Modulation of tension production by proctolin in the dorsal longitudinal muscles of the cricket Teleogryllus oceanicus.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 14:71-83 (1990) Modulation of Tension Production by Proctolin in the Dorsal Longitudinal Muscles of the Cricket, Teleogryllus oceanicus Bruce A. O'Gara Department of Zoology, Iowa State University, Ames, Iowa High-frequency electrical stimulation (-20 Hz) of the lateral nerve in abdominal segments of the cricket, Tileogryh oceanicus, caused an increase in tonus of the abdominal dorsal longitudinal muscle (DLM). This effect persisted for 1-5 min following stimulation. Application of the pentapeptide proctolin (threshold 1-10 nM) mimicked the increase in muscle tonus produced byelectrical stimulation. Individual twitches were unaffected or slightly reduced by proctolin. Low-frequencyelectrical stimulation (<7 Hz) of the lateral nerve counteracted a previously induced increase in muscle tonus, apparently by activation of an inhibitory motoneuron. y-Aminobutyric acid (CABA) mimicked the effect of low-frequency stimulation and reduced muscle tonus. Octopamine, in concentrations of ~ 0 . mM, 1 was inactive on the abdominal DLM when stimulated at low frequencies (0.5-2 Hz). Application of proctolin to the rnetathoracic DLM caused an increase in twitch amplitude but had little effect on basal tonus. In conjunction with the previously described responses of the metathoracic DLM to octopamine, these results show that the serially homologous abdominal and metathoracic DLMs have dissimilar responses to the modulators proctolin and octopamine. Key words: octopamine, CABA, differentiation, insect INTRODUCTION The abdominal dorsal longitudinal muscle of the cricket Teleogryllus oceanicus is specialized for relatively slow movements and postural control of the abdomen [l-41.In contrast to the abdominal dorsal longitudinal muscle (DLM)," *Abbreviations used: DLM = dorsal longitudinal muscle; DUM = dorsal unpaired median; EJP = excitatory junctional potential; GABA = y aminobutyricacid; IJP = inhibitory junctional potential. Acknowledgments: I thank Dr. C.D. Drewes and Dr. W.O. Friesen for critical comments on the manuscript. I also thank Dr. A.E. Kammer for a gift of proctolin. Received December 4,1989; accepted March 6,1990. Dr. Bruce O'Gara is now at Department of Biology, University of Virginia, Charlottesville, VA 22901. Address reprint requests there. Q 1990 Wiley-liss, Inc. 72 OCara the metathoracic DLM is specialized for the rapid contractions that power wing movements [5,6]. The metathoracic DLM responds to octopamine by producing increases in twitch amplitude, contraction rate, and relaxation rate. The receptor mediating these responses has been shown to be an octopaminel receptor [ 6 ] . Proctolin-responsive muscles show several distinct types of response to proctolin. One type of response is characterized by a proctolin-induced increase in basal tonus [7,8]. This maintained tension is also known as a lingering tension or “catchlikd’ contraction 191. In contrast, a second type of response to proctolin is characterized by increased twitch amplitude but little change in basal tonus [lo-121. Another important neuromodulatory substance, octopamine, is active on several arthropod neuromuscular systems (for review, see David and Coulon ). Octopamine increased the amplitude and relaxation rate of twitches induced by both slow  and fast motoneurons [6,15]. The apparent sources of octopamine for these muscles are (DUM) neurons. Although DUM neurons occur in the abdominal ganglion , it is not known whether the abdominal DLM is innervated by one of these octopaminergic neurons or whether tension production is modulated by octopamine. The purposes of this study were (1)to study the neural modulation of evoked twitches and basal tonus in the abdominal DLM; (2) to determine the effects of octopamine, proctolin, and GABA on tension production in the abdominal DLM; and (3) to examine the effects of proctolin on the metathoracic DLM. A brief report of some of these results has been previously published . MATERIALS AND METHODS Insects The care and feeding of Teleogryllus oceanicus have been previously described . Abdominal DLM