The Clp1 R140H mutation alters tRNA metabolism and mRNA 3' processing in mouse models of pontocerebellar hypoplasia.

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.

Visualizing the deep cerebellar nuclei using quantitative susceptibility mapping: An application in healthy controls, Parkinson's disease patients and essential tremor patients.

The visualization and identification of the deep cerebellar nuclei (DCN) (dentate [DN], interposed [IN] and fastigial nuclei [FN]) are particularly challenging. We aimed to visualize the DCN using quantitative susceptibility mapping (QSM), predict the contrast differences between QSM and T2* weighted imaging, and compare the DCN volume and susceptibility in movement disorder populations and healthy controls (HCs). Seventy-one Parkinson's disease (PD) patients, 39 essential tremor patients, and 80 HCs were enrolled. The PD patients were subdivided into tremor dominant (TD) and postural instability/gait difficulty (PIGD) groups. A 3D strategically acquired gradient echo MR imaging protocol was used for each subject to obtain the QSM data. Regions of interest were drawn manually on the QSM data to calculate the volume and susceptibility. Correlation analysis between the susceptibility and either age or volume was performed and the intergroup differences of the volume and magnetic susceptibility in all the DCN structures were evaluated. For the most part, all the DCN structures were clearly visualized on the QSM data. The susceptibility increased as a function of volume for both the HC group and disease groups in the DN and IN (p < .001) but not the FN (p = .74). Only the volume of the FN in the TD-PD group was higher than that in the HCs (p = .012), otherwise, the volume and susceptibility among these four groups did not differ significantly. In conclusion, QSM provides clear visualization of the DCN structures. The results for the volume and susceptibility of the DCN can be used as baseline references in future studies of movement disorders.

Cytokine enrichment in deep cerebellar nuclei is contributed by multiple glial populations and linked to reduced amyloid plaque pathology.

Alzheimer's disease (AD) pathology and amyloid-beta (Aß) plaque deposition progress slowly in the cerebellum compared to other brain regions, while the entorhinal cortex (EC) is one of the most vulnerable regions. Using a knock-in AD mouse model (App KI), we show that within the cerebellum, the deep cerebellar nuclei (DCN) has particularly low accumulation of Aß plaques. To identify factors that might underlie differences in the progression of AD-associated neuropathology across regions, we profiled gene expression in single nuclei (snRNAseq) across all cell types in the DCN and EC of wild-type (WT) and App KI male mice at age 7 months. We found differences in expression of genes associated with inflammatory activation, PI3K-AKT signalling, and neuron support functions between both regions and genotypes. In WT mice, the expression of interferon-response genes in microglia is higher in the DCN than the EC and this enrichment is confirmed by RNA in situ hybridisation, and measurement of inflammatory cytokines by protein array. Our analyses also revealed that multiple glial populations are responsible for establishing this cytokine-enriched niche. Furthermore, homogenates derived from the DCN induced inflammatory gene expression in BV2 microglia. We also assessed the relationship between the DCN microenvironment and Aß pathology by depleting microglia using a CSF1R inhibitor PLX5622 and saw that, surprisingly, the expression of a subset of inflammatory cytokines was increased while plaque abundance in the DCN was further reduced. Overall, our study revealed the presence of a cytokine-enriched microenvironment unique to the DCN that when modulated, can alter plaque deposition.

Electrophysiological alterations of the Purkinje cells and deep cerebellar neurons in a mouse model of Alzheimer disease (electrophysiology on cerebellum of AD mice).

