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The expansion of the neocortex, a hallmark of mammalian evolution 1 , 2 , was accompanied by an increase in cerebellar neuron numbers 3 . However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution. Single-nucleus RNA-sequencing data from the cerebellum of human, mouse and opossum is used to analyse the developmental dynamics of cell types and states in mammalian cerebellum and provide evolutionary insights.

To better understand the relationship between input and output connectivity for neurons of interest in specific brain regions, a viral-genetic tracing approach is used to identify input based on a combination of neurons’ projection and cell type, as illustrated in a study of locus coeruleus noradrenaline neurons. Noradrenaline circuit architecture New circuit tracing techniques have steadily increased our knowledge of the connectivity between brain regions and how such links may contribute to function and information processing. Here, Liqun Luo and colleagues expand this toolbox to include TRIO, a new strategy designed to characterize the input–output relationships between genetically specified populations of neurons. As a proof of concept, input–output tracing relationships and projection patterns were completed for the noradrenaline neurons of the locus coeruleus. Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 . However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input–output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The ‘tracing the relationship between input and output’ method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input–output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2- L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.

Both heterozygous loss and homozygous loss of Tsc1 in mouse cerebellar Purkinje cells (PCs) result in autistic-like behaviours, which can be prevented by treatment with the mTOR inhibitor, rapamycin; these findings demonstrate critical roles for PCs in autistic-like behaviours in mice. A novel mouse autism model Tuberous sclerosis is a rare tumour-causing genetic disorder that results from mutation of the genes TSC1 or TSC2 . Affected individuals often also have autism spectrum disorder associated with cerebellar pathology. Because clinical studies have implicated cerebellar dysfunction in the pathogenesis of autism, Mustafa Sahin and colleagues studied the functional consequences of disrupting the cerebellar Tsc1 gene in mice. The mutant mice exhibit pathological features common in patients with autism —reduced Purkinje cell numbers and increased markers of neuronal stress — and mice lacking Tsc1 in cerebellar Purkinje cells display autism-related behaviours. Both the cerebellar pathology and behavioural features are ameliorated by treating the mice with the mTOR inhibitor rapamycin. Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders 1 , but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss 2 , 3 , and isolated cerebellar injury has been associated with a higher incidence of ASDs 4 . However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs 5 that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology 6 and correlate cerebellar pathology with increased ASD symptomatology 7 , 8 . Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs 9 . However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Cerebellar ataxia is the primary manifestation of cerebellar degenerative diseases, and mitochondrial dysfunction in Purkinje cells (PCs) plays a critical role in disease progression. In this study, we investigated the feasibility of mitochondria transplantation as a potential therapeutic approach to rescue cerebellar neurodegeneration and elucidate the associated mechanisms. We constructed a conditional Drp1 knockout model in PCs (PCKO mice), characterized by progressive ataxia. Drp1 knockout resulted in pervasive and progressive apoptosis of PCs and significant activation of surrounding glial cells. Mitochondrial dysfunction, which triggers mitophagy, is a key pathogenic factor contributing to morphological and functional damage in PCs. Transplanting liver-derived mitochondria into the cerebellum of 1-month-old PCKO mice improved mitochondrial function, reduced mitophagy, delayed apoptosis of PCs, and alleviated cerebellar ataxia for up to 3 weeks. These findings demonstrate that mitochondria transplantation holds promise as a therapeutic approach for cerebellar degenerative diseases. Cerebellar ataxia, a hallmark of cerebellar degeneration (CBND), is driven by mitochondrial dysfunction in Purkinje cells. In this study, transplanting mitochondria into CBND mice improve mitochondrial function, reduce mitophagy, and reduce ataxia.

Control of synaptic transmission efficacy by neuronal activity and neuromodulators is pivotal for brain function. Synaptic suppression by cannabinoids activating CB1 receptors has been extensively studied at the molecular and cellular levels to understand the neuronal basis for effects of cannabis intake. Here, we focused on GPR55, a non-canonical type of cannabinoid receptor, which shows sensitivity to cannabidiol included in cannabis, aiming to highlight its actions on presynaptic function. Taking advantage of direct patch-clamp recordings from axon terminals of rat cerebellar Purkinje cells together with fluorescent imaging of vesicular exocytosis using synapto-pHluorin, we show that GPR55 suppresses synaptic transmission as CB1 receptor does, but through a distinct presynaptic modulation of release machinery. Activation of GPR55 reduced transmitter release by changing neither presynaptic action potential waveform nor Ca 2+ influx, but by making a large population of Ca 2+ -responsive synaptic vesicles insensitive to Ca 2+ influx through voltage-gated Ca 2+ channels, leading to substantial reduction of the readily releasable pool of vesicles. Thus, the present study identifies a unique mechanism to suppress presynaptic transmitter release by an atypical cannabinoid receptor GPR55, which would enable subtype-specific modulation of neuronal computation by cannabinoid receptors.

