All-optical strategies enable identification of functional neuronal ensembles with calcium imaging and replay/alter their spatiotemporal activity with optogenetics to decipher their behavioral implications. We previously developed a fiber-coupled microscope enabling two-photon (2P) functional imaging and 2P holographic photostimulation with near-single-cell resolution in freely moving mice: 2P-FENDO. Here, we present a significantly optimized 2P-FENDO-II system that achieves a four-times-larger field of view and a more homogeneous light distribution across the field of view, both for imaging and photostimulation, while achieving better flexibility and thus optimal adaptation to the study of freely moving mice. We demonstrate the performance and versatility of 2P-FENDO-II in experiments targeting the somatosensory cortex, the visual cortex, or the cerebellar cortex, in which we show concomitant calcium imaging with jGCaMP7s and optogenetic control with ChRmine. These enhancements establish 2P-FENDO-II as a groundbreaking tool for all-optical interrogation of neuronal circuits on large volume in naturalistic situations.

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.

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.

Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables the generation of precise spatiotemporal neuronal activity patterns and thus a broad range of experimental applications, such as high throughput connectivity mapping and probing neural codes for perception. Yet, current holographic approaches limit the resolution for tuning the relative spiking time of distinct cells to a few milliseconds, and the achievable number of targets to 100-200, depending on the working depth. To overcome these limitations and expand the capabilities of single-cell optogenetics, we introduce an ultra-fast sequential light targeting (FLiT) optical configuration based on the rapid switching of a temporally focused beam between holograms at kHz rates. We used FLiT to demonstrate two illumination protocols, termed hybrid- and cyclic-illumination, and achieve sub-millisecond control of sequential neuronal activation and high throughput multicell illumination in vitro (mouse organotypic and acute brain slices) and in vivo (zebrafish larvae and mice), while minimizing light-induced thermal rise. These approaches will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles.

We present a new micro-endoscope based on a fiber bundle and a GRIN lens, capable of 2P holographic photostimulation of multiple neurons with single cell precision and fast (100 Hz) 2P calcium imaging on fields of view of 250 µm 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.

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.

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.

Significance Neuronal subtypes are differentially affected by neuropathologies. For example, Purkinje cells, the principal neurons of the cerebellum, can be divided in subpopulations based on their sensitivity to pathological insult. However, the molecular mechanisms explaining why, among seemingly identical neurons, some will degenerate while others survive remain unknown. Here, we analyzed, in a disease model of cerebellar neurodegeneration, the metabolism of sphingolipids, complex lipids involved in cell apoptosis, and found that specific sphingolipids accumulate in the cerebellar region primarily affected by neurodegeneration. Preventing this accumulation by disrupting sphingolipid metabolism via genetic mutation caused a neuroprotective effect on subpopulations of Purkinje cells. Thus, our data indicate that sphingolipid metabolism is involved in the predisposition of neuronal subtypes to neurodegeneration.

Despite the canonical homogeneous character of its organization, the cerebellum plays differential computational roles in distinct sensorimotor behaviors. Previously, we showed that Purkinje cell (PC) activity differs between zebrin-negative (Z–) and zebrin-positive (Z+) modules (Zhou et al., 2014). Here, using gain-of-function and loss-of-function mouse models, we show that transient receptor potential cation channel C3 (TRPC3) controls the simple spike activity of Z–, but not Z+ PCs. In addition, TRPC3 regulates complex spike rate and their interaction with simple spikes, exclusively in Z– PCs. At the behavioral level, TRPC3 loss-of-function mice show impaired eyeblink conditioning, which is related to Z– modules, whereas compensatory eye movement adaptation, linked to Z+ modules, is intact. Together, our results indicate that TRPC3 is a major contributor to the cellular heterogeneity that introduces distinct physiological properties in PCs, conjuring functional heterogeneity in cerebellar sensorimotor integration.

Despite the canonical homogenous character of its organization, the cerebellum plays differential computational roles in distinct types of sensorimotor behaviors. However, the molecular and cell physiological underpinnings are unclear. Here we determined the contribution of transient receptor potential cation channel type C3 (TRPC3) to signal processing in different cerebellar modules. Using gain-of-function and loss-of-function mouse models, we found that TRPC3 controls the simple spike activity of zebrin-negative (Z-), but not of zebrin-positive (Z+), Purkinje cells. Moreover, in vivo TRPC3 also regulated complex spike firing and its interaction with simple spikes exclusively in Z- Purkinje cells. Finally, we found that eyeblink conditioning, related to Z- modules, but not compensatory eye movement adaptation, linked to Z+ modules, was affected in TRPC3 loss-of-function mice. Together, our results indicate that TRPC3 is essential for the cellular heterogeneity that introduces distinct physiological properties in an otherwise homogeneous population of Purkinje cells, conjuring functional heterogeneity in cerebellar sensorimotor integration.

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.