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Nature of the inclusions in the lumbosacral neurons of birds.

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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 completely unreactive to a variety of histochemical tests, including methods for demonstrating lipids, carotenoids, “wear and
tear” pigments, cholesterol and its esters,
glycerides, fatty acids, and glycogen. Cursory 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 discharge of the “secretory” granules through
the cell surface. With the aid of the phasecontrast 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 alcohol 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 technics; (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), western gulls ( L a r u s occidentalis), parakeets
(Melopsittacus undulatus), brown towhees (Pipilo 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
6-8805.
Present address: Department of ‘Zoology, University of Calcutta, Calcutta, India.
2
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. Paraldehydefuchsin with couterstains (cf. Clark, '55)
was also used on some sections. The thoracic area of the spinal cord of three twoand a half-year-old and three five-year-old
pigeons was also cursorily examined.
Standard cytochemical methods employed on paraffin-embedded tissues included (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 solutions 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 twoand 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 ( 0 4 ° C ) 1% osmium tetroxide in
saline solution buffered between pH 7.27.4 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, dehydrated in a graded series of ethanols, and
embedded in n-butyl methacrylate. Sections were cut on the Porter-Blum microtome with glass knives and were mounted
on formvar-coated copper grids stabilized
with carbon, Some of the grids were
stained with uranyl acetate (Watson, ' 5 8 ) .
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 attempts to modify neurohypophyseal neurosecretion (Hartmann, '58; Hartmann and
Fujita, '61). Histamine is, of course, an
acutely injurious agent.
OBSERVATIONS
Histology and cytology
Pigeon. 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 Mallory dye mixture; however, acid fuchsinstaining material was also evident. Stainable inclusions were present in motor
neurons regardless of cell size (ranging
AVIAN LUMBOSACRAL NEURON INCLUSIONS
195’
TABLE 1
Experimental manipulations of pigeons
years
5
1
Endocrine and “stress” experiments
2.5 mg deoxycorticosterone acetate (DCA) daily intra..
muscularly in sesame oil for ten days.
5
1
5 mg pellet of estradiol-17B implanted subcutaneously
in the neck region and maintained for ten days.
5
1
Subjected to restraint by tying legs together for ten days.
5
1
Maintained untreated for ten days.
3
2%
Deprived of water for 56 hours.
3
21/52
Deprived of water for 86 hours.
4
2%
Maintained untreated (normal food and water) for 86
hours.
4
5
4
5
Single intramuscular injection of histamine hydrochlo.
ride (10 mg/kg body weight); sacrificed 30 minutes
later.
4
5
Single intramuscular injection of adrenalin hydrochloride (1mg/kg body weight); sacrificed two hours later.
4
5
Single intramuscular injection of 0.5 ml of 0.9% NaCl
solution (volume of vehicle for drug injections); sacrificed two hours later.
Acute drug experiments
Single intramuscular injection of pilocarpine hydrochloride (5 mg/kg body weight); sacrificed 30 minutes
later.
from 17 X 351.1to 23 X 771.1in 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 micrographs of thoracic motor neurons also
showed infrequent inclusions of the Iumbosacral 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 distribution of the inclusions in the gull were
in all respects similar to that described
for the pigeon.
Chicken. None of the one- and fourmonth-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
-
A. GHOSH, H. A. BERN, I. GHOSH AND R. S . NISHIOKA
198
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 nonstaining (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 identically. 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 twoand 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 included 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
Inherent color
Mallory-Heidenhain
Paraldehyde-fuchsinwith
counterstains
Hematoxylin and eosin
Basophilia
Me tachromasia
PAS (after diastase)
Feulgen
Sudan black B
Mercury-bromphenol blue
Alkaline blue-tetrazolium
Schmorl
Droplets
Granules
Colorless
Mainly orange, some red
Sometimes slightly tinted
Orange, red, or unstained
Strongly orange G-staining
Eosinophilic
Strongly fuchsinophilic
Eosinophilic
Occasionally
-
- to +-
+
+
-
-
+
+ or strong +
Strong +
+
+
Variably+
AVIAN LUMBOSACRAL NEURON INCLUSIONS
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 electrondense material of homogeneous character
(figs. 17, 20-22). The diameter of some
of the larger bodies averaged 2.5 M, corresponding 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 cisternal area and possibly to add to the
volume of the inclusion (fig. 20). However, 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 irregularly-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 transition 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 mentioned above, these mitochondria1 inclusions also bear resemblance to small drop-
399
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-appearing 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, enormous 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.
