Nature of the inclusions in the lumbosacral neurons of birds

Nature of the Inclusions in the Lumbosacral Neurons of Birds’

ASOK GHOSH,a HOWARD A. BERN, IRA GHOSH2 AND

RICHARD S. NISHIOKA Department of Zoology and its Cancer Research Genetics LaboTatory, University of California, Berkeley

Sano (’54, ’55, ’58) has described as neurosecretory certain motor neurons in the lumbosacral region of the spinal cord of the chicken. The special neurons in the ventral horns contained distinct granules which stained with azocarmine, eosin, and phloxine. The inclusions proved com- pletely unreactive to a variety of histochemical tests, including methods for dem- onstrating lipids, carotenoids, “wear and tear” pigments, cholesterol and its esters, glycerides, fatty acids, and glycogen. Cur- sory observation indicated that lumbosacral cells of th,e pigeon and sparrow also contained characteristic granules. Sano called particular attention to the close association of blood capillaries with these cells, some of which showed capillaries penetrating the cytoplasm.

Tamiya et al. (’55) confirmed Sano’s observations on the chicken. Cells containing large numbers of large phloxinophil granules were predominantly present in the lumbar region of the spinal cord; in the sacral region the granules were more sparsely distributed and generally smaller. These workers found evidence for the dis- charge of the “secretory” granules through the cell surface. With the aid of the phase- contrast microscope, Takahashi et al. (’57) reported that, unlike hypothalamic neurosecretory granules, lumbosacral neuronal inclusions in the chicken did not lose their shape and stainability after al- cohol fixation. According to Fujita et al. (’57) the characteristic acidophil granules appear in the lumbosacral cells of chickens older than 15 days. The number of cells containing granules, as well as the number of granules per cell, increases with age. In 120-day-old chickens, cells containing granules were detected in every transverse

section passing through th,e lumbosacral crest of the spinal cord.

The aims of the present investigation were (i) to examine other avian species for the occurrence of lumbosacral neurons of the type described in the chicken; (ii) to relate ultrastructural and cytochemical properties of the inclusions with observations made with standard histologic tech- nics; (iii) to reconsider the reIation of age to the appearance of these granules; and (iv) to attempt to alter the number and appearance of these special neurons by chronic quasi-endocrine treatments and by acute phannacologic treatments.

MATERIALS AND METHODS

Birds. The birds used in this study were cockerels (Gallus domesticus), west- ern gulls (Larus occidentalis), parakeets (Melopsittacus undulatus) , brown towhees (Pipi lo f u s c u s ) , and silver king and white king pigeons (Columba Ziuia). The cockerels were killed at two ages: nine at 30 days and three at 120 days. The seven gulls and four parakeets were adults of both sexes; the five towhees were juveniles of both sexes. The pigeons were mainly males of several ages (numbers shown in parentheses): 35 days (5), 65 days ( 3 ) , 95 days (4), six months (3 ) , one year (3 ) , two and one-half years (5), and five years (four white kings). An additional 46 pigeons were used in the experimental series (table 1).

Cytology and cytochemistry. Birds were killed by cervical dislocation, and the lumbosacral portion of the spinal cord was dissected out and fixed immediately.

1 Aided by National Science Foundation grant

2 Present address: Department of ‘Zoology, Univer- 6-8805.

sity of Calcutta, Calcutta, India.

195

196 A. GHOSH, H. A. BERN, I. GHOSH AND R. S. NISHIOKA

For routine cytologic examination, paraffin sections of Bouin's-fixed tissues were stained with Cason's Mallory-Heidenhain combination ( GUIT, '53) and with Harris's alum-hematoxylin and eosin. Paraldehyde- fuchsin with couterstains (cf. Clark, '55) was also used on some sections. The thoracic area of the spinal cord of three two- and a half-year-old and three five-year-old pigeons was also cursorily examined.

