Background: Pyramidal neurons of the hippocampus are not homogeneous in their structure or function, yet the structure and function of individual neurons are well-correlated. Despite these well-known facts, few structural studies of pyramidal neuronal dendrites in the hippocampus have controlled for precise cellular position within the hippocampus along its dorsoventral (longitudinal), tangential (proximodistal), or radial axes. We believe this is largely due to technical limitations that limit positional information about the neuron, limit throughput, and limit the control of confounding variables. New method: Here, we present a simple approach to address these limitations effectively in mouse CA3, adapted from our prior work in the mouse cerebral cortex. Our approach tracks the dorsoventral, tangential, and radial positions of neurons within the hippocampus, increases the number of available neurons for analysis, and provides several new controls. Results: We demonstrate how topographic and morphological data are related to one another among mouse CA3 pyramidal neurons, in a pilot data set providing proof-of-concept. Comparison with existing methods: Other common methods of labeling neurons, such as biocytin fills or Golgi-Cox stains, are labor- or time-intensive. Here, we validate the use of the transgenic fluorescent Thy1-GFP-M line, so the labeling step is practically eliminated. Other methods section the hippocampus coronally, which distorts the dorsoventral, tangential, and radial axes. Here, we preserve all three axes. Some existing methods do not collect all sections in order, instead focusing on "more dorsal" or "more ventral" sections, for example. We preserve much more dorsoventral information. Recent work has shown that CA2 is better defined by immunohistochemical rather than neuroanatomical markers. We use that immunohistochemistry here to increase precision in defining tangential position. Other studies have not measured other, critical variables and relationships that could confound the results. Here, we assess many more of these variables and relationships to increase the rigor of our work. Conclusions: We developed a more rigorous method for preserving precise cellular positioning information among reconstructed mouse hippocampal pyramidal neurons. Although we provide several innovations in our method, each innovation identified here could independently improve the rigor and reproducibility of a wide variety of approaches for understanding the morphological heterogeneity of pyramidal neurons in the brain.

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In excitatory hippocampal pyramidal neurons, integrin β3 is critical for synaptic maturation and plasticity in vitro. Itgb3 is a potential autism susceptibility gene that regulates dendritic morphology in the cerebral cortex in a cell‐specific manner. However, it is unknown what role Itgb3 could have in regulating hippocampal pyramidal dendritic morphology in vivo, a key feature that is aberrant in many forms of autism and intellectual disability. We found that Itgb3 mRNA is expressed in the stratum pyramidale of CA3. We examined the apical dendritic morphology of CA3 hippocampal pyramidal neurons in conditional Itgb3 knockouts and controls, utilizing the Thy1‐GFP‐M line. We fully reconstructed the apical dendrite of each neuron and determined each neuron's precise location along the dorsoventral, proximodistal, and radial axes of the stratum pyramidale. We found a very strong effect for Itgb3 expression on CA3 apical dendritic morphology: neurons from conditional Itgb3 knockouts had longer and thinner apical dendrites than controls, particularly in higher branch orders. We also assessed potential relationships between pairs of topographic or morphological variables, finding that most variable pairs were free from any linear relationships to each other. We also found that some neurons from controls, but not conditional Itgb3 knockouts, had a graded pattern of overall diameter along the dorsoventral and proximodistal axes of the stratum pyramidale of CA3. Taken together, Itgb3 is essential for constructing normal dendritic morphology in pyramidal neurons throughout CA3.