Postnatal electrical and morphological abnormalities in lumbar motoneurons from transgenic mouse models of amyotrophic lateral sclerosis.

Antidromically identified lumbar motoneurons intracellularly recorded in the entire brainstem/spinal cord preparation isolated from SOD1(G85R) postnatal mice (P3-P10) were labelled with neurobiotin and fully reconstructed in 3D from serial sections in order to analyse their morphology. This staining procedure revealed differences between WT and SOD1(G85R) dendritic trees for most metric and topologic parameters analyzed. A highly complex morphology of SOD1(G85R) motoneurons dendrites (increased number of branching points and terminations) was found and the dendritic trees were longer compared to the WT motoneurons. These morphological changes observed in P8-P9 motoneurons mice occurred concomitantly with a decrease in the input resistance and gain. During electrophysiological recordings, four patterns of discharge were observed in response to ramp stimulations, that were equally distributed in WT and SOD1(G85R) motoneurons. In slice preparation, whole cell patch-clamp recordings made from developing motoneurons in SOD1(G85R) and double transgenic SOD1(G93A)/Hb9-eGFP mice showed that Riluzole, a blocker of persistent inward sodium conductance, altered the repetitive firing in a similar way for the 2 strains. These results show that the SOD1 mutations linked to familial ALS alter the development of the electrical and morphological properties of lumbar motoneurons.

Excitatory amino acids and synaptic transmission in embryonic rat brainstem motoneurons in organotypic culture.

We used brainstem motoneurons recorded in organotypic slice co-cultures maintained for more than 18 days in vitro, together with multibarrel ionophoretic applications of glutamate receptor agonists and bath applications of specific blocking agents, to study the responses of rat brainstem motoneurons to glutamate receptor activation, and the contribution of these receptors to synaptic transmission. Differentiated brainstem motoneurons in vitro are depolarized by glutamate, N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) iontophoresis, and express NMDA, AMPA and also specific kainate receptors, as evidenced by (+/-)2-amino-5-phosphonovaleric acid (APV)- and (-)1-(4-aminophenyl)-3-methyl-carbamoyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzo-diazepine [GYKI 53784 (LY303070)]-resistant depolarizations. Electrical stimulations applied to the dorsal part of the explant trigger excitatory synaptic potentials with latencies distributed in three regularly spaced groups. Excitatory postsynaptic potentials (EPSPs) in the earliest group have a similar latency and time course and correspond to monosynaptic activation. EPSPs in later groups have more scattered latencies and time courses and correspond to polysynaptic activation. Monosynaptic EPSPs are insensitive to the specific NMDA blocker APV, and are completely and reversibly suppressed by the non-competitive AMPA receptor antagonist GYKI 53784 (LY303070). Detailed analysis of the spontaneous excitatory synaptic activity shows that APV decreases the frequency of spontaneous EPSPs without modifying their shape or amplitude. We conclude that excitatory synapses on brainstem motoneurons in vitro are mainly activated through AMPA receptors (AMPA-Rs). NMDA receptors (NMDA-Rs) are present in the membrane, but are located either at extrasynaptic sites or silent synapses, and are not directly involved in synaptic transmission on motoneurons. On the contrary, NMDA receptors contribute to synaptic transmission within the premotor interneuronal network.

Developing rat brainstem motoneurones in organotypic culture express calcium permeable AMPA-gated receptors.

