Cerebellar Purkinje cells in male macaques combine sensory and motor information to predict the sensory consequences of active self-motion.

Accurate perception and behavior rely on distinguishing sensory signals arising from unexpected events from those originating from our own voluntary actions. In the vestibular system, sensory input that is the consequence of active self-motion is canceled early at the first central stage of processing to ensure postural and perceptual stability. However, the source of the required cancellation signal was unknown. Here, we show that the cerebellum combines sensory and motor-related information to predict the sensory consequences of active self-motion. Recordings during attempted but unrealized head movements in two male rhesus monkeys, revealed that the motor-related signals encoded by anterior vermis Purkinje cells explain their altered sensitivity to active versus passive self-motion. Further, a model combining responses from ~40 Purkinje cells accounted for the cancellation observed in early vestibular pathways. These findings establish how cerebellar Purkinje cells predict sensory outcomes of self-movements, resolving a long-standing issue of sensory signal suppression during self-motion.

Dynamic molecular network analysis of iPSC-Purkinje cells differentiation delineates roles of ISG15 in SCA1 at the earliest stage.

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.

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.

The olivary input to the cerebellum dissociates sensory events from movement plans.

Neurons in the inferior olive are thought to anatomically organize the Purkinje cells (P-cells) of the cerebellum into computational modules, but what is computed by each module? Here, we designed a saccade task in marmosets that dissociated sensory events from motor events and then recorded the complex and simple spikes of hundreds of P-cells. We found that when a visual target was presented at a random location, the olive reported the direction of that sensory event to one group of P-cells, but not to a second group. However, just before movement onset, it reported the direction of the planned movement to both groups, even if that movement was not toward the target. At the end of the movement if the subject experienced an error but chose to withhold the corrective movement, only the first group received information about the sensory prediction error. We organized the P-cells based on the information content of their olivary input and found that in the group that received sensory information, the simple spikes were suppressed during fixation, then produced a burst before saccade onset in a direction consistent with assisting the movement. In the second group, the simple spikes were not suppressed during fixation but burst near saccade deceleration in a direction consistent with stopping the movement. Thus, the olive differentiated the P-cells based on whether they would receive sensory or motor information, and this defined their contributions to control of movements as well as holding still.

Mesoscale simulations predict the role of synergistic cerebellar plasticity during classical eyeblink conditioning.

According to the motor learning theory by Albus and Ito, synaptic depression at the parallel fibre to Purkinje cells synapse (pf-PC) is the main substrate responsible for learning sensorimotor contingencies under climbing fibre control. However, recent experimental evidence challenges this relatively monopolistic view of cerebellar learning. Bidirectional plasticity appears crucial for learning, in which different microzones can undergo opposite changes of synaptic strength (e.g. downbound microzones-more likely depression, upbound microzones-more likely potentiation), and multiple forms of plasticity have been identified, distributed over different cerebellar circuit synapses. Here, we have simulated classical eyeblink conditioning (CEBC) using an advanced spiking cerebellar model embedding downbound and upbound modules that are subject to multiple plasticity rules. Simulations indicate that synaptic plasticity regulates the cascade of precise spiking patterns spreading throughout the cerebellar cortex and cerebellar nuclei. CEBC was supported by plasticity at the pf-PC synapses as well as at the synapses of the molecular layer interneurons (MLIs), but only the combined switch-off of both sites of plasticity compromised learning significantly. By differentially engaging climbing fibre information and related forms of synaptic plasticity, both microzones contributed to generate a well-timed conditioned response, but it was the downbound module that played the major role in this process. The outcomes of our simulations closely align with the behavioural and electrophysiological phenotypes of mutant mice suffering from cell-specific mutations that affect processing of their PC and/or MLI synapses. Our data highlight that a synergy of bidirectional plasticity rules distributed across the cerebellum can facilitate finetuning of adaptive associative behaviours at a high spatiotemporal resolution.

