Expression and Function of Two Nicotinic Subunits in Insect Neurons A. Vermehren, B.A. Trimmer Department of Biology, Tufts University, Medford, Massachusetts 02155
Received 13 February 2004; accepted 14 June 2004
ABSTRACT:
Nicotinic acetylcholine receptors (nAChRs) in insects are neuron-specific oligomeric proteins essential for the central transmission of sensory information. Little is known about their subunit composition because it is difficult to express functional insect nAChRs in heterologous systems. As an alternative approach we have examined the native expression of two subunits in neurons of the nicotinic-resistant, tobaccofeeding insect Manduca sexta. Both the ␣-subunit MARA1 and the -subunit MARB can be detected by in situ hybridization in the majority of cultured neurons with an overlapping, but not identical, distribution.
INTRODUCTION Nicotinic acetylcholine receptors (nAChRs) are a major family of pentameric ligand-gated channels expressed primarily in neurons and muscles. nAChRs are expressed at very high concentrations in the insect nervous system and have been used as a model system to study the function of centrally released ACh (Sattelle, 1988; Trimmer and Qazi, 1999). In addition, because of the importance of these receptors as targets for insecticides, extensive research has been carried out comparing the structural basis of ligand binding in insect and vertebrate receptors (Debnath et al., 2003; Correspondence to: B.A. Trimmer (
[email protected]). Contract grant sponsor: National Institute of Neurological Disorders and Stroke. Contract grant sponsor: NIH; contract grant number: NS30566. Contract grant sponsor: NSF; contract grant number: IBN0077812 (B.A.T.). © 2004 Wiley Periodicals, Inc. Published online 27 October 2004 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/neu.20088
Changes in intracellular Ca2ⴙ evoked by nicotinic stimulation are more strongly correlated to the expression of MARA1 than MARB and are independent of cell size. Unlike the previously reported critical role of MARA1 in mediating nicotinic Ca2ⴙ responses, down-regulation of MARB by RNA interference (RNAi) did not reduce the number of responding neurons or the size of evoked responses, suggesting that additional subunits remain to be identified in Manduca. © 2004 Wiley Periodicals, Inc. J Neurobiol 62: 289 –298, 2005
Keywords: neuronal cultures; Ca2ⴙ imaging; in situ hybridization; RNAi
Tomizawa and Casida, 2003; Shimomura et al., 2003; Zhang et al., 2002). Electrophysiological and radioligand binding studies on insect neuronal nAChRs suggest a diversity of nAChRs (Eastham et al., 1998; Lind et al., 1988, 2001), which is supported by the cloning and sequencing of multiple genes coding for insect nicotinic subunits (Schafer, 2002). However, the subunit composition and stoichiometry of insect nAChRs are poorly understood (Tomizawa and Casida, 2001), partly because functional expression in Xenopus oocytes has been difficult (Lansdell and Millar, 2000; Lansdell et al., 1997; Schulz et al., 2000). In most cases where functional heterologous expression of insect nAChR subunits has been achieved (Drosophila D␣2, D␣3, D␣4), it has required the introduction of a vertebrate subunit (chick 2, rat 2) (Bertrand et al., 1994; Lansdell and Millar, 2000; Sawruk et al., 1990; Schulz et al., 1998). An exception is the expression of the desert locust Schistocerca gregaria ␣L1 subunit, which produced functional homoligomeric receptors (Marshall et al., 1990). The recent identification and characterization of an atypical 289
