Insect NMDA receptors mediate juvenile hormone biosynthesis Ann-Shyn Chiang*†, Wei-Yong Lin*, Hsin-Ping Liu*, Maciej A. Pszczolkowski*, Tsai-Feng Fu*, Shu-Ling Chiu*, and Glenn L. Holbrook‡ *Department of Life Science, National Tsing Hua University, Hsinchu, 300 Taiwan, Republic of China; and ‡Department of Entomology, Pennsylvania State University, University Park, PA 16802 Edited by Bruce D. Hammock, University of California, Davis, CA, and approved October 29, 2001 (received for review June 23, 2001)
uvenile hormone (JH), a product of insect corpora allata, prevents metamorphosis and, in many species, has a reproductive role, being necessary for maturation of the oocytes. Synthesis of JH is a tightly regulated process, controlled by neurons and peptidergic neurosecretory cells, whose somata are located in the brain and project axons into the corpora allata (1). Several brain neuropeptides, termed allatostatins and allatotropins, have been shown to either stimulate (2) or inhibit (3–5) JH synthesis in short-term in vitro assays. Although the sequences of the receptors for these regulators are still uncharacterized, their existence has been demonstrated by photoaffinity labeling of corpora allata from the cockroach Diploptera punctata (6). In addition to the aforementioned neuropeptides, such neurotransmitters as octopamine (7) and dopamine (8) have been shown to influence JH synthesis, and receptors for the latter have been characterized pharmacologically in corpora allata of Manduca sexta (9). How neuropeptides and neurotransmitters ultimately affect JH synthesis has been investigated in Diploptera punctata. Allatostatins depress JH synthesis, apparently through a signal transduction pathway involving diacylglycerol and inositol 1,4,5trisphosphate (10), whereas octopamine induces concomitant changes in JH production and cAMP levels (7). Calcium is
www.pnas.org兾cgi兾doi兾10.1073兾pnas.012318899
Methods Reverse Transcription (RT)-PCR. Several nucleotide sequences were
used as specific primers to isolate cDNA: 5⬘-ATTCTCGAAGCCATTCAAATACCAAGG-3⬘ and 5⬘-GAGGAAGGCGGCCAGGTTG-3⬘ were used for putative Drosophila NMDAR1 (26); 5⬘-GAGTTCCACGAGTTT-3⬘ and 5⬘-CAGCGGATCCGAGGATTT-3⬘ were used for putative Drosophila NMDAR (EG:80H7.7) as predicted from computer analysis of the coding sequences of clone 80H7 in F LYBASE (27); and 5⬘-TTATGCAGCAGGGATGCGATATTAC-3⬘ and 5⬘-GTAGTAGATCCCGThis paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CA, corpus allatum; CC, corpus cardiacum; [Ca2⫹]i, cytosolic calcium concentration; JH, juvenile hormone; MK-801, (5R,10S)-(⫹)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine; NMDA, N-methyl-D-aspartate; NMDAR, the NMDA subtype of glutamate receptors; RT, reverse transcription. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF456127). †To
whom reprint requests should be addressed. E-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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another critical factor affecting JH production. The corpora allata of both Drosophila melanogaster and Diploptera punctata synthesize almost no JH in calcium-free medium (11, 12), and it is now widely accepted that elevated cytosolic calcium concentration (Ca2⫹i) in corpus allatum (CA) cells is essential for heightened JH production (13). Nevertheless, further investigation is needed on the role of calcium in transducing neuropeptide and neurotransmitter signals. Glutamate is a fast excitatory neurotransmitter in the central nervous system of both vertebrates and invertebrates. Glutamate binds to and activates postsynaptic receptors classified into metabotropic and ionotropic subtypes (14–16). Ionotropic glutamate receptors, named after their selective agonists—␣amino-3-hydroxy-5-methyl-4-isoxazolepropionate (kainate) and N-methyl-D-aspartate (NMDA)—are directly coupled to ion channels permeable to calcium (17, 18). The NMDA subtype of glutamate receptors (NMDAR) appears to have a role in neuronal development, synaptic plasticity, memory formation, and endocrine function of the pituitary in vertebrates (19–24). Thus far, functional NMDAR has not been identified in insects. Recently, we found that CA cells from an adult cockroach, Diploptera punctata, exhibited a Ca2⫹ influx and concomitant increase in JH synthesis on exposure to glutamate (25). Both responses were eliminated if the glutamate-treated cells were simultaneously exposed to a high concentration of Mg2⫹, a potent blocker of calcium channels coupled to NMDAR. This result suggested that functional NMDAR were present in CA cells. We now demonstrate mRNA transcripts and immunoreactive proteins of two NMDAR subunits, DNMDAR1 and DNMDAR2, in Drosophila brain neurons and CA cells. Functional analysis using cockroach corpora allata indicates that JH synthesis is elevated by an NMDA-induced influx of Ca2⫹ ions.
