Cerebellar Neurons Research Articles

The βγ subunit of heterotrimeric G proteins interacts with actin filaments during neuronal differentiation

The βγ subunit of heterotrimeric G proteins, a key molecule in the G protein-coupled receptors (GPCRs) signaling pathway, has been shown to be an important factor in the modulation of the microtubule cytoskeleton. Gβγ has been shown to bind to tubulin, stimulate microtubule assembly, and promote neurite outgrowth of PC12 cells. In this study, we demonstrate that in addition to microtubules, Gβγ also interacts with actin filaments, and this interaction increases during NGF-induced neuronal differentiation of PC12 cells. We further demonstrate that the Gβγ-actin interaction occurs independently of microtubules as nocodazole, a well-known microtubule depolymerizing agent did not inhibit Gβγ-actin complex formation in PC12 cells. A confocal microscopic analysis of NGF-treated PC12 cells revealed that Gβγ co-localizes with both actin and microtubule cytoskeleton along neurites, with specific co-localization of Gβγ with actin at the distal end of these neuronal processes. Furthermore, we show that Gβγ interacts with the actin cytoskeleton in primary hippocampal and cerebellar rat neurons. Our results indicate that Gβγ serves as an important modulator of the neuronal cytoskeleton by interacting with both microtubules and actin filaments, and is likely to participate in various aspects of neuronal differentiation including axon and growth cone formation.

19734 L-type calcium channels in cerebellar neuron development and motor learning

ABSTRACT IMPACT: We aim to understand how LTCCs impact cerebellar function. OBJECTIVES/GOALS: L-type calcium channels (LTCCs) are important in activity-dependent neurite outgrowth, which comprises neurite initiation and elongation. We used cerebellar granule neurons (CGNs) to differentiate between LTCC effects on neurite initiation vs elongation. We also tested cerebellar function in mice lacking specific LTCCs with behavioral assays. METHODS/STUDY POPULATION: CGNs were cultured from 129SvEv mouse pups at P4-P6. Potassum chloride (50mM) was used to stimulate neuronal cultures for 24 hours. Isradipine (20nM) was added to culture medium to inhibit all LTCCs for 1 hour. For Cav1.2 deletion, we crossed Cav1.2 conditional knockout mice (Cav1.2-cKO) to Syn-Cre mice (for deletion in most neurons) or Atoh1-Cre mice (for deletion in CGNs). The Cav1.2-cKO line was maintained on a 129SvEv background. For constitutive Cav1.3 deletion, mice were maintained on a C57BL/6NTac. Behavioral tasks included open field, rotarod, and Erasmus Ladder. Data were analyzed with sexes combined and separated to assess for sex as a biological variable. Studies were analyzed by one-way ANOVA, two-way ANOVA, or generalized linear mixed model, where appropriate. RESULTS/ANTICIPATED RESULTS: CGNs exhibited an increase in neurite initiation but not elongation when stimulated with potassium chloride, consistent with previous reports of activity-dependent neurite outgrowth in this cell type. LTCC inhibition with isradipine blunted KCl-induced neurite initiation. We observed no change in the length of either primary or secondary neurites with isradipine treatment with or without KCl stimulation. In our behavioral experiments, we observed no deficits in open field, rotarod, or Erasmus Ladder when Cav1.2 was deleted in most neurons (driven by Syn-Cre expression) or in cerebellar granule neurons (driven by Atoh1-Cre expression). In contrast, loss of Cav1.3 was associated with impaired motor learning in the rotarod task without evidence of ataxia on Erasmus Ladder. DISCUSSION/SIGNIFICANCE OF FINDINGS: We show a specific role for LTCCs in activity-dependent CGN neurite initiation. While loss of Cav1.2 does not affect motor learning, loss of Cav1.3 does impair motor learning. Our results help expand our understanding of LTCC function in cerebellar neurodevelopment and function.

Olig3 regulates early cerebellar development.

