Research ArticlePropriospinal Neurons of L3-L4 Segments Involved in Control of the Rat External Urethral Sphincter
Introduction
Coordination of the activity of the external urethral sphincter (EUS) striated muscle and the bladder (BL) smooth muscle which is essential for successful micturition is controlled by neural circuits located in the spinal cord and the brain stem (de Groat et al., 1981, de Groat et al., 2015, Hou et al., 2016). In the rat the main spinal circuits controlling EUS and BL activity are located in L6/S1 spinal segments (Chang et al., 2007). During urine storage bladder afferent activity induced by low intravesical pressures during bladder filling activates a spinal reflex pathway that induces tonic activity of the EUS to promote continence (the BL-to-EUS spinal reflex). On the other hand voluntary or reflex voiding in most species is mediated by supraspinal pathways involving the pontine micturition center in the brain stem that trigger a contraction of the bladder and simultaneous relaxation of the EUS (de Groat et al., 2015, Keller et al., 2018, Yao et al., 2019). However, EUS activity during voiding in rats and mice consists of rhythmic contractions separated by short periods of full relaxation of EUS muscle that functions as a pump to improve voiding efficiency (Yoshiyama et al., 2000, de Groat et al., 2001) and also to facilitate territorial scent marking (Cheng and de Groat, 2016, Kadekawa et al., 2016, Keller et al., 2018). In rats this activity termed EUS bursting is a prerequisite for efficient voiding because blocking EUS bursting with alpha bungarotoxin reduces voiding efficiency in rats (Yoshiyama et al., 2000). EUS electromyography (EUS EMG) during voiding in male and female rats exhibits bursting activity occurring at frequencies of ∼4–5 Hz. The activity consists of an active period (duration, 70–80 ms) and a silent period (duration, 100–200 ms) (Cheng and de Groat, 2004, de Groat and Yoshimura, 2015). After transection of the spinal cord at T8–T10 segments tonic EUS activity mediated by the BL-EUS spinal reflex pathway persists but the micturition reflex and EUS bursting is initially lost. However 3–6 weeks after spinal cord injury (SCI) reflex bladder contractions, EUS bursting and voiding return although voiding efficiency is reduced due to increased EUS tonic activity and changes in the EUS bursting pattern (Cheng and de Groat, 2004). Return of lower urinary tract function is attributed in part to reorganization of spinal reflex circuits (de Groat and Yoshimura, 2006, de Groat and Yoshimura, 2012, Tai et al., 2006) and plasticity in bladder afferent neurons (de Groat and Yoshimura, 2010, Kadekawa et al., 2017).
If the spinal cord is transected at L4 or more caudally, reflex bladder contractions return but EUS bursting and voiding do not return (Chang et al., 2007). Furthermore, targeted electrical stimulation of L3/L4 promotes voiding in humans and animals (Chang et al., 2019, Herrity et al., 2018). This suggests the existence at the level of the L3/L4 spinal segments of a second component of spinal cord circuitry that contributes to the emergence of EUS bursting and BL-EUS coordination after SCI (Chang et al., 2007). The L3/L4 compartment of the EUS-related circuit was named Lumbar Spinal Coordinating Center (LSCC) to indicate its role in EUS-BL coordination (Karnup et al., 2017).
In this study we examined the morphology and electrophysiological properties of neurons in the LSCC of L3/L4 spinal cord that may control EUS function during micturition. The neurons were identified in spinal cord slices by retrograde transsynaptic tracing methods in which pseudorabies virus (PRV) co-expressing a fluorescent marker (PRV-GFP or PRV-RFP) was injected into the EUS in juvenile (P18-P20) male rats 2 days prior to the experiments.
Section snippets
Experimental procedures
In this study all animal procedures were performed in accordance with University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) guidelines. A total of 28 juvenile male Sprague-Dawley rats (n = 13 for PRV-injected rats and n = 15 for control intact rats) between the ages of P18 and P20 were used because at this age the central circuitry underlying reflex voiding function is developed (Kruse et al., 1993, de Groat et al., 1981) and all major motor spinal circuits are fully
Location of EUS-related spinal neurons
The presence of GFP or RFP fluorescence in slices was examined on 1st, 2nd and 3rd days after injection of PRV-GFP/RFP into the EUS. Motoneurons innervating the EUS (EUS-MNs) located in the Onuf’s nucleus in the L6/S1 segments would be the first neurons infected by PRV; however, at 24 h post-injection they did not express GFP/RFP. On the 2nd day GFP or RFP fluorescence was observed in EUS-MNs as well as in smaller neurons in the L6/S1 dorsal commissure (DCM) dorsal to the central canal (CC) (
Discussion
This study examined the morphology and physiological properties of L3/L4 neurons presynaptic to EUS-MNs. We identified these neurons in slices using transneuronal tracing, recorded their activity with whole-cell patch clamp methods and then reconstructed their processes and soma location. Among the neurons studied we presume that there are at least two populations: propriospinal neurons (PSNs) and interneurons that represent major components of the recently identified L3/L4 Lumbar Spinal
Acknowledgements
This work was supported by a grant from the National Institutes of Health to W.C.deG. (DK-111382) and an NIDDK program project grant (P01DK-093424). We are thankful to Dr. L.W. Enquist who kindly provided us with PRV-Bartha (supported by his Virus Center grant # P40RR018604).
Disclosures
No conflict of interests, financial or otherwise, are declared by the authors.
Author contributions
W.C.de G. conception and design of research, edited the manuscript; S.V.K. performed experiments, analyzed data, prepared figures, drafted manuscript.
References (70)
- et al.
The central nervous system control of micturition in cats and humans
Behav Brain Res
(1998) - et al.
