Climbing fibers provide essential instructive signals for associative learning.

Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber (CF) inputs to the cerebellar cortex represent one of the strongest candidates in the vertebrate brain for conveying neural instructive signals. However, recent studies have shown that Purkinje cell stimulation can also drive cerebellar learning and the relative importance of these two neuron types in providing instructive signals for cerebellum-dependent behaviors remains unresolved. In the present study we used cell-type-specific perturbations of various cerebellar circuit elements to systematically evaluate their contributions to delay eyeblink conditioning in mice. Our findings reveal that, although optogenetic stimulation of either CFs or Purkinje cells can drive learning under some conditions, even subtle reductions in CF signaling completely block learning to natural stimuli. We conclude that CFs and corresponding Purkinje cell complex spike events provide essential instructive signals for associative cerebellar learning.

Structural and Functional Organization of Visual Responses in the Inferior Olive of Larval Zebrafish.

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.

The cerebellum.

The cerebellum, that stripey 'little brain', sits at the back of your head, under your visual cortex, and contains more than half of the neurons in your entire nervous system. The cerebellum is highly conserved across vertebrates, and its evolutionary expansion has tended to proceed in concert with expansion of cerebral cortex. The crystalline neuronal architecture of the cerebellar cortex was first described by Cajal a century ago, and its functional connectivity was elucidated in exquisite anatomical and physiological detail by the mid-20th century. The ability to clearly identify molecularly distinct cerebellar cell types that constitute discrete circuit elements is perhaps unparalleled among brain areas, even within the context of modern circuit neuroscience. Although traditionally thought of as primarily a motor structure, the cerebellum is highly interconnected with diverse brain areas and, as I will explain in this Primer, is well-poised to influence a wide range of motor and cognitive functions.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ('Myomatrix arrays') that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a 'motor unit,' during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and identifying pathologies of the motor system.

Synaptic mechanisms for associative learning in the cerebellar nuclei.

Associative learning during delay eyeblink conditioning (EBC) depends on an intact cerebellum. However, the relative contribution of changes in the cerebellar nuclei to learning remains a subject of ongoing debate. In particular, little is known about the changes in synaptic inputs to cerebellar nuclei neurons that take place during EBC and how they shape the membrane potential of these neurons. Here, we probed the ability of these inputs to support associative learning in mice, and investigated structural and cell-physiological changes within the cerebellar nuclei during learning. We find that optogenetic stimulation of mossy fiber afferents to the anterior interposed nucleus (AIP) can substitute for a conditioned stimulus and is sufficient to elicit conditioned responses (CRs) that are adaptively well-timed. Further, EBC induces structural changes in mossy fiber and inhibitory inputs, but not in climbing fiber inputs, and it leads to changes in subthreshold processing of AIP neurons that correlate with conditioned eyelid movements. The changes in synaptic and spiking activity that precede the CRs allow for a decoder to distinguish trials with a CR. Our data reveal how structural and physiological modifications of synaptic inputs to cerebellar nuclei neurons can facilitate learning.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ("Myomatrix arrays") that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a "motor unit", during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and in identifying pathologies of the motor system.

A step forward for stress-induced ataxia.

Patients with episodic ataxia type 2 (EA2) display attacks of severe incoordination and dystonia that can be triggered by stress. In a recent study, Snell, Vitenzon, Tara, and colleagues found a mechanistic pathway by which norepinephrine (NE) alters cerebellar Purkinje output to trigger attacks in a mouse model of EA2 and identified a pharmacological intervention that effectively reduces them.

Parallel locomotor control strategies in mice and flies.

Our understanding of the neural basis of locomotor behavior can be informed by careful quantification of animal movement. Classical descriptions of legged locomotion have defined discrete locomotor gaits, characterized by distinct patterns of limb movement. Recent technical advances have enabled increasingly detailed characterization of limb kinematics across many species, imposing tighter constraints on neural control. Here, we highlight striking similarities between coordination patterns observed in two genetic model organisms: the laboratory mouse and Drosophila. Both species exhibit continuously-variable coordination patterns with similar low-dimensional structure, suggesting shared principles for limb coordination and descending neural control.

