Purkinje Cell Dendritic Tree Research Articles

Models of Purkinje cell dendritic tree selection during early cerebellar development.

We investigate the relationship between primary dendrite selection of Purkinje cells and migration of their presynaptic partner granule cells during early cerebellar development. During postnatal development, each Purkinje cell grows more than three dendritic trees, from which a primary tree is selected for development, whereas the others completely retract. Experimental studies suggest that this selection process is coordinated by physical and synaptic interactions with granule cells, which undergo a massive migration at the same time. However, technical limitations hinder continuous experimental observation of multiple cell populations. To explore possible mechanisms underlying this selection process, we constructed a computational model using a new computational framework, NeuroDevSim. The study presents the first computational model that simultaneously simulates Purkinje cell growth and the dynamics of granule cell migrations during the first two postnatal weeks, allowing exploration of the role of physical and synaptic interactions upon dendritic selection. The model suggests that interaction with parallel fibers is important to establish the distinct planar morphology of Purkinje cell dendrites. Specific rules to select which dendritic trees to keep or retract result in larger winner trees with more synaptic contacts than using random selection. A rule based on afferent synaptic activity was less effective than rules based on dendritic size or numbers of synapses.

Models of Purkinje cell dendritic tree selection during early cerebellar development

Models of Purkinje cell dendritic tree selection during early cerebellar development

Models of Purkinje cell dendritic tree selection during early cerebellar development

Models of Purkinje cell dendritic tree selection during early cerebellar development

Serine/threonine kinase 17b (STK17B) signalling regulates Purkinje cell dendritic development and is altered in multiple spinocerebellar ataxias

Serine/threonine kinase 17b (STK17B, also known as DRAK2) is known to be a downstream effector of protein kinase C (PKC) in the immune system, in particular T lymphocytes. PKC activity also plays a critical role for dendritic development and synaptic maturation and plasticity in cerebellar Purkinje cells. We present evidence that STK17B is strongly expressed in mouse cerebellar Purkinje cells starting in the early postnatal period and remaining highly expressed throughout adult stages and that STK17B is a target of PKC phosphorylation in the cerebellum. STK17B overexpression potentiates the morphological changes of Purkinje cells seen after PKC activation, suggesting that it is a downstream effector of PKC. A phosphorylation mimetic STK17B variant induced a marked reduction of Purkinje cell dendritic tree size, whereas the inhibition of STK17B with the novel compound 16 (Cpd16) could partially rescue the morphological changes of the Purkinje cell dendritic tree after PKC activation. These findings show that STK17B signalling is an important downstream effector of PKC activation in Purkinje cells. Furthermore, STK17B was identified as a molecule being transcriptionally downregulated in mouse models of SCA1, SCA7, SCA14 and SCA41. The reduced expression of STK17B in these mouse models might protect Purkinje cell dendrites from the negative effects of overactivated PKC signalling. Our findings provide new insights in the role of STK17B for Purkinje cell dendritic development and the pathology of SCAs.

Amot and Yap1 regulate neuronal dendritic tree complexity and locomotor coordination in mice.

The angiomotin (Amot)–Yes-associated protein 1 (Yap1) complex plays a major role in regulating the inhibition of cell contact, cellular polarity, and cell growth in many cell types. However, the function of Amot and the Hippo pathway transcription coactivator Yap1 in the central nervous system remains unclear. We found that Amot is a critical mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. The conditional deletion of Amot and Yap1 in neurons led to a decrease in the complexity of Purkinje cell dendritic trees, abnormal cerebellar morphology, and impairments in motor coordination. Our results indicate that the function of Amot and Yap1 in dendrite growth does not rely on interactions with TEA domain (TEAD) transcription factors or the expression of Hippo pathway–dependent genes. Instead, Amot and Yap1 regulate dendrite development by affecting the phosphorylation of S6 kinase and its target S6 ribosomal protein.

