Åsa Thureson-Klein

Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: A possible mechanism for neuropeptide release

It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70-130 nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P less than 0.01) on the ipsilateral side 1-24 h after a unilateral skin lesion in the vibrissae area. A second increase (P less than 0.001) occurred 14-15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.

Exocytosis from Neuronal Large Dense-Cored Vesicles

Publisher Summary This chapter provides an overview of exocytosis from neuronal large dense-cored vesicles. Regulated exocytosis operates in cell types ranging from primitive protozoa to highly differentiated endocrine cells and neurons. Membrane-bounded vesicles, predestined for exocytosis, provide protective storage for substances that targets the intracellular metabolizing enzymes. In neurons, the secretory organelles that undergo exocytosis correspond to a variety of storage vesicles based on content. They fall into two general categories based on size: the large dense-cored vesicles (LDVs) measuring > 70 nm in diameter and the small vesicles in the range of 45–55 nm. The latter category, called “synaptic vesicles,” is familiar to cell biologists because of its role in the release of classical transmitter and amino acid transmitters. The aspects of the structure and contents of large and small vesicles are compared as they pertain to exocytosis. The differential recruitment of LDVs and small synaptic vesicles for exocytotic release is also emphasized when supported by ultrastructural correlates. Protein phosphorylation is important for LDV transport to and fusion with the plasmalemma.

Differential distribution of neuropeptides and serotonin in pig adrenal glands

A differential distribution of vasoactive neuropeptides and serotonin in chromaffin cells and nerve fibers within the adrenal glands of the pig (Sus scrofa) was found using immunohistochemical methods. Met- and leu-enkephalins, present at high levels in the medulla (measured by radioimmunoassay), occurred in adrenaline storing cells, some of which contained calcitonin gene-related peptide. Islets of chromaffin cells beneath the capsule also contained enkephalins and calcitonin gene-related peptide. Nerve fibers with enkephalin-like immunoreactivity were sparse, but many varicose fibers in the inner cortex and medulla showed calcitonin gene-related peptide immunofluorescence in a pattern similar to vasoactive intestinal polypeptide. Neuropeptide Y was mainly associated with perivascular fibers and neither neuropeptide Y nor vasoactive intestinal polypeptide immunoreactive chromaffin cells were detected. In contrast to the neuropeptides, most serotonin-like immunoreactivity coincided with noradrenaline histofluorescence. It is concluded that the distribution of nerve fibers with calcitonin gene-related peptide and vasoactive intestinal polypeptide would allow interactions between chromaffin and inner cortical cells. Stimuli activating noradrenaline chromaffin cells could release serotonin while stimulation of adrenaline storage cells would release enkephalin and, to a lesser extent, calcitonin gene-related peptide. Met-enkephalin, which occurs 3 4:1 over leu-enkephalin, is the most likely of the co-released peptides to reach distant receptors via the venous outflow.

Ultrastructural and biochemical correlates of the specificity of chlordecone‐potentiated carbon tetrachloride hepatotoxicity

Previous work demonstrated the specificity of chlordecone (Kepone) potentiated CCI4 hepatotoxicity in rats on the basis of functional, biochemical, and histochemical indices of liver damage. The present study was undertaken to elucidate mechanisms underlying the pathogenesis of the selectively potentiated hepatotoxic response to CCI4 after dietary exposure to low levels (10 ppm for 15 d) of chlordecone but not its structural analogs mirex and photomirex. On d 15 of pretreatment of male Sprague‐Dawley rats, either selected parameters of hepatic mixed‐function oxidase (MFO) activity were determined or corn oil or CCI4 (0.05 ml/kg in corn oll) was injected ip. Biochemical and ultrastructural assessment of liver damage was made 24 h after the injection. When given as the sole agents, photomirex, mirex, and chlordecone significantly increased microsomai pentobarbital hydroxylase and ethylmorphine demethylase activities. Both photomirex and mirex elevated microsomai cytochrome P‐450 content, while only photomir...

Ultrastructure of highly purified sympathetic nerve vesicles: Correlation between matrix density and norepinephrine content

Initial glutaraldehyde fixation followed by OsO4 or combined with potassium dichromate or picric acid preserves essentially all 3H-1-norepinephrine (NE) label in isolated sympathetic nerve vesicles (750 ) throughout preparation for electron microscopy. Cacodylate and s-collidine buffers increase the tendency to form contracted dense cores surrounded by a more translucid zone in vesicles, when compared to potassium phosphate buffer which maintains a homogeneous, dense, fine granular vesicle matrix. The typical contracted dense core appearance in nerve vesicles is considered to be an artifact. The loss of sedimentable NE content from vesicles under depleting conditions is roughly paralleled by a concurrent loss of dense staining matrix material. This process is at least partly reversible. The matrix density in vesicles is judged to approximate semi-quantitatively the NE content under the conditions tested, but all staining reactions appear to be indicative of the amount of complexing substance present, rather than of NE per se.

