Exploring atypical locations of mammalian neural stem cells: the human filum terminale.

Neurogenesis is a multifactorial event determined by local environmental cues, inherent cellular program as well as cellular milieu and may not necessarily be restricted to the SVZ and SGZ. NSCs have been isolated from or neurogenesis has been demonstrated in traditionally non neurogenic regions. This more permissive view of neurogenesis, however, is not widely accepted due to concerns regarding the methodologies used. Furthermore, it is compounded by the fact that the basal levels of increased neurogenesis in such regions has not been completely confirmed and thus precludes a paradigm shift. Were this non limited view of neurogenesis to be generally accepted after thorough investigation, it would open new avenues for regenerative medicine and stem cell therapy.

L-alpha-aminoadipate reduces glutamate release from brain tissue exposed to combined oxygen and glucose deprivation.

The effect of the glutamate analogue L-alpha-aminoadipate (alpha AA) on the release of glutamate and gamma-aminobutyric acid (GABA) from rat hippocampal slices was investigated in vitro. Oxygen/glucose deprivation caused a large release of glutamate and GABA. alpha AA added during energy deprivation reduced the glutamate release in a dose-dependent manner (56% reduction at 5 mM), whereas GABA release was unchanged. We speculate that ischemic glutamate release from the brain is mediated by a low affinity transport mechanism that is blocked by alpha AA.

Redistribution of glutamate and glutamine in slices of human neocortex exposed to combined hypoxia and glucose deprivation in vitro.

This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precursor, during cerebral ischemia. Slices (400-600 microns) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to "ischemic" conditions, were then fixed and embedded. Ultrathin sections were processed according to a postembedding immunocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloidal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose deprivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neuronal somata declined further to very low values, compared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose deprivation. This treatment was also associated with a reduced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmembrane ion concentrations and membrane potential probably contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be explained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded during ischemia.

Depolarization induces insulin-like growth factor binding protein-2 expression in vivo via NMDA receptor stimulation.

The effect of depolarization and N-methyl-D-aspartate (NMDA) receptor blockade on insulin-like growth factor-I (IGF-I), IGF binding protein-2 (IGFBP-2) and IGFBP-4 expression was analysed in vivo. Depolarization was induced in adult rat brains by applying 3 M KCl to the exposed cortex for 10 min. A subgroup of animals also received daily injections of MK-801. Four days after KCl exposure, the brains were analysed by in situ hybridization, immunohistochemistry and TUNEL. A significant upregulation of IGFBP-2 mRNA and protein was detected in astrocytes after KCl exposure This upregulation was reduced by MK-801 treatment. No alterations in IGF-I or IGFBP-4 mRNA levels were noted. We did not detect TUNEL positive cells, morphological signs of necrosis or apoptosis, or neuronal loss in the depolarized zone. Taken together, these findings indicate that upregulation of IGFBP-2 by depolarization is mediated by NMDA receptors, and, as no neuronal damage was detected, astrocytic NMDA receptors may be responsible for this upregulation.

Mechanisms concerned in the direct effect of isoflurane on rat hippocampal and human neocortical neurons.

The effect of isoflurane on postsynaptic neurons was studied by intracellular recordings from rat hippocampus and human neocortex in vitro. Isoflurane caused a hyperpolarization of the cell membrane. The hyperpolarization was reversed (although incompletely in some neurons) by increasing the membrane potential. The reversal potential was -80 +/- 12 mV (mean +/- S.D.) or 12 +/- 6 mV negative to the resting membrane potential. Potassium channel blockers reduced the isoflurane-induced hyperpolarization, while chloride loading was without effect. The transient depolarization preceding the hyperpolarization in some of the neurons was not reversed by hyperpolarization. The action potential was prolonged by 19 +/- 3% due to a slower rate of rise. The rise time was almost doubled. Firing threshold was increased by 4 +/- 3 mV (relative to the reference electrode). Subthreshold inward rectification was reduced or abolished. Some cells showed subthreshold outward rectification during isoflurane administration. These results suggest that isoflurane depressed neuronal excitability by (1) hyperpolarizing the cell membrane, at least partly by an increase in potassium conductance, (2) slowing the rate of rise of the action potential, presumably due to interference with the fast sodium channel, (3) decreasing subthreshold inward rectification and (4) increasing firing threshold.