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 estrogentreated birds.
Dehydration. No alteration of the inclusion-bearing neurons could be ascribed
to water deprivation.
Drug experiments. No consistent difference between the variously treated birds
and the saline-injected controls was noted.
On both light-microscope and ultrastructural levels, there was no sign of disintegration 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 fourmonth-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-mitochondria1in 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 viruslike material, which appears only in the
s. NISHIOKA
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 identical 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 conducted. 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, providing 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 mollusk 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 examples, 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-
perikaryon
and axon
hillock
perikaryon
and axon
hillock
perikaryon
and
neurite
Droplets
Granules
Globular
masses
LM*
EM*
up to 15 p
18
2-3.58
up to 15 p
(average)
0.7 X 1.2~
(average)
2.58
(Maximum diameter)
JlLB
01__
* LM = light microscope; EM = electron microscope.
in cell
Location
Inclusion
type
TABLE 3
spheroid
to ovoid
spheroid;
punctate
spheroid
LM
Shape
spheroid
to ovoid
irregular
spheroid
to ovoid
EM
orange G
paraldehydefuchsin
orange G
Stainabilitv
with
paraldehydefuchsin
mixture
associated with
lamellated
membrane systern; from
GoIgi(?) and/or
mitochondria( ?)
Origin
Possible
slight
basophilia
possibly
'lipofuscin"
composed of viruslike particles
in crystalline
array
within mitochondria(7);
from coales(PAScence of
positive;
mitostrongly
sudanophilic; chondria
occasionally
Schmorlpositive)
phospholipoprotein( 7)
specific
cytochermcal
features
Summary of characteristics of lumbosacral neuronal inclusions in the pigeon
2
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3
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0
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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 conceivable. 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 micrographs, 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 solvents; 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 apparent 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, Bondareff (’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 Hartmann (’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 ubiquity 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 1,0003,000 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 representatives of two vertebrate classes.
The lumbosacral neurons of at least
some species of birds are certainly involved
AVIAN LUMBOSACRAL NEURON INCLUSIONS
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. Noteworthy 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 paraldehydefuchsin and sudan black, their PAS reactivity, and their variable ability to reduce 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 specifically secretory. They do not appear to
form a system analogous or homologous
to that seen in the caudal spinal cord of
fishes.
20 3
LITERATURE CITED
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Bern, H. A., S. Nandi, R. A. Campbell and L. E
Pissott 1958 The effects of hormones and
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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.
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Bondareff, W. 1957 Genesis of intracellular
pigment in the spinal ganglia of senile rats
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364-369.
Clark, R. B. 1955 The posterior lobes of the
brain of N e p h t y s and the mucus-glands of the
prostomium. Quart. J. niicr. Sci., 96; 545-565.
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for electron microscopy. Anat. Rec., 121: 281.
(Abstract).
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des metastases pulmonaires des cancers mamniaires de la Souris. Proc. European Regional
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Fujita, H., and J. F. Hartmann 1961 Electron
microscopy of neurohypophysis in normal,
adrenaline-treated and pilocarpine-treated rabbits. 2. Zellforsch., 54: 734-763.
Fujita, H., N. Iwasa and H. Hiramatsu 1957
On the neurosecretory anterior horn cells in the
lumbosacral portion of the avian spinal cord.
IV. Ontogenetic observations. Arch. hist. jap.,
12: 465470.
