Background & Aims

Acute stress triggers endogenous analgesia. During stress, several brain areas are activated and send descending axons to suppress nociception at the spinal cord level. However, their spinal effector circuits and neurons are largely elusive. Here, we demonstrate that GABAergic interneurons of the superficial dorsal horn expressing the transcription factor Gbx1 are key elements of these circuits. Under resting conditions, their inhibition had little effect on nociception, but completely abolished swim stress-induced analgesia. Retrograde monosynaptic tracing revealed input from several brain areas (e.g. rostral ventromedial medulla). Optogenetic circuit tracing confirmed that this input is inhibitory, and that Gbx1 neurons in turn inhibit projection neurons targeting the lateral parabrachial nucleus, a key area in supraspinal pain relay. Our results thus identify GABAergic neurons of the superficial dorsal horn as key elements of a disinhibitory circuit for stress-induced analgesia.

Methods

To characterize spinal Gbx1 expression in adult mice, we conducted immunohistochemical and multiplex fluorescence in situ hybridization (mFISH) experiments in Gad67eGFP mice. To functionally manipulate Gbx1 neurons, Gbx1cre knock-in mouse line was generated by inserting an IRES cre expression cassette into the 3’ UTR of the Gbx1 gene. To investigate their function in spinal nociceptive control, we expressed excitatory (hM3Dq) or inhibitory (hM4Di) DREADDs in Gbx1 neurons and interrogated nociceptive behavior after chemogenetic activation or inhibition. To assess whether acute stress exposure activate dorsal horn Gbx1 neurons, forced swim stress and restraint stress test, together with nociceptive behaviors, were performed. To conclude, we investigated which brain areas provide monosynaptic input to spinal Gbx1 neurons employing modified rabies based retrograde trans synaptic tracing.

Results

Through immunohistochemical and mFISH experiments, we saw Gbx1-expressing neurons in the superficial dorsal horn (SDH) are purely GABAergic (both inhibitory galanin and NPY neurons), while deep dorsal horn Gbx1 neurons are predominantly glycinergic (only a subset expresses Pvalb). Injection of an AAV.flex.eGFP into the lumbar spinal cord of Gbx1cre mice efficiently labelled Gbx1-expressing neurons, showing nearly all eGFP neurons expressed Pax2 and none were Lmx1b positive. After their chemogenetic manipulation, Gbx1 neurons appear to not control acute nociception under resting conditions, but they cause pronounced analgesia when activated. In particular, chemogenetic inhibition of spinal Gbx1cre neurons in Gbx1cre mice, failed to develop stress-induced analgesia after CNO injection in the forced swim test compared to controls. We also demonstrated Gbx1 neurons activation inhibits spinal nociceptive output primarily via postsynaptic inhibition of lamina I projection neurons and that the majority of superficial dorsal horn Gbx1 neurons are under tonic inhibitory control by descending input from the RVM. Our data identify inhibitory neurons of the SDH as key modules of a stress-induced analgesia circuit that connects descending RVM neurons to spinal nociceptive output neurons.

Conclusions

Through Gbx1Cre mice and cre-dependent AAVs injections we targeted the majority of inhibitory neurons in the superficial dorsal horn (SDH). The intersectional experiments using Gbx1cre;GlyT2::dre mice together with Cre/Dre double dependent viral effector constructs indicate that SDH Gbx1 neurons are crucial for stress-induced analgesia. Chemogenetic silencing of Gbx1 neurons had little effect on nociceptive sensitivity in naïve mice but prevented swim stress-induced analgesia. Optogenetic experiments revealed almost exclusively inhibitory GABAergic but no glutamatergic input from the RVM to SDH Gbx1 neurons. These findings suggest that SDH Gbx1 neurons are normally silent but stress exposure activated them. Our results uncover a hitherto unknown disinhibitory circuit for acute stress-induced analgesia that spans from the RVM, via GABAergic SDH neurons, to nociceptive specific lamina I projection neurons.

References

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Presenting Author

Francesca Pietrafesa

Poster Authors

Francesca Pietrafesa

PhD Student

University of Zurich

Lead Author

Karen Haenraets

PhD

Lead Author

Robert P. Ganley

PhD

National Institutes of Health (NIH)

Lead Author

Sina Schalbetter

Lead Author

Fabienne Luzi

Lead Author

Donald I. MacDonald

Lead Author

Hendrik Wildner

PhD

University of Zurich, Institute of Pharmacology and Toxicology

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology