Rat Embryo Spinal Cord Research Articles

Ventral Root Boundary Cap Cells of Rat Spinal Cord Contain Connexin-43.

Boundary cap cells are a population of multipotent stem cells that have great potential for the use in the treatment of damaged nervous system. We studied the patterns of distribution of the gap junction protein connexin-43 (Cx43) in boundary cap cells of the ventral root of the spinal cord of rat embryos (E12-E20; n=40). It was found that Cx43 is expressed in ventral boundary cap cells at all stages of its existence during embryogenesis. At the early stages of prenatal development, the cytoplasmic distribution of Cx43 in the boundary cap cells predominates; at the later stages, Cx43-immunopositive punctate structures are identified. These puncta represent gap junction plaques between the cells. It can be assumed that during the early embryogenesis, Cx43 regulates the main histogenetic processes in boundary cap cells and only in the later stages of prenatal development, Cx43-mediated communications are formed between boundary cap cells.

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

Background: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. Objective: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. Methods: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. Results: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. Conclusion: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.

BMP7 is Downregulated in Lumbosacral Spinal Cord of Rat Embryos With Anorectal Malformation

BackgroundBone morphogenetic proteins (BMPs) comprise a highly conserved signaling protein family, which are involved in spinal cord formation, development and differentiation. Malformations of the lumbosacral spinal cord are associated with postoperation complications of anorectal malformation (ARM). However, the mechanism underlying the development of these malformations remains elusive. Materials and methodsEmbryonic rat ARM model induced by ethylenethiourea (ETU) was introduced to investigate BMP7 expression in lumbosacral spinal cord. BMP7 expression was analyzed by immunohistochemical staining, qRT-PCR, and Western blot analysis on embryonic (E) days 16, 17, 19, and 21. The expression of the neuronal marker neurofilament (NF) and pSmad1/5 was determined by immunofluorescence double staining and Western blot analysis during peak BMP7 expression. ResultsBMP7 mRNA (E16, 1.041 ± 0.169 versus 0.758 ± 0.0423, P < 0.05; E17, 1.889 ± 0.444 versus 1.601 ± 0.263, P < 0.05; E19, 2.898 ± 0.434 versus 1.981 ± 0.068, P < 0.01; and E21, 2.652 ± 0.637 versus 1.957 ± 0.09, P < 0.05;) and protein (E16, 1.068 ± 0.065 versus 0.828 ± 0.066, P < 0.01; E17, 1.728 ± 0.153 versus1.4 ± 0.148, P < 0.05; E19, 2.313 ± 0.141 versus 1.696 ± 0.21, P < 0.01; and E21, 2.021 ± 0.13 versus 1.43 ± 0.128, P < 0.01) were downregulated, and their expressions were specifically low in interneurons (IN) located in the dorsal horn of the lumbosacral spinal cord in embryos with ARM. On E19, Western blot analysis revealed reduced P-Smad1/5(1.13 ± 0.08 versus 0.525 ± 0.06, P < 0.01). ConclusionsAn implication of this study is the possibility that BMP7 downregulation contributes to maldevelopment of the lumbosacral spinal cord during embryogenesis in fetal rats with ARM, indicating that BMP7 may play an important role in ARM pathogenesis and the complications thereof.

The Expression of Shh, Ptch1, and Gli1 in the Developing Caudal Spinal Cord of Fetal Rats With Anorectal Malformations

BackgroundPostoperative incontinence and constipation still remain the major complications of anorectal malformations (ARMs), despite improvements in their treatment. One of the most important factors that affect postoperative anorectal function is malformations in the lumbosacral spinal cord. However, far too little attention has been paid to the underlying mechanism that produces these malformations. Materials and methodsThe levels of sonic hedgehog (Shh), patched homolog 1 (Ptch1), and zinc finger-containing transcription factors 1 (Gli1) expression were investigated in the lumbosacral spinal cord in ethylenethiourea-exposed rat fetus with ARMs, and Shh, Ptch1, and Gli1 expression was confirmed with immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot analyses during lumbosacral spinal cord development both in the ARMs and normal rat embryos. ResultsOur results have shown that Shh, Ptch1, and Gli1 expression in the lumbosacral spinal cord of rat embryos with ARMs was decreased at both the messenger RNA and protein levels, when compared with their expression levels in normal tissues (P < 0.05). ConclusionsThis study demonstrated that the expression of Shh, Ptch1, and Gli1 in lumbosacral spinal cord was remarkably reduced during late developmental stages in fetal rats with ARMs. These findings offered some important insights into the involvement of the Shh-Ptch1-Gli1 signaling pathway in the pathogenesis of lumbosacral spinal cord maldevelopment in rat fetus with ARMs, which leads to complications after procedures for ARMs.