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In vertebrates, the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. We have now demonstrated immunohistochemically glutamatergic nerve terminals in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibited elevated cytosolic [Ca2ⴙ], but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg2ⴙ. Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 M NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-dayold mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg2ⴙ. Having obtained substantive evidence of functioning NMDAR in insect corpora allata, we used reverse transcription PCR to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, we used the same primers for DNMDAR1 to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. Our results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution.
GCCACATTGC-3⬘ were used for Drosophila kainate receptor (28). Ring glands used in RT-PCR were from last-instar Drosophila at the wandering stage. Glands were washed three times in PBS and then transferred into 0.5-ml tubes containing 10 l of solution consisting of 0.15 M NaCl, 10 mM Tris, and 5 units兾l RNasin RNase inhibitor (Promega) at pH 8.0 (29). Tubes were frozen at ⫺80°C in a bottle containing precooled 95% ethanol. Samples were thawed rapidly at 37°C, and 1 l of each sample solution was run through a standard RT-PCR protocol (Access RT-PCR system, Promega) at an annealing temperature of 58°C; a Hybrid PCR Express thermal cycler (Hybraid Limited, Middlesex, U.K.) was used for this process. The direct RT-PCR method is efficient at detecting specific mRNA transcript in a small amount of tissue. The sequences of RT-PCR products were determined by dideoxynucleotide sequencing and verified by using F LYBASE. Immunohistochemical Labeling of Neurons. Brain-corpus cardiacum
(CC)-CA complexes were dissected, enzymatically digested, and fixed with microwave-aided procedures (30). Samples were blocked and penetrated overnight at 27°C in PBS-based blocking buffer (3 mM sodium azide兾2% Triton X-100兾10% normal goat serum, pH 7.4). Subsequently, the samples were incubated for 2 days at 27°C in 20 l of rabbit anti-glutamate antibody (Sigma) or rabbit anti-allatostatin antibody, diluted 1:1000 in PBS-based antibody diluent (0.02% sodium azide兾0.25% Triton X-100兾1% normal goat serum, pH 7.4). Immunolabeled tissue was washed three times in buffer (50 mM Tris base兾500 mM NaCl兾0.25% Triton X-100, adjusted to pH 7.4 with HCl) and then hybridized for 24 h at 27°C in 20 l of biotinylated goat anti-rabbit IgG (Molecular Probes) diluted 1:200 in antibody diluent. After three washes with buffer, each brain-CC-CA complex labeled with primary and biotinylated secondary antibodies was incubated with Cy-5-conjugated streptavidin (1 g/ml; Amersham Pharmacia) for 4 h at 27°C. In some cases, nuclei were counterstained overnight with 20 g/ml propidium iodide in PBS and then membranes were stained overnight with 0.435 mM 6-[(N-(7nitrobenz-2-oxa-1,3-diazol-4-yl)hexanonyl]sphingosine in DMSO. Immunohistochemical Labeling of Proteins. Proteins structurally
similar to rat NMDAR1A were localized in ring gland of D. melanogaster with a mouse monoclonal antibody against rat NMDAR1 (mab363, Chemicon, Temecula, CA). The antibody shows no crossreactivity with rat NMDAR2–5 and its epitope is between amino acids 660 and 811 of rat NMDAR1, which displays 62% amino acid identity to a putative D. melanogaster NMDAR protein (26). Ring glands from last-instar D. melanogaster were fixed in methanol for 30 min at ⫺20°C and then washed in PBS. The glands were permeabilized and blocked overnight at 4°C in PBS containing 1% Triton X-100 and 10% normal goat serum, and next incubated at 4°C for 48 h with mouse monoclonal NMDAR1 antibody diluted 1:750 in PBS. After three washes in PBS, specimens were incubated overnight at 4°C with biotinylated goat anti-mouse IgG diluted 1:200 in PBS. The tissues were washed and incubated at room temperature for 4 h with Oregon Green-conjugated streptavidin diluted 1:1000 in PBS. All immunohistochemically labeled samples were postfixed for 30 min at room temperature in PBS containing 4% paraformaldehyde. Thoroughly washed samples were directly cleared in the FocusClear solution (PacGene, Vancouver) for 2 h, mounted in a fresh drop of the same, and viewed with a Zeiss LSM 510 confocal microscope with a 40⫻ C-Apochromat waterimmersion objective lens. Quantifying Free Cytosolic Calcium. [Ca2⫹]i was monitored as
