Mouse Spinal Cord Cells Research Articles

Edge-Functionalized Graphene/Polydimethylsiloxane Composite Films for Flexible Neural Cuff Electrodes.

The design of neural electrodes has changed in the past decade, driven mainly by the development of new materials that open the possibility of manufacturing electrodes with adaptable mechanical properties and promising electrical properties. In this paper, we report on the mechanical and electrochemical properties of a polydimethylsiloxane (PDMS) composite with edge-functionalized graphene (EFG) and demonstrate its potential for use in neural implants with the fabrication of a novel neural cuff electrode. We have shown that a 200 μm thick 1:1 EFG/PDMS composite film has a stretchability of up to 20%, a Young's modulus of 2.52 MPa, and a lifetime of more than 10000 mechanical cycles, making it highly suitable for interfacing with soft tissue. Electrochemical characterization of the EFG/PDMS composite film showed that the capacitance of the composite increased up to 35 times after electrochemical reduction, widening the electrochemical water window and remaining stable after soaking for 5 weeks in phosphate buffered saline. The electrochemically activated EFG/PDMS electrode had a 3 times increase in the charge injection capacity, which is more than double that of a commercial platinum-based neural cuff. Electrochemical and spectrochemical investigations supported the conclusion that this effect originated from the stable chemisorption of hydrogen on the graphene surface. The biocompatibility of the composite was confirmed with an in vitro cell culture study using mouse spinal cord cells. Finally, the potential of the EFG/PDMS composite was demonstrated with the fabrication of a novel neural cuff electrode, whose double-layered and open structured design increased the cuff stretchability up to 140%, well beyond that required for an operational neural cuff. In addition, the cuff design offers better integration with neural tissue and simpler nerve fiber installation and locking.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Ca2+ signaling–mediated low-intensity pulsed ultrasound–induced proliferation and activation of motor neuron cells

Motor neuron diseases (MND) including amyotrophic lateral sclerosis and Parkinson disease are commonly neurodegenerative, causing a gradual loss of nerve cells and affecting the mechanisms underlying changes in calcium (Ca2+)-regulated dendritic growth. In this study, the NSC-34 cell line, a population of hybridomas generated using mouse spinal cord cells with neuroblastoma, was used to investigate the effect of low-intensity pulsed ultrasound (LIPUS) as part of an MND treatment model. After NSC-34 cells were seeded for 24 h, LIPUS stimulation was performed on the cells at days 1 and 3 using a non-focused transducer at 1.15 MHz for 8 min. NSC-34 cell proliferation and morphological changes were observed at various LIPUS intensities and different combinations of Ca2+ channel blockers. The nuclear translocation of Ca2+-dependent transcription factors was also examined. We observed that the neurite outgrowth and cell number of NSC-34 significantly increased with LIPUS stimulation at days 2 and 4, which may be associated with the treatment’s positive effect on the activation of Ca2+-dependent transcription factors, such as nuclear factor of activated T cells and nuclear factor-kappa B. Our findings suggest that the LIPUS-induced Ca2+ signaling and transcription factor activation facilitate the morphological maturation and proliferation of NSC-34 cells, presenting a promising noninvasive method to improve stimulation therapy for MNDs in the future.

Dorsal column and root stimulation at Aβ-fiber intensity activate superficial dorsal horn glutamatergic and GABAergic populations.

