A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity
© Bengtson et al; licensee BioMed Central Ltd. 2008
Received: 24 August 2007
Accepted: 24 January 2008
Published: 24 January 2008
Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic) may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death.
Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours) periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6%) reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting.
The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity-dependent neuroprotection. Other factors, in particular calcium signaling to the nucleus and the induction of survival promoting genes are more likely to mediate acquired neuroprotection.
Synaptic and extrasynaptic NMDA receptors are, respectively, coupled to survival and cell death pathways, which involves their opposing effects on the cAMP response element binding protein (CREB) [1–6] and their regulation of overlapping but distinct genomic programs recently revealed by a whole genome transcriptome analysis . The differential role of NMDA receptors provides a new concept explaining how the same receptor, dependent on its location, can couple to both survival and death. This concept represents an alternative to the "Ca2+ load" hypothesis, which attempts to assign a toxic threshold to Ca2+ influx associated with NMDA receptor activation [8, 9]. Precisely how NMDA receptors differentially regulate the activity of CREB or signaling molecules such as the extracellular signal-regulated kinases 1 and 2 (ERK1/2) is unknown, but differences in the NMDA receptor subunit composition and/or differences in signaling complexes associated with synaptic versus extrasynaptic NMDA receptors may be important [5, 10–13].
The toxic effects of extrasynaptic NMDA receptor activation can be counteracted to some extent by prior activation of synaptic NMDA receptors. For example, prolonged periods action potential (AP) bursting-induced with the GABAA receptor antagonist, bicuculline in cultured hippocampal networks robustly activates synaptic NMDA receptors, which protects against subsequent NMDA-induced excitotoxicity  as well as against pro-apoptotic stimuli such as serum deprivation  or staurosporine treatment . Similarly, minor ischemic events or preconditioning systemic doses of NMDA are neuroprotective [15–19]. The neuroprotective effects of preconditioning neurons with low concentrations of NMDA are mediated, at least in cultured hippocampal networks, via AP-induced stimulation of synaptic NMDA receptors . The molecules responsible for synaptic NMDA receptor-induced survival represent potential clinical targets to reduce neuron loss associated with pathological conditions including stroke and neurodegenerative diseases in which NMDA receptor-mediated excitotoxicity has been implicated [21–27].
NMDA receptor-mediated neuroprotection appears to involve multiple players including nuclear Ca2+ signaling, CREB, nuclear factor kappa B, ERK1/2, Akt1, phosphatidylinositol 3-kinase, protein kinase C epsilon, and brain-derived neurotrophic factor [6, 15–17, 19, 28, 29]. Given the central role of extrasynaptic NMDA receptors in cell death, it is also conceivable that signal-induced changes in surface expression or function of this pool of receptors could profoundly affect the susceptibility of neurons to toxic insults. The surface expression of NMDA receptors (presumably both synaptic and extrasynaptic receptors) is dynamic, whereby receptor endocytosis, exocytosis, and lateral movement are strongly regulated by activity [30–33]. The first step in determining whether changes in the relative distribution of NMDA receptors (synaptic versus extrasynaptic) are associated with and responsible for activity and NMDA receptor-induced neuronal survival, requires a method that allows the precise quantitative assessment of extrasynaptic NMDA receptor function in individual neurons.
Techniques for the identification of the extrasynaptic NMDA receptor pool in brain slices are emerging [34, 35]. However, considerable advances have been made in isolating extrasynaptic NMDA receptor function in cultured neurons. Such studies have employed a protocol, which specifically blocks synaptic NMDA receptors with MK-801. MK-801 is a use-dependent open channel NMDA receptor blocker, which enters the channel only after its activation but then becomes trapped inside the pore to "irreversibly" block the receptor as long as the receptor is not re-activated to release the blocker [36, 37]. Extrasynaptic NMDA receptor-mediated currents have been measured in single neurons isolated in micro-island cultures after blocking autaptic synapses with MK-801 during synaptic stimulation . Techniques for quantifying extrasynaptic NMDA receptor-mediated currents in cultures of neuronal networks have also been developed but the parameters necessary for use-dependent blockade of NMDA receptors require refinement. Mass activation of synaptic NMDA receptors in neuronal networks of hippocampal cultures can be achieved using the GABAA receptor antagonist, bicuculline, which initiates recurrent synchronous bursting [4, 5, 39]. MK-801 application during bicuculline-induced bursting in hippocampal cultures provides a use-dependent blockade of synaptic NMDA receptors allowing the extrasynaptic NMDA receptor population to be subsequently activated with bath applied NMDA [13, 40].
