Descending bulbospinal pathways and recovery of respiratory motor function following spinal cord injury

Respiratory Physiology & Neurobiology 169 (2009) 115–122

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Descending bulbospinal pathways and recovery of respiratory motor function following spinal cord injury夽 Stéphane Vinit a,∗ , Anne Kastner b,1 a b

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive, Madison, WI 53706-1102, USA Université Paul Cézanne Aix-Marseille III, UMR-CNRS 6231, CRN2 M, Avenue Escadrille Normandie Niemen, F-13397 Marseille Cedex 20, France

a r t i c l e

i n f o

Article history: Accepted 6 August 2009 Keywords: Bulbospinal respiratory pathways Phrenic response Premotor neurons

a b s t r a c t The rodent respiratory system is a relevant model for study of the intrinsic post-lesion mechanisms of neuronal plasticity and resulting recovery after high cervical spinal cord injury. An unilateral cervical injury (hemisection, lateral section or contusion) interrupts unilaterally bulbospinal respiratory pathways to phrenic motor neurons innervating the diaphragm and leads to important respiratory defects on the injured side. However, the ipsilateral phrenic nerve exhibits a spontaneous and progressive recovery with post-lesion time. Shortly after a lateral injury, this partial recovery depends on the activation of contralateral pathways that cross the spinal midline caudal to the injury. Activation of these crossed phrenic pathways after the injury depends on the integrity of phrenic sensory afferents. These pathways are located principally in the lateral part of the spinal cord and involve 30% of the medullary respiratory neurons. By contrast, in chronic post-lesion conditions, the medial part of the spinal cord becomes sufficient to trigger substantial ipsilateral respiratory drive. Thus, after unilateral cervical spinal cord injury, respiratory reactivation is associated with a time-dependent anatomo-functional reorganization of the bulbospinal respiratory descending pathways, which represents an adaptative strategy for functional compensation. © 2009 Elsevier B.V. All rights reserved.

1. Descending respiratory bulbospinal pathways to phrenic motor neurons Descending bulbospinal respiratory pathways innervate phrenic motor neurons located at the C3–C6 cervical level, which control the diaphragm in a somatotopic pattern (Mantilla and Sieck, 2008; Gordon and Richmond, 1990). Respiratory bulbospinal pathways also project to respiratory motor neurons at thoracic and lumbar levels controlling intercostal and abdominal muscles (Hilaire and Monteau, 1997). The respiratory premotor neurons are located in the brainstem, mostly in the rostral ventral respiratory group (rVRG) (Dobbins and Feldman, 1994; Onai et al., 1987). In addition to the rVRG, a dorsal respiratory group (located in the NTS area) is also an important respiratory center in some species (for instance in cats), but its contribution is minor in rodents (Hilaire and Pasaro, 2003; Monteau et al., 1989; Hilaire et al., 1990, Ellenberger et al., 1990). Respiratory premotor neurons receive inputs from the pre-Bötzinger complex in the medulla, considered

夽 This paper is part of a special issue entitled ‘Spinal cord injury—Neuroplasticity and recovery of respiratory function’, guest-edited by Gary C. Sieck and Carlos B. Mantilla. ∗ Corresponding author. Tel.: +1 608 263 5013; fax: +1 608 263 3926. E-mail addresses: [email protected] (S. Vinit), [email protected] (A. Kastner). 1 Tel.: +33 4 91 28 84 54; fax: +33 4 91 28 83 33. 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.08.004

to be a key respiratory rhythm generator (Gray et al., 2001; Hilaire and Pasaro, 2003; Tan et al., 2008). This feature represents a major difference between the respiratory motor system and the locomotor system. Thus, whereas for the locomotor system the central pattern generator (CPG) and premotor neurons are located in the spinal cord, the CPG and premotor neurons for respiration are located in the medulla. In addition, significant afferent feedback for respiration (e.g., lung mechanoreceptors and peripheral chemoreceptors) affect medullary premotor neurons, whereas significant proprioceptive feedback for the locomotor system affects premotor neurons in the spinal cord. According to anterograde and retrograde tracing analysis and electrophysiolgical cross-correlation analysis, respiratory neurons project to the phrenic motoneurons principally by direct pathways (Ellenberger et al., 1990; Ellenberger and Feldman, 1988; Yamada et al., 1988; Lipski et al., 1994; Dobbins and Feldman, 1994; Boulenguez et al., 2007; Onai et al., 1987; Tian and Duffin, 1996a,b; Juvin and Morin, 2005), in contrast to corticospinal and rubrospinal neurons that project principally to propriospinal premotor neurons in rodents (Courtine et al., 2007; Kastner and Gauthier, 2008). Most respiratory premotor neurons project bilaterally to the spinal cord. Thus, after application of an anterograde tracer in the rVRG area on one side, anterograde labelled respiratory axons are observed in both sides of the spinal cord, although ipsilateral projections appear denser than contralateral ones (Lipski et al., 1994; Ellenberger et al., 1990; Feldman et al., 1985). The bilateral projections of respiratory