Neurostimulation therapies are frequently used in patients with chronic pain conditions. They emerged from Gate Control Theory (GCT), which posits that Aβ-fiber activation recruits superficial dorsal horn (SDH) inhibitory networks to “close the gate” on nociceptive transmission, resulting in pain relief. However, the efficacy of current therapies is limited, and the underlying circuits remain poorly understood. For example, it remains unknown whether ongoing stimulation of Aβ-fibers is sufficient to drive activity in SDH neurons. We used multiphoton microscopy in spinal cords extracted from mice expressing the genetically encoded calcium indicator GCaMP6s in glutamatergic and GABAergic populations; activity levels were inferred from deconvolved calcium signals using CaImAn software. Sustained Aβ-fiber stimulation at the dorsal columns or dorsal roots drove robust yet transient activation of both SDH populations. Following the initial increase, activity levels decreased below baseline in glutamatergic neurons and were depressed after stimulation ceased in both populations. Surprisingly, only about half of GABAergic neurons responded to Aβ-fiber stimulation. This subset showed elevated activity for the entire duration of stimulation, while non-responders decreased with time. Our findings suggest that Aβ-fiber stimulation initially recruits both excitatory and inhibitory populations but has divergent effects on their activity, providing a foundation for understanding the analgesic effects of neurostimulation devices.Perspective: This article used microscopy to characterize the responses of mouse spinal cord cells to stimulation of non-painful nerve fibers. These findings deepen our understanding of how the spinal cord processes information and provide a foundation for improving pain-relieving therapies.

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

An efficient drug delivery vehicle for botulism countermeasure

BackgroundBotulinum neurotoxin (BoNT) is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. An effective medical countermeasure strategy would require developing a drug that could rescue poisoned neuromuscular synapses and include its efficient delivery specifically to poisoned presynaptic nerve terminals. Here we report a drug delivery strategy that could directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol.ResultsA targeted delivery vehicle was developed for intracellular transport of emerging botulinum neurotoxin antagonists. The drug delivery vehicle consisted of the non-toxic recombinant heavy chain of botulinum neurotoxin-A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted neurons. Our results indicated that the drug delivery vehicle entry into neurons was via BoNT-A receptor mediated endocytosis. Once internalized into neurons, the drug carrier component separated from the drug delivery vehicle in a fashion similar to the separation of the BoNT-A light chain from the holotoxin. This drug delivery vehicle could be used to deliver BoNT-A antidotes into BoNT-A intoxicated cultured mouse spinal cord cells.ConclusionAn effective BoNT-based drug delivery vehicle can be used to directly deliver toxin inhibitors into intoxicated nerve terminal cytosol. This approach can potentially be utilized for targeted drug delivery to treat other neuronal and neuromuscular disorders. This report also provides new knowledge of endocytosis and exocytosis as well as of BoNT trafficking.

A Targeted Therapeutic Rescues Botulinum Toxin-A Poisoned Neurons

AbstractBotulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. BoNT acts specifically by blocking neurotransmission primarily at peripheral nerve-muscle junctions causing severe flaccid muscle paralysis, which is fatal if proper medical care is not provided. The neurotoxin acts by specifically entering the presynaptic nerve endings where it interferes with the biochemical machinery involved in the process of neurotransmitter release, i.e., neuroexocytosis. Most serotypes of BoNT are known to remain active for weeks to months after entering the nerves, but BoNT/A is the most potent and long lasting in causing muscle paralysis. An effective medical countermeasure strategy requires developing a drug that could rescue poisoned neuromuscular synapses, and would include its efficient delivery specifically to presynaptic nerve terminals. Here we report rescuing of botulinum poisoned nerve cells by Mastoparan-7 (Mas-7), a peptide constituent of bee venom, that was delivered through a drug delivery vehicle (DDV) constructed from the non-toxic fragment of botulinum neurotoxin itself. We found that Mas-7 that was delivered into BoNT/A intoxicated cultured mouse spinal cord cells restored over 40% of stimulated neurotransmitter release. The rescue of the cell poisoning did not occur from inhibition of the endopeptidase activity of BoNT/A against its well known substrate, SNAP-25 that is mechanistically involved in the exocytosis process. Rather, Mas-7 induced a physiological host response apparently unrelated to SNAP-25, but linked to the phospholipase signal transduction pathway. In addition to providing the first effective antidote against botulism, our results open new avenues to study the mechanism of exocytosis, and also to examine an alternative cellular mechanism of botulinum neurotoxin action. An effective BoNT-based DDV can also be utilized for drug delivery against many neuronal and neuromuscular disorders.