described (25). Glands were enzymatically dissociated into suspensions of single cells, which were loaded with the Ca2⫹38 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.012318899
sensitive fluorescent indicator indo-1 (31). Individual cells were monitored with a dual-wavelength indo-1 ratiometric fluorescent system (PhoCal Pro, Life Science Resources, Cambridge, U.K.). The cells were superfused at about 0.8 ml/min in a standard solution nominally free of Mg2⫹, containing 152.8 mM NaCl, 12.22 mM KCl, 10 mM CaCl2, 23 mM trehalose, and 5 M glycine. The solution was buffered with 4 mM Hepes, titrated to pH 7.4 with NaOH, and adjusted to an osmolarity of 360 mOsm/kg with small amounts of NaCl and KCl. The solution was also augmented with 100 nM dopamine, which is known to induce a large reduction in basal [Ca2⫹]i in isolated rat melanotropes, thereby making the cells more sensitive to NMDA (32), though responsiveness of pituitary cells to NMDA is independent of dopamine (24). Our preliminary tests showed that dopamine induced a slight reduction of basal [Ca2⫹]i in CA cells. Chemicals were applied near cells by using a micropipette controlled by a hydraulic micromanipulator. Unless indicated otherwise, corpora allata were from mated 4-day-old adult females with basal oocytes 1.1–1.4 mm in length. Measuring JH Synthesis. A radiochemical assay, based on incor-
poration of [3H]methionine into JH III, was used to measure JH synthesis (33). Two culture media were used: L-15 (pH 7.4, 360 mOsm/kg, balanced with glucose, and supplemented with 2% Ficoll, prepared by Specialty Media, Lavalette, NJ) and minimum incubation medium (150 mM NaCl兾12 mM KCl兾4 mM Hepes兾100 nM dopamine兾5 M glycine兾2% Ficoll). We previously reported that removing dopamine from minimum incubation medium had no effect on JH synthesis (25). However, to maintain consistency with experiments in which we measured [Ca2⫹]i, we kept dopamine in the medium. In experiments with intact glands, the two members of a CA pair were split; one was incubated in medium containing agonists or antagonists of interest, and the other was used as a control and incubated without these agonists or antagonists (25). Results Glutamate-Immunoreactive Nerve Fibers in the CA of Diploptera punctata. To shed light on the physiological significance of
glutamate receptors in CA cells, we sought to identify glutamatergic nerves projecting to the CA. Brain-CC-CA complexes of 0-, 1-, 4-, and 5-day-old adult female cockroaches were immunoreacted with rabbit anti-glutamate antibody (n ⱖ 10 for each age). Confocal analysis of 57 optical sections at 2.1-m intervals through an entire CA showed that glutamate-immunoreactive nerve fibers were distributed throughout the gland (Fig. 1A). They were, however, less abundant than allatostatinergic nerves, which contact almost all CA cells (Fig. 1B). Nevertheless, glutamate-immunoreactive fibers were present in all examined glands. Although glutamatergic immunoreactivity varied among glands, this variation was age independent. Glutamateimmunoreactive nerve fibers were not seen in negative controls immunostained with the same protocol, but without antiglutamate. By contrast, in the positive control, several glutamatergic interneurons, and their varicosities in the antennal glomeruli, showed substantial immunoreactivity (Fig. 1C). A group of brain neurons in the lateral protocerebrum and several large neurosecretory cells in the pars intercerebralis were immunopositive (results not shown), but their projections were difficult to trace, and we are uncertain whether they send fibers to the CA. Regardless, at least some nerve fibers within the CA contained glutamate. It was therefore reasonable to pursue NMDAR. Variable Response of CA Cells of Diploptera punctata to NMDA.
Highly active corpora allata of 4-day-old mated females were more responsive to NMDA than the less active glands of younger and older females (Fig. 2). Indeed, the inclusion of 3 M NMDA in the incubation medium caused the glands of 4-day-old females Chiang et al.
