Abstract
Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I–III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.
Highlights
In the adult mammalian central nervous system (CNS) gap junctions provide for rapid metabolic and electrical intercellular communication between neurones and glia (Nagy and Rash 2000)
A Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception
Determining the identity and distribution of the connexins in the dorsal horn is of interest because recent data imply a role for gap junctions localized to neurones or glia in rhythmic activity manifest in rat dorsal horn circuitry in vitro (Asghar et al 2005; Chapman et al 2009) and gap junctions are implicated in pain facilitation and inflammation-induced nociceptive behaviours (Lan et al 2007; Qin et al 2006)
Summary
In the adult mammalian central nervous system (CNS) gap junctions provide for rapid metabolic and electrical intercellular communication between neurones and glia (Nagy and Rash 2000). Profiling studies in brain indicate that Cx subtypes are differentially localized to glia and neurones Cx36 and Cx45 are unambiguously present in neurones (Condorelli et al 1998; Maxeiner et al 2003; Hombach et al 2004) whereas Cx43 and Cx30 are widely present in astrocytes (Theis et al 2005). Understanding the identity and expression patterns of Cx proteins is a prerequisite to study the functional significance of gap junctions in the mature CNS. Few studies have described in detail the distribution of gap junctions in adult rat spinal cord, but freeze-fracture studies revealed an abundance of mixed electrical/chemical synapses distributed across the grey matter of laminae III–IX (Rash et al 1996). Regarding specific Cx subtypes, spinal cord expression profiles to date parallel those reported for brain. Determining the identity and distribution of the connexins in the dorsal horn is of interest because recent data imply a role for gap junctions localized to neurones or glia in rhythmic activity manifest in rat dorsal horn circuitry in vitro (Asghar et al 2005; Chapman et al 2009) and gap junctions are implicated in pain facilitation and inflammation-induced nociceptive behaviours (Lan et al 2007; Qin et al 2006)
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