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. 2010 Jul 14;30(28):9488-99.
doi: 10.1523/JNEUROSCI.4466-09.2010.

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

Carmen E Flores  1 , Roger Cachope, Srikant Nannapaneni, Smaranda Ene, Angus C Nairn, Alberto E Pereda
Affiliations

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

Carmen E Flores et al. J Neurosci. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
The Club endings: the striking synapses of the Mauthner cell. A, Diagram of the M-cell showing its axon cap, soma, and lateral dendrite. The large myelinated Club endings, referred to here as “Club endings”, are distributed along the distal portion of the lateral dendrite. These contacts belong to primary auditory afferents that establish mixed (electrical and chemical) synapses on the distal portion of the M-cell lateral dendrite. Right, The diagram summarizes the main structural features of CEs; specializations corresponding to chemical synapses are restricted to the periphery of the contact. The presynaptic and postsynaptic elements of the synapse are illustrated separately, for better appreciation of the PSDs. B, Projection image of the distal portion of the lateral dendrite obtained with laser scanning confocal microscopy, illustrating saccular afferents (green) terminating as CEs. Labeling of these afferents was obtained using a phospho-specific anti-Cx35 antibody (Cx35 phospho-Ser110), which is known to cross react to unknown high-molecular-weight phosphoproteins that appear to be expressed primarily in glial cells (Kothmann et al., 2007). Double labeling with monoclonal anti-Cx35/Cx36 antibody (red; Millipore Bioscience Research Reagents) reveals the area of contact between CEs and the distal portion of the M-cell lateral dendrite. C, Higher magnification of the labeled boxed region in B. The observation of multiple Cx35-labeled puncta is consistent with the presence of up to ∼200 gap junction plaques in individual CEs. D, Higher magnification of the labeled boxed region in B. Cx35 labeling is observed at the region of contact between two CEs and the M-cell dendrite. E, Image of a Club ending on the surface of the lateral dendrite obtained using DIC optics. These unusually large contacts are easily recognizable because of their big size and characteristic ring-like appearance caused by their prominent myelination.
Figure 2.
Figure 2.
Double immunolabeling of Cx35 and αCaMKII in the lateral dendrite of the Mauthner cells. A, Laser scanning confocal projection image of the distal portion of the M-cell lateral dendrite (average of 5 sections) using a polyclonal anti-αCaMKII antibody (G301; red). The G301 antibody also labeled neurofilaments (asterisks) in the cytoskeleton of the M-cell. B, Higher magnification of the labeled boxed region in A, illustrating αCaMKII labeling at individual CEs. C, Double labeling using the polyclonal anti-αCaMKII (G301; red) and anti-Cx35/Cx36 (green) antibodies, respectively. D, High magnification of the labeled boxed region in C.
Figure 3.
Figure 3.
Heterogeneity of αCaMKII labeling between contiguous Club endings. A, Confocal projection of the distal portion of the lateral dendrite showing immunolabeling for Cx35 using monoclonal anti-Cx35/Cx36 antibody. Note the regularity of Cx35 labeling between CEs. B, Confocal projection of a similar portion of the distal lateral dendrite showing immunolabeling for αCaMKII (G301 antibody). C, Higher magnification of the labeled boxed region in B. CaMKII distribution is highly variable between adjacent CEs (arrowheads, predominant in the terminal periphery; arrows, predominant on the entire surface of the contact). D–F, Examples of variability of CaM-KII labeling between CEs.
Figure 4.
Figure 4.
Intraterminal distribution of αCaMKII in Club endings. Confocal projections of individual Club endings showing the distribution of Cx35 (A), NR1 (B), and αCaMKII (C). A, B, Cx35 labeling is typically punctate and covers most of the surface contact area, whereas NR1 labeling is mostly restricted to the periphery of the synaptic contact [images in A and B were illustrated previously (Pereda et al., 2003)]. C, In contrast, αCaMKII labeling (G301 antibody) is more diffuse and highly variable between contiguous Club endings. Although αCaMKII labeling was always observed at the periphery, in which PSDs are located, it is also found at the center of the contact area of the CEs in some cases (compare top CEs with the bottom one). D, Quantification of the variability of αCaMKII distribution of CEs using a ratio between the labeling at periphery and center of each CE. Diagram of the regions selected for analysis: a peripheral “ring” area (in which PSDs are included) and a center area (in which gap junctions are predominant). A transition area (white) between periphery and center areas was not included in the analysis. The ratio between these areas was defined as the periphery/center index (for details, see Materials and Methods). Graph illustrates the distribution of this index for 43 Club endings.
