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1.  Cerebellar modules operate at different frequencies 
eLife  2014;3:e02536.
Due to the uniform cyto-architecture of the cerebellar cortex, its overall physiological characteristics have traditionally been considered to be homogeneous. In this study, we show in awake mice at rest that spiking activity of Purkinje cells, the sole output cells of the cerebellar cortex, differs between cerebellar modules and correlates with their expression of the glycolytic enzyme aldolase C or zebrin. Simple spike and complex spike frequencies were significantly higher in Purkinje cells located in zebrin-negative than zebrin-positive modules. The difference in simple spike frequency persisted when the synaptic input to, but not intrinsic activity of, Purkinje cells was manipulated. Blocking TRPC3, the effector channel of a cascade of proteins that have zebrin-like distribution patterns, attenuated the simple spike frequency difference. Our results indicate that zebrin-discriminated cerebellar modules operate at different frequencies, which depend on activation of TRPC3, and that this property is relevant for all cerebellar functions.
DOI: http://dx.doi.org/10.7554/eLife.02536.001
eLife digest
The cerebellum, located at the back of the brain underneath the cerebral hemispheres, is best known for its role in the control of movement. Despite its small size, the cerebellum contains more than half of the brain's neurons. These are organized in a repeating pattern in which cells called Purkinje cells receive inputs from two types of fibers: climbing fibers, which ascend into the cerebellum from the brainstem; and parallel fibers, which run perpendicular to the climbing fibers. This gives rise to a characteristic ‘crystalline’ structure.
As a result of this uniform circuitry, it was widely believed was that all Purkinje cells throughout the cerebellum would function the same way. However, the presence of distinct patterns of gene expression in different regions suggests that this is not the case. Molecules called zebrins, for example, are found in some Purkinje cells but not others, and this gives rise to a pattern of zebrin-positive and zebrin-negative stripes. A number of other molecules have similar distributions, suggesting that these differences in molecular machinery could underlie differences in cellular physiology.
Zhou, Lin et al. have now provided one of the first direct demonstrations of such physiological differences by showing that zebrin-positive cells generate action potentials at lower frequencies than zebrin-negative cells. This pattern is seen throughout the cerebellum, and is evident even when the positive and negative cells are neighbors, which indicates that these differences do not simply reflect differences in the locations of the cells or differences in the inputs they receive from parallel fibers. Additional experiments revealed that the distinct firing rates are likely not generated by zebrin itself, but rather by proteins that are expressed alongside zebrin, most notably those that work through an ion channel called TRPC3.
By showing that cells arranged in the same type of circuit can nevertheless have distinct firing rates, the work of Zhou, Lin et al. has revealed an additional level of complexity in the physiology of the cerebellum. In addition to improving our understanding of how the brain controls movement, these findings might also be of interest to researchers studying the increasing number of neurological and psychiatric disorders in which cerebellar dysfunction has been implicated.
DOI: http://dx.doi.org/10.7554/eLife.02536.002
doi:10.7554/eLife.02536
PMCID: PMC4049173  PMID: 24843004
cerebellum; cerebellar modules; Purkinje cells; zebrin II; TRPC3; mouse
2.  The Reelin Receptors Apoer2 and Vldlr Coordinate the Patterning of Purkinje Cell Topography in the Developing Mouse Cerebellum 
PLoS ONE  2008;3(2):e1653.
The adult cerebellar cortex is comprised of reproducible arrays of transverse zones and parasagittal stripes of Purkinje cells. Adult stripes are created through the perinatal rostrocaudal dispersion of embryonic Purkinje cell clusters, triggered by signaling through the Reelin pathway. Reelin is secreted by neurons in the external granular layer and deep cerebellar nuclei and binds to two high affinity extracellular receptors on Purkinje cells-the Very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer2). In mice null for either Reelin or double null for Vldlr and Apoer2, Purkinje cell clusters fail to disperse. Here we report that animals null for either Vldlr or Apoer2 individually, exhibit specific and parasagittally-restricted Purkinje cell ectopias. For example, in mice lacking Apoer2 function immunostaining reveals ectopic Purkinje cells that are largely restricted to the zebrin II-immunonegative population of the anterior vermis. In contrast, mice null for Vldlr have a much larger population of ectopic Purkinje cells that includes members from both the zebrin II-immunonegative and -immunopositive phenotypes. HSP25 immunoreactivity reveals that in Vldlr null animals a large portion of zebrin II-immunopositive ectopic cells are probably destined to become stripes in the central zone (lobules VI–VII). A small population of ectopic zebrin II-immunonegative Purkinje cells is also observed in animals heterozygous for both receptors (Apoer2+/−: Vldlr+/−), but no ectopia is present in mice heterozygous for either receptor alone. These results indicate that Apoer2 and Vldlr coordinate the dispersal of distinct, but overlapping subsets of Purkinje cells in the developing cerebellum.
doi:10.1371/journal.pone.0001653
PMCID: PMC2242849  PMID: 18301736
3.  Spatiotemporal Analysis of Purkinje Cell Degeneration Relative to Parasagittal Expression Domains in a Model of Neonatal Viral Infection▿  
Journal of Virology  2006;81(6):2675-2687.
