Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation ^1,2. While it is possible to study this system in vivo ³, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article⁴ we demonstrate how the STNS can be isolated from the stomach. Open in a separate windowClick here to view.^{(62M, flv)}     

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.Open in a separate windowClick here to view.^{(116M, flv)}     

Non-amidated and amidated members of the C-type allatostatin (AST-C) family are differentially distributed in the stomatogastric nervous system of the American lobster, <Emphasis Type="Italic">Homarus americanus</Emphasis>

The crustacean stomatogastric nervous system (STNS) is a well-known model for investigating neuropeptidergic control of rhythmic behavior. Among the peptides known to modulate the STNS are the C-type allatostatins (AST-Cs). In the lobster, Homarus americanus, three AST-Cs are known. Two of these, pQIRYHQCYFNPISCF (AST-C I) and GNGDGRLYWRCYFNAVSCF (AST-C III), have non-amidated C-termini, while the third, SYWKQCAFNAVSCFamide (AST-C II), is C-terminally amidated. Here, antibodies were generated against one of the non-amidated peptides (AST-C I) and against the amidated isoform (AST-C II). Specificity tests show that the AST-C I antibody cross-reacts with both AST-C I and AST-C III, but not AST-C II; the AST-C II antibody does not cross-react with either non-amidated peptide. Wholemount immunohistochemistry shows that both subclasses (non-amidated and amidated) of AST-C are distributed throughout the lobster STNS. Specifically, the antibody that cross-reacts with the two non-amidated peptides labels neuropil in the CoGs and the stomatogastric ganglion (STG), axons in the superior esophageal (son) and stomatogastric (stn) nerves, and ~ 14 somata in each commissural ganglion (CoG). The AST-C II-specific antibody labels neuropil in the CoGs, STG and at the junction of the sons and stn, axons in the sons and stn, ~ 42 somata in each CoG, and two somata in the STG. Double immunolabeling shows that, except for one soma in each CoG, the non-amidated and amidated peptides are present in distinct sets of neuronal profiles. The differential distributions of the two AST-C subclasses suggest that the two peptide groups are likely to serve different modulatory roles in the lobster STNS.     

Neuropilar Projections of the Anterior Gastric Receptor Neuron in the Stomatogastric Ganglion of the Jonah Crab,Cancer Borealis

Sensory neurons provide important feedback to pattern-generating motor systems. In the crustacean stomatogastric nervous system (STNS), feedback from the anterior gastric receptor (AGR), a muscle receptor neuron, shapes the activity of motor circuits in the stomatogastric ganglion (STG) via polysynaptic pathways involving anterior ganglia. The AGR soma is located in the dorsal ventricular nerve posterior to the STG and it has been thought that its axon passes through the STG without making contacts. Using high-resolution confocal microscopy with dye-filled neurons, we show here that AGR from the crab Cancer borealis also has local projections within the STG and that these projections form candidate contact sites with STG motor neurons or with descending input fibers from other ganglia. We develop and exploit a new masking method that allows us to potentially separate presynaptic and postsynaptic staining of synaptic markers. The AGR processes in the STG show diversity in shape, number of branches and branching structure. The number of AGR projections in the STG ranges from one to three simple to multiply branched processes. The projections come in close contact with gastric motor neurons and descending neurons and may also be electrically coupled to other neurons of the STNS. Thus, in addition to well described long-loop pathways, it is possible that AGR is involved in integration and pattern regulation directly in the STG.     

Mass spectrometric characterization and physiological actions of GAHKNYLRFamide, a novel FMRFamide-like peptide from crabs of the genus Cancer

