INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Acetylcholine (ACh) is a major and widespread transmitter in sensory neurons and interneurons of the insect brain (Gerschenfeld 1973; Sattelle 1985). As in vertebrates, nicotinic and muscarinic acetylcholine receptors (n- and mAChRs) can be distinguished (Breer 1981; Breer and Sattelle 1987; Pitman 1971); but in contrast to vertebrates, mAChRs generally occur in much lower concentration in insect nervous tissue than nAChRs (Knipper and Breer 1988).

mAChRs belong to a family of proteins with seven transmembrane domains that, on binding of ACh, activate heterotrimeric G proteins, thereby influencing the levels of intracellular second-messenger molecules. Five different subtypes (m1-m5) have been cloned from vertebrates that differ in their selectivity for agonists/antagonists, in their localization within the CNS, in their coupling to different G proteins, and thus in their effects on second messengers (reviewed by Bonner 1989; Felder 1995; Jones 1993). Typical effects evoked by muscarinic receptors are the inhibition of adenylate cyclase (AC), resulting in decreased intracellular levels of cAMP mediated by subtypes m2 and m4, and the activation of phospholipase C (PLC) mediated by the subtypes m1, m3, and m5, leading to the generation of the second-messenger inositol-1,4,5-triphosphate (IP₃) and diacylglycerine (DAG) (reviewed by Bonner 1989; Felder 1995; Jones 1993). Although less frequently, activation of the adenylate cyclase pathway by muscarinic agonists has also been documented in a few vertebrate preparations (Brown and Rietow 1981; Enyedi et al. 1982; Olianas and Onali 1992). Substantial cross-talk leading to the sequential activation of one of these pathways after the other has been confirmed in heterologous expression systems (Felder et al. 1989; Jones et al. 1991).

mAChRs have been found in various insect nervous systems (reviewed by Hannan and Hall 1993; Trimmer 1995), but only one insect mAChR, from the Drosophila brain, has been cloned to date (Onai et al. 1989; Shapiro et al. 1989). Its sequence displays a high degree of homology to vertebrate mAChRs and its heterologous expression in different vertebrate (Blake et al. 1993; Shapiro et al. 1989), and a Drosophila cell line (Millar et al. 1995) demonstrated an increase in cytosolic IP₃ and Ca²⁺ concentrations following activation by muscarinic agonists and a selectivity for antagonists that is similar to vertebrate m1 and m3 subtypes but distinct from m2 AChRs. Although detailed pharmacological classification of invertebrate mAChRs generally does not correlate well with that of vertebrates (reviewed by Hannan and Hall 1993), binding studies with muscarinic agonists and antagonists, evidence for coupling to different second-messenger pathways and heterogeneous physiological responses to muscarinic stimulation mediated by modulation of ligand- and voltage-gated ion channels (David and Pitman 1993, 1996a; Parker and Newland 1995) indicate that a variety of mAChR subtypes is also present in insects (Locusta migratoria synaptosomes: Knipper and Breer 1988; Periplaneta americana DUM-neurons: Lapied et al. 1992; honeybee, cockroach and housefly heads: Abdallah et al. 1991; Manduca sexta: Trimmer and Weeks 1993).

Evidence for the coupling of insect mAChRs to second-messenger pathways derives from studies on various species (reviewed by Trimmer 1995; Trimmer and Qazi 1996). In agreement with the results of the expression studies of the cloned Drosophila mAChR (Millar et al. 1995), an increase in phosphatidylinositol turnover was found in insect nervous systems (David and Pitman 1994; Duggan and Lunt 1988; Trimmer and Qazi 1996) in response to the application of muscarinic agonists. Membrane depolarization and/or increased excitability of the neurons following muscarinic stimulation seen in the same preparations (David and Pitman 1996a,b; Lapied et al. 1992; LeCorronc and Hue 1993; Trimmer 1994) could be mediated by activation of PLC, but the downstream mechanisms have not been described in detail and their potential link to the observed currents is difficult to prove. Hyperpolarizing currents that have been attributed to activation of presynaptically located mAChRs inhibit transmitter release from locust synaptosomes (Knipper and Breer 1988) acting via a reduction in cAMP-level. Similar effects on the synaptic release of ACh were also seen in cockroach and locust sensory afferents (LeCorronc and Hue 1993; Le Corronc et al. 1991; Parker and Newland 1995), presynaptic terminals of locust wing stretch receptor neurons (Leitch and Pitman 1995) and larvae of tobacco hawkmoths (Trimmer and Weeks 1993). The second-messenger cGMP participates in various behaviors of insects (chemosensory processing: Bicker 1998; eclosion behavior: Ewer and Truman 1996; modulation of photoreceptors: Schmachtenberg and Bicker 1999), but to date no direct coupling of this pathway to mAChRs has been confirmed (Trimmer and Qazi 1996).

