Serotonergic transmission after spinal cord injury.

J Neural Transm DOI 10.1007/s00702-014-1241-z

NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - REVIEW ARTICLE

Serotonergic transmission after spinal cord injury Raffaele Nardone • Yvonne Ho¨ller • Aljoscha Thomschewski • Peter Ho¨ller • Piergiorgio Lochner • Stefan Golaszewski • Francesco Brigo • Eugen Trinka

Received: 1 December 2013 / Accepted: 6 May 2014 Ó Springer-Verlag Wien 2014

Abstract Changes in descending serotonergic innervation of spinal neural activity have been implicated in symptoms of paralysis, spasticity, sensory disturbances and pain following spinal cord injury (SCI). Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury, including SCI. Current research suggests that serotonine (5-HT) release within the ventral horn of the spinal cord plays a critical role in motor function, and activation of 5-HT receptors mediates locomotor control. 5-HT originating from the brain stem inhibits sensory afferent transmission and associated spinal reflexes; by abolishing 5-HT innervation SCI leads to a disinhibition of sensory transmission. 5-HT denervation supersensitivity is one of the key mechanisms underlying the increased motoneuron excitability that occurs after SCI, and this hyperexcitability has been demonstrated to underlie the pathogenesis of spasticity after SCI. Moreover,

emerging evidence implicates serotonergic descending facilitatory pathways from the brainstem to the spinal cord in the maintenance of pathologic pain. There are functional relevant connections between the descending serotonergic system from the rostral ventromedial medulla in the brainstem, the 5-HT receptors in the spinal dorsal horn, and the descending pain facilitation after tissue and nerve injury. This narrative review focussed on the most important studies that have investigated the above-mentioned effects of impaired 5-HT-transmission in humans after SCI. We also briefly discussed the promising therapeutical approaches with serotonergic drugs, monoclonal antibodies and intraspinal cell transplantation.

R. Nardone Y. Ho¨ller A. Thomschewski P. Ho¨ller S. Golaszewski E. Trinka Department of Neurology, Christian Doppler Klinik, Paracelsus Medical University and Center for Cognitive Neuroscience, Salzburg, Austria

Introduction

R. Nardone (&) P. Lochner F. Brigo Department of Neurology, Franz Tappeiner Hospital, Via Rossini, 5, 39012 Merano (BZ), Italy e-mail: [email protected] R. Nardone Y. Ho¨ller A. Thomschewski P. Ho¨ller E. Trinka Spinal Cord Injury and Tissue Regeneration Center, Paracelsus Medical University, Salzburg, Austria F. Brigo Department of Neurological, Neuropsychological, Morphological and Movement Sciences, Section of Clinical Neurology, University of Verona, Verona, Italy

Keywords Serotonin Spinal cord injury Motor function Locomotion Spasticity Pain Monoclonal antibodies Cell therapy

Spinal cord injury (SCI) is a devastating condition, leading to loss of motor functions, including walking, especially caused by a damage to brain-derived axons that directly control voluntary limb movements through fast glutamate synaptic transmission (Thomas and Gorassini 2005; Jordan et al. 2008), and that represent the primary source of neuromodulators in the spinal cord, in particular serotonin (5-HT) and noradrenaline (NA) (Carlsson et al. 1963; Jacobs et al. 2002; Jordan et al. 2008). Cortico-, rubro-, vestibulo- and reticulospinal tracts and the important modulatory serotonergic, dopaminergic and noradrenergic fibre systems are interrupted by SCI. In particular, many of the neurons that normally coordinate rhythmic locomotor movements in humans and

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other mammals are located in the spinal cord (Grillner and Zangger 1979; Butt et al. 2002). These neurons require neuromodulators like 5-HT to function, setting them into a state of readiness for movement generation (Jacobs et al. 2002; Jordan et al. 2008). Thus, lack of 5-HT after SCI causes the spinal neurons caudal to the injury to remain in a state of relatively unexcitability. Numerous studies based on animal models illustrated that locomotor activity can be regained soon after spinal transection by means of the exogenous application of drugs that activate the neuromodulatory 5-HT, NA, and dopamine receptors (in vivo and in vitro) (Viala and Buser 1971; Cowley and Schmidt 1994; Chau et al. 1998; Kiehn and Kjaerulff 1996), including 5-HT2 and 5-HT7 receptor agonists (McEwen et al. 1997; Landry and Guertin 2004; Madriaga et al. 2004), or even transplants of 5-HT and NA producing cells into the spinal cord (Gimenez y Ribotta and Privat 1998; Ribotta et al. 2000), thus substantiating the critical relevance of these neuromodulators. An attempt to facilitate locomotion by administering different monoaminergic agonists revealed considerable modulation of particular parameters of hindlimb locomotion, especially after intrathecal injections of different specific serotonergic and noradrenergic agonists in the chronic phase after the SCI. Forelimb locomotion, in contrast, was found to be mostly unresponsive to these agonists. In this narrative review, we summarized the most important studies that have explored the effects of SCI on serotonergic transmission in humans. Moreover, we discussed the possible therapeutical implications by replacing or modulating this impaired neurotransmission.

Serotonin and raphe descending pathways Serotonergic pathways originate primarily at the raphe nuclei of the medulla oblongata, descend through the ventrolateral funiculus (VLF) and ventral funiculus (VF) to the ventral horn of the spinal cord and make to some extent contact with 5-HT-receptors of motoneurons (Skagerberg and Bjo¨rklund 1985; Holstege and Kuypers 1987; Sharma et al. 1997; Basbaum et al. 1988; Hornung 2003). Baseline levels at rest, during exercise, and post-exercise of 5-HT release in the period of motor function recovery following SCI have been assessed by means of treadmill exercise (Gerin et al. 2010). Stereotactical placement of microdialysis probes in laminae VII–IX of Rexed in the ipsilateral and caudal side to a lesion that was caused by sub-hemi-sections at the low spinal thoracic level allowed to sample 5-HT release over a month time period. Such a lesion interrupts the spinal neurons that project ipsilaterally from the medullary raphe magnus to

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the spinal cord (Skagerberg and Bjo¨rklund 1985; Basbaum et al. 1988) and which do not directly influence locomotor activity. In contrast, a lesion of lateral funiculus (LF) and VLF disrupts the medullary reticulospinal neuronal pathway running bilaterally within the ventral and lateral funiculi and terminating in laminae III–X, and leaves intact only fibres coming from the contralateral side and the medial part of the VF on the ipsilateral side, thus impairing the ipsilateral excitatory modulation on motoneurons. Moreover, serotonergic raphe spinal pathways from nuclei raphe obscurus and pallidus play a role in motor activity and terminate in the ventral horn which connects to motoneurons. The impairment of motor function modulation of the hindlimb ipsilateral to the lesion caused by disrupting this pathway hints that motor function and 5-HT release improvement after a lesion might be dependent upon neural reorganization within layers that have not been deprived of their original inputs, such as laminae X, VIII, the ventromedial part of lamina VI and the contralateral ventral horn. The lower general density of 5-HT fibers in the ipsilateral side suggests the involvement of regenerative compensatory release mechanisms such as increase of nonsynaptic junctions, or of 5-HT receptors onto motoneurons, interneuronal populations, and onto serotonergic terminals (Miller et al. 1996; Kim et al. 1999). 5-HT release during neural regeneration might play an important role in neuronal network re-arrangement, which later regulates locomotion. Variations of neurotransmitter release, especially of 5-HT, during long neurological recovery processes after a SCI can be monitored by microdialysis and detailed neuroanatomical studies. The results of Gerin et al. (2010) together with those of Dietz and Harkema (2004) suggest that in rats, within 2–3 weeks after SCI, locomotor exercise triggers neural plasticity and synaptic efficiency, and thus leads to a higher motor function 3–4 weeks after SCI (Kim et al. 1999; Gerin 2003). These effects can also be observed at a later point of time, in relation to the severity of the SCI. These results may explain the occasionally reported late recovery, even years after the SCI. Most importantly, these findings could yield therapeutical implications on future long-term chronic treatments of SCI patients such as administration of 5-HT release enhancing drugs, possibly in combination with treadmill exercise. In another recent study, Hentall and Gonzalez (2012) examined whether intermittent stimulation of the raphe magnus or its major midbrain input, the periaqueductal gray, influences recovery from incomplete SCI at thoracic level in rats. An increasing and lasting improvement of open-field motor performance and footprint and gridwalk performance can be observed after raphe magnus

