Influence of the vestibular system on the neonatal motor behaviors in the gray short-tailed opossum (Monodelphis domestica).

Marsupials are born very immature yet must be sufficiently autonomous to crawl on the mother's belly, find a teat and attach to it to pursue their development. Sensory inputs are necessary to guide the newborn to a teat and induce attachment. The vestibular system, which perceives gravity and head movements, is one of the senses proposed to guide newborns towards the teats but there are conflicting observations about its functionality at birth (postnatal day (P) 0). To test if the vestibular system of opossum newborns is functional and can influence locomotion, we used two approaches. First, we stimulated the vestibular apparatus in in vitro preparations from opossums aged from P1 to P12 and recorded motor responses: at all ages studied, mechanical pressures applied on the vestibular organs induced spinal roots activity whereas head tilts did not induce forelimb muscle contractions. Second, using immunofluorescence, we assessed the presence of Piezo2, a protein involved in mechanotransduction in vestibular hair cells. Piezo2 labeling was scant in the utricular macula at birth, but observed in all vestibular organs at P7, its intensity increasing up to P14; it seemed to stay the same at P21. Our results indicate that neural pathways from the labyrinth to the spinal cord are already in place around birth but that the vestibular organs are too immature to influence motor activity before the end of the second postnatal week in the opossum. It may be the rule in marsupial species that the vestibular system becomes functional only after birth.

Transient expression of NF200 by fibers in the nasal septum and rostral telencephalon of developing opossums (Monodelphis domestica).

Marsupials are born very immature and crawl on their mother's belly to attach to teats. Sensory information is required to guide the newborn and to induce attachment to the teat. Olfaction has been classically proposed to influence neonatal behaviors, but recent studies suggest that the central olfactory structures are too immature to account for them. In the newborn opossum, we previously described a fascicle of nerve fibers expressing neurofilament-200 (NF200, a marker of fiber maturity) from the olfactory bulbs to the rostral telencephalon. The course of these fibers is compatible with that of the terminal nerve that, during development, is characterized by the presence of neurons synthetizing gonadotropin hormones (GnRH). To evaluate if these fibers are related to the terminal nerve and if they play a role in precocious behaviors in opossums, we used immunohistochemistry against NF200 and GnRH. The results show that NF200-labeled fibers are present between P0 and P11, but do not reach much further caudally than the septal region. Only a few NF200-labeled fibers were found near the olfactory and vomeronasal epitheliums and they did not penetrate the olfactory bulbs. NF200-labeled fibers follow the same path as fibers labeled for GnRH. In contrast to the latter, NF200-labeled fibers are no longer visible at P15. These results suggest that these fibers are neither from the olfactory nor from the vomeronasal nerves but may be part of the terminal nerve. Their limited caudal extension does not support a role in the sensorimotor behaviors of the newborn opossum.

Influence of Temperature on Motor Behaviors in Newborn Opossums (Monodelphis domestica): An In Vitro Study.

External thermosensation is crucial to regulate animal behavior and homeostasis, but the development of the mammalian thermosensory system is not well known. We investigated whether temperature could play a role in the control of movements in a mammalian model born very immature, the opossum (Monodelphis domestica). Like other marsupials, at birth the opossum performs alternate and rhythmic movements with its forelimbs (FLs) to reach a teat where it attaches in order to continue its development. It was shown that FL movements can be induced by mechanical stimulation of the snout in in vitro preparations of newborns consisting of the neuraxis with skin and FLs intact. In the present study, we used puff ejections of cold, neutral (bath temperature) and hot liquid directed toward the snout to induce FL responses in such preparations. Either the responses were visually observed under a microscope or triceps muscle activity was recorded. Cold liquid systematically induced FL movements and triceps contractions, but neutral and hot temperatures were less potent to do so. Sections of the trigeminal nerves and removal of the facial skin diminished responses to cold and nearly abolished those to hot and neutral stimulations. Transient receptor potential melastatin 8 (TRPM8) being the major cold receptor cation channel in adult mammals, we employed immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to test for its expression, but found that it is not expressed before 13 postnatal days. Overall our results indicate that cold thermosensation exerts a strong influence on motor behaviors in newborn opossums.

Facial Mechanosensory Influence on Forelimb Movement in Newborn Opossums, Monodelphis domestica.

