Although it is a well-established metabolic pathway in the vertebrate brain, the necessity of the glutamate-glutamine cycle for excitatory neurotransmission remains controversial (Rothman et al., 2003
; Kam and Nicoll, 2007
). In this study we provide evidence that a fundamental part of the cycle, the transfer of astrocyte-derived glutamine to neurons, is essential for sustained epileptiform activity in acutely disinhibited neocortical slices. We demonstrate that the cycle contributes to excitatory neurotransmission in our slices and that exogenously supplied glutamate can enter the cycle. However, we find that in addition to the cycle, de novo
synthesis, which is occurring predominantly in astrocytes, is required for maintaining high levels of glutamate release in our in vitro
system. We also find, in contrast to previous suggestions, that the cycle can function independently of system A transporters and utilizes an, as of yet, unidentified neuronal glutamine transporter or transporters. Finally we find that the attenuation of network activity through inhibition of glutamine transport is associated with reduced frequency and amplitude of spontaneous events detected at the single cell level, suggesting that availability of glutamine influences neurotransmitter release at excitatory synapses during periods of intense network activity.
Although the majority of synaptically released glutamate is derived from the glutamate-glutamine cycle both in vivo
and in vitro
(Lieth et al., 2001
; Sibson et al., 2001
), glutamate can be derived from a number of other precursors which could explain the limited effect of disrupting the cycle pharmacologically or genetically on behavior and synaptic transmission (Blin et al., 2002
; Masson et al., 2006
; Kam and Nicoll, 2007
). While likely crucial for periods of increased neuronal activity (Oz et al., 2004
), these other pathways complicate the characterization of the glutamate-glutamine cycle, particularly in ex vivo
preparations. The acutely disinhibited rat neocortical slice provides us with a system in which the influence of the glutamate-glutamine cycle on excitatory synaptic transmission can be assessed in a physiologically intact system, and under conditions in which potentially confounding effects on metabolism of the inhibitory neurotransmitter GABA are rendered irrelevant.
Our finding of a marked reduction in epileptiform activity when slices are treated with MSO coupled with the minimal effect of MeAIB indicates that system A transport is not necessary for neuronal uptake of glutamine. This is consistent with previous studies that have suggested that the system A transporters SNAT1 and SNAT2 are unlikely to play a crucial role in the glutamate-glutamine cycle and evidence for system A-independent neuronal glutamine transport (Rae et al., 2003
; Conti and Melone, 2006
). However, a recent study suggests that MeAIB sensitive transport is a major pathway for neuronal glutamine uptake in vivo
(Kanamori and Ross, 2006
). In addition, we and others have demonstrated that in models of chronic hyperexcitability system A transport is required for sustained epileptiform activity (Tani et al., 2007
; Sandow et al., 2009
), suggesting that with chronic hyperexcitability, system A transporters contribute more significantly to the glutamate-glutamine cycle. The demonstration that MeAIB attenuates epileptiform activity in acutely disinhibited hippocampal slices (Bacci et al., 2002
), also suggests that the role of system A transport in the glutamate-glutamine cycle varies from brain region to brain region.
The molecular identity of the MeAIB insensitive transporter or transporters is unknown. Unfortunately, AIB, alanine and histidine, which all appear to disrupt the cycle, broadly inhibit glutamine transporters (Christensen, 1962
; Broer and Brookes, 2001
). MeAIB insensitive neuronal glutamine transport in neurons and synaptosomes has been described (Su et al., 1997
; Tamarappoo et al., 1997
; Rae et al., 2003
), but further biochemical and molecular studies will be needed to more completely characterize this step in the glutamate-glutamine cycle.
The rapid run down of evoked epileptiform fields in AOAA treated slices indicates that recycling alone cannot maintain the level of glutamate release required for epileptiform field generation in our system. The rescue of the epileptiform fields in these slices with glutamate does, however, confirm that the glutamate-glutamine cycle is intact in our slices. The far greater expression level of pyruvate carboxylase, an enzyme required for anaplerosis, in astrocytes compared to neurons suggests that most de novo
synthesis occurs in astrocytes and that the glutamate is converted to glutamine and then transferred to neurons (Shank et al., 1985
). However, if glutamine synthetase and the neuronal glutamine transport systems are completely inhibited by MSO and AIB, then the more complete response to AOAA (e. g.