The aim of this study was to investigate the possibility that the known neuroprotection afforded by synaptic NMDA receptor activation is mediated by changes in the surface expression and/or function of extrasynaptically localized NMDA receptors. A method based on previously used protocols was refined and verified in order to isolate extrasynaptic NMDA receptors and quantify their function in individual neurons that are part of a complex neuronal network. We found a surprisingly small neuron-to-neuron variation in extrasynaptic NMDA receptor function. Overnight network bursting, stimulating synaptic NMDA receptors, led to a measurable but very small loss of extrasynaptic NMDA receptor function, which seemed dwarfed by the dramatic neuroprotective effect of this treatment.
MK-801 treatment in bursting networks effectively isolates the extrasynaptic pool of NMDA receptors
Protective effects of AP bursting against NMDA toxicity
Ca2+responses to toxic NMDA treatment
Isolated extrasynaptic NMDA receptor-mediated currents and Ca2+signals are not affected by overnight AP bursting
Extrasynaptic NMDA receptors are coupled to a CREB shut-off mechanism and cell death pathways . We reasoned that a specific reduction in extrasynaptic NMDA receptor numbers or function could underlie the AP bursting-induced protective effects. NMDA receptors are known to be endocytosed from extrasynaptic sites adjacent to post synaptic densities in mature dendritic spines [33, 43, 44]. In our cultures at the time point of recordings (11–12 DIV) spines are present (see Additional file 1). To investigate whether such changes occur after prolonged AP bursting, extrasynaptic NMDA receptor-mediated whole cell currents and Ca2+ responses were measured at room temperature in single cells (see Figure 2 and text above) from overnight vehicle and AP bursting-treated neurons. All handling and medium change was performed in parallel to equalize non-specific effects. Activity of patched cells was assessed in cell-attached mode and in current clamp mode immediately after break-in (data not shown). All neurons in the bicuculline-induced AP bursting group showed regular bursts of APs in cell-attached and/or current clamp mode as well as bursting synaptic input (bursts of spontaneous EPSCs) in voltage clamp mode. 10 to 20% of cells in the vehicle-treated group showed evidence of weak but regular bursting and were discarded from the analysis.
Current and Ca2+ responses in the vehicle and AP bursting groups were equivalent.
607 ± 110
585 ± 82
peak current density
8.49 ± 1.08
9.41 ± 1.00
31.67 ± 6.60
25.95 ± 4.77
7.60 ± 1.14
8.21 ± 1.24
19.64 ± 3.49
16.92 ± 2.76
left half width
31.79 ± 3.73
39.27 ± 3.47
right half width
78.72 ± 13.94
77.15 ± 10.73
600 ± 133
540 ± 121
Ca2+ 10 min later
270 ± 179
149 ± 51
211 ± 79
152 ± 38
left half width
147 ± 101
105 ± 55
right half width
287 ± 150
159 ± 34
Comparison of AP bursting during MK-801 exposure in EPSC and bath NMDA experiments
Comparison of burst properties during MK-801 exposure in experiments measuring NMDA receptor-mediated EPSCs (EPSC) and those measuring whole cell current responses to bath NMDA after EPSC blockade (Extrasynaptic).
4.5 ± 0.7
4.9 ± 0.5
number of bursts
6.6 ± 1.4
6.2 ± 1.2
number of spikes
106 ± 34
97 ± 13
-60.9 ± 4.7
-58.2 ± 1.7
Vm during bursts
-50.0 ± 2.0
-53.7 ± 2.0
202.4 ± 73.6
187.6 ± 34.6
Large scale analysis of extrasynaptic NMDA receptor-mediated Ca2+responses
Validation of the bicuculline + MK-801 protocol to isolate extrasynaptic NMDA receptor function
In the presence of MK-801, NMDA receptors show a stepwise blockade in response to repeated synaptic activation. More than 90% blockade of synaptic receptors has been demonstrated in autaptic cultures after as few as 15 stimulations in 20 μM MK-801  or 60 stimuli in 5 μM MK-801  and similar blockade is seen in cortical brain slices after 30 stimulations in 40 μM MK-801 . The rate of blockade will depend on the probability of transmitter release, the postsynaptic membrane potential, the concentrations of MK-801 and glycine and the rate of insertion of any new NMDA receptors into the synapses.
