Two selected points (labeled with 1 and 2) of colocalization between the markers are at the crossing point of two lines indicating the planes through which orthogonal views of XCZ and YCZ projections were drawn. it is almost exclusively expressed by axon terminals and glial cells in laminae ICIIo. 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 PKC-containing excitatory neurons. Our results facilitate further thinking about the role of NKCC1 in spinal pain processing. strong class=”kwd-title” Subject terms: Cell biology, Pain Introduction Cation-chloride-cotransporters are crucially important in the regulation of intracellular and extracellular chloride concentrations. Although there are nine members of the cation-chloride cotransporter family1C3, Cl? gradient across the cell membranes of neurons is controlled by only two such proteins: K+/Cl? cotransporter 2 (KCC2) and Na+/K+/2Cl? cotransporter 1 (NKCC1)4C6. KCC2 extrudes chloride from the cytosol, whereas NKCC1 moves chloride ions into the cells7C11. Thus, NKCC1 is responsible for the intracellular accumulation of chloride12,13 and, acting alone or in an antagonistic relationship with KCC2, it sets the equilibrium potentials for GABAA and glycine receptor channels3. By serving as a primary regulator of the hyperpolarizing or depolarizing effects of GABAA and glycine receptor activation, NKCC1 acts as one of the key players in shaping complex neural network activity14,15, including spinal EIPA hydrochloride nociceptive information EIPA hydrochloride processing6,16C19. Although the pro-nociceptive role of NKCC1 in spinal pain processing has been convincingly demonstrated4,6,20, its effect on hyperalgesia and allodynia at the cellular level is still open to conflicting interpretation. The reason for this ambiguity is that inconsistent and contradictory results prevent generalization from being made about the cellular distribution of NKCC1. There is no general agreement on whether NKCC1 in the spinal Mouse monoclonal to CD41.TBP8 reacts with a calcium-dependent complex of CD41/CD61 ( GPIIb/IIIa), 135/120 kDa, expressed on normal platelets and megakaryocytes. CD41 antigen acts as a receptor for fibrinogen, von Willebrand factor (vWf), fibrinectin and vitronectin and mediates platelet adhesion and aggregation. GM1CD41 completely inhibits ADP, epinephrine and collagen-induced platelet activation and partially inhibits restocetin and thrombin-induced platelet activation. It is useful in the morphological and physiological studies of platelets and megakaryocytes cord is expressed by neurons and/or glial cells21,22. There are also contradictory results regarding whether this protein is distributed in all primary afferent neurons23C25 or only in certain subsets of them26. Despite much convincing evidence that at least some neurons in dorsal root ganglia express NKCC1, there have been no reports EIPA hydrochloride on the localization of the transporter EIPA hydrochloride in the spinal axon terminals of nociceptive afferents. We intended to contribute to this debate and provide experimental data on which the pro-nociceptive role of NKCC1 as well as its effect on hyperalgesia and allodynia can be more accurately interpreted. Thus, by using a very specific and highly sensitive antibody against NKCC1, we investigated the neuronal and glial localization of NKCC1 in the nociceptive recipient layers (laminae ICII) of the spinal dorsal horn by immunohistochemical techniques. Our results provide a set of valuable new data about the neuronal and glial localization of NKCC1 within the superficial spinal dorsal horn and thus facilitate further thinking about the role of NKCC1 in spinal pain processing. Results Specificity of the NKCC1 antibody To test the specificity of the antibody raised against NKCC1, we stained sections obtained from the spinal cord of NKCC1 knockout and wild type mice, and carried out a Western blot analysis. No specific staining was observed in sections obtained from NKCC1 knockout mice (Fig.?1b). The EIPA hydrochloride dorsal horn of wild type mice, however, was heavily stained, and the pattern of immunostaining was similar to that observed in the rat (Fig.?1 a, d). The Western blot analysis showed only one immunostained band at the molecular weight of the glycosylated NKCC1 protein (~?160?kDa; Fig.?1c). Open in a separate window Figure 1 Specificity of the anti-NKCC1 antibody and distribution of NKCC1 immunoreactivity in the spinal dorsal horn. aCb. Photomicrographs showing.