Recent studies suggest that calcium permeable excitatory amino acid receptors may play an important role in many developmental processes including dendritic differentiation, synaptogenesis and activity dependent plasticity or excitotoxicity related disorders. In this work, we investigate the existence of calcium permeable AMPA receptors in embryonic rat motoneurones in organotypic slice culture, which display an early sensitivity to AMPA and Kainate. We used excitatory amino acids induced cobalt uptake and show that developing motoneurones express Ca2+ permeable AMPA receptors. We demonstrate, as already described for other neuronal types in acute slices of adult animals, that the cobalt loading of motoneurones is not suppressed by blockade of synaptic activity by TTX. It is not induced by NMDA stimulation and does not result from the activation of voltage dependent calcium channels. It is specifically suppressed by the non-competitive AMPA antagonist GYKI 53784 (LY303070) and enhanced by the AMPA-receptor desensitization blocker Aniracetam. We conclude that cobalt loading results from the specific activation of AMPA receptors. We further show that, when cobalt loading is induced by threshold doses of Glutamate agonists, cobalt-sulfide deposits are found specifically in primary dendrites, dendritic spines and localized spots on the somatic and peripheral dendritic membrane. We suggest that this particular pattern of staining, different from the Golgi-like staining obtained with excitotoxic doses, may offer new information regarding the membrane density and distribution of calcium permeable AMPA receptors.

Synaptic inputs on rat brainstem motoneurones in organotypic slice culture.

To study the formation of target specific afferents on brain stem motoneurones of the rat, we used an organotypic co-culture of embryonic rat (E18) brain stem explants containing the facial or hypoglossal motor nuclei together with a tongue explant. The brain stem explants also contained known dorsal premotor structures such as lateral reticular nuclei and vestibular or spinal trigeminal nuclei. In cultures maintained in vitro for over 3 weeks, silver impregnation studies identified neurones in the dorsal sensory structures with axons arborizing within the motor nucleus. A double fluorescent labelling procedure demonstrated that axons originating from dorsal sensory regions come in close contact with identified motoneurones. Electrical stimulation of neurones in the dorsal regions induced monosynaptic and polysynaptic EPSPs and spikes in identified motoneurones together with muscle contraction. This work demonstrates that premotor structures in slice cultures develop organotypic functional synaptic connections with embryonic brain stem motoneurones.

Innervation of rat brainstem motoneurones in organotypic culture from a co-cultured sensory explant.

A co-culture procedure was used to investigate the formation of target-specific afferents on brainstem motoneurones of the rat in organotypic culture. Explants of brainstem motor nuclei together with a tongue explant were co-cultivated with explants obtained from vestibular, collicular or sensory trigeminal nuclei. Fiber connections between sensory and motor explants were observed after a few days in culture. Fluorescent labelling with carbocyanin dyes showed that fibers from the sensory explants invaded the motor explant, whereas no axon or dendrites from the motor explant were found penetrating the sensory structures. A double fluorescent labelling procedure, involving retrograde labelling of motoneurones and orthograde labelling of sensory fibers, demonstrated that sensory fibers come in close contact with labelled motoneurones. Electrical stimulation of the sensory explant induced muscle contraction, thus demonstrating that afferent fibers from the co-explanted structure establish functional synaptic connections with motoneurones.

Electrical properties of embryonic rat brainstem motoneurones in organotypic slice culture.

Organotypic co-cultures of embryonic E18-19 rat brainstem slices and tongue muscle maintained in vitro for more than 16 days were used to study the electrogenic properties of developing embryonic brainstem motoneurones derived from oculomotor, facial or hypoglossal nuclei. This preparation offers a unique opportunity to study the development of prenatal mammalian embryonic motoneurones. Our results show that embryonic rat brainstem motoneurones grown in organotypic culture with tongue muscle develop electrogenic membrane properties that can be compared with those described in newborn and adult animals in slice preparation. These motoneurones displayed a variety of sodium and calcium conductances. Two types of sodium conductances were present in all the recorded motoneurones. An Hodgkin-Huxley TTX-sensitive sodium conductance was involved in the spike potential, whereas a TTX-insensitive high threshold sodium conductance was uncovered when potassium and calcium currents were suppressed. Prominent calcium potentials contributed to the large delayed depolarizations following the spike potentials. High threshold calcium potentials were triggered with a slightly lower threshold than the sodium spike and contributed to the control of the pattern of discharge of the cell in response to slight shifts of membrane potentials. Low threshold calcium potentials were seen in 16% of the recorded motoneurones in hyperpolarized conditions. These calcium currents underlie the triggering of doublet spikes and rebound responses. Brainstem motoneurones in culture did not display differences that could be correlated with the origin of the motoneurone pool explanted and retained undifferentiated features suggesting that development of electrophysiological properties specific of each motoneurone pool are determined by presynaptic networks and target properties.

