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SOUTHERN SOCIETY OF ANATOMISTS
Twenty-first Annual Meeting
October 21-24, 1981
Columbia, South Carolina
Host
UNIVERSITY OF SOUTH CAROLINA
137
SOUTHERN SOCIETY OF ANATOMISTS
OFFICERS 1980-81
President
RICHARD J. WEYMOUTH
(South Carolina- Columbia)
President-Elect
RICHARD G. SKALKO
(East Tennessee)
Pas t-President
DALE E. BOCKMAN
(MedicalCollege of Georgia)
Secretary-Deasurer
GEORGE E. GOODE
(Eastern Virginia)
Councilors
MARILYN L. ZIMNY
(Louisiana State)
J. MICHAEL BARREW
(Medical College of Georgia)
MOHAMED SHARAWY
(MedicalCollege of Georgia)
D. LOUISE ODOR
(South Carolina- Columbia)
This meeting was dedicated to the memory of
HERBERT SCHAPIRO
Secretary-Tkeasurer of the Southern Society of Anatomists 1977-81
138
CORPORATE SPONSORS
The following firms have generously supported this, the 21st annual
Meeting of the Southern Society of Anatomists, by their participation in
the commercial exhibits andlor sponsoring of social events:
AMERICAN
OPTICAL
CORPORATION
BELLCO
GLASS,INC.
CAROLINA
BIOLOGICAL
SUPPLY
Co.
Du-PONT-SORV
ALL
ELECTRON
MICROSCOPY
SCIENCES,
INC.
GEORGIA
INSTRUMENTS,
INC.
HALCOINSTRUMENT
Co.
JEOL,USA, INC.
PHILIPS
ELECTRONICS
INSTRUMENTS
PHYSIMETRICS,
INC.
SOUTHERN
MICROINSTRUMENTS,
INC.
THEUPJOHN
COL.
V A S H A W SCIENTIFIC, INC.
WELCH-ALLYN
CARLZEISS~~LECTRON-OPTICAL
INSTRUMENT
DIVISION
139
OPENING SESSION
Dr. Roderick Macdonald, Dean
University of South Carolina, School of Medicine
Dr. Richard J . Weymouth, Professor and Chairman
Department of Anatomy, University of South Carolina
School of Medicine
President, Southern Society of Anatomists
A total of 45 papers were presented in 9 sessions as follows:
SESSION I
“Endocrinology”
(Chairperson: Carl R. Morgan)
SESSION I1
“Methods”
(Chairperson: Richard G. Skalko)
SESSION I11
“Reproductive Biology”
(Chairperson: Steven L. Quattropani)
SESSION IV
“Immunology”
(Chairperson: Dr. J . David Gangemi)
SESSION V
“Developmental Biology”
(Chairperson: Dr. Marilyn L. Zimny)
SESSION VI
“Cell Biology”
(Chairperson: Michael Barrett)
SESSION VII
“Oral Cavity Biology”
(Chairperson: Dr. James A. Hightower)
SESSION VIII
“Neurobiology I”
(Chairperson: Dr. Alvin J. Beitz)
SESSION IX
“Neurobiology II”
(Chairperson: Dr. Robert H. Brownson)
140
these hormones on embryonic c a r t i l a g e , however, have n o t been well documented. The present study investigated the e f f e c t s of PTH and
prostaglandin E 2 (PGE,) on cAMP metabolism in
isolated embryonic cartilage c e l l s from stage
26-28 of development. The production of cAMP
was increased t o similar levels by maximal
concentrations of each hormone during a 15minute period. Intracellular degradation of
CAMP, estimated as the difference in cAMP
levels in the presence and absence of the
potent phosphodiesterase inhibitor l-methyl3-isobutylxanthine (MIX), was also increased
by both hormones over the same concentration
ranges which resulted in the production of
CAMP. Other studies investigated the specif i c i t y of the cAMP response t o e i t h e r PTH o r
PGE2. The response of PTH was specific in
t h a t neither prolactin nor insulin resulted
in significant increases in CAMP. In addit i o n , neither PGF, nor PGF2, resulted in a
significant cAMP rgsponse, indicating t h a t
responsiveness of PGE, was specific. The
results demonstrate that both hormones
increase the production and degradation of
cAMP within the isolated embryonic cartilage
c e l l s . This study suggests t h a t PTH and PGE,
may regulate the metabolic a c t i v i t y of c a r t i lage in the embryonic s t a t e .
ANDREW$ Peter Michael, Department of Anatomy,
Georgetown
University
Medical
School,
Washington, D.C. Investigations of cytoplasmic
contractile and cytoskeletal elements i n the
kidney glomerulus.
Electron microscopy and in vitro techniques
were used to evaluate the
morphological
responses
of
glomerular
podocytes
and
glomerular
endothelial cells
to
compounds
which effect either cytoplasmic contractile
In
elements
or
cytoplasmic
rnicrotubules.
response to in vitro incubation in the presence
of cytochalasin D or B (compounds which inhibit
contraction
of
actin-like
microfilaments),
podocyte foot processes change in shape from
short processes with broad bases to taller
processes with narrow bases.
Coincident with
these shape changes, there is an increase in
the number of fully patent filtration slit
spaces between adjacent foot processes. In view
of these observations, it is proposed that
glornerular podocytes have the potential for
monitoring the filtration slit area available
for solute efflux across the glornerular wall by
modifying the shapes of their foot processes
(i.e.
expanding and contracting the bases of
these structures).
As in vitro incubation
continues
for
one
to
two
days,
the
cytochalasins inhibit the shape changes of
glornerular
podocytes,
the
loss
of
foot
processes,
filtration slits, endothelial pores
and
thickening of
the endothelium which
normally. occur during two days of in vitro
incubation.
The cytochalasins also inhibit the
loss of foot processes and filtration slits
which otherwise occur in response to enzymatic
removal of glornerular free surface sialic acid.
The in vitro depolymerization of glomerular
microtubules by any of a variety of drugs.
(e.g.
vinblastine
sulfate, colchicine) results
in the rounding up of podocyte cell bodies, the
thinning of pcdocyte major processes and a
partial
collapsing
of
the
glomerular
endothelial
walls.
These latter observations
suggest that microtubules play an important
cytoskeletal
role
in
maintaining
the
structural
integrety
of
these
cells.
"Supported by Grant No.AM25417 from NIH"
BEITZ*, Alvin J . , Department of Anatomy, Univers i t y of South Carolina, School of Medicine,
Columbia, South Carolina. (Introduced by James
A. Hightower). The Origin of Afferent Projections t o the Rodent Spinal Trigeminal Nucleus
with Special Emphasis on the Origin of Serotonergic Input.
The origin o f central afferent projections
t o the spinal trigeminal nucleus (STN) was
studied in eight r a t s using the retrograde
transport technique. Iontophoretic deposits
of HRP were stereotaxically placed in the STN
and 24-36 hours postoperatively the animals
were sacrificed by intracardiac perfusion with
a paraformaldehyde-glutaraldehyde fixative f o l lowed by a buffered sucrose solution. Brain
sections were subsequently reacted with Mesulam's (1978) tetramethyl benzidine procedure
and scanned for the presence of HRP positive
neurons. HRP labeled neurons were identified
in the periaqueductal gray ( P A G ) , nucleus cuneformis, dorsal raphe ( D R ) , parabrachial nuclei,
dorsolateral tegmental nucleus, raphe magnus,
locus coeruleus, A5 region of Dahlstrom and
Fuxe and the nucleus paragigantocellularis
(PGCL). The greatest number of labeled c e l l s
occurred in the lateral parabrachial nucleus,
the nucleus s o l i t a r i u s , the nucleus r e t i c u l a r i s
gigantocel l u l a r i s , the DR, the ventrolateral
PAG and the A5 and A8 areas of Dahlstrom and
BALLARD*, Timothy A . and David M. BIDDULPH,
Department of Anatomy, Bowman Gray School of
Medicine, Wake Forest University, WinstonSalem, North Carolina. (Sponsored by J . E .
Turner) Effects of parathyroid hormone and
prostaglandin E 2 on cycZic AMP metabolism i n
isolated embryonic cartilage c e l l s .
Both parathyroid hormone (PTH) and prostaglandins are known t o regulate the metabolic
a c t i v i t y of mature cartilage through a cyclic
AMP (CAMP) dependent mechanism. Effects of
141
HRP i n j e c t i o n s i n t o t h e STN a l s o r e s u l t e d
i n the presence of labeled c e l l s in spinal cord
lamina VI, VIII and X, in the pontine s a l v a t o r y
Fuxe.
