Bilateral Bulbospinal Projections to Pudendal Motoneuron Circuitry after Chronic Spinal Cord Hemisection Injury as Revealed by Transsynaptic Tracing with Pseudorabies Virus

Abstract

Complications of spinal cord injury in males include losing brainstem control of pudendal nerve-innervated perineal muscles involved in erection and ejaculation. We previously described, in adult male rats, a bulbospinal pathway originating in a discrete area within the medullary gigantocellularis (GiA/Gi), and lateral paragigantocellularis (LPGi) nuclei, which when electrically microstimulated unilaterally, produces a bilateral inhibition of pudendal motoneuron reflex circuitry after crossing to the contralateral spinal cord below T8. Microstimulation following a long-term lateral hemisection, however, revealed reflex inhibition from both sides of the medulla, suggesting the development or unmasking of an injury-induced bulbospinal pathway crossing the midline cranial to the spinal lesion. In the present study, we investigated this pathway anatomically using the transsynaptic neuronal tracer pseudorabies virus (PRV) injected unilaterally into the bulbospongiosus muscle in uninjured controls, and ipsilateral to a chronic (1-2 months) unilateral lesion of the lateral funiculus. At 4.75 days post-injection, PRV-labeled cells were found bilaterally in the GiA/Gi/LPGi with equal side-to-side labeling in uninjured controls, and with significantly greater labeling contralateral to the lesion/injection in lesioned animals. The finding of PRV-labeled neurons on both sides of the medulla after removing the mid-thoracic spinal pathway on one side provides anatomical evidence for the bilaterality in both the brainstem origin and the lumbosacral pudendal circuit termination of the spared lateral funicular bulbospinal pathway. This also suggests that this bilaterality may contribute to the quick functional recovery of bladder and sexual functions observed in animals and humans with lateral hemisection injury.

Effects of Lateral Funiculus Sparing, Spinal Lesion Level, and Gender on Recovery of Bladder Voiding Reflexes and Hematuria in Rats

“…In addition, the data showing that animals with lesions to only one side of the lateral funiculus recovered function faster than those with bilateral lesions of the lateral funiculus gives further support to the bilateral and, possibly, redundant nature of the ascending and descending pathways to pelvic organ circuitry, a conclusion reported in earlier electrophysiological [35][36][37] and pseudorabies virus tracing 47 studies of bilateral (crossed) connections in the lumbosacral GM from a spared lateral funiculus. In the present report, selective bilateral lesions of only the dorsal columns did not produce a significant effect on bladder voiding function, suggesting that whereas the dorsal column may transmit some degree of bladder sensation to the brain, the bilateral loss of those ascending pathways does not cause a significant loss of bladder voiding function or trigger the development of prolonged periods of lumbosacral reflex micturition.…”

“…[35][36][37]46,47 Following those terminal experiments, animals were euthanized with an overdose of urethane and transcardially perfused with 4% paraformaldehyde. Specific spinal cord segment blocks from the T6-T9 spinal cord were removed after careful confirmation of spinal segment number with cadaveric dissection of spinal nerves and vertebral segments.…”

“…Sections were stained with the standard Luxol fast blue (LFB) and cresyl violet procedure (Klüver-Barrera stain 48 ), which consisted of deparaffinization in xylene, dehydration in ethanol, overnight immersion in warm 0.1% LFB, two cycles of lithium carbonate with ethanol/distilled water rinse/wash, and 6-min immersion in warm cresyl violet. Precise reconstruction drawings were made of the injured cord to confirm the exact and total extent of the damage along the length of the entire lesion (in addition to the epicenter) by an observer blinded to both the surgical procedure and behavioral results, 47,49 thereby reducing the possibility of underestimating the size of the lesion while also verifying that specific spared WM regions were continuous through the mid-thoracic spinal cord. Lesions were then grouped into specific categories, eliminating those from analysis that did not fit the lesion extent criteria.…”

Identification of CNS Neurons Innervating the Levator Ani and Ventral Bulbospongiosus Muscles in Male Rats

“…The brain stem regions labeled with PRV after 4 days survival after LA muscle injections included the ventrolateral medulla, PP region, the LPGi, and the caudal raphe. Pudendal motoneurons are innervated by serotonergic projections that arise from these brain regions [34,56–62]. Serotonergic neurons in the LPGi exert a tonic descending inhibition of ejaculatory‐like reflexes, ex copula reflexes and male sexual behavior [57,58,63–66].…”

“…Pudendal motoneurons are innervated by serotonergic projections that arise from these brain regions [34,[56][57][58][59][60][61][62]. Serotonergic neurons in the LPGi exert a tonic descending inhibition of ejaculatory-like reflexes, ex copula reflexes and male sexual behavior [57,58,[63][64][65][66]. Genital sensitive neurons that respond to pain stimuli are present in the caudal raphe, including the raphe magnus, and ventrolateral medulla [67].…”