The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition, and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones in mice. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that suppresses the Purkinje cell differentiation program. Our work thus defines Olig3 as a key factor in early cerebellar development.

In Search of Molecular Markers for Cerebellar Neurons.

The cerebellum, the region of the brain primarily responsible for motor coordination and balance, also contributes to non-motor functions, such as cognition, speech, and language comprehension. Maldevelopment and dysfunction of the cerebellum lead to cerebellar ataxia and may even be associated with autism, depression, and cognitive deficits. Hence, normal development of the cerebellum and its neuronal circuitry is critical for the cerebellum to function properly. Although nine major types of cerebellar neurons have been identified in the cerebellar cortex to date, the exact functions of each type are not fully understood due to a lack of cell-specific markers in neurons that renders cell-specific labeling and functional study by genetic manipulation unfeasible. The availability of cell-specific markers is thus vital for understanding the role of each neuronal type in the cerebellum and for elucidating the interactions between cell types within both the developing and mature cerebellum. This review discusses various technical approaches and recent progress in the search for cell-specific markers for cerebellar neurons.

Amelioration of the abnormal phenotype of a new L1 syndrome mouse mutation with L1 mimetics.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.

Origins, Development, and Compartmentation of the Granule Cells of the Cerebellum.

Granule cells (GCs) are the most numerous cell type in the cerebellum and indeed, in the brain: at least 99% of all cerebellar neurons are granule cells. In this review article, we first consider the formation of the upper rhombic lip, from which all granule cell precursors arise, and the way by which the upper rhombic lip generates the external granular layer, a secondary germinal epithelium that serves to amplify the upper rhombic lip precursors. Next, we review the mechanisms by which postmitotic granule cells are generated in the external granular layer and migrate radially to settle in the granular layer. In addition, we review the evidence that far from being a homogeneous population, granule cells come in multiple phenotypes with distinct topographical distributions and consider ways in which the heterogeneity of granule cells might arise during development.

Abnormal cerebellar function and tremor in a mouse model for non-manifesting partially penetrant dystonia type 6.

Loss-of-function mutations in the Thap1 gene cause partially penetrant dystonia type 6 (DYT6). Some non-manifesting DYT6 mutation carriers have tremor andabnormal cerebello-thalamo-cortical signalling. We show that Thap1 heterozygote mice have action tremor, a reduction in cerebellar neuron number, andabnormal electrophysiological signals in the remaining neurons. These results underscore the importance of Thap1 levels for cerebellar function. These results uncover how cerebellar abnormalities contribute to different dystonia-associated motor symptoms. Loss-of-function mutations in the Thanatos-associated domain-containing apoptosis-associated protein 1 (THAP1) gene cause partially penetrant autosomal dominant dystonia type 6 (DYT6). However, the neural abnormalities that promote the resultant motor dysfunctions remain elusive. Studies in humans show that some non-manifesting DYT6 carriers have altered cerebello-thalamo-cortical function with subtle but reproducible tremor. Here, we uncover that Thap1 heterozygote mice have action tremor that rises above normal baseline values even though they do not exhibit overt dystonia-like twisting behaviour. At the neural circuit level, we show using in vivo recordings in awake Thap1+/- mice that Purkinje cells have abnormal firing patterns and that cerebellar nuclei neurons, which connect the cerebellum to the thalamus, fire at a lower frequency. Although the Thap1+/- mice have fewer Purkinje cells and cerebellar nuclei neurons, the number of long-range excitatory outflow projection neurons is unaltered. The preservation of interregional connectivity suggests that abnormal neural function rather than neuron loss instigates the network dysfunction and the tremor in Thap1+/- mice. Accordingly, we report an inverse correlation between the average firing rate of cerebellar nuclei neurons and tremor power. Our data show that cerebellar circuitry is vulnerable to Thap1 mutations and that cerebellar dysfunction may be a primary cause of tremor in non-manifesting DYT6 carriers and a trigger for the abnormal postures in manifesting patients.

