Autoradiographic Efficiency Research Articles

A new method for quenching correction leads to revisions of data in receptor autoradiography

Differential quenching of beta-emission affects strongly the analysis of receptor distribution patterns in quantitative receptor autoradiography with tritiated ligands. Different methods for the quenching correction have been described in the past, but some of these are of limited value, if a detailed anatomical parcellation is necessary. Other methods correct exclusively local variations in lipid concentration, which is an important, but only one of several factors causing quenching. A new method for the measurement of quenching (or autoradiographic efficiency) is presented, which permits an anatomically detailed and direct determination of the total quenching without lipid extraction procedures. This method is based on the measurement of autoradiographic efficiency in cryostat sections homogeneously labeled with tritiated formaldehyde by an underlying gelatine section containing this labeled compound. Regional and layer specific measurements of autoradiographic efficiency in cortical and subcortical regions of the human and rat brain are reported. A significant correlation was found between the density of myelin and autoradiographic efficiency but other factors were also shown to influence differential quenching. The use of the here presented correction procedure leads to revisions of the laminar distribution patterns reported for different receptors in human and rat cortical areas. Our results show, that a complete quenching correction is necessary for the mapping of receptor distributions with tritiated ligands.

Quantification of intracellular calcitonin gene transcripts in human medullary thyroid carcinoma (MTC) by in situ hybridization

The human calcitonin gene generates 2 distinct mature mRNAs by alternative RNA processing, encoding calcitonin (CT) in thyroid C-cells or a neuropeptide (CGRP) in the brain. We evaluated quantitatively by in situ hybridization the expression of the CT gene in tissue section of 5 MTCs (2 sporadic and 3 familial forms). The primary tumor of one MTC was compared to a brain metastasis. In situ hybridization was carried out with tritiated cDNA probes coding for CT and CGRP mRNA. After autoradiography the number of silver grains was counted in 400 cells by computerized analysis of digitized images and expressed as the labelling level (L.L. = grain area/cell area per day of autoradiographic exposure). This was used to calculate the relative abundance per cell of the specific messengers studied, which depends on the autoradiographic efficiency and the specific activity of the probe used. The CT mRNA content was 3.25-6.55 10(-10) micrograms equivalents in the 3 familial forms of MTC and 4.95-9.25 10(-10) micrograms equivalents for the 2 sporadic forms. The levels of CT mRNA in the brain metastasis and in the primary tumor were identical (4.10 10(-10) micrograms equivalents). CGRP mRNA expression was weaker in the sporadic and in the familial thyroid tumors (0.60-1.65 10(-10) micrograms equivalents). The content of mRNA CGRP in the brain metastasis (0.60 10(-10) micrograms equivalents) was lower than that in the primary tumor (1.05 10(-10) micrograms equivalents).(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative distribution of muscarinic receptors and choline acetyltransferase in rat medulla: examination of transmitter-receptor mismatch

Recent reports have identified discrepancies between the anatomical distribution of transmitters and their receptors, a phenomenon known as transmitter-receptor mismatch. However, quantitative determinations of transmitter activity and receptor density in individual brain regions have not been conducted in parallel. We therefore sought to determine quantitatively the relationship between muscarinic acetylcholine receptor density and the density of cholinergic innervation as reflected by activity of the biosynthetic enzyme for acetylcholine, choline acetyltransferase (ChAT). To assure sampling of equivalent regions using the two methods, an ‘electronic micropunch’ technique was developed to allow measurement of [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding within the corresponding cylinders of tissue obtained by the micropunch cannula. Nineteen regions of the rat medulla (1 mm diameter, 1 mm height) were studied. The micropunch region containing the gracile nucleus, the area postrema and the choroid plexus of the fourth ventricle contained the highest ChAT activity, but exhibited little [ 3H]QNB binding to muscarinic receptors. However, among the remaining 18 regions a strong correlation was obtained between uncorrected muscarinic receptor density and ChAT activity within each micropunched region ( r = 0.89, n = 18). Correction for autoradiographic efficiency weakened the overall relationship between receptor density and ChAT activity ( r = 0.58, n = 18). This was due to a relatively high density of receptors associated with fiber tract regions containing low ChAT activity. The presence of receptors within white matter is ordinarily obscured by high tritium quenching. This is consistent with the hypothesis that a portion of muscarinic receptors are located extrasynaptically and may be present within axons. When the 4 areas with the highest white matter content, as indicated by low autoradiographic efficiency, were considered separately, corrected muscarinic receptor density and ChAT activity were highly correlated in this group ( r = 0.86, n = 4) and in the remaining regions ( r = 0.70, n = 14). Thus, cholinergic innervation is a major determinant of muscarinic receptor density in both white matter and grey matter regions. We conclude that muscarinic receptor density is correlated with the extent of cholinergic innervation as indicated by ChAT activity. The transmitter-receptor mismatch which did occur could be accounted for by the existence of receptors undergoing axonal transport. These findings do not support the hypothesis that acetylcholine and other neurotransmitters diffuse across large distances to act on non-innervated receptors.

