Mouse Parthenogenetic Embryonic Stem Cells with Biparental-Like Expression of Imprinted Genes Generate Cortical-Like Neurons That Integrate into the Injured Adult Cerebral Cortex.

One strategy for stem cell-based therapy of the cerebral cortex involves the generation and transplantation of functional, histocompatible cortical-like neurons from embryonic stem cells (ESCs). Diploid parthenogenetic Pg-ESCs have recently emerged as a promising source of histocompatible ESC derivatives for organ regeneration but their utility for cerebral cortex therapy is unknown. A major concern with Pg-ESCs is genomic imprinting.

Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown.

The dependence on Ca2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca2+ concentrations were buffered by bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca2+ buffering on intracellular and extracellular Ca2+ concentration ([Ca2+]i and [Ca2+]e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form.

A modulation of glutamate-induced phosphoinositide breakdown by intracellular pH changes.

The influence of intracellular pH (pHi) changes on the formation of inositol phosphate metabolites (IPs) produced by glutamatergic stimulation was studied in 8-day-old rat brain synaptoneurosomes. For this purpose pHi was measured using 2',7'-bis-(2-carboxyl)-5,6-carboxyfluorescein (BCECF) fluorimetric assay in parallel with the basal and receptor-mediated formations of inositol monophosphate (IP1) and inositol bisphosphate (IP2). We found that glutamate (1 mM), which induces a transient acidification (delta pH = -0.

Kainate-induced status epilepticus leads to a delayed increase in various specific glutamate metabotropic receptor responses in the hippocampus.

Neuronal loss and gliosis were detected in the rat hippocampus soon after unilateral intra-amygdala injection of kainate (KA) (2.5 nmol) while solid mossy fiber sprouting could be seen only fourteen days after this injection. Using this experimental model, we examined the metabotropic glutamate receptor (mGluR)-induced inositol phosphate (IP) formation in hippocampal synaptoneurosomes and slices.

Ontogenesis of quisqualate-associated phosphoinositide metabolism in various regions of the rat nervous system.

The effect of postnatal age on phosphoinositide metabolism per se and on quisqualate-stimulated phosphoinositide metabolism was characterized in synaptoneurosomes prepared from nine different regions of the rat nervous system, namely the brainstem, cerebellum, cerebral cortex, colliculi, hippocampus, hypothalamus, olfactory bulb, spinal cord and striatum. In the hippocampus, striatum, cerebellum, cerebral cortex, brainstem, colliculus and spinal cord, the basal levels of inositol phosphate (inositol-1-phosphate+inositol-4,5-bisphosphate) formation were maximal two days after birth and declined steeply to steady-state levels from the age of 10 postnatal days. Similarly, in the olfactory bulb, basal inositol phosphate synthesis did not significantly change when measured during the period from postnatal day 10 to 42.

Effect of thiol reagents on phosphoinositide hydrolysis in rat brain synaptoneurosomes.

Some divalent ions, such as Cd2+ and Zn2+, are able to stimulate phosphoinositide (PI) breakdown and to inhibit receptor-mediated PI metabolism. These ions are also known to react with the free -SH groups of proteins. This prompted us to investigate the effects of more potent sulphydryl reagents, Hg2+ and p-chloromercuric benzosulphonic acid (PCMBS), on the inositol phosphate (IP) accumulation triggered by the neuroactive substances: glutamate, carbachol and K+, using synaptoneurosomes from 8-day-old rat forebrains.

Dithiotreitol specifically inhibits metabotropic responses of glutamate and depolarizing agents in rat brain synaptoneurosomes.

Dithiotreitol (DTT), a sulfhydryl reducing agent inhibits in a dose-dependent manner the inositol phosphates (IPs) accumulation responses evoked by glutamate and potassium without affecting that of carbachol in rat forebrain synaptoneurosomes. Furthermore, DTT neither provokes a depolarization of the membrane, nor increases the internal calcium concentration. Depolarization and internal calcium rise are known to stimulate IPs production.

