A transient outward current dependent on external calcium in rat cerebellar granule cells

Summary The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to anI _A-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca²⁺, but significantly inhibited by 3mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED₅₀=0.7mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca²⁺, Mn²⁺ and Mg²⁺. In 2.8mM Mn²⁺, the transient potassium conductance was comparable to that in 2.8mM Ca²⁺, while in 2.8mM Mg²⁺ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca²⁺ was present, Mg²⁺ up to 5mM had no effect. The transient current increased with increasing concentrations of external Ca²⁺, [Ca²⁺] _o , and the maximum conductancevs. [Ca²⁺] _o curve could be approximated by a one-site model. In addition, the current recorded with 5.5mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca²⁺ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.     

The toxin helothermine affects potassium currents in newborn rat cerebellar granule cells

Helothermine, a recently isolated toxin from the venom of the Mexican beaded lizard Heloderma horridum horridum was tested on K⁺ currents of newborn rat cerebellar granule cells. In whole-cell voltageclamp experiments, cerebellar granule neurons exhibited at least two different K⁺ current components: a first transient component which is similar to an I _A-type current, is characterized by fast activating and inactivating kinetics and blocked by 4-aminopyridine; a second component which is characterized by noninactivating kinetics, is blocked by tetraetylammonium ions and resembles the classical delayed-rectifier current. When added to the standard external solution at concentrations ranging between 0.1 and 2 m helothermine reduced the pharmacologically isolated I _A-type current component in a voltage- and dose-dependent way, with a half-maximal inhibitory concentration (IC₅₀) of 0.52 m. A comparison between control and nelothermine-modified peak transient currents shows a slowdown of activation and inactivation kinetics. The delayed-rectifier component inhibition was concentration dependent (IC₅₀ = 0.86 m) but not voltage dependent. No frequency-or use-dependent block was observed on both K⁺ current types. Perfusing the cells with control solution resulted in quite a complete current recovery. We conclude that helothermine acts with different affinities on two types of K⁺ current present in central nervous system neurons.     

Resting potential of rat cerebellar granule cells during early maturation in vitro

The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca²⁺ channels and a membrane potential that can respond to changes in external K⁺. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca²⁺ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from −40 mV at the first day in vitro to −60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca²⁺ influx through L-type and N-type voltage-activated Ca²⁺ channels, functional at day 0. Gross estimations of the permeabilities of Na⁺ and Cl⁻ were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K⁺ ions seems to underlie the evolution of the resting potential with time. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 11–21, 1997.     

Characterization of iodoacetate-mediated neurotoxicity in vitro using primary cultures of rat cerebellar granule cells

The neuroprotective efficacy of antioxidant molecules against iodoacetate (IAA) neurotoxicity in rat cerebellar granule cell (CGC) cultures was investigated. Transient exposure to IAA caused a concentration-dependent decrease in cell viability (ED50 = 9.8 microM). Dizocilpine maleate (MK-801), and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX), failed to prevent IAA toxicity. Certain antioxidant molecules were shown to be neuroprotective against IAA when combined with MK-801 but were ineffective when administered alone. (S)-(-)-Trolox, butylated hydroxytoluene (BHT), and U-83836E exhibited EC50 values of 78, 5.9, and 0.25 microM, respectively, in the presence of 10 microM MK-801. IAA also induced an increase in intracellular oxidative stress, which was quenched by the antioxidants (in the presence of MK-801) in cultures loaded with the oxidant sensitive dye 2'7'-dichlorodihydrofluorescein diacetate (DCFH-DA).     

Na-K-ATPase in rat cerebellar granule cells is redox sensitive

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.     

