Variability of distribution of Ca(2+)/calmodulin-dependent kinase II at mixed synapses on the mauthner cell: colocalization and association with connexin 35.

In contrast to chemical transmission, few proteins have been shown associated with gap junction-mediated electrical synapses. Mixed (electrical and glutamatergic) synaptic terminals on the teleost Mauthner cell known as "Club endings" constitute because of their unusual large size and presence of connexin 35 (Cx35), an ortholog of the widespread mammalian Cx36, a valuable model for the study of electrical transmission. Remarkably, both components of their mixed synaptic response undergo activity-dependent potentiation. Changes in electrical transmission result from interactions with colocalized glutamatergic synapses, the activity of which leads to the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII), required for the induction of changes in both forms of transmission. However, the distribution of this kinase and potential localization to electrical synapses remains undetermined. Taking advantage of the unparalleled experimental accessibility of Club endings, we explored the presence and intraterminal distribution of CaMKII within these terminals. Here we show that (1) unlike other proteins, both CaMKII labeling and distribution were highly variable between contiguous contacts, and (2) CaMKII was not restricted to the periphery of the terminals, in which glutamatergic synapses are located, but also was present at the center in which gap junctions predominate. Accordingly, double immunolabeling indicated that Cx35 and CaMKII were colocalized, and biochemical analysis showed that these proteins associate. Because CaMKII characteristically undergoes activity-dependent translocation, the observed variability of labeling likely reflects physiological differences between electrical synapses of contiguous Club endings, which remarkably coexist with differing degrees of conductance. Together, our results indicate that CaMKII should be considered a component of electrical synapses, although its association is nonobligatory and likely driven by activity.

An immunochemical marker for goldfish Mauthner cells.

The Mauthner (M-) cells are pair of large reticulospinal neurons that mediate tail-flip escape responses in fish. Exploring the co-localization of scaffold and gap junction proteins at mixed (electrical and chemical) synapses, we found that the use of a particular antibody against the scaffold protein Zonula Occludens-2 (ZO-2) resulted in labeling of these cells. We show here that this staining is restricted to the Mauthner cell and evenly distributed along its dendrites and axon, also prominent in small dendritic and axonal processes. Because the observed labeling is non-specific, we suggest that the antibody might recognize a soluble protein that is primarily expressed in the Mauthner cells. While the identity of this protein is presently unknown, the use of this antibody should facilitate the identification of the Mauthner cell and its fine processes during anatomical and immunohistochemical studies, which otherwise require intracellular injection of tracer molecules during electrophysiological recordings.