The stomatogastric ganglion (STG) and the cardiac ganglion (CG) of decapod crustaceans are modulated by neuroactive substances released locally and by circulating hormones released from neuroendocrine structures including the pericardial organs (POs). Using nanoscale liquid chromatography electrospray ionization quadrupole-time-of-flight tandem mass spectrometry and direct tissue matrix-assisted laser desorption/ionization Fourier transform mass spectrometry we have identified and sequenced a novel neuropeptide, GAHKNYLRFamide (previously misassigned as KHKNYLRFamide in a study that did not employ peptide derivatization), from the POs and/or the stomatogastric nervous system (STNS) of the crabs, Cancer borealis, Cancer productus and Cancer magister. In C. borealis, exogenous application of GAHKNYLRFamide increased the burst frequency and number of spikes per burst of the isolated CG and re-initiated bursting activity in non-bursting ganglia, effects also elicited by the FMRFamide-like peptides (FLPs) SDRNFLRFamide and TNRNFLRFamide. In the intact STNS (which contains the STG), exogenous application of GAHKNYLRFamide increased the frequency of the pyloric rhythm and activated the gastric mill rhythm, effects also similar to those elicited by SDRNFLRFamide and TNRNFLRFamide. FLP-like immunoreactivity in the POs and the STNS was abolished by pre-adsorption with the synthetic GAHKNYLRFamide. Different members of the FLP family exhibited differential degradation in the presence of extracellular peptidases. Taken collectively, the amino acid sequence of GAHKNYLRFamide, the blocking of FLP-like immunostaining, and its physiological effects on the CG and STNS suggest that this peptide is a novel member of the FLP superfamily.     

All-or-none control of the bursting properties of the pacemaker neurons of the labster pyloric pattern generator

In Crustacea the central pattern generator for the pyloric motor rhythm (filtration to the midgut) is known to be located within the stomatogastric ganglion (STG); its cycling activity is known to be organized by three endogenous burster neurons acting as pacemakers and driving 11 follower neurons. In Homarus, recordings from the isolated stomatogastric nervous system (Fig. 1) indicate that (1) the pyloric output can be generated only when the STG is afferented (i.e., connected to the more rostral oesophageal and commissural ganglia) (Fig. 2) and (2) the deafferntation of the STG results in a complete loss of the bursting properties of the pacemaker neurons (Fig. 4). Manipulation of the STG inputs responsible for unmasking the properties of the pacemakers strongly suggests that (1) they are not phasic inputs (Fig. 5) and (2) they are long-term acting inputs (Fig. 6). These results provide evidence for a neural all-or-none control of the bursting properties of the pacemaker neurons of a motor pattern generator.     

NO/cGMP Signaling and the Flexible Organization of Motor Behavior in Crustaceans

The basic elements of the NO/cGMP signaling pathway have beenidentified in the nervous systems of animals from nearly allof the major phyla. In crustaceans, the NO/cGMP pathway is associatedwith certain fundamental neuronal processes, including sensoryintegration and the organization and production of motor behavior.Here I review the evidence for NO synthesis and action in crustaceanneural networks, with an emphasis on the rhythmic motor circuitsof the crab stomatogastric ganglion (STG). In the STG, NO appearsto be released as an orthograde transmitter from descendingprojection neurons. NO's receptor, a cytopasmic isoform of guanylatecyclase (sGC), is expressed in a subset of the cells that participatein the gastric mill and pyloric central pattern generating networks.In spontaneously-active, in vitro preparations of the STG, pharmacologicalinhibitors of the NO/cGMP pathway cause the two rhythmic motorpatterns to collapse into a single conjoint rhythm. Parallelmotor output is restored when the ganglion is returned to normalsaline. Although precise mechanisms have yet to be determined,these data suggest that NO and cGMP play an important role inthe functional organization of STG networks. The STG, as wellas other crustacean models, provides a promising context forstudying the physiological and behavioral aspects of NO-mediatedsignaling in the nervous system.     

Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na⁺ saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a “borrowed transmitter”. Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron‐evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron‐evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave‐form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 380–392, 2003     

Stomatogastric nervous system of Galleria mellonella L. (Lepidoptera : Pyralidae): Changes during metamorphosis with special reference to FMRFamide neurons

Scanning electron microscopy and immunohistochemical staining for FMRFamide-like peptides revealed that the stomatogastric nervous system of Galleria mellonella (Lepidoptera : Pyralidae) includes 5 ganglia: the frontal ganglion with 4, the hypocerebral ganglion with 2, the ingluvial ganglion with 2–4, and each of the paired proventricular ganglia with 6–8 immunoreactive perikarya. Immunoreactivity was also found in axons to and within the corpora cardiaca, in the nerves connecting stomatogastric ganglia, as well as in 8 gastric nerves that extend along longitudinal midgut muscles. Adhesion of corpora cardiaca to the hypocerebral ganglion and partial merging and shortening of gastric nerves were the only conspicuous changes of the stomatogastric system that occurred during metamorphosis.     