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Drosophila subunit (D3) (Lansdell and Millar, 2002) and evidence from immunoprecipitation experiments in Drosophila and the peach-tomato aphid Myzus persicae suggest that the failure to form functional insect receptors from cloned genes results from inappropriate protein folding or missing factors in the expression systems, rather than a fundamental difference in the coassembly of insect ␣ and  subunits (Huang et al., 2000; Schulz et al., 2000). Although heterologous expression is useful in establishing subunit assembly it is limited to systems in which all the contributing genes are well characterized. Another way to assess the role of a particular subunit is to down-regulate its expression in cells that naturally express the receptor. This has been achieved using recombinant transgenes (knockouts) in mice lacking the 2 subunit (Picciotto et al., 1995), the ␣7 subunit (Orr-Urtreger et al., 1997), the ␣4 subunit (Marubio et al., 1999), and other subunits, as well as through antisense ablation (␣3) in chicken (Listerud et al., 1991). Another highly effective method is RNA interference (RNAi), in which double-stranded RNA (dsRNA) is introduced into cells to down-regulate cognate mRNA (Bass, 2000; Sharp and Zamore, 2000). Because of its high efficiency this method is a potentially powerful tool for manipulating gene expression in developing and fully developed organisms, and it could avoid many of the problems associated with developmental compensation or inefficient down-regulation. The present study examined the expression and putative roles of two nAChR subunits, MARA1 (Manduca acetylcholine receptor alpha-like subunit 1) and MARB (Manduca acetylcholine receptor betalike) (Eastham et al., 1998), using in situ hybridization (ISH), ratiometric Ca2⫹ imaging, and RNAi in neurons of Manduca sexta. The abdominal ganglia contain easily identified sensory to motoneuron synapses, which makes larval Manduca a useful model system for investigating the mechanisms of cholinergic signaling (Qazi et al., 1996; Trimmer and Weeks, 1991, 1993). The expression of MARA1 and MARB was found to differ between neurons, and Ca2⫹ signals evoked by ACh were more strongly correlated with the expression of MARA1 than MARB. These results provide evidence for differential subunit composition in natively expressed insect nAChRs.
MATERIALS AND METHODS All reagents were bought from Sigma (St. Louis, MO) unless otherwise noted.
Experimental Animals Tobacco hornworm larvae (M. sexta) were raised as a synchronous colony from eggs in individual plastic cups on an artificial wheatgerm-based diet (modified from Bell and Joachim, 1978) and maintained at 27°C in a light/dark cycle of 17:7 h. In all experiments, both males and females in their first day fifth instar were used.
Neuronal Cultures Neuronal cells were grown on glass coverslips coated with a Concanavalin A (200 g/mL)/laminin (2 g/mL) solution for 2 h at 37°C, rinsed with sterile water, and air-dried. Six first day fifth instar larvae were anesthetized by chilling for 30 min on ice. The abdominal nerve cord was exposed by making an incision along the dorsal midline, and removing the gut and pinning the animal in an elastomer-lined dish (Sylgard; Dow Corning, Edison, NJ) containing cold insect saline [IS; modified from Miyazaki (1980)] and containing (mmol l⫺1): NaCl 140, KCl 5, CaCl2 4, glucose 28, HEPES 5, pH 7.4. Abdominal ganglia (A1–A8) from six animals were individually removed and placed into 0.22 M filtered IS chilled on ice. After being transferred to an eppendorf tube and treated with collagenase (0.5 mg/mL)/dispase II (2 mg/mL; Roche, Switzerland) solution for 8 min at 37°C, the cells were disrupted with a micropipette. The remaining procedures were performed under aseptic conditions as described in Hayashi and Levine (1992) with some modifications: cells were washed twice through 5 mL of modified L15 culture medium (Hayashi and Levine, 1992) and centrifuged for 3 min at 5000 rpm. Neurons were then resuspended in 200 L modified L15 medium and 50 L aliquots of cell suspension (containing ⬇50,000 cells) were used per coverslip. The coverslips had a microgrid design (CELLocate, 175 m; Eppendorf, Westbury, NY) to help find the same groups of cells before and after ISH. The cells were allowed to settle and to adhere to the substrate for 2 h at room temperature and then the culture dishes were filled with 2 mL of modified L15 medium and incubated overnight at 27°C.
