Transcranial direct current stimulation of cerebellum alters spiking precision in cerebellar cortex: A modeling study of cellular responses

Transcranial direct current stimulation (tDCS) of the cerebellum has rapidly raised interest but the effects of tDCS on cerebellar neurons remain unclear. Assessing the cellular response to tDCS is challenging because of the uneven, highly stratified cytoarchitecture of the cerebellum, within which cellular morphologies, physiological properties, and function vary largely across several types of neurons. In this study, we combine MRI-based segmentation of the cerebellum and a finite element model of the tDCS-induced electric field (EF) inside the cerebellum to determine the field imposed on the cerebellar neurons throughout the region. We then pair the EF with multicompartment models of the Purkinje cell (PC), deep cerebellar neuron (DCN), and granule cell (GrC) and quantify the acute response of these neurons under various orientations, physiological conditions, and sequences of presynaptic stimuli. We show that cerebellar tDCS significantly modulates the postsynaptic spiking precision of the PC, which is expressed as a change in the spike count and timing in response to presynaptic stimuli. tDCS has modest effects, instead, on the PC tonic firing at rest and on the postsynaptic activity of DCN and GrC. In Purkinje cells, anodal tDCS shortens the repolarization phase following complex spikes (-14.7 ± 6.5% of baseline value, mean ± S.D.; max: -22.7%) and promotes burstiness with longer bursts compared to resting conditions. Cathodal tDCS, instead, promotes irregular spiking by enhancing somatic excitability and significantly prolongs the repolarization after complex spikes compared to baseline (+37.0 ± 28.9%, mean ± S.D.; max: +84.3%). tDCS-induced changes to the repolarization phase and firing pattern exceed 10% of the baseline values in Purkinje cells covering up to 20% of the cerebellar cortex, with the effects being distributed along the EF direction and concentrated in the area under the electrode over the cerebellum. Altogether, the acute effects of tDCS on cerebellum mainly focus on Purkinje cells and modulate the precision of the response to synaptic stimuli, thus having the largest impact when the cerebellar cortex is active. Since the spatiotemporal precision of the PC spiking is critical to learning and coordination, our results suggest cerebellar tDCS as a viable therapeutic option for disorders involving cerebellar hyperactivity such as ataxia.

Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.

Maturation of Purkinje cell firing properties relies on neurogenesis of excitatory neurons

Preterm infants that suffer cerebellar insults often develop motor disorders and cognitive difficulty. Excitatory granule cells, the most numerous neuron type in the brain, are especially vulnerable and likely instigate disease by impairing the function of their targets, the Purkinje cells. Here, we use regional genetic manipulations and in vivo electrophysiology to test whether excitatory neurons establish the firing properties of Purkinje cells during postnatal mouse development. We generated mutant mice that lack the majority of excitatory cerebellar neurons and tracked the structural and functional consequences on Purkinje cells. We reveal that Purkinje cells fail to acquire their typical morphology and connectivity, and that the concomitant transformation of Purkinje cell firing activity does not occur either. We also show that our mutant pups have impaired motor behaviors and vocal skills. These data argue that excitatory cerebellar neurons define the maturation time-window for postnatal Purkinje cell functions and refine cerebellar-dependent behaviors.

VTA-projecting cerebellar neurons mediate stress-dependent depression-like behavior

Although cerebellar alterations have been implicated in mental depression, the exact contribution of the cerebellum to depressive symptoms remains to be elucidated. Here, we demonstrated the crucial role of cerebellar neurons projecting to the ventral tegmental area (VTA) in the chronic stress-induced development of depression-like behavior. The combination of adeno-associated virus-based circuit mapping and electrophysiological recording identified network connections from crus I to the VTA via the dentate nucleus (DN) of the deep cerebellar nuclei. Chronic chemogenetic activation of inhibitory Purkinje cells in crus I suppressed c-Fos expression in the DN and depression-like behavior, which were triggered by chronic stress application. Furthermore, specific inhibition of neurons in the DN that project to the VTA prevented stressed mice from showing depression-like behavior, whereas specific activation of these neurons alone triggered depression-like behavior that was comparable with the one triggered by chronic stress application. Our results indicate that the VTA-projecting cerebellar neurons proactively regulate depression-like behavior, raising the possibility that cerebellum may be an effective target for the prevention of depressive disorders.

Transcriptome programs involved in the development and structure of the cerebellum

In the past two decades, mounting evidence has modified the classical view of the cerebellum as a brain region specifically involved in the modulation of motor functions. Indeed, clinical studies and engineered mouse models have highlighted cerebellar circuits implicated in cognitive functions and behavior. Furthermore, it is now clear that insults occurring in specific time windows of cerebellar development can affect cognitive performance later in life and are associated with neurological syndromes, such as Autism Spectrum Disorder. Despite its almost homogenous cytoarchitecture, how cerebellar circuits form and function is not completely elucidated yet. Notably, the apparently simple neuronal organization of the cerebellum, in which Purkinje cells represent the only output, hides an elevated functional diversity even within the same neuronal population. Such complexity is the result of the integration of intrinsic morphogenetic programs and extracellular cues from the surrounding environment, which impact on the regulation of the transcriptome of cerebellar neurons. In this review, we briefly summarize key features of the development and structure of the cerebellum before focusing on the pathways involved in the acquisition of the cerebellar neuron identity. We focus on gene expression and mRNA processing programs, including mRNA methylation, trafficking and splicing, that are set in motion during cerebellar development and participate to its physiology. These programs are likely to add new layers of complexity and versatility that are fundamental for the adaptability of cerebellar neurons.

Broad Influence of Mutant Ataxin-3 on the Proteome of the Adult Brain, Young Neurons, and Axons Reveals Central Molecular Processes and Biomarkers in SCA3/MJD Using Knock-In Mouse Model

Spinocerebellar ataxia type 3 (SCA3/MJD) is caused by CAG expansion mutation resulting in a long polyQ domain in mutant ataxin-3. The mutant protein is a special type of protease, deubiquitinase, which may indicate its prominent impact on the regulation of cellular proteins levels and activity. Yet, the global model picture of SCA3 disease progression on the protein level, molecular pathways in the brain, and neurons, is largely unknown. Here, we investigated the molecular SCA3 mechanism using an interdisciplinary research paradigm combining behavioral and molecular aspects of SCA3 in the knock-in ki91 model. We used the behavior, brain magnetic resonance imaging (MRI) and brain tissue examination to correlate the disease stages with brain proteomics, precise axonal proteomics, neuronal energy recordings, and labeling of vesicles. We have demonstrated that altered metabolic and mitochondrial proteins in the brain and the lack of weight gain in Ki91 SCA3/MJD mice is reflected by the failure of energy metabolism recorded in neonatal SCA3 cerebellar neurons. We have determined that further, during disease progression, proteins responsible for metabolism, cytoskeletal architecture, vesicular, and axonal transport are disturbed, revealing axons as one of the essential cell compartments in SCA3 pathogenesis. Therefore we focus on SCA3 pathogenesis in axonal and somatodendritic compartments revealing highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport and mitochondria was decreased. We demonstrate the accumulation of axonal vesicles in neonatal SCA3 cerebellar neurons and increased phosphorylation of SMI-312 positive adult cerebellar axons, which indicate axonal dysfunction in SCA3. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of various components of protein synthesis machinery in axons.