Effects of anoxia, hyperoxia, and salinity on neurons in the leech Nephelopsis obscura (Erpobdellidae): RNA redistribution by fluorescence histochemistry

JOURNAL

OF INVERTEBRATE

53, 93-101 (19891

PATHOLOGY

Effects of Anoxia, Hyperoxia, and Salinity on Neurons in the Leech Nephelopsis obscura (Erpobdellidae): RNA Redistribution by Fluorescence Histochemistry N. SINGHAL,**’

RAM *Division

HARVEY

of Ecology (Aquatic Ecology Paediatrics and Pathology,

B. SARNAT,?~

AND RONALD

Group), Department of Biological Universiry of Calgary. Calgary,

W. DAVIES*

Sciences and fDepartments Canada lI2N IN4

of

Received April 8, 1988; accepted June 14. 1988 The histochemical

distribution of cytoplasmic RNA in ganglion cells of the freshwater leech has been studied using the fluorochrome acridine orange as a marker of nucleic acids. Two series of experiments, employing 50 adult animals, involved changes in oxygen tension in the water and changes in salinity. Normal leech neurons exhibit finely granular orange fluorescence uniformly distributed throughout the cytoplasm, with a perinuclear ring of especially strong fluorescence. After exposure to anoxic (0% O,), hypoxic (20% O,), or hyperoxic (200% O?) conditions at 20°C for l-15 days, the orange cytoplasmic fluorescence is no longer uniformly distributed; the redistribution is generally toward the periphery, leaving the perinuclear zone without RNA fluorescence, but irregular zones of cytoplasm devoid of RNA also occur not as a gradient. Leeches exposed to salinity of, or greater than, 2.5 ppt for 15 days exhibit similar changes. These alterations are confirmed by electron microscopy. Seasonal fluctuations in oxygen tension and salinity of lake water affect the distribution and abundance of organisms. The acridine orange method provides one measure of stress to the nervous system in freshwater invertebrates that might be applicable to ecological studies as well as to metabolic studies of individual animals. Nephelopsis

obscura

k 1989 Academic KEY

WORDS:

Press. Inc.

acridine orange; anoxia; hyperoxia; hypoxia: leech: neuron; nucleic acids; RNA.

INTRODUCTION

hypoxic or even total anoxic conditions prevail at the substrate-water interface in particular (Baird et al., 1987). Less well recognized is the occurrence of short-term hyperoxia in the epilimnion during the spring and late summer because of the growth of oxygen-generating algae (Garey and Rahn, 1970). Neurons and other secretory cells may be selectively more vulnerable than other cells to changes in ambient oxygen tension, particularly if hypoxic or hyperoxic conditions are prolonged or recurrent. One cytological difference distinguishing secretory cells from other cells of the metazoan body is the abundance of cytoplasmic ribosomes, required for the continuous production of secretory products including neurotransmitters and the enzymes essential to their biosynthesis and degradation. The effects of anoxia, hypoxia, and hyperoxia on mammalian, and especially human, nervous systems are well documented

The freshwater leech Nephelopsis obAcura is a common inhabitant of ponds and lakes of North America (Davies, 1973; Sawyer, 1986), occurring in greatest numbers in Alberta (Davies et al., 1977), Alaska, and the northern United States (Klemm, 1985). The population ecology, feeding, reproduction, behavior, and physiology of N. obscura have all been investigated in Alberta (Davies and Everett, 1977; Davies et al., 1978, 1981, 1982a, b; Reynoldson and Davies, 1980; Linton et al., 1983a, b). In stratified temperate lakes, oxygen depletion occurs in the hypolimnion during summer and beneath surface ice in winter; ’ Present address: Department of Zoology, Kurukshetra University, Kurukshetra 132119, India. ’ To whom correspondence should be addressed at Alberta Children’s Hospital, 1820 Richmond Road S.W., Calgary, Alberta. Canada T2T 5C7. 93

0022-201 l/89 $1.50 Copyrghl All right,

0 1989 by Academic Prcsa. inc. of reproduction in any form reservf(l.