Standard cytochemical methods em- ployed on paraffin-embedded tissues in- cluded (1) mercury bromphenol blue technic (Mazia et al., '53) after Carnoy's fixation to visualize proteins; (2) alkaline blue tetrazolium reaction (Pearse, '60) after formalin fixation for protein-bound sulfhydryl (SH) and disulfide (SS) groups; (3) Sudan black B staining (McManus, '46) for protein-bound lipoidal materials; (4) periodic acid-SchS (PAS) technic (Glegg et al., '52) for polysaccharides and related substances, with and without prior exposure to diastase for the detection of glycogen; (5) 0.05% toluidine blue solu- tions at various pHs (cf. Montagna et al., '51) for basophilic and metachromatic materials (including ribonucleic acid (RNA) and mucopolysaccharides) ; (6) Feulgen method on Carnoy-fixed material for deoxyribonucleic acid (DNA) (Pearse, '60); ( 7 ) Schmorl's method (Pearse, '60) as an indication of possible lipofuscin.

Electron-microscope studies. Four two- and a half-year-old, three five-year-old, and four drug-treated pigeons provided material for electron-microscope examination. The lumbosacral region of the vertebral column was removed, and the spinal cord was exposed from the ventral side. Pieces of spinal cord at the anterior border of the glycogen body were excised and fixed in cold (04°C) 1% osmium tetroxide in saline solution buffered between pH 7.2- 7.4 with potassium dichromate (Dalton, '55). In a few cases, veronal-acetate buffer was used. Some pieces of spinal cord from the thoracic region were also taken. The tissues were fixed for two to three hours, rinsed with buffer solution, dehy- drated in a graded series of ethanols, and embedded in n-butyl methacrylate. Sec- tions were cut on the Porter-Blum micro- tome with glass knives and were mounted

on formvar-coated copper grids stabilized with carbon, Some of the grids were stained with uranyl acetate (Watson, ' 58) . The tissues were examined in an RCA EMU 3 electron microscope.

Experimental manipulations. Table 1 summarizes the experiments performed on pigeons, which were intended to alter the physiologic condition of the animals and thus possibly to modify the lumbosacral cells. Inasmuch as inclusions were virtually absent from one-year-old pigeons, but were present at two- and one-half-years and at older ages, experiments attempting to increase the amount or induce the appearance of stainable material were performed on younger (one-year-old) birds (silver king). Treatments with estrogen and corticoid were given because of their possible relation to ceroidogenesis (cf. Bern et al., '58). Restraint and water deprivation were aimed at determining if there were any consequences of "stress" of these kinds to be observed in these neurons. Experiments attempting to modify the inclusions already present were performed on five-year-old pigeons (white king). The drug treatments were selected because of their previous utilization in at- tempts to modify neurohypophyseal neurosecretion (Hartmann, '58; Hartmann and Fujita, '61). Histamine is, of course, an acutely injurious agent.

OBSERVATIONS

Histology and cytology None of the pigeons younger

than six months of age showed any stainable inclusions in the lumbosacral neurons. At six and 12 months of age one or two orange G-positive granules were visible in an occasional neuron.

At two and one-half years and five years of age all birds examined showed a large number of acidophil droplets and granules in at least some of the ventral horn cells in the lumbosacral region (figs. 1-3). These inclusions could be seen unstained with phase microscopy. They stained largely with the orange G component of the Mal- lory dye mixture; however, acid fuchsin- staining material was also evident. Stain- able inclusions were present in motor neurons regardless of cell size (ranging

Pigeon.

AVIAN LUMBOSACRAL NEURON INCLUSIONS 195’

TABLE 1 Experimental manipulations of pigeons

5

5

5

5

3

3

4

4

4

4

4

years Endocrine and “stress” experiments

1

1

1

1

2%

21/52

2%

5

5

5

5

2.5 mg deoxycorticosterone acetate (DCA) daily intra.. muscularly in sesame oil for ten days.

5 mg pellet of estradiol-17B implanted subcutaneously in the neck region and maintained for ten days.

Subjected to restraint by tying legs together for ten days.

Maintained untreated for ten days.

Deprived of water for 56 hours.

Deprived of water for 86 hours.

Maintained untreated (normal food and water) for 86 hours.

Acute drug experiments Single intramuscular injection of pilocarpine hydro-

chloride (5 mg/kg body weight); sacrificed 30 minutes later.