Dissection and Experimental Procedures For experiments involving the abdominal DLM, the cricket was decapitated and the thoracic segments discarded. The abdomen was opened with a middorsal incision, and the gut was removed. To reduce spontaneous movements, the nerve cord was removed in most experiments. The fat and air sacs overlying the abdominal DLM were carefully removed (see Carlson  for a description of the abdominal musculature of T. oceunicus). The exoskeletal attachment of the anterior end of the abdominal DLM was pinned to the bottom of the dissecting dish. To monitor muscle tension, a minutien pin, attached to a tension transducer (Narco Biosysterns F50, Houston, TX), was positioned to contact the posterior exoskeletal attachment of the abdominal DLM. The force transducer was positioned for optimally recording twitch amplitude. This method, involving semi-isometricmonitoring of tension, was found superior to attaching the transducer to the isolated posterior attachment of the DLM because the required isolation procedure greatly reduced the viability of the preparation. Experiments were performed using all abdominal segments except the first and the highly modified, fused posterior segments. No differences were noted in the responses of the abdominal DLM in any of the segments used. Modulationof Tension in the DLM 73 To study neurally evoked responses of the abdominal DLM, the lateral nerve was electrically stimulated with a suction electrode. Tension records were monitored on an oscilloscope or chart recorder (Brush RD 1684-00 or Gould 2200S, Cleveland, OH). Saline or drugs were usually superfused over the preparation at 0.75 mumin, a rate that exchanged the fluid volume covering the preparation every few seconds. During experiments using GABA (see Figs. 5 , 78),the perfusion system was not used, and drugs or saline changes were applied from a pipette. This method permitted more exact control of the timing of drug application. Metathoracic DLM Experimental Procedures The procedures used during experiments on the metathoracic DLM have been previously described . Briefly, the posterior muscle attachment was isolated from the exoskeleton and attached to the transducer, which measured isometric tension. Muscle twitches were evoked by stimulation of the four DLM motoneurons in mesothoracic nerve 6  with a suction electrode. Electrophysiology, Saline, and Drugs Intracellular electrical activity from the muscle was recorded using borosilicate glass microelectrodes filled with 1M K acetate (15-40 M a resistance). The composition of the saline was identical to that of OGara and Drewes . Drugs were purchased from Sigma (St. Louis, MO). The experimentswere conducted at room temperature (21-25°C). RESULTS Innervation of the Abdominal DLM Electrical stimuli applied to the lateral nerve evoked a large, overshooting response in DLM muscle fibers (Fig. 1A). The response was recruited in an all-or-none fashion and was accompanied by an observable twitch. In the absence of stimulation when the nerve cord was present, several smaller spontaneous EJP (Fig. 1B,C) and an inhibitory junctional potential (IJP) were also observed (Fig. 1D). The small EJPs often occurred during respiratory movements of the abdomen. When the lateral nerve was transected all spontaneous activity ceased (not shown). The pattern of innervation by the neurons producing these small junctional potentials was not studied in detail. However, the size and number of junctional potentials appeared similar to those in the grasshopper abdominal DLM 1191. Effects of Octopamine on the Abdominal DLM Application of octopamine at concentrations of 0.1-100 pM (n = 5) caused no detectable effect on twitches evoked by 0.5-2 Hz neural stimulation or on basal tonus at stimulation rates of 0-2 Hz (not shown). Aside from the absence of octopamine receptors, another explanation for this result is that octopaminergic neurons (presumably in the lateral nerve) may have been inadvertently stimulated. Thus, the abdominal DLM may have already been maximally facilitated before exogenous octopamine was applied. To test this idea, the octopamine antagonist metoclopramide 16,201 was applied to the preparation 74 Q’Cara D C -I Fig. 1. Synaptic