Calcium Imaging and Analysis of the Results Twenty-four to thirty hour neuronal cultures were individually incubated in 1 mL modified L15 medium containing 3 L of the Ca2⫹-sensitive dye FURA PE3 AM (Teflabs, Austin, TX) stock solution (1 mM in 20% pluronic F-127; Molecular Probes, Eugene, OR) for 90 min at room temperature in the dark. The slides were then placed in a perfusion chamber mounted on the stage of an Axioscope microscope (Zeiss, Germany), and the cells were thoroughly rinsed in supplemented insect saline (SIS; 100 mL insect saline containing 40 mg fructose, 67 mg DL malic acid, 37 mg ␣-keto glutaric acid, 6 mg succinic acid, and 55 mg pyruvic acid) for 10 –15 min. Relative Ca2⫹ levels were monitored ratiometrically using 350 and 380 nm excitation
Heterogeneity of nAChRs in Manduca wavelengths at 1 Hz (ARC lamp supply LPS 220; Ionoptix Corp.). After establishing stable recordings, the cells were perfused (bath exchange time 2.5–3 min) with SIS containing the muscarinic antagonist scopolamine (10 M, to block ACh-sensitive muscarinic receptors) for 2 min, followed by 5 min SIS containing ACh (100 M) in the presence of scopolamine. All Ca2⫹ levels are reported as relative ratios and no attempts were made to estimate the absolute intracellular calcium concentration ([Ca2⫹]i). The effect of cell size on the magnitude of Ca2⫹ signals was examined by measuring visible cell area in digital images of neurons identified during ratiometric imaging. Because the absolute magnitude of Ca2⫹ signals varied between cultures the Ca2⫹ ratios for combined data were normalized to the largest response in each culture. The amplitude of the Ca2⫹ change in responding cells was calculated as the difference between the fluorescence ratio during the response (first 50 s) and the ratio in the same cell before drug application (50 s, baseline). The three cells with the largest Ca2⫹ response amplitudes were used to calculate the mean maximum Ca2⫹ signal. Neurons were scored as Ca2⫹ responding cells when their responses were greater than 10% of the maximum signals. Values lower than 10% were not reliably different from background variations in ratio. A contingency table (chi-square) test was used to determine if the effects of the different treatments on the proportion of cells responding were significant. Statistical comparisons used a one-way analysis of variance (ANOVA) with repeated measures to determine the effects of multiple treatments on the response amplitude. A Bonferroni posthoc test was used to conduct pairwise comparisons between different treatment groups. Probability levels ⬍ 0.05 were deemed significant. All analyses were performed using SYSTAT 8.0 (Wilkinson).
In Situ Hybridization A nonradioactive detection method using digoxigenin (DIG)-labeled riboprobes was used for the ISH. The MARA1 and MARB sense and antisense riboprobes (accession numbers Y09795 and AJ007397; base pairs 301– 640 and 600 –1000 bp, respectively) were generated in the presence of DIG-UTP (Roche) using a T3 and T7 RNA polymerase (MEGAscript kit; Ambion, Austin, TX), respectively. MARA1 and MARB probes were about 340 and 400 bp in length when viewed on a 1% agarose gel. For the ISH all incubations were carried out at room temperature unless otherwise noted, and all the water used for the solutions was treated with diethyl pyrocarbonate (DEPC) to inactivate RNAses. Briefly, cultured neurons were fixed in 4% paraformaldehyde in PBS (0.8% NaCl, 0.2% KCl, 0.14% Na2HPO4, 0.02% KH2PO4) for 20 min and rinsed twice in PBS for 5 min. The cells were incubated in 0.2 N HCl for 8 min to inactivate alkaline phosphatases (APs), then washed in 0.1 M PBS containing 0.1% Triton X-100 (PBST) for 5 min at 37°C, and washed twice in PBS. Treatments with 0.1 M triethanolamine (TEA; 2 min) and 0.25% acetic anhydrate in 0.1 M TEA (10 min) were fol-