Serotonergic transmission after spinal cord injury

stimulation, starting 1–2 h after a moderate weight-drop injury at T8 level. Additionally, the authors observed an increased number of myelinated axons in perilesional white matter and 5-HTcontaining terminals in gray matter, quantified 14 weeks after SCI. The periaqueductal gray, an established and safe stimulation target in man, was found to similarly facilitate the recovery of motor performance and myelination (but not serotonergic terminals) when stimulated for 4–7 days.

Serotonin mapping Monoamines are key regulators of motoneuron and spinal neural circuit excitability in the spinal cord. In intact animals, the density of 5-HT and tyrosine hydroxylase (TH)-positive fibres is known to be 2–5 times higher in the lumbar than the cervical spinal cord (Hadjiconstantinou et al. 1984; Colado et al. 1988; Filli et al. 2011). Filli et al. (2011) found that the 5-HT and THpositive fibres in the cervical and lumbar hemicord reduced remarkably after a cervical unilateral hemisection. The authors reported no restoration of the ipsilesional 5-HT and TH fibre plexus up to 4 weeks after the lesion; moreover, a further reduction of the 5-HT and TH signals was detected from 4 to 28 days after the lesion. These findings are in agreement with other studies (Bregman 1987; Golder et al. 2001), where C2 and mid-thoracic unilateral hemisection were performed, but are in contrast to a study with a unilateral T8 hemisection (Saruhashi et al. 1996). At variance with Marlier et al. (1991a, b) in rats and Laporte et al. (1996) in humans, Perrin et al. (2011) found a significant presence of 5-HT1A receptors in the human ventral horns, mostly at lumbar level. This discrepancy may be explained by the different techniques that were used (immunodetection versus binding); moreover, Laporte et al. (1996) analyzed only aged human spinal cords. Ventral horn 5-HT innervation is involved in the control of locomotion (Jacobs 1991; Jacobs and Fornal 1993). The mapping of the spinal cord 5-HT innervation and its receptors is necessary to determine their involvement in specific functions. Perrin et al. (2011) performed a preliminary mapping of serotonergic processes and 5-HTlA receptors in thoracic and lumbar segments of the human spinal cord. 5-HT profiles in human spinal cord are present in the ventral horn, surrounding motoneurons, and also contact their presumptive dendrites at lumbar level, which is similar to what is known about rodents and non-human primates. 5-HTlA receptors are detectable in the same area, but are more densely expressed at lumbar level. 5-HT profiles are also present in the intermediolateral cell column (IMLC),

where 5-HTlA receptors are absent. Finally, Perrin et al. (2011) observed numerous serotonergic profiles and high levels of 5-HTlA receptors in the superficial part (equivalent of Rexed lamina II) of the dorsal horn.

Serotonin and motor function Several factors point to a direct role of 5-HT modulation in motor activity: its activation in the commissural region; its release variations within the ventral horn related to locomotor activity (Gerin et al. 1994; Schwartz et al. 2005); its increased release within descending serotonergic pathways in the lumbar spinal cord after treadmill exercise (Gerin et al. 1995); the improved locomotion in spinalized cats after systemic administration of 5-HT2A,2B,2C agonists (Barbeau and Rossignol 1991; Miller et al. 1996); 5-HT reuptake inhibitor treatment in newborn rats leading to improvement in locomotion (Kim et al. 1999). Understanding the motor impairments and the possible functional recovery of upper and lower extremities is of great importance. However, animal models investigating motor dysfunction following cervical SCI are rare. By using kinematic analysis, Filli et al. (2011) were able to demonstrate that adult rats that undergo an unilateral C4/ C5 hemisection showed a disproportionately better spontaneous recovery of hindlimb locomotion compared with forelimb locomotion. Also in primates and humans it is possible to achieve spontaneous motor recovery of the ipsilateral lower limb after cervical unilateral hemisection, while recovery remains marginal or absent for the upper extremity. In primates responsiveness to monoaminergic drugs may be absent in the forelimb and higher, although mainly functionally negative, in the hindlimbs. During inhibition of sensory transmission, brain stemderived 5-HT and NE usually facilitate motoneuron function (Schmidt and Jordan 2000; Hultborn et al. 2004; Li et al. 2004a; Heckman et al. 2005; Perrier and DelgadoLezama 2005). This facilitation is mediated by 5-HT2 and a1-adrenergic receptors that lower the sodium spike threshold and facilitate voltage-dependent persistent inward currents (PICs), in particular both persistent calcium (Ca PIC) and sodium (Na PIC) currents. PICs are essential for normal motoneuron functioning, including sustained firing in response to synaptic inputs (Perrier and Hounsgaard 2003; Gilmore and Fedirchuk 2004; Heckman et al. 2005; Harvey et al. 2006a). After SCI, motoneurons are rendered acutely unexcitable. This is mainly due to a lack of brain stem-derived 5-HT and NE innervation which is necessary for normal motoneuron functioning (Li et al. 2004a; Heckman et al. 2005). This is especially the case if the SCI includes the VF and VLF which contain most of

