of V0 and V6 CA, respectively (data not shown). At higher concentrations, norepi-nephrin had a statistically significant, but non-dose dependent, effect on JH acid biosynthesis by V6 CA (0.37±0.2 ng • 2 pr CA • 6 h vs 0.29±0.3 ng • 2 pr CA • 6 h at both 1025
and 1026
758 N.A. Granger et al. / Insect Biochemistry and Molecular Biology 30 (2000) 755–766
Table 1
Effect of dopamine and N-acetyl-dopamine on JH/JH acid biosynthesis in vitro by corpora allata from M. sexta fifth instars
Compound Stage Concentration (M) JH/JH acid synthesisa
Dopamine N-acetyl-dopamine
Dopamine V0b 0 0.19±0.021
1028 0.19±0.021
1027 0.25±0.021,2
1026 0.32±0.032
1025 0.42±0.033
V6b 0 0.59±0.041
1028 0.36±0.042
1027 0.23±0.023
1026 0.15±0.013,4
1025 0.10±0.024
N-acetyl-dopamine V0 0 0 0.14±0.011
1025 0 0.58±0.042
0 1027 0.14±0.021
0 1026 0.13±0.011
0 1025 0.18±0.031
V6b 0 0 0.57±0.061
1025 0 0.12±0.012
0 1027 0.23±0.043
0 1026 0.14±0.012,3
0 1025 0.11±0.012
ang JH (V0) or JH acid (V6) synthesis±SEM by 2 pr CA in 2 h per 0.1 ml. Synthesis measured by JH III RIA, n=6–7. V0=day 0 of the fifth
stadium; V6=day 6 of the fifth stadium. In each data set, mean values having different superscripts are significantly different (p#0.05).
b Significant effect noted at 1026M and higher.
phentolamine mesylate (Table 2). Interestingly, this compound effectively negated dopamine stimulation of JH biosynthesis by V0 CA, but it failed to block dopam-ine-stimulated cAMP formation. This result suggests that phentolamine mesylate could inhibit JH biosynthesis, but
Table 2
Effect of thea-adrenergic receptor antagonist phentolamine mesylate on JH/JH acid biosynthesis in vitro by corpora allata from M. sexta fifth instars
Stage Concentration (M) JH/JH acid synthesisa cAMP formed
Dopamine Phentolamine (pmol. h21)b
V0 0 0 0.16±0.011 1.84±0.081
1025 0 0.39±0.022 4.92±0.152,3
1025 10210 4.96±0.112,3
1025 1029 4.89±0.112,3
1025 1028 5.24±0.122
1025 1027 0.15±0.021 4.50±0.103
1025 1026 0.14±0.021 4.20±0.094
1025 1025 0.17±0.011 4.91±0.154
V6 0 0 0.46±0.031 3.92±0.151
1025 0 0.15±0.012 1.74±0.162,3
1025 10210 1.57±0.222
1025 1029 2.10±0.183,4
1025 1028 1.98±0.114
1025 1027 0.15±0.002 2.35±0.054
1025 1026 0.14±0.012 2.34±0.074
1025 1025 0.15±0.012 2.26±0.164
ang JH (V0) or JH acid (V6) synthesis±SEM by 2 pr CA in 2 h per 0.1 ml. Synthesis measured by JH III RIA, n=6–7. V0=day 0 of the fifth
stadium; V6=day 6 of the fifth stadium. In each data set, mean values having different superscripts are significantly different (p#0.05).
b cAMP formation measured by the adenylyl cyclase assay, n=3. In each data set, mean values having different superscripts are significantly
different (p#0.05).
homogenates of V6 glands. A subsequent examination of the adrenergic agonist synephrine and the cholinergic receptor agonist carbachol in this system demonstrated no effects on the activity of either V0 and V6 CA (data not shown).
3.3. Effects of dopamine receptor agonists and antagonists
The effects of the various agonists and antagonists on cAMP formation were tested with whole homogenates of V0 CA, and the results were expressed as EC50values.
In each case, the results mirror those obtained with the assay for JH biosynthesis in vitro.
