Abstract
Heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals from G protein-coupled receptors (GPCRs) to effector ion channels and enzymes Gαo, a member of the pertussis toxin-sensitive Gi/o family, is widely expressed in the brain, although its role within a neuronal context remains largely unknown. Using immunohistochemical and quantitative immunoelectron microscopy techniques, we have investigated the expression, cellular and subcellular localization of Gαo in the cerebellar cortex. Histoblot revealed that Gαo is expressed in many brain regions, including the cerebellum. At the cellular level, Gαo protein was distributed in Purkinje cells, basket cells, stellate cells, granule cells and Golgi cells. At the subcellular level, pre-embedding immunoelectron microscopy revealed mainly a postsynaptic localization of Gαo along the extrasynaptic plasma membrane of Purkinje cell dendritic shafts and spines, and dendrites of basket, stellate and granule cells. To a lesser extent, immunolabeling for Gαo was localized in different types of axon terminals establishing excitatory synapses. Moreover, post-embedding immunoelectron microscopy revealed the synaptic localization of Gαo on PSDs of glutamatergic synapses between Purkinje cell spines and parallel fiber terminals and its co-localization with GABAB1 in the same spines. Quantitative analysis of Gαo immunoparticles revealed they preferentially localized on the cytoplasmic face of the plasma membrane. Furthermore, the analysis revealed a high concentration of Gαo around excitatory synapses on Purkinje cell dendritic spines, but a uniform distribution in granule cell dendrites. These molecular-anatomical findings suggest that Gαo is a major signal transducer of specific GPCRs in different neuronal populations in the cerebellum.
Highlights
The interplay among neurons of the cerebellar cortex is key to achieving different functions, including fine motor control, maintenance of balance and posture, perception, memory and cognition (Ito, 2001, 2006)
Distribution of Gαo in the Cerebellum of the cerebellar cortex, extend their dendrites through the molecular layer where they receive inputs from climbing fibers. They receive inputs from parallel fibers originating in granule cells (GCs), which integrate sensory information arriving through the mossy fibers to modulate the activity of Purkinje cells (PCs) (Ito, 2001, 2006)
In addition to ion channels activation that cause the firing of neurons, the function of cerebellar cells depends on the signaling through G protein-coupled receptors (GPCRs) transducing stimuli across the plasma membrane (Oldham and Hamm, 2008; Weis and Kobilka, 2018)
Summary
The interplay among neurons of the cerebellar cortex is key to achieving different functions, including fine motor control, maintenance of balance and posture, perception, memory and cognition (Ito, 2001, 2006). Distribution of Gαo in the Cerebellum of the cerebellar cortex, extend their dendrites through the molecular layer where they receive inputs from climbing fibers They receive inputs from parallel fibers originating in granule cells (GCs), which integrate sensory information arriving through the mossy fibers to modulate the activity of PCs (Ito, 2001, 2006). Despite the involvement of Gαo in large part of the modulatory signaling in the brain (Wettschureck and Offermanns, 2005) and its large potential as therapeutic targets (Li et al, 2020), our understanding of Gαo distribution in different neuron populations and its organization in different neuronal compartments is limited To this end, we employed quantitative pre- and post-embedding immunoelectron microscopic techniques to unravel subcellular localization patterns of Gαo in different cerebellar cell types. We found that this G protein subunit exhibited distinct subcellular localization patterns throughout the cerebellar cortex, suggesting its involvement in regulating specific signaling pathways in postand pre-synaptic compartments
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