Reduced Purkinje cell size is compatible with near normal morphology and function of the cerebellar cortex in a mouse model of spinocerebellar ataxia

Spinocerebellar ataxia type 14 (SCA14) is a dominantly inherited neurodegenerative disease caused by diverse mutations in the Protein Kinase C gamma (PKCγ) gene which is one of the crucial signaling molecules of Purkinje cells. We have previously created a mouse model of SCA14 by transgenic expression of a mutated PKCγ gene causing SCA14 with a mutation in the catalytic domain. Purkinje cells from the mutated mice have a strong reduction of their dendritic tree in organotypic slice cultures typical for increased PKC activity. There was no overt degeneration of Purkinje cells in vivo and the cerebellum appeared morphologically normal with the exception of lobule 7 where abnormal Purkinje cells were present. Besides from mild motor deficits the mice have no major phenotype. We have now done a more extensive study of cerebellar morphology in these mice and show by rapid Golgi staining that there is a marked reduction of Purkinje cell dendritic tree size throughout the cerebellum. Despite this reduction in dendritic tree size, climbing fiber innervation of Purkinje cells as visualized by immunostaining for the vesicular glutamate transporter 2 (vGlut2) appeared normal in most parts of the cerebellum. The same was true for the expression of the activity and plasticity markers pS6, c-Fos and Arc. These finding suggest that the cerebellar cortex in the transgenic mice is functioning fairly normal and that the reduction of dendritic tree size and the increased PKC activity can be compensated in most Purkinje cells. Around cerebellar lobule 7 there was high transgene expression from the L7 promotor and Purkinje cells showed abnormal morphologies. Climbing fiber innervation as well as the expression of the activity and plasticity markers was strongly disturbed in this area. Our results show that there is substantial potential for functional compensation in the cerebellar cortex. In lobule 7, an area with high transgene expression, compensation failed resulting in Purkinje cell degeneration and dysfunction.

Altered Cerebellar Circuitry following Thoracic Spinal Cord Injury in Adult Rats.

Cerebellar function is critical for coordinating movement and motor learning. However, events occurring in the cerebellum following spinal cord injury (SCI) have not been investigated in detail. We provide evidence of SCI-induced cerebellar synaptic changes involving a loss of granule cell parallel fiber input to distal regions of the Purkinje cell dendritic tree. This is accompanied by an apparent increase in synaptic contacts to Purkinje cell proximal dendrites, presumably from climbing fibers originating in the inferior olive. We also observed an early stage injury-induced decrease in the levels of cerebellin-1, a synaptic organizing molecule that is critical for establishing and maintaining parallel fiber-Purkinje cell synaptic integrity. Interestingly, this transsynaptic reorganizational pattern is consistent with that reported during development and in certain transgenic mouse models. To our knowledge, such a reorganizational event has not been described in response to SCI in adult rats. Regardless, the novel results of this study are important for understanding SCI-induced synaptic changes in the cerebellum, which may prove critical for strategies focusing on promoting functional recovery.

Deletion of the GluRδ2 Receptor in the Hotfoot Mouse Mutant Causes Granule Cell Loss, Delayed Purkinje Cell Death, and Reductions in Purkinje Cell Dendritic Tree Area

Recent studies have found that in the cerebellum, the δ2 glutamate receptor (GluRδ2) plays a key role in regulating the differentiation of parallel fiber-Purkinje synapses and mediating key physiological functions in the granule cell-Purkinje cell circuit. In the hotfoot mutant or GluRδ2 knockout mice, the absence of GluRδ2 expression results in impaired motor-related tasks, ataxia, and disruption of long-term depression at parallel fiber-Purkinje cell synapses. The goal of this study was to determine the long-term consequences of deletion of GluRδ2 expression in the hotfoot mutant (GluRδ2 ho/ho ) on Purkinje and granule cell survival and Purkinje cell dendritic differentiation. Quantitative estimates of Purkinje and granule cell numbers in 3-, 12-, and 20-month-old hotfoot mutants and wild-type controls showed that Purkinje cell numbers are within control values at 3 and 12months in the hotfoot mutant but reduced by 20% at 20months compared with controls. In contrast, the number of granule cells is significantly reduced from 3months onwards in GluRδ2 ho/ho mutant mice compared to wild-type controls. Although the overall structure of Purkinje cell dendrites does not appear to be altered, there is a significant 27% reduction in the cross-sectional area of Purkinje cell dendritic trees in the 20-month-old GluRδ2 ho/ho mutants. The interpretation of the results is that the GluRδ2 receptor plays an important role in the long-term organization of the granule-Purkinje cell circuit through its involvement in the regulation of parallel fiber-Purkinje cell synaptogenesis and in the normal functioning of this critical cerebellar circuit.

Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2weeks of embryonic development points to a potential role of this transporter in the uptake of T3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may serve as a buffering mechanism against high T3 levels during early embryonic development, a hypothesis supported by the pattern of expression of a fluorescent TH reporter in this lineage. Overall, this study builds a picture of the TH dependency in multiple cerebellar cell types starting from early embryonic development.

β-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations.

Beta III spectrin is present throughout the elaborate dendritic tree of cerebellar Purkinje cells and is required for normal neuronal morphology and cell survival. Spinocerebellar ataxia type 5 (SCA5) and spectrin associated autosomal recessive cerebellar ataxia type 1 are human neurodegenerative diseases involving progressive gait ataxia and cerebellar atrophy. Both disorders appear to result from loss of β-III spectrin function. Further elucidation of β-III spectrin function is therefore needed to understand disease mechanisms and identify potential therapeutic options. Here, we report that β-III spectrin is essential for the recruitment and maintenance of ankyrin R at the plasma membrane of Purkinje cell dendrites. Two SCA5-associated mutations of β-III spectrin both reduce ankyrin R levels at the cell membrane. Moreover, a wild-type β-III spectrin/ankyrin-R complex increases sodium channel levels and activity in cell culture, whereas mutant β-III spectrin complexes fail to enhance sodium currents. This suggests impaired ability to form stable complexes between the adaptor protein ankyrin R and its interacting partners in the Purkinje cell dendritic tree is a key mechanism by which mutant forms of β-III spectrin cause ataxia, initially by Purkinje cell dysfunction and exacerbated by subsequent cell death.

Persistent Posttetanic Depression at Cerebellar Parallel Fiber to Purkinje Cell Synapses

Plasticity at the cerebellar parallel fiber to Purkinje cell synapse may underlie information processing and motor learning. In vivo, parallel fibers appear to fire in short high frequency bursts likely to activate sparsely distributed synapses over the Purkinje cell dendritic tree. Here, we report that short parallel fiber tetanic stimulation evokes a ∼7–15% depression which develops over 2 min and lasts for at least 20 min. In contrast to the concomitantly evoked short-term endocannabinoid-mediated depression, this persistent posttetanic depression (PTD) does not exhibit a dependency on the spatial pattern of synapse activation and is not caused by any detectable change in presynaptic calcium signaling. This persistent PTD is however associated with increased paired-pulse facilitation and coefficient of variation of synaptic responses, suggesting that its expression is presynaptic. The chelation of postsynaptic calcium prevents its induction, suggesting that post- to presynaptic (retrograde) signaling is required. We rule out endocannabinoid signaling since the inhibition of type 1 cannabinoid receptors, monoacylglycerol lipase or vanilloid receptor 1, or incubation with anandamide had no detectable effect. The persistent PTD is maximal in pre-adolescent mice, abolished by adrenergic and dopaminergic receptors block, but unaffected by adrenergic and dopaminergic agonists. Our data unveils a novel form of plasticity at parallel fiber synapses: a persistent PTD induced by physiologically relevant input patterns, age-dependent, and strongly modulated by the monoaminergic system. We further provide evidence supporting that the plasticity mechanism involves retrograde signaling and presynaptic diacylglycerol.

