This study successfully established a nocebo‐nausea animal model with dual mechanisms of observational learning and conditioning. When two mechanisms were combined, nausea responses increased much more than when conditioning was applied alone, though this benefit was only evident in terms of reduced grooming time. Twelve days after the initial induction, these nausea behaviors still exhibited an increasing trend, and the expectation of nausea was consistently intense. The investigation of potential mechanisms for nocebo nausea revealed the critical importance of the CCK and 5‐HT systems. Brain activations related to cognition, memory, and affection have been shown to contribute to developing nocebo. Our experiments illustrated the difficulty and complexity of eliciting nocebo effects in animals. Nonetheless, this animal model has made great progress and has distinct advantages.

This is the first animal model to try to simulate an actual clinical situation involving nocebo occurrence. A contextual mode that matched disease and treatment was used in our model. To date, most of the knowledge about nocebo stems from the side effects reported by patients receiving placebos, which were viewed as nocebo responses. ²⁷ Previous animal models mostly focused on a conditioned taste aversion model based on a procedure for nausea conditioning with sweet taste stimuli. ¹⁰ , ¹¹ It should be noted that a single conditioning mechanism is insufficient to account for nocebo effects. These models, on the other hand, are all based on healthy animals, possibly due to the difficulties of inducing nocebo effects in diseased animals. ²⁸ However, the fact is that nocebo effects are inextricably linked with illness states.

We also identified that, for the first time, the mere observation of peers' nausea could elicit social nausea transfer in rats. The social learning mechanism, which differs from conditioning, is thought to be a key mediator of expectations and nocebo effects. ²⁹ As in other unpleasant emotions (e.g., pain), our experiments demonstrated that rats have the ability to learn nausea from other rats. ¹³ However, nausea responses obtained through social learning were considerably weaker than those obtained through conditioning. Indeed, there has been little research to compare the evoking strengths of the two mechanisms in nocebo effects. Only a recent study ³⁰ investigated the role of distinct intensity conditioning methods in nocebo, and the results showed that more conditioning training is associated with stronger nocebo hyperalgesia. Thus, the difference in training days between the two inducing mechanisms was a possible explanation for the above phenomenon. The combination of different expectation induction procedures is thought to elicit significantly stronger nocebo effects. ⁵ We further made an attempt to combine observational learning with conditioning to evoke stronger nocebo nausea in rats. Just one behavioral indicator of nausea, however, was observed to induce stronger effects. It is unclear what complex interactional impact would be produced between the two inducing mechanisms. Still, the present animal model was considered to be more representative of the actual occurrence of nocebo.

Next, the underlying neurobiological mechanisms of nocebo nausea were examined. It has been proposed that anticipation anxiety and HPA axis activation are closely related to nocebo effects. ³¹ , ³² However, the HPA axis was not hyperactive in the current model. One crucial discovery was the considerable upregulation of serum CCK levels. As a physiological peptide produced from the intestine, CCK plays an important role in digestion and the control of appetite. ³³ Recent studies demonstrated that exogenous CCK at high dosages could directly trigger nausea in rodents, ³⁴ , ³⁵ and the process might be mediated by NTS that received gastrointestinal vagal afferent inputs. ³³ These findings identified the potential role of peripheral CCK in driving nocebo nausea and the possibility of serving as a novel serologic nocebo marker.

Additionally, a large body of research demonstrated that CCK functioned as a neuromodulator in the brain, controlling actions linked to feeding, mood, memory, and cognitive function. ³⁶ , ³⁷ , ³⁸ It has also been implicated as a crucial regulator of nocebo hyperalgesia. ³¹ , ³⁹ Similarly, we discovered significantly increased CCK mRNA expressions in the medulla when nocebo nausea occurred, which further determined the unique contribution of CCK to nocebo effects. However, the same change in CCK gene expression was found in rats only receiving LiCl injections. Thus, the complete exclusion of the impact of pharmacological actions on CCK alterations is difficult. Another potential explanation is that increased CCK is the product of nocebo mimicking the pharmacological effect of LiCl. Prior research has proven the specific imitation ability of nocebo for drugs. ⁴⁰ A representative example was that side effects reported by patients receiving placebos relied on side effects reported by patients receiving active medications. ⁴ We suggest future work is needed to further investigate the accurate source of CCK.

The signaling pathways connected to nausea, including the 5‐HT, cannabinoid, and dopamine receptor systems, were also investigated. ¹² The findings revealed considerable changes in 5‐HT receptor systems: the 5‐HT 2C receptor's mRNA expression had increased, while that of the 5‐HT 1A and 5‐HT 3A receptors had dropped. 5‐HT3 receptor antagonists have been shown in clinical trials to improve chemotherapy‐induced nausea and vomiting. ⁴¹ , ⁴² As an ion channel, the 5‐HT3 receptor mediates rapid depolarization of serotonergic neurons, resulting in the release of a variety of neurotransmitters associated with nausea and vomiting, including dopamine, CCK, 5‐HT, etc. ¹² Surprisingly, we discovered that the 5‐HT 3A receptor decreased rather than increased at the RNA level. Recent evidence also suggested that activation of the 5‐HT 2C receptor, rather than the 5‐HT3 receptor, was involved in nausea regulation, even though the latter was of major importance for vomiting. ⁴¹ Additionally, the activation of the 5‐HT 1A receptor was effective in inhibiting anticipatory nausea in rats by suppressing the release of 5‐HT. ⁴³ , ⁴⁴ These findings were consistent with our results regarding changes in 5‐HT 2C and 5‐HT 1A receptors. Notably, these differences seemed to be unique to the conditioning mechanism and were not affected by LiCl itself, implying that 5‐HT systems may play important roles in conditioned nausea.

