Purkinje Cell Dendritic Spines Research Articles

Functional regionalization of the differentiating cerebellar Purkinje cell population occurs in an activity-dependent manner.

The cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive. We show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer. Hence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Deficits in cerebellum-dependent learning and cerebellar morphology in male and female BTBR autism model mice.

Recently, there has been increased interest in the role of the cerebellum in autism spectrum disorders (ASD). To better understand the pathophysiological role of the cerebellum in ASD, it is necessary to have a variety of mouse models that have face validity for cerebellar disruption in humans. Here, we add to the literature on the cerebellum transgenic and induced mouse models of autism with the characterization of the cerebellum in the BTBR T+Itpr3tf/J (BTBR) inbred mouse strain, which has behavioral phenotypes that are suggestive of ASD in patients. When we examined both male and female BTBR mice in comparison to C57BL/6J (C57) controls, we noted that both sexes of BTBR mice showed motor coordination deficits characteristic of cerebellar dysfunction, but only the male mice showed differences in delay eyeblink conditioning, a cerebellum-dependent learning task that is also disrupted in ASD patients. Both male and female BTBR mice showed considerable expansion of and abnormal foliation in the cerebellum vermis--including significant expansion of specific lobules in the anterior cerebellum. In addition, we found a slight but significant decrease in Purkinje cell density in both male and female BTBR mice, irrespective of lobule. Furthermore, there was a marked reduction of Purkinje cell dendritic spines density in both male and female BTBR mice. These findings suggest that, for the most part, the BTBR mouse model successfully phenocopies many of the characteristics of the subpopulation of ASD patients that have a hypertrophic cerebellum. We discuss the significance of strain differences in the cerebellum as well as the importance of this first effort to identify both concordances and difference between male and female BTBR mice with regard to the cerebellum.

Movement disorder caused by FRRS1L deficiency may be associated with morphological and functional disorders in Purkinje cells

Ferric Chelate Reductase 1 Like (FRRS1L) protein has been identified as an auxiliary regulatory protein for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR). FRRS1L is highly expressed in the cerebellum and other brain regions associated with the control of motor function. Loss of FRRS1L has been shown to lead to impaired synaptic transmission via AMPARs and to movement disorders. We found that deletion of the FRRS1L gene causes hyperactivity, reduced muscle strength, impaired coordination, and ataxia in mice. Deletion also impairs Purkinje cell dendritic spine formation and AMPAR expression in the cerebellum and damages the electrophysiological discharge rhythm of Purkinje cells. Cerebrospinal fluid examination and oleic acid (OA)-induced lipid accumulation monitoring in FRRS1L-knockdown SH-SY5Y cells indicated that FRRS1L deficiency could lead to aberrant metabolism of amino acids, glucose, and lipids. In summary, we found that the deletion of FRRS1L leads to impaired motor coordination and cerebellar ataxia in mice, which might be related to the reduced expression of AMPARs, metabolic deviations, and dysplastic functional defects in Purkinje cells.

Cellular Diversity and Differential Subcellular Localization of the G-Protein Gαo Subunit in the Mouse Cerebellum.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals from G protein-coupled receptors (GPCRs) to effector ion channels and enzymes Gαo, a member of the pertussis toxin-sensitive Gi/o family, is widely expressed in the brain, although its role within a neuronal context remains largely unknown. Using immunohistochemical and quantitative immunoelectron microscopy techniques, we have investigated the expression, cellular and subcellular localization of Gαo in the cerebellar cortex. Histoblot revealed that Gαo is expressed in many brain regions, including the cerebellum. At the cellular level, Gαo protein was distributed in Purkinje cells, basket cells, stellate cells, granule cells and Golgi cells. At the subcellular level, pre-embedding immunoelectron microscopy revealed mainly a postsynaptic localization of Gαo along the extrasynaptic plasma membrane of Purkinje cell dendritic shafts and spines, and dendrites of basket, stellate and granule cells. To a lesser extent, immunolabeling for Gαo was localized in different types of axon terminals establishing excitatory synapses. Moreover, post-embedding immunoelectron microscopy revealed the synaptic localization of Gαo on PSDs of glutamatergic synapses between Purkinje cell spines and parallel fiber terminals and its co-localization with GABAB1 in the same spines. Quantitative analysis of Gαo immunoparticles revealed they preferentially localized on the cytoplasmic face of the plasma membrane. Furthermore, the analysis revealed a high concentration of Gαo around excitatory synapses on Purkinje cell dendritic spines, but a uniform distribution in granule cell dendrites. These molecular-anatomical findings suggest that Gαo is a major signal transducer of specific GPCRs in different neuronal populations in the cerebellum.

