Rat Cerebellar Cultures Research Articles

A Low Ethanol Dose Affects all Types of Cells in Mixed Long-Term Embryonic Cultures of the Cerebellum

The beneficial effect of the '1-drink-a-day' lifestyle is suggested by studies of cardiovascular health, and this recommendation is increasingly followed in many countries. The main objective of this study was to determine whether this pattern of ethanol use would be detrimental to a pregnant woman. We exposed a primary culture of rat cerebellum from embryonic day 17 (corresponding to second trimester in humans) to ethanol at a concentration of 17.6 mM which is roughly equivalent to one glass of wine. Acutely, there was no change in cell viability after 5 or 8 days of exposure relative to control. By 11 days, a reduction in the number of viable cells was observed without an accompanying change in caspase-3 activity (marker of apoptotic cell death), suggesting changes in cell proliferation. As the proportion of nestin-positive cells was higher in the ethanol-treated cultures after 5 days, we hypothesized that an increase in differentiation to neurons would compensate for the ongoing neuronal death. However, there were limits to this compensatory ability as the relative proportion of nestin-positive cells was decreased after 11 days. To further illustrate the negative long-term effects of this ethanol dose, cultures were exposed for 30 days. After this period, virtually no neurons or myelinating oligodendrocytes were present in the ethanol-treated cultures. In conclusion, chronic exposure to ethanol, even at small doses, dramatically and persistently affects normal development.

Cerebellar Purkinje cells incorporate immunoglobulins and immunotoxins in vitro: implications for human neurological disease and immunotherapeutics

BackgroundImmunoglobulin G (IgG) antibodies reactive with intracellular neuronal proteins have been described in paraneoplastic and other autoimmune disorders. Because neurons have been thought impermeable to immunoglobulins, however, such antibodies have been considered unable to enter neurons and bind to their specific antigens during life. Cerebellar Purkinje cells - an important target in paraneoplastic and other autoimmune diseases - have been shown in experimental animals to incorporate a number of molecules from cerebrospinal fluid. IgG has also been detected in Purkinje cells studied post mortem. Despite the possible significance of these findings for human disease, immunoglobulin uptake by Purkinje cells has not been demonstrated in living tissue or studied systematically.MethodsTo assess Purkinje cell uptake of immunoglobulins, organotypic cultures of rat cerebellum incubated with rat IgGs, human IgG, fluorescein-conjugated IgG, and rat IgM were studied by confocal microscopy in real time and following fixation. An IgG-daunorubicin immunotoxin was used to determine whether conjugation of pharmacological agents to IgG could be used to achieve Purkinje cell-specific drug delivery.ResultsIgG uptake was detected in Purkinje cell processes after 4 hours of incubation and in Purkinje cell cytoplasm and nuclei by 24-48 hours. Uptake could be followed in real time using IgG-fluorochrome conjugates. Purkinje cells also incorporated IgM. Intracellular immunoglobulin did not affect Purkinje cell viability, and Purkinje cells cleared intracellular IgG or IgM within 24-48 hours after transfer to media lacking immunoglobulins. The IgG-daunomycin immunotoxin was also rapidly incorporated into Purkinje cells and caused extensive, cell-specific death within 8 hours. Purkinje cell death was not produced by unconjugated daunorubicin or control IgG.ConclusionPurkinje cells in rat organotypic cultures incorporate and clear host (rat) and non-host (human or donkey) IgG or IgM, independent of the immunoglobulin's reactivity with Purkinje cell antigens. This property permits real-time study of immunoglobulin-Purkinje cell interaction using fluorochrome IgG conjugates, and can allow Purkinje cell-specific delivery of IgG-conjugated pharmacological agents. Antibodies to intracellular Purkinje cell proteins could potentially be incorporated intracellularly to produce cell injury. Antibodies used therapeutically, including immunotoxins, may also be taken up and cause Purkinje cell injury, even if they do not recognize Purkinje cell antigens.