Characterization of calcium/calmodulin-dependent protein kinase II activity in the nervous system of the lobster,Panulirus interruptus

Nervous system tissue fromPanulirus interruptus has an enzyme activity that behaves like calcium/calmodulin-dependent protein kinase II (CaM KII). This activity phosphorylates known targets of CaM KII, such as synapsin I and autocamtide 3. It is inhibited by a CaM KII-specific autoinhibitory domain peptide. In addition, this lobster brain activity displays calcium-independent activity after autophosphorylation, another characteristic of CaM KII. A cDNA from the lobster nervous system was amplified using polymerase chain reaction. The fragment was cloned and found to be structurally similar to CaM KII. Serum from rabbits immunized with a fusion protein containing part of this sequence immunoprecipitated a CaM KII enzyme activity and a family of phosphoproteins of the appropriate size for CaM KII subunits. Lobster CaM KII activity is found in the brain and stomatogastric nervous system including the commissural ganglia, commissures, stomatogastric ganglion and stomatogastric nerve. Immunoblot analysis of these same regions also identifies bands at an apparent molecular weight characteristic of CaM KII.     

The anatomy and physiology of the stomatogastric nervous system ofSquilla

Summary The stomatogastric nervous system of a mantis shrimp,Squilla oratoria, is described. The motor nerves of the stomatogastric ganglion (STG) and their innervation of muscles of the posterior cardiac plate (pcp) and pyloric systems are detailed.The STG contains more than 25 neurons. It sends out one pair of major output nerves. The pcp-pyloric cycle recorded from the motor axons in this nerve consists of rhythmic bursts of several units which fire with a characteristic phase relationship to each other. The rhythm is intrinsic to the STG itself, but it is modifiable.Recordings from the peripheral nerves reveal that identifiable cardiac plate, pyloric dilator and pyloric neurons control sequential contractions of the pcp and pyloric muscles to constrict or dilate a number of their attached ossicles.Several modulatory input fibres in the stomatogastric nerve, activated via stimulation of the superior or inferior oesophageal nerve (son, ion), prime or trigger the cyclic motor outputs. The son inputs induce distinct effects on the cardiac and pcp-pyloric pattern generators, while the ion inputs, via the oesophageal ganglion, excite only the pcp-pyloric generator.On the basis of anatomical and physiological observations, the possible functions of motor neurons involved in the pcp-pyloric cycle are described with reference to opening of the pcp and pyloric channels.This stomatogastric nervous system inSquilla is compared to that in decapods which has been well analyzed.Abbreviations CG commissural ganglion - ion inferior oesophageal nerve - lvn lateral ventricular nerve - OG oesophageal ganglion - pep posterior cardiac plate - son superior oesophageal nerve - STG stomatogastric ganglion - stn stomatogastric nerve - ivn inferior ventricular nerve     

Central nervous system projections to and from the commissural ganglion of the crab Cancer borealis

Higher-order inputs provide important regulatory control to motor circuits, but few cellular-level studies of such inputs have been performed. To begin studying higher-order neurons in an accessible model system, we have localized, in the supraesophageal ganglion (brain), neurons that are candidates for influencing the well-characterized motor circuits in the stomatogastric nervous system (STNS) of the crab Cancer borealis. The STNS is an extension of the central nervous system and includes four ganglia, within which are a set of motor circuits that regulate the ingestion and processing of food. These motor circuits are locally regulated by a set of modulatory neurons, most of which are located in the paired commissural ganglia (CoGs). These modulatory neurons are well-positioned to receive input from brain neurons because the circumesophageal commissures (CoCs) connect the brain with the CoGs. We have performed a series of CoC backfills to localize the brain neurons that are likely to innervate the CoGs and are, therefore, candidates for influencinng the STNS motor patterns. CoC backfill-labeled neuronal somata within the brain are clustered around a subset of anatomically defined neuropil regions. We have concomitantly localized many CoG neurons that project into the brain. This latter pathway presumably includes neurons that provide feedback regarding ongoing STNS activity. Interestingly, nearly all of these brain and CoG neurons project through the medial aspect of the CoC. This work provides an initial framework for future studies to determine the way that higher-order input regulates rhythmic motor patterns. This work was supported by a grant from the National Institute of Neurological Disorders and Strokes (NS42813 to M.P.N.) and a National Science Foundation Fellowship (DGE9616278 to M.S.K.).     

Phylogenetic,ontogenetic and adult adaptive plasticity of rhythmic neural networks: a common neuromodulatory mechanism?

