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Although the potential of adult neural stem cells to repair damage via cell replacement has been widely reported, the ability of endogenous stem cells to positively modulate damage is less well studied. We investigated whether medium conditioned by adult hippocampal stem/progenitor cells altered the extent of excitotoxic cell death in hippocampal slice cultures. Conditioned medium significantly reduced cell death following 24 h of exposure to 10 microM NMDA. Neuroprotection was greater in the dentate gyrus, a region neighboring the subgranular zone where stem/progenitor cells reside compared with pyramidal cells of the cornis ammonis. Using mass spectrometric analysis of the conditioned medium, we identified a pentameric peptide fragment that corresponded to residues 26-30 of the insulin B chain which we termed 'pentinin'. The peptide is a putative breakdown product of insulin, a constituent of the culture medium, and may be produced by insulin-degrading enzyme, an enzyme expressed by the stem/progenitor cells. In the presence of 100 pM of synthetic pentinin, the number of mature and immature neurons killed by NMDA-induced toxicity was significantly reduced in the dentate gyrus. These data suggest that progenitors in the subgranular zone may convert exogenous insulin into a peptide capable of protecting neighboring neurons from excitotoxic injury.

Recently, erythropoietin (EPO) and the nonerythropoietic derivative asialoEPO have been linked to tissue protection in the nervous system. In this study, we tested their effects in a model of neonatal hypoxia-ischemia (HI) in 7-day-old rats (unilateral carotid ligation and exposure to 7.7% O(2) for 50 min). EPO (10 U/g body weight = 80 ng/g; n = 24), asialoEPO (80 ng/g; n = 23) or vehicle (phosphate-buffered saline with 0.1% human serum albumin; n = 24) was injected intraperitoneally 4 h before HI. Both drugs were protective, as judged by measuring the infarct volumes, neuropathological score and gross morphological score. The infarct volumes were significantly reduced by both EPO (52%) and asialoEPO (55%) treatment, even though the plasma levels of asialoEPO had dropped below the detection limit (1 pm) at the onset of HI, while those of EPO were in the nanomolar range. Thus, a brief trigger by asialoEPO before the insult appears to be sufficient for protection. Proteomics analysis after asialoEPO treatment alone (no HI) revealed at least one differentially up-regulated protein, synaptosome-associated protein of 25 kDa (SNAP-25). Activation (phosphorylation) of ERK was significantly reduced in asialoEPO-treated animals after HI. EPO and the nonerythropoietic asialoEPO both provided significant and equal neuroprotection when administered 4 h prior to HI in 7-day-old rats. The protection might be related to reduced ERK activation and up-regulation of SNAP-25.

Activation of extracellular signal-related kinases (ERK1/2), also known as p42/44 mitogen-activated protein kinase (MAPK), is considered important for neuronal survival, cell proliferation and apoptosis. In the present study, activation (phosphorylation) of ERK1/2 (P-ERK) was investigated in brains of 7-day-oldrats after hypoxia-ischaemia (HI). In damaged areas, P-ERK-positive neurons appeared immediately after HI and the staining remained for at least 8 h. At later time points, 24 and 72 h post-HI, P-ERK-positive neurons were found in the core of the infarct and in the border zone to undamaged tissue. These cells also showed signs of DNA damage and calpain-induced fodrin breakdown, indicative of injury. At 72 h post-HI, P-ERK was also observed in microglia in the border zone to the damaged area and in astrocytes and oligodendrocytes in white matter of both hemispheres. P-ERK was strongly expressed in the subventricular zone in both hemispheres after HI at most time points, although the staining in the ipsilateral (damaged hemisphere) was stronger than in the contralateral (non-damaged hemisphere). In summary, ERK1/2 activation occurred early in neurons after HI in the neonatal brain, and mainly in cells displaying signs of damage.

Neurotoxicity in acute as well as chronic neurological diseases may be partly mediated by oxidative stress caused by overactivation of glutamate receptors. A key component of the cellular defense against oxidative stress is reduced glutathione. In our earlier work, we have shown that ischemia in brain induces increased efflux, elevated metabolism, and decreased tissue concentrations of glutathione. In this study, we have evaluated the effect of glutamate receptor activation on the efflux of glutathione from hippocampus in vitro. NMDA and kainate induced a delayed increase in glutathione, taurine, and phosphoethanolamine efflux. Extracellular glutathione was recovered mainly in the reduced form (85-95%); the efflux was dependent on extracellular calcium but unrelated to dantrolene-sensitive intracellular calcium release and independent of glutathione or NO synthesis. The NMDA-induced efflux of glutathione was enhanced by blockage of gamma-glutamyl transpeptidase, indicating an increased transpeptidation of glutathione after NMDA receptor activation. Our results suggest that increased efflux of glutathione could be a factor in initiating nerve cell death via a change in intracellular redox potential and/or a decrease in the intracellular capacity for inactivation of reactive oxygen species.

The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. L-Serine (2.5-10 mM), D-serine (10 mM), or ethanolamine (10 mM) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10-100 times in the extracellular fluid, L-serine caused a dose-dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, L-serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. D-Serine was as effective as L-serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not L-serine, also increased extracellular glutamate/aspartate levels in an MK-801-dependent fashion. A similar effect, but delayed in time, was observed with D-serine. These effects were inhibited by MK-801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.