COVID-19, stress, and inequities in (neuro)science.

The COVID-19 pandemic has posed major challenges for diversity, equity, and inclusion (DEI) efforts in research and academia. As chairs of the ALBA Network, we reflect on how the pandemic has exacerbated, and also shone a spotlight on, inequalities in science and society.

GluA4 facilitates cerebellar expansion coding and enables associative memory formation.

AMPA receptors (AMPARs) mediate excitatory neurotransmission in the central nervous system (CNS) and their subunit composition determines synaptic efficacy. Whereas AMPAR subunits GluA1-GluA3 have been linked to particular forms of synaptic plasticity and learning, the functional role of GluA4 remains elusive. Here, we demonstrate a crucial function of GluA4 for synaptic excitation and associative memory formation in the cerebellum. Notably, GluA4-knockout mice had ~80% reduced mossy fiber to granule cell synaptic transmission. The fidelity of granule cell spike output was markedly decreased despite attenuated tonic inhibition and increased NMDA receptor-mediated transmission. Computational network modeling incorporating these changes revealed that deletion of GluA4 impairs granule cell expansion coding, which is important for pattern separation and associative learning. On a behavioral level, while locomotor coordination was generally spared, GluA4-knockout mice failed to form associative memories during delay eyeblink conditioning. These results demonstrate an essential role for GluA4-containing AMPARs in cerebellar information processing and associative learning.

Cannabinoids modulate associative cerebellar learning via alterations in behavioral state.

Cannabinoids are notorious and profound modulators of behavioral state. In the brain, endocannabinoids act via Type 1-cannabinoid receptors (CB1) to modulate synaptic transmission and mediate multiple forms of synaptic plasticity. CB1 knockout (CB1KO) mice display a range of behavioral phenotypes, in particular hypoactivity and various deficits in learning and memory, including cerebellum-dependent delay eyeblink conditioning. Here we find that the apparent effects of CB1 deletion on cerebellar learning are not due to direct effects on CB1-dependent plasticity, but rather, arise as a secondary consequence of altered behavioral state. Hypoactivity of CB1KO mice accounts for their impaired eyeblink conditioning across both animals and trials. Moreover, learning in these mutants is rescued by walking on a motorized treadmill during training. Finally, cerebellar granule-cell-specific CB1KOs exhibit normal eyeblink conditioning, and both global and granule-cell-specific CB1KOs display normal cerebellum-dependent locomotor coordination and learning. These findings highlight the modulation of behavioral state as a powerful independent means through which individual genes contribute to complex behaviors.

Shared and specific signatures of locomotor ataxia in mutant mice.

Several spontaneous mouse mutants with deficits in motor coordination and associated cerebellar neuropathology have been described. Intriguingly, both visible gait alterations and neuroanatomical abnormalities throughout the brain differ across mutants. We previously used the LocoMouse system to quantify specific deficits in locomotor coordination in mildly ataxic Purkinje cell degeneration mice (pcd; Machado et al., 2015). Here, we analyze the locomotor behavior of severely ataxic reeler mutants and compare and contrast it with that of pcd. Despite clearly visible gait differences, direct comparison of locomotor kinematics and linear discriminant analysis reveal a surprisingly similar pattern of impairments in multijoint, interlimb, and whole-body coordination in the two mutants. These findings capture both shared and specific signatures of gait ataxia and provide a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies in mice.

Spatial and Temporal Locomotor Learning in Mouse Cerebellum.