Oxygen-sensitivity of potassium fluxes across plasma membrane of cerebellar granule cells

This study focuses on the oxygen-dependence of active and passive K⁺ fluxes across membranes of cerebellar granule cells of neonatal rats. Maximal Na⁺,K⁺-ATPase activity along with minimal passive K⁺ influx was observed within oxygen concentration range characteristic for neonatal rat cerebellum. Prolonged exposure to hypoxia as well as hyperoxia resulted in suppression of the Na⁺,K⁺-ATPase and activation of the passive K⁺ flux. Toxic effects of hypoxia could be partially prevented by inhibition of NO production with L-NAME. This was accomplished by suppression of Na⁺,K⁺-ATPase with subsequent reduction in ATP consumption concurrently with the reduction in passive K⁺ flux. Activation of the Na⁺,K⁺-ATPase by NO at physiological pO₂ could be abolished by inhibition of NO synthase by L-NAME or soluble guanylyl cyclase with ODQ. However, treatment of cells with activator of PKG Rp-8-CTP did not mimic normoxic activation of the active K⁺ influx. Oxygen-induced responses under normoxic conditions were differentially mediated by α1 isoform of the Na⁺,K⁺-ATPase catalytic subunit, whereas α2/3 isoform was predominantly active under conditions of severe hypoxia. We conclude that both hypoxia and hyperoxia trigger a gradual dissipation of transmembrane K⁺ gradient and loss of excitability of cerebellar neurons. The latter may be partially reversed by suppression of NO production under hypoxic conditions     

Voltage dependent sodium currents in cultured rat cerebellar granule cells

Sodium currents were studied in granule cells dissociated from rat cerebellum. Macroscopic currents were recorded using the patch-clamp technique. Sudium currents, which are TTX sensitive, reached a maximum peak value of 0.42±0.08 pA/m² at 18.4±2.2 mV (n=6). Activation and inactivation kinetics and steady-state properties were described in terms of Hodgkin and Huxley, parameters. The properties of sodium channels in cultured rat cerebellar granule cells are very similar to those reported for various neural preparations.     

Histamine excites rat cerebellar granule cells in vitro through H1 and H2 receptors.

The effects of histamine on the firing of cerebellar granule cells were investigated in vitro. Histamine predominantly produced excitatory (117/123, 95.1%) and in a few cases inhibitory (6/123, 4.9%) responses in granule cells. The histamine-induced excitation was not blocked by perfusing the slice with low Ca2+/high Mg2+ medium, supporting a direct postsynaptic action of histamine. The H1 receptor antagonists triprolidine and chlorpheniramine significantly diminished the histamine-induced excitation, but the H2 receptor antagonist ranitidine did not significantly reduce the excitation. On the other hand, the H2 receptor agonist dimaprit could elicit a weak excitation of granule cells. This dimaprit-induced excitation was blocked by ranitidine but not triprolidine. These results reveal that the excitatory effect of histamine on cerebellar granule cells is mediated by both H1 and H2 receptors with a predominant contribution of H1 receptors. The relevance of these findings to the possible function of the hypothalamocerebellar histaminergic fibers in cerebellum is discussed.     

Delayed rectifier outward K+ current mediates the migration of rat cerebellar granule cells stimulated by melatonin

Melatonin (MT) may work as a neuromodulator through the associated MT receptors in the central nervous system. Previously, our studies have shown that MT increased the I(K) current via a G protein-related pathway. In the present study, patch-clamp whole-cell recording, transwell migration assays and organotypic cerebellar slice cultures were used to examine the effect of MT on granule cell migration. MT increased the I(K) current amplitude and migration of granule cells. Meanwhile, TEA, the I(K) channel blocker, decreased the I(K) current and slowed the migration of granule cells. Furthermore, the effects of MT on the I(K) current and cell migration were not abolished by pre-incubation with P7791, a specific antagonist of MT(3)R, but were eliminated by the application of the MT(2)R antagonists K185 and 4-P-PDOT. I(K) current and cell migration were decreased by the application of dibutyryl cyclic AMP (dbcAMP), which was in contrast to the MT effect on the I(K) current and cell migration. Incubation with dbcAMP essentially blocked the MT-induced increasing effect. Moreover, incubation of isolated cell cultures in the MT-containing medium also decreased the cAMP immunoreactivity in the granule cells. It is concluded, therefore, that I(K) current, downstream of a cAMP transduction pathway, mediates the migration of rat cerebellar granule cells stimulated by MT.     