Stochastic reaction-diffusion modeling of calcium dynamics in 3D dendritic spines of Purkinje cells

Calcium (Ca2+) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression and long-term potentiation at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP3)-induced Ca2+ release. These Ca2+ transients happen in response to stimuli from parallel fibers (PFs) from granule cells and climbing fibers (CFs) from the inferior olivary nucleus. These events occur at low numbers of free Ca2+, requiring stochastic single-particle methods when modeling them. We use the stochastic particle simulation program MCell to simulate Ca2+ transients within a three-dimensional Purkinje cell dendritic spine. The model spine includes the endoplasmic reticulum, several Ca2+ transporters, and endogenous buffer molecules. Our simulations successfully reproduce properties of Ca2+ transients in different dynamical situations. We test two different models of the IP3 receptor (IP3R). The model with nonlinear concentration response of binding of activating Ca2+ reproduces experimental results better than the model with linear response because of the filtering of noise. Our results also suggest that Ca2+-dependent inhibition of the IP3R needs to be slow to reproduce experimental results. Simulations suggest the experimentally observed optimal timing window of CF stimuli arises from the relative timing of CF influx of Ca2+ and IP3 production sensitizing IP3R for Ca2+-induced Ca2+ release. We also model ataxia, a loss of fine motor control assumed to be the result of malfunctioning information transmission at the granule to Purkinje cell synapse, resulting in a decrease or loss of Ca2+ transients. Finally, we propose possible ways of recovering Ca2+ transients under ataxia.

Stochastic Reaction-Diffusion Modeling of Calcium Dynamics in 3D-Dendritic Spines of Purkinje Cells

Calcium (Ca2+) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression (LTD) and long-term potentiation (LTP) at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP3) induced Ca2+ release. These Ca2+ transients happen in response to stimuli from parallel fibers (PF) from granule cells and climbing fibers (CF) from the inferior olivary nucleus. These events occur at low numbers of free Ca2+ requiring stochastic single particle methods when modeling them.

Differential synapse density between Purkinje cell dendritic spine and parallel fiber varicosity in the rat cerebellum among the phylogenic lobules

The cerebellum is a region of the brain that plays an important role in motor control. It is classified phylogenetically into archicerebellum, paleocerebellum and neocerebellum. The Purkinje cells are lined in a row called Purkinje cell layer and it has a unique dendritic branches with many spines.The previous study reported that there is a difference of synapse density according to the lobules based on large two-dimensional data. However, recent study with high voltage electron microscopy showed there was no differences in dendritic spine density of the Purkinje cell according to its phylogenetic lobule. We analyzed Purkinje cell density in the II, VI and X lobules by stereological modules and synaptic density was estimated by double disector based on Purkinje cell density in the molecular layer of each lobule.The results showed that there was significant difference in the Purkinje cell density and synapse number according to their phylogenetic lobules. The number of Purkinje cell in a given volume was larger in the archicerebellum, but synapse density was higher in the neocerebellum.These data suggest that cellular and synaptic organization of the Purkinje cell is different according to their phylogenetic background.

Motor learning induces plastic changes in Purkinje cell dendritic spines in the rat cerebellum

IntroductionThe paramedian lobule of the cerebellum is involved in learning to correctly perform motor skills through practice. Dendritic spines are dynamic structures that regulate excitatory synaptic stimulation. We studied plastic changes occurring in the dendritic spines of Purkinje cells from the paramedian lobule of rats during motor learning. MethodsAdult male rats were trained over a 6-day period using an acrobatic motor learning paradigm; the density and type of dendritic spines were determined every day during the study period using a modified version of the Golgi method. ResultsThe learning curve reflected a considerable decrease in the number of errors made by rats as the training period progressed. We observed more dendritic spines on days 2 and 6, particularly more thin spines on days 1, 3, and 6, fewer mushroom spines on day 3, fewer stubby spines on day 1, and more thick spines on days 4 and 6. ConclusionThe initial stage of motor learning may be associated with fast processing of the underlying synaptic information combined with an apparent “silencing” of memory consolidation processes, based on the regulation of the neuronal excitability.

