The anterior cardiac plexus: an intrinsic neurosecretory site within the stomatogastric nervous system of the crab Cancer productus.

The stomatogastric nervous system (STNS) of decapod crustaceans is modulated by both locally released and circulating substances. In some species, including chelate lobsters and freshwater crayfish, the release zones for hormones are located both intrinsically to and at some distance from the STNS. In other crustaceans, including Brachyuran crabs, the existence of extrinsic sites is well documented. Little, however, is known about the presence of intrinsic neuroendocrine structures in these animals. Putative intrinsic sites have been identified within the STNS of several crab species, though ultrastructural confirmation that these structures are in fact neuroendocrine in nature remains lacking. Using a combination of anatomical techniques, we demonstrate the existence of a pair of neurosecretory sites within the STNS of the crab Cancer productus. These structures, which we have named the anterior cardiac plexi (ACPs), are located on the anterior cardiac nerves (acns), which overlie the cardiac sac region of the foregut. Each ACP starts several hundred micro m from the origin of the acn and extends distally for up to several mm. Transmission electron microscopy done on these structures shows that nerve terminals are present in the peripheral portion of each acn, just below a well defined epineurium. These terminals contain dense-core and, occasionally, electron-lucent vesicles. In many terminals, morphological correlates of hormone secretion are evident. Immunocytochemistry shows that the ACPs are immunopositive for FLRFamide-related peptide. All FLRFamide labeling in the ACPs originates from four axons, which descend to these sites through the superior oesophageal and stomatogastric nerves. Moreover, these FLRFamide-immunopositive axons are the sole source of innervation to the ACPs. Collectively, our results suggest that the STNS of C. productus is not only a potential target site for circulating hormones, but also serves as a neuroendocrine release center itself.

Immunocytochemical evidence for nitric oxide- and carbon monoxide-producing neurons in the stomatogastric nervous system of the crayfish Cherax quadricarinatus.

Nitric oxide (NO) and carbon monoxide (CO) have been shown to serve neuromodulatory roles in both vertebrates and invertebrates. Here, we use antibodies to their respective biosynthetic enzymes, nitric oxide synthase (NOS) and heme oxygenase 2 (HO-2), to map the distribution of putative gas-producing neurons in the stomatogastric nervous system (STNS) of the crayfish Cherax quadricarinatus. In this species, NOS immunolabeling is found in the neuropil of the stomatogastric ganglion (STG). This staining originates from two immunopositive axons that project to the STG through the superior oesophageal and stomatogastric nerves, presumably from cell bodies located in the commissural ganglia (CoGs). HO-2 immunoreactivity is present in small diameter fibers and varicosities in the periphery of nerves located in the anterior portion of the STNS. This labeling originates from approximately 12 somata in each CoG. Transmission electron microscopy done on the nerves of the anterior STNS shows they contain a neuroendocrine plexus. Collectively, our results indicate that NO- and CO-producing neurons are likely to exist in the crayfish STNS. Moreover, these gases appear to be produced by distinct subsets of the neurons present there. The localization of NO to the STG neuropil suggests that it serves as a locally released modulator or is involved in the local release of other substances within this ganglion. The presence of CO in the neurohemal plexus of the anterior STNS suggests that it serves as a circulating hormone or is involved in the control of neuroendocrine release from this plexus.

Stereotyped neuropil branching of an identified stomatogastric motor neuron.

Anatomical studies of the crab stomatogastric ganglion (STG) have suggested only minimal organization within the neuropil of this structure. Here, we present evidence that, for at least one intrinsic neuron type, the ventricular dilator (VD) neuron, a highly organized and stereotyped branching structure exists within the stomatogastric neuropil. Specifically, we show the morphology of the VD neuron consists of a single primary neurite that projects from the soma into the neuropil and bifurcates into a pair of subprimary neurites, which in turn exit the neuropilar region, one entering the left and the other the right medial ventricular nerve. Nearly all secondary neurite branching of the VD neuron is from the subprimary neurites. There are approximately 22 secondary branches/neuron (range 14-28), with no significant difference between the number of secondary branches off the right vs. the left subprimary neurite, although the ratio of secondary branches between subprimaries varies (range 0.4-1.6). The fine neurites that branch from the secondary processes segregate hemispherically within the neuropil, based on the subprimary neurite of origin. Within this hemispherical organization, another level of fine neurite segregation is present, namely, the fine neurites derived from each secondary branch are restricted to discrete regions of the hemisphere with only minimal overlap with those derived from other secondary branches. Monte Carlo simulations show that this segregation differs significantly from a random distribution. The organization of branching seen in the VD neuron may play a critical role in the electrotonic and local computational organization of this neuron and sets the stage for physiological experimentation addressing these issues.

Expression of nitric oxide synthase and nitric oxide-sensitive guanylate cyclase in the crustacean cardiac ganglion.

The cardiac ganglion is a simple central pattern-generating network that controls the rhythmic contractions of the crustacean heart. Enzyme assays and Western blots show that whole heart homogenates from the crab Cancer productus contain high levels of nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of arginine to citrulline with concomitant production of the transmitter nitric oxide (NO). Crab heart NOS is calcium-dependent and has an apparent molecular weight of 110 kDa. In the cardiac ganglion, antibodies to NOS and citrulline indicate the presence of a NOS-like protein and NOS enzymatic activity in the four small pacemaker neurons and the five large motor neurons of the cardiac network. In addition, all cardiac neurons label positively with an antibody to cyclic guanosine monophosphate (cGMP). The NO donor sodium nitroprusside (SNP, 10 mM) stimulates additional cGMP production in the isolated ganglion. This increase is blocked by [(1)H](1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ, 50 microM), an inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). Taken together, our data indicate that NO- and cGMP-mediated signaling pathways are enriched in the cardiac system relative to other crab tissues and that the cardiac network may be a target for extrinsic and intrinsic neuromodulation via NO produced from the heart musculature and individual cardiac neurons, respectively. The crustacean cardiac ganglion is therefore a promising system for studying cellular and synaptic mechanisms of nitrergic neuromodulation in a simple pattern-generating network.