A high density of muscarinic receptors in the rostral ventrolateral medulla of the rat is revealed by correction for autoradiographic efficiency

Previous reports of a low density of muscarinic receptors in the vicinity of the rostral ventrolateral reticular nucleus (RVL) conflict with the presence of a muscarinic cholinergic mechanism regulating arterial pressure, and may reflect low autoradiographic efficiency due to quenching of tritium emissions by white matter. Regional autoradiographic efficiency was determined using brains uniformly labeled with O-[ 3 H] methylglucose . Within the RVL, autoradiographic efficiency was only 50 ± 2% relative to gray matter. The uncorrected density of muscarinic receptors (M 2) labeled by [ 3H]quinuclidinyl benzilate in the RVL appeared to be low relative to cranial nerve nuclei, but the corrected muscarinic receptor density approximated that within the nucleus tractus solitarii. We conclude that a high density of M 2 muscarinic acetylcholine receptors is present in the RVL.

Histological aspects of in situ hybridization. Detection of poly(A) nucleotide sequences in mouse liver sections as a model system.

This study examined the detection of cellular poly(A) sequences in mouse liver sections by in situ hybridization using a 3H-labelled poly(dT) probe. Parameters examined included possible losses of target poly(A) sequences from sectioned cells, access of probe to target sequences, section thickness, hybridization conditions, autoradiographic efficiency, specific activity of probes and specificity of reaction. An improved protocol was devised that resulted in good preservation of histological detail in sectioned tissue blocks, and a calculated hybridization efficiency of 50%-100%. With the use of probes of defined sequence, the protocol should allow detection of unique mRNA sequences within single cells with an estimated sensitivity of 6-12 unique mRNA molecules per sectioned cell.

High-speed autoradiography of 3H-thymidine-labelled nuclei.

High-speed autoradiography with stripping film of 3H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20 degrees C, (b) exposure of cell preparations and blanks for various times at either -70 degrees C or +20 degrees C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time greater than or equal to 1 h at either temperature and relative humidity 20--30%. A linear relationship between autoradiographic efficiency and exposure time of 1--7 h was found at either temperature, although the efficiency of autoradiographs exposed at -70 degrees C was by approximately 30% higher than that of autoradiographs exposed at +20 degrees C. Background values of autoradiographs dried with a fan and exposed for 1/4--7 h at either -70 degrees C or +20 degrees C were 0.6--0.8 grain/100 micron2. Theoretical calculations and experimental data showed that high-speed autoradiographs are 30--50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.

Effect of exposure temperature on over-all efficiency of autoradiographs and on fogging of emulsion.

Systematic investigations of the influence of various temperatures of exposure on the autoradiographic efficiency and on the rate of fogging of Kodak AR 10 stripping film were carried out. The oxygen content and humidity of the exposed autoradiographs as well as the time of exposure were constant. Exposure of autoradiographs or storage of film at room temperature caused an increase of overall autoradiographic efficiency by approximately 40%, but although the rate of film fogging was also increased, it did not exceed the safe values of background. Both autoradiographic efficiency and rate of fogging were not altered significantly when a temperature of 4 C or -18 C for exposure was used. Therefore, room temperature of exposure is recommended for ordinary autoradiography. For a special autoradiographic technic when low temperature of exposure has to be used, the temperature -18 C may be safely applied without significant impairment of autoradiographic efficiency.

Techniques of Autoradiography

This book describes in detail the techniques of autoradiography as applied to biology and medicine. The last book on the subject was published by G. A. Boyd in 1955, and the progress made by autoradiography in the past 12 years has been such as to make Boyd's book obsolete. In the circumstances, the effort of Dr. Rogers is welcomed by all investigators using autoradiography to study life processes. The book includes 18 chapters dealing variously with both the theory and practice of autoradiography. A description of nuclear emulsions and of the photographic process is given in readable form, and after treating the problem of autoradiographic resolution and efficiency, Dr. Rogers describes in more detail some of the techniques in use. These include the dip-coating technique, the stripping-film technique, and autoradiography with the electron microscope. Two subjects are treated in more detail than can be found in articles or chapters dealing

Efficiency of Liquid Scintillation Counting and Autoradiography for Detecting Tritium in Spermatozoa

Assays by autoradiography (grain counts) and liquid scintillation (disintegrations per minute, dpm) were made on semen samples collected from 5 rabbits for 63 days following injection with H3-thymidine. The mean grain counts and mean dpm determined on semen samples containing radioactive spermatozoa were highly correlated (r = 0.93, P < 0.001). With this high relationship it was possible to calculate a meaningful regression of mean grain counts on dpm. It was found that 5.8 disintegrations were required per grain observed in NTB-3 emulsion for an autoradiographic efficiency of 17.3%. Utilizing direct sample preparation, liquid scintillation counting was demonstrated as a rapid method to detect initial appearance of H3-labeled spermatozoa. The correlation coefficient calculated for counts obtained on duplicate subsamples was 0.99. The efficiency of the procedure was estimated by means of external standards to range from 9.8 to 12.5%. Inclusion of thixotropic gel reduced errors in counting, but actual solid...

