Edouard Pearlstein

The in vitro neonatal rat spinal cord preparation: a new insight into mammalian locomotor mechanisms

The in vitro neonatal rat spinal cord preparation is the first mammalian nervous system isolated from the brainstem to the caudal end of the spinal cord. It permits the study of the cellular properties of mammalian locomotor networks and is unique in containing all the nervous structures related to locomotion. Although being a very immature system, this model has been considered as an adult preparation in which mammalian locomotor central pattern generators can be studied in detail. Nevertheless, one can also follow the development of locomotor functions during the perinatal period. Contrary to the adult, all neuroactive substances can directly reach the cellular structures in the brainstem-spinal cord preparation. When a neuroactive substance is applied to the bath, a single rhythmic activity is recorded along the cord. In fact, three rhythms can be isolated: one at the cervical level for the forelimbs, one at the lumbar level for the hind limbs and one in the sacrococcygeal region for the tail. Studies carried out on this preparation deal with three major areas: (1) relations between spontaneous activity and maturation of spinal network, (2) organisation of the different spinal networks, (3) key role of the descending pathways.

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.

Inhibitory connections between antagonistic motor neurones of the crayfish walking legs

The inhibitory relationship between two antagonistic groups of motor neurones (MNs) that control the second leg joint of the crayfish Procambarus clarkii, was investigated in an in vitro preparation of the ventral nerve cord. Paired intracellular recordings were used to test the hypothesis that reciprocal inhibitory connections between levator (Lev) and depressor (Dep) MNs are direct. The injection of depolarising current into a Lev MN induces a hyperpolarising response in the Dep MN. This inhibitory relationship does not require spikes in the presynaptic MN, because it persists when spikes are suppressed by the sodium channel blocker tetrodotoxin (TTX). This reciprocal inhibition is graded, and both the amplitude and the time constant of the hyperpolarising response increase with increasing amount of depolarising current injected into an antagonistic MN. Although this inhibition is slow (synaptic delay around 10 ms), it is probably supported by a direct glutamatergic synapse from the antagonistic glutamatergic MN because it persists in the presence of the γ‐amino‐butyric acid (GABA) synthesis inhibitor 3‐mercapto‐propionic acid (3‐MPA). This hypothesis is reinforced by the demonstration of close appositions between antagonistic MNs by using a confocal microscope, and by the presence of glutamate‐immunoreactive synapses on the neurites of MNs labelled for electron microscopy by intracellular injection of horseradish peroxidase. J. Comp. Neurol. 399:241–254, 1998.

Serotonin Enhances the Resistance Reflex of the Locomotor Network of the Crayfish through Multiple Modulatory Effects that Act Cooperatively

Serotonin (5HT) is an endogenous amine that modifies posture in crustacea. Here, we examined the mechanisms of action of 5HT on the resistance reflex in crayfish legs. This reflex, which counteracts movements imposed on a limb, is based on a negative feedback system formed by proprioceptors that sense joint angle movements and activate opposing motoneurons. We performed intracellular recordings from depressor motoneurons while repetitively stretching and releasing a leg joint proprioceptor in a resting in vitro preparation (i.e., a preparation that lacks spontaneous rhythmic activity). 5HT increased the amplitude of the depolarization during the release phase of the proprioceptor (corresponding to an upward movement of the leg) and the discharge frequency of the motoneurons. The 5HT-induced increase in the resistance reflex is caused, to a large extent, by polysynaptic pathways because it was very attenuated in the presence of high divalent cation solution. In addition to this activation of the polysynaptic pathways, 5HT also has postsynaptic effects that enhance the resistance reflex. 5HT causes a tonic depolarization, as well as an increase in the time constant and input resistance of motoneurons. We developed a simple mathematical model to describe the integrative properties of the motoneurons. The conclusion of this study is that the input frequency and the decay time constant of the EPSPs interact in such a way that small simultaneous changes in these parameters can cause a large effect on summation. Therefore, the conjunction of presynaptic and postsynaptic changes produces a strong cooperative effect on the resistance reflex response.

