Calcium neurotoxicity.
Explore the source record for details and available documents.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Biomedical subjects
W Paschen
Publications and source records attributed to W Paschen.
Changes in interferon-regulatory factor-1 mRNA levels after transient ischemia in rat brain.
To evaluate whether the interferon system in the brain is activated by a severe form of metabolic stress, and to elucidate the possible mechanism underlying this activation, changes in the interferon regulatory factor-1 (irf-1) mRNA levels were evaluated after transient cerebral ischemia, and after exposure of primary neuronal cells to experimental conditions resulting in a depletion of ER calcium stores. Following 30 min ischemia and 2 h recovery, irf-1 mRNA levels rose significantly and stayed high for up to 24 h of recovery. Irf-1 mRNA levels were also significantly increased in neurons in vitro after exposing cells to conditions resulting in ER calcium store depletion with or without a parallel increase in cytoplasmic calcium activity. It is concluded that transient cerebral ischemia induces activation of the interferon system and that disturbances of ER calcium homeostasis may play a role in this process.
Animals↗
Activation of gadd153 expression through transient cerebral ischemia: evidence that ischemia causes endoplasmic reticulum dysfunction.
The expression of the gene encoding the C/EBP-homologous protein (CHOP), which is also known as growth arrest and DNA-damage-inducible gene 153 (gadd153), has been shown to be specifically activated under conditions that disturb the functioning of the endoplasmic reticulum (ER). To investigate a possible role of ER dysfunction in the pathological process of ischemic cell damage, we studied ischemia-induced changes in gadd153 expression using quantitative PCR. Transient cerebral ischemia was produced in rats by four-vessel occlusion. In the hippocampus, ischemia induced a pronounced increase in gadd153 mRNA levels, peaking at 8 h of recovery (6.4-fold increase, p<0.01), whereas changes in the cortex were less marked (non-significant increase). To elucidate the possible mechanism underlying this activation process, gadd153 mRNA levels were also evaluated in primary neuronal cell cultures under two different conditions, both leading to a depletion of ER calcium pools in the presence or absence of an increase in cytoplasmic calcium activity. The first procedure, exposure to thapsigargin, an irreversible inhibitor of ER Ca2+-ATPase, caused a marked increase in gadd153 mRNA levels both in cortical and hippocampal neurons, peaking at 12-18 h after treatment. The second procedure, immersion of cells in calcium free medium supplemented with EGTA, caused only a transient increase in gadd153 mRNA levels, peaking at 6 h of recovery, indicating that a depletion of ER calcium stores in the absence of an increase in cytoplasmic calcium activity is sufficient to activate neuronal gadd153 expression. The results imply that transient cerebral ischemia disturbs the functioning of the ER and that these pathological changes are more pronounced in the hippocampus compared to the cortex.
Animals↗
Role of calcium in the activation of erp72 and heme oxygenase-1 expression on depletion of endoplasmic reticulum calcium stores in rat neuronal cell culture.
Endoplasmic reticulum (ER) calcium pool depletion was induced by 30 min exposure of primary neuronal cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. Twelve hours later, erp72 and heme oxygenase-1 (HO-1) mRNA levels were quantified by PCR. Protein synthesis was also measured. Transient Tg exposure of neurons induced a marked rise in mRNA levels (7-fold and a 21-fold increase in erp72 and HO-1 mRNA levels; P < 0.001). Loading of neurons with the calcium chelator 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetra(acetoxymethyl)ester (BAPTA-AM) prior to thapsigargin treatment had only a minor effect on the Tg-induced rise in gene expression. This small inhibitory effect may result from the severe suppression of protein synthesis caused by BAPTA-AM. The results suggest that the increase in stress gene expression induced by exposure of neurons to Tg is triggered by a decrease in ER calcium activity and not by the corresponding increase in cytoplasmic calcium activity.
Animals↗
Metabolic disturbances in hippocampal slices of fetal guinea pigs during and after oxygen-glucose deprivation: is nitric oxide involved?