Kit Ligand and Kit receptor tyrosine kinase sustain synaptic inhibition of Purkinje cells.

The cell-type-specific expression of ligand/receptor and cell-adhesion molecules is a fundamental mechanism through which neurons regulate connectivity. Here, we determine a functional relevance of the long-established mutually exclusive expression of the receptor tyrosine kinase Kit and the trans-membrane protein Kit Ligand by discrete populations of neurons in the mammalian brain. Kit is enriched in molecular layer interneurons (MLIs) of the cerebellar cortex (i.e., stellate and basket cells), while cerebellar Kit Ligand is selectively expressed by a target of their inhibition, Purkinje cells (PCs). By in vivo genetic manipulation spanning embryonic development through adulthood, we demonstrate that PC Kit Ligand and MLI Kit are required for, and capable of driving changes in, the inhibition of PCs. Collectively, these works in mice demonstrate that the Kit Ligand/Kit receptor dyad sustains mammalian central synapse function and suggest a rationale for the affiliation of Kit mutation with neurodevelopmental disorders.

Real-time field-programmable gate array-based closed-loop deep brain stimulation platform targeting cerebellar circuitry rescues motor deficits in a mouse model of cerebellar ataxia.

AIMS: The open-loop nature of conventional deep brain stimulation (DBS) produces continuous and excessive stimulation to patients which contributes largely to increased prevalence of adverse side effects. Cerebellar ataxia is characterized by abnormal Purkinje cells (PCs) dendritic arborization, loss of PCs and motor coordination, and muscle weakness with no effective treatment. We aim to develop a real-time field-programmable gate array (FPGA) prototype targeting the deep cerebellar nuclei (DCN) to close the loop for ataxia using conditional double knockout mice with deletion of PC-specific LIM homeobox (Lhx)1 and Lhx5, resulting in abnormal dendritic arborization and motor deficits. METHODS: We implanted multielectrode array in the DCN and muscles of ataxia mice. The beneficial effect of open-loop DCN-DBS or closed-loop DCN-DBS was compared by motor behavioral assessments, electromyography (EMG), and neural activities (neurospike and electroencephalogram) in freely moving mice. FPGA board, which performed complex real-time computation, was used for closed-loop DCN-DBS system. RESULTS: Closed-loop DCN-DBS was triggered only when symptomatic muscle EMG was detected in a real-time manner, which restored motor activities, electroencephalogram activities and neurospike properties completely in ataxia mice. Closed-loop DCN-DBS was more effective than an open-loop paradigm as it reduced the frequency of DBS. CONCLUSION: Our real-time FPGA-based DCN-DBS system could be a potential clinical strategy for alleviating cerebellar ataxia and other movement disorders.

PP2B-Dependent Cerebellar Plasticity Sets the Amplitude of the Vestibulo-ocular Reflex during Juvenile Development.

Throughout life, the cerebellum plays a central role in the coordination and optimization of movements, using cellular plasticity to adapt a range of behaviors. Whether these plasticity processes establish a fixed setpoint during development, or continuously adjust behaviors throughout life, is currently unclear. Here, by spatiotemporally manipulating the activity of protein phosphatase 2B (PP2B), an enzyme critical for cerebellar plasticity in male and female mice, we examined the consequences of disrupted plasticity on the performance and adaptation of the vestibulo-ocular reflex (VOR). We find that, in contrast to Purkinje cell (PC)-specific deletion starting early postnatally, acute pharmacological as well as adult-onset genetic deletion of PP2B affects all forms of VOR adaptation but not the level of VOR itself. Next, we show that PC-specific genetic deletion of PP2B in juvenile mice leads to a progressive loss of the protein PP2B and a concurrent change in the VOR, in addition to the loss of adaptive abilities. Finally, re-expressing PP2B in adult mice that lack PP2B expression from early development rescues VOR adaptation but does not affect the performance of the reflex. Together, our results indicate that chronic or acute, genetic, or pharmacological block of PP2B disrupts the adaptation of the VOR. In contrast, only the absence of plasticity during cerebellar development affects the setpoint of VOR, an effect that cannot be corrected after maturation of the cerebellum. These findings suggest that PP2B-dependent cerebellar plasticity is required during a specific period to achieve the correct setpoint of the VOR.