Bone morphogenetic protein 4 expression in the developing lumbosacral spinal cord of rat embryos with anorectal malformations

Although there are improvements in treatment of anorectal malformations (ARMs), patients can still develop fecal incontinence, constipation, and soiling with loss in quality of life. Recent evidence suggests that malformations in the lumbosacral spinal cord are one of the factors that affect postoperative anorectal function. However, the underlying mechanism that produces these malformations has yet to be elucidated. The bone morphogenetic proteins (BMPs) comprise a large group of highly conserved molecules that are involved in multiple processes and play important roles in the formation, development, and differentiation of the spinal cord. This study was designed to investigate the levels of BMP4 expression in the lumbosacral spinal cord in ARMs rat embryos induced by ethylenethiourea (ETU). Specifically, we assessed the association of BMP4 levels with the maldevelopment of the lumbosacral spinal cord and whether BMP4 acted through the canonical intracellular pathway in embryonic rats with ARMs. BMP4 expression was confirmed with immunohistochemical staining, RT-qPCR and western blot analyses of embryonic day (E) 16, E17, E19 and E21 embryos, moreover Smad1/5 and pSmad1/5 expression were confirmed with western blot analyses at peak time point of BMP4 expression. Our results reveal that BMP4 expression in the lumbosacral spinal cord of ARMs rat embryos is decreased at both the mRNA and protein levels and could decrease the phosphorylation of smad1/5, when compared with their expression levels in normal tissue. These results also suggest that reductions in BMP4 expression were possibly responsible for dysfunction of the lumbosacral spinal cord during late developmental stages in ARMs fetal rats. Taken together, we conclude a role for BMP4 in the pathogenesis of lumbosacral spinal cord maldevelopment in developing ARMs rats.

Reversible Integration of Microfluidic Devices with Microelectrode Arrays for Neurobiological Applications

The majority of current state-of-the-art microfluidic devices are fabricated via replica molding of the fluidic channels into PDMS elastomer and then permanently bonding it to a Pyrex surface using plasma oxidation. This method presents a number of problems associated with the bond strengths, versatility, applicability to alternative substrates, and practicality. Thus, the aim of this study was to investigate a more practical method of integrating microfluidics which is superior in terms of bond strengths, reversible, and applicable to a larger variety of substrates, including microfabricated devices. To achieve the above aims, a modular microfluidic system, capable of reversible microfluidic device integration, simultaneous surface patterning and multichannel fluidic perfusion, was built. To demonstrate the system’s potential, the ability to control the distribution of A549 cells inside a microfluidic channel was tested. Then, the system was integrated with a chemically patterned microelectrode array, and used it to culture primary, rat embryo spinal cord neurons in a dynamic fluidic environment. The results of this study showed that this system has the potential to be a cost effective and importantly, a practical means of integrating microfluidics. The system’s robustness and the ability to withstand extensive manual handling have the additional benefit of reducing the workload. It also has the potential to be easily integrated with alternative substrates such as stainless steel or gold without extensive chemical modifications. The results of this study are of significant relevance to research involving neurobiological applications, where primary cell cultures on microelectrode arrays require this type of flexible integrated solution.