Temperature sensitivity of thin unmyelinated fibers in rat hippocampal cortex.

The effect of changes in temperature on the activity of thin intracortical unmyelinated fibers was investigated in rat hippocampal slices. The amplitude of the presynaptic volley was increased by 40% when the temperature was raised from 23 degrees C to 30 degrees C, whereas the area of the presynaptic volley was reduced. Increasing the temperature above 30 degrees C was without further effect on the amplitude. The conduction velocity was 0.3 m.s-1 at 35 degrees C and 0.1 m.s-1 at 12 degrees C. The Q10 of velocity (25 degrees C-35 degrees C) was 1.6.

Confocal laser scanning microscopy used to monitor intracellular Ca2+ changes in hippocampal CA 1 neurons during energy deprivation.

An increase in intracellular calcium during cerebral ischemia has been proposed as a common final pathway underlying the events leading to neuronal death. Intracellular calcium has been measured with ion selective electrodes during energy deprivation (ED) in hippocampal slices and with fluorescent techniques in neuronal cultures. In the present study, we describe a novel method to visualize and quantify changes in intracellular calcium in brain slices using Confocal Laser Scanning Microscopy (CLSM). CA 1 pyramidal neurons in hippocampal slices were filled by intracellular injection with a 1:2 mixture of the fluorescent dyes Fluo 3 and Fura Red. The neurons were then visualized using CLSM, and the ratio of the fluorescence from each probe used to quantify intracellular calcium concentrations before and during ED. The free intracellular calcium concentration was 60 nM prior to ED and increased to 24 microM during ED. These results demonstrates that CLSM and fluorescent probes can be used in functional neuronal networks in addition to cell cultures as previously described.

Conductance changes and inhibitory actions of hippocampal recurrent IPSPs.

Intracellular recordings were obtained from CA1 pyramidal neurons in obliquely cut in vitro hippocampal slices. Recurrent IPSPs were elicited by antidromic stimulation of alvear fibers. The mechanisms by which IPSPs depress pyramidal cell excitability were investigated. Recurrent IPSPs could be reversed in sign by small hyperpolarizing currents applied through the recording electrode, indicating an increased membrane conductance. By using an AC bridge circuit it was found that the maximum impedance decrease usually occurred slightly before the peak of the IPSP. Otherwise the time course of the impedance change matched that of the IPSP itself. Inhibitory actions of the conductance increase were studied by adjusting the membrane potential to the IPSP equilibrium potential, thus allowing only the IPSP conductance to play an inhibitory role. Under these conditions non-linear summation of recurrent IPSPs with EPSPs originating in the apical dendrites could be demonstrated only during the initial 15--25 msec ofthe IPSP, which is the period of maximum conductance increase. The inhibition afforded by the hyperpolarization of the recurrent IPSP far outlasts the period of effective EPSP shunting by the inhibitory synaptic currents. The mechanisms of recurrent inhibition in the hippocampus thus appear similar to those operating in spinal motoneuron IPSPs.

Mechanisms of norepinephrine actions on hippocampal pyramidal cells in vitro.

Responses of pyramidal cells to topical application of norepinephrine (NE) were studied by intracellular recording in hippocampal slices in vitro. Norepinephrine hyperpolarized CA1 cells. Simultaneously, there was a decreased response to constant hyperpolarizing and depolarizing current pulses. The number of spikes evoked by constant depolarizing pulses was reduced. Spontaneous activity, when present, was reduced or abolished. The response to depolarizing current pulses was reduced more than the response to hyperpolarizing current pulses. The reduction of the depolarizing response was minimal for the first 6-8 msec of the pulse, whereafter it increased. The effects persisted after blocking synaptic transmission with low calcium-high magnesium concentrations in the incubation fluid. We conclude that the hyperpolarization is most likely due to a conductance increase. The mechanism behind the reduced response to depolarizing current pulses is discussed.

