Aeta neuropath. (Berl.) 31, 91 --96 (1975) 9 by Springer-Verlag 1975 O r i g i n a l a r b e i t e n 9 O r i g i n a l I n v e s t i g a t i o n s 9 Travauoe o r i g i n a u x

The Large Dense Core Vesicle: A Normal Organelle of the Central Nervous System Myelinated Axon* Frederick H. H a r v e y The Laboratories of Pathology and Neuropathology, University of Washington, Seattle, Washington Received May 30, 1974; Accepted November 1, 1974

Summary. Large dense core vesicles (LDCV) were ibund frequently enough in normal guinea pig central white matter, basis pontis and spinal cord posterior columns to be regarded as a normal organelle. A review of studies of LDCV and dense core particles (DCP), which have a different cytological localization and appear to be associated with a variety of diseases, suggests that they are the same, and hence remarkably ubiquitous. LDCV should no longer be regarded as a type of synaptie vesicle, but as an incompletely defined organelle common to many cell types. Key words: Dense Core Vesicles -- Organelles -- Electronic Microscopy. Introduction

Dense core vesicles (DCV) with mean diameters of roughly 50, 100 and 200 nanometers (nm) have been reported in the peripheral (Pellegrino De Iraldi and De Robertis, 1968), autonomic (Grillo and Palay), and central nervous system (CNS) (Peters et al.) of normal vertebrates. This report is concerned with the DCV with a mean diameter around 90 nm, the so-called large DCV (LDCV). L D C V have been reported in CNS neuronal perikarya of practically all neurons (Peters et al.) ; nerve endings (Duncan and Morales; F u x e et al. ; H6kfelt; Peters et al.); unmyelinated axons of the h y p o t h a l a m u s (Pellegrino De Iraldi et al., 1963), area postrema (Ishii and Nakamura), and l a m p r e y spinal cord (Smith et al.). T h e y have also been reported in peripheral myelinated and unmyelinated axons (Pellegrino De Iraldi a n d De Robertis) a n d in neuronal processes of the autonomic nervous system (Banks et al. ; Pick; Peters et al. ; Grillo and Palay). Although illustrated in optic nerve paranodal axoplasm b y Peters et al. LDOV have not been reported in CNS myelinated axons. This report describes L D C V in myelinated axons of normal guinea pig cerebral central white m a t t e r (CWM), cervical spinal cord posterior columns (PC) and basis pontis (BP). Since they/occurred relatively frequently in these r a n d o m l y selected sites t h e y are considered a normal organelle of the CNS myelinated axon. Their close resemblance to DCP, which are usually described in pathological states, is stressed. * Presented at the 71st Annual Joint Meeting of the American Association of Neuropathologists, American Association of Pathologists and Bacteriologists, and the Pediatric Pathology CIub, March 9, 1974, San Francisco, California. This research was supported in part by NIH GM-00100-14 and MINDS 5-t~01-NS03147-14. 7 Actaneuropath. (Berl.) Bd. 31

92

F.H. Harvey

Methods Eight random bred adult male (4) and female (4) guinea pigs (800 g) were anesthetized with 40 mg/kg sodium nembutal (tip.), and perfused for electron microscopy (EM) through either the ascending aorta or an internal jugular vein with 4--50/0 phosphate buffered glutaraldehyde (pit 7.4). Aortic (2 animals) : following induction of anesthesia a tracheostomy was performed and after opening the chest intermittent positive pressure respiration was maintained; the ascending aorta was cannulated and perfused in a standard manner (see below) with 5~ glutaraldehyde. Jugular (6 animals) : after induction of anesthesia a midline incision over the trachea was made and one internal jugular vein was cannulated and perfused with either 4o/0 (5 animals) or 5O/o (1 animal) glutaraldehyde. All animals were perfused from a height of 4 feet for 30--60 rain (300--400 cm3 of fixative) After perfusion tissue from CWM, BP and PC was removed, minced, washed with 7.5~ sucrose buffered with 0.2 M sodium eaeodylate and 1 M NaOH to pH 7.4, placed in buffered 1~ osmium tetroxide for 1 hr, dehydrated and embedded in epoxy resin (epon 812, Shell). Thin sections were cut with diamond knives, picked up on copper grids coated with carbon and parlodion (a purified pyroxylin: Mallinckrodt), stained with uranyl acetate and lead tartrate, and viewed in an Associated Electrical Industries, Ltd. 801 electron microscope. In one animal en bloc staining with 2% uranyl acetate (1 hr) was performed. 2--3 sections were made from each block and after the first LDCV was serendipidously encountered the original sections or photographs therefrom were studied in order to establish a rough incidence of ~ LDCV/section.

