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.

The Effect of Nucleo-Olivary Stimulation on Climbing Fiber EPSPs in Purkinje Cells.

Climbing fibers, connecting the inferior olive and Purkinje cells, form the nervous system's strongest neural connection. These fibers activate after critical events like motor errors or anticipation of rewards, leading to bursts of excitatory postsynaptic potentials (EPSPs) in Purkinje cells. The number of EPSPs is a crucial variable when the brain is learning a new motor skill. Yet, we do not know what determines the number of EPSPs. Here, we measured the effect of nucleo-olivary stimulation on periorbital elicited climbing fiber responses through in-vivo intracellular Purkinje cell recordings in decerebrated ferrets. The results show that while nucleo-olivary stimulation decreased the probability of a response occurring at all, it did not reduce the number of EPSPs. The results suggest that nucleo-olivary stimulation does not influence the number of EPSPs in climbing fiber bursts.

SK2 channels in cerebellar Purkinje cells contribute to excitability modulation in motor-learning-specific memory traces.

Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such "intrinsic plasticity" in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell-specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning.

Cerebellar plasticity and associative memories are controlled by perineuronal nets.

Perineuronal nets (PNNs) are assemblies of extracellular matrix molecules, which surround the cell body and dendrites of many types of neuron and regulate neural plasticity. PNNs are prominently expressed around neurons of the deep cerebellar nuclei (DCN), but their role in adult cerebellar plasticity and behavior is far from clear. Here we show that PNNs in the mouse DCN are diminished during eyeblink conditioning (EBC), a form of associative motor learning that depends on DCN plasticity. When memories are fully acquired, PNNs are restored. Enzymatic digestion of PNNs in the DCN improves EBC learning, but intact PNNs are necessary for memory retention. At the structural level, PNN removal induces significant synaptic rearrangements in vivo, resulting in increased inhibition of DCN baseline activity in awake behaving mice. Together, these results demonstrate that PNNs are critical players in the regulation of cerebellar circuitry and function.

Pavlovian eyeblink conditioning is severely impaired in tottering mice.

Cacna1a encodes the pore-forming α1A subunit of CaV2.1 voltage-dependent calcium channels, which regulate neuronal excitability and synaptic transmission. Purkinje cells in the cortex of cerebellum abundantly express these CaV2.1 channels. Here, we show that homozygous tottering (tg) mice, which carry a loss-of-function Cacna1a mutation, exhibit severely impaired learning in Pavlovian eyeblink conditioning, which is a cerebellar-dependent learning task. Performance of reflexive eyeblinks is unaffected in tg mice. Transient seizure activity in tg mice further corrupted the amplitude of eyeblink conditioned responses. Our results indicate that normal calcium homeostasis is imperative for cerebellar learning and that the oscillatory state of the brain can affect the expression thereof.NEW & NOTEWORTHY In this study, we confirm the importance of normal calcium homeostasis in neurons for learning and memory formation. In a mouse model with a mutation in an essential calcium channel that is abundantly expressed in the cerebellum, we found severely impaired learning in eyeblink conditioning. Eyeblink conditioning is a cerebellar-dependent learning task. During brief periods of brain-wide oscillatory activity, as a result of the mutation, the expression of conditioned eyeblinks was even further disrupted.

Cerebellar Learning Properties Are Modulated by the CRF Receptor.

Corticotropin-releasing factor (CRF) and its type 1 receptor (CRFR1) play an important role in the responses to stressful challenges. Despite the well established expression of CRFR1 in granular cells (GrCs), its role in procedural motor performance and memory formation remains elusive. To investigate the role of CRFR1 expression in cerebellar GrCs, we used a mouse model depleted of CRFR1 in these cells. We detected changes in the cellular learning mechanisms in GrCs depleted of CRFR1 in that they showed changes in intrinsic excitability and long-term synaptic plasticity. Analysis of cerebella transcriptome obtained from KO and control mice detected prominent alterations in the expression of calcium signaling pathways components. Moreover, male mice depleted of CRFR1 specifically in GrCs showed accelerated Pavlovian associative eye-blink conditioning, but no differences in baseline motor performance, locomotion, or fear and anxiety-related behaviors. Our findings shed light on the interplay between stress-related central mechanisms and cerebellar motor conditioning, highlighting the role of the CRF system in regulating particular forms of cerebellar learning.SIGNIFICANCE STATEMENT Although it is known that the corticotropin-releasing factor type 1 receptor (CRFR1) is highly expressed in the cerebellum, little attention has been given to its role in cerebellar functions in the behaving animal. Moreover, most of the attention was directed at the effect of CRF on Purkinje cells at the cellular level and, to this date, almost no data exist on the role of this stress-related receptor in other cerebellar structures. Here, we explored the behavioral and cellular effect of granular cell-specific ablation of CRFR1 We found a profound effect on learning both at the cellular and behavioral levels without an effect on baseline motor skills.