Alzheimer's disease is histopathologically well defined by the presence of amyloid deposits and tau-related neurofibrillary tangles in crucial regions of the brain. Interest is growing in revealing and determining possible pathological markers also in the cerebellum as its involvement in cognitive functions is now well supported. Despite the central position of the Purkinje cell in the cerebellum, its electrophysiological behaviour in mouse models of Alzheimer's disease is scarce in the literature. Our first aim was here to focus on the electrophysiological behaviour of the cerebellum in awake mouse model of Alzheimer's disease (APPswe/PSEN1dE9) and the related performance on the water-maze test classically used in behavioural studies. We found prevalent signs of electrophysiological alterations in both Purkinje cells and deep cerebellar nuclei neurons which might explain the behavioural deficits reported during the water-maze test. The alterations of neurons firing were accompanied by a dual (~16 and ~228 Hz) local field potential's oscillation in the Purkinje cell layer of Alzheimer's disease mice which was concomitant to an important increase of both the simple and the complex spikes. In addition, ß-amyloid deposits were present in the molecular layer of the cerebellum. These results highlight the importance of the output firing modification of the AD cerebellum that may indirectly impact the activity of its subcortical and cortical targets.

Deep Cerebellar Nuclei Functional Connectivity with Cerebral Cortex in Temporal Lobe Epilepsy With and Without Focal to Bilateral Tonic-Clonic Seizures: a Resting-State fMRI Study.

We aimed to explore the altered functional connectivity patterns within cerebello-cerebral circuits in temporal lobe epilepsy (TLE) patients with and without focal to bilateral tonic-clonic seizures (FBTCS). Forty-two patients with unilateral TLE (21 with and 21 without FBTCS) and 22 healthy controls were recruited. We chose deep cerebellar nuclei as seed regions, calculated static and dynamic functional connectivity (sFC and dFC) in the patients with and without FBTCS and healthy controls, and compared sFC and dFC among the three groups. Correlation analyses were used to assess relationships between the significantly altered imaging features and patient clinical parameters. Compared to the group without FBTCS, the FBTCS group showed decreased sFC between the right dentate nuclei and left hemisphere regions including the middle frontal gyrus, superior temporal gyrus, superior medial frontal gyrus and posterior cingulate gyrus, and significantly increased dFC between the right interposed nuclei and contralateral precuneus. Relative to HCs, the FBTCS group demonstrated prominently decreased sFC between the right dentate nuclei and left middle frontal gyrus. No significant correlations between the altered imaging features and patient clinical parameters were observed. Our results suggest that the disrupted cerebello-cerebral FC might be related to cognitive impairment, epileptogenesis, and propagation of epileptic activities in patients with FBTCS.

Endocannabinoids regulate cerebellar GABAergic transmission in a synapse type-dependent manner.

Endocannabinoids (eCBs) act as ubiquitous modulators of synaptic transmission via the activation of cannabinoid receptors (CBRs). Cerebellar Purkinje cells (PCs) make strong inhibitory synaptic contacts not only with neurons in the deep cerebellar nuclei (DCN) but also with Lugaro cells and globular cells, whose cell bodies are located underneath the PC layer. However, little is known about the modulatory actions of eCBs on GABA release from PC axon terminals. Here, we examined the effects of eCBs on the GABAergic transmission at PC-globular cell synapses and PC-large DCN neuron synapses electrophysiologically using mouse cerebellar slices. We showed that the types 1 and 2 CBR agonist WIN55212 did not affect either spontaneous or miniature inhibitory postsynaptic currents (IPSCs) in globular cells under control conditions and in a state of enhanced synaptic activity. By contrast, another Gi/o protein-coupled receptor agonist, baclofen, significantly reduced the miniature IPSC frequency in globular cells. WIN55212 had no effects on IPSCs in large DCN neurons. A type 2 CBR agonist, HU308, also had no effects on IPSCs in either globular cells or large DCN neurons. Moreover, the PCs' target neurons did not elicit depolarization-induced suppression of inhibition. These results suggest the lack of a functional role of CBRs at PCs' axon terminals. This is in sharp contrast to the fact that PCs receive abundant excitatory and inhibitory inputs that are under eCB-mediated presynaptic inhibitory modulation. The actions of eCBs are selective to distinct synapses and possibly contribute to information processes and rigorous signal transmission in the cerebellum.

Changes in membrane properties of rat deep cerebellar nuclear projection neurons during acquisition of eyeblink conditioning.

