Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis

Micromolar concentrations of the flavonoid kaempferol were found to efficiently block cerebellar granule cell (CGC) death through low K+-induced apoptosis, as demonstrated by prevention of the activation of caspase-3, internucleosomal DNA fragmentation, and chromatin condensation, without a significant rise in intracellular free Ca2+ concentration. Half of the maximum protection against CGC apoptosis was attained with 8 +/- 2 microM kaempferol. Reactive oxygen species (ROS) were monitored with 2',7'-dichlorodihydrofluorescein diacetate. Quantitative analysis of intracellularly and extracellularly oriented ROS production up to 3 h from the onset of low K+-induced CGC apoptosis was carried out with acquired digital fluorescence microscopy images of CGC in culture plates using a CCD camera, and also with fluorescence measurements of resuspended CGCs. In both cases, nearly 90% of ROS production by CGCs during the early stages (up to 3 h) after induction of low-K+ apoptosis occurs at the plasma membrane. Kaempferol, at concentrations that blocked CGC apoptosis, has been found to be a particularly potent blocker of extracellularly oriented ROS production by CGCs, and to inhibit the ascorbate-dependent NADH oxidase and superoxide anion production activities of the neuronal plasma membrane redox chain.     

In vivo analysis reveals different apoptotic pathways in pre- and postmigratory cerebellar granule cells of rabbit

Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation.     

In vivo analysis reveals different apoptotic pathways in pre‐ and postmigratory cerebellar granule cells of rabbit

Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly‐ADP‐ribose polymerase‐1 (PARP‐1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437–452, 2004     

Bone Morphogenetic Protein-6 Promotes Cerebellar Granule Neurons Survival by Activation of the MEK/ERK/CREB Pathway

Bone morphogenetic proteins (BMPs) have been implicated in the generation and postnatal differentiation of cerebellar granule cells (CGCs). Here, we examined the eventual role of BMPs on the survival of these neurons. Lack of depolarization causes CGC death by apoptosis in vivo, a phenomenon that is mimicked in vitro by deprivation of high potassium in cultured CGCs. We have found that BMP-6, but not BMP-7, is able to block low potassium–mediated apoptosis in CGCs. The neuroprotective effect of BMP-6 is not accompanied by an increase of Smad translocation to the nucleus, suggesting that the canonical pathway is not involved. By contrast, activation of the MEK/ERK/CREB pathway by BMP-6 is necessary for its neuroprotective effect, which involves inhibition of caspase activity and an increase in Bcl-2 protein levels. Other pathways involved in the regulation of CGC survival, such as the c-Jun terminal kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt/PKB, were not affected by BMP-6. Moreover, failure of BMP-7 to activate the MEK/ERK/CREB pathway could explain its inability to protect CGCs from low potassium–mediated apoptosis. Thus, this study demonstrates that BMP-6 acting through the noncanonical MEK/ERK/CREB pathway plays a crucial role on CGC survival.     

N-methyl-D-aspartate blocks activation of JNK and mitochondrial apoptotic pathway induced by potassium deprivation in cerebellar granule cells

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.     

Axonal branching pattern and coupling mechanisms of the cerebral giant neurones in the snail,lymnaea stagnalis

The axonal branching pattern of the two cerebral giant neurones (CGCs) of Lymnaea stagnalis was studied with intrasomatically applied horseradish peroxidase. The cells are symmetrical. Each CGC projects to the ipsilateral n. labialis medius and n. arteriae labialis, the subcerebral commissure, and to all ipsi- and contralateral buccal nerves. The contralateral buccal nerves are reached via the ipsilateral cerebro-buccal connective and the buccal commissure. The CGC fire action potentials 1:1 in a driver-follower relationship. Each cell is capable of both driving and following. The relationship depends on the membrane potentials of the somata. In driving CGC spikes are initiated in a cerebral spike trigger zone located near the soma. In following cells spikes are initiated in a distal zone located in the buccal ganglia. The buccal zone is only affected by the partner CGC. CGC are synchronized by three coupling mechanisms: mutual excitatory chemical synapses, electrotonic coupling, and common input. The chemical and electrotonic connections are located in the buccal ganglia. All spikes are relayed to the partner cell via the chemical synapses. The electrotonic coupling improves the efficiency of the chemical synapses. The dual connection selectively synchronizes the CGC-axonal spikes from each side of the buccal mass. Common excitatory input affects the cerebral spike trigger zones and can initiate simultaneous spikes in both cells. This results in bilateral synchrony of spikes in the CGC-axons in both the buccal and the lip nerves.     

