The neocortex is the main locus of cognition in the human brain and its expansion relative to that in non-human primates is a main anatomic distinction. Since the number of neocortical neurons is generally accepted to bear some relation with cognitive abilities, several stereological studies have quantified neocortical cell numbers in brains of adult humans and various species (Korbo et al., 1990; Pakkenberg and Gundersen, 1997; Jelsing et al., 2006; Christensen et al., 2007; Eriksen and Pakkenberg, 2007; Walløe et al., 2010; Fabricius et al., 2013). On the other hand, there have been few developmental studies of cell numbers in the human fetal and neonate forebrain (Winick, 1968; Dobbing and Sands, 1973; Rabinowicz et al., 1996), none of which employed stereological methods. This paucity may well reflect technical and ethical issues arising in studies of the fetal human brain. Furthermore, many earlier studies of brain cell numbers have been based upon assumptions that all cells (counting objects) are of uniform shape and size or are isotropically orientated in the specimen (Weibel and Gomez, 1962; Rose and Rohrlich, 1987). Violation of these assumptions give rise to significant bias in the estimation of cell numbers, but stereological methods accommodate these factors. Stereology is an application of spatial sampling that enables researchers to obtain three-dimensional information about the distribution of objects observed in a slice or section. In general, it is not possible to determine the exact cell numbers in large structures, but estimation of total cell numbers through application of stereological principles is efficient, i.e., “with a low variability after spending a moderate amount of time,” and unbiased, that is to say “without systematic deviation from the true value” (Gundersen et al., 1988a,b). Thus, the stereological methods yield reliable and efficient estimates of total cell numbers even in structures as complex and heterogenous as the human fetal brain. In order to support statistical comparison between groups, the stereological sampling frequency can be adjusted to give optimal precision without unwarranted effort (Gundersen et al., 1988a,b; West, 2012).

A series of stereological studies has contributed invaluable knowledge of the proliferation of total neocortical cell numbers in human fetal brain by use of the optical fractionator (Samuelsen et al., 2003, 2007; Larsen et al., 2006, 2008, 2010). The optical fractionator design is an ideal method to obtain total cell numbers in the fragile fetal brain due to the minimum of handling of the tissue. There follows a detailed and critical account of how to apply the optical fractionator scheme for robust estimation of cell numbers in the human fetal forebrain across normal and abnormal intrauterine development. Furthermore, there are some comments upon already published results. Since our histological methods did not distinguish between neurons and glial cells, the total cell numbers is a sum of future neurons and glial cells.

Main events in the development of the human brain include neuronal proliferation, migration, differentiation, synaptogenesis, pruning and myelination. Deviations from the normal developmental sequence due to genetic or environmental factors give rise to cerebral malformation. Severe misdevelopment of the brain can be diagnosed by post mortem examination or through macroscopic imaging techniques in utero. However, detecting subtle changes in cell numbers requires quantitative measurements by microscopy. We have focused our investigations on the total cell numbers in the maturing neocortex or in transient fetal zones destined to develop into neocortex (Kostovic and Judas, 1995). More than 40 years ago, the Boulder Committee, founded by the American Association of Anatomists, described and named these zones, which have no direct counterpart in the adult brain (The Boulder Committee, 1970). This description was revised by Bystron et al. (2008).

During human fetal weeks 3 and 4, the telencephalic wall contains only the ventricular zone (VZ). Neuroepithelial cells then constitute a homogenous cell population in the neural tube, and the major histogenic event is intensive symmetric proliferation where single progenitor cell first give rise to two progenitor cells before the onset of neurogenesis (Kornack, 2000). The first phase of neurogenesis starts at approximately fetal week 5, which occurs in conjunction with differentiation of the primordial neuroepithelial cells into radial progenitor cells. They provide a scaffold for migration of newborn cells to their predetermined position, while also acting as progenitor cells for more neurogensis (Malatesta et al., 2000; Miyata et al., 2001; Noctor et al., 2001; Heins et al., 2002). At fetal week 6, the subventricular zone (SVZ) arises on the border of the VZ through accumulation of outer radial glial cells and intermediate progenitors lacking an attachment to the ventricular surface. Even through early human postnatal life, immature neurons continue migrating from the SVZ to specific targets such as the olfactory bulb and prefrontal cortex (Sanai et al., 2011; Khodosevich et al., 2013; Wang et al., 2013; Paredes et al., 2016), but this process declines in early childhood (Sanai et al., 2011; Paredes et al., 2016).