Our recordings of evoked synaptic NMDA EPSCs showed an 80% blockade after 4 min of MK-801 exposure during which 5 to 6 network bursts occurred. A similar degree of blockade after 2 min of the MK-801 protocol has been previously reported . While further bursting may have produced a more complete blockade of synaptic NMDA receptors, it is necessary to limit the period of MK-801 application due to the presumed ongoing exchange of synaptic and extrasynaptic NMDA receptors through lateral movement . Such exchange is likely to add blocked receptors to the extrasynaptic receptor population and may prevent complete blockade of synaptic responses due to the continuous immigration of unblocked NMDA receptors into the synapse. The use of 4-aminopyridine to enhance bursting  could potentially avoid this problem by accelerating synaptic blockade. Higher concentrations of MK-801 are not recommended as they sometimes block burst activity in our cultures just as NMDA receptor antagonists can shut off rhythmic bursting in slices  and reduce synaptically-activated spikes in the hippocampus in vivo [for example ]. Alternatively, it remains possible that the residual 20% of EPSCs not blocked by the MK-801 and bicuculline protocol represent NMDA receptor-containing synapses which were not activated by bicuculline-induced AP bursting. A similar percentage of neurons in bicuculline-treated cultures did not show bursting in cell-attached and whole cell current clamp modes. Note that residual unblocked synaptic receptors will contribute to our estimates of the extrasynaptic pool of NMDA receptors. Considering that the synaptic pool represents about 50% of the total NMDA receptor pool based on estimates from similar hippocampal cultures , this implies that 17% of our estimate of extrasynaptic function may be generated by synaptic receptors. Our protocol effectively isolates the extrasynaptic NMDA receptor function in standard hippocampal neuronal cultures and is a robust method to selectively activate or quantify this receptor population. This can serve as a valuable tool to study extrasynaptic NMDA receptor function in vitro as we have done here to explore the involvement of trafficking in the protective effects of synaptic activity.
Overnight recurrent AP bursting only marginally alters extrasynaptic NMDA receptor function
The only significant difference found in extrasynaptic NMDA receptor function between vehicle and overnight bursting treatment groups was a slightly smaller Ca2+ response in the latter. The 6% difference between these groups was highly significant but seems unlikely to account for the dramatic reduction in cell death afforded by pretreating cells with overnight bursting. However, we cannot rule out the possibility that a critical threshold for toxicity exists and that this 6% difference in extrasynaptic receptor function straddles this threshold to produce a dramatic difference in cell death between control and AP bursting groups. However, it seems more likely that mechanisms other than reduced extrasynaptic NMDA receptor function – such as Ca2+ regulation of survival-promoting genes  – are primarily responsible for the protective effects of synaptic NMDA receptor stimulation with overnight AP bursting treatment. Our observation that extrasynaptic NMDA receptor numbers are only marginally affected by prolonged increases in synaptic activity parallel reports showing that extrasynaptic NMDA receptors in contrast to synaptic receptors are unaffected by prolonged depression of synaptic activity with ethanol .
Other potential mechanisms for activity-induced neuroprotection include an enhanced ability of pre-treated neurons to cope with a normally toxic Ca2+ load during NMDA treatment. Regular transient Ca2+ influx associated with AP bursting for an extended period might be expected to foster improved Ca2+ buffering, sequestration, or extrusion mechanisms in neurons. To the contrary, however, the normally toxic 10 min NMDA application produced a slightly higher average Ca2+ response in hippocampal neurons treated with overnight AP bursting (see Figure 4B). Thus prolonged AP bursting does not seem to promote neuroprotection by improving the dissipation of a toxic Ca2+ load.
