Perinatal development of lumbar motoneurons and their inputs in the rat

Brain Research Bulletin, Vol. 53, No. 5, pp. 635– 647, 2000 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter

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Perinatal development of lumbar motoneurons and their inputs in the rat Laurent Vinay,* Fre´de´ric Brocard, Jean-Franc¸ois Pflieger, Juliette Simeoni-Alias and Franc¸ois Clarac CNRS, De´veloppement et Pathologie du Mouvement, Marseille, France [Received 5 May 2000; Revised 31 July 2000; Accepted 7 August 2000] ABSTRACT: The rat is quite immature at birth and a rapid maturation of motor behavior takes place during the first 2 postnatal weeks. Lumbar motoneurons undergo a rapid development during this period. The last week before birth represents the initial stages of motoneuron differentiation, including regulation of the number of cells and the arrival of segmental and first supraspinal afferents. At birth, motoneurons are electrically coupled and receive both appropriate and inappropriate connections from the periphery; the control from supraspinal structures is weak and exerted mainly through polysynaptic connections. During the 1st postnatal week, inappropriate sensori-motor contacts and electrical coupling disappear, the supraspinal control increases gradually and myelin formation is responsible for an increased conduction velocity in both descending and motor axons. Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors are transiently overexpressed in the neonatal spinal cord. The contribution of non-NMDA receptors to excitatory amino acid transmission increases with age. Activation of ␥-aminobutyric acidA and glycine receptors leads to membrane depolarization in embryonic motoneurons but to hyperpolarization in older motoneurons. The firing properties of motoneurons change with development: they are capable of more repetitive firing at the end of the 1st postnatal week than before birth. However, maturation does not proceed simultaneously in the motor pools innervating antagonistic muscles; for instance, the development of repetitive firing of ankle extensor motoneurons lags behind that of flexor motoneurons. The spontaneous embryonic and neonatal network-driven activity, detected at the levels of motoneurons and primary afferent terminals, may play a role in neuronal maturation and in the formation and refinement of sensorimotor connections. © 2001 Elsevier Science Inc.

smooth movements of the trunk and the limbs, is present from the 16th postnatal day (P16, [140]). During the early postnatal period, spontaneous locomotor activity consists of crawling movements. Experimentally, locomotion can be evoked during this period in vivo by lifting the animal in the air (air stepping, [42,92]) or by placing it in a pool (swimming, [28,92,139]), two conditions in which postural constraints are reduced. In vitro, a fictive locomotor pattern can be elicited following bath application of excitatory amino acid agonists and serotonin (5HT) [29] or electrical stimulation of either some brainstem areas [8] or the ventrolateral funiculus of the spinal cord [89]. Altogether these observations demonstrate that the neural networks underlying locomotion are functional at birth (see [65] for the prenatal maturation of these networks). An immature posture is a limiting factor for spontaneous locomotion during the 1st postnatal week. A significant maturation of postural control occurs during this period [6,24]. This development depends on the maturation of several systems, such as the musculo-skeletal system, the sensori-motor networks, the higher brain centers, pathways descending to the spinal cord and the ascending tracts conveying sensory information and efference copy signals to supraspinal nuclei. The aim of this review is to give an updated picture of our understanding of the functional development of the lumbar motoneurons and their inputs. Although the conducting thread will be the spinal cord in the rat, some references will be made to other models which have proven useful for the investigation of neural development (Xenopus and chick spinal cord, hypoglossal motoneurons, hippocampus . . .). Each model having its own experimental advantages and limitations the hope is that a comparison between the recent advances provided by these research areas of developmental neurobiology will lead to a better understanding of the development of mammalian spinal cord.

KEY WORDS: Spinal cord maturation, Descending pathways, Sensori-motor pathways, Electrical properties, Posture, Locomotion.

MORPHOLOGICAL AND ELECTRICAL MATURATION OF MOTONEURONS INTRODUCTION

Motoneurons are produced on embryonic days (E) 11–12 (birth occurring at around E22) in the cervical spinal cord and E13–14 in the lumbo-sacral cord [5]. Roughly half of the motoneurons produced degenerate and die (67% in the mouse [86]). Oppenheim [106], using established procedures for determining the numerical loss of motoneurons during development in the rat, described a substantial loss of both brachial and lumbar motoneurons between

The rat is quite immature at birth and a rapid maturation of motor behavior takes place during the first 2 postnatal weeks [postnatal days (P) 0 –14]. It is only at the end of the 1st week after birth that animals become able to lift their trunk from the floor and to walk spontaneously [53,69]. The adult pattern of locomotion, characterized by an adduction of the hindlimbs during the stance phase and

* Address for correspondence: Dr. Laurent Vinay, CNRS, De´veloppement et Pathologie du Mouvement, 31 chemin Joseph Aiguier, F-13402 Marseille cedex 20, France. Fax: ⫹33-4-91-77-50-84; E-mail: [email protected]