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(Ahdab-Barmada et al., 1986), but this topic has received less attention in aquatic invertebrates experiencing oxygen fluctuations in their environment. The detection of morphological and biochemical alterations in the nervous system of the leech in conditions of stress is important not only for understanding the metabolism and behavior of the individual animal, but also for its ecological implications in explaining the effects on aquatic fauna of seasonal variations in dissolved oxygen and salinity in ponds and lakes. This study examines the effects of oxygen concentration and salinity on cytoplasmic ribonucleic acid (RNA) in ganglion cells of N. obscure using acridine orange (AO) fluorochrome as a histochemical marker of nucleic acids. Insoluble RNA (i.e., ribosomes) is perceived in the ultraviolet microscope as a brilliant orange-red fluorescence. DNA appears luminous yellow in color, and cytoplasmic proteins are pale green. Secretory cells are rich in ribosomes and nerve cells are among the cells of the body with the highest RNA content. A0 is thus a good stain for demonstrating neurons in the vertebrate nervous system (Von Bertalanffy and Bickis, 1956; Schmued et al., 1982; Sarnat, 1985; Fabian and Sarnat, 1987). Glial cells and the glial and neuronal fibers constituting the neuropi1 of the brain show only pale greenish background fluorescence (Sarnat, 1985). An intense orange-red AO-RNA fluorescence also is seen in ganglion cells of invertebrates, similar to the appearance of vertebrate neurons (Zeigler et al., 195 1; Sarnat and Sarnat, 1987). MATERIALS

AND METHODS

Large, mature specimens of N. obscuru (300-400 mg) were collected in May and June, 1986, from Stephenson’s Pond, described by Davies et al. (1987), located 4.5 km northwest of the city of Calgary, Alberta (114”16’W, 51”9’N), within the knob and kettle topography of the prairiefoothills transition zone (Legget, 1961). Ap-

AND

DAVIES

proximately 100 specimens were transported to the laboratory where they were held in aerated fresh water (salinity S = 0.5 ppt) under dim illumination for a period of 3 weeks prior to exposure to anoxic, hypoxic, or hyperoxic conditions, or to different salinities. The total number of animals studied including control specimens was 50. At the end of a 20-day acclimation to 20°C 100% oxygen saturation, and 0.5 ppt salinity, 50 preweighed active N. obscura were exposed, in two series of experiments, to anoxic (O%), hypoxic (20%), or hyperoxic (200%) oxygen tensions, or different salinities, at 20°C for 15 days. All oxygen tension experiments were conducted in 12-cm glass tubular (2 cm in diameter) flow-through respirometers (Wrona and Davies, 1984). Oxygen concentrations were maintained by bubbling oxygen or nitrogen through the water, and dissolved oxygen was measured with a Yellow Springs Instrument Model 51A oxygen meter and Model 5739 KC1 diffusion membrane chamber. Flow rates were adjusted to provide 8 to 10 gas volume changes per hour, with carbon dioxide concentration maintained at less than 0.5 ppt. Control animals were exposed to 100% oxygen at similar flow rates in identical flow-through respirometers. For salinity experiments, the stock solution of saline water (S = 5 ppt) containing chloride, sulfate, and bicarbonate salts of sodium, potassium, calcium, and magnesium was made following standard methods (APHA 1971) and kept in plastic containers, each with six leeches in 5 liters of aerated salt water. A control group of three containers was tilled with fresh water (S = 0.5 ppt), and the other groups of three containers each had 1.25, 2.5, and 5.0 ppt salt water, respectively. Each experiment of oxygen tension and salinity was in triplicate and repeated three times and run for 15 days. The animals were not fed during these experiments. Three animals from each series of experiments were sacrificed by fixation in 10%

LEECH

CYTOPLASMIC

buffered formalin. Transverse sections of the head and abdominal segments were embedded in paraffin and cut at 10 pm. They were stained with 0.02% acridine orange solution, following a previously published protocol (Sarnat, 1985; Sarnat and Samat, 1987; Fabian and Samat, 1987). The pH of the phosphate buffer was maintained at a constant 6.0. Sections were examined in a Leitz epifluorescence microscope and photographed using Kodak 35-mm ET film for tungsten light, ASA 160. Exposure time varied from 2 to 6 set, controlled by an automatic exposure meter. After examination of the A0 fluorescence, ganglion cell-containing sections of three animals of each group were incubated for I hr in bovine pancreatic ribonuclease, restained with AO, and reexamined in the fluorescence microscope for disappearance of the orange color of RNA. Other unstained paraffin sections also were examined for natural autofluorescence. Three other animals from each set of experiments were not immersed in formalin; cryostat sections of 6 p.m were prepared after freshly freezing in isopentane (2methylbutane) cooled to - 160°C in liquid nitrogen. The A0 technique was then applied to these sections, as with the formalin-fixed, paraffin-embedded sections, for comparison. Upon completion of the A0 techniques, sections were rinsed, restained with hematoxylin-eosin, and permanently mounted. RESULTS Examination of unstained frozen and paraffin-embedded sections in the ultraviolet microscope reveals only a faint greenish fluorescence of low intensity, the nonspecific characteristic of most proteincontaining tissues. No bright yellow autofluorescent granules, such as those emitted by lipofuscin, were identified in any of the animals examined. Ganglion cells in normal control leeches, whether in segmental or cerebral ganglia or as single peripheral nerve cells within so-

