Cerebellar target neurons provide a stop signal for afferent neurite extension in vitro.

The contributions of cell-cell interactions to the establishment of specific patterns of innervation within target brain regions are not known. To provide an experimental analysis of the regulation of afferent axonal growth, we have developed an in vitro assay system, based on the developing mouse cerebellum, in which afferent axons from a brainstem source of mossy fiber afferents, the basilar pontine nuclei, were cocultured with astroglia or granule neurons purified from the cerebellum. In the absence of cells from the cerebellum, pontine explants produced axons that fasciculated and extended rapidly on a culture surface treated with poly-lysine or laminin. When pontine neurites grew onto cerebellar astroglial cells, outgrowth was more abundant than on substrates alone, suggesting that glial cells provide a positive signal for axon extension. Time-lapse video microscopy indicated that the rate of neurite extension increased from less than 50 microns/hr to more than 100 microns/hr when axonal growth cones moved from the culture substratum onto an astroglial-cell surface. Acceleration of neurite extension was also observed as pontine neurites grew onto other pontine neurites. By contrast, when pontine neurites grew on granule neurons, the appropriate targets of mossy fibers, the length of pontine neurites was greatly reduced. As growing axons terminated on granule neurons, the target cells appeared to provide a "stop-growing signal" for axon extension. The length of pontine neurites decreased with increasing granule neuron density. Two lines of evidence suggested that the stop signal was contact mediated. First, video microscopy showed that pontine growth cones stopped extending after contacting a granule neuron. Second, the length of afferent axons was not reduced when pontine neurites grew at a distance from granule neurons. Competition experiments where both astroglia and granule neurons were plated together suggested that the growth arrest signal provided by granule neurons could override the growth-promoting signal provided by astroglial cells. These results suggest that specific cell-cell interactions regulate the growth of pontine afferent axons within their cerebellar target, with axoaxonal and axoglial interactions promoting axon extension and axon-target cell interactions interrupting axon extension.     

Nonlinear muscle recruitment during intramuscular and nerve stimulation

Future progress in neuromuscular prostheses will depend on developing techniques for stimulating paralyzed muscle, especially utilizing neuromuscular stimulation. We have found nonlinear force versus stimulus amplitude characteristic (recruitment) curves in the gastrocnemius-soleus-plantaris muscle group of the cat in response to stimulation of the tibial nerve near the muscle entry point. Such response characteristics are undesirable in neuromuscular control systems. Nonlinear recruitment curves usually consisted of two regions in which force increased linearly with stimulus amplitude, separated by a "plateau" region in which force was relatively constant. The two linear regions were associated with activation of separate neuromuscular compartments (lateral or medial gastrocnemius, plantaris, soleus, or subdivisions of those muscles). When the stimulated myoelectric responses from these compartments were plotted versus stimulus amplitude, the region of recruitment between threshold and saturation often did not appreciably overlap for different compartments, suggesting that the axons innervating those compartments were physically segregated within the nerve from axons innervating other compartments. Correlation coefficients between force and stimulated myoelectric response were very high (up to R2 = 0.99) when using a composite curve produced by averaging myoelectric response curves recorded from each of the active compartments. By dividing the tibial nerve into its component bundles or fascicles and stimulating each in turn, it was possible to show that individual bundles innervate non-overlapping groups of muscle compartments, and that recruitment of the nerve bundles over different threshold ranges could account for the nonlinear force/stimulus response curves initially observed. The presence of separate innervation of muscles or compartments by fascicles should be an important factor in designing functional neuromuscular stimulation (FNS) systems.     

Serodiagnosis of Trichomonas vaginalis infection by the indirect fluorescent antibody test.

The indirect fluorescent antibody test was used to detect antibodies to Trichomonas vaginalis in 200 antenatal patients. A total of 104 (52%) gave a positive reaction with antigen prepared from cultures of trichomonas isolated from seven of the patients. Antitrichomonal antibody was detected at a 1/4 dilution in 90% of patients with proven trichomoniasis, while the highest dilution at which antibody was detected was 1/32. IgG rather than IgM appeared to be the antibody class involved. Of those patients with no demonstrable trichomonal infection, 17% gave positive reactions at 1/4 dilution, while 64% had no demonstrable antibody. One of 30 children under 11 years of age gave a positive reaction.     

