Selective simulation of allelic expression: effectf antibodies to allotypic markers on lymphoid cells.

Peripheral blood leukocytes from rabbits which were heterozygous (b(5)/b(9)) for markers on their immunoglobulin light chains were maintained in vitro for up to 24 hours in the presence or absence of antibody to b9. After culture they were transferred into lethally irradiated b(4)/b(4)hosts. Recipients of cells exposed to antibodies to allotype markers showed a striking increase in concentration of circulating b9 molecules and number of b9 plasma cells in their spleens compared pared to control animals receiving untreated cells from the same donor. There was no appreciable difyerence between the two groups of recipients with respect to their content of b5 molecules and immunocytes.

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

Effects of dimethylsulfoxide on membrane currents of neuroblastoma x glioma hybrid cell.

Giga-ohm seal whole cell recording technique was used to examine ionic currents changes induced by dimethylsulfoxide (DMSO) in neuroblastoma X glioma hybrid NG 108-15 cells. DMSO (0.5-1%) reversible blocks sodium, potassium and calcium currents and shifts by about 6 mV the sodium inactivation curve towards more negative voltages.

Neurotropism of rabies virus. An in vitro study.

The relative susceptibility of neurons and glia, grown as monolayers in vitro, to rabies virus infection was explored. Established cell lines of neuronal or glial phenotype and primary cultures of cells derived from mouse dorsal root ganglia (DRC) or brain were used as homologues of the targets of rabies virus in the nervous system. Fixed rabies virus (CVS) strain was used in most experiments; other fixed rabies strains (PV, HEP, ERA) and a street rabies virus isolate were used in some. Virus-cell tropism was determined by immunofluorescence assay for rabies nucleocapsid antigen and cell permissivity was assessed by titration of virus yields. Neuronal cells always exhibited a much greater susceptibility to infection and a greater propensity to sustain viral growth. By immunofluorescence, 90-100% of neurons commonly had viral inclusion bodies, while doses of the virus three to four orders of magnitude higher still left greater than 99% of astrocytes, in brain cell cultures and 90 +/- 5% of the non-neuronal cells in DRG cultures without any obvious signs of rabies virus. Neuroblastoma cells (95 +/- 5% with viral antigens) produced viral yields about four orders of magnitude higher than glioma cells (10 +/- 5% with viral antigens). Though the overall infectivity of street virus was lower than that of fixed virus strains, a significantly higher viral tropism for neurons than for glia was maintained. Thus, primary neuronal cultures offer a means of exploring molecular events in rabies virus infection and their role in pathogenesis.

Localization of aldolase C mRNA in brain cells.

The expression of aldolase C and aldolase A mRNA was assessed by Northern blot hybridization using RNAs purified from cultured rat and mouse brain neurons and astroglial cells. Neurons were found to contain about 4-fold more aldolase C mRNA and about twice as much aldolase A mRNA than astroglia. Analysis of the cellular localization of aldolase C mRNA by in situ hybridization to brain slices showed a predominantly neuronal labeling with an irregular distribution. A strong signal was observed in Purkinje cell somata and a weaker signal in subpopulations of neurons in cerebral cortex, striatum, hippocampus, hypothalamic nuclei and primary olfactory cortex.

Expression of the transcripts initiated in the 62nd intron of the dystrophin gene.

The pattern of expression of two distal transcripts initiated in the 62nd intron of the dystrophin gene was investigated under different circumstances; (i) during the development of different rat tissues these transcripts and Dp71, a protein encoded by one of them, increased with brain development and decreased with muscle development; (ii) in cultured glial and neuronal cells, the distal promoter was coactivated with tissue-specific upstream promoters, the muscle-type promoter in glial cells and the brain-type promoter in neuronal cells, which suggests that activity of the upstream promoter does not interfere with activity of the distal promoter; (iii) in lymphoblasts of DMD patients with various deletions of the dystrophin gene, the most distal of which included the 56th intron, the production of the distal transcript was not perturbed.

A correlation between the appearance and the evolution of tetanus toxin binding cells and neurogenesis.