Insect mAChRs have been attributed with two main functions: the inhibition of transmitter release from sensory terminals and the regulation of the excitability of motoneurons or interneurons (e.g., LeCorronc and Hue 1993; Trimmer and Weeks 1989, 1993). Studies in Manduca sexta larvae suggested that mAChRs mediating increased excitability of proleg retractor motoneurons following strong sensory stimuli provide a form of "motor arousal" to increase the sensitivity for small sensory stimuli (Trimmer and Weeks 1993). In addition, central rhythm-generating circuits underlying various insect behaviors can be activated by global application of muscarinic agonists (locomotion: Büschges et al. 1995; Ryckebusch and Laurent 1993; pharyngeal movements: Gorczyca et al. 1991; chewing: Trimmer 1995), but the neurons directly affected by muscarinic stimulation remained unidentified.

Recently, our pharmacological studies in the protocerebrum of gomphocerine grasshoppers revealed a new functional role for mAChRs, showing them to be the basis for specific arousal underlying the selection and control of singing behavior (stridulation) appropriate for a certain behavioral situation (Heinrich et al. 2001a,b). Male grasshoppers perform a variety of different species- and context-specific songs that are used for intraspecific communication (reviewed by Elsner 1994). The muscles responsible for the hindleg movements underlying sound production are activated by thoracic pattern generating networks (Hedwig 1992; Ronacher 1989). These networks are controlled by the brain via descending command neurons with their dendritic arborizations in the protocerebrum dorsal and posterior to the central body complex (Hedwig 1994, 1995; Hedwig and Heinrich 1997). Microinjection of neuroactive substances into the dendritic area of these command neurons and into the upper or lower division of the central body complex revealed a central role of cholinergic activation in the control of stridulatory behavior (Heinrich et al. 1997, 2001a). Stimulation of both nAChRs and mAChRs by specific agonists induced stridulatory sequences similar to the natural behavior. However, the time courses of induced activity were clearly different, with activation of mAChRs leading to stridulation of slower developing intensity and longer duration when compared with the nicotinic effects. Following a single experimental application of ACh imitating the presynaptic release of the presumed natural transmitter, the muscarinic system is only weakly activated and stridulatory sequences are mainly triggered by activation of nAChRs (Heinrich et al. 1997, 2001b). The slower but longer-lasting activation of the muscarinic system accumulates excitation during long episodes of courtship stridulation. The level of activity of the second-messenger pathways initiated by mAChR activation determines the behavioral threshold to engage in singing behavior and the selection of stridulatory patterns according to the progress of courtship, culminating in an attempt to mate with the female (Heinrich et al. 2001a,b).

In this study, we extended the pharmacological investigations toward an analysis of the intracellular processes that translate the stimulation of mAChRs into excitation of brain neurons that control the activity of the command neurons. For this purpose, we used membrane permeable agents known to activate or inhibit the enzymes of the three second-messenger pathways cAMP, IP₃/DAG, and cGMP. The pharmacological agents were applied at sites where muscarine reliably elicited stridulatory behavior. Their ability to induce stridulation and their effect on muscarine-induced excitation were investigated. In this report, we present strong evidence that mAChRs in the cephalic control system for stridulatory behavior of grasshoppers mediate long-lasting excitation by activation of both AC leading to the formation of cAMP, and PLC generating the second messengers IP₃ and DAG. Because inhibition of each of these pathways can completely suppress muscarine-stimulated stridulation, they seem to be sequentially activated.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Adult specimen of the gomphocerine grasshoppers Omocestus viridulus (L. 1758) (O.v.) and Chorthippus biguttulus (L. 1758) (Ch.b.) were caught in the vicinity of Göttingen, Germany, and kept separately in the laboratory for up several weeks. Additional Ch.b. were reared from eggs that were collected in the previous summer and kept at 4°C for 4 mo. The nymphs hatched after ~1 wk at 26°C and were raised on wheat and supplemental food for crickets (Nekton, Pforzheim) at a 16/8 h light-dark-cycle. All experiments were conducted with male adults at temperatures of 20-25°C.