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lowed by two washes in 2X SSC. Hybridization with the probes was performed for 12–16 h at 46°C in Perfect HybTM plus hybridization buffer. Posthybridization washes included two in 2X SSC for 20 min each, one in 1X SSC, 50% formamide (at 46°C) for 20 min, two in 0.2X SSC for 15 min each, and a final wash in PBST. After blocking with 2% goat serum in PBST, slides were incubated with antiDIG antibodies coupled to AP (1:2000; Roche) at 4°C overnight. More washes included three in PBST for 15 min each, two in PBS for 15 min each, and a final wash with AP buffer (100 mM Trizma base, 100 mM NaCl, 5 mM MgCl2, 6 H2O) for 15 min. The AP reaction product was developed in the presence of NBT/BCIP (Promega, Madison, WI) for 1–2 h. Cells were photographed using a Magnafire SP camera (Mod. S99810; Optronics) mounted on an Olympus BX40 microscope at 10X and 40X magnifications. Finally, cells were dehydrated through an ethanol series (30 –100%), cleared with methyl salicylate, and mounted in cytoseal (Richard-Allan Scientific, Kalamazoo, MI). The colored digital pictures taken of the stained cells were transformed using Adobe Photoshop software into gray scale tiff images. The images were analyzed using gray absolute luminance values, and we made sure that all the images were compared using the same scale. The mean background intensity was subtracted from all images and the cell pixel intensity was compared between cultures. The procedure was identical for all comparisons. A circular region of interest was drawn around each cell and the mean pixel intensity values and cell area were obtained using the NIH Image/Scion imaging program (pixel intensity scale 1–256). Neurons with a mean pixel intensity of less than 30 in background-matched images were defined as unstained. Anti- DIG antibodies coupled to rhodamine (Roche) and fluorescein (Vector Labs, Burlingame, CA) were used at a 1:5000 dilution in blocking solution for double labeling ISH and the cells were washed in PBS and mounted in Vectashield mounting medium (Vector Labs). Digital pictures were taken using fluorescent filters for rhodamine and fluorescein.
dsRNA Treatments The MARA1 and MARB fragments (accession numbers Y09795 and AJ007397; base pairs 301– 640 and 600 – 1000 bp, respectively) were cloned into Litmus 28i vector (HiScribe RNAi kit; New England Biolabs, Beverly, MA), and dsRNA was obtained using T7 RNA polymerase following the instructions of the manufacturer. After RNA synthesis, 4 U of DNAse I was added and incubated for 15 min at 37°C. The RNA was subsequently extracted by adding an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitating with 1/6 volume of 4 M lithium chloride and 2 volumes of 100% ice cold isopropanol at ⫺80°C for 30 min. Following centrifugation at 14000 rpm for 20 min at 4°C, RNA samples were resuspended in annealing buffer (1 mM Tris pH 7.5, 1 mM EDTA, 2% PEG). dsRNA was generated by heating the tubes for 10 min at 70°C then incubating them for 3 h at room temperature. Samples were analyzed on a 1.5% aga-
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rose gel and stored at ⫺80°C. For the dsRNA treatments, 10 L containing 2 g dsRNA was mixed with 40 L modified L15 medium and added to neurons (just after settling) for 4 –5 h before flooding the dish with 2 mL of L15. As a control 10 L annealing buffer was used instead of dsRNA. Finally, dishes were filled with 3 mL of modified L15 medium and incubated for 24 –28 h at 27°C. An unrelated dsRNA sequence (MSF2, muscarinic acetylcholine receptor fragment 2) was used to control for nonspecific effects of the dsRNA [as described in Vermehren et al. (2001)].
RESULTS MARA1 and MARB Subunits The 5⬘ untranslated region and part of the 5⬘ region of the MARA1 subunit (including two introns) were initially cloned by Eastham et al. (1998) using genomic DNA (gDNA). The rest of the sequence (translated region including the 3⬘ untranslated region) was obtained from pupal brain complementary cDNA. The MARB subunit was also partially sequenced by Eastham et al. using RT-PCR with cDNA from 3 day-old eggs. We have now isolated and sequenced a 525 bp fragment using MARA1 specific primers, 5⬘-ATGAAGTTTGGCTCGTGGAC-3⬘ (forward) and 5⬘-CGAAGTCGGTGGAATGATCT-3⬘ (reverse), and larval gDNA. This fragment contains two novel introns (76 and 86 bp; accession number AY371500) upstream of the coding region for transmembrane region I of the ␣ subunit [Fig. 1(A)]. In addition, a 900 bp fragment of MARB has been isolated from gDNA using MARB-specific primers, 5⬘-TGATTTCATTTCTCTGCGTT-3⬘ (forward) and 5⬘-ATCGAAGTCTCGTTTTCCTG-3⬘ (reverse). This sequence contains three novel introns distributed throughout the regions coding for intracellular loops of the non-␣ subunit (82, 139, and 80 bp long; accession number AY371501) [Fig. 1(B)].