Etomidate enhances cerebellar CF-PC synaptic plasticity through CB1 receptor/PKA cascade in vitro in mice.

Etomidate (ET) is a widely used intravenous imidazole general anesthetic, which depresses the cerebellar neuronal activity by modulating various receptors activity and synaptic transmission. In this study, we investigated the effects of ET on the cerebellar climbing fiber-Purkinje cells (CF-PC) plasticity in vitro in mice using whole-cell recording technique and pharmacological methods. Our results demonstrated that CF tetanic stimulation produced a mGluR1-dependent long-term depression (LTD) of CF-PC excitatory postsynaptic currents (EPSCs), which was enhanced by bath application of ET (10 µM). Blockade of mGluR1 receptor with JNJ16259685, ET triggered the tetanic stimulation to induce a CF-PC LTD accompanied with an increase in paired-pulse ratio (PPR). The ET-triggered CF-PC LTD was abolished by extracellular administration of an N-methyl-(D)-aspartate (NMDA) receptor antagonist, D-APV, as well as by intracellular blockade of NMDA receptors activity with MK801. Furthermore, blocking cannabinoids 1 (CB1) receptor with AM251 or chelating intracellular Ca2+ with BAPTA, ET failed to trigger the CF-PC LTD. Moreover, the ET-triggered CF-PC LTD was abolished by inhibition of protein kinase A (PKA), but not by inhibition of protein kinase C inhibiter. The present results suggest that ET acts on postsynaptic NMDA receptor resulting in an enhancement of the cerebellar CF-PC LTD through CB1 receptor/PKA cascade in vitro in mice. These results provide new evidence and possible mechanism for ET anesthesia to affect motor learning and motor coordination by regulating cerebellar CF-PC LTD.

A machine-learning tool to identify bistable states from calcium imaging data.

Mapping neuronal activation using calcium imaging in vivo during behavioural tasks has advanced our understanding of nervous system function. In almost all of these studies, calcium imaging is used to infer spike probabilities because action potentials activate voltage-gated calcium channels and increase intracellular calcium levels. However, neurons not only fire action potentials, but also convey information via intrinsic dynamics such as by generating bistable membrane potential states. Although a number of tools for spike inference have been developed and are currently being used, no tool exists for converting calcium imaging signals to maps of cellular state in bistable neurons. Purkinje neurons in the larval zebrafish cerebellum exhibit membrane potential bistability, firing either tonically or in bursts. Several studies have implicated the role of a population code in cerebellar function, with bistability adding an extra layer of complexity to this code. In the present study, we develop a tool, CaMLSort, which uses convolutional recurrent neural networks to classify calcium imaging traces as arising from either tonic or bursting cells. We validate this classifier using a number of different methods and find that it performs well on simulated event rasters as well as real biological data that it had not previously seen. Moreover, we find that CaMLsort generalizes to other bistable neurons, such as dopaminergic neurons in the ventral tegmental area of mice. Thus, this tool offers a new way of analysing calcium imaging data from bistable neurons to understand how they participate in network computation and natural behaviours. KEY POINTS: Calcium imaging, compriising the gold standard of inferring neuronal activity, does not report cellular state in neurons that are bistable, such as Purkinje neurons in the cerebellum of larval zebrafish. We model the relationship between Purkinje neuron electrical activity and its corresponding calcium signal to compile a dataset of state-labelled simulated calcium signals. We apply machine-learning methods to this dataset to develop a tool that can classify the state of a Purkinje neuron using only its calcium signal, which works well on real data even though it was trained only on simulated data. CaMLsort (Calcium imaging and Machine Learning based tool to sort intracellular state) also generalizes well to bistable neurons in a different brain region (ventral tegmental area) in a different model organism (mouse). This tool can facilitate our understanding of how these neurons carry out their functions in a circuit.