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636 E15 and P0, but failed to detect a significant decrease after birth (see also [36]). However, other earlier studies had concluded that a significant motoneuron loss occurs within the first 5– 6 days after birth [18,102,112]. Changes in the Somatodendritic Morphology and Gap Junctions We will focus on one aspect of motoneuron differentiation, that of soma and dendrites, which is more related to the maturation of membrane properties and inputs; we will not discuss the differentiation of the axon, the axonal growth and the development of the neuromuscular junction (see [115] for review). The morphological development of motoneurons innervating several hindlimb muscles has been studied in the rat using retrograde labeling techniques [75,141] or intracellular injections [37]. There is a fourfold increase in the soma area of soleus (ankle extensor) motoneurons during the first 3 weeks after birth, with the greatest rate of growth occurring during the 2nd postnatal week [75,128,141]. At P21, soleus motoneurons have reached 70% of their adult size. The range of soma areas increases with postnatal age over this period, and the distribution becomes bimodal which may reflect a differential growth of ␣ and ␥ motoneurons (at around P7 for quadriceps femoris motoneurons [128] and at around P21 for soleus motoneurons [75,141]). The dendritic tree of motoneurons develops predominantly postnatally. Although the number of primary dendrites does not change postnatally [37], dendritic branches gradually increase in length during the first 2 postnatal months. At birth, the somatodendritic surface of the tibialis anterior and extensor digitorum longus motoneurons is covered by growth-associated spiny or hair-like appendages ([37] see also [35]). These disappear in a somatofugal direction, on the soma before P4 and on most proximal dendrites by P7 whereas they persist until the 2nd postnatal week on distal dendrites [35]. The complex longitudinal dendritic bundles of some motoneuron pools, such as the one innervating the soleus muscle, start to develop by P10 ([141] see also [13,14]). A period of dendritic retraction has been described to follow the period of dendritic extension during the development of motoneurons supplying flexor and extensor muscles of the forelimb [36]. A transient widespread electrotonic coupling is found between motoneurons in the perinatal spinal cord [138]. Graded ventral root stimulation after removal of Ca2⫹ from the bathing solution elicits electrotonic junctional potentials in lumbar motoneurons. Electrotonic coupling is quite specific and restricted to motoneurons innervating the same skeletal muscle [138]. Up to 18 (mean ⫽ 6) dye-labeled cells are observed following the injection of Neurobiotin into a single motoneuron at P0 [30]. However, electrical and dye coupling decrease with age and are no longer present after the 1st postnatal week. Motoneurons express five connexins : Cx36, Cx37, Cx43, Cx40 and Cx45 [30]. Although the latter two connexins are downregulated during development, the other gap junction proteins are expressed by a large proportion of adult motoneurons. Therefore, the disappearance of electrical and dye coupling may be due to a modulation (and not the loss) of junctional communication among motoneurons, by unknown mechanisms that would affect gap junction assembly, permeability or open state [30]. This coupling may be reestablished in certain circumstances such as following nerve injury [31]. Several roles for electrical coupling through gap junctions have been proposed [30,138]. An interesting hypothesis is related to specification of synapse formation: electrical coupling ensures that a common pattern of activity or subthreshold oscillations spread among motoneurons of a given motor pool (see below).

VINAY ET AL. Maturation of Membrane Properties Input resistance and rheobase. Marked age-related changes in electrophysiological properties of lumbar motoneurons have been described. Motoneurons are excitable at E15 [51]. The input resistance decreases with age [49,117]. This is consistent with the important increase in motoneuron size during postnatal development, although other factors, such as a decrease in membrane resistivity [132], may also contribute to this decrease in input resistance. Rheobase is negatively correlated with input resistance and increases more than five times during postnatal development [117]. Resting potential has been described to be similar in embryos and neonates [118,147] and to be unchanged during the postnatal period [117]. The lower rheobase and higher input resistance at early postnatal stages suggest a higher excitability to synaptic imputs [132], i.e., a smaller amount of synaptic current is needed to initiate cell firing, compared to later on during development. This increased excitability may account, at least partly, for the abundant spontaneous activity in spinal networks and for the motility of neonates. Development of sodium currents. From the very onset of motoneuron excitability (E15) in the rat, action potentials rely on sodium channel activation [51]. Such sodium-dependence of the action potentials at early developmental stages is not observed in spinal neurons of Xenopus embryos. In these neurons, action potentials are initially long in duration and primarily dependent on an inward calcium current whereas the mature action potentials are primarily sodium dependent [10,105,124]. Action potential properties change with age. Its threshold shifts towards more negative values in neonatal motoneurons (from ⫺35 mV at E15–16 to ⫺47 mV at P1⫺3; [51]) with little postnatal change. This shift is likely due to the threshold potential for INa activation 10 mV more negative in postnatal (⫺50 mV) than in embryonic motoneurons (⫺40 mV). Action potential amplitude increases whereas the duration decreases in neonatal motoneurons compared to embryonic motoneurons [51]. The duration decreases further slightly during postnatal development ([132] for rat hypoglossal motoneurons). Gao and Ziskind-Conhaim [51] have demonstrated that developmental changes in action potential properties of rat lumbar motoneurons result from large increase in the density of existing voltage-gated ion channels, and not from the loss of some channels or the expression of new channel types. The increase in amplitude is due to a larger inward sodium (INa) current, resulting from an increase in the number of sodium channels (Fig. 1B). The shortening of the action potential is due to higher rates of rise (shorter time to peak INa) and repolarization (increased potassium conductances, see below). Development of calcium currents. Action potentials in immature motoneurons are characterized by a pronounced after-depolarization (ADP), which is followed by a prolonged after-hyperpolarization (AHP; [49,137]). The ADP amplitude is strongly dependent on the resting membrane potential, being maximal when holding the cell membrane hyperpolarized [49,130]. The ADP can be large enough to trigger action potentials. The ADP is calciumdependent ([51,137]; see also [130] in neonatal hypoglossal motoneurons). The calcium current is particularly robust during the first few postnatal days and accounts for the tetrodotoxin-resistant spikes that can be elicited by direct stimulation [137]. By contrast, such calcium spikes can be elicited in animals older than 5 days only after decreasing potassium conductances by either intracellular cesium or extracellular tetraethylammonium (TEA). The ADP in neonatal hypoglossal motoneurons is caused by both an ␻-conotoxin-insensitive high voltage-activated (HVA) calcium conductance [131] and a low voltage-activated (LVA) conductance [130]. The HVA calcium current increases during perinatal develop-

