Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.     

Benchmark studies of the effectiveness of structural and internal materials as radiation shielding for the international space station

Accelerator-based measurements and model calculations have been used to study the heavy-ion radiation transport properties of materials in use on the International Space Station (ISS). Samples of the ISS aluminum outer hull were augmented with various configurations of internal wall material and polyethylene. The materials were bombarded with high-energy iron ions characteristic of a significant part of the galactic cosmic-ray (GCR) heavy-ion spectrum. Transmitted primary ions and charged fragments produced in nuclear collisions in the materials were measured near the beam axis, and a model was used to extrapolate from the data to lower beam energies and to a lighter ion. For the materials and ions studied, at incident particle energies from 1037 MeV/nucleon down to at least 600 MeV/nucleon, nuclear fragmentation reduces the average dose and dose equivalent per incident ion. At energies below 400 MeV/nucleon, the calculation predicts that as material is added, increased ionization energy loss produces increases in some dosimetric quantities. These limited results suggest that the addition of modest amounts of polyethylene or similar material to the interior of the ISS will reduce the dose to ISS crews from space radiation; however, the radiation transport properties of ISS materials should be evaluated with a realistic space radiation field.     

Cytosine arabinofuranoside-induced activation of astrocytes increases the susceptibility of neurons to glutamate due to the release of soluble factors

Activation of astrocytes occurs during many forms of CNS injury, but its importance for neuronal survival is poorly understood. When hippocampal cultures of neurons and astrocytes were treated from day 2–4 in vitro (DIV 2–4) with 1 μM cytosine arabinofuranoside (AraC), we observed a stellation of astrocytes, an increase in glial fibrillary acidic protein (GFAP) level as well as a higher susceptibility of the neurons to glutamate compared with cultures treated from DIV 2–4 with vehicle. To find out whether factors released into the culture medium were responsible for the observed differences in glutamate neurotoxicity, conditioned medium of AraC-treated cultures (MCMAraC) was added to vehicle-treated cultures and conditioned medium of vehicle-treated cultures (MCMvh) was added to AraC-treated cultures 2 h before and up to 18 h after the exposure to 1 mM glutamate for 1 h. MCMAraC increased glutamate neurotoxicity in vehicle-treated cultures and MCMvh reduced glutamate neurotoxicity in AraC-treated cultures. Heat-inactivation of MCMvh increased, whereas heat-inactivation of MCMAraC did not affect glutamate toxicity suggesting that heat-inactivation changed the proportion of factors in MCMvh inhibiting and exacerbating the excitotoxic injury. Similar findings were obtained using conditioned medium of pure astrocyte cultures of DIV 12 treated from DIV 2–4 with vehicle or 1 μM AraC suggesting that heat-sensitive factors in MCMvh were mainly derived from astrocytes. Treatment of hippocampal cultures with 1 mM dibutyryl-cAMP for 3 days induced an activation of the astrocytes similar to AraC and increased neuronal susceptibility to glutamate. Our findings provide evidence that activation of astrocytes impairs their ability to protect neurons after excitotoxic injury due to changes in the release of soluble and heat-sensitive factors.     

Specificity and Reversibility of the Inhibition by HgCl2 of Glutamate Transport in Astrocyte Cultures

Inhibition of glutamate transport is a potential indirect cause of excitotoxic damage by glutamate in the CNS. The mercuric ion, the form in which metallic mercury vapor is believed to exert its neurotoxic action, is a known inhibitor of amino acid transport. This study examines the specificity with which HgCl2 inhibits glutamate transport in mouse cerebral astrocytes by means of comparative measurements of 2-deoxyglucose uptake. Uptake of 2-deoxyglucose is an index of glucose utilization that reflects the function of Na+,K+-ATPase and hexokinase, and is sensitive to Na+ entry. The kinetic parameters, ionic dependence, and substrate specificity of glutamate transport in these astrocyte cultures were consistent with the commonly occurring system designated X-AG. Acute exposure to 0.5 microM HgCl2 inhibited by 50% the initial rate of glutamate transport but did not affect 2-deoxyglucose uptake. Glutamate transport was not detectably inhibited by Al2+, Pb2+, Co2+, Sr2+, Cd2+, or Zn2+ (10 microM as chlorides). The inhibitory action of 0.5 microM HgCl2 on glutamate transport was rapidly reversible. The action of 1-2 microM HgCl2 was progressive when exposures were extended to 1-3 h, and was more slowly reversible. These results suggest that Hg2+ can impair glial glutamate transport reversibly at exposure levels that do not compromise some other vital cell functions.     

