Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

ULTRASTRUCTURAL CHANGES IN THE MITOCHONDRIA OF CEREBELLAR PURKINJE CELLS OF NERVOUS MUTANT MICE

The maturation of cerebellar Purkinje cells of normal and nervous (nr/nr) mutant mice has been studied by light and electron microscopy. In the mutant, 90% of Purkinje cells selectively degenerate between postnatal days 23 and 50. Losses are greater in lateral than medial regions. Other cerebellar neurons appear normal. The first morphological abnormality recognized is the presence of rounded mitochondria in perikarya of some Purkinje cells of the mutant at 9 days after birth. By 15 days, all nr/nr Purkinje cells contain spherical mitochondria and begin to deviate from the normal maturational sequence. Elaboration of the extensive dendritic tree halts midway and newly formed axon collateral fibers degenerate. In the perikaryon, the basal polysomal accumulation and climbing fiber-somatic spine synapses are sometimes abnormally retained. Cisternae of the Golgi apparatus and rough endoplasmic reticulum cease to form aligned stacks, and decrease in number, while polysomes dissociate into free ribosomes. These changes are progressive, culminating in cell death. Although every nr/nr Purkinje cell demonstrates spherical mitochondria, some cells survive the critical period, retain a near-normal complement of organelles, and reacquire normal-appearing mitochondria. The disorder appears intrinsic to Purkinje cells since all major classes of synapses were identified before cell death.     

The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.     

Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts

A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.     

Evidence for neurotransmitter plasticity in vivo. II. Immunocytochemical studies of rat sweat gland innervation during development

Previous studies of the cholinergic sympathetic innervation of rat sweat glands provide evidence for a change in neurotransmitter phenotype from noradrenergic to cholinergic during development. To define further the developmental history of cholinergic sympathetic neurons, we have used immunocytochemical techniques to examine developing and mature sweat gland innervation for the presence of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and for two neuropeptides present in the mature cholinergic innervation, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). In 7-day old animals, intensely TH- and DBH-immunoreactive axons were closely associated with the forming glands. The intensity of both the TH and DBH immunofluorescence decreased as the glands and their innervation developed. Neither TH-IR nor DBH-IR disappeared entirely; faint immunoreactivity for both enzymes was reproducibly detected in mature animals. In contrast to noradrenergic properties, the expression of peptide immunoreactivities appeared relatively late. No VIP-IR or CGRP-IR was detectable in the sweat gland innervation at 4 or 7 days. In some glands VIP-IR first appeared in axons at 10 days, and was evident in all glands by 14 days. CGRP-IR was detectable only after 14 days. In addition to VIP-IR and CGRP-IR, we examined the sweat gland innervation for several neuropeptides which have been described in noradrenergic sympathetic neurons including neuropeptide Y, somatostatin, substance P, and leu- and met-enkephalin; these peptides were not evident in either developing or mature sweat gland axons. Our observations provide further evidence for the early expression and subsequent modulation of noradrenergic properties in a population of cholinergic sympathetic neurons in vivo. In addition, the asynchronous appearance during development of the two neuropeptide immunoreactivities raises the possibility that the expression of peptide phenotypes may be controlled independently.     

Acquisition of cholinergic and peptidergic properties by sympathetic innervation of rat sweat glands requires interaction with normal target

The sweat glands, a target of cholinergic sympathetic neurons, were replaced with parotid gland, a target of noradrenergic sympathetic neurons, in neonatal rats. This transplantation paradigm allowed sympathetic neurons that would normally innervate the sweat glands and develop a cholinergic phenotype to innervate the parotid gland instead. The innervation of the transplanted parotid gland did not develop a cholinergic phenotype, as assessed by choline acetyltransferase activity and acetylcholinesterase immunoreactivity, but continued to express intense catecholamine fluorescence. In addition, immunoreactivity for vasoactive intestinal peptide, normally expressed by the sympathetic innervation of the sweat glands but not the parotid, was observed in only a small percentage of the parotid-associated fibers. These results suggest that cellular interactions between neurons and their targets play an important role in the differentiation of mature neurotransmitter and neuropeptide phenotypes in vivo.     

Radioautographic visualization and biochemical identification of O- phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone

We injected NaH2(33)PO4 into normal 14-d-old embryonic chicks and examined the long bones by both radioautography and biochemical analyses from 10 to 240 min after the injection was completed. At 30 min, determination of the radiographic grain density revealed that 33P was concentrated principally in fibroblasts, preosteoblasts, and osteoblasts. With time, there was a progressive increase in the density of silver grains located over both the osteogenic cells and the regions of uncalcified (osteoid) and calcified extracellular organic matrices. Biochemical analyses identified 33P-O-phosphoserine as the major 33P component in glutaraldehyde-treated whole demineralized bone tissue and in EDTA-soluble, nondiffusible proteins extracted from the bones, both at the same time periods that 33P-induced silver grains were visualized by radioautography. 33P-O-phosphothreonine was also identified in experiments using a dosage of 10 mCi per embryo. The results provide the first combined direct biochemical and radioautographic identification that phosphoproteins are synthesized in bone and are located morphologically at the sites of mineralization. The data provide further evidence that phosphoproteins play a critical role in the biological calcification of vertebrate tissues.     

Spread of exotic plants in the landscape: the role of time,growth habit,and history of invasiveness

We investigated the relative contribution of minimum residence time, growth habit, and history of invasiveness to the spread of exotic plants in Michigan and California. Our data include minimum residence time as estimated by earliest herbarium collection records, growth habit, and history of invasiveness for over 2000 records from two herbaria (MI = 943, CA = 1131). Our data support the hypothesis that minimum residence time is highly associated with landscape spread, explaining 39–44% of variation in the number of counties invaded. In contrast, growth habit and history of invasiveness explained a small fraction of variation in spread in California but not Michigan. Over the past 30 years exotic plant species frequently became established in Michigan and California (≥50 species per decade), suggesting that many more species will become invasive over time. There is an urgent need to develop effective policies for exotic plant management. In both states we found significant positive correlations between minimum residence time and species occurrence on state invasive plant lists. Further, we found historical information on the pest status of a plant species introduced into a similar environment to be relevant in determining landscape spread of exotic plants. We conclude that efforts to predict exotic species spread based on biological characteristics may have limited success, and instead endorse pest risk analysis for proposed new imports coupled with rapid detection and early response for unintended and unwanted introductions.