Predictive neural computations in the cerebellum contribute to motor planning and faster behavioral responses in larval zebrafish.

The ability to predict the future based on past experience lies at the core of the brain's ability to adapt behavior. However, the neural mechanisms that participate in generating and updating predictions are not clearly understood. Further, the evolutionary antecedents and the prevalence of predictive processing among vertebrates are even less explored. Here, we show evidence of predictive processing via the involvement of cerebellar circuits in larval zebrafish. We presented stereotyped optic flow stimuli to larval zebrafish to evoke swims and discovered that lesioning the cerebellum abolished prediction-dependent modulation of swim latency. When expectations of optic flow direction did not match with reality, error signals arrive at Purkinje cells via the olivary climbing fibers, whereas granule cells and Purkinje cells encode signals of expectation. Strong neural representations of expectation correlate with faster swim responses and vice versa. In sum, our results show evidence for predictive processing in nonmammalian vertebrates with the involvement of cerebellum, an evolutionarily conserved brain structure.

Cerebellar Purkinje cell firing promotes conscious recovery from anesthesia state through coordinating neuronal communications with motor cortex.

Background: The neurobiological basis of gaining consciousness from unconscious state induced by anesthetics remains unknown. This study was designed to investigate the involvement of the cerebello-thalamus-motor cortical loop mediating consciousness transitions from the loss of consciousness (LOC) induced by an inhalational anesthetic sevoflurane in mice. Methods: The neural tracing and fMRI together with opto-chemogenetic manipulation were used to investigate the potential link among cerebello-thalamus-motor cortical brain regions. The fiber photometry of calcium and neurotransmitters, including glutamate (Glu), γ-aminobutyric acid (GABA) and norepinephrine (NE), were monitored from the motor cortex (M1) and the 5th lobule of the cerebellar vermis (5Cb) during unconsciousness induced by sevoflurane and gaining consciousness after sevoflurane exposure. Cerebellar Purkinje cells were optogenetically manipulated to investigate their influence on consciousness transitions during and after sevoflurane exposure. Results: Activation of 5Cb Purkinje cells increased the Ca2+ flux in the M1 CaMKIIα+ neurons, but this increment was significantly reduced by inactivation of posterior and parafascicular thalamic nucleus. The 5Cb and M1 exhibited concerted calcium flux, and glutamate and GABA release during transitions from wakefulness, loss of consciousness, burst suppression to conscious recovery. Ca2+ flux and Glu release in the M1, but not in the 5Cb, showed a strong synchronization with the EEG burst suppression, particularly, in the gamma-band range. In contrast, the Glu, GABA and NE release and Ca2+ oscillations were coherent with the EEG gamma band activity only in the 5Cb during the pre-recovery of consciousness period. The optogenetic activation of Purkinje cells during burst suppression significantly facilitated emergence from anesthesia while the optogenetic inhibition prolonged the time to gaining consciousness. Conclusions: Our data indicate that cerebellar neuronal communication integrated with motor cortex through thalamus promotes consciousness recovery from anesthesia which may likely serve as arousal regulation.

Implications of variable synaptic weights for rate and temporal coding of cerebellar outputs.