The ontogenesis of cells expressing surface membrane binding sites for tetanus toxin (Tt) was studied in the mouse nervous system. Cells were labeled shortly after the tissue dissociation and the toxin bound was revealed by immunofluorescence. In the brain, spinal cord and dorsal root ganglia the toxin binding cells (TBC) are found as of very early stages of nervous system organogenesis, i.e. at 10 days of gestation. There is a close temporal correlation between the pattern of emergence and accumulation of TBC and the known pattern of appearance of post-mitotic neurons in mouse cerebral cortex, cerebellum and spinal cord. The curves of TBC abundance as a function of fetal age in various nervous system areas are different. They show regional fluctuations in the proportion of TBC that reflect the cumulative changes in the dynamics of neuronal subpopulations. The results indicate that Tt can be used as an ontogenetically early marker of neuronal differentiation and that the acquisition of Tt receptors may represent one of the earliest detectable characteristics of the developing neurons.

Early appearance of cells bearing Na+ channels in developing mouse brain. A quantitative analysis using light microscopic autoradiography.

125I-alpha-Scorpion toxin (alpha-ScTx) binds to a component of the voltage-sensitive Na+ channel. We have previously shown that receptor capacity on dissociated mouse brain cells increases between days 12 and 19 of fetal life as does the expression of neurotoxin-sensitive 22Na+ influx. In the present study we have investigated the distribution of Na+ channels at the cellular level. Quantitative analysis by light-microscopic autoradiography was carried out on dissociated brain cells labeled with 125I-alpha-ScTx at 13, 15 and 18 fetal days. We have shown that at day 13 a large population of cells (39% of total) is alpha-ScTx-labeled, providing direct confirmation for a wide-spread presence of Na+ channels at an early stage of mouse brain development. The subsequent increase in receptor number with age is due both to an increase in alpha-ScTx-labeled cells (to 53% and 97% at days 15 and 18, respectively) and to an increase in the receptor density on these cells (10.9, 12.7 and 34.5 silver grains/1000 microns2 of cell surface for the 3 stages studied).

High-affinity binding of alpha-scorpion toxin: a neuronal property.

alpha-Scorpion toxin binding to its receptor--one component of the voltage-sensitive sodium channel--was studied in an attempt to define its phenotypic specificity. To this end we investigated the ability of neuronal, glial myogenic and fibroblastic cell lines to bind alpha-toxin II, purified from venom of the scorpion Androctonus australis Hector. A single class of saturable high-affinity (Kd congruent to 1 nM) binding sites, was present only in cell lines exhibiting some of the characteristics of normal neuronal cells, such as the N18, NIE-115, NS20, BN10-10, NG108-15 and T28 cell lines. NIA-103, which is an electrically non-excitable neuronal cell, gave negative results. In glial (G26-20, TR6B, C6) myogenic (T984) or fibroblastic (L) cell lines, we were unable to detect high-affinity binding sites for alpha-scorpion toxin. Primary cultures of rat skeletal muscle cells were also negative. Thus specific binding in the nanomolar range seems to be selectively associated with the neuronal phenotype. alpha-Scorpion toxin binding was tested before and after induction of neurites: in N18, NIE-115, NS20 cell lines, the differentiation brought on an increase in the number of binding sites but had little effect on the dissociation constant; in the hybrids NG108-15 and T28 high affinity saturable binding sites were detectable after but not prior to morphological differentiation.

Acetylcholine-activated currents in mouse neuroblastoma cells.

The nicotine and muscarinic responses of differentiated mouse neuroblastoma cells from the clonal line N1E 115 to applied cholinergic agents were recorded using single channel and whole cell patch clamp techniques. An inward macroscopic current induced by acetylcholine (ACh) at the resting potential was blocked by curare; cell-attached recordings revealed a single channel conductance of 18 pS and a lifetime of 36 ms at 30 degrees C, with 200 nM ACh. The zero current potential was close to 0 mV. The kinetics of these nicotinic currents were described by multiexponential functions for both the open and closed time distributions. An outward single channel current, present at resting and slightly depolarized potentials, was also observed and has been tentatively described as being dependent on muscarinic receptor activation, as it was usually blocked by atropine. Under our conditions of whole cell clamp, no macroscopic outward current sensitive to ACh was observed.

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

Neurotoxins as probes in the study of neuronal development.

