Connexin36 identified at morphologically mixed chemical/electrical synapses on trigeminal motoneurons and at primary afferent terminals on spinal cord neurons in adult mouse and rat

Abstract

Morphologically mixed chemical/electrical synapses at axon terminals, with the electrical component formed by gap junctions, is common in the CNS of lower vertebrates. In mammalian CNS, evidence for morphologically mixed synapses has been obtained in only a few locations. Here, we used immunofluorescence approaches to examine the localization of the neuronally expressed gap junction forming protein connexin36 (Cx36) in relation to the axon terminal marker vesicular glutamate transporter1 (vglut1) in spinal cord and trigeminal motor nucleus (Mo5) of rat and mouse. In adult rodents, immunolabelling for Cx36 appeared exclusively as Cx36-puncta, and was widely distributed at all rostro-caudal levels in most spinal cord laminae and in the Mo5. A high proportion of Cx36-puncta was co-localized with vglut1, forming morphologically mixed synapses on motoneurons, in intermediate spinal cord lamina, and in regions of medial lamina VII, where vglut1-containing terminals associated with Cx36 converged on neurons adjacent to the central canal. Unilateral transection of lumbar dorsal roots reduced immunolabelling of both vglut1 and Cx36 in intermediate laminae and lamina IX. Further, vglut1-terminals displaying Cx36-puncta were contacted by terminals labelled for glutamic acid decarboxylase65, which is known to be contained in presynaptic terminals on large diameter primary afferents. Developmentally, mixed synapses begin to emerge in the spinal cord only after the second to third postnatal week and thereafter increase to adult levels. Our findings demonstrate that axon terminals of primary afferent origin form morphologically mixed synapses containing Cx36 in broadly distributed areas of adult rodent spinal cord and Mo5.

Connexin36 expression in major centers of the auditory system in the CNS of mouse and rat: Evidence for neurons forming purely electrical synapses and morphologically mixed synapses

“…Though not evident at low magnification (Fig. 1), antibody labelling of Cx36 appears exclusively as immunofluorescence puncta (referred to here as Cx36-puncta), at least under the immunohistochemical protocols used here, which is qualitatively similar to the Cx36-puncta we detect in other CNS areas of mammals and fish (Curti et al, 2012; Nagy, 2012; Nagy et al, 2013; Bautista and Nagy, 2014; Bautista et al, 2014; Rash et al, 2014). Cx36-puncta are localized to the surface or periphery ( i.e., plasma membranes) of neuronal cell bodies and dendrites, as determined by confocal scanning through individual cells in the z-dimension.…”

“…The principle connexin component of neuronal gap junctions in mammalian systems is connexin36 (Cx36), which has been documented to occur in ultrastructurally-identified gap junctions between neurons (Rash et al, 2000, 2001), and which supports electrical synaptic transmission in many regions of the CNS (Bennett and Zukin, 2004; Connors and Long, 2004; Hormuzdi et al, 2004; Sohl et al, 2005; Meier and Dermietzel; 2006 Bautista et al, 2012). Immunohistochemical visualization of Cx36 in gap junctions at the ultrastructural level is well-correlated with its localization by immunofluorescence, at least in vivo (Rash et al, 2004, 2005, 2007a,b), a fortuitous feature arising from what appears to be immunolabelling and detection of Cx36 exclusively at gap junctions, with its other potential subcellular and intracellular sites apparently remaining masked and undetectable with currently available anti-Cx36 antibodies (Nagy, 2012; Nagy et al, 2013; Bautista and Nagy, 2014; Bautista et al, 2014). Thus, Cx36 represents at least one marker for allowing light microscopic immunofluorescence identification of Cx36-containing neuronal gap junctions and reveals their cellular localization.…”

“…Several methodological points, however, warrant emphasis. First, we have repeatedly noted that optimal immunofluorescence labelling with these antibodies is highly dependent on appropriate tissue fixation conditions, because detection of Cx36 appears to be notoriously sensitive to fixation (Nagy, 2012; Nagy et al, 2013; Bautista and Nagy, 2014; Bautista et al, 2014). Labelling of Cx36 is best with a 1% formaldehyde/picric acid fix, is significantly diminished with a 2% concentration of this fix, and is obliterated with a 4% concentration.…”

“…In mammalian CNS, evidence for mixed synapses has been found only at a few locations, including rat lateral vestibular nucleus (LVN) (Korn et al, 1973; Wylie, 1973; Sotelo and Korn, 1978; Nagy et al, 2013), spinal cord (Rash et al, 1996; Bautista et al, 2014) and hippocampus (Vivar et al, 2012; Nagy, 2012). The present findings suggest multiple additional sites where axon terminals form mixed synapses in the auditory system of mouse and rat.…”

Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

“…In view of the above, it is interesting to consider the possibility that the heterologous coupling between neurons seen so widely in goldfish hindbrain and spinal cord requires an asymmetric heterotypic configuration of connexins, whereas abundant homologous coupling at purely electrical synapse between similar neuronal classes in mammals may be a consequence of a single connexin being expressed in both sides of gap junctions in these neurons. While there is evidence for heterologous coupling ( i.e., between dissimilar neuronal classes) at a few sites in mammalian CNS, involving both mixed synapses (Korn et al, 1973; Nagy, 2012; Nagy et al, 2013; Bautista et al, 2014) and presumptive purely electrical synapses (Apostolides and Trussell, 2013), it is not yet clear whether the gap junctions at these heterologous couplings consist of homotypic or heterotypic junctions, and if homotypic, whether they have asymmetric molecular modifications of Cx36.…”