The opossum, Monodelphis domestica, is born very immature but crawls, unaided, with its forelimbs (FL) from the mother's birth canal to a nipple where it attaches to pursue its development. What sensory cues guide the newborn to the nipple and trigger its attachment to it? Previous experiments showed that low intensity electrical stimulation of the trigeminal ganglion induces FL movement in in vitro preparations and that trigeminal innervation of the facial skin is well developed in the newborn. The skin does not contain Vater-Pacini or Meissner touch corpuscles at this age, but it contains cells which appear to be Merkel cells (MC). We sought to determine if touch perceived by MC could exert an influence on FL movements. Application of the fluorescent dye AM1-43, which labels sensory cells such as MC, revealed the presence of a large number of labeled cells in the facial epidermis, especially in the snout skin, in newborn opossums. Moreover, calibrated pressure applied to the snout induced bilateral and simultaneous electromyographic responses of the triceps muscle in in vitro preparations of the neuraxis and FL from newborn. These responses increase with stimulation intensity and tend to decrease over time. Removing the facial skin nearly abolished these responses. Metabotropic glutamate 1 receptors being involved in MC neurotransmission, an antagonist of these receptors was applied to the bath, which decreased the EMG responses in a reversible manner. Likewise, bath application of the purinergic type 2 receptors, used by AM1-43 to penetrate sensory cells, also decreased the triceps EMG responses. The combined results support a strong influence of facial mechanosensation on FL movement in newborn opossums, and suggest that this influence could be exerted via MC.

Immunolocalization of cation-chloride cotransporters in the developing and mature spinal cord of opossums, Monodelphis domestica.

Spinal inhibition is required to generate coordinated outputs between antagonistic muscles during locomotion. It relies on low neuronal chloride concentration set by two cation-chloride cotransporters, NKCC1 and KCC2 which, respectively, pumps Cl(-) in or out of neurons. It is generally accepted that NKCC1 is gradually inactivated during development, while KCC2 is upregulated and activated, resulting in low intracellular [Cl(-)]. Newborn opossums are very immature but perform rhythmic and alternate movements of the forelimbs to crawl on the mother's belly and attach to a teat. Their hindlimbs are immobile. The alternation of the forelimbs suggests that mechanisms allowing spinal inhibition are present at birth. We studied the anatomical basis of inhibition in the spinal enlargements of postnatal opossums by immunolocalizing NKCC1 and KCC2. In some specimens, motoneurons and sensory afferents were labeled with TRDA prior to immunolabeling. At birth, both NKCC1 and KCC2 are detected in the presumptive gray and white matter of the ventral and the intermediolateral cord of both enlargements, but are sparse in the dorsal horn, where KCC2 is mostly seen on a small bundle of dendrites along primary afferents. KCC2 labeling is bright and has a mesh-like appearance in the gray matter and a radial appearance in the white matter, whereas NKCC1 is pale and diffuse. The subsequent expression of the cotransporters follows general ventrodorsal and mediolateral gradients, with the lumbar segments slightly lagging the cervical segments, until the mature pattern is observed around the 5th week. At all ages studied, KCC2 labeling is strong in the periphery of neurons. NKCC1 labeling decreases and becomes more uniformly distributed in the cells with age. Despite the significant anatomical and motor differences between the forelimbs and the hindlimbs of neonatal opossums, the maturation of KCC2 and NKCC1 is quite similar in both enlargements.

Distribution of the neuronal gap junction protein Connexin36 in the spinal cord enlargements of developing and adult opossums, Monodelphis domestica.

We use opossums Monodelphis domestica to study the development of mammalian motor systems. The immature forelimbs of the newborn perform rhythmic and alternating movements that are likely under spinal control. The hindlimbs start moving in the second week. Chemical synapses are scant in the spinal enlargements of neonatal opossums and the presence of electrochemical synapses has not been evaluated in this species or in other marsupials. As a first step aiming at evaluating the existence of such synapses in the neonatal spinal cord, we have investigated the presence of the exclusively neuronal gap junction protein connexin36 (Cx36) by immunohistochemistry in light microscopy. At birth, Cx36 immunoreactivity is moderate in the presumptive gray matter in both enlargements. Thereafter, it decreases gradually, except in the superficial dorsal horn where it increases to a plateau between P10 and P20. Cx36 labeling is detected in the presumptive white matter at birth, but then decreases except in the dorsal part of the lateral funiculus, where it is dense between P10 and P20. Cx36 has become virtually undetectable by P52. The presence of Cx36 in the spinal enlargements of postnatal opossums suggests that neurons might be linked by gap junctions at a time when chemical synapses are only beginning to form. The greater abundance of Cx36 observed transiently in the superficial dorsal horn suggests a stronger involvement of this protein in spinal sensory systems than in direct motor control of the limbs.

Developmental expression of spontaneous activity in the spinal cord of postnatal opossums, Monodelphis domestica: an anatomical study.

