Elsevier

Neuropharmacology

Volume 170, 15 June 2020, 107815
Neuropharmacology

Serotonergic modulation of sacral dorsal root stimulation-induced locomotor output in newborn rat

https://doi.org/10.1016/j.neuropharm.2019.107815Get rights and content

Highlights

  • Locomotor program activation by sacral afferents depends on the developmental stage.

  • 5-HT modulation become required to activate the spinal locomotor network.

  • 5-HT2A receptors activation mediates the pro-locomotor action of 5-HT.

  • 5-HT2A receptors are already expressed in the spinal cord at birth in the rat.

Abstract

Descending neuromodulators from the brainstem play a major role in the development and regulation of spinal sensorimotor functions. Here, the contribution of serotonergic signaling in the lumbar spinal cord was investigated in the context of the generation of locomotor activity. Experiments were performed on in vitro spinal cord preparations from newborn rats (0–5 days). Rhythmic locomotor episodes (fictive locomotion) triggered by tonic electrical stimulations (2Hz, 30s) of a single sacral dorsal root were recorded from bilateral flexor-dominated (L2) and extensor-dominated (L5) ventral roots. We found that the activity pattern induced by sacral stimulation evolves over the 5 post-natal (P) day period. Although alternating rhythmic flexor-like motor bursts were expressed at all ages, the locomotor pattern of extensor-like bursting was progressively lost from P1 to P5. At later stages, serotonin (5-HT) and quipazine (5-HT2A receptor agonist) at concentrations sub-threshold for direct locomotor network activation promoted sacral stimulation-induced fictive locomotion. The 5-HT2A receptor antagonist ketanserin could reverse the agonist's action but was ineffective when fictive locomotion was already expressed in the absence of 5-HT (mainly before P2). Although inhibiting 5-HT7 receptors with SB266990 did not affect locomotor pattern organization, activating 5-HT1A receptors with 8-OH-DPAT specifically deteriorated extensor phase motor burst activity. We conclude that during the first 5 post-natal days in rat, serotonergic signaling in the lumbar cord becomes increasingly critical for the expression of fictive locomotion. Our findings therefore further underline the importance of both descending serotonergic and sensory afferent pathways in shaping locomotor activity during postnatal development.

This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

Introduction

The control of locomotor behaviors relies on dynamic interactions between the supraspinal locomotor centers (from the cortex to brainstem), the spinal central pattern generators (CPGs) for locomotion and segmental sensory afferent information. In rat, the spinal locomotor network begins to develop during the embryonnic stage, is functional at birth but continues to mature up to 3 weeks of age (Brocard et al., 1999a, 1999b; Cazalets et al., 1990; Clarac et al., 2004; Vinay et al., 2002). During this postnatal period, neuromodulatory pathways originating from the brain progressively descend along the spinal cord and mature to reach their adult pattern of innervation between the second and fourth post-natal weeks (Vinay et al., 2000). Consequently, the operation of the locomotor network evolves over time, as does the repertoire of behaviors it produces, including the expression of adult locomotion from two post-natal weeks (Altman and Sudarshan, 1975; Clarac et al., 2004).

The isolated neonatal rat spinal cord preparation provides the opportunity to study the central genesis of locomotor outputs during the first few post-natal days, since this preparation continues to generate in vitro an array of rhythmical motor outputs, including locomotor-like activities, that the CNS produces in vivo. Activation methods include exogenous application of neurotransmitters and neuromodulators (Barrière et al., 2005, 2004; Beliez et al., 2014; Cazalets et al., 1992; Cowley and Schmidt, 1994; Juvin et al., 2005; Kiehn and Kjaerulff, 1996; Kiehn et al., 1999; Smith and Feldman, 1987; Smith et al., 1988; Sqalli-Houssaini and Cazalets, 2000) and electrical stimulation of specific brainstem areas (Atsuta et al., 1990; Juvin et al., 2007; Oueghlani et al., 2018; Zaporozhets et al., 2004), descending pathways (Iwahara et al., 1991; Magnuson et al., 1995; Magnuson and Trinder, 1997) or segmental sensory afferent pathways (Juvin et al., 2012; Lev-Tov et al., 2000; Marchetti et al., 2001; Smith and Feldman, 1987). At the lumbar level, where the hindlimb CPG for locomotion is located (Cazalets et al., 1995), rhythmical alternations of left-right segmental motor bursts as well as of ipsilateral flexor-like and extensor-like motor bursts recorded from the second (L2) and fifth (L5) lumbar ventral roots respectively, are a major hallmark of so-called “fictive locomotion” (Cazalets et al., 1992; Kiehn and Kjaerulff, 1996).