The aim of this study was to investigate the role of nitric oxide in metabolic disturbances induced in brain tissue of fetal guinea pigs by oxygen-glucose deprivation. Experiments were performed on hippocampal slices so as to exclude the effects of nitric oxide on the cardiovascular system. Metabolic disturbances were assessed by measuring changes in energy metabolism and protein synthesis after different periods of oxygen-glucose deprivation (OGD). Ten min after OGD of 40 min duration, the concentration of cGMP in tissue slices rose from 1.35 +/- 0.38 to 18.6 +/- 1.04 pmol/mg protein (P < 0.05). This rise was almost completely inhibited by the addition of 100 microM N-nitro-L-arginine (NNLA), indicating that NO-synthase was strongly activated after OGD in fetal brain tissue. However, addition of NNLA improved neither protein synthesis nor energy metabolism measured 12 h after OGD. Thus, nitric oxide does not appear to contribute directly to processes leading to metabolic disturbances induced by transient ischemia in immature brain tissue.
Adenosine Triphosphate↗
Effect of mild hypothermia during and after transient in vitro ischemia on metabolic disturbances in hippocampal slices at different stages of development
In the present study the neuroprotective effect of mild hypothermia (decrease of temperature from 37degreesC to 33degreesC) during and after transient ischemia in brain tissue at different stages of development was tested in vitro by measuring energy metabolism, glutamate release and protein biosynthesis rate (PSR) in hippocampal slices. Slices were taken from immature (E40) and mature (E60) guinea pig fetuses and adult guinea pigs. The slices were exposed to ischemia-like conditions (oxygen/glucose deprivation, OGD) for periods of between 10 to 40 min followed by a 2-h or 12-h recovery phase. During OGD, mild hypothermia slowed down the depletion of energy stores only in slices from immature fetuses, but had no effect on slices prepared from mature fetuses and adult animals. Hypothermia also reduced glutamate release significantly during oxygen/glucose deprivation. Lowering temperature to 33degreesC had no effect on energy metabolism and only a minor effect on PSR of slices from mature fetuses and adult animals subjected to 2 h of recovery. However, 12 h after OGD PSR was markedly improved by mild hypothermia in slices from mature animals and in slices from adults that had been exposed to OGD for only 20 or 30 min. The inhibition of PSR was more severe in the slices from adults than in those from mature fetuses subjected to the same duration of OGD. Age- and temperature-related differences in glutamate release during OGD did not fully agree with corresponding disparities in the values for PSR obtained 12 h after OGD. These results indicate that the neuroprotective effect of mild hypothermia was not mediated by a temperature-dependent retardation of the depletion of energy stores during OGD. Age-related disparities in the vulnerability of the brain to ischemia and the neuroprotective efficiency of mild hypothermia appear to be only partially reflected by the varying levels of glutamate release during ischemia but best reflected by the extent of PSR inhibition. It is concluded that mild hypothermia may be a suitable therapeutical intervention for the suppression of hypoxic-ischemic cell damage during birth.
Journal Article↗
Effect of mild hypothermia during and after transient in vitro ischemia on metabolic disturbances in hippocampal slices at different stages of development.
In the present study the neuroprotective effect of mild hypothermia (decrease of temperature from 37 degrees C to 33 degrees C) during and after transient ischemia in brain tissue at different stages of development was tested in vitro by measuring energy metabolism, glutamate release and protein biosynthesis rate (PSR) in hippocampal slices. Slices were taken from immature (E40) and mature (E60) guinea pig fetuses and adult guinea pigs. The slices were exposed to ischemia-like conditions (oxygen/glucose deprivation, OGD) for periods of between 10 to 40 min followed by a 2-h or 12-h recovery phase. During OGD, mild hypothermia slowed down the depletion of energy stores only in slices from immature fetuses, but had no effect on slices prepared from mature fetuses and adult animals. Hypothermia also reduced glutamate release significantly during oxygen/glucose deprivation. Lowering temperature to 33 degrees C had no effect on energy metabolism and only a minor effect on PSR of slices from mature fetuses and adult animals subjected to 2 h of recovery. However, 12 h after OGD PSR was markedly improved by mild hypothermia in slices from mature animals and in slices from adults that had been exposed to OGD for only 20 or 30 min. The inhibition of PSR was more severe in the slices from adults than in those from mature fetuses subjected to the same duration of OGD. Age- and temperature-related differences in glutamate release during OGD did not fully agree with corresponding disparities in the values for PSR obtained 12 h after OGD. These results indicate that the neuroprotective effect of mild hypothermia was not mediated by a temperature-dependent retardation of the depletion of energy stores during OGD. Age-related disparities in the vulnerability of the brain to ischemia and the neuroprotective efficiency of mild hypothermia appear to be only partially reflected by the varying levels of glutamate release during ischemia but best reflected by the extent of PSR inhibition. It is concluded that mild hypothermia may be a suitable therapeutical intervention for the suppression of hypoxic-ischemic cell damage during birth.