GABAA receptor-controlled 36Cl- influx in cultured rat cerebellar granule cells

gamma-Aminobutyric acid (GABA)-mediated and bicuculline-sensitive 36Cl- influx and bicuculline-sensitive [3H] GABA binding were demonstrated in cultures of rat cerebellar granule cells. The addition of 10(-5) M GABA produced a two-fold increase in 36Cl-influx over the basal level and the maximal increase was observed after approximately 20 sec. Progressive occupation of GABAA receptor by [3H]-(1S-9R)-bicuculline methiodide decreased 36Cl- influx activated by 10 microM GABA. The above results suggest that primary cultures of rat cerebellar granule cells provide a new and reliable model for studying the GABA activated chloride fluxes.     

Depressed transient outward potassium current density in catecholamine-depleted rat ventricular myocytes

The effect of catecholamine depletion (induced by prior treatment with reserpine) was studied in Wistar rat ventricular myocytes using whole cell voltage-clamp methods. Two calcium-independent outward currents, the transient outward potassium current (I(to)) and the sustained outward potassium current (I(sus)), were measured. Reserpine treatment decreased tissue norepinephrine content by 97%. Action potential duration in the isolated perfused heart was significantly increased in reserpine-treated hearts. In isolated ventricular myocytes, I(to) density was decreased by 49% in reserpine-treated rats. This treatment had no effect on I(sus). The I(to) steady-state inactivation-voltage relationship and recovery from inactivation remained unchanged, whereas the conductance-voltage activation curve for reserpine-treated rats was significantly shifted (6.7 mV) toward negative potentials. The incubation of myocytes with 10 microM norepinephrine for 7-10 h restored I(to), an effect that was abolished by the presence of actinomycin D. Norepinephrine (0.5 microM) had no effect on I(to). However, in the presence of both 0.5 microM norepinephrine and neuropeptide Y (0.1 microM), I(to) density was restored to its control value. These results suggest that the sympathetic nervous system is involved in I(to) regulation. Sympathetic norepinephrine depletion decreased the number of functional channels via an effect on the alpha-adrenergic cascade and norepinephrine is able to restore expression of I(to) channels.     

Meningeal cells stimulate and direct the migration of cerebellar external granule cells in vitro

The external granular layer is a secondary proliferative zone that arises from the caudolateral margin of the cerebellar ventricular zone and then spreads beneath the pial surface, eventually covering the entire cerebellar anlage. Here, both a part of the Bergmann glia and granule cells are generated. Selective destruction of the leptomeningeal cell layer during development in vivo disrupts the subpial extension of the external granular layer and the laminar deposition of its descendant cells. The mechanisms by which meningeal fibroblasts exert their controlling influence on cortical development have remained unclear but could involve diffusible factors and/or interactions mediated by direct cellular contacts. In order to test these assumptions, we have co-cultivated cerebellar slice explants with meningeal cells with and without interposition of a microfilter barrier. In this setup, meningeal cells by a diffusible factor stimulated the emigration of immature neurons exclusively from the external granular layer. This effect could also be elicited by fibroblasts from other tissues but not by nonfibroblastic cells such as, e.g., astroglia. In the Boyden chamber assay, the migration of undifferentiated neurons isolated from the external granular layer was chemotactically oriented towards the source of meningeal cell-conditioned media. In comparison, neurons from the internal granular layer did not respond to this stimulus. The attraction of immature neurons towards the pial surface could (1) represent a mechanism for the establishment of (subpial) secondary proliferative zones and (2) hypothetically also play a role in the outward-directed migration of postmitotic cells, e.g., in the isocortical anlage.     

Bioenergetic analysis of cerebellar granule neurons undergoing apoptosis by potassium/serum deprivation

Apoptosis induced by K+/serum deprivation (low K+) in cerebellar granule neurons has been extensively investigated. The mitochondria play a key role in apoptosis by releasing proapoptotic factors into the cytoplasm, and mitochondrial dysfunction has been proposed as an early or initiating event in this model. To directly test this hypothesis, cellular and mitochondrial bioenergetics were quantified by determining the respiratory parameters of coverslip-attached neurons. While oxidative phosphorylation rate decreased 39-49% in low K+, this was due to decreased cellular ATP demand rather than impaired ATP/ADP exchange or respiratory chain inhibition. From 3 to 5 h in low K+, apoptosis progressed from 13 to 40% despite no appreciable change in respiratory parameters. Changes in steady-state O2-, assessed with dihydroethidium, were seen in granule but not hippocampal neurons. The O2- change correlated with changes in [Ca2+]c, but not mitochondrial respiration. Thus, early mitochondrial dysfunction can be excluded in this common model of neuronal apoptosis.     