The intensive proliferation in the SVZ leads to formation of the ganglionic eminence in the basoventral part of the telencephalon. The ganglionic eminence later develops into the basal ganglia, but is in rodent also the source of neocortical inhibitory GABAergic interneurons, which migrate dorsally into the neocortex in a process known as tangential migration (Anderson et al., 1997; Tamamaki et al., 1997). Human neocortical interneurons are also derived from the ganglionic eminence (Letinic et al., 2002; Fertuzinhos et al., 2009), but the majority of neocortical interneurons arise in the VZ and SVZ of the dorsal telencephalon itself (Letinic et al., 2002).

The Boulder Committee originally suggested that before any post mitotic cells appear in the prospective cortex, the subpial processes of ventricular cells constitute a cell sparse layer which they termed the marginal zone (MZ). Increasing evidence has revealed that no such subpial layer exists prior to the tangential invasion of the first cortical neurons (Bystron et al., 2005, 2006), such that the term MZ is more properly employed after the formation of the cortical plate (Uylings, 2000; Bystron et al., 2008). A compartment consisting of heterogeneous post migratory cells and neuropil lying between the proliferative zones and the pial surface of the dorsal telencephalon before the appearance of the cortical plate is known as the preplate.

During fetal weeks 8 and 9, the first migratory wave of cells form the cortical plate (CP), a distinctive layer of tightly packed neurons. The CP divides the preplate into the MZ and the subplate/intermediate zone. The intermediate zone (IZ), which contains both radially and tangentially migrating cells together with axons, constitutes the future white matter. The subplate zone (SP) transiently appears between the IZ and the CP. The first phase of synaptogenesis is thought to occur in the SP, which is also a target for afferents arising from the brain stem, basal forebrain, thalamus and the ipsi- and contralateral cortices (Kostovic and Rakic, 1990). Together with the neighboring CP and the MZ, the SP will develop into the future cortical gray matter. The SP undergoes intensive reorganization and reaches its maximum thickness at 22 fetal weeks, begins to regress after fetal week 35, and ultimately disappears by the second postnatal year (Kostović et al., 2014).

Many recent studies have investigated the molecular background of neurogenesis, patterning of brain regions, and circuit formation in the developing human brain. However, very little has been known about the prenatal development of the human fetal brain in terms of growth in cell numbers. The first stereological study applying the optical fractionator to cell counting in human fetal brain appeared in 2003 (Samuelsen et al., 2003). In that study we estimated total cell numbers in selected zones of the fetal neocortical cerebral wall in 22 human fetuses aged 13 to 41 gestational weeks. Since our histological methods do not distinguish between neurons and glial cells, we report total cell numbers, i.e. the sum of future neurons and glial cells. We found that the growth in cell numbers in the human fetal forebrain appears to be two-phased, with an initial exponential phase from 13 to 20 gestational weeks, followed by linear phase from 22 weeks of gestation until term. From 13 to 20 weeks of gestation the total number of cells increased four-fold, from 3 × 10⁹ to 13 × 10⁹ cells, and increased by a further factor of 3 to 38 × 10⁹ cells at term (Figure 5). During the second half of gestation, we estimated 170 million new brain cells per day in the entire neocortical wall, equivalent to 2,000 new cells each second.