Subchronic exposure to acrylamide caused behaviour disorders and related pathological and molecular changes in rat cerebellum

Acrylamide (ACR) is a neurotoxin with moderate acute toxicity. Significant level of ACR exists in diet and drinking water. Occupational exposure causes motor function impairment, but the underlying mechanisms remain poorly defined. This study aims to explore whether microtubule-associated protein tau phosphorylation, excessive activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway and BDNF decline are involved in cerebellar neuron lesions and motor dysfunction after subchronic ACR exposure. The present results displayed that ACR caused gait abnormality and hind foot splay in rats. The HE and Nissl staining results revealed that ACR exposure aggravated cerebellar neuron lesions especially in purkinje cell layer. ACR markedly increased tau phosphorylation at Ser262 and Ser396/404 and inhibited the level of phosphorylation of glycogen synthase kinase 3β (P-GSK3β) at Ser9. The PERK-eukaryotic initiation factor-2α (eIF2α)-activating transcription factor 4 (ATF4) pathway was activated to promote CHOP expression and then to accelerate neuron lesions. Furthermore, ACR significantly decreased P-CREB at Ser133 and BDNF expression, which might be related to the inhibition of upstream signals from extracellular signal-related kinase (ERK) and protein kinase B (Akt). This work helps to elucidate the underlying mechanisms of ACR-induced neurotoxicity and present a potential target for prevention against the neurotoxicity.

Post-symptomatic Delivery of Brain-Derived Neurotrophic Factor (BDNF) Ameliorates Spinocerebellar Ataxia Type 1 (SCA1) Pathogenesis

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an abnormal expansion of CAG repeats in the Ataxin1 (ATXN1) gene. SCA1 is characterized by motor deficits, cerebellar neurodegeneration, and gliosis and gene expression changes. Expression of brain-derived neurotrophic factor (BDNF), growth factor important for the survival and function of cerebellar neurons, is decreased in ATXN1[82Q] mice, the Purkinje neuron specific transgenic mouse model of SCA1. As this decrease in BDNF expression may contribute to cerebellar neurodegeneration, we tested whether delivery of extrinsic human BDNF via osmotic ALZET pumps has a beneficial effect on disease severity in this mouse model of SCA1. Additionally, to test the effects of BDNF on established and progressing cerebellar pathogenesis and motor deficits, we delivered BDNF post-symptomatically. We have found that post-symptomatic delivery of extrinsic BDNF ameliorated motor deficits and cerebellar pathology (i.e., dendritic atrophy of Purkinje cells, and astrogliosis) indicating therapeutic potential of BDNF even after the onset of symptoms in SCA1. However, BDNF did not alter Purkinje cell gene expression changes indicating that certain aspects of disease pathogenesis cannot be ameliorated/slowed down with BDNF and that combinational therapies may be needed.

Cerebellar projections to the macaque midbrain tegmentum: Possible near response connections.

Since most gaze shifts are to targets that lie at a different distance from the viewer than the current target, gaze changes commonly require a change in the angle between the eyes. As part of this response, lens curvature must also be adjusted with respect to target distance by the ciliary muscle. It has been suggested that projections by the cerebellar fastigial and posterior interposed nuclei to the supraoculomotor area (SOA), which lies immediately dorsal to the oculomotor nucleus and contains near response neurons, support this behavior. However, the SOA also contains motoneurons that supply multiply innervated muscle fibers (MIFs) and the dendrites of levator palpebrae superioris motoneurons. To better determine the targets of the fastigial nucleus in the SOA, we placed an anterograde tracer into this cerebellar nucleus in Macaca fascicularis monkeys and a retrograde tracer into their contralateral medial rectus, superior rectus, and levator palpebrae muscles. We only observed close associations between anterogradely labeled boutons and the dendrites of medial rectus MIF and levator palpebrae motoneurons. However, relatively few of these associations were present, suggesting these are not the main cerebellar targets. In contrast, labeled boutons in SOA, and in the adjacent central mesencephalic reticular formation (cMRF), densely innervated a subpopulation of neurons. Based on their location, these cells may represent premotor near response neurons that supply medial rectus and preganglionic Edinger-Westphal motoneurons. We also identified lens accommodation-related cerebellar afferent neurons via retrograde trans-synaptic transport of the N2c rabies virus from the ciliary muscle. They were found bilaterally in the fastigial and posterior interposed nuclei, in a distribution which mirrored that of neurons retrogradely labeled from the SOA and cMRF. Our results suggest these cerebellar neurons coordinate elements of the near response during symmetric vergence and disjunctive saccades by targeting cMRF and SOA premotor neurons.