Spatio-temporal characterization of the pleiotrophinergic system in mouse cerebellum: Evidence for its key role during ontogenesis

The development of the central nervous system requires an appropriate micro-environment that is conditioned by a combination of various extracellular components. Most of the known signaling factors, such as neurotransmitters or neuropeptides, are soluble and diffuse into the extracellular matrix. However, other secreted molecules like proteoglycans or glycosaminoglycans anchor in the extracellular matrix to influence cerebral ontogenesis. As such, pleiotrophin (PTN), which binds the proteoglycans syndecan-3 (SDC3) and protein tyrosine phosphatase zeta (PTPζ), has been described as a pro-migratory and a pro-differentiating secreted cytokine on cortical neurons. In rat cerebellum, PTN is highly expressed during the first postnatal week, suggesting that this cytokine could participate to the development of the cerebellar cortex. According to this hypothesis, our spatio-temporal cartography of PTN, PTPζ and SDC3 indicated that, in mouse, the PTNergic system was present in the cerebellum at least from the first postnatal day (P0). Until P12, PTN was mainly expressed by granule cell precursors and located in the extracellular matrix, while SDC3 was expressed by Purkinje cells, Golgi cells and granule cell precursors, and PTPζ was present on Purkinje cells and Bergmann fibers. In vitro studies confirmed the presence of SDC3 on immature granule cells and demonstrated that PTN could stimulate directly their velocity in culture. In contrast, subarachnoidal injection of PTN in the cerebellum significantly reduced the rate of migration of granule cells, exacerbated their apoptosis and induced an atrophy of the Purkinje cell dendritic tree. Since differentiated granule cells did not express SDC3 or PTPζ, the PTN effect observed on migration and apoptosis may be indirectly mediated by Purkinje and/or Bergmann cells. From P21 to adulthood, the distribution of PTN, SDC3 and PTPζ changed and their expression dramatically decreased even if they were still detectable. PTN and SDC3 immunolabeling was restricted around Purkinje cell bodies and Golgi cells, whereas PTPζ was located around interneurons. These data suggested that, in the cerebellum of adult mice, PTN participates to the perineuronal nets that control neuronal plasticity. To conclude, the present work represents the first spatio-temporal characterization of the PTNergic system in the mouse cerebellum and indicates that PTN may contribute to cerebellum ontogenesis during the postnatal development as well as to neuronal plasticity at adulthood.

The Plasma Membrane Ca2+-ATPase2 (PMCA2) Is Involved in the Regulation of Purkinje Cell Dendritic Growth in Cerebellar Organotypic Slice Cultures

Purkinje cells are the principal neurons of the cerebellar cortex and have an extensive and elaborate dendritic tree. Chronic activation of type I metabotropic glutamate receptors inhibits Purkinje cell dendritic growth in organotypic cerebellar slice cultures. This effect is mediated by calcium influx through P/Q-type and T-type Ca2+ channels. We have now studied the role of the plasma membrane Ca2+-ATPase2 (PMCA2), a major calcium extrusion pump, for Purkinje cell dendritic development. We found that PMCA2 is strongly expressed in the plasma membrane and dendritic spines of Purkinje cells in organotypic slice cultures compatible with a role for controlling the local dendritic calcium equilibrium. Inhibition of PMCA2 activity by carboxyeosin resulted in a moderate reduction of Purkinje cell dendritic tree size indicating that the extrusion of calcium by PMCA2 is important for maintaining the dendritic calcium concentration and controlling dendritic growth. When inhibition of PMCA2 was combined with stimulation of type I metabotropic glutamate receptors, it partially rescued dendritic morphology. This protection can be explained by a compensatory inactivation of voltage-gated calcium channels in Purkinje cells after PMCA2 inhibition. Our results demonstrate that PMCA2 activity is an important regulator of the dendritic calcium equilibrium controlling Purkinje cell dendritic growth.