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FIG. 1. Maturation of electrical properties of lumbar motoneurons. (A) The development of firing properties is characterized by the transition from a single action potential (A1, upper trace) or a doublet (A1, middle trace, stronger current) to a sustained discharge (A2) in response to a prolonged depolarizing pulse. (B) Development of ionic currents underlying changes in action potential waveforms and repetitive firing behavior. Data for the sodium current density at postnatal day (P) 8 are from mouse motoneurons [52]; all the other values are from rat motoneurons [51]. (C) Open symbols: percentage of ankle extensor (E-MNs, squares) and flexor (F-MNs, circles) motoneurons capable of repetitive firing (such as in A2) at P0 –2 and P3–5. Filled symbols: steady state firing frequency in E-MNs (squares) and F-MNs (circles).

ment (Fig. 1B; [51]). It is mediated primarily (about 70%) by N-type Ca2⫹ channels and to a lesser extent by P/Q-type current (about 20%) and L-type current (10%). The relative contribution of the L-type Ca2⫹ channels to the HVA currents may increase with age, as suggested recently by Jiang et al. [70] who have shown that the L-type Ca2⫹ channel blocker nifedipine has no effect on a chemically induced rhythmic activity in the isolated mouse spinal cord at P2–P5, but reduces the motor output in animals older than P7. In agreement with these observations, experiments using immunohistochemical methods demonstrated that the labelling of the ␣1C and ␣1D subunits of the L-type calcium channels also increased with age, reaching a pattern similar to what is observed in the adult by P18 [70]. This may be of relevance for the development of posture and locomotion because L-type calcium channels have been shown to mediate plateau potentials in turtle motoneurons [60,64]. The LVA (T-type) calcium current was not considered in the study by Gao and Ziskind-Conhaim [51] on rat motoneurons. However, recordings from neurons cultured from the amphibian embryonic neural plate showed that T current has the lowest threshold among inward currents in young immature neurons and can depolarize cells and trigger action potentials [58]. These LVA currents are partly responsible for the spontaneous elevations of intracellular calcium in cultured neurons at early stages of differentiation. T currents are dominant at the earliest developmental stages of chick limb motoneurons; they decrease during development whereas N- and L-type calcium currents increase [91].

Development of potassium currents. The ability to fire repetitively, following a prolonged intracellular injection of depolarizing currents, also varies with age. Such pulses, in embryonic motoneurons, produce only one action potential followed by a long-lasting depolarization, which prevents generation of other action potentials very likely by prolonging Na2⫹ channel inactivation ([51] see also [113] in mouse retinal ganglion neurons). The same pulses trigger repetitive firing in most neonatal motoneurons (Fig. 1A; [51,144]. These two distinct stages in the development of repetitive firing have been described in other developing neurons (Xenopus spinal neurons [119,120], oculomotor neurons [129], retinal ganglion neurons [113], Cajal-Retzius cells [94], phrenic motoneurons [90]). An intermediate developmental stage consisting of a transient spike burst lasting 100 –200 ms has also been described [113,132b]. The development of potassium currents, by truncating the calcium currents, plays a major role in the shortening of the action potential, the development of AHP, the repolarization of the cell and thereby the generation of repetitive firing after birth. A large increase in noninactivating delayed rectifier-type current (IK) and calcium-dependent potassium current (IK(Ca)) has been observed after birth (Fig. 1B; [51]). An increase in the density of HVA Ca2⫹ currents may be necessary to activate the latter K⫹ conductance. A down-regulation of A-type K⫹ current (IA) may also be required for repetitive firing to occur [94]. Interestingly, IA, which is large in embryonic motoneurons, decreases postnatally (Fig. 1B; [51]). The extent of repetitive firing in embryonic amphibian