Calibration of stripping film autoradiography in sections of rat liver labelled with tritium

Four different doses of 3H- l-tyrosine were injected into the peritoneal cavity of 1-week-old rats, which were killed after 2 hr. Central parts of their livers were removed, fixed, embedded in paraffin, cut into sections of 1 to 10 μ thickness, and mounted on glass slides. The sections were coated with stripping film from Ilford nuclear research plates K-5, and exposed in dry air at 4 °C for periods varying from 1 to 17 days. After processing a 15 °C in complete darkness, the fixed films were rinsed, the underlying sections stained with hematoxylin-eosin, and mounted in balsam. The developed grains were counted over known areas of the sections, as well as over inactive sections mounted on the same slides, and the total areas of the sections were measured. From these data and from the time of exposure the net number of developable grains appearing in the emulsion per unit exposure time was computed. The glass slides carrying the sections were then crushed into appropriate reaction ampoules, the tritium in them was converted into gas, and its absolute activity was determined in metal cathode internal counters. The ratio of the total number of developable grains over a section to the total number of distintegrations in that section, multiplied by 100, was taken as the autoradiographic efficiency expressed in per cent. The relative grain yield was found to decrease with time; the nonlinearity in this relationship was evident, despite the random variation in the results, from the 11th day onward. Actually, the efficiency of grain production seemed to decrease, rather linearly and largely independently of both section thickness and amount of radioactivity in it, as the time of exposure increased from 1 to 17 days. Possible reasons for this are discussed with reference to earlier results, and “coincidence saturation” is excluded, while latent image regression and “charge backscattering” remain as potential explanations. The efficiency of grain production after 7 days of exposure declined in a non-linear fashion from about 14 per cent in sections of 1 μ thickness to about 2.5 per cent in 10 μ sections. The grain yields in sections of 5 and 10 μ thickness were approximately equal. The results are compared to those of previous authors, and backscattering, not considered by them, is taken into account in a rough calculation of the sensitivity of the stripping film on the basis of available data. The effect of an electrical charge on a surface on its backscattering properties, in addition to the atomic number of the material, is suggested as one possible explanation for the difference between the sensitivity obtained for the stripping film (70 per cent) and that given for the equivalent emulsion (100 per cent).

125I-DU (5-IODO-2′-deoxyuridine) in autoradiographic studies of cell proliferation

5- 125I-2′-deoxyuridine ( 125I-DU) was used as thymidine analogue for autoradiography of bone marrow cells of the rat. It was shown that 125I (gamma, 35 keV) gives an autoradiographic resolution sufficient for determining the relative labeling intensities of single cells. 125I-DU is incorporated, following a single injection, into the same type of bone marrow cells and yield labeling indices nearly identical to those observed with 3H-thymidine. A comparison of the relative labeling intensity of myelocytes and segmented neutrophiles at 1 hr 1 day and 3 days after injection of 125I-DU indicates that IDU is not lost from the DNA in these cells during division and subsequent maturation. The data obtained with 3H-thymidine is comparable to that obtained with 125I-DU. Since IDU is reutilized inefficiently or not at all on DNA catabolism in contrast to thymidine, single cell autoradiography can be used to study the effect of thymidine reutilization on measurements of cell proliferation and DNA renewal made with labeled thymidine. An investigation of the distribution of labeled cells of the megacaryocytic series at 1 hr and 3 days after a single injection of 125I-DU or of 3H-thymidine shows the effect of thymidine reutilization likely responsible for a near 100 per cent labeling of all megacaryocytic forms at day 3 after injection of 3H-thymidine. The data also indicate that a “quiescent stem cell” with regard to DNA synthesis cannot be assumed to be the only immediate precursor for megacaryoblasts. These precursor cells must synthesize DNA at least once within 3 days prior to differentiating into recognizable megacaryocytic elements. The overall mitotic index in the bone marrow did not change over a period of 3 days attesting the lack of recognizable radiation effects from 125I and of metabolic toxicity of IDU at the dose level used in the experiments. The autoradiographic efficiency of 125I in comparison with 3H under the conditions of the experiment is discussed.

Factors which affect efficiency of autoradiography with tritiated thymidine.

The overall efficiency of autoradiography with tritium-labeled thymidine has been found to be influenced by the following conditions: (1) exposure in an atmosphere of CO2 and the use of the stripping-film technique, both of which increase the autoradiographic efficiency when compared to exposure in air or to dip-coating technique; (2) latent image fading, which increases with increasing exposure. Up to 2 wk of exposure, however, this disadvantage is counterbalanced by the fading of the mechanical background produced during stripping or dip-coating; (3) the thickness of the inert coating interposed between the labeled locus and the sensitized emulsion. A layer of inert coating can be obtained that will arrest all beta particles from tritium, while having no effect on more energetic emitters like C14; (4) the amount of tritiated thymidine given, with relatively large amounts producing an increase in the mean grain count per labeled cell but not in the percentage of cells identifiable as labeled; and (5) the...