Tonic electrical stimulations of one of the sacral/coccygeal dorsal roots that notably convey sensory afferent inputs from the tail in rodent, is one relevant method for activating the lumbar locomotor networks in the isolated neonatal rat spinal cord (Lev-Tov et al., 2000). Activation of these networks is mediated by a sacral relay pathway that projects rostrally through the ventral funiculi (Cherniak et al., 2017; Etlin et al., 2010, 2014; 2013; Strauss and Lev-Tov, 2003). This pathway is also probably involved in the pro-locomotor action of perineal stimulations below a spinal cord lesion in cat and rat (Alluin et al., 2015; Barbeau and Rossignol, 1987; Fouad et al., 2000). Until now, however, no developmental investigation of this sacral ascending control of the lumbar network has been carried out. Thus, whether this sensory derived activation modality of the lumbar networks remains capable of triggering episodes of fictive locomotion during the first few days after birth, while the spinal neuromodulatory environment is evolving rapidly, remains to be established.

In the present work, we investigated the temporal organization of the rhythmic lumbar motor outputs triggered by a tonic electrical stimulation of a single sacral dorsal root of isolated spinal cord preparations from neonatal (postnatal day (P) 0–5) rats (Cazalets et al., 1992; Smith and Feldman, 1987). Specifically, we focused on the motor outputs recorded simultaneously from the L2 and L5 segments that convey motor commands to mostly flexor and extensor muscles, respectively. We found that the nature of motor output pattern induced by the sacral stimulation varies as a function of the developmental stage. Moreover, our data provide evidence that over time, serotonin (5-HT)-mediated modulatory signaling in the spinal cord becomes necessary to convert non-locomotor output into a fully organized locomotory pattern.

Section snippets

Material and methods

Experiments were performed on Sprague Dawley newborn rats (n = 101) of either sex aged from 0 to 5 days (P0–P5). The repartition of all animals was as followed: 5-HT only experiments (n = 14), partitionned spinal cord experiments (n = 9), quipazine experiments (n = 10), ketanserin experiments (n = 18), LP-44 experiments (n = 9), SB269970 experiments (n = 13), 8-OH-DPAT experiments n = 16, immunohistochemistry (n = 12 including 3 for antibody testing). All procedures were conducted in accordance

Characterization of sacral dorsal root stimulation-induced rhythmic activity

Unilateral tonic electrical stimulation (2Hz for 30s, 1 to 5x threshold) of a single sacral dorsal root which subsequently will be referred to as sacral DR stimulation (Lev-Tov et al., 2000), was very effective in triggering rhythmic motor outputs in ventral roots at the lumbar level. Bilateral alternation of motor bursts recorded from the L2 segment (lL2/rL2) was systematically expressed, although ipsilateral flexor- (L2)/extensor-like (L5) motor burst alternation was mainly obtained in

Discussion

The present work investigated the generation of fictive locomotor episodes elicited by unilateral tonic electrical stimulation of a single sacral (S2) dorsal root. We show that during the first 2 post-natal days (P0–P2), sacral DR stimulation triggers a complete locomotor output in most of the preparations tested. After P2, however, the same stimulation protocol mostly induces partial burst patterns and/or with inappropriate, non-locomotor phase relationships. However, at these later stages,

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

The authors are very grateful to Dr John Simmers for his constructive comments on this manuscript and its editing. The authors also thank Anne Fayoux for taking care of the animals and her management of breeding. The authors are also grateful to Aslak Grinsted for the matlab wavelet coherence package (http://noc.ac.uk/using-science/crosswavelet-wavelet-coherence) and Philipp Berens for the circular statistics toolbox.

References (117)

  • J.R. Cazalets et al.

    Variability as a characteristic of immature motor systems: an electromyographic study of swimming in the newborn rat

    Behav. Brain Res.

    (1990)
  • F. Clarac et al.

    The maturation of locomotor networks

    Prog. Brain Res.

    (2004)
  • F. Clarac et al.

    Role of gravity in the development of posture and locomotion in the neonatal rat

    Brain Res Brain Res Rev

    (1998)
  • J.W. Commissiong

    Development of catecholaminergic nerves in the spinal cord of the rat

    Brain Res.

    (1983)
  • J.W. Commissiong

    The development of catecholaminergic nerves in the spinal cord of rat. II. Regional development

    Brain Res.

    (1983)
  • K.C. Cowley et al.

    A comparison of motor patterns induced by N-methyl-D-aspartate, acetylcholine and serotonin in the in vitro neonatal rat spinal cord

    Neurosci. Lett.

    (1994)
  • J.P. Cummings et al.

    Prenatal and postnatal development of lamina IX neurons in the rat thoracic spinal cord

    Exp. Neurol.

    (1984)
  • K. Fouad et al.

    Treadmill training in incomplete spinal cord injured rats

    Behav. Brain Res.

    (2000)
  • C.A. Hinckley et al.

    Spinal locomotor circuits develop using hierarchical rules based on motorneuron position and identity

    Neuron

    (2015)
  • M. Iizuka et al.

    Development of the spatial pattern of 5-HT-induced locomotor rhythm in the lumbar spinal cord of rat fetuses in vitro

    Neurosci. Res.

    (1998)
  • D.A. Jackson et al.

    Receptor subtypes mediating facilitation by serotonin of excitability of spinal motoneurons

    Neuropharmacology

    (1990)
  • L.M. Jordan et al.

