A quantitative study of neuronal nitric oxide synthase expression in laminae I–III of the rat spinal dorsal horn

Nitric oxide produced by neuronal nitric oxide synthase (nNOS) in the spinal cord is required for development of hyperalgesia in inflammatory and neuropathic pain states. nNOS is expressed by some dorsal horn neurons, and an early study that used a histochemical method to identify these cells suggested that they were mainly inhibitory interneurons. We have carried out a quantitative analysis of nNOS-immunoreactivity in laminae I-III of the rat dorsal horn, to determine the proportion of inhibitory and excitatory neurons and axonal boutons that express the protein. nNOS was present in ∼5% of neurons in laminae I and III, and 18% of those in lamina II. Although most cells with strong nNOS immunostaining were GABA-immunoreactive, two-thirds of the nNOS-positive cells in lamina II and half of those in lamina III were not GABAergic, and some of these expressed protein kinase Cγ (PKCγ). We estimate that nNOS is present in 17-19% of the inhibitory interneurons in laminae I-II, and 6% of those in lamina III. However, our results suggest that nNOS is also expressed at a relatively low level by a significant proportion (∼17%) of excitatory interneurons in lamina II. nNOS was seldom seen in boutons that contained vesicular glutamate transporter 2, which is expressed by excitatory interneurons, but was co-localised with the vesicular GABA transporter (VGAT, a marker for GABAergic and glycinergic axons). nNOS was detected in 13% of VGAT boutons in lamina I and in 7-8% of those in laminae II-III. However, it was only found in 2-4% of the VGAT boutons that were presynaptic to PKCγ-expressing interneurons in this region. These results indicate that nNOS is more widely expressed than previously thought, being present in both inhibitory and excitatory neurons. They provide further evidence that axons of neurochemically defined populations of inhibitory interneuron are selective in their post-synaptic targets.