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents (3). Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells (11) are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period (4). We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

P/Q‐type and T‐type calcium channels, but not type 3 transient receptor potential cation channels, are involved in inhibition of dendritic growth after chronic metabotropic glutamate receptor type 1 and protein kinase C activation in cerebellar Purkinje cells

The development of a neuronal dendritic tree is modulated both by signals from afferent fibers and by an intrinsic program. We have previously shown that chronic activation of either type 1 metabotropic glutamate receptors (mGluR1s) or protein kinase C (PKC) in organotypic cerebellar slice cultures of mice and rats severely inhibits the growth and development of the Purkinje cell dendritic tree. The signaling events linking receptor activation to the regulation of dendritic growth remain largely unknown. We have studied whether channels allowing the entry of Ca(2+) into Purkinje cells, in particular the type 3 transient receptor potential cation channels (TRPC3s), P/Q-type Ca(2+) channels, and T-type Ca(2+) channels, might be involved in signaling after mGluR1 or PKC stimulation. We show that the inhibition of dendritic growth seen after mGluR1 or PKC stimulation is partially rescued by pharmacological blockade of P/Q-type and T-type Ca(2+) channels, indicating that activation of these channels mediating Ca(2+) influx contributes to the inhibition of dendritic growth. In contrast, the absence of Ca(2+) -permeable TRPC3s in TRPC3-deficient mice or pharmacological blockade had no effect on mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. Similarly, blockade of Ca(2+) influx through glutamate receptor δ2 or R-type Ca(2+) channels or inhibition of release from intracellular stores did not influence mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. These findings suggest that both T-type and P/Q-type Ca(2+) channels, but not TRPC3 or other Ca(2+) -permeable channels, are involved in mGluR1 and PKC signaling leading to the inhibition of dendritic growth in cerebellar Purkinje cells.

3D electron microscopic reconstruction of segments of rat cerebellar purkinje cell dendrites receiving ascending and parallel fiber granule cell synaptic inputs

Growing physiological evidence suggests that there are functional differences between synapses made by the ascending and parallel fiber segments of the granule axon on cerebellar Purkinje cells. Supporting this view, our previous electron microscopic studies suggested that these synapses also contacted different regions of the Purkinje cell dendrite, and in particular that ascending segment synapses are made exclusively on the smallest diameter Purkinje cell dendrites. In the current study we used serial electron microscopic techniques to reconstruct Purkinje cell dendritic segments up to almost 10 mum in length. Using a combination of anatomical and immunological labeling techniques we identified the ascending or parallel fiber origins of the excitatory synaptic inputs onto dendritic spines, as well as the location of inhibitory synapses made directly on the dendritic shaft. The results confirmed that there are regions of the Purkinje cell dendrite receiving exclusively ascending or parallel fiber synapses and that ascending segment synapses are only found on small-diameter dendrites. In addition, we describe for the first time small-diameter dendritic regions contacted by both types of excitatory synapses. While our data suggest that the majority of inhibitory inputs to the Purkinje cell tree are associated with parallel fiber synaptic inputs, we also found inhibitory inputs on dendritic regions with mixed ascending and parallel fiber inputs, or exclusively parallel fiber inputs. The finding that ascending and parallel fiber inputs can be segregated on the Purkinje cell dendritic tree provides further evidence that these excitatory granule cell synaptic inputs may be functionally distinct.

Distribution of granule cells projecting to focal Purkinje cells in mouse uvula-nodulus