Adenine↗
Effects of transient cerebral ischemia on hsp40 mRNA levels in rat brain.
Rats were subjected to transient cerebral ischemia by four-vessel occlusion of 30 min duration, followed by 2, 4, 8 or 24 h of recovery. Total RNA was isolated from the cerebral cortex and hippocampus, and reverse transcribed into cDNA. Hsp40 mRNA levels of samples were evaluated by quantitative PCR. Transient cerebral ischemia caused a marked increase in hsp40 mRNA levels to about 250% and 500% of control in the cortex and hippocampus respectively. Since hsp40 exerts a critical regulatory function in the HSC70/HSP70 ATPase cycle, an ischemia-induced rise of hsp40 mRNA levels could mark the onset of the recovery process after transient ischemia. On the other hand, the inhibitory action of hsp40 on P58 (a protein that activates protein synthesis by blocking the interferon-induced double-stranded RNA-activated protein kinase PKR) implies that the rise in hsp40 expression may equally well contribute to the post-ischemic suppression of protein synthesis.
Adenosine Triphosphatases↗
Erp72 expression activated by transient cerebral ischemia or disturbance of neuronal endoplasmic reticulum calcium stores.
Stress-induced activation of the expression of the endoplasmic reticulum (ER)-resident chaperon and member of the protein disulfide isomerase family erp72 was studied after transient cerebral ischemia in vivo using the four-vessel occlusion method and experimental depletion of ER calcium stores in primary neuronal cell cultures. After 8 days in vitro, neurons were exposed to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase, or the Tg solvent DMSO. In separate experiments neurons were pre-loaded with the cell-permeant calcium chelator BAPTA-AM before Tg exposure. Stress-induced changes in erp72 expression were analysed by quantitative PCR. Transient cerebral ischemia produced a significant increase in erp72 mRNA levels which rose to about 200% of control (hippocampus) or 300% of control (cortex). After depletion of ER calcium stores neuronal erp72 mRNA levels rose markedly, peaking at 12 h of recovery. Counteracting the Tg-induced rise in cytoplasmic calcium activity by preloading cells with the chelator BAPTA-AM did not influence erp72 expression significantly, suggesting that the activation of erp72 expression resulted from the depletion of ER calcium stores and not from the corresponding increase in cytoplasmic calcium activity. An activation of erp72 expression is indicative of a disturbance of ER function. The results of the present study therefore provide evidence to support the notion that transient cerebral ischemia induces disturbances of neuronal ER function, probably through a depletion of ER calcium stores.
Animals↗
Relation of neuronal endoplasmic reticulum calcium homeostasis to ribosomal aggregation and protein synthesis: implications for stress-induced suppression of protein synthesis.
Results from experiments performed with permanent non-neuronal cell lines suggest that endoplasmic reticulum (ER) calcium homeostasis plays a key role in the control of protein synthesis (PS). It has been concluded that disturbances in ER calcium homeostasis may contribute to the suppression of PS triggered by a severe metabolic stress (W. Paschen, Med. Hypoth., 47 (1996) 283-288). To elucidate how an emptying of ER calcium stores of these cells would effect PS and ribosomal aggregation of non-transformed fully differentiated cells, experiments were run on primary neuronal cell cultures. ER calcium stores were depleted by treating cells with thapsigargin (TG, a selective, irreversible inhibitor of ER Ca(2+)-ATPase), cyclopiazonic acid (CPA, a reversible inhibitor of ER Ca(2+)-ATPase), or caffeine (an agonist of ER ryanodine receptor). Changes in intracellular calcium activity were evaluated by fluorescence microscopy using fura-2-loaded cells. Protein synthesis was determined by measuring the incorporation of [3H]leucine into proteins. The degree of aggregation of ribosomes was evaluated by electron microscopy. TG induced a permanent inhibition of PS to about 10% of control which was only partially reversed within 2 h of recovery. CPA caused about 70% inhibition of PS, and PS recovered completely 60 min after treatment. Caffeine produced an inhibition of PS to about 50% of control. Loading cells with the calcium chelator BAPTA-AM (33.3 microM) alone suppressed PS without reversing TG- or caffeine-induced inhibition of PS, indicating that the suppression of PS was caused by a depletion of ER calcium stores and not by an increase in cytosolic calcium activity. TG-treatment of cells induced a complete disaggregation of polysomes which was not reversed within the 4 h recovery period following TG-treatment. After caffeine treatment of cells, we observed a heterogenous pattern of ribosomal aggregation: in some neurons ribosomes were almost completely aggregated while in other cells a significant portion of polyribosomes were disaggregated. The results indicate that a depletion of neuronal ER calcium stores disturbs protein synthesis in a similar way to the effects of transient forms of metabolic stress (ischemia, hypoglycemia or status epilepticus), thus implying that a disturbance in ER calcium homeostasis may contribute to the pathological process of stress-induced cell injury.
