(CCC) Colocalization of Cx36 and SCGN was associated with processes of the injected AII amacrine cell but not with CaMKII- immunoreactivity

(CCC) Colocalization of Cx36 and SCGN was associated with processes of the injected AII amacrine cell but not with CaMKII- immunoreactivity. retinas. We found that a Bendazac L-lysine polyclonal antibody was highly specific for CaMKII-. However, a monoclonal antibody (CB–1) recognized CaMKII- but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII- expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII- also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII- influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII–deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII- is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII- with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII- may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs. Keywords: CaMKII, gap junction, electrical synapse, connexin36, bipolar cell, retina, cross-reactivity, antibody Introduction Electrical synapses in the nervous system provide a fast route for intercellular signal transmission and fulfill several unique functions such as cell synchronization Bendazac L-lysine and network oscillations (Hormuzdi et al., 2001; Christie et al., 2005). Structurally, electrical synapses are gap junctions made of connexin proteins which belong to a gene family that comprises 20 different isoforms in the mouse. Amongst these isoforms, connexin36 (Cx36) is considered to be the main neuronal connexin due to its high abundance in the central nervous system (CNS), especially in the cerebellum, the olfactory bulb and the retina, where it couples a variety of different cell types (Bloomfield and V?lgyi, 2009). The vast majority of retinal Cx36 is expressed in AII amacrine cells (Feigenspan et al., 2001; Meyer et al., 2014), which integrate rod-generated signals and Bendazac L-lysine transmit them gap junctions into the cone pathway to enable scotopic vision (Gldenagel et al., 2001; Deans et al., 2002). Apart from the AII amacrine cell, Cx36 was also identified in photoreceptors (Feigenspan et al., 2004; Bolte et al., 2016), bipolar cells (Feigenspan et al., 2004; Han and Massey, 2005), ganglion cells (Schubert et al., 2005; Pan et al., 2010), and other amacrine cells (Brggen et al., 2015; Yadav et al., 2019). Accumulating evidence suggests that electrical and chemical synapses share striking similarities in terms of plasticity and may be regulated by the same key Bendazac L-lysine molecules (Pereda, 2014; Miller and Pereda, 2017; Alcam and Pereda, 2019). Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation, is capable of potentiating electrical coupling in an activity-dependent manner (Alev et al., 2008; del Corsso et al., 2012). This mechanism is quite conserved among species (e.g., rabbit: Kothmann et al., 2012; goldfish: Pereda Bendazac L-lysine et al., 1998; mouse: Turecek et al., 2014) and relies on activation of glutamatergic synapses that are situated in close proximity to neuronal gap junctions. Excitation of glutamate receptors in these synapses produces a Ca2+ influx that drives CaMKII activation and subsequent phosphorylation of Cx36, thereby enhancing electrical coupling (Alev et al., 2008; Flores et al., 2010; Kothmann et al., 2012). Recent reports indicate that this pathway operates in Mauthner cells in teleosts (Yang et Rabbit Polyclonal to PDK1 (phospho-Tyr9) al., 1990; Flores et al., 2010), neurons of the mammalian inferior olive (Turecek et.