Danger Ahead: Threat Signals Converge to Drive Pain, Hunger

Survival is the prime directive of every animal, and it takes work. Animals are under constant threat from starvation, thirst, extreme temperatures, and life-threatening injury and illness. But when multiple threat signals compete for attention, organisms must prioritize the most urgent among them and adjust behaviors accordingly.

 

Two recent papers tackle the encoding of pain, hunger, and other survival threats in the nervous system, with each study identifying neural signaling at the parabrachial nucleus (PBN). This is a key hindbrain structure where sensory signals are integrated with information from higher brain centers to assess threats and prioritize behaviors.

 

The first study, from Nicholas Betley and colleagues, University of Pennsylvania, Philadelphia, US, identifies a specific subpopulation of hypothalamic neurons that project to the PBN to dampen inflammatory—but not acute—pain during extreme hunger. The study was published March 22 in Cell.

 

Perry Fuchs, University of Texas at Arlington, US, who was not involved in the study, called it “a very important paper with broad implications for the field, beyond this circuit.”

 

The second study, from Richard Palmiter and colleagues, University of Washington, Seattle, US, identifies a population of neurons in the PBN that serve as a detector of multiple threats. The neurons responded to acute pain, itch, visceral pain, and even non-noxious stimuli that pose a potential threat, such as a novel food. The study appeared March 29 in Nature.

 

Reza Sharif-Naeini, who was not involved in either study, wrote in a comment for PRF, “The [Nature] study by [first author] Carlos Campos and colleagues follows a long series of outstanding research from the Palmiter group in their attempts to understand the physiological roles of subsets of neurons in the parabrachial nucleus.” (See full comment and accompanying figure from Sharif-Naeini below).

 

A threat hierarchy

Betley’s group has been studying neurons that control feeding behavior for years, but they wanted to study how hunger interacted with other stressors. “We wanted to see what other behaviors are changed with hunger, with either negative or positive consequences,” Betley told PRF. “We reasoned that individuals must prioritize the most acute threat to survival and behave accordingly,” Betley said.

 

So, the researchers paired two competing threats: pain and extreme hunger. Mice were deprived of food for 24 hours and then injected with formalin in the paw, a model of inflammatory pain with acute and persistent phases. Food-deprived mice displayed less paw licking in the 15 to 45 minutes following injection, whereas acute behaviors immediately after injection were similar to fed mice. The hungry mice’s responses to noxious mechanical and thermal stimuli mimicked those of fed mice, demonstrating that hunger attenuated persistent inflammatory pain but not acute pain. In addition, injection of complete Freund’s adjuvant (CFA), another model of inflammatory pain, produced hypersensitivity to thermal and mechanical stimuli in fed mice but not in food-restricted mice.

 

Hunger also reduced the affective component of inflammatory pain: Whereas fed mice exhibited conditioned place avoidance of formalin-paired cues, food-deprived mice did not. The hungry mice also displayed less formalin-induced immobility than fed mice.

 

“We didn’t expect that animals would have different responses to [acute and chronic] pain, but seeing this response specifically to longer-term pain—it made a lot of sense ethologically,” Betley said. This is because acute pain poses a high-priority threat, but an animal with persistent pain must forage and eat to avoid starvation.

 

Historically, researchers have looked at pain as a single entity, said Fuchs. “What’s exciting about this paper is that we are now looking at pain in a more holistic setting, as a homeostatic function. Pain is a threat to homeostasis just like hunger, so they probably activate very similar mechanisms. But how these systems interact with each other to engage goal-directed behaviors we really do not know. This study now provides an opening to understand these circuits.”

 

Hypothalamic neurons that link pain and hunger

To understand how hunger might dampen persistent inflammatory pain, Betley and colleagues turned to a population of hypothalamic neurons that express agouti-related peptide (AgRP), whose activation causes sated animals to eat, whereas inhibiting the neurons reduces eating in hungry mice.

 

“We started with the AgRP neurons because there is 20 years of work showing that their activity increases when animals are hungry, and that they are necessary and sufficient to regulate feeding. So they were the most likely suspects as an entry point to aspects of hunger that relate to pain,” Betley said.

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So first author Amber Alhadeff used optogenetics to investigate the AgRP neurons. When she photostimulated normally fed mice expressing channelrhodopsin-2 (ChR2) specifically in AgRP neurons, pain behaviors were dramatically reduced during the inflammatory phase after formalin injection, as was hypersensitivity after CFA injection, compared to mice without ChR2. Acute pain behaviors did not differ from controls. Photostimulation of ChR2-expressing AgRP neurons in mice during ongoing inflammatory pain led to reduced paw licking within minutes, suggesting AgRP neuron activation rapidly directed behavior.

 

Conversely, inhibiting AgRP neurons by using designer receptors exclusively activated by designer drugs (DREADD) technology in food-restricted mice reduced hunger’s analgesic effect. Together, the results indicated that AgRP neurons were necessary and sufficient to inhibit inflammatory pain.