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matic muscle, show uniformly bright orange cytoplasmic fluorescence when incubated with acridine orange (Figs. l-3). The intensity of the fluorescence is equally strong in large and small neurons, and the various neuronal types are not distinguished by this quality alone. The orange cytoplasmic fluorescence is generally confluent or finely granular and uniformly distributed throughout the cytoplasm. However, a ring of slightly more intense orange fluorescence is often seen surrounding the nucleus, particuiarly in large ganglion cells (Figs. 2, 3). Occasionally, concentric rings of orange fluorescence are detected between the nucleus and the limiting plasma membrane of the cell, but the zones between these rings never show complete clearing of the orange color. Rare neurons show an irregular distribution of AO-RNA fluorescence with zones of greater and lesser intensity not corresponding to either centripetal or centrifugal gradients. Orange fluorescence extends a short distance into the neuronal processes of most cells but more distal axonal segments and nerve fiber bundles exhibit only the green fluorescence of cytoplasmic proteins and membranous structures without ribosomes (Figs. 2-4). After exposure to anoxic and hypoxic conditions, ganglion cells show alterations in the distribution of orange cytoplasmic fluorescence. Intensity is perceived as reduced. The fluorescent material becomes more coarsely granular and redistributed more peripherally in the perikaryon, the perinuclear zone becoming green (Figs. 4, 5). Cytoplasmic zones cleared of orange fluorescence are irregular in contour and uneven in size. The residual orange fluorescence appears clumped or more confluent, though the margins are often diffuse and poorly demarcated. Large neurons, particularly Retzius cells, are more vulnerable to early change with mild hypoxia than are smaller neurons. Necrotic ganglion cells were seen only in animals exposed to total anoxia; these neurons completely lose their

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orange cytoplasmic fluorescence and are inconspicuous with A0 stain. The severity of alterations in individual ganglion cells and the proportion of neurons involved generally correlated with the severity of hypoxic stress. Hyperoxic conditions produce changes similar to those of hypoxia, but not as severe (Fig. 6). Necrotic and degenerating nerve cells were not found. Exposure to graded salinity produced alterations in the distribution of AO-RNA fluorescence indistinguishable from those of hypoxia or hyperoxia. At a salinity of 1.25 ppt, no changes were observed, but definite alterations were seen at 2.5 ppt (Fig. 7) and even more severe changes were found at 5.0 ppt. Digestion with bovine pancreatic ribonuclease completely abolished the orange fluorescence, when sections were restained with A0 and reexamined in the fluorescence microscope (Fig. 8), confirming that RNA is the source of the orange fluorescence in ganglion cells. The character of the AO-RNA fluorescence was fundamentally the same in freshly frozen, unfixed sections and in formalin-fixed, paraffin-embedded sections, although cytological detail was

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somewhat better defined in the paraffin sections . The redistribution and loss of cytoplasmic ribosomes disclosed by AO-RNA fluorescence in animals exposed to the various conditions of oxygen tension or sahnity, was confirmed by electron microscopy. These findings of decreased and peripheral redistribution of ribosomes, as well as other ultrastructural alterations of organelles, are discussed in detail in a separate paper (Singhal et al., 1988). DISCUSSION

Chromatolysis is a term applied to the eccentric displacement of the nucleus and redistribution of ribosomes to the periphery of the cytoplasm in mammalian neurons following axonal injury. Chromatolysis also is characteristic of hypoxic insults to the neuron, but hypoxia also decreases the total cytoplasmic RNA, unlike axotomy in which total RNA content remains stable (Edstrom and HydCn, 1943, HydCn 1962). Regeneration or recovery of neuronal function is associated with accumulation of RNA around the nucleus (Hyden, 1962). Both the centrifugal (i.e., chromatolytic) and centripetal redistributions of cytoplas-

