Normal Granule Cells Research Articles

Otx2 promotes granule cell precursor proliferation and Shh-dependent medulloblastoma maintenance in vivo

The developmental gene OTX2 is expressed by cerebellar granule cell precursors (GCPs), a cell population which undergoes massive expansion during the early postnatal period in response to sonic hedgehog (Shh). GCPs are thought to be at the origin of most medulloblastomas, a devastating paediatric cancer that arises in the developing cerebellum. OTX2 is overexpressed in all types of medulloblastomas, except in Shh-dependent type 2 medulloblastomas, although it has GCPs as cell-of-origin. This has led to the current view that OTX2 is not involved in tumorigenesis of this subgroup. How OTX2 might contribute to normal or tumoral GCP development in vivo remains unresolved. Here, we have investigated, for the first time, the physiological function of this factor in regulating proliferation and tumorigenesis in the developing mouse cerebellum. We first characterized Otx2-expressing cells in the early postnatal cerebellum and showed that they represent a unique subpopulation of highly proliferative GCPs. We next performed in vivo loss-of-function analysis to dissect out the role of Otx2 in these cells and identified a novel, Shh-independent, function for this factor in controlling postnatal GCP proliferation and cerebellum morphogenesis. Finally, we addressed the function of Otx2 in the context of type 2 medulloblastomas by directing Shh-dependent tumour formation in Otx2+ cells of the developing cerebellum and assessing the effects of Otx2 ablation in this context. We unravel an unexpected, mandatory function for Otx2 in sustaining cell proliferation and long-term maintenance of these tumours in vivo, therefore bringing unpredicted insight into the mechanisms of type 2 medulloblastoma subsistence. Together, these data pinpoint, for the first time, a crucial Shh-independent role for Otx2 in the control of proliferation of normal and tumoral granule cell precursors in vivo and make it an attractive candidate for targeted therapy in Shh-dependent medulloblastomas.

Synaptic Plasticity and Excitation-Inhibition Balance in the Dentate Gyrus: Insights from In Vivo Recordings in Neuroligin-1, Neuroligin-2, and Collybistin Knockouts.

The hippocampal dentate gyrus plays a role in spatial learning and memory and is thought to encode differences between similar environments. The integrity of excitatory and inhibitory transmission and a fine balance between them is essential for efficient processing of information. Therefore, identification and functional characterization of crucial molecular players at excitatory and inhibitory inputs is critical for understanding the dentate gyrus function. In this minireview, we discuss recent studies unraveling molecular mechanisms of excitatory/inhibitory synaptic transmission, long-term synaptic plasticity, and dentate granule cell excitability in the hippocampus of live animals. We focus on the role of three major postsynaptic proteins localized at excitatory (neuroligin-1) and inhibitory synapses (neuroligin-2 and collybistin). In vivo recordings of field potentials have the advantage of characterizing the effects of the loss of these proteins on the input-output function of granule cells embedded in a network with intact connectivity. The lack of neuroligin-1 leads to deficient synaptic plasticity and reduced excitation but normal granule cell output, suggesting unaltered excitation-inhibition ratio. In contrast, the lack of neuroligin-2 and collybistin reduces inhibition resulting in a shift towards excitation of the dentate circuitry.

Functional expression of calcium-permeable canonical transient receptor potential 4-containing channels promotes migration of medulloblastoma cells.

Key points The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells.OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues.Activation of TRPC4‐containing channels in DAOY cells, but not non‐transformed granule precursor cells, results in prominent increases in [Ca2+]i and promotes cell motility in wound healing and transwell migration assays.Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca2+]i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4.Our results suggest that OGR1 enhances expression of TRPC4‐containing channels that contribute to enhanced invasion and metastasis of granule precursor‐derived human medulloblastoma. Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilized in response to activation of cell surface receptors. Ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) is a proton‐sensing Gq‐coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We found that nine distinct human MB samples all expressed OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promoted expression of the proton‐potentiated member of the canonical transient receptor potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we found that all MB samples also expressed TRPC4. In DAOY cells, activation of TRPC4‐containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration were observed. Our results suggest that OGR1‐dependent increases in TRPC4 expression may favour formation of highly Ca2+‐permeable TRPC4‐containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB.