Using Sulforhodamine-101 (SR101) labeling and calcium imaging on in vitro preparations, we investigated the development of spontaneous activity in the spinal enlargements of a marsupial born more immature than eutherian mammals, the opossum Monodelphis domestica. Following the retrograde transport of Calcium Green dye from the limb nerves, we observed the occurrence of spontaneous calcium waves activating the motor columns of the cervical enlargement of opossums aged from P3 to P15 (day of birth: P0) and of the lumbar enlargement from at least P6 to P12. In other preparations, SR101 was added to the bath to identify the active cells. In P1 opossums, only a few SR101-labeled cells were observed in the cervical enlargement and none in the lumbar enlargement. At P5, their number increased cervically and they appeared in the lumbar enlargement. Motoneurons were the major cell type labeled by SR101 but dye leakage made their quantification inaccurate. SR101-labeled cells also occurred elsewhere in the ventral and dorsal grey. Their number increased until P12-14 in both enlargements and then decreased to disappear by P21, the last age examined. Thus in contrast to eutherian mammals, in which spontaneous activity is mostly prenatal, spontaneous activity occurs predominantly postnatally in opossums. It increases at the time when connections from the brain begin to impinge on spinal neurons and when the limbs, especially the hindlimbs, start moving and then decreases as the systems mature.

Inhibitory postsynaptic potentials in lumbar motoneurons remain depolarizing after neonatal spinal cord transection in the rat.

GABA and glycine are excitatory in the immature spinal cord and become inhibitory during development. The shift from depolarizing to hyperpolarizing inhibitory postsynaptic potentials (IPSPs) occurs during the perinatal period in the rat, a time window during which the projections from the brain stem reach the lumbar enlargement. In this study, we investigated the effects of suppressing influences of the brain on lumbar motoneurons during this critical period for the negative shift of the reversal potential of IPSPs (E(IPSP)). The spinal cord was transected at the thoracic level on the day of birth [postnatal day 0 (P0)]. E(IPSP), at P4-P7, was significantly more depolarized in cord-transected than in cord-intact animals (E(IPSP) above and below resting potential, respectively). E(IPSP) at P4-P7 in cord-transected animals was close to E(IPSP) at P0-P2. K-Cl cotransporter KCC2 immunohistochemistry revealed a developmental increase of staining in the area of lumbar motoneurons between P0 and P7 in cord-intact animals; this increase was not observed after spinal cord transection. The motoneurons recorded from cord-transected animals were less sensitive to the experimental manipulations aimed at testing the functionality of the KCC2 system, which is sensitive to [K(+)](o) and blocked by bumetanide. Although bumetanide significantly depolarized E(IPSP), the shift was less pronounced than in cord-intact animals. In addition, a reduction of [K(+)](o) affected E(IPSP) significantly only in cord-intact animals. Therefore influences from the brain stem may play an essential role in the maturation of inhibitory synaptic transmission, possibly by upregulating KCC2 and its functionality.

Perinatal development of the motor systems involved in postural control.

Motor behaviors of some species, such as the rat and the human baby, are quite immature at birth. Here we review recent data on some of the mechanisms underlying the postnatal maturation of posture in the rat, in particular the development of pathways descending from the brain stem and projecting onto the lumbar enlargement of the spinal cord. A short-lasting depletion in serotonin affects both posture and the excitability of motoneurons. Here we try to extrapolate to human development and suggest that the abnormalities in motor control observed in childhood--e.g. deficits in motor coordination--might have their roots in the prenatal period, in particular serotonin depletion due to exposure to several environmental and toxicological factors during pregnancy.

Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat.

The central pattern generators (CPGs) for locomotion, located in the lumbar spinal cord, are functional at birth in the rat. Their maturation occurs during the last few days preceding birth, a period during which the first projections from the brainstem start to reach the lumbar enlargement of the spinal cord. The goal of the present study was to investigate the effect of suppressing inputs from supraspinal structures on the CPGs, shortly after their formation. The spinal cord was transected at the thoracic level at birth [postnatal day 0 (P0)]. We examined during the first postnatal week the capacity of the CPGs to produce rhythmic motor activity in two complementary experimental conditions. Left and right ankle extensor muscles were recorded in vivo during airstepping, and lumbar ventral roots were recorded in vitro during pharmacologically evoked fictive locomotion. Mechanical stimulation of the tail elicited long-lasting sequences of airstepping in the spinal neonates and only a few steps in sham-operated rats. In vitro experiments made simultaneously on spinal and sham animals confirmed the increased excitability of the CPGs after spinalization. A left-right alternating locomotor pattern was observed at P1-P3. Both types of experiments showed that the pattern was disorganized at P6-P7, and that the left-right alternation was lost. Alternation was restored after the activation of serotonergic 5-HT(2) receptors in vivo. These results suggest that descending pathways, in particular serotonergic projections, control the strength of reciprocal inhibition and therefore shape the locomotor pattern in the neonatal rat.

