Nicoll

Journal Articles

Lu, Shi, Nicoll • 2009 • Cell - FullText

Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach

All WT AMPARs contain GluA2.

GluR1/2 heteromers compose >95% of somatic/extrasynaptic AMPARs 80% of synaptic AMPARs

GluR2/3 receptors contribute �20% to basal synaptic transmission

GluA2 deletion... No change in the mean amplitude of mEPSCs but a dramatic reduction in frequency. Suggests two processes occur during the loss of GluA2 1. 50% of synapses become devoid of AMPARs, 2. 50% of synapses fully repopulate with GluA2-lacking receptors. Implies there are two distinct populations of synapses, based on whether they can recruit GluA2-lacking receptors.

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We have used a single-cell genetic approach that combines the use of electrophysiology and conditional KO mice for GluA1, GluA2, and GluA3 (GRIA1fl/fl, GRIA2fl/fl, GRIA3fl/fl) to delete each of the GluA subunits, either alone or in combination, by expressing Cre recombinase in individual CA1 hippocampal pyramidal cells. The subunit composition of synaptic and extrasynaptic AMPARs was determined by simultaneous whole-cell recording from Cre-expressing cells and neighboring control cells, as well as recording from somatic outside-out patches (OOPs).

Two standard methods were used to determine whether surface AMPARs in WT mice contain or lack the GluA2 subunit. The first method was to measure the I/V relationship of glutamate- evoked AMPAR currents in OOPs pulled from the soma of CA1 pyramidal neurons in acute slices. The second method involved determining the sensitivity of the AMPAR response to the polyamine toxin, philanthotoxin 433 (PhTx-433). GluA2- lacking receptors are strongly and selectively blocked by PhTx- 433, whereas GluA2-containing receptors are not (Washburn and Dingledine, 1996).

Our results, combined with previous work, indicate that GluA1 homomers—and indeed any AMPAR complex lacking GluA2—are excluded from the surface of CA1 pyramidal cells from WT animals at the age of 2–4 weeks under basal conditions.

Since all surface AMPARs in CA1 pyramidal neurons contain GluA2, we were interested in how the cell responded to its loss. Following the transfection of Cre in slice cultures, the AMPAR EPSC amplitudes fell to �50% of control values at 6 days and then remained constant. rAAVCre-GFP experiments with GRIA2(fl/fl) mice also showed that loss of GluA2 caused an �50% loss of AMPAR EPSCs.

GluR2 DELETION

GRIA2(fl/fl) ORGANOTYPIC HIPPOCAMPAL SLICE CULTURE

Following the transfection of Cre in slice cultures, the AMPAR EPSC amplitudes fell to �50% of control values at 6 days and then remained constant. rAAVCre-GFP experiments with GRIA2fl/fl mice also showed that loss of GluA2 caused an �50% loss of AMPAR EPSCs.

Is the decrease in the evoked synaptic responses due to a uniform loss of receptors across the entire population of synapses, as in the case with GluA1 deletion? To address this question, we examined mEPSCs. Remarkably, there was no change in the mean amplitude of mEPSCs but a dramatic reduction in frequency. This suggests that two processes occur during the loss of GluA2; approximately half of the synapses become devoid of AMPARs, while in the other half of synapses, GluA2-containing receptors are gradually replaced by GluA2-lacking receptors. This implies that there are two distinct populations of synapses, based on whether they can recruit GluA2-lacking receptors.

We also examined the consequence of deleting GluA2A3 on the properties of mEPSCs. The results were similar to those for the GRIA2fl/fl, suggesting that there is an all-or-none silencing of a population of excitatory synapses.

This suggests a critical role for the GluA2 subunit in transferring extrasynaptic receptors to the synapse. This notion is all the more striking when one considers the all-or-none loss of mEPSCs upon deleting GluA2, which implies that one population of synapses requires the presence of GluA2 for any AMPAR trafficking, while another population does not.

GluR1 DELETION

GRIA1(fl/fl) ORGANOTYPIC HIPPOCAMPAL SLICE CULTURE

Following the expression of Cre in GRIA1fl/fl hippocampal slice cultures, there was a gradual decrease in AMPAR EPSC amplitudes that stabilized at �20% of control values at �14 days after transfection. The scatter plot shows that there was an 80% loss of the AMPAR EPSCs.

To analyze the mechanism for reduced AMPAR EPSCs, we recorded mEPSCs. There was a clear decrease in the amplitude of mEPSCs, as well as a decrease in frequency, indicating that there is a loss of AMPARs from all synapses.

These data suggest that GluR1/2 heteromers account for approximately 80% of synaptic AMPARs and GluR2/3 receptors contribute �20% to basal synaptic transmission.

Deletion of GluA1 resulted in a 95% loss of extrasynaptic receptors. The I/V of the remaining current was linear, indicating that the remaining receptors are primarily GluA2A3 heteromers. Compared to the profound loss of extrasynaptic current (�95%), synaptic currents were less impaired (80%) in GluA1-deleted cells, suggesting that synapses are capable of consolidating the few remaining GluA2A3 heteromers.

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Granger, Shi, Lu, Nicoll • 2011 • Nature - FullText

LTP requires a reserve pool of glutamate receptors independent of subunit type.

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