Researchers have developed innovative tools to investigate brain messenger proteins known as neuropeptides in the brains of active animals. Their findings suggest that neuropeptides are the main communicators in the fear circuits of murine brains, revealing that multiple neuropeptides collaborate in this process. This discovery helps explain why certain clinical trials focusing on just one neuropeptide have been unsuccessful. The insights gained from these tools and research can inform drug development for fear-related neurological disorders such as PTSD and anxiety, enhance pain management solutions, and shed light on other neural circuits in the brain.
Imagine the moment you accidentally grab the handle of a hot cast iron skillet—pain and apprehension hit you almost immediately. Sensory signals activate pain receptors in your finger, transmitting messages through your spinal cord to the brainstem. Here, specific neurons relay these pain signals to the amygdala, a brain region that regulates your emotional fear response and aids in remembering to steer clear of hot skillets in the future.
This rapid transformation of pain into a memory of threat led researchers to believe that fast-acting molecules called neurotransmitters were responsible for the process. However, a study conducted by Salk researchers revealed that larger, slower-acting molecules called neuropeptides serve as the main messengers in the fear circuit instead.
While neuropeptides are recognized for their role in brain communication, the intricacies remained elusive until the scientists developed the appropriate tools to study them in living animals. To investigate neuropeptides’ functions in this circuit, the team at Salk engineered two novel tools that enable real-time observation and modulation of neuropeptide release in live mice.
Their research, published in Cell on July 22, 2024, demonstrated that neuropeptides, not neurotransmitters, are the primary messengers in this fear circuit, with multiple neuropeptides working together in the process. These discoveries hold potential for creating more effective pain relief medications and new treatments for fear-related conditions such as PTSD and anxiety.
“There is so much more to learn about neuropeptides, but at Salk, we are building upon the legacy of Nobel Prize winner Roger Guillemin to emphasize their significance and further our research,” remarks senior author Sung Han, who serves as an associate professor and Pioneer Fund Development Chair at Salk. “To that end, we’ve developed two genetically encoded tools for monitoring and silencing neuropeptide release from nerve endings. We believe these advancements will greatly enhance neuropeptide research, and our findings regarding their role in fear processing are just the beginning.”
Processing and reacting to environmental stimuli require signals to traverse the body and brain, facilitated by neurons that create structured circuits to direct information flow. Neurons interact by sending and receiving substances like neurotransmitters and neuropeptides.
Traditionally, neuropeptides are seen as neuromodulators, assisting and regulating the functions of primary neurotransmitters. However, past researchers, including Roger Guillemin, proposed that neuropeptides could serve as primary transmitters themselves—a hypothesis that lacked rigorous testing due to the absence of tools for observing and manipulating neuropeptide release in active animals. The Salk team aimed to explore neuropeptides through the development of new tools to deepen understanding of their roles in brain circuits.
To focus specifically on neuropeptides, Han’s group exploited a unique feature: while neurotransmitters are contained in small packages called synaptic vesicles, neuropeptides are found in large dense core vesicles. By engineering specialized biochemical instruments to target these larger vesicles, they created neuropeptide sensors and silencers. The sensor attaches to large dense core vesicles with proteins that emit a glow upon release from the nerve endings, allowing scientists to observe neuropeptide release in real-time. The silencer selectively breaks down neuropeptides in these large vesicles, providing insights into brain activity in the absence of neuropeptides.
“We’ve introduced a groundbreaking method to track the movement and function of neuropeptides in living animals’ brains,” states Dong-Il Kim, the primary author of the study and a postdoctoral researcher in Han’s lab. “These innovations will enhance our comprehension of neuropeptide circuits and enable neuroscientists to investigate previously challenging questions.”
Using their newly developed neuropeptide tools alongside existing instruments for monitoring glutamate (the most prevalent neurotransmitter in the brain), the researchers examined how neuropeptides and glutamate reacted in live mice exposed to a mild stimulus intended to trigger the fear circuit. The results showed that neuropeptides, unlike glutamate, were released during the stimulus, and silencing neuropeptide release diminished fear responses, while glutamate silencing had no observable impact.
Han expressed both surprise and satisfaction that this brainstem fear circuit fundamentally relies on neuropeptides for messaging rather than glutamate. Moreover, their research also reaffirms their investigation of PACAP—a neuropeptide linked to modulating panic disorder.
“These groundbreaking tools and insights represent an important stride towards improved drug development for neurological disorders,” Han explains. “Our findings reveal that multiple neuropeptides are co-packaged within a single vesicle and released collectively in response to painful stimuli. This realization leads us to speculate that the failure of some clinical drugs targeting individual neuropeptides could be due to our previous underestimation of their collective action. Armed with this knowledge, we can now develop new drugs capable of addressing multiple neuropeptide receptors to enhance pain relief and treat fear-related disorders like PTSD more effectively.”
With their new neuropeptide toolkit, the research team plans to delve into other brain circuits and processes. Future research into neuropeptide signaling in varied brain regions, combined with their newfound understanding of the necessity to target multiple neuropeptides together, could drive the development of more effective drugs for various neurological conditions.
