Serotonin is one of the primary chemicals that the brain uses to influence mood and behavior, and is a key target for psychiatric drugs. But to aid in the design of improved drugs scientists need to know more about how serotonin affects brain cells and circuits in health and disease. Working in the simple model organism Caenorhabditis elegans, researchers at The Picower Institute for Learning and Memory at MIT used genetic analysis and whole-brain imaging techniques to generate a comprehensive account of how serotonin affects behavior, from the level of individual serotonin receptors, all the way to the animal’s whole brain.

The team, headed by senior author Steve Flavell, PhD, associate professor in The Picower Institute and MIT’s Department of Brain and Cognitive Sciences, described the findings in a paper in Cell, titled “Dissecting the functional organization of the C. elegans serotonergic system at whole-brain scale.” The study’s co-lead authors are Picower Institute postdoc Ugur Dag, PhD, MIT Brain and Cognitive Sciences graduate student Di Kang, and former research technician Ijeoma Nwabudike, who is now a MD-PhD student at Yale.

Serotonin signaling is involved in many aspects of behavior and cognition, and dysfunction of the serotonergic system is implicated in psychiatric disease, the authors noted. The serotonergic system is, consequently, the most common target of psychiatric drugs, and many psychotropic chemicals like psilocybin and LSD act on serotonin receptors. However,  as Flavell pointed out, “There have been major challenges in rationally developing psychiatric drugs that target the serotonergic system. The system is wildly complex. There are many different types of serotonergic neurons with widespread projections throughout the brain and serotonin acts through many different receptors, which are often activated in concert to change the way that neural circuits work.”

As the authors further stated, “The neurons that release serotonin are functionally diverse, project broadly throughout the brain, and exert their effects via 14 different receptors. Developing an integrative framework for serotonergic function that relates anatomy, receptors, circuits, and behavior across a whole nervous system would greatly aid our understanding of this important neuromodulatory system.”

The same complexities that scientists face when considering the serotonergic system in people are also relevant to the nematode worm C. elegans, but to a more manageably limited degree in this simple model organism. C. elegans has only 302 neurons and only six serotonin receptors. Moreover, all C. elegans neurons and their connections have been mapped out and its cells are accessible for genetic manipulation.

Flavell’s team has in addition developed imaging technologies that make it possible to track and map neural activity across the worm’s brain simultaneously. With this expertise in place the lab carried out a novel study revealing how the far-reaching molecular activity of serotonin changes brain-wide activity and behavior in the C. elegans model.

A study reported by Flavell in 2013 found that C. elegans uses serotonin to slow down when it reaches a patch of food, and the team traced its source to a neuron called NSM. In the new study, the team used more recently developed technologies to examine serotonin’s effects more comprehensively.

A wiring diagram of the C. elegans worm shows neurons and muscle cells (dots) that express receptors for serotonin. Each color denotes a specific receptor. Some neurons express more than one. The diagram appears as a figure in the research paper. [Di Kang/MIT Picower Institute]

First, the investigators focused on identifying the functional roles of the worm’s six serotonin receptors. To do that they created 64 different mutant strains covering the different combinations of knocking out the various receptors. For instance, one strain would have just one receptor knocked out while another strain would have all but that one missing and another would be missing three. In each of these worms the team stimulated serotonin release from the NSM neuron to prompt the characteristic slowing behaviors.

Analyzing their results indicated that three of the serotonin receptors—MOD-1, SER-4, and LGC-50—primarily drove the slowing behavior, and that the other three receptors— SER-1, SER-5 and SER-7—interacted with the receptors that drive slowing, and modulated how they functioned. Flavell suggested that these complex interactions between serotonin receptors in the control of behavior is likely to be directly relevant to psychiatric drugs that target these receptors, Flavell said.

The investigators also gained other important insights into serotonin’s actions. One was that different receptors respond to different patterns of serotonin release in live animals. For example, the SER-4 receptor only responded to sudden increases in serotonin release by the NSM neuron. In contrast, the MOD-1 receptor responded to continuous “tonic” changes in serotonin release by NSM. This suggested that different serotonin receptors are engaged at different times. “Understanding how a given serotonin receptor contributes to a behavioral output and how it interacts with the others will be critical to eventually target serotonin receptors in a rational way for therapeutics,” the team stated. “In mammals, different serotonin receptor types have been suggested to influence different aspects of behavior and cognition, and complex interactions likely shape these functional roles.”

C. elegans neurons
A 3D rendering of a C. elegans worm, mapping all of its neurons. [Steve Flavell/MIT Picower Institute]

Having teased out the roles of the serotonin receptors in the control of C. elegans behavior, the research team then used their imaging technologies to see how serotonin’s effects worked at a circuit level. For instance, they fluorescently tagged each receptor gene in each neuron across the brain so that they could see all the specific cells that expressed each receptor, providing a brain-wide map of where the serotonin receptors are located in C. elegans. They found that about half of the worm’s neurons express serotonin receptors, with some neurons expressing as many as five different types.

The team in addition used their ability to track all neuronal activity (based on calcium fluctuations) and all behaviors to investigate how the serotonergic neuron NSM affected other cells’ activity as the worms freely explored their surroundings. “Brain-wide calcium imaging in freely moving animals revealed how serotonin release is associated with changes in activity across the defined cell types of the animal’s brain,” the scientists stated. The results suggested that about half of the neurons across the worm’s brain changed activity when serotonin was released. Since the scientists knew which exact neurons they were recording from, they asked whether knowing which serotonin receptors each cell expressed could predict how they responded to serotonin. They found that the knowledge of which receptors were expressed in each neuron and its input neurons gave strong predictive power of how each neuron was impacted by serotonin.

All these findings shed light on the kinds of complexities and opportunities facing drug developers, Flavell noted. The study’s findings show how the effects of targeting one serotonin receptor could depend on how other receptors or the cell types that express them are functioning. In particular, the study highlights how the serotonin receptors act in concert to change the activity states of neural circuits.

“Here, we provide a global characterization of the serotonergic system that links these scales of analysis.,” the team noted. “Serotonin release by NSM alters locomotion in a manner that depends on all six serotonin Receptors … These results provide a global view of how serotonin acts on specific receptors at defined sites in a connectome to modulate brain-wide activity and behavior.”

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