 

PBN is the sole target for analgesia

The researchers then wanted to identify the downstream target of AgRP neurons that produced analgesia. In a previous study, Betley and colleagues (Betley et al., 2013) had shown that the axons of AgRP neurons project to their downstream targets with one-to-one architecture, with each neuron projecting to only one brain region.

 

In the current study, Alhadeff painstakingly photostimulated individual subpopulations of ChR2-expressing AgRP neurons that projected to the bed nucleus of the striatum (BNST), paraventricular thalamic nucleus (PVT), paraventricular hypothalamic nucleus (PVH), or the lateral hypothalamus (LH). Doing so evoked feeding but did not inhibit pain behaviors. Stimulation of AgRP neurons that projected to the periaqueductal gray (PAG) or the central nucleus of the amygdala (CeA) had no effect on feeding or pain behaviors. But activation of the 300 or so AgRP neurons projecting solely to the lateral PBN abolished animals’ pain responses to persistent inflammatory pain after formalin injection while leaving acute pain responses intact.

 

“They looked at all these targets to see which produced the analgesic effect. That’s a lot of work,” Sharif-Naeini told PRF. “This is pretty convincing evidence that the PBN is critical: They essentially shut off all the pain behavior when they activated terminals in the PBN as opposed to any other area.”

 

There was no reason to expect that one specific subpopulation alone would inhibit pain, Betley told PRF. “When we saw that just the subset projecting to the PBN regulated pain—that was exciting because it showed that the convergence of inflammatory pain and hunger is occurring at the PBN.”

 

The targets downstream from the PBN that inhibit inflammatory pain remain to be seen. Fuchs wonders whether the circuit might engage the hypothalamic-pituitary-adrenal (HPA) axis. “How much of the stress system is involved? We know about stress-induced analgesia. So teasing apart the different contributions will be important,” he told PRF.

 

In addition to AgRP, the neurons also express the neurotransmitters gamma-aminobutyric acid (GABA) and neuropeptide Y (NPY). To determine which was responsible for dampening inflammatory pain, fed mice received PBN microinjections of GABA, AgRP, or NPY a few minutes before formalin injection. NPY provided protection against inflammatory pain, whereas GABA and AgRP had no effect on pain behaviors. Delivering an antagonist of NPY Y1 receptors to the lateral PBN eliminated the analgesic effect of hunger in food-deprived mice as well as the analgesic effect of optogenetic stimulation of ChR2-expressing AgRP neurons in fed mice. This indicated that NPY signaling by the AgRP neurons at Y1 receptors links hunger and inflammatory analgesia.

 

Finally, the researchers showed that in contrast to persistent inflammatory pain, acute thermal pain (from a hotplate) decreased animals’ feeding behaviors—suggesting a rank-order hierarchy of threats headed up by acute pain. In vivo calcium signaling, used as a measure of neuronal activity, showed that the activity of AgRP neurons decreased in response to food presentation, as expected, but it also decreased in response to acute thermal pain. The results indicate that AgRP neurons are bidirectionally controlled by hunger and pain signals to direct behavior.

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Fasting is not a feasible way to curb pain in people, though, Betley said. After 24 hours without food, “a mouse loses 8 to 10 percent of its body weight, so that’s like five to six days without food for us. That’s massive hunger.” Instead, he said, once the endogenous pathway controlling hunger-induced analgesia is more fully characterized, it might be targeted independently of hunger, perhaps directly at the PBN or at a downstream target.

 

Most exciting, Betley said, “was the fact that we were able to dissociate responses to acute pain from chronic, inflammatory pain. And we demonstrated that the brain has the ability to [selectively inhibit inflammatory pain] without any drugs, so it’s up to our ingenuity to figure out how that happens and recapitulate it.”

 

The brain’s danger detector

The second study, led by Palmiter, focused on neurons in the lateral PBN that express calcitonin gene-related peptide (CGRP), which the group had previously shown are important drivers of appetite control. Several years ago, the group performed experiments in which they ablated hypothalamic AgRP neurons, which caused mice to starve (Luquet et al., 2005Carter et al., 2013).

 

“We discovered that AgRP neurons normally inhibited CGRP-expressing PBN neurons, but in their absence, PBN neurons became hyperactive and drove the starvation phenotype,” Palmiter said. “We knew that, when active, PBN neurons inhibited feeding behavior, but they were also activated by footshock. That led us to consider their role beyond feeding.”

 

So, Palmiter said, “the question became, is this one homogenous population of neurons, or is it a collection of different neurons that all happen to express CGRP, with some responding to different specific stimuli?”

 

To find out, first author Carlos Campos injected mice in the lateral PBN with a viral vector enabling expression of a fluorescent calcium indicator dye specifically in CGRP neurons. This allowed him to visually record activity in individual neurons. All CGRP-expressing neurons in the PBN from which he recorded showed transient calcium elevations in response to repeated tail pinches, but also to paw pinches, a warm metal rod to the lip, and injection of lipopolysaccharide (LPS), which causes visceral pain.