小脳神経細胞におけるシャペロン介在性オートファジーの活性低下は運動障害と神経変性を誘導する

小脳神経細胞におけるシャペロン介在性オートファジーの活性低下は運動障害と神経変性を誘導する

Возможные механизмы взаимозависимого участия базальных ганглиев и мозжечка в функционировании двигательных и сенсорных систем

The article describes a mechanism of functioning of the neural network that includes the neocortex, basal ganglia, cerebellum, and thalamus. This mechanism explains the role of the cerebellum in the performance of tasks previously associated with the basal ganglia and neocortex. Rules for long-term modifications of the efficacy of synaptic inputs to cerebellar neurons and spiny cells of the striatum, an input nucleus of the basal ganglia, have been formulated. These rules differ from those generally accepted. It follows from the proposed modulation rules that the activation of D1 receptors promotes the induction of LTP in synapses formed by mossy fibers carrying sensory information to cerebellar granule cells as well as neurons of the deep cerebellar nuclei (providing that they are inhibited by Purkinje cells). This results in the enhanced disynaptic (through the thalamic nuclei) excitation of the cerebellum target cells in the neocortex, striatum and dopaminergic structures. An increase in thalamo-striatal excitation, as well as the activation of D1 receptors on striatonigral cells and D2 receptors on striatopallidal cells, promotes the induction of LTP and LTD of the efficacy of cortical inputs to these neurons, respectively. As a consequence, synergistic disinhibition via the direct and indirect pathways through the basal ganglia is facilitated for those thalamic cells and associated neocortical neurons that were initially strongly activated by sensory stimuli. Simultaneously, the inhibition of the activity of remaining thalamic and neocortical neurons by the basal ganglia must be increased. The mechanism of functioning is similar for diverse the neuronal networks, each of which includes topographically connected areas of the neocortex, thalamus and basal ganglia. Since cerebellar granule cells as well as dopaminergic neurons respond to a conditioned sensory signal and reinforcing stimulus, it follows from the proposed mechanism that the cerebellum, together with the basal ganglia, may form the patterns of neuronal activity in neocortical areas determining sensory perception and choice of action.

Effects of Motor Learning on Motor Function and Synaptic Plasticity of Cerebellar Injuries

The purpose of this study was to investigate the effects of motor learning on motor function and synaptic plasticity in rats with cerebellar injuries caused by harmaline. The test subjects were divided into three groups, with 15 rats assigned to each group. Group I was a normal control group (induced by saline); Group II was a experimental control group (cerebellar injuries caused by harmaline); Group III was the group that received motor learning after being cerebellar injured by harmaline. In the motor function test, the outcome of group II was significantly lower than that of group III (p<0.001). In the histological findings, the dendrites and nuclei of the cerebellar neurons of the experimental groups were severely damaged. While group III showed a decrease in degeneration of the cerebellar neurons in comparison. The immunohistochemistric response of synaptophysin in the cerebellar cortex was lower in the experimental groups than group I. However group III’s expression of synaptophysin was higher than group II. In electron microscopy findings, the experimental groups showed degeneration of the Purkinje cell. These result suggest that improved motor function by motor learning, after harmaline induced cerebellar injuries, is associated with dynamically altered expression of synaptophysin in the cerebellar cortex and that it is related with synaptic plasticity.

EDR Peptide: Possible Mechanism of Gene Expression and Protein Synthesis Regulation Involved in the Pathogenesis of Alzheimer's Disease.