    Descending command systems for the initiation of locomotion in mammals

    Brain Res Brain Res Rev

    (2008)
  • L.M. Jordan et al.

    Propriospinal neurons involved in the control of locomotion: potential targets for repair strategies?

    Prog. Brain Res.

    (2002)
  • B. Kerai et al.

    Effect of transient neonatal muscle paralysis on the growth of soleus motoneurones in the rat

    Brain Res Dev Brain Res

    (1995)
  • E.S. Landry et al.

    Differential effects of 5-HT1 and 5-HT2 receptor agonists on hindlimb movements in paraplegic mice

    Prog. Neuro Psychopharmacol. Biol. Psychiatry

    (2004)
  • A. Lev-Tov et al.

    Pattern generation in non-limb moving segments of the mammalian spinal cord

    Brain Res. Bull.

    (2000)
  • T. Maeshima et al.

    The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat

    Brain Res.

    (1998)
  • D.S. Magnuson et al.

    Long-duration, frequency-dependent motor responses evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord

    Neurosci. Lett.

    (1995)
  • L. Marlier et al.

    Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord

    Brain Res.

    (1991)
  • H. Myoga et al.

    Postnatal development of locomotor movements in normal and para-chlorophenylalanine-treated newborn rats

    Neurosci. Res.

    (1995)
  • K. Nakajima et al.

    Prenatal administration of para-chlorophenylalanine results in suppression of serotonergic system and disturbance of swimming movements in newborn rats

    Neurosci. Res.

    (1998)
  • H. Nishimaru et al.

    5-Hydroxytryptamine-induced locomotor rhythm in the neonatal mouse spinal cord in vitro

    Neurosci. Lett.

    (2000)
  • S. Ozaki et al.

    Development of locomotor activity induced by NMDA receptor activation in the lumbar spinal cord of the rat fetus studied in vitro

    Brain Res Dev Brain Res

    (1996)
  • M.R. Pranzatelli et al.

    Plasticity and ontogeny of the central 5-HT transporter: effect of neonatal 5,7-dihydroxytryptamine lesions in the rat

    Brain Res Dev Brain Res

    (1992)
  • N. Rajaofetra et al.

    Pre- and postnatal development of noradrenergic projections to the rat spinal cord: an immunocytochemical study

    Brain Res Dev Brain Res

    (1992)
  • O. Alluin et al.

    Inducing hindlimb locomotor recovery in adult rat after complete thoracic spinal cord section using repeated treadmill training with perineal stimulation only

    J. Neurophysiol.

    (2015)
  • D.M. Antonino-Green et al.

    Neurons labeled from locomotor-related ventrolateral funiculus stimulus sites in the neonatal rat spinal cord

    J. Comp. Neurol.

    (2002)
  • M. Antri et al.

    5-HT1A receptors are involved in short- and long-term processes responsible for 5-HT-induced locomotor function recovery in chronic spinal rat

    Eur. J. Neurosci.

    (2003)
  • M. Antri et al.

    Locomotor recovery in the chronic spinal rat: effects of long-term treatment with a 5-HT2 agonist

    Eur. J. Neurosci.

    (2002)
  • Y. Atsuta et al.

    Characteristics of electrically induced locomotion in rat in vitro brain stem-spinal cord preparation

    J. Neurophysiol.

    (1990)
  • M. Barbu-Roth et al.

    Why does infant stepping disappear and can it be stimulated by optic flow?

    Child Dev.

    (2015)
  • G. Barrière et al.

    Neuromodulation of the locomotor network by dopamine in the isolated spinal cord of newborn rat

    Eur. J. Neurosci.

    (2004)
  • M. Beato et al.

    Serotonin-induced inhibition of locomotor rhythm of the rat isolated spinal cord is mediated by the 5-HT1 receptor class

    Proc. Biol. Sci.

    (1998)
  • L. Beliez et al.

    Multiple monoaminergic modulation of posturo-locomotor network activity in the newborn rat spinal cord

    Front. Neural Circuits

    (2014)
  • P. Berens

    CircStat: a matlab toolbox for circular statistics

    J. Stat. Softw.

    (2009)
  • D. Blivis et al.

    Differential effects of opioids on sacrocaudal afferent pathways and central pattern generators in the neonatal rat spinal cord

    J. Neurophysiol.

    (2007)
  • R. Bos et al.

    Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2

    Proc. Natl. Acad. Sci. U.S.A.

    (2013)
  • M.R. Brumley et al.

    The serotonergic agonists quipazine, CGS-12066A, and alpha-methylserotonin alter motor activity and induce hindlimb stepping in the intact and spinal rat fetus

    Behav. Neurosci.

    (2005)
  • G. Brüning et al.

    Prenatal development of the serotonin transporter in mouse brain

    Cell Tissue Res.

    (1997)
  • A.M. Cabaj et al.

    Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats

    J. Physiol. (Lond.)

    (2017)
  • 1

    These authors contributed equally to this work.

    View full text