Modulation of 7 T fMRI Signal in the Cerebellar Cortex and Nuclei During Acquisition, Extinction, and Reacquisition of Conditioned Eyeblink Responses.

Classical delay eyeblink conditioning is likely the most commonly used paradigm to study cerebellar learning. As yet, few studies have focused on extinction and savings of conditioned eyeblink responses (CRs). Saving effects, which are reflected in a reacquisition after extinction that is faster than the initial acquisition, suggest that learned associations are at least partly preserved during extinction. In this study, we tested the hypothesis that acquisition-related plasticity is nihilated during extinction in the cerebellar cortex, but retained in the cerebellar nuclei, allowing for faster reacquisition. Changes of 7 T functional magnetic resonance imaging (fMRI) signals were investigated in the cerebellar cortex and nuclei of young and healthy human subjects. Main effects of acquisition, extinction, and reacquisition against rest were calculated in conditioned stimulus-only trials. First-level ß values were determined for a spherical region of interest (ROI) around the acquisition peak voxel in lobule VI, and dentate and interposed nuclei ipsilateral to the unconditioned stimulus. In the cerebellar cortex and nuclei, fMRI signals were significantly lower in extinction compared to acquisition and reacquisition, but not significantly different between acquisition and reacquisition. These findings are consistent with the theory of bidirectional learning in both the cerebellar cortex and nuclei. It cannot explain, however, why conditioned responses reappear almost immediately in reacquisition following extinction. Although the present data do not exclude that part of the initial memory remains in the cerebellum in extinction, future studies should also explore changes in extracerebellar regions as a potential substrate of saving effects. Hum Brain Mapp 38:3957-3974, 2017. © 2017 Wiley Periodicals, Inc.

Cerebellar function and ischemic brain lesions in migraine patients from the general population.

Objective The objective of this article is to obtain detailed quantitative assessment of cerebellar function and structure in unselected migraine patients and controls from the general population. Methods A total of 282 clinically well-defined participants (migraine with aura n = 111; migraine without aura n = 89; non-migraine controls n = 82; age range 43-72; 72% female) from a population-based study were subjected to a range of sensitive and validated cerebellar tests that cover functions of all main parts of the cerebellar cortex, including cerebrocerebellum, spinocerebellum, and vestibulocerebellum. In addition, all participants underwent magnetic resonance imaging (MRI) of the brain to screen for cerebellar lesions. As a positive control, the same cerebellar tests were conducted in 13 patients with familial hemiplegic migraine type 1 (FHM1; age range 19-64; 69% female) all carrying a CACNA1A mutation known to affect cerebellar function. Results MRI revealed cerebellar ischemic lesions in 17/196 (8.5%) migraine patients and 3/79 (4%) controls, which were always located in the posterior lobe except for one control. With regard to the cerebellar tests, there were no differences between migraine patients with aura, migraine patients without aura, and controls for the: (i) Purdue-pegboard test for fine motor skills (assembly scores p = 0.1); (ii) block-design test for visuospatial ability (mean scaled scores p = 0.2); (iii) prism-adaptation task for limb learning (shift scores p = 0.8); (iv) eyeblink-conditioning task for learning-dependent timing (peak-time p = 0.1); and (v) body-sway test for balance capabilities (pitch velocity score under two-legs stance condition p = 0.5). Among migraine patients, those with cerebellar ischaemic lesions performed worse than those without lesions on the assembly scores of the pegboard task ( p < 0.005), but not on the primary outcome measures of the other tasks. Compared with controls and non-hemiplegic migraine patients, FHM1 patients showed substantially more deficits on all primary outcomes, including Purdue-peg assembly ( p < 0.05), block-design scaled score ( p < 0.001), shift in prism-adaptation ( p < 0.001), peak-time of conditioned eyeblink responses ( p < 0.05) and pitch-velocity score during stance-sway test ( p < 0.001). Conclusions Unselected migraine patients from the general population show normal cerebellar functions despite having increased prevalence of ischaemic lesions in the cerebellar posterior lobe. Except for an impaired pegboard test revealing deficits in fine motor skills, these lesions appear to have little functional impact. In contrast, all cerebellar functions were significantly impaired in participants with FHM1.

