Acetylcholine receptors of thoracic dorsal midline neurones in the cockroach Periplaneta americana.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 21 :289-301 (1992) Acetylcholine Receptors of Thoracic Dorsal Midline Neurones in the Cockroach, Periplaneta americana Donglin Bai, Heinrich Erdbrugger, Heinz Breer, and David B. Sattelle AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Cambridge, United Kingdom (0.B., D. B.S.); lnstitute of Zoophysiology, University of Stuttgart-Hohenheim, Stuttgart, Germany (H.E., H . B . ) The actions of acetylcholine and cholinergic ligands have been studied using dorsal midline neuron= from the rnetathoracic ganglion of the cockroachPeriplaneta americana. Both nicotine and oxotremorine depolarized dorsal midline neuronal cell bodies. Doseresponse curves for nicotine and oxotremorine saturated at different levels. Nicotine-induced depolarizations were completely or partially blocked by mecamylamine, d-tubocurarine, strychnine, and bicuculline, but were insensitive to a-bungarotoxin (100 nM), atropine (100 fl), scopolamine (10 pM), and pirenzepine (50 pM). Following pretreatment with collagenase, the dorsal midline neurones were sensitive to high doses of a-bungarotoxin (3 @). Oxotremorine-induced depolarizations were blocked by scopolamine (1 0 fl), atropine (100 pM), and pirenzepine (50 $4) and were insensitive to mecamylamine (10 pM) and d-tubocurarine(100 pM). The results indicate the coexistence of at least two distinct acetylcholine receptors on dorsal midline neuronal cell bodies in the cockroach metathoracic ganglion. o 1992 WiIey-Liss, Inc. Key words: a-bungarotoxin, nicotinic acetylcholine receptor, muscarinic acetylcholine receptor, mecamylamine, pirenzepine INTRODUCTION Insect nAChRs", in contrast to vertebrates and some invertebrates, appear to be confined to the nervous system and are present at relatively high Acknowledgments: The authors are indebted to Drs. Colin A. Leech and Nicola M. Anthony for helpful advice and discussion during the course of this work. Received March 23, 1992; accepted July 21, 1992. Address reprint requests to David B. Sattelle, AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U.K. *Abbreviations used: ACh = acetylcholine; CNS = central nervous system; Q = fast coxal depressor motor neurone; DM = dorsal midline neurone; DUM = dorsal unpaired median neurone; DUMETi = dorsal unpaired median neurone innervating the extensor tibiae muscle; EC50 = concentration required to produce 50% of maximum response; lCS0 = concentration requiredto inhibit response by 50%; mAChR = muscarinic acetylcholine receptor; nAChR = nicotinic acetylcholine receptor; R = resistance. 0 1992 Wiley-Liss, Inc. 290 Bai et al. concentrations [l].Many insect nicotinic receptors are sensitive to a-bungarotoxin . For example, this snake toxin blocks functional nicotinic receptors on identifiable interneurones [3-51 and motor neurones in the cockroach Peripluneta americana [4,6,7]. However, dorsal unpaired median neurone (DUMETi) of the locust Schistocerca nitens and unidentified DUM neurones in the third thoracic ganglion of cockroach respond to nicotine, but are less sensitive to a-bungarotoxin [8,9], Also, a depolarizing response to nicotine that is insensitive to a-bungarotoxin is present in the larval nervous system of Drosophila melanogaster [ 101. Insect muscarinic receptors, though less abundant overall, are also widely distributed in the nervous system of the locust Locusta migratoria [ll],cockroach [12-141, and Drosophila [10,15,16]. Dorsal unpaired median neurones have been characterized in several insect orders, such as cockroach , firefly , moth , and cricket . Located on the dorsal midline of the ganglion, they send axons symmetrically to both sides of peripheral nerve roots of the ganglion. The DUM neurones in the metathoracic ganglion of the cockroach have been classified into 6 different types on the basis of the branching of their axons 1171. A differential sensitivity to a-bungarotoxin between the identifiable fast coxal