Expression and distribution of all dopamine receptor subtypes (D 1–D 5) in the mouse lumbar spinal cord: A real-time polymerase chain reaction and non-autoradiographic in situ hybridization study

Dopamine is a catecholaminergic neuromodulatory transmitter that acts through five molecularly-distinct G protein–coupled receptor subtypes (D 1–D 5). In the mammalian spinal cord, dopaminergic axon collaterals arise predominantly from the A11 region of the dorsoposterior hypothalamus and project diffusely throughout the spinal neuraxis. Dopaminergic modulatory actions are implicated in sensory, motor and autonomic functions in the spinal cord but the expression properties of the different dopamine receptors in the spinal cord remain incomplete. Here we determined the presence and the regional distribution of all dopamine receptor subtypes in mouse spinal cord cells by means of quantitative real time polymerase chain reaction (PCR) and digoxigenin-label in situ hybridization. Real-time PCR demonstrated that all dopamine receptors are expressed in the spinal cord with strongly dominant D 2 receptor expression, including in motoneurons and in the sensory encoding superficial dorsal horn (SDH). Laser capture microdissection (LCM) corroborated the predominance of D 2 receptor expression in SDH and motoneurons. In situ hybridization of lumbar cord revealed that expression for all dopamine receptors was largely in the gray matter, including motoneurons, and distributed diffusely in labeled cell subpopulations in most or all laminae. The highest incidence of cellular labeling was observed for D 2 and D 5 receptors, while the incidence of D 1 and D 3 receptor expression was least. We conclude that the expression and extensive postsynaptic distribution of all known dopamine receptors in spinal cord correspond well with the broad descending dopaminergic projection territory supporting a widespread dopaminergic control over spinal neuronal systems. The dominant expression of D 2 receptors suggests a leading role for these receptors in dopaminergic actions on postsynaptic spinal neurons.

Primary cell culture for evaluation of botulinum neurotoxin antagonists

The actions of botulinum neurotoxin (BoNT) were studied on evoked release of the neurotransmitter glycine in primary mouse spinal cord cells. 3[H]-glycine was taken up by cells in physiological solution and released by depolarization with 56 mM K + in the presence of 2 mM Ca 2+. Release of 3[H]-glycine was found to be inhibited by BoNT serotypes A, B and E with similar potency ratios to those observed in the acutely isolated mouse diaphragm muscle. When spinal cord cultures were exposed to BoNT/A for 24 h, inhibition of 3[H]-glycine release was detected at toxin concentrations as low as 10 −14 M, and complete inhibition was observed at concentration ≥10 −12 M. Preincubation of BoNT/A with polyclonal equine antiserum led to antagonism of toxin-induced inhibition of 3[H]-glycine release in spinal cord cells and to protection of mice from the lethal effects of BoNT/A. It is concluded that spinal cord neurons are a useful model for studying botulinum intoxication and for evaluating BoNT antagonists.

The thrombin receptor mediates functional activity‐dependent neuromuscular synapse reduction via protein kinase C activation in vitro

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 369–381, 1999

The thrombin receptor mediates functional activity-dependent neuromuscular synapse reduction via protein kinase C activationin vitro

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system.

Selective block of N-methyl-D-aspartic acid (NMDA)-evoked whole-cell currents in mouse cultured spinal neurones by CGP 40116.