Preparation

For pharmacological stimulation, the grasshoppers' pronotum was attached with wax to a holder, and the head was fixed to the pronotum. The front cuticle of the head capsule was opened with a razor blade to expose the dorsal surface of the brain. The rest of the animal was left intact and capable of moving all its appendages freely, particularly its hindlegs used in stridulation. To record the stridulatory movements with two optoelectronic devices (Helversen and Elsner 1977), a piece of reflecting foil (Scotchlite 3 M, type 7610; 2 mm diam) was glued to the femur of each hindleg. The up and down movements of each hindleg were thus transformed into voltage signals proportional to the amplitude of movement. Additionally, the sounds produced during stridulation were recorded by a custom-made microphone.

Injection of drugs

The neuroactive substances [ACh, muscarine, 8-Br-cAMP, 8-Br-cGMP, 3-isobutyl-1-methylxanthine (IBMX) and forskolin obtained from Sigma-Aldrich; phorbol-12-myristate-13-acetate (PMA), SQ 22536, 2',5'-dideoxyadenosine (ddAdo), Rp-isomer of cAMPS (Rp-cAMPS), D-erythro-sphingosine, thapsigargin and zaprinast obtained from Calbiochem and H-89 and U-73122 obtained from Biomol] were dissolved in grasshopper saline (Clements and May 1974) to give concentrations of 10³ M. Forskolin, ddAdo, H-89, PMA, D-erythro-sphingosine, thapsigargin, U-73122, and zaprinast were first dissolved in dimethyl sulfoxide (DMSO) before saline was added to give a final concentration of 5% DMSO. Solutions of DMSO in saline had no effect, neither stimulatory when injected alone nor inhibitory when co-injected with muscarine, on the performance of stridulatory behavior. The agents were injected into a specific region of the brain using a pressure injection device (WPI, model PV 820). Double-barrel microcapillaries connected to a three-way stop-cock allowed application of approximately the same amount of two substances at the same site in the protocerebrum. Before the experiment, the tips of the capillaries were broken under visual control to produce tip diameters of ~10-15 µM. The pressure and pulse duration were then adjusted so that ~1-3 nl of a given substance was applied per injection. This had previously been confirmed by measuring the volumes of droplets injected into petroleum jelly (Hedwig and Heinrich 1997).

Data processing

The recorded signals were digitized on-line by means of a A/D-converter card (Real Time Devices AD3300) with the software Turbolab 4.3 (Bressner Technology, Germany) and stored as data files. The sampling rate for recording the stridulatory movements, the sound and the injection pulses was 3 kHz. The software NEUROLAB (Hedwig and Knepper 1992; Knepper and Hedwig 1996) was used for visual examination and filtering of the original data. Using the calculating program Excel (Microsoft), diagrams were generated and subsequently imported into the graphics program CorelDraw 7 (Microsoft), where they were assembled into figures.

Evaluation and statistical analysis of experimental results

When testing a substance for its ability to induce stridulation at a given site within the brain, experiments were defined as successful if stridulation was released in at least three successive trials. One injection of a stimulating drug usually released several song sequences that were separated by short pauses. The sum of the durations of all individual song sequences released by one stimulation was taken as the total duration of stridulation. When investigating putative inhibitory effects on muscarine-stimulated stridulation, the average duration of muscarine-induced songs was calculated from at least three trials, executed at intervals of 5 min. After application of the substance under investigation, experiments were defined as successful if the first pulse of muscarine following injection of that substance did not elicit stridulation or if the duration was below the range of 2 SDs of the average duration of muscarine-induced songs performed before inhibition. The inhibition was classified as irreversible if the duration of stridulation never recovered into that range again.