Neither the D1 agonists [R(+)-SKF 38393; (±)-SKF 38393 N-allyl; (±)-6-chloro APB] nor one D2 agonist [R(2)-TNPA] affected either JH biosynthesis or cAMP formation. Surprisingly, spiperone, a D2 receptor antag-onist, proved to be a potent antagonist of dopamine stimulation of JH biosynthesis and cAMP formation (EC50=291 nM; Fig. 1 and Table 6). A few other
antag-onists were marginally effective, with a rank order of potency of SCH23390, deschloro (EC50=2521
nM).fluphenazine (EC50=3162 nM)=R(+) eticlopride
(EC50=3162 nM).S(2) eticlopride (EC50=5230 nM).
clozapine (EC50=6921 nM).bulbocapnine (EC50=8465
nM) (Fig. 1; Table 6). All of the other compounds tested had no effect [R(+)SCH 23390, haloperidol, pimozide,
Fig. 1. Effect of dopamine D1 (SCH 23390, deschloro, HCl) and D2 receptor (spiperone HCl, and R(+) eticlopride HCl) antagonists on the production of cAMP in whole homogenates of V0 CA, in the presence (filled symbols) and absence (open symbols) of 1025 M dopamine.
Values are expressed as mean±SEM of pmol per hour, where n=3 or 4.
thiothixine, (±)sulpiride, pergolide methane sulfonate, butaclamol, and PD-128,970] (Table 6). Tests of the D2 receptor agonist (±) PPHT (Table 3) and a preliminary examination of the D3 receptor agonist (± )-7-hydroxy-DPAT revealed some interesting results. Rather than sti-mulating JH biosynthesis and cAMP production by V0 CA, both these compounds appeared to depress synthesis below control levels. Thus it appears that the Manduca D1-like receptor bears little similarity in its pharmaco-logical profile to vertebrate D1 receptors.
A very different pharmacological profile emerged for the D2-like receptor in V6 CA, one which indicated that this receptor bears some similarity to a vertebrate D2. Both D2 receptor agonists were potent effectors of cAMP pro-duction in gland homogenates [R(2)-TNPA: EC50=28
nM; (±)PPHT: EC50,100 nM], while three of the eight
D2 receptor antagonists tested [S(2)-eticlopride: EC50=
807 nM.spiperone: EC50=945 nM.pimozide: EC50=3162
nM] also had effects, although they were less potent than the agonists (Fig. 2; Table 6). As with the D1 receptor, agonists/antagonists of the other receptor sub-family also affected JH biosynthesis and cAMP formation. Two of the three D1 receptor agonists [(±)-SKF 38393 N-allyl: EC50=408 nM; (±)-6-chloro APB: EC50,100 nM] and
one of the two D1 receptor antagonists [R(+)SCH 23390, deschloro: EC50=534 nM] tested strongly affected cAMP
production (Fig. 2; Table 6). It was notable that the non-specific receptor agonists and antagonists (pergolide methane sulfonate, bulbocapnine, butaclamol, clozapine) and the D3-specific compounds [PD-128,970; (± )-7-hyd-roxy DPAT] had no effect, unlike the situation with V0 CA (Table 6). Thus it appears that the D2-like receptor in Manduca larval CA is similar in its pharmacological profile to vertebrate D2s, while still recognizing effectors of vertebrate D1 receptors.
3.4. Effect of EEDQ
EEDQ, a potent dopaminergic neurotoxin which works by binding irreversibly to vertebrate D1 and D2 receptors (Hamblin and Creese, 1983), was tested for its effect on JH/JH acid biosynthesis in the presence and absence of dopamine (Fig. 3). In the absence of exogenous dopamine, JH synthesis by V0 CA was maintained at control levels in the presence of concen-trations of EEDQ from 1027to 1025M. In the presence
of dopamine, EEDQ effectively blocked its stimulatory effect, but only at a concentration greater than 1026M.
With V6 CA, and in the absence of dopamine, EEDQ was a potent dopamine agonist, depressing the level of JH acid synthesis at 1027M to that obtained with 1025
M dopamine. In the presence of 1025 M dopamine,
760 N.A. Granger et al. / Insect Biochemistry and Molecular Biology 30 (2000) 755–766
Table 3
Effect of D2 dopamine receptor agonist (±)-PPHT HCl on JH biosynthesis in vitro and cAMP formation by corpora allata from day 0 fifth instars of M. sexta
Concentration (M) JH synthesisa cAMPb
Dopamine (±)-PPHT HCl (pmol h21)
0 0 0.09±0.011 2.24±0.351
1025 0 0.20±0.002 2.26±0.161
0 1029 2.42±0.201
0 1028 2.00±0.541,2
0 1027 0.08±0.011 1.60±0.172
0 1026 0.02±0.013 0.81±0.083
0 1025 0.03±0.003
ang JH synthesis±SEM by 2 pr CA in 2 h per 0.1 ml. Synthesis measured by JH III RIA, n=5–10. In each data set, mean values having different
superscripts are significantly different.
b cAMP formation measured by the adenylyl cyclase assay, n=3. In each data set, mean values having different superscripts are significantly
different (p#0.05).
