Abstract The nocebo effect, the evil twin of the better-known placebo effect, in which anticipation of harm worsens pain and other symptoms, is increasingly thought to be responsible for poor clinical outcomes. In humans, nocebo hyperalgesia (i.e., increased pain sensitivity) is blocked by proglumide, a cholecystokinin (CCK) receptor antagonist. Yet, the neural circuitry underlying nocebo hyperalgesia remains unidentified, largely due to a lack of appropriate animal models. Independently, our two laboratories developed unique animal models of CCK-dependent nocebo hyperalgesia in which the expectation of pain was elicited by environmental or social cues. We find that both nocebo paradigms share a neural circuit involving CCK release from neurons projecting from the anterior cingulate cortex to the lateral periaqueductal gray. This previously unrecognized pathway could represent a promising target for therapeutic interventions in pain-related disorders. One-Sentence Summary Pain expectations, whether environmentally conditioned or socially transmitted, are mediated by a shared neural circuit involving cholecystokinin (CCK) projections from the anterior cingulate cortex (ACC) to the lateral periaqueductal gray (PAG).
Strengths
Figures 1-4 link systemic CCK receptor blockade, PAG activation, local lPAG manipulation, ACC-to-lPAG tracing, and pathway-specific optogenetics into a coherent circuit argument. The use of both contextual conditioning and social observation strengthens the claim that the pathway is not tied to a single assay format.
↳ Results pp. 4-9; Figures 1-4
The study narrows the relevant site from broad PAG activation to the intermediate lPAG, where proglumide blocks hypersensitivity and CCK-8S induces pain behavior. RNAscope evidence for Fos and Cckbr co-expression further supports the receptor-level localization.
↳ Results pp. 5-7; Figure 3
The Discussion connects the mouse circuit findings to human proglumide evidence, PAG fMRI findings, and clinical nocebo concerns in pain care. This gives the preclinical mechanism a clearer translational trajectory than a purely discovery-oriented circuit study.
↳ Discussion pp. 10-13
Limitations
The mouse paradigms show conditioned and socially enhanced pain hypersensitivity, but they do not directly measure expectation. Claims that the circuit mediates nocebo hyperalgesia specifically therefore remain stronger than the behavioral construct evidence alone.
↳ Abstract; Main Text p. 3; Discussion pp. 9-13
The figure legends repeatedly state that full statistical details are in File S1, but those details are not in the supplied manuscript text. This limits independent verification of exact test outputs, multiplicity handling, and effect-size reporting.
↳ Figure legends pp. 21-23
The work is entirely in mice and the Discussion acknowledges that human nocebo effects are often larger in female subjects, whereas the mouse models did not show stark sex differences. Selective CCK-2 antagonist development is also presented as a future requirement rather than an available intervention.
↳ Discussion pp. 10-13
The adjudicated quality score reflects a strong preclinical circuit paper whose central evidence comes from convergent pharmacology, PAG localization, tracing, and optogenetic manipulation in Figures 1-4. Methodological Rigour was held below the top anchor because the supplied manuscript defers full statistical outputs to File S1, does not report power calculations, uses different von Frey methods across paradigms, and leaves some alternative anxiety or aversive-facilitation interpretations open. Contribution remains high because the study identifies a specific ACC-to-lPAG CCK pathway and tests necessity and sufficiency, but the mouse nocebo construct is still indirectly inferred. Impact potential is moderate because the work has a clear mechanistic link to pain care and CCK-2 targeting, while clinical application remains distant.
Nabu’s assessment, alongside the field’s view.
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Strong4.0
Confidence highThe paper makes a clear mechanistic contribution by linking two mouse nocebo-like pain paradigms to CCK signaling in an ACC-to-lPAG pathway. The score remains below the top anchor because the central nocebo construct is inferred from behavior rather than directly measured as expectation in mice.
both nocebo paradigms share a neural circuit involving CCK release
The design uses convergent pharmacology, c-Fos mapping, local PAG infusion, retrograde tracing, and optogenetic necessity and sufficiency tests, with random allocation and blinding declared. The score reflects strong execution tempered by deferred statistical details, unreported power justification, measurement heterogeneity across von Frey methods, and incomplete exclusion of broader aversive mechanisms.
Mice were allocated randomly to experimental groups and testing was conducted... blind to drug treatments.
The manuscript follows a clear sequence from systemic receptor blockade to regional localization and pathway-specific manipulation. The main clarity limitation is that terms such as nocebo, expectation, and shared mechanism are used more confidently than the mouse behavioral evidence alone can establish.
Using a combination of pharmacological, immunohistochemical, viral tracing, and optogenetic approaches
The paper situates the work in human CCK/nocebo pharmacology, PAG imaging, ACC-PAG pain circuitry, and prior animal social or contextual pain paradigms. It also discusses translational limitations including inconsistent proglumide clinical evidence and mismatch with human sex differences.
The human relevance of our findings is enhanced by the reverse translational approach used.
Lower confidence on Methodological Rigour — domain match limited.
Caveats4 of 4 checks
The experimental sequence is internally coherent and supports a circuit-level claim, but the construct label of nocebo is stronger than the direct behavioral evidence because expectation is inferred rather than measured. Statistical details are repeatedly deferred to File S1, limiting verification from the supplied manuscript alone.
Animal ethics approval, random allocation, and blinding are declared, but the supplied manuscript does not provide a protocol identifier or explicit data, code, preregistration, reporting-standard, or COI statements. These are transparency limitations rather than evidence of misconduct.
Flags: 1 declared / 5 total
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No retraction or expression of concern found in Crossref (includes Retraction Watch).
Sources: Crossref (incl. Retraction Watch) ✓
Where this paper’s evidence sits on the path from initial observation to real-world use.
The work provides mechanistic proof-of-concept in mice and builds on prior human proglumide evidence, but it does not provide human circuit data or clinical testing. The authors explicitly frame further drug development as needed before clinical translation.
Developing such targeted pharmacological tools will be critical to translating our preclinical findings.
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