Recent advances in light patterned optogenetic photostimulation in freely moving mice.

Optogenetics opened the door to a new era of neuroscience. New optical developments are under way to enable high-resolution neuronal activity imaging and selective photostimulation of neuronal ensembles in freely moving animals. These advancements could allow researchers to interrogate, with cellular precision, functionally relevant neuronal circuits in the framework of naturalistic brain activity. We provide an overview of the current state-of-the-art of imaging and photostimulation in freely moving rodents and present a road map for future optical and engineering developments toward miniaturized microscopes that could reach beyond the currently existing systems.

Purkinje cell microzones mediate distinct kinematics of a single movement.

The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the cerebellum, known to fine-tune multi-axial eye movements, is an ideal substrate for the study of potential functions of neuronal subpopulations. Here, we demonstrate that its recently identified subpopulations of 9+ and 9- Purkinje cells exhibit an intermediate Aldolase C expression and electrophysiological profile, providing evidence for a graded continuum of intrinsic properties among PC subpopulations. By identifying and utilizing two Cre-lines that genetically target these floccular domains, we show with high spatial specificity that these subpopulations of Purkinje cells participate in separate micromodules with topographically organized connections. Finally, optogenetic excitation of the respective subpopulations results in movements around the same axis in space, yet with distinct kinematic profiles. These results indicate that Purkinje cell subpopulations integrate in discrete circuits and mediate particular parameters of single movements.

A flexible two-photon fiberscope for fast activity imaging and precise optogenetic photostimulation of neurons in freely moving mice.

We developed a flexible two-photon microendoscope (2P-FENDO) capable of all-optical brain investigation at near cellular resolution in freely moving mice. The system performs fast two-photon (2P) functional imaging and 2P holographic photostimulation of single and multiple cells using axially confined extended spots. Proof-of-principle experiments were performed in freely moving mice co-expressing jGCaMP7s and the opsin ChRmine in the visual or barrel cortex. On a field of view of 250 µm in diameter, we demonstrated functional imaging at a frame rate of up to 50 Hz and precise photostimulation of selected groups of cells. With the capability to simultaneously image and control defined neuronal networks in freely moving animals, 2P-FENDO will enable a precise investigation of neuronal functions in the brain during naturalistic behaviors.

Sphingolipid metabolism governs Purkinje cell patterned degeneration in Atxn1[82Q]/+ mice.

Patterned degeneration of Purkinje cells (PCs) can be observed in a wide range of neuropathologies, but mechanisms behind nonrandom cerebellar neurodegeneration remain unclear. Sphingolipid metabolism dyshomeostasis typically leads to PC neurodegeneration; hence, we questioned whether local sphingolipid balance underlies regional sensitivity to pathological insults. Here, we investigated the regional compartmentalization of sphingolipids and their related enzymes in the cerebellar cortex in healthy and pathological conditions. Analysis in wild-type animals revealed higher sphingosine kinase 1 (Sphk1) levels in the flocculonodular cerebellum, while sphingosine-1-phosphate (S1P) levels were higher in the anterior cerebellum. Next, we investigated a model for spinocerebellar ataxia type 1 (SCA1) driven by the transgenic expression of the expanded Ataxin 1 protein with 82 glutamine (82Q), exhibiting severe PC degeneration in the anterior cerebellum while the flocculonodular region is preserved. In Atxn1[82Q]/+ mice, we found that levels of Sphk1 and Sphk2 were region-specific decreased and S1P levels increased, particularly in the anterior cerebellum. To determine if there is a causal link between sphingolipid levels and neurodegeneration, we deleted the Sphk1 gene in Atxn1[82Q]/+ mice. Analysis of Atxn1[82Q]/+; Sphk1-/- mice confirmed a neuroprotective effect, rescuing a subset of PCs in the anterior cerebellum, in domains reminiscent of the modules defined by AldolaseC expression. Finally, we showed that Sphk1 deletion acts on the ATXN1[82Q] protein expression and prevents PC degeneration. Taken together, our results demonstrate that there are regional differences in sphingolipid metabolism and that this metabolism is directly involved in PC degeneration in Atxn1[82Q]/+ mice.

Purkinje cell axonal swellings enhance action potential fidelity and cerebellar function.