Animals↗
Quantitative measurement of cerebral protein synthesis in vivo: theory and methodological considerations.
The true rate of cerebral protein synthesis can be calculated from the ratio of labeled proteins to integrated arterial plasma amino acid specific activity (SA) only when the fraction of amino acid precursor pool dilution is known. In the following, current experimental designs on the measurement of cerebral protein synthesis are discussed and compared to our own approach in which the determination of regional precursor pool dilution by recycled unlabeled leucine is combined with the quantitation of regional cerebral protein synthesis rates. For this purpose, a constant arterial plasma leucine SA level is maintained for 45 min by programmed intravenous infusion which is sufficient for complete equilibrium between tissue leucine pool SAs and plasma free leucine SA. In addition to the regional assessment of the precursor dilution factor, protein radioactivity can be determined in the same tissue sample or in parallel brain sections of the same animal by quantitative autoradiography. It is then possible to calculate the actual rate of protein synthesis using the correct fraction of precursor pool dilution. This renders our approach particularly suitable for the quantitative measurement of regional CPS under pathological conditions.
Amino Acids↗
Activation of heme oxygenase-1 expression by disturbance of endoplasmic reticulum calcium homeostasis in rat neuronal cell culture.
Evidence has been presented that disturbances of endoplasmic reticulum (ER) calcium homeostasis contribute to neuronal injury induced by transient cerebral ischemia. The present series of experiments was designed to study whether the expression of heme oxygenase-1 (HO-1), which is markedly increased after transient cerebral ischemia, is also activated by a disturbance of ER calcium homeostasis. ER calcium pools were depleted by a 30 min exposure of primary cortical and hippocampal neurons to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. In cortical neurons, HO-1 mRNA levels (analysed by quantitative polymerase chain reaction (PCR)) were significantly increased (22-fold) 12 h after exposure to Tg but had decreased again to only nine times control levels by 24 h after treatment. In hippocampal neurons, a significant increase in HO-1 mRNA levels was already apparent 4 h after treatment (8.3-fold over controls), levels rose further to 27-fold over controls after 6 h, and stayed high for up to 24 h after treatment (34-fold over controls). The similarity between the pattern of changes in HO-1 mRNA levels induced by transient ischemia and depletion of ER calcium stores suggests common underlying mechanisms.
Animals↗
No effect of glutamate on metabolic disturbances in hippocampal slices of mature fetal guinea pigs after transient in vitro ischemia.
The involvement of glutamate in the development of cerebral metabolic disturbances in mature fetuses after transient ischemia was studied using a hippocampal slice model. We investigated the effects of exogenously applied glutamate or glutamate antagonists on the recovery of energy metabolism and protein synthesis rate (PSR) in hippocampal slices of mature guinea pigs after in vitro ischemia. The slices were incubated in a thermostatically controlled flow-through chamber and gassed with carbogen (95% O2/5% CO2). In vitro ischemia was induced by transferring the slices to an aglycemic, artificial cerebrospinal fluid (aCSF) equilibrated with 95% N2/5% CO2. In a first set of experiments slices were exposed to 10 mM glutamate during a 20-40 min period of in vitro ischemia. In a second set slices were incubated in aCSF containing MK-801 (100 microM) or kynurenic acid (0.5 mM) 30 min before, during and 2 h after in vitro ischemia. After a 12 h recovery phase, the concentrations of adenylates in the slices were measured by HPLC after extraction with perchloric acid. PSR was calculated from the rate of incorporation of [14C]leucine into tissue proteins. Neither glutamate nor glutamate antagonists had any effect on the postischemic recovery of energy metabolism and PSR when applied during in vitro ischemia. It is therefore concluded that glutamate does not play a major role in the development of metabolic disturbances in hippocampal slices from mature guinea pig fetuses subjected to transient in vitro ischemia.
