The cerebellum fine-tunes motor activity via its Purkinje cell output. Purkinje cells produce two different types of spikes, complex spikes and simple spikes, which often show reciprocal activity: a periodical increase in complex spikes is associated with a decrease in simple spikes, and vice versa. This reciprocal firing is thought to be essential for coordinated motor behavior, yet how it is accomplished is debated. Here, we show in Ptf1a::cre;Robo3(lox/lox) mice that selectively rerouting the climbing fibers from a contralateral to an ipsilateral projection reversed the complex-spike modulation during sensory stimulation. Strikingly, modulation of simple spikes, which is supposed to be controlled by mossy fibers, reversed as well. Climbing fibers enforce this reciprocity in part by influencing activity of inhibitory interneurons, because the phase of their activity was also converted. Ptf1a::cre;Robo3(lox/lox) mice showed severe ataxia highlighting that climbing fiber input and its impact on reciprocity of Purkinje cell firing play an important role in motor coordination.

The brain must make sense of external stimuli to generate relevant behavior. We used a combination of in vivo approaches to investigate how the cerebellum processes sensory-related information. We found that the inferior olive encodes contexts of sensory-associated external cues in a graded manner, apparent in the presynaptic activity of their axonal projections (climbing fibers) in the cerebellar cortex. Individual climbing fibers were broadly responsive to different sensory modalities but relayed sensory-related information to the cortex in a lobule-dependent manner. Purkinje cell dendrites faithfully transformed this climbing fiber activity into dendrite-wide Ca2+ signals without a direct contribution from the mossy fiber pathway. These results demonstrate that the size of climbing-fiber-evoked Ca2+ signals in Purkinje cell dendrites is largely determined by the firing level of climbing fibers. This coding scheme emphasizes the overwhelming role of the inferior olive in generating salient signals useful for instructing plasticity and learning.

Functional neural circuits are formed by eliminating early-formed redundant synapses and strengthening necessary connections during development. In newborn mouse cerebellum, each Purkinje cell (PC) is innervated by multiple climbing fibers (CFs) with similar strengths. Subsequently, a single CF is selectively strengthened by postnatal day 7 (P7). We find that this competition among multiple CFs occurs on the soma before CFs form synapses along dendrites. Notably, in most PCs, the single CF that has been functionally strengthened (the "winner" CF) undergoes translocation to dendrites while keeping its synapses on the soma. Synapses of the weaker CFs (the "loser" CFs) remain around the soma and form "pericellular nests" with synapses of the winner CFs. Then most perisomatic synapses are eliminated nonselectively by P15. Thus, our results suggest that the selective translocation of the winner CF to dendrites in each PC determines the single CF that survives subsequent synapse elimination and persistently innervates the PC.

IP(3) signaling in Purkinje cells is involved in the regulation of cell functions including LTD. We have used a GFP-tagged pleckstrin homology domain to visualize IP(3) dynamics in Purkinje cells. Surprisingly, IP(3) production was observed in response not only to mGluR activation, but also to AMPA receptor activation in Purkinje cells in culture. AMPA-induced IP(3) production was mediated by depolarization-induced Ca(2+) influx because it was mimicked by depolarization and was blocked by inhibition of the P-type Ca(2+) channel. Furthermore, trains of complex spikes, elicited by climbing fiber stimulation (1 Hz), induced IP(3) production in Purkinje cells in cerebellar slices. These results revealed a novel IP(3) signaling pathway in Purkinje cells that can be elicited by synaptic inputs from climbing fibers.

Purkinje cell dendrites convert excitatory climbing fiber input into signals that instruct plasticity and motor learning. Modulation of instructive signaling may increase the range in which learning is encoded, yet the mechanisms that allow for this are poorly understood. We found that optogenetic activation of molecular layer interneurons (MLIs) that inhibit Purkinje cells suppressed climbing-fiber-evoked dendritic Ca2+ spiking. Inhibitory suppression of Ca2+ spiking depended on the level of MLI activation and influenced the induction of associative synaptic plasticity, converting climbing-fiber-mediated potentiation of parallel fiber-evoked responses into depression. In awake mice, optogenetic activation of floccular climbing fibers in association with head rotation produced an adaptive increase in the vestibulo-ocular reflex (VOR). However, when climbing fibers were co-activated with MLIs, adaptation occurred in the opposite direction, decreasing the VOR. Thus, MLIs can direct a continuous spectrum of plasticity and learning through their influence on Purkinje cell dendritic Ca2+ signaling.