Deletion of the \u03b1 subunit of the heterotrimeric Go protein impairs cerebellar cortical development in mice

Go is a member of the pertussis toxin-sensitive Gi/o family. Despite its abundance in the central nervous system, the precise role of Go remains largely unknown compared to other G proteins. In the present study, we explored the functions of Go in the developing cerebellar cortex by deleting its gene, Gnao. We performed a histological analysis with cerebellar sections of adult mice by cresyl violet- and immunostaining. Global deletion of Gnao induced cerebellar hypoplasia, reduced arborization of Purkinje cell dendrites, and atrophied Purkinje cell dendritic spines and the terminal boutons of climbing fibers from the inferior olivary nucleus. These results indicate that Go-mediated signaling pathway regulates maturation of presynaptic parallel fibers from granule cells and climbing fibers during the cerebellar cortical development.

Regional Regulation of Purkinje Cell Dendritic Spines by Integrins and Eph/Ephrins.

Climbing fibres and parallel fibres compete for dendritic space on Purkinje cells in the cerebellum. Normally, climbing fibres populate the proximal dendrites, where they suppress the multiple small spines typical of parallel fibres, leading to their replacement by the few large spines that contact climbing fibres. Previous work has shown that ephrins acting via EphA4 are a signal for this change in spine type and density. We have used an in vitro culture model in which to investigate the ephrin effect on Purkinje cell dendritic spines and the role of integrins in these changes. We found that integrins α3, α5 and β4 are present in many of the dendritic spines of cultured Purkinje cells. pFAK, the main downstream signalling molecule from integrins, has a similar distribution, although the intenstity of pFAK staining and the percentage of pFAK+ spines was consistently higher in the proximal dendrites. Activating integrins with Mg2+ led to an increase in the intensity of pFAK staining and an increase in the proportion of pFAK+ spines in both the proximal and distal dendrites, but no change in spine length, density or morphology. Blocking integrin binding with an RGD-containing peptide led to a reduction in spine length, with more stubby spines on both proximal and distal dendrites. Treatment of the cultures with ephrinA3-Fc chimera suppressed dendritic spines specifically on the proximal dendrites and there was also a decrease of pFAK in spines on this domain. This effect was blocked by simultaneous activation of integrins with Mn2+. We conclude that Eph/ephrin signaling regulates proximal dendritic spines in Purkinje cells by inactivating integrin downstream signalling.

Lesion-induced and activity-dependent structural plasticity of Purkinje cell dendritic spines in cerebellar vermis and hemisphere.

Neuroplasticity allows the brain to encode experience and learn behaviors, and also to re-acquire lost functions after damage. The cerebellum is a suitable structure to address this topic because of its strong involvement in learning processes and compensation of lesion-induced deficits. This study was aimed to characterize the effects of a hemicerebellectomy (HCb) combined or not with the exposition to environmental enrichment (EE) on dendritic spine density and size in Purkinje cell proximal and distal compartments of cerebellar vermian and hemispherical regions. Male Wistar rats were housed in enriched or standard environments from the 21st post-natal day (pnd) onwards. At the 75th pnd, rats were submitted to HCb or sham lesion. Neurological symptoms and spatial performance in the Morris water maze were evaluated. At the end of testing, morphological analyses assessed dendritic spine density, area, length, and head diameter on vermian and hemispherical Purkinje cells. All hemicerebellectomized (HCbed) rats showed motor compensation, but standard-reared HCbed animals exhibited cognitive impairment that was almost completely compensated in enriched HCbed rats. The standard-reared HCbed rats showed decreased density with augmented size of Purkinje cell spines in the vermis, and augmented both density and size in the hemisphere. Enriched HCbed rats almost completely maintained the spine density and size induced by EE. Both lesion-induced and activity-dependent cerebellar plastic changes may be interpreted as "beneficial" brain reactions, aimed to support behavioral performance rescuing.

