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Besides its role in the circadian rhythm, the pineal gland hormone melatonin (MLT) also possesses antiepileptogenic, antineoplastic, and cardioprotective properties, among others. The dosages necessary to elicit beneficial effects in these diseases often far surpass physiological concentrations. Although even high doses of MLT are considered to be largely harmless to humans, the possible side effects of pharmacological concentrations are so far not well investigated. In the present study, we report that pharmacological doses of MLT (3 mM) strongly altered the electrophysiological characteristics of cultured primary mouse cerebellar granule cells (CGCs). Using whole‐cell patch clamp and ratiometric Ca2+ imaging, we observed that pharmacological concentrations of MLT inhibited several types of voltage‐gated Na+, K+, and Ca2+ channels in CGCs independently of known MLT‐receptors, altering the character and pattern of elicited action potentials (APs) significantly, quickly and reversibly. Specifically, MLT reduced AP frequency, afterhyperpolarization, and rheobase, whereas AP amplitude and threshold potential remained unchanged. The altered biophysical profile of the cells could constitute a possible mechanism underlying the proposed beneficial effects of MLT in brain‐related disorders, such as epilepsy. On the other hand, it suggests potential adverse effects of pharmacological MLT concentrations on neurons, which should be considered when using MLT as a pharmacological compound. [ABSTRACT FROM AUTHOR]

Previous studies have shown the essential roles of O-GlcNAc transferase (Ogt) and O-GlcNAcylation in neuronal development, function and neurologic diseases. However, the function of Ogt and O-GlcNAcylation in the adult cerebellum has not been well elucidated. Here, we have found that cerebellum has the highest level of O-GlcNAcylation relative to cortex and hippo)campus of adult male mice. Specific deletion of Ogt in granule neuron precursors (GNPs) induces abnormal morphology and decreased size of the cerebellum in adult male Ogt deficient [conditional knock-out (cKO)] mice. Adult male cKO mice show the reduced density and aberrant distribution of cerebellar granule cells (CGCs), the disrupted arrangement of Bergman glia (BG) and Purkinje cells. In addition, adult male cKO mice exhibit aberrant synaptic connection, impaired motor coordina)tion, and learning and memory abilities. Mechanistically, we have identified G-protein subunit a12 (Ga12) is modified by Ogt-mediated O-GlcNAcylation. O-GlcNAcylation of Ga12 facilitates its binding to Rho guanine nucleotide exchange factor 12 (Arhgef12) and consequently activates RhoA/ROCK signaling. RhoA/ROCK pathway activator LPA can rescue the developmen)tal deficits of Ogt deficient CGCs. Therefore, our study has revealed the critical function and related mechanisms of Ogt and O-GlcNAcylation in the cerebellum of adult male mice. [ABSTRACT FROM AUTHOR]

Over the past decade, survival rates for extremely low gestational age neonates (ELGANs; <28 weeks gestation) has markedly improved. Unfortunately, a significant proportion of ELGANs will suffer from neurodevelopmental dysfunction. Cerebellar hemorrhagic injury (CHI) has been increasingly recognized in the ELGANs population and may contribute to neurologic dysfunction; however, the underlying mechanisms are poorly understood. To address this gap in knowledge, we developed a novel model of early isolated posterior fossa subarachnoid hemorrhage (SAH) in neonatal mice and investigated both acute and long-term-effects. Following SAH on postnatal day 6 (P6), we found significant decreased levels of proliferation with the external granular layer (EGL), thinning of the EGL, decreased Purkinje cell (PC) density, and increased Bergmann glial (BG) fiber crossings at P8. At P42, CHI resulted in decreased PC density, decreased molecular layer interneuron (MLI) density, and increased BG fiber crossings. Results from both Rotarod and inverted screen assays did not demonstrate significant effects on motor strength or learning at P35-38. Treatment with the anti-inflammatory drug Ketoprofen did not significantly alter our findings after CHI, suggesting that treatment of neuro-inflammation does not provide significant neuroprotection post CHI. Further studies are required to fully elucidate the mechanisms through which CHI disrupts cerebellar developmental programming in order to develop therapeutic strategies for neuroprotection in ELGANs. [ABSTRACT FROM AUTHOR]

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome. [ABSTRACT FROM AUTHOR]

Introduction: Tramadol abuse is increasing among male and female teenagers with a history of substance abuse and anxiety. Many adolescent addicts take tramadol as substitute for other narcotics. Tramadol uptake is associated with many side effects. Thyme has been presumed to have a neuroprotective effect against brain damage. Aim of the work: To assess the harmful effects of tramadol on the pre and postnatal development of the rat cerebellar granule cells and to evaluate the possible ameliorative effect of thyme if being administered with tramadol simultaneously. Material & Methods: Three sets of forty-eight mature female albino rats were randomly organized into three equal groups; control (G1), tramadol treated (G2) and tramadol+thyme treated (G3). After pregnancy, the pregnant rats of each group were categorized into a group designed to be sacrificed at the gestational ages 13th, 16th and 19th for the prenatal study. In the other pregnant rat group, their offspring were further subdivided according to their ages into 3 subgroups (newborn, 10th and 20th postnatal day) for postnatal evaluation. G1 was not given any treatment. Tramadol HCL (40 mg/Kg/day) dissolved in tab water was given orally to G2. Tramadol (40 mg/Kg/day) and thyme extract (500 mg/kg/day) were given orally to G3. A light, electron microscopic examination and morphometric analysis were performed in the study. Results: By light and electron microscopic examination, there were degenerative and apoptotic changes in the cerebellar cortex of G2. G3 showed improvement in histological changes. Morphometric results showed a decrease in the cerebellar cortex thickness between the G1 and G2 which was significant and an increase in the cerebellar cortex thickness G3 when compared with that of G2. Conclusion: The study assigns that tramadol administration induces adverse effects on the prenatal and postnatal development of the rat cerebellar cortex. It also highlights an improving role of thyme in tramadol-induced cerebellar cortex injury of the pre and postnatal development. [ABSTRACT FROM AUTHOR]

