Association of connexin36 with adherens junctions at mixed synapses and distinguishing electrophysiological features of those at mossy fiber terminals in rat ventral hippocampus.

BACKGROUND: Granule cells in the hippocampus project axons to hippocampal CA3 pyramidal cells where they form large mossy fiber terminals. We have reported that these terminals contain the gap junction protein connexin36 (Cx36) specifically in the stratum lucidum of rat ventral hippocampus, thus creating morphologically mixed synapses that have the potential for dual chemical/electrical transmission. METHODOLOGY: Here, we used various approaches to characterize molecular and electrophysiological relationships between the Cx36-containing gap junctions at mossy fiber terminals and their postsynaptic elements and to examine molecular relationships at mixed synapses in the brainstem. RESULTS: In rat and human ventral hippocampus, many of these terminals, identified by their selective expression of vesicular zinc transporter-3 (ZnT3), displayed multiple, immunofluorescent Cx36-puncta representing gap junctions, which were absent at mossy fiber terminals in the dorsal hippocampus. In rat, these were found in close proximity to the protein constituents of adherens junctions (i.e., N-cadherin and nectin-1) that are structural hallmarks of mossy fiber terminals, linking these terminals to the dendritic shafts of CA3 pyramidal cells, thus indicating the loci of gap junctions at these contacts. Cx36-puncta were also associated with adherens junctions at mixed synapses in the brainstem, supporting emerging views of the structural organization of the adherens junction-neuronal gap junction complex. Electrophysiologically induced long-term potentiation (LTP) of field responses evoked by mossy fiber stimulation was greater in the ventral than dorsal hippocampus. CONCLUSIONS: The electrical component of transmission at mossy fiber terminals may contribute to enhanced LTP responses in the ventral hippocampus.

Patterns of connexin36 and eGFP reporter expression among motoneurons in spinal sexually dimorphic motor nuclei in mouse.

BACKGROUND: Sexually dimorphic spinal motoneurons (MNs) in the dorsomedial nucleus (DMN) and dorsolateral nucleus (DLN) as well as those in the cremaster nucleus are involved in reproductive behaviours, and the cremaster nucleus additionally contributes to testicular thermoregulation. It has been reported that MNs in DMN and DLN are extensively linked by gap junctions forming electrical synapses composed of connexin36 (Cx36) and there is evidence that subpopulation of MNs in the cremaster nucleus are also electrically coupled by these synapses. METHODOLOGY: We used immunofluorescence methods to detect enhanced green fluorescent protein (eGFP) reporter for Cx36 expression in these motor nuclei. RESULTS: We document in male mice that about half the MNs in each of DMN and DLN express eGFP, while the remaining half do not. Further, we found that the eGFP+ vs. eGFP- subsets of MNs in each of these motor nuclei innervate different target muscles; eGFP+ MNs in DMN and DLN project to sexually dimorphic bulbocavernosus and ischiocavernosus muscles, while the eGFP- subsets project to sexually non-dimorphic anal and external urethral sphincter muscles. Similarly, eGFP+ vs. eGFP- cremaster MNs were found to project to anatomically distinct portions of the cremaster muscle. By immunofluorescence, nearly all motoneurons in both DMN and DLN displayed punctate labelling for Cx36, including at eGFP+/eGFP+, eGFP+/eGFP- and eGFP-/eGFP- cell appositions. CONCLUSIONS: Most if not all motoneurons in DMN and DLN are electrically coupled, including sexually dimorphic and non-dimorphic motoneurons with each other, despite absence of eGFP reporter in the non-dimorphic populations in these nuclei that have selective projections to sexually non-dimorphic target muscles.

Connexin36 localization along axon initial segments in the mammalian CNS.

Electrical synapses formed by gap junctions occur at a variety of neuronal subcellular sites in the mammalian central nervous system (CNS), including at somatic, dendritic and axon terminal compartments. Numerous electrophysiological studies using mice and rats, as well as computer modelling approaches, have predicted the additional occurrence of electrical synapses between axons near their emergence from neuronal somata. Here, we used immunofluorescence methods to search for localization of the neuronal gap junction-forming protein connexin36 (Cx36) along axon initial segments (AISs) labelled for the AIS marker ankyrinG. Immunofluorescent Cx36-puncta were found to be associated with AISs in several CNS regions of mice, including the spinal cord, inferior olive and cerebral cortex. Localization of Cx36-puncta at AISs was confirmed by confocal single scan and 3D imaging, immunofluorescence intensity profiling and high resolution structured illumination microscopy (SIM). AISs measuring up to 30 µm in length displayed typically a single Cx36-punctum and the incidence of these long AISs displaying Cx36-puncta ranged from 3% to 7% in the inferior olive and in various layers of the cerebral cortex. In the inferior olive, the gap junction associated protein zonula occludens-1 (ZO-1) was found to be co-localized with Cx36-puncta on AISs, indicating that these puncta have some of the molecular constituents of gap junctions. Our results add to the neuronal subcellular locations at which Cx36 is deployed, and raise possibilities for its involvement in novel functions in the AIS compartment.