Expression of MARA1 and MARB Transcripts in Cultured Neurons The expression of MARA1 and MARB transcripts in neuronal cultures was assessed by ISH using alkalinephosphatase-coupled probes that were highly specific for each subunit. MARA1 was detected in 64% (n ⫽ 103, four separate cultures) of the cultured neurons and MARB in 74% (n ⫽ 147, five separate cultures) of neurons in parallel cultures. The possible colocalization of these two subunits was examined using a double-antisense probe ISH procedure with rhodamine (MARA1) and fluorescein
Figure 1 The location of introns in MARA1 and MARB genomic sequences. The relative positions of introns (numbered thin lines), exons (thick lines), and transmembrane regions (open boxes) are shown in the gDNA sequence of MARA1 (A) and MARB (B). The amino acids at the apparent exon boundaries (* indicates the relative position of the introns) are shown for each of the corresponding introns in MARA1 (1– 4) and MARB (5–7). The intron boundaries of MARA1 are compared with those found in Drosophila D␣4. There are no introns reported in Drosophila genes that correspond to the MARB sequences. Numbers in parentheses indicate the size of the Manduca introns. Introns 1 and 2 (MARA1) were obtained by Eastham et al. (1998), and introns 3, 4 (MARA1), and 5–7 (MARB) have not previously been reported.
(MARB) labels in four separate neuronal cultures. Fluorescence levels detected using control sense probes were indistinguishable from cultures with no probes at all (using DIG antibodies) [Fig. 2(A)]. Large differences in the expression of these subunits were found in individual cells. Some neurons expressed relatively large amounts of MARA1 relative to MARB (18/50), some neurons expressed more MARB than MARA1 (11/50), and some neurons expressed an overlap in MARA1 and MARB subunit expression (11/50). About 10/50 neurons showed no staining for either MARA1 or MARB [Fig. 2(C)].
Calcium Entry through nAChRs After loading the cells with the Ca2⫹ indicator FURA PE 3 AM, approximately 40% (n ⫽ 192, nine separate cultures) of the neurons challenged with 100 M ACh in the presence of 10 M scopolamine showed a readily detectable increase in fluorescence. The typical Ca2⫹ response was a rapid increase (time to peak 16.08 ⫾ 7.26 s, n ⫽ 98) followed by a slow decline to the resting levels within 5 min after ACh wash out. The amplitude of the responses ranged from 0.2–10 ratio units with durations between 25 and 50 s. Preincubation with the nicotinic receptor antagonist
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Figure 2 The expression of MARA1 and MARB subunits in cultured neurons. (A) Control sense probes corresponding to conserved regions of MARA1 (rhodamine, top) and MARB (fluorescein, bottom). Probes were hybridized to fixed 48 h cultured Manduca neurons. (B) Antisense probes corresponding to conserved regions of MARA1 and MARB were labeled with rhodamine and fluorescein, respectively, and then hybridized to fixed 48 h cultured Manduca neurons. A representative field of view is shown using rhodamine filters (MARA1 subunit, top) and fluorescein filters (MARB subunit, bottom). (C) The relative intensity of fluorescence for the visible field is shown as a 3D surface plot for each set of conditions to illustrate the cell-specific differences in expression of MARA1 (top) and MARB (bottom). (D) Merged picture of MARA1 and MARB (B). White numbers indicate a cell expressing mainly MARA1 (1), a cell expressing mainly MARB (2), and a group of cells expressing both MARA1 and MARB subunits (3). Scale bar: 50 m.
mecamylamine (10 M) blocked the ACh-induced [Ca2⫹]i increase (data not shown).