Fig. 1. Anti-glutamate immunoreactivity in the corpora allata and antennal lobe of 4-day-old adult mated females of Diploptera punctata. (A) Glutamateimmunoreactive nerve fibers (green) in a CA and nuclei (red) counterstained with propidium iodide. Fibers are distributed throughout the gland but fail to reach every cell. (B) Allatostatin-immunoreactive nerve fibers in the CA. Depth code with different colors indicates distance of an object from the surface. (C) Glutamate-immunoreactive interneurons (green) in the antennal lobe sending projections into individual glomeruli (purple). Nuclei (red) are counterstained with propidium iodide. (Scale bar ⫽ 50 m in all pictures.)
to produce about 70% more JH, whereas glands of lower basal activity produced just 10–20% more JH. What is more, the glands of 8-day-old females showed no response at all to 3 M NMDA. Nonetheless, NMDA had a clear impact on cellular
production of JH, and given this, it was reasonable to hypothesize the presence of functional NMDAR in insects. We therefore sought to characterize putative insect NMDAR, which, in mammals, are calcium-permeable, glutamate-gated ion channels whose function can be disrupted with Mg2⫹, MK-801, and conantokin T. Cockroach NMDAR Are Mg2ⴙ-Gated Calcium Channels. The action of
the putative NMDA receptor, or receptors, was characterized by monitoring changes in [Ca2⫹]i. We examined corpora allata from adult females of Diploptera punctata because their sizable glands could be readily dissociated into single cells retaining high rates of JH synthesis in vitro (34). On brief exposure to 60 M NMDA, most CA cells exhibited a rapid increase in fluorescence intensity of calcium-bound indo-1, and a symmetric decrease in fluorescence intensity of calcium-unbound indo-1 (Fig. 3A), indicating that NMDA induced an increase in free cytosolic [Ca2⫹]i (Fig. 3B). Within 5 min, cytosolic calcium returned to basal levels. Of 43 cells, 77% showed this response (Fig. 3C), achieving an average maximum calcium concentration of 865.69 ⫾ 120.65 nM (n ⫽ 33). The increase in [Ca2⫹]i was, however, largely abolished when cells were exposed to Mg2⫹ or MK-801, specific blockers of channels in NMDAR. Just 14% of 57 cells in 3 mM Mg2⫹ responded to 60 M NMDA, and only 6% of 48 cells in 1 M MK-801. When cells were incubated in nominally calcium-free superfusion solution containing 1 mM EGTA, the NMDAinduced response was also nearly abolished (6% of cells responded). Thus, increased cytosolic [Ca2⫹]i, stimulated by NMDA, appears a result of Ca2⫹ influx from the extracellular environment rather than Ca2⫹ release from intracellular stores.
Chiang et al.
determine how the NMDA-mediated Ca2⫹ influx affected JH synthesis, CA function was examined both in the presence and absence of NMDA. In glutamate-free L-15 medium, NMDA had a dose-dependent effect on JH production (Fig. 4A Upper); 60 M NMDA and 1 mM NMDA increased JH synthesis by 44 ⫾ 5% (P ⬍ 0.001, Student’s paired t test) and 58 ⫾ 19% (P ⬍ 0.01, Student’s paired t test), respectively. However, because L-15 is a complex medium containing several components that might interact with NMDAR, we incubated glands in a minimal PNAS 兩 January 8, 2002 兩 vol. 99 兩 no. 1 兩 39
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Dose- and Mg2ⴙ-Dependent NMDA Stimulation of JH Synthesis. To
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Fig. 2. Developmental changes in CA sensitivity to NMDA. (A) Basal (E) and NMDA-stimulated (F) rates of JH synthesis by CA of differently aged females mated on day 0. (B) The level of stimulation expressed as a percent value, calculated from data in A. The members of a CA pair were incubated separately, one in minimum incubation medium with 3 M NMDA and the other in the same medium but without NMDA. Data points for the glands of a pair are placed at the same horizontal position along the x axis. Percent stimulation was calculated as 100 ⫻ (stimulated rate ⫺ basal rate)兾basal rate of JH synthesis of the two members of same CA pair. Bars represent mean ⫾ SEM. n ⫽ 10 gland pairs for each day. **, ***, a significant difference (P ⬍ 0.01 and P ⬍ 0.001, respectively) between stimulated and basal rates of JH synthesis on the same day.
