Fluorescent Based Tracing of Sensory Nerve Subtypes in Brainstem

Abstract

Processing vagal and trigeminal afferent inputs in the brainstem is essential for regulating reflexes including those from the respiratory tract. Afferent nerves can be categorized into several subtypes depending on the ganglionic origin of cell bodies (placodal vs. neural crest), conduction velocity and response patterns to stimuli. These various nerve subtypes convey different types of information which is integrated at the brainstem to control respiratory reflexes. Vagal afferent nerve subtypes express particular ion channels/receptors which are stimulated by various stimuli resulting in different information being relayed to the brainstem. For example, stimulation of vagal jugular C‐fiber nociceptors evokes cough, whereas stimulation of vagal nodose C‐fiber nociceptors inhibits cough. Vagal afferents are known to innervate the nucleus of the solitary tract in the dorsal medulla. However, the innervation patterns may vary depending on the subsets of vagal afferents. The variation makes it difficult to understand how distinct peripheral vagal afferent inputs are processed in the central nervous system.In the current study, we focused on developing a neuroanatomical map of various sensory afferent nerve subtypes using a transgenic mouse model system. Cre‐LoxP reporter system allows us to target the specific ion channel/receptor positive sensory nerve subtypes enabling tracking of afferent nerve subtypes throughout medulla. Transient receptor potential vanilloid 1 (Trpv1), tachykinin 1 (Tac1) and 5‐hydroxytriptamine receptor 3 (5HT3) Cre strains were bred with Floxed Ai9 ROSA tdTomato; thus, expressing red fluorescent specific to Trpv1, Tac1 and 5HT3 positive cells. Mice (6 to 8‐week‐old) were euthanized and tissue was collected. Brainstem were cryosectioned at 30 μm thickness and slide mounted sequentially. Additionally, we used a passive tissue clearing technique (CLARITY‐PACT) to visualize the sensory nerve projections in dissected whole medulla. Images were taken using Nikon Eclipse microscope and Olympus FV1200 confocal microscope. The 3D images were processed with Imaris software.As a result, we were able to identify each sensory nerve subtype in the medulla and define their projections to different regions of medulla ‐ these include the nucleus of solitary tract (NTS), paratrigeminal nucleus (Pa5) and spinal trigeminal tract (sp5). While brainstem projection of the Trpv1, Tac1 and 5ht3 Cre/Rosa strains showed similarities in gross regional expression, the patterns of these projections were distinct depending on the strains. For example, Trpv1‐tdTomato projections were found in the commissural NTS but not the medial NTS, whereas Tac1‐tdTomato projections were largely absent from the commissural NTS but were abundantly present in the medial NTS. The only overlap of Trpv1 and Tac1 projections was not in the NTS, but rather in Pa5 and sp5, suggesting that vagal jugular C‐fiber nociceptors may terminate in Pa5 as opposed to the NTS. The cleared whole medulla from each strain were consistent with the medulla sections and were used to yield 3D maps of the sensory afferent nerve projections. Based on these observations, a genetic tool using Cre‐LoxP reporter system allowed us to define the neuronal circuitry of major vagal sensory afferent nerve subtypes. This information may be useful to study functionalities of each sensory nerve subtypes to better understand the mechanism of respiratory reflexes.Support or Funding InformationNIH Common Fund SPARC OT2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.