Abstract
Gap junctions between neurons serve as electrical synapses, in addition to conducting metabolites and signaling molecules. During development, early-appearing gap junctions are thought to prefigure chemical synapses, which appear much later. We present evidence for this idea at a central, glutamatergic synapse and provide some mechanistic insights. Loss or reduction in the levels of the gap junction protein Gjd2b decreased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in cerebellar Purkinje neurons (PNs) in larval zebrafish. Ultrastructural analysis in the molecular layer showed decreased synapse density. Further, mEPSCs had faster kinetics and larger amplitudes in mutant PNs, consistent with their stunted dendritic arbors. Time-lapse microscopy in wild-type and mutant PNs reveals that Gjd2b puncta promote the elongation of branches and that CaMKII may be a critical mediator of this process. These results demonstrate that Gjd2b-mediated gap junctions regulate glutamatergic synapse formation and dendritic elaboration in PNs.
Highlights
The formation of synapses is an elaborate multi-step process involving several classes of signaling molecules and electrical activity (Waites et al, 2005; McAllister, 2007)
gap junction delta 2b protein (Gjd2b) is expressed at high levels in the cerebellum of larval zebrafish beginning at 4 dpf, a stage at which cerebellar neurons have been specified but chemical synaptic connections are still forming (Bae et al, 2009; Jabeen and Thirumalai, 2013) (Figure 1-figure supplement 1A)
Purkinje neurons (PNs) were electroporated with neurobiotin or serotonin, one or two PNs along with several non-PNs were detected (Figure 7-figure supplement 1, Supplementary file 1), indicating that PNs are likely to be coupled to other cerebellar cell types via rectifying junctions.To determine if the observed dendritic growth deficits in gjd2b-/- mutant PNs are due to lack of Gjd2b in PNs we introduced Gjd2b tagged to mCherry into single PNs in gjd2b-/- fish (Figure 7A)
Summary
The formation of synapses is an elaborate multi-step process involving several classes of signaling molecules and electrical activity (Waites et al, 2005; McAllister, 2007). Neurons show increased gap junctional connectivity early on in development at stages that precede chemical synapse formation (Montoro and Yuste, 2004; Marin-Burgin et al, 2008; Jabeen and Thirumalai, 2013). Gap junctions could mediate chemical synaptogenesis by increasing correlations in activity, by transmitting synaptogenic signaling molecules, or by providing enhanced mechanical stability at junctional sites between connected pairs. It is not clear which of these functions of gap junctions are critical for synaptogenesis. We set out to investigate whether gap junctions regulate structural and functional synaptic development of cerebellar Purkinje neurons (PNs) and if yes, what mechanisms may be involved using larval zebrafish as our model system. Using knock-down and knock-out approaches, we show here that Gjd2b is required for the formation of glutamatergic synapses and for normal dendritic arbor growth of PNs
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