Conservation of Glutamate Receptor 2-Containing AMPA Receptors during Long-Term Potentiation

In this paper, Adesnik and Nicoll show that hippocampal LTP can be expressed in the absence of a change in the GluR2 subunit composition of the AMPA receptor. The data in this paper are convincing. The findings are in contrast to our recent paper (Plant et al., 2006); however, we feel that our study is also convincing. Taken together, these findings indicate that LTP can cause the rapid incorporation of GluR2-lacking AMPA receptors, but that LTP can also be expressed without using this mechanism. Here I, on behalf of all the authors of our paper, would like to make a number of points with regards some of the issues raised in the Adesnik and Nicoll study.

1. The Adesnik and Nicoll paper cannot explain the following three observations that we made in Plant et al. A) When making whole-cell recordings in the absence of added spermine in the intracellular solution, no change in the rectification of the EPSC was observed with LTP (Figure 1e of Plant et al.). This ‘no spermine’ experiment is a standard control for labs that study polyamine-dependent rectification of AMPA receptors e.g. (Rozov and Burnashev, 1999). This is an important control: it shows that the rectification of the EPSC we observe immediately after LTP induction is dependent upon the presence of intracellular polyamines. This strongly suggests that the rectification of the EPSC is caused by the presence of GluR2-lacking AMPA receptors. B) We can detect an increase in the peak amplitude of the EPSC measured at a holding potential of +40 mV at 25 minutes after LTP induction (Figure 1d in Plant et al). This is essentially an internal positive control showing we are able to detect increases in EPSC amplitude at +40 mV when they occur. C) We show that an increase in the peak amplitude of the pharmacologically-isolated AMPA receptor-mediated EPSC (in the absence of an NMDA receptor-mediated component) is observed at 25 minutes after LTP induction compared to that 3 minutes after LTP (Figure 1f, Plant et al). This is in the absence of a potentially confounding NMDA receptor-mediated component at +40 mV and strongly supports our contention that under our experimental conditions inwardly rectifying AMPA receptors are initially expressed at synapses immediately after LTP, but are then replaced with non-rectifying AMPA receptors by 25 minutes. D) Consistent with our observations on the change in inward rectification of the EPSC, we show in Plant et al that the polyamine toxin philanthotoxin (that blocks GluR2-lacking AMPA receptors) selectively blocks EPSCs only in the LTP pathway and only when applied at 3 or 10 minutes after LTP induction (Figure 2a-d, Plant et al). This is a very reliable finding for our experimental conditions that has been observed by two different experimenters in different labs at different times (Plant in the Isaac lab; Pelkey in the McBain lab).

In the Adesnik and Nicoll paper it is suggested that we induced some LTP when collecting EPSCs at +40 mV during the baseline period immeidately prior to applying the LTP induction protocol. This is not the case as can readily be seen for the control pathway used in all our experiments of this type. For the control pathway, EPSCs were also collected at +40 mV during the baseline and no LTP was induced as is clearly shown in these data sets (e.g. Fig. 1a in Plant et al). Therefore, collecting a few EPSCs at +40 mV did not induce LTP in our experiments.

3. Adesnik and Nicoll suggest that measuring the peak of the mixed AMPA and NMDA receptor-mediated EPSC at +40 mV is not a reliable measure of the AMPA receptor-mediated component because it is almost entirely dominated by the NMDA component (Figure 2 C). Further they use an initial slope measurement of the EPSC to analyze changes in the AMPA component with minimal contamination from the NMDA receptor-mediated component at +40 mV. We have now reanalyzed our original data (from Plant et al) using the same early slope measurements of the dual component EPSC. Using this measure we still find that there is no significant increase in the AMPA component of the EPSC recorded at a holding potential of +40 mV for up to 15 minutes after LTP induction. For example, for our data originally shown in Figure 1c of Plant et al, EPSC initial slope (measured at 0 -1.4ms after EPSC onset) at -70 mV = 170 ± 12 % of baseline (P <0.001, n = 12), and at +40 mV = 125 ± 13 % of baseline (P = 0.1, n = 12). This confirms that in our experiments rectifying AMPA receptors are incorporated at synapses during the earliest phase of LTP expression.

4. Adesnik and Nicoll cite other work to suggest that our findings in Plant et al are inconsistent with previous studies. The studies referred to were performed on hippocampal slices or organotypic slice culture from animals of various ages and used a variety of LTP induction protocols. It is clear from the literature that LTP induction and expression mechanisms are developmentally regulated. For example the GluRA (GluR1) knock out exhibits hippocampal LTP early in development but not in adult (Jensen et al., 2003). Also, the role of different kinases changes for LTP induction during development, with PKA dominating early in development and CaMKII being the major mechanism in the adult, see for example, (Wikstrom et al., 2003; Yasuda et al., 2003). Thus it is a reasonable possibility that the switch in GluR2 subunit composition we report is not a mechanism used during LTP under all conditions. It will be of great interest to determine what variables influence the expression mechanisms of LTP, particularly with regards the role for GluR2-lacking AMPA receptors.

References:

Jensen V, Kaiser KM, Borchardt T, Adelmann G, Rozov A, Burnashev N, Brix C, Frotscher M, Andersen P, Hvalby O, Sakmann B, Seeburg PH, Sprengel R (2003) A juvenile form of postsynaptic hippocampal long-term potentiation in mice deficient for the AMPA receptor subunit GluR-A. J Physiol 553:843-856.

Plant K, Pelkey KA, Bortolotto ZA, Morita D, Terashima A, McBain CJ, Collingridge GL, Isaac JT (2006) Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation. Nat Neurosci 9:602-604.

Rozov A, Burnashev N (1999) Polyamine-dependent facilitation of postsynaptic AMPA receptors counteracts paired-pulse depression. Nature 401:594-598.

Wikstrom MA, Matthews P, Roberts D, Collingridge GL, Bortolotto ZA (2003) Parallel kinase cascades are involved in the induction of LTP at hippocampal CA1 synapses. Neuropharmacology 45:828-836.

Yasuda H, Barth AL, Stellwagen D, Malenka RC (2003) A developmental switch in the signaling cascades for LTP induction. Nat Neurosci 6:15-16.