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the 5-HT that innervate the ventral horn (Schmidt and Jordan 2000), and therefore the spinal cord becomes areflexic immediately after SCI, despite the exaggerated sensory afferent transmission. However, over weeks after SCI, in the so-called chronic spinal state, with the reemergence of large Ca PICs and Na PICs, motoneurons spontaneously regain their excitability. At that time, the exaggerated sensory transmission, especially the prolonged excitatory post-synaptic potentials (EPSP), trigger the PICs, which ultimately determine the occurrence of many-second lasting muscle spasms in humans (Gorassini et al. 2002; Norton et al. 2008) and rats (Bennett et al. 2004; Li et al. 2004a). In the last decade the understanding of spontaneous recovery of motoneuron functioning in subjects with chronic SCI has made significant progress (Gorassini et al. 2002; Bennett et al. 2004; Hultborn et al. 2004; Harvey et al. 2006b; Murray et al. 2010). In the weeks after spinal transection, 5-HT2 and a1-receptors on motoneurons become spontaneously active (Harvey et al. 2006b, Murray et al. 2010), due to ‘‘constitutive receptor activity’’, thus activity in the absence of 5-HT or any other ligand (Harvey et al. 2006b; Murray et al. 2010). In fact, such receptors have a tendency to become active without 5-HT presence. Recently they have been shown to be of functional relevance in brain areas where 5-HT is present, including the normal intact striatum and cortex (Gurevich et al. 2002; De Deurwaerdere et al. 2004). This spontaneous receptor activity leads to reemergence of large PICs that render the motoneurons permanently excitable (Harvey et al. 2006b; Murray et al. 2010). Similar plasticity is also likely to occur in the 5-HT1 receptors that normally inhibit sensory transmission, because 5-HT1 receptors can exhibit constitutive activity in single-cell cloned receptor systems (Selkirk et al. 1998). However, since general inhibition is not restored in the chronic state after SCI, and in particular the exaggerated long EPSPs that trigger muscle spasms remain in that state, this kind of plasticity might lack of functional relevance (Baker and Chandler 1987; Li et al. 2004a). Although after SCI animals and humans sometimes have substantial PICs facilitated by brainstem-derived 5-HT and NE (Gorassini et al. 2004; Udina et al. 2010; Murray et al. 2011a, b), these PICs do not cause uncontrolled motoneuron firing. In fact, motoneurons are directly hyperpolarized by postsynaptic inhibition arising from glycinergic and GABAergic neurons in the spinal cord and brain (Holstege and Bongers 1991; Jankowska 1992; Rekling et al. 2000; Nielsen et al. 2007). Thus, the postsynaptic inhibition appropriately terminates the voltage-dependent PICs (Bennett et al. 1998; Heckman et al. 2005). In contrast, partly because of loss of 5-HT and NE (Jankowska et al. 2000), making motoneuron firing PICs difficult to voluntarily terminate, there is a reduction in such

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postsynaptic inhibitory currents (Nielsen et al. 2007; Boulenguez et al. 2010).

Serotonine and locomotion Central pattern generator Animal models revealed that locomotion results from the activation of a spinal central pattern generator (CPG) (Grillner 2002). This CPG has been described in many animal species, including lampreys, cats, rodents, and other vertebrates (Grillner and Wallen 1985; Buchanan and Grillner 1987; Dietz and Harkema 2004). A spinal CPG may also exists in humans (Dimitrijevic et al. 1998). The CPG is conceptualized as self-sustained neural network, which produces locomotor-like neural activity, and is controlled and modulated by sensory afferents as well as supraspinal commands (Grillner and Wallen 1985). In rats, the CPG has been localized in the lumbar cord (Ribotta et al. 2000). The descending 5-HT system in lampreys (Harris-Warrick and Cohen 1985) and rats (Schmidt and Jordan 2000) was found to be involved in the activation of the spinal CPG. On the other hand, the spinal cord and particularly its ventral horns are profusely innervated by 5-HT in rats (Marlier et al. 1991a, b; Skagerberg and Bjo¨rklund 1985) and monkeys (Rajaofetra et al. 1992). Chronic microdialysis of the rat’s spinal cord has revealed a massive release of 5-HT during treadmill-driven locomotion (Gerin et al. 1995). Therefore, it is assumed that 5-HT plays a central role in motor control throughout phylogenies. The investigation of 5-HT neuron transplantation (Ribotta et al. 2000) revealed that 5-HT input to the CPG located at lumbar level in the rat is mandatory for locomotor functioning. Electrical stimulation of the lumbar cord in humans (Dimitrijevic et al. 1998; Minassian et al. 2007) leads to alternative leg movements in paraplegic patients, evidenciating the existence of a CPG in humans. Moreover, Antri et al. (2003) and Guertin (2008) observed that treatment with the 5-HT1A agonist 8-OH-DPAT elicited reflex locomotion on a treadmill in spinal-cordtransected rats. In summary, these findings suggest that 5-HT receptors—among other factors—could trigger the CPG in the lumbar spinal cord in humans. Recovery of locomotor function Rudimentary locomotor-like movements spontaneously emerge over the weeks after SCI, and improve with training (Kuhn and Macht 1948; Barbeau and Rossignol 1991; de Leon et al. 1998). This could lead to the idea that neuromodulatory receptor systems (in particular


5-HT2 receptors) are somehow re-activated in the absence of 5-HT. The exact mechanisms of compensation for lost brain stem 5-HT are still unknown, but might be related with certain isoforms of the 5-HT2 receptor, the abovementioned ‘‘constitutively active receptors’’ (Westphal and Sanders-Bush 1994; Herrick-Davis et al. 1999; Niswender et al. 1999; Chanrion et al. 2008). It is important to note that the constitutively active 5-HT2 receptor isoforms are upregulated with reduced 5-HT in cortical neurons (Gurevich et al. 2002), suggesting that loss of 5-HT after SCI might have similar effects. Indeed, this has been demonstrated for motoneurons after SCI (Murray et al. 2010). The spontaneous recovery of locomotor activity especially is important in animals and humans with incomplete SCI, that is, sparing part of the descending connection from the brain. These cases can learn to use these spared connections to produce substantial recovery of functional walking, especially with training (Barbeau et al. 2002; Thomas and Gorassini 2005; Wirz et al. 2005; Ballermann and Fouad 2006). It is known that lesioned corticospinal axons sprout above the level of SCI and form new connections, such as relaying descending inputs around the injury through spared propriospinal pathways (Fouad et al. 2001; Bareyre et al. 2004; Vavrek et al. 2006; Courtine et al. 2008). This process correlated well with recovery in animal models (Weidner et al. 2001; Ballermann and Fouad 2006). These spared and new connections become functional in case that the locomotor-related neurons below the SCI are ‘‘primed’’ to respond to the enhanced or recovered descending inputs. However, in most of the incomplete lesion models this hypothesis is difficult to be tested. In fact, it is difficult to disentangle recovery resulting from changes at the spinal level from plasticity in spared descending signals, including neuromodulatory 5-HT inputs. To address this issue, the staggered hemisection model of SCI (Jane et al. 1964; Courtine et al. 2008; Cowley et al. 2008) is ideal. In this model, most of the direct descending inputs, including 5-HT, are ablated, whereas spared local propriospinal neurons relay descending signals around the lesion site, unlike in transected animals. This allows spontaneous recovery of independent, fully functional locomotion in the absence of most 5-HT (Courtine et al. 2008; Cowley et al. 2008). A staggered hemisection injury model in rats has been performed because these rodents showed a loss of most descending axons, but also a robust recovery of locomotor function (Fouad et al. 2010). Immunolabeling for 5-HT showed little remaining 5-HT below the SCI. Moreover, locomotor ability was not correlated with the amount of residual 5-HT. Locomotion was not affected by blocking the 5-HT2 receptors with an intrathecal (IT) application of the neutral antagonist SB242084. This indicates that residual 5-HT below the injury does not contribute to

