Receptor Autoradiography
Overview
Autoradiography can be used to produce a visual image of the
distribution of receptor binding sites (known or unknown)
for a radiolabeled drug in a tissue. Cyostat-cut tissue sections
can be incubated directly in the radiolabeled compound and subsequently
imaged (in
vitro autoradiography) or the radiolabeled compound can be given to the live animal
followed by sectioning of the tissue and imaging (ex
vivo autoradiography).
Protocol summary:
(i) In vitro autoradiography Cryostat-cut tissue sections are incubated with the radiolabeled drug until equilibrium is reached. The sections are washed in fresh buffer to remove unbound radiotracer, dried and imaged using either phosphorimaging or beta imaging.
(ii) Ex vivo autoradiography The radiolabeled drug is administered to the animal via a tail vein. The animal is then sacrificed and the target organ(s) dissected free and snap-frozen. Sections are cut using a cryostat, dried and exposed to a phosphor screen together with autoradiography standards.
Examples:
Fig 1
Binding of [3H]MK801 to NMDA
receptors in coronal sections in the rat brain. Upper
two sections are total binding and lower two sections
non-specific binding, defined using unlabeled MK801 (10
µM).
Fig 2
In vitro binding of [3H]LRRK2-IN-1 to
the LRRK2 enzyme in rat kidney. Mutations causing constitutive
activity in this enzyme in the brain have been implicated in cases of
familial Parkinson's disease. Non-specific binding was defined
using unlabeled LRRK2-IN-1 (10 µM).
Fig 3
[35S]GTPγS binding in rat brain slices.
In the presence of the 5-HT1a agonist, 8-hydroxy-DPAT (10
µM), [35S]GTPγS binding was increased relative to basal levels in the hippocampus,
ventral cortex and hypothalamus,
reflecting the distribution of agonist-activated receptors. Non-specific [35S]GTPγS binding was defined using unlabeled GTPγS (10
µM)
Fig 4
Ex
vivo autoradiography in brain from a mouse administered the
radioiodinated cannabinoid CB1 receptor radioligand,
[125I]AM2233. Sections (coronal) imaged using phosphor
imaging. Radioactivity distribution is consistent with
selective binding to the CB1 receptor.