Fig. 2. Effect of dopamine D1 (6-chloro-APB HBr; SCH 23390, deschloro, HCl) and D2 receptor (TNPA HBr, spiperone HCl) agonists and antagonists on the production of cAMP in whole homogenates of V6 CA, in the presence (filled symbols) and absence (open symbols) of 1025M dopamine. Values are expressed as mean±SEM of pmol
per hour, where n=3 or 4.
3.5. Indentification of dopamine receptors in the brain and ventral nerve cord of Manduca fifth instars
Dopamine was found to have the same stage-specific effects on the formation of cAMP in homogenates of brains from days 0 and 6 as it did with CA from these two stages, in approximately the same concentration range (Table 4). However, the differential effects of
Fig. 3. Effect of the dopaminergic neurotoxin EEDQ on the synthesis of JH and JH acid by V0 and V6 CA, respectively, in the presence (black bars) and absence (gray bars) of 1025M dopamine. Values are
expressed as mean±SEM of ng JH/JH acid synthesis by 2 pr (V0) or 1 pr (V6) CA in 2 h per 0.1 ml of incubation medium, where n=6–7.
Table 4
Effect of dopamine on cAMP production in whole homogenates of brain and ventral ganglia from M. sexta fifth instars
Stage Tissue Dopamine (M) cAMP formed
(pmol. ug protein21h21)a
V0 Brain 0 0.008±0.0031
1028 0.021±0.0052
1027 0.046±0.0033
1026 0.104±0.0194
1025 0.259±0.0295
Thoracic nerve cord 0 0.349±0.1071
1025 0.648±0.0802
V6 Brain 0 0.070±0.0051
1028 0.055±0.0072
1027 0.041±0.0112,3
1026 0.035±0.0083
1025 0.014±0.0014
Thoracic nerve cord 0 0.141±0.0221
1025 0.583±0.0492
Abdominal nerve cord 0 0.079±0.0091
1025 0.184±0.0212
aV0 brain=8 per assay tube; V0 thoracic ganglia=10; V6 brain=6; V6 thoracic ganglia=10; V6 abdominal ganglia=10.
Table 5
Effects of dopamine receptor agonists and antagonists on cAMP formation in whole homogenates of brains of M. sexta fifth instars
Type Compound Stage EC50[nM]a
D1 receptor agonist (±)-6-chloro APB V0 .10,000
V6 680
D1 receptor antagonist R(+) SCH 23390 deschloro HCl V0 .10,000
V6 312
D2 receptor agonist TNPA HBr V6 94
D2 receptor antagonist spiperone V0 136
V6 156
(±) sulpiride V6 8371
aCalculated by log probit regression analysis from cAMP assay data. V0=brains taken from day 0 of the fifth stadium; V6=brains taken on
day 6 of the fifth stadium.
4. Discussion
The results of this study support the existence of two distinct dopamine-like receptors in the CA and also the brain of fifth stadium Manduca larvae, on the following bases: (1) the stage-specific and differential effect of dopamine on both CA biosynthetic activity and on aden-ylyl cyclase activity in the CA and brain; (2) the strik-ingly different pharmacological profiles obtained on days 0 and 6 with the same dopamine receptor agonists and antagonists (Table 6); (3) the lack of such effects with other neurotransmitters; (4) and the lack of other neurotransmitters in the CA itself (Granger et al., 1996). Although norepinephrin slightly inhibited JH acid biosynthesis by V6 CA, the a-adrenergic antagonist phentolamine mesylate did not, and only marginally antagonized the inhibition of cAMP formation by dopa-mine. This antagonist did appear to block the dopamine stimulation of JH biosynthesis by dopamine but had no effect on cAMP formation. Thus it was concluded that
the effects of these two compounds are probably not mediated via dopamine receptors. While the list of other neurotransmitters examined is not extensive, the
Mand-uca dopamine receptors do appear to be fairly specific
for dopamine. By contrast, norepinephrin was found to be an effective agonist of receptor-mediated increases in cAMP levels in Xenopus oocytes expressing the
Droso-phila DopR99B dopamine receptor, although dopamine
was two orders of magnitude more potent (Reale et al., 1997). In an extensive study of the pharmacology of dopamine receptors in the honey bee brain, an overlap-ping affinity of the D2-like receptor for phenolaminergic compounds was discovered (Table 6; Kokay and Mer-cer, 1996).
