Morphologically mixed chemical–electrical synapses formed by primary afferents in rodent vestibular nuclei as revealed by immunofluorescence detection of connexin36 and vesicular glutamate transporter-1

Abstract

Axon terminals forming mixed chemical/electrical synapses in the lateral vestibular nucleus of rat were described over forty years ago. Because gap junctions formed by connexins are the morphological correlate of electrical synapses, and with demonstrations of widespread expression of the gap junction protein connexin36 (Cx36) in neurons, we investigated the distribution and cellular localization of electrical synapses in the adult and developing rodent vestibular nuclear complex, using immunofluorescence detection of Cx36 as a marker for these synapses. In addition, we examined Cx36 localization in relation to that of the nerve terminal marker vesicular glutamate transporter-1 (vglut-1). An abundance of immunolabelling for Cx36 in the form of Cx36-puncta was found in each of the four major vestibular nuclei of adult rat and mouse. Immunolabelling was associated with somata and initial dendrites of medium and large neurons, and was absent in vestibular nuclei of Cx36 knockout mice. Cx36-puncta were seen either dispersed or aggregated into clusters on the surface of neurons, and were never found to occur intracellularly. Nearly all Cx36-puncta were localized to large nerve terminals immunolabelled for vglut-1. These terminals and their associated Cx36-puncta were substantially depleted after labyrinthectomy. Developmentally, labelling for Cx36 was already present in the vestibular nuclei at postnatal day 5, where it was only partially co-localized with vglut-1, and did not become fully associated with vglut-1-positive terminals until postnatal day 20 to 25. The results show that vglut-1-positive primary afferent nerve terminals form mixed synapses throughout the vestibular nuclear complex, that the gap junction component of these synapses contain Cx36, that multiple Cx36-containing gap junctions are associated with individual vglut-1 terminals and that the development of these mixed synapses is protracted over several postnatal weeks.

Immunocytochemical Evidence for Electrical Synapses in the Dorsal Descending and Dorsal Anterior Octaval Nuclei in the Goldfish, <b><i>Carassius auratus</i></b>

“…As mentioned previously, the overlap of eighth nerve terminals with Cx35 puncta in dDO and dAO requires further study, but it is consistent with such observations on the goldfish lateral dendrite of the Mauthner neuron [Pereda et al, 2003] and in rodents, where unilateral labyrinthectomy results in almost complete homolateral depletion of Cx36 puncta in the ventral cochlear nucleus as well as in primary vestibular nuclei [Nagy et al, 2013;Rubio and Nagy, 2015]. This observation is also consistent with other morphological and/or physiological reports of mixed synapses between eighth nerve fibers and vestibular nuclei in agnathan and gnathostome fishes [Stefanelli and Caravita, 1970;Hinojosa, 1973;Rovainen, 1974;Korn et al, 1977], frogs [Precht et al, 1974;Babalian and Shapovalov, 1984], lizards [Richter Brain Behav Evol 2019;93:34-49 DOI: 10.115993:34-49 DOI: 10.…”

“…Although the seven cell populations in the goldfish dDO, some of which contain morphologically varied neurons, have yet to be functionally analyzed, the localization of Cx35, and thus presumed electrical synapses, to just 3 of them parallels the restriction of purely electrical and mixed synapses within the primary auditory nuclei of rodents and rhesus monkeys Rubio, 2009, 2011;Apostolides and Trussell, 2013;Rubio and Nagy, 2015]. Finally, recent protocols that optimize visualization of purely electrical and mixed synapses in the CNS [Nagy et al, 2013;Rash et al, 2015;Rubio and Nagy, 2015], applied to species from a variety of taxa, may reveal undiscovered shared features that underlie important processing principles in the central auditory system across vertebrates.…”

“…Thus, much more information is needed to determine the extent to which inputs from the saccule, utricle, and additionally the lagena form mixed synapses in dDO and dAO. Since unilateral labyrinthectomy in rodents results in almost complete homolateral depletion of Cx36 puncta in cochlear and vestibular nuclei [Nagy et al, 2013;Rubio and Nagy, 2015], transection of individual otolithic end organ branches in fish could reveal the afferent source of mixed synapses in dDO and dAO. Additional supportive morphological evidence for the presence of mixed synapses in dDO and dAO would derive from transmission electron microscopy and immunohistological colocalization of Cx35 with vesicular glutamate transporter 1 (vglut1).…”

“…If auditory and other octaval neurons are electrically coupled by collateral branches of individual eighth nerve fibers, these neurons could be receiving a specific type of afferent input [Nagy et al, 2013]. Computations for sound localization, for example, likely require the comparison of information from afferents innervating specific hair cell orientation populations within and possibly among otolithic end organs.…”

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

“…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.…”