We also estimated the total number of cells in the developing ganglionic eminence in 13 human fetuses aged 22 to 29 fetal weeks with use of the optical fractionator (Larsen, 2010; Larsen et al., 2010). From 11 to 20 fetal weeks, the total number of cells in the ganglionic eminence increased six-fold from 0.47 × 10⁶ to 2.86 × 10⁶, whereafter total cell numbers declined until the structure finally disappeared around term. In that study, we demonstrated that the ganglionic eminence, like the neocortex, exhibits an exponential development of total cell numbers from 10 to 20 fetal weeks. Our stereological data are in agreement with the volumetric data from Huang et al. (2009), who used diffusion tensor imaging of post mortem human fetal brains to chart the increasing volume of the ganglionic eminence during the second trimester. Previous studies have identified the ganglionic eminence as an important source of neocortical inhibitory interneurons and oligodendrocyte progenitor cells (Anderson et al., 1997; Tamamaki et al., 1997; Letinic et al., 2002; Rakic and Zecevic, 2003; Ma et al., 2013), which may well drive the exponential development in total cell numbers lasting until mid-gestation. Although the ganglionic eminence disappears around birth, the production of neocortical inhibitory interneurons continues into early postnatal life (Arshad et al., 2016), and remnants of the SVZ produce cells until early infancy (Del Bigio, 2011).

In the final study to investigate the normal developing brain in term infants, we estimated total cell numbers in 10 normal neonate brains within the CP/MZ, the SP, the IZ and the SVZ/VZ (Larsen et al., 2006) by applying the optical fractionator. The gestational ages ranged from 38 to 42 weeks. In that study, the total number of cells in the entire neocortical part was almost 33 × 10⁹ in the human neonate forebrain, and around 24 × 10⁹ in the CP/MZ. Other studies of the adult neocortex indicate a total of 50 × 10⁹ cells in females and 65 × 10⁹ in males (Pelvig et al., 2008), of which 19 × 10⁹ are neurons in females and 23 × 10⁹ are neurons in males (Pakkenberg and Gundersen, 1997). Compiling these studies indicates that the total number of neurons in neonates equals the total number in adults, whereas glial cells continue to proliferate in postnatal development. Indeed, the first stereological study of total cell numbers during neocortical development of rat pups found similar growth trajectories of neurons and glial cells (Behnam-Rassoli et al., 1991). In agreement with these findings, two recent stereological studies (Kjær et al., 2016; Sigaard et al., 2016) showed linear increases in both oligodendrocyte and astrocyte numbers during the first 3 years of human life in humans, and that the number of neocortical neurons has already attained the adult population at least at term.

The growth in fetal brain cell numbers is so complex and rapid that any disruption is apt to derail the normal growth program away from delicately predetermined interrelationships, likely resulting in anatomic and functional deficits persisting to maturation. With this in mind, we have investigated total cell numbers in the forebrain in fetuses with Down syndrome (DS) and in intrauterine growth-restricted (IUGR) fetuses, which constitute relative common conditions of abnormal fetal development. Alterations of brain development and intellectual disabilities occur in DS (Schmidt-Sidor et al., 1990; Devenny et al., 1996, 2000; Krinsky-McHale et al., 2002; Nelson et al., 2005). We used the optical fractionator to compare the total cell numbers in forebrain of 4 DS fetuses aged 19 weeks of gestation to 8 normal control fetuses The total cell number was 34% lower in the neocortical part of the cerebral wall of DS fetuses (6.85 × 10⁹) compared to normal controls (10.4 × 10⁹). We suppose that the intellectual disability often occurring in DS may arise from a structural deficit in the human fetal brain already present in the second trimester (Larsen et al., 2008).

We also used the optical fractionator to to estimate the cell numbers in the forbrain of nine severely affected IUGR fetuses and 15 control fetuses with gestational ages ranging from 19 to 41 weeks. The total cell number in the CP/MZ was significantly lower in IUGR fetuses compared to controls, and the daily increase rate in the CP/MZ of IUGR fetuses was only half of the controls; whereas control fetuses acquired an average of 173 million CP/MZ cells per day from midgestation to term, the IUGR fetuses acquire only 86 million new cells per day (Samuelsen et al., 2007).

