Accumulation and identification of lipofuscin-like pigment in the neurons of Bulla gouldiana (gastropoda: opisthobranchia)

Mechanisms of Ageing and Development, 7 (1978) 53--64 © Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

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ACCUMULATION AND IDENTIFICATION OF LIPOFUSCIN-LIKE PIGMENT IN THE NEURONS OF BULLA GOULDIANA (GASTROPODA: OPISTHOBRANCHIA)

L A U R A J. ROBLES* Department of Biological Science, California State College, Dominguez Hills, Dominguez Hills, California 90747 fU.S.A.) and Department of Biological Sciences, University of CaliJbrnia, Santa Barbara, Santa Barbara, California 93106 (U.S.A.)

(Received March 22, 1977;in revised form May 8, 1977)

SUMMARY A few reports suggest that pigmented granules found in molluscan neurons accumulate with age as do lipofuscin granules in vertebrate cells; however, no reports on molluscan neurons include detailed descriptions of granule accumulation or histochemical tests to identify the pigment as lipofuscin-like. In this study light microscope observations of living ganglia from 1.7, 2.7, and 3.0 cm and larger (shell length) sized Bulla gouldiana showed an increasing accumulation of orange-red pigment in the perikaryon corresponding to increasing shell size (i.e. age). With the electron microscope similar results were obtained, and lipofuscin-like granules were seen in the nerve cell cytoplasm of veliger larvae and in all adult sized Bulla. Staining with Sudan black B, Nile blue, chrome alum hematoxylin, PAS reagents, and exposure of the neurons to u.v. light to observe subsequent autofluorescence, yielded positive results in the areas of pigmented granule accumulation. Thus, the brillant orange-red granules that accumulate with age in the peripheral cytoplasm of adult Bulla neurons, and which are probably also present in larval stages, chemically resemble the lipofuscin granules of vertebrates. Similarities and differences between molluscan pigmented granules and vertebrate lipofuscin granules, in relation to structure and mechanisms of development and accumulation, are discussed.

INTRODUCTION

Lipofuscin pigment granules accumulate in a variety of aging vertebrate cells including neurons [1-16]. Among the molluscs, pigmented granules also occur in nerve

*Present address: Department of Biological Science, California State College, Dominguez Hills, 1000 East Victoria Street, Dominguez Hills, California 90747 (U.S.A.).

54 cells and the morphology and formation of these granules have been described in several gastropods [17-28]. The morphological similarity between gastropod pigmented granules and vertebrate lipofuscin granules, as well as the accumulation of the pigmented granules in larger (i.e. older) gastropods, have been suggested [22, 26, 29]. In addition, the localization of acid phosphatase by electron microscopy shows that both the vertebrate lipofuscin and gastropod pigmented granules are of possible lysosomal origin [7, 14, 27, 28, 30-39]. Although similar to vertebrate lipofuscin granules in morphology and origin, the pigmented granules in molluscan neurons have not been chemically shown to contain a lipofuscin-like pigment and descriptions of the accumulation of these granules within neuronal cell bodies are lacking. In this study histochemical tests and u.v. light absorption were used to identify lipofuscin pigment and light and electron microscope observations were employed to show the accumulation of pigment containing granules in the neurons of different aged Bulla gouMiana; the results of these studies indicate that the pigmented granules found in Bulla neurons have chemical properties similar to vertebrate lipofuscin granules and that molluscan granules also accumulate with age. Possible mechanisms for the development and accumulation of this pigment within Bulla neurons are suggested.