Infection of newborn Lewis rats with Borna disease virus (neonatal Borna disease [NBD]) results in cerebellar damage without the cellular inflammation associated with infections in later life. Purkinje cell (PC) damage has been reported for several models of early-life viral infection, including NBD; however, the time course and distribution of PC pathology have not been investigated rigorously. This study examined the spatiotemporal relationship between PC death and zonal organization in NBD cerebella. Real-time PCR at postnatal day 28 (PND28) revealed decreased cerebellar levels of mRNAs encoding the glycolytic enzymes aldolase C (AldoC, also known as zebrin II) and phosphofructokinase C and the excitatory amino acid transporter 4 (EAAT4). Zebrin II and EAAT4 immunofluorescence analysis in PND21, PND28, PND42, and PND84 NBD rat cerebella revealed a complex pattern of PC degeneration. Early cell loss (PND28) was characterized by preferential apoptotic loss of zebrin II/EAAT4-negative PC subsets in the anterior vermis. Consistent with early preferential loss of zebrin II/EAAT4-negative PCs in the vermis, the densities of microglia and the Bergmann glial expression of metallothionein I/II and the hyaluronan receptor CD44 were higher in zebrin II/EAAT4-negative zones. In contrast, early loss in lateral cerebellar lobules did not reflect a similar discrimination between PC phenotypes. Patterns of vermal PC loss became more heterogeneous at PND42, with the loss of both zebrin II/EAAT4-negative and zebrin II/EAAT4-positive neurons. At PND84, zebrin II/EAAT4 patterning was abolished in the anterior cerebellum, with preferential PC survival in lobule X. Our investigation reveals regional discrimination between patterns of PC subset loss, defined by zebrin II/EAAT4 expression domains, following neonatal viral infection. These findings suggest a differential vulnerability of PC subsets during the early stages of virus-induced neurodegeneration.
doi:10.1128/JVI.02245-06
PMCID: PMC1865998  PMID: 17182680
4.  Processing of visual signals related to self-motion in the cerebellum of pigeons 
In this paper I describe the key features of optic flow processing in pigeons. Optic flow is the visual motion that occurs across the entire retina as a result of self-motion and is processed by subcortical visual pathways that project to the cerebellum. These pathways originate in two retinal-recipient nuclei, the nucleus of the basal optic root (nBOR) and the nucleus lentiformis mesencephali, which project to the vestibulocerebellum (VbC) (folia IXcd and X), directly as mossy fibers, and indirectly as climbing fibers from the inferior olive. Optic flow information is integrated with vestibular input in the VbC. There is a clear separation of function in the VbC: Purkinje cells in the flocculus process optic flow resulting from self-rotation, whereas Purkinje cells in the uvula/nodulus process optic flow resulting from self-translation. Furthermore, Purkinje cells with particular optic flow preferences are organized topographically into parasagittal “zones.” These zones are correlated with expression of the isoenzyme aldolase C, also known as zebrin II (ZII). ZII expression is heterogeneous such that there are parasagittal stripes of Purkinje cells that have high expression (ZII+) alternating with stripes of Purkinje cells with low expression (ZII−). A functional zone spans a ZII± stripe pair. That is, each zone that contains Purkinje cells responsive to a particular pattern of optic flow is subdivided into a strip containing ZII+ Purkinje cells and a strip containing ZII− Purkinje cells. Additionally, there is optic flow input to folia VI–VIII of the cerebellum from lentiformis mesencephali. These folia also receive visual input from the tectofugal system via pontine nuclei. As the tectofugal system is involved in the analysis of local motion, there is integration of optic flow and local motion information in VI–VIII. This part of the cerebellum may be important for moving through a cluttered environment.
doi:10.3389/fnbeh.2013.00004
PMCID: PMC3569843  PMID: 23408161
optic flow; cerebellum; vestibulocerebellum; zebrin; accessory optic system; pretectum; oculomotor cerebellum
5.  Parasagittal Zones in the Cerebellar Cortex Differ in Excitability, Information Processing, and Synaptic Plasticity 
Cerebellum (London, England)  2012;11(2):10.1007/s12311-011-0347-1.
At the molecular and circuitry levels, the cerebellum exhibits a striking parasagittal zonation as exemplified by the spatial distribution of molecules expressed on Purkinje cells and the topography of the afferent and efferent projections. The physiology and function of the zonation is less clear. Activity-dependent optical imaging has proven a useful tool to examine the physiological properties of the parasagittal zonation in the intact animal. Recent findings show that zebrin II-positive and zebrin II-negative zones differ markedly in their responses to parallel fiber inputs. These findings suggest that cerebellar cortical excitability, information processing, and synaptic plasticity depend on the intrinsic properties of different parasagittal zones.