The EDR peptide (Glu-Asp-Arg) has been previously established to possess neuroprotective properties. It activates gene expression and synthesis of proteins, involved in maintaining the neuronal functional activity, and reduces the intensity of their apoptosis in in vitro and in vivo studies. The EDR peptide interferes with the elimination of dendritic spines in neuronal cultures obtained from mice with Alzheimer’s (AD) and Huntington’s diseases. The tripeptide promotes the activation of the antioxidant enzyme synthesis in the culture of cerebellum neurons in rats. The EDR peptide normalizes behavioral responses in animal studies and improves memory issues in elderly patients. The purpose of this review is to analyze the molecular and genetics aspects of the EDR peptide effect on gene expression and synthesis of proteins involved in the pathogenesis of AD. The EDR peptide is assumed to enter cells and bind to histone proteins and/or ribonucleic acids. Thus, the EDR peptide can change the activity of the MAPK/ERK signaling pathway, the synthesis of proapoptotic proteins (caspase-3, p53), proteins of the antioxidant system (SOD2, GPX1), transcription factors PPARA, PPARG, serotonin, calmodulin. The abovementioned signaling pathway and proteins are the components of pathogenesis in AD. The EDR peptide can be AD.

A bionic cerebellar motion control model and its application in arm control

How to realize the control of limb movement and apply it to intelligent robot systems at the level of cerebellar cortical neurons is a hot topic in the fields of artificial intelligence and rehabilitation medicine. At present, the cerebellar model usually used is only for the purpose of controlling the effect, borrowing from the functional mode of the cerebellum, but it ignores the structural characteristics of the cerebellum. In fact, in addition to being used for controlling purposes, the cerebellar model should also have the interpretability of the control process and be able to analyze the consequences of cerebellar lesions. Therefore, it is necessary to establish a bionic cerebellar model which could better express the characteristics of the cerebellum. In this paper, the process that the cerebellum processes external input information and then generates control instructions at the neuron level was explored. By functionally segmenting the cerebellum into homogeneous structures, a novel bionic cerebellar motion control model incorporating all major cell types and connections was established. Simulation experiments and force feedback device control experiments show that the bionic cerebellar motion control model can achieve better control effect than the currently widely used cerebellar model articulation controller, which verifies the effectiveness of the bionic cerebellar motion control model. It has laid the foundation for real brain-like artificial intelligence control.

Treatment‐responsive primary autoimmune cerebellar ataxia in a patient with IgG and IgM anticerebellar antibodies

Primary autoimmune cerebellar ataxia (PACA) in the absence of another triggering disease represents an emerging category of neurological illness. We report such a case whose ataxia was markedly responsive to plasma exchange. We analyzed patient serum for the presence of IgM or IgG anticerebellar neuronal antibodies. Case presentation: rat cerebellar slice cultures incubated with patient sera were studied for IgG and IgM antibody uptake, intracellular binding, and neuronal death. Patient serum was evaluated for anti-myelin associated glycoprotein (anti-MAG) and associated anti-glycolipid antibodies. Antibodies were taken up by viable cerebellar neurons and bound to intracellular antigens. Uptake and predominantly nuclear binding of IgG were seen in granule cells whereas cytoplasmic binding of IgM was observed predominantly in Purkinje cells. Intracellular antibody accumulation was not accompanied by neuronal death, consistent with the patient's excellent clinical response to plasma exchange. Anti-MAG or other associated anti-glycolipid antibodies were not detected. PACA may be associated with both IgG and IgM antibodies reactive with cerebellar neuronal antigens. Our patient's response to plasma exchange supports a role for antineuronal antibodies in disease pathogenesis and emphasizes the need for rapid diagnosis and treatment.