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axons are also shown by the bilateral labelling of the rVRG neurons obtained after injection of retrograde tracer into one phrenic area (Onai et al., 1987; Boulenguez et al., 2007), indicating that respiratory neurons travel on both sides of the spinal cord. The descending respiratory pathways from the medulla are located in the lateral and ventromedial funiculi of the cervical cord, as shown by anterograde tracing analysis (Feldman et al., 1985; Lipski et al., 1994; Lane et al., 2008a,b). However, a unilateral C2 lesion of the spinal cord restricted to the ventrolateral area, which leaves the ventromedial area intact, is sufficient to abolish ipsilateral phrenic nerve (PN) and hemidiaphragm activity (Vinit et al., 2006) indicating that PN activity in intact rats is controlled principally by these lateral pathways. According to anterograde tracing, medial respiratory fibers appear to project preferentially to intercostal motor neurons (in cats) (Feldman et al., 1985), although axon collaterals connected to phrenic motor neurons have also been reported in rats (Lipski et al., 1994). Although direct monosynaptic projections from the rVRG neurons to phrenic motor neurons may dominate, it has been shown that some bulbospinal neurons project to upper-cervical inspiratory neurons (Tian and Duffin, 1996a) and that respiratory phrenic motor neurons may also be innervated by cervical interneurons present at different locations in the cervical spinal cord (Lipski et al., 1993, 1994; Tian and Duffin, 1996b; Lane et al., 2008b; Juvin and Morin, 2005; Lu et al., 2004). Some cervical interneurons may act as phrenic premotor neurons (Lipski et al., 1994, 1993; Tian and Duffin, 1996a; Lu et al., 2004; Qin et al., 2002; Lane et al., 2008a,b), but these interneurons do not appear to mediate inspiratory drive in the absence of supraspinal descending inputs (Vinit et al., 2006; Gauthier et al., 2006). This is different from the locomotor system in which central pattern generators (CPG), consisting of a network of phasically active spinal interneurons, directly innervate local motor neurons and evoke rhythmic muscle contractions (Rossignol et al., 2008). A feature of the respiratory network is that phrenic motor neurons receive inputs from contralateral descending pathways crossing the spinal midline at the C3–C6 cervical level, designated “crossed phrenic pathways” (Goshgarian et al., 1991; Goshgarian, 2003; Moreno et al., 1992; Boulenguez et al., 2007). According to the current model, such crossed phrenic pathways are normally not of adequate strength to drive phrenic motor neurons, but can be activated in some specific conditions of respiratory stress (see next section). These crossed phrenic pathways are located mainly in the lateral part within the cervical spinal cord. Indeed, their contribution to PN activity is completely suppressed by a C1 /C2 section restricted to the lateral side (Vinit et al., 2007) and moreover a phrenic response in hemisected rats can be evoked by stimulation in the C2 ventrolateral funiculus of the contralateral side (Ling et al., 1995). Therefore, these crossed phrenic pathways may follow in line with the main descending pathways that innervate contralateral phrenic motor neurons. An important question is whether the innervation of phrenic motor neurons by such crossed phrenic pathways involves direct projections of respiratory axons running contralaterally to the lesion and crossing the spinal cord midline at the level of the phrenic nuclei. Axons crossing the midline at the cervical level were indeed observed using anterograde labelling from the rVRG (Goshgarian et al., 1991) but not in another study using intracellular injection of neurobiotin in rVRG neurons (Lipski et al., 1994). Injection of a monosynaptic retrograde tracer (fluorogold) in the phrenic area beneath a spinal hemisection (at C2 level) lead to the labelling of 30% of the contralateral rVRG neurons (Boulenguez et al., 2007). In these experiments, the tracer did not diffuse across the midline or across the lesion site, excluding the possibility that it may be captured by respiratory axons innervating contralateral phrenic motor neurons or by axotomized neurons. The labelled neurons may therefore correspond to rVRG neurons that project to

