A polyclonal antibody against allatostatin 1 (AST-1) of cockroach Diploptera punctata was used to investigate the distribution of AST-like immunoreactivity within the abdomen of the locust, Schistocerca gregaria. In the abdominal ganglia, AST-like immunoreactivity was found in both cell bodies and neuropile. In ganglia 6 and 7, staining was found in serial homologous cell bodies in anterior dorsolateral and dorsomedial, and posterior ventrolateral and ventromedial locations. In the terminal ganglion, the numerous immunoreactive somata were smaller in size than those in the unfused ganglia. The combination of backfill experiments with immunocytochemistry showed that, in abdominal ganglion 7, one neuron of the ventromedian cell body cluster and two of the ventrolateral cluster innervated the oviduct, which itself was covered with a dense mesh of AST-immunoreactive varicosities. Combining electron microscopy with immunohistochemistry revealed AST-like immunoreactivity in dense-core vesicles located in neurohaemal-like terminals lacking structures normally found in synapses. A mesh of AST-immunoreactive varicosities was also found on the muscles of the spermatheca and the spermathecal duct. In addition, a mesh of strongly stained varicosities was present in the distal perisympathetic organs (neurohaemal organs in the abdomen) and on the lateral heart nerves (a putative neurohaemal release zone). These data indicate that AST is an important neuroactive substance that is probably involved in multiple tasks in the control of the locust abdomen.

Neuropeptides are peptides with profound effects on the nervous system. The function of neuropeptides can be studied in detail in the stomatogastric nervous system (STNS). Neuropeptides are ubiquitously distributed in the STNS and it contains well‐studied neural circuits that are strongly modulated by neuropeptides. The STNS controls the movements of the foregut in crustaceans and has been studied intensively in a variety of decapod crustaceans including crayfish. This article reviews our knowledge of neuropeptides in the crayfish STNS. Within crayfish, peptides reach the circuits of the STNS as neurohormones released by neurohaemal organs or by putative neurohemal zones located within the STNS. As transmitters, neuropeptides are present in identified motoneurons, interneurons, and sensory neurons (mainly shown by immunocytochemistry), indicating a multiple role of peptides in the plasticity of neural networks. Neuropeptides are not only present in varicosities within the neuropil of ganglia, but also in varicosities on muscles and within small neuropil patches along nerves. This suggests that the muscles of the stomach are under a more direct modulatory control than previously thought, and that information processing can also occur within nerves. In addition to anatomical studies, biochemical and electrophysiological methods were used. For example, MALDI‐TOF MS (matrix‐assisted laser desorption ionization time of flight mass spectrometry) revealed the presence of four different peptides of the orcokinin family within a single neuron, and electrophysiological experiments demonstrated that the networks of the STNS are not only under excitatory but also inhibitory peptidergic influence. Comparing the similarities and differences between the STNS of crayfish and that of other decapod crustaceans has already contributed to our knowledge about peptides and will further help to unravel peptide function in the plasticity of neural circuits. For example, the identified neurons in the STNS can be used to study co‐transmission because neuropeptides are co‐localized with classical transmitters, biogenic amines, or other peptides in these neurons. Microsc. Res. Tech. 60:302–312, 2003. © 2003 Wiley‐Liss, Inc.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).

The stomatogastric nervous system (STNS) of decapod crustaceans has long been used to study the modulation of small neural circuits. Profiles in the sheath of the nerves and ganglia of the STNS, which contain only dense‐core vesicles, have been described in electron microscopical studies (Friend [1976] Cell Tissue Res. 175:369–380; Kilman and Marder [1997] Soc Neurosci Abstr. 23:477; Skiebe and Ganeshina [2000] J Comp Neurol 420:373–397). These profiles resemble those found in neurohemal organs and suggest the presence of neurohemal release zones in the STNS. To map these putative neurohemal release zones, a combination of two antibodies was used in the present study. A synapsin antibody recognizing vesicle proteins of clear vesicles was combined with a synaptotagmin antibody recognizing vesicle proteins of clear and dense‐core vesicles. Exclusive synaptotagmin‐like staining, therefore, indicated the regions with only dense‐core vesicles. Such a staining was found in a mesh in the perineural sheath of nerves in the STNS of all three species investigated. In the crayfish Cherax destructor and the lobster Homarus americanus , the stained mesh was located in the sheath of nerves connecting all four ganglia of the STNS, whereas in the crab Cancer pagurus it was found on different nerves, which are more directly exposed to the hemolymph in this species. Exclusive synaptotagmin‐like staining was also found in a putative neurohemal release zone in the sheath of the circumoesophageal connectives and the postoesophageal commissure in C. destructor . These data suggest that an important source of modulation of the networks and the muscles of the stomach is a compartmentalized release of neurohormones from zones in the STNS. J. Comp. Neurol. 453:280–291, 2002. © 2002 Wiley‐Liss, Inc.

