A Toolbox of Criteria for Distinguishing Cajal–Retzius Cells from Other Neuronal Types in the Postnatal Mouse Hippocampus

Abstract

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.

Glutamate released by Cajal-Retzius cells impacts specific hippocampal circuits and behaviors

“…First, we verified that the genetic manipulations introduced in KOs did not affect hippocampal CR densities. CRs were identified by their typical tadpole-like shape, location in the molecular layers (i.e., molecular layer of the dentate gyrus and stratum lacunosum moleculare of the hippocampus proper), and by their specific EYFP and p73 expression ( Anstötz and Maccaferri, 2020 ; Figures 1A – 1D ). As shown in Figures 1C and 1D , the quantification of CRs (by p73 immunoreactivity) did not reveal significant differences in their linear densities along the hippocampal fissure, either between ΔNp73-control and ΔNp73-KO or between PDE1c-control and PDE1c-KO mice ( Table S1 ).…”

“…Cajal-Retzius cells were identified and quantified by their p73 immunoreactivity ( Anstötz and Maccaferri, 2020 ) using a Zeiss Axioscope2 (Carl Zeiss Microscopy, LLC, White Plains, NY) equipped with Neurolucida 11 software suite (MBF bioscience). Hippocampal molecular layers (molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the CA1 subfield of the hippocampus proper) were chosen as regions of interest and linear cellular densities were calculated as previously described in detail ( Anstötz et al, 2016 , 2018a ).…”

“…Post hoc morphometric data were analyzed with Neurolucida Explorer software ( Anstötz and Maccaferri, 2020 ). Sholl analysis was performed by measuring dendritic lengths within a 50 μm initial radius and in the circular crowns obtained by 50 μm increments for granule cells, interneurons, and basal dendrites of pyramidal cells.…”

Analysis of the Expression Pattern of Cajal-Retzius Cell Markers in the <b><i>Xenopus laevis</i></b> Forebrain

“…Its loss or the loss of the hem expression of Wnt3a, implicated in the induction of self-renewal of radial glia and the early differentiation of cortical intermediate progenitors [Munji et al, 2011], results in loss of the hippocampus [Lee et al, 2000;Yoshida et al, 2006;Caronia-Brown et al, 2014], whereas ectopic hems are capable of inducing ectopic hippocampus [Mangale et al, 2008]. Particularly in the hippocampus, CR cells are small non-GABAergic neurons that are tightly associated with the hippocampal fissure, that selectively express the proteins p73 and Calretinin [Anstötz and Maccaferri, 2020]. The molecular mechanisms by which the hem acts during development in the formation of the rest of the cortex and fate specification of the cortical primordium has been the focus of in-depth analysis in recent years.…”

Characterization of Oxytocin Receptor Expression Within Various Neuronal Populations of the Mouse Dorsal Hippocampus

“…Further, we may have simply counted other non-GABAergic neurons expressing INMs. For example, as Figure 13 shows, a sizable proportion of the DG Calb2 (calretinin) neurons, especially in the inner part of the granule cell layer, do not express GABA, as noted by others (Liu et al, 1996;Jinno, 2011;Anstotz and Maccaferri, 2020). These were included in our analyses and one should be more cautious about the DG numbers with regard to Calb2 as interneurons.…”