Cholinergic control of the walking network in the crayfish Procambarus clarkii

The output of a neuronal network results generally from both the properties of the component neurons and their synaptic relationships. This article aims at synthesizing various results obtained on the neural network generating locomotion in vitro. In the preparation used, consisting of the last three thoracic ganglia (3-5) along with motor nerves from the 5th leg ganglion to the promotor, remotor, levator and depressor muscles, motor nerve recordings generally revealed only tonic activity in several different motoneurons (MNs). However, rhythmic activity can be obtained by the use of cholinergic agents such as the oxotremorine (Oxo) superfused in the bath (5 x 10(-5) M). If Oxo is pressure-ejected locally in the ganglion, it is possible, depending upon the locus where the drug is applied, to elicit a rhythmic activity restricted to a group of antagonistic MNs. To analyze how cholinergic agents are able to induce such rhythmic activity, very small volumes of drug (50-200 pl), were applied close to the recording electrode. Two types of depolarizing response occurred: a fast large amplitude depolarization (5-20 mV) and a long lasting (10s to several minutes) low amplitude depolarization (1-3 mV). These responses persisted in the presence of TTX and Co(2)+. The transient initial depolarization is a mixed nicotinic and muscarinic voltage-independent response during which the input resistance decreases by 20 to 40%. In contrast, the long lasting component is voltage-dependent, exclusively muscarinic and associated to a 5-10% increase of input resistance due to the closing of a K+ conductance that is active at the resting Vm, and totally suppressed at holding potentials below -70 mV. More generally, K+ currents activated at resting potential are responsible for membrane potential stability. The injection of TEA, a blocker of the K+ currents, through the recording electrode is able to unmask plateaus above a threshold depolarization. These plateaus are TTX-sensitive but persist in the presence of Ca(2)+ channel blockers. Moreover, in 10% of TEA-filled MNs a spontaneous pacemaker activity was revealed. The organization of the locomotor network is also based upon connections between MNs and INs. Within a MN pool, connections are only loosely established, appearing to consist mainly of electrical coupling. Inhibitory synaptic connections between MNs of opposite pools are mediated by chloride channels. However, the neurotransmitter involved could be either GABA or glutamate. Therefore, at the level of a given joint, a basic rhythm occurs due to both motoneuronal membrane properties and motoneuronal connectivity. However, the coordination of all MNs of an entire leg during fictive walking activity requires the involvement of INs. Based upon these data, we propose a two-stage model of the locomotor network organization: a joint motoneuronal level and a whole leg interneuronal level.

Neuromodulation of reciprocal glutamatergic inhibition between antagonistic motoneurons by 5-hydroxytryptamine (5-HT) in crayfish walking system

In an in vitro preparation of the crayfish thoracic locomotor system, paired intracellular recordings were performed from antagonistic depressor (Dep) and levator (Lev) motoneurons (MNs) that control the second joint of walking legs. Connections between these two groups of MNs consist mainly of inhibitory connections and weak electrotonic synapses. Injection of depolarizing current into a Lev MN results in a hyperpolarization in a Dep MN, and vice versa. This reciprocal glutamatergic inhibition, is not changed in the presence of the sodium channel blocker tetrodotoxin (TTX) and therefore is likely supported by a direct connection between MNs. By contrast, reciprocal inhibition is largely reduced in the presence of 5-hydroxytryptamine (5-HT; 10 microM). Direct micro-application of glutamate pressure-ejected close to an intracellularly recorded MN, evoked an inhibitory response in that MN, accompanied by a decrease of input resistance. These two effects were dramatically reduced in the presence of 5-HT. Thus 5-HT could be involved in mechanisms of dynamic reconfigurations of the neural network controlling leg movements in crayfish.

GABA and glutamate-like immunoreactivity at synapses on depressor motorneurones of the leg of the crayfish, Procambarus clarkii

To investigate their synaptic relationships, depressor motorneurones of the crayfish leg were impaled with microelectrodes, intracellularly injected with horseradish peroxidase, and prepared for electron microscopy. Post‐embedding immunogold labelling with antibodies against γ‐aminobutyric acid (GABA) or glutamate was carried out either alone or together on the same section and allowed the identification of three classes of input synapses: 51% were immunoreactive for glutamate and contained round agranular vesicles, 31% were immunoreactive for GABA and contained pleomorphic agranular vesicles, and the remainder were immunoreactive for neither and also predominantly contained pleomorphic agranular vesicles. Output synapses were abundant in some of the motorneurones but were not seen in others, suggesting that members of the motor pool differ in their connectivity. J. Comp. Neurol. 422:510–520, 2000.