The networks of the stomatogastric nervous system (STNS) have long been model systems to investigate how circuit dynamics relevant for behavior depend on peptide modulation. One focus in the system has been on peptides as cotransmitters released within the neuropil of the ganglia (see Marder et al., this Vol.). In addition, however, both anatomical and physiological data suggest that peptides also act on the networks and muscles of the STNS as neurohormones released by neurohemal organs and neurohemal release zones. The release zones in the STNS are located on the surface of connectives, commissures, nerves, and ganglia and contain dense-core vesicles. A paracrine release of peptides within the neuropil of the STG has also been suggested in two species. Peptides are also present in neuropil patches located in the core of particular nerves, suggesting that these nerves are not only important for information transfer, but may also be involved in modulation. These findings suggest that neuromodulation by peptides within the crustacean STNS is even more complex than previously assumed.

The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated orcokinin-like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM-c6), and a sensory neuron (anterior gastric receptor neuron). Five orcokinin-related peptides were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) post source decay fragmentation in samples of either the stomatogastric ganglion or the pericardial organs. Four of these peptides are identical to peptides derived from the cloned Procambarus clarkii precursor (Yasuda-Kamatani and Yasuda [2000] Gen. Comp. Endocrinol. 118:161-172), including the original [Asn(13)]-orcokinin (NFDEIDRSGFGFN, [M+H](+) = 1,517.7 Da), [Val(13)]-orcokinin ([M+H](+) = 1,502.7 Da), [Thr(8)-His(13)]-orcokinin ([M+H](+) = 1,554.8 Da), and FDAFTTGFGHS ([M+H](+) = 1,186.5 Da). The fifth peptide is a hitherto unknown orcokinin variant: [Ala(8)-Ala(13)]-orcokinin ([M+H](+) = 1,458.7 Da). The masses of all five peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other orcokinin-like immunoreactive neurons. In the oesophageal nerve, in which all the orcokinin-like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four orcokinin masses were detected in the anterior median nerves, in which all the orcokinin-like immunoreactivity derives from the AM-c6 neuron. This study therefore lays the groundwork to investigate the function of the orcokinin peptide family using single identified neurons in a well-studied system.

The stomatogastric nervous system (STNS) controls the movements of the foregut and the oesophagus of decapod crustaceans and is a good example for demonstrating that peptides are ubiquitously distributed chemical mediators in the nervous system. The stomatogastric ganglion (STG), one of the four ganglia of the STNS, contains the most intensively investigated neuronal circuits. The other ganglia, including the two commissural ganglia (CoGs) and the oesophageal ganglion (OG), are thought to be modulatory control centres. Peptides reach the STNS either as neurohormones or are released as transmitters. Peptide neurohormones can be released either from neurohaemal organs or from local neurohaemal release zones located on the surface of nerves and connectives. There were thought to be no peptidergic neurones with cell bodies in the STG itself. However, some have recently been described in adults of four species, in addition to a transient expression of peptides during development in two species. None of these peptidergic neurones has been investigated physiologically, in contrast to peptidergic neurones that project to the STG and have cell bodies in either the CoGs or the OG. It has been shown that neurones containing the same peptide elicit different motor patterns, that the peptide transmitter and the classical transmitter are not necessarily co-released and that the effect of a peptidergic neurone depends on its firing frequency and on which other modulatory neurones are co-active. The activity of modulatory projection neurones can be elicited by sensory neurones, and their activity can depend on the firing frequency of the sensory neurone. In addition to being found within the neuropile of ganglia, peptides are present in neuropile patches located within the nerves of the STNS, suggesting that these nerves can integrate as well as transfer information. Furthermore, sensory neurones and muscles exhibit peptide-like immunoreactivity and are modulated by peptides. Bath-applied peptides elicit peptide-specific motor patterns within the STG by targeting subsets of neurones. This divergence is contrasted by a convergence at the level of currents: five different peptides modulate a single current. Peptides not only induce motor patterns but can also switch the alliance of neurones from one network to another or are able to fuse different networks. In general, peptides are the most abundant group of modulators within the STNS; they are ubiquitously present, indicating that they play multiple roles in the plasticity of neural networks.