1. CGP 40116 is the active (R)-enantiomer of the most potent N-methyl-D-aspartic acid (NMDA) receptor antagonist presently available: 2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849). In this study, we describe the effect of CGP 40116 on whole-cell currents induced by excitatory amino acids in cultured mouse spinal cord cells by use of the whole-cell patch-clamp technique. 2. We found that application of CGP 40116 in the nM range, concentration-dependently inhibited whole-cell current evoked by 20 microM NMDA in mouse cultured spinal neurones (IC50 +/- s.e. mean 48 +/- 8 nm CGP 40116). 3. The compound appeared to be highly selective for the NMDA current. At concentrations as high 1 microM, currents evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid were not affected by CGP 40116. The threshold concentration for antagonism of NMDA-induced responses was 10 nM suggesting a selectivity ratio of > or = 100 fold for NMDA receptors versus AMPA or kainate receptors. 4. CGP 40116 produced a parallel rightward displacement of the NMDA log concentration-current curve indicating competitive antagonism at the transmitter recognition site of the NMDA receptor complex. An apparent dissociation constant for the antagonist was calculated from the displacement of the agonist concentration-current curve: 117 +/- 53 nM CGP 40116 (estimated Kd +/- s.e.). Like other competitive NMDA antagonists, CGP 40116 blocked NMDA-evoked current in a voltage-independent manner.

Effects of methylprednisolone on lesioned and uninjured mammalian spinal neurons: viability, ultrastructure, and network electrophysiology.

An in vitro investigation was undertaken to provide information regarding the effectiveness of methylprednisolone sodium succinate (MPSS) as a treatment for the primary mechanical injury of spinal cord (SC) trauma. Exposure of uninjured mouse SC cells to MPSS for 24 h caused neuronal stress when the concentration exceeded 150 micrograms/mL; neuronal death occurred at concentrations above 600 micrograms/mL. The concentration range for MPSS protection of SC neurons subjected to a defined physical injury (laser microbeam transection of a primary dendrite 100 microns from the perikaryon) was very narrow: survival in the 30 micrograms/mL group differed significantly from the untreated control group (68.5% +/- 14.1 vs. 47.1% +/- 14.1), treatment with 20 or 60 micrograms/mL MPSS did not increase survival, and treatment with 100 micrograms/mL MPSS accelerated ultrastructural deterioration and increased the likelihood of death. Enhanced survival of lesioned neurons was observed when 30 micrograms/mL MPSS was applied within 15 min of dendrotomy but not when MPSS was administered 2 h after lesioning. Multimicroelectrode plate (MMEP) studies of SC network electrical activity indicated that MPSS associated readily with neuronal membranes. This finding was consistent with the hypothesis that MPSS may protect lesioned neurons by stabilizing damaged membranes, enhancing lesion resealing, and limiting the spread of ion-mediated damage. However, comparisons of neurite die-back 24 h after dendrotomy found no significant difference between MPSS-treated and control neurons. Application of 30 or 100 micrograms/mL MPSS increased the spontaneous burst activity of SC networks grown on MMEPs, however, there was no evidence that the increased excitability at these concentrations was the result of specific actions of MPSS on GABA or NMDA synapses.

Protective effects of tirilazad mesylate and metabolite U-89678 against blood-brain barrier damage after subarachnoid hemorrhage and lipid peroxidative neuronal injury

