AMPA Receptor

Determining ad hoc experimental parameters for quantitative autoradiography of AMPAR using [³H]AMPA ligand

Defining the parameters for a quantitative autoradiography experiment can be done three ways: (1) determined experimentally (not necessary for us), (2) taken from an existing protocol (which we did for our first experiment), (3) defined ad hoc using binding affinity data (which I've done below). After surveying the literature and consulting several databases, I've concluded that the binding affinity (dissociation constant K_d) for [3H]AMPA in rodents is ~28 nM. This means that a buffer containing a 28 nM concentration of [3H]AMPA will result in 50% of the total expressed AMPAR being bound to the radioligand. In the first experiment we used a 15 nM concentration; under the law of mass action this should result in 35% of expressed AMPAR being bound to ligand. The calculations for converting radioactivity (specific activity in Curies) to molecular concentrations is theoretically concrete, but converting this to film exposure days is only estimable.

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The [3H]AMPA from Sigma is manufactured with the given parameters

• Total Amount

- 25 uCi or 250 uCi

• Specific Activity

- 60 Ci/mmol

• Concentration

- 1 mCi/mL

• Molecular Weight

- 186.2 g/mol

Radioactivity to Molecular Concentration 1 mCi/ml * 1 mmol/60 Ci * 1 Ci/1000 mCi = 1.67e-5 mmol/mL = 16.7 uM

Sigma sends us a 16.7 uM concentration of [3H]AMPA and we can order either 25 uL ($300) or 250 uL ($1100). We want to determine how many mice brains can be analyzed if we order 25 uL or 250 uL [3H]AMPA. When the tissue is cut using the cryostat it is placed on glass slides. The number of slides in the experiment is key. Using the drop technique (the best way to conserve our precious radioligand) each slide requires 1 mL of incubation buffer (e.g. the Tris buffer containing the [3H]AMPA). All the brain regions of interest can be fit onto 3 slides; therefore there are 3 slides per subject.

Now to compare protocols...

Protocol A -- 15 nM [3H]AMPA

• 15 nM / (15 nM + 28 nM) = 35% bound receptors
• 16.7 uM * x mL = .015 uM * 1 mL
• x = 0.898 uL [3H]AMPA / mL Tris
• 25 uL / 0.898 uL = 28 slides or 10 subjects
• 250 uL / 0.898 uL = 278 slides or 92 subjects

At this concentration we are exposing film for 30 days.

Protocol B -- 42 nM [3H]AMPA

• 42 nM / (42 nM + 28 nM) = 60% bound receptors
• 16.7 uM * x mL = .042 uM * 1 mL
• x = 2.5 uL [3H]AMPA / mL Tris
• 25 uL / 2.5 uL = 10 slides or 3 subjects
• 250 uL / 2.5 uL = 100 slides or 33 subjects

At this concentration we can estimate a film exposure time of ~21 days

Protocol C -- 100 nM [3H]AMPA

• 100 nM / (100 nM + 28 nM) = 78% bound receptors
• 16.7 uM * x mL = .1 uM * 1 mL
• x = 6.0 uL [3H]AMPA / mL Tris
• 25 uL / 6.0 uL = 4 slides or 1 subjects
• 250 uL / 6.0 uL = 41 slides or 14 subjects

At this concentration we can estimate a film exposure time of ~16 days

Thus, I conclude that protocol B would be optimal given we order 250 uCi and protocol A will remain optimal given we order 25 uCi of [3H]AMPA.

Binding Assay 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptors

PROCEDURES
Radioligand	Pre-Incubation	Incubation	Wash
3H-AMPA 15 nM	50 mM Tris-HCl 100 mM KCl pH 7.4 10 min	15 nM 3H-AMPA Pre-Incubation Buffer 60 min	Pre-Incubation Buffer 10 min x2 baths 1 dip diH2O

Experimental determination of receptor-ligand binding affinity for [3H]AMPA

10 nM [³H]AMPA bound 32% of receptors in rat. Frozen-thawed tissue in buffer with KCl bound 36% of receptors. Specific binding curve extrapolation estimates the Kd of AMPA to be ~28 nM (Olsen et al 1987).

Experimental Goals

There is some behavioral evidence that PKMzeta (PKMz) is associated with long term memory maintenance. Molecular studies have shown that PKMz interacts with the GluR2 subunit of AMPA receptors (AMPAR), and with the help of NSF proteins, mediate the trafficking of AMPAR to the membrane.

In a modular fashion, PKMz is localized in dendritic spines. Once activated, PKMz remains constitutively active (much like a prion). In this way, AMPAR can be sustained in high densities at relevant synapses.

ZIP is a designer molecule that has been shown to inactivate PKMz. There are several high-profile reports of successfully using ZIP to abolish a learned behavior. These experiments have failed replication on several accounts. If ZIP inactivates PKMz, this should be reflected by a relatively long-lasting decrease in AMPAR density. This is what I am interested in exploring. I want to know how to effectively use ZIP as a tool to understand memory maintenance and network properties of memory engrams.