From the recorded data, the latencies between injection pulses and onsets of stridulation, and the total duration of the released stridulatory activity (see preceding text) were determined. Analyses were performed using relative values calculated by setting the longest latency and longest duration of all songs stimulated during a given experiment as 100%, except for the experiments shown in Fig. 2C, middle, where the duration resulting from the first injections of each series was set to 100%. We used the Wilcoxon-Mann-Whitney test to determine significant differences in the latency and duration of stridulation released by two different substances (Figs. 1A and 2, left and middle). Potential changes in the duration of muscarine-induced stridulation following the injection of another substance (Figs. 2, right, 3-5) were evaluated by a nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test. This nonparametric test was chosen because we generally included all experiments, whether successful interference with muscarine stimulation occurred or not, into the statistical analysis, and therefore a normal distribution of the stimulated durations of stridulation could not be assumed. An unbiased inclusion of all experimental data into the analysis results in comparatively conservative statements about significances, allowing only strong and/or robust effects to alter the control parameters significantly. Potential changes in the latency and the duration of songs released by a series of pulses of muscarine were analyzed by the ANOVA test for repeated measures (Fig. 1, B and C) in combination with Tukey's multiple comparison test. To provide an estimate of the variance of the measured parameters, SDs of the means were calculated and included into the histograms. All statistical analysis was performed using the program Prism 2.01 (GraphPad Software). As we found no differences in the reaction of the two species, O.v. and Ch.b., used in the experiments (see Fig. 2, A-C), the results were pooled for certain analyses.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Stimulation of grasshopper stridulation by cholinergic agonists. A: injections of acetylcholine (ACh; ) and muscarine () into the protocerebrum elicited species-specific stridulatory hindleg movements after distinct latencies and of distinct durations. B: increased volumes of muscarine used for stimulation led to prolonged stridulatory behavior () that started after progressively shorter latencies (). C: repeated application of identical muscarine injections at regular intervals of 5 min elicited stridulation of gradually increasing durations () and decreasing latencies (). Left: 1 experiment. Middle and right: average of 27 experiments. RHL/LHL: movements of the right/left hindleg; error bars: SD; *P < 0.05; **P < 0.001; ***P < 0.0001.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Stimulation of the cAMP second-messenger pathway. Average relative duration (left) and latency (middle) of stridulation stimulated by muscarine in comparison to forskolin (A), 8-Br-cAMP (B), and 3-isobutyl-1-methylxanthine (IBMX; C). Right: effects on stridulation, induced by injections of muscarine at regular intervals of 5 min. O.v., Omocestus viridulus; Ch.b., Chorthippus biguttulus. All substances were prepared at a concentration of 103 M and were injected with identical pulse parameters to the same site within the protocerebrum where stridulation was stimulated with muscarine. Error bars: SD; *P < 0.05; **P < 0.001; ***P < 0.0001.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Stimulation of mAChRs

MUSCARINE-STIMULATION RELEASES STRIDULATION SIMILAR TO THE NATURAL BEHAVIOR. Microinjection of small amounts of muscarine into the central protocerebrum of O.v. and Ch.b. lead to the release of long-lasting stridulation, divided into separate song sequences, that is indistinguishable from natural behavior (Heinrich et al. 1997, 2001a). In O.v., calling and courtship songs can be elicited. Pharmacologically stimulated courtship songs follow a gradual increase in intensity and duration of individual song sequences that is similar to spontaneous courting (Hedwig and Heinrich 1997; Heinrich et al. 1997). In Ch.b., calling and courtship songs differ only slightly in their movement and sound patterns (Elsner 1974; Helversen 1972) and the species-specific movements and activity/pause patterns can reliably be reproduced by pharmacological stimulation (Heinrich et al. 1998).

AMOUNT OF MUSCARINE INJECTED DETERMINES THE TIME COURSE OF STIMULATED STRIDULATION. In a series of preparations, the amount of muscarine injected to a fixed site in the protocerebrum was altered by varying the duration of the pressure pulse used for its ejection from the capillary. The average relative duration of the released stridulatory behavior increased with the duration of pressure pulses from 34.2 ± 16.9% (n = 8) after pulses of 50 ms to 75.8 ± 12.0% (n = 8) following muscarine pulses of 300 ms (Fig. 1B, ). Further increasing the pulse duration evoked no additional increase in the duration of the induced stridulation. In contrast, the latency between injection pulse and onset of stridulation was inversely related to the amounts of muscarine used for stimulation (Fig. 1B, ). The mean relative latency decreased gradually from 93.9 ± 10.0% (n = 8) after pulses of 50 ms to 56.1 ± 17.9% (n = 9) after muscarine pulses of 200 ms and remained unchanged with larger volumes of muscarine.