It is possible that the relative contribution of synaptic and extrasynaptic NMDA receptors to the Ca2+ load activated by an NMDA insult determines its toxicity and that the relative contribution of these two receptor populations is altered by activity. The Ca2+ response to bath applied NMDA arises from a mixed source including both synaptic and extrasynaptic NMDA receptor populations and VOCCs, which differentially couple to CREB function and mitochondrial membrane potential breakdown [4, 20]. The larger Ca2+ transients in cells pretreated with AP bursting (see Figure 4B) may result from an enhanced survival-promoting synaptic NMDA receptor component, which more than compensates for the observed slightly reduced extrasynaptic NMDA receptor function in this group. NMDA receptor exocytosis and synaptic function can be up-regulated by synaptic activity leading to long-term potentiation [50, 51]. AP bursting in hippocampal cultures also potentiates synaptic transmission . Any enhancement of synaptic NMDA receptor number or function should promote the protective effect of this receptor population.
It remains possible that overnight bursting accelerates the development of spines and synapses containing NMDA receptors thus increasing the synaptic NMDA receptor pool in a given neuron. Ca2+ signaling from synaptic NMDA receptors is known to enhance dendritic outgrowth  and synaptic delivery of NMDA receptors occurs within hours of synaptic activation [53–55]. This scenario predicts an increase in the total (synaptic plus extrasynaptic) NMDA receptor pool consistent with the slightly higher Ca2+ response to bath applied NMDA in cultures exposed to overnight AP bursting (see Figure 4B). The sprouting and/or growth of new NMDA receptor-containing synapses would also strengthen the relative contribution of synaptic receptors to the response to bath applied NMDA. This would help counteract the extrasynaptic NMDA receptor-mediated toxic effects and facilitate neuroprotection in cultures treated with overnight AP bursting. Regardless of any (minor) alteration in surface expression or distribution of NMDA receptors, the opposition of synaptic activity to NMDA receptor-mediated death converges at a level downstream of the receptor, through opposing effects on CREB function and target gene activation [2, 4, 7].
We have developed and validated a technique for the isolation and quantitative functional assessment of the extrasynaptic NMDA receptor pool in cultured hippocampal neurons participating in neuronal networks. With this method we have shown that prolonged periods of AP bursting, which protects neurons from subsequent toxic insults, causes little change in the function of the extrasynaptic NMDA receptor pool, a receptor population linked to neuron death.
Hippocampal Cell Culture
Hippocampal neurons from new-born Sprague Dawley rats were prepared as described  except that growth media was supplemented with B27 (Gibco/BRL or Invitrogen, San Diego, CA) 3% rat serum and 1 mM glutamine. Neurons were plated onto 12 mm glass coverslips or plastic 4-well dishes at a density between 400 and 600 cells per mm2. All stimulations and recordings were done after a culturing period of 10 to 12 days during which hippocampal neurons develop a rich network of processes, express functional NMDA-type and AMPA/kainate-type glutamate receptors, and form synaptic contacts [3, 4, 57].
The induction of network bursting and the cell death assay
Bursts of AP firing throughout the neuronal network was induced by treatment of the neurons with 50 μM bicuculline [3, 39]. Bicuculline was dissolved in DMSO which did not exceed a final concentration of 0.05%. Cells with or without 16 h bicuculline pretreatment were subjected to 20 μM NMDA for 10 min at 37°C to induce cell death. After washout of NMDA cells were incubated for a further 5 h at 37°C before fixation with paraformaldehyde (4%) and stained with Hoechst 33528. Cell death was evaluated at a light microscope (Leica DM IRBE) with 40× magnification by counting condensed nuclei in 20 fields of view for every condition in each experiment. Pictures of representative areas were taken with a CCD camera (Spot Insight2; Visitron Systems, Puchheim, Germany).
Patch clamp recordings
Whole-cell patch clamp recordings were made from cultured hippocampal neurons plated on coverslips secured with a platinum ring in a recording chamber (PM-1, Warner Instruments, Hamden, CT, USA) mounted on a fixed-stage upright microscope (BX51WI, Olympus, Hamburg, Germany). Differential interference contrast optics, infrared illumination and a CCD camera (Photometrics Coolsnap HQ, Visitron Systems, Puchheim, Germany) were used to view neurons on a computer monitor using a software interface (Metamorph, Universal Imaging Systems, Downington PA, USA).