We have investigated the expression of surface membrane binding sites for tetanus toxin and alpha-scorpion toxin (AaHII) on cells of the in vivo developing mouse nervous system. There is a close temporal correlation in the pattern of emergence and accumulation of tetanus toxin binding cells (TBC) and that of post-mitotic neurons. In different nervous system areas, the fluctuations in relative TBC abundance reflect regional changes in the dynamics of neuronal subpopulations. The results indicate that the acquisition of membrane tetanus toxin binding sites may represent one of the earliest detectable characteristics of nascent neurons. The Na+ channel-associated scorpion toxin become detectable in fetal mouse brain two days after the appearance of TBC. Their density increases with fetal age without change in receptor properties. At all stages, scorpion toxin binds to a single class of noninteracting sites with a KD = 0.1 - 0.5 nM. The affinity of binding is voltage-dependent. Studies on brain cells and various cell lines grown in vitro suggest a selective association of the high affinity scorpion toxin receptors with neuronal phenotype. In culture, as in vivo, there is a time dependent increase in receptor density. These results indicate that both tetanus toxin and scorpion toxin can be used as qualitative markers of neuronal differentiation; moreover, estimates of the density of scorpion toxin binding sites provide a quantitative index of neuronal maturation.

Phenotypic modulation in mouse neuroblastoma cells: the case of acetylcholinesterase.

Mouse neuroblastoma clone NIE-115 contains two species of acetylcholinesterase (AChE) which sediment at 4S and 11S. The former is predominant in growing cells (70%), whereas the 11S is more abundant in cells with neurites (55--65%). When cell replication is arrested by sodium butyrate, there is an increase in AChE activity but no modification in the proportion of 4S:11S species. Inhibition of protein synthesis by cycloheximide provokes a slow increase in the 11S form and a parallel decrease in 4S. Neurite retraction in presence of colchicine or excess serum does not lead to a concomitant reversion in the high 11S proportion. We postulate that the 11S AChE is formed by a conversion of the 4S and that the shift in the S-value ratio of the enzyme reflects changes in membrane remodeling during neurite extension.

Ionic channels in mouse astrocytes in culture.

We observed Na, K, and Cl voltage-dependent currents in a patch-clamp study of mouse brain astrocytes. In whole-cell recordings, depolarizations activated inward currents that were identified as Na currents since they were blocked by TTX, although complete block required high concentrations (greater than 1 microM). The corresponding single-channel Na currents were observed in outside-out patches. The channels were opened by a depolarizing pulse applied from a holding potential identical to the resting potential (-70 to -80 mV). Therefore, they may be considered functional Na channels. After addition of veratridine and an alpha-scorpion toxin, the decay of Na currents in whole-cell recordings was slower than observed under control conditions. At the single-channel level, the channels appeared to open in bursts. Depolarization did not increase the duration of the bursts, but inside each burst, increased the time spent in the open state. The K currents observed in the whole-cell recording mode were separated into inactivating and noninactivating currents. The inactivating current resembled the A current in its kinetics, its insensitivity to tetraethylammonium, and its sensitivity to 4-aminopyridine. At the single-channel level, at least 3 classes of K channels were observed at steady depolarized potentials. They resembled the K channels found in chromaffin cells by Marty and Neher (1985). Large conductance channels (385 pS) activated around 0 mV were identified as Cl channels.

Neuronal acquisition of tetanus toxin binding sites: relationship with the last mitotic cycle.

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Dev. Brain Res. 5, 139-147). Using a combined approach of immunocytochemistry and [3H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 +/- 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.

Na+-channel-associated scorpion toxin receptor sites as probes for neuronal evolution in vivo and in vitro.