Single intramuscular injection of histamine hydrochlo. ride (10 mg/kg body weight); sacrificed 30 minutes later.

Single intramuscular injection of adrenalin hydrochlo- ride (1 mg/kg body weight); sacrificed two hours later.

Single intramuscular injection of 0.5 ml of 0.9% NaCl solution (volume of vehicle for drug injections); sacrificed two hours later.

from 17 X 351.1 to 23 X 771.1 in area). Both the fine granules and the larger spheroidal droplets measuring up to 3.5 I.I in diameter were restricted to the perikaryon, generally concentrated toward the periphery, and were entirely absent from the axon, other than the axon hillock. Only some cells were closely associated with capillaries (fig. 3), and no invasion of capillaries into the perikaryon was encountered, In a few animals occasional droplets were greater than 3.5 p in diameter; these represent a third type of inclusion (see below).

Examination of the thoracic spinal cord revealed a small number of cells, in three out of six older pigeons, containing fewer granules and droplets than were seen in the lumbosacral regions. Electron micro- graphs of thoracic motor neurons also showed infrequent inclusions of the Iumbo- sacral types. The data make it clear that inclusion formation is a phenomenon not restricted solely to neurons of the lumbo-

sacral region, although it is considerably more extensive there.

Gull. Acidophil droplets and granules were seen in the lumbosacral motor cells in all gulls (fig. 13), although the number of stainable cells was less than in the older pigeons. The morphology and distri- bution of the inclusions in the gull were in all respects similar to that described for the pigeon.

Chicken. None of the one- and four- month-old cockerels showed cells with acidophil droplets or granules.

Parakeet. A thorough examination of the lumbosacral region of these adult birds failed to reveal any acidophil granules.

Towhee. No granules or droplets were seen in the lumbosacral spinal cord of the juveniles of this species.

Cytochemistry The two principal kinds of inclusions in

pigeon neurons - granules and droplets - appear to be cytochemically distinct in

198 A. GHOSH, H. A. BERN, I. GHOSH AND R. S . NISHIOKA

regard to certain of their features. Whereas the granules were occasionally basophilic and consistently PAS-positive (fig. 4), the droplets were unreactive. The PAS reaction of the granules was not abolished by prior treatment with diastase and hence is not due to glycogen. The granules reacted intensely with the sudan stain, whereas the droplets were virtually non- staining (fig. 5).

Some of the granules were moderately reactive in the Schmorl test (reduction of ferricyanide), indicating their possibly lipofuscin nature, but the droplets were entirely negative. Both inclusions reacted intensely for protein (fig. 6 ) and for protein-bound sulfhydryl (SH) and disulfide (SS) groups (fig. 7). The alkaline blue tetrazolium method is not highly specific for SH and SS groups, and it is possible that the positive reaction of the granules reflected their lipofuscin nature.

With non-cytochemical staining methods, both inclusions are strongly acidophilic. However, the granules prove to be paraldehyde fuchsin-positive (figs. 8 and 9), whereas the droplets react strongly with orange G.

The inclusions from the gull neurons (fig. 13) react in much the same way as do those from the pigeon, but not identi- cally. Both kinds of inclusion are intensely PAS-positive (fig. 14). The granular inclusions of the gull were intensely reactive with the Schmorl method (fig. 15) and

with the alkaline blue tetrazolium method for SH and SS (fig. 16). However, the droplets in the gull neurons do not appear to contain SH and SS groups, unlike those of the pigeon.

Ultrastructure The electron-microscope observations

were made on lumbosacral cells of two- and one-half and five-year-old pigeons. Along with motor neurons of usual appearance, some cells were characterized by the presence of appreciable numbers of inclusions, which appeared to correspond to the cells containing acidophil inclusions on the light-microscope level. The ultrastructural characteristics of th,ese neurons are given below.

Nucleus. The nucleus, bounded by the characteristic double membrane, contained an irregular, electron-dense nucleolus.

Nissl substance. The ultrastructure of the Nissl bodies in these cells was similar to that described for mammalian nerve cells (e.g., Hess, '55), and consisted of tubular and lamellar structures associated with typical ribonucleoprotein granules. The granular membrane system often in- cluded cisternae of considerable size.