Essential involvement of the NMDA receptor in ethanol preconditioning‐dependent neuroprotection from amyloid‐βin vitro

In several epidemiological studies, moderate ethanol consumption has been associated with reduced risks of cognitive decline or Alzheimer's dementia. Of potential relevance is that brain cultures preconditioned with moderate ethanol concentrations are resistant to neurotoxic Alzheimer's amyloid-beta (Abeta) peptides. Using rat cerebellar mixed cultures we investigated whether certain membrane receptors were early 'sensors' in moderate ethanol preconditioning (MEP). In a 6-day MEP protocol (30 mM ethanol), neuroprotection from Abeta25-35 was undiminished by antagonism during the first 3 days of either adenosine A(1) or Galpha(i/o) protein-coupled receptors. However, similar cotreatment with memantine or DL-2-amino-5-phosphono-pentanoic acid (AP-5), antagonists of NMDA receptors (NMDAR), abolished neuroprotection, indicating key early involvement of this ionotropic glutamate receptor. Also in these cultures, directly activating NMDAR using subexcitotoxic NMDA preconditioning prevented Abeta neurotoxicity. By day 2 of MEP, we observed increased levels of NMDAR subunits NR1, NR2B, and NR2C that persisted through day 6. Interestingly, memantine co-exposure blocked elevations in the obligatory NR1 subunit. Furthermore, 2 days of MEP significantly increased two indicators of synaptic NMDAR localization, NR2B phospho-Tyr1472, and post-synaptic density 95 scaffolding protein. The results indicate that ethanol preconditioning-dependent neuroprotection is associated with early increases in NR subunits concomitant with enhancement of synaptic localization and activity of NMDAR.

Involvement of independent mechanism upon poly(ADP‐ribose) polymerase (PARP) activation in methylmercury cytotoxicity in rat cerebellar granule cell culture

Poly(ADP-ribose) polymerase (PARP) activation plays a role in repairing injured DNA, while its overactivation is involved in various diseases, including neuronal degradation. In the present study, we investigated the use of a PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), whether methylmercury-induced cell death in the primary culture of cerebellar granule cells involved PARP activation. DPQ decreased the methylmercury-induced cell death in a dose-dependent manner. Unexpectedly, this protective effect was DPQ specific; none of the other PARP inhibitors--1,5-dihydroxyisoquinoline, 3-aminobenzamide, or PJ34--affected neuronal cell death. Methylmercury-induced cell death involves the decrease of glutathione (GSH) and production of reactive oxygen species. Therefore, to understand the mechanism by which DPQ inhibits cytotoxicity, we first studied the effect of DPQ on buthionine sulfoximine- or diethyl maleate-induced death of primary cultured cells and human neuroblastoma IMR-32 cells, both of which are mediated by GSH depletion. DPQ inhibited the cell death of both cultured cells, but it did not restore the decrease of cellular GSH by buthionine sulfoximine to the control level. Second, we evaluated the antioxidant activity of PARP inhibitors by methods with ABTS (2-2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) or DPPH (1,1-diphenyl-2-picrylhydrazyl) used as a radical because antioxidants also efficiently suppress methylmercury-induced cell death. The antioxidant activity of DPQ was the lowest among the tested PARP inhibitors. Taken together, our results indicate that DPQ effectively protects cells against methylmercury- and GSH depletion-induced death. Furthermore, they suggest that DPQ exerts its protective effect through a mechanism other than PARP inhibition and direct antioxidation, and that PARP activation is not involved in methylmercury-induced neuronal cell death.

Astroglia growth retardation and increased microglia proliferation by lithium and ornithine decarboxylase inhibitor in rat cerebellar cultures: Cytotoxicity by combined lithium and polyamine inhibition