doi:10.1007/s12311-011-0347-1
PMCID: PMC3856581  PMID: 22249913
Parasagittal zones; Cerebellar cortex; Parallel fibers; Purkinje cells; Zebrin II
6.  Decreased EAAT2 protein expression in the essential tremor cerebellar cortex 
Genetic polymorphisms in Solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2) have been linked with essential tremor. SLC1A2 encodes excitatory amino acid transporter type 2 (EAAT2), which clears glutamate from the synaptic cleft. One postulated mechanism for essential tremor is the over-excitation of glutamatergic olivo-cerebellar climbing fibers, leading to excitotoxic death of Purkinje cells. Other glutamatergic excitatory signals are transmitted to Purkinje cells via parallel fibers of cerebellar granule neurons. Therefore, the expression level of glutamate transporters could be important in essential tremor pathogenesis. Using Western blotting, we compared the expression levels of the two main glutamate transporters in the cerebellar cortex, EAAT1 and EAAT2, in postmortem tissue from 16 essential tremor cases and 13 age-matched controls. We also studied the localization of EAAT1 and EAAT2 using immunohistochemistry in 10 essential tremor cases and 12 controls. EAAT1 protein levels were similar in cases and controls (1.12 ± 0.83 vs. 1.01 ± 0.69, p =0.71) whereas EAAT2 protein levels in essential tremor cases were only 1/3 of that in controls (0.35 ± 0.23 vs. 1.00 ± 0.62, p < 0.01). Interestingly, EAAT2, but not EAAT1, was expressed in astrocytic processes surrounding the Purkinje cell axon initial segment, a region of previously observed pathological changes in essential tremor. Our main finding, a significant reduction in cerebellar cortical EAAT2 protein levels in essential tremor, suggests that Purkinje cells in essential tremor might be more vulnerable to excitotoxic damage than those of controls.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0157-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s40478-014-0157-z
PMCID: PMC4239402  PMID: 25391854
Essential tremor; EAAT1; EAAT2; Excitotoxicitiy; Purkinje cell; Neurodegenerative
7.  Patterned Neuroprotection in the Inpp4awbl Mutant Mouse Cerebellum Correlates with the Expression of Eaat4 
PLoS ONE  2009;4(12):e8270.
The weeble mutant mouse has a frame shift mutation in inositol polyphosphate 4-phosphatase type I (Inpp4a). The phenotype is characterized by an early onset cerebellar ataxia and neurodegeneration, especially apparent in the Purkinje cells. Purkinje cell loss is a common pathological finding in many human and mouse ataxic disorders. Here we show that in the Inpp4awbl mutant, Purkinje cells are lost in a specific temporal and spatial pattern. Loss occurs early in postnatal development; however, prior to the appearance of climbing fibers in the developing molecular layer, the mutant has a normal complement of Purkinje cells and they are properly positioned. Degeneration and reactive gliosis are present at postnatal day 5 and progress rapidly in a defined pattern of patches; however, Inpp4a is expressed uniformly across Purkinje cells. In late stage mutants, patches of surviving Purkinje cells appear remarkably normal with the exception that the climbing fibers have been excessively eliminated. Surviving Purkinje cells express Eaat4, a glutamate transporter that is differentially expressed in subsets of Purkinje cells during development and into adult stages. Prior to Purkinje cell loss, reactive gliosis and dendritic atrophy can be seen in Eaat4 negative stripes. Our data suggest that Purkinje cell loss in the Inpp4awbl mutant is due to glutamate excitotoxicity initiated by the climbing fiber, and that Eaat4 may exert a protective effect.
doi:10.1371/journal.pone.0008270
PMCID: PMC2788419  PMID: 20011524
8.  Purkinje cell compartmentalization in the cerebellum of the spontaneous mutant mouse dreher 
Brain structure & function  2012;219(1):35-47.
The cerebellar morphological phenotype of the spontaneous neurological mutant mouse dreher (Lmx1adr-J) results from cell fate changes in dorsal midline patterning involving the roof plate and rhombic lip. Positional cloning revealed that the gene Lmx1a, which encodes a LIM homeodomain protein, is mutated in dreher, and is expressed in the developing roof plate and rhombic lip. Loss of Lmx1a causes reduction of the roof plate, an important embryonic signaling center, and abnormal cell fate specification within the embryonic cerebellar rhombic lip. In adult animals, these defects result in variable, medial fusion of the cerebellar vermis and posterior cerebellar vermis hypoplasia. It is unknown whether deleting Lmx1a results in displacement or loss of specific lobules in the vermis. To distinguish between an ectopic and an absent vermis, the expression patterns of two Purkinje cell specific compartmentation antigens, zebrin II/aldolase C and the small heat shock protein HSP25, were analyzed in dreher cerebella. The data reveal that despite the reduction in volume and abnormal foliation of the cerebellum, the transverse zones and parasagittal stripe arrays characteristic of the normal vermis are present in dreher, but may be highly distorted. In dreher mutants with a severe phenotype, zebrin II stripes are fragmented and distributed non-symmetrically about the cerebellar midline. We conclude that although Purkinje cell agenesis or selective Purkinje cell death may contribute to the dreher phenotype, our data suggest that aberrant anlage patterning and granule cell development lead to Purkinje cell ectopia, which ultimately causes abnormal cerebellar architecture in dreher.
doi:10.1007/s00429-012-0482-6
PMCID: PMC4140632  PMID: 23160833
whole mount immunohistochemistry; HSP25; zebrin II; cerebellar development; Lmx1a
9.  Cerebellar Zonal Patterning Relies on Purkinje Cell Neurotransmission 
The Journal of Neuroscience  2014;34(24):8231-8245.
Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity.
doi:10.1523/JNEUROSCI.0122-14.2014
PMCID: PMC4051975  PMID: 24920627
ataxia; circuitry; connectivity; gene expression; inhibition; physiology
10.  Vesicular GABA release delays the onset of the Purkinje cell terminal depolarization without affecting tissue swelling in cerebellar slices during simulated ischemia 
Neuroscience  2010;168(1):108-117.