Expression Patterns of MARA1 and MARB in Ca2ⴙ-Responding Neurons To identify how nicotinic Ca2⫹ responses correlated with the expression of each subunit neurons were cultured on coverslips with etched grids that provided microscopic landmarks. Cells were loaded with FURA PE3, their Ca2⫹ responses monitored, and their location noted before the cells were fixed and processed for ISH. A large percentage (64%) of cultured neurons expressing MARA1 (n ⫽ 66, four separate cultures) had detectable nicotinic Ca2⫹ responses but
Figure 6
Figure 6 Down-regulation of MARB subunit transcripts in neuronal cultures by RNAi does not significantly affect the proportion of cells responding to 100 M ACh (in 10 M scopolamine) or the magnitude of their Ca2⫹ response. (A) Neurons in culture expressing MARB (left) and MARB dsRNA-treated cells (right). (B) MARB down-regulation did not significantly affect the proportion of cells responding to ACh (2 ⫻ 2 contingency table; G ⫽ 0.56, p ⬎ 0.1). (C) Box plot showing that down-regulation of MARB transcripts did not significantly reduce Ca2⫹ responses to ACh [two-way ANOVA F(1, 1) ⫽ 2.236, p ⫽ 0.146]. Dots represent 5th and 95th percentiles.
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staining was examined as a function of the measured cell area. Most cultured neurons were small, 8 –16 m in diameter (50 –200 m2), with a few larger cells ranging between 30 –36 m (300 –1000 m2). Overall, there was no strong relationship between cell size and the size of the nicotinic Ca2⫹ responses (MARA1 r2 ⫽ 0.0112; MARB r2 ⫽ 0.0352; Fig. 3). Although most of the large cells strongly expressed MARA1 or MARB there was no strong relationship between the size of these neurons and the intensity of ISH staining (MARA1 r2 ⫽ 0.1087; MARB r2 ⫽ 0.0621; Fig. 4). Therefore, staining intensity is not a function of cell thickness and the amount of the subunit transcripts is not confined to a single class of neuron. There was no strong correlation between the magnitude of the nicotinic responses and the intensity of staining for MARA1 (r2 ⫽ 0.1500; n ⫽ 103) or MARB (r2 ⫽ 0.1662; n ⫽ 147), but there were differences in the distribution of the responses
Figure 3 The amplitude of nicotinic Ca2⫹ responses is not a function of neuron size (area, m2). The size of neurons was estimated by measuring the area of each neuron in the same field of view as that used for Ca2⫹ imaging. Each point represents the Ca2⫹ response of an individual neuron normalized to the maximum fluorescence ratio change in each culture (see Materials and Methods). A dashed line separates responding cells (region II) from nonresponding cells (region I). No significant correlations between Ca2⫹ response and cell size were observed (r2 ⫽ 0.0112; n ⫽ 103 for MARA1 and r2 ⫽ 0.0352; n ⫽ 147 for MARB).
only 23% of MARB-positive neurons responded similarly (n ⫽ 109, five separate cultures). Because staining intensity and the detection of Ca2⫹ responses could vary with cell size (large cells might appear more densely stained in transmission microscopy, epifluorescence intensity could be greater), the distribution of Ca2⫹ responses and ISH
Figure 4 The intensity of ISH staining is not a function of neuron size (area, m2). The intensity of ISH staining using the MARA1 antisense probe (A) or the MARB antisense probe (B) was measured in background-matched images of neurons and plotted as a function of neuron area. Each point represents the staining of an individual neuron. Dashed lines separate neurons that express each subunit (region II) from cells that do not express the subunits (region I). No significant correlations between the intensity of ISH (expression of transcripts) and cell size were observed (r2 ⫽ 0.1087; n ⫽ 103 for MARA1 and r2 ⫽ 0.0621; n ⫽ 147 for MARB).
