Using a new approach for studying live embryonic mouse brains at single-cell resolution, researchers headed by a team at the Institute of Molecular and Clinical Ophthalmology Basel (IOB) have identified an active multi-layer pyramidal-to-pyramidal neuronal circuit that forms in the cortex during an unexpectedly early stage of development. Perturbing this circuit genetically, the team showed, led to changes similar to those seen in the brains of people with autism.

Layer 5 pyramidal neurons in normal mice (left) compared with mice with autism gene knocked out (right), showing a patch of disorganized cortex. [Institute of Molecular and Clinical Ophthalmology Basel (IOB)]

“Understanding the detailed development of cell types and circuits in the cortex can provide important insights into autism and other neurodevelopmental diseases,” said Botond Roska, PhD, IOB director and corresponding author of the team’s published paper in Cell. “This is what our findings confirm.”

In their report, titled “Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex,” Roska and colleagues concluded, “… pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.”

Autism has long been associated with faulty circuits in the cortex, which is the part of the brain that governs sensory perception, cognition, and other high-order functions. Most of the cortex is composed of excitatory cells called pyramidal neurons (PNs). The IOB team wanted to study when and how these neurons assemble into the first active circuits in the cortex, but that posed a difficult challenge. As the authors explained, while the activity and communication within PN-to-PN circuits have been intensively investigated, in vivo, in the adult, when and how the first active PN circuits assemble in vivo isn’t known.” And while answers to such questions will be key to increasing what is understood about cortical circuit development, the team continued, “… since neurodevelopmental disorders are associated with defects within cortical circuits, insights into pyramidal circuit formation may also be relevant for understanding the mechanisms of diseases such as autism spectrum disorder … patches of disorganized cortical tissue have been observed in brains of both children with autism as well as in mouse models of the disorder, around the time of birth.” However, it remains unknown if mutations in autism-related genes may perturb the development of PN-to-PN circuits during embryonic development.”

Pyramidal neurons measure only a tenth of the width of a human hair, and any movement during experimental procedures might lead to inaccurate recordings of activity. For their research, the team devised a surgical solution for keeping the neurons stable. Embryos were secured inside agar-filled 3D holding devices within the mother’s abdominal cavity, so that normal embryonic blood flow and temperature could be maintained. “Here, we developed a method with sufficient mechanical stability to perform both two-photon imaging from individual neurites and two-photon targeted patch clamp recordings, in healthy living mouse embryos connected to the dam …” they wrote.

The prevailing view is that the cortex develops in an “inside-out fashion,” with the deepest of its six layers appearing first. According to this current view, the investigators wrote, “During cortical development, PNs migrate to their final locations in neocortex, with layers forming in an inside-out fashion … PNs that will populate layers 5 and 6 (L5-PNs and L6-PNs) are born first. In mice, these neurons appear between embryonic day (E) 11.5 and E14.5 and start to migrate into the developing cortex from E12.5 onwards.”

In this way, pyramidal neurons were thought to slowly become active as they migrate to their final locations in the cortex and form connections with each other. However, commented co-lead author Arjun Bharioke, PhD, a systems neuroscientist in IOB’s Central Visual Circuits Group, “we actually detected a very different activity pattern.” Focusing specifically on mouse embryonic Rbp4-Cre pyramidal neurons, the team’s studies, using techniques including in vivo patch clamp recordings and two-photon calcium imaging, discovered a very early transient circuit that was already highly active and correlated even before the six-layer cortex had formed.

Diagrammatic representation of embryonic circuit development, showing the new, early embryonic circuit, compared to the later formation of layer 5. [Institute of Molecular and Clinical Ophthalmology Basel (IOB)]

This indicated that the neurons were already connected prior to their migration to form layer 5. The transient circuit initially had two layers: a deep layer and a superficial layer. Later, the superficial layer became silent and vanished, while the classical layer-by-layer cortical development resumed, with a third intermediate layer forming layer 5.

“We also wanted to understand how this circuit changes in an autism model,” noted co-lead author Martin Munz, PhD, an IOB developmental biologist in the Central Visual Circuits Group. Working with knock-out mouse lines missing one or both alleles of two autism-associated genes, Chd8 and Grin2b, the team made a key finding. The absence of these genes is known to cause significant autism in children. And in both homozygous and heterozygous knockout mice, the superficial layer remained active as a developmental remnant. “Throughout embryonic development, it never disappeared,” Munz says. Moreover, the knockout mouse brains contained patchy areas of cortical disorganization similar to those seen in people with autism.

“… First, we found that these genetic manipulations interfere with the transition phase of the biphasic PN activity pattern during embryonic development,” the team noted. “… Second, we found that the genetic manipulations resulted in the perturbation of the multi-layered transient circuits … the patchy disorganization of mouse cortex that we report is reminiscent of the patchy disorganization of cortical tissue observed in children with autism.”

The findings suggest that the spatial organization of pyramidal neurons is regulated by the newly-found circuit, and that “changes to embryonic circuits play a role in dysfunctions associated with neurodevelopmental disorders, including autism spectrum disorder,” Bharioke said.

Noting limitations of their research, the authors concluded, “Our work identifying embryonic PN-to-PN active circuit motifs, together with the in vivo imaging and recording methods that we developed, provide an opportunity to study the effects of genes associated with neurodevelopmental disorders, and in particular with autism spectrum disorder, on identified circuits in the living embryo.”

In future research, IOB researchers will “carefully look at the superficial and deep layers of this early circuitry and independently manipulate them,” Roska commented. “This will be instructive for learning about the etiology of neurodevelopmental diseases.”

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