potentials in the abdominal DLM. (A) A single stimulus delivered to the lateral nerve elicited a fast muscle spike. Spontaneous excitatory responses (B,C) and IJPs (D) were also recorded. The largest spikes in B were apparently evoked by the same motor neuron activated in A. Voltage scale: (A), (B), (D) 10 mV; (C) 5 mV. Time scale: (A) 5 ms; (B), (D)100 ms; (C)50 ms. at a concentration of 0.1 mM for periods of 630 min. Metoclopramide produced no reduction of neurally induced twitches (n = 6), suggesting that no inadvertent octopaminergic modulation of the abdominal DLM had occurred. Neural Modulation of Basal Tonus The possibility that basal tonus could be predictably modulated by neural stimulation was examined by repetitive stirnulation of the lateral nerve at various frequencies. As shown in Figure 2, stimulation at 20 Hz for 3 s caused an increase in basal tonus which outlasted the neural stimulation for up to 5 min. As shown in Figure 2B, the increase in tonus was dependent upon stimulus frequency. In most preparations, the lowest stimulation frequency which increased tonus was -7 Hz, corresponding approximately to the frequency that just caused incomplete tetanus (ix., the frequency of stimulation at which the muscle did not return to resting tension between twitches). Lower stimulus frequencies (i.e., those producing no fusion of individual twitches) caused a reduction of tonus (Fig. 2). Muscle membrane potential during neurally induced increases in basal tonus was usually unchanged, but occasionally a slight depolarization of <5 mV occurred (several fibers from each of six preparations were sampled) (Fig. 3). Since these small depolarizationswere unusual, they may be the result of movement artifacts. In a few preparations, distinct stimulus voltage thresholds were found for increasing or decreasing tonus (Fig. 4). In these preparations, low-frequency (2-Hz)stimulation produced substantialincreases or decreases in tonus depending on stimulation voltage (Fig. 4B). The intracellular electrical correlate for the decrease in tonus was a threshold-dependent hyperpolarizing potential that followed the fast spike (Fig. 4C). The amplitude and duration of this potential appeared similar to those of the spontaneously occurring IJPs (Fig. 1D). This hyperpolarizing potential can also be seen in Figure 3 (trace i) as a small deflection below baseline during the stimulation-induced reduction in tonus. Modulation of Tension in the DLM 75 A 2 0 Hz 2 Hz I 8 2OHz 2Hz Fig. 2. Modulation of tonus by stimulation of the lateral nerve. (A) Stimulation at 20 H t for 3 s (bar) produced a large phasic contraction. After termination of stimulation, muscle tonus was elevated. Near the end of the record, stimulation at 2 Hz evoked twitches and a decrease of tonus. (B)At the beginning of the record the lateral nerve was stimulated at 20 Hz for 3 s (bar). Tonus was then reduced by2-Hz stimulation. Stimulation at 30 Hz for 3 s evoked a larger increase of tonus compared to 20-Hz stimulation. The increase of tonus was then counteracted by 3-Hz stimulation of the lateral nerve. The variation in the width of the trace was caused by vibration. Time scale: 15 s . Tension scale: 75 mg. t A - Fig. 3. Membrane potential during neurally induced changes of tonus. The bottom trace (t) shows tension of the abdominal DLM. Muscle tonus was increased by 20-Hz stimulation for 3 s (bar) and counteracted by 2-Hz stimulation. The top trace (i)shows muscle membrane potential during changes of tonus. A series of fast muscle spikes (peaks clipped by amplification), evoked during 20-Hz stimulation, was followed by -5 mV depolarization that accompanied the increase of tonus. This was the largest sustained shift of membrane potential in any preparation. Voltage scale: 10 mV. Time scale: 5 s. Tension scale: 75 rng. 76 O'Cara A B Fig. 4. Thresholds for changing tonus. (A) The first and third twitches were elicited by a single pulse and were followed by a reduction of tonus. The second twitch, elicited by a slightly lower stimulus voltage, was followed by an increase of basal tonus. (B) Large increases or decreases of tonus were produced during 2-HZ stimulation by alternating between the stirnulusvoltages in A. (C) The