Previous studies have shown changes in membrane properties of neurons in rat deep cerebellar nuclei (DCN) as a function of development, but due to technical difficulties in obtaining viable DCN slices from adult animals, it remains unclear whether there are learning-related alterations in the membrane properties of DCN neurons in adult rats. This study was designed to record from identified DCN cells in cerebellar slices from postnatal day 25-26 (P25-26) rats that had a relatively mature sensory nervous system and were able to acquire learning as a result of tone-shock eyeblink conditioning (EBC) and to document resulting changes in electrophysiological properties. After electromyographic electrode implantation at P21 and inoculation with a fluorescent pseudorabies virus (PRV-152) at P22-23, rats received either four sessions of paired delay EBC or unpaired stimulus presentations with a tone conditioned stimulus and a shock unconditioned stimulus or sat in the training chamber without stimulus presentations. Compared with rats given unpaired stimuli or no stimulus presentations, rats given paired EBC showed an increase in conditioned responses across sessions. Whole-cell recordings of both fluorescent and nonfluorescent DCN projection neurons showed that delay EBC induced significant changes in membrane properties of evoked DCN action potentials including a reduced after-hyperpolarization amplitude and shortened latency. Similar findings were obtained in hyperpolarization-induced rebound spikes of DCN neurons. In sum, delay EBC produced significant changes in the membrane properties of juvenile rat DCN projection neurons. These learning-specific changes in DCN excitability have not previously been reported in any species or task.

Novel Missense CACNA1G Mutations Associated with Infantile-Onset Developmental and Epileptic Encephalopathy.

The CACNA1G gene encodes the low-voltage-activated Cav3.1 channel, which is expressed in various areas of the CNS, including the cerebellum. We studied two missense CACNA1G variants, p.L208P and p.L909F, and evaluated the relationships between the severity of Cav3.1 dysfunction and the clinical phenotype. The presentation was of a developmental and epileptic encephalopathy without evident cerebellar atrophy. Both patients exhibited axial hypotonia, developmental delay, and severe to profound cognitive impairment. The patient with the L909F mutation had initially refractory seizures and cerebellar ataxia, whereas the L208P patient had seizures only transiently but was overall more severely affected. In transfected mammalian cells, we determined the biophysical characteristics of L208P and L909F variants, relative to the wild-type channel and a previously reported gain-of-function Cav3.1 variant. The L208P mutation shifted the activation and inactivation curves to the hyperpolarized direction, slowed the kinetics of inactivation and deactivation, and reduced the availability of Ca2+ current during repetitive stimuli. The L909F mutation impacted channel function less severely, resulting in a hyperpolarizing shift of the activation curve and slower deactivation. These data suggest that L909F results in gain-of-function, whereas L208P exhibits mixed gain-of-function and loss-of-function effects due to opposing changes in the biophysical properties. Our study expands the clinical spectrum associated with CACNA1G mutations, corroborating further the causal association with distinct complex phenotypes.

Perineuronal Nets in the Deep Cerebellar Nuclei Regulate GABAergic Transmission and Delay Eyeblink Conditioning.

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.

Altered synaptic and firing properties of cerebellar Purkinje cells in a mouse model of ARSACS.

KEY POINTS: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative human disease characterized in part by ataxia and Purkinje cell loss in anterior cerebellar lobules. A knock-out mouse model has been developed that recapitulates several features of ARSACS. Using this ARSACS mouse model, we report changes in synaptic input and intrinsic firing in cerebellar Purkinje cells, as well as in their synaptic output in the deep cerebellar nuclei. Changes in firing are observed in anterior lobules that later exhibit Purkinje cell death, but not in posterior lobules that do not. Our results show that both synaptic and intrinsic alterations in Purkinje cell properties likely contribute to disease manifestation in ARSACS; these findings resemble pathophysiological changes reported in several other ataxias. ABSTRACT: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease that includes a pronounced and progressive cerebellar dysfunction. ARSACS is caused by an autosomal recessive loss-of-function mutation in the Sacs gene that encodes the protein sacsin. To better understand the cerebellar pathophysiology in ARSACS, we studied synaptic and firing properties of Purkinje cells from a mouse model of ARSACS, Sacs-/- mice. We found that excitatory synaptic drive was reduced onto Sacs-/- Purkinje cells, and that Purkinje cell firing rate, but not regularity, was reduced at postnatal day (P)40, an age when ataxia symptoms were first reported. Firing rate deficits were limited to anterior lobules that later display Purkinje cell death, and were not observed in posterior lobules where Purkinje cells are not lost. Mild firing deficits were observed as early as P20, prior to the manifestation of motor deficits, suggesting that a critical level of cerebellar dysfunction is required for motor coordination to emerge. Finally, we observed a reduction in Purkinje cell innervation onto target neurons in the deep cerebellar nuclei (DCN) in Sacs-/- mice. Together, these findings suggest that multiple alterations in the cerebellar circuit including Purkinje cell input and output contribute to cerebellar-related disease onset in ARSACS.