The effect of the volatile anesthetic isoflurane on Ca(2+)-dependent glutamate release from rat cerebral cortex.

A major effect of volatile anesthetics is to reduce excitatory synaptic transmission. In the present study the stimulated release of glutamate under the influence of increasing concentrations of isoflurane was studied in vitro by utilizing hippocampal slices from Wistar rats. Ca(2+)-dependent release was calculated by subtracting stimulated release with blocked synaptic transmission (50 mM K+, 0 mM Ca2+ and 4 mM Mg2+) from total evoked release (50 mM K+, 2 mM Ca2+ and 1 mM Mg2+). Isoflurane 0.5, 1.5 and 3% reduced Ca(2+)-dependent release of glutamate to 69, 58 and 49%, respectively (P < 0.05 for all related to control). These results are in agreement with the possibility of reduced release of transmitter as a mechanism of action of volatile anesthetics.

Calcium-dependent release of glutamate from human cerebral cortex.

The release of the excitatory amino acids glutamate and aspartate from human neocortex was investigated in vitro by utilizing brain tissue removed during anterior temporal lobectomies for tumor or epilepsy. Depolarization (50 mM K+) increased the glutamate release to 291% of control (809 pmol/mg/min) during blocked synaptic transmission and to 669% (1859 pmol/mg/min) when synaptic transmission was not blocked. Aspartate release increased to 141% (326 pmol/mg/min) and 178% (412 pmol/mg/min) respectively. The difference between release with and without blocked synaptic transmission was statistically significant only for glutamate (P less than 0.01). These data provides evidence for a Ca(2+)-dependent release of glutamate, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

Depletion of intracellular Ca2+ stores or lowering extracellular calcium alters intracellular Ca2+ changes during cerebral energy deprivation.

Cytoplasmatic calcium concentrations are elevated two to three fold during cerebral ischemia. In order to determine the role of calcium-release from intracellular stores vs. calcium entry from the extracellular space, intracellular stores were depleted by the use of thapsigargin and calcium was removed from the incubation fluid prior to energy deprivation (ED). CA 1 pyramidal neurons in hippocampal rat slices were filled with a 1:2 mixture of Fluo-3 and Fura Red by intracellular injection. The neurons were visualized in a Confocal Laser Scanning Microscope (CLSM) and the fluorescence ratio from the probe mixture was used to quantify the calcium concentration. Intracellular calcium concentration was monitored before and during ED. The intracellular calcium concentration was 55 nM prior to ED and increased to 25 microM during ED. The resting levels were the same in the experimental groups, but the increase during ED was significantly lower in the intervention groups. The increase in the calcium free group was to 1 microM and in the thapsigargin group to 5 microM. In the last experimental group, thapsigargin treatment and removal of extracellular calcium, the intracellular calcium increased to 630 nM. These results demonstrate that the increased intracellular calcium seen during ED originates from several sources. Calcium-release from intracellular stores may be of major importance in calcium-related neuronal injury during cerebral ischemia.

Calcium dependent release of gamma-aminobutyric acid (GABA) from human cerebral cortex.

The release of the amino acids GABA, taurine, glycine, glutamine and leucine from human neocortex was investigated in vitro by utilizing brain tissue removed during 8 standard temporal lobectomies for epilepsy or tumor. Slices (0.5 mm thick) were cut from each biopsy and randomly placed in three different chambers. After 90 min preincubation, the three sets of slices were incubated for 60 s in wells containing, respectively, (A) regular ACSF (control), (B) ACSF with 50 mM K+ (to depolarize the cell membrane) and (C) ACSF with 50 mM K+, 0 mM Ca2+ and 4 mM Mg2+ (depolarization during blocked synaptic transmission). The content of amino acids in the wells was determined by high-performance liquid chromatography after pre-column derivatization of the amino acids with o-phthalaldehyde. Membrane depolarization (well B) increased the GABA release to 650% (620 pmol/mg) of control (well A, 95 pmol/mg). Blocking synaptic transmission (well C) reduced the evoked release by 50% (360 pmol/mg). The release of glycine, taurine, glutamine and leucine during membrane depolarization was not significantly different from the control values. The data provide evidence for a Ca(2+)-dependent release of GABA, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

Zinc and glycine do not modify low-magnesium-induced epileptiform activity in the immature neocortex in vitro.