Neurosteroids that can enhance GABAA receptor sensitivity protect cerebellar Purkinje cells against transient episodes of global brain ischemia, but little is known about how ischemia affects GABAergic transmission onto Purkinje cells. Here we use patch-clamp recording from Purkinje cells in acutely prepared slices of rat cerebellum to determine how ischemia affects GABAergic signaling to Purkinje cells. In voltage-clamped Purkinje cells, exposing slices to solutions designed to simulate brain ischemia caused an early, partial suppression of the frequency of spontaneous inhibitory post synaptic currents (sIPSCs), but after 5-8 minutes GABA accumulated in the extracellular space around Purkinje cells, generating a large (~17 nS), sustained GABAA receptor-mediated conductance. The sustained GABAA conductance occurred in parallel with an even larger (~117 nS) glutamate receptor-mediated conductance, but blocking GABAA receptors did not affect the timing or magnitude of the glutamate conductance, and blocking glutamate receptors did not affect the timing or magnitude of the GABAA conductance. Despite the lack of interaction between GABA and glutamate, blocking GABAA receptors significantly accelerated the onset of the Purkinje cell “ischemic” depolarization (ID), as assessed with current-clamp recordings from Purkinje cells or field potential recordings in the dendritic field of the Purkinje cells. The Purkinje cell ID occurred ~2 minutes prior to the sustained glutamate release under control conditions and a further 1-2 minutes earlier when GABAA receptors were blocked. Tissue swelling, as assessed by monitoring light transmittance through the slice, peaked just after the ID, prior to the sustained glutamate release, but was not affected by blocking GABAA receptors. These data indicate that ischemia induces the Purkinje cell ID and tissue swelling prior to glutamate release, and that blocking GABAA receptors accelerates the onset of the ID without affecting tissue swelling. Taken together these data may explain why Purkinje cells are one of the most ischemia sensitive neurons in the brain despite lacking NMDA receptors, and why neurosteroids that enhance GABAA receptor function protect Purkinje cells against transient episodes of global brain ischemia.
doi:10.1016/j.neuroscience.2010.03.009
PMCID: PMC4007153  PMID: 20226232
11.  Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse) 
PLoS ONE  2014;9(4):e94327.
The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.
doi:10.1371/journal.pone.0094327
PMCID: PMC3983142  PMID: 24722417
12.  Interaction of plasticity and circuit organization during the acquisition of cerebellum-dependent motor learning 
eLife  2013;2:e01574.
Motor learning occurs through interactions between the cerebellar circuit and cellular plasticity at different sites. Previous work has established plasticity in brain slices and suggested plausible sites of behavioral learning. We now reveal what actually happens in the cerebellum during short-term learning. We monitor the expression of plasticity in the simple-spike firing of cerebellar Purkinje cells during trial-over-trial learning in smooth pursuit eye movements of monkeys. Our findings imply that: 1) a single complex-spike response driven by one instruction for learning causes short-term plasticity in a Purkinje cell’s mossy fiber/parallel-fiber input pathways; 2) complex-spike responses and simple-spike firing rate are correlated across the Purkinje cell population; and 3) simple-spike firing rate at the time of an instruction for learning modulates the probability of a complex-spike response, possibly through a disynaptic feedback pathway to the inferior olive. These mechanisms may participate in long-term motor learning.
DOI: http://dx.doi.org/10.7554/eLife.01574.001
eLife digest
Practice makes perfect in many areas of life, such as playing sport or even just drinking coffee from a cup without spilling any. Our brains can learn and improve these motor skills through trial, error and learning, with such “motor learning” depending on the cerebellum, a part of the brain that helps to coordinate all kinds of movements.
Motor learning is a product of the organization of the cerebellar circuit, which is well understood, and the “plasticity” in the synapses that determine how cerebellar neurons interact with each other. The cerebellum contains cells called Purkinje cells that receive distinctive inputs from two pathways: a pathway involving inputs from many parallel fibers, which convey signals related to sensory events or motor commands; and a pathway involving input from a single climbing-fiber, which conveys signals from a part of the brain called the inferior olive nucleus.
Research on slices of brain has revealed many sites and forms of cerebellar plasticity that could participate in motor learning. In one form of plasticity, the strength of the synapses between the parallel fibers and the Purkinje cell can be changed when a signal sent along the climbing fiber arrives the Purkinje cell.
Yang and Lisberger have now taken the next step by studying the cerebellum of a monkey as it performs a motor learning task. Remarkably these experiments show that the climbing fiber inputs cause plasticity of Purkinje cell activity, just as happens in the experiments on brain slices. Further, some learning in the cerebellum restricts further learning, so that the cerebellum puts boundaries on its own learning. Overall the results make clear how learning is a property of groups of neurons working together in a circuit, rather than simply of changes in the strength of specific synapses.
By shedding light on what happens in the cerebellum during short-term motor learning, the work of Yang and Lisberger will benefit efforts to understand how the cerebellum is involved in motor learning on all time scales.
DOI: http://dx.doi.org/10.7554/eLife.01574.002
doi:10.7554/eLife.01574
PMCID: PMC3871706  PMID: 24381248
non-human primate; smooth pursuit eye movements; climbing fiber; cerebellar learning; trial-over-trial learning; floccular complex; Other
13.  Neuronal glutamate transporters regulate glial excitatory transmission 
In the central nervous system, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuro-glial signaling.
doi:10.1523/JNEUROSCI.5232-11.2012
PMCID: PMC3532047  PMID: 22302796
synaptic transmission; Purkinje cell; parallel fiber; EAAT4
14.  Excitability and Synaptic Alterations in the Cerebellum of APP/PS1 Mice 
PLoS ONE  2012;7(4):e34726.
