Glutamate mediates several modes of neurotransmission in the central nervous system including recently discovered retrograde signaling from neuronal dendrites. We have previously identified the system N transporter SN1 as being responsible for glutamine efflux from astroglia and proposed a system A transporter (SAT) in subsequent transport of glutamine into neurons for neurotransmitter regeneration. Here, we demonstrate that SAT2 expression is primarily confined to glutamatergic neurons in many brain regions with SAT2 being predominantly targeted to the somatodendritic compartments in these neurons. SAT2 containing dendrites accumulate high levels of glutamine. Upon electrical stimulation in vivo and depolarization in vitro, glutamine is readily converted to glutamate in activated dendritic subsegments, suggesting that glutamine sustains release of the excitatory neurotransmitter via exocytosis from dendrites. The system A inhibitor MeAIB (alpha-methylamino-iso-butyric acid) reduces neuronal uptake of glutamine with concomitant reduction in intracellular glutamate concentrations, indicating that SAT2-mediated glutamine uptake can be a prerequisite for the formation of glutamate. Furthermore, MeAIB inhibited retrograde signaling from pyramidal cells in layer 2/3 of the neocortex by suppressing inhibitory inputs from fast-spiking interneurons. In summary, we demonstrate that SAT2 maintains a key metabolic glutamine/glutamate balance underpinning retrograde signaling by dendritic release of the neurotransmitter glutamate.

Activity-dependent changes in ionotropic glutamate receptors at the postsynaptic membrane are well established and this regulation plays a central role in the expression of synaptic plasticity. However, very little is known about the distributions and regulation of ionotropic receptors at presynaptic sites. To determine if presynaptic receptors are subject to similar regulatory processes we investigated the localisation and modulation of AMPA (GluR1, GluR2, GluR3) and kainate (GluR6/7, KA2) receptor subunits by ultrasynaptic separation and immunoblot analysis of rat brain synaptosomes. All of the subunits were enriched in the postsynaptic fraction but were also present in the presynaptic and non-synaptic synaptosome fractions. AMPA stimulation resulted in a marked decrease in postsynaptic GluR2 and GluR3 subunits, but an increase in GluR6/7. Conversely, GluR2 and GluR3 increased in the presynaptic fraction whereas GluR6/7 decreased. There were no significant changes in any of the compartments for GluR1. NMDA treatment decreased postsynaptic GluR1, GluR2 and GluR6/7 but increased presynaptic levels of these subunits. NMDA treatment did not evoke changes in GluR3 localisation. Our results demonstrate that presynaptic and postsynaptic subunits are regulated in opposite directions by AMPA and NMDA stimulation.

Neuronal gap junctional hemichannels, composed of pannexin-1 subunits, have been suggested to play a crucial role in epilepsy and brain ischaemia. After a few minutes of anoxia or ischaemia, neurons in brain slices show a rapid depolarization to ∼−20 mV, called the anoxic depolarization. Glutamate receptor blockers can prevent the anoxic depolarization, suggesting that it is produced by a cation influx through glutamate-gated channels. However, in isolated hippocampal pyramidal cells, simulated ischaemia evokes a large inward current and an increase in permeability to large molecules, mediated by the opening of pannexin-1 hemichannels. N-methyl-d-aspartate is also reported to open these hemichannels, suggesting that the activation of N-methyl-d-aspartate receptors, which occurs when glutamate is released in ischaemia, might cause the anoxic depolarization by evoking a secondary ion flux through pannexin-1 hemichannels. We tested the contribution of pannexin hemichannels to the anoxic depolarization in CA1 pyramidal cells in the more physiological environment of hippocampal slices. Three independent inhibitors of hemichannels—carbenoxolone, lanthanum and mefloquine—had no significant effect on the current generating the anoxic depolarization, while a cocktail of glutamate and gamma-aminobutyric acid class A receptor blockers abolished it. We conclude that pannexin hemichannels do not generate the large inward current that underlies the anoxic depolarization. Glutamate receptor channels remain the main candidate for generating the large inward current that produces the anoxic depolarization.

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. AbstractPrevious studies have indicated that presynaptic -amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) contribute to the regulation of neurotransmitter release. In hippocampal synapses, the presynaptic surface expression of several AMPAR subunits, including GluA2, is regulated in a ligand dependent manner. However, the molecular mechanisms underlying the presynaptic trafficking of AMPARs are still unknown. Here, using bright-field immunocytochemistry, western blots, and quantitative immunogold electron microscopy of the hippocampal CA1 area from intact adult rat brain, we demonstrate the association of AMPA receptors with the presynaptic active zone and with small presynaptic vesicles, in Schaffer collateral synapses in CA1 of the hippocampus. Furthermore, we show that GluA2 and protein interacting with C kinase 1 (PICK1) are colocalized at presynaptic vesicles. Similar to postsynaptic mechanisms, overexpression of either PICK1 or pep2m, which inhibit the N-ethylmaleimide sensitive fusion protein (NSF)-GluA2 interaction, decreases the concentration of GluA2 in the presynaptic active zone membrane. These data suggest that the interacting proteins PICK1 and NSF act as regulators of presynaptic GluA2-containing AMPAR trafficking between the active zone and a vesicle pool that may provide the basis of presynaptic components of synaptic plasticity.

Glutamate, the main excitatory neurotransmitter in the brain, may cause excitotoxic damage through excessive release during a number of pathological conditions. We have developed an immunocytochemical assay to investigate the mechanisms and regulation of glutamate release from intact, cultured neurons. Our results indicate that cultured hippocampal neurons have a large surplus of glutamate available for release upon chemically induced depolarization. Long incubations with high K(+)-concentrations, and induction of repetitive action potentials with the K(+)-channel blocker 4-aminopyridine (4-AP), caused a significant reduction in glutamate labeling in a subset of boutons, demonstrating that transmitter release exceeded the capacity for replenishment. The number of boutons where release exceeded replenishment increased continuously with time of stimulation. This depletion was Ca(2+)-dependent and sensitive to bafilomycin A1 (baf), indicating that it was dominated by vesicular release mechanisms. The depletion of glutamate from cell bodies and dendrites was also Ca(2+)-dependent. Thus, under the present conditions, cytosolic glutamate is taken up in vesicles prior to release, and the main escape route for the amino acid is through vesicular exocytosis. Depolarization with lower concentrations of K(+) caused sustainable release of glutamate, i.e., without full depletion.