Alterations in glutamate transport are commonly observed as a consequence of a variety of adverse conditions such as hypoxia or exposure to ROS generators. Transporter dysfunction is believed to be a precursor of neurotoxic cascades. We investigated the effects of particle radiation on isolated and mixed cell types of the CNS. Neurons and astrocytes produced reciprocal glutamate transport profiles when exposed to 250 MeV protons, 290 MeV/nucleon carbon ions, and 1000 MeV/nucleon iron ions. Exposure of neuronal transporters resulted in increased transporter activity, whereas astrocyte transporters responded with a decrease in transporter activity. These results resemble those we reported previously with γ rays. This strongly suggests that alterations in glutamate transporter activity are a common response to ionizing radiations. Based on observations by other investigators, particle radiation is known to have a greater impact on biological end points of neurotoxicity in NT2 cells (25
) and oxidative stress in neural precursors (26
). We expected to see responses that would reflect the differences in radiation quality and LET. We did not observe a clear LET dependence with protons and heavy ions. However, consistent with observations made by other investigators, we observed a longer biological effect with particle radiation than with γ rays (17
The increase in glutamate uptake in neurons after exposure to protons and carbon and iron ions was surprising because glutamate transporters localized on neurons are generally believed not to participate significantly in the clearance of extracellular glutamate. It is not currently understood what the main function is of the transporters expressed on neurons. However, it has been suggested that their function may involve regulation of cellular oxidative stress and synaptic remodeling, because neuronal glutamate transport is associated with cysteine uptake (27
), and the activity and expression of EAAT3 have been shown to be highly regulated by neuronal activity (29
). The increase in transporter activity we measured in neurons after particle exposure was not unexpected because we had previously noted this response using γ rays (17
). However, the changes in neuron transporter activity were much greater than those measured in astrocytes, and the changes observed in neurons were greater at all times and for all types of radiation used here. We did not detect a simple LET dependence of modulated transporter activity in neurons. In astrocytes, the general response to protons and carbon- and iron-ion radiation was a decrease in transporter activity similar to that observed using γ rays. In astrocytes at 3 h, differences after exposure could be attributed to fluence. However, unlike the recovery time observed previously with γ radiation, transporter activity in astrocytes after particle irradiation did not return to baseline by 2 days after exposure. The alterations in astrocyte transporter activity were evident at 2 days after exposure to all three radiations and were still evident at 7 days after carbon- and iron-ion irradiation. Overall, the biological response of neurons and astrocytes measured in terms of glutamate uptake was more pronounced and longer-lasting with HZE-particle radiation than with γ rays. These findings are consistent with those of other investigators in which exposure of neural precursors to particle radiation resulted in more rapid and persistent changes (26
In the brain, neurons and glia do not exist in isolation. Rather they comprise a functional unit. To better understand the collective response resulting from neuroglial interactions, we plated mixed cultures of neurons and astrocytes at similar ratios to those found in the brain. Coculturing of neurons and astrocytes produced cultures that exhibited a robust and radioresistant phenotype in which the uptake response did not differ from that of unirradiated mixed cultures in response to proton and carbon-ion radiation. However, irradiation of cultures with high-LET iron ions produced an initial change in transporter activity at 3 h after exposure.
We used 250 MeV protons with a corresponding LET of 0.4 keV/μm, which is not numerically that different than 0.25 keV/μm for γ rays. However, irradiation with protons resulted in changes in transporter activity that were more dramatic at 3 h compared to γ radiation and that persisted at later times that were not detectable after γ irradiation (). A similar response pattern was observed with carbon and iron ions. This appears to indicate that track structure with more clustering of ions is significant in transporter activity. The magnitude of change for a given dose decreased with time. Iron-ion irradiation of astrocytes failed to produce initial changes in uptake at 3 h; however, at 2 days after exposure, astrocytes exhibited decreased uptake and continued to do so at 7 days after 10 and 50 cGy. It may be that the subtle differences in the response patterns of neurons and astrocytes to different ions result from the complex interplay of track structure for a particular ion species and cytoarchitectural influence (32
Glutamate uptake is regulated at several levels, primarily via expression of transporter protein and functional activity of the expressed transporters. Previous investigations led us to conclude that changes in glutamate uptake after radiation exposure are not regulated by changes in transporter protein expression levels but rather are regulated at the level of transporter activity (17
). Glutamate transporters appear to be susceptible to changes in cellular redox state, because they possess redox-sensing cysteine residues that regulate transport rate via thiol-disulfide redox interconversion. Three transporter subtypes [EAAT-1 (GLAST), EAAT-2 (GLT1), and EAAT-3] have been shown to be equally inhibited by oxidants (33
). If radiation were in fact modulating transporter activity principally via an ROS-mediated mechanism, then it seems logical that any ROS stimulus could initiate this mechanism to modulate transporter activity. Chemically increasing ROS levels using Paraquat failed to evoke long-term uptake changes or to mirror the changes in transporter activity observed after radiation exposure. Therefore, the significant and persistent changes in transporter activity observed in astrocytes and neurons after irradiation are not principally mediated by redox modulatory mechanisms. As such, it does not appear that secondary reactive processes, like increased oxidant levels after exposure, regulate transporter activity long term, nor do they account for the reciprocal responses in these particular cell types in the context of radiation-induced ROS.