depressor motor neurone Df and dorsal midline neurones of the cockroach metathoracic ganglion has been detected . Recently, using dissociated, cultured, dorsal midline neurones from the cockroach terminal abdominal ganglion, Lapied et al.  detected more than one type of cholinergic receptor, including one that is a-bungarotoxin-insensitive. Although a detailed pharmacological profile has been determined for the nicotinic type of receptors on motor neurone Df in situ , little is known of the cholinergic pharmacology of in situ dorsal midline neurones. Neuronal nicotinic receptors that exhibit differences in sensitivity to a-bungarotoxin and K-bungarotoxin are present in the nervous systems of vertebrates [22,23], but less is known of the diversity of nicotinic receptors in insects. Recently, cloning and sequencing of putative insect nicotinic receptor subunits have revealed both d i k e and non-a-like polypeptides 126273. Subtypes of muscarinic receptors, which share some pharmacological properties with the Mi and M2 receptors of vertebrates, have been detected in the insect nervous system [ZS]. Also, two putative muscarinic receptors have been cloned and sequenced from Drosophila 129,301. In the present study, pharmacological data suggest the coexistence of nicotinic receptors and pirenzepinesensitive muscarinic receptors on cell bodies of thoracic dorsal midline neurones of the cockroach (Periplanefa a7sericana). MATERIALS AND METHODS Electrophysiology Experiments were performed at room temperature (21-24°C) on adult male cockroaches. The methathoracic ganglion was removed from the animal, desheathed, and then transferred to a Perspex experimental chamber, perfused continuously with cockroach saline at a rate of 1ml/min. Drug applica- nAChRs of Dorsal Midline Neurones 291 tion was via this bath perfusion system, either using a pipette to inject 100 pl agonist directly into the chamber, or using a syringe pump (Raze1A-99; Raze11 Scientific Instruments, Inc., Stanford, CT) to inject agonists into the experimental chamber. Cells were located using a binocular microscope. Intracellular recordings were made from neuronal cell bodies located in the dorsal midline region of the ganglion, using glass microelectrodes (R = 20-50M a) filled with 2 M potassium acetate solution and connected to a high-impedance DC amplifier. Cockroach saline consisted of 214 mM NaC1,3.1 mM KCl, 9 mM CaClz, 50 mM sucrose, 10 mM TES (adjusted to pH 7.2 with NaOH). The DM cells investigated are a group of four large-diameter DUM neurones. Chemicals Nicotine, strychnine hydrochloride, d-tubocurarine chloride, mecamylamine, oxotremorine, scopolamine, atropine sulfate, collagenase type V, and hyaluronidase type I-S were obtained from Sigma Chemical Co. (St. Louis, MO). The purified snake toxins a-bungarotoxin and K-bungarotoxin were obtained from Biotoxins (St. Cloud, FL). Pirenzepine was obtained from Dr. Karl Thomae GmbH, Biberach a.d. Riss, Germany. Bicuculline methiodide was purchased from Cambridge Research Biochemicals (Cambridge UK). RESULTS Depolarization of dorsal midline cells was detected following exposure to certain nicotinic and muscarinic cholinergic agonists. Nicotine, a potent nicotinic acetylcholine receptor agonist, depolarized the cell body membrane of dorsal midline neurones at concentrations above 10 nM. Peak depolarization (about 30 mV) was observed at 10 p M nicotine, and was accompanied by an increase in membrane conductance. The EC50 value for nicotine on dorsal midline neurones (0.2 pM) reveals that these cell bodies are slightly more sensitive to this agonist than the cell body membrane of motor neurone Df (EC50 = 3 pM) in the same ganglion . A typical depolarization response induced by syringe pump application of nicotine is shown in Figure 1. Oxotremorine, a muscarinic agonist, also depolarized cell bodies of dorsal midline neurones. Compared to nicotine-induced responses, the oxotremorine-induced depolarizations displayed a slower onset and longer duration 2 min Fig. 1. Depolarization of a dorsal midline neuronal cell body by bath-applied nicotine (10 pM, arrowhead indicates onset) and oxotremorine (100 pM, filled circle indicates onset). The voltage responses were recorded intracellularly from the same cell. Agonists were injected via a syringe pump (10 s application) into the continuously flowing saline. Resting membrane potential was -64 rnV. 292 I -0 F 30 - 20 - I I Nicotine 0-0 4Q - I I - Carbarnylcholine I oxotremorine A-A E Y .