Figure 5.
Figure 5.
Connexin35 and αCaMKII colocalize at Club endings and associate in goldfish brain. A, Confocal z-section of a single terminal showing colocalization (yellow) of Cx35 (green) and αCaMKII (red). Labeling of Cx35 and αCaMKII colocalizes at the periphery and the center of the synaptic contact. B, Higher magnification of the labeled boxed region in B. αCaMKII and Cx35 colocalize at some Cx35 puncta (arrowheads) but is absent at others (asterisks). C, Sequence alignment of mouse Cx36 cytoplasmic loop and C-terminal αCaMKII binding site regions (Alev et al., 2008) reveals a high degree of similarity with perch Cx35 (86 and 92%, respectively). D, Immunoblot detection of Cx35 in two different samples (lanes 2 and 3) with monoclonal Cx35/Cx36 antibody after IP of αCaMKII from goldfish hindbrain with G301 antibody. The Cx35 immunoblot exhibits three identifiable bands.
Figure 6.
Figure 6.
αCaMKII associates with ZO-1 in goldfish brain. A, Extensive colocalization of Cx35 (green) and ZO-1 (red) at CEs in the M-cell lateral dendrite. B, Immunoblot detection of ZO-1 (lane 2) with mouse ZO-1 antibody after IP of αCaMKII from goldfish hindbrain with G301 antibody.
Figure 7.
Figure 7.
Electrical synapses between neighboring Club endings coexist at different degrees of conductance on the M-cell lateral dendrite. A, Differences in gap junctional conductance between CEs evidenced by multiple simultaneous recordings in the same M-cell dendrite. The recordings of unitary synaptic potentials (red top traces) were obtained sequentially from the same dendritic position, and it cannot be explained by differences in the amplitude of the presynaptic action potentials (black bottom traces), in which variability was much lower. Only two of these nine unitary synaptic potentials exhibit a clear chemical component (e.g., “chemical” in recording a). B, Variability in the amplitude of unitary coupling potentials does not represent variability in the electrotonic distances from the recording site, because a similar diversity in coupling strength can be revealed using Neurobiotin dye coupling (Smith and Pereda, 2003). Transfer of Neurobiotin from the M-cell to neighboring CEs differs dramatically (dark, white arrowheads; unlabeled, black arrowheads), indicating that junctions differ in permeability. View of the bifurcation of the lateral dendrite (dark branches) obtained with DIC optics and revealing differences of labeling between neighboring CEs (only a small number of CEs are generally labeled after injection of Neurobiotin into the M-cell, indicating that gap junction permeability at CEs is generally low). Inset, A single terminal distinguished solely by the use of DIC optics. C, Variability of coupling coefficients among individual CEs in five different fish. Inner circle represents the M-cell lateral dendrite. The length of each line is proportional to the coupling coefficient of that CE. For calibration, the circle represents a coupling potential of 0.020. D, Histogram showing the periphery/center index distribution of the CaMKII distribution at single terminals (n = 43). E, Histogram showing the amplitude distribution of the unitary electrical EPSPs (n = 92).
Figure 8.
Figure 8.
Potential mechanism for differences in CaMKII labeling between contiguous Club endings. Electrical synapses at adjacent CEs coexist at different degrees of conductance (indicated by different colors). The extensive colocalization of Cx35 with ZO-1 suggests that this scaffold protein could constitute a structural component of gap junctions at these terminals. Activity of neighboring chemically transmitting regions within the terminal trigger changes in junctional conductance, via a PSD-mediated mechanism (arrows) (Pereda and Faber, 1996; Pereda et al., 1998) promoting the association of CaMKII to Cx35 and ZO-1. The association of CaMKII to electrical synapses would be thus nonobligatory and driven by synaptic activity. For convenience, a simplified gap junction is illustrated; the diagram does not indicate whether the association is exclusively presynaptic or postsynaptic.
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