Transcriptional cascade from Math1 to Mbh1 and Mbh2 is required for cerebellar granule cell differentiation

Cerebellar granule cells (CGCs) are the most abundant neuronal type in the mammalian brain, and their differentiation is regulated by the basic helix-loop-helix gene, Math1. However, little is known about downstream genes of Math1 and their functions in the cerebellum. To investigate them, we have here established an electroporation-based in vivo gene transfer method in the developing mouse cerebellum. Misexpression of Math1 ectopically induced expression of Bar-class homeobox genes, Mbh1 and Mbh2, which are expressed by CGCs. Conversely, their expression was repressed in CGCs by knockdown of Math1. These findings, taken together with chromatin immunoprecipitation assays, suggest that Math1 directly regulates the Mbh genes in CGCs. Furthermore, a dominant-negative form of the Mbh proteins disrupted proper formation of the external granule layer and differentiation of CGCs, whereas misexpression of the Mbh genes ectopically induced expression of a CGC marker in nonneuronal cells, indicating that the Mbh proteins are required for the differentiation of CGCs.     

Multiple subtypes of serotonin receptors in the feeding circuit of a pond snail

In the central nervous system of the pond snail Lymnaea stagnalis, serotonergic transmission plays an important role in controlling feeding behavior. Recent electrophysiological studies have claimed that only metabotropic serotonin (5-HT(2)) receptors, and not ionotropic (5-HT(3)) receptors, are used in synapses between serotonergic neurons (the cerebral giant cells, CGCs) and the follower buccal motoneurons (the B1 cells). However, these data are inconsistent with previous results. In the present study, we therefore reexamined the serotonin receptors to identify the receptor subtypes functioning in the synapses between the CGCs and the B1 cells by recording the compound excitatory postsynaptic potential (EPSP) of the B1 cells evoked by a train of stimulation to the CGC in the presence of antagonists: cinanserin for 5-HT(2) and/or MDL72222 for 5-HT(3). The compound EPSP amplitude was partially suppressed by the application of these antagonists. The rise time of the compound EPSP was longer in the presence of MDL72222 than in that of cinanserin. These results suggest that these two subtypes of serotonin receptors are involved in the CGC-B1 synapses, and that these receptors contribute to compound EPSP. That is, the fast component of compound EPSP is mediated by 5-HT(3)-like receptors, and the slow component is generated via 5-HT(2)-like receptors.     

Effects of halothane on feeding motor activity in the snail Lymnaea stagnalis

Snails exposed to the general anaesthetic halothane show an increase in biting plus mouthing movements. Perfusion of the isolated CNS with halothane leads to a period of increased spiking activity, followed by suppression of activity in identified feeding motoneurones in the buccal ganglia. Synaptic inputs to motoneurones from interneurones of the buccal feeding pattern generator are differentially affected. Possible mechanisms underlying the generation of motoneuronal bursting in the presence of halothane are examined.     

Responses of a bursting pacemaker to excitation reveal spatial segregation between bursting and spiking mechanisms

Central pattern generators (CPGs) frequently include bursting neurons that serve as pacemakers for rhythm generation. Phase resetting curves (PRCs) can provide insight into mechanisms underlying phase locking in such circuits. PRCs were constructed for a pacemaker bursting complex in the pyloric circuit in the stomatogastric ganglion of the lobster and crab. This complex is comprised of the Anterior Burster (AB) neuron and two Pyloric Dilator (PD) neurons that are all electrically coupled. Artificial excitatory synaptic conductance pulses of different strengths and durations were injected into one of the AB or PD somata using the Dynamic Clamp. Previously, we characterized the inhibitory PRCs by assuming a single slow process that enabled synaptic inputs to trigger switches between an up state in which spiking occurs and a down state in which it does not. Excitation produced five different PRC shapes, which could not be explained with such a simple model. A separate dendritic compartment was required to separate the mechanism that generates the up and down phases of the bursting envelope (1) from synaptic inputs applied at the soma, (2) from axonal spike generation and (3) from a slow process with a slower time scale than burst generation. This study reveals that due to the nonlinear properties and compartmentalization of ionic channels, the response to excitation is more complex than inhibition.     

Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.     

Persistent sodium current is a nonsynaptic substrate for long-term associative memory

Although synaptic plasticity is widely regarded as the primary mechanism of memory [1], forms of nonsynaptic plasticity, such as increased somal or dendritic excitability or membrane potential depolarization, also have been implicated in learning in both vertebrate and invertebrate experimental systems [2], [3], [4], [5], [6] and [7]. Compared to synaptic plasticity, however, there is much less information available on the mechanisms of specific types of nonsynaptic plasticity involved in well-defined examples of behavioral memory. Recently, we have shown that learning-induced somal depolarization of an identified modulatory cell type (the cerebral giant cells, CGCs) of the snail Lymnaea stagnalis encodes information that enables the expression of long-term associative memory [8]. The Lymnaea CGCs therefore provide a highly suitable experimental system for investigating the ionic mechanisms of nonsynaptic plasticity that can be linked to behavioral learning. Based on a combined behavioral, electrophysiological, immunohistochemical, and computer simulation approach, here we show that an increase of a persistent sodium current of this neuron underlies its delayed and persistent depolarization after behavioral single-trial classical conditioning. Our findings provide new insights into how learning-induced membrane level changes are translated into a form of long-lasting neuronal plasticity already known to contribute to maintained adaptive modifications at the network and behavioral level [8].     

Aryl hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse

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 EC(50) 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 α-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.     

