Estimation of the Number of Synapses in the Cerebral Cortex: Methodological Considerations

Abstract

In the present work we discuss several sampling procedures commonly used for counting synapses in the cerebral cortex. We compare, within the same tissue, two frequently used sterereological methods for determining the numerical density of synapses per unit volume, using as an example the estimation of the number of types of synapses by layers in the neuropil of the adult human temporal neocortex. These two methods are a size-frequency method (formula N(A)/d) and the disector method (sigmaQ-/a x h). Since the size-frequency method is assumption-based and the disector method is considered to be an unbiased method, the latter is often recommended for the quantification of synapses and other objects. We obtained, however, similar estimates for the numerical density of the different types of synapses using both methods, although they presented different technical difficulties and statistical properties. In addition, we show that the size-frequency method is more efficient and easier to apply than the disector method. Nevertheless, there are other methods for quantification which may also be valid, depending on the aim of the research; but the data reported in many articles are often complicated, which makes it very difficult for the reader to follow all the steps of the calculation. If certain basic information were given, this would facilitate the interpretation and sharing of important information with other laboratories, regardless of the method used for quantification. Finally, based on our present results and previous literature, we propose a simple general protocol for estimating the numerical synaptic density by volume in the neuropil of the cerebral cortex.

Gender differences in human cortical synaptic density

“…At least 30 micrographs at a magnification of ϫ30,000 were obtained per layer and case (for a detailed description on the estimation of synapses see ref. 23). …”

“…23). The density of synapses per unit volume of the neuropil was calculated by using the formula N V ϭ NA/d where NA is the number of synaptic profiles per unit area and d is the average cross-sectional length of synaptic junctions.…”

Synapses are lost during aging in the primate prefrontal cortex

“…Sterio, 1984;Calverley et al, 1988). In comparisons of the data generated by these two methods, both DeFelipe et al (1999) and we (Peters et al, 2001) have shown that the methods give comparable results, and that when the effect of age on the numerical density of a population of synapses is being estimated, both methods reveal the same trends in changes with age (Peters et al, 2001). This being so, we have chosen to use the sizefrequency method to make our estimations.…”

Barrel Pattern Formation Requires Serotonin Uptake by Thalamocortical Afferents, and Not Vesicular Monoamine Release

“…Synaptic density per unit area was estimated from 10 electron microscopic samples of neuropil from layer IV per animal (DeFelipe et al, 1999). In wt animals, we sampled neuropil only from the centers or hollows of the barrels, the region of termination of thalamic afferents (White, 1979), whereas in ko mice the neuropil was sampled from regions within layer IV that showed lower cellular density.…”