Previous work has shown that NMDARs can be important in cell firing, both in the visual system (Miller et al., 1989
; Sillito et al., 1990
; Blitz and Regehr, 2003
) and in the dentate gyrus of hippocampus (Abraham and Mason, 1988
; Burgard et al., 1989
; Dahl et al., 1990
). The consequences of NMDAR inhibition on action potential generation in hippocampal neurons using LTP-inducing stimulation, however, had not been previously studied. Here, we show that NMDARs play a particularly important role in determining whether neurons will fire in response to synaptic stimulation, including LTP-inducing stimulation such as 100 Hz. This role in action potential generation has important implications regarding the interpretation of pharmacological studies looking at cell-wide ERK activation; blockade of NMDARs results in a loss of p-ERK staining that can now be directly tied to a blockade of action potentials. Two conditions where action potentials were not blocked in the presence of antagonists, bicuculline or TBS, show that when action potentials are preserved, ERK activation remains intact.
Because the effect of these antagonists can be rapidly reversed on washout, NMDAR blockade appears to be an acute effect, distinct from the NMDAR-dependent persistence of adenosine-mediated bursting in CA3 (Thummler and Dunwiddie, 2000
). Given our results, it is also interesting to note that rhythmic oscillations and bursts evoked with synaptic stimulation are inhibited with NMDAR antagonists (Bonansco et al., 2002
). Curiously, our dose-response curves for the effect of NMDAR antagonists on action potential generation () are shifted rightward by nearly a factor of ten when compared to published affinities of these drugs for the NMDAR (Benveniste et al., 1990
). This suggests that a significant number of NMDARs need to be blocked before any partial effects can be seen on action potential number. Maximal effects on action potentials (at 5 and 100 Hz) occur at concentrations that appear to be in the selective ranges of the drugs (i.e. no apparent effect on AMPA receptors, Supplementary Figure 2), and therefore cannot be explained simply as non-selective effects of the drugs on AMPA receptors. Effects on presynaptic NMDARs can also be ruled out, given our observation that intracellular MK801 also blocked action potentials generation. In addition, non-specific drug effects on sodium channels are also unlikely, given that bicuculline can restore action potential generation in the continued presence of NMDAR antagonists. Repetitive stimulation such as that used here is apparently unnecessary for the role of NMDARs in action potential firing, given that single population spikes are also sensitive to NMDAR antagonists in the dentate gyrus (Abraham and Mason, 1988
; Burgard et al., 1989
; Dahl et al., 1990
Using mice instead of rats, one study found that action potentials (population spikes) induced with 5 Hz stimulation were insensitive to APV at 100μM (Thomas et al., 1998
). Given our observation that higher stimulation intensities reduced the effects of NMDAR antagonists (Supplementary Figure 4), it is likely that this discrepancy is due to differences in the relative effectiveness of electrical stimulation between mice and rats, or perhaps in stimulus intensity used in the two studies. This is supported by the observation that 80% of the EPSPs recorded with 5 Hz stimulation in Thomas, et al. evoked spikes (Thomas et al., 1998
), whereas less than 50% of the EPSPs evoked action potentials in our study. If the AMPA receptor component of the EPSPs were sufficiently large, NMDARs would not be necessary for action potential firing.
Interestingly, although bicuculline has no effect on basal ERK activation (), we do see increased staining with stimulation whenever bicuculline is used. We find this to be wholly consistent with our assertion that action potentials are important for ERK activation in that as more action potentials are evoked, a larger number of cells and wider area are stained. An increase in intensity could simply be due to the cells firing more action potentials for a given stimulus. If NMDARs were contributing something substantial in addition to calcium, we would expect to see a larger effect of the antagonists, at least in the dendrites; we do not.
Why are action potentials induced with TBS resistant to blockade of NMDARs? A likely explanation, which is supported by our finding that short within-burst intervals are important for antagonist resistance, is that the temporal summation of the AMPA receptor currents occurring during the (100 Hz) bursts is sufficient to support action potential firing without NMDAR involvement (Figures and ). Action potentials induced with TBS occurred in response to approximately 1 out of every 2 bursts, both with and without NMDAR antagonists, and thus do not appear at rates higher than those induced with 5 Hz synaptic stimulation. Other mechanisms, such as inhibitory tone, neuromodulatory action, or repetitive stimulation are more likely to be responsible for any bursting of post-synaptic CA1 neurons that may occur during the theta rhythm (present, but not observed consistently in our study). An interesting contrast can be made with another LTP-inducing stimulation, 100 Hz, where typically only 1-2 spikes are fired, if any, during NMDAR blockade. The difference is likely to be due to the fact that synaptic responses typically run down during a 1-second episode at 100 Hz, perhaps preventing consistent temporal summation beyond the first few pulses. NMDARs, then, seem to boost the signal of fatiguing presynaptic terminals to drive action potential firing postsynaptically. Similarly, NMDARs might also boost the signal of lower frequencies of presynaptic activity, such as 5 Hz, which lack the rapid, repetitive and summating presynaptic activity of TBS.