Results LDCV, defined as spheroidal bodies composed of a n osmiophilic spheroidal core separated from a n external limiting u n i t m e m b r a n e b y a narrow u n s t a i n e d space or shell, were initially f o u n d serendipidously i n CNS m y e l i n a t e d axons i n a s t u d y p u r p o r t i n g to produce b e t t e r white m a t t e r fixation. Sections (2--3/block) or photographs were t h e n searched for a d d i t i o n a l LDCV i n order to arrive a t a rough estimate of their incidence per section. I t is emphasized t h a t to find LDCV one m u s t scan a t 2 0 0 0 0 • although t h e y can be f o u n d i n p r i n t s from 7 5 0 0 •

negatives. 25 LDCV were found in 13 blocks from the 8 guinea pigs and 4 blocks were negative. It is not thought that an exact measurement of the frequency of LDCV (~/axon or 4~/unit length axon) can be extracted from this data, however from 1--6 LDCV/positive section were found. Breaking the incidence down by region: 19 LDCV were found in 8 blocks of CWM, i LDCV from 1 block of BP, and 5 LDCV from 3 blocks of PC giving a rough incidence of 2 LDCV/positive section regardless of the a n a t o m i c site sampled. The incidence was n o t influenced b y the m e t h o d of perfusion. Table 1 shows the dimensions of the LDCV encountered. T h e y are usually spherical (Figs. 1 a n d 2), however 6 were 10--30~ non-spherical. The outer m e m b r a n e has a u n i t m e m b r a n e structure w h e n "en bloc" staining with u r a n y l acetate is performed (Fig.4), otherwise it appears as a single osmiophilic line (Fig. 3).

Fig.3. LDCV from a myelinated axon stained routinely; • 200000, bar represents 50 nm Fig, 4. LDCV from a myelinated axon en bloc stained with uranyl acetate prior to osmication; • 200 000, bar ~epresents 50 nm

Dense Core Vesicle

93

Fig. 1. Two LDCV's (large arrows) within adjacent myelinated (My) axons (Ax); mitochondria (Mit); • 143000, bar represents 100 n m Fig. 2. Two LDCV's in myelinated axons; • 213500, bar represents 1000 n m 7*

94

F. H. Harvey Table 1. LDCV dimensions (nm)

Vesicle diameter a, a Core diameter a Membrane thickness Space (peri-corc) thickness

Mean

Range

@ LDCV measured

91.6 59 7.1 7.2

67-- 140 33-- 113.5 6.3-- 8.3 6.3-- 7.7

25 22 b 3c 3e

a 6 LDCV cores were 10--30~ non-spherical and the mean diameter was used. b 4 LDCV cores had indistinct margins and were not measured. c Measurements were restricted to LDCV with magnifications of 60000--150000x (negative). d There was no significant difference (1~ between vesicle or core diameters of 5 highly magnified LDCV (20000--150000• ) compared with 20 LDCV at 7500x. Table 2. Conditions associated with DCP Acute necrotizing encephalitis (Bouteille et al., 1967) Astrocytoma (Gonatas et al.) Diffuse degeneration of unknown cause (Gonatas et al.) Diffuse disseminated sclerosis (Andrews and Andrews) Jakob-Creutzfeldt disease (Bots et al.) Metachromatie leucodystrophy (Gonatas et al.) Normal and reactive rat astrocytes (Andrews and Vernand) Progressive supranuclear palsy (Powell et al.) Schilder's disease (Suzuki and Grover; Bouteille et al., 1968; Bouteille et al., 1966; Elizan et al.) Schilder's disease with spongioblastoma (Boyazis et al.) Subacute sclerosing encephalitis (Gonatas and Shy; Telez-Nagel et al. ; Gonatas et al.) Tay-Sachs disease (Gonatas et al.) Table 3. Cytologic localization of DCP Astrocytes (Suzuki and Grover; Andrews; Andrews and Vernand; Gonatas et al.; Andrews and Andrews; Gonatas and Shy; Bouteille et al., 1967; Bouteille et al., 1968; Bots et al.; Boutcille et al., 1966) Axons (Gonatas et al. ; Bouteille et al., 1968) Mononuclear phagocytes (Gonatas et al.) Neuronal perikarya (Bouteille et al., 1968 ; Powell et al.) Synaptic endings (Bouteille et al., 1968) Unidentified cells (Elizan et al.) Unidentified cell processes (Gonatas et al.)