Adenosine Triphosphate↗
Developmental changes of RNA editing of glutamate receptor subunits GluR5 and GluR6: in vivo versus in vitro.
In the present series of experiments we compared the up-regulation of GluR5 and GluR6 mRNA editing during the transition from the embryonic to the adult state with changes in the extent of editing during neuronal development in vitro. RNA was isolated from rats, from the cerebral cortex, hippocampus and cerebellum of embryonic brains (E19) and adult brains (2 months old), as well as from neurons prepared from the cortex, hippocampus and cerebellum of embryonic brains (E19) and held in tissue culture for 2, 8 or 16 days. Quantification of mRNA editing was achieved by using standards prepared from plasmids with cDNA inserts derived from the edited and unedited state of both GluR5 and GluR6 mRNA. In addition, GluR5 mRNA levels were determined in brain tissue and neuronal cells in culture by quantitative PCR. Developmental changes in the extent of GluR5 and GluR6 mRNA editing were different in vivo compared to in vitro. For GluR5 mRNA editing these differences were most pronounced in cerebellar neurons compared to cerebellar tissue: the extent of GluR5 mRNA editing found in vivo at E19 was significantly down-regulated in cerebellar neurons during the first 8 days in culture, and after 16 days in vitro the extent of editing was still about 50% of that found in the adult state in vivo. For GluR6 mRNA editing these differences were most pronounced in hippocampal neurons compared to the hippocampus in vivo: the extent of GluR6 mRNA editing found in vivo at E19 was significantly down-regulated in vitro during the whole culturing period, most pronounced after 8 days in vivo (to below 40% of that found at E19 and to below 30% of that found in adult hippocampus). GluR5 mRNA levels increased markedly from E19 to the adult brain. However, we could not find any specific pattern of changes in mRNA levels which might account for the development changes in the profile of GluR5 mRNA editing. Comparing developmental changes in the extent of mRNA editing of glutamate receptor subunits may help to elucidate the molecular and regulatory mechanisms of this important editing reaction. Strict control and clear indication of the age of primary neuronal cell cultures used should be required in accounts of electrophysiological or neurotoxicological studies as this would increase comparative usefulness of such experiments, since calcium fluxes through glutamate receptor ion channels are likely to influence the system significantly.
Analysis of Variance↗
Temporal analysis of changes in neuronal c-fos mRNA levels induced by depletion of endoplasmic reticulum calcium stores: effect of clamping cytoplasmic calcium activity at resting levels.
Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. Tg-induced rise in [Ca2+]i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca2+]i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c-fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c-fos mRNA levels (approximately 10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c-fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c-fos. Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.
Animals↗
Changes in endoplasmic reticulum Ca(2-)-ATPase mRNA levels in transient cerebral ischemia of rat: a quantitative polymerase chain reaction study.
Transient cerebral ischemia was produced in rats using the four-vessel occlusion model. After 30 min ischemia and 2, 4, 8, or 24 h of recirculation, total RNA was isolated from the cortex, striatum and hippocampus and reverse transcribed into cDNA. Endoplasmic reticulum (ER) calcium-ATPase (SERCA, subunit 2b) cDNA was amplified using appropriate primers. Ischemia-induced changes in SERCA mRNA levels were analyzed by quantitative polymerase chain reaction (PCR). For quantification, each PCR reaction was run in the presence of an internal standard. In control brains SERCA mRNA levels amounted to 392 +/- 43,431 +/- 86, and 409 +/- 21 micrograms mRNA/g total RNA in the cortex, striatum and hippocampus, respectively. SERCA mRNA levels did not change significantly during the first 8 h of recovery. After 24 h of recovery, however, SERCA mRNA levels decreased sharply in the hippocampus and striatum (P < 0.001 versus control) but not in the cortex. It is concluded that in vulnerable brain structures a post-ischemic disturbance in ER calcium homeostasis may limit the recovery of neurons from metabolic stress.
