Elsevier

Neuroscience

Volume 425, 15 January 2020, Pages 12-28
Neuroscience

Research Article
Propriospinal Neurons of L3-L4 Segments Involved in Control of the Rat External Urethral Sphincter

https://doi.org/10.1016/j.neuroscience.2019.11.013Get rights and content

Highlights

  • External urethral sphincter-related (EUS-R) spinal neurons were labeled by transneuronal virus tracing.

  • One group of EUS-R neurons was identified in the L3-L4 spinal segments near the central canal.

  • Properties of L3-L4 EUS-R neurons were studied in spinal slices with patch clamp and intracellular filling methods.

  • EUS-R propriospinal neurons projecting into the ventral funiculus and interneurons were identified.

  • L3-L4 EUS-R neurons are thought to have a role in coordinating bladder and EUS function during voiding.

Abstract

Coordination of activity of external urethral sphincter (EUS) striated muscle and bladder (BL) smooth muscle is essential for efficient voiding. In this study we examined the morphological and electrophysiological properties of neurons in the L3/L4 spinal cord (SC) that are likely to have an important role in EUS-BL coordination in rats. EUS-related SC neurons were identified by retrograde transsynaptic tracing following injection of pseudorabies virus (PRV) co-expressing fluorescent markers into the EUS of P18-P20 male rats. Tracing revealed not only EUS motoneurons in L6/S1 but also interneurons in lamina X of the L6/S1 and L3/L4 SC. Physiological properties of fluorescently labeled neurons were assessed during whole-cell recordings in SC slices followed by reconstruction of biocytin-filled neurons. Reconstructions of neuronal processes from transverse or longitudinal slices showed that some L3/L4 neurons have axons projecting toward and into the ventro-medial funiculus (VMf) where axons extended caudally. Other neurons had axons projecting within laminae X and VII. Dendrites of L3/L4 neurons were distributed within laminae X and VII. The majority of L3/L4 neurons exhibited tonic firing in response to depolarizing currents. In transverse slices focal electrical stimulation (FES) in the VMf or in laminae X and VII elicited antidromic axonal spikes and/or excitatory synaptic responses in L3/L4 neurons; while in longitudinal slices FES elicited excitatory synaptic inputs from sites up to 400 μm along the central canal. Inhibitory inputs were rarely observed. These data suggest that L3/L4 EUS-related circuitry consists of at least two neuronal populations: segmental interneurons and propriospinal neurons projecting to L6/S1.

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)

  • W.C. de Groat et al.

    Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury

    Prog Brain Res

    (2006)
  • W.C. de Groat et al.

    Plasticity in reflex pathways to the lower urinary tract following spinal cord injury

    Exp Neurol

    (2012)
  • W.C. de Groat et al.

    Anatomy and physiology of the lower urinary tract

    Handb Clin Neurol

    (2015)
  • L. Deng et al.

    Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment

    Exp Neurol

    (2016)
  • A.D. Dobberfuhl et al.

    Identification of CNS neurons innervating the levator ani and ventral bulbospongiosus muscles in male rats

    J Sex Med

    (2014)
  • H. Gao et al.

    Morphological and electrophysiological features of motor neurons and putative interneurons in the dorsal vagal complex of rats and mice

    Brain Res

    (2009)
  • M.N. Kruse et al.

    Spinal pathways mediate coordinated bladder/urethral sphincter activity during reflex micturition in decerebrate and spinalized neonatal rats

    Neurosci Lett

    (1993)
  • I. Nadelhaft et al.

    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

    (2001)
  • S. Ratté et al.

    Impact of neuronal properties on network coding: roles of spike initiation dynamics and robust synchrony transfer

    Neuron

    (2013)
  • W.R. Reed et al.

    Inter-enlargement pathways in the ventrolateral funiculus of the adult rat spinal cord

    Neuroscience

    (2006)
  • M.D. Staudt et al.

    A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections

    J Sex Med

    (2012)
  • A.E. Stepien et al.

    Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells

    Neuron

    (2010)
  • K. Sugaya et al.

    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

    (1997)
  • X.Q. Sun et al.

    Spinal neurons involved in the control of the seminal vesicles: a transsynaptic labeling study using pseudorabies virus in rats

    Neuroscience

    (2009)
  • C. Xu et al.

    Identification of lumbar spinal neurons controlling simultaneously the prostate and the bulbospongiosus muscles in the rat

    Neuroscience

    (2006)
  • M. Yoshiyama et al.

    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

    (2000)
  • E.M. Abud et al.

    Spinal stimulation of the upper lumbar spinal cord modulates urethral sphincter activity in rats after spinal cord injury

    Am J Physiol Renal Physiol

    (2015)
  • J. Allard et al.

    Spinal cord control of ejaculation

    World J Urol

    (2005)
  • L. Anglister et al.

    Ascending pathways that mediate cholinergic modulation of lumbar motor activity

    J Neurochem

    (2017)
  • B. Alstermark et al.

    Motor command for precision grip in the macaque monkey can be mediated by spinal interneurons

    J Neurophysiol

    (2011)
  • C. Bellardita et al.

    Spatiotemporal correlation of spinal network dynamics underlying spasms in chronic spinalized mice

    Elife

    (2017)
  • J.D. Breton et al.

    Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord

    Mol Pain

    (2009)
  • H.-Y. Chang et al.

    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

    (2007)
  • H.H. Chang et al.

    Spinal cord stimulation ameliorates detrusor over-activity and visceromotor pain responses in rats with cystitis

    Neurourol Urodyn

    (2019)
  • C.L. Cheng et al.

    Effect of orchiectomy and testosterone replacement on lower urinary tract function in anesthetized rats

    Am J Physiol Renal Physiol

    (2016)
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