Periodic Reporting for period 4 - TYPEWIRE (Reconstructing wiring rules of in vivo neural networks using simultaneous single-cell connectomics and transcriptomics)
Reporting period: 2024-07-01 to 2025-06-30
The brain performs sophisticated functions and complex behaviours, orchestrated by highly specialized cells. Neurons are the cells at the core of the nervous system’s computational capabilities. In recent years, we and others have advanced single-cell RNA-sequencing to reveal their extraordinary molecular diversity in transcriptome-based cell-type taxonomies. It is the unique combinations of circuits that these different neuronal types form – within a practically unlimited space of possible implementations – that encode the large functional repertoire of the nervous system. However, little is known about the basic organizational principles of cells within the circuits – the ‘wiring rules’: What is the topology of the networks of single cells? What is the relation between network topologies and function? And finally, how do cell types and gene expression determine wiring?
Answering these questions will help understand nervous system computation at the level of its cellular building blocks. But we face a conceptual challenge to measure connectivity on a large scale, with resolution to single cells. The vision of this proposal was to develop and apply new approaches that allow us to investigate neuronal connectivity. Our work measured synaptic input connections, for thousands of single neurons, in the mouse cortex. We found that each neuron is highly connected within its immediate neighborhood, but also receives parallel inputs from multiple regions spanning the full brain. Our continued efforts have the potential to systematically address basic functional questions in neuroscience. They can expand our understanding of neuronal circuits in health and disease, to an unprecedented resolution and scale.
Answering these questions will help understand nervous system computation at the level of its cellular building blocks. But we face a conceptual challenge to measure connectivity on a large scale, with resolution to single cells. The vision of this proposal was to develop and apply new approaches that allow us to investigate neuronal connectivity. Our work measured synaptic input connections, for thousands of single neurons, in the mouse cortex. We found that each neuron is highly connected within its immediate neighborhood, but also receives parallel inputs from multiple regions spanning the full brain. Our continued efforts have the potential to systematically address basic functional questions in neuroscience. They can expand our understanding of neuronal circuits in health and disease, to an unprecedented resolution and scale.
We have established several of the challenging techniques required to measure neuronal connections both by fluorescence microscopy, and by RNA-sequencing. Based on our initial, sometimes surprising results, we were also inspired to develop other, parallel approaches that have already helped address some quantitative aspects of neuronal connectivity. We targeted regions in the brain concerned with somatosensation, or spatial navigation, learning and memory, or social behaviors. We have learned of some differences between wiring in male and female mice, and begun to explore how wiring architectures differ by brain region. Our work revealed that each single neuron receives not only countless local inputs, but parallel inputs from many regions across the brain. Finally, studying hundreds of neuronal networks per experiment has also revealed how each network appears to be dominated by inputs of just one region, which remains to be explored in more detail.
We developed complex and uniform libraries of genetic barcodes to faithfully distinguish input networks of hundreds of single cells in a single experiment, using RNA-sequencing. This has allowed us to begin to build a type of network statistic for different brain regions in the mouse brain, and can be further applied to describe subtle differences in neuronal wiring between individuals, in health and disease.