Evolutionary neurotoxicology of manganese (Mn) and its implications for Mn‐induced mixed dementia

AbstractBackgroundManganese (Mn) is an essential element for all living systems, involved in several biological processes, but chronic and/or acute exposure for this metal can lead to adverse outcome pathways, especially neurotoxicity. In this manner; we hypothesized that if these alterations are conserved cross‐species, this must improve the understanding of the toxicological mechanisms of this metal. Thus, this study aimed to investigate the evolutionary neurotoxicology mechanisms of the manganese.MethodsDifferent biological models (Saccharomyces cerevisiae ‐ yeast, Danio rerio ‐ zebrafish, Mus musculus ‐ cerebellar granular neurons and Homo sapiens ‐ human neuroblastoma SH‐SY5Y cell line) were exposed for chmical species of Mn and studied through different bioanalytical approaches such as cell viability‐MTT‐assay; sensitivity‐gene‐screening assay; b‐galactosidase assay; ribosome profiles; translation protein efficiency assay; semi‐quantitative proteomics ‐ tandem mass tag; metallomics; transcriptomics (human gene expression microarray kit and qRT‐PCR); bioinformatics (gene ontology and prediction of protein‐protein interaction, which were conducted at the String database and the comparative toxicogenomic database respectively. The results were expressed as mean ± SEM of at least 3 experiments; LC50 and statistically significant differences were estimated by ANOVA (analysis of variance followed by Bonferroni’s tests), using the GraphPad Prism 4.0 Software Inc, San Diego, CA, USAResultWe revealed that chemical species of Mn2+ (0.001‐1.5 mM) disrupt metal homeostasis (K, Ca, Fe and Cu); protein metabolism, including translation, post‐translation, and protein degradation as well as associated pathways such as those involved in energetics, cell signaling, cell cycle and neurotransmitter metabolism, in a suite of organisms, from yeast to mammalian. Collectively these cross‐talking alteration of cell pathways potentially can explain with more precision the mechanism of neurotoxicity of this metal. Additionally, analysis in the comparative toxicogenomic database suggested that Mn‐induced neurotoxicity shares mechanism with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease; but sometimes involving different genes or proteins, which needs to be considered before drawing comparisons to humans.ConclusionThe evolutionary neurotoxicology of the manganese showed in this study represent a new paradigm for Mn‐induced neurotoxicity and neurodegeneration and open a new avenue to study the role of other essential trace elements in similar pathogenic processes.

Intrinsic plasticity of cerebellar stellate cells is mediated by NMDA receptor regulation of voltage-gated Na+ channels.

We show that NMDA receptors (NMDARs) elicit a long-term increase in the firing rates of inhibitory stellate cells of the cerebellum NMDARs induce intrinsic plasticity through a Ca2+ - and CaMKII-dependent pathway that drives shifts in the activation and inactivation properties of voltage-gated Na+ (Nav ) channels An identical Ca2+ - and CaMKII-dependent signalling pathway is triggered during whole-cell recording which lowers the action potential threshold by causing a hyperpolarizing shift in the gating properties of Nav channels. Our findings open the more general possibility that NMDAR-mediated intrinsic plasticity found in other cerebellar neurons may involve similar shifts in Nav channel gating. Memory storage in the mammalian brain is mediated not only by long-lasting changes in the efficacy of neurotransmitter receptors but also by long-term modifications to the activity of voltage-gated ion channels. Activity-dependent plasticity of voltage-gated ion channels, or intrinsic plasticity, is found throughout the brain in virtually all neuronal types, including principal cells and interneurons. Although intrinsic plasticity has been identified in neurons of the cerebellum, it has yet to be studied in inhibitory cerebellar stellate cells of the molecular layer which regulate activity outflow from the cerebellar cortex by feedforward inhibition onto Purkinje cells. The study of intrinsic plasticity in stellate cells has been particularly challenging as membrane patch breakthrough in electrophysiology experiments unintentionally triggers changes in spontaneous firing rates. Using cell-attached patch recordings to avoid disruption, we show that activation of extrasynaptic N-methyl-d-aspartate receptors (NMDARs) elicits a long-term increase in the firing properties of stellate cells by stimulating a rise in cytosolic Ca2+ and activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). An identical signalling pathway is triggered during whole-cell recording which lowers the action potential threshold by causing a hyperpolarizing shift in the gating properties of voltage-gated sodium (Nav ) channels. Together, our findings identify an unappreciated role of Nav channel-dependent intrinsic plasticity in cerebellar stellate cells which, in concert with non-canonical NMDAR signalling, provides the cerebellum with an unconventional mechanism to fine-tune motor behaviour.