The present work relates to the possibility that the ATP-independent enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2), which has been postulated to be part of an amino acid uptake system, is active during cerebral ischemia. This was evaluated in the ischemic rat striatum by determination of intra- and extracellular concentrations of gamma-glutamyl dipeptides (the products of the transpeptidation) and glutathione (the physiological gamma-glutamyl donor). An ischemic period (0-30 and 31-60 min) resulted in prominent increases in the respective concentration of extracellular gamma-glutamylglutamate (24- and 67-fold), gamma-glutamyltaurine + gamma-glutamylglycine (5.8- and 19-fold), and gamma-glutamylglutamine (2.6- and 6.8-fold) as revealed using in vivo microdialysis. The changes coincided with increased respective extracellular concentrations of glutamate (83- and 115-fold), taurine (17- and 25-fold), glycine (4.6- and 6.1-fold), and glutamine (1.7- and 2.1-fold). Furthermore, under anoxic conditions in vitro (0-30 and 0-60 min), respective striatal tissue concentrations were increased for gamma-glutamylglutamate (20- and 17-fold), gamma-glutamyltaurine (6.7- and 11-fold), gamma-glutamylglutamine (1.7- and 1.2-fold), and gamma-glutamylglycine (14- and 18-fold), whereas glutathione levels were, on an average, decreased by approximately 350 microM. In summary, gamma-glutamyl transpeptidase is involved in de novo dipeptide synthesis in the mammalian brain during anoxic conditions, indicating transport of amino acids such as glutamate.

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.

Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.

Tissue slices from the superior colliculi (SC) of the rabbit were superfused and investigated 1 week after unilateral eye removal. Amino acid levels were determined both in the tissue slices and in the medium after chemical depolarisation (56 mM K). The amino acid determinations were done fluorimetrically by precolumn derivation and HPLC separation. Colliculi contralateral to the enucleation exhibited a 16% reduction in glutamate compared with the ipsilateral colliculi. The Ca-dependent release of glutamate or other amino acids tested was not appreciably affected by enucleation. However, both the total and the Ca-independent release of glutamate was lower from contralateral SC slices compared with the ipsilateral slices. The results do not favour glutamate as the major optic nerve transmitter in the rabbit, but do not rule out glutamate as a transmitter in a minor population of retinal fibres.

The effect of severe insulin-induced hypoglycemia on the extracellular levels of endogenous amino acids in the rat striatum was examined using the brain microdialysis technique. A characteristic pattern of alterations consisting of a 9-12-fold increase in aspartate (Asp), and more moderate increases in glutamate (Glu), taurine (Tau), and gamma-aminobutyric acid (GABA), was noted following cessation of electroencephalographic activity (isoelectricity). Glutamine (Gln) levels were reduced both during and after the isoelectric period and there was a delayed increase in extracellular phosphoethanolamine (PEA) content. The effects of decortication and excitotoxin lesions on the severe hypoglycemia-evoked efflux of endogenous amino acids in the striatum were also examined. Decortication reduced the release of Glu and Asp both 1 week and 1 month post-lesion. The efflux of other neuroactive amino acids was not affected significantly. In contrast, GABA, Tau, and PEA efflux was attenuated in kainate-lesioned striata. Glu and Asp release was also reduced under these conditions, and a smaller decrease in extracellular Gln was noted. These data suggest that GABA, Glu, and Asp are released primarily from their transmitter pools during severe hypoglycemia. The releasable pools of Tau and PEA appear to be located in kainate-sensitive striatal neurons. The significance of these results is discussed with regard to the excitotoxic theory of hypoglycemic cell death.

Hypoglycemia-evoked changes in levels of extracellular excitatory and inhibitory amino acids were studied using the microdialysis technique. A newly designed dialysis probe was inserted stereotaxically into the rat hippocampus. Animals were then subjected to insulin-induced hypoglycemia; then blood glucose levels were restored by glucose injections after a 30-min period of isoelectric electroencephalography. Dialysates were collected before, during, and after the isoelectric period. Amino acids in the dialysates were analyzed by liquid chromatography and fluorescence detection following automatic precolumn derivatization with o-phthaldialdehyde. During the isoelectric phase, the concentration of aspartate increased 15-fold, whereas glutamate, gamma-amino-butyric acid, taurine, and phosphoethanolamine levels were elevated three- to sixfold. Smaller increases were observed for nonneuroactive amino acids such as asparagine, alanine, and phenylalanine. In contrast to all other amino acids, the glutamine content was reduced to less than 30% of preisoelectric values. The concentrations of the neuroactive amino acids were restored to normal in the post-isoelectric phase. These data demonstrate that there is an extracellular overflow of neuroactive amino acids, especially aspartate, during severe hypoglycemia.

The chemically evoked efflux of endogenous amino acids from perfused rabbit superior colliculus (SC) slices was studied. Amino acids in the perfusates were determined fluorimetrically with a precolumn derivatisation method and subsequent separation on an HPLC column. Potassium-induced depolarisation caused a calcium-dependent release of beta-alanine, GABA, glutamate, and aspartate. Veratridine-evoked efflux was essentially similar in selectivity and could be blocked by tetrodotoxin (TTX). The results are indicative of a neurotransmitter role of beta-alanine, GABA, glutamate, and aspartate in the SC of the rabbit.