Dysfunctional dendritic arborization is a key feature of many developmental neurological disorders. Across various human brain regions, basal dendritic complexity is known to increase along a caudal-to-rostral gradient. We recently discovered that basal dendritic complexity of layer II/III cortical pyramidal neurons in the mouse increases along a caudomedial-to-rostrolateral gradient spanning multiple regions, but at the time, no molecules were known to regulate that exquisite pattern. Integrin subunits have been implicated in dendritic development, and the subunit with the strongest associations with autism spectrum disorder and intellectual disability is integrin β3 (Itgb3). In mice, global knockout of Itgb3 leads to autistic-like neuroanatomy and behavior. Here, we tested the hypothesis that Itgb3 is required for increasing dendritic complexity along the recently discovered tangential gradient among layer II/III cortical pyramidal neurons. We targeted a subset of layer II/III cortical pyramidal neurons for Itgb3 loss-of-function via Cre-loxP-mediated excision of Itgb3. We tracked the rostrocaudal and mediolateral position of the targeted neurons and reconstructed their dendritic arbors. In contrast to controls, the basal dendritic complexity of Itgb3 mutant neurons was not related to their cortical position. Basal dendritic complexity of mutant and control neurons differed because of overall changes in branch number across multiple branch orders (primary, secondary, etc.), rather than any changes in the average length at those branch orders. Furthermore, dendritic spine density was related to cortical position in control but not mutant neurons. Thus, the autism susceptibility gene Itgb3 is required for establishing a tangential pattern of basal dendritic complexity among layer II/III cortical pyramidal neurons, suggesting an early role for this molecule in the developing brain.

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.

Background Autism spectrum disorder (ASD) is characterized by repetitive behaviors, deficits in communication, and overall impaired social interaction. Of all the integrin subunit mutations, mutations in integrin β3 (Itgb3) may be the most closely associated with ASD. Integrin β3 is required for normal structural plasticity of dendrites and synapses specifically in excitatory cortical and hippocampal circuitry. However, the behavioral consequences of Itgb3 function in the forebrain have not been assessed. We tested the hypothesis that behaviors that are typically abnormal in ASD—such as self-grooming and sociability behaviors—are disrupted with conditional Itgb3 loss of function in forebrain circuitry in male and female mice. Methods We generated male and female conditional knockouts (cKO) and conditional heterozygotes (cHET) of Itgb3 in excitatory neurons and glia that were derived from Emx1-expressing forebrain cells during development. We used several different assays to determine whether male and female cKO and cHET mice have repetitive self-grooming behaviors, anxiety-like behaviors, abnormal locomotion, compulsive-like behaviors, or abnormal social behaviors, when compared to male and female wildtype (WT) mice. Results Our findings indicate that only self-grooming and sociability are altered in cKO, but not cHET or WT mice, suggesting that Itgb3 is specifically required in forebrain Emx1-expressing cells for normal repetitive self-grooming and social behaviors. Furthermore, in cKO (but not cHET or WT), we observed an interaction effect for sex and self-grooming environment and an interaction effect for sex and sociability test chamber. Limitations While this study demonstrated a role for forebrain Itgb3 in specific repetitive and social behaviors, it was unable to determine whether forebrain Itgb3 is required for a preference for social novelty, whether cHET are haploinsufficient with respect to repetitive self-grooming and social behaviors, or the nature of the interaction effect for sex and environment/chamber in affected behaviors of cKO. Conclusions Together, these findings strengthen the idea that Itgb3 has a specific role in shaping forebrain circuitry that is relevant to endophenotypes of autism spectrum disorder.

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.

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