-e0 1 m .- N -m0 L n al a - IQ 0 - -8 -6 -7 Loglo ILigandl -5 -4 -3 (MI Fig. 2. Dose-response curves for depolarizing actions of bath-applied cholinergic agonists on dorsal midline neurones. The mean peak amplitude of depolarization i s recorded for various concentrations of bath-applied nicotine (0)carbarnylcholine oxotrernorine (A). Vertical bars represent twice t h e standard error of the mean (n = 3). (m), (Fig. 1).Dose-response curves for the depolarizing actions of bath-perfused nicotine, oxotremorine, and carbamylcholine were constructed (Fig. 2). Depolarizing responses to nicotine and carbamylcholine saturated at a higher level than responses to oxotremorine. All were more effective than acetylcholine. The presence of the anticholinesterase neostigmine (1 pM) enhanced sensitivity to acetylcholine.This was further increased by a 5 min pretreatment of the tissue with a mixture of the enzymes collagenase (0.5 mg/ml) and hyaluronidase (0.5 mglml) (Fig. 3). Thus, the low sensitivity of dorsal midline cells to exogenous acetylcholine appears to be due to enzymatic breakdown of the neurotransmitter, and physical barriers. Actions of nAChR Antagonists Cholinergic antagonists were used to test for the existence of subpopulations of cholinergic receptors on dorsal midline neurones. Mecamylamine and d-tubocurarine. Mecamylamine is a potent blocker of vertebrate ganglionic nicotinic receptors  and nicotinic receptors of the cockroach motor neurone Df . When bath-applied at a concentration of 10 pM, mecamylamine was normally ineffective (n = 6 ) on dorsal midline neurones, but following an application at 100 pM it blocked 90% the response to nicotine (n = 8; Fig. 4a). Thus responses to nicotine on dorsal midline neuronal cell bodies were less sensitive to mecamylamine than those of the cell body of motor neurone Db for which an IC50 of 2.5 pM was estimated . Mecamylamine (10 pM n = 4;100 pM, n = 6 )failed to block oxotremorine-in- nAChRs of Dorsal Midline Neurones ACh ACh+Neostigmine ACh + NeoQtgmine + E I T Z ~ ? S * 0-0 40 - *-* &-A 30 - t- 20 - 8 10- 293 Y z 0 3 w 0 0 0 - I I I 1 I -7 -6 -5 -4 -3 Fig. 3. Dose-response curves for depolarizing actions of bath-applied acetylcholine on dorsal midline neurones. The desheathed preparations are bathed in saline (O), saline containing 1 fl neostigmine (O), or saline containing neostigmine after pretreatment of the preparation with a mixture of collagenase (0.5 mg/ml) and hyaluronidase (0.5 mg/ml) for about 5 min (A).In the presence of the enzymes and the anticholinesterase neostigrnine, dorsal midline cells respond to lower concentrations of acetylcholine. +Mecamylamine (1.0~10-4~) 15 mln (b) Control +Mecamylamlne 11.0~104~1 21 mln Fig. 4. Actions of bath-applied mecamylamine on nicotine-induced (1 0 KM nicotine, arrowheads indicate onset) and oxotremorine-induced (1 00 pM oxotremorine, filled circles indicate onset) depolarizations of dorsal midline neurones. Mecamylamine (1 00 p,M) totally blocked the nicotineinduced depolarizations (a) and was without effect on oxotremorine-induced depolarizations (b). Resting membrane potential was -85 mV. 294 Bai eta!. duced depolarization of dorsal midline neurones (Fig. 4b). Another nAChR antagonist, d-tubocurarine (100 kM, n = 4), was found to block the depolarizing response to nicotine, but failed to block oxotremorine-induced responses of the same neurones. a-Bungarotoxin and K-bungarotoxin. a-Bungarotoxin, a blocker of vertebrate muscle nicotinic acetylcholine receptors, is effective on insect nicotinic receptors of motor neurone Df . K-Bungarotoxin ( = neuronal bungarotoxin) is