In Alzheimer's disease (AD), the severity of cognitive symptoms is better correlated with the levels of soluble amyloid-beta (Aβ) rather than with the deposition of fibrillar Aβ in amyloid plaques. In APP/PS1 mice, a murine model of AD, at 8 months of age the cerebellum is devoid of fibrillar Aβ, but dosage of soluble Aβ1–42, the form which is more prone to aggregation, showed higher levels in this structure than in the forebrain. Aim of this study was to investigate the alterations of intrinsic membrane properties and of synaptic inputs in Purkinje cells (PCs) of the cerebellum, where only soluble Aβ is present. PCs were recorded by whole-cell patch-clamp in cerebellar slices from wild-type and APP/PS1 mice. In APP/PS1 PCs, evoked action potential discharge showed enhanced frequency adaptation and larger afterhyperpolarizations, indicating a reduction of the intrinsic membrane excitability. In the miniature GABAergic postsynaptic currents, the largest events were absent in APP/PS1 mice and the interspike intervals distribution was shifted to the left, but the mean amplitude and frequency were normal. The ryanodine-sensitive multivescicular release was not altered and the postsynaptic responsiveness to a GABAA agonist was intact. Climbing fiber postsynaptic currents were normal but their short-term plasticity was reduced in a time window of 100–800 ms. Parallel fiber postsynaptic currents and their short-term plasticity were normal. These results indicate that, in the cerebellar cortex, chronically elevated levels of soluble Aβ1–42 are associated with alterations of the intrinsic excitability of PCs and with alterations of the release of GABA from interneurons and of glutamate from climbing fibers, while the release of glutamate from parallel fibers and all postsynaptic mechanisms are preserved. Thus, soluble Aβ1–42 causes, in PCs, multiple functional alterations, including an impairment of intrinsic membrane properties and synapse-specific deficits, with differential consequences even in different subtypes of glutamatergic synapses.
doi:10.1371/journal.pone.0034726
PMCID: PMC3325253  PMID: 22511962
15.  Desynchronization of multivesicular release enhances Purkinje cell output 
Neuron  2011;70(5):991-1004.
Summary
The release of neurotransmitter-filled vesicles following action potentials occurs with discrete time courses: sub-millisecond phasic release that can be desynchronized by activity followed by ‘delayed release’ that persists for tens of milliseconds. Delayed release has a well established role in synaptic integration, but it is not clear whether desynchronization of phasic release has physiological consequences. At the climbing fiber to Purkinje cell synapse, the synchronous fusion of multiple vesicles is critical for generating complex spikes. Here we show that stimulation at physiological frequencies drives the temporal dispersion of vesicles undergoing multivesicular release, resulting in a slowing of the EPSC on the millisecond time scale. Remarkably, these changes in EPSC kinetics robustly alter the Purkinje cell complex spike in a manner that promotes axonal propagation of individual spikelets. Thus, desynchronization of multivesicular release enhances the precise and efficient information transfer by complex spikes.
doi:10.1016/j.neuron.2011.03.029
PMCID: PMC3148031  PMID: 21658590
synaptic transmission; Purkinje cell; climbing fiber; vesicle fusion
16.  Rapid development of Purkinje cell excitability, functional cerebellar circuit, and afferent sensory input to cerebellum in zebrafish 
The zebrafish has significant advantages for studying the morphological development of the brain. However, little is known about the functional development of the zebrafish brain. We used patch clamp electrophysiology in live animals to investigate the emergence of excitability in cerebellar Purkinje cells, functional maturation of the cerebellar circuit, and establishment of sensory input to the cerebellum. Purkinje cells are born at 3 days post-fertilization (dpf). By 4 dpf, Purkinje cells spontaneously fired action potentials in an irregular pattern. By 5 dpf, the frequency and regularity of tonic firing had increased significantly and most cells fired complex spikes in response to climbing fiber activation. Our data suggest that, as in mammals, Purkinje cells are initially innervated by multiple climbing fibers that are winnowed to a single input. To probe the development of functional sensory input to the cerebellum, we investigated the response of Purkinje cells to a visual stimulus consisting of a rapid change in light intensity. At 4 dpf, sudden darkness increased the rate of tonic firing, suggesting that afferent pathways carrying visual information are already active by this stage. By 5 dpf, visual stimuli also activated climbing fibers, increasing the frequency of complex spiking. Our results indicate that the electrical properties of zebrafish and mammalian Purkinje cells are highly conserved and suggest that the same ion channels, Nav1.6 and Kv3.3, underlie spontaneous pacemaking activity. Interestingly, functional development of the cerebellum is temporally correlated with the emergence of complex, visually-guided behaviors such as prey capture. Because of the rapid formation of an electrically-active cerebellum, optical transparency, and ease of genetic manipulation, the zebrafish has great potential for functionally mapping cerebellar afferent and efferent pathways and for investigating cerebellar control of motor behavior.
doi:10.3389/fncir.2014.00147
PMCID: PMC4271617  PMID: 25565973
Purkinje cell; patch clamp; cerebellum; zebrafish; parallel fiber; climbing fiber; visual input
17.  Coding of stimulus strength via analog calcium signals in Purkinje cell dendrites of awake mice 
eLife  2014;3:e03663.
The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial.
DOI: http://dx.doi.org/10.7554/eLife.03663.001
eLife digest
A region of the brain known as the cerebellum plays a key role in learning how to anticipate an event. For example, if you know that a puff of air is going to be directed at your eye, it's a good idea to close it in advance. However, how much you need to close it depends on how strong that puff of air is. A very strong puff might require closing the eye completely to protect it. In contrast, it is probably better to only partially close the eye if you know a lighter puff of air is coming, so that you can still see.