intracellular correlate (i)for the decrease of tonus was a hyperpolarizing potential that follows the fast muscle spike. Threshold for the hyperpolarization was identical to the threshold that produced a decrease of tonus in A and B. In the tension record (t) a slightly faster muscle relaxation occurred following the hyperpolarizingpotential. Voltage scale: 10 mV. Time scale: (A) I s; (B)2 s; (C) 50 ms. Tension scale: 150 mg. Role of GABA in Regulating Tonus If the hyperpolarization that accompanied the decrease in tonus was caused by the inhibitory neurotransmitter GABA, application of GABA should cause a similar decrease in tonus. In each of eight preparations, GABA application caused marked reductions of tonus (Fig. 5A). Further evidence that neurally induced reductions of tonus were mediated by GABA was obtained by application of the GABA antagonist picrotoxin. Exposure of the abdominal DLM to 1mM picrotoxin for 10 min blocked the reduction of basal tonus normally produced by 2-Hz stimulation of the lateral nerve (n = 10) (Fig. 58). Taken together, these results suggest that the reductions of tonus in the abdominal DLM are mediated by the release of GAM from an inhibitory motoneuron. ROLE OF PROCTOLIN IN REGULATING TONUS Proctolin applied to the abdominal DLM caused increased tonus (Fig. 6), thus mimicking the increase of tonus produced by repetitive neural stimulation (see Fig. 2). The effects of proctolin were dose dependent at concentrations between 1nMand 1pM (n = 6) (Fig. 6). Concentrations >1pM were not used. Proctolin-inducedincreases of tonus were also antagonized by lateral nerve stimulation at 2 Hz (n = 3) (Fig. 7A). However, 2-Hz lateral nerve stimulation did not cause relaxation of the abdominal DLM to baseline tension. Application of 1mM GABA completely antagonized the effect of 1 pM proctolin (n = 5 ) (Fig. 7B). Following GABA application, tonus remained reduced for at least 5 min, even though proctolin was still present in the bath. Y Modulation of Tension in the DLM A 77 r i 20Hz f Saline + ------- GABA B i - 24H 1 20Hz Fig. 5. Effects of GABA in modulating tonus. (A) Muscle tonus was increased by stimulating the lateral nerve at 20 Hz for 5 s (bar). Addition of 0.3 ml saline had no effect, but 0.3 ml of 1 mM GABA produced a reduction of tonus. (B) Neurally induced changes of tonus were blocked by picrotoxin. During a control period (1)an increase of tonus was produced by stirnulation at 20 Hz for 5 s (bar) and a reduction of tonus was induced by 2 Hz stimulation. Ten minutes after picrotoxin (1 mM) treatment (2), an increase in tonus was produced by 20-Hz stimulation (bar) but the normal reduction of tonus by 2-Hz stimulation was blocked. Time scale: 30 s. Tension scale: 100 mg. IO-~M t IO-~M 10-8M V 1Om6M Fig. 6. Effects of proctolin on tonus of the abdominal DLM. Proaolin was applied at the first arrow and was washed off at the second arrow. A dose-dependent increase of tonus was evident. Bars on each panel indicate equivalent amounts of tension. Time scale: 5 rnin. Tension scale 150 mg. 78 O’Cara B 4 Proctolin 4 2 Hz 4 Proctolin 4 Saline 4 GABll Fig. 7. Termination of proctolin-induced increases of tonus. (A) An increase of tonus was produced by addition of 0.3 rnl of 1 FM proctolin. Muscle tonus was then reduced by 2-Hz stimulation of the lateral nerve. (6)The proctolin-induced (1 FM) increase of tonus was reversed by GABA (0.3 rnl of 1 rnM) but was unaffected by saline (0.3 rnl). Time scale: 2 min. Tension scale: 200 mg. Effect of Proctolin on Neurally Induced Twitches To examine the effects of proctolin on twitches of the abdominal DLM, the lateral nerve was stimulated at 0.5 Hz, a frequency that normally caused no change of basal tonus. When proctolin was applied to the preparation, tonus increased and twitches were superimposed on this increase of tonus (Fig. 8C). Comparison of twitches, before and after proctolin application, showed either no effect (2 of 5 preparations) (Fig. $A) or a slight reduction in amplitude (3 of 5 preparations) (Fig. 8B). The maximal effect of proctolin in any preparation was a 23%reduction in twitch amplitude (Fig. 8B,C). Effect of Proctolin on the Metathoracic DLM In contrast to the metathoracic DLM , the abdominal