Allatostatin-like immunoreactivity in the abdomen of the locust Schistocerca gregaria.

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 in the crayfish stomatogastric nervous system.

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.

Putative neurohemal release zones in the stomatogastric nervous system of decapod crustaceans.

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.

Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor.

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.