Dual-Pseudorabies Viral Tracing for Spinal Tyrosine Hydroxylase Interneurons Involved in Segmental Micturition Reflex Circuitry in Spinal Cord Injured Rats

Abstract

Traumatic spinal cord injury (SCI) often leads to urinary dysfunction. Although an involuntary micturition reflex can be established to elicit voiding with time, complications arise in the form of bladder hyper-reflexia and detrusor-sphincter dyssynergia that cause incontinence and inefficient expulsion of urine. To date, the neuronal mechanisms that underlie regulation of micturition after SCI are not well understood. We recently observed an increase of a population of tyrosine hydroxylase (TH)+ cells in the rat lumbosacral cord post-SCI, which contribute to the sustention of a low level of dopamine that modulates the recovered bladder reflex. To identify whether spinal TH+ cells are involved in the micturition reflex pathway post-SCI, two isoforms of the trans-synaptic retrograde tracer, pseudorabies virus encoding green fluorescent protein (GFP; PRV-152) or red fluorescent protein (RFP; PRV-614), were injected into the bladder detrusor or the external urethral sphincter (EUS), respectively, 3 weeks after a spinal cord transection at the 10th thoracic level (T10) in rats. Immunohistochemistry was performed to examine infected TH+ cells in the caudal cord at both 48 and 72?h post-injection. As a result, double-labeled TH+/GFP+ and TH+/RFP+ cells could be found in the superficial dorsal horn, parasympathetic nuclei, and dorsal gray commissure (lamina X) at both time points. More importantly, a shared population of TH+ interneurons (TH+/GFP+/RFP+) exists between bladder and EUS circuitry. These results suggest that spinal TH+ interneurons may coordinate activity of the bladder and EUS that occurs during micturition reflexes post-SCI.

Keywords

• bladder
• external urethral sphincter
• spinal cord transection
• tyrosine hydroxylase
• trans-synaptic tracing