DLM was insensitive to octopamine. Thus it was of interest to determine if the two muscles also differed in their responses to proctolin. Within 1min of application of proctolin to the metathoracic DLM, a clearly observable increase in twitch tension occurred (Fig. 9). Twitch amplitude continued to increase for as long as proctolin was applied (S10 min).The threshold concentrationwhich produced an increase in twitch amplitude was -0.1 pM. At 0.1 pM, twitch amplitude was increased by8.9 k 3.5%(mean k SEM) (n = 3) while contractionand relaxationrates were unchanged. At 1FM twitch amplitude was increased by92.3 2 18.6%,contraction rate by 50.6 k 13.3%,and relaxation rate by 31.2 21.1% (n = 3). These effects persisted for at least 2 h after exposure to 1pM proctolin. * DISCUSSION The pharmacological responses of the abdominal DLM contrast markedIy with those of the metathoracic DLM. While proctolin application to the abdom- Modulationof Tension in the DLM 79 J C 4 + Fig. 8. Effect of proctolin on twitches of the abdominal DLM.(A$) Two superimposed twitches are shown, one prior to proctolin application and the other near the end of 7-to 8-min proctolin exposure. Baselines were adjusted to nullify the proctolin effect on muscle tonus. (C) The effects of 0.1 pM proctolin on both basal tonus and twitches are shown. Proctolin was applied at the first arrow and washed off at the second arrow. The deflection at the second arrow i s an artifact of saline change. In some preparations, proctolin either had little or no effect on twitches (A) or in other preparations produced a decrease of twitch amplitude (6,C). (C)Time scale: 50 ms (A$), 2.5 min (C). Tension scale: 15 mg (A$), 45 mg (0. inal DLM produced an increase of tonus (Fig. 6),it did not greatly affect twitch amplitude. However, in contrast to the abdominal DLM, proctolin application to the metathoracic DLM caused an increase of twitch amplitude (Fig. 9). The concentrations of proctolin necessary to produce measurable effects on the metathoracic DLM were relatively high for a peptide neuromodulator. However, the physiological relevance of the proctolin response is suggested by the finding that the proctolin-induced facilitation of twitch amplitude (at 1 pM) was similar to the maximal amount of facilitation produced by octopamine or its agonists . High-frequency stimulation of the lateral nerve produced a "catchlike" tension in the abdominal DLM (see Fig. 2). Unfortunately, the use of the term "catch' has become imprecise in the literature [21-241. Such increases of tonus may not be called catch contractions but may be termed only "catchlike" because these contractures have not met the criteria fordefining catch . Catch 80 O’Cara 2 1 / Fig. 9. The effect of proctolin on the metathoracic DLM. (A) Traces I show twitch tension (lower) and differentiated twitches (upper) prior to proctolin application. Contraction rate is indicated by downward deflections and relaxation rate is indicated by upward deflections. Traces 2 show these parameters after application of 1 IJ.Mproctolin. (B)The response of the same preparation to 1 M proctolin was recorded at a slow paper speed. Due to the slow paper speed individual contractions are not evident in either the tension (t) or differentiated (d) records. Note that the differentiated signal is inverted compared with A. Contraction rate is indicated by deflections above the white line, and relaxation rate is indicated by deflections below the line. In this preparation, twitch amplitude was increased by 105.9% and contraction rate by 52.4%, but relaxation rate was not facilitated. Time scale : (A) 20 ms; (6)2 rnin. Tension scale: 0.4 g. Differentiated traces: (A) 2 8.s-I; (6) 1 g.s-l. is characterized by an extremely strong resistance to stretch during and after initial shortening. Once the muscle is in catch no further shortening occurs. It is unknown whether the abdominal DLM exhibits these properties of catch. Until a preparation (e.