Quantitative autoradiography
In this method, thin tissue sections of the brain are incubated with a specific radioligand, the unbound ligand removed by washing and the resulting tissue section placed on a sensitive photographic film. The sites where the radioligand binds to the tissue fog the film, and following its development grain counts, densitometric analysis and photometric techniques can be used to quantify the extent of the binding of the radioligand to specific cellstructures. More recently, the application of computerized image analysis has simplified the problem of visualization and quantification. The system most widely used consists of a television camera linked to an IBM personal computer. This system can colour code images to enhance contrast, modify images, subtract one image from another and average densities in specific regions of the visual field. The ability of this system to include and process autoradiographic standards enables the density of the radioligand binding to be quantified. Finally, the system can tabulate, store and calculate the Bmax and Kd values of the radioligand. One of the most important uses of autoradiography in psychopharmacology lies in enabling the sites in the brain where a drug acts to be identified. For example, the brain region or specific neuronal circuit that is affected by a drug may be visualized. Since most psychotropic drugs have multiple effects in the brain, autoradiographic methods have helped to explain the complexity of the effects by identifying the receptors and their distribution in the brain. Changes in receptor density may reflect neuronal function. Receptor mapping has therefore been applied to biopsy and autopsy samples from patients with Parkinsonism, Alzheimer’s disease, schizophrenia and depression. The results of such studies have already begun to throw light on the possible biochemical changes that underlie such diseases. There are practical limitations to the autoradiographic technique however. The resolution using the light microscope is limited and it is seldom possible to readily identify specific cell structures that contain the receptors or to distinguish between functional and non-functional receptors to which the ligand is bound. It should be emphasized that this is also a problem with conventional radioligand techniques as applied to membrane preparations or to tissue homogenates. To overcome the problem of resolution, electron microscopic autoradiography may eventually prove to be of value, particularly when this is combined with immunohistochemical techniques to improve the resolution.
In this method, thin tissue sections of the brain are incubated with a specific radioligand, the unbound ligand removed by washing and the resulting tissue section placed on a sensitive photographic film. The sites where the radioligand binds to the tissue fog the film, and following its development grain counts, densitometric analysis and photometric techniques can be used to quantify the extent of the binding of the radioligand to specific cellstructures. More recently, the application of computerized image analysis has simplified the problem of visualization and quantification. The system most widely used consists of a television camera linked to an IBM personal computer. This system can colour code images to enhance contrast, modify images, subtract one image from another and average densities in specific regions of the visual field. The ability of this system to include and process autoradiographic standards enables the density of the radioligand binding to be quantified. Finally, the system can tabulate, store and calculate the Bmax and Kd values of the radioligand. One of the most important uses of autoradiography in psychopharmacology lies in enabling the sites in the brain where a drug acts to be identified. For example, the brain region or specific neuronal circuit that is affected by a drug may be visualized. Since most psychotropic drugs have multiple effects in the brain, autoradiographic methods have helped to explain the complexity of the effects by identifying the receptors and their distribution in the brain. Changes in receptor density may reflect neuronal function. Receptor mapping has therefore been applied to biopsy and autopsy samples from patients with Parkinsonism, Alzheimer’s disease, schizophrenia and depression. The results of such studies have already begun to throw light on the possible biochemical changes that underlie such diseases. There are practical limitations to the autoradiographic technique however. The resolution using the light microscope is limited and it is seldom possible to readily identify specific cell structures that contain the receptors or to distinguish between functional and non-functional receptors to which the ligand is bound. It should be emphasized that this is also a problem with conventional radioligand techniques as applied to membrane preparations or to tissue homogenates. To overcome the problem of resolution, electron microscopic autoradiography may eventually prove to be of value, particularly when this is combined with immunohistochemical techniques to improve the resolution.
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