Analysis of Variance↗
Ontogenetic differences in energy metabolism and inhibition of protein synthesis in hippocampal slices during in vitro ischemia and 24 h of recovery.
The present study was designed to clarify whether ontogenetic differences in the vulnerability of the brain towards hypoxic-ischemic insults are only caused by the low cerebral energy demand of immature animals or whether there are additional mechanisms, such as protein synthesis (PSR), that may be involved in this phenomenon. We therefore measured tissue levels of adenylates and PSR in hippocampal slices from immature (E40) and mature (E60) guinea pigs fetuses and from adult guinea pigs during in vitro ischemia and 24 h of recovery using a recently modified method. Hippocampal slices were incubated in a temperature controlled flow-through chamber, gassed with 95% O2/5% CO2. In vitro ischemia was induced by transferring slices to a glucose-free artificial cerebrospinal fluid (aCSF) equilibrated with 95% N2/5% CO2. The duration of ischemia ranged from 10 to 40 min. Adenylates were measured by HPLC after extraction with perchloric acid. PSR was evaluated as the incorporation rate of [14C]leucine into proteins. Under control conditions, tissue levels in adenylates did not change, whereas PSR increased slightly in hippocampal slices from mature fetuses and adult animals during a 24-h control incubation period. In slices from immature fetuses ATP levels were only maintained for 2 h. During in vitro ischemia the decline in ATP, total adenylate pool, and adenylate energy charge was much slower in slices from immature fetuses than in slices from mature fetuses or adults. After in vitro ischemia, ATP and the total adenylate pool did not completely recover in mature fetuses and adults, whereas adenylate energy charge almost returned to control values independently of the developmental stage. Two hours after in vitro ischemia PSR was undisturbed in slices from immature fetuses, but severely inhibited in slices from mature fetuses and adults. With ongoing recovery, PSR in mature fetuses returned to control values, while in adults it was still inhibited even 24 h after in vitro ischemia. From these results we conclude that hippocampal slices prepared from mature guinea pig fetuses as well as from adult guinea pigs can be held metabolically stable during long-term incubation using a recently modified technique. However, in slices from immature fetuses a stable energy state could not be maintained for more than 2 h. We further conclude that postischemic disturbances in PSR closely reflect the ontogenetic changes in the vulnerability of the brain to ischemia and that low energy metabolism is certainly not the only cause of the increased vulnerability of the fetal brain to ischemia.
Adenosine Triphosphate↗
Regional analysis of developmental changes in the extent of GluR6 mRNA editing in rat brain.
The extent of mRNA editing of the kainate receptor subunit GluR6 was evaluated in the cortex, hippocampus and cerebellum of embryonic brains at days 14 and 19 of gestation, in brains of animals aged 4, 25 days, or 3 months, and in hippocampal neurons isolated from embryonic brains at day 19 of gestation and held in tissue culture for 2 or 8 days. Total RNA was isolated and reverse transcribed into cDNA, which was used as template for PCR across the edited base A in TMII of GluR6. The extent of editing was evaluated by restriction digest of PCR products with Bbv 1, gel electrophoresis and image analysis of bands. In all brain structures studied the extent of editing was significantly upregulated during development (P < 0.001). The most pronounced increase in the extent of editing was observed between embryonic days 14 and 19. Highest levels were reached 4 days (94 +/- 1.3%) or 3 months after birth (95 +/- 1.7%) in the cortex and hippocampus, respectively. Notably, in hippocampal neurons held in tissue culture editing was sharply reduced to 67 +/- 3.1% and 29 +/- 3.1% after 2 or 8 days in culture (P < 0.001 vs. the embryonic and adult state). The results illustrate that moderate but significant regional differences exist in the regulation of GluR6 mRNA editing during development (cortex vs. hippocampus and cerebellum). Comparing developmental changes in the extent of editing of AMPA/kainate receptor subunits in vivo and in vitro may help to elucidate the molecular mechanisms of the editing process.
Animals↗