Mitochondria and Golgi apparatus. On the ultrastructural level, the mitochondria and the Golgi apparatus showed their usual features: cristae, and cisternae associated with lamellated agranular membrane systems, respectively. These organ-

TABLE 2

Cytochemical and staining reactions of inclusions in the lumbosacral ventral horn cells of the pigeon

Reaction of Test

Granules Droplets

Inherent color Mallory-Heidenhain Paraldehyde-fuchsin with

Hematoxylin and eosin Basophilia Me tachromasia PAS (after diastase) Feulgen Sudan black B Mercury-bromphenol blue Alkaline blue-tetrazolium Schmorl

counterstains

Colorless Mainly orange, some red

Strongly orange G-staining Eosinophilic

- -

- to +- + + -

Sometimes slightly tinted Orange, red, or unstained

Strongly fuchsinophilic Eosinophilic

Occasionally + + or strong +

-

Strong + + + Variably+

AVIAN LUMBOSACRAL NEURON INCLUSIONS 399

elles are discussed below in relation to the possible genesis of spheroid and irregular bodies.

Cytoplasmic inclusions (fig. 17). The two principal types of inclusion were evident ultrastructurally as ( i ) spheroid bodies (droplets) and (ii) irregular bodies (granules). Small and large globular masses (iii) of virus-like particles were also encountered.

(i) Droplets (spheroid bodies). These structures were spheroid or ovoid, and were filled with a moderately electron- dense material of homogeneous character (figs. 17, 20-22). The diameter of some of the larger bodies averaged 2.5 M, corre- sponding to the size of the droplets seen with the light microscope. These inclusions were often surrounded by complexes of membranes, multilayered and often thick. In some instances, a close association of the droplet with the Golgi apparatus was suggested; occasionally large Golgi cisternae had the same appearance as the limiting membrane system of the droplet, only without its contents (fig. 24). Double membranes seemed to encapsulate the cis- ternal area and possibly to add to the volume of the inclusion (fig. 20). How- ever, there was also some evidence for the formation of these droplets within mitochondria (fig. 25), where dense homogeneous spheroids of various sizes can be occasionally found.

(ii) Granules (irregular bodies). Many of the cells contained numerous irregu- larly-shaped bodies (figs. 17-19, 22, 24). Some of these were of size and shape comparable to those of mitochondria; others were larger (1.2 p X 0.7 p). Unlike the spheroid bodies, these cytoplasmic inclusions were not homogeneous. Few to many vesicular areas were present, along with occasional suggestions of internal membranes resembling modified cristae mitochondriales (fig. 19). Series of transi- tion stages between normal mitochondria and these irregular bodies can be conceived (fig. 23). The initial sign of transformation of mitochondria may be represented by the accumulation of electron-dense material inside the organelle, although, as men- tioned above, these mitochondria1 inclusions also bear resemblance to small drop-

lets. Sometimes the entire dense granule resembled a transformed mitochondrion; however, most of these structures showed cavities and electron-lucent areas (figs. 19 and 22) and appeared to arise from the coalescence of metamorphosed mitochondria (fig. 22).

(iii) Virus-like particles. Small and large masses of virus-like particles were present along with spheroid and irregular bodies (figs. 17 and 22) in many lumbosacral neurons of three of the seven untreated two and one-half and five-year-old pigeons examined and in all four of the drug-treated birds examined. The average particle measured about 600 A in diameter and consisted of an electron-dense rim (about 100 A thick) surrounding a less opaque core (“doughnut-like”). They were arranged into crystalline clusters in the perikaryon, which sometimes attained a maximum diameter of 15 p; occasionally masses were also present in the axons. The presence of virus-like particles is not a constant feature of the secretory-appear- ing cells; however, their occurrence in large accumulations - simulating secretion masses - is of vital significance to the interpretation (or misinterpretation) of evidence for neurosecretory activity.