Figure 7. Summary of whole-cell ERK activation by synaptic activity.Different presynaptic stimulation (5 Hz, 100 Hz, or TBS) results in action potentials that are either dependent (5 Hz and 100 Hz) or independent (TBS) on NMDARs. ERK is activated under either condition, (more ...)
Whether the firing patterns of TBS and 100 Hz differ in the resulting nuclear biochemistry is unknown, although both activate ERK equally (but see (Raymond and Redman, 2002
; Selcher et al., 2003
)). TBS, though, is very effective at inducing late-LTP with stimulation thresholds very similar to those seen with ERK activation (Dudek, unpublished observations), between 60 and 80 presynaptic pulses (Dudek and Fields, 2001
). Action potentials initiated near the cell body can back-propagate into apical dendrites (Spruston et al., 1995
; Johnston et al., 1996
), and recent studies on spike-timing dependent plasticity (STDP) suggest that dendritic action potentials may provide an additional means of achieving the postsynaptic depolarization needed to activate and open NMDAR channels to induce LTP or LTD (Balaban et al., 2004
; Dan and Poo, 2004
). Accordingly, induction of LTP and/or LTD at the synapse in vivo
may have little requirement for postsynaptic firing other than the timing of the presynaptic activity with respect to the postsynaptic firing or bursting. Nuclear events, in contrast, may depend critically on the number and firing rate of post-synaptic action potentials. Synapse-specificity would be achieved with the LTP- or LTD-specific tagging of synapses (Sajikumar and Frey, 2004
One advantage of regulating transcription with action potentials is that large amounts of signal could be generated that are stoichiometrically favorable for transcription. It is unlikely that transcription factors such as NFκB, for example (Meffert et al., 2003
), or a synaptic cargo carried by importin/karyopherin (Thompson et al., 2004
), would be produced in quantities sufficient for transcription if produced by only a few synapses, without either the potentiation of many synapses or of an unknown amplifying mechanism. Cell-wide activation of signaling, such as that seen for ERK induced with action potentials, can provide sufficient quantities of active transcription factors on a timescale that supports the rapid induction of some genes (within 5 minutes for arc
(Guzowski et al., 1999
), for example) and within the time-window where late-phase-LTP is sensitive to RNA synthesis inhibitors (Nguyen et al., 1994
; Frey et al., 1996
). What is the evidence that a synapse-to-nucleus signal is necessary for late-phase LTP? While some recent data shows that importin translocation from synaptic compartments occurs in response to a chemically-induced LTP (Thompson et al., 2004
), the presence of such a signal in LTP has largely been inferred based on studies showing that nuclear signaling is sensitive to NMDAR antagonists (Deisseroth et al., 1996
; Steward et al., 1998
; Matsuo et al., 2000
). If the relevant gene expression (transcription and/or translation) relies on action potentials, the effects of NMDAR antagonists could give the mistaken impression of a signal from the synapse if action potentials were inadvertently blocked. Our data support the hypothesis that NMDARs can play a role in nuclear events through their effect on action potential generation. Previous evidence has shown that signaling pathways associated with late-LTP can be activated in hippocampal CA1 neurons without synaptic activity; somatic action potentials induced by backfiring the cells are sufficient for phosphorylation of ERK and the cAMP response element-binding protein, as well as induction of Zif268 protein (Dudek and Fields, 2002
). Furthermore, evidence in support of the idea of action potential-dependent transcriptional events comes from data showing that action potentials alone, in the absence of synaptic activity, are sufficient for the rescue of early-LTP in a synaptic tagging-type experiment (Frey and Morris, 1997
; Dudek and Fields, 2002
). Although those studies clearly implicated the L-type voltage sensitive calcium channels in these processes, we find that our stimulation-dependent, NMDAR-independent ERK staining is not entirely blocked with nifedipine when bicuculline is used (data not shown). We expect that because bicuculline allows for a greater number of action potentials (or action potentials are better controlled), additional sources of calcium are recruited that are sufficient for ERK activation, such as calcium from internal stores or non-L-type calcium channels. We interpret this to mean that the source of calcium is not critical for ERK activation.
We have now demonstrated that action potentials are not only sufficient, but are also necessary for nuclear signaling, in the form of ERK activation, in physiological contexts. Based on these findings, we believe that there are biochemical consequences to blocking NMDARs that are directly related to the role of NMDARs in action potential generation. These findings therefore have important implications for the interpretation of studies showing NMDAR-dependent and -independent forms of learning (Bannerman et al., 1995
) and their biochemistries (Cammarota et al., 2000
). In the future, the use of theta-burst stimulation or bicuculline could help to distinguish the specific roles that NMDARs have independent from their role in action potential generation.