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABAA receptor populations in cerebellar granule cells, we took advantage of the two‐photon uncaging method as this technique allows to stimulate the cell locally in well‐identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF‐16, a 16 amino acids long peptide obtained from the amino‐terminal end of the AF protein. After the treatment with AF‐16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF‐16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABAA receptors, containing the α6 subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell. [ABSTRACT FROM AUTHOR]

The discovery by Altman and coworkers of adult-born microneurons in the olfactory bulb and dentate gyrus has triggered a long stream of studies and many attempts to harness adult neurogenesis, promote regeneration after injury, and contrast cognitive decline in the elderly. Likewise, the discovery of postnatal neurogenesis in the cerebellum has provided the framework for many subsequent molecular studies, including investigations of developmental processes and the assessment of GC progenitor (GCP) clonal expansion in the context of human disease. Here, I will briefly discuss some of the discoveries made in the field of cerebellar development over the years building upon the findings of Altman and his colleagues, touching upon signaling pathways that regulate granule cell neurogenesis and their involvement in developmental and neoplastic disorders of the cerebellum. [ABSTRACT FROM AUTHOR]

Store-operated calcium entry (SOCE) is a unique Ca2+ entry pathway that is specifically activated by the depletion of Ca2+ from the endoplasmic reticulum (ER). This process is ubiquitous in non-excitable cells; in contrast, the role of SOCE in neurons is still poorly understood because of the complexity of Ca2+ influx mechanisms existing in these cells. However, growing evidence indicates that SOCE plays an important role in the maintenance of intracellular Ca2+ homeostasis, neuronal signaling, and plasticity, and this emphasizes the necessity to include SOCE in mathematical models of the calcium dynamics. Here we extend our previously published models of the calcium dynamics by incorporating the data on Ca2+-induced calcium release and SOCE based on the experimental results obtained mostly on cerebellar slices. Our simulations have shown that SOCE in neurons is probably necessary to maintain the calcium levels in the cytosol and ER and may influence repetitive Ca2+ signals in the case of significant store depletion. Modeling of SOCE, however, remains problematic because of the difficulty of experimental isolation of this process. [ABSTRACT FROM AUTHOR]

Cerebellar granule cells (GCs) relay mossy fiber (MF) inputs to Purkinje cell dendrites via their axons, the parallel fibers (PFs), which are individually located at a given sublayer of the molecular layer (ML). Although a certain degree of heterogeneity among GCs has been recently reported, variability of GC responses to MF inputs has never been associated with their most notable structural variability, location of their projecting PFs in the ML. Here, we utilize an adeno-associated virus (AAV)-mediated labeling technique that enables us to categorize GCs according to the location of their PFs, and compare the Ca2+ responses to MF stimulations between three groups of GCs, consisting of either GCs having PFs at the deep (D-GCs), middle (M-GCs), or superficial (S-GCs) sublayer. Our structural analysis revealed that there was no correlation between position of GC soma in the GC layer and location of its PF in the ML, confirming that our AAV-mediated labeling was important to test the projection-dependent variability of the Ca2+ responses in GCs. We then found that the Ca2+ responses of D-GCs differed from those of M-GCs. Pharmacological experiments implied that the different Ca2+ responses were mainly attributable to varied distributions of GABAA receptors (GABAARs) at the synaptic and extrasynaptic regions of GC dendrites. In addition to GABAAR distributions, amounts of extrasynaptic NMDA receptors appear to be also varied, because Ca2+ responses were different between D-GCs and M-GCs when glutamate spillover was enhanced. Whereas the Ca2+ responses of S-GCs were mostly equivalent to those of D-GCs and M-GCs, the blockade of GABA uptake resulted in larger Ca2+ responses in S-GCs compared with D-GCs and M-GCs, implying existence of mechanisms leading to more excitability in S-GCs with increased GABA release. Thus, this study reveals MF stimulation-mediated non-uniform Ca2+ responses in the cerebellar GCs associated with the location of their PFs in the ML, and raises a possibility that combination of inherent functional variability of GCs and their specific axonal projection contributes to the information processing through the GCs. [ABSTRACT FROM AUTHOR]