In two histamine-treated pigeons, enor- mous numbers of virus-like particles in crystalline array were noted. Adjacent thick sections of methacrylate-embedded tissue revealed massive inclusions under phase microscopy. Ordinary staining of paraffin sections of the spinal cord from these birds showed that these masses were acidophilic (orange G-staining and eosinophilic) (figs. 9-12). A slight basophilia with toluidine blue was also noted, and the masses were Feulgen-negative. These inclusions attained a size considerably greater than the similarly-staining droplets, as large as 15 c1 in diameter in some cases. Occasionally they protruded from the surface of the perikaryon (figs. 9 and 11); smaller masses extended into the axons (fig. 12). The usual inclusions were also present (fig. 10).

Effect of experimental manipulations

Hormone and “stress” experiments. The controls of this series showed rare orange

200 A. GHOSH, H. A. BERN, r. GHOSH AND R. s. NISHIOKA

G-staining granules in a small number of lumbosacral neurons. No change was noted after DCA treatment or after restraint. However, a perceptible increase in granular inclusions was noted in the estrogen- treated birds.

Dehydration. No alteration of the inclusion-bearing neurons could be ascribed to water deprivation.

Drug experiments. No consistent dif- ference between the variously treated birds and the saline-injected controls was noted. On both light-microscope and ultrastructural levels, there was no sign of disinte- gration or concentration of either the granule or the droplet type of inclusion. The massive virus aggregations in the two histamine-treated birds were not considered to be a result of the drug treatment.

DISCUSSION

Acidophilic inclusions similar to those described in the chicken by Sano and his co-workers have been found in motor neurons of the lumbosacral spinal cord of the pigeon and gull. Sano (’54) had also previously recorded their occurrence in the pigeon. Examination of the thoracic region of the pigeon spinal cord shows that these inclusions are not limited to the lumbosacral region, although they are found in much greater numbers in the latter region. Other birds examined, including four- month-old chickens, failed to show inclusions.

The inclusions as seen in the light microscope appear to fall into three categories: ( 1) huge globular masses, (2) droplets, and (3) granules. Ultrastructural and cytochemical data indicate that these three categories represent different entities and not a continuous spectrum of inclusion varying only in size. The principal distinguishing features of these inclusions are summarized in table 3. It is possible that additional types of inclusions also exist. Thus, in figure 23 the more homogeneous granules, near-mitochondria1 in size and shape, could be lysosomes, rather than stages in granule formation.

The large globules are unquestionably composed of virus-like particles of unknown significance. There is little to suggest any cytopathogenic effect of the virus- like material, which appears only in the

cytoplasm. In this respect they resemble the inclusions described in mouse mammary tumor cells (David-Ferreira, ’60; Smoller et al., ’61). The prominent crystalline arrangement, however, is characteristic of the neuronal masses. These inclusions need not be further considered herein, but it should be emphasized that their reactions to standard stains is iden- tical with that of the smaller droplets. Accordingly, on the light-microscope level this latter category certainly would include some smaller virus-like masses as well. The presence in neurons of acidophilic droplets actually composed of virus-like particles might inadvertently be adduced as evidence for “Gomori-negative” neurosecretory material, were further examination (largely ultrastructural) not con- ducted. The “viroid” inclusions occur also in neurites and hence could simulate axonal transport of neurosecretion. Whether the huge colloid inclusions described by Bargmann and Jakob (’52) in hypothalamic neurosecretory neurons of the pigeon are related to these virus-like inclusions remains problematical. The chrome-hematoxylin staining (along with some evidence of phloxinophilia) of the hypothalamic masses may be ascribable to the neurosecretory material intermingled with virus-like particles.

The two smaller categories of inclusions are present in neurons and in pigeons where the virus-like masses were found and aIso where they were not found. The droplets are conspicuous elements, provid- ing the major support for the suggestion of neurosecretory activity in these cells. Their significance is unknown. There is no evidence for their axonal transport, and their cytochemical properties are suggestive of similar inclusions seen in a variety of neurons. The neurons of the gastropod mol- lusk Aplysia, for example, are characterized by similar elements which do, however, enter the axons (Bern, ’62; Simpson, Bern and Nishioka, unpubl.). Large inclusions similar to these are seen along with typical neurosecretory granules (as ex- amples, in cells of the goldfish preoptic nucleus by Palay, ’60, of the cockroach pars intercerebralis by Bern et al., ’61, and of the teleost caudal neurosecretory system by Bern and Nishioka, unpubl. ). Drop-