Stable and efficient locomotion requires the precise coordination of movement across the limbs and body. Learned changes in interlimb coordination can be induced by exposure to a split-belt treadmill that imposes different speeds under each side of the body. Here, we demonstrate locomotor learning on a split-belt treadmill in mice. Mouse locomotor adaptation is specific to measures of interlimb coordination, has spatial and temporal components that adapt at different rates, and is context specific. The many similarities between human and mouse locomotor adaptation suggest that this form of locomotor learning is highly conserved across vertebrates. Using a variety of approaches, we demonstrate that split-belt adaptation in mice specifically depends on the intermediate cerebellum but is insensitive to large lesions of the cerebral cortex. Finally, cell-type-specific chemogenetics combined with quantitative behavioral analysis reveals that spatial and temporal components of locomotor adaptation are dissociable on the circuit level. VIDEO ABSTRACT.

Locomotor activity modulates associative learning in mouse cerebellum.

Changes in behavioral state can profoundly influence brain function. Here we show that behavioral state modulates performance in delay eyeblink conditioning, a cerebellum-dependent form of associative learning. Increased locomotor speed in head-fixed mice drove earlier onset of learning and trial-by-trial enhancement of learned responses that were dissociable from changes in arousal and independent of sensory modality. Eyelid responses evoked by optogenetic stimulation of mossy fiber inputs to the cerebellum, but not at sites downstream, were positively modulated by ongoing locomotion. Substituting prolonged, low-intensity optogenetic mossy fiber stimulation for locomotion was sufficient to enhance conditioned responses. Our results suggest that locomotor activity modulates delay eyeblink conditioning through increased activation of the mossy fiber pathway within the cerebellum. Taken together, these results provide evidence for a novel role for behavioral state modulation in associative learning and suggest a potential mechanism through which engaging in movement can improve an individual's ability to learn.

Transient inhibition and long-term facilitation of locomotion by phasic optogenetic activation of serotonin neurons.

Serotonin (5-HT) is associated with mood and motivation but the function of endogenous 5-HT remains controversial. Here, we studied the impact of phasic optogenetic activation of 5-HT neurons in mice over time scales from seconds to weeks. We found that activating dorsal raphe nucleus (DRN) 5-HT neurons induced a strong suppression of spontaneous locomotor behavior in the open field with rapid kinetics (onset ≤1 s). Inhibition of locomotion was independent of measures of anxiety or motor impairment and could be overcome by strong motivational drive. Repetitive place-contingent pairing of activation caused neither place preference nor aversion. However, repeated 15 min daily stimulation caused a persistent increase in spontaneous locomotion to emerge over three weeks. These results show that 5-HT transients have strong and opposing short and long-term effects on motor behavior that appear to arise from effects on the underlying factors that motivate actions.

A quantitative framework for whole-body coordination reveals specific deficits in freely walking ataxic mice.

The coordination of movement across the body is a fundamental, yet poorly understood aspect of motor control. Mutant mice with cerebellar circuit defects exhibit characteristic impairments in locomotor coordination; however, the fundamental features of this gait ataxia have not been effectively isolated. Here we describe a novel system (LocoMouse) for analyzing limb, head, and tail kinematics of freely walking mice. Analysis of visibly ataxic Purkinje cell degeneration (pcd) mice reveals that while differences in the forward motion of individual paws are fully accounted for by changes in walking speed and body size, more complex 3D trajectories and, especially, inter-limb and whole-body coordination are specifically impaired. Moreover, the coordination deficits in pcd are consistent with a failure to predict and compensate for the consequences of movement across the body. These results isolate specific impairments in whole-body coordination in mice and provide a quantitative framework for understanding cerebellar contributions to coordinated locomotion.

A matter of trial and error for motor learning.

Climbing fiber inputs to cerebellar Purkinje cells are thought to carry error signals that can trigger motor learning across multiple time scales. A new study by Kimpo et al. finds that the potency of climbing fibers as instructive signals for adaptation of the vestibulo-ocular reflex depends on task conditions.

All Purkinje cells are not created equal.

Although the wiring of the cerebellar cortex appears to be uniform, the neurons in this region of the brain behave more differently from each other than previously thought.