Because of t h e l a r g e c e r e b r a l c o r t e x of
dolphin b r a i n a s well a s i t s a b i l i t i e s i n communication and echo l o c a t i o n , t h i s animal provides a unique model t o study the c e r e b r a l c o r t i c a l c y t r o a r c h i t e c t u r e of regions reported t o
be involved i n sensory-motor, communicative
a s s o c i a t i o n and p o s s i b l e l e a r n i n g a r e a s . However because of the Sea Mammal P r o t e c t i o n Act of
1972 t h e s e zpecimens a r e n o t r e a d i l y a v a i l a b l e .
Nonetheless deceased specimens have been donated
by p r i v a t e and public oceanaria and c o l l e c t e d
from beached animals along t h e P a c i f i c Coast.
Because of t h e a v a i l a b i l i t y of m u l t i p l e sample
s i t e s from b r a i n s of two dolphin genera (Turs i o p s and Delphinus), t o t a l i n g f i v e animals, a
study of c e r e b r a l c o r t i c a l l a y e r thickness between r i g h t and l e f t hemispheres was p o s s i b l e .
An e a r l i e r r e p o r t on "Brain s i z e and Asymmetry"
Ridgway and Brownson, 1979, e s t a b l i s h e d presence
of a s i g n i f i c a n t l y l a r g e r c o r t i c a l s u r f a c e a r e a
on t h e r i g h t s i d e of t h e Tursiops b r a i n . The
a u t h o r s asked the following question: How does
this r i g h t s u r f a c e dominance r e l a t e t o c o r t i c a l
t h i c k n e s s e s and do c o r t i c a l thicknesses d i f f e r
in v a r i o u s f u n c t i o n a l r e g i o n s ? I n t h i s study
comparisons were made of t h e c o r t i c a l l a y e r s
between regions; r i g h t and l e f t hemispheres and
between genera. Although many comparisons between these two genera were t a b u l a t e d , a
synopsis would include the following f i n d i n g s :
1. C o r t i c a l t h i c k n e s s was found t o be symmetrical f o r a l l regions studied.
2. Asymmetric c o r t i c a l a r e a s a s previously r e ported do not d i c t a t e a concomitant asymmetry
of thickness.
3 . Visual c o r t e x i s c o n s i s t e n t l y t h i n . (Vision
i n sea mammals i s considered marginal).
4. The molecular l a y e r of t h e parahippocampal
gyrus i s t h i c k e r than t h e molecular l a y e r of
a l l o t h e r c o r t i c a l a r e a s studied i n t h e Tursiops
brain.
5. The a u d i t o r y c o r t e x i n Tursiops brain i s
t h i c k e r than t h e a u d i t o r y c o r t e x i n Oelphinus
brain.
6. The t o t a l c o r t i c a l t h i c k n e s s i n a l l a r e a s
s t u d i e d ranged from 1.30 mm ( v i s u a l ) t o 1.76 mm
(motor).
nucleus, i n t h e c o n t r a l a t e r a l p r i n c i p a l sensory
nucleus of V , i n t h e intermediate l a y e r of the
s u p e r i o r c o l l i c u l u s and the dorsal column nuc l e i . I n o r d e r t o determine t h e s p e c i f i c o r i g i n of s e r o t o n i n p r o j e c t i o n s t o STN, an addit i o n a l 5 r a t s were i n j e c t e d with HRP i n t o t h i s
a r e a and t h e animals were subsequently processed u t i l i z i n g the combined r e t r o g r a d e t r a n s p o r t immunohistochemical technique of Bowker e t a l . ,
(1981). Neurons which contain s e r o t o n i n (5-HT)
and p r o j e c t t o t h e STN were i d e n t i f i e d by t h e
presence of both brown and black r e a c t i o n product i n t h e same c e l l . The h e a v i e s t concentrat i o n o f double-labeled c e l l s were found i n the
nucleus raphe magnus. Double-labeled c e l l s were
a l s o found i n the PGCL and t h e DR. Many of the
above p r o j e c t i o n s a r i s e from nuclei of t h e endogenous a n a l g e s i a system and may serve t o modul a t e n o c i c e p t i v e o r o f a c i a l input. Supported
by NSF ENS 7906456.
BOCKMAN, Dale E . , William R. BOYDSTON and
Marion C. ANDERSON*, Department of Anatomy,
Medical College of Georgia, Augusta, Georgia,
and Department of Surgery, Medical University
of South C a r o l i n a , Charleston, South Carolina.
Origin of t u b u l a r complexes i n human chronic
pancreatitis.
T i s s u e samples from seven p a t i e n t s w i t h
chronic p a n c r e a t i t i s were s t u d i e d by l i g h t
and e l e c t r o n microscopy, using samples from
t h e pancreas of seven donors a s a r e f e r e n c e
group. Tubular complexes were observed i n
four of t h e p a t i e n t s w i t h chronic pancreat i t i s . Tubular complexes, which have been
i n t e r p r e t e d a s r e s u l t i n g from "ductular
r e d u p l i c a t i o n " i n o t h e r s t u d i e s of chronic
p a n c r e a t i t i s , and "ductular p r o l i f e r a t i o n " i n
s t u d i e s of p a n c r e a t i c adenocarcinoma, were
s t u d i e d c a r e f u l l y t o t r y t o determine t h e i r
o r i g i n . Extensive r e t r o g r e s s i v e changes i n
a c i n a r c e l l s l e a d i n g t o diminished zymogen
g r a n u l e s , decreased c e l l h e i g h t , and concomm i t a n t i n c r e a s e i n luminal diameter were cons i s t e n t w i t h t h e i n t e r p r e t a t i o n t h a t phenot y p i c modulation of a c i n a r c e l l s t o t a k e on
t h e c h a r a c t e r i s t i c s of d u c t u l a r c e l l s produced t h e t u b u l e s . I t was thus concluded
t h a t i n c h r o n i c p a n c r e a t i t i s , a s has been
shown f o r p a n c r e a t i c adenocarcinoma, t h e
t u b u l a r complexes o r i g i n a t e from a c i n a r c e l l s
r a t h e r than from p r o l i f e r a t i o n of p r e e x i s t i n g
d u c t u l e s . F i b r o s i s and thickening of t h e
b a s a l lamina of exocrine p a n c r e a t i c c e l l s and
c a p i l l a r i e s were c o n s i s t e n t w i t h an a l t e r e d
c a p a b i l i t y f o r transmission of m a t e r i a l
between blood v e s s e l s and exocrine c e l l s .
(Supported by Grant No. CA22063 from t h e
National Cancer I n s t i t u t e , DHEW, through t h e
National P a n c r e a t i c Cancer P r o j e c t . )
COFFEY*, Aline Kirtland and Peter Michael
ANDREWS*, Department of Anatomy, Georgetown
University
Medical School, Washington,
D.C.
of
Factors which
improve t h e preservation
nephron morphology during cold storage.
Light and electron microscopy were used to
evaluate the nephron morphology of rat kidneys
which were perfused and stored a t 0-2 C for up
to 48 hours in preservation solutions which (1)
contained different osmotic agents; ( 2 ) were
adjusted to different levels of osmolality; and
(3) which had different ionic compositions (e.g.
intracellular-like,
extracellular-like).
Storage in an enriched phosphate buffered
isotonic
culture media which contained very little effect i v e osmotic agent(i.e.Mediurn
199), results in
significant necrosis of glomeruli and uriniferous tubules within I 2 to 24 hours.
Storage i n
Collins solution which has an intracellularlike ionic
composition
and
which
contains
dextrose as an osmotic agent, results in significantly better preservation of kidney nephrons.
When
the
dextrose i n Collins solution
is
replaced with mannitol or sucrose, there is
BROWNSON, Robert H . , RIDGWAY, Sam and A l b e r t
KLEIN, Department of Anatomy, Eastern Virginia
Medical School, Norfolk, V i r g i n i a ; and Naval
Ocean Systems Center, San Dieqo, C a l i f o r n i a .
Regionai C o r t i c a l Layer Thicknesses i n the
Dolphin Brain.
142
r a t , suggesting t h a t ovarian maturation or
c y c l i c i t y a r e weight/age r e l a t e d .
(Supported
in p a r t by a grant from T h e Rockefeller
Foundation and NIH Grant HD-06899).
dramatic improvement in the preservation of
glomeruli and uriniferous tubules.
Overall,
sucrose appears to be the most effective osmotic
agent in preventing degeneration of the nephron
during cold storage.
Further improvement in
nephron preservation is seen when t h e osrnolality
of
Collins
solution
is
raised
(i.e.400
to
600mOsm) by adding more of the osmotic agents.
