Whole cell current clamp recordings were made from CA1 pyramidal neurons in acute hippocampal slices. The cells were filled through the patch pipette with the morphological dye Alexa Fluor 594 (15 µM) and the Ca2+
indicator Fluo-5F (300 µM). To determine if ambient glutamate levels are high enough to bind significant numbers of NMDARs, we measured Ca2+
transients in both spines and dendritic shafts evoked by back-propagating action potentials 
(bAP; ). As NMDARs are expressed synaptically and extrasynaptically 
transients evoked by bAPs in the two cellular compartments may be mediated by synaptic and extrasynaptic NMDARs bound by ambient glutamate as well as by VGCCs. As has been shown previously 
, however, pharmacological block of NMDARs did not alter the Ca2+
transients in either compartment (; spine: 88.0±6.90%, dendrite: 90.5±5.71%; p>0.1; n
11). Though NMDAR activation by exogenous glutamate boosts bAP-elicited Ca2+
, the present results, and those of others 
, suggest that there is little tonic activation of NMDARs in either spines or dendritic shafts. Furthermore, there appears to be no developmental shift in the tonic activation of NMDARs since D-AP5 also failed to alter the Ca2+
transients from spines (; 94.4±5.27%; p>0.3; n
17) and dendrites (; 90.7±4.78%; p>0.05; n
13) of CA1 pyramidal neurons from older animals (P33–40).
NMDAR antagonism has no differential effect on bAP-evoked Ca2+ signals in dendrites or spines.
This experiment may not be sensitive enough to detect low level activation of NMDARs because of infrequent channel gating and because bAPs may be too short to engage the slow components of NMDAR Mg2+
. In addition, Ca2+
influx through infrequently open NMDARs during a bAP may be small relative to the Ca2+
contribution from VGCCs. To increase the potential contribution of NMDARs to the Ca2+
transient, pyramidal cells were voltage clamped at −65 mV and stepped to +5 mV for 40 ms () in the presence of mibefradil and nimodipine (both at 20 µM), antagonists of the predominant VGCCs on pyramidal cell dendrites and spines 
, along with TTX (0.5 µM). Subsequent application of D-AP5 did not affect the voltage step-evoked Ca2+
transient (; p>0.1 for both spine and dendrite). To ensure that this technique was sensitive enough to detect NMDAR activation, we applied 5 µM NMDA to the superfusate (equivalent to ~250 nM glutamate) 
following washout of D-AP5. NMDA significantly increased the voltage step-evoked Ca2+
signal (; spine: 7.54 fold increase, p<0.001; dendrite: 2.46 fold increase, p<0.01; n
11). Data from both apical and basal dendrites were pooled since no differences were observed between these two regions. Taken together, these data reinforce the notion that ambient glutamate is maintained at low concentrations, producing minimal NMDAR activation in both synaptic and extrasynaptic compartments.
Ambient glutamate concentrations are too low to generate significant Ca2+ influx through NMDARs in dendrites or spines.
We approached the issue of transporter distribution and preferential synaptic protection by blocking glutamate uptake. If the extrasynaptic glutamate concentration is higher than that in the cleft because transporters prevent diffusion of glutamate into the synapse, blocking transporters should result in a large Ca2+
increase in the spine as extrasynaptic glutamate rushes into the cleft and activates synaptic NMDARs. Spines exhibited a Ca2+
increase during a 40 ms depolarization with iontophoresis of the glutamate transporter substrate and NMDAR agonist, L-aspartate (; black and gray traces), confirming the presence of NMDARs. However, TBOA (100 µM) did not increase the Ca2+
transient in the same spines during the 40 ms depolarization when compared to the control voltage step without L-aspartate iontophoresis (, compare green and red traces; 20.6±13.62%; p>0.5; n
5;). TBOA was effective in blocking transporters, however, as the NMDAR-mediated Ca2+
signal evoked by iontophoresis of L-aspartate was increased in the presence of TBOA (). This result indicates that glutamate transporters do not normally generate a concentration gradient of ambient glutamate between extrasynaptic and synaptic extracellular compartments.
Transporter blockade does not reveal an ambient glutamate concentration gradient between extracellular compartments.