an antagonist of many vertebrate neuronal nicotinic acetylcholine receptors. This toxin also blocks the nicotinic receptors of motor neurone Df . On dorsal midline neurones, a-bungarotoxin (0.1 pM) and K-bungarotoxin (0.1 pM) failed to block the depolarizing actions of nicotine (n = 3 for each toxin; Fig. 5 ) . A higher concentration of a-bungarotoxin (0.5 pM, n = 1) also failed to block nicotine-induced depolarization even after a 107 min exposure. However, following pretreatment with collagenase (0.5 mg/ml) and hyaluronidase (0.5 mg/ml), a higher dose of a-bungarotoxin (3 pM) did block responses to nicotine. So the dorsal midline cells are much less sensitive to a-bungarotoxin than Df, but are not completely insensitive. Actions of "Non-nAChR" Antagonists Bicuculline. Bicuculline, a specific antagonist for vertebrate GABAA receptors , was found to be an antagonist at acetylcholine receptors on cell body membranes of dissociated locust neurones , and cockroach motor neurone Df in situ (S.D. Buckingham and D.B. Sattelle, unpublished observations), On cockroach dorsal midline neurones, a high dose of bicuculline (100 p M ) partially and reversibly blocked nicotinic responses (Fig. 6), while a lower concentration of bicuculline was without effect. Strychnine. Low concentrations of a vertebrate glycine receptor antagonist , strychnine (10 pM), failed to block nicotine-induced depolarizations on dorsal midline neurones (n = 5). However, following perfusion with 100 Control +a-Bungarotoxin 1.ox10 - 7 ~ 127 mln Control +K-Bungarotoxin 1.ox10 - 7 ~ 145 min I Fig. 5. Effects of bath-applied a-bungarotoxin and K-bungarotoxin on nicotine-induced responses of dorsal midline neurones in desheathed metathoracic ganglia. Perfusion with saline containing a-bungarotoxin (0.1 FM) and K-bungarotoxin (0.1 IJM) is without effect on the nicotine-induced responses (arrowheads indicate the onset of 10 pM nicotine). Resting membrane potentials were about -75 mV for both experiments. nAChRs of Dorsal Midline Neurones Control +Bicuculline Wash (1.0~10-4~) 26 min 26 min A Control A A +Strychnine 1.ox10 - 4 ~ 18 min 295 JL A Wash 19 min 10 mln Fig. 6. The effects of bicuculline and strychnine on the nicotine (1 0 pM)-induceddepolarizations on the dorsal midline neurones. Bicuculline (100 pM) partially and reversibly blocks the nicotine-induced depolarization. Strychnine (1 00 p.M) reversibly blocks the response to nicotine. Resting membrane potentials were -70 mV (upper traces) and -72 rnV (lower traces). Arrowheads indicate onset of nicotine application. pM strychnine the response to nicotine was almost totally blocked. Nicotineinduced responses recovered from strychnine block after rebathing with normal saline (n = 4; Fig. 6). Actions of mAChR Antagonists Scopolamine and atropine. The muscarinic antagonist scopolamine (10 pM) blocked the depolarizing actions of oxotremorine (100 pM, n = 6), but failed to modify the response to 10 pM nicotine (n = 6; see Fig. 7). Following block by scopolamine, the response to oxotremorine sometimes recovered on rebathing the preparation in normal saline, but in other preparations recovery was incomplete even after a prolonged (2 h) wash. Thus, scopolamine blocked only the slower, oxotremorine-induced depolarization. Another muscarinic antagonist atropine (1 rnM, n = 1; 100 pM, n = 5) reversibly blocked oxotremorine-induced depolarization. Atropine (100 pM) failed to block the response of dorsal midline neurones to nicotine, in contrast with the findings for motor neurone Dr . Pirenzepine. Pirenzepine is a specific blocker for the Mi subtype of muscarinic acetylcholine receptors . Bath-applied pirenzepine (50 yM) blocked the response of dorsal midline neurones to oxotremorine, but not to nicotine. Rebathing with normal saline resulted in partial recovery of the response (Fig. 8). Thus, pirenzepine-sensitive mAChRs are present on dorsal midline neuronal cell bodies. 