A Connectomic Analysis of a Developing Brain Undergoing Neurogenesis

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PROJECT SUMMARY / ABSTRACT Over the course of the development and into adulthood, the human brain builds neural circuits composed of thousands of types of neurons. As new neurons are born, they are incorporated into developing and existing circuits making connections to neurons that are nearby as well as neurons that are in distant parts of the brain. Many neurological conditions are related to the improper growth of networks in the brain. Yet, we lack a basic understanding of how neural circuits change as new neurons join. To address this question, this proposal uses a novel animal model, the mollusc, Berghia stephanieae, in which it is possible to construct a cellular- and synaptic-level wiring diagram of the entire brain at several juvenile stages as well as the adult. Using these whole brain connectomes, the project will track the changes in specific neurons, in neural circuits, and in whole brain networks as the number of neurons in the brain increases by over 40-fold. Neurons will be identified by intersectional labeling of gene expression using sets of up to five in situ hybridization chain reaction probes that label different mRNA sequences. Overlapping sets of probes will used so that individually identifiable neurons and neuron types can be distinguished based on their patterns of gene expression combined with their soma location and size. Additionally, in adult animals, neurons will be labeled using fluorescent tracers applied to nerves emanating from the brain. Machine learning (ML) will be employed to classify neuronal types based on all of these features. ML classifications of neurons across developmental stages will be corrected by humans to enhance the predictive power of the ML. A series of connectomes of the brains of an adult and juveniles from four stages will be constructed. The brain will be serially sectioned. Each of the 30 nm thick sections will be imaged using a 61 beam scanning electron microscope. The sections will be aligned and all neurons will be automatically reconstructed in 3D. The reconstructions will include all axons, dendrites, and synapses. Again, humans will proofread the results to correct the ML algorithm. The result will be five complete brain connectomes spanning from the early juvenile with 500 neurons to the mature adult with over 23,000 neurons. The developmental series will be analyzed to test hypotheses about the organization and development of neurons, neural circuits, and entire brain networks. Changes in neural structure of identified neurons will be tracked over development. Comparisons will be made between neural types as new neurons are added. Complete neural circuitry for visual, olfactory, and motor systems will be determined. Finally, the project will determine whether hubs develop around the oldest neurons or whether the network scales without concentrating connectivity at particular hubs. The results will provide an unprecedented look at how the synaptic networks of neurons across an entire brain change as new neurons are added.