638 neurons differentiating in culture decreases with time, during a period in which IA is acquired by all neurons [110]. Application of the K⫹ channel blocker 4-aminopyridine (4-AP) causes sustained repetitive spiking in neonatal rat mesencephalic trigeminal neurons that previously were unable to fire more than one action potential [38]. After the first spike evoked from hyperpolarized levels in these neurons, subsequent action potentials are prevented by the contributions of both a transient (similar to IA) and a sustained (I4AP) 4-AP-sensitive outward currents. Mathematical modeling of these cells indicated that a reduction of the maximal conductance of IA by 60 –90% (to mimic the inactivation of this current by depolarization to ⫺50 mV) causes the cell model to fire a transient spike burst [38]. However, I4AP is able to prevent sustained firing of action potentials; its reduction by 90% in the model elicits repetitive firing. Whether such currents are inhibiting repetitive firing also in immature spinal motoneurons in the rat is unknown. Noninactivating K⫹ currents are reduced by 60 –75% by TEA ([51], see also [127]); it would be of interest to determine whether 4-AP abolishes the remaining component and if so, how it develops with age. In conclusion, the switch from a single action potential to repetitive firing during development appears to result from the combination of several factors. Changes in the relative contribution of Ca2⫹-independent and Ca2⫹-dependent K⫹ currents may be crucial in this process [126]. Maturation of firing properties of motoneurons is associated with the gradual differentiation of muscle fiber types. This simultaneous development gradually leads to the match between the properties of motoneurons and those of the target muscle fibers observed in the adult. The maintenance of this match is then largely dependent on the maintenance of functional innervation. Differentiation into slow and fast muscle units starts around the end of the 1st postnatal week ([66,108], for review see [98]). Differential Maturation of Motoneurons Belonging to Different Motor Pools Most of the studies on the maturation of motoneurons in the rat considered the whole population of motoneurons, irrespective of their functional identity, i.e., the muscle they innervate. However, maturation may not proceed simultaneously in the motor pools innervating, for instance, flexor and extensor muscles or proximal and distal muscles. The development of the dendritic fields of motoneurons innervating the extensor muscles of the distal forelimb lags behind that of antagonistic motoneurons [36]. To investigate this further, we compared recently the changes in excitability of motoneurons innervating antagonistic muscles such as the ankle flexors (F-MNs) and the anti-gravity ankle extensors (E-MNs). Data showed that age-related changes in rheobase during the first five postnatal days are much less pronounced for E-MNs than for F-MNs [23,132b]. The fraction of motoneurons exhibiting a sustained firing in response to a prolonged depolarizing pulse (Fig. 1A2) is much higher at birth in F-MNs than in E-MNs (Fig. 1C, empty circle and square, respectively). This percentage increases to reach 100% in F-MNs and only 71% in E-MNs at P3–5. The steady state firing frequency of the motoneurons with repetitive discharge is higher in F-MNs than in E-MNs (Fig. 1C, filled circle and square, respectively). Part of this disparity can be attributed to differences in AHP duration between the two types of motoneurons. The AHP duration is shorter in F-MNs than in E-MNs at birth; it decreases with age in both groups of motoneurons as previously described for hypoglossal motoneurons [132]. These results demonstrate that the development of repetitive firing of E-MNs lags behind that of F-MNs. Similarly, elimination of polyneuronal innervation, starts later in the soleus muscle (around P8 [26]) than in the flexor muscles (P3 [11]). Differences therefore

VINAY ET AL. exist in the rate of maturation of membrane properties between motor pools innervating antagonistic muscles, and E-MNs are more immature than F-MNs at birth. This is functionally adapted to the position of the embryo in utero, whose limbs, in flexion, do not need to counteract gravity. DEVELOPMENT OF INPUTS TO LUMBAR MOTONEURONS Segmental Inputs The development of dorsal root projections to the lumbar spinal cord has been studied in the rat using morphological and physiological techniques [84,122,145]. Collaterals from dorsal root fibres start to reach the dorsal part of the dorsal horn at E15.5, the intermediate region at E16.5 and the motor nuclei at E17.5. The number of collaterals entering the ventral horn then increases with age [84]. The percentage of collaterals exhibiting growth cones decreases from 75% at E17.5 to 15% at around birth. Electron microscopic examination demonstrated that immature synapses with few synaptic vesicles and synaptic densities are present in the ventral horn at E17 [145]. Dorsal root stimulation evokes long latency excitatory postsynaptic potentials (EPSPs) conveyed by polysynaptic pathways at E15 [145]. Short-latency EPSPs, which are likely monosynaptic, are evoked in most motoneurons at E17, i.e., 1–2 days after the formation of long-latency polysynaptic connections [145]. The stretch reflex appears at E19 –20 [83]. The magnitude of the monosynaptic response increases until about P2 [84]. After this age it has been described either to decrease [84] or be constant (from P1–3 to P7–9 [117]; from P2 to P4 [25]). The initial monosynaptic connections are not all specific; at E19 –21, 29% of the motoneurons receive monosynaptic innervation from primary afferents of antagonistic muscle [118]. The proportion of such inappropriate contacts peaks at P0 –2 and then decreases; the majority of monosynaptic connections are appropriate within 1 week after birth. Whether inappropriate contacts become silent or are physically eliminated is not known. However, the pattern of connections has been described to be specific with only weak connections with antagonistic motoneurons at P0 –7 in the mouse ([93]; see [55] for review). The apparent discrepancy between these results might be due to different developmental stages reached by the rat and mouse central nervous systems at birth, and/or the pattern of neonatal connections to motoneurons innervating distal muscles being more immature than that to motoneurons innervating proximal muscles. Further experiments using other techniques are required to answer the question of specificity of the earliest monosynaptic connections. Altogether these results suggest that at least two slightly overlapping stages may be distinguished in the development of monosynaptic connections between primary afferents and motoneurons: (1) outgrowth of dorsal root fibres and formation of monosynaptic contacts until about P2; (2) from P2 onwards stabilization and refinement of connections with elimination of redundant and possible inappropriate synapses. These phases may also be identified in the case of the development of cutaneous afferent fiber inputs. Recordings from single cutaneous primary afferent units in the dorsal root ganglion showed that all the major cutaneous receptor types are developed at birth in the rat, although peak firing frequency and ability to follow high frequency electrical stimulation are low [46]. However, recordings from dorsal horn cells showed that the synaptic coupling between cutaneous afferents and central cells is quite immature in the early postnatal period [45]. Receptive field areas are large at birth and nociceptive withdrawal reflexes are often misdirected, bringing the stimulated area of the skin towards the stimulus instead of away [62]. The size of receptive fields de-

