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

• Cx36 is abundant in the vestibular nuclei (VN) of rat and mouse.
• Cx36 is localized to sites of contact between axon terminals and VN neurons.
• Axon terminals displaying Cx36 in VN are of primary afferent origin.
• Mixed synapses in the VN had a relatively late appearance developmentally.

Axon terminals forming mixed chemical/electrical synapses in the lateral vestibular nucleus of rat were described over 40 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 immunolabeling for Cx36 in the form of Cx36-puncta was found in each of the four major vestibular nuclei of adult rat and mouse. Immunolabeling 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 immunolabeled for vglut-1. These terminals and their associated Cx36-puncta were substantially depleted after labyrinthectomy. Developmentally, labeling 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 20–25. 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 contains 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.