We find that there is not a steep concentration gradient of glutamate between the synaptic and extrasynaptic space and, consequently, that the synaptic compartment is not preferentially shielded by glutamate transporters. We conclude that ambient glutamate is not significantly compartmentalized but rather is universally low throughout the neuropil of the hippocampus. The various techniques may Nortriptyline measure glutamate in different extracellular compartments such that, for example, NMDAR-mediated currents mainly report synaptic glutamate levels whereas microdialysis and amperometry measure extrasynaptic glutamate. Indeed, the distribution of ambient glutamate within the extracellular space is an issue of debate and a steep concentration gradient between extrasynaptic and synaptic regions of the neuropil has been proposed. We report that in area CA1 of the hippocampus, however, low ambient glutamate concentrations are maintained throughout the neuropil. Any differences in glutamate concentrations across the neuropil must be quite modest. We observed NMDAR-mediated Ca2+ elevations in the dendrite and spine only in response to exogenous application of NMDAR agonists, suggesting NMDARs on both structures are rarely bound by ambient glutamate. In whole cell recordings, a small NMDAR-mediated current is activated by ambient glutamate and is increased by inhibiting transport. However, this tonic current represents the activity of only a small fraction of the total number of NMDARs expressed by a neuron. Detection of such a small fractional activation in a single spine, which expresses at least 1000-fold fewer NMDARs than the whole cell, would be unlikely, despite the sensitivity of 2PLSM. Pantoprazole sodium Detecting ambient glutamate in the extrasynaptic space using NMDAR Ca2+ influx may be problematic if the expression of these receptors is low in this compartment and if the dendritic signal is contaminated by bound calcium indicator diffusing from activated spines. Such contamination could also result in slower or delayed Ca2+ signals in dendritic shafts. As an alternative test for high extrasynaptic glutamate concentrations, we monitored Ca2+ in spines while blocking glutamate transporters. This should collapse any existing extracellular glutamate gradient and allow synaptic NMDARs to respond to extrasynaptic levels of glutamate. Because the synaptic cleft volume is small, relative to the volume of the extrasynaptic space, extracellular glutamate in the synapse will rapidly approach the concentration in the extrasynaptic compartment once the gradient is disrupted. Therefore, if extrasynaptic levels are in the micromolar range, transporter block should cause large Ca2+ elevations in spines. However, TBOA did not increase the spine Ca2+ signal. As 5 mM NMDA activates large NMDAR-mediated Ca2+ transients in spines, the ineffectiveness of TBOA suggests that extrasynaptic levels of glutamate must be substantially lower than 250 nM.