Immunocytochemical localization of calretinin-containing neurons in the rat periaqueductal gray and colocalization with enzymes producing nitric oxide: a double, double-labeling study.

The pattern of distribution and colocalization of the calcium-binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH-diaphorase (NADPH-d) histochemistry and with NOS immunocytochemistry, respectively. Cal-immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl). Double-labeled neurons were found only in PAG-dl. The first experimental approach indicated that 33-41% of the NADPH-d-positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH-d activity appeared in 19-26% of the Cal-immunopositive (Cal(IP) ) neurons. Two-color immunofluorescence revealed that ∼39-43% of NOS-immunoreactive (NOS(IR) ) neurons were double-labeled with Cal and ∼23% of Cal(IP) neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG-dl, showed that Cal(IP) neurons had a cross-sectional area of 94.7 µm², whereas Nadph+ neurons and double-labeled neurons were slightly smaller, having a cross-sectional area of 90.5 and 91.4 µm², respectively. On electron microscopy, Cal(IP) axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially Cal(IP) dendrites. These experiments suggest that PAG-dl is characterized by a high degree of heterogeneity.