Cerebellar projections to the ventral lateral geniculate nucleus and the thalamic reticular nucleus in the cat.

The ventral lateral geniculate nucleus (LGNv) is a retinorecipient part of the ventral thalamus and in cats, it consists of medial (M), medial intermediate (IM), lateral intermediate (IL), lateral (L), and dorsal (D) subdivisions. These subdivisions can be differentiated not only by their cytoarchitecture, but also by their connectivity and putative functions. The LGNv may play a role in visuomotor gating, in that there is evidence of cerebellar afferent projections to the intermediate subdivisions. The cerebellar posterior interpositus (IP) and lateral (LC) nuclei are known to project to IM and IL, but the specifics of these projections are unclear. We hypothesized that the IP and LC project differentially to IM and IL. To evaluate LGNv innervation by the deep cerebellar nuclei, we injected the tract-tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into several different regions of the LGNv and cerebellar nuclei of adult cats in either sex. Small injections into the middle and posterior LGNv retrogradely labeled cells in the ventral part of the IP. However, injections in the anterior regions of the LGNv, with or without diffusion into the thalamic reticular nucleus (Re), retrogradely labeled cells in the ventral part of both the IP and the LC. Confirmatory injections into the IP and LC produced terminal-like labeling distributed in IM, IL, and Re; injections mostly localized to the LC resulted in labeling mainly in IM and Re. We concluded that the IP projects to IL whereas the LC projects to IM and Re.

Facilitation of mossy fibre-driven spiking in the cerebellar nuclei by the synchrony of inhibition.

KEY POINTS: Large premotor neurons of the cerebellar nuclei (CbN cells) integrate synaptic inhibition from Purkinje neurons and synaptic excitation from mossy fibres to generate cerebellar output. We find that mossy fibre inputs to CbN cells generate unitary AMPA receptor EPSCs of ∼1 nS that decay in ∼1 ms and mildly voltage-dependent NMDA receptor EPSCs of ∼0.6 nS that decay in ∼7 ms. A few hundred mossy fibres active at a few tens of spikes s-1 must converge on CbN cells to generate physiological CbN spike rates (∼60 spikes s-1 ) during convergent inhibition from spontaneously active Purkinje cells. Dynamic clamp studies in cerebellar slices from weanling mice demonstrate that synaptic excitation from mossy fibres becomes more effective at increasing the rate of CbN cell spiking when the coherence (synchrony) of convergent inhibition is increased. ABSTRACT: Large projection neurons of the cerebellar nuclei (CbN cells), whose activity generates movement, are inhibited by Purkinje cells and excited by mossy fibres. The high convergence, firing rates and strength of Purkinje inputs predict powerful suppression of CbN cell spiking, raising the question of what activity patterns favour excitation over inhibition. Recording from CbN cells at near-physiological temperatures in cerebellar slices from weanling mice, we measured the amplitude, kinetics, voltage dependence and short-term plasticity of mossy fibre-mediated EPSCs. Unitary EPSCs were small and brief (AMPA receptor, ∼1 nS, ∼1 ms; NMDA receptor, ∼0.6 nS, ∼7 ms) and depressed moderately. Using these experimentally measured parameters, we applied combinations of excitation and inhibition to CbN cells with dynamic clamp. Because Purkinje cells can fire coincident simple spikes during cerebellar behaviours, we varied the proportion (0-20 of 40) and precision (0-4 ms jitter) of synchrony of inhibitory inputs, along with the rates (0-100 spikes s-1 ) and number (0-800) of excitatory inputs. Even with inhibition constant, when inhibitory synchrony was higher, excitation increased CbN cell firing rates more effectively. Partial inhibitory synchrony also dictated CbN cell spike timing, even with physiological rates of excitation. These effects were present with ≥10 inhibitory inputs active within 2-4 ms of each other. Conversely, spiking was most effectively suppressed when inhibition was maximally asynchronous. Thus, the rate and relative timing of Purkinje-mediated inhibition set the rate and timing of cerebellar output. The results suggest that increased coherence of Purkinje cell activity can facilitate mossy fibre-driven spiking by CbN cells, in turn driving movements.