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DISCUSSION Diversity of Insect nAChRs
Figure 5 Most MARB-expressing neurons do not generate nicotinic Ca2⫹ responses. Nicotinic Ca2⫹ responses are grouped according to their expression (stained, unstained) of MARA1 (gray bars) or MARB (black bars). The percentage of neurons with or without Ca2⫹ responses (response, no response) is shown on the x axis. Most neurons expressing MARA1 (51/66) generated nicotinic Ca2⫹ responses but most neurons expressing MARB (26/109) did not respond. A small subset of unstained neurons produced nicotinic Ca2⫹ responses (6/37 for MARA1 and 3/38 for MARB).
(Fig. 5). Approximately 77% of the MARA1-expressing neurons (n ⫽ 66) produced Ca2⫹ responses, while 23% of the strongly stained MARB-expressing neurons (n ⫽ 109) did not have detectable Ca2⫹ responses. In addition, a very small subset of unstained neurons produced nicotinic Ca2⫹ responses (16% of the non-MARA1 expressing cells, n ⫽ 37, and 18% of the non-MARB expressing neurons, n ⫽ 38).
Role of MARA1 and MARB nAChR Subunits As reported previously, treatment of cultured neurons with dsRNA decreases the expression of MARA1 and significantly reduces both the proportion of AChresponding neurons and the size of the nicotinic Ca2⫹ signals (Vermehren et al., 2001). A similar treatment of cultured neurons with dsRNA corresponding to MARB blocked the expression of MARB measured by ISH [Fig. 6(A)]. Treatment with dsRNA corresponding to the muscarinic fragment MSF2 had no effect on Ca2⫹ responses to ACh under these conditions (Vermehren et al., 2001). The down-regulation of the MARB transcript did not affect the general shape or size of the cultured neurons, the proportion of ACh-responding neurons [Fig. 6(B)], or the size of the Ca2⫹ signals in normal [Fig. 6(C)] or low sodium saline (not shown).
The relatively small number of neurons and stereotypical organization of the insect CNS provides an opportunity to understand the functional role of receptor diversity. Although the number of genes coding for insect nAChR subunits is slightly smaller than for vertebrates (there are 10 identified in Drosophila), additional receptor diversity can be achieved through alternate splicing, A-to-I pre-mRNA editing (Sattelle et al., 2002), and possibly by post-translational modification. Only two Manduca subunits have been identified to date, but the results presented here suggest that they might contribute to a larger number of products. The positions of the four introns identified in MARA1 (Fig. 1) are conserved in the Drosophila D␣4 gene and correspond to separate exons 1/2, 2/3, 4/5, and 5/6 (Lansdell and Millar, 2000). It has been shown that alternate splice variants of D␣4 are produced in Drosophila, containing alternate exon 4 versions, or lacking exons 2 or 4. Alternate splicing and pre-mRNA editing is also found in Drosophila gene D␣6, which has editing sites conserved in the ␣7-2 nicotinic subunit of the Lepidoptera tomato budworm, Heliothis virescens (Grauso et al., 2002). It is therefore expected that MARA1 is similarly processed to produce more ␣ subunits. With regard to the non-␣ nicotinic subunits, it has been shown that both large immature mRNA and smaller transcripts of the Drosophila ARD gene are expressed in the CNS (Hermans-Borgmeyer et al., 1989). The presence of the three introns identified in MARB (Fig. 1) suggests that alternate transcripts could be produced in Manduca, but this possibility has not been further explored.