296 Bai et at. Control +Scopolamine (1.0~10-5~) 14 min +Scopolamine (1.0~105~) 17 min Control A Fig. 7 . Actions of scopolamine on the oxotremorine (100 pM)-induced and nicotine (10 pM)-induced depolarizations of a dorsal midline neurone are illustrated. The response to oxotremorine application (filled circles) i s totally blocked by scopolamine (10 pM), while the response of the same cell to nicotine (arrowheads) i s unaffected. Resting membrane potential was -80 rnV. DISCUSSION Acetylcholine receptors on dorsal midline neuronal cell bodies in the cockroach have been described. Electrophysiological studies have shown that both nicotine and oxotremorine depolarize these cells. The response to nicotine rapidly follows the onset of bath-application of the ligand and sometimes induces a short burst of action potentials. In contrast, the response to oxotremorine develops more slowly, resulting in a prolonged depolarization. In vertebrates there are two quite distinct forms of cholinergic receptor which serve quite distinct functions . Fast-acting nicotinic receptors of muscle and CNS [38,39] are normally associated with an increase in cation (mainly Na', K', and Ca+ +) conductance, though recently a nicotinic response coupled to a K+-selective channel has been detected . By contrast, a modulatory role is often attributed to vertebrate muscarinic recepControl +Pirenz ine 50x10&P M 15 min Wash 14 min i0mV 0 0 1 0 Fig. 8 . Pirenzepine, an MI subtype-specific muscarinic antagonist (50 pM), blocked oxotrernorine (1 00 pM, filled circles)-induced depolarizations. After rebathing the preparation in normal saline, the oxotremorine-induced depolarizations partially recovered. Resting membrane potential was -80 mV. nAChRs of Dorsal Midline Neurones 297 tors which can control the rate and amount of information transfer in selected pathways in the nervous system. Muscarinic actions of acetylcholine may involve a variety of conductance mechanisms and involve a number of different receptor subtypes , including activation or depression of certain (particularly K + ) cation conductances. One interpretation of our results is that a cholinergic receptor with "mixed" (i.e., muscarinichicotinic) pharmacological properties is present on dorsal midline neurones. Binding studies have indicated that putative receptors with a mixed pharmacology may be present in insect nervous tissue . Adopting this model to account for all the findings for the dorsal midline cells, it is difficult to reconcile the scopolamine, atropine, and pirenzepine block of the slower oxotremorine-induced depolarization with the absence of any action of these antagonists on nicotine-induced depolarization. Also, the nicotinic antagonists mecamylamine and d-tubocurarine blocked nicotine-induced depolarization, but did not impair the depolarization in response to oxotremorine recorded from the same cell. Thus, the cholinergic pharmacology of dorsal midline cells strongly indicates the coexistence of distinct muscarinic and nicotinic receptor types on the same neurone. Other observations support this view, namely the striking differencesin the rise-time and duration of the depolarizing actions of nicotine and oxotremorine. Also the dose-response curves for nicotine and oxotremorine saturated at different levels. Three pharmacologically distinct acetylcholine receptors have been detected on identified neurones in the pleural ganglion of Aplysia californica .They are distributed in different combinations on functionallydistinct neurones. Benson has shown the coexistence of two distinct cholinergic receptors on unidentified dissociated locust neurones corresponding to nicotinic and muscarinic subtypes. Recently, dual muscarinic and nicotinic actions have been seen on Drosophila neurones [lo], though it remains to be determined whether or not they are present on the same cell. The responses to nicotine of thoracic dorsal midline cells differ from those of motor neurone Df (Table 1).The nicotinic receptors of dorsal midline cells appear less sensitive to nicotinic antagonists, such as mecamylamine and d-tubocurarine, and in particular a-bungarotoxin. In view of the effects of