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Table 6
Pharmacology of M. sexta D1- and D2-like dopamine receptors compared to other insect dopamine receptors
Type of compounds Manduca V0 Manduca V6 Cockroach Honey bee D2 Honey bee D1 Drosophila Drosophila Mosquito
Compound name CA EC50nM CA EC50nM brain EC50nMa brain KinMb brain KinMc D1 EC50nMd D1/5 EC50nM EC50nM
Neurotransmittor
dopamine 3000 .10,000 300–500 500 1000
serotonin .10,000 .10,000 .10,000 .10,000 .10,000 NE NE
norepinephrin .10,000 .10,000 7000
octopamine .10,000 .10,000 18 .10,000 NE NE 5500
D1 agonist
R(+)-SKF38393 .10,000 .10,000 NE
(±)-SKF38393, N-allyl HCl .10,000 408
(±)-6-chloroAPB .10,000 ,100
D1 antagonist
R(+)-SCH23390 .10,000 6816 3772 456 9.5 NB
R(+)-SCH233909deschloro HCl 2521 534
D2 agonist
TNPA HBr .10,000 28 .10,000
(±)-PPHT HCl inhibition? ,100
D2 antagonist
haloperidol .10,000 .10,000 .10,000
spiperone 291 945 49 0.17 .10,000 NB
pimozide .10,000 3162
fluphenazine 3162 .10,000 20 788
thiothixine .10,000 .10,000
S(2)-eticlopride 5230 807
R(+)-eticlopride 3162 .10,000
(±) sulpiride .10,000 .10,000 3772 .10,000
D2/D1 antagonist
butaclamol .10,000 .10,000 16 56 .10,000
bulbocapnine 8465 .10,000
D1/D2 antagonist
pergolide methane sulfonate .10,000 .10,000
D3 agonist
PD-128,970 HCl .10,000 .10,000
(±)-7-hydroxy DPAT HBr inhibition? .10,000 NB
D4/D3/D2 antagonist
clozapine 6921 .10,000
Other
phentolamine .10,000
synephrine .10,000
carbachol .10,000
N-acetyl-dopamine NE
a Calculated from K
ivalues.
b [3H]-spiperone as the competing ligand in a ligand binding assay.
c [3H]-SCH23390 as the competing ligand in a ligand binding assay
profiles do not fit the stereotypical profile of vertebrate D1 receptors since in most cases, compounds that bind to either D1 or D2 vertebrate dopamine receptors will bind to the insect D1-like receptors (Table 6). This was demonstrated more than a decade ago in the first binding studies of the dopamine receptor in the cockroach brain, where antagonists of serotonin, adrenergic, and octopa-mine receptors, as well as several D2 receptor antagon-ists, all were found to inhibit dopamine-stimulated cAMP accumulation (Orr et al., 1987). The most potent antagonists were selective for neither D1 nor D2 recep-tors. In the well-studied honey bee brain, the saturable, high affinity binding of the D1 antagonist SCH23390 to a D1-like receptor was inhibited by D2 receptor antagon-ists and non-selective dopamine receptor antagonantagon-ists (Kokay and Mercer, 1996).
The two different dopamine receptors cloned in
Dro-sophila are also D1-like, in that they stimulate adenylyl
cyclase when activated. The most recently cloned (DopR99B; Feng et al., 1996) shows a significant level of pharmacological similarity to D1 vertebrate receptors, since agonists and antagonists with D1 selectivity were generally more effective than those with D2-like recep-tor selectivity. However, a significant number of agonists/antagonist selective for D2 receptors as well as the neurotransmitters epinephrin and norepinephrin also exerted effects. Furthermore, while the DopR99B recep-tor is directly coupled to the activation of adenylyl cyclase (Feng et al., 1996), it is also linked to the initiation of a transient intracellular Ca2+signal, and the
two responses can be activated selectively by different synthetic agonists (Reale et al., 1997).
The “D1/5-like” Drosophila receptor cloned by Gotzes et al. (1994) has 99% base sequence identity to the receptor subsequently reported by Sugamori et al. (1995) and thus can be considered as the same receptor. Gotzes et al. (1994) found that this receptor did not bind other neurotransmitters tested (tyramine, octopamine, serotonin) and had relatively low affinity for SKF 38393, a D1 agonist which binds with high affinity to vertebrate D1 receptors. This agonist was only one third as potent as dopamine at an equivalent concentration. Sugamori et al. (1995) also observed the lack of binding by other neurotransmitters and found that this D1-like receptor bound certain D1 and D2 antagonists, with a rank order of potency of butaclamol.SCH23390.flupenthixol. spiperone (Table 6). The receptor had a poor affinity for benzazepines such as SKF 38393 and did not recognize the dopamine metabolite N-acetyl-dopamine.
The pharmacological profile of the Manduca D1-like receptor demonstrates that this receptor is substantially different even from other insect D1-like receptors. Only one compound of all those tested — the D2 receptor antagonist spiperone — strongly inhibited the dopamine-stimulated cAMP production. Neither D1 nor D2
agon-ists had any effect, and marginal effects were observed with a few other widely divergent compounds.