Key points: A T258F mutation of the glycine receptor increases the receptor affinity to endogenous agonists, modifies single‐channel conductance and shapes response decay kinetics.Glycine receptors of cerebellar granule cells play their functional role not continuously, but when the granule cell layer starts receiving a high amount of excitatory inputs.Despite their relative scarcity, tonically active glycine receptors of cerebellar granule cells make a significant impact on action potential generation and inter‐neuronal crosstalk, and modulate synaptic plasticity in neural networks; extracellular glycine increases probability of postsynaptic response occurrence acting at NMDA receptors and decreases this probability acting at glycine receptors.Tonic conductance through glycine receptors of cerebellar granule cells is a yet undiscovered element of the biphasic mechanism that regulates processing of sensory inputs in the cerebellum.A T258F point mutation disrupts this biphasic mechanism, thus illustrating the possible role of the gain‐of‐function mutations of the glycine receptor in development of neural pathologies. Functional glycine receptors (GlyRs) have been repeatedly detected in cerebellar granule cells (CGCs), where they deliver exclusively tonic inhibitory signals. The functional role of this signalling, however, remains unclear. Apart from that, there is accumulating evidence of the important role of GlyRs in cerebellar structures in development of neural pathologies such as hyperekplexia, which can be triggered by GlyR gain‐of‐function mutations. In this research we initially tested functional properties of GlyRs, carrying the yet understudied T258F gain‐of‐function mutation, and found that this mutation makes significant modifications in GlyR response to endogenous agonists. Next, we clarified the role of tonic GlyR conductance in neuronal signalling generated by single CGCs and by neural networks in cell cultures and in living cerebellar tissue of C57Bl‐6J mice. We found that GlyRs of CGCs deliver a significant amount of tonic inhibition not continuously, but when the cerebellar granule layer starts receiving substantial excitatory input. Under these conditions tonically active GlyRs become a part of neural signalling machinery allowing generation of action potential (AP) bursts of limited length in response to sensory‐evoked signals. GlyRs of CGCs support a biphasic modulatory mechanism which enhances AP firing when excitatory input intensity is low, but suppresses it when excitatory input rises to a certain critical level. This enables one of the key functions of the CGC layer: formation of sensory representations and their translation into motor output. Finally, we have demonstrated that the T258F mutation in CGC GlyRs modifies single‐cell and neural network signalling, and breaks a biphasic modulation of the AP‐generating machinery. Key points: A T258F mutation of the glycine receptor increases the receptor affinity to endogenous agonists, modifies single‐channel conductance and shapes response decay kinetics.Glycine receptors of cerebellar granule cells play their functional role not continuously, but when the granule cell layer starts receiving a high amount of excitatory inputs.Despite their relative scarcity, tonically active glycine receptors of cerebellar granule cells make a significant impact on action potential generation and inter‐neuronal crosstalk, and modulate synaptic plasticity in neural networks; extracellular glycine increases probability of postsynaptic response occurrence acting at NMDA receptors and decreases this probability acting at glycine receptors.Tonic conductance through glycine receptors of cerebellar granule cells is a yet undiscovered element of the biphasic mechanism that regulates processing of sensory inputs in the cerebellum.A T258F point mutation disrupts this biphasic mechanism, thus illustrating the possible role of the gain‐of‐function mutations of the glycine receptor in development of neural pathologies. [ABSTRACT FROM AUTHOR]

A glutamate model of stroke was analyzed from the standpoint of the development of a typical pathological process that is thought to occur when major regulatory mechanisms are violated. The analysis made it possible to isolate the main mechanisms that underlie a transition from normal physiological processes to common pathological changes. This review considers a generalizing concept of how the pathological process develops. Following the concept, the typical pathological process is based on nonspecific distortion of cyclic regulatory processes and arises when reactive nitrogen species (RNS) and reactive oxygen species (ROS) increase simultaneously. Once RNS and ROS concentrations are beyond the regulatory capabilities of biochemical antioxidant systems, nitric oxide and superoxide anion radical cycles are disrupted. In the context of the concept, damage to cell membranes and subcellular structures in glutamate toxicity arises because the above alterations lead to the generation of nitrogen dioxide, which is a highly reactive compound, is involved in free radical chain reactions, and oxidizes the main biochemical components of living organisms: DNA/RNA (guanines primarily), fatty acids (unsaturated fatty acids that are components of phospholipid membranes), and proteins (the SH groups of sulfur-containing amino acids and the OH groups of tyrosine residues to produce nitrotyrosine). The concept agrees well with the ideas that every disease starts with a failure of regulatory mechanisms (R. Virkhov) and that dysregulatory pathology forms its basis (G.N. Kryzhanovsky). The mechanisms of the toxic effects of glutamate- and NO-generating compounds as a model of stroke made it possible to suggest methods to reduce their damaging effects. The methods have already been used as part of therapy for ischemic and hemorrhagic strokes, hemorrhages, and head injuries. [ABSTRACT FROM AUTHOR]

A recently reported rapid potentiation of NMDA receptors by Group I metabotropic glutamate receptors (mGluRls) via a Homer protein link is distinct from the classical, relatively slow inhibitory G-protein-associated signaling triggered by mGluRl activation. The relationship between these two mechanisms remains unknown. Here, we focused on the mGluRI-dependent modulation of NMDAR response in hippocampal dentate gyrus granule cells and cerebellar granule cells of C57BL6-J mice and found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. At a shorter time interval (units of millisecond), the Homer-mediated signal from mGluRls prevails, causing upregulation of NMDAR function, in both dentate gyrus granule cells and cerebellar granule cells. Our results shed light on the possible mechanisms of anti-schizophrenia drugs that disrupt Homer-containing protein link. [ABSTRACT FROM AUTHOR]

Objective: The aim of the present study was to investigate the role of ionic homeostasis in cisplatin (cis-diamminedichloroplatinum (II), CDDP)-induced neurotoxicity. CDDP is a severely neurotoxic antineoplastic agent that causes neuronal excitotoxicity. According to some studies, calcium influx increases, whereas potassium efflux decreases neuronal death. Nimodipine and glibenclamide were used to analyze the role of ionic flows in CDDP-induced neurotoxicity in rat primary cerebellar granule cell (CGC) culture. Materials and Methods: CGC culture was prepared from the cerebella of Sprague Dawley 5-day-old pups. The submaximal concentration of CDDP was determined and then given with 1, 10, or 50 µM of drugs into culture. Neurotoxicity was investigated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. One-way analysis of variance, Kruskal-Wallis H test, and Tukey test were applied for statistical analysis. Results: CDDP induced neurotoxicity in a concentration-dependent manner. Neither nimodipine nor glibenclamide was able to protect CGCs against CDDP neurotoxicity. Conclusion: By blocking L-type voltage-gated calcium channels, nimodipine did not prevent CDDP neurotoxicity in CGCs. Ca2+ influx via these channels seemed to be insufficient to cause a change in CDDP-induced neurotoxicity. Similarly, glibenclamide failed to prevent CDDP neurotoxicity. Further studies are needed to elucidate the mechanisms of these preliminary results. [ABSTRACT FROM AUTHOR]