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.

Independently specified Atoh1 domains define novel developmental compartments in rhombomere 1.

The rhombic lip gives rise to neuronal populations that contribute to cerebellar, proprioceptive and interoceptive networks. Cell production depends on the expression of the basic helix-loop-helix (bHLH) transcription factor Atoh1. In rhombomere 1, Atoh1-positive cells give rise to both cerebellar neurons and extra-cerebellar nuclei in ventral hindbrain. The origin of this cellular diversity has previously been attributed to temporal signals rather than spatial patterning. Here, we show that in both chick and mouse the cerebellar Atoh1 precursor pool is partitioned into initially cryptic spatial domains that reflect the activity of two different organisers: an isthmic Atoh1 domain, which gives rise to isthmic nuclei, and the rhombic lip, which generates deep cerebellar nuclei and granule cells. We use a combination of in vitro explant culture, genetic fate mapping and gene overexpression and knockdown to explore the role of isthmic signalling in patterning these domains. We show that an FGF-dependent isthmic Atoh1 domain is the origin of distinct populations of Lhx9-positive neurons in the extra-cerebellar isthmic nuclei. In the cerebellum, ectopic FGF induces proliferation while blockade reduces the length of the cerebellar rhombic lip. FGF signalling is not required for the specification of cerebellar cell types from the rhombic lip and its upregulation inhibits their production. This suggests that although the isthmus regulates the size of the cerebellar anlage, the downregulation of isthmic FGF signals is required for induction of rhombic lip-derived cerebellar neurons.

Electrical Stimulation Normalizes c-Fos Expression in the Deep Cerebellar Nuclei of Depressive-like Rats: Implication of Antidepressant Activity.

The electrical stimulation of specific brain targets has been shown to induce striking antidepressant effects. Despite that recent data have indicated that cerebellum is involved in emotional regulation, the mechanisms by which stimulation improved mood-related behaviors in the cerebellum remained largely obscure. Here, we investigated the stimulation effects of the ventromedial prefrontal cortex (vmPFC), nucleus accumbens (NAc), and lateral habenular nucleus on the c-Fos neuronal activity in various deep cerebellar and vestibular nuclei using the unpredictable chronic mild stress (CMS) animal model of depression. Our results showed that stressed animals had increased number of c-Fos cells in the cerebellar dentate and fastigial nuclei, as well as in the spinal vestibular nucleus. To examine the stimulation effects, we found that vmPFC stimulation significantly decreased the c-Fos activity within the cerebellar fastigial nucleus as compared to the CMS sham. Similarly, there was also a reduction of c-Fos expression in the magnocellular part of the medial vestibular nucleus in vmPFC- and NAc core-stimulated animals when compared to the CMS sham. Correlational analyses showed that the anxiety measure of home-cage emergence escape latency was positively correlated with the c-Fos neuronal activity of the cerebellar fastigial and magnocellular and parvicellular parts of the interposed nuclei in CMS vmPFC-stimulated animals. Interestingly, there was a strong correlation among activation in these cerebellar nuclei, indicating that the antidepressant-like behaviors were possibly mediated by the vmPFC stimulation-induced remodeling within the forebrain-cerebellar neurocircuitry.

