Quantitative in vitro phosphor imaging using [3H] and [18F] radioligands: the effects of chronic desipramine treatment on serotonin 5-HT2 receptors
Introduction
Receptor autoradiography is a widely used technique to determine the characteristics and distribution of neurotransmitter receptors. Traditionally, in vitro receptor binding assays are performed on tissue slices with tritiated ligands and X-ray film, or with membrane homogenates and liquid scintillation counting. Phosphor imaging autoradiography has recently emerged as a sensitive and widely available alternative with several advantages over X-ray film autoradiography.
The most important advantage of phosphor imaging over film autoradiography is temporal. The high sensitivity of phosphor screens allows exposure times to be reduced to one-tenth of X-ray film exposure times (Johnston et al., 1990), allowing data to be collected and analyzed in hours or days instead of weeks. Another advantage of phosphor screens over film is economy. Multisensitive phosphor screens can be used repeatedly, by erasing any latent image remaining after scanning by direct exposure to white light. With the added care and precautions reported here for the use of tritium-sensitive phosphor screens, we have been able to use these screens at least seven times to obtain reliable quantitative data. Phosphor imaging also provides a practical alternative, as tritium-sensitive X-ray film is becoming increasingly difficult to obtain.
Historically, autoradiography with positron emission tomography (PET) ligands has been very difficult to perform due to the short half-lives of positron-emitting isotopes and the low sensitivity of X-ray film. Phosphor screens have higher sensitivity, and good, quantifiable images can be obtained from PET tracers within the naturally short exposure times. In vitro autoradiography with PET ligands is an important adjunct technique for corroborating in vivo PET data, and the ability to use the exact same ligand for in vitro studies as is used in vivo permits closer comparisons. Recently, PET groups have begun to add in vitro phosphor imaging with [11C]- or [18F]-labelled compounds as an adjunct to in vivo imaging, and the technique has an important role in tracer discovery (Bergstrom et al., 2004). Qualitative anatomical distribution studies are common, often performed ex vivo, where the animal is injected with the radiotracer, sacrificed after tracer uptake, and tissue slices are apposed to the storage phosphor screens (e.g. Besret et al., 1998, Passchier et al., 2000, Doze et al., 2002). Phosphor imaging with PET tracers has also been used with live tissue preparations, where the tissue is removed from the animal but maintained in biological buffer solutions with oxygenation during incubation with the PET compound (Matsumura et al., 1995, Sasaki et al., 2002). Very few studies have used in vitro phosphor imaging with PET tracers for quantitative autoradiography (Gatley et al., 1998, Nikolaus et al., 2001, Nikolaus et al., 2003).
We have used a routine pharmacological challenge to demonstrate and validate the use of PET tracers and phosphor imaging for quantitative in vitro receptor autoradiography. Chronic antidepressant treatment consistently leads to decreased binding to cortical serotonin 5-HT2 receptors. This effect has most often been demonstrated with the tricyclic antidepressant desipramine hydrochloride (DMI), which decreases 5-HT2 receptor binding in both humans and animals (Bergstrom and Kellar, 1979, Goodnough and Baker, 1994, Yatham et al., 1999). The effect is robust and has been detected with various 5-HT2 ligands, both in vivo by PET, and in vitro using membrane preparations. To the best of our knowledge, however, decreased 5-HT2 binding as a result of chronic DMI treatment has not been investigated by slice autoradiography, which has much higher spatial resolution than homogenate binding assays. Here, using the effects of chronic DMI treatment on 5-HT2 binding as the validation end-point, we describe aspects of the method critical to the acquisition of accurate quantitative autoradiographic phosphor imaging data in vitro, using both the traditional, tritiated 5-HT2 ligand, [3H]ketanserin, and the positron-emitting ligand [18F]setoperone.
Section snippets
Subjects
Adult male Sprague-Dawley rats (n = 5 per group), 300 g at the start of the study, were the subjects of this experiment. Rats were housed on a 12:12 light–dark cycle, with lights on at 6:00 a.m. They were housed in dyads with food and water available ad libitum. The rats were given 15 mg/kg i.p. Desipramine hydrochloride (Sigma) or a similar volume of vehicle (sterile water) daily for 17 days. On day 18, the animals were decapitated, and their brains were quickly removed and frozen in isopentane
Fit and reproducibility of [18F]standard curves: the spillover effect
Displaying the image relative to the spot in question (“spillover correction”) when determining the optical density of a particular point on the standard curve is critical to the production of accurate standard curves with excellent goodness of fit and high reproducibility. Because the standards are created on a two-dimensional surface, where the known value is the amount of activity in each spot, and because the shapes of the standard spots and distribution of activity within the spot can
Conclusions
In conclusion, we have described several ways to exploit the inherently high resolution and sensitivity of storage phosphor screens to obtain quantitative receptor binding data that show good reliability and reproducibility with either tritiated or positron-emitting ligands. The temporal, economical, and practical advantages of phosphor imaging make it an excellent technique to replace traditional X-ray film tritiated ligand autoradiography. In particular, quantitative in vitro phosphor imaging
Acknowledgements
The authors would like to thank the staff of the UBC/TRIUMF PET group. We are especially grateful to Dr. T. Ruth for his suggestions during the development of the PET autoradiographic assay. We would also like to thank Paul Piccioni for his help in creating the point sources for the resolution experiment. This work was funded by the Canadian Institutes of Health Research MPO 14535 and a Natural Sciences and Engineering Research Council Postgraduate Scholarship to E.M.S.
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