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.

Potential implications of a monosynaptic pathway from mossy cells to adult-born granule cells of the dentate gyrus.

The dentate gyrus (DG) is important to many aspects of hippocampal function, but there are many aspects of the DG that are incompletely understood. One example is the role of mossy cells (MCs), a major DG cell type that is glutamatergic and innervates the primary output cells of the DG, the granule cells (GCs). MCs innervate the GCs as well as local circuit neurons that make GABAergic synapses on GCs, so the net effect of MCs on GCs – and therefore the output of the DG – is unclear. Here we first review fundamental information about MCs and the current hypotheses for their role in the normal DG and in diseases that involve the DG. Then we review previously published data which suggest that MCs are a source of input to a subset of GCs that are born in adulthood (adult-born GCs). In addition, we discuss the evidence that adult-born GCs may support the normal inhibitory ‘gate’ functions of the DG, where the GCs are a filter or gate for information from the entorhinal cortical input to area CA3. The implications are then discussed in the context of seizures and temporal lobe epilepsy (TLE). In TLE, it has been suggested that the DG inhibitory gate is weak or broken and MC loss leads to insufficient activation of inhibitory neurons, causing hyperexcitability. That idea was called the “dormant basket cell hypothesis.” Recent data suggest that loss of normal adult-born GCs may also cause disinhibition, and seizure susceptibility. Therefore, we propose a reconsideration of the dormant basket cell hypothesis with an intervening adult-born GC between the MC and basket cell and call this hypothesis the “dormant immature granule cell hypothesis.”

Abstract 707: Distinct roles of Na,K-ATPase function and expression in medulloblastoma

Abstract Medulloblastoma, which usually arises in the cerebellum, is the most common malignant brain cancer in children and is still associated with substantial mortality and survivors often suffer from serious life-long therapy-related side effects. In recent years several medulloblastoma subtypes with distinct developmental origins, genetic profiles, pathway signatures, and clinicopathological features have been identified. The current consensus divides medulloblastoma tumors into four subgroups, WNT, sonic hedgehog (Shh), Group 3 and Group 4 allowing for different targeted therapeutic approaches. Not specifically restricted to a defined subgroup, aberrant activation of the epidermal growth factor receptor family (EGFR/ErbB) has been reported in a large percentage of medulloblastoma and has been associated with poor outcome. The Shh pathway is an important signaling pathway involved in cerebellar development. Shh is secreted from the Purkinje cells, and acts as a mitogen for cerebellar granule precursor (CGP) cells. Mutations leading to hyperactive Shh signaling have been associated with improper migration and differentiation of CGP cells and medulloblastoma formation. Still, the molecular targets contributing to Shh-mediated proliferation in these cells are poorly understood. Na,K-ATPase is a membrane protein that maintains intracellular ion homeostasis, and is responsible for generating ion gradients across cell membranes. Consisting of a catalytic alpha subunit and a beta subunit, the enzyme not only pumps sodium ions out and potassium ions into the cell using ATP but also functions a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and expression have been reported in various cancers and may contribute to tumor development and progression. We now show that the Na,K-ATPase beta1-subunit is drastically reduced in medulloblastoma tumors of mice with aberrant activation of Shh signaling. In addition, Shh activation prevented the upregulation of the beta1-subunit in primary cultures of normal cerebellar granule cells and the polycomb transcription factor Bmi1 that is induced upon activation of Shh signaling repressed beta1-subunit levels. Furthermore, shRNA-mediated knockdown of the beta1-subunit increased cell proliferation and tumorigenicity of medulloblastoma cells. Nevertheless, cardiac glycosides that inhibit the pump function of Na,K-ATPase inhibited EGF-induced signaling and cell motility suggesting that Na,K-ATPase alpha and beta subunit may have dual functions in CGP cells and medulloblastoma. Citation Format: Zhiqin Li, Alisa Litan, Seung Joon Lee, Bruce Graves, Sonali P. Barwe, Sigrid A. Langhans. Distinct roles of Na,K-ATPase function and expression in medulloblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 707. doi:10.1158/1538-7445.AM2015-707

Dentate Gyrus Status and Seizure Outcome: Does a Relationship Exist or Not?