Glegg, R. E., Y. Clermont and C. P. Leblond 1952
The use of lead tetraacetate, benzidine, o-dianisidine and a “film test” in investigating the
periodic-acid-Schiff technic. Stain Technol.,
27: 277-305.
Gurr, E. 1953 A Practical Manual of Medical
and Biological Staining Techniques. Interscience, New York.
Gusek, W., and A. Santoro 1961 Zur Ultrastruktur der Epiphysis cerebri der Ratte. Endokrinologie, 41; 105-129.
Hager, H. 1961 Ergebnisse der Elektronenmikroskopie am zentralen, peripheren und
vegetativen Nervensystem. Ergebn. Biol., 24:
106-154.
Hartmann, J. F. 1958 Electron microscopy of
the neurohypophysis in normal and histaminetreated rats. Z. Zellforsch., 48: 291-308.
Hess, A. 1955 The fine structure of young and
old spinal ganglia. Anat. Rec., 123: 399-423.
Hudson, G., and J. F. Hartmann 1961 The
relationship between dense bodies and mito-
204
A. GHOSH, H. A. BERN, I. GHOSH AND R. S. NISHIOKA
chondria in motor neurones. Z. Zellforsch.,
54: 147-157.
Mazia, D., P. A. Brewer and M. Alfert 1953
The cytochemical staining and measurement of
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Montagna, W., H. B. Chase and H. P. Melaragno
1951 Histology and cytochemistry of human
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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. ~ 3 1 0 .
2
Lumbar motor neuron froin two and one-half-year-old male pigeon to
show droplets ( D ) , granules ( G ) , and Nissl bodies ( N ) . Mallory.
>: 920.
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.
5
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.
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.
8
Lumbar motor neuron of five-year-old male pigeon to show paraldehyde fuchsin-staining of granules. X 880.
9
Lumbar motor neuron of histamine-treated five-year-old male pigeon
to show paraldehyde fuchsin-staining granules ( G ) and clear globular
masses ( V ) . ( I n this preparation orange G-staining was kept to a
minimum.) x 880.
10
Lumbar motor neuron of histamine-treated five-year-old male pigeon
to show acidophilic droplets ( D ) and globular masses ( V ) . Mallory.
x 975.
11
As in figure 10. Note large cytoplasmic masses.
x
975.
12 As in figure 10. Note globular mass ( V ) i n axon. (Cells shown i n
figures 10-12 were taken from three different pigeons.) x 975.
Abbreviatioiis
C, capillary
CR, cristae
D, droplet
F, myelinated nerve fiber
G, granule
GA, Golgi apparatus
206
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 5!
207
PLATE 3
EXPLANATION O F FIGURES
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
EXPLANATION O F FIGURES
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).
18
Electron micrograph from five-year-old male pigeon to show irregular
granules. Note vesicular areas in some ( V S ) .
Abbreviations
C, capillary
CR, cristae
D, droplet
F, myelinated nerve fiber
G, granule
GA, Golgi apparatus
210
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 4
211
PLATE 5
EXPLANATION O F FIGURES
19
Electron micrograph of irregular granules from two and one-halfyear-old male pigeon, Note vesicular nature of granules; suggestion
cf cristae (CR) in lower granule. Possible mitochondria1 origin of
granules is iIlustrated.
20
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.
21
Electron micrograph of nearly spheroid droplets from two and onehalf-year-old male pigeon to show homogeneous nature and external
membranes.
Abbreviations
C, capillary
CR, cristae
D, droplet
F, myelinated nerve fiber
G, granule
GA, Golgi apparatus
212
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 5
213
PLATE 6
EXPLANATION OF FIGURES
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 i n 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
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 6
215
PLATE 7
EXPLANATION OF FIGURES
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.
25
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.
Abbreviations
C, capillary
CR, cristae
D, droplet
F, myelinated nerve fiber
G, granule
GA, Golgi apparatus
216
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 7
217
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