Embryonic Rat Motoneurons Research Articles

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells.

BackgroundNeuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function.MethodsThis report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development.ResultsThis unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions.ConclusionThis coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.

Rat Cortical Oligodendrocyte–Embryonic Motoneuron Co-Culture: An <I>In Vitro</I> Axon-Oligodendrocyte Interaction Model

Mechanisms that control the differentiation and function of oligodendrocytes in the central nervous system are complex and involve multiple inputs from the surrounding environment, including localized concentrations of growth factors and the extracellular matrix. Dissection and analysis of these inputs are key to understanding the pathology of central nervous system demyelinating diseases such as multiple sclerosis, where the differentiation of myelinating oligodendrocytes from their precursors underlies the remission phase of the disease. In vitro co-culture models provide a mechanism for the study of factors that regulate differentiation of oligodendrocyte precursors but have been difficult to develop due to the complex nature of central nervous system myelination. This study describes development of an in vitro model that merges a defined medium with a chemically modified substrate to study aspects of myelination in the central nervous system. We demonstrate that oligodendrocyte precursors co-cultured with rat embryonic motoneurons on non-biological substrate (diethylenetriamine trimethoxy-silylpropyldiethylenetriamine), can be induced to differentiate into mature oligodendrocytes that express myelin basic protein, using a serum-free medium. This defined and reproducible model of in vitro myelination could be a valuable tool for the development of treatments for demyelinating diseases such as multiple sclerosis.

Tissue engineering the monosynaptic circuit of the stretch reflex arc with co-culture of embryonic motoneurons and proprioceptive sensory neurons

The sensory circuit of the stretch reflex arc is composed of intrafusal muscle fibers and their innervating proprioceptive neurons that convert mechanical information regarding muscle length and tension into action potentials that synapse onto the homonymous motoneurons in the ventral spinal cord which innervate the extrafusal fibers of the same muscle. To date, the in vitro synaptic connection between proprioceptive sensory neurons and spinal motoneurons has not been demonstrated. A functional in vitro system demonstrating this connection would enable the understanding of feedback by the integration of sensory input into the spinal reflex arc. Here we report a co-culture of rat embryonic motoneurons and proprioceptive sensory neurons from dorsal root ganglia (DRG) in a defined serum-free medium on a synthetic silane substrate (DETA). Furthermore, we have demonstrated functional synapse formation in the co-culture by immunocytochemistry and electrophysiological analysis. This work will be valuable for enabling in vitro model systems for the study of spinal motor control and related pathologies such as spinal cord injury, muscular dystrophy and spasticity by improving our understanding of the integration of the mechanosensitive feedback mechanism.

Weighted least-squares approach for identification of a reduced-order adaptive neuronal model.

This brief is focused on the parameter estimation problem of a second-order adaptive quadratic neuronal model. First, it is shown that the model discontinuities at the spiking instants can be recast as an impulse train driving the system dynamics. Through manipulation of the system dynamics, the membrane voltage can be obtained as a realizable model that is linear in the unknown parameters. This linearly parameterized realizable model is then utilized inside a prediction error-based framework to design a dynamic estimator that allows for rapid estimation of model parameters under a persistently exciting input current injection. Simulation results show the feasibility of this approach to predict multiple neuronal firing patterns. Results using both synthetic data (obtained from a detailed ion-channel-based model) and experimental data (obtained from in vitro embryonic rat motoneurons) suggest directions for further work.

Modulation of synaptic transmission and analysis of neuroprotective effects of valproic Acid and derivates in rat embryonic motoneurons.

Amyotrophic lateral sclerosis is a devastating motoneuron disorder for which no effective treatment exists. There is some evidence for neuroprotective effects of valproic acid (VPA). The beneficial effects, however, are limited due to the adverse effects of VPA. To overcome this problem, a number of VPA derivates with fewer side effects have been synthesized. In the present study, we investigated the viability of highly purified embryonic motoneurons cultured on glial feeder layers, composed of either astrocytes or Schwann cells, or in monoculture, in presence of VPA and its three derivates 3-propyl-heptanoic acid (3-PHA), PE-4-yn enantiomers (R- and S-PE-4-yn). An excitotoxic stimulus, kainate (KA), was added at day in vitro 9 (DIV9) and the neuroprotective effect of either simultaneous incubation (DIV9) or pre-incubation (DIV1) of VPA and its derivates was tested. The survival of motoneurons under simultaneous application of KA and VPA derivates was not remarkably increased. Pre-incubation with VPA and even more with the derivates before the addition of KA, however, significantly reduced their vulnerability against the KA-induced neurotoxic effect. Our data suggest that the neuroprotective capacities of VPA and its three derivates tested here drastically increase when they are added several days before KA. Most prominent neuroprotective effects were seen for the PE-4-yn enantiomers. Patch-clamp experiments revealed an antiexcitotoxic effect of the S-PE-4-yn enantiomer that reduces the frequency of postsynaptic currents and enhances the inhibitory postsynaptic transmission dependent on the co-culture condition.

