Developing a chemogenetic strategy to target spinal sympathetic preganglionic neurons after spinal cord injury

Traumatic spinal cord injury (SCI) produces profound autonomic cardiovascular dysfunction that can prevent full participation in rehabilitation. A major clinical barrier is orthostatic hypotension, which occurs when descending brainstem sympathetic drive cannot effectively drive spinal sympathetic preganglionic neurons (SPNs). Reduced SPN activity limits vasoconstriction, promoting venous pooling in the lower extremities, and can cause dizziness or syncope upon postural change. Although spinal electrical stimulation can improve orthostatic tolerance after SCI, the cellular and circuit mechanisms responsible for these benefits remain poorly defined. In particular, it is unclear whether stimulation improves cardiovascular control by directly activating SPNs or indirectly by recruiting propriospinal networks that synapse onto SPNs. A preparation that enables selective, focal control of SPNs would provide a powerful way to define spinal mechanisms of cardiovascular recovery and guide more targeted therapeutic strategies. We hypothesize that chemogenetic stimulation of SPNs after SCI will improve the recovery of sympathetic regulation of blood pressure. In this study, we describe a gene delivery and chemogenetic approach designed to selectively target SPNs for activation using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). To restrict expression to SPNs, we established a colony of transgenic Long Evans ChAT::Cre rats, in which Cre recombinase expression is driven by the choline acetyltransferase (ChAT) promoter. We then used a retrograde delivery strategy intended to reach SPNs via peripheral injection. Specifically, we injected ~400 nL into the celiac ganglia of either a retrograde tracer control (FluoroGold, FG) or a Cre-dependent retrograde viral vector (retro-DIO-CAG-hM3Dq-mCherry). Thoracic spinal cord tissue was processed for immunofluorescence to assess labeling (FG or mCherry) and colocalization with ChAT+ SPNs. FG produced clear, unilateral labeling localized to the ipsilateral sympathetic region of the spinal cord, demonstrating that the celiac ganglion injection route provides reliable retrograde access to SPNs. In contrast, the hM3Dq mCherry viral vector produced minimal detectable SPN expression under the conditions tested, despite successful targeting with FG in control rats. Furthermore, central injections of the same virus showed successful retrograde labelling of central pathways, indicating that the viral vector and reporter protein are functional. These findings suggest that the current limitation is viral transduction or expression rather than anatomical access. Plausible contributing factors include vector performance in this pathway or insufficient promoter cassette-driven expression in SPNs following retrograde transport. Our ongoing work is focused on optimizing retrograde viral expression in SPNs. Once robust expression is achieved, we will activate DREADDs using clozapine-N-oxide and quantify cardiovascular effects with radiotelemetry-based measurements of arterial pressure in SCI and control rats. This platform will enable mechanistic tests of whether selective SPN activation can restore cardiovascular stability and will help distinguish direct SPN recruitment from indirect circuit-mediated effects after SCI. This study was supported by the National Institutes of Health (R03NS140993 to MZ) and East Tennessee State University’s Student Faculty Collaborative Grant and ETSU Research Enhancement Grant. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.