Fig. 3. Effects of channel blockers on cockroach NMDAR. Changes in [Ca2⫹]i in response to 60 M NMDA in a representative CA cell, measured with indo-1 ratiometric microfluorimetry. (A) Changes in fluorescence intensity for bound (F⬘ ⫽ 405 nm) and unbound (F⬘ ⫽ 490 nm) calcium after NMDA administration. (B) Changes in [Ca2⫹]i derived from the ratio of fluorescence intensities (F⬘ ⫽ 405 nm兾F⬘ ⫽ 490 nm) of data presented in A (25). Horizontal bars in A and B indicate the period of NMDA superfusion. (C) Percentage of cells responding to 60 M NMDA in superfusing solution containing standard components, standard components minus Ca2⫹, or standard components with 3 mM Mg2⫹ and 1 M MK-801. The number of examined cells is shown in parentheses above each bar.
medium lacking amino acids (25). The results were similar, but the response was Mg2⫹-dependent (Fig. 4A Lower). In the absence of Mg2⫹, the highest level of stimulation was achieved with just 3 M NMDA, and greater concentrations of the compound were less stimulatory, or even inhibitory. By contrast, in the presence of Mg2⫹, low concentrations of NMDA (1–3 M) did not stimulate JH synthesis, but much higher concentrations (60–100 M) did. NMDAR Can Be Blocked with Specific Antagonists. Elevated intracellular Ca2⫹ concentration appears necessary for high levels of JH synthesis (25), but excess cytosolic Ca2⫹ has inhibitory effects. Thus, Mg2⫹ could either inhibit JH synthesis by blocking Ca2⫹ influx through ligand-bound NMDAR or stimulate JH synthesis by preventing excessive Ca2⫹ influx. A functional role of NMDA-like receptors was demonstrated by blocking the receptors with specific antagonists in Mg2⫹-free minimum incubation medium (Fig. 4B). When the two members of a CA pair were incubated in the same medium, with or without NMDA, they produced similar amounts of JH. However, when one gland was incubated in medium containing 3 M NMDA, it always produced more JH than its partner not exposed to NMDA. When NMDAR antagonists—Mg2⫹, MK-801 or conantokin T—were added, along with NMDA, the stimulation in JH synthesis was significantly reduced (Fig. 4B). NMDAR mRNA in Drosophila Brain and Ring Gland. Two genes from
D. melanogaster encoding sequences with high homology to rat
40 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.012318899
Fig. 4. Effects of NMDA and NMDAR antagonists on JH synthesis. (A Upper) Dosage effect of NMDA on glands in glutamate-free L-15 medium containing 1.8 mM Mg2⫹ and 1.2 mM Ca2⫹. (Lower) Dosage effect of NMDA on glands in minimum incubation medium without Mg2⫹ (F) or with 5 mM Mg2⫹ (E); trehalose was reduced in the latter to maintain osmotic pressure at 360 mOsm. Percentage stimulation is the ratio of rates of JH synthesis of experimental (with NMDA) glands over control (without NMDA) glands, the glands being members of the same CA pair. (B) Effects of NMDAR antagonists on JH synthesis in Mg2⫹-free minimum incubation medium (saline). Stimulation of JH synthesis by NMDA was reduced by including 1.8 mM Mg2⫹, 7.4 M MK-801, or 1 M conantokin T (ConT) in the medium. The data show the effect of the treatments on individual members, experimental (F) and control (E), of the same gland pair. The glands of a pair produced similar JH when both were incubated in minimal medium (saline), or in the same medium with 3 M NMDA (P ⬎ 0.05, Student’s paired t test). In all other cases, experimental glands produced significantly more or less JH than control glands (***, P ⬍ 0.001, Student’s paired t test). Each bar represents the mean ⫾ SEM for the ratio of JH synthesis of 10 gland pairs.
NMDAR1 and NMDAR2 had previously been cloned and characterized, and were shown to be expressed in the central nervous system (26, 35). Because their cellular localization and functional roles have not yet been clarified, these genes are classified as putative subunits of Drosophila NMDAR, named DNMDAR1 and DNMDAR2. Having found evidence for functioning NMDAR in a cockroach, we probed for expression of DNMDAR mRNA in the ring gland and brain of last-instar D. melanogaster by using analytical direct RT-PCR (Fig. 5A). The sequences of RT-PCR products were verified by dideoxynucleotide sequencing. The procedure yielded a 385-bp cDNA fragment identical in sequence to intact DNMDAR1 and a 462-bp cDNA fragment identical to intact DNMDAR2. The RT-PCR method revealed that neither the brain nor the ring gland of last instars contained mRNA transcripts of kainate receptors (Fig. Chiang et al.