Current knowledge of transporter regulation suggests that the transporters are differentially regulated wherein each transporter subtype is responsive to multiple and at times opposing factors. An example of this is PKC activation, which is known to increase EAAT-1 and EAAT-2 expression but decrease EAAT3 trafficking to the cell surface (34
). Furthermore, the predominant transporters EAAT-1 and EAAT-2 can be reciprocally regulated by the same stimulus. Examples of this are the up-regulation EAAT-1 but not EAAT-2 with cAMP and the opposing effects of glutamate on EAAT-1 and EAAT-2 expression (35
). When assessing transporter activity, we must keep in mind that we are measuring the collective uptake activity of all transporter subtypes present on a given cell type. Astrocytes and neurons can express any combination of transporter subtypes at any given time. Given this, it is probable that radiation may be activating a signaling cascade that preferentially activates a molecular target with opposing effects in neurons and astrocytes. Such a mechanism appears more likely to involve regulation of glutamate transporter activity rather than a strictly redox mechanism and also helps explain the reciprocal effects observed in the two cell types.
Other investigators have proposed that cell type-specific metabolic and signaling pathways may be responsible for differences in the glial uptake response after injury (36
). Astrocytes express several enzymes involved in glutamate metabolism and transport that neurons do not. An example of this is that astrocytes preferentially metabolize acetate compared to neurons. These cell-specific differences become evident when a toxin such as fluoroacetate, a selective inhibitor of the TCA cycle in astrocytes, impairs not only synthesis of glutamate precursor but also glutamate uptake (38
). Such differences in the cellular metabolic machinery could account for the uptake response observed in cultures of isolated neurons and astrocytes.
In our mixed cultures we measured low uptake activity in both irradiated and unirradiated mixed cultures. Cocultures of neurons and astrocytes exhibited an uptake response distinct from that observed in isolated neurons or astrocytes. The basal transporter activity in mixed cultures was generally low. In unirradiated mixed cultures, neurons seemed to influence the uptake response given the relatively low level of transporter activity and ratio of neurons to astrocytes. In trying to understand this response pattern we cannot ignore the bidirectional crosstalk that takes place between neurons and astrocytes. Nakajima et al
. investigated the influence of neuronal conditioned medium on the activity and level of glutamate transporters in rat microglia. They demonstrated that a neuronal stimulus appeared to promote the uptake of glutamate in activated microglia (40
). Similar to this finding, we observed that coculturing neurons and astrocytes produced a basal uptake that was low and that appeared to be influenced by neurons. When mixed cultures were irradiated, we did not see characteristic changes in uptake that were predominantly neuron- or astrocyte-driven. That is, irradiated mixed cultures did not exhibit uptake responses expected of either irradiated astrocytes or neurons. Because the response profile observed in irradiated mixed cultures did not differ significantly from unirradiated cell cultures, this suggests a resistance to perturbations such as radiation. We believe the blunted response was indicative of a homeostatic effect composed of interactive neuron-astrocyte responses. This effect can be interpreted to mean that the overall uptake of glutamate from the extracellular space is not expected to be compromised after radiation exposure. Furthermore, when the individual response of each cell type was examined, we found that in neuron cultures uptake increased after exposure, whereas in astrocytes uptake decreased but was not completely abolished. Therefore, it seems reasonable to surmise that radiation-induced neurotoxic cascades would not be initiated by altered glutamate uptake at the level of glutamatergic receptor overactivation because the net concentration of extracellular glutamate should be the same in irradiated and unirradiated mixed cultures. Alterations in glutamate uptake, however, could result in abnormal intracellular glutamate concentrations. Intracellular changes in neurotransmitter concentrations could potentially trigger aberrant signaling between neurons and astrocytes because intercellular signaling between these cells is partly mediated by glutamate and calcium. Calcium-dependent glutamate release from astrocytes is known to modulate neuronal excitability (41
) and synaptic transmission (42
). Changes in these fundamental CNS functions may ultimately translate to dysfunction in cognitive and behavioral output. Our data indicate that glutamate is involved in the radioresponse of the CNS, perhaps not at the level of receptor-mediated excitotoxicity but rather as a signaling mediator of radiation injury.
An additional component to consider in the regulation of glutamate-mediated transmission through neuron-glial interactions is the role of the vasculature, which is paramount in this process by supplying the basic energy components required to sustain such interactions. Increased neuronal activity places an energetic demand that is met by a continuous vascular supply of glucose and oxygen (43
). Data suggest that glutamate coordinates both metabolic and vascular responses to neuronal activity, with astrocytes playing a key role in coupling synaptic activity to glucose use (44
). Several mechanisms have been suggested to be involved in neurometabolic and neurovascular coupling during activation. These models are supported by anatomical studies that demonstrate that, in addition to being surrounded by astrocytic processes, blood vessels are also in direct contact with nerve terminals in some cases. Different subtypes of receptors for select neurotransmitters and neuropeptides have been identified on specific cellular components of the microvascular unit (i.e., endothelial and smooth cells and their associated perivascular astrocytes) (43
). Furthermore, bidirectional calcium signaling can occur between astrocytes and endothelial cells via gap junctions and paracrine signaling (48
). Clearly, a significant degree of intercellular signaling takes place among the various CNS cell types. The relationship between radiation-induced glutamate changes and neuroglial and vascular intercellular signaling represents a potential area of study in understanding radiation-induced pathophysiology within the CNS.