PSA mimetic 5-nonyloxytryptamine protects cerebellar neurons against glutamate induced excitotoxicity: An in vitro perspective

PSA-NCAM is a molecule of therapeutic interest for its key role in promoting neuritogenesis and synaptic plasticity. The current study was aimed to investigate the neuroregenerative potential of 5-nonyloxytryptamine (5-NOT) as PSA mimetic compound against glutamate induced excitotoxicity. 2D and 3D cultures of cerebellar neurons challenged with glutamate were used to ascertain the effect of 5-NOT on neurite outgrowth, migration and expression of neuronal plasticity markers. Glutamate excitotoxicity is one of the major underlying pathological factor responsible for neurodegeneration in various neurological disorders such as trauma, stroke, ischemia, epilepsy and neurodegenerative diseases.5-NOT treatment was observed to promote axonal growth and defasiculation in glutamate challenged neurons as well as promoted the migration of cerebellar neurons in both wound scratched area and cerebellar explant cultures. Further, 5-NOT treatment upregulated the expression of synaptic plasticity and cell survival pathway proteins which showed reduced expression after glutamate induced excitotoxicity. Thus, this preliminary data reveals thatPSA-mimetic,5-NOT may prove to be a potential neuroprotective candidate for neurodegenerative diseases.

Effects of a human-based mixture of persistent organic pollutants on the in vivo exposed cerebellum and cerebellar neuronal cultures exposed in vitro

Exposure to persistent organic pollutants (POPs), encompassing chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) compounds is associated with adverse neurobehaviour in humans and animals, and is observed to cause adverse effects in nerve cell cultures. Most studies focus on single POPs, whereas studies on effects of complex mixtures are limited. We examined the effects of a mixture of 29 persistent compounds (Cl + Br + PFAA, named Total mixture), as well as 6 sub-mixtures on in vitro exposed rat cerebellar granule neurons (CGNs). Protein expression studies of cerebella from in vivo exposed mice offspring were also conducted. The selection of chemicals for the POP mixture was based on compounds being prominent in food, breast milk or blood from the Scandinavian human population. The Total mixture and sub-mixtures containing PFAAs caused greater toxicity in rat CGNs than the single or combined Cl/Br sub-mixtures, with significant impact on viability from 500x human blood levels. The potencies for these mixtures based on LC50 values were Br + PFAA mixture > Total mixture > Cl + PFAA mixture > PFAA mixture. These mixtures also accelerated induced lipid peroxidation. Protection by the competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) indicated involvement of the NMDA receptor in PFAA and Total mixture-, but not Cl mixture-induced toxicity. Gene-expression studies in rat CGNs using a sub-toxic and marginally toxic concentration ((0.4 nM-5.5 µM) 333x and (1 nM-8.2 µM) 500x human blood levels) of the mixtures, revealed differential expression of genes involved in apoptosis, oxidative stress, neurotransmission and cerebellar development, with more genes affected at the marginally toxic concentration. The two important neurodevelopmental markers Pax6 and Grin2b were downregulated at 500x human blood levels, accompanied by decreases in PAX6 and GluN2B protein levels, in cerebellum of offspring mice from mothers exposed to the Total mixture throughout pregnancy and lactation. In rat CGNs, the glutathione peroxidase gene Prdx6 and the regulatory transmembrane glycoprotein gene Sirpa were highly upregulated at both concentrations. In conclusion, our results support that early-life exposure to mixtures of POPs can cause adverse neurodevelopmental effects.