Intracellular Ca 2+ regulation in rat motoneurons during development

Changes in intracellular Ca 2+ concentration ([Ca 2+] i) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca 2+ stores, via both ryanodine-sensitive and IP 3-sensitive intracellular Ca 2+ channels control [Ca 2+] i homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca 2+] i increased progressively from days 1–7 in culture. These responses were blocked by ryanodine and nicardipine but not by ω-conotoxin-GVIA or ω-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca v1 Ca 2+ channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca 2+ oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca 2+ stores from the endoplasmic reticulum, induced an increase in [Ca 2+] i in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5–6 days in vitro. Xestospongin-C, a membrane-permeable IP 3 receptor inhibitor blocked the CPA-induced [Ca 2+] i response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP 3 Ca 2+ channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca 2+] i homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca 2+ channel responsible for a Ca 2+-induced Ca 2+ release in embryonic motoneurons following voltage-dependent Ca 2+ entry via L-type Ca 2+ channels.

Rat embryonic motoneurons in long-term co-culture with Schwann cells—a system to investigate motoneuron diseases on a cellular level in vitro

Investigations of motoneuron diseases on a cellular and molecular level require long-term cultivation of primary cells. Here we present a new culture system in which matured motoneurons interact with their physiological partners like interneurons, astroglia and peripheral glia cells. This enables motoneuron-maturation for up to 3 weeks, while motoneurons consistently reached large diameters of their somata of 30–45 μm, occasionally more than 80 μm. Dissociated rat embryonic ventral spinal cord cells were enriched for motoneurons by density gradient centrifugation and seeded on a non-confluent mono-layer of highly enriched neonatal rat Schwann cells. Immunocytochemical visualization of neuron specific βIII-tubulin in all neurons and of motoneuron specific non-phosphorylated neurofilament H/M, respectively, revealed that after 3 days in vitro >70% of all neurons were motoneurons. After 20 days in vitro , a motoneuron fraction of 12% was maintained. Motoneurons were susceptible to transient transfection with green fluorescent protein cDNA when liposomal transfection and an enhancer substance were combined. Synaptic connections enabled formation of spontaneously active neuronal networks which provide a culture model to study glutamate excitotoxicity and calcium deregulation on a molecular level. Both mechanisms are implied in the pathophysiology of amyotrophic lateral sclerosis, a neurodegenerative motoneuron disorder.

Inkjet printing of viable mammalian cells

The purpose of this study was to explore the use of a commercial thermal printer to deposit Chinese Hamster Ovary (CHO) and embryonic motoneuron cells into pre-defined patterns. These experiments were undertaken to verify the biocompatibility of thermal inkjet printing of mammalian cells and the ability to assemble them into viable constructs. Using a modified Hewlett Packard (HP) 550C computer printer and an HP 51626a ink cartridge, CHO cells and rat embryonic motoneurons were suspended separately in a concentrated phosphate buffered saline solution (3×). The cells were subsequently printed as a kind of “ink” onto several “bio-papers” made from soy agar and collagen gel. The appearance of the CHO cells and motoneurons on the bio-papers indicated an healthy cell morphology. Furthermore, the analyses of the CHO cell viability showed that less than 8% of the cells were lysed during printing. These data indicate that mammalian cells can be effectively delivered by a modified thermal inkjet printer onto biological substrates and that they retain their ability to function. The computer-aided inkjet printing of viable mammalian cells holds potential for creating living tissue analogs, and may eventually lead to the construction of engineered human organs.

Activation of P-type calcium channel regulates a unique thapsigargin-sensitive calcium pool in embryonic motoneurons.

By regulating voltage-dependent Ca2+ influx and intracellular Ca2+ homeostasis, electrical activity plays a central role in motoneuron development. Dissociated cultures of purified embryonic rat motoneurons were used to explore the molecular mechanisms by which Ca2+ influx control [Ca2+]i transients in these neurons. Thapsigargin (250 nm) and cyclopiazonic acid (10 micro m), which deplete Ca2+ stores in the endoplasmic reticulum, decrease by 30% the depolarization-induced [Ca2+]i transients in motoneurons without affecting voltage-activated calcium currents. This thapsigargin-sensitive intracellular Ca2+ pool differs from other previous described Ca2+ stores that are sensitive to ryanodine or caffeine, inositol triphosphate, insulin and from mitochondrial Ca2+ pools. Thapsigargin affected the Cav2.1 P-type Ca2+ channel component of the depolarization-induced [Ca2+]i transient in motoneurons but spared [Ca2+]i transient induced by Cav1 L-type and Cav2.2 N-type Ca2+ channel components, suggesting a close functional relationship between Cav2.1 subunit and this unique thapsigargin-sensitive Ca2+ store. Altogether the present results demonstrate a new pathway, used by embryonic motoneurons, to regulate Ca2+ signalling through voltage-activated (Cav2.1) Ca2+ channels.