Discussion Since LDCV were e n c o u n t e r e d relatively f r e q u e n t l y in m y e l i n a t e d CNS axons i n 8 n o r m a l guinea pigs t h e y are i n t e r p r e t e d as being a n o r m a l organelle. I t is likely t h a t t h e y exist in m y e l i n a t e d axons t h r o u g h o u t the CNS a n d in other vertebrates. W h e t h e r the LDCV represent a smaller p o p u l a t i o n of lysosomes, p r o t e i n b o u n d lipid inclusions, neurosecretory or n e u r o t r a n s m i t t e r material, are derived from microtubules (Pellegrino De I r a l d i a n d De Robertis) etc. is a moot question (Peters et al.). Their significance i n other locations has been considered i n

Dense Core Vesicle

95

several studies with inconclusive results (Fuxe et al. ; I-I6kfelt; Pellegrino De Iraldi and Eteheverry). The m e m b r a n e thickness of these L D C V corresponds to t h a t of the smooth surfaced c y t o m e m b r a n e s (Sj6strand) and this could mean L D C V are derived from the Golgi apparatus. Logically (and morphologically) L D C V in myelinated CNS axons are related to those in CNS nerve endings, where t h e y are p r o b a b l y more amenable to further study, however LDCV (of identical morphology) m a y have different functions in different sites. A morphologically identical b o d y (but see Andrews and Andrews) called a dense core particle (DCP) has been found predominantly in astrocytes (Table 3) and usually in pathological h u m a n tissue. The underlying disease states are diverse (Table 2) and the relationship between the D C P and these various diseases is unknown. Although D C P are usually associated with astrocytes in h u m a n pathological conditions, and L D C V with neurons (and their processes) in normal animals, it is a reasonable hypothesis t h a t t h e y are the same. Hence it would be likely t h a t t h e y would have shared cellular functions such as intracellular digestion rather t h a n those related to neurotransmission. These processes might be accelerated in certain disease states, especially in astrocytes, offering an explanation for the enigmatic difference between where L D C V and D C P are usually found. Certain viruses (MeCraeken and Dow; Andrews and Andrews) closely resemble L D C V and perhaps this report will aid those interested in EM detection of viruses in distinguishing t h e m from these normal organelles. McCraeken and Dow state t h a t since Aujesky's virus, a herpes virus, and L D C V in peripheral nerve can be morphologieMly identical one m u s t d o c u m e n t budding before identifying a viral agent. Aclcnowledgement. The author gratefully acknowleges the skillful assistance of the technicians of the Electron Microscope Laboratory, Department of Pathology, University of Washington, Seattle, and of Johsel Namkung for the photographic illustrations. References Andrews, J. M.: The ultrastructtlral neuropathology of multiple sclerosis, 23--52. In: Multiple sclerosis immunology, virology and ultrastrueture, F. Wolfgram, G. W. Ellison, J. G. Stevens and J. M. Andrews, (Eds.). New York: Academic Press 1962 Andrews, J. M., Andrews, M. A. : The significance of dense core particles in subacute demyelinating disease in an adult. Lab. Invest. 28, 236--243 (1973) Andrews, J. M., Vernand, R. L. : Basic concepts of ultrastructural anatomy. Bull. Los Angeles neurol. Soc. 36, 131 --155 (1971) Banks, P., Mayor, D., Tomlinson, I). R.: Further evidence for the involvement of microtubules in the intra-axonal movement of noradrenMine storage granules. J. Physiol. (Lond.) 219, 755--761 (1971) Bots, G. T., Man, J. C. de, Verjaal, A. : Virus-like particles in brain tissue from two patients with Creutzfeldt-Jakob disease. Acta neuropath. (Berl.) 18, 267 -- 270 (1971 ) Bouteille, M., Guazzi, G. C., Martin, J.J., Masselin, S., ttoudart, R., Delarue, J. : Un cas d'enc6phalite pgriaxilc diffuse de Schilder : II. Etude nltrastructurale. Ann. Anat. path. 13, 55--68 (1968) Bouteille, M., Gaazzi, G. C., Masselin, S., Houdart, 1~., Delarue, J. : Particules d'aspect viral obscrv@es au microscope ~lectronique dans une biopsie c4r4brale d'enc@halite p~ri-axile diffuse de Schilder. Presse m@d. 74, 2353--2354 (1966) Bouteille, M., Vedrenne, C., Dupuy-Coin, A.M., Dclarue, J. : Inclusions intranucl~aires particulibres observ@es au microscope ~lectronique. dans un cas d'enc@halitee aigu6 n~crosante. Ann. Anat. path. 12, 403--410 (1967)