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202 A. GHOSH, H. A. BERN, I. GHOSH AND R. S . NISHIOKA

lets have been described in the chicken by Tamiya et al. (’55) as being discharged from the surf ace of the perikaryon. We are not convinced that this occurs in our pigeon or gull material; the few possibly extracellular droplets seen could easily represent an artifact. The droplets may arise from transformation of the Golgi apparatus; however, a sequence of stages leading from mitochondria is also conceiv- able. Formation of pigment droplets from mitochondria in rat pineal cells has been proposed by Gusek and Santoro (’61). Nevertheless, we are hesitant to conclude anything definite from our electron micro- graphs, inasmuch as they can be arbitrarily arranged in a sequence to support almost any one of the several possible paths of droplet formation.

The third category of inclusions, the granules, conceivably is equivalent to a ceroid-type lipofuscin (cf. Pearse, ’60). The lack of a definite inherent coloration contraindicates this conclusion, although ceroid passes through a variety of chemical and tinctorial stages during its formation, and some colorless “ceroid has been observed in fishes (Wood and Yasatake, ’56). The insolubility of the granules in fat sol- vents; their positive reaction to paraldehyde-fuchsin, PAS, and sudan black; the occasional positive reaction in the Schmorl ferricyanide reduction test - all suggest ceroidal material. The colored pigments described in ganglion cells (Sulkin, ’53; Pearse, ’60) are similar to the virtually colorless granules observed in the pigeon neurons. The suggestive increase in the number of granules after estrogen treatment and the lack of a response to DCA treatment are consonant with what is known about ceroidogenesis in other tissues (Alpert, ’53; Bern et al., ’58).

The fuchsinophilic nature of the granular material could be interpreted as evidence for “Gomori-positive” neurosecretion, along with the “Gomori-negative” droplets. The electron microscope reveals no “elementary neurosecretory granules” in the 1,000-3,000 A size range, generally characteristic of established neurosecretory systems (cf. Bern, ’62). Instead, one finds changes in mitochondria suggestive of their involvement in production of the

granular material. Dense bodies within mitochondria, homogeneous inclusions of mitochondrial size and shape, and the ap- parent fusion of partially altered mitochondria with the irregular granule masses, all support a probable mitochondrial origin of the granular material. Hess (’55) claims a mitochondrial origin for neuronal pigment in old guinea pigs; however, Bon- dareff (’57) disputes this and suggests that lipofuscin in senile rat spinal ganglion may arise from the Golgi apparatus. In a recent review, Hager (’61 ) has emphasized that the relation between so-called lysosomes (resembling dense mitochondria) and liposomes (pigment bodies) is still uncertain. Furthermore, Hudson and Hart- mann (’61) suggest that mitochondria originate from the dense bodies rather than vice versa.

The non-neurosecretory status of the droplet and granule inclusions is supported by the electron-microscope evidence already described. The lack of response to drugs known to alter neurohypophyseal neurosecretion (Hartmann, ’58; Fujita and Hartmann, ’61) may provide further evidence. The absence of axonal transport and the lack of a neurohemal organ also are inconsistent with a neurosecretory system. In addition, we have not found these inclusions in younger pigeons (nor in younger chickens, unlike the Sano group), nor in older birds of another species (parakeet). One would expect that a true neurosecretory system not be one that develops in association with aging of the animal (as it appears to in the pigeon).

Sano (’62), in a review of the caudal neurosecretory system of fishes, suggests that the avian lumbosacral cells may be homologous or analogous to the teleost caudal neurosecretory cells. The ubi- quity of the caudal system and its presence throughout the postembryonic life of all teleosts, the possession of a neurohemal organ or area, and the ultrastructural evidence (elementary granules in the 1,000- 3,000 A range, organized by the Golgi apparatus, and transported down axons), all point to a lack of relation between these superficially similar cell groups in the rep- resentatives of two vertebrate classes.