Purified neurotoxin II of the scorpion Androctonus australis Hector (ScTx) has previously been shown to bind specifically to the Na+-ionophore-associated, voltage-sensitive receptor sites of excitable cells. We have conducted binding studies, using high-specific-activity 125I-labeled ScTx, to detect and quantify the Na+-channel receptors on cells of the developing fetal mouse brain. In vivo, the onset of detectable specific binding is at 12 fetal days. The rate of receptor appearance is initially slow but increases sharply as of the 16th day of mouse ontogenesis. The mean number of receptors at 12 and 19 days is 120 and 20,000 per cell, respectively (i.e., 0.5 and 80 per square micrometer). When corrected for the fraction of cell population corresponding to putative neuroblasts and neurons, identified by immunofluorescence as tetanus toxin binding cells, these values are, respectively, 1040 and 33,900 ScTx receptors per tetanus toxin binding cell or 4.2 and 136 per square micrometer. At all stages, the toxin binds to a single class of noninteracting sites; Kd = 0.1-0.5 nM. Similar findings in terms of ScTx-receptor properties and quantitative evolution were obtained in vitro. Specific 125I-labeled ScTx binding the presence of tetanus toxin binding cells. In cultures of central nervous system glia without neurons, only nonspecific low-level ScTx binding was detected. These results suggest that the high-affinity scorpion toxin receptors may be used as quantitative markers of neuronal differentiation.

Neurotoxin-sensitive sodium channels in neurons developing in vivo and in vitro.

Fetal mouse brain cells were investigated by 22Na+ flux assays with the aim to determine the ontogenetic time course of appearance of functional voltage-sensitive sodium channels. Their pharmacological properties were assessed by measurement of the response to known neurotoxins, acting at site 1, 2, or 3 of the Na+ channel. Brain cell suspensions, prepared at 11-19 d of prenatal development in vivo, and fetal brain neurons in culture were explored. In vivo neurotoxin-sensitive Na+ influx becomes detectable at 12 d of gestation, in concordance with the time of appearance of saturable binding sites for alpha-scorpion toxin (alpha-ScTx) and saxitoxin. Progression in fetal age or in time in vitro is accompanied by an increase in the initial rate and in the amplitude of Na+ uptake stimulated by batrachotoxin or veratridine. The general pharmacological properties of developing Na+ channels are very similar to the known properties of voltage-dependent Na+ channels in adult nerve: Batrachotoxin acts as a full channel agonist and veratridine as a partial agonist. Their respective apparent affinities are increased in presence of alpha-ScTx, in agreement with the known positive cooperativity of toxins acting at sites 2 and 3 of the Na+ channel. alpha-ScTx alone induces a small increase in Na+ permeability; its effect is greatly amplified in the presence of batrachotoxin or veratridine. The apparent affinity of alpha-ScTx is reduced by cell depolarization. Tetrodotoxin and saxitoxin block the increase in Na+ permeability induced by batrachotoxin, veratridine, and alpha-ScTx.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of polyglutamylated alpha- and beta-tubulin in mouse brain neurons.

Polyglutamylation is an important posttranslational modification of tubulin that is very active in nerve cells, where it accounts for the main factor responsible for tubulin heterogeneity. In the present work, we have analyzed quantitative and qualitative changes in glutamylated alpha- and beta-tubulin occurring during neuronal differentiation in culture. Glutamylated alpha- and beta-tubulin both markedly accumulate during this process with a time course remarkably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation of the two subunits are not exactly the same. Glutamylated alpha-tubulin is already abundant in very young neurons and displays, at this stage, a wide range of its degree of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated beta-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into alpha- and beta-tubulin decreases during differentiation, as well as the rate of the reverse deglutamylation reaction, suggesting that accumulation of glutamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin. Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable alpha-tubulin, which can occur in combination with polyglutamylation and contributes to increase the complexity of tubulin in mature neurons.

Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells.

Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low-abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co-reverse-transcribed and co-amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments. Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.

Dystrophin gene transcribed from different promoters in neuronal and glial cells.

It has been shown that the dystrophin gene, which is defective in patients with Duchenne and Becker muscular dystrophy (reviewed in ref. 1), is transcribed in brain from a specific promoter that is different from the one used in muscle, and so the two types of transcripts differ at least in their first exon. We recently found that the dystrophin gene is expressed at a higher level in primary cultures of neuronal cells than in astro-glial cells derived from adult mouse brain. Here we investigate the use of two different promoters in each cell type. Our results demonstrate that the brain-type promoter of the dystrophin gene is highly specific to neurons, in which there is a significant increase in the amount of brain-specific messenger RNA during the course of in vitro maturation. By contrast, the muscle-type promoter is active in a wider range of cell types, including not only striated and smooth muscle, but also glial cells to a lesser extent, and probably neurons.