Quantitative assessment of specificity in immunoelectron microscopy.

In immunoelectron microscopy (immuno-EM) on ultrathin sections, gold particles are used for localization of molecular components of cells. These particles are countable, and quantitative methods have been established to estimate and evaluate the density and distribution of "raw" gold particle counts from a single uncontrolled labeling experiment. However, these raw counts are composed of two distinct elements: particles that are specific (specific labeling) and particles that are not (nonspecific labeling) for the target component. So far, approaches for assessment of specific labeling and for correction of raw gold particle counts to reveal specific labeling densities and distributions have not attracted much attention. Here, we discuss experimental strategies for determining specificity in immuno-EM, and we present methods for quantitative assessment of (1) the probability that an observed gold particle is specific for the target, (2) the density of specific labeling, and (3) the distribution of specific labeling over a series of compartments. These methods should be of general utility for researchers investigating the distribution of cellular components using on-section immunogold labeling.

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy.

Reelin is a glycoprotein ( approximately 400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.

The use of nonparametric statistics in quantitative electron microscopy.

Parametric statistical methods assume samples that have a normal distribution and representative sample sizes (i.e. n >20). Quantitative electron microscopy is inherently restricted to small sample sizes and a priori there is no way to know if the expression of the ligand being studied has a normal distribution. Thus to make statistical inferences based on data generated by quantitative electron microscopy using parametric methods may not be justified. Nonparametric statistical methods offer a tool for the evaluation of data that do not meet the criteria for analysis by parametric methods. In this report I show the utility of using nonparametric statistical methods for the analysis of data generated by quantitative electron microscopy.