The 21-aminosteroid lipid-peroxidation inhibitor, tirilazad mesylate (U-74006F), recently was shown in a large multinational Phase III clinical trial to decrease mortality and improve neurological recovery in patients 3 months after onset of aneurysmal subarachnoid hemorrhage (SAH). A major tirilazad metabolite in animals and man, U-89678 is formed when the 4-5 double bond in the A-ring is reduced and has been postulated to contribute significantly to tirilazad's neuroprotective effects. In the first experiment of the present study, the authors compared the effects of tirilazad and U-89678 on acute blood-brain barrier (BBB) damage in rats subjected to SAH via injection of 300 microliters of autologous nonheparinized blood under the dura of the left cortex. The rats were treated by intravenous administration of either 0.3 or 1.0 mg/kg of tirilazad or U-89678 10 minutes before and 2 hours after SAH, and BBB damage was quantified according to the extravasation of the protein-bound Evans' blue dye into the injured cortex 3 hours post-SAH. The results revealed that 0.3 and 1.0 mg/kg tirilazad significantly reduced SAH-induced BBB damage 35.2% (p < 0.05) and 60.6% (p < 0.0001), respectively, in comparison to treatment with vehicle. The 0.3- and 1.0-mg/kg doses of U-89678 also decreased injury by 39.1% (p < 0.05) and 21.3% (not significant), respectively. In the second experiment, the investigators assessed the relative abilities of tirilazad and U-89678 to protect cultured neurons from iron-induced lipid peroxidative injury. Fetal mouse spinal cord cells were pretreated with 3, 10, or 30 microM tirilazad or U-89678 for 1 hour and then exposed to 200 microM ferrous ammonium sulfate (FAS) for 40 minutes. Cell viability was measured in terms of the uptake of [3H]alpha-(methyl)-aminoisobutyric acid 45 minutes after the FAS treatment. Both compounds enhanced neuronal survival in a concentration-dependent fashion. Although the two were equally efficacious, U-89678 was slightly more potent than its parent. On the basis of these findings, the authors conclude that the tirilazad metabolite, U-89678, possesses vaso- and neuroprotective properties that are essentially equivalent to the parent 21-aminosteroid. Hence, U-89678 probably contributes to the protective effects of tirilazad in SAH and other insults to the central nervous system.

Mechanism for the channel-opening reaction of strychnine-sensitive glycine receptors on cultured embryonic mouse spinal cord cells.

The strychnine-sensitive glycine receptor, a member of a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system, forms an anion-specific transmembrane channel in response to glycine binding. A rapid-reaction technique, a cell-flow method with a 10-ms time resolution, was adapted for measurements with cultured embryonic mouse spinal cord cells containing glycine receptors. Whole-cell current responses resulting from the opening of glycine receptor channels were measured at pH 7.4, 22-24 degrees C, and transmembrane voltages of -40 and -75 mV. Two different receptor forms, A alpha and A beta, were detected. At saturating glycine concentrations, an average of 70% of the whole-cell current amplitude was associated with form A alpha and 30% with A beta. The constants pertaining to the minimum mechanisms that account for the concentration of the two open-channel receptor forms over a 100-fold range of glycine concentration were determined by cell-flow measurements of the current amplitudes and of the falling (desensitizing) rate of the current. The dissociation constant of the site controlling channel opening was 220 microM on the basis of three binding sites for A alpha and 380 microM on the basis of two binding sites for A beta. The channel-opening equilibrium constant, phi-I, was 170 for A alpha and 110 for A beta. The rate coefficients for desensitization, alpha and beta, associated with these two forms have maximum values of 0.7 and 0.1 s-1, respectively. The rates at which the receptors recovered from desensitization were also measured, using a double-flow mixing device, and were found to be 0.06 s-1 for A alpha and 0.02 s-1 for A beta. In the presence of 100 microM glycine, the apparent dissociation constant for the inhibitor picrotoxinin from receptor form A alpha was 80 microM, and that from A beta was 460 microM. This suggests that A beta contains beta-subunits (58 kD), because this subunit confers picrotoxinin insensitivity to glycine receptors (Pribilla, I., et al. (1992) EMBO J. 11, 4305). In the case of one receptor form (A alpha), the chemical mechanism and its constants led to two measurements that could be assessed by an independent method, the single-channel current-recording technique: (i) the fraction of receptor channels open at a given glycine concentration (ALn)o and (ii) the rate coefficient for desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.

Method to improve the sensitivity of flow cytometric membrane potential measurements in mouse spinal cord cells.

We have developed a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa-C4(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell size and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log oxonol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov-Smirnov analysis of pre- vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique.