the ipsilateral phrenic motor neurons by direct contralateral pathways crossing the spinal cord at the phrenic level (Boulenguez et al., 2007). In another study (Moreno et al., 1992), injection of a transynaptic tracer (WGA-HRP) in the paralysed hemidiaphragm in hemisected rats resulted in the labelling of rVRG neurons. The labelled rVRG premotor neurons in this study may also putatively represent neurons connected to ipsilateral phrenic motor neurons by direct crossing axons, but, since the tracer was transynaptic, one cannot exclude the possibility of indirect pathways from these labelled neurons (via cervical interneurons for instance). Moreover, it has been shown that some phrenic motor neurons exhibit dendrites crossing the midline (Lindsay et al., 1991; Prakash et al., 2000; Boulenguez et al., 2007) that may receive respiratory inputs from non-crossing contralateral axons. 2. Respiratory motor function following acute or sub-chronic SCI Traumatic spinal cord injuries (SCI) in humans can lead to devastating motor and autonomic disabilities, one of which is impaired breathing (Zimmer et al., 2008; Winslow and Rozovsky, 2003). Such ventilatory deficits are particularly severe in persons with injuries at the cervical level, which are frequently encountered in humans (Winslow and Rozovsky, 2003). Respiratory failure is the principal cause of mortality and morbidity after such cervical injury (DiMarco, 2005) and the survival of patients with respiratory insufficiency often requires mechanical ventilatory assistance (Oo et al., 1999), at least during the acute phase. Rodents models are employed in most studies of SCI effects and treatments. High cervical spinal cord hemisection is the classic paradigm to analyze respiratory deficits and recovery in rodents (Zimmer et al., 2008; Kastner and Gauthier, 2008; Lane et al., 2008a). Hemisection interrupts respiratory pathways projecting to phrenic motor neurons on the injured side, leading to complete ipsilateral hemidiaphragmatic paralysis. Because bulbospinal respiratory pathways are bilateral, the contralateral pathways remain intact. Thus, the hemisected animals can maintain adequate respiratory function for survival (Golder et al., 2003; Polentes et al., 2004; Zimmer et al., 2008). Indeed, cervical spinal cord hemisection in rats does not affect minute ventilation due to compensatory mechanisms that increase breathing frequency (Goshgarian et al., 1986; Fuller et al., 2006), although ventilatory deficit can be revealed during conditions of respiratory stress (Fuller et al., 2008). Creating a lesion that will occupy less space within the spinal cord can increase morphological and cellular mechanisms contributing to compensatory post-lesion plasticity in this model, as demonstrated in the locomotor system (Schucht et al., 2002). Therefore, we have developed a model of injury restricted to the lateral part of the cervical spinal cord, thereby sparing the whole medial region (Vinit et al., 2006). This experimental model of incomplete unilateral section is sufficient to abolish ipsilateral phrenic nerve (PN) and hemidiaphragm activity (Vinit et al., 2006) and offers the advantage of preserving several critical functions such as locomotor and sensory functions. Moreover, the spared tissue (medial and contralateral part of the spinal cord) still contains uninjured respiratory pathways that could putatively play a role in the recovery of ipsilateral PN activity. These features validate the use of the lateral cervical section model to study post-lesion processes that may rely on spared tissue. One may wonder whether these rodent models of SCI reflect the clinical physiopathology of SCI in humans (Courtine et al., 2007; Kastner and Gauthier, 2008; Lane et al., 2008a). Complete or partial sections of the spinal cord are almost never encountered in humans. Therefore, these rodent models may not be the most relevant for translation to humans, and contusion models may be more representative of human injuries. Cervical contusion