When the nocebo occurs, significant changes in brain function are observed; however, no definitive conclusion has been reached. The available evidence is primarily based on human neuroimaging studies of nocebo hyperalgesia, and some brain regions have been suggested to be clearly activated, such as the thalamus, insula, hippocampus, ACC, PAG, amygdala, etc. ⁴⁵ , ⁴⁶ , ⁴⁷ We noted that few studies had examined changes in brain activation in nocebo nausea. Being a transcription factor encoded by the immediate early gene family, c‐Fos has been widely used as a marker of neuronal activity. ⁴⁸ , ⁴⁹ Here, we further investigated the brain maps of positive c‐Fos neurons to uncover the underlying neural mechanisms of nocebo nausea. Our findings revealed similar activated brain regions in nocebo nausea, particularly those primarily responsible for integrating cognition, emotion, and nausea messages, such as the ACC, insula, BLA, PVN, PVT, PAG, DRD, and NTS. This was in line with a recent study that detected c‐Fos expression in different brain regions after inducing conditioned nausea in male rats, with more activated neurons in the frontal cortex, insula, and PVN. ⁵⁰ The ACC and insula, the key brain regions identified in our study, could integrate emotional and cognitive stimuli to directly trigger nausea. ¹² , ⁵¹ Strong evidence also suggested that they were associated with the production of aversion emotions. ⁵² , ⁵³ The amygdala played an important role in the execution of emotion and motivation functions, as well as in the pairing of a conditioning stimulus with aversive or rewarding outcomes. ⁵⁴ Recent studies also reported that PVT activation pushed the establishment of the association between novel stimuli and expected punitive cues, suggesting it was important in aversive association learning. ⁵⁵ These brain activations partially explained the sustained aversion to saccharin solution in nocebo rats.

Furthermore, we confirmed that PVN had a unique effect on nausea caused by conditioning rather than observational learning. PVN is known to regulate autonomic and neuroendocrine functions, which establishes a wide connection to the limbic system, NTS, and DMV. ⁵⁶ , ⁵⁷ Oxytocin (OT) released by OT neurons in PVN, in particular, regulates a variety of neural functions, including recognition, learning, emotion, reward, and memory. ⁵⁸ Previous research found that it was critical not only in the mediation of conditioned taste avoidance but also in the expression of socially mediated conditioned nausea in rats. ⁵⁹ , ⁶⁰ More importantly, we also observed the unique changes of CCK neurons in PVN at the same time, which were not related to LiCl administrations. Additional studies are needed to investigate in more detail how PVN is involved in nocebo nausea and what role CCK plays.

As noted previously, the importance of 5‐HT systems in nausea has been shown, and we also observed that brain regions that predominantly contained serotonergic neurons, including PAG and DRD, were strongly activated. Asides from directly eliciting nausea responses, 5‐HT has recently been indicated to participate in the associative learning of conditioned and unconditioned stimulus by regulating synaptic plasticity. ⁶¹ Somewhat unexpectedly, CCK was obviously reduced in these brain regions. It has been unclear how CCK is associated with 5‐HT systems yet, and this is a significant target for future studies. Our findings also demonstrated that nocebo nausea was accompanied by the activation of the vomiting center – NTS. ⁶² Importantly, such neurobiological alterations could still be evoked after 12 days of initial nocebo nausea responses.

Some limitations should be pointed out. First, sex hormones have been suggested to broadly regulate the functions of the neural systems, including mood and cognition. ⁶³ Only male rats were used in our study to avoid the fluctuation of hormone levels in female rats. Although no definite conclusions could be drawn, there was some evidence that females were more susceptible to nocebo effects. ⁴ , ⁶⁴ Future studies should, therefore, further assess the role of sex differences in nocebo behaviors. Second, the current rat model did not fully represent the condition of nocebo nausea seen in humans. Large differences existed between animals and humans, whether at structural or physiological levels. Humans produce the expectation of nocebo based on prior experience as well as complicated cognitive and psychological processes, while the difficulty of acquiring identical expectations has been shown in animals. ² Thus, the discovery revealed in the animal model needed to be further allied with human research. Third, our preliminary exploration identified the potential of CCK and related brain regions in the regulation of nocebo nausea. However, the present study cannot completely exclude the impact of the pharmacological actions of LiCl on CCK release. There was a clear need to further investigate whether and how CCK played a detailed role in developing nocebo.