Effects of insulin-like growth factors on organotypic cocultures of embryonic rat brainstem slices and skeletal muscle fibers.

Embryonic rat brainstem slices including the facial and hypoglossal motor nuclei were maintained in organotypic cocultures with explants of embryonic tongue or post-natal skeletal muscle for periods up to 3 weeks. Survival and differentiation of motoneurones were dependent both on the type of muscle explant and its position relative to the brainstem. Tongue explants induced a more important glial outgrowth, a motoneurone migration towards the muscle, earlier muscular contractions and a more complete neuronal and muscular differentiation. Since the foetal tongue contains IGF levels as high as foetal liver, these effects might be due in part to diffusion of IGF from the explanted muscle. Indeed, foetal liver explants or crude foetal liver extracts induced effects similar to those of tongue explants. These effects can be reproduced by addition of IGF-1 or IGF-2, or both, into the culture medium. Although IGF-1 and IGF-2 had similar effects, IGF-1 induced a more pronounced muscular differentiation and IGF-2 promoted neuronal differentiation. Our results suggest that IGFs are good candidates as muscle-derived neurotrophic factors promoting survival and differentiation of rat cranial motoneurones. These results also stress the importance of neuroglial trophic interactions and target development.

Barium-induced bistability in rat ocular motoneurones in vitro.

Intracellular recordings were obtained from adult rat oculomotor neurones in an in vitro brainstem slice preparation. In motoneurones hyperpolarized to levels below -90 mV, depolarizing current injections induced voltage-dependent low-threshold plateau potentials. These potentials were triggered at rest when charge transfer through Ca2+ channels was increased by Ba2+ substitution. In such conditions, the membrane displayed voltage-dependent bistable properties similar to those described in other preparations and shown in vivo to be under neurotransmitter control. These results show that increasing inward currents in adult oculomotor neurones of the rat induce functionally different responses to slight shifts of membrane potential.

An Organotypic Co-culture of Embryonic Rat Brainstem and Tongue or Skeletal Muscle.

Transverse sections of rat embryo brainstem (embryonic age 18 - 19 days) containing the brainstem motor nuclei were explanted, together with a small piece of tongue or skeletal muscle, in a plasma clot and maintained in culture for 2 - 3 weeks using the roller tube technique. The results show that brainstem motoneurons survived, differentiated and innervated newly formed multinucleated myotubes which displayed large synchronized contractions after 1 week in culture. Muscle fibre contractions could be induced by excitatory amino acid applications and suppressed by curarization. Muscle fibres differentiated normally. During the first week they showed diffuse acetylcholinesterase positivity and multi-innervation. During the second and third weeks the number of motor end-plates was greatly reduced and transverse striation was observed. In the presence of muscle fibres, the brainstem thinned out and spread, becoming one or two cell layers thick, and the motoneurons tended to migrate towards the muscle fibres, becoming clearly observable in the living culture. When the muscle explant was not present, the brainstem explant did not spread and remained several cell layers thick, while acetylcholinesterase-positive cells, presumed to be motoneurons, tended to disappear. The preparation described is well suited for electrophysiological studies of differentiating motoneurons and offers direct access to their dendritic tree, a most desirable feature for patch-clamp or multisite optical recording.

Electrical activity of rat ocular motoneurons recorded in vitro.

The electrophysiological properties of rat oculomotor neurons were studied in an in vitro slice preparation. Motoneurons were identified by their antidromic response to third nerve rootlet stimulation, and by their orthodromic responses to medial longitudinal fasciculus and reticular stimulations. Passive membrane properties showed the existence of an inward rectification mechanism in all the recorded motoneurons. The action potential is comprised of several distinct components. The fast initial spike, composed of an initial segment spike and a somatodendritic spike, is followed by a delayed depolarization, an afterhyperpolarization and a late afterdepolarization. The afterhyperpolarization has a maximum duration of 55 ms. The late afterdepolarization is a voltage-dependent mechanism that produces an oscillatory behavior in depolarized cells. Two types of motoneurons were distinguished on the basis of their response to long-lasting depolarizing current pulses. The intensity-frequency curves show the existence of a primary and secondary range of discharge and the study of the interspike intervals points to specific properties of the conductance underlying the afterhyperpolarization. It is concluded that large, stellate motoneurons of the brainstem maintained in vitro retain specific electrophysiological properties, comparable to those described in vivo and which differentiate the ocular motoneurons from spinal motoneurons.