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models developed in rats have been shown to lead to a substantial impairment of ipsilateral diaphragmatic function, although such deficits are not as complete as in the case of a lateral section (El Bohy et al., 1998; Baussart et al., 2006). This contusion model may be appropriate for analysing potential treatments that have been shown to be effective after cervical hemisection (Gauthier et al., 2002; 2006; Polentes et al., 2004; Li et al., 2003). Thus, both contusion injury and C2 hemisection may be valid and complementary models for human cervical injuries. The respiratory system is endowed with a substantial level of neuroplasticity (Baker-Herman et al., 2004; Morris et al., 2003; Feldman et al., 2003; Mitchell and Johnson, 2003). A well-studied process of post-lesion plasticity is the “crossed phrenic phenomenon” (CPP) (Porter, 1895; Goshgarian, 2003; Mitchell and Johnson, 2003). It designates, in animals with a unilateral cervical SCI, the partial recovery of the ipsilateral phrenic motor neuron activity reported under specific experimental conditions, through inputs from contralateral descending respiratory pathways crossing the midline at the level of the phrenic nuclei (“crossed phrenic pathways”, see previous section) (Goshgarian et al., 1991; Moreno et al., 1992; Boulenguez et al., 2007). The expression of the CPP in hemisected animals is normally inhibited, in part via a GABA-A mediated receptor mechanism (Zimmer and Goshgarian, 2007) that may operate at the level of phrenic motor neurons or premotor neurons. The activation of the CPP is dependent on serotonergic transmission (Ling et al., 1994; Zhou et al., 2001) and requires an increase in the central respiratory drive induced by a respiratory stress, such as contralateral phrenicotomy (performed several hours after cervical hemisection) (Lewis and Brookhart, 1951; Goshgarian, 2003) or chronic intermittent hypoxia (Fuller et al., 2003; see Vinit et al. in this issue for a review). Although crossed phrenic pathways are located mainly in the lateral part within the contralateral cervical spinal cord, CPP intensity may be reinforced by regulatory pathways located medially in the spinal cord, since the CPP evoked by contralateral phrenicotomy in hemisected mice is stronger if the hemisection spares the ventromedial funiculus (Minor et al., 2006). Activation of the CPP in hemisected rats also occurs spontaneously several days following SCI in the absence of respiratory stress (i.e. contralateral phrenicotomy), since a partial restoration of ipsilateral PN activity has been reported (Vinit et al., 2007; Fuller et al., 2006, 2008; Golder and Mitchell, 2005). However, although neuromuscular transmission has been found to be improved after a C2 hemisection (Mantilla et al., 2007; Prakash et al., 1999; Rowley et al., 2005; Miyata et al., 1995), an hemidiaphragm EMG activity has never been detected after a short post-lesion time lapse (Vinit et al., 2006; Miyata et al., 1995; Nantwi et al., 1999; Mantilla et al., 2007; Alilain and Goshgarian, 2008). This apparent discordance between PN activity and diaphragm recorded activity is not clearly understood but it is possible that, due to the low level of PN activity, some weak diaphragm activity may have been missed in the mentioned studies. After cervical unilateral section, the affected hemidiaphragm will sustain both a severe reduction of motor neuron inputs and the mechanical loading imposed by the residual activity of contralateral hemidiaphragm; this latter may however not have a major physiological incidence on the affected hemidiaphragm, since it has been shown the recorded length changes in the paralyzed (denervated) hemidiaphragm did not result from continued contractions of the contralateral side but rather from motor denervation (Zhan et al., 1995). The diaphragm contains very few muscle spindles compared to limb muscle. Despite the paucity of diaphragmatic afferent feedback, several studies in uninjured animals have shown that stimulation of sensory afferents within the PN can elicit a ventilatory response (Hussain et al., 1990; Yu and Younes, 1999; Road, 1990; Butler et al., 2003). Although such afferent feedback has a limited role during normal breathing, it may play a more