Lithium, the most prevalent treatment for manic-depressive illness, might have a neuroprotective effect after brain injury. In culture, lithium can exert neurotoxic effects associated with reduction in polyamine synthesis but neuroprotective effects as cultured neurons mature. Cumulative evidence suggests that lithium may exert some of its effects on neurons indirectly, by initially acting on glial cells. We used rat cerebellar cultures to ascertain the effects of lithium on ornithine decarboxylase (ODC) activity, the enzyme catalyzing the first step in polyamine synthesis, and to compare effects of lithium with those of the ODC inhibitor alpha-difluoromethylornithine (DFMO) on neuron survival and glial growth. Switching cultures from high (25 mM) to low (5 mM) KCl concentrations served as the traumatic neuronal insult. The results indicate the following. 1) Whereas high depolarizing KCl concentration enhances neuron survival, it inhibits astroglial growth. 2) Lithium (LiCl; 1-5 mM) enhances neuronal survival but inhibits astroglial growth. 3) Lithium treatment leads to reduced ODC activity. 4) DFMO enhances neuron survival but inhibits astroglial growth. 5) Lithium and DFMO lead to transformation of astroglia from epithelioid (flat) to process-bearing morphology and to increased numbers of microglia. 6) Combined lithium plus DFMO treatment is cytolethal to both neurons and glia in culture. In conclusion, lithium treatment results in growth retardation and altered cell morphology of cultured astroglia and increased microglia proliferation, and these effects may be associated with inhibition of polyamine synthesis. This implies that direct effects on astrocytes and microglia may contribute to the effects of lithium on neurons.

Expression of C/EBPα and C/EBPβ in glial cells in vitro after inducing glial activation by different stimuli

We studied the involvement of two members of the CCAAT/enhancer binding proteins (C/EBPs) family of transcription factors, C/EBPα and C/EBPβ, in glial activation induced by different stimuli in rat cerebellar neuronal-glial cultures. Glial activation was induced by two types of neuronal death – excitotoxic and apoptotic. We had previously reported that these two induction mechanisms resulted in different patterns of glial activation (K. Pérez-Capote, J. Serratosa, C. Solà, Excitotoxic and apoptotic neuronal death induce different patterns of glial activation in vitro, J. Neurochem. 94 (2005) 226–237), only the former involving an inflammatory response. Glial activation was also induced by lipopolysaccharide (LPS) from E. coli, an inflammatory agent with a known direct effect on glial cells. Using immunocytochemical techniques, here we examined whether changes in C/EBPα and C/EBPβ expression are selectively associated with a determinate pattern of glial activation. Excitotoxic neuronal death increased glial C/EBPβ expression in the absence of alterations in C/EBPα expression, while no effect was observed following neuronal apoptosis. LPS treatment decreased C/EBPα and increased C/EBPβ expression in glial cells. These results implicate C/EBPs in glial activation, although these two factors appear to have different roles. C/EBPα expression decreases only in response to LPS, while C/EBPβ expression is increased by both LPS and excitotoxic neuronal death, although not by neuronal apoptosis. These results show that C/EBPβ plays an active role in glial activation, but only when this involves an inflammatory reaction, suggesting a role for C/EBPβ in neuroinflammation.

Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression

Parkinson disease (PD) is the second-most common age-related neurodegenerative disease and is characterized by the selective destruction of dopaminergic neurons. Increasing evidence indicates that oxidative stress plays a crucial role in the pathogenesis of idiopathic PD. Anti-oxidant agents including catalase, manganese porphyrin and pyruvate confer cytoprotection to different cell cultures when challenged with 6-hydroxydopamine (6-OHDA). Herein we used rat cerebellar granular cell cultures to ascertain the plausible cellular pathways involved in pyruvate-induced cytoprotection against 0.1 mM 6-OHDA. Pyruvate provided cytoprotection in a concentration-dependent manner (2-10 mM). Consistent with its well-established anti-oxidant capacity, pyruvate (10 mM) prevented 6-OHDA-induced lipid peroxidation by blocking the rise in intracellular peroxides and maintaining the intracellular reduced glutathione (GSH) levels. Further experiments revealed that pyruvate increased Akt, but not extracellular signal-regulated kinase phosphorylation. Moreover, phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated pyruvate-induced cytoprotection indicating that PI3K-mediated Akt activation is necessary for pyruvate to induce cytoprotection. On the other hand, pyruvate also up-regulated glutathione peroxidase mRNA levels, but not those of the anti-oxidant enzymes superoxide dismutase-1 and -2, catalase or the anti-apoptotic oncogenes Bcl-2 or Bcl-xL. In summary, our results strongly suggest that pyruvate, besides the anti-oxidant properties related to its structure, exerts cytoprotective actions by activating different anti-apoptotic routes that include gene regulation and Akt pathway activation.