Exposure of neocortical slices from immature rats to saline containing no added magnesium induced spontaneous epileptiform activity that consisted of bursts of low-amplitude isolated discharges lasting 50-90 sec, recurring every 90-300 sec. Bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-7-phosphonoheptanoic acid led to a rapid, reversible suppression of epileptiform activity, indicating involvement of NMDA receptors. Perfusion with zinc or glycine, putative modulators of the NMDA receptor, with suppressive and enhancing properties, respectively, had no effect on the frequency or duration of the epileptiform discharges. These results indicate that in the immature neocortex in vitro, application of zinc or glycine does not modulate NMDA receptor-mediated, low-magnesium-induced epileptiform discharges.

An experimental study of the effect of isoflurane on epileptiform bursts.

The effect of isoflurane on penicillin- and picrotoxin-induced epileptiform activity was tested using hippocampal slice preparations. Isoflurane reduced both the frequency of spontaneous epileptiform bursts and the number of population spikes within each burst in a dose-dependent manner. The last population spikes in the burst were most sensitive to the anesthetic, whereas the first 4-6 spikes were quite resistant and persisted until spontaneous activity was abolished at 3% isoflurane. Isoflurane increased the stimulus current required to evoke epileptiform bursts and shifted the relationship between stimulus current and population spike amplitude to the right. At 3% isoflurane, a dose that usually causes iso-electric EEG and abolishes all spontaneous epileptiform activity, responses could still be evoked, and then invariably had an epileptiform pattern. The maximum response was reduced compared to control and 1.5% isoflurane. With isoflurane there was a reduced tendency for activity to be transmitted from one region within the hippocampus to the other. This effect was also dose-dependent. However, transmitted activity always retained a typical epileptiform character, although the number of population spikes within a train to some extent decreased with increasing concentrations of isoflurane.

Epileptogenic effect of antibiotic drugs.

The epileptogenicity of antibiotic drugs represents a clinical problem, and it is well known that the use of penicillin and certain other preparations can induce seizures. In the present study, the authors investigated the epileptogenic properties of different concentrations of 12 commonly used antibiotic medications belonging to seven separate groups. The drugs were tested in the hippocampus, which has a low threshold for the development of epileptiform activity. The hippocampal slice technique, using rat tissue, was employed since absence of the blood-brain barrier allows administration of the drugs in known concentrations. The preparation was exposed to antibiotics in known concentrations and the amplitude and number of population spikes were recorded. Penicillin G was used as a reference substance. Cloxacillin (> or = 1 gm/liter), cephalothin (> or = 1 gm/liter), gentamicin (> or = 80 mg/liter), chloramphenicol (> or = 1 gm/liter), ciprofloxacin (> or = 50 mg/liter), erythromycin (> or = 1 gm/liter), and ampicillin (> or = 1 gm/liter) showed moderate to marked epileptogenic effects, whereas cefuroxime, clindamycin, cefotaxime, vancomycin, and tobramycin had no epileptogenic effects.

Cytotoxic effect of Ca++ released from intracellular stores during cerebral energy deprivation.

The increase in cytoplasmatic calcium concentration during cerebral ischemia has been proposed as a key event leading to neuronal death. In order to investigate a possible role of calcium-release from intracellular stores in ischemic neuronal injury, intracellular calcium pools were depleted prior to ischemia by the use of thapsigargin. Evoked activity (population spike) in rat hippocampal slices was monitored during a 30 min control period, 9 min of energy deprivation and 60 min of recovery. The population spike recovered to 27% (17-33) (median and 95% confidence interval) following energy deprivation in normal calcium, to 56% (50-58) in calcium-free incubation fluid and to 83% (75-88) in slices pretreated with 1 microM thapsigargin. Combining calcium removal and thapsigargin pretreatment did not improve recovery further. Both removal of extracellular calcium and emptying intracellular calcium stores prior to energy deprivation thus improved functional recovery following energy deprivation, however the latter was more effective. These results suggest that calcium release from intracellular stores may be of major importance in calcium-related neuronal injury during cerebral ischemia.

