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

Abstract

Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at “large myelinated club ending” synapses on Mauthner cells of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical “pre-potentials” immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified “mixed” (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with Cx35 restricted to axon terminal hemiplaques and Cx34.7 restricted to apposing Mauthner cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of Cx36 on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins.

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

“…The current study focuses on the distribution of Cx35 in dDO and dAO and confirms previous reports of electrical synapses in MG, T, and RF [Hinojosa, 1973;Korn et al, 1977;Rash et al, 2015]. Cx35 puncta are restricted to a subset of auditory projection neurons in three of the seven dDO cell populations previously identified in goldfish [McCormick and Wallace, 2012]: Ld-rf, Ld, and Lv.…”

“…The report by Rash et al [2015] that Cx35-positive neurons are present in medullary vestibular nuclei and in the reticular formation in goldfish is consistent with the localization of such neurons in vDO, vAO, MG, T, and RF in the current study. Straka and Baker [2013] provide an overview and evolutionary perspective of the primary vestibular nuclei in fish.…”

“…The observation that conductance favors retrograde flow has been correlated with molecular asymmetry in the gap junction hemiplaques, in that Cx35 is expressed on the presynaptic hemiplaque, whereas Cx34.7 is expressed on the postsynaptic hemiplaques . Such heterotypic gap junctions were subsequently reported in glutamatergic mixed synapses in areas of the goldfish hindbrain, midbrain, and forebrain [Rash et al, 2015].…”

“…observations] was qualitatively identical to that in a previous report [Pereda et al, 2003], it is possible that the 24-hour fixation period used in this study led to underrepresentation of Cx35 distribution in dDO and dAO. Rash et al [2015] noted that fixation limited to 60-90 min in paraformaldehyde picric acid fixative facilitated observation of Cx35 immunostaining in the goldfish. In the current study, the consistent observation of Cx35 in only some dDO cell populations and on neurons of particular morphology within those populations likely means that Cx35 is expressed only in these locations, although the fixation protocol may have diminished the density of observable Cx35 puncta and possibly the number of Cx35-positive neurons.…”

“…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). Mixed synapses in teleost fish and mammals have only been identified at glutamatergic synapses [reviewed in Rash et al, 2015].…”