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FIG. 2. Development of descending pathways in the rat. (A) Schematic transverse sections of the brain stem (adapted from [99]) showing the location of some of the nuclei known to be important for the control of posture and locomotion. The major neurotransmitters are indicated for most nuclei [99]. The arrival times of the first descending fibers in the upper and lower lumbar cord are adapted from [85] taking the nuclear definitions into account. Abbreviations: ACh, acetycholine; 5HT, serotonin; SP, substance P; Glu, glutamate; GABA, ␥-aminobutyric acid; NA, noradrenaline. (B) Gradual arrival of descending fibers in the lumbar enlargement. Data on NA, 5HT and vestibulospinal projections are from [34,128,87], respectively. (C) Location of the motor pools innervating the different muscles of the hindlimb, in the lumbar enlargement; adapted from [100].

creases with age, and from the 3rd postnatal week, adequate withdrawal reflexes can be elicited, suggesting that some inappropriate connections become depressed or eliminated. Inputs From Supraspinal Structures Anatomical data. The development of descending projections from the brain to the lumbar cord has been studied in the rat using anatomical techniques ([9,81,87]; for review and thorough description see Lakke [85]). Neurons in most spinal projecting brainstem nuclei are generated from E11 to E15 [1– 4]. The earliest projections are first detected in the cervical cord at E13–14, at lower thoracic levels at E14 –15 and at lower lumbar levels before birth (Fig. 2A; [85]). Cells of origin are located in the medullary and pontine reticular formation, in the interstitial nucleus of the medial longitudinal fasciculus and in the lateral vestibular nucleus. However, the projections arising from several brainstem structures, such as the large-celled nucleus ventral to gigantocellularis referred to here as nucleus reticularis (n.r.) magnocellularis pars beta ([99] corresponding to the n.r. gigantocellularis pars alpha and n.r. gigantocellularis ventral in Lakke [85]) and the nucleus raphe pallidus, reach the upper lumbar cord slightly before birth and the lower lumbar cord during the first postnatal days (Fig. 2A). The

earliest corticospinal projections arrive by the end of the 1st postnatal week [40,116]. Three important points should be considered when interpreting these anatomical data. First, a brainstem nucleus may exert influences upon a given level of the spinal cord through polysynaptic connections before any axon arising from this structure has reached this level [25,48,134]. Second, although the first axons of most descending pathways are present within the white matter at lumbar levels at birth, a considerable number of axons continue to arrive in the lumbar enlargement postnatally. For instance, the adult projection pattern from the lateral vestibular nucleus is established only during the 3rd postnatal week (Fig. 2B, continuous line [87]). Similarly, only a few 5HT terminals and varicosities are seen in close apposition with only half of quadriceps femoris motoneurons at birth whereas at P5, such appositions are found with all the motoneurons examined and their number continues to increase (Fig. 2B, dashed line; [128]; see also the development of noradrenergic innervation quantified by the increase in concentration of norepinephrine in the whole spinal cord, Fig. 2B, dotted line [34]). The adult pattern of 5HT innervation in the lumbar cord is reached only at P21 [22,109]. These anatomical data on the arrival of descending pathways are confirmed by the expression of

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FIG. 3. Development of ventral funiculus input to lumbar motoneurons. (A) Average L5 ventral root potentials evoked by stimulation of the ventrolateral funiculus at the C1 level of the spinal cord at the same intensity (2.5 times threshold) at two ages (postnatal days (P) 0 and P4; [25]). Labels 1 and 2 indicate the two components of the response. **p ⬍ 0.01; ***p ⬍ 0.001. (B) Intracellular recordings from ankle extensor motoneurons showing a typical response to a supramaximal stimulation of the ventrolateral funiculus. Percentages of ankle extensor motoneurons firing in response to such a stimulation (P0 –2, n ⫽ 37; P3–5, n ⫽ 23). (C) Post-stimulus time histogram of the discharge elicited in the L4 ventral root following a supramaximal stimulation of the ventrolateral funiculus.

the growth-associated protein GAP-43, which is detected by immunocytochemistry in the white matter of the lumbar spinal cord until about P10, except in the areas of the corticospinal tracts where staining remains until P29 [47]. Third, all the anatomical studies mentioned above described the growth of axons through the white matter. After the initial arrival of fibers at a given segment of the spinal cord, there may be a waiting period during which the axons change their direction and enter the adjacent gray matter as shown for corticospinal axons (2 days [72]). The growth into the ventral horn or other gray target area and the formation of collateral branches may take another day. A period of at least 3– 4 days should therefore be added to the arrival time of the first descending fibres in the lumbar cord, to allow functional synapses between these fibres and target interneurons or motoneurons. Functionally, the time-table of descent should be considered altogether with the distribution of the motor pools innervating the different hindlimb muscles (Fig. 2C; [100]). Apart from motoneurons innervating the semitendinosus muscle (hip extensor and knee flexor) which are located caudally in the lumbar cord (L4 – 6), motoneurons of distal hindlimb muscles tend to be most caudal in the cord. This is particularly true for extensor motoneurons. This means that, for instance, these motoneurons as well as semitendinosus motoneurons or L3–5 interneurons do not receive any “functional” direct inputs from the later developed descending pathways until about P5. The supraspinal control at birth would therefore be exerted primarily on proximal muscles whereas the control over distal muscles would increase during the first days after birth. This would account for the proximodistal gradient in the maturation of