Na+-dependent high-affinity glutamate transport in macrophages

Excessive accumulation of glutamate in the CNS leads to excitotoxic neuronal damage. However, glutamate clearance is essentially mediated by astrocytes through Na+-dependent high-affinity glutamate transporters (excitatory amino acid transporters (EAATs)). Nevertheless, EAAT function was recently shown to be developmentally restricted in astrocytes and undetectable in mature astrocytes. This suggests a need for other cell types for clearing glutamate in the brain. As blood monocytes infiltrate the CNS in traumatic or inflammatory conditions, we addressed the question of whether macrophages expressed EAATs and were involved in glutamate clearance. We found that macrophages derived from human blood monocytes express both the cystine/glutamate antiporter and EAATs. Kinetic parameters were similar to those determined for neonatal astrocytes and embryonic neurons. Freshly sorted tissue macrophages did not possess EAATs, whereas cultured human spleen macrophages and cultured neonatal murine microglia did. Moreover, blood monocytes did not transport glutamate, but their stimulation with TNF-alpha led to functional transport. This suggests that the acquisition of these transporters by macrophages could be under the control of inflammatory molecules. Also, monocyte-derived macrophages overcame glutamate toxicity in neuron cultures by clearing this molecule. This suggests that brain-infiltrated macrophages and resident microglia may acquire EAATs and, along with astrocytes, regulate extracellular glutamate concentration. Moreover, we showed that EAATs are involved in the regulation of glutathione synthesis by providing intracellular glutamate. These observations thus offer new insight into the role of macrophages in excitotoxicity and in their response to oxidative stress.     

Effects of very low fluences of high-energy protons or iron ions on irradiated and bystander cells

In space, astronauts are exposed to radiation fields consisting of energetic protons and high atomic number, high-energy (HZE) particles at very low dose rates or fluences. Under these conditions, it is likely that, in addition to cells in an astronaut's body being traversed by ionizing radiation particles, unirradiated cells can also receive intercellular bystander signals from irradiated cells. Thus this study was designed to determine the dependence of DNA damage induction on dose at very low fluences of charged particles. Novel techniques to quantify particle fluence have been developed at the NASA Space Radiation Biology Laboratory (NSRL) at Brookhaven National Laboratory (BNL). The approach uses a large ionization chamber to visualize the radiation beam coupled with a scintillation counter to measure fluence. This development has allowed us to irradiate cells with 1 GeV/nucleon protons and iron ions at particle fluences as low as 200 particles/cm(2) and quantify biological responses. Our results show an increased fraction of cells with DNA damage in both the irradiated population and bystander cells sharing medium with irradiated cells after low fluences. The fraction of cells with damage, manifest as micronucleus formation and 53BP1 focus induction, is about 2-fold higher than background at doses as low as ～0.47 mGy iron ions (～0.02 iron ions/cell) or ～70 μGy protons (～2 protons/cell). In the irradiated population, irrespective of radiation type, the fraction of damaged cells is constant from the lowest damaging fluence to about 1 cGy, above which the fraction of damaged cells increases with dose. In the bystander population, the level of damage is the same as in the irradiated population up to 1 cGy, but it does not increase above that plateau level with increasing dose. The data suggest that at fluences of high-energy protons or iron ions less than about 5 cGy, the response in irradiated cell populations may be dominated by the bystander response.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

Induction of micronuclei in human fibroblasts across the Bragg curve of energetic heavy ions

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LEpsilonTau gamma or X rays, the presence of shielding does not always reduce the radiation risks for energetic charged-particle exposure. The dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak. However, the Bragg curve does not necessarily represent the biological damage along the particle path since biological effects are influenced by the track structures of both primary and secondary particles. Therefore, the "biological Bragg curve" is dependent on the energy and the type of the primary particle and may vary for different biological end points. Here we report measurements of the biological response across the Bragg curve in human fibroblasts exposed to energetic silicon and iron ions in vitro at two different energies, 300 MeV/nucleon and 1 GeV/nucleon. A quantitative biological response curve generated for micronuclei per binucleated cell across the Bragg curve did not reveal an increased yield of micronuclei at the location of the Bragg peak. However, the ratio of mono- to binucleated cells, which indicates inhibition of cell progression, increased at the Bragg peak location. These results confirm the hypothesis that severely damaged cells at the Bragg peak are more likely to go through reproductive death and not be evaluated for micronuclei.     

Proton-HZE-particle sequential dual-beam exposures increase anchorage-independent growth frequencies in primary human fibroblasts

The radiation field in deep space contains high levels of high-energy protons and substantially lower levels of high-atomic-number, high-energy (HZE) particles. Calculations indicate that cellular nuclei of human space travelers will be hit during a 3-year Mars mission by approximately 400 protons and approximately 0.4 HZE particles. Thus most cells in astronauts will be hit by a proton(s) before being hit by an HZE particle. To investigate effects of dual ion irradiations on human cells, we irradiated primary human neonatal fibroblasts with protons (1 GeV/nucleon, 20 cGy) followed from 2.5 min to 48 h later by iron or titanium ions (1 GeV/nucleon, 20 cGy) and then measured clonogenic survival and frequency of anchorage-independent growth. This frequency depends on the interval between hydrogen- and iron-ion irradiation, with a critical window between 2.5 min and 1 h producing about three times more anchorage-independent colonies per survivor than expected from simple addition of the two ions separately. The hydrogen-titanium-ion dual-beam irradiation produced similar increases that persisted to approximately 6 h. At longer intervals, anchorage-independent growth frequencies were similar to those expected for additivity. However, irradiation of cells with either an iron or a titanium particle first followed by protons produced only additive levels.     