Histochemical identification of two fibre types in the retractor bulbi muscle of the cat.

In adult cats, the fibre population of the retractor bulbi muscle (RB) was analysed, using the histochemical reactions of ATPases. The muscle was found to contain type-2 fibres only, of which 70% were 2a and 30% 2b. Such ATPase profiles, corresponding to fast-twitch fibers, are in agreement with the mechanical properties of the muscle. Both types, 2a and 2b, included fibres in which the oxidative enzyme content was high and fibres in which it was low. The glycogen content of all fibers in the RB was uniformly low.

Resistance to glycogen depletion of motor units in the cat rectus lateralis muscle.

In nembutal anesthetized adult cats, intracellular stimulation of single abducens motoneurones was used to elicit glycogen depletion of their muscle units. Stimulation by short trains (13 pulses at 40 Hz) delivered once a second, was applied for 20 to 110 min. The activation of the motor unit was monitored by intracellular recording of motoneurone action potentials and by EMG. After the end of stimulation, the muscle was excised and frozen to be cut in serial sections that were processed for demonstration of either glycogen, ATPases or SDH. In two experiments, a motor unit could be histochemically identified because 10-15 fibres showed zones of complete glycogen depletion measuring about 5 mm in length. All the depleted fibres had the same histochemical profile: ATPases reactions gave dark staining with alkaline preincubation and light staining with acid preincubation whereas SDH activity was low. In other experiments, prolonged stimulation produced either no depletion at all or very limited zones of partial depletion in a few muscle fibres.

Ultrastructural and electrophysiological properties of accessory abducens nucleus motoneurones: an intracellular horseradish peroxidase study in the cat.

The physiological and morphological (light and electron microscopy) properties of six retractor bulbi motoneurones were analysed using the technique of intracellular recording and intracellular labelling with horseradish peroxidase. The retractor bulbi motoneurones were identified by antidromic invasion and orthodromic responses following stimulation of trigeminal afferents were studied. Two of these motoneurones were examined ultrastructurally. Terminal boutons forming synapses with labelled soma, labelled proximal and distal dendrites were characterized. Serial sections allowed the axon hillock to be analyzed and the initial segment of a presumed motoneurone to be observed in the section where the injected motoneurone was described. The ultrastructure of unidentified elements observed in the accessory abducens nucleus is stressed.

Comparison of antidromic and orthodromic action potentials of identified motor axons in the cat's brain stem.

Recordings were made from identified central axons at a known distance from their somata, to compare the action potentials resulting from antidromic and synaptic excitation. By taking advantage of the anatomical configuration within the brain stem of the motoneurones innervating the retractor bulbi muscle in the orbit, their axons were penetrated in the VIth nucleus and labelled by electrophoretic injection of horseradish peroxidase. Excitatory post-synaptic potentials recorded in the retractor bulbi axons at about 3 mm from the soma were six times smaller than in the soma. The space constant of the axonal segment between the retractor bulbi and the abducens nucleus was estimated to be 1.7 mm. When the axons propagated action potentials the attenuation was increased to eighteen times due to the nodes of Ranvier intercalated between the soma and the site of recording. Antidromic action potentials displayed stepwise changes in amplitude and shape when stimuli were applied at intervals decreasing from 5 ms to 0.7 ms. The changes were related to the different lengths of refractoriness of the soma, initial segment and axon. Orthodromic action potentials evoked by synaptic excitation displayed similar changes in amplitude and shape. These observations lead to the conclusion that the soma, initial segment and neighbouring nodes of Ranvier contribute significantly to the shape of the action potential. Contrary to the generally accepted view, it appears that the efferent discharge along motor axons can be initiated without a simultaneous activation of the somato-dendritic or even the initial segment membrane, as revealed by the lack of somato-dendritic and/or initial segment contribution to the shape of the synaptically evoked action potentials.