posture [6,24,121]. The L1–2 rhythmogenic networks which have been shown to be of primary importance for generation of locomotion [27,78,79] may not receive inputs from n.r. magnocellularis pars beta until P1. Interestingly, the latter nucleus has been shown to be important in the control of posture and locomotion in the rat and the cat (see [99] for review). Development of the ventral funiculus-evoked input to motoneurons. Few studies have investigated the functional development of descending pathways (see [8,48,134] for stimulations applied in the brainstem and [41,136] for stimulations of the spinal cord at a thoracic level which may activate both descending and propriospinal pathways). We have investigated the development of ventral funiculus (VF)-evoked input to lumbar motoneurons in in vitro isolated brainstem/spinal cord preparation of the neonatal rat [25]. The VF was stimulated at the cervical C1 level to activate some of the reticulospinal and vestibulospinal fibers and to reduce the contribution of the propriospinal system. The magnitude of the evoked L5 ventral root potentials increases significantly during the first days after birth (Fig. 3A). This is likely due to the arrival of descending pathways. The latency of responses decreases markedly with age (see also [48,135]). Myelination, which starts in the VF at birth [114], in addition to the increase in axon diameter may be responsible for this decrease in latency. More recently, we recorded from ankle extensor motoneurons and observed that a supramaximal VF stimulation was able to trigger an action potential in only 3% of the cells at birth (Fig. 3B). By contrast, the response was large enough to reach firing threshold in 35% of the motoneurons recorded at P3–5.

DEVELOPMENT OF LUMBAR MOTONEURONS IN THE RAT Recording of both ventral root potentials (Fig. 3A) and ventral root discharges (Fig. 3C) revealed that VF-evoked responses are made of two components whose relative magnitude changes with age: the first component, which is smaller than the second one at P0, becomes larger at the end of the 1st postnatal week. The influence of VF axons on lumbar motoneurons may therefore be exerted at birth more through polysynaptic connections than through direct connections [25,48]. These may develop during the early postnatal period. Altogether, the latter data indicate that VF input to lumbar motoneurons is rather weak at birth. Its gradual increase during the 1st postnatal week and the recruitment of ankle extensor motoneurons may account for the increased use of the ankle joint in postural reactions during this period [24]. Development of Inhibitory and Excitatory Amino Acid Transmission

␥-Aminobutyric acid (GABA)A and glycine receptors. In embryonic and neonatal rat lumbar motoneurons, application of GABA and glycine induces membrane depolarization. Activation of GABAA and glycine receptors causes the opening of Cl⫺ channels with consequent outward movement of Cl⫺ because the intracellular Cl⫺ concentration is maintained at a relatively high level by an inwardly directed Cl⫺ active transport mechanism coupled to Na⫹ and K⫹ (see [7] for review of the mechanisms underlying GABAA-receptor-mediated primary afferent depolarizations). This efflux transiently drives the transmembrane potential toward the equilibrium potential for chloride (ECl) which is above the resting membrane potential in immature motoneurons. The subsequent reduction in the amplitude of glycine- and GABAinduced depolarization reported during development [143] is likely due to a shift of ECl toward more negative potentials. GABA and GABAA receptor subunit mRNAs are transiently expressed in abundance in the majority of embryonic neurons [88]. The relative importance of glycine as compared to GABA increases during spinal cord maturation, as shown by the measurement of glycineand GABA-gated currents during the short-pulse ejection of the aminoacids from pipettes positioned close to motoneuron somata [50]. The density of the GABA current is larger than that of the glycine current before birth. The density of the glycine current increases significantly after birth, whereas there is little change, if any, in the density of the GABA current [50]. GABA and glycine might be considered as excitatory neurotransmitters if their application on the spinal cord induces burst discharges in motoneurons, as shown for glycine at E15.5 [101]. However, the large increase in membrane conductance associated with GABA-gated (and very likely glycine-gated) depolarizations causes a shunt of incoming excitatory currents in motoneurons and reduces their excitability ([50] for GABA; see [146] for review). Therefore, insofar as the depolarization does not reach the firing threshold, i.e., there is no propagation of electrical activity, GABA functions as an inhibitory transmitter in immature motoneurons during late fetal and early postnatal periods. A weaker subthreshold activation of GABAA receptors may nevertheless be functionally excitatory by removing the voltage-dependent block of Nmethyl-D-aspartate (NMDA) receptors by Mg2⫹ (see [17] in the hippocampus). NMDA and non-NMDA receptors. Both NMDA [73] and nonNMDA receptors [67] are transiently expressed at high levels in the rat spinal cord ventral horn during early postnatal period, and the level of NMDA receptors falls in the 2nd and 3rd postnatal weeks. The magnitude of NMDA-induced currents in motoneurons has been described to either increase [107] or decrease [63] during the first 2 postnatal weeks. This discrepancy may be due to