The therapeutic effect of memantine through the stimulation of synapse formation and dendritic spine maturation in autism and fragile X syndrome

Although the pathogenic mechanisms that underlie autism are not well understood, there is evidence showing that metabotropic and ionotropic glutamate receptors are hyper-stimulated and the GABAergic system is hypo-stimulated in autism. Memantine is an uncompetitive antagonist of NMDA receptors and is widely prescribed for treatment of Alzheimer's disease treatment. Recently, it has been shown to improve language function, social behavior, and self-stimulatory behaviors of some autistic subjects. However the mechanism by which memantine exerts its effect remains to be elucidated. In this study, we used cultured cerebellar granule cells (CGCs) from Fmr1 knockout (KO) mice, a mouse model for fragile X syndrome (FXS) and syndromic autism, to examine the effects of memantine on dendritic spine development and synapse formation. Our results show that the maturation of dendritic spines is delayed in Fmr1-KO CGCs. We also detected reduced excitatory synapse formation in Fmr1-KO CGCs. Memantine treatment of Fmr1-KO CGCs promoted cell adhesion properties. Memantine also stimulated the development of mushroom-shaped mature dendritic spines and restored dendritic spine to normal levels in Fmr1-KO CGCs. Furthermore, we demonstrated that memantine treatment promoted synapse formation and restored the excitatory synapses to a normal range in Fmr1-KO CGCs. These findings suggest that memantine may exert its therapeutic capacity through a stimulatory effect on dendritic spine maturation and excitatory synapse formation, as well as promoting adhesion of CGCs.     

Increased bursting glutamatergic neurotransmission in an auditory forebrain area of the zebra finch (Taenopygia guttata) induced by auditory stimulation

The caudomedial nidopallium (NCM) is a telencephalic area involved in auditory processing and memorization in songbirds, but the synaptic mechanisms associated with auditory processing in NCM are largely unknown. To identify potential changes in synaptic transmission induced by auditory stimulation in NCM, we used a slice preparation for path-clamp recordings of synaptic currents in the NCM of adult zebra finches (Taenopygia guttata) sacrificed after sound isolation followed by exposure to conspecific song or silence. Although post-synaptic GABAergic and glutamatergic currents in the NCM of control and song-exposed birds did not present any differences regarding their frequency, amplitude and duration after song exposure, we observed a higher probability of generation of bursting glutamatergic currents after blockade of GABAergic transmission in song-exposed birds as compared to controls. Both song-exposed males and females presented an increase in the probability of the expression of bursting glutamatergic currents, however bursting was more commonly seen in males where they appeared even without blocking GABAergic transmission. Our data show that song exposure changes the excitability of the glutamatergic neuronal network, increasing the probability of the generation of bursts of glutamatergic currents, but does not affect basic parameters of glutamatergic and GABAergic synaptic currents.     

Ionic mechanisms underlying spontaneous CA1 neuronal firing in Ca2+-free solution

Hippocampal CA1 neurons exposed to zero-[Ca(2+)] solutions can generate periodic spontaneous synchronized activity in the absence of synaptic function. Experiments using hippocampal slices showed that, after exposure to zero-[Ca(2+)](0) solution, CA1 pyramidal cells depolarized 5-10 mV and started firing spontaneous action potentials. Spontaneous single neuron activity appeared in singlets or was grouped into bursts of two or three action potentials. A 16-compartment, 23-variable cable model of a CA1 pyramidal neuron was developed to study mechanisms of spontaneous neuronal bursting in a calcium-free extracellular solution. In the model, five active currents (a fast sodium current, a persistent sodium current, an A-type transient potassium current, a delayed rectifier potassium current, and a muscarinic potassium current) are included in the somatic compartment. The model simulates the spontaneous bursting behavior of neurons in calcium-free solutions. The mechanisms underlying several aspects of bursting are studied, including the generation of triplet bursts, spike duration, burst termination, after-depolarization behavior, and the prolonged inactive period between bursts. We show that the small persistent sodium current can play a key role in spontaneous CA1 activity in zero-calcium solutions. In particular, it is necessary for the generation of an after-depolarizing potential and prolongs both individual bursts and the interburst interval.     

Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms

Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high-K⁺ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K⁺ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K⁺ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.     

Effect of halothane on CO2 production from glucose, fructose and pyruvate in rat cerebral cortical slices

Abstract— The effect of halothane on rat cerebral cortical metabolism was studied by measuring ¹⁴CO₂ production from various [¹⁴C]-labelled substrates. Glucose metabolism was depressed by clinically-used concentrations of halothane (0.35 mm ; 1.65 MAC) which did not significantly affect the metabolism of fructose or pyruvate. We concluded that halothane blocked an early step(s) in glycolysis preceding phosphofructokinase. Hexokinase was considered unlikely as the site of blockade, leaving glucose uptake or the glucose phosphate isomerase step as the most likely site(s). Higher concentrations of halothane (0.7 mm ; 3.3 MAC) were required to block the metabolism of fructose or pyruvate to CO₂. This action of halothane was attributed to the known inhibition by halothane of electron transport processes.