Axonal plasticity allows neurons to control their output, which critically determines the flow of information in the brain. Axon diameter can be regulated by activity, yet how morphological changes in an axon impact its function remains poorly understood. Axonal swellings have been found on Purkinje cell axons in the cerebellum both in healthy development and in neurodegenerative diseases, and computational models predicts that axonal swellings impair axonal function. Here we report that in young Purkinje cells, axons with swellings propagated action potentials with higher fidelity than those without, and that axonal swellings form when axonal failures are high. Furthermore, we observed that healthy young adult mice with more axonal swellings learn better on cerebellar-related tasks than mice with fewer swellings. Our findings suggest that axonal swellings underlie a form of axonal plasticity that optimizes the fidelity of action potential propagation in axons, resulting in enhanced learning.

Region-specific preservation of Purkinje cell morphology and motor behavior in the ATXN1[82Q] mouse model of spinocerebellar ataxia 1.

Purkinje cells are the primary processing units of the cerebellar cortex and display molecular heterogeneity that aligns with differences in physiological properties, projection patterns, and susceptibility to disease. In particular, multiple mouse models that feature Purkinje cell degeneration are characterized by incomplete and patterned Purkinje cell degeneration, suggestive of relative sparing of Purkinje cell subpopulations, such as those expressing Aldolase C/zebrinII (AldoC) or residing in the vestibulo-cerebellum. Here, we investigated a well-characterized Purkinje cell-specific mouse model for spinocerebellar ataxia type 1 (SCA1) that expresses human ATXN1 with a polyQ expansion (82Q). Our pathological analysis confirms previous findings that Purkinje cells of the vestibulo-cerebellum, i.e., the flocculonodular lobes, and crus I are relatively spared from key pathological hallmarks: somatodendritic atrophy, and the appearance of p62/SQSTM1-positive inclusions. However, immunohistological analysis of transgene expression revealed that spared Purkinje cells do not express mutant ATXN1 protein, indicating the sparing of Purkinje cells can be explained by an absence of transgene expression. Additionally, we found that Purkinje cells in other cerebellar lobules that typically express AldoC, not only display severe pathology but also show loss of AldoC expression. The relatively preserved flocculonodular lobes and crus I showed a substantial fraction of Purkinje cells that expressed the mutant protein and displayed pathology as well as loss of AldoC expression. Despite considerable pathology in these lobules, behavioral analyses demonstrated a relative sparing of related functions, suggestive of sufficient functional cerebellar reserve. Together, the data indicate that mutant ATXN1 affects both AldoC-positive and AldoC-negative Purkinje cells and disrupts normal parasagittal AldoC expression in Purkinje cells. Our results show that, in a mouse model otherwise characterized by widespread Purkinje cell degeneration, sparing of specific subpopulations is sufficient to maintain normal performance of specific behaviors within the context of the functional, modular map of the cerebellum.

The basal interstitial nucleus (BIN) of the cerebellum provides diffuse ascending inhibitory input to the floccular granule cell layer.

The basal interstitial nucleus (BIN) in the white matter of the vestibulocerebellum has been defined more than three decades ago, but has since been largely ignored. It is still unclear which neurotransmitters are being used by BIN neurons, how these neurons are connected to the rest of the brain and what their activity patterns look like. Here, we studied BIN neurons in a range of mammals, including macaque, human, rat, mouse, rabbit, and ferret, using tracing, immunohistological and electrophysiological approaches. We show that BIN neurons are GABAergic and glycinergic, that in primates they also express the marker for cholinergic neurons choline acetyl transferase (ChAT), that they project with beaded fibers to the glomeruli in the granular layer of the ipsilateral floccular complex, and that they are driven by excitation from the ipsilateral and contralateral medio-dorsal medullary gigantocellular reticular formation. Systematic analysis of codistribution of the inhibitory synapse marker VIAAT, BIN axons, and Golgi cell marker mGluR2 indicate that BIN axon terminals complement Golgi cell axon terminals in glomeruli, accounting for a considerable proportion ( > 20%) of the inhibitory terminals in the granule cell layer of the floccular complex. Together, these data show that BIN neurons represent a novel and relevant inhibitory input to the part of the vestibulocerebellum that controls compensatory and smooth pursuit eye movements.