Different populations of parvalbumin‐ and calbindin‐D28k‐immunoreactive neurons contain GABA and accumulate 3H‐D‐aspartate in the dorsal horn of the rat spinal cord

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Abstract
The colocalization of parvalbumin (PV), calbindin‐D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H‐D‐aspartate selectively were investigated in neurons of laminae I‐IV of the dorsal horn of the rat spinal cord. Following injection of 3H‐D‐aspartate into the basal dorsal horn (laminae IV‐VI), perikarya selectively accumulating 3H‐D‐aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H‐D‐aspartate were found exclusively in laminae I–III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H‐D‐aspartate‐labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H‐D‐aspartate. A high proportion of PV‐immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP‐immunoreactive perikarya were GABA‐negative. GABA‐immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I–IV. A significant proportion of the GABA‐negative but PV‐immunoreactive neurons also showed CaBP‐immunoreactivity in laminae IL and IV. These results show that out of the two calcium‐binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium‐binding proteins in the dorsal horn of the rat spinal cord.