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.     

Long-term pathomorphologic changes in the neurons of the rat brain following irradiation with carbon ions and gamma rays

Quantitative and qualitative morphological changes in neurons and glia of rat brain were studied one month after exposure to accelerated carbon ions 4 GeV/nucleon (LET-76 MeV cm2.r-1) and gamma-radiation (137Cs, 0.25 to 4.0 Gy). There were certain peculiarities in the structural changes induced by the effect of carbon ions that possessed a higher relative biological effectiveness.     

An assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation

Conditioned taste aversion was used to evaluate the behavioral toxicity of exposure to high-energy iron particles (56Fe, 600 MeV/amu) in comparison to that of gamma photons (60Co), high-energy electrons, or fission neutrons. Exposure to high-energy iron particles (5-500 cGy) produced a dose-dependent taste aversion with a maximal effect achieved with a dose of 30 cGy. Gamma photons and electrons were the least effective stimuli for producing a conditioned taste aversion, with a maximal aversion obtained only after exposure to 500 cGy, while the effectiveness of fission neutrons was intermediate to that of photons and iron particles, and a maximal aversion was obtained with a dose of 100 cGy. In the second experiment, rats with lesions of the area postrema were exposed to iron particles (30 cGy), but failed to acquire a taste aversion. The results indicate that (1) high-energy iron particles are more toxic than other qualities of radiation and (2) similar mechanisms mediate the behavioral toxicity of gamma photons and high-energy iron particles.     

The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by manganese

In the central nervous system (CNS), extracellular concentrations of amino acids (e.g., aspartate, glutamate) and divalent metals (e.g., zinc, copper, manganese) are primarily regulated by astrocytes. Adequate glutamate homeostasis and control over extracellular concentrations of these excitotoxic amino acids are essential for the normal functioning of the brain. Not only is glutamate of central importance for nitrogen metabolism but, along with aspartate, it is the primary mediator of excitatory pathways in the brain. Similarly, the maintenance of proper Mn levels is important for normal brain function. Brain glutamate is removed from the extracellular fluid mainly by astrocytes via high affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of Mn on specific glutamate transporters have yet to be determined. As a first step in this process, we examined the effects of Mn on the transport of [D-2, 3-3H]D-aspartate, a non-metabolizable glutamate analog, in Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) or GLT-1 (EAAT2). Mn-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was pronounced in both the GLT-1 and GLAST transfected cells. This resulted in a statistically significant inhibition (p<0.05) of glutamate uptake compared with transfected control in the absence of Mn treatment. These studies suggest that Mn accumulation in the CNS might contribute to dysregulation of glutamate homeostasis.     

Neoplastic transformation in vitro by mixed beams of high-energy iron ions and protons

The radiation environment in space is complex in terms of both the variety of charged particles and their dose rates. Simulation of such an environment for experimental studies is technically very difficult. However, with the variety of beams available at the National Space Research Laboratory (NSRL) at Brookhaven National Laboratory (BNL) it is possible to ask questions about potential interactions of these radiations. In this study, the end point examined was transformation in vitro from a preneoplastic to a neoplastic phenotype. The effects of 1?GeV/n iron ions and 1?GeV/n protons alone provided strong evidence for suppression of transformation at doses ≤5?cGy. These ions were also studied in combination in so-called mixed-beam experiments. The specific protocols were a low dose (10?cGy) of protons followed after either 5-15?min (immediate) or 16-24?h (delayed) by 1?Gy of iron ions and a low dose (10?cGy) of iron ions followed after either 5-15?min or 16-24?h by 1?Gy of protons. Within experimental error the results indicated an additive interaction under all conditions with no evidence of an adaptive response, with the one possible exception of 10?cGy iron ions followed immediately by 1?Gy protons. A similar challenge dose protocol was also used in single-beam studies to test for adaptive responses induced by 232?MeV/n protons and (137)Cs γ radiation and, contrary to expectations, none were observed. However, subsequent tests of 10?cGy of (137)Cs γ radiation followed after either 5-15?min or 8?h by 1?Gy of (137)Cs γ radiation did demonstrate an adaptive response at 8?h, pointing out the importance of the interval between adapting and challenge dose. Furthermore, the dose-response data for each ion alone indicate that the initial adapting dose of 10?cGy used in the mixed-beam setting may have been too high to see any potential adaptive response.     

