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NOS immunoreactivity was assayed in CNS and peripheral tissues of the sea slugs Pleurobranchaea californica, Tritonia diomedea and Aplysia californica using different antisera against mammalian nitric oxide synthase in Western blots. Polyclonal anti-nNOS labeled at 250, 185, 170, 155, 100, 75, and 65 kD in extracts of Pleurobranchaea CNS, salivary gland and esophagus but not of gills or muscle. The labeling pattern for Tritonia in bands at 250, 200, 120/110, 100, 69, 65, and 60 kD differed somewhat. Anti-nNOS labeling in Aplysia was markedly different, with bands labeled only at 69 and 60 kD in CNS extracts, and at 200, 190, 69 and 60 kD in salivary and esophagus extracts. The wide variation in NOS immunoreactivity is consistent with species differences in tissue localization and biochemical properties of molluscan NOS isoforms., (Copyright 1999 Academic Press.)

Putative nitric oxide synthase (NOS) activity was assayed in molluscan CNS through histochemical localization of NADPH-diaphorase and through measurement of L-arginine/L-citrulline conversion. Several hundreds of NADPH-dependent diaphorase-positive neurons stained consistently darkly in the nervous system of the predatory opisthobranch Pleurobranchaea californica, whereas stained neurons were relatively sparse and/or light in the other opisthobranchs (Philine, Aplysia, Tritonia, Flabellina, Cadina, Armina, Coriphella, and Doriopsilla sp.) and cephalopods (Sepia and Rossia sp.). L-Arginine/L-citrulline conversion was beta-NADPH dependent, insensitive to removal of Ca2+, inhibited by the calmodulin blocker trifluoperazine, and inhibited by the competitive NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) but not D-NAME. Inhibitors of arginase [L-valine and (+)-S-2-amino-5-iodoacetamidopentanoic acid)] did not affect L-citrulline production in the CNS. NOS activity was largely associated with the particulate fraction and appeared to be a novel, constitutive Ca(2+)-independent isoform. Enzymatic conversion of L-arginine/L-citrulline in Pleurobranchaea and Aplysia CNS was 4.0 and 9.8%, respectively, of that of rat cerebellum, L-Citrulline formation in gill and muscle of Pleurobranchaea was not significant. The localization of relatively high NOS activity in neuron somata in the CNS of Pleurobranchaea is markedly different from the other opisthobranchs, all of which are grazers. Potentially, this is related to the animal's opportunistic predatory lifestyle.

1. The ventral white cells (VWC's) of the buccal ganglion of Pleurobranchaea, so named for their position and color, are a bilateral pair of neuron somata. Each sends a single axon out its contralateral stomatogastric nerve and has a dendritic field originating close to the soma. 2. The vwcs exhibit spontaneous episodes of prolonged depolarization (duration 1--4 min) accompanied by repetitive action-potential activity and separated by regular intervals of 3--30 min. Such prolonged burst episodes can be triggered by short pulses of depolarizing current. During the repetitive activity of the spontaneous bursts or that driven by imposed depolarization, the action potentials progressively broaden to 5--16 times their initial duration. 3. During spontaneous bursting or activity driven by imposed depolarization, the cyclic motor output of the feeding network is initiated or accelerated with a latency corresponding with the development of appreciable VWC spike broadening. When broadening of antidromic VWC spikes is suppressed by imposed hyperpolarization of the soma, the frequency of feeding cycles is significantly lower than when broadened spikes are allowed to develop. When trains of spikes are driven by depolarizing current, the motor output of the feeding network is not initiated until the VWC spikes have broadened to a repeatable "threshold" duration, regardless of the intensity of the depolarizing current. 4. The endogenous production of prolonged burst episodes, triggered by depolarizing current pulses, and progressive spike broadening can be demonstrated in the surgically isolated VWC soma. 5. The paired VWCs are strongly electrically coupled and display highly synchronous activity. They receive synaptic inputs from many previously identified interneurons of the feeding network and are thus reciprocally coupled within the network. 6. These results demonstrate that the capacity of this neuron to generate broadened action potentials during repetitive activity confers the ability to command coordinated motor-network output. The appropriate repetitive activity can be produced endogenously in the form of prolonged bursts of spikes.