Extensive research has focused on how neurons in and around the cerebellum work together to achieve this goal. When an event—such as a puff of air—occurs, signals are sent to large neurons in the cerebellum, called Purkinje cells, by ‘climbing fibers’. However, climbing fibers were thought to be able to respond in only two ways: either they fire in a single burst to signal that an event has occurred, or they don't fire. It was therefore unclear how the finer details of the event (for example, the strength of the puff of air) are transmitted to the cerebellum.
Najafi et al. imaged the level of calcium in the cerebellum of mice, as this indicates how active the neurons are. When a puff of air was directed at the eyes of the mice, Najafi et al. saw that the size of the response of the Purkinje cells corresponded with how big the puff of air was. Najafi et al. show that the size of this response, which is based mostly on input from the climbing fibers, is also influenced by input from an additional unknown source.
These findings show that Purkinje cells of the cerebellum receive detailed information about the nature of an event, such as a puff of air. What remains to be seen is whether the cerebellum uses this information to learn the correct response, that is how hard to blink to avoid the expected puff.
DOI: http://dx.doi.org/10.7554/eLife.03663.002
doi:10.7554/eLife.03663
PMCID: PMC4158287  PMID: 25205669
cerebellum; plasticity; climbing fiber; motor learning; unconditioned stimulus; neural coding; mouse
18.  AMPA receptor-mediated presynaptic inhibition at cerebellar GABAergic synapses: a characterization of molecular mechanisms 
The European journal of neuroscience  2004;19(9):2464-2474.
A major subtype of glutamate receptors, AMPA receptors (AMPARs), are generally thought to mediate excitation at mammalian central synapses via the ionotropic action of ligand-gated channel opening. It has recently emerged, however, that synaptic activation of AMPARs by glutamate released from the climbing fibre input elicits not only postsynaptic excitation but also presynaptic inhibition of GABAergic transmission onto Purkinje cells in the cerebellar cortex. Although presynaptic inhibition is critical for information processing at central synapses, the molecular mechanisms by which AMPARs take part in such actions are not known. This study therefore aimed at further examining the properties of AMPAR-mediated presynaptic inhibition at GABAergic synapses in the rat cerebellum. Our data provide evidence that the climbing fibre-induced inhibition of GABA release from interneurons depends on AMPAR-mediated activation of GTP-binding proteins coupled with down-regulation of presynaptic voltage-dependent Ca2+ channels. A Gi/o-protein inhibitor, N-ethylmaleimide, selectively abolished the AMPAR-mediated presynaptic inhibition at cerebellar GABAergic synapses but did not affect AMPAR-mediated excitatory actions on Purkinje cells. Furthermore, both Gi/o-coupled receptor agonists, baclofen and DCG-IV, and the P/Q-type calcium channel blocker ω-agatoxin IVA markedly occluded the AMPAR-mediated inhibition of GABAergic transmission. Conversely, AMPAR activation inhibited action potential-triggered Ca2+ influx into individual axonal boutons of cerebellar GABAergic interneurons. By suppressing the inhibitory inputs to Purkinje cells, the AMPAR-mediated presynaptic inhibition could thus provide a feed-forward mechanism for the information flow from the cerebellar cortex.
doi:10.1111/j.0953-816X.2004.03347.x
PMCID: PMC3387903  PMID: 15128400
AMPA-type glutamate receptor; cerebellum; GABAergic inhibitory synapse; presynaptic inhibition; rat
19.  Reevaluation of the Beam and Radial Hypotheses of Parallel Fiber Action in the Cerebellar Cortex 
The Journal of Neuroscience  2013;33(28):11412-11424.
The role of parallel fibers (PFs) in cerebellar physiology remains controversial. Early studies inspired the “beam” hypothesis whereby granule cell (GC) activation results in PF-driven, postsynaptic excitation of beams of Purkinje cells (PCs). However, the “radial” hypothesis postulates that the ascending limb of the GC axon provides the dominant input to PCs and generates patch-like responses. Using optical imaging and single-cell recordings in the mouse cerebellar cortex in vivo, this study reexamines the beam versus radial controversy. Electrical stimulation of mossy fibers (MFs) as well as microinjection of NMDA in the granular layer generates beam-like responses with a centrally located patch-like response. Remarkably, ipsilateral forepaw stimulation evokes a beam-like response in Crus I. Discrete molecular layer lesions demonstrate that PFs contribute to the peripherally generated responses in Crus I. In contrast, vibrissal stimulation induces patch-like activation of Crus II and GABAA antagonists fail to convert this patch-like activity into a beam-like response, implying that molecular layer inhibition does not prevent beam-like responses. However, blocking excitatory amino acid transporters (EAATs) generates beam-like responses in Crus II. These beam-like responses are suppressed by focal inhibition of MF-GC synaptic transmission. Using EAAT4 reporter transgenic mice, we show that peripherally evoked patch-like responses in Crus II are aligned between parasagittal bands of EAAT4. This is the first study to demonstrate beam-like responses in the cerebellar cortex to peripheral, MF, and GC stimulation in vivo. Furthermore, the spatial pattern of the responses depends on extracellular glutamate and its local regulation by EAATs.