Fuzzy neuronal model of motor control inspired by cerebellar pathways to online and gradually learn inverse biomechanical functions in the presence of delay.

Contrary to forward biomechanical functions, which are deterministic, inverse biomechanical functions are generally not. Calculating an inverse biomechanical function is an ill-posed problem, which has no unique solution for a manipulator with several degrees of freedom. Studies of the command and control of biological movements suggest that the cerebellum takes part in the computation of approximate inverse functions, and this ability can control fast movements by predicting the consequence of current motor command. Limb movements toward a goal are defined as fast if they last less than the total duration of the processing and transmission delays in the motor and sensory pathways. Because of these delays, fast movements cannot be continuously controlled in a closed loop by use of sensory signals. Thus, fast movements must be controlled by some open loop controller, of which cerebellar pathways constitute an important part. This article presents a system-level fuzzy neuronal motor control circuit, inspired by the cerebellar pathways. The cerebellar cortex (CC) is assumed to embed internal models of the biomechanical functions of the limb segments. Such neural models are able to predict the consequences of motor commands and issue predictive signals encoding movement variables, which are sent to the controller via internal feedback loops. Differences between desired and expected values of variables of movements are calculated in the deep cerebellar nuclei (DCN). After motor learning, the whole circuit can approximate the inverse function of the biomechanical function of a limb and acts as a controller. In this research, internal models of direct biomechanical functions are learned and embedded in the connectivity of the cerebellar pathways. Two fuzzy neural networks represent the two parts of the cerebellum, and an online gradual learning drives the acquisition of the internal models in CC and the controlling rules in DCN. As during real learning, exercise and repetition increase skill and speed. The learning procedure is started by a simple and slow movement, controlled in the presence of delays by a simple closed loop controller comparable to the spinal reflexes. The speed of the movements is then increased gradually, and output error signals are used to compute teaching signals and drive learning. Repetition of movements at each speed level allows to properly set the two neural networks, and progressively learn the movement. Finally, conditions of stability of the proposed model as an inverter are identified. Next, the control of a single segment arm, moved by two muscles, is simulated. After proper setting by motor learning, the circuit is able to reject perturbations.

Integration of Purkinje cell inhibition by cerebellar nucleo-olivary neurons.

Neurons in the cerebellar cortex, cerebellar nuclei, and inferior olive (IO) form a trisynaptic loop critical for motor learning. IO neurons excite Purkinje cells via climbing fibers and depress their parallel fiber inputs. Purkinje cells inhibit diverse cells in the cerebellar nuclei, including small GABAergic nucleo-olivary neurons that project to the IO. To investigate how these neurons integrate synaptic signals from Purkinje cells, we retrogradely labeled nucleo-olivary cells in the contralateral interpositus and lateral nuclei with cholera toxin subunit B-Alexa Fluor 488 and recorded their electrophysiological properties in cerebellar slices from weanling mice. Nucleo-olivary cells fired action potentials over a relatively narrow dynamic range (maximal rate, ∼ 70 spikes/s), unlike large cells that project to premotor areas (maximal rate, ∼ 400 spikes/s). GABA(A) receptor-mediated IPSCs evoked by electrical or optogenetic stimulation of Purkinje cells were more than 10-fold slower in nucleo-olivary cells (decay time, ∼ 25 ms) than in large cells (∼ 2 ms), and repetitive stimulation at 20-150 Hz evoked greatly summating IPSCs. Nucleo-olivary firing rates varied inversely with IPSP frequency, and the timing of Purkinje IPSPs and nucleo-olivary spikes was uncorrelated. These attributes contrast with large cells, whose brief IPSCs and rapid firing rates can permit well timed postinhibitory spiking. Thus, the intrinsic and synaptic properties of these two projection neurons from the cerebellar nuclei tailor them for differential integration and transmission of their Purkinje cell input.

Alterations in cerebellar physiology are associated with a stiff-legged gait in Atcay(ji-hes) mice.