96

F . H . Harvey

Boyazis, R.M., Martin, L., Bouteille, M., Guazzi, G.C., Manacorda, A.: Images histochimiques et ultrastrncturales duns un cas de scl6rose en plaque associ@e s un spongioblastome. Riv. Pat. herr. ment. 88, 1--20 (1967) Duncan, D., Morales, 1%.: Location of large core synaptic vesicles in the dorsal gray matter of the cat and dog spinal cord. Amer. J. Anat. 186, 123--127 (1973) Elizan, T.S., Schwarty, J., Kott, E., Pedersen, B.: Schilder's Disease: virology studies concerning dense core particles. Neurology (~inneap.) 24, 428--430 (1974) :Fuxe, K., tt6kfelt, T., Nilsson, O. : A fluorescence and elcctronmicroscopic study on certain brain regions rich in monoamine terminals. Amer. J. Anat. 117, 33--46 (1965) Gonatas, N. K., Martin, J., Evangelista, I. : The osmophilic particles of astrocytes. Viruses, lipid droplets or products of secretion ? J. Neuropath. exp. Neurol. 26, 369--376 (1967) Gonatas, N. K., Shy, G. M. : Virus-like particles in subacute sclerosing encephalitis. Nature (Lond.) 208, 1338--1339 (1965) Grillo, M., Palay, S. L.: Granule-containing vesicles in the autonomic nervous system. In: Electron microscopy, S. S. Breese, Jr. (Ed.). New York: Academic Press 1963 HSkfelt, T.: On the ultrastrnctural localization of noradrenaline in the central nervous system of the rat. Z. Zellforsch., Abt. Histochem. 79, 110--117 (1967) H6kfelt, T.: The possible ultrastrnctural identification of tubero-infundibular dopamine containing nerve endings in the median eminence of the rat. Brain Res. 5, 121--123 (1967) Ishii, S., :Nakamura, I. : Electron microscopic study on the large granulated vesicles in enlarged axons of the area postrema. J. Electron Mic. 21, 85--88 (1972) McCracken, R.M., Dow, C.: An electron microscopic study of Aujesky's Disease. Acta neuropath. (Berl.) 25, 207--219 (1973) Pellegrino De Iraldi, A., De Robertis, E.: The neurotubular system of the axon and the origin of granulated and nongranulated vesicles in regenerating nerves. Z. Zellforsch., Abt. Histoehem. 87, 330--344 (1968) Pellegrino De Iraldi, A., Duggan, H. F., De P~obertis, E. : Adrenergic synaptic vesicles in the anterior hypothalamus of the rat. Anat. l%ec. 145, 521--531 (1963) Pellegrino De Iraldi, A., Etcheverry, G. J. : Ultrastrnetural changes in the nerve endings of the median eminence after nialamide-DOPA administration. Brain Res. 6, 614--618 (1967) Peters, A., Palay, S. L., Webster, H. De F. : The fine structure of the nervous system. :New York-Evanston-London: Harper and Row 1970 Pick, J. : On the submicroscopic organization of the myelinated sympathetic nerve fiber in the frog (Rana pipiens). Anat. Rec. 144, 295--325 (1962) Powell, It. C., London, G. W., Lampert, P. W. : Neurofibrillary tangles in progressive supranuclear palsy. J. :Neuropath. exp. :Neurol. 38, 98--106 (1974) Sj6strand, F. S.: Ultrastructure and function of cellular membranes, 151--210. In: The membranes, A. J. Dalton and F. Haguenau (Eds.). New York-London: Academic Press 1968 Smith, D. S., Jarlfors, U., Beranek, R. : The organization of synaptic axoplasm in the lamprey (Petromyzon marinus) central nervous system. J. Cell Biol. 46, 199--219 (1970) Suzuki, :N., Grover, W. D.: Ultrastructural and biochemical studies of Schilder's disease: I. Ultrastrncture. J. Neuropath. exp. :Neurol. 29, 392--404 (1970) Telez-Nagel, I., Hurter, D. H., Johnson, A. B., Carver, D. : Virns-like particles in subaeute sclerosing encephalitis: Electron microscopic, neurological and histochemical studies. J. Neuropath. exp. Neurol. 26, 121 (abstract) (1967) Frederick g . Harvey, M.D. P. O. BOX 2148 Santa Fe, New Mexico 87501 U.S.A.

The large dense core vesicle: a normal organelle of the central nervous system myelinated axon.

Large dense core vesicles (LDCV) were found frequently enough in normal guinea pig central white matter, basis pontis and spinal cord posterior column...
948KB Sizes 0 Downloads 0 Views