Chloride influx during cerebral energy deprivation.

The purpose of the present study was to investigate the possible role of chloride influx and GABA release during cerebral energy deprivation (ED). The functional activity measured by evoked activity (population spike) in hippocampal slices was recorded during nine minutes of ED and 60 minutes recovery. Treatment groups were exposed to ED following administration of the GABAA antagonist penicillin G (pc G) or substitution of extracellular chloride. The release of glutamate and GABA was measured by HPLC. The efflux of 36Cl from preloaded slices was measured during ED with and without blocking the GABAA receptor. The population spike disappeared during ED, and there was a marked release of GABA and glutamate. During recovery the population spike recovered partially. Both application of pc G and substitution of extracellular chloride during ED improved population spike recovery. Uptake of radiolabeled chloride was significantly reduced by pc G. Glutamate release, but not GABA, was significantly reduced by chloride substitution. These results indicate a possible role of chloride mediated injury during ED, and suggest that chloride entry may partly occur through ligand-operated channels. Furthermore there may be an early chloride dependent release of glutamate during cerebral ischemia, whereas later release seems to be chloride independent.

Changes in brain amino acid content induced by hyposmolar stress and energy deprivation.

The changes in endogenous amino acids in brain extracellular and intracellular compartments evoked by hyposmotic stress and energy deprivation were compared. Tissue content and release of ten amino acids were measured simultaneously in rat hippocampal slices by means of high performance liquid chromatography. Hyposmotic stress induced a large release of taurine (25568 pmol mg-1 protein), and a smaller release of glutamate, accompanied by an inverse change in tissue content. Adding mannitol to correct osmolarity, blocked these changes. Energy deprivation caused an increase in the release of all amino acids except glutamine. The release was particularly large for glutamate and GABA (31141 and 13282 pmol mg-1, respectively). The intracellular concentrations were generally reduced, but the total amount of the released amino acids increased In contrast to the effect seen during hyposmolar stress, mannitol enhanced the changes due to energy deprivation. The results show that hyposmolar stress and energy deprivation cause different content and release profiles, suggesting that the mechanisms involved in the two situations are either different or modulated in different ways. The intracellular amino acid depletion seen during energy deprivation shows that increased outward transport is probably a primary event, and increased amino acid formation likely secondary to this release.

Changes in amino acid release and membrane potential during cerebral hypoxia and glucose deprivation.

Excessive release of glutamate is believed to play a major role in the susceptibility of neurons to ischaemia. Whether the glutamate release is the primary event or occurs in response to electrophysiologic alterations has not been clarified. In the present study, the amino acid release was therefore correlated to changes in electrophysiological parameters and energy status during conditions of low oxygen tension and varying glucose concentrations in rat hippocampal slices. Plain hypoxia failed to produce glutamate release. All neurons underwent, however, a slow depolarization causing most of the neurons to lose their membrane potential within 10 minutes. By restoring the membrane potential to resting level by current injection, the neurons could still be activated synaptically and respond to transmitter application. Following reoxygenation most of the cells regained their resting membrane potential, but showed reduced excitability. When the slices were exposed to hypoxia combined with glucose deprivation (simulated ischaemia), there was a pronounced increase in the glutamate release. This glutamate release was always preceded by a fast anoxic depolarization. Whereas hypoxia reduced the ATP content only to approximately 50%, ATP was depleted in slices exposed to simulated ischaemia. The results demonstrate that although the neurons lose their membrane potential completely during hypoxia, there is no glutamate release. A fast anoxic depolarization provoked by simulated ischaemia, however, is always followed by glutamate release, probably due to a more severe ATP depletion.