With regard to D2-like insect dopamine receptors, that of the honey bee brain was identified by the high affinity binding of [3H]-spiperone to bee brain homogenates
(Table 6). Since tyramine was more effective at displac-ing [3H]-spiperone than dopamine, and octopamine also
displayed high affinity for3[H]-spiperone binding sites,
the authors concluded that the identification of the [3
H]-spiperone binding sites as D2-like receptors was not cer-tain. However, the clear identification of a D1-like recep-tor with spacial and temporal distributions totally differ-ent from that of [3H]-spiperone binding (Kokay and
Mercer, 1997; Kokay et al., 1998), strongly suggests the existence of a non-D1-like binding site with high affinity for dopamine in the honey bee brain. The Manduca D2-like receptor is the first to be identified in an insect by dopamine inhibition of adenylyl cyclase activity, but it, too, defies classification. While a variety of D2 receptor agonists and antagonists interacted with this receptor, it also recognized compounds defined as D1 receptor agon-ists and antagonagon-ists in vertebrate systems.
On the basis of what is now a considerable body of pharmacological data, it must be concluded that trying to classify insect dopamine receptors as D1- or D2-like based on vertebrate pharmacology is difficult, if not impossible. As proposed by Downer (1990), the pharma-cological characteristics of these receptors are so distinct from those of vertebrate D1 and D2 receptors, that they must comprise a totally new class of dopamine receptors. Certainly, a comparison of sequence data for Drosophila and vertebrate D1-like receptors bears this out. For example, the C-terminal tails of the Drosophila receptors are intermediate in length (62 and 64 amino acids: Gotzes et al., 1994; Sugamori et al., 1995) between those of vertebrate D1 (113–117 amino acids) and D2 (16–18 amino acids) receptors (Feng et al., 1996). In addition, the sequence of the hydrophobic core region of the
Dro-sophila D1-like receptor (Gotzes et al., 1994; Sugamori
et al., 1995) is only 46–48% identical to that of ver-tebrate D1s, and even less to the DopR99B receptor (43%; Feng et al., 1996). So not only do insect receptors fall into a unique class of their own, but there can also exist distinctive sub-classes of receptor within the same insect.
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reported the absence of this metabolite in corpus car-diacum–corpus allatum complexes on day 5. Neverthe-less, N-acetyl-dopamine is present in fairly constant lev-els in the brain throughout the fifth stadium, with sharply defined peaks on days 2 and 9 (Sparks and Geng, 1992; Geng et al., 1993; Sparks et al., 1997), and thus the brain could serve as a source of this compound. The inhibitory effect of N-acetyl-dopamine on JH acid biosynthesis by V6 CA was equivalent to that of dopamine, providing the first evidence of a possible function for an acetylated biogenic amine.
The results of the EEDQ experiments are puzzling. On the one hand, it is clear that without exogenous dopa-mine, V0 CA are not affected by EEDQ, a potent neuro-toxin that binds irreversibly to vertebrate D1 and D2 receptors. In the presence of dopamine, EEDQ is effec-tive in blocking the binding of dopamine to its receptors and the resulting stimulation of JH synthesis by V0 CA, but only at a concentration equivalent to that of dopam-ine (1025 M). Based on its weak antagonistic activity, it
appears that EEDQ has the ability to bind to the V0 dopamine receptor, perhaps allosterically; based on what is known of the Drosophila D1-like receptors, it is entirely possible that the ligand binding site is different from that of a vertebrate D1 receptor. On the other hand, EEDQ is an effective inhibitor of JH acid biosynthesis by V6 CA in the absence of exogenous dopamine, while also blocking the effect of exogenous dopamine at a con-centration equal to that of dopamine. One possible expla-nation is that the allatostatic action of EEDQ on the V6 CA is totally unrelated to dopamine receptor binding. However, it also appears able to bind to the D2-like receptors, since it can partially override the inhibition by 1025 M dopamine. An alternative explanation is that in
the absence of dopamine, EEDQ is affecting the function of endogenous dopamine, which is fairly high on day 6 (4.9 pg per gland) compared to day 0 (1.4 pg per gland) (Granger et al., 1996). In vivo, there are two other poss-ible sources of dopamine available to the CA: the hemo-lymph and cerebral dopaminergic cells innervating the CA (Granger et al., 1996). Knowledge of the source or sources of dopamine affecting CA activity, the ligand binding kinetics of the receptors, as well as their struc-tures, should provide some answers as to actual effect of EEDQ.