Combined Application of Neutrophin-3 Gene and Neural Stem Cells is Ameliorative to Delay of Denervated Skeletal Muscular Atrophy after Tibial Nerve Transection in Rats

Examination of the therapeutic efficacy of neural stem cells (NSCs) has recently become the focus of much investigation. In this study we present an insight of the effects of combined application with neurotrophin-3 (NT-3) and NSCs that derived from rat embryo spinal cord on delaying denervated skeletal muscular atrophy after tibial nerve was severed. NT-3 gene was amplified by PCR and subcloned into lentiviral vector pWPXL-MOD to construct a lentiviral expression vector pWPXL-MOD-NT-3. A positive clone expressing NT-3 (named NSCs-NT-3) was obtained and used for differentiation in vitro and transplantation. Sixty adult rats, whose tibial nerves were sectioned, were divided into two groups: one grafted with NSCs-NT-3 (experimental group, n = 30) and the other with NSCs transfected by pWPXL-MOD (control group, n = 30). The cell survival and differentiation, NT-3 gene expression, and effect of delaying denervated skeletal muscular atrophy were examined through immunohistostaining, RT-PCR, Western blot, electrophysiological analysis, and mean cross-sectional area (CSA) of gastrocnemius, respectively. The results show that the NT-3 gene, which is comprised of 777 bp, was cloned and significantly different expression were detected between NSCs and NSCs-NT-3 in vitro. Quantitative analysis of the choline acetyltransferase (ChAT) immunopositive cells revealed a significant increase in experimental group compared to the control group 4 weeks after implantation (p < 0.01). Twelve weeks after transplantation, the ChAT immunopositive cells were detected near the engrafted region only in experimental group. Furthermore, the effect in delaying denervated skeletal muscular atrophy is indicated in the EMG examination and mean CSA of gastrocnemius. These findings suggest that the neural stem cells expressing NT-3 endogenously would be a better graft candidate for the delay of denervated skeletal muscular atrophy.

Receptors to Steroid Hormones and Aromatase Are Expressed by Cultured Motoneurons but Not by Glial Cells Derived from Rat Embryo Spinal Cord

The aim of this study was to examine the expression of aromatase and receptors to steroid hormones in cultured motoneurons (MNs). We first developed an original method for obtaining rat MN cultures. Dissociated E15 rat spinal cords were purified using metrizamide and bovine serum albumin density gradients, and cells were then seeded on the culture substratum. We optimized the culture parameters and found that simple addition of rat muscle extract (ME) and conditioned culture medium (CM) from glial cell lines (GCL) derived from spinal cord were sufficient to obtain almost pure MN cultures. MNs were characterized by the presence of specific MN markers and electrophysiology. MNs could be kept alive for 2 weeks. We demonstrate that ME and CM are essential for MN development and survival respectively. Immunocytochemistry and aromatase activity assay indicated the presence of androgen and estrogen receptors as well as aromatase in MNs but not in GCL. This is the first report demonstrating the presence of both female and male sex hormone receptors and a key enzyme in steroid hormone metabolism in MNs and its absence in GCL, at least in our culture conditions. This in vitro model appears to be valuable for elucidating the impact of the sex hormone circuit in neuronal maturation. The relevance of this model for the comprehension of neurodegenerative diseases is discussed.

0311 Effect of mercuric chloride on development of the spinal cord of rat embryos

0311 Effect of mercuric chloride on development of the spinal cord of rat embryos

Adenosine triphosphate inhibits cytokine release from lipopolysaccharide-activated microglia via P 2y receptors

Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor α (TNF-α), interleukin 1β and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10–300 μM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P 2y receptor agonists (ADPβS and 2-methylthio-ATP) on LPS-induced TNF-α release. The rank order of inhibitory potency of endogenous purines on TNF-α release was: ATP>ADP>>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 μM 2-methylthio-ATP showed a potency similar to 100 μM ATP. The addition of ADPβS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P 2y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.