The antisecretory factor is an endogenous protein found in all mammalian tissues investigated so far. It acts by counteracting intestinal hypersecretion and various forms of inflammation, but the detailed mechanism of antisecretory factor (AF) action is unknown. We tested neuronal GABAA receptors by means of AF-16, a potent AF peptide derived from amino acids 36-51 from the NH2 part of AF. Cultured rat cerebellar granule cells were used, and the effects on the GABA-mediated chloride currents were determined by whole-cell patch clamp. Both the neurotransmitter GABA and AF-16 were added by perfusion of the experimental system. A 3-min AF-16 preincubation was more efficacious than 30 s in significantly elevating the rapidly desensitizing GABA-activated chloride current. No effect was found on the tonic, slowly desensitizing current. The GABA-activated current increase by AF-16 demonstrated a low k of 41 pM with a maximal increase of 37% persisting for some minutes after AF washout, independent from GABA concentration. This indicates an effect on the maximal stimulation (E%Max) excluding an altered affinity between GABA and its receptor. An immunocytochemical fluorescence approach with anti ² subunit antibodies demonstrated an increased expression of GABAA receptors. Thus, both the electrophysiological and the immunofluorescence approach indicate an increased appearance of GABAA receptors on the neuronal membrane. The rationale of the experiments was to test the effect of AF on a defined neuronal population of GABAA receptors. The implications of the results on the impact of AF on the enteric nervous system or on brain function are discussed. [ABSTRACT FROM AUTHOR]

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (cGK) signaling pathway regulates the clustering and the recruitment of proteins and vesicles to the synapse, thereby adjusting the exoendocytic cycle to the intensity of activity. Accordingly, this pathway can accelerate endocytosis following large-scale exocytosis, and pre-synaptic cGK type II (cGKII) plays a major role in this process, controlling the homeostatic balance of vesicle exocytosis and endocytosis. We have studied synaptic vesicle recycling in cerebellar granule cells from mice lacking cGKII under strong and sustained stimulation, combining imaging techniques and ultrastructural analyses. The ultrastructure of synapses in the adult mouse cerebellar cortex was also examined in these animals. The lack of cGKII provokes structural changes to synapses in cultured cells and in the cerebellar cortex. Moreover, endocytosis is slowed down in a subset of boutons in these cells when they are stimulated strongly. In addition, from the results obtained with the selective inhibitor of cGKs, KT5823, it can be concluded that cGKI also regulates some aspects of vesicle cycling. Overall, these results confirm the importance of the cGMP pathway in the regulation of vesicle cycling following strong stimulation of cerebellar granule cells. [ABSTRACT FROM AUTHOR]

The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route. [ABSTRACT FROM AUTHOR]

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1- pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors ( CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C ( PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2′- O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule ( RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2′- O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/ PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing. [ABSTRACT FROM AUTHOR]

Acute injury of central nervous system (CNS) starts a cascade of morphological, molecular, and functional changes including formation of a fibrotic scar, expression of transforming growth factor beta 1 (TGF-β1), and expression of extracellular matrix proteins leading to arrested neurite outgrowth and failed regeneration. We assessed alteration of electrophysiological properties of cerebellar granule cells (CGCs) in two in vitro models of neuronal injury: (i) model of fibrotic scar created from coculture of meningeal fibroblasts and cerebral astrocytes with addition of TGF-β1; (ii) a simplified model based on administration of TGF-β1 to CGCs culture. Both models reproduced suppression of neurite outgrowth caused by neuronal injury, which was equally restored by chondroitinase ABC (ChABC), a key disruptor of fibrotic scar formation. Voltage-dependent calcium current was not affected in either injury model. However, intracellular calcium concentration could be altered as an expression of inositol trisphosphate receptor type 1 was suppressed by TGF-β1 and restored by ChABC. Voltage-dependent sodium current was significantly suppressed in CGCs cultured on a model of fibrotic scar and was only partly restored by ChABC. Administration of TGF-β1 significantly shifted current-voltage relation of sodium current toward more positive membrane potential without change to maximal current amplitude. Both transient and sustained potassium currents were significantly suppressed on a fibrotic scar and restored by ChABC to their control amplitudes. In contrast, TGF-β1 itself significantly upregulated transient and did not change sustained potassium current. Observed changes of voltage-dependent ion currents may contribute to known morphological and functional changes in injured CNS. [ABSTRACT FROM AUTHOR]

The recycling of synaptic vesicle ( SV) proteins and transmitter release occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the adaptor protein 1 and 3 complex ( AP-1/ AP-3). As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A ( BFA) to inhibit AP-1/ AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 min after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone ( AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/ AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (VGSCs) are responsible for the action potential generation in excitable cells including neurons and involved in many physiological and pathological processes. Scorpion toxins are invaluable tools to explore the structure and function of ion channels. BmK NT1, a scorpion toxin from Buthus martensii Karsch, stimulates sodium influx in cerebellar granule cells (CGCs). In this study, we characterized the mode of action of BmK NT1 on the VGSCs and explored the cellular response in CGC cultures. BmK NT1 delayed the fast inactivation of VGSCs, increased the Na currents, and shifted the steady-state activation and inactivation to more hyperpolarized membrane potential, which was similar to the mode of action of α-scorpion toxins. BmK NT1 stimulated neuron death (EC = 0.68 µM) and produced massive intracellular Ca overloading (EC = 0.98 µM). TTX abrogated these responses, suggesting that both responses were subsequent to the activation of VGSCs. The Ca response of BmK NT1 was primary through extracellular Ca influx since reducing the extracellular Ca concentration suppressed the Ca response. Further pharmacological evaluation demonstrated that BmK NT1-induced Ca influx and neurotoxicity were partially blocked either by MK-801, an NMDA receptor blocker, or by KB-R7943, an inhibitor of Na/Ca exchangers. Nifedipine, an L-type Ca channel inhibitor, slightly suppressed both Ca response and neurotoxicity. A combination of these three inhibitors abrogated both responses. Considered together, these data ambiguously demonstrated that activation of VGSCs by an α-scorpion toxin was sufficient to produce neurotoxicity which was associated with intracellular Ca overloading through both NMDA receptor- and Na/Ca exchanger-mediated Ca influx. [ABSTRACT FROM AUTHOR]

Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the EDs and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity. [ABSTRACT FROM AUTHOR]

In mammals, excitatory synapses contain two major types of ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl- d-aspartate receptors ( NMDARs). Both receptor types are comprised of several subunits that are post-translationally modified by N-glycosylation. However, the precise N-glycans that are attached to these receptor types are largely unknown. Here, we used biochemistry to confirm that native NMDARs are extensively N-glycosylated; moreover, we found that the NMDAR GluN2B subunit differs from GluN1 subunits with respect to endoglycosidase H sensitivity. Next, we used a complete panel of lectins to determine the glycan composition of NMDARs in both cerebellar tissue and cultured cerebellar granule cells. Our experiments identified 23 lectins that pulled down both the GluN1 and GluN2B NMDAR subunits. We then performed an electrophysiological analysis using representative lectins and found that pre-incubating cerebellar granule cells with the AAL, WGA, or ConA alters the receptor's biophysical properties; this lectin-mediated effect was eliminated when the cells were deglycosylated with peptide-N-glycosidase F. Similar lectin-mediated effects were observed using HEK293 cells that express recombinant GluN1/GluN2B receptors. Finally, using mutant recombinant GluN subunits expressed in HEK293 cells, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data provide new insight into the role that N-glycosylation plays in regulating the function of NMDA receptors in the central nervous system. All animal experiments were performed in accordance with relevant institutional ethics guidelines and regulations with respect to protecting animal welfare. [ABSTRACT FROM AUTHOR]

The calcium-sensing receptor (CaSR) is a widely expressed homodimeric G-protein coupled receptor structurally related to the metabotropic glutamate receptors and GPRC6A. In addition to its well characterized role in maintaining calcium homeostasis and regulating parathyroid hormone release, evidence has accumulated linking the CaSR with cellular differentiation and migration, brain development, stem cell engraftment, wound healing, and tumor growth and metastasis. Elevated expression of the CaSR in aggressive metastatic tumors has been suggested as a potential novel prognostic marker for predicting metastasis, especially to bone tissue where extracellular calcium concentrations may be sufficiently high to activate the receptor. Recent evidence supports a model whereby CaSR-mediated activation of integrins promotes cellular migration. Integrins are single transmembrane spanning heterodimeric adhesion receptors that mediate cell migration by binding to extracellular matrix proteins. The CaSR has been shown to form signaling complexes with the integrins to facilitate both the movement and differentiation of cells, such as neurons during normal brain development and tumor cells under pathological circumstances. Thus, CaSR/integrin complexes may function as a universal cell migration or homing complex. Manipulation of this complex may be of potential interest for treating metastatic cancers, and for developmental disorders pertaining to aberrant neuronal migration. [ABSTRACT FROM AUTHOR]

Cerebellar granule cells precursors are derived from the upper rhombic lip and migrate tangentially independent of glia along the subpial stream pathway to form the external germinal zone. Postnatally, granule cells migrate from the external germinal zone radially through the Purkinje cell layer, guided by Bergmann glia fibers, to the internal granular cell layer. Medulloblastomas (MBs) are the most common malignant childhood brain tumor. Many of these tumors develop from precursor cells of the embryonic rhombic lips. Four main groups of MB are recognized. The WNT group of MBs arise primarily from the lower rhombic lip and embryonic brainstem. The SHH group of MBs originate from cerebellar granule cell precursors in the external germinal zone of the embryonic cerebellum. The cellular origins of type 3 and type 4 MBs are not clear. Several ubiquitin ligases are revealed to be significant factors in development of the cerebellum as well as in the initiation and maintenance of MBs. Proteasome dysfunction at a critical stage of development may be a major factor in determining whether progenitor cells which are destined to become granule cells differentiate normally or become MB cells. We propose the hypothesis that proteasomal activity is essential to regulate the critical transition between proliferating granule cells and differentiated granule cells and that proteasome dysfunction may lead to MB. Proteasome dysfunction could also account for various mutations in MBs resulting from deficiencies in DNA checkpoint and repair mechanisms prior to development of MBs. Data showing a role for the ubiquitin ligases β-TrCP, FBW7, Huwe1, and SKP2 in MBs suggest the possibility of a classification of MBs based on the expression (over expression or under expression) of specific ubiquitin ligases which function as oncogenes, tumor suppressors or cell cycle regulators. [ABSTRACT FROM AUTHOR]

The effects of a classical 1,4-benzodiazepine agonist, such as diazepam, its catabolite N-desmethyl-diazepam (nordiazepam), and 1,5-benzodiazepines such as clobazam and RL 214 (a triazolobenzodiazepine previously synthesized in our labs) were evaluated on native GABA receptors of cerebellar granule cells in culture. The parameter studied was the increase of GABA-activated chloride currents caused by these substances. The contributions of α6 β2/3 γ2 and α1 α6 β2/3 γ2 receptor subtypes to the increase of GABA-activated chloride current were investigated by comparing the effects of such substances in the presence vs. the absence of furosemide. Furosemide is in fact able to block such receptors. It was found that the percent enhancement of peak GABA-activated current doubled for diazepam, clobazam, and RL 214. However, it did not change for N-desmethyl-diazepam. These results indicate that diazepam, clobazam, and RL 214 interact exclusively with α1 β2/3 γ2 receptors, while N-desmethyl-diazepam seems to interact with not only α1- but also α6-containing receptors. [ABSTRACT FROM AUTHOR]