REPETITIVE MUSCARINE STIMULATION ALTERS THE TIME COURSE OF RELEASED SONGS. Repeated injections of muscarine, applied to the same site at regular intervals of 5 min, induced stridulation of gradually increasing duration. As illustrated by one typical experiment with Ch.b. (Fig. 1C, left), this increase in duration saturated after the third or fourth stimulation and was associated with a decrease in the latency to the onset of singing. The statistical analysis of 27 experiments revealed a significant increase of the duration from 100% following the first muscarinic stimulation to 191 ± 106.2% (P = 0.001) after the fourth pulse of muscarine (Fig. 1C, middle). In the same experiments, the latency decreased significantly from 100 to 49.5% ± 26.2% (P = 0.001; Fig. 1C, right). The intervals between individual muscarine stimuli could be extended to 10 min and still produce the alterations of durations and latencies, although they were weaker with longer intervals. This demonstrated that the experimentally evoked muscarinic excitation not only outlasts the period of muscarine-induced stridulation (usually 1-2 min) but persists for 10 min.

Activation of the cAMP pathway

ACTIVATION OF AC OR PKA STIMULATES STRIDULATION. At those sites in the grasshopper brain where muscarine successfully elicited stridulation, activation of AC by injection of forskolin (Arrese et al. 1999) and activation of PKA by microinjection of 8-Br-cAMP (Lundquist and Nässel 1997; Smith et al. 1984) reliably released long-lasting stridulatory behavior. With respect to the movement patterns and the coordination of the hindlegs, no differences were seen between muscarine-induced songs and songs stimulated with forskolin or 8-Br-cAMP (data not shown). Similar amounts of forskolin and muscarine induced stridulatory activity of about the same durations (Fig. 2A, left). In contrast, the duration of stridulation released by 8-Br-cAMP was significantly shorter when compared with muscarinic stimulation (Fig. 2B, left). For O.v., the average relative duration of muscarine-induced songs was 70.8 ± 25.7% (n = 14), whereas injection of 8-Br-cAMP was followed by 21.6 ± 18.9% (n = 19; P = 0.001) relative duration of stridulation. For Ch.b., the mean relative duration of stridulation after injection of muscarine (72.5 ± 22.7%, n = 15) was also significantly (P = 0.05) longer than stridulation induced by 8-Br-cAMP (47.2 ± 28.5%, n = 12). Because we found no differences in the results obtained with the two different grasshopper species, O.v. and Ch.b., in these or any other experiments (see also Fig. 2, A and C), the results, after calculating the relative values, were pooled for all further analysis. Comparison of the latencies of muscarine-induced stridulation with latencies after injection of forskolin (Fig. 2A, middle) or 8-Br-cAMP (Fig. 2B, middle) revealed no significant differences.

INHIBITION OF PHOSPHODIESTERASE (PDE) RELEASES STRIDULATION. Inhibition of cyclic nucleotide-dependent phosphodiesterases, e.g., by IBMX, has been shown to cause accumulation of the cyclic nucleotides cAMP and cGMP in insect neurons (Trimmer and Qazi 1996). Injection of IBMX into the grasshopper brain elicited coordinated stridulatory movements that were indistinguishable from muscarine-stimulated stridulation induced at the same site within the brain (data not shown). However, the time course of the released stridulation differed in that the average relative duration of stridulatory behavior after injection of IBMX was significantly shorter than following injection of muscarine (Fig. 2C, left). In O.v. it decreased from 46.6 ± 31.6% (n = 23) for muscarine to 17.4 ± 9.5% (n = 26; P = 0.001) for IBMX. In Ch.b., muscarine-induced songs (62.4 ± 23.1%, n = 22) were significantly (P = 0.001) longer than songs released by IBMX (38.3 ± 15.5%, n = 23). In both species, the latency between the injection pulse and the onset of stridulation was longer after injection of IBMX (96.2 ± 60.9%, n = 49), compared with latencies seen after injection of muscarine (52.5 ± 30.4%, n = 45; P = 0.001; Fig. 2C, middle).