In the last years an increasing interest in dentate gyrus (DG) abnormalities has emerged. Several studies investigated morphologic features of granular cell pathology (GCP) [1-3] and neurogenesis in the human DG, considering that aberrant growth of neural stem cells could be involved in different neurologic and psychiatric diseases. In particular, in 2009 [1], a classification system has been elaborated for GCP, recognizing three different histologic patterns: no-GCP (normal granule cell layer), GCP type 1 (substantial granule cell loss) and GCP type 2 (various architectural abnormalities in granule cell layer, mainly granule cell dispersion (GCD)). Since then, some efforts to investigate a possible association between the postsurgical seizure outcome and GCP have been made [2-4] but no unambiguous results have been reported, and attempts to clarify this issue could be useful. Some of these papers [2, 3] provided evidence that GCD does not appear to have any influence on outcome following surgery. Thus, they demonstrated that a statistically significant correlation between seizure outcome and GCD, that is a subtype of GCP type 2, has not been observed. On the contrary, in those studies in which the overall subtypes of DG abnormalities (GCP type 1 and GCP type 2) have been considered [1, 4] in comparison to cases with normal granule cell layer, the authors observed that “the association between favorable postsurgical outcome and granule cell pathology (Type 1 or Type 2) was statistically significant (P = 0.028)” [1]. On the other hand, if we want to understand the role of DG in temporal lobe epilepsy, we cannot ignore GCP type 1 (characterized by significant granule cell loss), considering the significant correlation between granule cell loss in the DG and the same patient’s regenerative capacity in vitro, as well as the same patient’s ability to store and recall new memories. These considerations are not meant to be a criticism of indepth studies, but an overly restrictive approach, distinguishing, for example, GCD from bilamination of granule cell layer, could remain a matter of debate among neuropathologists, rather than offering a new useful tool to neurologists or neurosurgeons. In keeping with the histologic demonstration of an abnormal granule cell layer, the status of DG should no longer be considered as an accessory morphologic finding without significance in predicting epileptogenic outcome. In conclusion, further investigations are needed, but the observations already available argue significantly for the association between favorable postsurgical outcome and granule cell pathology (type 1 and type 2). Therefore, the status of DG could help clinicians in predicting the postoperative outcome and planning a better follow-up schedule.

Effect of Sonic hedgehog activation on CXCR7 expression and CXCR4 signaling in medulloblastoma.

e13537 Background: Stratification of pediatric medullolastoma into molecular subtypes elevates hopes that subtype-specific therapy can be more efficacious and less toxic. To realize this goal, the molecular targets that distinguish each subtype must be defined. We found that Sonic hedgehog (Shh)-subtype medulloblastoma can be further divided by CXCR4 expression and that CXCR4 inhibition has potent anti-tumor effects. Here we present data that Shh regulates CXCR4 activity through CXCR7, whose differential expression between normal granule cells and medulloblastoma cells makes its function an ideal therapeutic target. Methods: Single-cell suspensions were plated with or without Shh or cyclopamine followed by treatment with CXCL12. SmoA1 brain tumors were dissociated into single cells and transplanted into nude mice and treated with vehicle, AMD3100, GDC-0449, or both. Tumors were measured and harvested at the conclusion of treatment. Results: Using primary cultures of Shh-driven medulloblastoma we found that Shh activation enhances CXCR4 surface localization and promotes CXCL12-induced signaling, similar to effects of Shh on normal granule cells. We evaluated expression of CXCR4 internalization regulators and found that Shh activation altered the levels of GRKs, arrestins and CXCR7. Among these, CXCR7 expression exhibited the greatest differential response to Shh pathway activation between normal cells and medulloblastoma: CXCR7 expression was highly expressed in normal cells despite Shh effects, but almost completely absent in tumor cells. Conclusions: We conclude that activation of the Shh pathway in medulloblastomas promotes CXCR4 surface localization through inhibition of internalization mediators. This cross-talk is also evident in developing GPCs but with one notable difference: CXCR7 expression (high in normal cells, absent in tumor cells). This distinction suggests that CXCR7 antagonists, which are moving towards clinical trials, may be detrimental to normal cerebellar development and inactive against medulloblastoma. We propose that restoring CXCR7 activity would have negligible effects on granule cells and potently block CXCR4 function in medulloblastoma.