Aims: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. Methods: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA ß1/2/3 subunits was carried out by Western blot analysis. Results: Upon incubation of neurons with PGE2 (1 μM) for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA β1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. Conclusion: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate (NMDA) receptors mediate fast excitatory synaptic transmission in the mammalian central nervous system. The activation of NMDA receptors plays a key role in brain development, synaptic plasticity, and memory formation, and is a major contributor to many neuropsychiatric disorders. Here, we investigated the mechanisms that underlie the trafficking of GluN1/GluN2C receptors. Using an approach combining molecular biology, microscopy, and electrophysiology in mammalian cell lines and cultured cerebellar granule cells, we found that the surface delivery of GluN2C-containing receptors is reduced compared to GluN2A- and GluN2B-containing receptors. Furthermore, we identified three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits that are required for proper intracellular processing and surface delivery of NMDA receptors. These results shed new light on the regulation of NMDA receptor trafficking, and these findings can be exploited to develop new strategies for treating some forms of neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs. [ABSTRACT FROM AUTHOR]

From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, wedemonstrate that presynapticcGKtype II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells. [ABSTRACT FROM AUTHOR]

Although melatonin ( MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field ( ELF- EMF)-induced Nav activity in rat cerebellar granule cells ( GCs). Exposing cerebellar GCs to ELF- EMF for 60 min. significantly increased the Nav current ( INa) densities by 62.5%. MT (5 μM) inhibited the ELF- EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF- EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF- EMF. ELF- EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF- EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF- EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca2+ level, but it significantly elevated the intracellular Ca2+ level evoked by the high K+ stimulation in cerebellar GC that were either exposed or not exposed to ELF- EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF- EMF exposure through Ca2+ influx-induced Ca2+ release. [ABSTRACT FROM AUTHOR]

The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variableNterminus (β4e ) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4 variants (β4a and β4b ), β4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear-targeting properties (β4bβ4a>β4e ). Together, these findings support isoform-specific functions of β4 splice variants in neurons, with β4b playing a dual role in channel modulation and gene regulation, whereas the newly detected β4e variant serves exclusively in calcium-channel-dependent functions. [ABSTRACT FROM AUTHOR]

GABA A receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABA A receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABA A receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 β2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn 2+ which block α6 containing and incomplete receptors. [ABSTRACT FROM AUTHOR]

ABSTRACT Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20 months of age were exposed to methylmercury by feeding a total dose of 5.0 mg kg−1 body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Soluble cytosolic carbonic anhydrases (CAs) are well known to participate in pH regulation of the cytoplasm of mammalian cells. Membrane-bound CA isoforms—such as isoforms IV, IX, XII, XIV, and XV—also catalyze the reversible conversion of carbon dioxide to protons and bicarbonate, but at the extracellular face of the cell membrane. When human CA isoform IV was heterologously expressed in Xenopus oocytes, we observed, by measuring H+ at the outer face of the cell membrane and in the cytosol with ion-selective microelectrodes, not only extracellular catalytic CA activity but also robust intracellular activity. CA IV expression in oocytes was confirmed by immunocytochemistry, and CA IV activity measured by mass spectrometry. Extra- and intracellular catalytic activity of CA IV could be pharmacologically dissected using benzolamide, the CA inhibitor, which is relatively slowly membrane-permeable. In acute cerebellar slices of mutant mice lacking CA IV, cytosolic H+ shifts of granule cells following CO2 removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CA IV contributes robust catalytic activity intracellularly, and that this activity participates in regulating H+ dynamics in the cytosol, both in injected oocytes and in mouse neurons. [ABSTRACT FROM AUTHOR]

Background Congenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2- CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2- CDG, however, remains elusive. Results Herein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation. Conclusions Our results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons' inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients. [ABSTRACT FROM AUTHOR]

Cultured cerebellar granule cells were co-loaded with Ca-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (ΔΨ). A 60-min incubation of cells in glucose-free solution containing 2-deoxy- D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca]. This sMD was insensitive to a removal of external Ca or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6-8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]) and ΔΨ changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP] and ΔΨ. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ΔΨ despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion. [ABSTRACT FROM AUTHOR]

The role of genetic inheritance in brain development has been well characterized, but little is known about the contributions of natural environmental stimuli, such as the effect of light-dark cycles, to brain development. In this study, we determined the role of light stimuli in neuronal cell migration to elucidate how environmental factors regulate brain development. We show that in early postnatal mouse cerebella, granule cell migration accelerates during light cycles and decelerates during dark cycles. Furthermore, cerebellar levels of insulin-like growth factor 1 (IGF-1) are high during light cycles and low during dark cycles. There are causal relationships between light-dark cycles, speed of granule cell migration, and cerebellar IGF-1 levels. First, changes in light-dark cycles result in corresponding changes in the fluctuations of both speed of granule cell migration and cerebellar IGF-1 levels. Second, in vitro studies indicate that exogenous IGF-1 accelerates the migration of isolated granule cells through the activation of IGF-1 receptors. Third, in vivo studies reveal that inhibiting the IGF-1 receptors decelerates granule cell migration during light cycles (high IGF-1 levels) but does not alter migration during dark cycles (low IGF-1 levels). In contrast, stimulating the IGF-1 receptors accelerates granule cell migration during dark cycles (low IGF-1 levels) but does not alter migration during light cycles (high IGF-1 levels). These results suggest that during early postnatal development light stimuli control granule cell migration by altering the activity of IGF-1 receptors through modification of cerebellar IGF-1 levels. [ABSTRACT FROM AUTHOR]

Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundamental to re-establishing conditions for synaptic transmission. As there are distinct endocytotic pathways that each differ in their efficiency to generate releasable synaptic vesicles, we used the dye FM1-43 to track vesicle recycling, and to determine whether nerve terminals use multiple pathways of endocytosis. We identified two types of synaptic boutons in cultured cerebellar granule cells that were characterized by weak or strong FM1-43-unloading profiles. Decreasing the extent of exocytosis dramatically increased the proportion of synaptic boutons that exhibited strong FM1-43-unloading and dramatically reduced the number of endosome-like structures. Hence, we concluded that efficient recycling of synaptic vesicles is concomitant with the formation of non-releasable endosomes in both types of synaptic boutons, although to different extents. Furthermore, cell maturation in culture increased the proportion of synaptic boutons that were capable of an intense release response, whereas the chronic blockage of synaptic activity diminished the capacity of boutons to release dye. [ABSTRACT FROM AUTHOR]

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR)/)) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC50 of 127 ± 21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and a-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS. [ABSTRACT FROM AUTHOR]

The biochemical pathways that mediate the degeneration of dopaminergic neurons in the substantia nigra of patients with Parkinson’s disease are largely unknown. Recently, aberrant cell cycle events have been shown to be associated with neuronal death in several neurodegenerative diseases. In the present study, we investigated the role of DNA polymerases (DNA pols) in 1-methyl-4-phenylpyridinium (MPP+)-induced neuronal apoptosis in cerebellar granule cells. After exposure to MPP+, the neurons entered S phase of the cell cycle. Neuronal cell cycle re-entry and apoptosis were attenuated by flavopiridol, which is a broad inhibitor of cyclin-dependent kinases (CDKs). MPP+ exposure significantly increased the expression of DNA pol-β and primase but did not affect the expression of the canonical replicative DNA pols, including DNA pol-δ and pol-ε. Dideoxycytidine, which is a pharmacological inhibitor of DNA pol-β, attenuated the neuronal apoptosis mediated by MPP+. In a similar manner, the expression of a dominant negative form of DNA pol-β was also neuroprotective. These results suggest that DNA pol-β may have a causal role in MPP+-induced neuronal apoptosis. [ABSTRACT FROM AUTHOR]

Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here, we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase, isozyme 1 (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen peroxide or tert-butyl peroxide and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake, and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated with increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants, ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (1) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (2) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides. [ABSTRACT FROM PUBLISHER]

The mechanisms of protective effect of N-methyl-D-aspartate (NMDA) receptor stimulation on apoptosis of neurons at their early stage of development are poorly understood. In the present study, we investigated the effects of NMDA on staurosporine (St)- and low-potassium (LP)-evoked apoptotic cell death in primary cerebellar granule cell (CGC) cultures at 7 days in vitro (DIV). We found that NMDA (200 μM) attenuated the St (0.5 μM)- and LP (5 mM KCl)-induced neuronal cell death in 7 but not 12 DIV CGC as confirmed by LDH release and MTT reduction assays. Moreover, NMDA attenuated St-and LP-evoked DNA fragmentation and cytosolic apoptosis inducing factor (AIF) protein level but not caspase-3 activation induced by both pro-apoptotic factors. Neuroprotective effects of NMDA on St-induced apoptosis in CGC were attenuated by inhibitors of ERK/MAPK-signaling, PD 98059 and U0126 but not by NMDA receptor antagonists, AP-5 (100 μM) and MK-801 (1 μM) or by inhibitors of PI3-K/Akt pathway (LY 294002 and wortmannin). In contrast to staurosporine model of apoptosis, AP-5 and MK-801 but not inhibitors of PI3-K/Akt and MAPK/ERK1/2 prevented the NMDA-mediated neuroprotection in LP-induced apoptosis of CGC. In separate experiments, we observed also the anti-apoptotic action of NMDA on St (0.5 μM)- and salsolinol (250 μM)-evoked cell death in human neuroblastoma SH-SY5Y cells without its influence on caspase-3 activity, induced by these pro-apoptotic factors. These data indicate that neuroprotection evoked by NMDA in CGC strongly depends on used pro-apoptotic agent and could engage NMDA channel function or be connected with the activation of pro-survival MAPK/ERK1/2 pathway. It is also suggested that anti-apoptotic effects of NMDA is connected with inhibition of fragmentation of DNA via caspase-3-independent mechanism. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain. The localization of GABA receptors type A (GABAARs) at strategically located domains of the neuronal membrane is of vital importance for fast inhibitory synapse transmission efficacy. We have shown before that the lateral mobility of GABAARs depends on subunit composition of the complex. To study the lateral mobility of GABAARs in living, cultured neurons, we transfected cerebellar granule cells with either the complete α1 GABAAR subunit or with a truncation of the α1 subunit that lacks the major intracellular loop (M3/M4). We examined the location and lateral mobility of receptors containing both versions of the α1 subunit in living neurons. From fluorescence recovery after photobleaching experiments we present novel evidences that the intracellular M3/M4 loop of the α1 subunit restricts the lateral mobility of GABAARs when expressed in neurons. In addition, our immunocytochemical studies suggested that receptors containing the truncated subunit seem to be unable to reach synaptic localizations. Here we show for the first time that the α1 intracellular loop (M3/M4) domain has a relevant role in controlling the lateral mobility of GABAARs in neurons, and we believe that this is a novel and important contribution in neurobiology of GABAA receptors. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In the postnatal forebrain, the extracellular matrix protein reelin is expressed and secreted by subsets of GABAergic neurons, whereas in the cerebellum reelin is detected in glutamatergic cells of the granule cell layer. Thus, various regions of the postnatal brain present different patterns of reelin expression, whose significance remains unknown. We combined immunocytochemical and pharmacological approaches to characterize the phenotypic and temporal profiles of reelin expression in dissociated cultures of cerebellar granule neurons. A single type of reelin immunoreactivity, identified by a punctate labelling, was present in the somata of the majority of neurons. This immunoreactivity was observed throughout maturation and was exclusively present in glutamatergic neurons expressing the vesicular glutamate transporter 1. Neurons containing the reelin receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr) represented about 80% of cerebellar neurons. The vast majority of reelin-positive neurons coexpressed Apoer2, suggesting that reelin immunoreactivity resulted in part from receptor-bound reelin. Inhibition of protein synthesis with cycloheximide completely abolished reelin immunoreactivity. In contrast, blocking protein secretion with brefeldin A did not affect the proportion of punctate neurons but revealed a subpopulation of neurons characterized by a solid reelin staining. These data show for the first time that a homogeneous population of glutamatergic neurons can synthesize and secrete reelin in cerebellar granule cells in vitro. [ABSTRACT FROM PUBLISHER]