Highly purified splenic nerve vesicles: Early post-mortem effects on ultrastructure

SummaryElectron microscopic analyses of whole pellet depths confirm the claim that large dense-core vesicles (750Å) can be isolated from bovine splenic nerve at a routine purity of 80–90%. After a minimal 10 min post-mortem delay at the slaughterhouse, essentially all vesicles in cold control preparations possess a homogeneous, finely granular, electron-dense matrix. This appearance is maintained after brief incubation with ATP at 30 °C, even though a newly discovered ATP-insensitive norepinephrine (NE) pool (20%) is rapidly and completely lost. Subsequent depletion of the remaining NE (80%) in the slower ATP-sensitive pool is paralleled by proportional decrements in vesicle matrix density.In contrast, cold control vesicles in our earlier preparation with a 20–30 min post-mortem delay appear relatively depleted, but gain electron density after brief incubation with ATP at 30 °C. This effect can be duplicated in the present preparation by several procedures designed to mimic the additional post-mortem delay.Ultrastructural events associated with NE depletion and vesicle degeneration begin with random dense granulation of the original finely granular matrix, followed by aggregation to form very electron-dense 200Å granules. The latter occur intravesicularly at first, but are released in increasing numbers as vesicles swell and rupture. Clusters of the 200Å granules give the appearance of dense cores both intra- and extravesicularly, particularly after certain fixation and staining procedures.

Are NPY and enkephalins costored in the same noradrenergic neurons and vesicles

Separate studies show that NPY and enkephalins are widely distributed in peripheral noradrenergic neurons. In the present study, the subcellular costorage and release in response to intense sympathetic stimulation and reserpine at near therapeutic doses (0.05 mg/kg every other day) were examined. In young pig arteries and vas deferens, enkephalin and D beta H immunofluorescence show consistent but not total overlap. Also NPY is colocalized with D beta H in many fibers but with VIP (nonnoradrenergic) in others. Ultrastructural immunogold labeling indicates that individual terminals contain large dense cored vesicles (LDVs) which store either NPY or enkephalins, even though costorage of both peptides occurs. Some LDVs costore NPY and VIP, especially in the middle cerebral artery and in the lamina propria of vas deferens. Acute CNS ischemia depletes enkephalins and norepinephrine in all tissues analyzed without parallel loss of NPY. Reserpine depletes norepinephrine 70-85% but does not deplete NPY or enkephalins. The latter is in contrast to commonly used high doses known to produce nonspecific, detergent-like effects. In fact, low doses of reserpine induce a time-dependent new synthesis and processing of NPY precursor peptides in vas deferns. Contrasting effects of reserpine on NPY and enkephalin contents, new synthesis and apparent processing, and a differential response to acute CNS ischemia were found in every tissue studied. Activation of precursor neuropeptide processing occurred immediately upon intense sympathetic stimulation in most tissues. Dual localization of NPY in noradrenergic and nonnoradrenergic fibers and differences in subcellular LDV storage help explain why enkephalin correlates better than NPY with norepinephrine loss in response to acute CNS ischemia. Furthermore, the costorage of NPY and enkephalins in distinct subpopulations of noradrenergic fibers, which varies according to tissue, is likely to be under separate CNS control.

Morphological effects of osmolarity on purified noradrenergic vesicles

SummaryLarge dense core vesicles (LDV) were purified from bovine splenic nerve homogenates by the sucrose-D2O density gradient method. Vesicles were subjected to a 50% increase and decrease in osmolarity from the control 330 mosmol 1−1 by adjusting sucrose or potassium phosphate buffer during pre- and/or postfixation. Control vesicles with a mean diameter of 717 Å readily swelled to ∼ 1050 Å and shrunk to ∼ 600 Å in the hypotonic and hypertonic media, respectively, with either sucrose or phosphate buffer. The dense core responded similarly but to a lesser degree. Prefixation in glutaraldehyde had little effect on vesicle sensitivity to subsequent tonicity change, nor did the fixativeper se exert an obvious osmotic effect. Thus, final vesicle size was largely determined by the OsO4 postfixation medium and principally by the vehicle rather than the fixative. In controls there was a mixture of spherical to oblate vesicles mostly filled with an electron-dense matrix. Upon swelling, more vesicles became spherical and nearly all had a prominent translucent halo between core and membrane. Upon shrinking, more vesicles became oblate, the halo was obliterated and the electron-density of the matrix increased. Frequency distributions of vesicle diameters at different tonicity clearly indicated that the diameter of LDV could overlap the 400–500 Å range characteristic of small dense core vesicles; however, there was no suggestion of a population of the latter in the purified LDV fraction. Implications are discussed concerning the biochemical and morphological identification of ‘light’ and ‘heavy’ density peaks of noradrenaline and dopamine β-hydroxylase from mixed vesicle populations and the possible relevance of changes in vesicle shape to a functional statein situ.

Lipid accumulation in mouse hepatocytes after morphine exposure.

After morphine injection lipid accumulation in mouse hepatocytes begins within 2 h and continues for 24 h when most hepatocytes are filled with lipid droplets. In spite of morphine maintenance the liver recovers as the accumulated lipids are coupled with protein and subsequently transported and released into the perisinusoidal space of Disse.

Substance P activates leukocytes and platelets in rabbit microvessels

The effect of substance P on leukocytes and platelets in rabbit skeletal muscle microvasculature was studied by intravital microscopy and electron microscopy. Local application of substance P caused vasodilatation and formation of aggregates of platelets and leukocytes in postcapillary venules with subsequent migration of leukocytes through the vessel wall. Many neutrophils were partially degranulated. Aggregate formation induced by substance P could not be prevented by autacoid antagonists. However, superfusion with calcitonin gene-related peptide prior to challenge with substance P greatly inhibited aggregate formation and leukocyte extravasation.