Molecular determinants of emerging excitability in rat embryonic motoneurons.

Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na(+) current is supported by Nav1.2 and/or Nav1.3 alpha-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Nav beta 2.1 transcript was not detected, whereas the Nav beta 1.1 transcript was present. Even in the absence of Nav beta 2.1, alpha-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K(+) currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K(+) current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv alpha-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K(+) current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na(+) and K(+) channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.

Localization of transforming growth factor beta in association with neuromuscular junctions in adult human muscle.

Transforming growth factors-beta 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF-beta subclasses for motoneurons has been shown in vitro. TGF-beta 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF-beta 1 stimulates proliferation of rat Schwann cells. Recently, TGF-beta 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF-beta 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF-beta 1; 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF-beta 1 and TGF-beta 3. Intense immunoreactivity of TGF-beta 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF-beta 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF-beta with neuromuscular junctions may suggest a significant function in neuromuscular communication.

Ionic mechanisms underlying excitatory effects of serotonin on embryonic rat motoneurons in long-term culture

The actions of serotonin were investigated on motoneurons isolated from embryonic day 14 rat spinal cord and enriched by metrizamide density gradient centrifugation. Trophic support was provided by a spinal cord glial monolayer, ciliary neurotrophic factor and heat-inactivated serum. Cultures were maintained for 17–83 days and investigated using whole-cell patch-clamp recording. Serotonin evoked slow depolarizations (6.2±0.7 or 9.3±1.3 mV in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione and strychnine, ec 50 8.2 nM), which were reversibly blocked by 0.1 μM ketanserin. Serotonin generated synaptic potentials in motoneurons, lowered the threshold for repetitive firing and changed the slope of the current intensity–firing frequency relationship. The inward current evoked by serotonin (−147±15.2 pA) was ascribed to a complex ionic mechanism, which varied amongst neurons in the sampled population. It was due to closure of barium-sensitive potassium channels, effects on I h and increase in a separate mixed cation current which comprised both transient voltage-sensitive and sustained components. We conclude that serotonergic responses develop in motoneurons cultured under these conditions in the absence of serotonergic input, sensory neurons or many interneurons.

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca 2+ influx in motoneuron development raises important questions about the regulation of Ca 2+ signalling induced by voltage-dependent Ca 2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca 2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca 2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca 2+ current activated at 0 mV from a holding potential of −100 mV, approximatively 45% was omega-conotoxin-GVIA (1 μM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca 2+ currents. Based on this pharmacology, changes in intracellular free Ca 2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K + depolarization, the intracellular Ca 2+ transient induced by the activation of each type of Ca 2+ channel appeared to be quantitatively proportional to their Ca 2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K +-induced intracellular free calcium concentration transients indicating both the absence of Ca 2+-gated Ca 2+-release channels and of a Ca 2+-induced Ca 2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca 2+ channels that function as important regulators of intracellular Ca 2+ signalling and may be involved in their development.

Motoneuron Apoptosis Is Blocked by CEP-1347 (KT 7515), a Novel Inhibitor of the JNK Signaling Pathway

Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24-48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50 of 20 +/- 2 nM. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50 of CEP-1347 for JNK1 activation was the same as the EC50 for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons.

Muscle-derived factors are known to be important for the survival of developing spinal motoneurons, but the molecules involved have not been characterized. Hepatocyte growth factor/scatter factor (HGF/SF) plays an important role in muscle development and motoneuron axon outgrowth. We show that HGF/SF has potent neurotrophic activity (EC50=2 pM) for a subpopulation (40%) of purified embryonic rat motoneurons. Moreover, HGF/SF is an essential component of muscle-derived support for motoneurons, since blocking antibodies to HGF/SF specifically inhibited 65% of the trophic activity of media conditioned by C2/C7 skeletal myotubes, but did not inhibit the trophic activity secreted by Schwann cell lines. High levels of expression of the HGF/SF receptor c-Met in the spinal cord are restricted to subsets of motoneurons, mainly in limb-innervating segments. Consistent with this distribution, cultured motoneurons from limb-innervating brachial and lumbar segments showed a more potent response to HGF/SF than did thoracic motoneurons. By the end of the period of motoneuron cell death, levels of c-Met mRNA in motoneurons were markedly reduced, suggesting that the effects of HGF/SF may be limited to the period of motoneuron cell death. HGF/SF may play an important role during motoneuron development as a muscle-derived survival factor for a subpopulation of limb-innervating motoneurons.

