Science Spotlight

Connect-seq: A new neuron detective around the block

From the Buck lab, Basic Sciences Division
“Everybody knows Da Vinci's iconic Vitruvian Man, even if they don't know it's called the Vitruvian Man. Less well known is the Vitruvian Mouse, which shows the hidden geometry in our rodent friends. This may help explain the long symbiotic relationship we have had with mice over the ages, as they have taken part in our folklore, genetics, pharmaceuticals, astronautics, and literary symbolism, among other things. In return, we have given them an environment in which to breed like, well, rats.”
“Everybody knows Da Vinci's iconic Vitruvian Man, even if they don't know it's called the Vitruvian Man. Less well known is the Vitruvian Mouse, which shows the hidden geometry in our rodent friends. This may help explain the long symbiotic relationship we have had with mice over the ages, as they have taken part in our folklore, genetics, pharmaceuticals, astronautics, and literary symbolism, among other things. In return, we have given them an environment in which to breed like, well, rats.” David Deen, 2006, with permission.

If you think understanding the mouse brain is an easy business, think twice! The average mouse brain contain 75 million neurons, all interconnected in a vast array of seemingly convoluted circuits. Our understanding of this complex circuitry, particularly when it comes to specific neuronal components of most circuits has been limited. This is largely because with the current toolset, a neuroscientist used to have to chose between mapping, or tracing, neuronal circuits or peering at the signaling molecules at the single cell level with cells ostracized from their circuits. The picture remained incomplete.

Researchers from the Buck lab (Basic Sciences Division), recently developed a new technique to overcome this hurdle. This new technique, called Connect-seq, combines the powerful technology of retrograde viral tracing with state-of-the-art single cell transcriptomics. Using Connect-seq in mice, the researchers were able to probe the molecular identities of individual neurons connected in a circuit. They published their work in a recent issue of the Proceedings of the National Academy of Sciences. The study was led by Drs. Naresh Hanchate and Eun Jeong Lee, postdocs in the Buck lab.

To test their strategy, the authors targeted an engineered virus to corticotropin-releasing hormone neurons (CRHNs) inside the mouse brain. CRHNs are located in the hypothalamus and are involved in physiological responses to fear and stress. The engineered virus is able to travel retrogradely across synapses thereby allowing the research team to trace the incoming neural circuits. The authors then isolated virus-infected neurons and conducted single-cell transcriptomics which allowed them to define the transcriptomes of individual upstream neurons.

The analysis uncovered a diverse array of neurotransmitter and neuromodulator signaling molecules in neurons directly upstream of CRHNs, including more than 40 different neuropeptides. As such, it affords a detailed understanding of the hypothalamic neurons that control physiological responses to stress.

The authors found that several of these signaling molecules were expressed at once within individual neurons, contrary to traditional paradigm of one neuron, one signaling molecule.

The study, provides an unprecedent opportunity to overlay the molecular map of a specific circuit of neurons on an anatomical map, which permits molecular and genetic interrogation of the molecular circuitry underlying neural responses. This level of understanding of how individual neurons function as part of a neuronal circuit may guide the future development of medical interventions that reduce the negative effects of stressful situations, according to the researchers.

Hanchate N, Lee, Ellis A, Kondoh K, Kuang D, Basom R,Trapnell C, Buck LB. 2020. Connect-seq to superimpose molecular on anatomical neural circuit maps. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.1912176117

This work was made possible through funding from the Millen Literary Trust, the Howard Hughes Medical Institute, the National Institutes of Health, the Paul G. Allen Frontiers Foundation, and the Alfred P. Sloan Foundation.