The lumbosacral neurons of at least some species of birds are certainly involved

AVIAN LUMBOSACRAL NEURON INCLUSIONS 20 3

in the production of prominent intracellular bodies. However, from our observations on the pigeon, there appears to be no basis for ascribing any secretory significance to these inclusions. They appear with increase in age of the animal (evidently in the thoracic as well as in the lumbosacral region) and may represent the transformation of worn-out cell organelles. Note- worthy tinctorial evidence alone again appears to be an inadequate criterion for the delineation of neurosecretory activity of neurons.

SUMMARY AND CONCLUSIONS

1. Acidophilic inclusions are described in the lumbosacral motor neurons of the pigeon and the gull.

2. Inclusions were not encountered in younger chickens or towhees, nor in adult parakeets.

3. The inclusions in the pigeon were of two principal kinds: droplets and granules. The granules differ from the larger droplets in their affinity for paraldehyde- fuchsin and sudan black, their PAS re- activity, and their variable ability to re- duce ferricyanide in the Schmorl test. The granules appear to be lipofuscin in nature. The gull inclusions differed only slightly from those in the pigeon.

4. In the electron microscope, the droplets appear as homogeneous spheroids or ovoids with complex limiting membranes. The granules are irregular masses of variable electron density.

5. The possible origin of the droplets from either the Golgi apparatus or mitochondria is indicated; the granules appear to represent transformed and coalesced mitochondria.

6. A third type of cytoplasmic inclusion, consisting of masses of virus-like particles in crystalline array, was also encountered. These masses were acidophilic and often simulated secretory material.

7. The non-viroid inclusions in the lumbosacral neurons apparently represent transformed cell organelles. There is little basis for considering these cells specifi- cally secretory. They do not appear to form a system analogous or homologous to that seen in the caudal spinal cord of fishes.

LITERATURE CITED Alpert, M. 1953 Hormonal induction of deposi-

tion of ceroid pigment in the mouse. Anat. Rec., 116: 469493.

Bargmann, W., and K. Jakob 1952 Uber Neuro- sekretion im Zwischenhirn der Vogel. Z. Zell- forsch., 36: 556-562.

Bern, H. A. 1962 Properties of neurosecretory cells. Proc. 111 International Symposium on Comparative Endocrinology. Gen. Comp. Endo. Suppl., 1: 117-132.

Bern, H. A., S. Nandi, R. A. Campbell and L. E Pissott 1958 The effects of hormones and other agents on weight changes and on ceroid deposition induced by estrogen administration and by hypophysectomy in the adrenal glands of BALB/cCrgl mice. Acta endocr., 31: 349-383.

Bern, H. A., R. S. Nishioka and I. R. Hagadorn 1961 Association of elementary neurosecre. tory granules with the Golgi complex. J. Ultra- structure Res., 5: 311-320.

Bondareff, W. 1957 Genesis of intracellular pigment in the spinal ganglia of senile rats A n electron microscopic study. J. Geront., 12: 364-369.

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Photomicrography by Victor Duran

PLATE 1

EXPLANATION O F FIGURES

1 Parasagittal section of ventral horn area of lumbar spinal cord of five-year-old male pigeon to show large motor neurons containing numerous acidophilic inclusions. Mallory. ~310.

Lumbar motor neuron froin two and one-half-year-old male pigeon to show droplets ( D ) , granules ( G ) , and Nissl bodies (N) . Mallory. >: 920.

2

3 As in figure 2.Note capillary near cell. X 700.

4 Lumbar motor neuron from five-year-old male pigeon to show positive PAS reaction of granular inclusions. X 975.

Lumbar motor neuron from two and one-half-year-old male pigeon to show sudanophilia of granular inclusions indicated by arrow (droplets are unstained i n this cell). >: 920.

5

Abbreviations

C, capillary CR, cristae D, droplet F, myelinated nerve fiber G, granule GA, Golgi apparatus

M, mitochondrion N, Nissl substance P, polystyrene particle V, viroid inclusion VS, vesicular area

AVIAN LUMBOSACRAL NEURON INCLUSIONS Asok Ghosh, Howard A. Bern, Ira Ghosh and Richard S. Nishioka

PLATE IL

205

PLATE 2

EXPLANATION OF FIGURES

6 Lumbar motor neuron of two and one-half-year-old male pigeon to show proteinaceous nature of inclusions. Bromphenol blue. x 920.