Chicken cerebellar granule neurons rapidly develop excitotoxicity in culture

Rat cerebellar granule cell culture is widely used as a model to study factors that control neuronal differentiation and death (e.g. excitotoxicity). However, a main drawback of this model is its dependence on depolarizing culture condition (25 mM potassium). In addition, it is quite expensive to maintain and requires animal facilities. Here we report that cerebellar granule neuron cultures from chicken may be used as an alternative model to study excitotoxicity. Surprisingly, fetal chicken cells may be grown in a physiological potassium concentration (5 mM potassium). They develop excitotoxicity rapidly in culture (fully developed at 3 days in vitro), and respond to glutamate excitotoxicity similar to rat cultures (ROS production and activation of caspase-3).

Synthesis, antioxidant activity and structure–activity relationships for a new series of 2‐(N‐acylaminoethyl)indoles with melatonin‐like cytoprotective activity

5-Methoxy-2-(N-acetylaminoethyl)indole (5d), a melatonin analogue derived from the transposition of the acetylaminoethyl side chain from C3 to C2 of the indole nucleus, had been previously characterized as a low affinity antagonist at MT1 and MT2 membrane receptors; this molecule is endowed with good in vitro antioxidant and cytoprotective potency in rat cerebellar cell cultures, comparable to or better than those of melatonin. In order to further investigate the role of structure-antioxidant activity relationships in cytoprotection, the structure of 5d was systematically modulated to design a new series of compounds. The 5-methoxy group was replaced by substituents with different electronic and lipophilic properties and it was moved to a different position on the indole ring. Other modifications of the lead structure involved the methylation of the indole nitrogen or its replacement by a sulfur atom. The side chain was also modified either increasing its lipophilicity or introducing an ionisable acid group. The antioxidant activity of this set of compounds was evaluated by the ABTS and conjugated dienes (CD) assays, while their cytoprotection was evaluated against kainate-induced cytotoxicity in cultured cerebellar neurons. In both antioxidant assays, the shift of the 5-methoxy group to the 4-position of the indole nucleus led to the most active radical scavenger (9), more potent than the parent compound and melatonin in the antioxidant tests, but much less effective as a cytoprotectant. Sharp structure-activity relationships were registered for cytoprotection, where the maintenance of the 5-alkoxy-2-(N-acylaminoethyl)indole scaffold appeared as the key feature to confer both antioxidant and cytoprotective activity to the structure. Some derivatives of the set, however, together with the most potent 5d, maintained a significant antioxidant and cytoprotective effect and could be employed as tools for in vivo pharmacological investigations on neuroprotective efficacy of melatonin-related indoles.

Robust axonal sprouting and synaptogenesis in organotypic slice cultures of rat cerebellum exposed to increased potassium chloride

Organotypic slices of the rat cerebellum, cultured in physiological levels [K +] o (5 mM) for 14 days, loose the majority of granule cells in the anterior lobe resulting in few axons and atypical Purkinje cell dendrites with vacant spines. When the culture medium was switched from 5 mM to 20, 30 or 40 mM [K +] o during the last 7 days of cultures, slices developed axons with numerous vesicle-filled boutons that made synaptic contact with Purkinje cell spines. Most boutons had one or two spine profile contacts, while some were unusually large. Enlarged boutons abutted Purkinje cell somata or their dendrites, causing intervening spines to invaginate terminals to form rosette synaptic complexes. Calbindin immuno-labeling excluded Purkinje cell axonal collaterals as the source of rosette boutons and suggested a granule cell origin. Quantification of vacant spines as compared to those on boutons revealed a threshold for potassium, between 10 and 20 mM, where the number of synaptic spines increased and vacant spines decreased drastically. These findings suggest that elevated [K +] o triggers an activity-dependent plasticity in rat cerebellar slice cultures by promoting axonal sprouting with formation of vesicle-filled boutons and synaptogenesis on open receptor sites of Purkinje cell spines.

Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation

Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, α-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca 2+ concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca 2+ concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5–p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity.

Excitotoxic and apoptotic neuronal death induce different patterns of glial activation in vitro

We have studied glial activation in rat cerebellar neuronal-glial cultures after inducing neuronal death using various stimuli. Cultures were exposed to 100 microm glutamate for 20 min, which induces excitotoxic neuronal death, or to potassium/serum deprivation, which induces apoptosis of granule neurons. We evaluated alterations in several parameters related to glial activation: nuclear factor-kappaB activation, nitric oxide and tumour necrosis factor-alpha production, which are associated with a pro-inflammatory response, glial proliferation and phagocytic activity. Although the two experimental models of neuronal damage resulted in the death of most neuronal cells within 24 h, differences were observed in the response of the various glial parameters evaluated. While nitric oxide production was not detected in any case, tumour necrosis factor-alpha production, nuclear factor-kappaB activation and glial proliferation were only induced in the presence of excitotoxic neuronal death. However, phagocytosis was induced in both cases, although earlier in the case of apoptotic neuronal death. These results show that glial cells respond to excitotoxic neuronal death with an inflammatory response associated with proliferation and phagocytosis. In contrast, whilst glial cells do not produce pro-inflammatory molecules in the presence of apoptotic neuronal death, phagocytic activity is rapidly induced.

Chronic interleukin‐6 exposure alters metabotropic glutamate receptor‐activated calcium signalling in cerebellar Purkinje neurons

Chronic central nervous system expression of the cytokine interleukin-6 (IL-6) is thought to contribute to the histopathological, pathophysiological, and cognitive deficits associated with various neurological disorders. However, the effects of chronic IL-6 expression on neuronal function are largely unknown. Previous studies have shown that chronic IL-6 exposure alters intrinsic electrophysiological properties and intracellular Ca2+ signalling evoked by ionotropic glutamate receptor activation in cerebellar Purkinje neurons. In the current study, using primary cultures of rat cerebellum, we investigated the effects of chronic IL-6 exposure on metabotropic glutamate receptor (mGluR)-activated Ca2+ signalling and release from intracellular Ca2+ stores. Chronic exposure (6-10 days) of Purkinje neurons to 500 units/mL IL-6 resulted in elevated resting Ca2+ levels and increased intracellular Ca2+ signals evoked by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) compared to untreated control neurons. Chronic IL-6 treatment also augmented Ca2+ signals evoked by brief 100 mm K+ depolarization, although to a lesser degree than responses evoked by DHPG. Depleting intracellular Ca2+ stores with sarcoplasmic-endoplasmic reticulum ATPase inhibitors (thapsigargin or cyclopiazonic acid) or blocking ryanodine receptor-dependent release from intracellular stores (using ryanodine) resulted in a greater reduction of DHPG- and K+-evoked Ca2+ signals in chronic IL-6-treated neurons than in control neurons. The present data show that chronic exposure to elevated levels of IL-6, such as occurs in various neurological diseases, alters Ca2+ signalling involving release from intracellular stores. The results support the hypothesis that chronic IL-6 exposure disrupts neuronal function and thereby may contribute to the pathophysiology associated with many neurological diseases.

HIV-1 coat protein gp120 decreases NO-dependent cyclic GMP accumulation in rat brain astroglia by increasing cyclic GMP phosphodiesterase activity

The human immunodeficiency virus type-1 (HIV-1) coat glycoprotein gp120 has been proposed as a likely etiologic agent of HIV-associated dementia (HAD). The pathogenic mechanisms underlying HAD have not yet been fully elucidated, but different evidences indicate that glial cells play an essential role in the development and amplification of the disease. The NO/cyclic GMP (cGMP) system is a widespread signal transduction pathway in the CNS involved in numerous physiological and pathological functions. Increased expression of NO synthase has been reported in the brain of AIDS patients and in cultured rodent glial cells exposed to gp120. The aim of this study was to investigate if gp120 could cause alterations in the metabolism of the NO physiological messenger cGMP that could contribute to the pathogenesis of HAD. Here, we show that long-term treatment (more than 24 h) of rat cerebellar astrocyte-enriched cultures with gp120 (10 nM) induces changes in the cultured cells—astrocyte stellation and proliferation of ameboid microglia—compatible with the acquisition of a reactive phenotype and reduces the capacity of the astrocytes to accumulate cGMP in response to NO in a time-dependent manner (maximal after 72 h). Measurements in cell extracts show that gp120 enhances Ca 2+-independent cGMP phosphodiesterase activity by 80–100% without significantly affecting soluble guanylyl cyclase (sGC). Experiments in whole cells using specific phosphodiesterase inhibitors indicate that the viral protein increases the activity of cGMP specific phosphodiesterase 5.

A Genistein-Sensitive Na+/Ca2+ Exchange Is Responsible for the Resting [Ca2+]i and Most of the Ca2+ Plasma Membrane Fluxes in Stimulated Rat Cerebellar Type 1 Astrocytes

The differential role of Na+/Ca2+ exchange in the regulation of intracellular ionized calcium ([Ca2+]i) in immunological and pharmacologically identified type 1 astrocytes and Purkinje cells was studied in rat cerebellar culture, using Ca2+ (Fluo-3, Fura-2) and Na+ (SBFI) fluorescence measurements. The mean resting [Ca2+]i was significantly higher (191 +/- 8 nM, n=25) in type 1 astrocytes than in Purkinje cells (92 +/- 2.5 nM, n=35). In contrast to Purkinje cells, in unstimulated cerebellar type 1 astrocytes, forward and reverse Na+/Ca2+ modes operate under resting physiological conditions, being responsible for most of the total Ca2+ transplasma membrane fluxes. Four observations support this hypothesis: (1) under resting conditions of temperature and ionic composition, Na+o removal causes a remarkable increase in [Ca2+]i, being inhibited by 2',4' dichlorobenzamil (DCB), and 2-[2-[4-(nitrobenzilloxiphenyl ethyl] isothiourea metanesulfonate (KB-R7943); (2) Ca2+o removal in the presence of Na+o causes an important drop in [Ca2+]i, which is absent in Li+o or NMG+o (N-methyl-D-glucamine) containing medium; (3) the reverse mode exchange inhibitor KB-R7943 mimics the removal of Ca2+o only in the presence of Na+o; and (4) under loaded [Na+]i conditions (ouabain or the activation of taurine-Na+-cotransport), reverse mode exchange increases in both astrocytes and Purkinje cells. In type 1 astrocytes stimulated with endothelin-3 (ET-3), the recovery of the Ca2+i signal occurs largely through the Na+/Ca2+ exchanger. Genistein, a tyrosine kinase inhibitor, completely and reversibly blocks all exchange activity, but not its inactive analogue daidzein, thus suggesting that the Na+/Ca2+ exchanger of cerebellar type 1 astrocytes may be modulated by phosphorylation. Our main conclusion is that in rat cerebellar type 1 astrocytes under resting physiological conditions, most of the total transplasma membrane Ca2+ fluxes take place through the Na+/Ca2+ exchanger, thus accounting for the resting [Ca(2+)]i.

Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum.