Although melatonin affects developing neurons and is neuroprotective, a role of melatonin receptors termed MT1 and MT2 in these actions is unclear. We investigated the effects of melatonin on the levels of the brain derived neurotrophic factor (BDNF) in the developing cerebellum and cerebellar granule cells (CGC) of wild-type (WT), MT1- and MT2-knockout mice. A model of low-potassium CGC toxicity was used to evaluate neuroprotection. A 14-day-old pups and CGC cultures were treated with melatonin; 0.01 mg/kg intraperitoneally and 1 nM in vitro, respectively. Treatment of WT pups and CGC with melatonin did not alter BDNF levels. The absence of MT2 but not MT1 receptors enabled melatonin to increase cerebellar and CGC BDNF content. Nanomolar melatonin was neuroprotective in MT2-knockout but not WT CGC. We propose that CGC from MT2-knockout mice could serve as a model for studying the influence of melatonin on human CGC, which express MT1 but not MT2 receptors. [ABSTRACT FROM AUTHOR]

In the developing rat cerebellum functional NMDA receptors (NMDARs) expressing the NR2C subunit have been identified on or after postnatal day 19. We obtained primary cultured cells from 19- to 35-day-old rat cerebellum that expressed few oligodendrocytes or astrocytes. Cultured cells were immunoreactive for neuron-specific proteins thus indicating a neuronal population. The primary neuron present was the granule cell as indicated by immunofluorescence for the GABAA alpha 6 subunit. Whole-cell patch-clamp experiments indicated that functional NMDARs were present. Functional characteristics of NMDARs expressed in cerebellar granule cells (CGCs) obtained from adolescent animals were similar to those previously reported for NMDARs expressed in CGCs obtained from neonatal rats. Cultured CGCs obtained from older animals contained NMDARs that were inhibited by EtOH and were less sensitive to the NR2B subunit-specific antagonist Ro 25-6981. Furthermore, NMDA-induced currents were smaller than those observed in CGCs. Western blot analysis indicated the presence of the NMDA NR2A and NR2C subunits, but not the NR2B in cultures obtained from the adolescent rats. CGCs obtained from adolescent rats express functional NMDARs consistent with a developmental profile observed in vivo. [ABSTRACT FROM AUTHOR]

AMPA receptors (AMPAR) mediate the majority of fast excitatory neurotransmission in the central nervous system (CNS). Transmembrane AMPAR regulatory proteins (TARPs) have been identified as a novel family of proteins which act as auxiliary subunits of AMPARs to modulate AMPAR trafficking and function. The trafficking of AMPARs to regulate the number of receptors at the synapse plays a key role in various forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Expression of the prototypical TARP, stargazin/TARPγ2, is ablated in the stargazer mutant mouse, an animal model of absence epilepsy and cerebellar ataxia. Studies on the stargazer mutant mouse have revealed that failure to express TARPγ2 has widespread effects on the balance of expression of both excitatory (AMPAR) and inhibitory receptors (GABAA receptors, GABAR). The understanding of TARP function has implications for the future development of AMPAR potentiators, which have been shown to have therapeutic potential in both psychological and neurological disorders such as schizophrenia, depression and Parkinson's disease. [ABSTRACT FROM AUTHOR]

Abstract: Glycogen synthase kinase-3 (GSK-3) is involved in the pathogenesis of several neurodegenerative diseases. In addition, as oxidative stress has been implicated in all neurodegenerative disorders, the inhibition of both pathways offers a potential strategy for preventing or delaying neurodegeneration. We examined the cytoprotective effects of lithium and SB-415286, two inhibitors of GSK-3, using a rat B65 cell line and also in cerebellar granule cells (CGN). H2O2 decreased the inactive form of GSK-3 (phospho-GSK-3 at Ser9), as measured by immunoblot experiments involving an antibody against the inactive form of the enzyme. Moreover, lithium inhibited this effect. While SB-415286 exerted a protective effect, lithium did not attenuate the toxic effects of H2O2 (1mM). We then examined those mechanisms implicated in the protective effects of SB-415286. When we analyzed reactive oxygen species (ROS) production using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate in B65 cells, as well as in CGN, we found that SB-415286 strongly reduced DCF fluorescence. Lithium, however, did not exhibit any antioxidant properties. We conclude that the GSK-3 inhibitor SB-415286 has antioxidant properties, which may explain the cytoprotective effects against H2O2 damage. Furthermore, inhibition of GSK-3 activity was not involved in this protective effect. [Copyright &y& Elsevier]

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c- jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide. [ABSTRACT FROM AUTHOR]