One aim of this study was to assess the comparative effects of con-G, con-R[1-17], and con-T on their abilities to inhibit ion flow through whole cell and synaptic NMDARs in developing hippocampal neurons and to assess mechanistic consequences of their activities on the cells. Con-G is a NR2B subunit-selective NMDAR inhibitor, with inhibition efficacy decreasing in receptors containing NR1/NR2B > NR1/NR2A/NR2B
NR1/NR2A, whereas con-R[1-17] and con-T potently inhibit both NR2A and NR2B-containing NMDARs. Thus, this study also provides an assessment of the importance of ion flow in specific subunit combinations of NMDARs in the neuron population under development.
We initially confirmed, both in whole cells and in synaptic locations, that the NR2B/NR2A ratio is higher in immature neurons than in mature neurons. Thus, con-G, con-R[1-17], and con-T should be effective inhibitors at all developmental stages. Using whole cell recordings, we offer evidence that con-T and con-R[1-17], which inhibit both NR2A and NR2B-containing NMDARs, are more effective inhibitors of current flow than the NR2B-selective con-G at all stages of development. The differences in efficacy between con-T/con-R[1-17] and con-G become larger as the neurons age because of the developmental increases in the NR2A/NR2B ratio, resulting in relative decreases of inhibition by con-G. This is also the case in the synaptic NMDARs, wherein con-T and con-R[1-17] more effectively inhibited the amplitudes of sEPSCs, relative to con-G, DIV 19 neurons, as compared to DIV 13 neurons. Our data are consistent with other work (Alex et al., 2006
; Barton et al., 2004l
), and demonstrate that inhibition of NMDA-evoked currents and NMDAR-mediated sEPSCs by con-G is dampened in mature neurons.
Developmental changes in efficacy of the conantokins are also noted in the ability of these conantokins to inhibit inward flow of Ca2+ into neurons, with stronger inhibition of Ca2+ influx by con-R[1-17] and con-T than con-G at all developmental stages, except in the oldest neurons, where the inhibitory strengths equalized. These data suggest that comparative inhibition by con-G, con-R[1-17], and con-T can be employed to assess functional contribution of NR2B and NR2A in neurons.
The phosphorylation/dephosphorylation of the transcription factor, CREB, is one of the upstream events that regulates Ca2+
-mediated signaling events, e.g., neuronal apoptosis. Also, events that increase P-CREB levels in neurons enhance pro-survival genes and neuronal plasticity (Sassone-Corsi et al., 1988
; Sauerwald et al., 1990
). Our data show that activation of NMDARs is one of the initiating events leading to P-CREB, since addition of con-G, con-R[1-17], or con-T, to DIV 9 neurons leads to severe attenuation of Ca2+
flow into these cells, coupled with the lack of appearance of P-CREB, thus linking Ca2+
influx via the NMDAR to P-CREB formation. From previous studies (Sala et al., 2000
), and from our observations, it is apparent that phosphorylation/dephosphorylation of CREB in neurons is related to the developmental stage of the neurons. Diminished levels of P-CREB in DIV 14 neurons in the presence of NMDA has been correlated to NMDAR-dependent stimulation of a dephosphorylation pathway involving phosphatase PP1 (Cho et al., 1992
; Sala et al., 2000
). In the presence of an NMDAR inhibitor, antagonism of this PP1 pathway may be "shut-off" allowing for enhanced P-CREB levels. Our original hypothesis linking iCa2+
to downstream signaling events was confirmed in immature neurons, where inhibition of CREB phosphorylation paralleled inhibition of iCa2+
influx. However, this linkage did not hold true in DIV 16 neurons where robust inhibition of Ca2+
influx by conantokins did not correlate with diminishment of P-CREB. It has been shown that high levels of iCa2+
in postsynaptic neurons activates the calmodulin-CaMKII axis, which, in-turn, could phosphorylate CREB, whereas lower levels of iCa2+
results in calmodulin-induced activation of one of its targets PP2B, which potentiates the PP1 pathway (Bito et al., 1996
; Groth et al., 2003
), perhaps ultimately leading to dephosphorylation of CREB, or another protein necessary for CREB phosphorylation. Thus, while it may be speculated that diminishment of iCa2+
influx by conantokins in immature neurons may be triggering activation of phosphatases, thus allowing for CREB dephosphorylation, an event that does not occur in mature neurons, we find that levels of the relevant and ubiquitous eukaryotic protein phosphatases, e.g.,
PP1, PP2A, and PP2B, are not affected by conantokin treatments in either DIV 9 and DIV 16 neurons. Similarly, levels of activated (P-Thr286)CaMKII and (P-Thr196)CaMKIV, main regulators of neuronal CREB phosphorylation, are also not affected by conantokins, despite their ability to inhibit Ca2+
influx. A direct correlation between Ca2+
influx and P-CREB has been published (Deisseroth et al., 1996
), wherein it was observed that spatial location of Ca2+
entry is an important determinant of CREB phosphorylation. Emigrated Ca2+
binds with high affinity to calmodulin at the synaptic sub-membrane, resulting in CREB activation. In , it is observed that the levels of iCa2+
in DIV 7 and in DIV 16 neurons are similar. Therefore, the difference in P-CREB levels in DIV 9 and DIV 16 neurons could be due to developmental regulation of the Ca2+
threshold for CREB phosphorylation.