Patches of peptide-immunoreactive varicosities have been found in nerves of the stomatogastric nervous system (STNS) of decapod crustaceans. In the present study, these patches were examined in detail in the stomatogastric nerve (stn) and in the superior oesophageal nerve (son) of the crayfish Cherax destructor by using whole-mount immunocytochemical techniques combined with confocal microscopy and, in addition, electron microscopy. Double-labeling experiments with antibodies generated against the peptides allatostatin, FMRFamide and proctolin, combined with an antibody generated against the small vesicle protein synapsin, suggest that each patch contains small synaptic vesicles in addition to all three peptides. The neuropil regions of the ganglia of the STNS were also strongly stained by the synapsin antibody. Synapsin-like immunoreactivity was also studied in the crab Cancer pagurus and the lobster Homarus americanus. A similar pattern of staining was found for all three species, but the distribution within the stn varied. In H. americanus, a lightly stained weblike structure was found on the surface of nerves including the inferior oesophageal nerve, the son, and the anterior stn. By using electron microscopy, synapses were found in the core of the stn-son junction of C. destructor, in the same region where the synapsin-like and the peptide staining was localized. In addition, putative neurohemal release sites were found in the peripheral sheath of the stn. The presynaptic profiles found in the core of the stn seem to correspond to the types of presynaptic profiles found in the neuropil of the stomatogastric ganglion. These findings demonstrate that synaptic neuropil is present in the nerves of the STNS of a decapod crustacean.

To compare the stomatogastric nervous system of the crayfish Cherax destructor with those of other decapod species, the distribution of FLRF (Phe-Leu-Arg-Phe) amide-, proctolin- and crustacean cardioactive peptide (CCAP)-like immunoreactivities was studied in the stomatogastric nervous system and in neurosecretory structures by using wholemount immunocytochemical techniques and confocal microscopy. In addition, the number of cells in the stomatogastric ganglion (19-24) and axon profiles in the stomatogastric nerve (157-165) were counted. FLRFamide-like immunoreactivity was present within numerous cell bodies and neuropil of the commissural ganglia, in the neuropil of the stomatogastric ganglion, and in one cell body of the esophageal ganglion. FLRFamide-like immunoreactivity was also found in two cell bodies at the junction of the stomatogastric nerve with the superior esophageal nerve and in two cell bodies in the inferior ventricular nerve. Proctolin-like immunoreactivity was present in numerous cell bodies and neuropil of the paired commissural ganglia and in the neuropil of the stomatogastric ganglion. CCAP-like immunoreactivity was found in the neuropil and in one to four cell bodies in the commissural ganglia. Both proctolin- and CCAP-immunoreactive varicosities occurred on the surface of the circumesophageal connectives and on the postesophageal commissure, indicating a neurohemal source within the stomatogastric nervous system, which was verified by electron microscopy. The pericardial organs showed FLRFamide-, proctolin-, and CCAP-like immunoreactivity. This staining pattern suggests that FLRFamide-like and proctolin-like peptides are used as neurohormones and as neuromodulators in the stomatogastric nervous system of the crayfish C. destructor, whereas CCAP-like peptides may only affect the stomatogastric ganglion as a neurohormone. (C) 1999 Wiley-Liss, Inc.

The distribution of allatostatin (AST)-like immunoreactivity was studied in the stomatogastric nervous system (STNS) and the neurosecretory pericardial organs (PO) of four decapod crustacean species by using wholemount immunocytochemical techniques and confocal microscopy. AST-like immunoreactivity was found within the STNS of all four species; its distribution in each was unique. In all four species, AST-like immunoreactivity was present in the paired commissural ganglia (CoG), in the esophageal ganglion (OG), in the stomatogastric ganglion (STG), and in their connecting nerves. Within the CoGs, numerous cell bodies and neuropil were stained. In the OG, two cell bodies were immunoreactive, although their branching pattern varies between species. In the STG of C. pagurus and H. americanus, neuropil was stained extensively, but no labeled cell bodies were found. Surprisingly, in C. destructor and P. clarkii, cell bodies were stained in the STG, one brightly stained cell body in both species and an additional two to five weakly stained cell bodies in P. clarkii. In all four species, stained gastropyloric receptor cells were present. In contrast to the variable staining within the STNS, all four species have a similar pattern of AST-like immunoreactivity within the PO. Only in C. destructor, AST-immunoreactive varicosities occur on the surface of the circumesophageal connectives and on the postesophageal commissure and suggest another neurohaemal source for AST-Like peptides in this species. The pattern of this staining suggests that AST-like peptides are likely utilized as both neurohormones and as neuromodulators in the STNS of decapod crustacea. J. Comp. Neurol. 403:85-105, 1999. (C) 1999 Wiley-Liss, Inc.