A large number of stereological studies have used the stereological ratio estimators (density multiplied by reference volume) to quantify total cell numbers in the human brain (Pakkenberg and Gundersen, 1997; Andersen et al., 2003; Pelvig et al., 2008; Karlsen and Pakkenberg, 2011; Karlsen et al., 2014). In that approach, the number of cells is counted in optical disectors, knowing the dimensions of the disector and the total volume (see e.g., Pakkenberg and Gundersen, 1997). The method of multiplying density by the reference volume is obviously a good choice when volume and total cell numbers are both matters of interest. It is then possible to obtain reliable mean estimates of both parameters for making comparisons by group or treatment. The adult brain suffers less unpredictable dimensional change during tissue processing than typically occurs in fetal material.

Due to the unpredictable shrinkage and distoration during fixation of fetal brain, it is of questionable value to attempt an estimation of volumes and densities. However, the optical fractionator method for estimating total cell numbers is the obvious choice for fetal brain studies, because results are robust to deformation, shrinkage, and swelling of the reference space. Furthermore, it is easier to apply the optical fractionator, because no reference volume estimate is required.

Despite the advantages of the optical fractionator method, some pitfalls should be considered.

If these recommendations and stipulations are satisfied, total cell number estimates with the optical fractionator are indeed “unbiased for all practical purposes” (Gundersen et al., 1988a,b). Correct use of the optical fractionator requires some expertise with stereological tools, and therefore might not be the preferred method for researchers with no access to expert guidance. In such circumstances, multiplying the density by the reference volume method might be more appropriate, as it is more robust and less vulnerable to sampling design. In the present context, there is a particular need to minimize handling of fragile fetal tissue and when all requirements are observed, the optical fractionator method is the perfect choice to estimate total cell numbers in fetal brain.

In the three decades since the landmark study showing how to obtain unbiased estimation of individual particle numbers was published (Sterio, 1984), the optical disector method has matured to a general tool for quantifying the number of cells in brain and other organs. Subsequently, the optical fractionator method has made it possible to overcome important methodological problems arising from unpredictable shrinkage during fixation of the fetal brain and furthermore, several user-friendly software programs exist which can assist the un-experienced researcher with the calculations and evaluation of the estimate quality.

Despite the widespread knowledge of the advantages of unbiased stereological methods, many quantitative studies continue to use cell densities in sections. However, the volume/reference trap should be avoided. When using the density multiplied by reference volume, the reference volume and the numerical cell density both contribute to the final readout of total cell counts. To avoid bias, stereologists should always report the numerator and denominator instead of simply reporting cell density. It is better to graph the explicit number or length as a function of the volume. Density, a ratio between two variables, is of limited value unless used to obtain total cell numbers. Alternatively, one may use the optical fractionator to obtain directly the total cell numbers.

Present histological methods do not distinguish between neurons and glial cells until term. Thus, we estimate total cell numbers and net proliferation, although certain transient features can be seen to decline in size and cell counts during late gestation. In future studies the use of an apoptotic marker in combination with stereological methods in the fetal brain could contribute to a more exact knowledge of the growth in cell numbers. Furthermore, other cell-labeling techniques could eventually give separate estimates of neurons and glial cells during human fetal brain development.

In summary, we have applied the optical fractionator to investigate total cell numbers of the developing human fetal and neonate forebrain. Remarkably, the production of billions of cells, many of which are neurons, occurs during a rather brief span of intense and exponential proliferation. Furthermore, we have demonstrated a reduced total cell number in the forebrain in DS fetuses at midgestation and in IUGR fetuses during the third trimester.

The author confirms being the sole contributor of this work and approved it for publication.