MATERIALS AND METHODS

Bulla gouldiana were collected and maintained in the laboratory as previously described [28]. Light microscope observations of whole living ganglia were made with a Zeiss Universal Research microscope. For electron microscopy, ganglia from different sized Bulla (1.7, 2.7, and 3.0 cm and larger in shell lengths) and veliger larvae contained within egg-strings deposited in laboratory aquaria were fixed by immersion for 1-2 hours in 3% glutaraldehyde buffered in 0.1 M sodium cacodylate, pH 7.4. The tissues were then postfixed for one hour in 2% buffered OsO4 followed by dehydration in a graded ethanol series. Next, the ganglia were embedded in Araldite, sectioned, stained, and examined in a Siemens Elmiskop IA or a Phillips EM 300 [28]. Light microscopic stains including Sudan black B, alternative Nile blue, chrome alum hematoxylin, and PAS reagents were used to identify lipofuscin pigment. Ganglia fixed in 10% formalin in sea water and unfixed ganglia were stained in a saturated solution of Sudan black B in 70% ethanol for 15 rain [40], rinsed in distilled water and examined. Sections (10 /2m) from ganglia fixed in 10% formalin in sea water and embedded in paraffin were stained with Nile blue and chrome alum hematoxylin [41]. The PAS reaction [42] was performed on 1 ~m sections of tissue fixed and embedded for electron microscopy [28]. To test for lipofuscin autofluorescence, whole ganglia were fixed in 10% formalin in sea water for two days, frozen and cut into 10 ~m sections in an Ames Lab-Tek cryostat, and the frozen sections were examined immediately in a Zeiss fluorescent microscope (Osram HBO-200 light source, BG 38, BG 12, and UG 5 filters) [9, 12].

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Fig. 1. Lipofuscin-like granule (arrow) in developing ganglion (g) of a veliger larvae. X 18,700. RESULTS

Pigmented granule accumulation Ganglia from veliger larvae of Bulla gouldiana examined by electron microscopy (EM) show occasional large granules in the developing neuronal cytoplasm which resemble the granules seen in adult neurons (Figs. 1, 3 and 5). When examined by light microscopy, the ganglia of 1.7 cm Bulla appear light orange in color, with the pigment concentrated around the cell periphery (Fig. 2). When examined by EM, these neurons are found to have granules in the cell periphery corresponding in position to the orangered pigment seen in living ganglia (Fig. 3). In 2.7 cm and larger Bulla the neurons are deep red and more pigment is concentrated in the cell periphery, forming a cap-like structure (Fig. 4). Electron microscopic observations of 3 cm and larger Bulla reveal accumulations of the presumed pigment-containing granules in the neuronal cytoplasm (Fig. 5). Lipofuscin identification Staining procedures used in the identification of lipofuscin pigment in Bulla neurons all yield positive results. With Sudan black B only the areas in the neuronal

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Fig. 2. Light micrograph of a living ganglion of a 1.7 cm Bulla showing pigment (arrow) contained in neuronal cell bodies. The pigment is predominately located in the cell periphery, x 750. cytoplasm containing accumulations of pigmented granules stain black. Also, the pigmented granules stain green with Nile blue, blue black with chrome alum hematoxylin, and red with PAS reagents. When excited with u.v. light, the areas of Bulla neurons containing the orange-red pigment fluoresce yellow (Fig. 6).

DISCUSSION The results o f this study indicate that the pigmented granules in Bulla neurons accumulate with age, stain with various chemical agents, and exhibit autofluorescent

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Fig. 3. Electron micrograph of a neuron from a 1.7 cm Bulla. A few pigmented granules (arrows) are seen in the neuronal cytoplasm. × 21,250.

properties as do lipofuscin granules found in aging vertebrate cells [ 1-16, 30, 31, 35, 38, 40, 46]. Based on these common characteristics, the pigmented granules in the neurons of Bulla gouldiana may be described as lipofuscin-like. Aside from these characteristics, molluscan pigmented granules and vertebrate lipofuscin granules have other similarities, and differences, in both structure and mechanism of development and accumulation of the mature granule. In mice, Sekhon and Maxwell [14] described the appearance of various granular structures within the cytoplasm of aging neurons; L1 granules, primary lysosome-like structures, and I-,2 granules, autophagic vacuoles or multivesicular bodies, were the earliest types of granules present in the neurons. Within both of these granule types, signs of