Our studies indicate that NMDA-induced P-CREB levels in immature neurons were robust and that the NMDAR-specific antagonists, conantokins and MK-801, attenuated phosphorylation of CREB. In these DIV 9 neurons, inhibition of phosphatases PP1, PP2A, and PP2B did not prevent deactivation of CREB when co-treated with the NMDA/NMDAR antagonists. This suggests that in immature neurons PP1, PP2A, and PP2B are not involved in modification of CREB activation in the presence of these inhibitors, although in another study it was reported that in other cAMP-responsive cells maintenance of P-CREB levels is controlled by its phosphatases (Bito et al., 1996
; Hagiwara et al., 1992
). In mature neurons, differential effects on P-CREB were observed that were dependent on the type of conantokin utilized. According to the immunoblots of , selective inhibition of PP1 allows for sustained activation of CREB when con-G was incubated with the neurons. In neurons treated with con-G, inhibition of PP2A and PP2B dramatically decreased P-CREB levels. On the other hand, inhibition of PP2B in neurons co-treated with NMDA/con-R[1-17] and NMDA/con-T caused increased levels of P-CREB in DIV 16 neurons, indicating that while the end-effects of various NMDAR antagonists are the same, i.e., enhanced P-CREB levels in mature neurons, the phosphatases that gate the dephosphorylation of CREB are different.
Our observations further indicate that treatment of DIV 9 neurons with NMDA/con-G increased NR2B at extrasynaptic sites, as observed by their presence in the neuron bodies. It has been shown that reduction of NR2B at synaptic sites (dendrites), relative to extrasynaptic sites, after inhibition of CaMKII effectively diminished CREB phosphorylation (Gardoni et al., 2009
). Additionally, glutamate- or hypoxia-induced Ca2+
entry via extrasynaptic NMDARs triggered dephosphorylation of CREB, whereas Ca2+
influx through the synaptic complex promoted CREB phosphorylation (Hardingham and Bading, 2002
). Herein, we show that although NMDA-stimulated iCa2+
influx is decreased in both immature and mature neurons by con-G, accompanying changes in the cellular location of the NR2B subunit depends on neuron maturity. Increased levels of NR2B subunits in the extrasynaptic sites of neuron bodies in DIV 9 neurons in the presence of NMDA/con-G implies that extrasynaptic activation of the NMDAR is leading to a CREB shut-off pathway. However, treatment of DIV 16 neurons with NMDA/con-G does not lead to an increase in NR2B subunit in neuron bodies, as observed in DIV neurons, thus allowing CREB phosphorylation to occur, implying sustained synaptic stimulation in more mature neurons.
In conclusion, we show conantokin-mediated inhibition of synaptic current flow by NMDARs in developing neurons reflects the subunit composition of these receptors and that inhibition of current flow in NMDARs in immature and mature neurons is directly linked to Ca2+ mobilization via these channels and to signaling events that occur downstream of P-CREB generation. Thus, conantokins can be employed as non-channel blocking alternative pharmacological and, potentially, therapeutic tools to mechanistically isolate extrasynaptic and synaptic events in neurons that relate to NMDAR channels of specific subunit composition.