Distribution of transferrin, ferritin, and lactoferrin in human tissues.

An immunoperoxidase staining technique was used for detecting three major iron-binding proteins (transferrin, ferritin, and lactoferrin) in routine histological paraffin sections of human tissue. Transferrin was found mainly in hepatocytes, a variety of epithelial and myoepithelial cells, renal tubular cells, and histiocytes. Ferritin was most readily found in histiocytes and liver cells, with weaker reactions seen in epithelial cells. Lactoferrin was found in lactating breast tissue, bronchial glands, polymorphs, and gastric and duodenal epithelial cells. The technique is potentially valuable for investigating abnormal iron states.     

Labial salivary gland biopsy in Sjögren''s disease

A labial biopsy technique is described and was used to study 40 patients with connective tissue disease and 60 postmortem subjects. More than one focus of lymphocytes per 4 sq mm of minor salivary tissue was found to be a consistent finding in patients with Sjögren''s disease. The labial biopsy is shown to be a further valuable investigative procedure in such patients.     

Interaction between rod and cone systems in dichoptic visual masking

The brightness of a brief flash of light is reduced by the suitable presentation of a second flash in an adjacent region of the visual field. This making effect (metacontrast) can be induced dichoptically, that is with the test flash presented to one eye and the masking flash to the other. By a suitable choice of wavelengths and conditioning field, the test flash may be arranged to effectively stimulate only rod receptors and the masking flash only cone receptor. A dichoptic masking effect is still obtained.     

Use of serum-free hormonally defined media to evaluate the effects of growth factors and inhibitors on proliferation of estrogen-responsive mammary and pituitary tumor cells in culture

Summary Serum-free defined media have been developed for assay of the mitogenic effects of growth factors on human MCF-7, human T-47D, and mouse COMMA-D mammary cells as well as for identification of mitogens and inhibitors of GH₄C₁ rat pituitary tumor cell growth. These lines were shown to grow in vivo in response to a variety of hormones including estrogens and thyroid hormones. With mammary cells, complete hormonally and nutritionally defined media were established that supported continuous passage at 50 to 90% of the serum stimulated rate. The strategy used to measure mitogens for mammary cells was to identify nutritional conditions where the growth rate was reduced greatly without impairing the response to picomolar to nanomolar concentration of growth factors. The effects of polypeptide growth factors and tissue extracts were estimated by their addition to basal medium and measuring cell number increases or labeled thymidine incorporation into DNA. In a variation of this methodology, the MTW9/PL2 rat mammary cells were used to identify secreted autocrine growth factors; nutritionally defined conditions were sought for growth of these rat cells in the complete absence of exogenous growth factors. The factors secreted into the medium were detected by bioassays with COMMA-D or MCF-7 mammary cell lines. The effects of growth factors-inhibitors on pituitary cells were measured by a related method; the GH₄C₁ cells were grown at less than optimal rates in a defined medium designated TRM-1. Addition of mitogens to TRM-1 stimulated pituitary cell growth whereas addition of inhibitors caused reduced levels of growth. The methods described in this report offer new means of assaying growth factors-inhibitors for a range of mammary and pituitary tumor cells.     

Effect of spleen cell populations on resolution of Cryptosporidium parvum infection in SCID mice.

Persistent infection was established in SCID mice given 10(7) Cryptosporidium parvum oocysts. Nine groups of infected SCID mice were inoculated with 10(6), 10(5), or 10(4) total spleen cells, CD8-depleted spleen cells, or CD4-depleted spleen cells from naive BALB/c donors. Infection was significantly reduced in all treatment groups. The most profound effect occurred with spleen cell preparations containing CD4 T lymphocytes but depleted of CD8 T lymphocytes.