Here, we show that a nitric oxide synthase (NOS) pseudogene is expressed in the CNS of the snail Lymnaea stagnalis. The pseudo-NOS transcript includes a region of significant antisense homology to a previously reported neuronal NOS (nNOS)-encoding mRNA. This suggested that the pseudo-NOS transcript acts as a natural antisense regulator of nNOS protein synthesis. In support of this, we show that both the nNOS-encoding and the pseudo-NOS transcripts are coexpressed in giant identified neurons (the cerebral giant cells) in the cerebral ganglion. Moreover, reverse transcription-PCR experiments on RNA isolated from the CNS establish that stable RNA–RNA duplex molecules do form between the two transcripts in vivo. Using an in vitro translation assay, we further demonstrate that the antisense region of the pseudogene transcript prevents the translation of nNOS protein from the nNOS-encoding mRNA. By analyzing NOS RNA and nNOS protein expression in two different identified neurons, we find that when both the nNOS-encoding and the pseudo-NOS transcripts are present in the same neuron, nNOS enzyme activity is substantially suppressed. Importantly, these results show that a natural antisense mechanism can mediate the translational control of nNOS expression in the Lymnaea CNS. Our findings also suggest that transcribed pseudogenes are not entirely without purpose and are a potential source of a new class of regulatory gene in the nervous system.

The mollusc, Lymnaea stagnalis, has been used as a model to study the mechanisms of nitric oxide (NO)-dependent processes in the CNS. Putative NO-containing neurones in Lymnaea are localized in the buccal ganglia, predominantly in areas where sensory neurones known to regulate feeding are found. The NO-generating substance, S-nitrosocysteine (S-NC, 5 x 10⁻⁵–10⁻³ M) activates feeding movements of the buccal mass and modulates the activity of buccal motoneurones. An inhibitor of NO synthase, N^G-methyl-L-arginine (10⁻⁴ M) decreases the frequency of background buccal movements and has opposite effects to S-NC on the buccal motoneurones. We suggest that NO is a messenger in the CNS of Lymnaea and may be involved in coordination of feeding motor patterns.