Harrahill, A. | bioRxiv Science

bioRxiv2024

Reverse engineering placebo analgesia

Placebo analgesia is a widely observed clinical phenomenon. Establishing a robust mouse model of placebo analgesia is needed for careful dissection of the underpinning circuit mechanisms. However, previous studies failed to observe consistent placebo effects in rodent models of chronic pain. We wondered whether strong placebo analgesia can be reverse engineered using general anesthesia-activated neurons in the central amygdala (CeAGA) that can potently suppress pain. Indeed, in both acute and chronic pain models, pairing a context with CeAGA-mediated pain relief produced robust context-dependent analgesia, exceeding that induced by morphine in the same paradigm. We reasoned that if the analgesic effect was dependent on reactivation of CeAGA neurons by conditioned contextual cues, the analgesia would still be an active treatment, rather than a placebo effect. CeAGA neurons indeed receive monosynaptic inputs from temporal lobe areas that could potentially relay contextual cues directly to CeAGA. However, in vivo imaging showed that CeAGA neurons were not re-activated in the conditioned context, despite mice displaying a strong analgesic phenotype, supporting the notion that the cue-induced pain relief is true placebo analgesia. Our results show that conditioning with activation of a central pain-suppressing circuit is sufficient to engineer placebo analgesia, and that purposefully linking a context with an active treatment could be a means to harness the power of placebo for pain relief.

neuroscience↗

bioRxiv2023

Brainstem premotor mechanisms underlying vocal production and vocal-respiratory coordination

Speech generation critically depends on precise controls of laryngeal muscles and coordination with ongoing respiratory activity. However, the neural mechanisms governing these processes remain unknown. Here, we mapped laryngeal premotor circuitry in adult mice and viral-genetically identified excitatory vocal premotor neurons located in the retroambiguus nucleus (RAmVOC) as both necessary and sufficient for driving vocal-cord closure and eliciting mouse ultrasonic vocalizations (USVs). The duration of RAmVOC activation determines the lengths of USV syllables and post-inspiration phases. RAmVOC-neurons receive inhibitory inputs from the preBotzinger complex, and inspiration needs can override RAmVOC-mediated vocal-cord closure. Ablating inhibitory synapses in RAmVOC-neurons compromised this inspiration gating of laryngeal adduction, resulting in de-coupling of vocalization and respiration. Our study revealed the hitherto unknown circuits for vocal pattern generation and vocal-respiratory coupling. One-Sentence SummaryIdentification of RAmVOC neurons as the critical node for vocal pattern generation and vocal-respiratory coupling.

neuroscience↗

bioRxiv2023

Transgenic Targeting of Fcrls Creates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity

Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell-types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines are able to achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specific Fcrls gene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis in Fcrls-2A-Cre mice. Crossing Fcrls-2A-Cre mice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border associated macrophages (BAMs) are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line, Cx3cr1-CreM (MMRRC). Fcrls-2A-Cre mice are immediately available (JAX Stock #036591) and serve as a valuable addition to the communitys microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable. Significance StatementThe microglia toolbox is continuously growing with more transgenic lines and most recently even viral tools becoming available. When selecting a Cre driver line, investigators must weigh relative strengths and weaknesses of available lines and carefully make the best choice for their given application. These tradeoffs include (1) availability and ease of employment, (2) chromosomal positioning of Cre with respect to the floxed allele (should not be on the same chromosome for conditional knockout studies), (3) activity level of a given Cre line and thus completeness of recombination across the microglia population, (4) specificity with respect to acceptable off-target cell types and tissues, (5) temporal aspects including earliest onset of Cre expression or inducibility, (6) robustness in disease contexts, and (7) potential perturbation of microglia homeostasis through Cre itself or disruption of the targeting locus. When selecting a mouse line, it is evident that there may not be a one-size-fits all solution but an application-based preference and choice from the diverse repertoire of microglia tools. Fcrls-2A-Cre mice are an excellent addition to this toolbox.

neuroscience↗