doi:10.1523/JNEUROSCI.0711-13.2013
PMCID: PMC3724546  PMID: 23843513
20.  Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map 
The spatial organization of the cerebellar afferent map has remarkable correspondence to two aspects of intrinsic patterning within the cerebellum embodied by a series of lobules and Purkinje cell (PC) striped gene expression. Using male and female mice we tested whether the Engrailed (En) homeobox genes are a common genetic substrate regulating all three systems, since they are expressed in spatially restricted domains within the cerebellum and are critical for patterning PC gene expression and foliation. Indeed, we discovered that En1/2 are necessary for the precise targeting of mossy fibers to distinct lobules, as well as their subsequent resolution into discrete parasagittal bands. Moreover, each En gene coordinately regulates afferent targeting and the striped pattern of PC protein expression (e.g. ZebrinII/AldolaseC) independent of regulating foliation. We further found that En1/2, rather than the presence of a full complement of lobules are critical for generating PC protein stripes and mossy fiber bands, and that PC striped gene expression is determined prior to afferent banding. Thus, the En transcription factors not only regulate cerebellum circuit topography, but they also link afferent and efferent neurons precisely enough that alterations in PC protein expression can be used as a read out for underlying defects in circuitry. In summary, our data suggests that En1/2 are master regulators of 3-dimensional organization of the cerebellum and coordinately regulate morphology, patterned gene expression and afferent topography.
doi:10.1523/JNEUROSCI.0653-10.2010
PMCID: PMC2921890  PMID: 20668186
mossy fiber targeting; molecular code; foliation; Gli2
21.  Detailed Expression Pattern of Aldolase C (Aldoc) in the Cerebellum, Retina and Other Areas of the CNS Studied in Aldoc-Venus Knock-In Mice 
PLoS ONE  2014;9(1):e86679.
Aldolase C (Aldoc, also known as “zebrin II”), a brain type isozyme of a glycolysis enzyme, is expressed heterogeneously in subpopulations of cerebellar Purkinje cells (PCs) that are arranged longitudinally in a complex striped pattern in the cerebellar cortex, a pattern which is closely related to the topography of input and output axonal projections. Here, we generated knock-in Aldoc-Venus mice in which Aldoc expression is visualized by expression of a fluorescent protein, Venus. Since there was no obvious phenotypes in general brain morphology and in the striped pattern of the cerebellum in mutants, we made detailed observation of Aldoc expression pattern in the nervous system by using Venus expression in Aldoc-Venus heterozygotes. High levels of Venus expression were observed in cerebellar PCs, cartwheel cells in the dorsal cochlear nucleus, sensory epithelium of the inner ear and in all major types of retinal cells, while moderate levels of Venus expression were observed in astrocytes and satellite cells in the dorsal root ganglion. The striped arrangement of PCs that express Venus to different degrees was carefully traced with serial section alignment analysis and mapped on the unfolded scheme of the entire cerebellar cortex to re-identify all individual Aldoc stripes. A longitudinally striped boundary of Aldoc expression was first identified in the mouse flocculus, and was correlated with the climbing fiber projection pattern and expression of another compartmental marker molecule, heat shock protein 25 (HSP25). As in the rat, the cerebellar nuclei were divided into the rostrodorsal negative and the caudoventral positive portions by distinct projections of Aldoc-positive and negative PC axons in the mouse. Identification of the cerebellar Aldoc stripes in this study, as indicated in sample coronal and horizontal sections as well as in sample surface photos of whole-mount preparations, can be referred to in future experiments.
doi:10.1371/journal.pone.0086679
PMCID: PMC3903578  PMID: 24475166
22.  Migration, early axonogenesis, and Reelin-dependent layer-forming behavior of early/posterior-born Purkinje cells in the developing mouse lateral cerebellum 
Neural Development  2010;5:23.
Background
Cerebellar corticogenesis begins with the assembly of Purkinje cells into the Purkinje plate (PP) by embryonic day 14.5 (E14.5) in mice. Although the dependence of PP formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate the PP. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated.
Results
We show that Purkinje cells generated on E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially, after only transiently migrating radially, towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12.5. After their somata reach the outer/dorsal region by E13.5, they change 'posture' by E14.5 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate. In reeler cerebella, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly extended axon-like fibers until E13.5, but then fail to form the PP due to lack of the posture-change step.
Conclusions
Previously unknown behaviors are revealed for a subset of Purkinje cells born early in the posteior lateral cerebellum: tangential migration; early axonogenesis; and Reelin-dependent reorientation initiating PP formation. This study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.
doi:10.1186/1749-8104-5-23
PMCID: PMC2942860  PMID: 20809939
23.  Gating of neural error signals during motor learning 
eLife  2014;3:e02076.
Cerebellar climbing fiber activity encodes performance errors during many motor learning tasks, but the role of these error signals in learning has been controversial. We compared two motor learning paradigms that elicited equally robust putative error signals in the same climbing fibers: learned increases and decreases in the gain of the vestibulo-ocular reflex (VOR). During VOR-increase training, climbing fiber activity on one trial predicted changes in cerebellar output on the next trial, and optogenetic activation of climbing fibers to mimic their encoding of performance errors was sufficient to implant a motor memory. In contrast, during VOR-decrease training, there was no trial-by-trial correlation between climbing fiber activity and changes in cerebellar output, and climbing fiber activation did not induce VOR-decrease learning. Our data suggest that the ability of climbing fibers to induce plasticity can be dynamically gated in vivo, even under conditions where climbing fibers are robustly activated by performance errors.