Inhibition of the cAMP-pathway

INHIBITION OF AC SUPPRESSES MUSCARINE-STIMULATED STRIDULATION. We used ddAdo (Fryer and Zucker 1993) and SQ 22536 (Zhang et al. 1999) to inhibit AC. Injection of ddAdo led to a reversible decrease in the duration of muscarine-stimulated stridulatory activity in 7 experiments, whereas in another 14 experiments, the subsequent muscarine-stimulated song duration remained within the range of two times the SD of the average of the control stimulations (see METHODS, section for criteria that define successful experiments). In the representation of all the experiments performed with ddAdo, the average relative duration of muscarine-induced songs of 69.2 ± 14.2% (n = 21) was significantly (P = 0.05) reduced to 38.8 ± 35.8% (n = 10) during a period of 5 min after the injection of ddAdo (Fig. 3A). The durations then gradually increased to reach control levels between 10 and 15 min after the injection of ddAdo (72.1 ± 22.6%, n = 14).

View larger version (45K): [in this window] [in a new window]   Fig. 3. Inhibition of the cAMP second-messenger pathway. Average relative duration of muscarine-induced stridulation before () and after () application of 2',5'-dideoxyadenosine (ddAdo; A), SQ 22536 (B), H-89 (C), and the Rp-isomer of cAMP (Rp-cAMPS; D). All substances were prepared at a concentration of 103 M and were injected with identical pulse parameters to the same site within the protocerebrum where stridulation was stimulated with muscarine at regular intervals of 5 min. Error bars: SD; *P < 0.05; **P < 0.001; ***P < 0.0001. Only the significant differences to muscarine stimulation before Rp-cAMPs treatment are indicated in D.

INHIBITION OF PKA SUPPRESSES MUSCARINE-STIMULATED STRIDULATION. To inhibit PKA activity, we injected either H-89 (Nagano et al. 1998; Smith et al. 1996) or Rp-cAMPS (Lundquist and Nässel 1997; Smith et al. 1996) to the same sites in the protocerebrum where muscarine stimulated stridulation. In six experiments, injections of H-89 led to a reversible inhibition of muscarine-induced stridulation, and in two other trials, the inhibition was irreversible. The average relative duration of muscarine-induced songs of all 16 experiments conducted was significantly reduced after injection of H-89 (Fig. 3C). The duration decreased from 67.5 ± 16.0% (n = 16) before to 20.9 ± 35.7% (n = 7; P = 0.01) within 5 min and 33.5 ± 26.4% (n = 12; P = 0.05) between 5 and 10 min after application of H-89. Beyond 10 min after H-89 injections, no inhibitory effects on muscarine-induced stridulation remained.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Interference with the inositol-1,4,5-triphosphate (IP3)/diacylglycerine (DAG) second-messenger pathway. Average relative duration of muscarine-induced stridulation before () and after () application of U-73122 (A), neomycin (B), thapsigargin (C), and D-erythro-sphingosine (D). All substances were prepared at a concentration of 103 M and were injected with identical pulse parameters to the same site within the protocerebrum where stridulation was stimulated with muscarine at regular intervals of 5 min. Error bars: SD; *P < 0.05; **P < 0.001; ***P < 0.0001.

Activation/inhibition of other second-messenger pathways

IP₃/DAG-MEDIATED PATHWAY IS NECESSARY TO MEDIATE THE EXCITATORY EFFECTS OF MACHRS. To investigate a possible involvement of IP₃ or DAG in mediating the muscarinic excitation, we injected U-73122 (Vroemen et al. 1997) to inhibit PLC, the initial enzyme of this pathway. In 8 of 12 experiments, a reversible reduction in the duration of muscarine-induced stridulation was found. The average relative duration of songs elicited by muscarine in all experiments decreased significantly (P = 0.01) from 73.5 ± 15.1% (n = 12) to 26.7 ± 27.3% (n = 10) in the period of 5 min after injection of U-73122 (Fig. 4A). This effect dropped below significance level in the following 5-min interval and appeared to be fully reversed in the time period between 10 and 15 min after the application of the PLC inhibitor (62.3 ± 32.3%, n = 12). Employment of neomycin (Shibanaka et al. 1993), a substance that reduces PLC activity by binding to the enzyme's substrate PIP₂, produced a gradually reversing reduction of muscarine-stimulated stridulation from 69.9 ± 15.4% (n = 32) before to 47.3 ± 23.9% (n = 11; P = 0.01) after neomycin application (Fig. 4B), very similar to the effects of U-73122. Thus two substances known to diminish the activity of PLC by different mechanisms affected mAChR-mediated excitation in a similar way.

