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Recent data have emphasized the neurochemically distinct nature of subterritories in the accumbens part of the rat ventral striatum termed the core, shell, and rostral pole. In order to gain a more comprehensive understanding of how afferents are distributed relative to these subterritories, immunohistochemical detection of retrogradely transported Fluoro-Gold was carried out following iontophoretic injections intended to involve selectively one of the subterritories. The data revealed that a number of cortical afferents of the medial shell and core originate in separate areas, i.e., the dorsal peduncular, infralimbic, and posterior piriform cortices (to medial shell) and the dorsal prelimbic, anterior agranular insular, anterior cingulate, and perirhinal cortices (to core). The lateral shell and rostral pole are innervated by cortical structures that also project either to the medial shell or core. The orbital, posterior agranular insular, and entorhinal cortices, hippocampus, and basal amygdala were observed to innervate the accumbens in a topographic manner. Following core injections, strong bilateral cortical labeling was observed. Few labeled cortical cells were observed contralaterally following injections in the medial shell. Intermediate numbers of labeled neurons were observed in contralateral cortices following lateral shell injections. Robust subcortical labeling in a variety of structures in the ventral forebrain, lateral hypothalamus, deep temporal lobe, and brainstem was observed after shell injections, particularly those that involved the caudal dorsomedial extremity of the shell, i.e., its "septal pole." Selective ipsilateral labeling of subcortical structures in the basal ganglia circuitry was observed following injections in the core and, to a lesser extent, lateral shell. It was concluded that a number of afferent systems exhibit varying degrees of segregation with respect to the accumbal subterritories.
We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.
The ELF alkaline phosphate substrate can be used to fluorescently label a wide variety of biological targets. This substrate yields a bright, photostable yellow-green fluorescent precipitate at the site of enzymatic activity. ELF labeling can be as much as 40 times as bright and hundreds of times as photostable as labeling with conventional fluorophores and yields signals capable of very fine submicroscopic resolution. Signal development is also extremely rapid, making the signal amplification technology well suited for applications such as RNA in situ hybridization.
Two discrete areas in frontal cortex are involved in generating saccadic eye movements--the frontal eye field (FEF) and the supplementary eye field (SEF). Whereas FEF represents saccades in a topographic retinotopic map, recent evidence indicates that saccades may be represented craniotopically in SEF. To further investigate the relationship between these areas, the topographic organization of afferents to FEF from SEF in Macaca mulatta was examined by placing injections of distinct retrograde tracers into different parts of FEF that represented saccades of different amplitudes. Central FEF (lateral area 8A), which represents saccades of intermediate amplitudes, received afferents from a larger portion of SEF than did lateral FEF (area 45), which represents shorter saccades, or medial FEF (medial area 8A), which represents the longest saccades in addition to pinna movements. Moreover, in every case the zone in SEF that innervated lateral FEF (area 45) also projected to medial FEF (area 8A). In one case, a zone in rostral SEF projected to both lateral area 8A from which eye movements were evoked by microstimulation as well as medial area 8A from which pinna movements were elicited by microstimulation. This pattern of afferent convergence and divergence from SEF onto the retinotopic saccade map in FEF is indicative of some sort of map transformation between SEF and FEF. Such a transformation would be necessary to interconnect a topographic craniotopic saccade representation in SEF with a topographic retinotopic saccade representation in FEF.
In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.
An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.
These studies were designed to determine the pattern of initial afferent fiber ingrowth into the prenatal spinal gray matter and the establishment of the topographic organization of the presynaptic neuropil in the dorsal horn. A total of 113 lumbar dorsal root ganglia were labeled with carbocyanine fluorescent dye DiI or DiA in 67 rat embryos and neonatal pups aged embryonic day 13 to postnatal day 0 (E13-P0). The initial fiber penetration of the lumbar spinal gray began at E15 and was restricted to the segments of entry. Subsequent growth of fibers into gray matter of adjacent segments began approximately one day later, and this delay was continued, about one day for each successive segment. A second wave of ingrowth of putative small-diameter afferents into the substantia gelatinosa began at E19 and also displayed the same rostrocaudal delay. Fiber ingrowth was specific and occupied the somatotopic area appropriate for the adult, from the earliest stages (E18) in which dorsal horn laminae could be adequately defined. The somatotopic organization of the presynaptic neuropil in laminae III and IV did not change significantly throughout embryonic development as the amount of overlap between adjacent and non-adjacent ganglion projections remained constant throughout embryonic development. In addition, it was found that fibers innervating the proximal and distal hindlimb entered the spinal gray simultaneously at E15 before the innervation of the distal toes was established. The results of these studies indicate that the somatotopic organization of the presynaptic neuropil is established very early in development and requires little refinement to match that seen in the adult. The simultaneous penetration of the fibers originating from the proximal and distal areas of the limb before innervation is complete suggests that this ingrowth may be independent of the establishment of specific peripheral connections.
Development of the peripheral innervation patterns of the L1-S1 lumbosacral ganglia and motor segments in embryonic day 12-17 (E12-17) rat embryos was examined using carbocyanine dyes. Individual dorsal root ganglia (DRGs) and/or isolated ventral horn (VH) segments, or individual peripheral nerves, were isolated in rat embryos fixed at different stages and filled with one of three carbocyanine dyes; DiI, DiA, and DiO. Individual experimental preparations included labeling of 1) single DRGs; 2) multiple DRGs with alternating dyes, DiO, DiI, and DiA; 3) single isolated VH segments; 4) multiple VH segments with alternating dyes; 5) single VH segments and the corresponding segmental DRGs with different dyes; and 6) two or more individual peripheral nerves labeled with different dyes. Results from these preparations have shown that the first fibers exited the lumbar ventral horn and DRGs at E12. At E13 major nerve trunks (e.g., femoral and sciatic) were visible as they exited the plexus region. By E14 afferent fibers were present in the epidermis of the proximal hindlimb, and the major nerve trunks extended into the leg. Fibers originating from L3 to L5 (DRG and VH) reached the paw by E14.5-E15, and the epidermis of the most distal toes was innervated by E16-E16.5. While afferent fibers and motor axons of the same segmental origin mixed extensively in the spinal nerve, fibers of different segmental origin combined in the plexus and major nerve trunks with little or no interfascicular mixing. Dermatomes observed at E14 were in general spotty and non-contiguous. However, by E16 the dermatomes resembled mature forms with substantial overlap only between adjacent ones. Thus the adult pattern of spatial relationships between cutaneous afferent fibers in the periphery is established early in development.
Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [2H4]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH2C6F5)3-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z430/434. Twenty samples can be assayed in 2 days. Precision of the assay is +/- 2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100-500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.
TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.