AMPA-evoked acetylcholine release from cultured spinal cord motoneurons and its inhibition by GABA and glycine

The release of [ 3H]acetylcholine evoked by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and its inhibition mediated by GABA A and glycine receptors were studied in superfused cultured rat embryo spinal cord motoneurons prelabeled with [ 3H]choline. AMPA elicited tritium release, possibly representing [ 3H]acetylcholine release in a concentration-dependent manner. The release was external Ca 2+-dependent and was sensitive to Cd 2+ ions, ω-conotoxin GVIA and ω-conotoxin MVIIC, but not to nifedipine, suggesting the involvement of N-, P/Q-, but not L-type Ca 2+ channels. The AMPA effect was insensitive to tetrodotoxin. The glutamate receptors involved are AMPA type since the AMPA-evoked [ 3H]acetylcholine release was blocked by LY303070 and was potentiated by the antidesensitizing agent cyclothiazide. Muscimol inhibited completely the AMPA effect on [ 3H]acetylcholine release; muscimol was potentiated by diazepam and antagonized by SR95531, indicating the involvement of benzodiazepine-sensitive GABA A receptors. Glycine, acting at strychnine-sensitive receptors, also inhibited the effect of AMPA, but only in part. The inhibitory effects of muscimol and glycine are additive. We conclude that glutamate can act at AMPA receptors sited on spinal motoneurons to evoke release of acetylcholine. GABA and glycine, possibly released as cotransmitters from spinal interneurons, inhibit glutamate-evoked acetylcholine release by activating GABA A and glycine receptors on motoneurons.

Prenatal expression of pituitary adenylate cyclase activating polypeptide (PACAP) in autonomic and sensory ganglia and spinal cord of rat embryos.

Prenatal expression of pituitary adenylate cyclase activating polypeptide (PACAP) in autonomic and sensory ganglia and spinal cord of rat embryos.

Reversal in concentrations of actived CaMKII and calcineurin for long lasting stimulation

The release of [3H]acetylcholine evoked by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and its inhibition mediated by GABAA and glycine receptors were studied in superfused cultured rat embryo spinal cord motoneurons prelabeled with [3H]choline. AMPA elicited tritium release, possibly representing [3H]acetylcholine release in a concentration-dependent manner. The release was external Ca2+-dependent and was sensitive to Cd2+ ions, ω-conotoxin GVIA and ω-conotoxin MVIIC, but not to nifedipine, suggesting the involvement of N-, P/Q-, but not L-type Ca2+ channels. The AMPA effect was insensitive to tetrodotoxin. The glutamate receptors involved are AMPA type since the AMPA-evoked [3H]acetylcholine release was blocked by LY303070 and was potentiated by the antidesensitizing agent cyclothiazide. Muscimol inhibited completely the AMPA effect on [3H]acetylcholine release; muscimol was potentiated by diazepam and antagonized by SR95531, indicating the involvement of benzodiazepine-sensitive GABAA receptors. Glycine, acting at strychnine-sensitive receptors, also inhibited the effect of AMPA, but only in part. The inhibitory effects of muscimol and glycine are additive.We conclude that glutamate can act at AMPA receptors sited on spinal motoneurons to evoke release of acetylcholine. GABA and glycine, possibly released as cotransmitters from spinal interneurons, inhibit glutamate-evoked acetylcholine release by activating GABAA and glycine receptors on motoneurons.

Effect of dalargin on the activity of cytochrome oxidase and glutamate dehydrogenase in rat spinal cord dissociated cultured neurons

Dissociated cultured neurons from the rat embryo spinal cord were grown for six days in the presence of dalargin, the synthetic analog of leu-enkephalin. Then the activities of two enzymes of energy metabolism, cytochrome oxidase (CO) and glutamate dehydrogenase (GDH), were studied in these neurons using quantitative cytochemical technique. Dalargin, which possesses the properties of nerve growth factor, enhanced the nerve cell growth and increased the activity of the above enzymes, with GDH activity being increased more significantly. According to the classical standpoint, increased GDH activity under conditions of acute energy deflciency favors the invoivement of some amino acids in a citric acid cycle for subsequent reproduction. One can suggest, in this relation, that the increased energy production caused by the enhanced nerve cell growth in the presence of dalargin was partially compensated by the amino acid splitting. The results allow us to suggest that the effect of dalargin (growth factor) on the nerve cells is similar to the effects of the extremal factors, and requires additional energy to be supplied.

