Localization and age-related changes of nitric oxide- and ANP-mediated cyclic-GMP synthesis in rat cervical spinal...

An immunocytochemical technique was used to study the localization and developmental aspects of cyclic GMP (cGMP)-synthesizing structures in the cervical spinal cord of 2-week and 3-month-old Lewis rats in response to the nitric oxide (NO) donor sodium nitroprusside (SNP) and/or atrial natriuretic peptide (ANP). By using cell-specific markers, the cell structures involved were investigated. To visualize cGMP, a combined technique of low- and high-power magnification, using a confocal laser scanning microscope was used. NOS-mediated cGMP synthesis was observed in the cervical spinal cord in laminae I, II and III in 14-day-old rats, which activity was mainly absent at the age of 3 months. The involvement of NO in the NMDA-mediated increase in cGMP immunostaining (cGMP-IS) was demonstrated by the absence of cGMP-IS in slices incubated in the presence of NMDA together with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This NO-mediated effect of NMDA on cGMP-IS was completely absent in the 3-month-old rats. ANP-mediated cGMP synthesis resulted in an increase in cGMP in laminae I and II, which was generally similar at both ages. Astrocytes in both white and gray matter were found to be cGMP-IS in the basal, NO- and ANP-stimulated conditions. Using confocal laser microscopy, NO-mediated cGMP synthesis was observed in large cholinergic terminals nearby motor neurons in the ventral horn. An extensive colocalization between NO-stimulated cGMP synthesis and parvalbumin-positive (GABAergic) neurons and fibers was observed in all laminae. In the ANP-stimulated condition, a colocalization with parvalbumin structures was found in laminae II and III. No NO- or ANP-mediated cGMP synthesis was found in fibers immunopositive for the presynaptic glutamate transporter, serotonin, or tyrosine hydroxylase.