A specific transduction mechanism for the glutamate action on phosphoinositide metabolism via the quisqualate metabotropic receptor in rat brain synaptoneurosomes: I. External Na+ requirement.

The characteristics of the transduction mechanism(s) activated by glutamate (Glu) via the quisqualate metabotropic receptor, as well as by depolarizing agents, to trigger formation of inositol phosphates (IPs) were investigated in 8-day-old rat forebrain synaptoneurosomes. The replacement of external Na+ by various compounds (Li+, Tris+, N-methyl-D-glucamine+, and sucrose) induces an increase in basal accumulation of IPs and depolarizes synaptoneurosome membranes. Under these conditions, Glu- and K(+)-induced accumulations of IPs are inhibited, whereas the carbachol (Carb)-elicited response of IPs parallels the basal one.

Pharmacological characterization of the quisqualate receptor coupled to phospholipase C (Qp) in striatal neurons.

A detailed pharmacological characterization of the quisqualate (QA) receptor coupled to phospholipase C (Qp) was performed in striatal neurons. The experiments were carried out in the presence of the ionotropic antagonists MK-801 (1 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM), concentrations that block N-methyl-D-aspartate (NMDA) or alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in these cells. QA, ibotenate and trans-1-aminocyclopentyl-1,3-dicarboxylate (ACPD) evoked dose-dependent inositol phosphate formations with EC50 values of 0.

The glutamate receptor of the Qp-type activates protein kinase C and is regulated by protein kinase C.

In striatal neurons in primary culture quisqualate potently stimulated the formation of inositol phosphates via a metabotropic receptor we recently termed Qp in order to distinguish it from the classical ionotropic quisqualate receptor termed Qi. Here we show that 10 microM of quisqualate activated in a rapid and transient manner protein kinase C as assessed by its translocation from the cytosolic to the membrane fraction. As 10 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), the Qi specific agonist, was without effect, this translocation was most probably mediated by the Qp receptor.

K+ differentially affects the excitatory amino acids- and carbachol-elicited inositol phosphate formation in rat brain synaptoneurosomes.

K+, excitatory amino acids (EAAs) and carbachol (Carb) were tested separately or in pairs for their ability to stimulate inositol phosphate (IPs) formation in rat forebrain synaptoneurosomes. K+ ions per se, stimulate IPs synthesis (158% of the control value) as well as EAAs and Carb. The glutamate (Glu)- and quisqualate (QA)-elicited IPs formation is not additive with that evoked by K+.

Developmental changes in the chemosensitivity of rat brain synaptoneurosomes to excitatory amino acids, estimated by inositol phosphate formation.

The evolution of excitatory amino acids-(EAA) stimulated inositol phosphates (IPs) turnover during postnatal development was investigated in synaptoneurosomes prepared from rat forebrains. The two main EAA agonists which induce the IPs synthesis were quisqualate (QA) and N-methyl-D-aspartate (NMDA). The QA and NMDA stimulations of IPs formation present a particular developmental pattern, characterized by an active phase during rat synaptogenesis.

A new quisqualate receptor subtype (sAA(2)) responsible for the glutamate-induced inositol phosphate formation in rat brain synaptoneurosomes.

Inositol phosphate synthesis elicited by excitatory amino acids was measured in rat forebrain synaptoneurosomes in presence of Li(+). Quisqualate (QA) was the most potent excitatory amino acid inducing inositol phosphate formation. This QA action was not blocked by any of the usual antagonists [glutamate-amino-methyl-sulphonate (GAMS); glutamate-diethyl-ester (GDEE); ?-d-glutamyl-glycine (?-DGG)] known to inhibit the QA-induced depolarization.

Characterization of subtypes of excitatory amino acid receptors involved in the stimulation of inositol phosphate synthesis in rat brain synaptoneurosomes.

The action of excitatory amino acids (EAA) on inositol phosphates (IPs) synthesis was examined in forebrain synaptoneurosomes of Long Evans rats (6-9 days old). Glutamate (GLU) (EC50: 23 microM) and quisqualate (QA) (EC50: 0.12 microM) enhanced IPs turnover.