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Fig. 4. Light micrograph of a living ganglion from a 2.7 cm Bulla. Note dense pigmentation in cell periphery (arrows). × 300.

pigmentation included the appearance of bands, dense granules, and vacuoles. The L3 granules, or mature lipofuscin granules, were prevalent in aged mice and were characterized as being larger, irregular in outline, and as containing numerous bands, granular material, and large, peripherially located vacuoles. Similar appearing mature granules have been described repeatedly in neurons and other vertebrate cell types [47, 48]. Sekhon and Maxwell [14] concluded that the lysosome-like Lx and L2 granules in mice gave rise to L3 granules as a result of gradual structural and chemical alterations over time. Other reports have demonstrated acid phosphatase activity in structures resembling the L~, L2, and L3 granules in various cell types [7, 12, 30, 31, 37, 38]. These results, therefore, suggest that, because of their acid hydrolase content and lysosome-like morphology,

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Fig. 5. Electron micrograph of the accumulation of lipofuscin-like pigmented granules in a neuron of the cerebral ganglion (3.0 cm Bulla). vertebrate lipofuscin granules may be considered as lysosomes of the residual body variety [7, 16]. The lysosomal origin and mechanism of development of the pigmented, lipofuscinlike granules found in Bulla neurons is similar to that described for vertebrate lipofuscin bodies [28]. As in vertebrates, small, dense, uniformly granular bodies are apparent in the neuronal cytoplasm and some of these dense lysosome-like granules contain lamellar and lipid inclusions. In addition, multivesicular bodies are numerous and contain vesicular and lamellar substances suggesting an autophagic origin [28]. The large pigmented granules found in all adult Bulla neurons, whose accumulation a n d chemical characteristics are described in this study, are circular, ovoid or more irregular in outline, and contain dense lipid, granular and lamellar materials, and vesicular inclusions. Electron microscope

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Fig. 6. Light micrograph of a fluorescing neuron in the visceral ganglion of Bulla gouldiana. Fluorescing region occurs in area of pigmented granule accumulation (arrow). × 1200. enzyme localizations in Bulla neurons also show the small dense bodies, multivesicular bodies and the larger, mature pigmented granules to be acid phosphatase positive [28]. Intermediate sized granules with varying amounts of inclusions are also acid phosphatase positive; therefore, it seems reasonable to suggest that the pigmented, lipofuscin-like granules in Bulla neurons arise from the gradual alteration of lysosomal structures as do lipofuscin granules of vertebrate cells. The mechanism of accumulation of the pigmented granules in Bulla neurons, and thus the accumulation of lipofuscin pigment within the neuronal perikaria, is probably also similar to the mechanism in vertebrates. Mature vertebrate neurons are post-mitotic [49] and residual or lipofuscin bodies formed during normal autophagic processes [14] are not diluted between successive generations of daughter cells, but instead, accumulate within the cytoplasm. Mature invertebrate neurons are also post-mitotic [49] and in molluscs, the nerve cells increase in both cell body and nuclear size with age [26]. This study and previous studies [28] demonstrate that normal autophagic processes also occur in molluscan neurons, resulting in the accumulation of pigmented, lipofuscin-like granules within the cytoplasm. Additional factors probably also contribute to the rate of pigment formation and granule accumulation in both vertebrate and invertebrate neurons, including the ability of the lysosomal enzymes to degrade intracellular membranes, the rate of peroxidation of lipid substances within the lysosomes, exocytosis of lysosomal contents, and cell damage with age leading to increased autophagocytosis and to accumulation of lipofuscin granules within the perikaria [ 16]. In addition to the above similarities in their mechanisms of development and accumulation with the neuronal cytoplasm, lipofuscin-like granules in Bulla neurons and