the absence of intermittent epileptiform events) suggests in addition that some de novo
synthesis occurs in neurons where glutamate can be packaged directly into vesicles without going through a glutamine intermediate. This interpretation would be consistent with the capacity of cultured neurons to sustain glutamate release in the absence exogenous glutamine (Kam and Nicoll, 2007
Our field recording data suggest that disruption of the glutamate-glutamine cycle attenuates glutamatergic neurotransmission. This presumably results from a limited availability of glutamate. With less glutamate available for vesicular packaging it follows that the amount in each vesicle (e. g.
quantal size) will be reduced and, in the extreme case, vesicles will contain no detectable glutamate (Wolosker et al., 1996
). This would be tested most directly by measuring miniature excitatory post-synaptic potentials in the presence of tetrodotoxin (Katz and Miledi, 1965
). However, the requirement for frequent activation of neuronal networks in our system precludes the use of tetrodotoxin. Our alternative approach, analyzing spontaneous synaptic events with whole cell voltage clamp recordings, revealed a reduction in amplitude and a far greater decrease in frequency of events. The reduction in amplitude of spontaneous events is consistent with a decrease in miniature excitatory post synaptic events and therefore quantal size (Min and Appenteng, 1996
). However, the small reduction (~10%) would not explain the more marked effect on epileptiform events or even the monosynaptic early evoked responses recorded from the same cells.
The reduction in spontaneous EPSC frequency (~75%) with AIB is more consistent with the effect on epileptiform events and the reduction in the early evoked responses. The simplest explanation for the reduction in frequency of spontaneous events is a decreased probability of release (Katz, 1962
). Unfortunately, the need for consistent intermittent stimulation in our system precludes the use of standard measures of release probability and vesicle fusion such as pair-pulsed ratio and FM1-43 destaining kinetics (Katz and Miledi, 1968
; Pyle et al., 1999
). Further, it is not directly obvious how AIB would cause a reduction in probability of vesicle fusion. An alternative explanation for the reduced frequency of spontaneous events is that some terminals that form synapses onto the recorded cell are recruited into the activated network and are readily depleted of glutamate when neuronal glutamine transport is blocked. Vesicles from these terminals would continue to fuse with the same probability, but no detectable glutamate would be released. The spontaneous events and persisting monosynaptic evoked responses that are detected in the presence of AIB could arise from terminals that are perhaps less activated by the recurrent activity of the epileptiform event.
Can disrupting the glutamate-glutamine cycle be used to treat epilepsy? Our data and that of others suggest that it can (Bacci et al., 2002
; Tani et al., 2007
; Eid et al., 2008
). However, recent work has demonstrated that cycle activity is impaired in epileptic tissue (Pan et al., 2008
). These contradictory findings may be explained, in part, by the system being studied and which step in the cycle is disrupted. Blocking the cycle after the synaptic release of glutamate but before synthesis of glutamine would lead to a paradoxical increase in excitability as synaptic glutamate will not be efficiently cleared from the extracellular space (Rothstein et al., 1996
; Tanaka et al., 1997
). Indeed inhibiting astrocytic glutamate transporters increases epileptiform activity (Dulla et al., 2008
). In addition, MSO leads to seizures when given to rodents (Rowe and Meister, 1970
), and cases of human temporal lobe epilepsy have been associated with a reduction in astrocytic expression of glutamine synthetase (Eid et al., 2004
). The absence of a similar effect in our slices treated with MSO may reflect the fact that synaptic glutamate does not readily build up in perfused brain slices. Disrupting the cycle after glutamine synthesis, but before glutamate release (either at the intercellular transfer of glutamine, synthesis of glutamate, or vesicular packaging and release), should lead to a decrease in synaptic levels of the neurotransmitter. Inhibiting neuronal glutamine uptake is particularly appealing as the primary metabolic effect would be an increase in extracellular glutamine. While blocking neuronal glutamine uptake might also inhibit synthesis of GABA (Liang et al., 2006
; Fricke et al., 2007
), an undesirable effect when trying to treat hyperexcitability, our data suggest that MeAIB sensitive transporters, which appear to be important in GABA synthesis, are not essential to the glutamate-glutamine cycle. Thus identifying and targeting the MeAIB insensitive neuronal glutamine transporters in the cycle may be of particular importance.