Electrical consequences of spine dimensions in a model of a cortical spiny stellate cell completely reconstructed from serial thin sections

We built a passive compartmental model of a cortical spiny stellate cell from the barrel cortex of the mouse that had been reconstructed in its entirety from electron microscopic analysis of serial thin sections (White and Rock, 1980). Morphological data included dimensions of soma and all five dendrites, neck lengths and head diameters of all 380 spines (a uniform neck diameter of 0.1 m was assumed), locations of all symmetrical and asymmetrical (axo-spinous) synapses, and locations of all 43 thalamocortical (TC) synapses (as identified from the consequences of a prior thalamic lesion). In the model, unitary excitatory synaptic inputs had a peak conductance change of 0.5 nS at 0.2 msec; conclusions were robust over a wide range of assumed passive-membrane parameters. When recorded at the soma, all unitary EPSPs, which were initiated at the spine heads, were relatively iso-efficient; each produced about 1 mV somatic depolarization regardless of spine location or geometry. However, in the spine heads there was a twentyfold variation in EPSP amplitudes, largely reflecting the variation in spine neck lengths. Synchronous activation of the TC synapses produced a somatic depolarization probably sufficient to fire the neuron; doubling or halving the TC spine neck diameters had only minimal effect on the amplitude of the composite TC-EPSP. As have others, we also conclude that from a somato-centric viewpoint, changes in spine geometry would have relatively little direct influence on amplitudes of EPSPs recorded at the soma, especially for a distributed, synchronously activated input such as the TC pathway. However, consideration of the detailed morphology of an entire neuron indicates that, from a dendro-centric point of view, changes in spine dimension can have a very significant electrical impact on local processing near the sites of input.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

The transport of morphologically abnormal sperm in the female reproductive tract of mice

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.     

Influence of the concentration of sperm on the percentage of eggs fertilized for three strains of mice

This study was conducted to determine the optimal concentration of sperm to use for the insemination of females to detect differences among strains of mice in the percentage of eggs fertilized. Female ICR mice were inseminated with sperm of concentrations ranging from 0.25 to 8 × 10⁶/50 μl from males of either DBA/2N, CF1, or C57BL/6N strains. Differences among strains were detected only when approximately 50% of the eggs were fertilized but not when each of the strains fertilized either a high or low percentage of eggs. The optimal concentration of sperm therefore was the concentration that gave approximately 50% fertilized eggs.     

Characterization of a stable,anchorage-dependent clone obtained from a spontaneously transformed mouse cell line

Summary A variant nontransformed clone, I21, was selected from the spontaneously transformed mouse fibroblast line, IT22. Selection was done by plating IT22 in methylcellulose and picking single cells after 2 d. Cultures derived from these single cells were selected again and one clone, I21, derived from the second round of selection was characterized extensively. I21 and IT22 have the same plating efficiency (PE) on plastic, but in agarose they differ by 1000-fold. In comparison to IT22, I21 has a normal morphological appearance, a lower saturation density, a higher viability in stationary phase, an increased doubling time, an increased chromosome content, and is unable to form tumors in nude mice. I21 has remained remarkably stable in culture and has not reverted to the transformed phenotype for at least 300 generations in culture. Over 100 clones of I21, expanded to 10⁶ cells, failed to show an increased PE in agarose. Even expansion of the rare colonies of I21 that grow in agarose failed to produce clones similar to IT22. The research was supported by the Medical Research Council and the National Cancer Institute of Canada. R. Godbout was supported by a 1967 Science Scholarship and by an MRC Studentship. B. L. Gallie is a Research Associate of the Ontario Cancer Treatment and Research Foundation.     

The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

Determination of the glucosidase-stimulating proteins by competitive enzyme-linked immunoassay

A procedure for the immunoassay of cohydrolase sphingolipid-I in mouse tissue is described. This cohydrolase (actually a mixture of at least four related proteins) stimulates or activates the beta-glucosidase which hydrolyzes ceramide glucoside, a widely occurring glycosphingolipid. The method involves extraction of cohydrolase from tissue homogenate with a salt-buffer solution, removal of proteins by adjustment to pH 6, further removal of proteins by heating, and removal of interfering materials with a small size exclusion column. Antibodies were raised to bovine cohydrolase in rabbits and purified with an affinity column made from cohydrolase. The immunoassay involves binding of antibody by the cohydrolase sample (20-200 pg) in competition with cohydrolase that has been chemically linked to horseradish peroxidase. The mixture is treated with particle-linked second antibody and centrifuged; the pellet is then assayed fluorometrically for peroxidase content. Initial application of the method showed that cohydrolase was present in all mouse tissues studied and that its concentration paralleled that of glucocerebrosidase relatively closely. Changes with age (14 and 92 days) occurred in a similar fashion for the two substances.     

Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)