Shubhro Pal

Long-term alteration of calcium homeostatic mechanisms in the pilocarpine model of temporal lobe epilepsy

The pilocarpine model of temporal lobe epilepsy is an animal model that shares many of the clinical and pathophysiological characteristics of temporal lobe or limbic epilepsy in humans. This model of acquired epilepsy produces spontaneous recurrent seizure discharges following an initial brain injury produced by pilocarpine-induced status epilepticus. Understanding the molecular mechanisms mediating these long lasting changes in neuronal excitability would provide an important insight into developing new strategies for the treatment and possible prevention of this condition. Our laboratory has been studying the role of alterations in calcium and calcium-dependent systems in mediating some of the long-term neuroplasticity changes associated with epileptogenesis. In this study, [Ca(2+)](i) imaging fluorescence microscopy was performed on CA1 hippocampal neurons acutely isolated from control and chronically epileptic animals at 1 year after the induction of epileptogenesis with two different fluorescent dyes (Fura-2 and Fura-FF) having high and low affinities for [Ca(2+)](i). The high affinity Ca(2+) indicator Fura-2 was utilized to evaluate [Ca(2+)](i) levels up to 900 nM and the low affinity indicator Fura-FF was employed for evaluating [Ca(2+)](i) levels above this range. Baseline [Ca(2+)](i) levels and the ability to restore resting [Ca(2+)](i) levels after a brief exposure to several glutamate concentrations in control and epileptic neurons were evaluated. Epileptic neurons demonstrated a statistically significantly higher baseline [Ca(2+)](i) level in comparison to age-matched control animals. This alteration in basal [Ca(2+)](i) levels persisted up to 1 year after the induction of epileptogenesis. In addition, the epileptic neurons were unable to rapidly restore [Ca(2+)](i) levels to baseline following the glutamate-induced [Ca(2+)](i) loads. These changes in Ca(2+) regulation were not produced by a single seizure and were not normalized by controlling the seizures in the epileptic animals with anticonvulsant treatment. Peak [Ca(2+)](i) levels in response to different concentrations of glutamate were the same in both epileptic and control neurons. Thus, glutamate produced the same initial [Ca(2+)](i) load in both epileptic and control neurons. Characterization of the viability of acutely isolated neurons from control and epileptic animals utilizing standard techniques to identify apoptotic or necrotic neurons demonstrated that epileptic neurons had no statistically significant difference in viability compared to age-matched controls. These results provide the first direct measurement of [Ca(2+)](i) levels in an intact model of epilepsy and indicate that epileptogenesis in this model produced long-lasting alterations in [Ca(2+)](i) homeostatic mechanisms that persist for up to 1 year after induction of epileptogenesis. These observations suggest that altered [Ca(2+)](i) homeostatic mechanisms may underlie some aspects of the epileptic phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.

In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons.

Calcium ions and calcium-dependent systems have been implicated in the pathophysiology of status epilepticus (SE). However, the dynamics of intracellular calcium ([Ca2+]i) levels during SE has not yet been studied. We have employed the hippocampal neuronal culture (HNC) model of in vitro SE that produces continuous epileptiform discharges to study spatial and dynamic changes in [Ca2+]i levels utilizing confocal laser scanning microscopy and the calcium binding dye, indo-1. During SE, the average [Ca2+]i levels increased from control levels of 150-200 nM to levels of 450-600 nM. This increased [Ca2+]i was maintained for the duration of SE. Following SE, [Ca2+]i levels gradually returned to basal values. The duration of SE was shown to affect the ability of the neuron to restore resting [Ca2+]i levels. Both N-methyl-D-aspartate (NMDA) receptor-gated and voltage-gated Ca2+ channels (VGCCs) contributed to the increased calcium entry during SE. Moreover, this elevation in [Ca2+]i occurred in both the nucleus and cytosol. These results provide the first dynamic measurement of [Ca2+]i during prolonged electrographic seizure discharges in an in vitro SE model and suggest that prolonged epileptiform discharges give rise to abnormal sustained increases in [Ca2+]i levels that may play a role in the neuronal cell damage and long-term plasticity changes associated with SE.