Silencing the Majority of Cerebellar Granule Cells Uncovers Their Essential Role in Motor Learning and Consolidation

Cerebellar granule cells (GCs) account for more than half of all neurons in the CNS of vertebrates. Theoretical work has suggested that the abundance of GCs is advantageous for sparse coding during memory formation. Here, we minimized the output of the majority of GCs by selectively eliminating their CaV2.1 (P/Q-type) Ca(2+) channels, which mediate the bulk of their neurotransmitter release. This resulted in reduced GC output to Purkinje cells (PCs) and stellate cells (SCs) as well as in impaired long-term plasticity at GC-PC synapses. As a consequence modulation amplitude and regularity of simple spike (SS) output were affected. Surprisingly, the overall motor performance was intact, whereas demanding motor learning and memory consolidation tasks were compromised. Our findings indicate that a minority of functionally intact GCs is sufficient for the maintenance of basic motor performance, whereas acquisition and stabilization of sophisticated memories require higher numbers of normal GCs controlling PC firing.

Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory

Deep brain stimulation (DBS) is an established therapeutic modality for the treatment of movement disorders and an emerging therapeutic approach for the treatment of disorders of mood and thought. For example, recently we have shown that DBS of the fornix may ameliorate cognitive decline associated with dementia. However, like other applications of DBS, the mechanisms mediating these clinical effects are unknown. As DBS modulates neurophysiological activity in targeted brain regions, DBS might influence cognitive function via activity-dependent regulation of hippocampal neurogenesis. Using stimulation parameters analogous to clinical high-frequency DBS, here we addressed this question in mice. We found that acute stimulation of the entorhinal cortex (EC) transiently promoted proliferation in the dentate gyrus (DG). Cells generated as a consequence of stimulation differentiated into neurons, survived for at least several weeks, and acquired normal dentate granule cell (DGC) morphology. Importantly, stimulation-induced promotion of neurogenesis was limited to the DG and not associated with changes in apoptotic cell death. Using immunohistochemical approaches, we found that, once sufficiently mature, these stimulation-induced neurons integrated into hippocampal circuits supporting water-maze memory. Finally, formation of water-maze memory was facilitated 6 weeks (but not 1 week) after bilateral stimulation of the EC. The delay-dependent nature of these effects matches the maturation-dependent integration of adult-generated DGCs into dentate circuits supporting water-maze memory. Furthermore, because the beneficial effects of EC stimulation were prevented by blocking neurogenesis, this suggests a causal relationship between stimulation-induced promotion of adult neurogenesis and enhanced spatial memory.

Morphometry of hilar ectopic granule cells in the rat

Granule cell (GC) neurogenesis in the dentate gyrus (DG) does not always proceed normally. After severe seizures (e.g., status epilepticus [SE]) and some other conditions, newborn GCs appear in the hilus. Hilar ectopic GCs (EGCs) can potentially provide insight into the effects of abnormal location and seizures on GC development. Additionally, hilar EGCs that develop after SE may contribute to epileptogenesis and cognitive impairments that follow SE. Thus, it is critical to understand how EGCs differ from normal GCs. Relatively little morphometric information is available on EGCs, especially those restricted to the hilus. This study quantitatively analyzed the structural morphology of hilar EGCs from adult male rats several months after pilocarpine-induced SE, when they are considered to have chronic epilepsy. Hilar EGCs were physiologically identified in slices, intracellularly labeled, processed for light microscopic reconstruction, and compared to GC layer GCs, from both the same post-SE tissue and the NeuroMorpho database (normal GCs). Consistently, hilar EGC and GC layer GCs had similar dendritic lengths and field sizes, and identifiable apical dendrites. However, hilar EGC dendrites were topologically more complex, with more branch points and tortuous dendritic paths. Three-dimensional analysis revealed that, remarkably, hilar EGC dendrites often extended along the longitudinal DG axis, suggesting increased capacity for septotemporal integration. Axonal reconstruction demonstrated that hilar EGCs contributed to mossy fiber sprouting. This combination of preserved and aberrant morphological features, potentially supporting convergent afferent input to EGCs and broad, divergent efferent output, could help explain why the hilar EGC population could impair DG function.

