Activity In Organotypic Cultures Research Articles

Ceramide Kinase Inhibition Blocks IGF-1-Mediated Survival of Otic Neurosensory Progenitors by Impairing AKT Phosphorylation.

Sphingolipids are bioactive lipid components of cell membranes with important signal transduction functions in health and disease. Ceramide is the central building block for sphingolipid biosynthesis and is processed to form structurally and functionally distinct sphingolipids. Ceramide can be phosphorylated by ceramide kinase (CERK) to generate ceramide-1-phosphate, a cytoprotective signaling molecule that has been widely studied in multiple tissues and organs, including the developing otocyst. However, little is known about ceramide kinase regulation during inner ear development. Using chicken otocysts, we show that genes for CERK and other enzymes of ceramide metabolism are expressed during the early stages of inner ear development and that CERK is developmentally regulated at the otic vesicle stage. To explore its role in inner ear morphogenesis, we blocked CERK activity in organotypic cultures of otic vesicles with a specific inhibitor. Inhibition of CERK activity impaired proliferation and promoted apoptosis of epithelial otic progenitors. CERK inhibition also compromised neurogenesis of the acoustic-vestibular ganglion. Insulin-like growth factor-1 (IGF-1) is a key factor for proliferation, survival and differentiation in the chicken otocyst. CERK inhibition decreased IGF-1-induced AKT phosphorylation and blocked IGF-1-induced cell survival. Overall, our data suggest that CERK is activated as a central element in the network of anti-apoptotic pro-survival pathways elicited by IGF-1 during early inner ear development.

Development of Cortical Pyramidal Cell and Interneuronal Dendrites: a Role for Kainate Receptor Subunits and NETO1.

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.

Midazolam is effective to reduce cortical network activity in organotypic cultures during severe cholinergic overstimulation with soman

Intoxication with organophosphorus compounds can result in life-threatening organ dysfunction and refractory seizures. Sedation or hypnosis is essential to facilitate mechanical ventilation and control seizure activity. The range of indications for midazolam includes both hypnosis and seizure control. Since benzodiazepines cause sedation and hypnosis by dampening neuronal activity of the cerebral cortex, we investigated the drug’s effect on action potential firing of cortical neurons in brain slices. Extensive cholinergic overstimulation was induced by increasing acetylcholine levels and simultaneously treating the slices with soman to block acetylcholinesterase activity. At control conditions midazolam reduced discharge rates (median/95% confidence interval) from 8.8 (7.0–10.5) Hz (in the absence of midazolam) to 2.2 (1.4–2.9) Hz (10 μM midazolam) and 1.6 (0.9–2.2) Hz (20 μM midazolam). Without midazolam, cholinergic overstimulation significantly enhanced neuronal activity to 13.1 (11.0–15.2) Hz. Midazolam attenuated firing rates during cholinergic overstimulation to 6.5 (4.8–8.2) Hz (10 μM midazolam) and 4.1 (3.3–6.0) Hz (20 μM midazolam), respectively. Thus, high cholinergic tone attenuated the drug’s efficacy only moderately. These results suggest that midazolam is worth being tested as a promising drug to induce sedation and hypnosis in patients suffering from severe organophosphorous intoxication.

Chemoprevention of Colon and Small Intestinal Tumorigenesis in APCmin/+ Mice By SHetA2 (NSC721689) without Toxicity