NMDAR-Like Protein Expression in Drosophila CA Cells and Brain Neurons. The ring gland comprises three types of endocrine cells:
large ecdysteroid-producing cells occupying most of the gland; small CA cells clustered into a single group at the gland’s distal portion; and small CC cells situated opposite the CA, close to the brain. Immunohistochemical labeling using a mouse monoclonal antibody against rat NMDAR1 revealed that only the CA cells expressed rat NMDAR1-like immunoreactive protein (Fig. 5B). The antibody also labeled two brain neurons in the lateral protocerebrum, one per hemisphere (Fig. 5C). Projections of these neurons extended horizontally toward the pars intercerebralis, where extensive terminal arborizations were observed. A Putative NMDAR in the CA of Diploptera punctata. We used the
Fig. 5. Localization of NMDAR in the brain and ring gland of last-instar D. melanogaster. (A) Analytical direct RT-PCR yielded a 385-bp cDNA fragment of DNMDAR1 and a 462-bp cDNA fragment of DNMDAR2 in the brain and ring gland; mRNA transcripts of Drosophila kainate receptors (610 bp) (17) were, however, not detected. Genomic DNA fragments for DNMDAR1 (590 bp with an intron), DNMDAR2 (888 bp), and kainate (795 bp) were also identified and sequenced. DNA molecular size standards (M) are shown from 100 to 1,000 bp by 100-bp steps. (B) Immunohistochemically labeled whole-mount of rat NMDAR1-like proteins in the ring gland. The frontal view of the entire ring gland was reconstructed from 16 confocal image series at 1.6-m z axis intervals. Anti-NMDAR1 monoclonal antibody labeled only CA cells (arrow), clustered into a single group in the distal portion of the ring gland. These cells have smaller nuclei than neighboring ecdysteroid-producing cells. Corpus cardiacum cells also have small nuclei but are located opposite the CA, close to the brain (arrowhead). (Scale bar, 50 m.) (C) Frontal view of two NMDAR1immunoreactive neurons in the brain. The image was derived from the projection of 68 confocal image series at 1-m z axis intervals. (Scale bar, 50 m.)
5A), an expected result that served as a negative control; kainate receptors are highly expressed in late embryos but undetectable in last-instar (28). Genomic DNA fragments of DNMDAR1 (590 bp), DNMDAR2 (888 bp), and kainate (795 bp) matched sequences reported in F LYBASE (27). Note that each of these DNA fragments contains an intron.
primers for DNMDAR1 to probe for the presence of a putative NMDAR in the CA of Diploptera punctata. Direct RT-PCR yielded a 435-bp cDNA fragment (Table 1). The amino acid sequence deduced from the fragment indicated that the projected translational product would share certain similarity with the glutamate receptors of other animals (analyzed by BLASTX in the National Center for Biotechnology Information). For example, the amino acids likely encoded by the base-pair 237– 142 region of the cloned cDNA would be expected to have 46% similarity to amino acids in the glutamate receptor subunit of snail Lymnaea stagnalis (EMBL database accession no. CAA42683). Moreover, the amino acids encoded by the basepair 276–151 region of the cDNA fragment would be expected to share up to 37% of their identity with amino acids of the ␣ -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective receptor subunit of the squid Loligo opalescens (EMBL database accession no. CAB65182). Discussion The current results lend credence to our earlier hypothesis that ionotropic NMDA-subtype glutamate receptors are present in cells of the insect CA (25). This hypothesis arose from our finding that Mg2⫹ blocked the stimulatory effect of glutamate on JH synthesis by corpora allata of Diploptera punctata. We have now demonstrated mRNA transcripts and immunoreactive proteins of two NMDAR subunits, DNMDAR1 and DNMDAR2, in brain neurons and CA cells of Drosophila (Fig. 5). We used the same primers for DNMDAR1 and found a putative NMDA receptor fragment in CA of Diploptera punctata. And an in vitro assay with dissociated cells from Diploptera punctata CA has shown that JH synthesis is regulated by glutamate-induced calcium influx through NMDAR (Fig. 3). Together, these results provide experimental evidence indicating the existence of functional NMDAR or NMDA-like receptors in insects. Insect NMDAR appear to exhibit both striking similarities to and important differences from like receptors in vertebrates. In both groups, the receptors are glutamate-gated ion channels with high calcium permeability, whose function is disrupted by Mg2⫹, MK-801, and Conantokin T. Mammalian NMDAR are tetra-
Table 1. Sequence of the 435-bp cDNA fragment produced by direct RT-PCR of Diploptera punctata corpora allata using primers derived from the cDNA sequence of Drosophila NMDAR1
Chiang et al.
