Could anyone provide me with the details of the calculation of AMPAR/NMDAR ratio? I have read that it is calculated at two different holding potentials -70 and +40 mV. But I did not understand the reason for these specific numbers? In some cases people have used AP-5 and CNQX but others do not. What is the exact interpretation of this number?
Hi Anu,
An AMPAR/NMDAR ratio gives you a measure of the relative expression of AMPAR and NMDAR at the synapse. In order to culculate this ratio, you need a value (amplitude) for both of them to a certain stimulus. You can get the value for AMPAR at -70mV, since NMDAR do not pass current at that potential. If you depolarize to +40mV, the NMDAR do pass current and contribute to your response. Therefore, you should calculate the amplitude when there is no contribution from the AMPAR (we usually analyze the average amplitude from 60ms to 65ms after stimulation, since AMPAR-current is (almost) zero). Alternatively, you can isolate NMDAR-current by blocking AMPAR with CNQX.
The value of the ratio itself does not mean anything, you can only use it to compare between different conditions, and see if the relative contribution of AMPAR to NMDAR is altered. The holding potentials of -70mV and +40mV are not fixed, they are just used because they give a driving force large enough to record a nice response.
Hope this helps you.
Dear Anu,
To differentia these currents, apart from voltage, your pipette solution is important too. There are also complications when you just rely on voltage-clamp, such as space clamp issues. For example, in motoneurons I can record both NMDA and AMPA currents at -60 mV holding, most likely due to the fact that the holding potential at distal dendrites and spines are different than the command potential you are applying at the soma. At +40 mV you will also be recording outward Cl- currents gated by GABA/glycine, to distinguish you will need to block GABA/glycine receptors and/or use ions such as methylsulphanate/gluconate that do not permeate via Cl channels.
best wishes, Refik
Hi Refik,
Thank you for your addition. This is really helpful. I will keep your point in mind about the Cl- current.
Not my field, but I presume you are familiar with this article in Nature:A
FIGURE 3 | Synaptic strength measured using the AMPAR/NMDAR ratio.
FROM THE FOLLOWING ARTICLE:
Synaptic plasticity and addiction
Julie A. Kauer & Robert C. Malenka
Nature Reviews Neuroscience 8, 844-858 (November 2007)
doi:10.1038/nrn2234
The basal strength of excitatory synapses is difficult to compare between different cells and preparations. Calculating the ratio of -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR)-mediated synaptic currents to N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic currents of a population of stimulated synapses is a normalization procedure that facilitates such comparisons because it is independent of parameters such as the positioning of electrodes or the number of synapses that are activated. It is commonly calculated by holding the membrane potential of cells at positive potentials (for example, +40 mV), to completely relieve the block of NMDARs by magnesium, and measuring the amplitude of AMPAR excitatory postsynaptic currents (EPSCs) and NMDAR EPSCs. A typical procedure is to record a dual component EPSC (mediated by both AMPARs and NMDARs; not shown) and then apply the NMDAR antagonist D-APV to isolate the AMPAR EPSC. The NMDAR EPSC is obtained by digital subtraction of the AMPAR EPSC from the dual component EPSC. a | A synapse is illustrated in the basal or control state. The isolated AMPAR and NMDAR components of the EPSC are shown below the synapse diagram. b | After LTP induction, if AMPARs are inserted into the postsynaptic membrane, the AMPAR component of the EPSC is enhanced, whereas the NMDAR component remains unchanged, increasing the ratio. This method assumes that there are no significant changes in the proportion of rectifying (Ca2+-permeable) AMPARs that would confound measurements made at +40 mV.
Hi Frederick,
Thanks for the concise explanation and the review link. I should certainly read it.
Be sure to download the attachment. I repeats the text, but has a good figure.
NMDARs express their full conductance at +40mV because this is the potential at which Mg2+ is fully unblocked. At any other valuer you would have partial block of NMDARs and an error in the ration estimation :)