Expression of MARA1 and MARB Our experiments show that approximately threefourths of Manduca neurons in culture express either MARA1 or MARB. However, this number might be an underestimate of the expression of nicotinic receptors because the distributions of MARA1 and MARB do not always overlap, and additional subunits probably remain to be identified. Because of its molecular similarities to human ␣7 and locust L␣1 subunits (Amar et al., 1995), it is possible that the MARA1 subunit is capable of forming functional homoligomeric receptors that are highly Ca2⫹ permeable (Delbono et al., 1997). This possibility is consistent with the results of the double ISH labeling experiments in which cells were detected expressing MARA1 but not
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MARB (Fig. 2), and with the prevalence of MARA1 in neurons with nicotinic Ca2⫹ responses (Fig. 5). Previous results show that small nicotinic Ca2⫹ responses can be evoked in isolated Manduca neurons even when cholinergic voltage changes are prevented by maintaining neurons in a saline containing very little Na⫹ (Vermehren et al., 2001; Zayas et al., 2002). Hence, although voltage-gated Ca2⫹ entry contributes to the normal nicotinic responses some Ca2⫹ enters directly through the nAChRs. Another interesting result is that some populations of neurons express MARB but not MARA1. If these subunits form functional receptors they must do so by combining with another non-MARA1 ␣ subunits. The possibility of additional Manduca ␣ subunits is supported by Eastham and colleagues, who showed, using Southern blots, that a probe based on the locust ␣L1 detected an unknown nicotinic-like band (Eastham et al., 1998). The results reported here are from neurons that were maintained in culture for at least 24 h. Although many cells behave differently in culture, isolated Manduca neurons maintain many of their normal morphological, developmental, and receptor-response properties (Hoffman and Weeks, 1998; Levine and Weeks, 1996; Melville et al., 2003; Streichert et al., 1997; Streichert and Weeks, 1994). It is our eventual goal to confirm the diversity of Manduca nAChRs by examining their expression in identified neurons both in culture and in the living ganglion.
Functional Analysis of nAChRs by RNAi The study of insect nAChRs has been hampered by the lack of subunit specific ligands and because attempts to make functional receptors in heterologous expression systems have largely been unsuccessful. Hence, despite the potential advantages of studying receptor heterogeneity in identified neurons, relatively little is known about the functions of different subunits in insect nAChRs. Subtype-specific antibodies have been developed for Drosophila receptors and used to examine location and co-assembly of ALS, D-␣3, and ARD proteins in the CNS (Chamaon et al., 2000; Schloss et al., 1991; Schuster et al., 1993). In addition, a polyclonal antibody recognizes Locust nAChRs (Leitch et al., 1993), but these immunoreagents have not been used in functional assays and their specificity in other insects has not been tested. Our own attempts to examine MARA1 protein expression in Manduca neurons using commercial antibodies (anti- rat ␣7; Santa Cruz, CA) were uninformative. In Western blots the antibodies detected an ␣ subunit from rat brain extracts but nothing in
Manduca CNS extracts and there was no specific staining of fixed Manduca neuronal cultures (data not shown). Presumably, the rat ␣7 epitope is not present or accessible in Manduca receptors. As an alternative approach, we have tested the ability of cognate dsRNA to disrupt the expression and function of native nAChRs in Manduca neurons in culture. This method is particularly useful for subunits that contribute to easily monitored responses such as Ca2⫹ gating (e.g., MARA1), but it can also provide useful information about structural subunits such as MARB whose activity requires the presence of the ACh-binding ␣ subunits. The results presented here and in a previous report (Vermehren et al., 2001) are the first to explore the roles of insect nAChR subunit genes using RNAi. A major finding is that some Manduca nAChRs couple directly to Ca2⫹ entry (the nicotinic channel is probably permeable to Ca2⫹), and that MARA1 is an important subunit of these receptors. Hence, most neurons that express high levels of MARA1 also have strong ACh-evoked Ca2⫹ responses. In contrast, most neurons with high levels of MARB do not exhibit Ca2⫹ responses. Furthermore, there is no change in the proportion of neurons producing Ca2⫹ responses, or the average size of these responses, when MARB expression is strongly suppressed by RNAi. Therefore, MARB is not essential for the majority of AChevoked Ca2⫹ responses. Presumably, MARB is primarily associated with low Ca2⫹ permeability nAChRs and MARA1 forms homomeric receptors or heteromeric receptors with unknown subunits. The functional characterization of these different nAChRs is currently being investigated through down-regulation of MARA1 and MARB in identified neurons in culture and in the intact larval nervous system. A key question these experiments plan to address is how different nAChR subtypes are functionally specialized for the transmission of sensory information into the CNS. We thank Dr. Adrian Wolstenholme (University of Bath, England) for providing the MARA1 nAChR subunit clone and the MARB nAChR subunit partial sequence, and Dr. Sara Lewis (Tufts University) for help with the statistics.
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