GAGGAAGGCG GGCATGCCGG ACGCTTGCCG GTCAAAGGAG TTTCGCAGGA GTAAACATAC CCAGTCTTCG CTGGCCGCCT
GCCAGGTTGC GCTTTTCTAT CCTTTGTAAG ATATCGCCGG TCAGCAGAGT GGTGCGTAAA TTGACTTTCT TCCTC
GCACATGTGC GTGCACGCTG TGTACAGCGT TGACACCCTT TAGCCTGCTT TCTGCACTTC TCTGAGCATC
TGCATTGTGT CTCGGATTAC CAGTGTGCCA GTATTTGATT CACCGCTTCT TACGCCGAAT CAGAACACCG
TTGCATCAAA TTCGTGACAG TCCTTGATGT TTCTTCAGTG ACCATTGTCA TTCTTTTTGT CCTGCTTCAG
AGAAAAGCCC TCAGCAGATC CGCCTTTATC CTGGCAGGTA TGACAGCGTC AAGCTGCTTT CGCAGACAAC
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1 61 121 181 241 301 361 421
Sequence
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Position no.
mers consisting of two type 1 (NMDAR1) and two type 2 (NMDAR2) subunits (36). Computer analysis indicates that cDNA sequences of DNMDAR1 and DNMDAR2 display high homology to vertebrate NMDAR1 and NMDAR2, respectively, so it is reasonable to assume that DNMDAR1 and DNMDAR2 coassemble into a heteromeric receptor protein, especially because both co-occur in the same embryonic tissues (26, 35). But, unlike rat NMDAR1 subunit, the putative DNMDAR1 fails to form functional homomeric receptors when it is expressed in Xenopus oocytes (26). High homology of amino acid sequences and similarity of pharmacological responses to various agonists and antagonists, together, suggest that insect and vertebrate NMDAR share structural and functional properties and that ionotropic glutamate receptors may have become specialized early in animal evolution. It remains to be tested whether insect NMDAR, like mammalian ones, have a role in cellular mechanisms of learning. The regulation of JH synthesis by both neurohormones (allatostatins and allatotropins) and neurotransmitters (octopamine and dopamine) has been extensively investigated (1, 37). In Diploptera, allatostatins depress JH synthesis, apparently through a signal transduction pathway involving diacylglycerol and inositol 1,4,5-trisphosphate (10), whereas octopamine induces concomitant changes in JH production and cAMP levels (7). Calcium is another critical factor affecting JH production. The corpora allata of both D. melanogaster and Diploptera punctata synthesize almost no hormone in calcium-free medium (11, 12), and it is now generally accepted that elevated [Ca2⫹]i in CA cells is essential for heightened JH production. Our recent
finding that L-glutamate both increases [Ca2⫹]i in CA cells and stimulates JH synthesis in vitro provides yet more insight into regulation of the CA in Diploptera (25). Indeed, glutamatergic control over CA function seems a real phenomenon given the impact of L-glutamate on intracellular JH synthesis and the presence of anti-glutamate immunoreactivity in nerve terminals within the corpora allata (Fig. 1). L-Glutamate induces a calcium influx into CA cells, but how the agonist’s activity is regulated in vivo is not certain. It is possible that release of L-glutamate from presynaptic axons is tightly controlled. Alternatively, L-glutamate receptor number or responsiveness may largely determine the agonist’s impact on JH synthesis. The former hypothesis loses support in light of our immunocytochemical observations on glutamatergic terminals within the CA of female Diploptera in the first 5 days of adulthood. During this time, JH synthesis increases dramatically, yet glutamatergic nerve fibers remain largely unchanged. It therefore seems likely that the effect of L-glutamate is regulated at the receptor level, especially because isolated CA in vitro exhibit temporal changes in their responsiveness to L-glutamate (Fig. 2). How L-glutamate interacts with other regulatory factors in controlling reproduction of Diploptera, and other insects, remains to be examined.
