Understanding the mechanisms underlying generation of neuronal variability and complexity remains a basic challange to neuroscience. Structural variation in the human genome is likely to be one important mechanism for neuronal diversity and brain disease. A combination of multiple different forms of aneuploid cells due to loss or gain of whole chromosomes (mosaic aneuploidy) giving rise to cellular diversity at the genomic level have been described in neurons of the normal and diseased adult human brain [1–11]. However, the incidence and regional distribution of neuronal aneuploidy in the human brain, whether it affects all chromosomes to the same extent and its impact on brain development and function still remain obscure.

Aneuploidy is defined as the loss and/or gain of chromosomes giving raise to a numerical deviation from haploid genome multiples [12]. While aneuploid cells have been typically associated with pathophysiological conditions such as cancer [13], Down's syndrome [14], Turner's syndrome [15] and mosaic variegated aneuploidy [16] and idiopathic autism [17], cells in normal individuals have basically been assumed to contain identical euploid genomes [18]. Still, earlier hypotheses suggested that a number of mammalian somatic tissues are populated by polyploid cells. Adult neurons of mammals were assumed to be postmitotic cells characterized to some extent by a polyploid chromosome complement. Testing this hypothesis in the past through histochemical methods, however, yielded controversial results through technical limitations [19,20]. However, with the recent development of molecular cytogenic techniques, aneuploid cells in the normal developing and mature brain have clearly been identified, indicating that the maintenance of aneuploid neurons in the adult CNS is a widespread, if not universal, property of organisation [1–11].

Recent studies of the embryonic brain have shown that about approximately one-third of the dividing cells that give rise to the cerebral cortex have genetic variability, manifested as chromosome aneuploidy [3,7,21]. Neurons that comprise the adult cerebral cortex arise from mitotic neural progenitor cells in the ventricular zone, a proliferating region where aneuploid cells appear to be generated through various chromosome segregation defects initially [7,22]. While a portion of these aneuploid cells apparently die during development [7,23,24], aneuploid neurons have been identified in the mature brain in all areas assayed [3–8,11,25] indicating that aneuploidy does not necessarily impair viability [26]. Aneuploid neurons in the adult have been shown to make distant connections and express markers associated with neural activity which indicates that these neurons can be integrated into brain circuitry [18].

Neurodegenerative changes in AD are not randomly distributed throughout the cerebral cortex but rather follow a defined pattern of progression that provides the basis for the neuropathological staging [77]. Both, certain cortical areas and types of neurons are earlier and more constanly involved than others. Neurofibrillary degeneration in the cerebral cortex preferentially affects lamina III and V pyramidal neurons, leaving interneurons unaffected. Further, degeneration preferentially affects the limbic cortex and cortical association areas while primary sensory and motor areas are largely spared. The reasons for this selective neuronal vulnerability are unkown.

Neurofibrillary degeneration in the cerebral cortex, moreover, typically occurs in clusters. Degeneration predominantly involves pyramidal neurons within supra- and infragranular layers with the clustering in these layers often being in register with each other [78] indicating a columnar organisation of degeneration [79]. This NFT clustering is highly correlated with symptoms [80]. Also plaques appear to show a degree of vertical clustering between apical dendritic clusters [81].

The columnar structure is disorganised in cortical areas affected by pathology in AD [82]. Cortical atrophy in AD, moreover, is due to a decrease in cortical length indicating a shrinkage or loss of columns while cortical thickness remains unaffected [83,84]. Also during normal aging, minicolumn width shinks in those cortical regions that are potentially vulnerable to degeneration in AD [85]. A study on trisomy 21 Down's patients, furthermore, suggest that children with the disorder exhibit early adult-like neuronal mini-column spacing [86]. This may relate to the apparently accelerated aging and early onset of AD in Down's syndrome.

This issue of columnar organisation and clustering has been pursued with respect to other pathological structures. Disruption of the columnar organisation of glial processes has been reported for AD [87]. Lewy bodies and Pick bodies [88,89] also exhibit clustered distribution which reflect a modular structure. Taken together, available evidence on the distribution of cortical pathology indicates a determination of the pattern of pathology through the modular organisation of the cortex [90] which basically is a structural reflection of its ontogeny.

AD is a disorder that selectively affects the human brain. It has been suggested that AD is specific to man because of molecular genetic events which promoted a rapid evolution of the hominid brain [91,92]. The phylogenetic expansion of the hominid neocortex most likely arose through a rapid multiplication of the fundamental columnar building blocks.

During embryonic development of the cerebral cortex, cells migrate towards the surface and form mini-columns of cells. They appear to be grouped into larger macro-columns, which form the basis of the mapping of functions across the brain's surface.

Pyramidal neurons, potentially vulnerable to neurofibrillary degeneration in AD, according to the radial unit hypothesis, derive from radially migrating neurons that originate in the ventricular zone of the pallium (cortex). In the primate brain, they find their way to the distant cortex by using radially oriented glial fibres as guides. On the contrary, cortical interneurons, unaffected by degeneration in AD, are born outside the cortex in the ganglionic eminence and reach their final position through tangential migration [93,94].

