Microcomputer-based three-dimensional reconstruction of in situ hybridization autoradiographs.

A number of software routines were written for the public domain Macintosh-based video-densitometry program, Image, to facilitate in situ hybridization analysis. These routines utilize fiducial marks drilled in unused portions of the brain to rotate high-magnification images of individual sections so that they may be placed in register. The regions of interest within the registered sections are outlined and intermediate sections interpolated to produce a data set representing a three-dimensional volume. This shell is then filled with the original density data obtained with one or more in situ hybridization probes. The final object can be viewed in various degrees of translucency and examined from different angles or computationally resectioned to yield new information concerning activity changes within the region and relationships between reactive sites. These static images can be collected and rapidly displayed as a movie. These routines have been used in this paper to display the differential hybridization of three different mRNA probes in the hypothalamic paraventricular nucleus.

Laminin supports neurite outgrowth from explants of axotomized adult rat retinal neurons.

The influence of laminin on neurite outgrowth from explants of adult rat retina and its distribution in normal and lesioned rat optic nerves were examined. Neurite outgrowth required the presence of laminin in the substratum, and as with a goldfish retinal explant system, was markedly stimulated by prior axotomy. Except for blood vessels and the nerve sheath, normal rat optic nerve was devoid of laminin immunoreactivity. Unlike results seen in the goldfish optic nerve, injury to the rat optic nerve induced no observable increase in laminin content or change in its distribution. The differences in the in vivo regenerative capacities of these two species may in part be related to the differences in their abilities to provide a proper substratum for axon regrowth.

A microcomputer-based image analyzer for quantitating neurite outgrowth.

An automated device that recognizes and quantitates neurite outgrowth from goldfish retinal explants is described. The system components include a video camera-inverted microscope and an Apple II microcomputer with an attached Motorola 68000 coprocessor. Values for neurite outgrowth were generated within 30 s, required no user input other than defining the microscopic field, and correlated well with a subjective measure, the nerve growth index.

Quantitation of thin-layer chromatograms with an Apple II computer-based videodensitometer.

An inexpensive, high-speed densitometer made from an Apple II computer, a black-and-white video camera, and an image digitizing board is described. By supplementing the computer with a very fast coprocessor, one can obtain a measurement of a typical thin-layer spot in 30-40 s. Only minimal electronic expertise is required for assembling the system from the commercially available components. Coefficients of variation for multiple readings of a single 500-ng spot of charred lipid were about 0.5%. For lipid spots weighing 250 to 2000 ng, a second-order relationship between weight and density reading was obtained with a correlation coefficient of 0.998.

Laminin and optic nerve regeneration in the goldfish.

Previous work from our laboratory had shown that goldfish retinal fragments explanted onto a polylysine substratum 1 to 2 weeks following optic nerve crush exhibit a striking clockwise pattern of neuritic outgrowth. In the present study, however, when the basal lamina component laminin was used as a substratum, neurites grew out as uncurved spokes, were less fasciculated, and had an increased rate of elongation. When laminin was combined with polylysine in the substratum, the degree of fasciculation and rate of elongation were similar to those seen on laminin alone, whereas the tendency for clockwise outgrowth was even more pronounced than that observed with polylysine alone. These results suggest that regenerating neurites have an affinity for laminin. Using an antibody to murine Engelbreth-Holm-Swarm sarcoma laminin, which cross-reacted with basal lamina in goldfish tissue sections, we studied the histochemical distribution of laminin in the goldfish visual system. Immunoperoxidase staining for laminin showed a characteristic scalloped pattern of staining in cross-sections of optic nerve bundles. Following optic nerve crush, the reaction product became much more diffuse and intense, especially distal to the crush site. When the retinal ganglion cell bodies were eliminated by removing the eye, the degenerating optic nerve stump still showed the intensive staining. We interpret these results to indicate that optic nerve glia are responsible in large part for the formation of laminin. Taken together, these in vivo and in vitro findings suggest that laminin plays a role in nerve regeneration in the goldfish central nervous system.

An inexpensive, microcomputer-based, video densitometer for quantitating thin-layer chromatographic spots.

An inexpensive video densitometer based on an Apple II microcomputer using commercially available components was developed. The device was used to quantitate charred lipid and colored spots on thin-layer chromatography plates. The method is useful for lipid samples weighing 0.1 to 4 micrograms on high-performance plates and 1 to 20 micrograms on ordinary plates. The software automatically corrects for baseline drift due to dirt or nonuniformity in the plate.