Electrical stimulation of the sacral dorsal grey commissure evokes relaxation of the external urethral sphincter in the cat
Neurosci Lett
(1998) - et al.
Ejaculation elicited by microstimulation of lumbar spinothalamic neurons
Eur Urol
(2008) - et al.
Ascending and descending propriospinal pathways between lumbar and cervical segments in the rat: evidence for a substantial ascending excitatory pathway
Neuroscience
(2013) - et al.
The role of capsaicin-sensitive afferent fibers in the lower urinary tract dysfunction induced by chronic spinal cord injury in rats
Exp Neurol
(2004) - et al.
Neurochemical excitation of thoracic propriospinal neurons improves hindlimb stepping in adult rats with spinal cord lesions
Exp Neurol
(2015) - et al.
Identification of early RET+ deep dorsal spinal cord interneurons in gating pain
Neuron
(2016) - et al.
Electrophysiological and morphological properties of neurons in the substantia gelatinosa of the mouse trigeminal subnucleus caudalis
Pain
(2009) - et al.
Developmental and injury induced plasticity in the micturition reflex pathway
Behav Brain Res
(1998) - et al.
Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine
J Auton Nerv Syst
(1981)
Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury
Prog Brain Res
Plasticity in reflex pathways to the lower urinary tract following spinal cord injury
Exp Neurol
Anatomy and physiology of the lower urinary tract
Handb Clin Neurol
Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment
Exp Neurol
Identification of CNS neurons innervating the levator ani and ventral bulbospongiosus muscles in male rats
J Sex Med
Morphological and electrophysiological features of motor neurons and putative interneurons in the dorsal vagal complex of rats and mice
Brain Res
Spinal pathways mediate coordinated bladder/urethral sphincter activity during reflex micturition in decerebrate and spinalized neonatal rats
Neurosci Lett
Separate urinary bladder and external urethral sphincter neurons in the central nervous system of the rat: simultaneous labeling with two immunohistochemically distinguishable pseudorabies viruses
Brain Res
Impact of neuronal properties on network coding: roles of spike initiation dynamics and robust synchrony transfer
Neuron
Inter-enlargement pathways in the ventrolateral funiculus of the adult rat spinal cord
Neuroscience
A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections
J Sex Med
Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells
Neuron
The central neural pathways involved in micturition in the neonatal rat as revealed by the injection of pseudorabies virus into the urinary bladder
Neurosci Lett
Spinal neurons involved in the control of the seminal vesicles: a transsynaptic labeling study using pseudorabies virus in rats
Neuroscience
Identification of lumbar spinal neurons controlling simultaneously the prostate and the bulbospongiosus muscles in the rat
Neuroscience
Influences of external urethral sphincter relaxation induced by alpha-bungarotoxin, a neuromuscular junction blocking agent, on voiding dysfunction in the rat with spinal cord injury
Urology
Spinal stimulation of the upper lumbar spinal cord modulates urethral sphincter activity in rats after spinal cord injury
Am J Physiol Renal Physiol
Spinal cord control of ejaculation
World J Urol
Ascending pathways that mediate cholinergic modulation of lumbar motor activity
J Neurochem
Motor command for precision grip in the macaque monkey can be mediated by spinal interneurons
J Neurophysiol
Spatiotemporal correlation of spinal network dynamics underlying spasms in chronic spinalized mice
Elife
Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord
Mol Pain
Serotonergic drugs and spinal cord transections indicate that different spinal circuits are involved in external urethral sphincter activity in rats
Am J Physiol Renal Physiol
Spinal cord stimulation ameliorates detrusor over-activity and visceromotor pain responses in rats with cystitis
Neurourol Urodyn
Effect of orchiectomy and testosterone replacement on lower urinary tract function in anesthetized rats
Am J Physiol Renal Physiol
Cited by (21)
Neuro-urology research: a comprehensive overview
2023, Neuro-Urology Research: A Comprehensive OverviewVoluntary versus reflex micturition control
2023, Neuro-Urology Research: A Comprehensive OverviewMild thermal stimulation of the buttock skin increases urinary voiding efficiency in anesthetized rats
2022, Autonomic Neuroscience: Basic and ClinicalSlow development of bladder malfunction parallels spinal cord fiber sprouting and interneurons' loss after spinal cord transection
2022, Experimental NeurologyCitation Excerpt :Although voiding appears like a simple turn on and off action, it is achieved by a complex interaction of the somatic and autonomic nervous system (de Groat et al., 2015). Imaging studies in humans, and electrophysiological and viral tracing studies in animals have shown that the neuronal circuitry controlling lower urinary tract function involves the peripheral nervous system, spinal cord nuclei, brainstem structures, as well as multiple forebrain regions (Karnup and de Groat, 2020; Marson, 1997; Nadelhaft and Vera, 2001). An insult to the spinal cord that destroys large parts of the white matter will result in neurogenic lower urinary tract dysfunction (Schöps et al., 2015).
Spinal interneurons of the lower urinary tract circuits
2021, Autonomic Neuroscience: Basic and ClinicalCitation Excerpt :In this review we will concentrate on exclusively spinal LUT-related circuits and, in particular, on interneurons modulating activity of motoneurons (for EUS) and preganglionic neurons (for the bladder). We will also present some relevant data which were obtained in the course of previous work (Karnup and de Groat, 2020a; Karnup and De Groat, 2020b), but have not been published. Descending pathways relay PMC output signal to the lumbo-sacral spinal cord causing activation of the bladder parasympathetic neurons, inhibition of the motorneurons innervating the EUS (EUS-MNs) and modulation of activity of relevant spinal interneurons (reviewed in (de Groat et al., 2015; de Groat and Yoshimura, 2006)).