Synaptic Targets of Glycinergic Neurons in Laminae I-III of the Spinal Dorsal Horn.

A great deal of evidence supports the inevitable importance of spinal glycinergic inhibition in the development of chronic pain conditions. However, it remains unclear how glycinergic neurons contribute to the formation of spinal neural circuits underlying pain-related information processing. Thus, we intended to explore the synaptic targets of spinal glycinergic neurons in the pain processing region (laminae I-III) of the spinal dorsal horn by combining transgenic technology with immunocytochemistry and in situ hybridization accompanied by light and electron microscopy. First, our results suggest that, in addition to neurons in laminae I-III, glycinergic neurons with cell bodies in lamina IV may contribute substantially to spinal pain processing. On the one hand, we show that glycine transporter 2 immunostained glycinergic axon terminals target almost all types of excitatory and inhibitory interneurons identified by their neuronal markers in laminae I-III. Thus, glycinergic postsynaptic inhibition, including glycinergic inhibition of inhibitory interneurons, must be a common functional mechanism of spinal pain processing. On the other hand, our results demonstrate that glycine transporter 2 containing axon terminals target only specific subsets of axon terminals in laminae I-III, including nonpeptidergic nociceptive C fibers binding IB4 and nonnociceptive myelinated A fibers immunoreactive for type 1 vesicular glutamate transporter, indicating that glycinergic presynaptic inhibition may be important for targeting functionally specific subpopulations of primary afferent inputs.

Morphological and neurochemical characterization of glycinergic neurons in laminae I-IV of the mouse spinal dorsal horn.

A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor ß (RORß), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing.

Differential expression of Na+/K+/Cl- cotransporter 1 in neurons and glial cells within the superficial spinal dorsal horn of rodents.

Although convincing experimental evidence indicates that Na+/K+/Cl- cotransporter 1 (NKCC1) is involved in spinal nociceptive information processing and in the generation of hyperalgesia and allodynia in chronic pain states, the cellular distribution of NKCC1 in the superficial spinal dorsal horn is still poorly understood. Because this important piece of knowledge is missing, the effect of NKCC1 on pain processing is still open to conflicting interpretations. In this study, to provide the missing experimental data, we investigated the cellular distribution of NKCC1 in the superficial spinal dorsal horn by immunohistochemical methods. We demonstrated for the first time that almost all spinal axon terminals of peptidergic nociceptive primary afferents express NKCC1. In contrast, virtually all spinal axon terminals of nonpeptidergic nociceptive primary afferents were negative for NKCC1. Data on the colocalization of NKCC1 with axonal and glial markers indicated that it is almost exclusively expressed by axon terminals and glial cells in laminae I-IIo. In lamina IIi, however, we observed a strong immunostaining for NKCC1 also in the dendrites and cell bodies of PV-containing inhibitory neurons and a weak staining in PKCγ-containing excitatory neurons. Our results facilitate further thinking about the role of NKCC1 in spinal pain processing.