641 different concentrations of magnesium ions and glycine used in these experiments, which may strongly influence the amplitude of NMDA-induced currents. In addition, the NMDA receptor affinity for magnesium, which is high in the spinal cord [107], and the voltage dependence of the block may change with age, as shown in the hippocampus [16,77]. The relative distribution and conductance of single NMDA receptor channel openings remain unchanged during this period, suggesting that the subunit composition of NMDA receptors remains about the same [107]. The amplitude of kainate and AMPA currents does not change during the early postnatal period [63]. The composition of GluR1 through GluR4 (GluR-A-GluR-D) subunits and the proportion of the flip and flop splice variants for these subunit transcripts change with age [68]. Because these are important factors determining the functional properties and the Ca2⫹ permeability of AMPA receptors [71], it has been suggested that the non-NMDA receptors expressed by neonatal rat motoneurons show greater sensitivity to agonists (i.e., produce higher levels of depolarization) and allow Ca2⫹ entry [68] as shown in cultured Xenopus spinal neurons [54,111]. Respective contributions in synaptic transmission. The polysynaptic transmission from dorsal roots to motoneurons in the E16 –17 embryo is blocked by antagonists of either of NMDA [145] or glycine or GABAA receptors [143]. This suggests that glutamate-mediated synapses are formed in series with GABAergic and glycinergic synapses and that NMDA receptors mediate the excitation of GABAergic and glycinergic interneurons [146]. The monosynaptic EPSPs evoked by stimulation of dorsal roots are mediated by both NMDA and non-NMDA receptors [145]. Responses evoked by VF stimulation are also mediated by these two receptors at birth (Brocard, Vinay and Clarac, unpublished observations). However, their respective contributions to synaptic transmission changes with age: blocking NMDA receptors suppresses VF-evoked ventral root discharges in P0 –5 rats but not at P6 –7 demonstrating that from this age the non-NMDA component is large enough to reach action potential threshold (see [17] for the sequential development of NMDA and AMPA receptor-mediated glutamatergic synaptic transmission in the hippocampus). SPONTANEOUS ACTIVITY AND MATURATION OF SENSORY-MOTOR NETWORKS What is the Relationship Between Spontaneous Activity and Locomotion? Embryonic spontaneous motility has been observed in many species (see [97] for the rat). Although most studies of spontaneous activity in the developing nervous system have focused on the embryonic and fetal stages, it is likely that a continuity exists between prenatal and postnatal spontaneous behaviors [21,97]. The neural activity underlying the myoclonic twitching that is commonly associated with active sleep in neonates is produced, at least in part, in the spinal cord [20]. Prenatal and postnatal spontaneous activities may subserve similar developmental functions. Lumbar motoneurons in rat fetuses (E13.5–18.5) in vitro exhibit spontaneous activity consisting of bursts that are synchronized on the right and left sides of the spinal cord [96,101]. This activity is synaptically mediated and relies on glycine transmission at the earliest stages (E14.5). Excitatory amino acid transmission becomes dominant at E17.5 as shown by the fact that kynurenate abolishes the activity at this stage. GABAA receptors regulate the level of excitation since bicuculline reduces the amplitude of the bursts but does not block them [101]. Spontaneous motoneuronal activity can also be recorded in the neonatal rat spinal cord (Fig. 4A; [44]). Although a thorough comparison should be made, the magnitude of this activity appears to be lower after birth than at

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FIG. 4. Spontaneous activity in motoneurons and primary afferent terminals. (A) Simultaneous recordings from two ventral and one dorsal root on the same side of a postnatal day 2 rat in vitro. Modified from [44]. (B) The antidromic activity recorded from the dorsal root disappears in the presence of picrotoxin, a ␥-aminobutyric acidA receptor antagonist. (C) Sub-threshold postsynaptic potentials in neonatal motoneurons in phase with the antidromic dorsal root activity. Steady depolarization of the cell allowed to identify these potentials as excitatory (giving rise to action potential, asterisk) and inhibitory (hyperpolarizations).

fetal stages. When present, the motor bursts in the L3 and L5 ventral roots on the same side are not in antiphase as it is the case when the central pattern generator for locomotion is active ([29, 76]; L2/L3 and L5 represent the flexor-extensor alternation). A distinction should therefore be made between spontaneous activity and locomotion until the precise relationships between the networks driving these activities be elucidated (see also [12] for discussion of this point). Many authors confuse the two phenomena, probably because spontaneous bursting occurs synchronously on both sides of the spinal cord in the rat fetus at E15.5, a stage at which chemically-induced rhythmic bursting is also synchronous in the left and right ventral roots as well as in the L2 and L5 ventral roots on the same side [82]. The chemically induced activity is alternating at birth, whereas the spontaneous motor activity is still in phase in L3 and L5 ventral roots. Rather than representing the antecedent of locomotion, spontaneous rhythmic motor activity in the spinal cord (see also [57] in Xenopus, [32,95,104] in the chick) may reflect the general rhythmogenic property of developing networks (for recent reviews see [43,103]). This property has been described in other parts of the central nervous system, such as the trigeminal system [61], the hippocampus [15] and the visual system [142]. Spontaneous GABAergic Input Onto Primary Afferents An intense spontaneous bursting activity can be recorded from dorsal roots in the neonatal rat spinal cord in vitro (Figs. 4A–C; [44,80,133]). These bursts are generated by afferent terminal depolarizations reaching firing threshold and are conducted antidromically along peripheral nerves. Antidromic action potentials are more numerous in saline solutions with chloride concentration reduced to 50% of control and are blocked by picrotoxin or bicuculline (Fig. 4B; [44,133,135]). They are therefore mediated by an activation of GABAA receptors. Depolarizations most often