Closed-loop circuitries between cortical and subcortical regions can facilitate precision of output patterns, but the role of such networks in the cerebellum remains to be elucidated. Here, we characterize the role of internal feedback from the cerebellar nuclei to the cerebellar cortex in classical eyeblink conditioning. We find that excitatory output neurons in the interposed nucleus provide efference-copy signals via mossy fibers to the cerebellar cortical zones that belong to the same module, triggering monosynaptic responses in granule and Golgi cells and indirectly inhibiting Purkinje cells. Upon conditioning, the local density of nucleocortical mossy fiber terminals significantly increases. Optogenetic activation and inhibition of nucleocortical fibers in conditioned animals increases and decreases the amplitude of learned eyeblink responses, respectively. Our data show that the excitatory nucleocortical closed-loop circuitry of the cerebellum relays a corollary discharge of premotor signals and suggests an amplifying role of this circuitry in controlling associative motor learning.

We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.

Neurotransmitter spillover represents a form of neural transmission not restricted to morphologically defined synaptic connections. Communication between climbing fibers (CFs) and molecular layer interneurons (MLIs) in the cerebellum is mediated exclusively by glutamate spillover. Here, we show how CF stimulation functionally segregates MLIs based on their location relative to glutamate release. Excitation of MLIs that reside within the domain of spillover diffusion coordinates inhibition of MLIs outside the diffusion limit. CF excitation of MLIs is dependent on extrasynaptic NMDA receptors that enhance the spatial and temporal spread of CF signaling. Activity mediated by functionally segregated MLIs converges onto neighboring Purkinje cells (PCs) to generate a long-lasting biphasic change in inhibition. These data demonstrate how glutamate release from single CFs modulates excitability of neighboring PCs, thus expanding the influence of CFs on cerebellar cortical activity in a manner not predicted by anatomical connectivity.

Ca2+-activated ion channels shape membrane excitability and Ca2+ dynamics in response to cytoplasmic Ca2+ elevation. Compared to the Ca2+-activated K+ channels, known as BK and SK channels, the physiological importance of Ca2+-activated Cl- channels (CaCCs) in neurons has been largely overlooked. Here we report that CaCCs coexist with BK and SK channels in inferior olivary (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for the control of motor learning and timing. Ca2+ influx through the dendritic high-threshold voltage-gated Ca2+ channels activates CaCCs, which contribute to membrane repolarization of IO neurons. Loss of TMEM16B expression resulted in the absence of CaCCs in IO neurons, leading to markedly diminished action potential firing of IO neurons in TMEM16B knockout mice. Moreover, these mutant mice exhibited severe cerebellar motor learning deficits. Our findings thus advance the understanding of the neurophysiology of CaCCs and the ionic basis of IO neuron excitability.

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.

Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.

Neurexins are recognized as key organizers of synapses that are essential for normal brain function. However, it is unclear whether neurexins are fundamental building blocks of all synapses with similar overall functions or context-dependent specifiers of synapse properties. To address this question, we produced triple cKO (conditional knockout) mice that allow ablating all neurexin expression in mice. Using neuron-specific manipulations combined with immunocytochemistry, paired recordings, and two-photon Ca2+ imaging, we analyzed excitatory synapses formed by climbing fibers on Purkinje cells in cerebellum and inhibitory synapses formed by parvalbumin- or somatostatin-positive interneurons on pyramidal layer 5 neurons in the medial prefrontal cortex. After pan-neurexin deletions, we observed in these synapses severe but dramatically different synaptic phenotypes that ranged from major impairments in their distribution and function (climbing-fiber synapses) to large decreases in synapse numbers (parvalbumin-positive synapses) and severe alterations in action potential-induced presynaptic Ca2+ transients (somatostatin-positive synapses). Thus, neurexins function primarily as context-dependent specifiers of synapses.

The importance of neuronal morphology in brain function has been recognized for over a century. The broad applicability of "digital reconstructions" of neuron morphology across neuroscience subdisciplines has stimulated the rapid development of numerous synergistic tools for data acquisition, anatomical analysis, three-dimensional rendering, electrophysiological simulation, growth models, and data sharing. Here we discuss the processes of histological labeling, microscopic imaging, and semiautomated tracing. Moreover, we provide an annotated compilation of currently available resources in this rich research "ecosystem" as a central reference for experimental and computational neuroscience.