The examination of the effect of dopamine on brain adenylyl cyclase activity strongly suggests the existence of the dopamine receptors in the Manduca larval brain that are substantially similar, if not identical, to those of the CA. These results contrast with those of a previous study of adenylyl cyclase activity in the brain of
Mand-uca on day 5 of the fifth stadium (Combest et al., 1985).
In that study, in three separate experiments, dopamine had effects ranging from none to a three-fold stimulation of cAMP formation in three separate experiments. It is not clear why this difference was observed, especially
since the assay system used in this and two previous studies (Granger et al., 1995, 1996) is based on that of Combest et al. (1985).
A limited test of selected dopamine D1 and D2 recep-tor agonists and antagonists on the production of cAMP in brain homogenates revealed effects similar to those obtained with CA on days 0 and 6, supporting the exist-ence of two different dopamine receptors in the larval brain. In addition, a preliminary immunohistological study of sections of V0 and V6 brains has revealed the binding of antibodies to both vertebrate D1 and D2 receptors in discrete areas (Granger and Brighton, unpublished). There does not appear to be stage-specific populations of D1- and D2-like receptors in the ventral nerve cord, since homogenates of both V0 and V6 thoracic/abdominal sections of the nerve cord only responded to the presence of dopamine with an increase in cAMP production. Thus, in addition to the temporal appearance of these receptors, there may also be clearly defined spatial localizations of these receptors within the nervous system. Both age-related changes in dopamine receptor densities and specific spatial distributions of dopamine receptors have been found in the honey bee brain (Kokay and Mercer, 1997; Kokay et al., 1998).
In Manduca, an allatotropin isolated from pharate adult heads was found to stimulate only adult CA (Kramer et al., 1991), and no other allatotropic neuro-peptide has been found for the larvae of this species. However, recent studies have shown that the Manduca allatotropin is a functional moiety in several other Lepi-doptera, including larvae of the tomato moth, Lacanobia
oleracea (Audsley et al., 2000) (see Stay, 2000; Gilbert
et al., 2000, for reviews). Thus in Manduca larvae, the larval allatotropin may not be a neuropeptide, but rather an aminergic neurotransmitter, dopamine, since it stimu-lates JH synthesis early in the fifth instar when the JH titers are high (Granger et al., 1996). By contrast, the allatostatin isolated from the heads of pharate adult
Man-duca by Kataoka et al. (1989) can inhibit the
biosyn-thetic activity of larval CA. Dopamine also inhibits
Man-duca larval CA, beginning on day 3 of the fifth instar
(Granger et al., 1996), concomitant with increasing pre-dominance of JH acid biosynthesis and release by the CA (Janzen et al., 1991). Thus the respective roles of the allatostatin and dopamine in inhibiting the CA during the critical reprogramming period of the fifth stadium need to be defined. Clearly the roles of dopamine in the development of Manduca, particularly during larval– pupal metamorphosis, need further investigation and would be facilitated by the molecular characterization of the dopamine receptors.
Acknowledgements
Indianapolis, IN. The authors thank Drs L.I. Gilbert and Anna Rachinsky for their reviews of the manuscript and gratefully acknowledge the contribution of Louise Stud-ley in the rearing and staging of the animals.
References
Arakawa, S., Gocayne, J.D., McCombie, W.R., Urquhart, D.A., Hall, L.M., Fraser, C.M., Venter, J.C., 1990. Cloning, localization, and permanent expression of a Drosophila octopamine receptor. Neu-ron 2, 343–354.
Audsley, N., Weaver, R.J., Edwards, J.P., 2000. Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia
oleracea, and regulation by Manduca sexta allatostatin and
allato-tropin. Insect. Biochem. Mol. Biol. 30, 681–689.
Bell, R.A., Joachim, F.G., 1976. Techniques for rearing laboratory col-onies of tobacco hornworms and pink bollworms. Ann. Entomol. Soc. Amer. 69, 365–373.
Combest, W.L., Sheridan, D., Gilbert, L., 1985. The brain adenylate cyclase system of the tobacco hornworm, Manduca sexta. Putative control by biogenic amines. Insect Biochem. 15, 579–588. Creese, I., Sibley, D.R., Hamblin, M.W., Leff, S.E., 1983. The
classi-fication of dopamine receptors: relationship to radioligand binding. Ann. Rev. Neurosci. 6, 43–71.
Downer, R.G., 1990. Octopamine, dopamine, and 5-hydroxytrytamine in the cockroach nervous system. In: Huber, I., Masler, E.P., Rao, B.R. (Eds.), Cockroaches as Models for Neurobiology: Appli-cations in Biomedical Research, Vol 2. CRC Press, Baton Rouge, FL, pp. 103–124.