accompanied the dorsal root bursts in motoneurons at resting membrane potential (Fig. 4C). Tonic depolarization of the motoneuron shows the presence of both excitation (asterisks) and inhibition. Excitatory input may be explained by at least two mechanisms. The first one involves common neuronal connections coactivating the motoneurones and the primary afferent terminals. The second mechanism may involve single depolarization of primary afferent terminals by GABAergic interneurons, the antidromic discharges may have postsynaptic effects in lumbar motoneurons and first-order interneurons. Calcium-Mediated Regulation of Neuronal Development Spontaneous activity may be associated with Ca2⫹ oscillations in motoneurons as well as in other neurons. Calcium may enter into cells through different routes. Whatever the receptors involved (GABAA, glycine, non-NMDA), the spontaneous activity results in membrane depolarization and thereby to Ca2⫹ entry through voltage-dependent Ca2⫹ channels. Ca2⫹ entry is enhanced by NMDA receptor activation and possibly by the Ca2⫹ ion permeability of non-NMDA receptors (see above). Influx of calcium triggers release from intracellular stores that amplifies the calcium signal (see [19] for recent review). A central role for intracellular calcium in the promotion of neuronal survival [33] and the regulation of neuronal differentiation has been documented by many investigators. Spinal cells of the Xenopus embryo exhibit spontaneous elevations of intracellular calcium [57,125]. Both the calcium entry and calcium-induced calcium release are necessary to promote neuronal differentiation [39]. Calcium influx through voltage-dependent channels is necessary at an early stage of the development to allow maturation of the delayed rectifier potassium current of embryonic Xenopus spinal neurons [39,59]. The maturation of these potassium channels plays an important role in the developmental conversion of the ionic dependence of action po-

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FIG. 5. Summary of the different changes occurring in the spinal cord during perinatal development. (A) Age-related changes normalized to adult (100%). Some data such as the amplitude of the monosynaptic reflex at postntatal day (P) 7, compared to adult, are hypothetical and indicated by question marks. (B) Schematic representation of the development of the supraspinal control over lumbar motoneurons and the relative functional importance of excitatory and inhibitory amino acid receptors (the darker the symbol the more important the receptor). The influence of supraspinal structures is exerted mainly through unmyelinated fibers and polysynaptic pathways at around birth; more direct connections are present at the end of the first postnatal week (note also that fibers are partly myelinated). Electrical coupling (but not gap junctions) disappears during development. Activation of glycine and ␥-aminobutyric acid (GABA)A receptors leads to membrane depolarization at birth and to hyperpolarization from the 2nd postnatal week onwards. The importance of glycine as a major inhibitory neurotransmitter increases during maturation. The contribution of AMPA receptors to excitatory amino acid transmission also increases.

tentials in embryonic Xenopus spinal neurons (i.e., from calciumdependent to sodium-dependent spikes [10]) and in the shortening of action potential duration in neonatal rat motoneurons (see above). The contribution of calcium to the developmental regulation of neurotransmitter synthesis has also been demonstrated. The appearance of GABA immunoreactivity in Xenopus spinal neurons in dissociated cell culture indeed depends on spontaneous calcium influx at early stages of development [59,123]. It is blocked by transcriptional inhibitors, suggesting that GABA expression is regulated by calcium through the regulation of transcription of GABA synthetic enzymes [123]. Development of Reflex Arcs Spontaneous bursting activity in primary afferent terminals may be important for the development of sensorimotor connections, in particular, axon extension (see [56,74] for the contribution of intracellular calcium to the regulation of neurite elongation), central pathfinding and synapse formation. It is indeed known that neural activity plays a major role in the establishment of appropriate connectivity in the central nervous system. At an early stage in the development of reflex arcs, the spontaneous synchronized oscillatory activity observed in motoneurons and primary afferent terminals may serve as a recognition signal between the pre- and postsynaptic elements. This could help ensure that motoneurons of

a given motor pool receive similar synaptic inputs. Once reflex arcs are formed, the activity in primary afferent terminals would return to motoneurons as afferent signals which will serve to stabilize the synapse. Whether the spontaneous activity in the immature rat spinal cord also regulates neuronal differentiation or neuritic extension is unknown. CONCLUSIONS This review has demonstrated that the rat spinal cord undergoes a significant continuous transformation during perinatal development. All these modifications should be taken into account by neuroscientists using this preparation and pooling the data obtained over a time window of only a few days. The rapid development that rat motoneurons undergo during this period may be schematically summarized as follows (Figs. 5A,B). The last week before birth represents the initial stages of motoneuron development including regulation of the number of cells and the arrival of segmental and first supraspinal afferents. At around birth, motoneurons have a low rheobase which, in addition to the overexpression of glutamate receptors and the depolarizing action of the main neurotransmitters (including the “inhibitory” aminoacids GABA and glycine), leads to abundant spontaneous activity; they receive both appropriate and inappropriate connections from the

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periphery; the control from supraspinal structures is relatively weak and exerted mainly through polysynaptic connections over the whole motor pool, due to electrical coupling; extensor motoneurons are more immature than flexor motoneurons; neurons are weakly myelinated. One week after birth, inappropriate segmental connections have disappeared; the influence exerted by descending pathways is more important both quantitatively and qualitatively, in particular because of the increased magnitude of the non-NMDA component and of the absence of electrical coupling. Myelination of descending pathways and peripheral nerves is likely to play a crucial role in the development of motor control by increasing conduction velocity, thereby reducing the delay of the motor command. The combination of all these factors contributes to the maturation of posture which occurs during the early postnatal period, from a prone position to quadrupedal stance.

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