FIG. 1. Transverse section through brain of control specimen of Nephelopsis obscuru. Strong orange AO-RNA fluorescence is seen throughout the cytoplasm of both large and small neurons, while nerve fibers appear green. Acridine orange. Bar = 10 urn. FIGS. 2 and 3. Transverse sections through brain (cerebral ganglion) of two specimens of Nephldopsis obscura. Large and small ganglion cells exhibit uniform cytoplasmic distribution of AO-RNA fluorescence that often shows a finely granular texture. An especially prominent ring of strong orange color surrounds the nucleus, particularly in the larger neurons (arrow). Acridine orange. Bar = 10 pm. FIGS. 4 and 5. Brains of two specimens of Nephelopsis obscuru exposed to anoxic conditions for 36 hr. The orange cytoplasmic fluorescence has become less intense and very unevenly redistributed as irregular aggregates in the periphery of the cytoplasm of most neurons, or irregular zones of cytoplasm are depleted of RNA fluorescence (arrows). Acridine orange. Bar = 10 pm. FIG. 6. Ganglion of Nephelopsis obscura exposed to 200% oxygen for 15 days. Redistribution of AO-RNA fluorescence is seen, with clearing of orange color from perinuclear or circumscribed peripheral zones of cytoplasm (arrows). Acridine orange. Bar = 10 pm. FIG. 7. Brain of Nephelopsis obscura exposed to 2.5 ppt salinity for 10 days. The chromatolysis or peripheral redistribution of AD-RNA fluorescence in neurons and particularly the perinuclear zone lacking orange fluorescence (arrow) is similar to that seen after hypoxic or hyperoxic stresses. Acridine orange. Bar = 10 pm. FIG. 8. Same section as Figure 7, incubated with ribonuclease for I hr and restained with AO. The disappearance of the orange color confirms that RNA was the origin of this fluorescence. Bar = 10 urn.

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mic RNA have been demonstrated in neurons of human neonates following birth asphyxia, using the acridine orange fluorochrome technique (Sarnat, 1987). The reason for redistribution is that RNA is encoded and produced in the nucleus and normally moves peripherally in the cytoplasm. Chromatolysis represents inhibition of new nuclear RNA synthesis while previously formed ribosomes become detached from the endoplasmic reticulum (i.e., degranulation) and continue to move outward; aggregation of RNA around the nuclear membrane results from a resumption or intensification of new RNA synthesis. In insects, the coarse granular basophilic Nissl bodies characteristic of mammalian motor neurons are not normally seen because ribosomes are more uniformly distributed in the cytoplasm with less tendency to become associated with stacked membranes of endoplasmic reticulum. After axonal injury of insect ganglion cells, the nucleus becomes eccentric as with mammals, and cytoplasmic RNA forms a dense band around the nucleus, as demonstrated with basic pyronine dyes (Cohen, 1967). This band or ring eventually moves peripherally. Radioautography shows that the perinuclear ring is indeed newly synthesized RNA, when tritiated uridine is injected around the ganglion 24 hr before axonal injury (Cohen, 1967). Hyperoxia produces neuronal reactions resembling those of hypoxia and even progressing to cellular necrosis in the rat (Ahdab-Barmada et al., 1986). Excessive oxygen is probably toxic to invertebrate neurons as well. The decrease in mitochondrial oxidative enzymatic activity after even mild or intermittent hypoxia Marzatice et al., 1986) is probably related to impaired RNA transcription, essential to ribosomal translation in the synthesis of enzymes and other proteins. Our studies provide evidence that the leech ganglion cell does not differ fundamentally from the mammalian neuron in its vulnerability to impaired RNA synthesis induced by

AND

DAVIES

changes in the availability of oxygen and increased salinity. Many of the identified neurons of leeches are defined electrophysiologically and in terms of specific transmitters secreted (Belanger and Orchard, 1986; Gerasimov, 1967; Nicholls and Baylor, 1968; Walker, 1967). Each segmental ganglion in leeches contains about 350 nerve cells, including a pair of large octopaminergic Leydig cells and a pair of giant serotoninergic Retzius cells whose long axons extend peripherally to the somatic musculature that they inhibit. It is not known whether simple amino acids such as aspartic acid and glutamic acid mediate neurotransmission in annelids as they do in vertebrates. Such amino acidsecreting neurons in the brain of newts (Turicha torosa and Ambystoma tigrinium) show much less AO-RNA fluorescence than do neurons synthesizing acetylcholine, monoamines, serotonin, or neuropeptides (Fabian and Sarnat, 1987). The A0 method we employed, and particularly the incubation pH of 6.0 rather than the more traditional pH 2 to 4, permits visualization of insoluble (i.e., ribosomal and nucleolar) RNA. While nerve fibers contain a considerable amount of soluble and mitochondrial RNAs, they lack ribosomes and only the nonspecific green fluorescence of proteins is seen in axoplasm, while orange fluorescence is intense in the cytoplasm of the neuronal cell body. The validity of the A0 method in nerve cells of the leech exposed to hypoxic and hyperoxic conditions is confirmed by ultrastructural studies of ribosomal redistribution (Singhal et al., 1988). An additional subjective change noted in association with the redistribution of AORNA fluorescence after exposure to anoxic conditions in particular was that the intensity of the fluorescence was perceived to be diminished. However, quantitation of such phenomena is difficult in histochemical preparations of any type because of nonuniform distribution within the microscopic field and many technical variables that