1. Gilbert, L. I., Granger, N. A. & Roe, R. M. (2000) Insect Biochem. Mol. Biol. 30, 617–644. 2. Kataoka, H., Toschi, A., Li, J. P., Carney, R. L., Schooley, D. A. & Kramer, S. J. (1989) Science 243, 1481–1483. 3. Woodhead, A. P., Stay, B., Seidel, S. L., Khan, M. A. & Tobe, S. S. (1989) Proc. Natl. Acad. Sci. USA 86, 5997–6001. 4. Pratt, G. E., Farnsworth, D. E., Fok, K. F., Siegel, N. R., McCormack, A. L., Shabanowitz, J., Hunt, D. F. & Feyereisen, R. (1991) Proc. Natl. Acad. Sci. USA 88, 2412–2416. 5. Donly, B. C., Ding, Q., Tobe, S. S. & Bendena, W. G. (1993) Proc. Natl. Acad. Sci. USA 90, 8807–8811. 6. Cusson, M., Prestwich, G. D., Stay, B. & Tobe, S. S. (1991) Biochem. Biophys. Res. Commun. 181, 736–742. 7. Thompson, C. S., Yagi, K. J., Chen, Z. F. & Tobe, S. S. (1990) J. Comp. Physiol. B 160, 241–249. 8. Granger, N. A., Sturgis, S. L., Ebersohl, R., Geng, C. & Sparks, T. C. (1996) Arch. Insect Biochem. Physiol. 32, 449–466. 9. Granger, N. A., Ebersohl, R. & Sparks, T. C. (2000) Insect Biochem. Mol. Biol. 30, 755–766. 10. Rachinsky, A., Zhang, J. & Tobe, S. S. (1994) Mol. Cell. Endocrinol. 105, 89–96. 11. Richard, D. S., Applebaum, S. W. & Gilbert, L. I. (1990) Mol. Cell. Endocrinol. 68, 153–161. 12. Kikukawa, S., Tobe, S. S., Solowiej, S., Rankin, S. M. & Stay, B. (1987) Insect Biochem. 17, 179–187. 13. Rachinsky, A. & Tobe, S. S. (1996) Arch. Insect Biochem. Physiol. 33, 259–282. 14. Monaghan, D. T., Bridges, R. J. & Cotman, C. W. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 365–402. 15. Gasic, G. P. & Hollmann, M. (1992) Annu. Rev. Physiol. 54, 507–536. 16. Littleton, J. T. & Ganetzky, B. (2000) Neuron 26, 35–43. 17. Mayer, M. L. & Westbrook, G. L. (1987) J. Physiol. (London) 394, 501– 527.
18. Schneggenburger, R., Zhou, Z., Konnerth, A. & Neher, E. (1993) Neuron 11, 133–143. 19. Choi, D. W. (1988) Neuron 1, 623–634. 20. Gustafsson, B. & Wigstrom, H. (1988) Trends Neurosci. 11, 156–162. 21. Kaczmarek, L., Kossut, M. & Skangiel-Kramska, J. (1997) Physiol. Rev. 77, 217–255. 22. Olney, J. W. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 47–71. 23. Meeker, R. B., Greenwood, R. S. & Hayward, J. N. (1994) Endocrinology 134, 621–629. 24. Villalobos, C., Nunez, L. & Garcia-Sancho, J. (1996) FASEB J. 10, 654–660. 25. Pszczolkowski, M. A., Lee, W. S., Liu, H. P. & Chiang, A. S. (1999) Mol. Cell. Endocrinol. 158, 163–171. 26. Ultsch, A., Schuster, C. M., Laube, B., Betz, H. & Schmitt, B. (1993) FEBS Lett. 324, 171–177. 27. The FlyBase Consortium (1996) Nucleic Acids Res. 24, 53–56. 28. Ultsch, A., Schuster, C. M., Laube, B., Schloss, P., Schmitt, B. & Betz, H. (1992) Proc. Natl. Acad. Sci. USA 89, 10484–10488. 29. Klebe, R. J., Grant, G. M., Grant, A. M., Garcia, M. A., Giambernardi, T. A. & Taylor, G. P. (1996) BioTechniques 21, 1094–1100. 30. Chiang, A. S., Liu, Y. C., Chiu S. L., Hu, S. H., Huang, C. Y. & Hsieh, C. H. (2001) J. Comp. Neurol. 440, 1–11. 31. Grynkiewicz, G., Poenie, M. & Tsien R. Y. (1985) J. Biol. Chem. 260, 3440–3450. 32. Giovannucci, D. R. & Stuenkel, E. L. (1995) Neuroendocrinology 62, 111–122. 33. Feyereisen, R. & Tobe, S. S. (1981) Anal. Biochem. 111, 372–375. 34. Chiang, A. S., Holbrook, G. L., Cheng, H. W. & Schal, C. (1998) Invertebr. Reprod. Dev. 33, 25–34. 35. Volkner, M., Lenz-Bohme, B., Betz, H. & Schmitt, B. (2000) J. Neurochem. 75, 1791–1799. 36. McBain, C. J. & Mayer, M. L. (1994) Physiol. Rev. 74, 723–760. 37. Stay, B. (2000) Insect Biochem. Mol. Biol. 30, 653–662.
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We thank R. Feyereisen for providing antibody to allatostatin, P. Iyu for providing conantokin T, and C. Schal for comments on the manuscript. This work was funded by grants from the National Science Council (Taiwan), the VTY Joint Research Program, and the Program for Promoting Academic Excellence of Universities.
Chiang et al.
