New brain-mapping tool may be the 'START' of next-generation therapeutics

Using the technique, the researchers became the first to identify the patterns of connectivity made by transcriptomic subtypes of inhibitory neurons in the cerebral cortex. They say having this ability to map the connectivity of neuronal subtypes will drive the development of novel therapeutics that can target certain neurons and circuits with greater specificity. Such treatments could be more effective and produce fewer side effects than current pharmacological approaches.

The study, published on September 30, 2024, in Neuron, is the first to resolve cortical connectivity at the resolution of transcriptomic cell types.

"When it comes to treating neurological and neuropsychiatric disorders, we've essentially been trying to fix a machine without fully understanding its parts," says senior author Edward Callaway, professor and Vincent J. Coates Chair in Molecular Neurobiology at Salk. "START is helping us create a detailed blueprint of the brain's many parts and how they all connect."

It's like trying to repair a car without knowing what an engine or an axle is, he says. But if you had a diagram of the car's parts, you could start to understand how they might work together to make the wheels spin and the car move. That knowledge would then make it much easier to spot a problem in the system and figure out which tools you'll need to fix it.

When describing a brain's parts, neurons are initially grouped into two broad classes: excitatory (those that stimulate brain activity) and inhibitory (those that suppress activity) -- similar to the accelerator and brake in a car. From there they can be further sorted into subclasses: Excitatory neurons are categorized by the layer of the brain they're in while inhibitory neurons are identified by the marker proteins they express.

Recent advances in transcriptomics now allow these subclasses to be broken down even further. Using single-cell RNA sequencing, scientists can now group cells with similar gene expression patterns and define each cluster as a specific neuronal subtype.

"Defining a cell type is complicated because you might group cells differently depending on which method you're using to look at them," Callaway says. "Two cells can have slightly different gene expression patterns but perform a similar function, or two cells with similar gene expression could be further separated based on their anatomy, connectivity, or physiology. If you only consider one of those features, you could end up over-splitting or under-splitting the groups. START helps us understand what level of categorization may be most meaningful to circuit function, and that will inform which cells to target with new therapeutics."

To create START, the Callaway lab engineered a way to combine single-cell RNA sequencing with another technique they had developed previously: monosynaptic rabies virus tracing. The approach lets a modified virus hop from one cell type of interest to only the cells directly connected to it. By detecting where the virus ends up, the researchers can map which cells are connected to which.

The researchers first used their new tool to explore connectivity patterns in the mouse visual cortex. START was able to resolve around 50 different subtypes of inhibitory neurons in this region and map their connections to excitatory neurons in each layer of the cortex. The researchers' findings identified distinct connectivity patterns across various transcriptomic subtypes of inhibitory neurons that could not have been distinguished using previous methods.

"People often treat all inhibitory neurons as a single uniform group, but they're actually very diverse, and trying to study or clinically target them as one group can obscure important differences that are critical to brain function and disease," says first author Maribel Patiño, a former graduate student in Callaway's lab and current psychiatry resident at UC San Diego School of Medicine.

START revealed that each cortical layer of excitatory neurons received selective input from specific transcriptomic subtypes of Sst, Pvalb, Vip, and Lamp5 inhibitory cells. Each subtype's unique connectivity helps establish sophisticated microcircuits that likely contribute to specialized brain functions.

For example, the researchers were able to resolve an inhibitory subtype called Sst Chodl cells, which are thought to be associated with sleep regulation. Using START, they found that Chodl cells were the cell type most densely connected to layer 6 excitatory neurons, which are known to project to the thalamus to coordinate sleep rhythms.

This unprecedented resolution will allow neuroscientists to continue uncovering how specific neuronal subtypes shape the brain's circuitry to produce our thoughts, perceptions, emotions, and behaviors.

The researchers' next steps are to create viral vectors and gene-editing technologies that target each individual cell subtype. In the future, these tools could be adapted into novel therapeutics that selectively modify the specific neuron populations contributing to conditions such as autism, Rett syndrome, and schizophrenia.

"We don't know exactly how this information is going to be used 10 or 20 years from now, but what we do know is that technologies are changing rapidly, and the way the brain is treated today with drugs is not the way the brain will be treated in the future," says Callaway. "START can help drive this innovation, so the viruses and resources are all freely available for the entire neuroscience community to use."

Other authors include Marley A. Rossa, Willian Nuñez Lagos, and Neelakshi S. Patne of the Salk Institute.

The work was supported by the National Institutes of Health (R34 NS116885, T32 GM007198, P30 014195, S10 OD023689) and the Paul and Daisy Soros Fellowship for New Americans.