generation of locomotion. The application of SB242084 antagonizes the muscle activity induced by IT application of the 5-HT2 receptor agonists alpha-methyl-5-HT. Conversely, the blockade of the constitutively active 5-HT2 receptor with the potent inverse agonist SB206553 eliminates most hindlimb weight support and reduces the locomotor score in both hind legs, thus markedly impairing stepping. However, Fouad et al. (2010) reported that even in the most severely impaired animals, rhythmic sweeping movements of the hindlimb feet were still visible during forelimb locomotion. Thus, it is most likely that the 5-HT receptor antagonist SB206553 did not completely eliminate locomotor drive to the motoneurons or motoneuron excitability. Moreover, the same application of SB206553 had no effect on stepping in normal rats. Therefore, while normal rats can compensate for loss of 5-HT2 receptor activity after severe SCI, the constitutive activity in these 5-HT2 receptors is mandatory for locomotor recovery. We can conclude that the 5-HT2 receptors become constitutively active replace the spinal cord’s dependence on brain-stem-derived 5-HT and ultimately play an important role in walking and general muscle activity after SCI. This constitutive receptor activity has previously been demonstrated for motoneurons after SCI (Murray et al. 2010), and the results of Fouad et al. (2010) extend these findings to the involvement of constitutive receptor activity in the locomotor output. The constitutively active 5-HT2 receptors contribute to functional walking along with many other mechanisms, including other receptors (Jordan et al. 2008) and propriospinal relays (Courtine et al. 2008; Cowley et al. 2008). Bharne et al. (2011) investigated the effects of 5-HT transmission on induced neuronal regeneration in swissalbino mice. The animals were subjected to experimental SCI at thoracic (T 10–12) level by compression method and thereafter treated with ritanserin, a 5-HT antagonist, alone or in combination with alpha-melanocyte stimulating hormone (a-MSH). The motor function score (0–10) of each mouse was monitored prior to, and on days 1, 4, 7, 10 and 14 following SCI. Untreated animals almost completely regained hind limb motor function on day 14. Animals given a-MSH or ritanserin recovered already on day 10. Animals which were treated with both agents showed a similar degree of recovery on day 4. Histological examination of the spinal cord following the experimental SCI showed demyelination, necrosis and cyst formation. Treatment with ritanserin, alone and in combination with a-MSH, significantly prevented the tissue damage. Concluding, it is highly likely that the neurotropic and locomotor recovery activity of a-MSH may be potentiated by early antagonism of 5-HT(2A/2C) receptors. Depletion of 5-HT input can determine adaptive alterations of the 5-HT2C and 5-HT2A-receptor (R) function

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via changes in mRNA editing or protein expression, respectively. After a complete spinal cord transection at the thoracic (T10) level 5-HT2A-R mRNA expression may be upregulated below the site of SCI without signs of changes in 5-HT2C-R mRNA editing or expression (Navarrett et al. 2012). Therefore, it seems unlikely that 5-HT2C-R editing is regulated by extracellular 5-HT levels. Instead, it is more plausible that the editing process is just one of the ways in which excitability of motoneurons can be restored following SCI. To confirm this hypothesis, the influence of excitatory locomotor circuits on motoneurons in the thoracic spinal cord of rats needs further investigation. Locomotor recovery after SCI can be impaired by denervation-induced plastic changes and hyperexcitability of spinal motoneurons. To further investigate the plastic responses of locomotor network interneurons (INs) after SCI in an adult mouse SCI model, Husch et al. (2012) analyzed the effects of complete SCI on the intrinsic electrophysiological properties, excitability, and neuromodulatory responses to 5-HT in lumbar V2a spinal INs, which contribute to coordinate left–right alternation during locomotion. Despite a reduced input resistance, V2a Ins showed no significant changes in baseline excitability or action potential properties 4 weeks after SCI. However, V2a INs became 100- to 1,000-fold more sensitive to 5-HT. Immunocytochemical analysis revealed that SCI caused a coordinated loss of 5-HT fibers and the 5-HT transporter (SERT). Blocking SERT with citalopram in intact mice did not increase 5-HT sensitivity to the level seen after SCI. SCI also determined an increase in 5-HT2C R cluster number and intensity, suggesting that several plastic changes cooperate in increasing 5-HT sensitivity. It can be concluded that different components of the spinal neuronal network responsible for coordinating locomotion are differentially affected by SCI. In addition, the summarized results highlight the importance of understanding these changes when considering therapies targeted at functional recovery.

Serotonin and sensory function Descending brainstem systems innervating the spinal cord, especially those releasing 5-HT and NE, determine a marked inhibition of sensory transmission to spinal motoneurons and ascending tracts. The consequences of SCI are attenuation of both segmental reflexes and sensory perception (Lundberg 1982; Schmidt and Jordan 2000; Millan 2002; Yoshimura and Furue 2006). There is a direct inhibition by 5-HT and NE on sensory transmission. The inhibition acts by inhibitory Gi protein-coupled receptors, such as 5-HT1A, 5-HT1B, 5-HT1D, and a2-adrenergic

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receptors, on sensory afferent terminals (including lowthreshold group I and II muscle and skin afferents and high-threshold pain afferents) and/or excitatory spinal neurons that are involved in polysynaptic reflexes and ascending sensory transmission (Lundberg 1982; Jankowska et al. 1993, 1994; Manuel et al. 1995; Clarke et al. 1996, 2002; Singer et al. 1996; Di Pasquale et al. 1997; Rekling et al. 2000; Schmidt and Jordan 2000; Millan 2002; Li et al. 2004b; Yoshimura and Furue 2006; Jordan et al. 2008). Another indirect inhibition by 5-HT and NE targets at sensory transmission by activating excitatory Gqcoupled 5-HT2 and a1-adrenergic receptors located on inhibitory interneurons (but not on afferents). This facilitates inhibitory interneurons, such as those involved in group Ia reciprocal and Ib non-reciprocal inhibition (Jankowska et al. 2000; Hammar and Jankowska 2003) and pain transmission (Obata et al. 2004; Yoshimura and Furue 2006).

Serotonin and spasticity Spasticity complicates daily living in many individuals with SCI. After SCI, hyperreflexia and general spasticity are a common disturbing condition (Kuhn and Macht 1948; Ashby and McCrea 1987; Maynard et al. 1990; Noth 1991; Dietz and Sinkjaer 2007; Nielsen et al. 2007). These consequences may result from a disinhibition of brain stem 5-HT-mediated transmission especially if SCI includes the dorsal lateral funiculus (DLF), where most of the 5-HT innervation of the dorsal horn arises (Lundberg 1982; Heckman 1994; Taylor et al. 1999; Schmidt and Jordan 2000). Murray et al. (2011a, b) investigated this hypothesis in a rat model of SCI, where a pronounced muscle spasticity can be observed (Bennett et al. 1999, 2004), with similar characteristics to what is known about human muscles after injury (Kuhn and Macht 1948; Maynard et al. 1990). In this experimental model, SCI lead to the occurrence of unusually prolonged EPSPs on motoneurons, lasting up to 1 s. These EPSPs are triggered by lowthreshold cutaneous and muscle afferents (Baker and Chandler 1987). A similar exaggerated synaptic transmission was seen in spastic humans with SCI using motor unit recordings (Norton et al. 2008). In both rats and humans, spasms (long-lasting muscle contractions lasting several seconds) are initiated by long EPSPs, while before injury the same stimulation mainly determines inhibition of ongoing muscle activity (Bennett et al. 1999, 2004; Norton et al. 2008). Exogenously applied 5-HT or NE can inhibit muscle spasms in these rats (Li et al. 2004a), essentially replacing lost brain stem 5-HT. However, it is still unknown where this inhibition occurs (pre- or