Finally, storage in simple sodium phosphate
buffered solutions containing either dextrose,
rnannitol, or sucrose results in the same quality
of
morphological preservation as seen with
Collins
intracellular-like
solutions
containing
similar amounts of these osmotic agents.
It
appears, therefore, that the selection of an
effective
osmotic
agent
(e.g.sucrose)
and
making
the
preservation
solution
hypertonic
with this osmotic agent are of primary importance
in
preserving
nephron
morphological
integrity during cold storage. The presence of
an intracellular-like
ionic composition in t h e
preservation solution, however, does not appear
to influence morphological preservation.
The
effects of the above variables on the ultrastructural morphology of different segments of
the uriniferous tubules and t h e renal corpuscles
a r e described and illustrated.
"Supported by
Grant No. AM25417 from NIH."
DONALDSON,
Donald J . and Mary K. Dunlap,
Department
of
Anatomy,
University
of
Tennessee Center f o r t h e Health Sciences,
Memphis,
Tennessee and S c h o o l of D e n t a l
Medicine,
Southern
Illinois
University,
Edwardsville,
Illinois.
Epidermal
M i g r a t i o n During Attempted C l o s u r e of S k i n
Wounds
Observations
- i n t h e A d u l t Newt:
Based on C y t o c h a l a s i n T r e a t m e n t a n d S c a n n i n g
E l e c t r o n Microscopy.
Cell
-_
Epidermal c l o s u r e of s k i n wounds on newt
l i m b e x p l a n t s was i n h i b i t e d to e q u a l d e g r e e s
by c y t o c h a l a s i n s R , D a n d d i h y d r o c y t o c h a l a s i n
B ( H 2 C B ) . The c y t o c h a l a s i n s o l v e n t , d i m e t h y l s u l f o x i d e (DMSO), had no e f f e c t on m i g r a t i o n
a t t h e low c o n c e n t r a t i o n p r e s e n t i n t h e
c y t o c h a l a s i n and c o n t r o l s o l u t i o n s . However,
5% DMSO
solution
completely
blocked
a
mobility.
Wounds on limb e x p l a n t s and l i m b s
i n s i t u responded s i m i l a r l y t o c y t o c h a l a s i n
treatment.
I n h i b i t i o n of m i g r a t i o n by H 2 C ~
was r e v e r s i b l e even when p r o t e i n s y n t h e s i s
w a s reduced by 73%.
Scanning e l e c t r o n
microscopy of wound e p i t h e l i u m m i g r a t i n g on
nucleopore
filters
revealed
extensive
l a m e l l i p o d i a on m a r g i n a l c e l l s and t h e f i r s t
row of
submarginal c e l l s .
Cytochalasin
t r e a t m e n t produced p l i c a t i o n s i n t h e upper
s u r f a c e and f r e e edge of t h e n o r m a l l y smooth
lamellipodia.
T h i s d i s t u r b a n c e of t h e f r e e
edge revealed
f o c a l adhesions
with
the
substratum.
The f a c t t h a t m i g r a t i o n w a s
i n h i b i t e d by CD a n d HZCB ( t w o c y t o c h a l a s i n s
w i t h an a f f i n i t y f o r c o n t r a c t i l e p r o t e i n s
b u t w i t h o u t some of t h e s i d e e f f e c t s of CB)
l e a d s u s t o conclude t h a t epidermal c e l l s
u t i l i z e a c t i n or a c t i n - l i k e p r o t e i n s during
wound c l o s u r e .
These r e s u l t s i n c r e a s e t h e
l i k l i h o o d t h a t t i s s u e c e l l s of a l l t y p e s
w h e t h e r i n v i t r o o r i n v i v o , s h a r e a common
b i o c h e m i c a l b a s i s f o r cell movement.
Curry, Thomas E . , David R . G a r r i s , and
I . Lawrence, J r . , Department of Anatomy, East
Carolina U n i v e r s i t y School of Medicine,
G r e e n v i l l e , NC 27834. C o r r e l a t i o n s between
o v a r i a n s t a t e and ovarian biood-fioi ~.~
, ..
t h e pseudopregnant r a t was s t u d i e d in order t o
determine i f growth r e l a t e d changes occur in
the ovarian v a s c u l a r supply. Adult, female
r a t s were maintained under c o n t r o l l e d
environmental c o n d i t i o n s and examined d a i l y
f o r reproductive c y c l i c i t y . Pseudopregnancy
was induced in r a t s weighing between 170 and
3209 by c e r v i c a l s t i m u l a t i o n on t h e afternoon
of p r o e s t r u s and morning of e s t r u s . Day 1 of
pseudopregnancy was denoted by an i n f l u x o f
leukocytes i n t o the vaginal lavage. On e i t h e r
Day 7 o r 8 of pseudopregnancy, r a t s were
a n e s t h e t i z e d and prepared f o r ovarian blood
flow determinations using an electromagnetic
blood flow monitor. The ovarian branch of t h e
utero-ovarian a r t e r y was i s o l a t e d , an
a p p r o p r i a t e l y s i z e d and c a l i b r a t e d e l e c t r o d e
placed around the exposed v e s s e l , and a 10-20
minute recording of OBF c o l l e c t e d and analyzed
f o r mean flow r a t e (ml/min). No s t a t i s t i c a l
d i f f e r e n c e s in OBF e x i s t e d between
measurements made on Days 7 o r 8. A highly
s i g n i f i c a n t ( r = 0.985; p < 0.001)
c o r r e l a t i o n c o e f f i c i e n t was found t o e x i s t
between body weight (200-2709) and OBF r a t e s ,
which was not a t t r i b u t a b l e t o d i f f e r e n c e s i n
ovarian weights o r corpus luteum number. A
s i m i l a r , b u t l e s s s i g n i f i c a n t , c o r r e l a t i o n was
found t o e x i s t between body weight and OBF in
r a t s ranging from 170 t o > 3209. When
c o n t r o l l e d f o r body weighT d i f f e r e n c e s , OBF
was found t o remain f a i r l y c o n s t a n t during
pseudopregnancy. These d a t a i n d i c a t e t h a t a
p o s i t i v e c o r r e l a t i o n e x i s t s between body
weight (growth) and OBF in the pseudopregnant
DUNCAN*, Thane E . , W . Keith O'STEEN and Jan E.
OONNELLY, Department of Anatomy, Bowman Gray
School of Medicine, Wake F o r e s t U n i v e r s i t y ,
Winston-Salem, North C a r o l i n a . S t r e s s and
Light-induced r e t i n a l degeneration.
Exposure of a l b i n o r a t s t o h i g h i n t e n s i t y
l i g h t r e s u l t s i n r a p i d l o s s o f photoreceptor
c e l l s (Noel1 e t a l . 1966). The hormonal s t a t u s
and age of a r a t a t exposure i n f l u e n c e t h e
degree of photoreceptor damage (O'Steen 1979).
Preliminary s t u d i e s have i n d i c a t e d t h a t combined e t h e r / s u r g i c a l s t r e s s p r i o r t o exposure
i n c r e a s e s the s u s c e p t i b i l i t y of t h e r e t i n a t o
damage by l i g h t (O'Steen, i n p r e s s ) . When r a t s
are stressed, prolactin secretion increases a s
does ACTH r e l e a s e , s t i m u l a t i n g t h e adrenal t o
r e l e a s e c o r t i c o s t e r o i d s . Adrenalectomized r a t s
have t h i c k e r o u t e r nuclear l a y e r (ONL) measurements than sham-operated animals. S t r e s s e d
r a t s have more s e v e r e l y damaged r e t i n a s w i t h
c y s t i c degeneration and s i g n i f i c a n t l y reduced
ONL t h i c k n e s s measurements a s comDared t o
143
i n c r e a s e d i n d i a b e t i c hamsters b u t t h e l u t e a l
c e l l s e x h i b i t e d a s i g n i f i c a n t ( p < 0.01)
r e d u c t i o n i n l i p i d c o n t e n t . The reduced l i p i d
corresponded w i t h a marked decrease i n serum
p r o g e s t e r o n e l e v e l s i n d i a b e t i c hamsters
compared t o matched c o n t r o l s . Based on t h e s e
r e s u l t s , i t i s suggested t h a t t h e
hypothalamic-ovarian axis i s both
m o r p h o l o g i c a l l y and f u n c t i o n a l l y i m p a i r e d i n
t h e d i a b e t i c hamster. (Supported i n p a r t b y
The Upjohn Company and NIH Grant AM-21933).
r e t i n a s o f u n s t r e s s e d and adrenalectomized r a t s .