Chapter 35 Neurotrophic factors for experimental treatment of motoneuron disease

Publisher Summary This chapter focuses on the neurotrophic factors for experimental treatment of motoneuron disease. Experiments designed to elucidate the pharmacokinetics, binding proteins, and availability of these factors for motoneurons are necessary in order to improve strategies for planning clinical trials with such factors, and thus to provide a basis for evaluating the potential usefulness of these trophic factors for the treatment of human disease. Motoneurons express insulin-like growth factor (IGF)-receptors, and it has been observed that the expression of IGFR is increased in motoneurons of patients suffering from amyotrophic lateral sclerosis. These data suggest that indeed IGF could play a role in the maintenance of motoneurons during postnatal life. Experimental treatment of patients with motoneuron disease has shown that subcutaneous injection of IGF has beneficial effects. Insulin-like growth factor-I (IGF-I) has been shown to be a potent survival factor for embryonic chicken and rat motoneurons. At maximal concentrations, its potency in serum-free cultures of rat motoneurons is similar to that of brain-derived neurotrophic factor (BDNF). Interestingly, although IGF-I is also active on lesioned facial motoneurons in newborn rats, the effects are significantly lower than those observed with CNTF or BDNF. It could be that this is due to the presence of inhibitory IGF binding proteins in nerve tissue.

GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.

For survival, embryonic motoneurons in vertebrates depend on as yet undefined neurotrophic factors present in the limb bud. Members of the neurotrophin family are currently the best candidates for such neurotrophic factors, but inactivation of their receptor genes leads to only partial loss of motoneurons, which suggests that other factors are involved. Glial cell line-derived neurotrophic factor (GDNF), originally identified as a trophic factor specific for dopaminergic neurons, was found to be 75-fold more potent than the neurotrophins in supporting the survival of purified embryonic rat motoneurons in culture. GDNF messenger RNA was found in the immediate vicinity of motoneurons during the period of cell death in development. In vivo, GDNF rescues and prevents the atrophy of facial motoneurons that have been deprived of target-derived survival factors by axotomy. GDNF may therefore be a physiological trophic factor for spinal motoneurons. Its potency and specificity in vitro and in vivo also make it a good candidate for treatment of motoneuron disease.

Actions of CNTF and neurotrophins on degenerating motoneurons: Preclinical studies and clinical implications

Spinal motoneurons innervating skeletal muscle were amongst the first neurons shown to require the presence of their target cells to develop appropriately. Isolated embryonic chick and rat motoneurons have been used to identify neurotrophic factors and cytokines capable of supporting the survival of developing motoneurons. Such factors include ciliary neurotrophic factor (CNTF), which is present physiologically in high amounts in myelinating Schwann cells of peripheral nerves, and brain-derived neurotrophic factor (BDNF) which is synthesized in skeletal muscle and, after peripheral nerve lesion, in Schwann cells. These factors have been further analyzed for their physiological significance in maintaining motoneuron function in vivo, and for their potential therapeutic usefulness in degenerative motoneuron disease. Both CNTF and BDNF are capable of rescuing injured facial motoneurons in newborn rats. Furthermore, CNTF prolongs survival and improves motor function of pmn mice, an animal model for degenerative motoneuron disease, by preventing degeneration of motoneuron axons and somata. Thus treatment of human motoneuron disease with neurotrophic factors should be possible, provided that rational means for application of these factors can be established considering also the appearance of potential side effects.

Rapid purification of embryonic rat motoneurons: An in vitro model for studying MND/ALS pathogenesis

Rapid purification of embryonic rat motoneurons: An in vitro model for studying MND/ALS pathogenesis

Members of several gene families influence survival of rat motoneurons in vitro and in vivo.

The survival and functional maintenance of spinal motoneurons, both during the period of developmental cell death and in adulthood, have been shown to be dependent on trophic factors. In vitro experiments have previously been used to identify several survival factors for motoneurons, including CNTF, LIF, and members of the neurotrophin, FGF, and IGF gene families. Some of these factors have also been shown to be active in vivo, either on chick motoneurons during embryonic development or on lesioned facial and spinal motoneurons of the newborn rat. Here we demonstrate that lesioned newborn rat facial motoneurons can be rescued by NT-4/5, IGF-I, and LIF. Furthermore, in contrast to chick motoneurons, the survival of isolated embryonic rat motoneurons can be maintained by the neurotrophins BDNF, NT-3, and NT-4/5. IGF-I and FGF-5 were also active in this system, each supporting more than 50% of the originally plated neurons. The responsiveness of motoneurons to multiple factors in vitro and in vivo suggests that motoneuron survival and function are regulated by the coordinated actions of members of different gene families.