Feng, G., Hannan, F., Reale, V., Hon, Y.Y., Kousky, C.T., Evans, P.D., Hall, L.M., 1996. Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster. J. Neu-rosci. 16, 3925–3933.
Geng, C., Sparks, T.C., Skomp, J.R., Gajewski, R.P., 1993. Biogenic amines in the brain of Manduca sexta during larval–pupal meta-morphosis. Comp. Biochem. Physiol. 106C, 275–284.
Gilbert, L.I., Granger, N.A., Roe, R.M., 2000. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem. Mol. Biol. 30, 617–644.
Gingrich, J.A., Caron, M.G., 1993. Recent advances in the molecular biology of dopamine receptors. Ann. Rev. Neurosci. 16, 299–321. Gotzes, F., Balfanz, S., Baumann, A., 1994. Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human D1/5receptors. Recep. Chann. 2, 131–141.
Granger, N.A., Allen, L.G., Sturgis, S.L., Combest, W., Ebersohl, R., 1995. Corpora allata of the larval tobacco hornworm contain a calcium/calmodulin-sensitive adenylyl cyclase. Arch. Insect Biochem. Physiol. 30, 149–164.
Granger, N.A., Goodman, W.G., 1988. Radioimmunoassays: juvenile hormones. In: Miller, T.A., Gilbert, L.I. (Eds.), Immunological Techniques: Arthropods. Springer-Verlag, New York, pp. 215–251. Granger, N.A., MacDonald, J.D., Menold, M., Ebersohl, R., Hiruma, K., Cann, C.L., Riddiford, L.M., 1994. Evidence of a stimulatory role for cyclic AMP in the regulation of corpus allatum activity in
Manduca sexta. Molec. Cell Endocrinol. 103, 73–80.
Granger, N.A., Sturgis, S.L., Ebersohl, R., Geng, C., Sparks, T.C., 1996. Dopaminergic control of corpora allata activity in the larval tobacco hornworm, Manduca sexta. Arch. Insect Biochem. Physiol. 32, 449–466.
Hamblin, M.W., Creese, I., 1983. Behavioral and radioligand binding evidence for irreversible dopamine receptor blockade by N-ethoxy-carbonyl-2-ethoxy-1,2,-dihydroquinolone. Life Sci. 32, 2247–2255. Han, K.-A., Millar, N.S., Groteweil, M.S., Davis, R.L., 1996. DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies. Neuron 16, 1127–1135.
Janzen, W.P., Menold, M., Granger, N.A., 1991. Effects of endogenous esterases and an allatostatic factor on the products of Manduca
sexta corpora allata in vitro. Physiol. Entomol. 16, 283–293.
Kaatz, H., Eichmu¨ller, S., Kreissle, S., 1994. Stimulatory effect of dopamine on juvenile hormone biosynthesis in honey bees (Apis
mellifera): physiological and immunocytochemical evidence. J.
Insect Physiol. 40, 865–872.
Kataoka, H., Toschi, A., Li, J.P., Carney, R.L., Schooley, D.A., Kramer, S.J., 1989. Identification of an allatotropin from adult
Manduca sexta. Science 243, 1481–1483.
Kokay, I.C., McEwan, J., Mercer, A., 1998. Autoradiographic localiz-ation of [3H]-SCH23390 and [3H]-spiperone binding sites in honey
bee brain. J. Comp. Neurol. 394, 29–37.
Kokay, I.C., Mercer, A.R., 1996. Characterization of dopamine recep-tors in insect (Apis mellifera) brain. Brain Res. 706, 47–56. Kokay, I.C., Mercer, A.R., 1997. Age-related changes in dopamine
receptor densities in the brain of the honey bee, Apis mellifera. J. Comp. Physiol. A181, 415–423.
Kramer, S.J., Toschi, A., Miller, C., Kataoka, H., Quistad, G.B., Li, J.P., Carney, R.L., Schooley, D.A., 1991. Identification of an allato-statin from the tobacco hornworm Manduca sexta. Proc. Natl. Acad. Sci. USA 88, 9458–9462.
Krueger, R.R., Kramer, K.J., Hopkins, T.L., Speirs, R.D., 1990. N-b -Alanyldopamine and N-acetyl dopamine occurrence and synthesis in the central nervous system of Manduca sexta. Insect Biochem. 20, 605–610.
Lafont-Cazal, M., Baehr, J.C., 1988. Octopaminergic control of corpora allata activity in an insect. Experiencia 44, 895–896. Meller, V., Combest, W.L., Smith, W.A., Gilbert, L.I., 1988. A
calmo-dulin-sensitive adenylyl cyclase in the prothoracic glands of the tobacco hornworm, Manduca sexta. Molec. Cell. Endocrinol. 59, 67–76.