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significant role in conditions of respiratory stress (Forster, 2003; Erickson et al., 1994; Corda et al., 1965; Teitelbaum et al., 1993; Jammes and Balzamo, 1992). In rats with a unilateral cervical C2 section, removal of phrenic sensory afferents by an acute (Vinit et al., 2007) or chronic (Golder et al., 2003) ipsilateral PN section completely suppressed the PN restorative activity; This effect of ipsilateral phrenicotomy was prevented additionally by removal of the dorsal root ganglia (Vinit et al., 2007), indicating that it may be linked to the axotomy of sensory afferents rather than the axotomy of motor neurons. In addition, muscle paralysis by nicotinic receptor inhibition that will indirectly inactivate sensory afferent pathways, leads to an effect similar to ipsilateral phrenicotomy, i.e. a complete decline of ipsilateral PN activity (Vinit et al., 2007). These data support the view that CPP activation may depend on the activity of ipsilateral phrenic afferents. In fact, ipsilateral and contralateral phrenic afferents may have opposite modulatory effects on CPP activity. Thus, an acute dorsal rhizotomy at the contralateral cervical level a few days after the cervical C2 hemisection is sufficient to induce the CPP (Goshgarian, 1981). It suggests that the induction of CPP activity by contralateral phrenicotomy may not only be related to the respiratory stress engendered by the diaphragm denervation, but also to the interruption of inhibitory contralateral phrenic sensory afferents. 3. Bulbospinal pathways involved in the recovery of respiratory motor function following chronic cervical SCI In patients with high cervical SCI who need assisted ventilation, post-traumatic diaphragm recovery does occur in some cases (in 12/33 patients according to Oo et al., 1999) and some of these patients can be fully weaned from ventilatory assistance several months after injury (Oo et al., 1999; Winslow and Rozovsky, 2003; DiMarco, 2005), thus suggesting that important processes of respiratory plasticity may occur after cervical trauma. In rats, several studies have reported a spontaneous increase in ipsilateral phrenic activity in the months following unilateral C2 cervical injury, revealing a progressive spontaneous respiratory recovery in relation to the post-lesion time (Nantwi et al., 1999; Golder et al., 2001a,b; Golder and Mitchell, 2005; Fuller et al., 2006; Vinit et al., 2006, 2008). For instance, we have found that the intensity of ipsilateral PN activity 3 months after lateral cervical SCI reaches around 40% of the value in intact animals compared to only 25% in rats analyzed 7 days after SCI (Vinit et al., 2008). This may reflect an increase in descending phrenic drive or an effect of the injury on ipsilateral phrenic motor neurons. It is unlikely that this long-term PN reactivation relies on chronic respiratory stress induced by the long-term chronic injury, as several studies have shown that respiratory blood gases, pH, and blood pressure were not modified several months after cervical hemisection (Goshgarian et al., 1986; Golder et al., 2001a,b; Golder and Mitchell, 2005; Doperalski and Fuller, 2006; Fuller et al., 2006). Moreover, the role of activating ipsilateral phrenic afferents may be less essential after long-term chronic injury (Vinit et al., 2008). This suggests that distinct mechanisms of post-lesion respiratory recovery occur after a short-term sub-chronic and long-term chronic SCI. Despite substantial recovery of PN activity, the level of diaphragm recovery after chronic C2 injury remains less clear; according to some studies (Goshgarian, 1981; Polentes et al., 2004; Vinit et al., 2006, 2008) hemidiaphragm EMG signal remains very weak or undetectable in a large majority of hemisected animals, whereas two other studies reported an hemidiaphragm recovery in all C2 injured rats (Nantwi et al., 1999) or in 4/6 of them (Alilain and Goshgarian, 2008). However, these studies did not attempt to quantify EMG signal intensity, so that the various results in the reported timing of diaphragm recovery may reflect, at least partially, different threshold levels that were used to assess whether