Male SD r a t s were housed w i t h m i n i m a l h a n d l i n g
i n c o n t r o l l e d i l l u m i n a t i o n (14 hours 1 i g h t : l O
hours darkness) a t 22 t 2OC f o r one week. Sham
b i l a t e r a l a d r e n a l e c t o m i e s were performed under
ether. A t various time periods a f t e r stress,
animals were exposed t o 24 h o u r s o f c o n t i n u o u s
f l u o r e s c e n t l i g h t (380 ft. c a n d l e s ) a t 30°C.
The r a t s were t h e n housed i n c y c l i c l i g h t f o r
t h r e e weeks u n t i l autopsy. L i g h t m i c r o s c o p i c
e x a m i n a t i o n o f t h e h i s t o p a t h o l o g y was performed
as w e l l as measurement o f t h e r e s u l t i n g r e t i n a l
t h i c k n e s s by o c u l a r micrometer. The d a t a sugg e s t t h a t t h e age o f t h e r a t a t t h e t i m e o f
s t r e s s may p o s s i b l y i n f l u e n c e t h e degree o f
s u s c e p t i b i l i t y o f t h e r e t i n a t o subsequent
l i g h t exposure. Younger a d u l t male r a t s (mean
body w e i g h t = 340 gms) showed l e s s severe damage as w e l l as a d i f f e r e n t r e t i n a l p a t t e r n o f
d e g e n e r a t i o n t h a n d i d o l d e r male r a t s (mean
body w e i g h t = 640 gms). The s e v e r i t y o f r e t i n a l damage v a r i e s w i t h t h e t i m e p e r i o d a f t e r
s t r e s s , b u t t h e p e r i o d o f maximal damage has
n o t y e t been e l u c i d a t e d . T h i s i n f o r m a t i o n may
p r o v i d e a m e t a b o l i c t i m e frame i n which t o
search f o r t h e mechanism by which s t r e s s a f f e c t s the death o f r e t i n a l photoreceptors
induced by l i g h t exposure.
GOODE, G.E., and M.A. Clendenin, Department o f
Anatomy, E a s t e r n V i r g i n i a M e d i c a l School, Norf o l k , VA 23501 M o r p h o l o g i c a n a l y s i s o f neurons
o f the c a t substantia gelatinosa i n t r a c e l l u l a r l y stained w i t h horseradish peroxidase.
Small neurons o f t h e s u b s t a n t i a g e l a t i n o s a
(SG) i n t h e lumbar d o r s a l h o r n o f t h e c a t were
t a r g e t s f o r i n t r a c e l l u l a r r e c o r d i n g s and
enzyme l a b e l l i n g . Some c e l l s were l a b e l l e d by
i n t r a c e l l u l a r iontophoresis while other
neurons were s t u d i e d f o l l o w i n g i n c o r p o r a t i o n
o f e x t r a c e l l u l a r marker p r o t e i n . No more t h a n
t h r e e " i n j u r y f i l l e d c e l l s " appeared i n o u r
s e r i a l r e c o n s t r u c t i o n s of 100 m i c r o n Vibratome
s e c t i o n s r e a c t e d w i t h DAB, i n c o n t r a s t t o
i n t r a c e l l u l a r i n j e c t i o n s when t h e enzyme was
l i m i t e d t o t h e c e l l from which p h y s i o l o g i c r e c o r d i n g s were made. Two d i f f e r e n t c e l l t y p e s
were l a b e l l e d i n t h e s u p e r f i c i a l zone o f t h e
s u b s t a n t i a g e l a t i n o s a , The c e l l s n e a r t h e
marginal-SG i n t e r f a c e have s m a l l o b l o n g o r
rounded soma. T h e i r p r o x i m a l d e n d r i t e s r a d i a t e
t o form s m a l l s p h e r i c a l f i e l d s w i t h i n a
hundred m i c r o n s o f t h e p e r i k a r y o n , whereas
t h e i r d i s t a l dendrites form l o n g i t u d i n a l arrays
f o r s e v e r a l hundred m i c r o n s w i t h i n a c o r d segment. Proximal d e n d r i t e s a r e t o r t u o u s , h i g h l y
branched a r b o r s w i t h d i s t i n c t beaded d i l a t i o n s
a l o n g t h e i r l e n g t h . A v a r i e t y o f complexes o f
s h o r t s p i n e s as w e l l as l o n g t h i n p e d i c u l e s
w i t h s p i n e heads p r o j e c t f r o m t h e s e p r o x i m a l
d e n d r i t e s . The l a r g e r diameter, l o n g l o n g i t u d i n a l d i s t a l d e n d r i t e s o f these c e l l s a r e
r e l a t i v e l y spine f r e e u n t i l a t t h e i r d i s t a l
t i p s t h e y branch i n t o t h i n p e d i c u l e s w i t h
m u l t i p l e s p i n e heads.
The morphology o f these neurons i n t h e
s u p e r f i c i a l SG i s i n c o n t r a s t t o l a b e l l e d
c e l l s i n lamina I V . Their l a r g e r c e l l bodies
are s t e l l a t e , t h e i r dendrites form l a r g e r
s p h e r i c a l f i e l d s p r o x i m a l l y , and t h e i r l a r g e
d i s t a l dendrites p r o j e c t d o r s a l l y through
lamina 111, I 1 and I i n t o t h e d o r s a l r o o t
e n t r y zone. These d e n d r i t e s a r e r e l a t i v e l y
aspinous. The morphology o f l a b e l l e d neurons
w i l l be d i s c u s s e d w i t h r e g a r d s t o t h e i r p h y s i
o l o g y and u l t r a s t r u c t u r e i n an a t t e m p t t o uncover a common denominator f o r t h e m o d u l a t i o n
of n o c i c e p t i v e i n f o r m a t i o n i n t h e i n t e r n e u r o ( S u p p o r t by USPHS Genera
n a l p o o l o f t h e SG.
Research Support G r a n t 218).
Supported by G r a n t No. EY02359 f r o m N a t i o n a l
Eye I n s t i t u t e , NIH.
G a r r i s , O.R., A.R. D i a n i , D. Davis*, G. A l l e n * ,
C. Smith* and G.C. G e r r i t s e n * , E a s t C a r o l i n a
U n i v e r s i t y School o f Medicine, G r e e n v i l l e , NC
27834 and t h e Upjohn Company, Kalamazoo, M I
Aqflnl
Hypothalamic and o v a r i a n a l t e r a t i o n s i n t h e
k e t o n u r i c , d i a b e t i c Chinese hamster.
The r e l a t i o n s h i p between d i a b e t e s and t h e
associated morphological a l t e r a t i o n s i n both
hypothalamic and o v a r i a n t i s s u e s was
i n v e s t i g a t e d i n t h e l o n g term, K e t o n u r i c
d i a b e t i c Chinese hamster. Matched d i a b e t i c and
n o n d i a b e t i c c o n t r o l hamsters were i n s p e c t e d for
r e p r o d u c t i v e c y c l i c i t y b y v a g i n a l lavage f o r
two weeks. D u r i n g d i e s t r u s , each animal was
perfused, t h e hypothalamus and o v a r i e s
c o l l e c t e d , s e c t i o n e d and p r e p a r e d f o r e i t h e r
l i g h t o r e l e c t r o n microscopy. The m i d - p o i n t of
each v e n t r a l hypothalamic n u c l e u s (VMH) was
l o c a t e d , photographed and e n l a r g e d f o r
morphometric a n a l y s i s . R e p r e s e n t a t i v e s e c t i o n s
o f o v a r i e s were t r e a t e d i n a s i m i l a r manner f o r
analysis of size, f o l l i c l e population densities
and l u t e a l s t r u c t u r e . Serum samples were
analyzed f o r p r o g e s t e r o n e c o n c e n t r a t i o n s .
Examination o f t h e VMH o f d i a b e t i c hamsters
r e v e a l e d t h a t b o t h t h e area and a b s o l u t e number
o f neurons/nucleus was reduced compared t o
matched c o n t r o l v a l u e s ( p < 0.01). The d e n s i t y
o f neurons p e r u n i t area o f VMH remained
c o n s t a n t between groups. A l l d i a b e t i c hamsters
were observed t o be a c y c l i c . The o v a r i e s o f
d i a b e t i c hamsters c o n t a i n e d s i g n i f i c a n t l y more
a t r e t i c f o l l i c l e s than t h o s e o f matched
c o n t r o l s . There were no d i f f e r e n c e s between
t h e a b s o l u t e numbers o f p r i m a r y , secondary o r
t e r t i a r y ( g r a a f i a n ) f o l l i c l e s i n t h e groups.