enzyme treatment on the acetylcholine dose-response curve, poor accessibility of chemicals onto the cell surface of dorsal midline neurones may account for this reduced sensitivity, though intrinsic differencesin receptor pharmacology cannot be ruled out . Also, dorsal midline neurones in situ [present study] appear less sensitive to cholinergic ligands than isolated cells , which may be due to improved drug access to the neuronal membrane in dissociated, enzyme-treated cells. Pharmacologically distinct subtypes of muscarinic receptors, termed MI and M2, have been established in both vertebrates  and in insects [ll]. Pirenzepine, a novel muscarinic antagonist, binds with high affinity to the MI type receptor, and can be employed to distinguish between the two receptor types. In insects, muscarinic binding sites in synaptic membranes display a low affinity for pirenzepine, indicating that M2 receptors predominate in nerve terminals. Conversely, pirenzepine-sensitive receptor subtypes are 298 Bai et at. TABLE 1. Pharmacology of Nicotinic Receptors of Functionally Distinct Cockroach Neurones Studied In Situ* Cell type Nicotine Carbamylcholine a-Bungarotoxin K-BUngarOtOXin Dihydro-P-erythroidine d-Tubocurarine Mecamylamine Bicuculline Strychnine Atrovine Motor Motor Interneurone Dorsal ,.,idline + + -1 -I + + t Block Block Block Block Block Block Block Block Block n.t. n.t. Block n.t. n.t. n.t. Block Block n.t. n.t. Block n.t. Blocka n.t. n.t. f -a -a n.t. Block Block Block Block Sensorv + + n.t. n.t. n.t. n.t. Block n.t. n.t. *Abbreviations: + = agonist; - = ineffective blocker at concentrations up to 100 $4; -a = ineffective blocker at concentrations up to 100 nM; DM = dorsal midline neurone; D, = slow extensor motor neurone; GI2 = giant interneurone 2; LFHSN = lateral filiform hair sensory neurone; n.t. = not tested. All other abbreviations are as in the text. "S.D. Buckingham and D.B. Sattelle, unpublished observations. more abundant on the cell bodies [ll].In a brief report, Benson  has confirmed the existence of Mi-like receptors on isolated, unidentified locust neuronal somata, which are blocked by QNB, scopolamine, and pirenze ine The EC5o for pirenzepine in these experiments was 7.1 ? 4.5 x 10- M, whereas rnethoctramine, an M2 type mAChR blocker, was without effect. Recent binding studies on mAChRs from 3 insect species, including cockroach, have shown high affinity for 4-DAMP, an M3-selective antagonist . Furthermore, there is a preferential coupling of vertebrate MI and M2 receptors to the regulation of phosphoinositide hydrolysis and adenylate cyclase systems respectively . In insects there is evidence that M2 type receptor may be involved in autoregulation of ACh release and they may be negatively linked to adenylate cyclase [113. It will be of interest to extend these studies, using a uniquely identifiable dorsal midline neurone (DUM5rl) recently described in the cockroach metathoracic ganglion . Further experiments are needed to characterize the different nicotinic and muscarinic receptors and their associated signal transduction mechanisms in the nervous systems of insects, but here we provide evidence for the coexistence of representatives of each class on dorsal unpaired median neurones of the cockroach metathoracic ganglion. Y . LITERATURE CITED 1. Sattelle DB: Acetylcholine receptors of insects. Adv Insect Physiol15, 215 (1980). 2. Breer H, Sattelle DB: Molecular properties and functions of insect acetylcholine receptors. J Insect Physiol33, 771 (1987). nAChRs of Dorsal Midline Neurones 299 3. Harrow ID, Hue B, Gepner JI, Hall LM, Sattelle DB: An a-bungarotoxin-sensitive acetylcholine receptor in the CNS of the cockroach, Periplaneta americana (L.). In: Insect Neurobiology and Pesticide Action. Rickett FE, ed. Society of Chemical Industry, London, pp 137-144 (1980). 4. Sattelle DB, David JA, Harrow ID, Hue B: Actions of a-bungarotoxin on identified insect central neurones. In: Receptors for Neurotransmitters, Hormones, and Pheromones in Insects. Sattelle DB, Hall LM, Hildebrand JG, eds. Elsevier, North Holland, Amsterdam, pp 125-139 (1980). 