Retrograde Optogenetic Fingerprinting Reveals a Unique Somatostatin‐Sensitive Neuronal Group in Kölliker‐Fuse Nucleus that Sets the Postinspiratory Rhythm

Multiscale fingerprinting is a novel neurotracing technique for testing the connectivity of two functionally identified neuronal populations in a network. By using this powerful technique we have recently identified a pontomedullary postinspiratory (post‐I) pathway whereby a group of NMDA receptor‐dependent post‐I driver neurons in Kölliker‐Fuse nucleus (KFN) send excitatory inputs to downstream post‐I interneurons and motoneurons in medulla 1 . Because NMDA receptor is likely also expressed in other respiratory‐related neurons in KFN, it per se is not a perfect cell type‐specific marker of post‐I driver neurons and must be combined with other (timing‐ and response‐specific) markers in order to form a tripartite ‘fingerprint’ that uniquely identifies these neurons. In the present series of studies aimed to identify a more selective singular marker of KFN post‐I driver neurons, we found firstly that microinjection of the neuropeptide somatostatin (SST) into the KFN also markedly suppressed post‐I activity recorded from the cervical vagal efferent nerve. However, compared with microinjections of other neuromodulatory agents (including an antagonist for NMDA receptor or agonists for μ‐opioid or GABA A receptors), SST microinjection caused the least disturbances in other phases of the respiratory rhythm. This observation at the network level indicates that the presence of SST receptors was probably unique to post‐I driver neurons among other neurons in KFN that modulate the respiratory rhythm. To verify this at the cellular level, we picoinjected SST at neurons in KFN while recording their activities extracellularly. Recordings from a variety of neurons revealed that among all respiratory‐related neurons in KFN, only post‐I neurons and a few inspiratory neurons were sensitive (inhibited) to SST. To further dissect these neurons, we microinjected a retrograde HSV vector encoding EYFP‐hChR2 into the Bötzinger complex (BötC) and the hypoglossal nucleus (nXII) in order to retrogradely transduce neurons that send axonal projections to the injection sites. Neurotracing using this retrograde optogenetic fingerprinting technique led to the following results. (1) In rats with HSV injection at BötC, photostimulation at KFN increased both the amplitude and duration of vagal efferent post‐I discharge and prolonged the expiratory duration without affecting phrenic discharge. Sustained 15‐sec photostimulation caused post‐stimulation potentiation of vagal post‐I activity similar to that observed after hypoxic chemostimulation. (2) In rats with HSV injection at nXII, photostimulation at KFN increased the amplitude of hypoglossal nerve inspiratory phasic discharge and extended the duration of hypoglossal inspiratory discharge to encroach into the late expiratory/pre‐inspiratory phase without affecting the amplitude and inspiratory phase duration of phrenic discharge. We conclude that the pontomedullary post‐I pathway from KFN to BötC is selectively driven by KFN SST‐sensitive post‐I driver neurons while the KFN to nXII pathway is selectively driven by KFN expiratory‐inspiratory phase‐spanning neurons involved in the control of hypoglossal activity. Support or Funding Information HL093225, HL127258 and NS094178