We have previously shown that progesterone (PROG) is synthesized by Schwann cells and promotes myelin formation in the peripheral nervous system (PNS). We now report that this neurosteroid also stimulates myelination in organotypic slice cultures of 7-day-old (P7) rat and mouse cerebellum. Myelination was evaluated by immunofluorescence analysis of the myelin basic protein (MBP). After 7 days in culture (7DIV), we found that adding PROG (2(-5) x 10(-5) M) to the culture medium caused a fourfold increase in MBP expression when compared to control slices. The effect of PROG on MBP expression involves the classical intracellular PROG receptor (PR): the selective PR agonist R5020 significantly increased MBP expression and the PR antagonist mifepristone (RU486) completely abolished the effect of PROG on this MBP expression. Moreover, treatment of P7-cerebellar slice cultures from PR knockout (PRKO) mice with PROG had no significant effect on MBP expression. PROG was metabolized in the cerebellar slices to 5alpha-dihydroprogesterone (5alpha-DHP) and to the GABAA receptor-active metabolite 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP, allopregnanolone). The 5alpha-reductase inhibitor L685-273 partially inhibited the effect of PROG, and 3alpha,5alpha-THP (2(-5) x 10(-5) M) significantly stimulated the MBP expression, although to a lesser extent than PROG. The increase in MBP expression by 3alpha,5alpha-THP involved GABAA receptors, as it could be inhibited by the selective GABAA receptor antagonist bicuculline. These findings suggest that progestins stimulate MBP expression and consequently suggest an increase in CNS myelination via two signalling systems, the intracellular PR and membrane GABAA receptors, and they confirm a new role of GABAA receptors in myelination.

Regulation of the Na+-dependent glutamate/aspartate transporter in rodent cerebellar astrocytes.

The regulation of the Na+-dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with L-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca2+/diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [3H]-D-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the L-glutamate-dependent [3H]-D-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the L-glutamate effect. In sharp contrast, in cortical astrocytes, both L-glutamate and TPA pre-exposure result in an augmentation of the [3H]-D-aspartate uptake. These findings suggest that the Na+-dependent glutamate uptake GLAST undergoes a region-specific regulation.

Ceramide-induced cell death in primary neuronal cultures: upregulation of ceramide levels during neuronal apoptosis.

Ceramide is a sphingolipid that has been implicated both in apoptosis and protection from cell death. We show that in both rat cerebellar granule cells and cortical neuronal cultures application of C(2)-ceramide causes cell death in a dose- and time-dependent manner. Similar effects were observed with the exogenous application of bacterial sphingomyelinase, which hydrolyzes sphingomyelin located on the outer leaflet of the plasma membrane and leads to endogenous ceramide accumulation. Furthermore, endogenous ceramide levels were increased during apoptosis induced by nutrient deprivation or etoposide treatment. These findings suggest that upregulation of ceramide levels, which may be generated through activation of sphingomyelinase, contributes to neuronal apoptosis.

Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABA A receptors

The preservation of the neuronal circuitry in rat cerebellar slice cultures provides an advantage in monitoring the development and characterizing the pharmacology of GABA A receptor subtypes. Sprague–Dawley rats, 8–11 days of age, were decapitated, their cerebella were cut into 400-μm slices and transferred into culture dishes. Cell viability and organotypic cerebellar organization of the culture remained well preserved up to 3 weeks. Autoradiographic procedures were introduced in these advanced culture technique and employed [ 3H]Ro 15-4513 in the absence and presence of 10 μM diazepam to visualize all benzodiazepine (BZD) and diazepam-insensitive (DIS) binding sites, respectively. Since expression of the α6 subunit variant of the GABA A/BZD receptor is restricted to the cerebellar granule cells and the BZD receptor agonist diazepam has very low affinity for this subunit, changes in DIS [ 3H]Ro 15-4513 binding sites during cultivation time can be attributed to changes in α6 subunit expression. A time-dependent development of total and DIS [ 3H]Ro 15-4513 binding sites were observed in the culture with a trend towards an increase in GABA A receptor α6 subunit levels during the first week. These findings suggest that explant preparations can be used to examine morphological changes in rat cerebellar slices. In addition, these preparations can be utilized to study the pharmacological effects of GABA A/BZD selective drugs on postnatal development of GABA A receptors in rat cerebellum.

Selective and AMPA Receptor-Dependent Astrocyte Death Following Prolonged Blockade of Glutamate Reuptake in Rat Cerebellar Cultures

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 μM), whereas MK-801 (10 μM), a noncompetitive blocker of N-methyl-d-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1–4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.