Spinocerebellar ataxia type 6 protein aggregates cause deficits in motor learning and cerebellar plasticity.

Spinocerebellar ataxia type 6 (SCA6) is linked to poly-glutamine (polyQ) within the C terminus (CT) of the pore-forming subunits of P/Q-type Ca(2+) channels (Cav2.1) and is characterized by CT protein aggregates found in cerebellar Purkinje cells (PCs). One hypothesis regarding SCA6 disease is that a CT fragment of the Cav2.1 channel, which is detected specifically in cytosolic and nuclear fractions in SCA6 patients, is associated with the SCA6 pathogenesis. To test this hypothesis, we expressed P/Q-type channel protein fragments from two different human CT splice variants, as predicted from SCA6 patients, in PCs of mice using viral and transgenic approaches. These splice variants represent a short (CT-short without polyQs) and a long (CT-long with 27 polyQs) CT fragment. Our results show that the different splice variants of the CTs differentially distribute within PCs, i.e., the short CTs reveal predominantly nuclear inclusions, whereas the long CTs prominently reveal both nuclear and cytoplasmic aggregates. Postnatal expression of CTs in PCs in mice reveals that only CT-long causes SCA6-like symptoms, i.e., deficits in eyeblink conditioning (EBC), ataxia, and PC degeneration. The physiological phenotypes associated specifically with the long CT fragment can be explained by an impairment of LTD and LTP at the parallel fiber-to-PC synapse and alteration in spontaneous PC activity. Thus, our results suggest that the polyQ carrying the CT fragment of the P/Q-type channel is sufficient to cause SCA6 pathogenesis in mice and identifies EBC as a new diagnostic strategy to evaluate Ca(2+) channel-mediated human diseases.

Cerebellar cortex and cerebellar nuclei are concomitantly activated during eyeblink conditioning: a 7T fMRI study in humans.

There are controversies whether learning of conditioned eyeblink responses primarily takes place within the cerebellar cortex, the interposed nuclei, or both. It has also been suggested that the cerebellar cortex may be important during early stages of learning, and that there is a shift to the cerebellar nuclei during later stages. As yet, human studies have provided little to resolve this question. In the present study, we established a setup that allows ultra-high-field 7T functional magnetic resonance imaging (fMRI) of the cerebellar cortex and interposed cerebellar nuclei simultaneously during delay eyeblink conditioning in humans. Event-related fMRI signals increased concomitantly in the cerebellar cortex and nuclei during early acquisition of conditioned eyeblink responses in 20 healthy human subjects. ANOVAs with repeated-measures showed significant effects of time across five blocks of 20 conditioning trials in the cortex and nuclei (p < 0.05, permutation corrected). Activations were most pronounced in, but not limited to, lobules VI and interposed nuclei. Increased activations were most prominent at the first time the maximum number of conditioned responses was achieved. Our data are consistent with a simultaneous and synergistic two-site model of learning during acquisition of classically conditioned eyeblinks. Because increased MRI signal reflects synaptic activity, concomitantly increased signals in the cerebellar nuclei and cortex are consistent with findings of learning related potentiation at the mossy fiber to nuclear cell synapse and mossy fiber to granule cell synapse. Activity related to the expression of conditioned responses, however, cannot be excluded.

Axonal sprouting and formation of terminals in the adult cerebellum during associative motor learning.