7 Lumbar motor neuron of two and one-half-year-old male pigeon to show reaction of inclusions with alkaline blue tetrazolium, possibly indicative of protein-bound SH and SS groups. x 920.

Lumbar motor neuron of five-year-old male pigeon to show paraldehyde fuchsin-staining of granules. X 880.

Lumbar motor neuron of histamine-treated five-year-old male pigeon to show paraldehyde fuchsin-staining granules (G) and clear globular masses (V) . ( In this preparation orange G-staining was kept to a minimum.) x 880.

Lumbar motor neuron of histamine-treated five-year-old male pigeon to show acidophilic droplets ( D ) and globular masses ( V ) . Mallory. x 975.

8

9

10

11 As in figure 10. Note large cytoplasmic masses. x 975.

12 As in figure 10. Note globular mass ( V ) in axon. (Cells shown in figures 10-12 were taken from three different pigeons.) x 975.

Abbreviatioiis



206


PLATE 5!

207

PLATE 3


13 Lumbar motor neuron from adult gull. Mallory. X 920.

14 As in figure 13. Both droplets and granules are PAS-positive. x 750.

15 As in figure 13. To show possible lipofuscin nature of granules. Schmorl method. x 750.

16 As in figure 13. Only the granules appear to react with alkaline blue tetrazolium. x 920.

AVIAN LUMBOSACRAL NEURON INCLUSIONS Asok Ghosh, Howard A. Bern, Ira Ghosh and Richard S . Nishioka

PLATE 3

209

PLATE 4


17 Low-power electron micrograph from two and one-half-year-old male pigeon to show three principal types of inclusion : membrane-limited droplets (D) , irregular granules (G) , mass of virus-like particles ( V ) . Note fine granular nature of cytoplasm (Nissl).

Electron micrograph from five-year-old male pigeon to show irregular granules. Note vesicular areas in some ( V S ) .

18

Abbreviations



210


PLATE 4

211

PLATE 5


19 Electron micrograph of irregular granules from two and one-half- year-old male pigeon, Note vesicular nature of granules; suggestion cf cristae (CR) in lower granule. Possible mitochondria1 origin of granules is iIlustrated.

Electron micrograph of ovoid droplet from two and one-half-year-old male pigeon. Note resemblance of part of external membrane to Golgi lamellae containing electron-dense material, suggesting possible origin of droplets from Golgi apparatus. However, cf. figure 25.

Electron micrograph of nearly spheroid droplets from two and one- half-year-old male pigeon to show homogeneous nature and external membranes.

20

21

Abbreviations



212


PLATE 5

213

PLATE 6


22 Electron micrograph of inclusions from two and one-half-year-old male pigeon. Note membrane-limited droplet ( D ) ; “vacuolated” irregular granules ( G ) ; mass of virus-like particles ( V ) . MG is mitochondrion apparently attached to irregular granule, again suggesting a mitochondria1 origin for the latter.

23 Electron micrograph from two and one-half-year-old male pigeon to show possible stages in the formation of irregular granules. 1, 2, 3 , 4: possible transformation of mitochondria into dense body which later becomes “vacuolated.” l’, 2’, 3, 4: possible formation of increasingly large dense mass within mitochondrion.

Abbreviations

C, capillary CR, cristae D, droplet F, myelinated nerve fiber G , granule GA, Golgi apparatus


214


PLATE 6

215

PLATE 7


24 Electron micrograph of inclusions from two and one-half-year-old male pigeon. D, droplets; G, granules; GA, Golgi apparatus to show resemblance of large cisterna to externum of droplets.

Electron micrograph from five-year-old adrenalin-treated male pigeon. Note possible stages in the formation of droplets (D) within mitochondria and the transformation of mitochondria1 membranes into limiting membranes of the droplet.

25

Abbreviations



216


PLATE 7

217

Documents

Nature of the inclusions in the lumbosacral neurons of birds