The number o f c o r p o r a l u t e a l o v a r y was
c o n s i s t e n t i n b o t h d i a b e t i c and c o n t r o l
hamsters. L u t e a l area was s i g n i f i c a n t l y
-TI,
A.K., Izbi-atoy Of Neurochemistry,
NIKDS. National Institutes of H e a l t h . Bethesda
Marylid. Ccenparisun of Various A g e n t s of Immunosuppression in Sciiwann, Nerve and Muscle
Cell Transplantation.
144
s k i n which lacks epidermal placodes and dermal
condensations, anchor filaments a r e fewer and
e suggest t h a t
d i s t r i b u t e d randomly. W
these t r a n s i e n t f i bronecti n-associated
anchor filaments play a major r o l e i n
the i n i t i a l establishment of the f e a t h e r
rudiment.
Supported by ACS Grant #lN-l07F and
NSF Grant #PCM-S011745.
I n recent years transplatation of nervous
and muscular tissues has received great attention. Hawever, while a u t q r a f t s survive, allg r a f t s do not because they e l i c i t and are destroyed by an irnnune reaction. In the present
investigation, an analysis was made, to determine whether treatment with various -suppressive drugs muld prevent the rejection of
Schwann, nerve and muscle c e l l s in allografts.
IsOgenic strains of Brown Norway (BN), Fisher
(FR) and k w i s (LE) r a t s whose tissues differ
i n major and minor and only minor transplantation antigens (FR and LE) were used. FR r a t s
served a s hosts and received following LE and/
or F3N tissues: (1) A 4 an segnwt of ne?xe
joined to host peroneal nerve ( 2 ) A nodose
ganglion i q l a n t e d into each sterncmastoid muscle (3) A extensor d i g i t o m longus muscle
placed i n the site of host EDL. Hosts were divided into groups and received no treatment or
daily treatment with one of the following drugs
cyclophosphamide ( 5 nq/kg) ; azathioprine (Az,
5 nq/kg: AZ and prednisolone (5 q / k g each) :
a n t i l y q h q t e sennn (m);
a new drug Cyclosp r i n A (Cy.A, 5 or 25 nq/kg). The untreated
rats rejected a l l a l l q e n i c tissues within 2-4
wks. cyclophosphamide treatment prolonged the
survival of allografts but was toxic t o the
animals (animals l o s t weight and appeared sick).
TZ alone was not effective i n preventing rejection. Hmver, Az and prednisolone together were imnmosuppressive but the mnbination
was also toxic. ALS was effective i n preventing rejection but again pro3uced toxic effects.
C~.A
was very effective i n preventing the rejection a t a l m dose ( 5 nq/kg) with r
m side
effects. However, high dose was toxic to the
animals (25 q / k g over 30 days). It is evident
that C ~ . A is the best inmumsuppressive agent
for preventing the rejection of allqenic
Schwann, nerve am3 muscle cells.
HENKEL, Craig K. and John W . WHITLEY*, Department of Anatomy, Bowman Gray School of Medicine,
Winston-Salem, North Carolina. (Sponsored by
W. K. 0 ' Steen) A pre-eolZicuZar auditory
projection t o the medial geniculate nucleus
i n the eat.
I n general the i n f e r i o r c o l l i c u l u s i s an
o b l i g a t o r y r e l a y i n auditory pathways t o the
thalamus. However, i n this study we r e p o r t
findings w i t h both the anterograde autoradiographic and retrograde horseradish peroxidase
t r a c i n g methods of a b i l a t e r a l projection from
pontine auditory s t r u c t u r e s t o the medial
g e n i c u l a t e nucleus. I n 23 c a t s t r i t i a t e d
leucine i n j e c t i o n s were placed in p a r t s of t h e
s u p e r i o r o l i v a r y complex and nuclei of t h e
l a t e r a l lemniscus. Four cases i n which the
i n j e c t i o n s i t e involved the posteromedial subd i v i s i o n of t h e ventral nucleus of t h e l a t e r a l
lemniscus c l e a r l y labeled a f a s c i c l e of f i b e r s
on each s i d e of the pons t h a t ended i n the
medial divison of the medial geniculate
nucleus. T h e labeled f a s c i c l e s coursed medial
t o the lemniscus, bypassed the i n f e r i o r
c o l l i c u l u s and continued r o s t r a l l y through the
midbrain ventromedial t o t h e brachium of the
i n f e r i o r c o l l i c u l u s . Preliminary HRP r e s u l t s
i n which the i n j e c t i o n s i t e f i l l e d p a r t s of t h e
medial and dorsal subdivisions of the medial
geniculate nucleus confirmed t h a t t h e o r i g i n
of these f a s c i c l e s was mainly l a r g e , multipolar
c e l l s i n the posteromedial subdivision of t h e
ventral nucleus of the l a t e r a l lemniscus.
Additional HRP labeled c e l l s were s c a t t e r e d in
the adjacent a n t e r o l a t e r a l and dorsomedial
p e r i o l i v a r y nuclei. Together the findings
i n d i c a t e t h a t several p r e - c o l l i c u l a r regions
p r o j e c t d i r e c t l y t o thalamo-cortical channels
without relaying in t h e i n f e r i o r c o l l i c u l u s .
HAAKE, Anne R.*and SAWYER, Roger H.* Department
o f Biology, University of South Carolina,
Columbia, S.C. 29208 (Introduced by Or. D.L.
Odor). D i s t r i b u t i o n of f i b r o n e c t i n during
normal and abnormal f e a t h e r morphogenesis.
The avian mutant, s c a l e l e s s , undergoes abnormal f e a t h e r morphogensis. Since f i b r o n e c t i n
i s proposed t o function i n morphogenesis, adhesion, and c e l l u l a r m o t i l i t y , we have compared
i t s d i s t r i b u t i o n during f e a t h e r morphogenesis
i n normal and s c a l e l e s s backskin. I n d i r e c t i m munofluorescence and imnunoferritin e l e c t r o n
microscopy were used t o l o c a l i z e t h e fibronect i n . I t i s absent from the epidermis, b u t
present along t h e basement membrane of both
normal and mutant backskins. However, s i g n i f i c a n t d i f f e r e n c e s a r e seen in the dermal d i s t r i b u t i o n when comparing normal and s c a l e l e s s
backskins. Anchor filaments, which a r e continuous w i t h the basement membrane, extend
deep i n t o t h e dermis during normal f e a t h e r
morphogenesis. Surprisingly, these anchor
filaments g i v e i n t e n s e fluorescence and a r e
ferritin-positive w i t h anti-fibronectin
antibody. They a r e e s p e c i a l l y prominent beneath f e a t h e r placodes, but a l s o occur i n
interplacode regions. The anchor filaments
a r e no longer seen beneath elevated f e a t h e r
germs. Although present i n s c a l e l e s s back-
Supported in p a r t by Grant No. BNS 7918832
from the National Science Foundation.
HIGHTOWER, James A . , and J. David GANGEMI*,
Departments of Anatomy and Microbiology & Immunology, School of Medicine, University of South
Carolina, Columbia, South Carolina. An Autoradiographic Analysis of Murine Lung, Liver and
Spleen C e l l s Following Treatment w i t h a T r i t i m Labeled Irmnunopotentiating Agent (CP-46,665).
Clearance and orqan l o c a l i z a t i o n of t h e svnt h e t i c l i p o i d a l a m i k CP-46,665 was examinedin C3H mice following intravenous inoculation.
Autoradiographs of lung cytocentrifuge preparat i o n s revealed a moderate accumulation of the
drug in a l v e o l a r macrophages w i t h i n 2 hours,
heavier accumulation by 24 hours and p e r s i s t a n c e
a t lower l e v e l s f o r 7 days o r longer. Concomi-
145
v e r t e b r a t e c e n t r a l nervous system. Anatomical
and biochemical c h a r a c t e r i s t i c s of t h e r e t i n a l
ganglion c e l l response t o an o p t i c nerve crush
or axotomy have been well defined in vivo. To
examine in v i t r o conditions which promote t h e
regeneraton of r e t i n a l ganglion c e l l processes,
we varied the preparation of goldfish r e t i n a s
f o r c e l l and t i s s u e c u l t u r e and measured neur i t e outgrowth. Four t o s i x r e t i n a s from common goldfish (Carassius a u r a t u s ) were d i s s o c i ated f o r c e l l c u l t u r e . T h e e f f e c t of a conditioning lesion was determined by t h e administ r a t i o n of an o p t i c nerve crush e i t h e r 7 o r 14
days p r i o r t o the preparation of c u l t u r e s .