The occurrence of intestinal polyps in people at high risk for developing colorectal cancer provides an opportunity to test the efficacy of chemoprevention agents. In this situation of treating otherwise healthy people, the potential for toxicity must be minimal. The small-molecule flexible heteroarotinoid (Flex-Het), called SHetA2, has chemoprevention activity in organotypic cultures in vitro and lack of toxicity at doses capable of inhibiting xenograft tumor growth in vivo. The objective of this study was to evaluate SHetA2 chemoprevention activity and toxicity in the APC(min/+) murine model. Oral administration of SHetA2 at 30 and 60 mg/kg five days per week for 12 weeks significantly reduced development of intestinal polyps by 40% to 60% depending on the dose and sex of the treatment group. Immunohistochemical and Western blot analysis of polyps showed reduced levels of cyclin D1 and proliferating cell nuclear antigen in both SHetA2 treatment groups. Western blot analysis also showed SHetA2 induction of E-cadherin, Bax, and caspase-3 cleavage along with reduction in Bcl-2, COX-2, and VEGF, consistent with SHetA2 regulation of apoptosis, inflammation, and angiogenesis. Neither dose caused weight loss nor gross toxicity in APC(min/+) or wild-type littermates. Magnetic resonance imaging (MRI) of cardiac function showed no evidence of SHetA2 toxicity. SHetA2 did not alter left ventricular wall thickness. In summary, SHetA2 exerts chemoprevention activity without overt or cardiac toxicity in the APC(min/+) model. SHetA2 modulation of biomarkers in colon polyps identifies potential pharmacodynamic endpoints for SHetA2 clinical trials.

RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

BackgroundEarly inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood.Methodology/Principal FindingsBy using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation.Conclusions/SignificanceWe conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.

Cyclooxygenase-2 is a neuronal target gene of NF-κB

BackgroundNF-κB is implicated in gene regulation involved in neuronal survival, inflammmatory response and cancer. There are relatively few neuronal target genes of NF-κB characterized.ResultsWe have identified the neuronal cyclooxygenase-2 (COX-2) as a NF-κB target gene. In organotypic hippocampal slice cultures constitutive NF-κB activity was detected, which was correlated with high anti-COX-2 immunoreactivity. Aspirin a frequently used painkiller inhibits neuronal NF-κB activity in organotypic cultures resulting in a strong inhibition of the NF-κB target gene COX-2. Based on these findings, the transcriptional regulation of COX-2 by NF-κB was investigated. Transient transfections showed a significant increase of COX-2 promoter activity upon stimulation with PMA, an effect which could be obtained also by cotransfection of the NF-κB subunits p65 and p50. In the murine neuroblastoma cell line NB-4, which is characterized by constitutive NF-κB activity, COX-2 promoter activity could not be further increased with PMA or TNF. Constitutive promoter activity could be repressed upon cotransfection of the inhibitory subunit IκB-α. EMSA and mutational analysis conferred the regulatory NF-κB activity to the promoter distal κB-site in the human COX-2 promoter.ConclusionsNF-κB regulates neuronal COX-2 gene expression, and acts as an upstream target of Aspirin. This extends Aspirin's mode of action from a covalent modification of COX-2 to the upstream regulation of COX-2 gene expression in neurons.

Activity-dependent development of spontaneous bioelectric activity in organotypic cultures of rat occipital cortex

The development of spontaneous bioelectric activity (SBA) in organotypic tissue cultures (OTCs) from rat occipital cortex was studied by means of extracellular recording techniques in OTCs grown normally for 6–51 days in vitro (DIV), and in OTCs in which SBA had been silenced from DIV 4 on for 2 to 3 weeks by elevating the Mg 2+ levels in the growth medium. The proportions of spontaneously active neurones increased from about 25% at 6–14 DIV to more than 80% beyond the third week in vitro. Mature neurones discharged at shorter intervals and more vigorously than immature neurones; the developmental increase in firing rate was not significant, however. In OTCs 6–14 DIV the majority of spontaneously active neurones fired sluggishly in a regular manner. The remaining neurones fired action potentials in the form of discrete bursts resembling interictal activity in vivo. The proportions of these neurones increased from about 40% at 6–14 DIV to more than 80% beyond the third week in vitro. During development in vitro the mean burst duration increased from 3.5 s to about 8 s whereas the mean burst rate (between 0.7–1 bursts/min) remained constant. Activity-deprived neurons had low firing rates and fired action potentials in the form of discrete bursts with a mean burst rate of 0.4/min. The proportions of spontaneously active neurons, the variability of neuronal firing and the viability of the explants either were not altered by the activity blockade or had recovered to control values after 5–6 days in normal growth medium. We conclude that in OTCs of rat neocortex the absence of SBA during development in vitro delays the maturation of excitatory mechanisms responsible for the developmental increase in firing intensity. The development of burst firing modes is less affected by activity blockade.