5. Sattelle DB, Harrow ID, Hue B, Pelhate M, Gepner JI, Hall LM: a-Bungarotoxin blocks excitatory synaptic transmission between cercal sensory neurones and giant interneurone 2 of the cockroach Periplaneta americuna. J Exp Biol 207, 473 (1983). 6. Cam CE, Fourtner CR: Pharmacological analysis of a monosynaptic reflex in the cockroach Periplaneta umericana. J Exp Biol 86, 259 (1980). 7. David JA, Sattelle DB: Actions of cholinergic pharmacological agents on the cell body membrane of the fast coxal depressor motoneurone of the cockroach (Periplaneta americana) J Exp Biol 108, 119 (1984). 8. Goodman CS, Spitzer NC: Embryonic development of neurotransmitter receptors in grasshoppers. In: Receptors for Neurotransmitters, Hormones and Pheromones in insects. Sattelle DB, Hall LM, Hildebrand JG, eds. Elsevier/North-Holland, Amsterdam, pp 195-207 (1980). 9. Lane NJ, Swales LS, David JA, Sattelle DB: Differential accessibility to two insect neurones does not account for differences in sensitivity to a-bungarotoxin. Tissue Cell 14,489 (1982). 10. Gorczyca MG, Budnik V, White K, Wu CF: Dual muscarinic and nicotinic action on a motor program in Drosophila. J NeurobiolZ2, 391 (1991). 11. h i p p e r M, Breer H: Subtypes of muscarinic receptors in insect nervous system. Comp Biochem Physiol [Cl 90, 275 (1988). 12. Lummis SCR, Sattelle DB: Binding of N-[propionyl-3H]propionylated a-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta arnericana. Comp Biochem Physiol [Cl80, 75 (1985). 13. Lumrnis SCR, Sattelle DB: [N-methyl-'H]Scopolamine binding sites in the central nervous system of the cockroach Periplaneta americana. Arch Insect Biochem Physiol3,339 (1986). 14. Hue B, Lapied B, Malecot CO: Do presynaptic muscarinic receptors regulate ACh release in the CNS of the cockroach Periplaneta americana? J Exp BiolZ42, 447 (1989). 15. Dudai Y, Ben-Barak J: Muscarinic receptor in Drosophila melanagaster demonstrated by binding of [ 3H]quinuclidinylbenzilate. FEBS Lett 81, 134 (1977). 16. Hairn N, Nahum S, Dudai Y: Properties of a putative muscarinic cholinergic receptor from Drosophila melanogaster. J Neurochem 32, 543 (1979). 17. Tanaka Y, Washio H: Morphological and physiological properties of the dorsal umparied median neurons of the cockroach rnetathoracic ganglion. Comp Biochem Physiol A 91, 37 (1988). 18. Christensen TA, Carlson AD: The neurophysiology of larval firefly luminescence: Direct activation through four bifurcating (DUM) neurons. J Comp Physiol 148, 503 (1982). 300 Bai et al. 19. Casaday GB, Carnhi J M Metamorphosis of flight motor neurones in the moth, Munducu sextu. J Comp Physiol 112, 143 (1976). 20. Clark R: Structural and functional changes in an identified cricket neurone after separation from the soma. 1: Structural changes. J Comp Neurol 170, 253 (1976). 21. Lapied B, Corronc HL, Hue B: Sensitive nicotinic and nicotinic-muscarinicreceptors in insect neurosecretory cells. Brain Res 553, 132 (1990). 22. Whiting P, Esch F, Shimasaki S, Lindstrom J: Neuronal nicotinic acetylcholine receptor a-subunit is cooled for by the cDNA clone n4. FEBS Lett 219, 459 (1987). 23. Deneris ES, Connolly J, Rogers SW, Duvoisin R: Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Neurosci 12, 34 (1991). 24. Hermans-Borgmeyer I, Hoffmeister S, Sawruk E, Betz H, Schmitt B, Gundelfinger ED: Neuronal acetylcholine receptors in Drosopkila: Mature and immature transcripts of the ard gene in the developing central nervous system. Neuron 2, 1147 (1989). 25. Bossy B, Ballivet M, Spierer P: Conservation of neural nicotinic acetylcholine receptors from Drosopkila to vertebrate central nervous systems. EMBO J 7, 611 (1988). 26. Marshall J, David JA, Darlison MG, Barnard EA, Sattelle DB: Pharmacology, cloning and expression of insect nicotinic acetylcholine receptors. In: Nicotinic Acetylcholine Receptors in the Nervous System. Clementi F, Gotti C, Sher E, eds. NATO AS1 Series H, SpringerVerlag, Herdelberg, Vol. 25, pp 257-281 (1988). 