Retinas were dissociated i n e i t h e r pronase
or trypsin a t various concentrations. Suspended c e l l s were seeded on 35 mm p l a s t i c
dishes coated w i t h e i t h e r a poly-L-lysine,
polyornithine o r collagen s u b s t r a t e s . Leibouv i t z n u t r i e n t media was supplemented w i t h 10%
f e t a l c a l f serum, 20 mM HEPES and gentomycin
s u l f a t e . C e l l s were grown i n media supplemented w i t h or without methyl c e l l u l o s e . Suspended c e l l s were measured i n several hemocytometer counts. C e l l s i n c u l t u r e were counted
f o r n e u r i t e outgrowth and examined by phase
c o n t r a s t l i g h t and e l e c t r o n microscopy. After
one week in c u l t u r e , d i s s o c i a t e d r e t i n a s which
received a 14-day conditioning crush showed a
4-fold increase i n t h e percentage of ganglion
c e l l s w i t h outgrowth over counterparts which
received no p r i o r l e s i o n . Pronase d i s s o c i a t i o n
produced a 2-fold increase i n c e l l survival and
t h e percentage of neurons w i t h n e u r i t e outgrowth when compared t o t r y p s i n i z a t i o n methods.
After 2 weeks i n c u l t u r e 84% of the attached
r e t i n a l ganglion c e l l s had regenerated processes. Nutrient media which was not supplemented w i t h methyl c e l l u l o s e did not show t h i s
capacity t o support c e l l survival and outgrowth.
Outgrowth was a l s o favored by amino acid subs t r a t e s over c u l t u r e s on collagen s u b s t r a t e s .
Supported by N I H Grant No. NS12070. J.E.T. i s
also the recipient o f N I H Research Career
Development Award NS00338.
t a n t l y , label was observed i n hepatocytes,
Kupffer c e l l s and a v a r i e t y of c e l l types i n
the red pulp of the spleen including macrophages, neutrophils, lymphocytes and developing
erythrocytes. Neither s p l e n i c white pulp nor
draining lymph nodes were heavily labeled.
v i t r o suppression of tumor c e l l growth and synthesis of prostaglandin Ez(PGE2) by a l v e o l a r
macrophages recovered from t r e a t e d mice suggests t h a t CP-46,665 a c t i v a t e s these c e l l s more
quickly ( 2 days versus 5-9 days) and a t lower
dosages (0.3 mg/kg versus 6 mg/kg) than t h e
c l a s s i c a l b a c t e r i a l agent, C. parvum. These
data suggest t h a t the novel s y n t h e t i c l i p o i d a l
amine CP-46,665 i s localized i n a number of
organs and c e l l types and t h a t one o f t h e consequences i s the rapid a c t i v a t i o n of murine
a l v e o l a r macrophages. T h i s work was supported
in p a r t by P f i z e r Grant 5108.
Hougland, M.W. and S k a l k o , R.G.,
Department of
Anatomy, Q u i l l e n - D i s h n e r C o l l e g e of M e d i c i n e ,
y,
E a s t Tennessee S t a t e U n i v e r s i t y
- ,. J o h n s o n C i t ~.
T e n n e s s e e . The Ontogeny of A c e t y l c h o l i n e s t e r ase and Neuromuscular J u n c t i o n s i n F o r e l i m h
M u s c u l a t u r e of Mice.
A c e t y l c h o l i n e s t e r a s e (AChE) a c t i v i t y was
l o c a l i z e d , h i s t o c h e m i c a l l y (Wenger and Wenger,
1 9 7 8 ) , i n t h e f o r e l i m b m u s c l e s of mouse embryos
The r e s u l t s i n d i c a t e
and f e t u s e s from E10-El7.
t h a t t h e s e q u e n c e of change i n AChE l o c a l i z a t i o n i s c o n s t a n t b u t t h a t t h e a p p e a r a n c e and
d i s t r i b u t i o n of r e a c t i o n p r o d u c t i s muscles p e c i f i c . On E13, AChE a c t i v i t y i s d i s t r i b uted d i f f u s e l y over myoblasts i n t h e postaxial
b r a c h i a l r e g i o n (e.g. t r i c e p s b r a c h i i , l a t e r a l
h e a d ) . A wide t r a n s v e r s e band of more i n t e n s e
enzyme a c t i v i t y a p p e a r s o v e r t h e area of muscle
a t t h e s i t e of t h e f u t u r e e n d p l a t e r e g i o n o f
t h e n e u r o m u s c u l a r j u n c t i o n (E15, t r i c e p s b r a c h i i ) . The p a t t e r n of b a n d i n g and t h e l o c a t i o n
o f t h e band is c h a r a c t e r i s t i c f o r e a c h muscle.
F i n a l l y , a t h i n i r r e g u l a r l i n e of enzyme a c t i v i t y a p p e a r s i n t h e c e n t e r of t h e t r a n s v e r s e
b a n d , i n d i c a t i n g t h e p o s i t i o n of t h e d e v e l o p i n g
motor e n d p l a t e .
I n a s e p a r a t e experiment, s e r i a l s e c t i o n s
of f o r e 1 imbs were s t a i n e d t o d e m o n s t r a t e n e r v e s
and n e r v e t e r m i n a l s . On E l l , n e r v e p r o c e s s e s
a r e p r e s e n t i n t h e p e r i p h e r y of t h e limb mesenchyme, e x t e n d to t h e t i p of t h e limb bud by E l 2
and a r b o r i z e among t h e m y o b l a s t s .
As different i a t i o n p r o g r e s s e s (E14-E17), t h e l o c a l i z a t i o n
of t h e s i t e s of n e u r o m u s c u l a r j u n c t i o n s become
d e f i n i t i v e w i t h nerve f i b e r s appearing r e g u l a r l y
a l i g n e d a c r o s s t h e w i d t h of t h e muscle where
AChE a c t i v i t y is most i n t e n s e .
KANTNER*, R o b e r t M. and M a r g a r e t L . KIRBY, Dep a r t m e n t of Anatomy, M e d i c a l C o l l e g e o f G e o r g i a ,
Augusta, G e o r g i a . Changes i n d o r s a l h o r n a c i d
phosphatase a c t i v i t y i n r e s p o n s e t o p a i n .
T r e a t m e n t w i t h c a p s a i c i n h a s b e e n shown t o
d e p l e t e s u b s t a n c e P and a c i d p h o s p h a t a s e (AP)
a c t i v i t y i n t h e s u b s t a n t i a g e l a t i n o s a (SG).
C a p s a i c i n a l s o c a u s e s d e g e n e r a t i o n of axon
t e r m i n a l s t h a t p r e f e r e n t i a l l y mediate chemical
I t h a s been
p a i n s t i m u l i (C f i b e r s ) i n r a t s .
p o s t u l a t e d t h a t AP a c t i v i t y i s i n v o l v e d i n some
a s p e c t of n e u r o t r a n s m i t t e r s y n t h e s i s a n d l o r
d e g r a d a t i o n m e d i a t i n g n o c i c e p t i v e i n p u t . On
t h e 1 6 t h and 1 7 t h d a y s o f g e s t a t i o n , r a t s were
i n j e c t e d w i t h e i t h e r 50 mg/kg of c a p s a i c i n i n
1 O X a l c o h o l , 10% Tween i n 0.9% s a l i n e o r v e h i c l e
( c o n t r o l ) . AP a c t i v i t y w a s s t u d i e d i n t h e SG
o f 15 day o l d o f f s p r i n g o f t h e s e p r e t r e a t e d
dams a f t e r e i t h e r s u b c u t a n e o u s (s.c.) i n j e c t i o n
of formalin (a chronic pain stimulus) o r s a l i n e
i n t o t h e d o r s a l a s p e c t o f the r i g h t f o r e p a w .
S i x t y minutes a f t e r S.C. i n j e c t i o n s , t h e animals
were d e c a p i t a t e d . S e c t i o n s from c e r v i c a l and
lumbar e n l a r g e m e n t s of t h e s p i n a l c o r d were
t r e a t e d by t h e Gomori method f o r a c i d phospha-
S u p p o r t e d by N.I.H. Biomedical R e s e a r c h Development G r a n t III-SO8-RR 091 71-02.
JOHNSON*, James E. and James E. TURNER,
Department of Anatomy, Bowman Gray School of
Medicine, Wake Forest University, WinstonSalem, North Carolina. In v i t r o conditions
which promote the regeneration of goldfish
r e t i n a l ganglion c e l l processes.