Spontaneous neuronal activity in organotypic cultures of mouse dorsal root ganglion leads to upregulation of calcium channel expression on remote Schwann cells

It is well established that neurons regulate the properties of both central and peripheral glial cells. Some of these neuro-glial interactions are modulated by the pattern of neuronal electrical activity. In the present work, we asked whether blocking the electrical activity of dorsal root ganglion (DRG) neurons in vitro by a chronic treatment with tetrodotoxin (TTX) would modulate the expression of the T-type Ca2+ channel by mouse Schwann cells. When recorded in their culture medium, about one-half of the DRG neurons spontaneously fired action potentials (APs). Treatment for 4 days with 1 μM TTX abolished both spontaneous and evoked APs in DRG neurons and in parallel significantly reduced the percentage of Schwann cells expressing Ca2+ channel currents. On the fraction of Schwann cells still expressing Ca2+ channel currents, these currents had electrophysiological parameters (mean amplitude, mean inactivation time constant, steady-state inactivation curve) similar to those of control cultures. Co-treatment for 4 days with 1 μM TTX and 2 mM CPT-cAMP, a cAMP analogue that induces the expression de novo of Ca2+ channel currents in Schwann cells deprived of neurons, maintained the percentage of Schwann cells expressing Ca2+ channel currents, showing that TTX does not directly affect the expression of Ca2+ channel currents by Schwann cell. We conclude that blocking spontaneous activity of DRG neurons in vitro downregulates Ca2+ channel expression by Schwann cells. These results strongly suggest that DRG neurons upregulate Ca2+ channel expression by Schwann cells via the release of a diffusible factor whose secretion is dependent on electrical activity. GLIA 29:281–287, 2000. © 2000 Wiley-Liss, Inc.

Spontaneous neuronal activity in organotypic cultures of mouse dorsal root ganglion leads to upregulation of calcium channel expression on remote Schwann cells

It is well established that neurons regulate the properties of both central and peripheral glial cells. Some of these neuro-glial interactions are modulated by the pattern of neuronal electrical activity. In the present work, we asked whether blocking the electrical activity of dorsal root ganglion (DRG) neurons in vitro by a chronic treatment with tetrodotoxin (TTX) would modulate the expression of the T-type Ca(2+) channel by mouse Schwann cells. When recorded in their culture medium, about one-half of the DRG neurons spontaneously fired action potentials (APs). Treatment for 4 days with 1 microM TTX abolished both spontaneous and evoked APs in DRG neurons and in parallel significantly reduced the percentage of Schwann cells expressing Ca(2+) channel currents. On the fraction of Schwann cells still expressing Ca(2+) channel currents, these currents had electrophysiological parameters (mean amplitude, mean inactivation time constant, steady-state inactivation curve) similar to those of control cultures. Co-treatment for 4 days with 1 microM TTX and 2 mM CPT-cAMP, a cAMP analogue that induces the expression de novo of Ca(2+) channel currents in Schwann cells deprived of neurons, maintained the percentage of Schwann cells expressing Ca(2+) channel currents, showing that TTX does not directly affect the expression of Ca(2+) channel currents by Schwann cell. We conclude that blocking spontaneous activity of DRG neurons in vitro downregulates Ca(2+) channel expression by Schwann cells. These results strongly suggest that DRG neurons upregulate Ca(2+) channel expression by Schwann cells via the release of a diffusible factor whose secretion is dependent on electrical activity.

The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus

Intracellular recordings were made from CA1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N- methyl- d-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist d-2-amino-5-phosphonovalerate ( d-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.