DOI: http://dx.doi.org/10.7554/eLife.02076.001
eLife digest
The cerebellum (or ‘little brain’) is located underneath the cerebral hemispheres. Despite comprising around 10% of the brain’s volume, the cerebellum contains roughly half of the brain’s neurons. Many of the functions of the cerebellum are related to the control and fine-tuning of movement, and people whose cerebellum has been damaged have problems with balance and coordination, and with learning new motor skills.
One of the roles of the cerebellum is to control a reflex known as the vestibulo-ocular reflex, which enables us to keep our gaze fixed on an object as we turn our heads. The cerebellum relays information about head movements to the muscles that control the eyes, instructing the eyes to move in the opposite direction to the head. This keeps the image of the object we are looking at stable on the retina.
The vestibulo-ocular reflex is controlled by a circuit that includes Purkinje cells (which are the main output cells of the cerebellum) and climbing fibres (which originate in the brainstem). Any failure of the vestibulo-ocular reflex to fully compensate for head movements generates an error signal that activates the climbing fibres. These in turn modify the output of Purkinje cells, leading ultimately to adjustments in eye movements.
However, Kimpo et al. have now obtained evidence that Purkinje cells can modulate their response to the instructions they receive from climbing fibres. Monkeys sat in a rotating chair while a visual object they were trained to track with their eyes was moved to induce errors in the vestibulo-ocular reflex. When the object was moved so that a bigger reflexive eye movement was required to stabilize the image, the activation of the climbing fibres in response to the error led to a change in the response of the Purkinje cells, as expected. However, when a smaller reflexive eye movement was needed, the error-driven responses of the climbing fibres did not alter the responses of Purkinje cells. Similar results were obtained using pulses of light to artificially activate climbing fibres and thus simulate error signals.
The work of Kimpo et al. indicates that the cerebellum does not blindly follow the instructions it receives from the brainstem, but can instead modulate its responses to incoming information about performance errors. Further work is now required to identify factors that influence the responsiveness of the cerebellum: such information could ultimately be used to improve learning of motor skills and recovery from injury.
DOI: http://dx.doi.org/10.7554/eLife.02076.002
doi:10.7554/eLife.02076
PMCID: PMC3989583  PMID: 24755290
rhesus macaque; climbing fibers; cerebellum; motor learning; vestibulo-ocular reflex; supervised learning; mouse; other
24.  Remodeling of Monoplanar Purkinje Cell Dendrites during Cerebellar Circuit Formation 
PLoS ONE  2011;6(5):e20108.
Dendrite arborization patterns are critical determinants of neuronal connectivity and integration. Planar and highly branched dendrites of the cerebellar Purkinje cell receive specific topographical projections from two major afferent pathways; a single climbing fiber axon from the inferior olive that extend along Purkinje dendrites, and parallel fiber axons of granule cells that contact vertically to the plane of dendrites. It has been believed that murine Purkinje cell dendrites extend in a single parasagittal plane in the molecular layer after the cell polarity is determined during the early postnatal development. By three-dimensional confocal analysis of growing Purkinje cells, we observed that mouse Purkinje cells underwent dynamic dendritic remodeling during circuit maturation in the third postnatal week. After dendrites were polarized and flattened in the early second postnatal week, dendritic arbors gradually expanded in multiple sagittal planes in the molecular layer by intensive growth and branching by the third postnatal week. Dendrites then became confined to a single plane in the fourth postnatal week. Multiplanar Purkinje cells in the third week were often associated by ectopic climbing fibers innervating nearby Purkinje cells in distinct sagittal planes. The mature monoplanar arborization was disrupted in mutant mice with abnormal Purkinje cell connectivity and motor discoordination. The dendrite remodeling was also impaired by pharmacological disruption of normal afferent activity during the second or third postnatal week. Our results suggest that the monoplanar arborization of Purkinje cells is coupled with functional development of the cerebellar circuitry.
doi:10.1371/journal.pone.0020108
PMCID: PMC3105010  PMID: 21655286
25.  Functional Relations of Cerebellar Modules of the Cat 
The Journal of Neuroscience  2010;30(28):9411-9423.
The cerebellum consists of parasagittal zones that define fundamental modules of neural processing. Each zone receives input from a distinct subdivision of the inferior olive (IO)—activity in one olivary subdivision will affect activity in one cerebellar module. To define functions of the cerebellar modules, we inactivated specific olivary subdivisions in six male cats with a glutamate receptor blocker. Olivary inactivation eliminates Purkinje cell complex spikes, which results in a high rate of Purkinje cell simple spike discharge. The increased simple spike discharge inhibits output from connected regions of the cerebellar nuclei. After inactivation, behavior was evaluated during a reach-to-grasp task and during locomotion. Inactivation of each subdivision produced unique behavioral deficits. Performance of the reach-to-grasp task was affected by inactivation of the rostral dorsal accessory olive (rDAO) and the rostral medial accessory olive (rMAO) and, possibly, the principal olive. rDAO inactivation produced paw drag during locomotion and a deficit in grasping the handle during the reach-to-grasp task. rMAO inactivation caused the cats to reach under the handle and produced severe limb drag during locomotion. Inactivation of the dorsal medial cell column, cell group β, or caudal medial accessory olive produced little deficit in the reach-to-grasp task, but each produced a different deficit during locomotion. In all cases, the cats appeared to have intact sensation, good spatial awareness, and no change of affect. Normal cerebellar function requires low rates of IO discharge, and each cerebellar module has a specific and unique function in sensory–motor integration.
doi:10.1523/JNEUROSCI.0440-10.2010
PMCID: PMC3865504  PMID: 20631170

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