The goldfish o p t i c nerve i s an e s t a b l i s h e d
model f o r t h e study of regeneration i n t h e
146
tase. Differences between formalin and saline
groups of capsaicin and control rats for AP
activity were quantified by microdensitometric
readings (Zeiss, 1.0 mm aperture) of SG for the
right and left sides of cervical and lumbar
areas of each animal. Saline-injected control
animals showed much greater AP activity in
cervical and lumbar SG than saline-injected
capsaicin animals. In animals injected with
formalin (forepaw) the AP activity in capsaicin
and control animals was identical. However AP
activity in the lumbar region was decreased in
capsaicin animals. Microdensitometry reflected
the visual differences: formalin treated capsaicin animals had significantly greater AP
activity than saline treated capsaicin. Control and capsaicin animals given s . ~ .formalin
had no significant differences in the cervical
SG/AP activity. These preliminary results
indicate that a functional relationship exists
between chronic chemical pain stimulus and AP
activity of the S G . The exact role of AP in
the spinal cord is unclear; however, changes in
its activity appear to reflect the transmission
of nociceptive input.
Supported by PHS grant DA 02060
KNAPP, L.W.*and SAWYER, R.H.* Department o f
B i o l o g y , U n i v e r s i t y o f South Carolina,
Columbia, S.C. 29208 ( I n t r o d u c e d by D r .
L. T e r r a c i o ) . Morphogenetic c e l l aggregat i o n and k e r a t i n i z a t i o n o f d i s s o c i a t e d
skin cells.
E p i t h e l ial-mesenchvmal i n t e r a c t i o n s a r e
u b i q u i t o u s i n vertebFate embryogenesis. The
morphogenesis and biochemical d i f f e r e n t i a t i o n
o f a v i a n s k i n r e q u i r e s progressive, r e c i p r o c a l
t i s s u e i n t e r a c t i o n s between embryonic epidermis
and dermis (Sawyer 1979 Dev. B i o l . 68, 1-15).
Regional d i f f e r e n c e s i n the types o f e p i d e r m a l s p e c i f i c k e r a t i n p r o t e i n s synthesized a l s o
depend on these t i s s u e i n t e r a c t i o n s . The
morphogenetic p o t e n t i a l o f t h e t i s s u e s o f t h e
s k i n r e f l e c t t h e p r o p e r t i e s and c h a r a c t e r i s t i c s
o f t h e c e l l s o f which they a r e composed.
Therefore, i n v e s t i g a t i o n of t h e morphogenetic
and b i o s y n t h e t i c c a p a c i t y o f s k i n c e l l s d i s s o c i a t e d from i s o l a t e d normal and ' s c a l e l e s s '
mutant embryos (Abbott and Asmundson 1957,
-J._Hered.
_ _ 48, 63-70) i s analysed i n aggregates
by i n d i rec't-imnunofl uroescence u s i n g a n t i sera
produced a g a i n s t alpha- and b e t a - k e r a t i n s .
F s u l t s show t h a t c e l l u l a r aggregates o f
s c a l e l e s s ' and normal s k i n r e o r g a n i z e i n t o
epidermal s t r u c t u r e s which synthesize both
t y p e s o f k e r a t i n s . E l a b o r a t i o n o f alpha-kerat i n s predominates i n aggregates, w h i l e betak e r a t i n s a r e formed o n l y i n subperidermal
c e l l s . Epidermal d i f f e r e n t i a t i o n and r e g u l a t i o n o f k e r a t i n s y n t h e s i s i n v i t r o a l l o w s us t o
analyse t h e r o l e o f t i s s u e q p e c i f i c c e l l
i n t e r a c t i o n s i n s k i n d i f f e r e n t i a t i o n and
m a t u r a t i o n which epitomize e p i t h e l i a l mesenchymal i n t e r a c t i o n s .
Supported by NSF PCM 8011745 t o RHS and
N I H Biomedical Research Support Grant
2 SO7 RR07160 t o LWK.
Morphometry o f C e l l u l a r D i s t r i b u t i o n W i t h i n
the Rat Adenohypophysis.
Althouah t h e r e have been several
q u a l i t a t i v e - 1 i g h t microscopic s t u d i e s
describing the d i s t r i b u t i o n o f
adenohypophyseal c e l l types, t h e c u r r e n t study
quantifies t h i s d i s t r i b u t i o n using electron
microscopy and morphometry. F i v e female
Sprague-Dawley r a t s were c y c l e d and s a c r i f i c e d
a t 9 AM on diestrus-1.
The p i t u i t a r i e s were
c a r e f u l l y removed and t h e a n t e r i o r p i t u i t a r y
was dissected by a c r o s s - s e c t i o n a l c u t through
t h e isthmus. Each h e m i p i t u i t a r y was f u r t h e r
d i v i d e d i n t o q u a r t e r s w i t h 2 b l o c k s coming
from t h e isthmus r e g i o n ( c e n t r a l ) and 2 blocks
from t h e p e r i p h e r y ( i . e . 8 b l o c k s per gland).
The t i s s u e was prepared f o r E.M. and subjected
t o a morphometric a n a l y s i s u s i n g standard
p o i n t counting methods. Three l e v e l s o f
sampling were considered i n c l u d i n g t h e animal
l e v e l , t h e block ( r e g i o n a l ) l e v e l , and t h e
g r i d l e v e l . A nested a n a l y s i s o f variance was
used t o a s c e r t a i n which l e v e l c o n t r i b u t e d t h e
most t o t h e variance w i t h i n t h e system. For
example, i n mammotropes t h e b l o c k ( r e g i o n a l )
l e v e l o f sampling was r e s p o n s i b l e f o r
approximately 78% o f t h e variance as opposed
t o t h e animal (9%) and t h e g r i d (13%) l e v e l s .
The c e l l types were i d e n t i f i e d u s i n g c l a s s i c a l
morphologic features, and i t was found t h a t
t h e r e were more mammotropes i n t h e periphery
than t h e c e n t r a l area (554,470 f 73,511
cells/mn3 t i s s u e vs. 412,158 f 68,130
c e l l s/mn3). There was 1it t l e r e g i onal
d i f f e r e n c e i n t h e somatotropes, and
s u r p r i s i n g l y l i t t l e i n t h e t o t a l gonadotropes.
However t h e r e were more Type1 gonadotropes i n
c e n t r a l regions (58,626 f 10 004 c e l l s / m 3
The number
vs. 35,314 f 10,000 c e l l s / m J ) .
o f c o r t i c o t r o p e s was s l i g h t l y g r e a t e r i n the
c e n t r a l p o r t i o n (80,121 f 10 911 cells/mm3
vs. 67,848
11,993 c e l l s / m J ) .
This study
confirms many o f t h e e a r l i e r q u a l i t a t i v e
observations although t h e magnitude o f t h e
r e g i o n a l d i s t r i b u t i o n appears t o be l e s s than
p r e v i o u s l y thought. (Supported by N.C. United
Way and N I H HD 15259-01.)
Kwasigroch, T.E. and Skalko, R.G., Departmentof
Anatomy, Quillen-Dishner College of Medicine,
East Tennessee State University, Johnson City
Tennessee. The Use of Limb Culture and Image
Analysis in Teratology.
Organ culture of embryonic mouse limbs is a
valuable tool when used to study the mechanisms
of drug action. Its use in screening drugs f o r
teratogenic hazard has not, to date, been validated.Studies are being conducted to evaluate
the potential of using a submerged limb culture
method in screening. Pregnant mice were randomly assigned to one of four treatment groups.
Group 1 was untreated. Group 2 received 250 mg/
kg of hydroxyurea ( H U ) ; group 3 , 500 mg/kg 5bromodeoxyuridine (BrdU) and group 4, 250 mg/kg
HU followed in 3h by 500 mg/kg BrdU. Treatments
were on Embryonic day 11. On E-12, forelimbs
were cut off and cultured in 2 ml of chemicallydefined medium (75% Biggers medium + 25% of a
solution of 7.0g NaC1,0.42g KCland 0.67gCaC12/
1Hp). Limbs were cultured 6 days with medium
changes at days 2 and 4. Cultures were flushed
daily with a gas mixture of 50% 02, 5% C02, 45%
and M.C. Poole. Department
Kornegay, W.D.*,
o f Anatomy, School o f Medicine, East C a r o l i n a
U n i v e r s i t y , G r e e n v i l l e , N.C.
27834.
147
.
neurons vhose dendrites have a very dense
covering of spines. Neurons in CM and CI have
longer, more r e c t i l i n e a r dendrites with fewer
dendritic spines. This study suggests t h a t Ce
consists of a t l e a s t four subdivisions; the
relationship of these subdivisions to neuropeptide localization and connectivity of Ce
remains t o be determined. (Supported, in p a r t ,
by University of South Carolina Grant
11040F411) .
N
Hindlimbs were cultured similarly, but with
26% 02, 5% C02 75% N on days 1 and 2 and 50%
0 on days 3 to 6. &bs
were then fixed in
Biuin's, stained with Alcian blue, mounted on
slides and examined for defects and changes in
areaof limb-hone anlagen using image analysis.
