Stress affects the physiological functions and behaviors of individuals, yielding both positive and negative outcomes. On the positive side, stressors trigger the fight and flight response that is crucial to the performance and survival under threats. On the negative side, impaired adaptation and resistance to stressful events can precipitate the onset or relapse of symptoms in a range of psychiatric and behavioral disorders, including anxiety, depression, hyperphagia or hypophagia, and even posttraumatic stress disorder (PTSD). A balance between two opposing stress and anti-stress mechanisms determines the overall impact of stress. The former renders organisms more sensitive and vulnerable to stress-related disorders, while the later imparts resilience and resistance. Thus, identifying mechanisms that make individuals resistant or less vulnerable to stressful stimuli is an important approach for the prevention of stress-related disorders. Recent studies showed that many brain areas (such as the amygdala, hypothalamus and hippocampus) are implicated in initiating or mediating stress-induced changes in emotion and behavior. However, relatively little is known about the neuronal mechanisms of anti-stress, especially at the neural circuit level.
Neuropeptide Y (NPY) is a 36-amino-acid peptide that plays important roles in the control of many basic physiological functions and behaviors, including vasoconstriction, energy metabolism, and feeding behavior. In addition, a number of reports indicate that NPY has anti-stress properties. Direct administration of NPY into the brain ventricle or multiple brain areas reduces anxiety, and high cerebral levels of NPY can prevent the development of stress-induced behavior disruption and freezing. Consistently, NPY knockout mice are more anxious, and individuals with lower cerebral levels of NPY are more vulnerable to trauma-induced diseases such as PTSD. Given the potent anxiolytic effects of NPY, it was recently under investigation in clinical trials as a therapeutic option for managing stress-related psychiatric disorders. NPY neurons are widely distributed throughout the brain, including the cortex, hypothalamus, thalamus, hippocampus, and brainstem. However, which population of NPY neurons exerts anxiolytic effects, whether they are stressor-specific and how they interact with stress-related circuits remain unknown.
In the present study, employing a combination of immediate early gene activation and in vivo fiber photometry recording of neuronal activity, we successfully identify a population of brainstem NPY neurons that responds quickly and strongly to various stress stimuli. Manipulating these NPY neurons using chemogenetic and optogenetic approaches ameliorate acute stress-induced hypophagia and anxiety levels through inhibitory neural circuits. Our study provides mechanistic insights into how the brain actively resists stress and help with the development of novel therapeutic strategies for stress-related disorders.
To search for anxiolytic NPY neurons, we adopted an acute novelty stress paradigm, in which group-housed mice were individually transferred into new empty cages without padding materials for 24 hs. Novelty stress significantly suppressed feeding in the first 4 hs, followed by a food intake increase during 4-24 hs, which may represent a compensatory effect aimed at restoring homeostasis in the body. Despite a slightly increase, the total 24-h food intake during the whole stress period was not significantly altered. Based on this observation, the food intake over a 24-h period during novelty stress was measured in the following experiment, as 24 hs is sufficiently to capture the entire dynamic process of feeding inhibition and subsequent recovery. Moreover, 1-h novelty stress significantly increased serum levels of corticosterone, a prevalent marker for stress level. Elevated plus maze (EPM) and open field test (OFT) are common anxiety-assessment behavior tests, in which anxiety level inversely correlates with the time animals spend in the open arms and the time that animals explored the center of the arena, respectively. We examined how acute novelty stress influences behaviors in EPM and OFT. 2-h novelty stress significantly decreased time mice spent on the open arm of the EPM, as well as time spent and distance traveled in the center of the OFT. Of note, total distance in the open field test didn’t differ between groups, meaning that novelty stress didn’t influence locomotor activity. Collectively, these results suggested that novelty stress induces acute hypophagia and increases stress levels.
Fig. 1: Acute novelty stress decreases food intake, induces anxiety and activates NPY DRN/vlPAG neurons.
a Acute novelty stress paradigm. Group-housed mice were individually transferred into new cages without padding to induce acute novelty stress. 4-h, 4–24-h and 24-h food intake under acute novelty stress and non-stress conditions. n = 10 cages (50 mice) for non-stress group and n = 50 mice for novelty stress group. In this study, the food intake of group-housed mice was calculated by dividing the total food per cage by mice number in the cage. In b Two-way ANOVA with post-hoc Šídák’s multiple comparisons test. In c Two-sided unpaired Student’s t test. d The levels of serum corticosterone before and 1 h after novelty stress. Two-sided paired Student’s t test. e Representative elevated plus maze (EPM) traces of non-stress and novelty stress groups. The gray shades indicate the closed arms. f Percentages of open-arm time in the EPM. Two-sided unpaired Student’s t test with Welch’s correction. g Representative open field test (OFT) traces of non-stress and novelty stress groups. The gray shades indicate the center arena. h Percentages of time, travel distance in the center area and total travel distance of OFT. Two-sided unpaired Student’s t test. i 2-h novelty stress induced Fos (purple) signals in NPY DRN/vlPAG neurons (green) from Npy GFP mice. Representative images and quantitative data. In i, blue represents DAPI staining and arrows indicate Fos+NPY+ neurons. The rightmost panels are magnified images. In j, for percentage among Fos+ neurons, two-sided unpaired Student’s t test; For percentage among NPY+ neurons, two-sided unpaired Student’s t test with Welch’s correction. k Fiber photometry recording setup. l NPY DRN/vlPAG calcium signals in mice individually transferred to a novel cage without padding for 1 min and back to home cages. Two-way ANOVA with post-hoc Šídák’s multiple comparisons test. Right: Average calcium changes when mice were in new cages (20–40 s) and home cages (80–100 s). Two-sided paired Student’s t test. Data are shown as mean ± SEM. Unless specified, ‘n’ refers to mice number. Source data are provided as a Source Data file.
We then performed whole-brain Fos immunostaining to identify neurons activated by acute novelty stress. After 2-h novelty stress, mice were sacrificed and whole-brain Fos expression was visualized by immunofluorescence. Consistent with previous reports, we observed significantly increased Fos signaling in multiple stress-related brain regions, such as the medial preoptic area/medial preoptic nucleus (MPA/MPO), paraventricular thalamic nucleus (PVT), paraventricular hypothalamic nucleus (PVN), and basolateral amygdaloid nucleus (BLA). In addition, there were significantly more Fos+ neurons in the dorsal raphe nucleus and ventrolateral periaqueductal gray region (DRN/vlPAG) of stress-exposed mice than in control mice. Notably, a considerable proportion of Fos+ neurons in the DRN/vlPAG region are NPY neurons, suggesting that NPY DRN/vlPAG neurons were activated by acute novelty stress.
To further uncover the dynamic activity of NPY DRN/vlPAG neurons in response to stress, we performed fiber photometry recording of NPY DRN/vlPAG neuronal calcium.
