Everyone has experienced a change in their pace and depth of breathing in an anxious or painful situation. Yet, the neural circuitry between this tight physiological coordination was not clear. A team of scientists from the Salk Institute in La Jolla, California, has now uncovered the neural network that coordinates breathing rhythm with emotions.

Deciphering this neural circuitry can contribute to pain management, insights on psychological theories of anxiety, investigations into the nature of pain, and the development of analgesics that would prevent opioid-induced respiratory depression (OIRD).

Opioids are widely prescribed to treat both acute and chronic pain. This has led to an opioid crisis. Death due to opioid abuse can be caused by OIRD, a condition that represses breathing as well as pain.

The new findings are reported in an article published in the journal Neuron on December 17, titled “Divergent brainstem opioidergic pathways that coordinate breathing with pain and emotions.” This work was funded by the National Institute of Mental Health, the Brain Research Foundation, the Mary K. Chapman Foundation, the Jesse & Caryl Philips Foundation, the National Institutes of Health-National Cancer Institute and the Waitt Foundation.

Sung Han, PhD, (left) is senior author of the paper and Shijia Liu, (right) a graduate student in Han’s lab is first author of the paper. [Salk Institute]
The researchers focused on a group of neurons in the brainstem called the lateral parabrachial nucleus (PBL). They found, neurons at the core of the PBL project to the amygdala—a region of the brain important in emotional processing such as fear and pain, and neurons from the outer shell of the PBL project to a region in the medulla called the pre-Bötzinger complex that generates breathing patterns. The authors show, neurons in the core and shell of the PBL influence each other according to inputs from the amygdala and the pre-Bötzinger complex, to make our breaths quick and shallow when we experience pain or anxiety.

“We are the first group to demonstrate how the lateral parabrachial nucleus coordinates breathing and pain,” says Sung Han, PhD, assistant professor in Salk’s Clayton Foundation Laboratories for Peptide Biology and senior author of the paper. “By understanding the circuits in this brain region, we may be able to tease apart breathing regulation and pain regulation to develop a medication that inhibits feelings of pain without repressing breathing, like OIRD.”

“This appears to be a very well-done study, which is consistent with a number of other recent papers that focus on the lateral parabrachial nucleus as containing neurons that make the mu opioid receptor and which cause arousal responses as well as increased breathing,” says Clifford Saper MD, PhD, professor at the Department of Neurology at Harvard University, who was not involved in this study. “This work dovetails nicely with the other papers by Kevin Yackle and Elle Levitt that indicate that the inhibition of this circuitry by opiate drugs probably plays a major role in the epidemic of opioid-induced respiratory depression.”

Earlier work from Han’s team had uncovered that morphine and other opiates inhibit neurons that synthesize a receptor protein called mu opioid receptor (MOR). This inhibition in turn represses breathing rhythms.  They also showed that reactivating the cells that express MOR can reverse OIRD.

The current study suggests other strategies for preventing OIRD, possibly by inhibiting neurons at the core of the PBL to blunt amygdala-induced effects, or exciting neurons in the outer shell of the PBL to support breathing rhythms.

“We have identified two anatomically distinct neuronal populations expressing mu-opioid receptors in the core and shell of the lateral parabrachial nucleus. Core neurons regulate pain by projecting to the central amygdala, one of the pain controlling centers in the brain, and shell neurons regulate breathing by projecting to the medullary breathing centers,” says Han. “These results suggest that opioids’ analgesic effect, and respiratory depression may be mediated by different group of neurons in the PBL, which may make inroad in developing safer analgesics, or cures for opioid overdose.”

Shell neurons (green) project to the breathing center and core neurons (red) project to the pain/emotion center from the PBL. [Salk Institute]
In this study, the researchers used light and chemical agents to prove that turning MOR-expressing neurons on or off in the lateral parabrachial nucleus changes breathing rate in mice. Using fluorescent tracers to map the inputs and outputs of the MOR-expressing neurons of the PBL, the authors show neurons clustered in the core of the region project to the central amygdala, while neurons clustered in the surrounding shell project to the pre-Bötzinger complex.

The researchers then stimulated one population of neurons while recording electrical signals from the other to show subpopulations of the core and shell neurons are reciprocally connected with an excitatory network between them. This excitatory network coordinates electrical signals of fear and pain with breathing rhythms.

“We simultaneously recorded breathing rhythm (using thermistor implanted in a nasal cavity that detects temperature difference between inhaled/exhaled air in the nose) and neural activity (using fiber photometry calcium imaging) in awake mice. That enabled us to test the role of PBL opioid receptor neurons in regulating breathing rhythm,” says Han.

Shijia Liu, a graduate student in Han’s lab and first author of the paper, says, “We have found very intricate circuits involving upstream and downstream input to these neurons. By uncovering this circuit mechanism, we can better explain why breathing can often be coordinated with pain and anxiety.”

Han says, “The biggest problem these days is that opioids reduce pain but also reduce breathing. So, people die. By understanding those two mechanisms in our research, maybe we can manipulate certain populations of neurons by pharmacological intervention so that we can control pain without changing the breathing.”

Han’s team is working on genetic analyses of neurons in the core and shell of the PBL. “We plan to identify functional markers exclusively expressed in core or shell populations to find therapeutic targets that selectively alleviate pain with minimal effects on breathing,” says Han.

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