 

These findings indicated that the neurons responded to various noxious stimuli regardless of the bodily site of stimulation. The neurons also encoded stimulus intensity; they responded with increasing calcium activity to more intense electric shocks to the tail and hotter noxious thermal stimuli.

 

CGRP-expressing neurons in the PBN responded to non-nociceptive threats as well, including subcutaneous injection of chloroquine, which causes itch. Campos then manipulated these neurons so that they could no longer release neurotransmitters. Mice with silenced CGRP-expressing PBN neurons exhibited less scratching behavior than controls in response to chloroquine and fewer swipes attempting to remove an adhesive sticker from the neck, another itch-related behavior.

 

“These neurons don’t care about the modality of a stimulus,” said Greg Dussor, University of Texas at Dallas, US, who was not involved in the study. “They simply signal a potentially dangerous situation.”

 

The researchers next restricted food overnight and recorded neuronal activity upon food presentation. When animals are in a hungry state, CGRP-expressing PBN neurons are already inhibited by AgRP inputs, but calcium signals fell further upon mice visualizing the chow.

 

“We noticed that their activity changed depending on cues. They were inhibited during feeding—an appetitive signal—demonstrating bidirectional control of these neurons by sensory stimuli,” Campos said. “They seem to be activated by negative valence signals and inhibited by something positive happening.” As feeding progressed, the neurons became more active, and eating declined, demonstrating their role as satiety sensors to prevent overeating.

 

The CGRP-expressing PBN neurons act as a general danger sensor without revealing the nature of the threat, which Palmiter likened to a home alarm. “The alarm goes off while you’re away, and you don’t know if it’s a broken window, an intruder, or a fire—you just know that something bad has happened.”

 

The actual and the possible

Interestingly, even the possibility of a threat activated these neurons. Although the cells were inhibited by the positive cue of familiar chow pellets, presentation of a novel, high-fat pellet enhanced calcium activity during the first exposure to the pellets.

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“Even a palatable novel food—something that ultimately might become your favorite food—is scary at first,” Palmiter told PRF. “Novelty sends a warning: Taste it gingerly; don’t poison yourself.”

 

With subsequent exposures to the tasty food, however, the CGRP-expressing PBN neurons ceased to activate, instead becoming inhibited just as during normal feeding. Presentation of a novel inedible object—a marble—also initially triggered neuronal activity, which ebbed away with continued exposure. The marble, however, did not lead to neuronal inhibition like food did. “So these neurons get activated not only by real scary things, but also by things that are potentially scary,” Palmiter said.

 

The ability of CGRP-expressing PBN neurons to adapt based on repeated exposure to the novel cues showed they were sensitive to positive learning, so the researchers tested whether they were also sensitive to negative associations. Mice were conditioned to create a fear memory in a paradigm pairing a footshock with a neutral tone. The next day, the tone produced freezing—a fear behavior—along with activation of the CGRP-expressing PBN neurons, as did being placed in the shock chamber. These findings indicated the cells respond not only to a primary noxious experience but also to recall of the event. In mice with silenced CGRP-expressing PBN neurons, freezing behavior in response to the conditioned tone was rapidly extinguished.

 

Food for thought

Dussor described the PBN’s emerging role in pain. “The somatosensory pain pathway tells us, Where is the stimulus coming from, and what kind of pain is it? But parallel to that pathway, activity in the PBN and the amygdala seems to encode the emotional response to pain.

 

“It’s interesting to think about what’s actually activating these CGRP-expressing neurons, what’s upstream,” Dussor said. “Sensory inputs from the spinal cord feed into the circuit, but the fact that you can activate them with just a potentially threatening stimulus—upstream circuits from the cortex have to process the fact that the animal has never seen this before.”

 

Dussor also wondered how the CGRP-expressing PBN neurons might respond to chronic pain.

 

“If you looked at these neurons, say, a month after nerve injury, are they continuing to drive the emotional states in chronic pain? And if you silenced them to get rid of the emotional response to pain, does that take away your protective fear response to danger? You would need to selectively turn off their response to pain but not to other stimuli, and this paper suggests you can’t. Therapeutically, it’s challenging to think about. From a neuroscience perspective, it’s a very interesting observation.”

 

A key question remains: Are the PBN neurons described by the two studies the same or different populations? Palmiter told PRF that the CGRP neurons may well be involved, but they probably are not the primary targets of the AgRP neurons that mediate the analgesic effect.

 

“Our initial explorations suggest that it will likely be different neuron populations,” Betley told PRF, but they have not yet investigated the question specifically.

 

One way to find out, Sharif-Naeini said, would be to look in PBN neurons for CGRP and the Y1 NPY receptor to see if they are expressed in overlapping or distinct sets of cells.

 

Sharif-Naeini also told PRF that new technology such as optogenetics “has opened the door to map these circuits linking supraspinal processing centers—this is just the beginning. It’s an exciting time to be in neuroscience.”

 

Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.

 

Image credit: wetzkaz/123RF Stock Photo

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