Plasticity of Astroglial Glutamate and γ-Aminobutyric Acid Uptake in Cell Cultures Derived from Postnatal Mouse Cerebellum

Abstract: The plasticity of astroglial glutamate and γ-aminobutyric acid (GABA) uptakes was investigated using mouse cerebellar cell cultures. The influence of external factors, such as different sera and/or the presence of neurons, was examined. Control autoradiography experiments showed that after short-term exposure to radioactive amino acids, granule cells took up neither glutamate nor GABA, and β-alanine predominantly inhibited astroglial GABA uptake. Astroglial uptake was quantified by measuring the radioactivity taken up by the cells in the culture and relating this measurement to the number of glial fibrillary acidic protein-positive cells present. Glutamate uptake was investigated in astroglial cultures and subcultures and in neuro-nal-astroglial cultures derived from postnatal day 4 mouse cerebella. In the absence of neurons, glutamate uptake increased during the first 9 days after plating and then leveled off. At 14 days in vitro in horse serum, which favors the differentiation of fibrous-like astrocytes, glutamate uptake related to astrocyte number was twice as high as in fetal calf serum. In the presence of cerebellar neurons, this rate was even higher. The specificity of the responsiveness of astrocytes to neurons with respect to glutamate uptake was investigated by comparing GABA uptake in the different culture conditions. Neurons also increased the rate of GABA uptake by astrocytes. Another component of the astroglial plasma membrane, the density of β-adrenergic receptors, was, however, not markedly affected by the presence of neurons. Hence, these results showed that in astrocytes plated from postnatal day 4 mouse cerebella, the level of neuro-transmitter uptake can be regulated in vitro by factors present in sera and by cerebellar neurons in the culture. However, this plasticity declined during development because astrocytes plated from postnatal day 8 cerebella and cultured under identical conditions were less active in glutamate uptake and were insensitive to the presence of horse serum. The latter observation suggested that the metabolic plasticity of astrocytes is restricted to a period defined early in cerebellar development and is no longer evident by postnatal day 8.     

The response of tissue-equivalent proportional counters to heavy ions

The paper presents a theoretical model for the response of a tissue-equivalent proportional counter (TEPC) irradiated with charged particles. Heavy ions and iron ions in particular constitute a significant part of radiation in space. TEPCs are used for all space shuttle and International Space Station (ISS) missions to estimate the dose and radiation quality (in terms of lineal energy) inside spacecraft. The response of the tissue-equivalent proportional counters shows distortions at the wall/cavity interface. In this paper, we present microdosimetric investigation using Monte Carlo track structure calculations to simulate the response of a TEPC to charged particles of various LET (1 MeV protons, 2.4 MeV alpha particles, 46 MeV/nucleon 20Ne, 55 MeV/nucleon 20Ne, 45 MeV/nucleon 40Ar, and 1.05 GeV/nucleon 56Fe). Data are presented for energy lost and energy absorbed in the counter cavity and wall. The model calculations are in good agreement with the results of Rademacher et al. (Radiat. Res. 149, 387-389, 1998), including the study of the interface between the wall and the sensitive region of the counter. It is shown that the anomalous response observed at large event sizes in the experiment is due to an enhanced entry of secondary electrons from the wall into the gas cavity.     

Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.     

mBAND analysis of chromosomal aberrations in human epithelial cells exposed to low- and high-LET radiation

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to (137)Cs gamma rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and gamma rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.     

Alterations in Uptake and Release Rates for GABA, Glutamate, and Glutamine During Biochemical Maturation of Highly Purified Cultures of Cerebral Cortical Neurons, a GABAergic Preparation

This study demonstrates that virtually homogenous cultures of mouse cerebral neurons, obtained from 15-day-old embryos, differentiate at least as well as cultures which in addition contain astrocytes. This was indicated by glutamate decarboxylase activity which within 2 weeks rose from a negligible value to twice the level in the adult mouse cerebral cortex, and by a gamma-aminobutyric acid (GABA) uptake rate which quadrupled during the second week in culture and reached higher values than in brain slices. Within the same period, the GABA content increased four to five times to 75 nmol/mg protein, and a potassium-induced increase in [14C]GABA efflux became apparent. Although the development was faster than in vivo, optimum differentiation required maintenance of the cultures beyond the age of 1 week. Uptake and release rates for glutamate and glutamine underwent much less developmental alteration. At no time was there any potassium-induced release of radioactivity after exposure to [14C]glutamate, and the glutamate uptake was only slightly increased during the period of GABAergic development. This indicates that exogenous glutamate is not an important GABA precursor. Similarly, glutamine uptake was unaltered between days 7 and 14, although a small potassium-induced release of radioactivity after loading with glutamine suggests a partial conversion to GABA.