postsynaptic) and which receptors are involved. It is reasonable to hypothesise that 5-HT1 (or 5-HT2) receptors could mediate this inhibition. To address these questions, Murray et al. (2011a) applied selective 5-HT receptor agonists while recording spasms and associated EPSPs. The target of this research was developing novel antispastic drugs to replace lost 5-HT innervation. The emergence of unusually long EPSPs in motoneurons play a crucial role in triggering long polysynaptic reflexes (LPRs) that produce muscles spasms. The authors found that EPSPs and associated LPRs recorded in vitro were inhibited by 5-HT1B or 5-HT1F receptor agonists, including zolmitriptan [5-HT1B/1D/1F and LY344864 (5-HT1F)], after chronic spinal transection in rats. The effects of 5-HT receptor agonists were highly correlated with their binding affinity only to 5-HT1B and 5-HT1F receptors. Zolmitriptan also inhibited the LPRs and general muscle spasms as revealed by in vivo recordings in awake, chronic spinal injured rats. The 5-HT1B receptor antagonists SB216641 and GR127935 and the inverse agonist SB224289 reduced the inhibition of LPRs by 5-HT1B agonists (zolmitriptan) while, when applied alone, they had no effect on the LPRs. This leads to the assumption that 5-HT1B receptors remain silent and do not adapt to chronic injury. After reducing EPSPs with zolmitriptan, a large glycinemediated inhibitory postsynaptic current (IPSC) becomes evident. Most importantly, zolmitriptan does not change motoneuron properties. Thus, 5-HT1B/1F agonists, such as zolmitriptan, can restore inhibition of sensory transmission after SCI without affecting general motoneuron function. This fiinding gives rise to the hope that such agonists may serve as a novel class of antispastic drugs. In a successive study, Murray et al. (2011b) aimed at evaluating which 5-HT receptor subtypes influence Ca PICs that aid functional recovery but also contribute to uncontrolled muscle spasms after SCI. In this study, spasms were quantified by recording the long-lasting reflexes (LLRs) on ventral roots in response to dorsal root stimulation. Ca PICs were quantified by intracellular recording in synaptically isolated motoneurons. The results showed that selective agonists to 5-HT2B and 5-HT2C receptors cause dose dependent increases in the LLRs and associated Ca PICs. On the other hand, selective antagonists to 5-HT2B (e.g., RS127445) and 5-HT2C (SB242084) receptors inhibit the agonist-induced increase in LLR. Moreover, specifically neutral antagonists at 5-HT2B/C receptors (e.g., RS127445) do not influence LLRs and CA PICS, thus suggesting that these receptors are not activated by residual 5-HT in the spinal cord. Inverse agonists (such as SB206553) that block constitutive activity at 5-HT2B or

5-HT2C receptors were found to markedly reduce the LLRs. Again, this finding further confirms the presence of constitutive activity in these receptors. Motoneuron hyperexcitability is a key mechanism in the pathogenesis of spasticity after SCI. In particular, denervation supersensitivity is thought to underlie the increased motoneuron excitability. 5-HT supersensitivity may be caused by 5-HT receptor up-regulation. To verify this hypothesis, Kong et al. (2011) investigated changes in levels of 5-HT2A receptor immunoreactivity (5-HT2ARIR) following a spinal transection in the sacral spinal cord of rats. No significant changes were found in 5-HT2AR-IR density in the spinal segments below the lesion in the motoneurons up to 16 h following the transection. After 1 day, the density levels of 5-HT2AR-IR increased in the motoneurons and their dendrites, and became 3.4-fold higher in spinalized rats than in sham-operated rats. The upregulation progressed until a maximal level was reached 28 days after SCI. Concurrently, 5-HT and 5-HT transporter expressions were down-regulated. We may conclude that the upregulation of 5-HT2ARs could at least partly underlie the development of 5-HT denervation supersensitivity in spinal motoneurons following SCI which in turn supports the view that this supersensitivity is important for the pathogenesis of the subsequent development of spasticity.

Serotonine and pain The spinal control of nociception in rats involves serotonergic projections to the dorsal horn through 5-HT receptors (Bardin et al. 2003; Suzuki et al. 2004; Kayser et al. 2007). Specific descending projections to lamina II are involved in controlling nocicepive transmission both in animals (Marlier et al. 1991a, b) and humans (Mayer 1984). 5-HT is relevant for transmission, processing, and control of nociceptive signals (Eide and Hole 1993; Millan 2002; Kayser et al. 2010). Most studies have focused on 5-HT1A, 5-HT1B/1D, 5-HT2A/2C and 5-HT3 receptors (Eide and Hole 1993; Oyama et al. 1996; Obata et al. 2000; Kayser et al. 2002, 2010; Ahn and Basbaum 2006; Colpaert 2006; Pichon et al. 2010), but the investigation of the exact contribution of other receptors, in particular the 5-HT7 receptors, has just begun. Emerging evidence indicates that upregulation of the 5-HT2A receptors of neurons in the dorsal horn promotes spinal hyperexcitation and impairs spinal l-opioid mechanisms during neuropathic pain. Aira et al. (2012) investigated the involvement of spinal glutamate receptors, including metabotropic glutamate receptors (mGluR) and NMDA, in 5-HT2A receptor-

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induced hyperexcitability after spinal nerve ligation (SNL) in rats. The authors found that high-affinity 5-HT2A receptor agonist methylamine hydrobromide (TCB-2) increased C-fiber-evoked dorsal horn potentials after SNL. This enhancement was prevented by a group I mGluR antagonist AIDA but not by a group II mGluR or a NMDA antagonist. Before SNL, pharmacological stimulation of 5-HT2A receptor with TCB-2 lead to rapid bilateral upregulation of mGluR expression in cytoplasmic and synaptic fractions of spinal cord homogenates, which was attenuated by protein kinase C inhibitor chelerythrine. In addition, the stimulation enhanced evoked potentials during co-stimulation of mGluR with 3,5-DHPG. In the examined animals, 5-HT2A receptors and mGluR1 were coexpressed in postsynaptic densities in dorsal horn neurons. After SNL, a bilateral upregulation of mGluR1 in 5-HT2AR-containing postsynaptic densities was observed. However, upregulation of mGluR1 in synaptic compartments was partially prevented by chronic administration of a selective 5-HT2AR antagonist, confirming that 5-HT2AR-mediated control of mGluR1 upregulation is triggered by SNL. Changes in thermal and mechanical pain thresholds following SNL were increasingly reversed over the days after injury by chronic 5-HT2AR blockade. Thus, 5-HT2AR may play a role in hyperexcitation and pain after nerve injury. Additionally, mGluR1 upregulation can be seen as a feedforward activation mechanism contributing to 5-HT2AR-mediated facilitation. To elucidate the role played by peripheral versus central 5-HT7 receptors in nociceptive processing, Brenchat et al. (2012) investigated the respective contribution of both receptors in the modulation of mechanical hypersensitivity under two different sensitizing neurogenic/neuropathic conditions. In the first set of experiments, administration of a selective 5-HT7 receptor agonist reduced capsaicininduced mechanical hypersensitivity dependent on the dose. In addition, it inhibited mechanical hypersensitivity secondary to capsaicin injection. In contrast, a dosedependent enhancement of capsaicin-induced mechanical hypersensitivity was observed after local intraplantar injection of the 5-HT7 receptor agonist. In a second set of experiments rats were submitted to neuropathic pain (spared nerve injury model). A significant inhibition of nerve injury-induced mechanical hypersensitivity was found after intraperitoneal as well as intrathecal administration of the same agonist. These studies provide evidence that, under experimental pain, activation of 5-HT7 receptors leads to antinociceptive effects at the level of the spinal cord as well as pronociceptive effects at the periphery. The antinociceptive effect mediated by central 5-HT7 receptors seems to predominate over the pronociceptive effect at the periphery when a selective 5-HT7