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an EMG signal was present or not. In rats with chronic cervical SCI, a substantial hemidiaphragm reactivation can be generated during conditions of respiratory stress evoked by acute contralateral phrenicotomy (Vinit et al., 2006, 2008), what shows moreover that neuromuscular transmission or muscular efficiency are preserved after chronic spinal injury. This hemidiaphragm recovery induced by contralateral phrenicotomy was sufficient to allow animal survival in standard air ventilatory conditions (Vinit et al., 2006, 2008). Ipsilateral spontaneous phrenic restorative activity may play a role in the return of ventilatory function in chronically injured rats as supported by evidence that a chronic ipsilateral phrenicotomy in C2 hemisected rats leads to decreased tidal volume 2 months after this dual injury, but little change in rats with a sole C2 hemisection (Golder et al., 2003). An important question is whether this restoration of ipsilateral PN activity after chronic SCI relies on crossed phrenic pathways that are initially present (see Section 1) or on the recruitment/development of other alternative descending pathways. In rats with a chronic lateral C2 injury, we found that ipsilateral PN activity was minimally affected by an additional lateral C2 section on the contralateral side interrupting the main crossed phrenic pathways (Vinit et al., 2008). By contrast, a complete section of the medial spinal part suppressed ipsilateral PN activity (Vinit et al., 2008). Thus, long-term phrenic restorative activity may not be triggered by the crossed phrenic pathways (located in the lateral part of the contralateral spinal cord), but rather by alternative descending pathways, located principally in the medial part of the spinal cord. Thus, restoration of PN activity may operate by distinct mechanisms and pathways after acute/sub-chronic and chronic injury. Further evidence of distinct pathways is suggested by the fact that, although intensity of PN activity increases in relation to post-lesion time, electrical stimulation of the contralateral ventrolateral area (where crossed phrenic pathways are located) did not lead to higher ipsilateral phrenic evoked responses after chronic injury (Fuller et al., 2006). It may also explain why rats with a chronic unilateral C2 injury, showed an ipsilateral PN activity only when the C2 section spared the medial region, but not when the hemisection was complete (Li et al., 2003). The functional contribution of medial pathways is also supported by the observation of a lower respiratory frequency and a greater tidal volume during hypercapnic challenge in hemisected rats (1 month post-SCI) with ventromedial tissue sparing compared to rats with complete hemisection (Fuller et al., 2009). Thus, although respiratory medial pathways make only a minor functional contribution to phrenic motor neuron activity in intact rats and are not sufficient to trigger a detectable PN activity after an acute or sub-chronic lateral injury (Vinit et al., 2006, 2007), they become able to sustain a substantial phrenic restorative activity after a chronic lateral injury. Therefore, these medial restorative pathways can be considered as “new” functional pathways innervating the ipsilateral phrenic nucleus. Chronic partial cervical SCI may lead to an anatomo-functional reorganization of the respiratory network (Vinit et al., 2008). Thus, recruitment or reinforcement of medial pathways after chronic lateral injury may involve important anatomical and morphological plasticity processes. It may include synaptic remodelling from axon terminals that were already connected to the phrenic motor neurons (Goshgarian, 2003) or the development of new collaterals to ipsilateral phrenic motor neurons from axons that were initially targeted to other motor neurons (for instance, intercostal ones). It is also possible that respiratory propriospinal neurons may contribute to the development of new relay pathways after chronic injury. Long-term phrenic reactivation after chronic SCI may also rely on strengthening of excitatory glutamatergic drive to phrenic motoneurons through the up-regulation of glutamate receptors (NR2A subunit and GluR1) (Alilain and Goshgarian, 2008) and on enhanced serotonergic neurotransmission through increased 5-

HT2A receptors and 5-HT levels (Golder and Mitchell, 2005; Fuller et al., 2006).

4. Comparison of plasticity processes in the corticospinal locomotor and bulbospinal respiratory pathways in rodents Partial SCI may induce substantial morphological changes in the neural networks affected by the injury. Thus, in the locomotor system, functional recovery after partial chronic injury of the main corticospinal tract has been related to collateral sprouting of alternative spinal pathways resulting in the reinnervation of deafferented target motor neurons (Weidner et al., 2001; Fouad et al., 2001; Bareyre et al., 2004, Bareyre, 2007). For example, a section of the dorsal (principal) corticospinal tract can induce sprouting of ventral corticospinal fibers (initially very minor) that will establish new connections with the deafferented motor neurons and sustain motor recovery a few months after SCI (Weidner et al., 2001; Steward et al., 2008). Such reorganization between principal and accessory pathways after chronic partial SCI also applies for the respiratory system, since the interruption of the main respiratory pathways (lateral) can be compensated for by an enhanced contribution of medial pathways that play only a minor role in intact rats (see previous section). Thus, in both the respiratory and locomotor system after partial chronic SCI, a strategy to by-pass the injury will consist of redistribution and rewiring of the descending pathways within the spinal cord through the development of accessory pathways (Figs. 1 and 2). SCI-related anatomical reorganization in the corticospinal locomotor system depends in great part on the development of new polysynaptic relay pathways that involve spinal premotor neurons (Bareyre et al., 2004; Courtine et al., 2008). However, corticospinal neurons already project principally to propriospinal premotor neurons in intact rodents; therefore, SCI may reinforce this initial organization rather than modify it (Courtine et al., 2007). The contribution after chronic SCI of relay pathways through propriospinal neurons may be quite different for the respiratory system, since respiratory neurons project to the phrenic motor neurons principally by direct pathways in intact animals (Kastner and Gauthier, 2008). However, spinal respiratory interneurons projecting to phrenic motor neurons are present at different locations in the cervical spinal cord (Lipski et al., 1993, 1994; Tian and Duffin, 1996a,b; Lane et al., 2008b; Lu et al., 2004). The whole number of respiratory interneurons projecting to the ipsilateral phrenic nucleus was not altered 2 weeks after C2 hemisection (Lane et al., 2008b), but one can suppose that these cells could play a more important role in the transmission of respiratory drive to motor neurons after long-term chronic SCI. In this case, the post-lesion network organization may look like the neonates’ respiratory network, which has many more polysynaptic respiratory descending projections (Juvin and Morin, 2005). Axonal injury can induce a cell body response consisting of the up-regulation of regeneration-associated genes such as growthassociated protein 43 (GAP-43) and c-Jun, which trigger axon growth and sprouting. The ability of a cell to respond to injury depends on the distance between the site of axon injury and the cell body (Jenkins et al., 1993). After cervical SCI in rodents, cell bodies of bulbospinal (and rubrospinal) neurons respond to the injury by up-regulating regeneration-associated genes (Vinit et al., 2005, 2009; Jenkins et al., 1993; Houle et al., 1998), whereas those of corticospinal neurons, that are more distant from injury site, do not (Mason et al., 2003). Consequently, one can expect that the potential for axonal growth and plasticity, as related to the cell body response to injury, will be better for bulbospinal and rubrospinal neurons than for corticospinal ones. Such differential plasticity potential after SCI between corticospinal and bulbospinal neurons

