Scientists have introduced a groundbreaking tool named START, which allows for highly precise mapping of the brain’s complex neuronal connections. This innovative technology will enable researchers to pinpoint the connectivity of various neuron types, facilitating the creation of new therapeutics that can specifically target certain neurons and circuits, leading to enhanced effectiveness and reduced side effects.
Researchers at the Salk Institute have launched a new neurotechnology for brain mapping, called Single Transcriptome Assisted Rabies Tracing (START). This advanced tool merges two sophisticated methods—monosynaptic rabies virus tracing and single-cell transcriptomics—to achieve exceptional precision in mapping the intricate connections within the brain.
With this method, scientists have become pioneers in identifying the connectivity patterns of transcriptomic subtypes of inhibitory neurons located in the cerebral cortex. They believe this mapping will pave the way for innovative therapies that can target specific neuron types and circuitry more accurately, resulting in more effective treatments that may also have fewer adverse effects compared to existing pharmacological options.
The findings, which appeared in the September 30, 2024, issue of Neuron, represent the first instance of clarifying cortical connectivity at the granularity of transcriptomic cell types.
“In the realm of treating neurological and neuropsychiatric issues, we’ve often tried to resolve problems without a solid understanding of the components involved,” notes Edward Callaway, the study’s senior author and professor at Salk. “START is enabling us to craft a detailed map of the brain’s various components and their interconnections.”
He likens this to attempting to fix a car without knowing what its engine or axle does. However, having a diagram showing how all the parts work together would make it much easier to identify issues and determine what tools would be needed for repairs.
When understanding the brain’s components, neurons are initially divided into two broad types: excitatory neurons, which trigger brain activity, and inhibitory neurons, which dampen it—similar to how an accelerator and brake function in a vehicle. These can then be identified in more specific subclasses based on their location in the brain and the proteins they produce.
Recent advancements in transcriptomics enable even further classification of these subclasses. By employing single-cell RNA sequencing, scientists can now categorize cells with comparable gene expression patterns, defining each group as a unique neuronal subtype.
“Determining a cell type is complex, as classification can vary based on the methods used for analysis,” Callaway explains. “Two cells may have slightly different gene expression profiles but fulfill similar roles, or conversely, similar expressions may differ based on anatomy, connectivity, or physiology. If we focus on just one of these features, we risk either oversimplifying or overcomplicating our classifications. START helps clarify what categorization is most significant for circuit functions, thereby guiding us on which cells to focus on for new therapeutics.”
To develop START, the Callaway team ingeniously merged single-cell RNA sequencing with a previously developed method: monosynaptic rabies virus tracing. This approach allows a customized virus to move from one targeted cell type to only those cells directly linked to it. By tracking the virus’s location, researchers can delineate which cells are interconnected.
The research team initially applied this novel tool to examine connectivity in the mouse visual cortex. START successfully identified around 50 distinct subtypes of inhibitory neurons in the region and mapped their connections to excitatory neurons across each cortical layer. The results revealed unique connectivity patterns across various transcriptomic subtypes of inhibitory neurons that previous techniques could not distinguish.
“Many researchers tend to view all inhibitory neurons as a single homogenous group, yet they are quite diverse. Studying or targeting them as one entity can obscure crucial differences vital for understanding brain function and disease,” states Maribel Patiño, the lead author and a former graduate student in Callaway’s lab, now a psychiatry resident at UC San Diego School of Medicine.
START demonstrated that each cortical layer of excitatory neurons received selective input from specific subtypes of inhibitory cells: Sst, Pvalb, Vip, and Lamp5. The unique connectivity of each subtype contributes to complex microcircuits that likely underpin specialized brain functions.
For instance, the research identified a subtype called Sst Chodl cells, linked to sleep regulation. Through START, the team discovered that these Chodl cells had the highest density of connections with layer 6 excitatory neurons, which project to the thalamus to help regulate sleep cycles.
This unprecedented level of detail will allow neuroscientists to further understand how specific neuronal subtypes influence the brain’s circuitry to shape our thoughts, perceptions, emotions, and behaviors.
In the future, the researchers plan to develop viral vectors and gene-editing technologies that specifically target individual cell subtypes. These innovations could evolve into new therapeutic strategies that selectively modify neuron populations involved in conditions such as autism, Rett syndrome, and schizophrenia.
“We may not know how this information will be utilized a decade or two from now, but it’s clear that technology is advancing rapidly. The methods by which we treat the brain with drugs will ultimately change,” Callaway predicts. “START could spearhead this evolution, ensuring that the viral techniques and resources remain accessible to the whole neuroscience community.”
Other contributors to this research include Marley A. Rossa, Willian Nuñez Lagos, and Neelakshi S. Patne from the Salk Institute.
This research was funded by the National Institutes of Health (R34 NS116885, T32 GM007198, P30 014195, S10 OD023689) and the Paul and Daisy Soros Fellowship for New Americans.