The standard extracellular solution contained (in mM) NaCl 140, KCl 5.3, MgCl2 1, CaCl2 2, HEPES 10, glycine 0.01, glucose 30, Na-pyruvate 0.5. Patch electrodes (3–4 MΩ) were made from borosilicate glass (1.5 mm, WPI, Sarasota, FL, USA) and filled with a potassium methylsulphate based intracellular solution (containing in mM: KCH3SO4, 135; NaCl, 8; KCl, 12; HEPES, 10; K2-phosphocreatine, 10; Mg2-ATP, 4; Na3-GTP, 0.3; pH 7.35 with KOH). Recordings were made with a Multiclamp 700B amplifier, digitized through a Digidata 1322A A/D converter, acquired and analysed using pClamp software (Axon Instruments, Union City, CA, USA). Access (range: 10 – 28 MΩ) was monitored regularly during voltage clamp recordings and data was rejected if changes greater than 20% occurred. All membrane potentials have been corrected for the calculated junction potential of -11 mV (JPCalc program by Dr. Peter H. Barry).
Measurement of NMDA receptor-mediated eEPSCs
Synaptic NMDA receptor-mediated currents were recorded at a holding potential of +40 mV in the presence of 10 μM glycine, 1 mM extracellular Mg2+, -(-)bicuculline (50 μM, Sigma), and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10 μM, Tocris). In some recordings, extracellular Ca2+ concentration was reduced to 0.2 mM to reduce Ca2+ mediated inactivation of the NMDA receptor . Evoked excitatory post-synaptic currents (eEPSCs) were recorded in response to single 100 μs long constant current pulse stimuli (80 to 200 μA) from an A365 stimulus isolator using either a tungsten stereotrode (World Presicion Instuments, Sarasota, FL, USA) or 2 glass electrodes whose tips were positioned in contact with the tissue matrix on the surface of the coverslip, on either side of the recorded cell and at a separation of 100 to 200 μm.
MK-801 protocol to isolate extrasynaptic NMDA receptors
Extrasynaptic NMDA receptor-mediated currents were recorded following blockade of synaptic NMDA receptors using MK-801 application during recurrent network bursting activity. 10 μM glycine was used in all our measurements of extrasynaptic NMDA receptor function to minimize glycine dependent NMDA receptor desensitization  and does not prime cells for NMDA receptor internalization . Patched neurons were held in current clamp mode and bicuculline was applied. Once regular bursting was established (2–4 min), MK-801 (10 μM, Tocris Cookson Ltd, Bristol, UK) was added for a further 3–10 min of bursting activity. The MK-801/bicuculline solution was then washed out for 4–6 min with a solution containing no glycine and 1 μM TTX (Tocris) to halt all action potentials and NMDA receptor activation thus preventing unblocking. The cell was then voltage clamped at -71 mV and a zero Mg2+ solution containing glycine (10 μM, 2 mins, 6 ml/min) was washed on for 2 min before bath application of NMDA (100 μM, 30 s, 6 ml/min). Current and calcium responses were used to quantify the total functional pool of extrasynaptic NMDA receptors for each cell. NMDA current responses typically showed an initial peak followed by decay which did not reach steady state within this application period. Ca2+ responses did not reach a peak or steady state plateau within this period.
Alternatively, to test for the blockade of NMDA-receptor-mediated EPSCs following the MK-801/bicuculline protocol, bicuculline was not removed during the MK-801 washout period and CNQX (10 μM) was added instead of TTX. CNQX was just as rapid and effective as TTX in blocking all burst activity.
Where F represents the average fluorescence intensity in a somatic ROI, Fmax represents the maximal F after incubation in ionomycin (50 μM), Fmin represents the minimal F after subsequent application of EGTA (30 mM) or a saturated manganese solution (1:200). NMDA responses were normalized to Fmax (i.e. FNMDA/Fmax). All data are expressed as mean ± standard error of the mean.
We would like to thank Iris Bünzli-Ehret for cell culture preparations and Daniela Mauceri who provided the confocal image of spines shown in the Additional file 1. We also wish to specially thank Malte Wittmann and Giles Hardingham who helped establish the protocol for the cell death assay. This work was supported by the Alexander von Humboldt-Foundation (Wolgang-Paul-Prize to H.B.), EU Project GRIPANNT, the EU Network of Excellence NeuroNE, and the Sonderforschungsbereich (SFB) 488.
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