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Many calcium-binding proteins are expressed in a region-and cell-type specific manner in the mammalian hippocampus. Neuronal calcium-binding proteins (NECABs) are also expressed in hippocampal neurons, but few species have been investigated, with partly controversial findings. We here describe NECAB1, NECAB2 and NECAB3 as well as parvalbumin, calbindin, and calretinin in the European mole, and compare staining patterns of these proteins with those in mouse and other species. While subtle differences are present, NECAB staining in the European mole was generally similar to those in mouse. Common to European moles, mice, and other species we investigated, large hilar polymorphic cells, likely to represent mossy cells, were positive for all three NECABs. NECAB1 and 2 are suitable as markers for these cells along the entire septotemporal axis of the hippocampus. In the European mole, parvalbumin, calbindin and calretinin showed traits that have been described in other species before, albeit in a unique combination. In summary, we provide the first description of distribution of these proteins in the hippocampus of the European mole. This subterranean, insectivorous, and solitary living species belongs to the Order of Eulipotyphla. Despite many similarities with other subterranean species from the rodent order in terms of lifestyle, its hippocampus is cytoarchitecturally much more elaborated than in, e.g., mole-rats. It remains an open question if the hippocampal structure of the European mole reflects evolutionary constraints or ecology. Our descriptive study highlights the diversity in hippocampal cytoarchitecture even in small mammalian species.

The hippocampus of many mammals contains a histoarchitectural region that is not present in laboratory mice and rats—the reflected blade of the CA3 pyramidal cell layer. Pyramidal cells of the reflected blade do not extend dendrites into the hippocampal molecular layer, and recent evidence indicates that they, like the proximal CA3 pyramids in laboratory rats and mice, partially integrate functionally with the dentate circuitry in pattern separation. Quantitative assessments of phylogenetic or disease-related changes in the hippocampal structure and function treat the reflected blade heterogeneously. Depending on the ease with which it can be differentiated, it is either assigned to the dentate hilus or to the remainder of CA3. Here, we investigate the impact that the differential assignment of reflected blade neurons may have on the outcomes of quantitative comparisons. We find it to be massive. If reflected blade neurons are treated as a separate entity or pooled with dentate hilar cells, the quantitative makeup of hippocampal cell populations can differentiate between species in a taxonomically sensible way. Assigning reflected blade neurons to CA3 greatly diminishes the differentiating power of all hippocampal principal cell populations, which may point towards a quantitative hippocampal archetype. A heterogeneous assignment results in a differentiation pattern with little taxonomic semblance. The outcomes point towards the reflected blade as either a major potential player in hippocampal functional and structural differentiation or a region that may have cloaked that hippocampi are more similarly organized across species than generally believed.

Sampling is a critical step in procedures that generate quantitative morphological data in the neurosciences. Samples need to be representative to allow statistical evaluations, and samples need to deliver a precision that makes statistical evaluations not only possible but also meaningful. Sampling generated variability should, e.g., not be able to hide significant group differences from statistical detection if they are present. Estimators of the coefficient of error (CE) have been developed to provide tentative answers to the question if sampling has been “good enough” to provide meaningful statistical outcomes. We tested the performance of the commonly used Gundersen-Jensen CE estimator, using the layers of the mouse hippocampal dentate gyrus as an example (molecular layer, granule cell layer and hilus). We found that this estimator provided useful estimates of the precision that can be expected from samples of different sizes. For all layers, we found that a smoothness factor (m) of 0 generally provided better estimates than an m of 1. Only for the combined layers, i.e., the entire dentate gyrus, better CE estimates could be obtained using an m of 1. The orientation of the sections impacted on CE sizes. Frontal (coronal) sections are typically most efficient by providing the smallest CEs for a given amount of work. Applying the estimator to 3D-reconstructed layers and using very intense sampling, we observed CE size plots with m = 0 to m = 1 transitions that should also be expected but are not often observed in real section series. The data we present also allows the reader to approximate the sampling intervals in frontal, horizontal or sagittal sections that provide CEs of specified sizes for the layers of the mouse dentate gyrus.