Nitric oxide (NO) in Melibe

Nitric oxide is a gaseous neurotransmitter or neuromodulator produced by the enzyme nitric oxide synthase, in response to calcium. In Melibe NOS is found in two neurons in the brain and these cells project to the pedal ganglia, where many neurons controlling locomotion and swimming are located. Treatment of isolated brains, which spontaneously produce a swimming rhythm, with NO donors, slows and inhibits the rhythm. Stimulation of the NOS neurons has similar effects. These data are summarized, with figures, below. Currently, we are trying to determine the normal role played by these NOS cells and how they produce their effects on follower neurons and circuits. Most of the work described below was carried out in collaboration with Jim Newcomb, a graduate students in the laboratory and has been submitted for publication.

Anatomy I. Diaphorase staining

Histochemical staining of the Melibe brain using the NADPH-diaphorase method reveals only two bilaterally symmetricl neurons in the cerebropleural ganglion (Figure 1). These two cells do not stain if the appropriate controls are performed, or if NOS is blocked.

Interestingly, Melibe tentacles also stain for NOS, although the significance of the observation is not clear at the present time.

Anatomy II. Immunohistochemistry
Immunohistochemical staining of Melibe brains with anti-NOS antibodies results in staining of the same two neurons that are revealed with diaphorase histochemistry (Figure 2).

Anatomy III. Lucifer yellow injections
Injection of the NOS neurons with lucifer yellow revealed more detail about their structure. Double labelling with diaphorase confirmed that we injected the correct cells. See Figure 3.

Pharmacology and Physiology of NO in Melibe
Treatment of isolated Melibe brains with a NO-donor, such as sodium nitroprusside (SNP) alters the normal swim rhythm. It slows considerably and becomes more erratic (Figure 4). In intact animals this manifests itself as very slow swimming. The effect of NO donors is eliminated by perfusion with the NO scavenger hemoglobin. Theses same effects were obtained with cGMP analogues.

Stimulation of the NOS neuron also inhibits the swim rhythm (Figure 5).