Circuit reorganization in cerebellar cultures after injury

As elaborated in a recent review (Seil, 2014), we used an easily manipulable cerebellar culture system to study injury-induced reorganizational changes in a simplified central nervous system (CNS) model. The structural and functional interrelationships of the neurons of the rodent cerebellar cortex are well known (Eccles et al., 1967). There are five major neuronal types, including Purkinje, granule, basket, stellate and Golgi cells. Purkinje cells are inhibitory and are the only cortical neurons to project to other parts of the CNS, their axons terminating mainly in the underlying deep cerebellar nuclei. Purkinje cell axons also emit collaterals that project to other cortical neurons, including other Purkinje cells. Granule cells are the only excitatory cortical neurons and they project axons as bundles of parallel fibers to the dendrites of all other cortical neurons and form numerous synapses with Purkinje cell dendritic spines. The basket, stellate and Golgi cells are all inhibitory interneurons, their axons confined to the cortex. All of these neuronal types are represented in organotypic cerebellar cultures derived from newborn mice and allowed to develop in vitro (Seil, 1979).

HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice.

Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset.

Reduced Purkinje cell dendritic arborization and loss of dendritic spines in essential tremor

Based on accumulating post-mortem evidence of abnormalities in Purkinje cell biology in essential tremor, we hypothesized that regressive changes in dendritic morphology would be apparent in the Purkinje cell population in essential tremor cases versus age-matched controls. Cerebellar cortical tissue from 27 cases with essential tremor and 27 age-matched control subjects was processed by the Golgi-Kopsch method. Purkinje cell dendritic anatomy was quantified using a Neurolucida microscopic system interfaced with a motorized stage. In all measures, essential tremor cases demonstrated significant reductions in dendritic complexity compared with controls. Median values in essential tremor cases versus controls were: 5712.1 versus 10 403.2 µm (total dendrite length, P=0.01), 465.9 versus 592.5 µm (branch length, P=0.01), 22.5 versus 29.0 (maximum branch order, P=0.001), and 165.3 versus 311.7 (number of terminations, P=0.008). Furthermore, the dendritic spine density was reduced in essential tremor cases (medians=0.82 versus 1.02 µm(-1), P=0.03). Our demonstration of regressive changes in Purkinje cell dendritic architecture and spines in essential tremor relative to control brains provides additional evidence of a pervasive abnormality of Purkinje cell biology in this disease, which affects multiple neuronal cellular compartments including their axon, cell body, dendrites and spines.

Mutant β-III spectrin causes mGluR1α mislocalization and functional deficits in a mouse model of spinocerebellar ataxia type 5.

Spinocerebellar ataxia type 5 (SCA5), a dominant neurodegenerative disease characterized by profound Purkinje cell loss, is caused by mutations in SPTBN2, a gene that encodes β-III spectrin. SCA5 is the first neurodegenerative disorder reported to be caused by mutations in a cytoskeletal spectrin gene. We have developed a mouse model to understand the mechanistic basis for this disease and show that expression of mutant but not wild-type β-III spectrin causes progressive motor deficits and cerebellar degeneration. We show that endogenous β-III spectrin interacts with the metabotropic glutamate receptor 1α (mGluR1α) and that mice expressing mutant β-III spectrin have cerebellar dysfunction with altered mGluR1α localization at Purkinje cell dendritic spines, decreased mGluR1-mediated responses, and deficient mGluR1-mediated long-term potentiation. These results indicate that mutant β-III spectrin causes mislocalization and dysfunction of mGluR1α at dendritic spines and connects SCA5 with other disorders involving glutamatergic dysfunction and synaptic plasticity abnormalities.

Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4–5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Deletions in GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze in humans

To identify the genetic cause of a syndrome causing cerebellar ataxia and eye movement abnormalities. We identified 2 families with cerebellar ataxia, eye movement abnormalities, and global developmental delay. We performed genetic analyses including single nucleotide polymorphism genotyping, linkage analysis, array comparative genomic hybridization, quantitative PCR, and Sanger sequencing. We obtained eye movement recordings of mutant mice deficient for the ortholog of the identified candidate gene, and performed immunohistochemistry using human and mouse brain specimens. All affected individuals had ataxia, eye movement abnormalities, most notably tonic upgaze, and delayed speech and cognitive development. Homozygosity mapping identified the disease locus on chromosome 4q. Within this region, a homozygous deletion of GRID2 exon 4 in the index family and compound heterozygous deletions involving GRID2 exon 2 in the second family were identified. Grid2-deficient mice showed larger spontaneous and random eye movements compared to wild-type mice. In developing mouse and human cerebella, GRID2 localized to the Purkinje cell dendritic spines. Brain MRI in 2 affected children showed progressive cerebellar atrophy, which was more severe than that of Grid2-deficient mice. Biallelic deletions of GRID2 lead to a syndrome of cerebellar ataxia and tonic upgaze in humans. The phenotypic resemblance and similarity in protein expression pattern between humans and mice suggest a conserved role for GRID2 in the synapse organization between parallel fibers and Purkinje cells. However, the progressive and severe cerebellar atrophy seen in the affected individuals could indicate an evolutionarily unique role for GRID2 in the human cerebellum.

Conserved cellular distribution of the glutamate receptors GluA2/3, mGlu1a and mGlu2/3 in isolated cultures of rat cerebellum

To what extent the intrinsic glutamatergic system of the cerebellum is able to keep normal features in the absence of mossy and climbing fibres, is at present not known. To answer this question, immunocytochemistry for light and high resolution electron microscopy was used to reveal the cellular and subcellular distribution of glutamate receptors in isolated cerebellar cultures. The localization of the ionotropic glutamate receptor subunits GluA2/3 and the metabotropic glutamate (mGlu) 1a and mGlu2/3 receptor subtypes was carried out in 0 to 9-day-old rat parasagittal slices developed in vitro for 20–40 days.The typical localization of GluA2/3, mGlu1a and mGlu2/3 observed in Purkinje cells, granule cells, Golgi cells and unipolar brush cells was maintained in the organotypic cultures. Furthermore, the subcellular distribution of mGlu1a showed the characteristic in vivo perisynaptic position in Purkinje cell dendritic spines receiving parallel fibre synapses.We conclude that the cellular and subcellular localization of the studied ionotropic and metabotropic glutamate receptors is not affected by the removal of the two extrinsic cerebellar glutamatergic inputs, the mossy and climbing fibres.

A Study of the Spatial Protein Organization of the Postsynaptic Density Isolated from Porcine Cerebral Cortex and Cerebellum

Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ∼ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.

Precise localization of the voltage-gated potassium channel subunits Kv3.1b and Kv3.3 revealed in the molecular layer of the rat cerebellar cortex by a pre-embedding immunogold method

A proper motor activity relies on a correct cerebellar function. The Kv3.1 and Kv3.3 voltage-gated potassium channels are key proteins involved in cerebellar function and dysfunction, as the lack of these causes severe motor deficits. Both channel subunits are coexpressed in granule cells and are rapidly activated at relatively positive potentials to support the generation of fast action potentials. However, the contribution of each subunit to the molecular architecture of the parallel fibers, the granule cell axons, is so far unknown. The goal of this study was to elucidate the relative distribution of Kv3.1b and Kv3.3 in specific compartments of the rat parallel fibers by using a pre-embedding immunocytochemical method for electron microscopy. Numerous Kv3.1b and Kv3.3 silver-intensified gold particles were associated with membranes of parallel fiber synaptic terminals and their intervaricose segments. Kv3.1b was found in about 85% of parallel fiber synaptic terminals and in about 47% of their intervaricose portions. However, only 28% of intervaricosities and 23% of parallel fiber presynaptic boutons were Kv3.3 immunopositive. The analysis also revealed that 54% of Purkinje cell dendritic spines localized Kv3.3. Although both potassium channel subunits share localization in the same presynaptic parallel fiber compartments, the present results with the method used indicate that there are a higher percentage of parallel fibers labeled for Kv3.1b than for Kv3.3, and that the labeling intensity for each subunit is higher in specific subcompartments analyzed than in others.