Mossy and climbing fibers convey a broad array of signals from vestibular end organs to Purkinje cells in the vestibulo-cerebellum. We have shown previously that Purkinje cell simple spikes (SSs) and climbing fiber–evoked complex spikes (CSs) in the mouse uvula-nodulus are arrayed in 400 μm wide sagittal climbing fiber zones corresponding to the rotational axes of the vertical semicircular canals. It is often assumed that mossy fibers modulate a higher frequency of SSs through the intermediary action of granule cells whose parallel fibers course through the Purkinje cell dendritic tree. This assumption is complicated by the diffuse topography of vestibular primary afferent mossy fiber projections to the uvula-nodulus and the dispersion of mossy fiber signals along folial axes by parallel fibers. Here we measure this parallel fiber dispersion. We made microinjections of neurobiotin into the molecular layers of different folia within the mouse vestibulo-cerebellum and measured the distribution of granule cells retrogradely labeled by the injected neurobiotin. Sixty-two percent of labeled granule cells were located outside a 400 μm sagittal zone flanking the injection site. The dispersion of labeled granule cells was ∼2.5 mm along folial axes that were 2.7–2.9 mm wide. Our data suggest that topographic specificity of SSs could not be attributed to the topography of vestibular primary afferent mossy fiber–granule cell projections. Rather the response specificity of SSs must be attributed to other mechanisms related to climbing fiber–evoked Purkinje cell and interneuronal activity.

Spontaneous electrical activity and dendritic spine size in mature cerebellar Purkinje cells

Previous experiments have shown that in the mature cerebellum both blocking of spontaneous electrical activity and destruction of the climbing fibres by a lesion of the inferior olive have a similar profound effect on the spine distribution on the proximal dendrites of the Purkinje cells. Many new spines develop that are largely innervated by parallel fibers. Here we show that blocking electrical activity leads to a significant decrease in size of the spines on the branchlets. We have also compared the size of the spines of the proximal dendritic domain that appear during activity block and after an inferior olive lesion. In this region also, the spines in the absence of activity are significantly smaller. In the proximal dendritic domain, the new spines that develop in the absence of activity are innervated by parallel fibers and are not significantly different in size from those of the branchlets, although they are shorter. Thus, the spontaneous activity of the cerebellar cortex is necessary not only to maintain the physiological spine distribution profile in the Purkinje cell dendritic tree, but also acts as a signal that prevents spines from shrinking.

Purkinje cell dendrites grow in alignment with Bergmann glia

The pattern of growth of Purkinje cell dendrites has been analyzed and related to their interactions with Bergmann glial radial processes. In cerebellar slice cultures from mice expressing green fluorescent protein (GFP) under the glial fibrillary acidic protein (GFAP) promoter, Purkinje cells were transfected and imaged with two-photon microscopy over 2 days. We report that while the Purkinje cell dendritic tree grows, individual dendrites increase or decrease in length. Importantly, we demonstrate that vertical growth of Purkinje cell dendrites occurs primarily in alignment with radial glial processes. These findings suggest that radial glial processes provide a structural substrate for the directional growth of Purkinje cell dendrites, thus influencing the shape of the dendritic tree.

Low threshold calcium currents in rat cerebellar Purkinje cell dendritic spines are mediated by T‐type calcium channels

The functional role of low voltage activated (LVA) calcium channels in the cerebellar Purkinje cell dendritic tree is not completely understood. Since the localization of these channels will influence their possible roles in dendritic integration and induction of plasticity, we set out to characterize the LVA calcium current in Purkinje cell dendrites in acute cerebellar slices of young rats. Using a combination of electrophysiological recordings and two-photon laser scanning microscopy, we show that LVA calcium current recorded at the soma can be correlated with voltage-dependent calcium transients in Purkinje cell dendritic spines. Blocking sodium and potassium conductances allowed us to isolate and characterize a fast inactivating inward current activated positive to -55 mV. Activation and steady-state inactivation kinetics, voltage-dependent deactivation kinetics, and pharmacological experiments (using omega-agatoxin-IVA, mibefradil and nickel) show that this current is carried by T-type calcium channels. Furthermore, the LVA calcium transient observed in the dendritic spines of the Purkinje cell is well correlated with the current recorded at the soma, suggesting that T-type calcium channels are the main component of the LVA calcium input in spines. The fast rising phase of the calcium transient in spines and the absence of delay between the onset in the spine and the parent dendrite show that T-type calcium channels are present both in spines and dendrites of the Purkinje cell.