O’Dowd, B.F., 1993. Structures of dopamine receptors. J. Neurochem. 60, 804–816.
Orr, G., Gole, J.W.D., Notman, H.J., Downer, R.G.H., 1987. Pharma-cological characterization of the dopamine-sensitive adenylyl cyclase in cockroach brain: evidence for a distinct dopamine recep-tor. Life Sci. 41, 2705–2715.
Osborne, R.H., 1996. Insect neurotransmission: neurotransmitters and their receptors. Pharmacol. Ther. 69, 117–1142.
Pastor, D., Piulachs, M.-D., Cassier, P., Andre, M., Belles, X., 1991. Etude in vivo et in vitro de l’action de la dopamine sur la croissance des ovocytes et la production d’hormone juvenile chez Blattela
ger-manica (L.). CR Acad. Sci. Paris 313, 207–212.
Rachinsky, A., 1994. Octopamine and serotonin influence corpora allata activity in honey bee (Apis mellifera) larvae. J. Insect Phy-siol. 40, 549–554.
Reale, V., Hannan, F., Hall, L.M., Evans, P.D., 1997. Agonist-specific coupling of a cloned Drosophila melanogaster D1-like dopamine rceptor to multiple second messenger pathways by synthetic agents. J. Neurosci. 17, 6545–6553.
Robb, S., Cheek, T.R., Hannan, F.L., Hall, L.M., Midgley, J.M., Evans, P.D., 1994. Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger sys-tems. EMBO J. 13, 1325–1330.
Roeder, T., 1994. Biogenic amines and their receptors in insects. Comp. Biochem. Physiol. 107C, 1–12.
Saudou, F., Boschert, U., Amlaiky, N., Plassat, J.-L., Hen, R., 1992. A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns. EMBO J. 11, 7–17. Sparks, T.C., Geng, C., 1992. Analysis of biogenic amines in the cen-tral nervous system of the tobacco hornworm by high performance liquid chromatography with 16-sensor electrochemical detection. Anal. Biochem. 205, 319–325.
Par-766 N.A. Granger et al. / Insect Biochemistry and Molecular Biology 30 (2000) 755–766
vez, H., Parvez, S. (Eds.), Progress in HPLC–HPCE: Coulometric Electrode Array Detectors for HPLC, Vol 6. VSP, Utrecht, Nether-lands, pp. 145–168.
Stay, B., 2000. A review of the role of neurosection in the control of juvenile hormone synthesis: A tribute to Berta Scharrer. Insect. Biochem. Mol. Biol. 30, 653–661.
Stay, B., Fairbairn, S., Yu, C.G., 1996. Role of allatostatins in the regulation of juvenile hormone synthesis. Arch. Insect Biochem. Physiol. 32, 287–298.
Stay, B., Tobe, S.S., Bendena, W.G., 1994. Allatostatins: identification, primary structures, functions, and distribution. Adv. Insect Physiol. 25, 267–337.
Strader, C.D., Fong, T.M., Graziano, M.P., Tota, M.R., 1995. The fam-ily of G-protein-coupled receptors. FASEB J. 9, 745–754. Sugamori, K.S., Demchyshyn, L.L., McConkey, F., Forte, M.A.,
Niz-nik, H.B., 1995. A primordial dopamine D1-like adenylyl cyclase-linked receptor from Drosophila melanogaster displaying poor affinity for benzazepines. FEBS Lett. 362, 131–138.
Thompson, C.S., Yagi, K.J., Chen, Z.F., Tobe, S.S., 1990. The effects
of octopamine on juvenile hormone biosynthesis, electrophysiol-ogy, and cAMP content of the corpora allata of the cockroach,
Diploptera punctata. J. Comp. Physiol. B160, 241–249.
Vanden Broeck, J., Vulsteke, V., Huybrechts, R., De Loof, A., 1995. Characterization of a cloned locust tyramine receptor cDNA by functional expression in permanently transformed Drosophila S2 cells. J. Neurochem. 64, 2387–2395.
Von Nickisch-Rosenegk, E., Krieger, J., Kubick, S., Laage, R., Strobel, J., Strotmann, J., Breer, H., 1996. Cloning of biogenic amine recep-tors from moths (Bombyx mori and Heliothis virescens). Insect Biochem. Molec. Biol. 26, 817–827.
Witz, P., Amlaiky, N., Plassat, J.-L., Maroteaux, L., Borrelli, E., Hen, R., 1990. Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase. Proc. Natl. Acad. Sci. USA 8, 8940–8944.