Caspase-12 is a caspase family member for which functions in regulating cell death and inflammation have previously been suggested. In this study, we used caspase-12 lacZ reporter mice to elucidate the expression pattern of caspase-12 in order to obtain an idea about its possible in vivo function. Strikingly, these reporter mice showed that caspase-12 is expressed explicitly in Purkinje neurons of the cerebellum. As this observation suggested a function for caspase-12 in Purkinje neurons, we analyzed the brain and behavior of caspase-12 deficient mice in detail. Extensive histological analyses showed that caspase-12 was not crucial for establishing cerebellum structure or for maintaining Purkinje cell numbers. We then performed behavioral tests to investigate whether caspase-12 deficiency affects memory, motor, and psychiatric functions in mice. Interestingly, while the absence of caspase-12 did not affect memory and motor function, caspase-12 deficient mice showed depression and hyperactivity tendencies, together resembling manic behavior. Next, suggesting a possible molecular mechanistic explanation, we showed that caspase-12 deficient cerebella harbored diminished signaling through the brain-derived neurotrophic factor/tyrosine kinase receptor B/cyclic-AMP response binding protein axis, as well as strongly enhanced expression of the neuronal activity marker c-Fos. Thus, our study establishes caspase-12 expression in mouse Purkinje neurons and opens novel avenues of research to investigate the role of caspase-12 in regulating psychiatric behavior.

The maintenance of metabolic homeostasis relies on the ability to flexibly transit between catabolic and anabolic states in response to insulin signaling. Here we show insulin-activated ATM is a critical mediator of this process, facilitating the swift transition between catabolic-and-anabolic fates of glucose by regulating the functional status of PKM2 and HIF1α. In Ataxia-Telangiectasia (A-T), these mechanisms are disrupted, resulting in intrinsic insulin resistance and glucose intolerance. Consequently, cells exhibit a compensatory dependence on glutamine as an alternative metabolite for energy metabolism. Cerebellar degeneration, a hallmark of A-T, is characterized by the pronounced vulnerability of Purkinje cells, attributed to their unexpected sensitivity to insulin. Supplementation with α-ketoglutarate, the α-keto acid backbone of glutamine, has demonstrated potentials in alleviating glutamine dependence and attenuating Purkinje cell degeneration. These findings suggest that peripheral metabolic deficiencies may contribute to sustained neurodegenerative changes in A-T, underscoring the importance of screening, monitoring and addressing these metabolic disruptions in patients. Insulin-activated ataxia-telangiectasia mutated (ATM) regulates glucose metabolism. Here the authors report that its disruption in a mouse model of ataxia-telangiectasia leads to insulin resistance, glutamine dependence, and selective Purkinje cell degeneration, while α-Ketoglutarate supplementation shows promise in mitigating neurodegeneration.

Due to the uniform cyto-architecture of the cerebellar cortex, its overall physiological characteristics have traditionally been considered to be homogeneous. In this study, we show in awake mice at rest that spiking activity of Purkinje cells, the sole output cells of the cerebellar cortex, differs between cerebellar modules and correlates with their expression of the glycolytic enzyme aldolase C or zebrin. Simple spike and complex spike frequencies were significantly higher in Purkinje cells located in zebrin-negative than zebrin-positive modules. The difference in simple spike frequency persisted when the synaptic input to, but not intrinsic activity of, Purkinje cells was manipulated. Blocking TRPC3, the effector channel of a cascade of proteins that have zebrin-like distribution patterns, attenuated the simple spike frequency difference. Our results indicate that zebrin-discriminated cerebellar modules operate at different frequencies, which depend on activation of TRPC3, and that this property is relevant for all cerebellar functions. The cerebellum, located at the back of the brain underneath the cerebral hemispheres, is best known for its role in the control of movement. Despite its small size, the cerebellum contains more than half of the brain's neurons. These are organized in a repeating pattern in which cells called Purkinje cells receive inputs from two types of fibers: climbing fibers, which ascend into the cerebellum from the brainstem; and parallel fibers, which run perpendicular to the climbing fibers. This gives rise to a characteristic ‘crystalline’ structure. As a result of this uniform circuitry, it was widely believed was that all Purkinje cells throughout the cerebellum would function the same way. However, the presence of distinct patterns of gene expression in different regions suggests that this is not the case. Molecules called zebrins, for example, are found in some Purkinje cells but not others, and this gives rise to a pattern of zebrin-positive and zebrin-negative stripes. A number of other molecules have similar distributions, suggesting that these differences in molecular machinery could underlie differences in cellular physiology. Zhou, Lin et al. have now provided one of the first direct demonstrations of such physiological differences by showing that zebrin-positive cells generate action potentials at lower frequencies than zebrin-negative cells. This pattern is seen throughout the cerebellum, and is evident even when the positive and negative cells are neighbors, which indicates that these differences do not simply reflect differences in the locations of the cells or differences in the inputs they receive from parallel fibers. Additional experiments revealed that the distinct firing rates are likely not generated by zebrin itself, but rather by proteins that are expressed alongside zebrin, most notably those that work through an ion channel called TRPC3. By showing that cells arranged in the same type of circuit can nevertheless have distinct firing rates, the work of Zhou, Lin et al. has revealed an additional level of complexity in the physiology of the cerebellum. In addition to improving our understanding of how the brain controls movement, these findings might also be of interest to researchers studying the increasing number of neurological and psychiatric disorders in which cerebellar dysfunction has been implicated.

In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.