nNOS-expressing neurons in the vmPFC transform pPVT-derived chronic pain signals into anxiety behaviors

Anxiety is common in patients suffering from chronic pain. Here, we report anxiety-like behaviors in mouse models of chronic pain and reveal that nNOS-expressing neurons in ventromedial prefrontal cortex (vmPFC) are essential for pain-induced anxiety but not algesia, using optogenetic and chemogenetic strategies. Additionally, we determined that excitatory projections from the posterior subregion of paraventricular thalamic nucleus (pPVT) provide a neuronal input that drives the activation of vmPFC nNOS-expressing neurons in our chronic pain models. Our results suggest that the pain signal becomes an anxiety signal after activation of vmPFC nNOS-expressing neurons, which causes subsequent release of nitric oxide (NO). Finally, we show that the downstream molecular mechanisms of NO likely involve enhanced glutamate transmission in vmPFC CaMKIIα-expressing neurons through S-nitrosylation-induced AMPAR trafficking. Overall, our data suggest that pPVT excitatory neurons drive chronic pain-induced anxiety through activation of vmPFC nNOS-expressing neurons, resulting in NO-mediated AMPAR trafficking in vmPFC pyramidal neurons.

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October 2018
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All the data supporting the findings of this study are available within the paper and its supplementary information file and from the corresponding author upon reasonable request. Source data for all figures are provided with the paper online.
No sample-size calculation was performed. Sample size was 12-16 mice for animal behavioral tests, 3-6 mice for biological measurements and 10-33 neurons from 4-6 mice for electrophysiological recordings. According to previously reported studies in which these experiments were conducted, such sample sizes were sufficient (Science 363: 276-281 (2019); Nat Commun 6: 7660 (2015). doi: 10.1038/ncomms8660; Neuron 87: 605-620 (2015)). Our prior experience and preliminary experimental data also ensured the sufficiency. We have done many works on depression and anxiety behaviors in rodents and data were published (Proc Natl Acad Sci USA 109: 14224-14229 (2010); Nat Med 20: 1050-1054 (2014); J Neurosci 30: 2433-2441 (2010)). In this study, we have obtained positive results in most of our experiments, suggesting the sample size was sufficient to show the difference between groups. Regarding the few negative results, similar sample sizes were used with the experiments in which positive results were obtained. More importantly, our claims were always demonstrated with multiple experiments. The results from different experiments were consistent with each other, indicating that the sample size was sufficient to give true results. False positive or negative results at random could hardly always agree with each other.
For biological measurements and electrophysiological recordings, no data were excluded from the analysis unless the data were not successfully obtained for technical or accidental reasons. For behavioral tests, animals were excluded from the analysis if accidental events occured, such as escape, fall from the open arms of elevated plus maze, or restless movement in hyperalgesia measurement. The data from the mice whose brain region was not been correctly targeted in microinjection were also excluded. Exclusion criteria were pre-established.
The accurate target of desired brain regions has been replicated with 10 mice in 10 independent experiments. Representative micrographs shown without statistical quantification were always conducted in triplicate with 3-6 mice and similar results were observed. For animal behavioral tests and other quantified experiments, none was replicated with the same condition except some preliminary experiments for animal behavioral tests. All the findings in the preliminary experiments were successfully replicated by the formal experiments. In the formal experiments, animals or samples were always divided into 2-3 cohorts (each cohort included equal number of animals or samples from all groups) for big sample sizes and received treatments or measurements on different days. Therefore, replication was successfully achieved in once experiment. Additionally, in this study, the most of experimental findings were verified with different methods. For example, the anxietylike behaviors in OF and EPM were confirmed with NSF and LDB.
Random number table was used to randomly allocate animals to different groups.
The investigators were blinded to group allocation during data collection and analysis.