Plastic changes in the efficacy of synapses are widely regarded to represent mechanisms underlying memory formation. So far, evidence for learning-dependent, new neuronal wiring is limited. In this study, we demonstrate that pavlovian eyeblink conditioning in adult mice can induce robust axonal growth and synapse formation in the cerebellar nuclei. This de novo wiring is both condition specific and region specific because it does not occur in pseudoconditioned animals and is particularly observed in those parts of the cerebellar nuclei that have been implicated to be involved in this form of motor learning. Moreover, the number of new mossy fiber varicosities in these parts of the cerebellar nuclei is positively correlated with the amplitude of conditioned eyelid responses. These results indicate that outgrowth of axons and concomitant occurrence of new terminals may, in addition to plasticity of synaptic efficacy, contribute to the formation of memory.

Automated high-throughput mouse transsynaptic viral tracing using iDISCO+ tissue clearing, light-sheet microscopy, and BrainPipe.

Transsynaptic viral tracing requires tissue sectioning, manual cell counting, and anatomical assignment, all of which are time intensive. We describe a protocol for BrainPipe, a scalable software for automated anatomical alignment and object counting in light-sheet microscopy volumes. BrainPipe can be generalized to new counting tasks by using a new atlas and training a neural network for object detection. Combining viral tracing, iDISCO+ tissue clearing, and BrainPipe facilitates mapping of cerebellar connectivity to the rest of the murine brain. For complete details on the use and execution of this protocol, please refer to Pisano et al. (2021).

Young Domestic Pigs (Sus scrofa) Can Perform Pavlovian Eyeblink Conditioning.

Introduction: Pigs have been an increasingly popular preclinical model in nutritional neuroscience, as their anatomy, physiology, and nutrition requirements are highly comparable to those of humans. Eyeblink conditioning is one of the most well-validated behavioral paradigms in neuroscience to study underlying mechanisms of learning and memory formation in the cerebellum. Eyeblink conditioning has been performed in many species but has never been done on young pigs. Therefore, our aim here was to develop and validate an eyeblink conditioning paradigm in young pigs. Method: Eighteen intact male pigs were artificially reared from postnatal day 2-30. The eyeblink conditioning setup consisted of a sound-damping box with a hammock that pigs were placed in, which allowed the pig to remain comfortable yet maintain a typical range of head motion. In a delay conditioning paradigm, the conditional stimulus (CS) was a 550 ms blue light-emitting diode (LED), the unconditional stimulus (US) was a 50 ms eye air-puff, the CS-US interval was 500 ms. Starting at postnatal day 14, pigs were habituated for 5 days to the eyeblink conditioning setup, followed by 5 daily sessions of acquisition training (40 paired CS-US trials each day). Results: The group-averaged amplitude of conditioned eyelid responses gradually increased over the course of the 5 days of training, indicating that pigs learned to make the association between the LED light CS and the air-puff US. A similar increase was found for the conditioned response (CR) probability: the group-averaged CR probability on session 1 was about 12% and reached a CR probability of 55% on day 5. The latency to CR peak time lacked a temporal preference in the first session but clearly showed preference from the moment that animals started to show more CRs in session 2 and onwards whereby the eyelid was maximally closed exactly at the moment that the US would be delivered. Conclusion: We concluded that 3-week-old pigs have the capability of performing in a cerebellar classical conditioning task, demonstrating for the first time that eyeblink conditioning in young pigs has the potential to be a valuable behavioral tool to measure neurodevelopment.

Homologous organization of cerebellar pathways to sensory, motor, and associative forebrain.

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here, we quantify pathways between the cerebellum and forebrain by using transsynaptic tracing viruses and a whole-brain analysis pipeline. With retrograde tracing, we find that most descending paths originate from the somatomotor cortex. Anterograde tracing of ascending paths encompasses most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei. In the neocortex, sensorimotor regions contain the most labeled neurons, but we find higher densities in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlate with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of the cerebellar cortex to diverse forebrain targets. We conclude that shared areas of the cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.

Differential spatiotemporal development of Purkinje cell populations and cerebellum-dependent sensorimotor behaviors.

