Mónica L. Fiszman

GABA INDUCES PROLIFERATION OF IMMATURE CEREBELLAR GRANULE CELLS GROWN IN VITRO

The presence of GABA and its receptors early in rodent nervous system development has lead to speculation on the role of this transmitter system in neuroblast proliferation, migration and differentiation. We studied the effect of GABA and GABA agonists on immature cerebellar granule cell proliferation and survival. Cerebellar granule cell suspensions were obtained from 6-8-day-old rats and grown in culture for up to 7 days in serum-containing or serum-free medium. The addition of GABA (0.1-100 microM) or muscimol (0.01-10 microM) 2 h after inoculation and harvested 22 h later, lead to an increase in 3H-thymidine incorporation over control samples with the correspondent increase in granule cells number assayed 48 h later. The effect on cell proliferation exerted by GABAA agonists was blocked by MgCl2 and nifedipine, as well as by the chloride channel blocker, picrotoxin (50 microM), and the GABAA receptor specific blocker, bicuculline (50 microM). The increase on cell proliferation induced by GABA also was blocked by PD98059 (75 microM), a specific inhibitor of the mitogen-activated protein kinase kinase (MAPKK). GABAA receptor-mediated proliferation was consistently seen in cells inoculated in serum-containing medium supplemented with 25 mM KCl but not seen in serum-free medium, with 5 mM or 25 mM KCl. The presence of serum did not enhance the survival of cerebellar granule cells grown for 7 days in either 5 mM or 25 mM KCl. Additionally, neither GABA nor muscimol applied from day 2 to day 7 in vitro affected cell survival in any culture condition. We conclude that GABA and GABAA receptor agonists influence granule cell proliferation but not survival and that this effect is mediated by a calcium influx via voltage-dependent calcium channel activation, with a subsequent activation of the MAPK cascade.

GABA-induced neurite outgrowth of cerebellar granule cells is mediated by GABA(A) receptor activation, calcium influx and CaMKII and erk1/2 pathways.

During neuronal development, GABAA‐mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1–1000 µm) induces an increase in the intracellular calcium concentration through the activation of GABAA receptors and voltage‐gated calcium channels of the L‐subtype. Perforated patch‐clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to − 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro. On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium‐dependent mechanism triggered by voltage‐gated calcium channel activation. Specific blockers of calcium‐calmodulin kinase II and mitogen‐activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium‐dependent kinases.

NMDA Receptors Increase the Size of GABAergic Terminals and Enhance GABA Release

In developing cerebellar interneurons, NMDA increases spontaneous GABA release by activating presynaptic NMDA receptors. We investigated the role of these receptors on differentiating basket/stellate cells in cerebellar cultures grown under conditions allowing functional synaptic transmission. Presynaptic GABAergic boutons were visualized either by GAD65 immunostaining or by using cells derived from GAD65-enhanced green fluorescent protein (eGFP) transgenic mice, in which cerebellar basket/stellate cells express eGFP. After the first week in culture, whole-cell recordings from granule cells reveal that acute application of NMDA increases miniature IPSC (mIPSC) frequency. Interestingly, after 2 weeks, the mIPSC frequency increases compared with the first week but is not modulated by NMDA. Furthermore, in cultures chronically treated with NMDA for 1 week, the size of the GABAergic boutons increases. This growth is paralleled by increased mIPSC frequency and the loss of NMDA sensitivity. Direct patch-clamp recording from these presynaptic terminals reveals single NMDA-activated channels, showing multiple conductance levels, and electronic propagation from the somatodendritic compartment. Our results demonstrate that NMDA receptors alter GABAergic synapses in developing cerebellar cultures by increasing the size of the terminal and spontaneous GABA release. These findings parallel changes in inhibitory synaptic efficacy seen in vivo in developing GABAergic interneurons of the molecular layer of the cerebellum.

Contribution of Ca2+ calmodulin‐dependent protein kinase II and mitogen‐activated protein kinase kinase to neural activity‐induced neurite outgrowth and survival of cerebellar granule cells

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long‐term survival of cerebellar granule cells. The effect of voltage‐gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin‐dependent protein kinase II and mitogen‐activated protein kinase kinase (MEK1), to the effects of high [K+]e under serum‐free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24–48 h under low‐density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin‐dependent protein kinase II. This also produced an increase in long‐term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin‐dependent protein kinase II and of the extracellular signal‐regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin‐dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

Role of calcium and kinases on the neurotrophic effect induced by γ-aminobutyric acid

An increasing body of evidence supports a trophic action of γ‐aminobutyric acid (GABA) during nervous system development. The purported mediator of these trophic effects is a depolarizing response triggered by GABA, which elicits a calcium influx in immature CNS cells. This Mini‐Review focuses on the neurotrophic role of neural activity and GABA and some of the most common intracellular cascades activated by depolarization and trophic factors. Several biological effects induced by GABA in the developing nervous system are reviewed, with particular emphasis on what is known about calcium‐dependent neurotrophic effects induced by GABA and its intracellular mechanisms.

