Abstract
The inferior olive (IO) is a nucleus located in the brainstem and it is part of the olivo-cerebellar loop. This circuit plays a fundamental role in generation and acquisition of coherent motor patterns and it relies on synchronous activation of groups of Purkinje cells (PC) in the cerebellar cortex. IO neurons integrate their intrinsic oscillatory activity with excitatory inputs coming from the somatosensory system and inhibitory feedback coming from the cerebellar nuclei. Alongside these chemical synaptic inputs, IO neurons are coupled to one another via connexin 36 (Cx36) containing gap junctions (GJs) that create a functional syncytium between neurons. Communication between olivary neurons is regulated by these GJs and their correct functioning contributes to coherent oscillations in the IO and proper motor learning. Here, we explore the cellular pathways that can regulate the coupling between olivary neurons. We combined in vitro electrophysiology and immunohistochemistry (IHC) on mouse acute brain slices to unravel the pathways that regulate olivary coupling. We found that enhancing the activity of the protein kinase A (PKA) pathway and blocking the Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway can both down-regulate the size of the coupled network. However, these two kinases follow different mechanisms of action. Our results suggest that activation of the PKA pathway reduces the opening probability of the Cx36 GJs, whereas inhibition of the CaMKII pathway reduces the number of Cx36 GJs. The low densities of Cx36 proteins and electrical synapses in βCaMKII knock-out mice point towards an essential role for this protein kinase in regulating the density of GJs in the IO. Thus, the level of olivary coupling is a dynamic process and regulated by a variety of enzymes modulating GJs expression, docking and activity.
Highlights
The olivocerebellar system is essential for coordination of movements and is tuned by synchronous firing between bands of Purkinje cells (PC) in the cerebellar cortex (Welsh, 2002; Shin et al, 2007)
The generation of action potentials (AP) of inferior olive (IO) neurons depends on subthreshold oscillations of the membrane potential and their synchronization is promoted by Connexin 36 (Cx36) containing gap junctions (GJs; i.e., electrical synapses)
Immunohistochemical as well as ultrastructural analysis of GJs was performed on olivary slices of αCaMKII and βCaMKII knock-out mice to investigate the importance of these enzymes for the insertion of Cx36 proteins and the formation of electrical synapses
Summary
The olivocerebellar system is essential for coordination of movements and is tuned by synchronous firing between bands of Purkinje cells (PC) in the cerebellar cortex (Welsh, 2002; Shin et al, 2007). Disruption of olivary coupling affects the firing behavior of olivary neurons, increasing the occurrence of doublets and thereby corrupting the ability to respond with acute motor reflexes or acquire new motor behaviors (Van Der Giessen et al, 2008; Bazzigaluppi et al, 2012; De Gruijl et al, 2014) All these results suggest that the coupling between IO neurons is fundamental for correct functioning of the olivocerebellar system and for proper execution of motor commands (Llinás, 2011). In the IO Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is able to phosphorylate Cx36 (Alev et al, 2008; Siu et al, 2016), is required for NMDARdependent strengthening of electrical coupling between olivary neurons (Turecek et al, 2014) Taken together, these lines of evidence led us to hypothesize a role for both PKA and CaMKII in regulating the coupling between olivary neurons. The latter was confirmed in βCaMKII knock-out mice, but not αCaMKII knock-out mice, suggesting involvement of the actin cytoskeleton in the olivary glomerulus
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