Neuromodulatory inputs are known to play a major role in the adaptive plasticity of rhythmic neural networks in adult animals. Using the crustacean stomatogastric nervous system, we have investigated the role of modulatory inputs in the development of rhythmic neural networks. We found that the same neuronal population is organised into a single network in the embryo, as opposed to the two networks present in the adult. However, these adult networks pre-exist in the embryo and can be unmasked by specific alterations of the neuromodulatory environment. Similarly, adult networks may switch back to the embryonic phenotype by manipulating neuromodulatory inputs. During development, we found that the early established neuromodulatory population display alteration in expressed neurotransmitter phenotypes, and that although the population of modulatory neurones is established early, with morphology and projection pattern similar to adult ones, their neurotransmitter phenotype may appear gradually. Therefore the abrupt switch from embryonic to adult network expression occurring at metamorphosis may be due to network reconfiguration in response to changes in modulatory input, as found in adult adaptive plasticity. Strikingly, related crustacean species express different motor outputs using the same basic network circuitry, due to species-specific alteration in neuromodulatory substances within homologous projecting neurones. Therefore we propose that alterations within neuromodulatory systems to a given rhythmic neural network displaying the same basic circuitry may account for the generation of different motor outputs throughout development (ontogenetic plasticity), adulthood (adaptive plasticity) and evolution (phylogenetic plasticity).Abbreviations CoG Commissural ganglion - OG Oesophageal ganglion - STG Stomatogastric ganglion - STNS Stomatogastric nervous system     

Identification, physiological actions, and distribution of TPSGFLGMRamide: a novel tachykinin-related peptide from the midgut and stomatogastric nervous system of Cancer crabs

In most invertebrates, multiple species-specific isoforms of tachykinin-related peptide (TRP) are common. In contrast, only a single conserved TRP isoform, APSGFLGMRamide, has been documented in decapod crustaceans, leading to the hypothesis that it is the sole TRP present in this arthropod order. Previous studies of crustacean TRPs have focused on neuronal tissue, but the recent demonstration of TRPs in midgut epithelial cells in Cancer species led us to question whether other TRPs are present in the gut, as is the case in insects. Using direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation, we found that at least one additional TRP is present in Cancer irroratus, Cancer borealis, Cancer magister, and Cancer productus. The novel TRP isoform, TPSGFLGMRamide, was present not only in the midgut, but also in the stomatogastric nervous system (STNS). In addition, we identified an unprocessed TRP precursor APSGFLGMRG, which was detected in midgut tissues only. TRP immunohistochemistry, in combination with preadsorption studies, suggests that APSGFLGMRamide and TPSGFLGMRamide are co-localized in the stomatogastric ganglion (STG), which is contained within the STNS. Exogenous application of TPSGFLGMRamide to the STG elicited a pyloric motor pattern that was identical to that elicited by APSGFLGMRamide, whereas APSGFLGMRG did not alter the pyloric motor pattern.     

Localization of stomatogastric IV neuron cell bodies in lobster brain

Summary The cell bodies of the inferior ventricular nerve (IVN) through-fibers of the lobster stomatogastric nervous system were located using cobalt chloride backfills and intracellular recordings. Following backfills of the IVN, two cell bodies in the supraesophageal ganglion (or brain) were stained with cobalt. These cells, each approximately 30 m in diameter, were located at the base of the IVN, just inside the connective tissue sheath surrounding the brain, and were identifiable on the basis of their close proximity to the IVN.In order to record from the cells, an in vitro preparation was made which included the cell bodies, their axons in the IVN and the stomatogastric nervous system. Intracellular recordings showed that the axons projected to the stomatogastric ganglion and made synaptic connections onto identified neurons. The axon trajectories and synaptic connections correlated with those previously described for the IVN through-fibers using extracellular stimulation and recording techniques.Abbreviations IVN inferior ventricular nerve - SN stomatogastric nerve     

Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na(+) saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a "borrowed transmitter". Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron-evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron-evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave-form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult.     