Morphological and electrophysiological properties of trigeminal neurones projecting to the accessory abducens nucleus of the cat.

Horseradish peroxidase was injected into the somata or axons of neurones located in the nucleus oralis of the spinal trigeminal complex and projecting to the accessory abducens nucleus. A group of 43 axons with electrophysiologically identified responses to the stimulation of three different areas of the face were studied. The latencies of their orthodromic responses following trigeminal stimulation and their pattern of discharges were compared to those of secondary trigeminal neurones and retractor bulbi motoneurones. Labelled trigeminal axons were found to generate collaterals for the accessory abducens nucleus. Terminal ramifications are present in the rostro-caudal part of the motor nucleus where the dendritic arborization of the motoneurones has been described. Connections to facial and trigeminal motor nuclei were also present, suggesting that secondary trigeminal axons distributed the information to the three motor targets at the brain stem level. It is suggested that the trigemino-retractor bulbi reflex is part of facial reflexes involved in orienting reactions, and protective responses resulting in coordinated movements of the facial musculature.

The vibrissal pad as a source of sensory information for the oculomotor system of the cat.

Responses from lateral rectus, medial rectus and retractor bulbi nerves were obtained following electrical stimulation of the vibrissal pad of the cat. Discharges in afferent fibres dissected from the infraorbital nerve were recorded during movements of the vibrissae and following electrical stimulation of the vibrissal pad. Both stimuli activated the same population of A alpha fibers. Intracellular records were obtained from lateral rectus motoneurones identified antidromically in the principal abducens nucleus and from retractor bulbi motoneurones similarly identified in the accessory abducens nucleus. EPSPs (3 mV) were recorded in lateral rectus motoneurones following electrical stimulation of the ipsilateral vibrissal pad at a latency of 3.5 ms. Large-amplitude disynaptic EPSPs (15 mV) were recorded in retractor bulbi motoneurones following the same vibrissal stimulation. The synaptic excitation evoked in both lateral rectus and retractor bulbi motoneurones through stimulation of the ipsilateral vibrissal pad induced an early retraction followed by an abduction of the eye ball. The hypothesis is that the vibrissal message might complement other sensory modalities in the generation of patterned eye movements.

[Oculomotor reflex evoked by vibrassae stimulation in the cat]. / Réflexe oculomoteur déclenché par la stimulation des vibrisses chez le chat.

The oculomotor effects of vibrissae stimulation have been studied in the "encéphale isolé" non-anaesthetized Cat. Intracellular recording of lateral rectus motoneurones showed an excitatory projection via the ipsilateral infraorbital nerve to these motoneurones. The afferent fibers are type A alpha, recuited with a low threshold (0.2-0.5 mA) and convey information about vibrissae displacement.

Anatomical and electrophysiological identification of motoneurones supplying the cat retractor bulbi muscle.

Motoneurones innervating the retractor bulbi muscle in the cat have been identified by retrograde labelling with horseradish peroxidase, by intracellular recording and by intracellular staining with horseradish peroxidase. Their somata are found in an accessory abducens nucleus, analogous to that described in some other species, which consists of a narrow column of cells situated in the lateral tegmental reticular field, above the superior olive and medial to the facial nerve. The column of cells extends over approximately 1.5 mm from P 5.5 to P 7. The retractor bulbi motoneurones number from 80 to 120 and have large, elongated somata which give rise to five or six major dendrites. Their axons cross the reticular formation in a dorso-medial direction to pass through the principal abducens nucleus before turning to leave the brain stem in the 6th nerve. Antidromic latencies ranged from 0.4 to 0.7 ms. Some retractor bulbi motoneurones could also be activated antidromically by stimulation of the lateral rectus muscle nerve.