Purkinje cell (PC) synapses onto cerebellar nuclei (CbN) neurons allow signals from the cerebellar cortex to influence the rest of the brain. PCs are inhibitory neurons that spontaneously fire at high rates, and many PC inputs are thought to converge onto each CbN neuron to suppress its firing. It has been proposed that PCs convey information using a rate code, a synchrony and timing code, or both. The influence of PCs on CbN neuron firing was primarily examined for the combined effects of many PC inputs with comparable strengths, and the influence of individual PC inputs has not been extensively studied. Here, we find that single PC to CbN synapses are highly variable in size, and using dynamic clamp and modeling we reveal that this has important implications for PC-CbN transmission. Individual PC inputs regulate both the rate and timing of CbN firing. Large PC inputs strongly influence CbN firing rates and transiently eliminate CbN firing for several milliseconds. Remarkably, the refractory period of PCs leads to a brief elevation of CbN firing prior to suppression. Thus, individual PC-CbN synapses are suited to concurrently convey rate codes and generate precisely timed responses in CbN neurons. Either synchronous firing or synchronous pauses of PCs promote CbN neuron firing on rapid time scales for nonuniform inputs, but less effectively than for uniform inputs. This is a secondary consequence of variable input sizes elevating the baseline firing rates of CbN neurons by increasing the variability of the inhibitory conductance. These findings may generalize to other brain regions with highly variable inhibitory synapse sizes.

Human Purkinje cells outperform mouse Purkinje cells in dendritic complexity and computational capacity.

Purkinje cells in the cerebellum are among the largest neurons in the brain and have been extensively investigated in rodents. However, their morphological and physiological properties remain poorly understood in humans. In this study, we utilized high-resolution morphological reconstructions and unique electrophysiological recordings of human Purkinje cells ex vivo to generate computational models and estimate computational capacity. An inter-species comparison showed that human Purkinje cell had similar fractal structures but were larger than those of mouse Purkinje cells. Consequently, given a similar spine density (2/µm), human Purkinje cell hosted approximately 7.5 times more dendritic spines than those of mice. Moreover, human Purkinje cells had a higher dendritic complexity than mouse Purkinje cells and usually emitted 2-3 main dendritic trunks instead of one. Intrinsic electro-responsiveness was similar between the two species, but model simulations revealed that the dendrites could process ~6.5 times (n = 51 vs. n = 8) more input patterns in human Purkinje cells than in mouse Purkinje cells. Thus, while human Purkinje cells maintained spike discharge properties similar to those of rodents during evolution, they developed more complex dendrites, enhancing computational capacity.

Development and Optimization of a Multilayer Rat Purkinje Neuron Culture.

Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.

Olov Oscarsson's Description of Afferent Pathways to the Cerebellum: Excellent Physiology, Base for Anatomy, and Road Toward Understanding Function.

Olov Oscarsson's review on the functional organization of spinocerebellar paths is a prime demonstration of the great skills and huge knowledge base of the electrophysiologists of his era working on communication systems in the brain. Oscarsson describes and characterizes in detail no less than ten different communication lines between the spinal cord and the cerebellum. As such, his work proved to be a highly fertile basis for ongoing physiological and anatomical research. However, even after 50 years of continuing cerebellar research, many questions are still open and even care must be taken that the differentiation in spinocerebellar paths, so carefully demonstrated by Oscarsson, is not lost in present-day research.

Sepsis Impairs Purkinje Cell Functions and Motor Behaviors Through Microglia Activation.

The most common clinical manifestation of sepsis-related encephalopathy (SAE) is the deterioration of cognitive function. Besides, increasing evidence shows that SAE patients exhibit coordination and sensorimotor dysfunctions, suggesting that SAE affects motor function with unclear mechanism. In the present work, we explored the effects of SAE on cerebellar Purkinje cells (PCs) using cecal ligation and perforation (CLP), a standard model for inducing sepsis symptoms similar to those in human patients. Our results show that the sepsis can activate microglia in the cerebellum and promote the secretion of inflammatory factor TNF-α, which increases intrinsic excitability and synaptic transmission of PCs, inhibits the synaptic plasticity of PCs, and impairs motor learning of mice. These findings address how SAE changes PC functions, and thereby are of great significance to reveal pathophysiological feathers of human patients suffering from SAE.

Reduction of Microvessel Number and Length in the Cerebellum of Purkinje Cell Degeneration Mice.

