Axonal mapping of the giant peptidergic neurons VD 1 and RPD 2 located in the CNS of the pond snail Lymnaea stagnalis, with particular reference to the innervation of the auricle of the heart

Abstract

VD 1 and RPD 2 are two giant neuropeptidergic neurons located respectively in the visceral and right parietal ganglion of the central nervous system (CNS) of the pond snail Lymnaea stagnalis. They are the most prominent representatives of a system of neurons expressing a gene that is similar to the gene expressed in R 15 of Aplysia californica. Both neuronal systems are involved in the regulation of cardiorespiratory phenomena. In the present study the axonal branches of VD 1 and RPD 2 were mapped using immunocytochemical and tracer studies. To this end the α1-antiserum (directed to one of the VD 1/RPD 2 neuropeptides) was used in combination with Lucifer yellow (LY) and Ni-lys tracers. In whole mount preparations of the CNS, immunostained axons of VD 1 and RPD 2 were observed to run to the pleural, cerebral and pedal ganglia and in several nerves. Upon LY injection of VD 1 thin axon branches were observed in the internal right parietal nerve. These run to the skin in the mantle area near the pneumostome and osphradium. The skin of the lips appeared to receive a similar innervation via the lip nerves. Thick LY filled axons of VD 1 and RPD 2 were observed in the intestinal nerve. They could be traced to the heart region. The pericardial branch of the intestinal nerve innervates the pericardium and heart (Ni-lys tracing). Immunocytochemically, using the α1-antiserum, it was demonstrated that this nerve branch carries the axons of VD 1 and RPD 2 to the venous side of the auricle, where they enter the pericardial cavity and ramify in the auricle (but not in the ventricle). Many immunopositive varicosities were observed on the auricular muscle fibers. Comparison of the stainings in the auricle with those obtained with antisera to the cardioactive neuropeptides FMRFamide and SCP showed no colocalization. The functional significance of the axonal distribution patterns of VD 1 and RPD 2 and the morphological and functional homology of the neurons to R 15 of Aplysia are discussed.