Following the last mitotic division, immature neurons of the ventricular and sub-ventricular zones attach to an adjacent set of glial guidance fibres. Thus, clonally related neurons generated serially in time at the same locus in the germinal epithelium migrate sequentially along the same or adjacent set of glial guidance fibres, and settle in the inside to outside pattern aligned in a single vertical column [95–97]. Neurons of this radial column form an ontogenetic unit, the fundamental building block in the developing neocortex [98]. Thus, the basic columnar organisation of the cortex reflects its mode of generation [99–101].

The evolutionary expansion of neocortical size in mammals which is particularly prominent in anthropoid primates (i.e. monkeys, apaes, humans) reflects an increased number of cortical cells. According to the radial unit hypothesis, this phylogenetic expansion in cortical surface area results from an increase in the number of founder cells prior to neurogenesis. This increase in the number of cells is generated by two mechanisms: (i) an increase in the size of the embryonic subventricular zone and (ii) a prolonged period of cell cycle activity of progenitor cells during neurogenesis [102]. In macaque monkey, the period during which cell division occurs is 10 times longer than in rodent and the cell cycle duration is two to five times longer than in mouse [103]. Substantially more total rounds of cell divisions elapse during the prolonged neurogenetic period of the monkey cortex, providing the basis for increased cell proliferation. Moreover, unlike the progressive slowing that occurs during cortical development in rodents, cell division accelerates during neurogenesis of the enlarged cortical layers in monkeys. These findings suggest that evolutionary modifications of the duration and number of progenitor cell divisions contribute to both the expansion and laminar elaboration of the primate neocortex [104]. The enlarged cortex of great apes reflects a longer period of neuronal formation during pre-natal development, so that each dividing progenitor cell undergoes more cell cycles before stopping cell division [104]. Cortical progenitors undergo 11 rounds of cell division in mice [105], at least 28 in the macaque [104], and probable four more in human [106]. This phylogenetic prolongation of mitotic activity provides the basis for a potentially higher rate of accumulating mitotic errors during neurogenesis. It is unclear, however, whether different mammalian species differ with respect to the frequency of aneuploid neurons in the CNS.

Until recently, the DNA amounts of defined single neurons have been difficult to study under conditions of preserved tissue architecture. Although chromosome mapping on interphase nuclei by fluorescence in situ hybridisation (FISH) has made considerable progress in recent years [4,7,21,107], it still suffers from major technical limitations. These limitations mainly arise from the application of FISH to isolated cellular nuclei which prevents from clearly attributing cytogenetic changes to defined subsets of cells and to study these changes in a cytoarchitectonic context of preserved tissue architecture. These technical restrictions make it difficult to study the potential link between cytogenetic individuality and selective neuronal vulnerability and, thus, require the implementation of novel techniques.

Therefore, we have developed a novel approach for the DNA quantification of single identified neurons in brain slices based on a combination of slide-based cytometry (SBC) after DNA staining with propidiumiodide, chromogenic in situ hybridisation (CISH) with chromosome-specific probes and laser microdissection followed by quantitative PCR (qPCR) of alu repeats [5,108]. All three methods are applicable to tissue sections and can be combined with immunocytochemical detection of specific marker proteins which allow for further specification of cellular identity in the context of preserved tissue architecture. This is a clear advantage when analysing tissue sections containing different cell types (e.g. neurons versus glia) and modes of pathological affections (e.g. cytoplasmic inclusions, neurofibrillary degeneration).

To confirm our experimental approach we cross validated the three methods, i.e SBC, CISH and qPCR through subsequent application one by one to a defined subset of microscopically identified neurons and obtained a remarkably high inter-method reliability (Figure 1). Obviously, this combination of the three methods in a row can be easily adapted to other issues. When the PI intergral (SBC) is plotted against the number of hybridisation spots (CISH), a highly significant correlation will be obtained (Figure 1A). Similarly, the PI integral shows a highly significant correlation with the DNA content determined by PCR amplification of alu repeats (Figure 1B). The DNA amount determined by PCR amplification of alu repeats also correlates highly significantly with the number of hybridisation spots (Figure 1C).

Based on these correlations, the DNA amount (mean ± SD) of a single diploid neuron identified by two hybridisation spots can be determined as 2.52 ± 0.87 pg, while the DNA content of a neuron with four spots amounts to 5.94 ± 1.28 pg. For comparison, the mean single cell DNA content (±SD) of lymphocytes treated identically to brain tissue and used for control experiments was determined at 2.07 pg ± 0.6 pg.

As in our studies the DNA content of single neurons determined by SBC is far better correlated to CISH (Figure 1A) than is the qPCR measurement (Figures 1B/C), we conclude that SBC is a very reliable technique for future studies on the single cell DNA content in tissue sections.

The results of the present study on the DNA amount of identified cortical neurons, obtained through a combination of slide-based cytometry, chromogenic in situ hybridization and PCR amplification of alu-repeats, indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons of the normal human brain might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication. The combination of cytometry with molecular biological characterisation of single microscopically identified neurons as outlined here might be a promising approach to study molecular neuronal individuality in the context of preserved tissue architecture. It reflects the concept of cytomics and will forward our understanding of the molecular architecture and functionality of neuronal systems [109,110].