&.,the abdominal DLM) has satisfied the formal critiera of catch, alterations of maintained tension would be more productively referred to as alterations of basal tonus. Low-frequency stimulation caused decreases of muscle tonus (see Fig. 2). Modulation of Tension in the DLM 81 The intracellular event that correlated with reductions of abdominal DLM tonus was a hyperpolarizing potential (Fig. 4C), similar in size and duration to spontaneous inhibitory junctional potentials. GABA mimicked the effects of low frequency stimulation and reduced muscle tone, regardless of whether the increase of tonus was induced by proctolin or lateral nerve stimulation (Figs. 5A, 7B). Likewise, GABA antagonizes proctolin-induced increases of tonus in lobster skeletal muscle . These results suggest that decreases of tonus in the abdominal DLM were produced by an inhibitory motoneuron. Similar actions of inhibitory motoneurons have been described in other arthropod muscles [8,21-22,241. Increases of tonus in the cricket abdominal DLM were induced by highfrequency stimulation, but with little change in muscle membrane potential. Similar effects of high-frequency stimulation have been seen in the cockroach coxal depressor muscle  and locus extensor tibiae 1221. More recent evidence indicates that both muscles are innervated by motoneurons that use 1-glutamateand proctolin as co-transmitters, with 1-glutamateinitiating twitches and proctolin causing increased tonus [7,8,26]. A similar situation may exist in the abdominal DLM. Proctolin usually had no effect or slightly reduced the amplitude of abdominal DLM twitches without affecting twitch duration (Fig. 8). This contrasts with the prolongation of the relaxation phase of twitches in the cockroach coxal depressor treated with proctolin . This apparent difference between the abdominal DLM and the cockroach coxal depressor may be an artifact. Inhibitory motoneurons are known to shorten the relaxation phase of muscle contractions [27-291. In most abdominal DLM preparations, an inhibitory motoneuron was unavoidably stimulated along with the unit(s) that produced the twitches and increases of tonus, since both units had similar thresholds (see Fig. 4). In those abdominal DLM preparations in which activation of the inhibitor could be avoided (see Fig. 4), twitch duration was increased as compared to when inhibitory motoneurons were activated. Although it is unknown whether the abdominal DLM motoneurons contain proctolin, several neurons in the same general location as the abdominal DLM motoneurons exhibit proctolin-like immunoreactivity in the cockroach . An additional indication that proctolin is involved in abdominal muscle function is the localization of a proctolin-like peptide in abdominal muscles of the Colorado potato beetle , cockroach ,and Drosophila . Further work is needed to determine if the T. oceanicus abdominal DLM motoneurons contain and release proctolin. The abdominal DLM was unresponsive to octopamine at concentrations of CO.1 mM when stimulated at frequencies that do not produce tetanus. This contrasts with the high sensitivity of the serially homologous metathoracic DLM to octopamine . The absence of octopamine sensitivity in the abdominal DLM may be explained in several ways. First, octopaminergic innervation of the abdominal DLM may not exist. Although octopaminergic DUM neurons exist in the abdominal ganglia, the peripheral sites of innervation by these DUM neurons are unknown . Second, octopaminergic inputs onto the abdominal DLM may exist but may have been stimulated during capture and dissection [ZO], or by electrical stimulation. Since the octopamine antagonist 82 O’Cara metoclopramide did not reduce twitch amplitude, this suggests twitch amplitude potentiation had not occurred prior to octopamine application. However, since the antagonistic effect of metoclopramide is only due to competition for binding sites, it would not antagonize a long-lasting process induced by endogenous octopamine before the application of metoclopramide. It remains possible that if the abdominal DLM were activated by stimulation of single identified motoneurons rather than by stimulation of the whole lateral nerve, specific octopamine effects may be revealed. The results presented in this paper as well as those of OGara and Drewes  show that these two serially homologous muscles have differential pharmacological sensitivities to octopamine and proctolin. 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