LEECH

CYTOPLASMIC

must be controlled. Fluorescence histochemistry poses the additional problem of photodecomposition or decay of the fluorescence upon continued exposure to ultraviolet light. Exposure times of less than 30 set do not produce quenching of A0 fluorescence detectable to the human eye (Sarnat, 1985). Special quantitative techniques, such as flow cytometry, are developed to address the problem of measuring fluorescence in individual cells, but were not employed’in the present study, and we are therefore reluctant to draw conclusions regarding intensity of fluorescence, except for noting its total loss in degenerated or necrotic cells. Focal infarction or necrosis of tissue is common in vertebrates and usually results from ischemia in the territory of a particular blood vessel, at times associated with hypoxia. Such infarcts are rare among invertebrates because of a more open vascular system and increased potential for oxygen transport to tissues through the integument (Sparks, 1972). However, selective vulnerability or selective resistance of various tissues to changes in oxygen tension occur in both vertebrates and invertebrates because of metabolic differences. Additional observations in our AO-RNA studies of N. obscura exposed to hypoxia indicate that nerve cells are more vulnerable than are most other cells of the body, including intestinal epithelium, integument, and gonadal cells. The characteristic histological appearance of coagulation necrosis in vertebrates is similar in the oyster Crassotrea gigas (Sparks and Pauley, 1964). Cytological details of cell death are generally more prominent in the nucleus than in the cytoplasm: nuclear chromatin condenses and the nucleus shrinks (pyknosis), chromatin ruptures into the cytoplasm (karyorhexis), or there is loss of basophilic staining and disappearance of chromatin without rupture of the nuclear membrane (karyolysis). Cytoplasm, particularly of neurons and other secretory cells, becomes acidophilic and

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more amorphous, but these changes are subtle (Sparks, 1972). The A0 technique allows another means of studying cytoplasmic changes during degenerative processes by demonstrating loss and redistribution of RNA. Lipofuscin pigment increases in mammalian neurons with aging and is highly autofluorescent (Sarnat, 1968). There are at least six ultrastructurally distinct forms of neuronal lipofuscin in the human brain (Boellaard and Schlote, 1986). This pigment also occurs in many invertebrates, including insects (Miquel et al., 1974; Sohal et al., 1984) and turbellarians (H. B. Sarnat and F. J. Wrona, unpublished data). Our inability to demonstrate autofluorescent lipfuscin granules in N. obscura may be due to its dispersal as a more diffuse form, or because the cytoplasm does not contain remnants of degenerating membranous material during the physiological turnover of organelles (Fawcett, 1966). Knowledge of RNA redistribution in the cytoplasm of leech neurons exposed to varying salinities is of ecological importance in assessing the distribution of a species and its impact on ecosystems. Some of the secretory cells of the tegmentum are found to function as osmoregulators under neural control (Sawyer, 1986). One explanation for the reduction in AO-RNA fluorescence with increasing salinity is that the leech is dependent upon endogenous supplies of energy for metabolic processes, and an osmotic pressure of the external medium exceeding a critical threshold requires the apportioning of larger amounts of energy to maintain the ionic and osmotic regulatory equilibrium, thereby limiting energy available for other functions. Our study implies that decreased synthesis of ribosomes may be one such sacrificed function of the nerve cell. The similar responses of ganglion cells to a variety of different stresses of oxygen tension and salinity further suggests that neurons of leeches are capable of only a limited number of reactions to diverse merabolic insults.

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We conclude that the acridine orange fluorochromic method is a sensitive technique for demonstrating the distribution of cytoplasmic RNA in ganglion cells of the leech. Further studies are in progress to relate the various identified nerve cells to sensitivity of inhibition of RNA transcription, protein synthesis, and chromatolytic change after exposure to anoxic or hyperoxic conditions. AO-stained sections may be rinsed and then restained with specific immunoperoxidases to identify neurotransmitters and their associated enzymes. The functional and metabolic integrity of the nervous system is assessed in the individual animal by the A0 technique, and an ecological impact of environmental factors on freshwater invertebrates also may be inferred. ACKNOWLEDGMENTS We thank Linda Hines, Yvonne Smink, Pauline Orton, Cathy Jackson, and Ibolyn Gedeon for their technical assistance. The manuscript was typed by Barbara Cooper. This work was supported by a research grant to H.B.S. from the Alberta Children’s Hospital Foundation and to R.W.D. from the Natural Science and Engineering Council of Canada.

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