CGMP-MEDIATED PATHWAY IS NOT INVOLVED IN MEDIATING THE EXCITATORY EFFECTS OF MACHRS. In analogy to the experiments carried out with the rather unspecific PDE inhibitor IBMX (compare Fig. 2C), we also tested for the involvement of cGMP-specific PDE by injection of the specific inhibitor zaprinast (Paupardin-Tritsch et al. 1986). At sites where muscarine reliably elicited stridulation, injection of zaprinast never induced stridulatory activity according to the criteria for a successful elicitation (see METHODS) (Fig. 5A, left). In addition, latencies (not shown) and the durations of muscarine-induced stridulation were not altered by preceding injections of zaprinast (Fig. 5A, right). This suggested that the increased excitation following injections of IBMX was based on the reduced hydrolysis of cAMP rather than of cGMP. This was supported by the result that direct increase of cGMP levels through injections of 8-Br-cGMP (Schmachtenberg and Bicker 1999), at sites in the grasshoppers brain where muscarine reliably elicited stridulation neither induced stridulatory activity (Fig. 5B, left) nor altered the time course of muscarine-stimulated stridulation (Fig. 5B, right). Because pulses of muscarine retained their activating effects throughout the course of the experiments and 8-Br-cAMP, a substance with very similar membrane permeability, was effective when tested in the same way, this may suggest that there is no cGMP-dependent process contributing to the muscarinic excitation in the cephalic control system of stridulation.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Stimulation of the cGMP second messenger pathway. Following injection to the same sites where muscarine reliably stimulated stridulatory behavior, zaprinast (A) and 8-Br-cGMP (B) neither elicited stridulation by themselves (left) nor had an effect on stridulation, induced by muscarine at regular intervals of 5 min (right). The concentration of all substances was 103 M. Error bars: SD; *P < 0.05; **P < 0.01; ***P < 0.0001.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The aim of this study was to determine the signal transduction pathway activated by the mAChRs in the cephalic control circuits for stridulation in grasshoppers. Experiments were performed with restrained but functionally intact grasshoppers. The singing behavior that is controlled by the brain regions under study was used as a monitor for the level of excitation induced by small volumes of injected neuroactive agents. Although pharmacological studies on fully intact and behaving preparations face certain limitations compared with studies on isolated cells, on tissue preparations or on heterologous expression systems, the physiological significance of any cellular or subcellular mechanism observed can be directly determined. Our recent studies (Heinrich et al. 2001b) suggested that mAChR-mediated excitation lowers the behavioral threshold to perform stridulation in response to relevant sensory stimuli and determines the selection of song patterns associated with a particular behavioral situation. The results presented in this publication support this view and reveal that mAChRs in the central protocerebrum of grasshoppers mediate long-lasting and temporally accumulating excitation by sequential activation of the AC/cAMP/PKA and the PLC/IP₃/DAG second-messenger pathways.

CONTRIBUTION OF MACHRS IN THE CENTRAL PROTOCEREBRUM TO THE CONTROL OF STRIDULATION IN GRASSHOPPERS. Various studies (reviewed by Elsner 1994) suggested that the species- and behavioral context-specific stridulation patterns of grasshoppers are generated by metathoracic rhythm-generating circuits. The performance of a particular pattern is initiated and maintained by tonic activating input from cephalothoracic command neurons, three types of which have been identified in the species O. viridulus (Hedwig 1994, 1995; Hedwig and Heinrich 1997). All command neurons identified so far in various species extend most of their dendrites within a small brain neuropil close to the posterior and dorsal border of the central body complex without extending any arborizations into this prominent structure implicated with sensory integration and motor control (Homberg 1993; Huber 1960a,b; Strauss and Heisenberg 1993). However, these command neurons may only relay the excitation that is initially generated by yet unidentified presynaptic neuropils involved in the initiation and selection of stridulation. Since in various species injections of cholinergic agonists into the two brain regions mentioned elicited stridulation that was composed of different patterns performed in the correct sequence (Heinrich et al. 1997, 2001a), the circuits concerned with the decision when and which pattern to sing may be, at least in part, located within these areas.