In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.

The modulation of the pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis, by crustacean cardioactive peptide (CCAP) is described. CCAP activated pyloric rhythms in most silent preparations, and altered the phase relationships of pyloric motor neuron firing in all preparations. In CCAP, the pyloric rhythms were characterized by long lateral pyloric (LP) neuron bursts of action potentials. The threshold for CCAP action was ∼10−10 M, with increasing effects at higher CCAP concentrations. The changes in motor pattern evoked by CCAP produced significant changes in LP-innervated muscle movement. These movements were additionally potentiated by CCAP applications to isolated nerve–muscle preparations. Thus, enhanced motor neuron firing and increase of the gain of the neuromuscular junctions are likely to operate coordinately in response to hormonally released CCAP. High CCAP concentrations sometimes resulted in modification of the normal 1:1 alternation between the pyloric dilator (PD) and LP neurons to patterns of 2:1, 3:1, or 4:1 alternation. CCAP seems to activate slow intrinsic oscillations in the LP neuron, as well as enhance faster oscillations in the pacemaker group of PD/anterior burster (AB) neurons. Simulations of fast and slow oscillators with reciprocal inhibitory coupling suggest mechanisms that could account for the mode switch from 1:1 alternation to multiple PD bursts alternating with one LP neuron burst.

The crustacean stomatogastric ganglion, which is situated in the ophthalmic artery, can be modulated by both intrinsically released molecules and hormones. In the crab Cancer borealis, over a dozen neuroactive compounds have been identified in the input axons that project into the stomatogastric neuropil. However, little is known about the modulator content of the two major neurohemal organs, the sinus glands and the pericardial organs, in this crab. We now report the results of a series of immunocytochemical experiments designed to identify putative neurohormones in these tissues. We find that the majority of modulators present in the input axons of the stomatogastric ganglion are also present in at least one of the neurohemal organs. Specifically, allatostatin-like, buccalin-like, cholecystokinin-like, FLRFamide-like, GABA-like, locustatachykinin-like, myomodulin-like, proctolin-like, red pigment concentrating hormone-like and serotonin-like immunoreactivities are all present in both the stomatogastric neuropil and at least one of the neurohemal organs. Thus, these substances are likely to serve a dual role as both local and hormonal modulators of the stomatogastric network. Two other substances, beta-pigment dispersing hormone and crustacean cardioactive peptide, are not present in the stomatogastric neuropil, but beta-pigment dispersing hormone immunoreactivity is present in the sinus glands and crustacean cardioactive peptide immunoreactivity is present in the pericardial organs. It is likely that crustacean cardioactive peptide exerts its influence on the stomatogastric neural circuit via hormonal pathways. Double-labeling experiments show that the patterns of modulator co-localization present in the stomatogastric neuropil are different from those in the neurosecretory organs, suggesting that few rules of colocalization hold across these tissues.

The effects of four Diploptera punctata allatostatin peptides on the stomatogastric nervous system of the crab Cancer borealis were studied. All of the peptides had similar actions on the activity of neurons involved in rhythmic movements of the pyloric region of the stomach, decreasing the frequency of the pyloric rhythm in a dose-dependent manner. Diploptera allatostatin 3 (D-AST-3) was slightly more effective than the others. The absolute change in the frequency of the pyloric rhythm depended on the starting frequency, demonstrating that the effect of D-AST-3 depends on the preceding physiological state of the preparation. The largest decreases were observed when the starting frequency was slower than 0.8 Hz. Whole-mount immunocytochemistry with anti-Diploptera allatostatin 1 antibodies demonstrated the presence of allatostatin-like peptides in the paired commissural ganglia, the unpaired oesophageal ganglion and the stomatogastric ganglion, and in their connecting and motor nerves. Dense processes were labeled in the stomatogastric ganglion, 12-19 cell bodies and neuropil staining were found in each commissural ganglion, two cell bodies were stained in the oesophageal ganglion and two pairs of cell bodies, the gastropyloric receptor neurons, were stained in peripheral nerves.

The omega neurons of crickets are connected with each other by reciprocal inhibition. This inhibition could be mimicked by bath-applied histamine and blocked by histamine H1-antagonists. Histamine, like ON1, also influenced the ascending interneuron AN2, so that its response pattern more closely reflected the temporal structure of the calling song. This evidence strongly suggests that histamine is the inhibitory transmitter of the ON1s.