Different Neuroligands and Signal Transduction Pathways Stimulate CREB Phosphorylation at Specific Developmental Stages Along Oligodendrocyte Differentiation

Abstract : We have shown previously that the pattern of expression of the transcription factor CREB (cyclic AMP‐response element binding protein) in developing oligodendrocytes (OLGs) suggests a role during a period that precedes the peak of myelination in rat brain. We have now investigated the signaling pathways that could be responsible for activating CREB by phosphorylation at different stages along OLG maturation. CREB phosphorylation was studied in short‐term cultures of immature OLG precursor cells and young OLGs isolated from 4‐ and 11‐day‐old rat cerebrum, respectively. The results indicated that at both developmental stages, CREB phosphorylation could be stimulated by either increased concentrations of cyclic AMP and cyclic AMP‐dependent protein kinase activation or increased Ca2+ levels and a protein kinase C activity. The results also showed that CREB phosphorylation in immature OLG precursor cells could be up‐regulated by treatment with histamine, carbachol, glutamate, and ATP (neuroligands known to increase Ca2+ levels in these cells), by signaling cascade(s) that involve a protein kinase C activity, as well as the mitogen‐activated protein kinase pathway. In contrast, in cells isolated from 11‐day‐old rats, at a developmental stage that immediately precedes the beginning of the active period of myelin synthesis, CREB phosphorylation was only stimulated by treatment with the β‐adrenergic agonist isoproterenol in a process that appears to be mediated by a cyclic AMP/cyclic AMP‐dependent protein kinase‐dependent pathway. These results support the idea that CREB could be a mediator of neuronal signals that, coupled to specific signal transduction cascades, may play different regulatory roles at specific stages along OLG differentiation.

Hippocampal neurons exhibit both persistent Ca2+ influx and impairment of Ca2+ sequestration/extrusion mechanisms following excitotoxic glutamate exposure.

Exposure of neurons to glutamate is an essential element of neuronal function, producing transient elevations in free intracellular calcium ([Ca2+]i) that are required for normal physiological processes. However, prolonged elevations in [Ca2+]i have been observed following glutamate excitotoxicity and have been implicated in the pathophysiology of delayed neuronal cell death. In the current study, we utilized indo-1 and fura-2ff Ca2+ imaging techniques to determine if glutamate-induced prolonged elevations in [Ca2+]i were due to persistent influx of extracellular Ca2+ or from impairment of neuronal Ca2+ extrusion/sequestration mechanisms. By experimentally removing Ca2+ from the extracellular solution following glutamate exposure, influx of Ca2+ into the neurons was severely attenuated. We observed that brief glutamate exposures (<5 min, 50 microM glutamate) resulted in a Ca2+ influx that continued after the removal of glutamate. The Ca2+ influx was reversible, and the cell was able to effectively restore [Ca2+]i to resting levels. Longer, excitotoxic glutamate exposures (> or = 5 min) generated a Ca2+ influx that continued for the duration of the recording period (>1 h). This persistent Ca2+ influx was not primarily mediated through traditionally recognized Ca2+ channels such as glutamate receptor-operated channels or voltage-gated Ca2+ channels. In addition to the persistent Ca2+ influx, longer glutamate exposures also produced a lasting disruption of Ca2+ extrusion/sequestration mechanisms, impairing the ability of the neuron to restore resting [Ca2+]i. These data suggest that glutamate-induced protracted [Ca2+]i elevations result from at least two independent, simultaneously occurring alterations in neuronal Ca2+ physiology, including a persistent Ca2+ influx and damage to Ca2+ regulation mechanisms.

Effect of Nerve Growth Factor and Forskolin on Glycosyltransferase Activities and Expression of a Globo‐Series Glycosphingolipid in PC12D Pheochromocytoma Cells

Abstract: The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK‐dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo‐series neutral GSL, Galα1‐3Galα1‐4Galβ1‐4Glcβ1‐1′Cer (GalGb3), and the activity of GalGb3‐synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)‐, GM3‐, GD1a‐, and GM2‐synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti‐GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3‐synthase activity may be closely related to the cellular differentiation process in this cell line.

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: Relationship with neuronal development

Previous studies from this laboratory demonstrated the presence of a UDP-galactose:Gb3Cer α1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Galα1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the α-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Galα1-3Gb3Cer in DRGN, we examined the expression of Galα1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Galα1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after, 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cell bodies and neurites. The expression of Galα1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Galα1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development.