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media.

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H2O2 exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H2O2 to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 μM) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.

Extracellular potassium concentration regulates proliferation of immature cerebellar granule cells

The present study examines the effect of depolarizing potassium concentrations on the proliferation of immature rat cerebellar neurons. Cells inoculated in serum free medium and 5 mM KCl (5 K) showed a high degree of 3H-thymidine incorporation that decreased 24-48 h after plating as differentiation began. During the first 24 h after inoculation, cells grown in high potassium (25 K), showed a 34 +/- 3% increase (mean +/- S.E.M., n = 12) in 3H-thymidine incorporation as compared with the values observed in 5 K. After 24 h in vitro, cells grown in 25 K showed 23 +/- 3% (mean +/- S.E.M., n = 3) less DNA synthesis than those inoculated in 5 K. The increase in DNA synthesis due to 25 K was blocked by MgCl2 and nifedipine, but not by omega-conotoxin GVIA, suggesting that it is mediated by a Ca2+ influx via voltage-gated calcium channels (VGCC) of the L-subtype. High potassium-induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor (PD98059, 75 microM). The number of neurons counted after 48 h in vitro in 25 K was 35-100% above of the number obtained with 5 K and this increase also was blocked by MgCl2 and nifedipine. These data support the hypothesis that depolarizing activity during neurogenesis plays a role in the modulation of cerebellar granule cells proliferation.

Cu/Zn superoxide dismutase activity at different ages in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive disorder resulting from degeneration of motor neurons in the brain and spinal cord. Sporadic ALS (SALS) accounts for the majority of patients and the familial form (FALS) represents fewer than 10% of all cases. Since it was found that there are Cu/Zn superoxide dismutase (SODI) gene mutations in 20% of FALS patients and that FALS and SALS patients show similar clinical features, it has been postulated that both may share a common physiopathological mechanism. We studied Cu/Zn SOD1 activity in cytosolic extracts of erythrocytes from 125 normal individuals and 40 SALS patients. We found that enzyme activity does not change with age in control subjects and tends to decrease in most SALS patients older than 60 years. A subpopulation of five SALS patients had significantly increased SOD1 activity; four of these patients over 70 years old. There was no correlation between enzyme activity and time of onset of the disease, or clinical forms of the illness. The variation in SOD1 activity in ageing SALS patients compared with younger patients suggests that they may undergo an oxidative disbalance contributing to the development of the disease.

In vitro neurotoxic properties and excitatory aminoacids concentration in the cerebrospinal fluid of amyotrophic lateral sclerosis patients. Relationship with the degree of certainty of disease diagnoses.

Fiszman ML, Ricart KC, Latini A, Rodríguez G, Sica REP. In vitro neurotoxic properties and excitatory aminoacids concentration in the cerebrospinal fluid of amyotrophic lateral sclerosis patients. Relationship with the degree of certainty of disease diagnoses.
Acta Neurol Scand: 2010: 121: 120–126.
© 2009 The Authors Journal compilation

The effect of X-radiation on cerebellar granule cells grown in culture. Ganglioside GM1 neuroprotective activity.

In this paper we describe the effects of X-radiation on the viability of cerebellar granule cells grown in culture. Cell cultures were exposed to X-rays 2 h after plating and then grown for 1-7 days. Two days after X-ray exposure with a dose-range of 0.1-2 Gy (acute effect), a significant decrease in neuronal number was observed. The magnitude of the lethal effect was directly correlated to the dose of X-ray applied. When the interval between plating and irradiation was increased, the acute lethal effect of X-rays decreased. 3H-thymidine incorporation was maximal during the first 24 h in vitro and decreased to nearly blank levels, after 72 h. In some experiments, cells present in each culture dish were counted at day 2 and at day 7. We observed that the number of cells present in sham-irradiated cultures decreased from day 2 to day 7, reflecting cell death after several days in vitro. The cell loss observed in X-irradiated cultures was significantly greater as compared with sham-irradiated cultures, confirming the deleterious effect of X-ray on cell survival. This effect was completely prevented by GM1 (6.5, 10 and 30 microM) added 48 h after X-ray exposure, but not 1 h after plating. We conclude that X-rays induce two different effects: an acute effect related to impaired DNA synthesis which is very active during the first 24 h in vitro, and a long-term effect owing to a sublethal damage in the surviving neuronal population.