Distinct populations of Purkinje cells (PCs) with unique molecular and connectivity features are at the core of the modular organization of the cerebellum. Previously, we showed that firing activity of PCs differs between ZebrinII-positive and ZebrinII-negative cerebellar modules (Zhou et al., 2014; Wu et al., 2019). Here, we investigate the timing and extent of PC differentiation during development in mice. We found that several features of PCs, including activity levels, dendritic arborization, axonal shape and climbing fiber input, develop differentially between nodular and anterior PC populations. Although all PCs show a particularly rapid development in the second postnatal week, anterior PCs typically have a prolonged physiological and dendritic maturation. In line herewith, younger mice exhibit attenuated anterior-dependent eyeblink conditioning, but faster nodular-dependent compensatory eye movement adaptation. Our results indicate that specific cerebellar regions have unique developmental timelines which match with their related, specific forms of cerebellum-dependent behaviors.

AMPAR Auxiliary Protein SHISA6 Facilitates Purkinje Cell Synaptic Excitability and Procedural Memory Formation.

The majority of excitatory postsynaptic currents in the brain are gated through AMPA-type glutamate receptors, the kinetics and trafficking of which can be modulated by auxiliary proteins. It remains to be elucidated whether and how auxiliary proteins can modulate synaptic function to contribute to procedural memory formation. In this study, we report that the AMPA-type glutamate receptor (AMPAR) auxiliary protein SHISA6 (CKAMP52) is expressed in cerebellar Purkinje cells, where it co-localizes with GluA2-containing AMPARs. The absence of SHISA6 in Purkinje cells results in severe impairments in the adaptation of the vestibulo-ocular reflex and eyeblink conditioning. The physiological abnormalities include decreased presence of AMPARs in synaptosomes, impaired excitatory transmission, increased deactivation of AMPA receptors, and reduced induction of long-term potentiation at Purkinje cell synapses. Our data indicate that Purkinje cells require SHISA6-dependent modification of AMPAR function in order to facilitate cerebellar, procedural memory formation.

TRPC3 is a major contributor to functional heterogeneity of cerebellar Purkinje cells.

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.

Caffeine has no effect on eyeblink conditioning in mice.

Caffeine is one of the most widely used drugs in the world. In the brain, caffeine acts as an antagonist for the adenosine A1 and A2B receptors. Since A1 receptors are highly concentrated in the cortex of the cerebellum, we hypothesized that caffeine could potentially affect learning tasks that require the cerebellar cortex, such as eyeblink conditioning. To test this hypothesis, we examined the effect of low (5mg/kg) and high (50mg/kg) doses of caffeine, injected intraperitoneally before training, on eyeblink conditioning in mice. The results show that, at the dosages we used, caffeine affects neither the rate of acquisition, nor the timing of the onset or peak of the conditioned blink responses. Therefore, we conclude that caffeine neither improves nor worsens performance on eyeblink conditioning.

Impact of parallel fiber to Purkinje cell long-term depression is unmasked in absence of inhibitory input.

Pavlovian eyeblink conditioning has been used extensively to study the neural mechanisms underlying associative and motor learning. During this simple learning task, memory formation takes place at Purkinje cells in defined areas of the cerebellar cortex, which acquire a strong temporary suppression of their activity during conditioning. Yet, it is unknown which neuronal plasticity mechanisms mediate this suppression. Two potential mechanisms include long-term depression of parallel fiber to Purkinje cell synapses and feed-forward inhibition by molecular layer interneurons. We show, using a triple transgenic approach, that only concurrent disruption of both these suppression mechanisms can severely impair conditioning, highlighting that both processes can compensate for each other's deficits.

Dynamic modulation of activity in cerebellar nuclei neurons during pavlovian eyeblink conditioning in mice.

While research on the cerebellar cortex is crystallizing our understanding of its function in learning behavior, many questions surrounding its downstream targets remain. Here, we evaluate the dynamics of cerebellar interpositus nucleus (IpN) neurons over the course of Pavlovian eyeblink conditioning. A diverse range of learning-induced neuronal responses was observed, including increases and decreases in activity during the generation of conditioned blinks. Trial-by-trial correlational analysis and optogenetic manipulation demonstrate that facilitation in the IpN drives the eyelid movements. Adaptive facilitatory responses are often preceded by acquired transient inhibition of IpN activity that, based on latency and effect, appear to be driven by complex spikes in cerebellar cortical Purkinje cells. Likewise, during reflexive blinks to periocular stimulation, IpN cells show excitation-suppression patterns that suggest a contribution of climbing fibers and their collaterals. These findings highlight the integrative properties of subcortical neurons at the cerebellar output stage mediating conditioned behavior.