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receptor agonist is systemically administered. Still, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to pain facilitation after nerve injury remain unclear. In another study (Gu et al. 2011), intrathecal injection of a selective 5-HT3 receptor agonist induced spinal glial hyperactivity, neuronal hyperexcitability, and pain hypersensitivity in rats. Neuron-tomicroglia signalling via chemokine fractalkine, microglia to astrocyte signalling via the cytokine IL-18, astrocyte to neuronal signalling by IL-1b, and enhanced activation of GluN (NMDA) receptors in the spinal dorsal horn were detected. Moreover, brain-derived neurotrophic factorinduced descending pain facilitation was accompanied by enhanced expression of spinal cluster of differentiation molecule 11B (CD11B, CD11b) and glial fibrillary acid protein (GFAP) in the spinal dorsal horn. These effects were significantly prevented by functional blockade of spinal 5-HT3 receptors. Molecular depletion of the descending 5-HT system reduced upregulation of spinal CD11b and GFAP. Lin et al. (2012) have demonstrated that injection of fibronectin into the spinal dorsal column immediately after SCI inhibits the development of mechanical allodynia (but not thermal hyperalgesia) over an 8-month observation period following spinal cord dorsal column crush. Interestingly, fibronectin treatment blocked the reduction of 5-HT in the superficial dorsal horn. Therefore, treatment with fibronectin provides a potential therapeutic intervention to treat chronic pain following SCI.

Serotonine and therapy Pharmacological application of monoamines Local pharmacological application of monoamins (5-HT, dopamine, NA, or their agonists) has been shown to partially substitute missing global excitatory inputs. Animals of different species (including mice, rats, cats, and monkeys) that are completely spinalized showed remarkable transient activation of hindlimb central pattern generators and locomotion (Forssberg and Grillner 1973; Barbeau and Rossignol 1991; Fedirchuk et al. 1998; Feraboli-Lohnherr et al. 1999; Antri et al. 2003, 2005; Guertin 2004; Landry and Guertin 2004). From fish to men, monoaminergic bulbospinal projections are important for initiation and modulation of locomotion (Grillner 2003). After severe SCI, uvarious monoaminergic receptors are upregulated, which then can be pharmacologically targeted by locally applied monoaminergic drugs (Giroux et al. 1999; Lee et al. 2007; Hayashi et al. 2010). Powerful physiological effects in the hindlimb of animals with complete SCI were reported after application of monoaminergic agonists, but


very little is known about their potential in animals with partial SCI. Voluntary hindlimb locomotion was not improved by injection of direct 5-HT agonists after severe thoracic contusion injury in spinalized rats (Feraboli-Lohnherr et al. 1999; Antri et al. 2003, 2005). However, locomotor improvement was achieved by application of the 5-HT precursor 5-hydroxytryptophan (Hayashi et al. 2010). In cats, responses to monoaminergic agonists seem to depend on type and extent of the lesion (Brustein and Rossignol 1999). It has been hypothesised that postsynaptic receptor changes are induced at a certain threshold of denervation of monoaminergic fibres (Hayashi et al. 2010). Upregulation of lumbar 5-HT2C receptors was only seen after severe, but not moderate contusion injury in the thoracic spinal cord. Moreover, monoaminergic drugs applied after complete or large ventral spinal cord injury act exclusively on postsynaptic receptors. In contrast, for moderate lesions, the drugs can affect presynaptic (unlesioned terminals) and postsynaptic receptors. Since the function of these receptors could depend on their location (i.e. negative feedback role for presynaptic receptors), the differing pre-/postsynaptic receptor balance is likely to affect the pharmacological effects on locomotion. Obviously, there are fundamental differences between fore- and hindlimb spinal motor circuitries and their functional dependence on remaining descending inputs and exogenous spinal excitation. Future therapeutic strategies to improve upper and lower limb function in patients with incomplete cervical SCI could be designed according to these differences. An incomplete SCI results in profound impairments in reflex excitability and volitional strength, which contribute to loss of function. Human and animal models suggest that disturbed monoaminergic input, particularly 5HT, from supraspinal centers moderates impaired motor function following SCI. Thompson and Hornby (2013) investigated the effects of 5HT medications on motor function in individuals with chronic SCI. Srength, spasticity/spasms, and walking ability were assessed clinically in 12 individuals with chronic ([1 year) incomplete SCI following acute administration of either 8 mg cyproheptadine, a 5HT antagonist, or 10 mg escitalopram, a selective 5HT reuptake inhibitor (SSRI), in a double-blinded, randomized, crossover design. This study revealed that 5HT medications modulate both volitional and reflexive behaviors with little change in walking performance. 5HT antagonist medications depressed clinical measures of strength and spasticity/spasms whereas SSRIs augmented them. These results are consistent with the dysregulation of 5HT-sensitive spinal neurons following SCI. As a consequence, clinicians should be more aware of the motor consequences of 5HT medications.

Monoclonal antibodies A focal trauma to the brain or spinal cord is known to release several endogenous neurodestructive agents, resulting in adverse cellular reactions. Sharma and Sharma (2012) identifyed 5-HT, dynorphin A (Dyn A 1-17), nitric oxide synthase (NOS), and tumor necrosis factor-a (TNF-a) as potential neurodestructive signals in the CNS injury caused by trauma or hyperthermia. Using monoclonal antibodies (mAB) as countermeasures for these neurochemicals and other vasoactive agents results in neutralization of the above-mentioned agents, marked neuroprotection and increased posttraumatic repair mechanisms. Furthermore, a suitable combination of mAB, for example, NOS and TNF-a, when applied 60–90 min after trauma, was found to be capable to enhance neuroprotective ability and thwart cell and tissue injury after SCI. It seems that mAB could be a suitable therapeutic agents in CNS injuries to achieve neuroprotection and/or neurorepair. Rats were treated acutely by administration of a mAB raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after SCI. This treatment lead to improved neurological outcomes, which could be attributed to the reduced infiltration of neutrophils into the injured spinal cord. More recently, Geremia et al. (2012) demonstrated that neutrophil infiltration into the injured mouse spinal cord microenvironment could be reduced by anti-CD11d treatment. Additionally, the authors observed increased white matter sparing and reductions in myeloperoxidase (MPO) activity, reactive oxygen species, lipid peroxidation, and scar formation. The described improvement was associated with increased 5-HT immunoreactivity below the level of the lesion and improved locomotor recovery. Cell treatment Cell therapy to release antinociceptive agents near the site of the SCI is a logical next step in the development of treatment modalities for SCI. A few clinical trials, especially for chronic pain, have tested the potential of transplant of cells. In a number of animal studies, a 5-HT rat cell line (Eaton et al. 1997; Hains et al. 2001a, b, 2003a) or 5-HT raphe transplants (Feraboli-Lohnherr et al. 1997; Gimeńez Y Ribotta et al. 2000) has been used to ameliorate the functional impairments that may be associated with SCI. Supplemental cell therapy after SCI can create a spinal environment that can relieve local damage and promote regenerative responses in multiple axonal populations, including descending spinal 5-HT fibers (Ramer et al. 2004). In addition, cell therapy may reverse