Contribution of 5-HT to locomotion – the paradox of Pet-1−/− mice

Serotonin (5‐HT) plays a critical role in locomotor pattern generation by modulating the rhythm and the coordinations. Pet‐1, a transcription factor selectively expressed in the raphe nuclei, controls the differentiation of 5‐HT neurons. Surprisingly, inactivation of Pet‐1 (Pet‐1−/− mice) that causes a 70% reduction in the number of 5‐HT‐positive neurons in the raphe does not impair locomotion in adult mice. The goal of the present study was to investigate the operation of the locomotor central pattern generator (CPG) in neonatal Pet‐1−/− mice. We first confirmed, by means of immunohistochemistry, that there is a marked reduction of 5‐HT innervation in the lumbar spinal cord of Pet‐1−/− mice. Fictive locomotion was induced in the in vitro neonatal mouse spinal cord preparation by bath application of N‐methyl‐d,l‐Aspartate (NMA) alone or together with dopamine and 5‐HT. A locomotor pattern characterized by left–right and flexor–extensor alternations was observed in both conditions. Increasing the concentration of 5‐HT from 0.5 to 5 μm impaired the pattern in Pet‐1−/− mice. We tested the role of endogenous 5‐HT in the NMA‐induced fictive locomotion. Application of 5‐HT2 or 5‐HT7 receptor antagonists affected the NMA‐induced fictive locomotion in both heterozygous and homozygous mice although the effects were weaker in the latter strain. This may be, at least partly, explained by the reduced expression of 5‐HT2AR as observed by means of immunohistochemistry. These results suggest that compensatory mechanisms take place in Pet‐1−/− mice that make locomotion less dependent upon 5‐HT.

Development of Interlimb Coordination in the Neonatal Rat

Locomotion is a type of motor behaviour that is produced by spinal neuronal networks associated with the different limbs. The rat in which development lasts three weeks in utero and continues during the first three post-natal weeks appears to be a very attractive model for the study of interlimb coordination maturational mechanisms. An early expression of rhythmic locomotor activity is observed at birth in vitro. Neuroactive substances like excitatory amino acid or amines (5-HT...) applied on the entire brainstem/spinal cord preparation induce a unique rhythmical activity over the cervical and the lumbar generators. This strict coordination results from a mutual interaction between generators since both bursting frequencies are altered significantly after functional isolation, with a slowing down of the isolated cervical network and a significant acceleration of the lumbar burst generator. However the rat does not walk at birth due to an absence of postural regulations. During the first two weeks, the neonatal rat is able to swim or to produce “air stepping” with an ipsilateral and a contralateral alternation between the different limbs. Adult walking occurs only during the third postnatal week. At least two mechanisms are able to control this early interlimb coordination . Numerous studies showed that 5-HT has ubiquitous topic and trophic effects on the early development of neurons and synapses. Studies on “PCPA” treated and “spinal” neonatal rats demonstrated strong deficits in locomotor movements. The second mechanism concerns the sensory afferents after birth; proprioceptive peripheral loops feed the central network and adapt its activation in a coordinated manner.

Aminergic Modulation of Sensory-Motor Integration in the Walking System of the Crayfish

Locomotion is a motor act that is centrally generated by specialized neural networks called central pattern generators. Though capable of generating a patterned motor activity, these networks remain under control of both superior command center and sensory inputs (Grillner and Dubuc 1988; Rossignol and Dubuc 1994; Cazalets et al. 1998; Jordan 1998). Indeed, numerous sensory cues are able (in both vertebrates and invertebrates) to trigger, modulate or even stop locomotion (El Manira et al. 1991a; Laurent 1991; Elson et al. 1992; Viana Di Prisco et al. 1997). This allows the animal to adapt its motor acts to its environment in order to produce an optimized behavior. Nevertheless, depending on the ongoing behavior of the animal and/or on the event appearing in its environment, sensory feedback can have a different significance or may sometimes be not pertinent. Therefore it is not surprising that the nervous system has developed a number of mechanisms that ensure fine modulations of the sensory-motor organization. Such modifications may be classified depending on the time scale of their effects: some are phasic and involve classical neurotransmission and receptor channels, as is the case of presynaptic inhibition, where interneurons inhibit the sensory information at the presynaptic level, while others exert a more continuous control involving metabotropic receptors and/or neuromodulators.