Recent evidence suggests that dystonia, a movement disorder characterized by sustained involuntary muscle contractions, can be associated with cerebellar abnormalities. The basis for how functional changes in the cerebellum can cause dystonia is poorly understood. Here we identify alterations in physiology in Atcay(ji-hes) mice which in addition to ataxia, have an abnormal gait with hind limb extension and toe walking, reminiscent of human dystonic gait. No morphological abnormalities in the brain accompany the dystonia, but partial cerebellectomy causes resolution of the stiff-legged gait, suggesting that cerebellar dysfunction contributes to the dystonic gait of Atcay(ji-hes) mice. Recordings from Purkinje and deep cerebellar nuclear (DCN) neurons in acute brain slices were used to determine the physiological correlates of dystonia in the Atcay(ji-hes) mice. Approximately 50% of cerebellar Purkinje neurons fail to display the normal repetitive firing characteristic of these cells. In addition, DCN neurons exhibit increased intrinsic firing frequencies with a subset of neurons displaying bursts of action potentials. This increased intrinsic excitability of DCN neurons is accompanied by a reduction in after-hyperpolarization currents mediated by small-conductance calcium-activated potassium (SK) channels. An activator of SK channels reduces DCN neuron firing frequency in acute cerebellar slices and improves the dystonic gait of Atcay(ji-hes) mice. These results suggest that a combination of reduced Purkinje neuron activity and increased DCN intrinsic excitability can result in a combination of ataxia and a dystonia-like gait in mice.

Subtle microstructural changes of the cerebellum in a knock-in mouse model of DYT1 dystonia.

The dystonias are a group of disorders characterized by involuntary twisting and repetitive movements. DYT1 dystonia is an inherited form of dystonia caused by a mutation in the TOR1A gene, which encodes torsinA. TorsinA is expressed in many regions of the nervous system, and the regions responsible for causing dystonic movements remain uncertain. Most prior studies have focused on the basal ganglia, although there is emerging evidence for abnormalities in the cerebellum too. In the current studies, we examined the cerebellum for structural abnormalities in a knock-in mouse model of DYT1 dystonia. The gross appearance of the cerebellum appeared normal in the mutant mice, but stereological measures revealed the cerebellum to be 5% larger in mutant compared to control mice. There were no changes in the numbers of Purkinje cells, granule cells, or neurons of the deep cerebellar nuclei. However, Golgi histochemical studies revealed Purkinje cells to have thinner dendrites, and fewer and less complex dendritic spines. There also was a higher frequency of heterotopic Purkinje cells displaced into the molecular layer. These results reveal subtle structural changes of the cerebellum that are similar to those reported for the basal ganglia in the DYT1 knock-in mouse model.

Modulation of semaphorin3A in perineuronal nets during structural plasticity in the adult cerebellum.

In the adult central nervous system (CNS) subsets of neurons are enwrapped by densely organized extracellular matrix structures, called perineuronal nets (PNNs). PNNs are formed at the end of critical periods and contribute to synapse stabilization. Enzymatic degradation of PNNs or genetic deletion of specific PNN components leads to the prolongation of the plasticity period. PNNs consist of extracellular matrix molecules, including chondroitin sulfate proteoglycans, hyaluronan, tenascins and link proteins. It has been recently shown that the chemorepulsive axon guidance protein semaphorin3A (Sema3A) is also a constituent of PNNs, binding with high affinity to the sugar chains of chondroitin sulfate proteoglycans. To elucidate whether the expression of Sema3A is modified in parallel with structural plasticity in the adult CNS, we examined Sema3A expression in the deep cerebellar nuclei of the adult mouse in a number of conditions associated with structural reorganization of the local connectivity. We found that Sema3A in PNNs is reduced during enhanced neuritic remodeling, in both physiological and injury-induced conditions. Moreover, we provide evidence that Sema3A is tightly associated with Purkinje axons and their terminals and its amount in the PNNs is related to Purkinje cell innervation of DCN neurons, but not to glutamatergic inputs. On the whole these data suggest that Sema3A may contribute to the growth-inhibitory properties of PNNs and Purkinje neurons may directly control their specific connection pattern through the release and capture of this guidance cue in the specialized ECM that surrounds their terminals.