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neuropathic pain by reducing hyperexcitability in the dorsal horn (Hains et al. 2003a). The severe contusive SCI model with a weight drop device has been employed to examine both motor dysfunction (van Meeteren et al. 2003; Gruner 1992; Eaton et al. 2008) and pain mechanisms (Lindsey et al. 2000; Hains et al. 2003b) in many studies. Among other mechanisms, these models induce changes in intraspinal biochemistry. These changes are due to loss of 5-HT modulation by the releasing interneurons in the spinal cord and a loss of supraspinal control of voluntary locomotor activity. On the other hand, examinations of denervation supersensitivity to 5-HT following SCI corroborate behavioral studies showing the effectiveness of 5-HT in reducing allodynia and hyperalgesia after SCI (Hains et al. 2003c). Intraspinal transplants of hNT2.19 cells, an immortalized human neuronal cell line which actively secretes 5-HT, were used to enhance 5-HT levels near lumbar motor pools (Eaton et al. 2008) and to improve motor function in the rat following severe contusive SCI. Eaton et al. (2012) found that some cell lines derived from the human neuronal NT2 cell line parentage, hNT2.19 and hNT2.17 lines, which synthesize and release 5HT and the neurotransmitters gamma-aminobutyric acid (GABA), respectively, potently and permanently reverse behavioral hypersensitivity near the spinal sensory cell centers of the lumbar dorsal horn. Most importantly, this reversal occurs without inducing tumors or other complications after grafting and, therefore, could provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain. In another study, a unique neuronal cell line that synthesizes and secretes/releases 5HT was isolated (Eaton et al. 2008). Hind paw tactile allodynia and thermal hyperalgesia induced by severe contusive SCI were potently reversed by lumbar subarachnoid transplant of differentiated cells. However, this treatment had no effect on open field motor scores, stride length, foot rotation, base of support, or gridwalk footfall errors associated with the SCI. The authors thus concluded that the human neuronal serotonergic hNT2.19 cells could be used as a biologic minipump for antinoception, but this would likely require intraspinal grafts for motor recovery. Myelin or axon growth inhibition Axonal growth and functional recovery could be promoted by several therapeutic approaches when they are initiated shortly after central nervous system injury. Wang et al. (2011) examined the efficacy of blockade of myelinderived inhibitors with soluble Nogo receptor (NgR1, RTN4R) decoy protein after spinal cord hemisection and contusion injury.

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In NgR1 decoy-treated animals raphe spinal axon density increased caudal to the injury, as detected by immunohistology and by positron emission tomography using a 5-HT reuptake ligand. Recovery from experimental refractory condition of chronic SCI is promoted by antagonizing myelin-derived inhibitors signaling with NgR1 decoy. In contrast with previous reports, Lee et al. (2010) reported that attenuating myelin or inhibition of axon growth fail to promote regeneration of injured axons and in particular 5-HT axon regeneration through a spinal cord transection. The authors analyzed raphe spinal 5-HT axon regeneration in mice deficient in two major myelin inhibitors, Nogo and MAG, and their common receptor NgR1 (or NgR). There was no significant enhancement of 5-HT axon regeneration beyond the injury site in either Nogo/ MAG/NgR1 triple mutants or NgR1 single mutants after a complete transection SCI. The authors assessed if class 3 Semaphorins that are expressed by glial fibrillary acidic protein (GFAP)-negative meningeal fibroblasts at the injury site contribute to the regeneration of 5-HT by analyzing mice deficient in PlexinA3 and PlexinA4, two key receptors for class 3 Semaphorins, with or without additional NgR1 deletion. However, they detected no enhanced regeneration of 5-HT or corticospinal axons in PlexinA3/ PlexinA4 double mutants or PlexinA3/PlexinA4/NgR1 triple mutants.

Serotonin and phrenic nerve Many cases of SCI occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. SCIrelated death is often an effect of impaired breathing. So that restoring respiratory activity is the ultimate ambition. After cervical SCI, the chondroitin sulfate proteoglycans (CSPGs), which are a key component of the perineuronal net (PNN), are up-regulated around phrenic motor neurons. CSPGs powerfully inhibit plasticity, sprouting and regeneration (Davies et al. 1997; Massey et al. 2006). Plasticity of spared tracts and restoration of limited activity to the paralyzed diaphragm can be promoted by digestion of these potently inhibitory extracellular matrix molecules with Chondroitinase ABC (ChABC). As a consequence, some function can be restored through increased regeneration of severed axons, as well as enhanced sprouting and/or improved conduction of spared fibers (Garcia-Alias et al. 2009; Hunanyan et al. 2010). Alilain et al. (2011) demonstrated that ChABC treatment in combination with application of a peripheral nerve autograft results in lengthy regeneration of 5-HT axons and other bulbospinal fibers with marked recovery of diaphragm function. Following recovery and initial transection of the


bridge, there occurs an unusual diaphragmatic EMG activity which is overall increased and tonic. Thus, a considerable remodelling of spinal cord circuitry after regeneration could be assumed. Next, the restored activity is completely eliminated, proving that regeneration is critical for the return of function. Overall, these results show how it could be possible to use plasticity of spared tracts and regeneration of essential pathways in order to profoundly restore function of a single muscle following debilitating CNS trauma. Following lateral hemisection at the level of the upper cervical spinal cord (C2), there is a rapid upregulation of the PNN related proteolgycans. This change can be detected in the vicinity of the lesion and distally at the level of the phrenic nucleus (C3–C6). An increase of the CSPGs far from the lesion site has been reported first in the deafferented dorsal column nuclei. Massey et al. (2006) reported a similar phenomenon around denervated motoneurons. Recently, it was discovered that the upregulation of CSPGs at the lesion site is governed by extravasation of a fibrinogen TGF-beta complex through the open blood– brain barrier which triggers inhibitory matrix release by reactive astrocytes. Following SCI at C(2) (SH hemisection model) phrenic activity progressively recovers. Functional recovery may be contributed by neuroplasticity in the postsynaptic expression of neurotransmitter receptors. Phrenic motoneurons express multiple 5-HT and glutamatergic receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH was unclear. In a recent study, Mantilla et al. (2012) were the first to systematically document changes in motoneuron expression of the neurotransmitter receptors involved in regulation of motoneuron excitability. Using quantitative realtime RT-PCR in LCM samples, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B in adult male rats. The time course of changes in 5-HT and glutamatergic receptors mRNA expression was determined up to 21 days after SH. Phrenic motoneuron expression of 5-HTR2a increased *8fold (relative to control) 14 days after SH, whereas NMDA expression increased *16-fold 21 days after SH.

Serotonin and autonomic functions In experimental animals as well as in humans especially after severe upper thoracic SCI an exaggerated spinal sympathetic activation often leads to autonomic dysreflexia. In order to investigate the contribution of 5-HT bulbospinal axons to the development of autonomic dysreflexia

after SCI, Cormier et al. (2010) induced episodic hypertension in rats with varying severity of SCI. The authors performed immunohistochemical analysis for 5-HT and choline acetyl transferase on T8–T12 spinal segments. They wanted to assess 5-HT-containing axons in the intermediolateral cell column (IMLC), and to identify sympathetic preganglionic neurons, respectively. Mean arterial pressure (MAP) was measured at rest and after colon distension-induced hypertension to test autonomic dysreflexia. In this study, the authors found a correlation between the magnitude of the pressor response seen after colon distension, SCI severity, and density of 5-HTimmunoreactive axons in the IMLC. Moreover, intrathecal administration of the 5-HT2A agonist dimethoxy-4iodamphetamine lead to a higher resting MAP and blocked colon distension-induced hypertension. In contrast, the 5-HT2A antagonist ketanserin decreased resting MAP and was permissive to the colon distension-induced pressor response. Thus, SCI-induced loss of 5-HT inputs into the spinal cord IMLC seems to be proportional to the pathogenesis of autonomic dysreflexia and hypotension seen after SCI. Therefore, sparing of 5-HT axons beyond a critical threshold may preserve cardiovascular regulation and prevent the development of autonomic dysreflexia.

