The study of brain circuits depends on a clear understanding of the role played by different neuronal populations. Therefore, the unambiguous identification of different cell types is essential for the correct interpretation of experimental data. Here, we emphasize to the broader neuroscience community the importance of recognizing the persistent presence of Cajal–Retzius cells in the molecular layers of the postnatal hippocampus, and then we suggest a variety of criteria for distinguishing Cajal–Retzius cells from other neurons of the hippocampal molecular layers, such as GABAergic interneurons and semilunar granule cells. The toolbox of criteria that we have investigated (in male and female mice) can be useful both for anatomical and functional experiments, and relies on the quantitative study of neuronal somatic/nuclear morphology, location and developmental profile, expression of specific molecular markers (GAD67, reelin, COUP-TFII, calretinin, and p73), single cell anatomy, and electrophysiological properties. We conclude that Cajal–Retzius cells are small, non-GABAergic neurons that are tightly associated with the hippocampal fissure (HF), and that, within this area of interest, selectively express the proteins p73 and calretinin. We highlight the dangers of using markers such as reelin or COUP-TFII to identify Cajal–Retzius cells or GABAergic interneurons because of their poor specificity. Lastly, we examine neurons of the postnatal hippocampal molecular layers and show cell type-specific differences in their dendritic/axonal morphologies and density distributions, as well as in their membrane properties and spontaneous synaptic inputs. These parameters can be used to distinguish biocytin-filled and/or electrophysiologically recorded neurons and should be considered to avoid interpretational mistakes.
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