Towards a clinico-pathological classification of granule cell dispersion in human mesial temporal lobe epilepsies

The dentate gyrus (DG) plays a pivotal role in the functional and anatomical organization of the hippocampus and is involved in learning and memory formation. However, the impact of structural DG abnormalities, i.e., granule cell dispersion (GCD), for hippocampal seizure susceptibility and its association with distinct lesion patterns in epileptic disorders, such as mesial temporal sclerosis (MTS) remains enigmatic and a large spectrum of pathological changes has been recognized. Here, we propose a clinico-pathological classification of DG pathology based on the examination of 96 surgically resected hippocampal specimens obtained from patients with chronic temporal lobe epilepsy (TLE). We observed three different histological patterns. (1) A normal granule cell layer was identified in 11 patients (no-GCP; 18.7%). (2) Substantial granule cell loss was evident in 36 patients (referred to as granule cell pathology (GCP) Type 1; 37.5%). (3) Architectural abnormalities were observed in 49 specimens, including one or more of the following features: granule cell dispersion, ectopic neurons or clusters of neurons in the molecular layer, or bi-lamination (GCP Type 2; 51%). Cell loss was always encountered in this latter cohort. Seventy-eight patients of our present series suffered from MTS (81.3%). Intriguingly, all MTS patients displayed a compromised DG, 31 (40%) with significant cell loss (Type 1) and 47 (60%) with GCD (Type 2). In 18 patients without MTS (18.7%), seven displayed focally restricted DG abnormalities, either cell loss (n = 5) or GCD (n = 2). Clinical histories revealed a significant association between DG pathology patterns and higher age at epilepsy surgery (p = 0.008), longer epilepsy duration (p = 0.004), but also with learning dysfunction (p < 0.05). There was no correlation with the extent of pyramidal cell loss in adjacent hippocampal segments nor with postsurgical seizure relief. The association with long-term seizure histories and cognitive dysfunction is remarkable and may point to a compromised regenerative capacity of the DG in this cohort of TLE patients.

Reduced excitability in the dentate gyrus network of βIV‐spectrin mutant mice in vivo

The submembrane cytoskeletal meshwork of the axon contains the scaffolding protein betaIV-spectrin. It provides mechanical support for the axon and anchors membrane proteins. Quivering (qv(3j)) mice lack functional betaIV-spectrin and have reduced voltage-gated sodium channel (VGSC) immunoreactivity at the axon initial segment and nodes of Ranvier. Because VGSCs are critically involved in action potential generation and conduction, we hypothesized that qv(3j) mice should also show functional deficits at the network level. To test this hypothesis, we investigated granule cell function in the dentate gyrus of anesthetized qv(3j) mice after electrical stimulation of the perforant path in vivo. This revealed an impaired input-output relationship between stimulus intensity and granule cell population spikes and an enhanced paired-pulse inhibition of population spikes, indicating a reduced ability of granule cells to generate action potentials and decreased network excitability. In contrast, the input-output curve for evoked field excitatory postsynaptic potentials (fEPSPs) and paired-pulse facilitation of fEPSPs were unchanged, suggesting normal excitatory synaptic transmission at perforant path-granule cell synapses in qv(3j) mutants. To corroborate our findings, we analyzed the influence of VGSC density reduction on dentate network activity using an established computational model of the dentate gyrus network. This in silico approach confirmed that the loss of VGSCs is sufficient to explain the electrophysiological changes observed in qv(3j) mice. Taken together, our findings demonstrate that betaIV-spectrin is required for normal granule cell firing and for physiological levels of network excitability in the mouse dentate gyrus in vivo.