Discussion and conclusions Serotonergic innervation of the spinal cord in mammals plays multiple roles in the control of motor, sensory, and visceral functions. In particular 5HT is naturally present in the dorsal and ventral horns of the spinal cord and in spinal pathways mediating nociceptive and motor function. The projections to the superficial layers of the dorsal horn, leading through non-synaptic projections, correspond to the nociceptive pathway, whereas the minority of synaptic contacts, present mostly in deepest layers, could be involved in locomotion. Following SCI, descending axons are known to sprout and form new connections. Neurons caudal to the injury are capable of rhythmic locomotor-related activity, that can form the basis for substantial functional recovery of walking even if there is substantial loss of crucial brain stem-derived neuromodulators like 5-HT. Yet, it has to be shown that the results obtained in the rat model are applicable to the human spinal cord, especially with respect to the ultrastructural organization of the spine. Understanding and promoting this plasticity in spared connections has been an important focus of spinal cord research. Recovery after SCI is influenced by brainstem regions with descending axons, thus presenting potential targets for treatment. Specifically, neurons in the hindbrain raphe

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magnus respond to sensory and chemical concomitants of trauma (e.g., pain, circulating cytokines) and release trophic substances (including 5-HT) in widespread spinal regions. However, the relationship between 5-HT release in the ventral horn and motor function recovery after SCI in exercising animals is still a matter of research. Specific neural pathways leading to motor function improvement need to be identified in order to administer specific corresponding neurochemical treatments to SCI patients. Additionally, we need further information about neuroplasticity of receptors expressed in a pool of motoneurons that is compromised by SCI to identify appropriate pharmacological targets to alter motoneuron excitability. In particular, determining the mechanism by which serotonergic fibers persist and sprout after a lesion could lead to therapeutic strategies for SCI. A recovery of motoneurons after spinal cord transection may depend on adaptive alterations of the serotonin 2C (5-HT2C) and 2A (5-HT2A) receptor function. 5-HT2B and 5-HT2C receptors on motoneurons become constitutively active after injury and ultimately contribute to recovery of motoneuron function and emergence of spasms. The results of the study of Fouad et al. (2010) demonstrate that functional benefits are not only related to plasticity or regeneration of descending input. But that they instead can be caused by changing the activity of receptors and neurons below the level of SCI. Enhancing functional recovery experimentally could focus with a clear design on altering 5-HT2 receptor activity, whether by direct 5-HT agonist application or 5-HT cell transplants to increase 5-HT receptor activity (Ribotta et al. 2000; Guertin 2004) or by inverse agonists or genetic manipulations to alter expression of constitutively active 5-HT receptors, or other relevant receptor systems, like 5-HT7 receptors. Recent studies indicate that the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation. There are cellular and molecular mechanisms at the spinal level which are responsible for descending 5-HTmediated pain facilitation during the development of persistent pain after tissue and nerve injury. A future target of new pain therapies should be in future studies the descending facilitation-induced glial involvement, and in particular the blocking of intercellular signalling transduction between neurons and glia (Gu et al. 2011). Numerous neurochemicals and other vasoactive agents actively contribute the development of posttraumatic brain pathology and/or repair mechanisms. For example, mAB could represent suitable therapeutic agents to achieve neuroprotection and/or neurorepair in CNS injuries. A promising approach to treat pain and paralysis after SCI is

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the transplantation of cells which produce 5-HT biologic agents that can modulate the sensory and motor responses. Interestingly, 5-HT receptors located on phrenic motoneurons also play a critical role in the neuroplasticity following SCI. Thus, a human 5-HT neuronal cell line that can restore the function(s) of a damaged spinal cord, and be genetically manipulated, stored, and expanded would have the potential to be extremely useful for clinical applications. In rats, for example, a substitutive transplantation of 5-HT neurons or regeneration under the trophic influence of grafted stem cells can reduce the functional consequences of SCI at thoracic level. Local specific therapies could involve cellular and/or pharmacological tools targeting the serotonergic system. Furthermore, several observations suggest that raphe magnus neurons mediate restorative feedback in acute SCI. For possible treatments in humans, they could be activated directly or indirectly (via periaqueductal gray).After nervous system injury, 5-HT is not likely to be present in adequate amounts to effectively modulate the sensory/ motor imbalance that induces neuropathic pain and motor impairments. Therefore, replacement or supplementation of endogenous 5-HT for sensory and motor recovery may be a reasonable approach. In fact, its loss after SCI is dependent on injury severity (Novakovic et al. 1998) and correlates with loss of motor function (Young 1996) and the alterations in the sensory system that provide an environment conducive of neuropathic pain (Yaksh and Wilson 1979). In conclusion, we summarized the most important studies that have highlighted the critical role of 5-HT transmission in the restoration of the impaired functions after SCI. 5-HT, together with other neurochemicals, or serotoninergic drugs may promote posttraumatic repair mechanisms. Monoclonal antibodies and cell therapy also represent promising therapeutical approaches. Further studies are warranted to corroborate and extend the initial findings.

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Interactive Effects Between Exercise and Serotonergic Pharmacotherapy on Cortical Reorganization After Spinal Cord Injury.

Urethral hypotonicity after suprasacral spinal cord injury.

Percutaneous suprapubic cystostomy after spinal cord injury.

Lymphocytes and autoimmunity after spinal cord injury.

Prevention of thromboembolism after spinal cord injury.

Intrathecal Pressure After Spinal Cord Injury.

Urodynamic patterns after traumatic spinal cord injury.

Electrophysiologic changes in lumbar spinal cord after cervical cord injury.

Trigemino-cervical-spinal reflexes after traumatic spinal cord injury.

Effects of serotonergic medications on locomotor performance in humans with incomplete spinal cord injury.

Curcumin improves the integrity of blood-spinal cord barrier after compressive spinal cord injury in rats.

Review of Epidural Spinal Cord Stimulation for Augmenting Cough after Spinal Cord Injury.

Spinal cord transplants enhance the recovery of locomotor function after spinal cord injury at birth.

Spinal cord injury rehabilitation.

Spinal cord injury rehabilitation.

Dopamine is produced in the rat spinal cord and regulates micturition reflex after spinal cord injury.

Spinal cord injury pain.

Acute spinal-cord injury.

Screening for neuropathic pain after spinal cord injury with the spinal cord injury pain instrument (SCIPI): a preliminary validation study.

Cecal bascule after spinal cord injury: A case series report.

Colonoscopy after spinal cord injury: a case-control study.

Comprehensive assessment of walking function after human spinal cord injury.

Exercise awareness and barriers after spinal cord injury.

Bimanual reach to grasp movements after cervical spinal cord injury.