61 vertebrate lipofuscin granules differ in several ways including the morphology of the mature granule and the color of the contained pigment. The large pigmented granules in Bulla neurons are not as irregular in outline and do not have large peripherially located vesicles as do vertebrate lipofuscin granules [14, 28, 47, 48]. Also, the color of the pigmented granules in Bulla neurons is orange-red compared to the usual yellow to brown color of vertebrate lipofuscin [41]. The bright red pigmentation of Bulla granules could arise from the presence of carotenoids [28, 41] or could be due to a lipofuscin-like pigment that is chemically different from that found in vertebrates. The pigmented lipofuscin-like granules in molluscan neurons may have several roles in the functioning of the nerve cells, although the influence of accumulated lipofuscin on vertebrate cell physiology is poorly understood [16]. Herkart [50] demonstrated a light induced change in the ultrastructure of pigmented granules in Aplysia neurons. In darkness, the material contained within these structures was granular in appearance, but upon illumination, rearranged into membrane-like lamellae. Brown and Brown [51, 52] and Brown et al. [53] confirmed this morphological change in the granules and also showed that the granules stored Ca 2÷. Upon illumination this Ca 2÷ was released from the granules and presumably mediated the increased conductance of the cell membrane to K ÷, resulting in membrane hyperpolarization. This hyperpolarization was best elicited at 490 nm, and could involve a carotenoid pigment [52]. Bulla neurons also hyperpolarize when illuminated, and preliminary results seem to indicate that pigmented granules are possible sites of Ca 2÷ storage (Robles and Fisher, in preparation). Pigmented granules in molluscan neurons may therefore function as a type of primitive photoreceptor. It is possible that the lipofuscin could participate in the formation of membranous whorls within the granules and upon illumination provide a membrane surface into which the chromophore, a carotenoid, could reside. The relationships between the lipofuscin, carotenoid pigment and Ca 2÷ within the granules are unknown but could be analogous to the arrangement of similar molecules in the outer segments of vertebrate photoreceptors. The pigment contained in the lipofuscin granules of molluscs may also assume the role of an electron acceptor during periods of anoxia to maintain energy production [44, 54-57]. The redox potential in the neurons of Anodonta has been measured under anaerobic conditions [56] and results suggest that a special electron acceptor present in the ganglia prevents a rapid decrease in the redox potential even after total exhaustion of the oxygen content of the incubation medium. In addition, this substance is present in the pigmented granules [56]. The granules of Anodonta also show an ultra-structural alteration with mass proliferation of membranes inside the cytochromes (pigmented granules) during anoxic periods, an event which may be of importance in electron transport and the arrangement of enzymes necessary for this function [57]. High acid phosphatase activity exhibited in the granules during these conditions may be necessary for the liberation of substances within the granules in order to form new membranes. Data also show that species of molluscs with good anoxibiotic tolerance contain numerous and large pigmented granules [55] and since Bulla gouldiana inhabits mudflat areas and is often found burrowed beneath the surface of the mud, an electron acceptor other than oxygen may be advantageous [58].

62 This s t u d y suggests h i s t o c h e m i c a l and time d e p e n d e n t similarities b e t w e e n the pigmented, vertebrate

lipofuscin-like granules in Bulla n e u r o n s and t h e lipofuscin granules o f tissues. Bulla

neurons

may

be useful in f u r t h e r e l u c i d a t i n g the origin,

a c c u m u l a t i o n , a n d i n f l u e n c e o f lipofuscin-like granules o n n e r v o u s tissue.

ACKNOWLEDGEMENTS T h e a u t h o r wishes to t h a n k Dr. S. K. Fisher for his assistance and Dr. J a m e s C r o n s h a w for t h e use o f his EM facilities. T h i s investigation was s u p p o r t e d b y USPHS g r a n t E Y 0 0 8 8 8 to Dr. S. K. Fisher at UC S a n t a Barbara.

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