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Fig. 1. Respiratory network reorganization following a C2 sub-chronic and chronic SCI. (A) C2 lateral SCI (representative transverse section to left of diagram) interrupts the main ipsilateral (ipsi) respiratory pathways (originating from the rostral ventral respiratory group -rVRG- in the brainstem) and results in an inactivation of the ipsi phrenic nerve (PN). 7 days post-lesion, a slight ipsi PN activity has reappeared; an example of rhythmic PN discharges (recorded on intact PNs) is represented beneath the corresponding PNs. An acute contralateral (contra) SCI in the lateral side (grey rectangle) leads to an inactivation of both ipsi and contra PN activities, indicating that ipsi PN activity is due to contra crossed phrenic pathways located laterally (thick line). (Upper inset) rVRG neurons labelled by a retrograde dye (Fluorogold) from the ipsi phrenic area (beneath the injury). Scale bar: 100 ␮m. (B) 3 months after SCI, ipsi PN activity has partially recovered. After an additional acute contra SCI in the lateral side (grey rectangle), the ipsi PN activity is not affected, suggesting that ipsi PN activity may be due principally to medial respiratory pathways (thick line), which do not contribute substantially to PN activity after acute/sub-chronic SCI (fine line in (A)). (Upper inset) VRG labelled neurons with fluorogold from the ipsi phrenic area. Scale bar: 100 ␮m.

has been observed after peripheral nerve graft in the vicinity of the injury (Decherchi and Gauthier, 2002; Gauthier et al., 2002; Ye and Houle, 1997; Blits et al., 2000) and in other experimental conditions designed to promote axon growth (Kim et al., 2004; Blesch et al., 2004; Wakabayashi et al., 2001; López-Vales et al., 2007; Vavrek et al., 2007). Whereas the pattern generator for respiration is located in the medulla, that for the locomotor system (CPG) is located in the spinal

cord (see Section 1). Thus, after spinal cord section above the level of the motoneurons, the locomotor CPG may generate rhythmic alternating contractions of flexor and extensor muscles (Rossignol ´ ´ et al., 2008; Majczynski and Sławinska, 2007; Barrière et al., 2008). Proprioceptive sensory feedback for the locomotor system affects propriospinal neurons and appears to be crucial for the proper function of these locomotor CPG. Thus, treadmill training or epidural stimulation leads to facilitation of stepping in spinally injured

Fig. 2. Role of phrenic nerve (PN) afferents after a C2 SCI. (A) Schematic representation of the protocol. After a post-lesion time lapse of 7 days or 3 months, ipsi PN activity is recorded on intact PN (connected to the diaphragm) and after an ipsi phrenicotomy (1). In some animals 7 days post-SCI, the cervical dorsal root ganglia were removed. The contra PN is connected to the contra diaphragm. (B) Ipsi PN activity at 7 days and 3 months post-SCI. In rats 7 days post-SCI, ipsi phrenicotomy (1) leads to a inactivation of ipsi PN. This effect is prevented by a removal of the dorsal root ganglia (2), suggesting that it depends on sensory afferents. In rats 3 months post-SCI, ipsi phrenicotomy does not affect ipsi PN activity.

