Glutamine from Glial Cells Is Essential for the Maintenance of the Nerve Terminal Pool of Glutamate: Immunogold Evidence from Hippocampal Slice Cultures

Abstract

The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of L-glutamine synthetase [L-glutamate :ammonia ligase (ADP-forming) ; EC 6 .3 .1 .2] . In control cultures, glutamate-like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells . Glutamine-like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals . After inhibition of glutamine synthetase with L-methionine sulfoximine, glutamate-like immunoreactivity was reduced by 52% in terminals and increased nearly fourfold in glia . Glutamine-like immunoreactivity was reduced by 66% in glia following L-methionine sulfoximine, but changed little in other compartments . In cultures that were treated with both L-methionine sulfoximine and glutamine (1 .0 mM), glutamate-like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate-like immunoreactivity was increased and glutamine-like immunoreactivity was decreased to a similar extent as in cultures treated with L-methionine sulfoximine alone . We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate ; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient . Abbreviations used : GLN-LT, glutamine-like immunoreactivity ;Glu-LI, glutamate-like immunoreactivity; MSO, i .-methionine sulfoximine.

Block of Glutamate-Glutamine Cycle Between Astrocytes and Neurons Inhibits Epileptiform Activity in Hippocampus

“…6). Therefore by removing glutamate from the synaptic cleft, astrocytes might also replenish the neuronal pool of neurotransmitter (Laake et al 1995). In this study, we demonstrate that the functional integrity of the glutamate-glutamine cycle is indeed required for supporting epileptiform activity in hippocampus.…”

“…In this experimental model, long bursts of action potentials, coincident with calcium oscillations, commonly occur either spontaneously (Bacci et al 1999b;Verderio et al 1999) or on removal of magnesium ions from the external medium (Abele et al 1990). This activity, which has been considered as an "in vitro" model of epilepsy (De Lorenzo et al 1998;Furshpan and Potter 1989;Segal 1994;Segal and Furshpan 1990), relies on glutamatergic neurotransmission (Abele et al 1990;Bacci et al 1999b;Lawrie et al 1993;Murphy et al 1992;Nunez et al 1996) and requires the removal of glutamate from the synaptic environment by adjacent astrocytes ). Once taken up by glial transporters, glutamate is converted by FIG.…”

“…To directly test the functional significance of the astrocyte-dependent glutamate-glutamine pathway, cultures were exposed to 100 M methionine sulfoximine (MSO), a selective inhibitor of the enzyme glutamine synthetase, which is specifically expressed by glial cells (Martinez-Hernandez et al 1977). This experimental treatment has been previously found to result in a block of glutamine production in astrocytes (Derouiche and Rauen 1995;Laake et al 1995;Ronzio et al 1969). One hour of treatment with MSO was sufficient to produce either a complete block of epileptiform activity, leaving basal synaptic activity only ( Fig.…”

Evolution of the Neurochemical Profile after Transient Focal Cerebral Ischemia in the Mouse Brain

“…In vivo 1 H-MRS of ischemic evolution in mice H Lei et al are likely to retain sufficient energy for the uptake of extracellular Glu to convert into Gln, both of which are major steps to sustain Glu/Gln homeostasis in Gln-Glu cycling (Rossi et al, 2007) and require ATP (Laake et al, 1995). Glu released from neurons after ischemia, leading to extracellular accumulation and excitotoxicity (Benveniste et al, 1984), is cleared by the astrocytes and converted into Gln.…”

The Astrocytic Contribution in ALS: Inflammation and Excitotoxicity

“…These transporters internalise glutamate, eg. into the astrocyte, for conversion to glutamine that is returned to the pre-synaptic neuron to be release again as glutamate (Laake et al, 1995). Decreased glutamate uptake and EAAT2 protein levels are a common feature in both fALS and sALS and both in vitro and in vivo model systems (Staats and Van Den Bosch, 2009).…”