27. Marshall J, Buckingham SD, Shingai R, Lunt GG, Goosey MW, Darlison MG, Sattelle DB, Barnard EA: Sequence and functional expression of a single a-subunit of an insect nicotinic acetylcholine receptor. EMBO J 9, 4391 (1990). 28. Sattelle DB, Breer H: Cholinergic nerve terminals in the central nervous system of insects: Molecular aspects of structure, function and regulation. J Neuroendocrinol 2, l(1990). 29. Onai T, FitzGerald MG, Aradawa S, Gocayne JD, Urquhart DA, Hall LM, Fraser CM, McCombie WR, Venter JC: Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor. FEBS Lett 255,219 (1989). 30. Shapiro RA, Wakimoto BT, Subers EM, Nathanson NM: Characterization and functional expression in mammalian cells of genomic and cDNA clones encoding a Drosophila muscarinic acetylchorine receptor. Proc Natl Aead Sci USA 86, 9039 (1989). 31. Volle RL, Koelle GB: Ganglionic stimulating and blocking agents. In: The Pharmacological Basis of Therapeutics. Goodman LS, Gilman A, eds. Macmillan, London, 5th ed. pp 565574 (1975). 32. Pinnock RD, Lummis SCR, Chiappinelli VA, Sattelle DB: k-Bungarotoxin blocks an ru-bungarotoxin-sensitive nicotinic receptor in the insect central nervous system. Brain Res 458, 45 (1988). 33. Hill DR, Bowery NG: 3H-baclofenand 3H-GABAbind to bicuculline-insensitive GABABsites in rat brain. Nature 290, 149 (1981). 34. Benson JA:Bicucullineblocks the response to acetylcholineand nicotine but not to muscarine or GABA in isolated insect neuronal somata. Brain Res 458, 65 (1988). nAChRs of Dorsal Midline Neurones 301 35. Aprison MH, Eerman R: The distribution of glycine in cat spinal cord and roots. Life Sci 4, 2075 (1965). 36. Hammer R: Pirenzepine distinguishes between different subclasses of muscarinic receptor. Nature 283, 90 (1980). 37. Nicoll RA, Malenka RC, Kauer JA: Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol Rev 70, 513 (1990). 38. Colquhoun D, Ogden DC, Mathie A: Nicotinic acetylcholine receptors of nerve and muscle: Functional aspects. Trends Pharmacol Sci 8, 465 (1987). 39. Mathie A, Cull-Candy SG, Colquhoun D: The mammalian neuronal nicotinic receptor and its block by drugs. In: Molecular Basis of Drug &Pesticide Action. (Neurotox 88). Lunt, GG, ed. Elsevier Science Publishers BV, Amsterdam, pp 393403 (1988). 40. Wong LA, Gallagher JP: A direct nicotinic receptor-mediated inhibition recorded intracellularly in vitro. Nature 341, 439 (1989). 41. Eldefrawi AT, OBrien RD: Binding of muscarone by extracts of housefly brain. J Neurochem 17, 1287(1970). 42. Mansour NA, Eldefrawi ME, Eldefrawi AT: Isolation of putative acetylcholine receptor proteins from housefly brain. Biochemistry16, 4126 (1977). 43. Jewess PJ, Clark BS, Donnellan JF: Isolation of a housefly head protein fraction that exhibits high affinity binding of cholinergic ligands. Croat Chem Acta 47, 459 (1975). 44. Kehoe JS: Three acetylcholine receptors in Aplysiu neurones. J Physiol225, 115 (1972). 45. Benson JA: Transmitter receptors on insect neuronal somata: GABAergic and cholinergic pharmacology. In: Molecular Basis of Drug and Pesticide Action (Neurotox '88). Lunt GG, ed. Elsevier Science Publishers BV, Amsterdam, pp 193-206 (1988). 46. Benson JA: M I-like muscarinic receptors mediated cholinergic activation of an inward current in isolated neuronal somata from locust thoracic ganglia. SOCNeurosci (Abstracts) 25, 365 (1989). 47. Abdallah EA, Eldefrawi ME, Eldefrawi AT: Pharmacologic characterization of muscarinic receptors of insect brains. Arch Insect Biochem Physiol17, 107 (1991). 48. Elia AJ, Gardner DR: Some morphological and physiological characteristics of an identifiable dorsal umpaired median neurone in the metathoracic ganglion of the cockroach, Periptuneta amevicanu (L.). Comp Biochem Physiol [Cl 95, 55 (1990). 49. Harrow ID, Sattelle DB: Acetylcholine receptors on the cell body membrane of giant interneurone 2 in the cockroach, Periplaneta umericuna. J Exp Biol105, 339 (1983). 50. Blagburn JM, Sattelle DB: Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach, Periplaneta umericuna. J Comp Physiol162, 215 (1987).