Computational modeling of reduced excitability in the dentate gyrus of betaIV-spectrin mutant mice

Event Abstract Back to Event Computational modeling of reduced excitability in the dentate gyrus of betaIV-spectrin mutant mice Peter Jedlicka1*, Raphael Winkels1, Felix K. Weise1, Christian Schultz2, Thomas Deller1 and Stephan W. Schwarzacher1 1 Goethe University, NeuroScience Center, Clinical Neuroanatomy, Germany 2 Justus Liebig University, Institute of Anatomy and Cell Biology, Germany The submembrane cytoskeletal meshwork of the axon contains the scaffolding protein betaIV-spectrin. It provides mechanical support for the axon and anchors membrane proteins. Quivering (qv3j) mice lack functional betaIV-spectrin and have reduced voltage-gated sodium channel (VGSC) immunoreactivity at the axon initial segment and nodes of Ranvier. Because VGSCs are critically involved in action potential generation and conduction, we hypothesized that qv3j mice should also show functional deficits at the network level. To test this hypothesis, we investigated granule cell function in the dentate gyrus of anesthetized qv3j mice after electrical stimulation of the perforant path in vivo. This revealed an impaired input-output (IO) relationship between stimulus intensity and granule cell population spikes and an enhanced paired-pulse inhibition (PPI) of population spikes, indicating a reduced ability of granule cells to generate action potentials and decreased network excitability. In contrast, the IO curve for evoked field excitatory postsynaptic potentials (fEPSPs) and paired-pulse facilitation of fEPSPs were unchanged, suggesting normal excitatory synaptic transmission at perforant path-granule cell synapses in qv3j mutants.To better understand the influence of betaIVspectrin and VGSC density changes on the dentate gyrus network activity, we employed computational modeling approach. We used a recently developed and highly detailed computational model of the dentate gyrus network (Santhakumar et al., J Neurophysiol 93:437–453, 2005). The network model is based on realistic morphological and electrophysiological data and consists of perforant path inputs and connections of granule, mossy, basket and hilar cells. The role of VGSCs in network excitability was analyzed by systematically varying their densities in axosomatic compartments. This in silico approach confirmed that the loss of VGSCs is sufficient to explain the electrophysiological changes observed in qv3j mice. Computer simulations of the IO and PPI test indicated that in the dentate circuit with altered VGSCs, network excitability decreases owing to impaired spike-generator properties of granule cells and subsequent relative increase of GABAergic inhibitory control over granule cell firing.Taken together, our in vivo and in silico data demonstrate that the destabilization of VGSC clustering in qv3j mice leads to a reduced spike-generating ability of granule cells and considerably decreased network excitability in the dentate circuit. This provides the first evidence that betaIV-spectrin is required for normal granule cell firing and for physiological levels of network excitability in the mouse dentate gyrus in vivo. Conference: Bernstein Conference on Computational Neuroscience, Frankfurt am Main, Germany, 30 Sep - 2 Oct, 2009. Presentation Type: Poster Presentation Topic: Information processing in neurons and networks Citation: Jedlicka P, Winkels R, Weise FK, Schultz C, Deller T and Schwarzacher SW (2009). Computational modeling of reduced excitability in the dentate gyrus of betaIV-spectrin mutant mice. Front. Comput. Neurosci. Conference Abstract: Bernstein Conference on Computational Neuroscience. doi: 10.3389/conf.neuro.10.2009.14.054 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Aug 2009; Published Online: 26 Aug 2009. * Correspondence: Peter Jedlicka, Goethe University, NeuroScience Center, Clinical Neuroanatomy, Frankfurt, Germany, jedlicka@em.uni-frankfurt.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Peter Jedlicka Raphael Winkels Felix K Weise Christian Schultz Thomas Deller Stephan W Schwarzacher Google Peter Jedlicka Raphael Winkels Felix K Weise Christian Schultz Thomas Deller Stephan W Schwarzacher Google Scholar Peter Jedlicka Raphael Winkels Felix K Weise Christian Schultz Thomas Deller Stephan W Schwarzacher PubMed Peter Jedlicka Raphael Winkels Felix K Weise Christian Schultz Thomas Deller Stephan W Schwarzacher Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Blockade of Estrogen Receptor Signaling Inhibits Growth and Migration of Medulloblastoma