Degenerative effects of nerve tissues are often accompanied by changes in vascularization. In this regard, knowledge about hereditary cerebellar degeneration is limited. In this study, we compared the vascularity of the individual cerebellar components of 3-month-old wild-type mice (n = 8) and Purkinje cell degeneration (pcd) mutant mice, which represent a model of hereditary cerebellar degeneration (n = 8). Systematic random samples of tissue sections were processed, and laminin was immunostained to visualize microvessels. A computer-assisted stereology system was used to quantify microvessel parameters including total number, total length, and associated densities in cerebellar layers. Our results in pcd mice revealed a 45% (p < 0.01) reduction in the total volume of the cerebellum, a 28% (p < 0.05) reduction in the total number of vessels and a lower total length, approaching 50% (p < 0.001), compared to the control mice. In pcd mutants, cerebellar degeneration is accompanied by significant reduction in the microvascular network that is proportional to the cerebellar volume reduction therefore does not change density of in the cerebellar gray matter of pcd mice.

Whisker kinematics in the cerebellum.

The whisker system is widely used as a model system for understanding sensorimotor integration. Purkinje cells in the crus regions of the cerebellum have been reported to linearly encode whisker midpoint, but it is unknown whether the paramedian and simplex lobules as well as their target neurons in the cerebellar nuclei also encode whisker kinematics and if so which ones. Elucidating how these kinematics are represented throughout the cerebellar hemisphere is essential for understanding how the cerebellum coordinates multiple sensorimotor modalities. Exploring the cerebellar hemisphere of mice using optogenetic stimulation, we found that whisker movements can be elicited by stimulation of Purkinje cells in not only crus1 and crus2, but also in the paramedian lobule and lobule simplex; activation of cells in the medial paramedian lobule had on average the shortest latency, whereas that of cells in lobule simplex elicited similar kinematics as those in crus1 and crus2. During spontaneous whisking behaviour, simple spike activity correlated in general better with velocity than position of the whiskers, but it varied between protraction and retraction as well as per lobule. The cerebellar nuclei neurons targeted by the Purkinje cells showed similar activity patterns characterized by a wide variety of kinematic signals, yet with a dominance for velocity. Taken together, our data indicate that whisker movements are much more prominently and diversely represented in the cerebellar cortex and nuclei than assumed, highlighting the rich repertoire of cerebellar control in the kinematics of movements that can be engaged during coordination. KEY POINTS: Excitation of Purkinje cells throughout the cerebellar hemispheres induces whisker movement, with the shortest latency and longest duration within the paramedian lobe. Purkinje cells have differential encoding for the fast and slow components of whisking. Purkinje cells encode not only the position but also the velocity of whiskers. Purkinje cells with high sensitivity for whisker velocity are preferentially located in the medial part of lobule simplex, crus1 and lateral paramedian. In the downstream cerebellar nuclei, neurons with high sensitivity for whisker velocity are located at the intersection between the medial and interposed nucleus.

Heterogeneous encoding of temporal stimuli in the cerebellar cortex.

Local feedforward and recurrent connectivity are rife in the frontal areas of the cerebral cortex, which gives rise to rich heterogeneous dynamics observed in such areas. Recently, similar local connectivity motifs have been discovered among Purkinje and molecular layer interneurons of the cerebellar cortex, however, task-related activity in these neurons has often been associated with relatively simple facilitation and suppression dynamics. Here, we show that the rodent cerebellar cortex supports heterogeneity in task-related neuronal activity at a scale similar to the cerebral cortex. We provide a computational model that inculcates recent anatomical insights into local microcircuit motifs to show the putative basis for such heterogeneity. We also use cell-type specific chronic viral lesions to establish the involvement of cerebellar lobules in associative learning behaviors. Functional heterogeneity in neuronal profiles may not merely be the remit of the associative cerebral cortex, similar principles may be at play in subcortical areas, even those with seemingly crystalline and homogenous cytoarchitectures like the cerebellum.