COUPLING OF MACHRS IN THE BRAIN OF GRASSHOPPERS TO INTRACELLULAR SIGNALING PATHWAYS. To determine the second-messenger pathways that mediate the excitatory effects of mAChR activation in the protocerebrum of grasshoppers, membrane-permeable substances that have been demonstrated to interfere with intracellular signaling cascades in insects were injected to sites within the brain, where muscarine elicited a certain duration of stridulation. Because there was no way of measuring the accumulation of these substances inside the neurons that are relevant for the control of stridulation, each experiment was analyzed with respect to the effectiveness of a certain volume of muscarine to stimulate stridulation at the same injection site. The results therefore are based on the assumption that both muscarine and the test substance acted on the same neurons. However, only relatively strong effects obtained in a relatively high number of experiments reached significance level in the evaluation of all experiments with a given substance. Interpretation was difficult when drugs had little or no effect because stridulation is known to be influenced by various specific and unspecific stimuli and sufficient accumulation of a substance within neurons and its actions on the specific set of enzymes expressed in these cells are not certain. To minimize unspecific effects on the signaling pathways in question, usually two or more drugs that activated or inhibited a certain enzyme or mechanism were used to confirm the respective results.

EVIDENCE FOR SEQUENTIAL ACTIVATION BY MACHRS OF THE AC- AND THE PLC- INITIATED SECOND-MESSENGER PATHWAYS. Complete suppression of muscarine-stimulated stridulation by inhibitors of AC and PKA (Fig. 3) and by two inhibitors of PLC with different mechanisms of action (Fig. 4, A and B) suggested that both pathways are present in the same neurons and necessary to generate the excitatory effects of muscarine. Stridulation could be stimulated by sole activation of AC or PKA or by inhibition of PDE activity. Also, forskolin-stimulation of AC, the key enzyme that initiates this signaling pathway, evoked stridulation with similar latencies and durations as those induced by muscarine when injected to the same sites in the brain (Fig. 2A). Taken together, these results suggest that both signaling pathways, AC/cAMP/PKA and PLC/IP₃/DAG, are most likely sequentially activated instead of being initiated independently by different subtypes of mAChRs. A parallel effect on cAMP and IP₃ second-messenger pathways has been demonstrated in studies on cultured neuronal cell bodies (Lapied et al. 1992), on cell lines transfected with muscarinic receptor subtypes (Peralta et al. 1988), and on isolated cilia of rat olfactory neurons (Vogl et al. 2000). However, muscarinic agonists evoked opposing effects in these preparations, e.g., stimulation of phosphoinositol hydrolysis and inhibition of AC or opposing hyper- and depolarizing currents, indicating a differential regulation or even a mutual inhibition (Vogl et al. 2000) of both signaling pathways. Sequential activation of AC and PLC has also been reported from various studies, e.g., on human neuroblastoma cells (Baumgold and Fishman 1988) and various transfected cells (Felder et al. 1989; Jones et al. 1991; Peralta et al. 1988). A physiological relevance for neuronal function remained unclear because relatively high agonist concentrations were needed to substantially stimulate both pathways in a given preparation (Felder et al. 1989; Jones et al. 1991) and high levels of expression of a given receptor can lead to an increase in amplitude of the signal transduction response and a loss of selectivity (Schwarz et al. 1993).

AC/CAMP/PKA PATHWAY ACTIVATES PLC. In principle, PLC could be stimulated by phosphorylation, as it has been demonstrated for PLC subtypes implicated with growth and differentiation processes (reviewed by Rhee and Choi 1992). However, PKA-mediated phosphorylation of PLC subtypes contributing to neuronal signaling had either no or inhibitory effects on PLC activity (Liu and Simon 1996; Rhee and Choi 1992).

PLC/IP₃/DAG PATHWAY ACTIVATES AC. At least two mechanisms could account for activation of AC by PLC-initiated signaling pathways. DAG, generated by PLC-mediated phosphoinositol hydrolysis could activate PKC, which has been shown to phosphorylate and activate the AC2 subtype (Bol et al. 1997; Zimmermann and Taussig 1996). Other subtypes such as AC1 (Vorherr et al. 1993; Wu et al. 1993) and an AC found in Drosophila (Levin et al. 1992; Livingstone et al. 1984) can be activated by Ca²⁺. This mechanism has already been described in connection with mAChR stimulation, although high agonist concentrations or conditions that inhibited cAMP degradation by PDEs called into question its potential physiological relevance (Baumgold and Fishman 1988; Felder et al. 1989; Jones et al. 1991; Peralta et al. 1988).