Medulloblastoma (MD) is the most common malignant brain tumor in children. These invasive neuroectodermal tumors arise from cerebellar granule cell-like precursors. In the developing cerebellum, estrogen influences growth and viability of granule cell precursors that transiently express elevated levels estrogen receptor-beta (ERbeta) during differentiation. Immunoanalysis revealed that ERbeta was expressed in the maturing human cerebellum, in all 22 primary MD tumors analyzed, and in two MD-derived cell lines (D283Med and Daoy). Very low levels of ERalpha-like proteins were detected in each cell line and 41% of tumor samples. Physiological concentrations of the 17beta-estradiol- or the ERbeta-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile diarylpropionitrile dose-dependently increased MD growth and cellular migration. In contrast, the ERalpha-selective agonist (4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol did not influence MD growth. Similar to previous studies in normal cerebellar granule cell precursors, these studies demonstrate that the physiological actions of estrogens in MD are mediated by ERbeta. Preclinical studies assessing the therapeutic efficacy of antiestrogen chemotherapeutics for treating human MD were performed. It was found that pharmacological inhibition of ER-mediated signaling with the ER antagonist drug Faslodex (ICI182,780) blocked all estrogen-mediated effects in both cell culture and xenograft models of human MD. These studies have revealed that functional ERbeta expression is a fundamental aspect of MD biology and has defined antiestrogen therapy as a potentially efficacious clinical approach to improve the long-term outcomes for MD patients.

Targeted calretinin expression in granule cells of calretininnull mice restores normal cerebellar functions

Ca2 binding proteins such as calretinin, characterized by the presence of EF-hand motifs that bind Ca2+ ions, are involved in the shaping of intraneuronal Ca2+ fluxes. In the cerebellar cortex, information processing tightly relies on variations in intracellular Ca2+ concentration in Purkinje and granule cells. Calretinin-deficient (Cr-/-) mice present motor discoordination, suggesting cellular and network cerebellar dysfunctions. To determine the cell specificity of these alterations, we constructed transgenic Cr-/- mice exhibiting a selective reexpression of calretinin in granule cells through the promoter function of the GABAA receptor alpha6 subunit gene. Normal granule cell excitability and wild-type Purkinje cell firing behavior in awake mice were restored while the emergence of high-frequency oscillations was abolished. Behavioral analysis of these calretinin-rescue mice revealed that normal motor coordination was restored as compared with Cr-/- mice. These results demonstrate that calretinin is required specifically in granule cells for correct computation in the cerebellar cortex and indicate that the finetuning of granule cell excitability through regulation of Ca2+ homeostasis plays a crucial role for information coding and storage in the cerebellum.

Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures

Granule cells in the dentate gyrus are born throughout life, and various stimuli can affect their development in the adult brain. Following seizures, for instance, neurogenesis increases greatly, and some new cells migrate to abnormal (ectopic) locations, such as the hilus. Previous electrophysiological studies of this population have shown that they have intrinsic properties that are similar to normal granule cells, but differ in other characteristics, consistent with abnormal integration into host circuitry. To characterize the response of ectopic hilar granule cells to perforant path stimulation, intracellular recordings were made in hippocampal slices from rats that had pilocarpine-induced status epilepticus and subsequent spontaneous recurrent seizures. Comparisons were made with granule cells located in the granule cell layer of both pilocarpine- and saline-treated animals. In addition, a few ectopic hilar granule cells were sampled from saline-treated rats. Remarkably, hilar granule cells displayed robust responses, even when their dendrites were not present within the molecular layer, where perforant path axons normally terminate. The evoked responses of hilar granule cells were similar in several ways to those of normally positioned granule cells, but there were some differences. For example, there was an unusually long latency to onset of responses evoked in many hilar granule cells, especially those without molecular layer dendrites. Presumably this is due to polysynaptic activation by the perforant path. These results indicate that synaptic reorganization after seizures can lead to robust activation of newly born hilar granule cells by the perforant path, even when their dendrites are not in the terminal field of the perforant path. Additionally, the fact that these cells can be found in normal tissue and develop similar synaptic responses, suggests that seizures, while not necessary for their formation, strongly promote their generation and the development of associated circuits, potentially contributing to a lowered seizure threshold.