Blockade of calcium entry accelerates arsenite-mediated apoptosis in rat cerebellar granule cells

Arsenical exposure can cause defects in the central nervous system, yet the underlying cellular and molecular mechanisms are largely unknown. We have recently demonstrated that sodium arsenite induces apoptosis of cultured cortical and cerebellar neurons, suggesting that arsenite-induced neuronal apoptosis may contribute to at least some of its neurotoxic effects. Here we investigated the effect of Ca2+ on arsenite-mediated cerebellar granule neuron death. Sodium arsenite induced apoptosis in cerebellar neurons which were maintained in the presence of serum and depolarizing concentrations of potassium chloride (25 mM KCI). Under these conditions, inhibition of calcium entry by N-methyl-D-aspartate (NMDA) receptor blocker DL-aminophosphonovalerate (APV) or calcium channel antagonist nifedipine increased arsenite-induced apoptosis, while APV or nifedipine alone had little effect on cell viability. In cortical neurons or cerebellar neurons maintained at low potassium (5 mM), arsenite also induced apoptosis. However, the addition of APV or nifedipine did not alter levels of arsenite-induced apoptosis. These data suggest that arsenite-mediated apoptosis is regulated by intracellular calcium levels.     

Pycnogenol and vitamin E inhibit ethanol-induced apoptosis in rat cerebellar granule cells

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol-induced activation of caspase-3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure.     

Antiapoptotic effects of roscovitine in cerebellar granule cells deprived of serum and potassium: a cell cycle-related mechanism

Neuronal apoptosis may be partly due to inappropriate control of the cell cycle. We used serum deprivation as stimulus and reduced potassium from 25 to 5mM (S/K deprivation), which induces apoptosis in cerebellar granule neurons (CGNs), to evaluate the direct correlation between re-entry in the cell cycle and apoptosis. Roscovitine (10 microM), an antitumoral drug that inhibits cyclin-dependent kinase 1 (cdk1), cdk2 and cdk5, showed a significant neuroprotective effect on CGNs deprived of S/K. S/K deprivation induced the expression of cell cycle proteins such as cyclin E, cyclin A, cdk2, cdk4 and E2F-1. It also caused CGNs to enter the S phase of the cell cycle, measured by a significant incorporation of BrdU (30% increase over control cells), which was reduced in the presence of roscovitine (10 microM). On the other hand, roscovitine modified the expression of cytochrome c (Cyt c), Bcl-2 and Bax, which are involved in the apoptotic intrinsic pathway induced by S/K deprivation. We suggest that the antiapoptotic effects of roscovitine on CGNs are due to its anti-proliferative efficacy and to an action on the mitochondrial apoptotic mechanism.     

Event-driven simulation of cerebellar granule cells

Around half of the neurons of a human brain are granule cells (approximately 10(11)granule neurons) [Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science. McGraw-Hill Professional Publishing, New York]. In order to study in detail the functional role of the intrinsic features of this cell we have developed a pre-compiled behavioural model based on the simplified granule-cell model of Bezzi et al. [Bezzi, M., Nieus, T., Arleo, A., D'Angelo, E., Coenen, O.J.-M.D., 2004. Information transfer at the mossy fiber-granule cell synapse of the cerebellum. 34th Annual Meeting. Society for Neuroscience, San Diego, CA, USA]. We can use an efficient event-driven simulation scheme based on lookup tables (EDLUT) [Ros, E., Carrillo, R.R., Ortigosa, E.M., Barbour, B., Ags, R., 2006. Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics. Neural Computation 18 (12), 2959-2993]. For this purpose it is necessary to compile into tables the data obtained through a massive numerical calculation of the simplified cell model. This allows network simulations requiring minimal numerical calculation. There are three major features that are considered functionally relevant in the simplified granule cell model: bursting, subthreshold oscillations and resonance. In this work we describe how the cell model is compiled into tables keeping these key properties of the neuron model.     

Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.