A specific effect of muscle cells on the distribution of presynaptic proteins in neurites and its absence in a C2 muscle cell variant

The distribution of neurofilament (NF) and synaptic vesicle (SV) proteins in neurites cultured in vitro was visualized with immunocytochemical methods. NF and SV proteins were detected in neurites from both embryonic mouse spinal cord and chick ciliary ganglion neurons. NF proteins generally occupied more proximal, unbranched neurite segments while SV proteins were most often found in highly branched terminal segments. Neurites from mouse spinal cord cells showed a striking segregation of the NF and SV proteins into distinct domains; neurites from chick ciliary ganglion cells exhibited a similar, though less pronounced segregation. In cocultures of neurons and muscle cells, the neurite segments in contact with myotubes more often stained for SV than for NF while the opposite was true for neurites not in contact with myotubes. The preferential association of SV neurites with myotubes was also observed when the myotubes were previously fixed with paraformaldehyde. This association was absent in neurites growing over Chinese hamster ovary cells, suggesting that the effect is specific for muscle cells. Coculture of neurons with variant strains of C2 myotubes that are deficient in AChR (1R-) or proteoglycans (S27) revealed a preferential association of SV neurites with 1R- myotubes but not with S27 myotubes. Thus, proteoglycans on the surface of C2 myotubes may influence the growth and/or differentiation of presynaptic neurons.

Characteristics and specific localization of receptors for atrial natriuretic peptides at non-neuronal cells in cultured mouse spinal cord cells

Characteristics of atrial natriuretic peptide receptors were determined in cultured mouse spinal cord cells. Saturation and competition experiments demonstrated the presence of a single class of atrial natriuretic peptide binding sites with high affinity (K D = 0.054 nM) and a density of 1.92 fmoles/10 6 cells. A similar affinity (K D = 0.070 nM) was observed in rat spinal cord membrane preparations. These atrial natriuretic peptide binding sites were functional receptors since the treatment of cells with atrial natriuretic peptide increased cyclic guanosine monophosphate levels within these cells in a classical time-dependent manner. When atrial natriuretic peptide was applied onto the cell body of intracellularly recorded spinal cord neurons, this peptide did not evoke a change of the input resistance or of the resting membrane potential value. Light-microscopic autoradiography studies showed that no atrial natriuretic peptide binding could be detected on typical birefringent neurons but it could be located on astroglial and epithelial cells as identified by immunocytochemical markers. These results show that functional atrial natriuretic peptide receptors with high affinity exist in cultured mouse spinal cord cells and are not located on neurons. The presence of atrial natriuretic peptide receptors on astrocytes suggests that this neuropeptide might be a good candidate for neuron-glial communication. As the atrial natriuretic peptide binding sites previously shown in epithelia responsible for maintaining fluid and electrolyte gradients, the atrial natriuretic peptide receptors on epithelial cells in these spinal cord cultures may be involved in vivo in the control of water balance in the central nervous system.

Tetanus toxin binding to mouse spinal cord cells: an evaluation of the role of gangliosides in toxin internalization

Studies on the binding of 125I-labelled tetanus toxin to mouse spinal cord cell cultures revealed two classes of toxin acceptor, one a high-affinity protease-sensitive site, the other a lower affinity protease-resistant site. The latter was shown to be sialidase-sensitive and may represent binding to gangliosides. Tetanus toxin internalization by spinal cord cells is rapid and coated pits have been implicated in the process (Parton et al. J. Neurochem., 49 (1987) 1057–1068). To investigate the role of gangliosides in the internalization process, trisialoganglioside was incorporated into the plasma membrane of Balb/c 3T3 cells which lack endogenous toxin-binding activity. 125I-labelled tetanus toxin bound specifically to ganglioside treated cells at 4°C. On warming cells to 37°C, a proportion of bound toxin was degraded indicating that some internalization of toxin had occurred. The mechanism of internalization was studied using tetanus toxin adsorbed to colloidal gold. At 4°C, toxin-gold was concentrated in non-coated cell surface membrane invaginations. On warming cells to 37°C, toxin gold was internalized via non-coated vesicles and accumulated within multivesicular bodies and lysosomes. The relative roles of coated and non-coated vesicular uptake systems in the mechanism of action of tetanus toxin are discussed.