Rb and p107 are required for normal cerebellar development and granule cell survival but not for Purkinje cell persistence.

The involvement of the retinoblastoma gene product (Rb) and its family members (p107 and p130) in cell cycle exit and terminal differentiation of neural precursor cells has been demonstrated in vitro. To investigate the roles of Rb and p107 in growth, differentiation and apoptosis in the developing and mature cerebellum, we selectively inactivated either Rb alone or in combination with p107 in cerebellar precursor cells or in Purkinje cells. In our mouse models, we show that (1) Rb is required for differentiation, cell cycle exit and survival of granule cell precursors; (2) p107 can not fully compensate for the loss of Rb function in granule cells; (3) Rb and p107 are not required for differentiation and survival of Purkinje cells during embryonic and early postnatal development; (4) Rb function in Purkinje cells is cell autonomous; and (5) loss of Rb deficient CNS precursor cells is mediated by p53-independent apoptosis.

Protoporphyrin IX production and its photodynamic effects on glioma cells, neuroblastoma cells and normal cerebellar granule cells in vitro with 5-aminolevulinic acid and its hexylester.

5-Aminolevulinic acid (ALA) has shown promising in photodynamic detection and therapy of brain tumor. However, the knowledge on selective accumulation of ALA-induced protoporphyrin IX (PpIX) in brain tumor tissue is still fragment. In the present study, the rat C6 glioma cells, human SK-N-SH neuroblastoma cells, and rat normal cerebellar granule cells (RCG) were used to investigate the PpIX production and photocytotoxicity in vitro. The C6 cells and SK-N-SH cells showed a similar kinetics of PpIX accumulation after exposure to ALA or ALA hexyl ester (ALA-H), with an initial increase up to 6-8 h and then saturated. In the case of RCG cells, the PpIX accumulation slowly increased until 12 h studied. However the cellular PpIX content was more than 10 times higher in the C6 and SK-N-SH cells than that in the normal RCG cells. The intracellular localization of PpIX measured by cofocal laser scanning microscopy was in same pattern in the C6 glioma cells and RCG normal cells with a diffuse cytoplasm distribution. The sensitivity of the C6 cells and SK-N-SH cells to ALA or ALA-H PDT was similar. It appears that ALA-H could achieve similar or slightly better results than ALA with respect to PpIX production and photoinactivation of cells, although a 10 times lower concentration of ALA-H was used.

Impaired postnatal development of hippocampal neurons and axon projections in the Emx2-/- mutants.

The specification and innervation of cerebral subregions is a complex layer-specific process, primed by region-specific transcription factor expression and axonal guidance cues. In Emx2-/- mice, the hippocampus fails to form a normal dentate gyrus as well as the normal layering of principal neurons in the hippocampus proper. Here, we analyzed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking Emx2 expression in vitro. As these mutants die perinatally, we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In late embryonic Emx2-/- cultivated hippocampi, both the perforant path as well as the distribution of calretinin-positive cells are affected. Traced entorhinal afferents in co-cultures with hippocampus from embryonic Emx2-/- mice terminate diffusely in the prospective dentate gyrus in contrast to the layer-specific termination of co-cultures from wild-type littermates. In addition, in brain slice cultures from null mutants the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. Our data indicate that Emx2 is essential for the terminal differentiation of granular cells and the correct formation of extrinsic and intrinsic hippocampal connections.