Neurotrophins increase motoneurons' ability to innervate skeletal muscle fibers in rat spinal cord-human muscle cocultures

Neurotrophins, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-5 (NT-5) and brain-derived neurotrophic factor (BDNF), were studied in vitro in a coculture model of human skeletal muscle myotubes and rat embryo spinal cord explants, which enables the different steps of functional innervation to be followed, including neurite outgrowth, synapse formation and induction of contractile activity. We found that NT-3, NT-5, BDNF, but not NGF simultaneously induced a significant increase in the number and length of neurites emerging from spinal cord explants, the number of endplates per muscle fiber, and the area of innervated muscle fibers around each spinal cord explant. These results suggest that neurotrophins NT-3, NT-5 and BDNF enhance spinal cord motoneurons potential of innervation.

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria® substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria® dishes have a modified plastic surface with a net positive charge.) On both polylysine and Primaria® substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria® substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Membrane-bound Ca2(+)-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells. The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca2(+)-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca2(+)-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca2(+)-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca2(+)-ATPase, postulating for the activities of Ca2(+)-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.

NMDA receptors mediate poly- and monosynaptic potentials in motoneurons of rat embryos

We determined the contribution of glutamate receptor subtypes to developing excitatory synaptic transmission in isolated spinal cord of rat embryos. Using electrophysiological and morphological techniques, we studied the pattern of development of synapses between dorsal root afferents and motoneurons in lumbar spinal cords of 15- to 21-d-old rat embryos. Motoneuron dendritic fields and afferent projections onto motoneurons were identified by labeling with HRP. Afferents first entered the gray matter at Day 15 of gestation, and by Day 16 they terminated close to motoneuron dendritic trees. Afferent axons projected onto motoneuron dendritic fields at Day 17, when boutons were detected on motoneuron dendrites that were crossed by afferent axons. To determine the time course of formation of functional sensorimotor synapses and their pharmacological properties, a dorsal root was stimulated while recording intracellularly from segmental motoneurons. At Day 16, excitatory postsynaptic potentials (EPSPs) with long latencies, slow rates of rise, and long durations were recorded. The amplitudes of these EPSPs increased with membrane depolarization and in the absence of extracellular Mg2+. These EPSPs were blocked by D-2-amino-5-phosphonovalerate (APV) and ketamine, which are selective antagonists of N-methyl-D-aspartate (NMDA) receptors. These findings suggest that initial synaptic transmission in embryonic motoneurons is mediated solely by NMDA receptors. Short-latency EPSPs with fast rates of rise were first recorded in most motoneurons by Day 17. These EPSPs were composed of fast- and slow-rising potentials. The slow component was blocked by APV, while the fast component was eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione and kynurenate. This indicates that the short-latency EPSPs are mediated by both NMDA and non-NMDA receptors. Dose-response curves of motoneurons to L-glutamate, NMDA, and kainate demonstrated that motoneurons are sensitive to these agonists prior to the formation of synapses between afferents and motoneurons. Motoneuron responses to NMDA and kainate increased immediately after the onset of short-latency EPSPs. This increased sensitivity could be due to extracellular factors influenced by growing sensory axons or intrinsic properties of differentiating motoneurons.

Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria® and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4–5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP-positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin-positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.

Electrical properties of motoneurons in the spinal cord of rat embryos

Electrical properties of immature motoneurons were studied in vitro using isolated segments of spinal cords of rat embryos aged 14–21 days of gestation. Stable resting potentials and evoked synaptic potentials were recorded for more than 9 hr, indicating that motoneurons remain viable for many hours. Motoneurons are electrically excitable at 14 days of gestation and from the onset of excitability the action potentials are Na +-dependent but slow rising long-duration Ca 2+-dependent action potentials can be evoked if K + conductance is reduced. Thus, during embryonic development the regenerative potential inward current is Na +- and Ca 2+-dependent. During motoneurons' differentiation there are some changes in their electrical properties: resting membrane potential increases, input resistance decreases, input capacitance increases, threshold for action potential decreases, and maximum rate of rise of action potential increases. Afferent motoneuron contacts are formed at 16–18 days of gestation when excitatory synaptic potentials can first be evoked in response to dorsal root stimulation. The changes in input capacitance and threshold for action potential occur at the onset of functional afferent motoneuron contacts, but it is not known whether these changes are autonomous or are influenced by the newly formed sensory inputs.