Postural modifications and neuronal excitability changes induced by a short-term serotonin depletion during neonatal development in the rat.

Serotonin (5-HT) plays an important role both in the development and in the recovery of locomotion after spinalization in vertebrates. We investigated the contribution of the serotonergic system to the maturation of the lumbar motoneurons and networks in the neonatal rat. A 5-HT synthesis inhibitor, p-chlorophenylalanine (PCPA), was administered daily from the first postnatal day (P0) onward. This protocol depleted serotonin in the spinal cord within 3-4 d, as demonstrated by immunohistochemistry. PCPA-treated rats exhibited postural changes characterized by lesser flexion at the knee and ankle levels and lesser extension of the hip. Posture was asymmetric, suggesting possible deficits in the interlimb coordination. Intracellular recordings were made at P3-5 from motoneurons innervating different hindlimb muscles, using the in vitro brainstem-spinal cord-nerve-attached preparation. In PCPA-treated rats, the conduction velocity of motoneurons was increased, and their excitability was decreased (because of higher rehobase and input conductance) compared with sham animals. In accordance with postural observations, changes were more pronounced in hip extensor/knee flexor than in ankle extensor motoneurons. The maturation of repetitive firing properties was stopped by PCPA treatment, although PCPA, applied in vitro, had no effect on membrane properties. The spontaneous endogenously generated activity, which is a characteristic of immature networks, was increased in PCPA-treated rats, suggesting that developing lumbar networks are sensitive to 5-HT levels. Serotonin may play a critical role during development in regulating the balance between the excitability of motoneurons and that of interneurons. Interneuronal excitability is crucial for the activity-dependent development of spinal cord networks.

Picrotoxin and bicuculline have different effects on lumbar spinal networks and motoneurons in the neonatal rat.

Bicuculline is the most commonly used GABA(A) receptor antagonist to investigate the contribution of these receptors in motor control. However, this compound has been shown recently to potentiate the burst firing of neurons in various brain regions by blocking a calcium-activated potassium current underlying the spike after-hyperpolarization (AHP). This effect may distort our understanding of the role of GABA(A) receptors at the network level. In vitro brainstem-spinal cord preparations isolated from neonatal rats were used to compare the effects of bicuculline methiodide (bicuculline-M) and picrotoxin (PTX), another GABA(A) receptor antagonist, on the AHP of lumbar motoneurons as well as on spontaneous and locomotor-like motor activities. Intracellular recordings of lumbar motoneurons showed that bicuculline-M (20 microM) reduced the AHP to 57% of control whereas PTX (20-60 microM) had no significant effect. Bath-application of increasing concentrations of PTX caused an increase in spontaneous ventral root activity, which further increased significantly when bicuculline-M was added. The effects of both antagonists were tested on fictive locomotion. The left-right alternation was disrupted in the presence of bicuculline-M. A slow synchronous bursting activity of large amplitude also appeared in the presence of PTX. This slow rhythm was superimposed on a faster rhythm which still exhibited some degree of left-right alternation. These data demonstrate that bicuculline-M may not reveal accurately the contribution of GABA(A) receptors in motor control and the intrinsic properties of disinhibited networks.

Development of posture and locomotion: an interplay of endogenously generated activities and neurotrophic actions by descending pathways.

The adult pattern of locomotion is observed at the end of the second postnatal week in the rat. The in vitro spinal cord isolated from immature rats has served as a valuable preparation to study the mechanisms underlying the development of locomotion. Although the rat is unable to walk at birth, because of an immature posture, its spinal cord networks can generate at least two kinds of motor patterns in vitro. One activity is called 'fictive locomotion' because it shares several common features with locomotion observed in vivo. This fictive locomotor pattern is rarely observed spontaneously and its release requires either pharmacological or electrical stimulation of the spinal cord. A second endogenously generated activity observed in this preparation occurs spontaneously and exhibits phase relationships between motor outputs that are quite different from the fictive locomotor pattern. Here we review some of the developmental functions this spontaneous activity may subserve. It is likely a major trigger for the maturation of lumbar networks in the fetus, at a stage when inputs from both the periphery and supraspinal structures are weak. Pathways descending from the brainstem arrive in the lumbar enlargement during the last week in utero and the first two postnatal weeks. These pathways, through the neurotransmitters they contain, especially monoamines, are essential for the expression of some neuronal properties and may regulate several ongoing developmental processes.