Molecular cloning and characterization of crustacean type-one dopamine receptors: D1alphaPan and D1betaPan

Dopamine (DA) differentially modulates identified neurons in the crustacean stomatogastric nervous system (STNS). While the electrophysiological actions of DA have been well characterized, little is known about the dopaminergic transduction cascades operating in this system. As a first step toward illuminating the molecular underpinnings of dopaminergic signal transduction in the crustacean STNS, we have cloned and characterized two type-one DA receptors (DARs) from the spiny lobster (Panulirus interruptus): D(1alphaPan) and D(1betaPan). We found that the structure and function of these arthropod DARs are well conserved across species. Using a heterologous expression system, we determined that DA, but not serotonin, octopamine, tyramine or histamine activates these receptors. When stably expressed in HEK cells, the D(1alphaPan) receptor couples with Gs, and DA elicits an increase in [cAMP]. The D(1betaPan) receptor responds to DA with a net increase in [cAMP] that is mediated by Gs and Gz.     

Neurons with histaminelike immunoreactivity in the segmental and stomatogastric nervous systems of the crayfishPacifastacus leniusculus and the lobsterHomarus americanus

Summary We used a polyclonal antiserum against histamine to map histaminelike immunoreactivity (HLI) in whole mounts of the segmental ganglia and stomatogastric ganglion of crayfish and lobster. Carbodiimide fixation permitted both HRP-conjugated and FITC-conjugated secondary antibodies to be used effectively to visualize HLI in these whole mounts. Two interneurons that send axons through the inferior ventricular nerve (ivn) and the stomatogastric nerve to the stomatogastric ganglion had strong HLI, both in crayfish and in lobster. These ivn interneurons were known from other evidence to be histaminergic. The neuropil of the stomatogastric ganglion in both crayfish and lobster contained brightly labeled terminals of axons that entered the ganglion from the stomatogastric nerve. No neuronal cell bodies in this ganglion had HLI. Each segmental ganglion contained at least one pair of neurons with HLI. Some neurons in the subesophageal ganglion and in each thoracic ganglion labeled very brightly. Axons of projection interneurons with strong HLI occurred in the dorsal lateral tracts of each segmental ganglion, and sent branches to the lateral neuropils and tract neuropils of each ganglion. All the labeled neurons were interneurons; no HLI was observed in peripheral nerves.     

Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus

Summary The distribution of substance P-like immunoreactivity in the stomatogastric nervous systems of three decapod crustacean species, Cancer borealis, Homarus americanus, and Panulirus interruptus, was studied. The stomatogastric ganglion showed dense staining in the neuropil, but none in the somata. A single neuron stained in the esophageal ganglion. Lucifer yellow backfills and intracellular injections followed by incubation with the substance P antibody showed that the axons of this neuron project into the inferior esophageal nerves towards the paired commissural ganglia. The commissural ganglia showed a pronounced projection from a large bundle of fibers in the anterior medial portion of the circumesophageal connective. Additionally, less dense neuropil and stained somata were seen in the commissural ganglia. Staining was completely blocked by preabsorption with authentic substance P, physalaemin, eledoisin, and substance K. These data suggest that in the nervous system of crustacean species a molecule with C-terminal homology to substance P and other tachykinins is released as a neuroregulator in the stomatogastric ganglion.     

Neuromodulation for behavior in the locust frontal ganglion

Neuromodulators orchestrate complex behavioral routines by their multiple and combined effects on the nervous system. In the desert locust, Schistocerca gregaria, frontal ganglion neurons innervate foregut dilator muscles and play a key role in the control of foregut motor patterns. To further investigate the role of the frontal ganglion in locust behavior, we currently focus on the frontal ganglion central pattern generator as a target for neuromodulation. Application of octopamine, a well-studied insect neuromodulator, generated reversible disruption of frontal ganglion rhythmic activity. The threshold for the modulatory effects of octopamine was 10^–6 mol l^–1, and 10^–4 mol l^–1 always abolished the ongoing rhythm. In contrast to this straightforward modulation, allatostatin, previously reported to be a myoinhibitor of insect gut muscles, showed complex, tri-modal, dose-dependent effects on frontal ganglion rhythmic pattern. Using a novel cross-correlation analysis technique, we show that different allatostatin concentrations have very different effects not only on cycle period but also on temporal characteristics of the rhythmic bursts of action potentials. Allatostatin also altered the frontal ganglion rhythm in vivo. The analysis technique we introduce may be instrumental in the study of not fully characterized neural circuits and their modulation. The physiological significance of our results and the role of the modulators in locust behavior are discussed.Abbreviation CPG central pattern generator - FG frontal ganglion - JH juvenile hormone - STNS stomatogastric nervous system