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rats that depends on both the CPGs activity and feedback sensory inputs (Frigon and Rossignol, 2006; Lavrov et al., 2008). In addition, cutaneous sensory inputs from the hindpaw are necessary for locomotor recovery in spinalized cats (Bouyer and Rossignol, 2003). In the respiratory system, although sensory feedback from the respiratory muscle may improve phrenic restorative activity after sub-chronic injury (see Section 2, Vinit et al., 2007) it may not act directly on spinal respiratory interneurons; moreover spinal interneurons do not appear to function as CPG nor mediate inspiratory drive in the absence of supraspinal descending inputs (Vinit et al., 2006; Gauthier et al., 2006). Thus the specific features of the locomotor and respiratory networks in intact animals may in part determinate the different strategies of anatomical plasticity that will take place after spinal cord injury. 5. Conclusion The respiratory system is an adequate model to study the plasticity processes occurring after a partial cervical spinal cord injury and the resulting recovery. Chronic cervical SCI can indeed induce substantial modifications in the respiratory neural network. Further studies will be necessary to specify the axonal and synaptic reorganizations supporting the restored respiratory activity and the involved cellular mechanisms. Despite the anatomo-functional reorganization of respiratory pathways, the spontaneous respiratory recovery is still limited and needs to be enhanced by using appropriate treatments (i.e. cell transplantation, intermittent hypoxia,. . .) known to improve axonal sprouting or other plasticity processes. Acknowledgements The authors gratefully acknowledge Courtney H. Guenther and Dr James A. Windelborn from the Mitchell lab for their helpful advice and critical comments of this manuscript. Dr. Stéphane Vinit is supported by a Craig H. Neilsen Foundation Fellowship. References Alilain, W.J., Goshgarian, H.G., 2008. Glutamate receptor plasticity and activityregulated cytoskeletal associated protein regulation in the phrenic motor nucleus may mediate spontaneous recovery of the hemidiaphragm following chronic cervical spinal cord injury. Exp. Neurol. 212, 348–357. Baker-Herman, T.L., Fuller, D.D., Bavis, R.W., Zabka, A.G., Golder, F.J., Doperalski, N.J., Johnson, R.A., Watters, J.J., Mitchell, G.S., 2004. BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia. Nat. Neurosci. 7, 48–55. Bareyre, F.M., 2007. Neuronal repair and replacement in spinal cord injury. J. Neurol. Sci. 265 (1–2), 63–72. Bareyre, F.M., Kerschensteiner, M., Raineteau, O., Mettenleiter, T.C., Weinmann, O., Schwab, M.E., 2004. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat. Neurosci. 7, 269–277. Barrière, G., Leblond, H., Provencher, J., Rossignol, S., 2008. Prominent role of the spinal central pattern generator in the recovery of locomotion after partial spinal cord injuries. J. Neurosci. 28, 3976–3987. Baussart, B., Stamegna, J.C., Polentes, J., Tadié, M., Gauthier, P., 2006. A new model of upper cervical spinal contusion inducing a persistent unilateral diaphragmatic deficit in the adult rat. Neurobiol. Dis. 22, 562–574. Blesch, A., Yang, H., Weidner, N., Hoang, A., Otero, D., 2004. Axonal responses to cellularly delivered NT-4/5 after spinal cord injury. Mol. Cell. Neurosci. 27, 190–201. Blits, B., Dijkhuizen, P.A., Boer, G.J., Verhaagen, J., 2000. Intercostal nerve implants transduced with an adenoviral vector encoding neurotrophin-3 promote regrowth of injured rat corticospinal tract fibers and improve hindlimb function. Exp. Neurol. 164 (1), 25–37. Boulenguez, P., Gauthier, P., Kastner, A., 2007. Respiratory neuron subpopulations and pathways potentially involved in the reactivation of phrenic motoneurons after C2 hemisection. Brain Res. 1148, 96–104. Bouyer, L.J.G., Rossignol, S., 2003. Contribution of cutaneous inputs from the hindpaw to the control of locomotion. II. Spinal cats. J. Neurophysiol. 90, 3640–3653. Butler, J.E., McKenzie, D.K., Gandevia, S.C., 2003. Reflex inhibition of human inspiratory muscles in response to contralateral phrenic nerve stimulation. Resp. Physiol. Neurobiol. 138, 87–96. Corda, M., von Euler, C., Lennerstrand, G., 1965. Proprioceptive innervation of the diaphragm. J. Physiol. (Lond.) 178, 161–177.

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