Preproenkephalin mRNA is expressed in a subpopulation of GABAergic neurons in the spinal dorsal horn of the GAD67-GFP knock-in mouse

GABA (gamma-aminobutyric acid)ergic neurons in the spinal dorsal horn have been reported to be divided into distinctive populations, with different cotransmitters and neuropeptides. In this study, we examined the colocalization of enkephalin (ENK) mRNA with GABA in the spinal dorsal horn using the glutamic acid decarboxylase (GAD)(67)-green fluorescence protein (GFP) knock-in mouse. Our approach was to perform in situ hybridization histochemistry to detect mRNA for preproenkephalin (PPE, the precursor protein for ENK), combined with immunohistochemistry for GFP to reveal GABAergic neurons. Quantitative analysis indicated that more than 44.4% (2967/6681) of GFP-immunoreactive neurons showed signals for PPE mRNA in the spinal dorsal horn. While 53.9% (2967/5501) of PPE mRNA-expressing neurons were immunoreactive for GFP. The double-labeled neurons were observed throughout the spinal dorsal horn, although they had a preferential localization in superficial layers. The present results provide a detailed morphological evidence that ENK and GABA colocalized in a subpopulation of neurons in the spinal dorsal horn, which are likely to represent local inhibitory dorsal horn interneurons involved in the modulation of pain transmission.