No defects were produced by HU. BrdU produced
both forelimb (6.1%) and hindlimb (24.5%) ectrodactyly and syndactyly (34.7% and 51.0%).
With the HU-3h-BrdU regimen HU had a synergistic effect upon BrdU. Both ectrodactyly (forelimb = 8.2; hindlimb = 38.8%) and syndactyly
(38.7% and 98.0Dincreased. Limb defects using
this in vivolin vitro approach were analogous
to, but 2.5 to 10 fold more common than, what
occured in parallel teratology. Area data of
bone-anlagen were used to compute limb area and
percentage of the limb occupied by long bones
or paw. Forelimb area was reduced 48.5% and
hindlimb area 28% after HU-3h-BrdU. There was
also a comparative reduction in paw area. This
study shows that the in vivolin vitro-image
analysis approach is useful in determining the
type and severity of limb defects and may be
sufficiently quick, easy, sensitive and reproducible to be useful in teratogenicity screening. Supported by N.I.H. Biomedical Research
Development Grant #l-SOB-RR 09171-02.
MCKINNEY, R.V.; SINGH, B.B.; STEFLIK, D.E.; and
SCHAFFNER, D.L.
Department of Oral Pathology,
Medical College of Georgia, Augusta.
Morphological -Observations of Deer (Artiodactyl a)
Tongue Filiform Papillae.
The purpose of t h i s investiqation was toexamine
the keratinization pattern of deer tongue f i l i form papillae and compare i t with that of another ungulate, the cow.previously reported from
our laboratories. Specimens f o r the current
study were taken from the dorsal surface of the
tongues of adult deer and processed f o r l i g h t
microscopy as well as transmission and scanning
electron microscopy. SEM showed mu1 t i p l e posteriorly curved filiform papillae admixed w i t h a
few fungiform papillae. The filiform papilla
apparently consisted of a primary papilla and a
number of smaller secondary projections originating frcm a comnon "bulb-like" structure. The
surface epithelium of the papillae contained
extensive mi croridges . Histological examination
of the primary filiform papillae revealed two
d i s t i n c t components. These included a principle
hard keratin cell l i n e and a clearly demarkated
s o f t keratin cell l i n e , However, the external
projection of the papilla consisted of onlyhard
keratin. The s o f t keratin differentiated from
granular c e l l s containing a plethora o f eosinophilic and a lesser amount of basophilic keratohyalin granules (KHG). The KHG often appeared
as the doninent c e l l u l a r component a t timesdisplacing the nuclei toward the cell periphery.
The hard keratin cell l i n e , which culminates int o the f u l l y keratinized external projection,
differentiated from a nongranular cell l i n e .
U1 trastructurally, the massive KHG exhibited
variable electron density and morphology. Many
KHG appeared to be composites of f i n e granular
and course granular components. Some granules
also exhibited a filamentous component. The
KHG were not limited by membranes and were
often associated with tonofilaments and/orpolyribosomes. This study shows t h a t the deer
tongue filiform papilla has a f u l l y keratinized
external projection similar t o t h a t of cow.
However, the deer filiform papilla has a more
prominant differentiating s o f t keratin cell l i n e
than the cow where the s o f t keratin contributes
only a minor sheath t o the hard keratin core.
Furthermore, the keratinized external projections of deer and cow have marked s i m i l a r i t i e s
to the cortex of the mamnalian hair shaft.
McDONALD*, Alexander J . , Department of Anatomy
University of South Carolina School of Medicine,
Columbia, South Carolina (Introduced bv James
R. Augustine) Cytoarchitecture of the i e n t r a l
amygdaloid nucleus in the r a t .
The central nucleus of the amvadala (Ce)
contains numerous neuropeptides and projects t o
several important subcortical regions involved
in visceral function. Since preliminary reports
indicate t h a t d i s t i n c t neuropeptides and projections are associated with discrete portions
of Ce, a detailed knowledge of the cytoarchitecture of t h i s nucleus should contribute t o an
understanding of i t s overall organization. The
cytoarchitecture of Ce was studied using Nissl,
Kluver-Barrera, and Golgi techniques. Nissl
preparations reveal that Ce consists of several
d i s t i n c t subdivisions. The medial subdivision
(CM) contains a heterogeneous population of
neurons which are larger t h a n c e l l s of more
l a t e r a l portions of Ce. CM i s found medial t o
the s t r i a terminalis caudally; i t increases in
size a t the rostral pole of Ce where i t merges
with the substantia innominata and anterior
amygdaloid area. Adjacent t o CM throughout
most of i t s extent i s CL, the lateral subdivision of Ce. CL appears spherical in coronal
sections and consists of round, medium-sized
neurons. Between CL and the putamen i s a region
of low cell density which contains neurons
similar t o those of CL and the putamen. This
region, which encapsulates the lateral aspect
of CL, i s termed the lateral capsular subdivisi o n of Ce (CLC). Rostrally, CLC i s divided into
dorsal and ventral parts by a laminar extension
of the putamen which extends medially t o become
contiguous with CL. In the rostral portion of
Ce a cell-dense area composed of verticallyoriented neurons i s interposed between CL and
CM; t h i s small area i s termed the intermediate
subdivision of Ce (CI). Golgi preparations
reveal t h a t CL and CLC contain medium-sized
R. Moir*, C. Smith* and
McNeill, M.E.,
C.R. Morgan, Department of Anatomy, School of
kdicne, East Carolina University, Greenville,
North Carolina. The Neurohypophysis in
Alloxan-Diabetic Rats.
148
Arginine vasopressin (AVP; a n t i d i u r e t i c
hormone, ADH) i s produced i n t h e hypothalamus
and r e l e a s e d from axons i n t h e neurohypophysis
( N H ) . Deficiency of ADH i s involved i n t h e
p o l y u r i a and p o l y d i p s i a of d i a b e t e s i n s i p i d u s .
Though a b n o r m a l i t i e s of water metabolism a r e
c m o n i n d i a b e t e s m e l l i t u s , abnormal
f u n c t i o i n of t h e NH i s r a r e l y considered.
There a r e e l e v a t e d ADH l e v e l s i n human
d i a b e t i c s w i t h s e v e r e hyperglycemia (Zerbe,
1981). W
e a r e i n v e s t i g a t i n g t h e NH t o
determine whether t h e r e a r e changes i n t h i s
gland i n a l l o x a n - d i a b e t i c ( A D ) r a t s .
The animals were perfused with
g l u t a r a l d e h y d e and t h e NH was embedded i n
Durcupan f o r LM and TEM study. Semithin c r o s s
s e c t i o n s of the entire NH were s t a i n e d with
t o l u i d i n e b l u e and examined w i t h t h e LM. The
most s t r i k i n g d i f f e r e n c e was t h e increased
l i p i d c o n t e n t of t h e p i t u i c y t e s . The point
counting method of Weibel and Bolender (1973)
was used t o q u a n t i f y l i p i d i n semithin
sections.
N = 3
AD RATS
CONTROLS
STUDENT
TI --TlELCITl
355.33 g
509.00 g
<0.01
Body
t25.86
t9.71
Weight
B1 ood
361.33 mg/dl 116.33 mg/dl <0.001
Sugar
t13.62
t9.33
<O.OOl
L i p i d Content 31.03%
13.7%
( P i t u i c y t e s ) t1.13
t.93
These d a t a suggest t h a t NH f u n c t i o n i s
a l t e r e d i n AD r a t s and t h a t t h e p i t u i c y t e s a r e
involved i n ADH r e l e a s e o r storage. Our
ongoing s t u d y of t h i n s e c t i o n s w i l l provide
more information on the t e r m i n a l s t h a t r e l e a s e
ADH. Immunocytochemical s t u d i e s a r e planned.
The NH i n d i a b e t e s m e l l i t u s deserves a t t e n t i o n
s i n c e d i a b e t i c s a r e markedly more prone t o
renal f a i l u r e t h a n a r e nondiabetics.
MELOAN, S. N., H. PUCHTLER and F. S.
WALDROP, Department of Pathology, Medical
College of Georgia. Effects of d i e structureL
substituents and complex formation on infrared
fluorescence.
Previous investigations in this laboratory
demonstrated infrared (IR) fluorescence of
certain triarylmethane dyes. W e extended these
studies t o other dye classes and t o effects of
metals, phosphomolybdic and picric acid on IR
fluorescence of bound dyes.
Blue and green azo, oxazine, anthraquinone
and triarylmethane dyes with major absorption
bands in the 600-700 nm region were employed.
1R fluorescence was recorded with Kodak
Ektachrorne infrared film as already described
(Histochemistry 5 3 2 1 1-230, 1980).
Ring closure in dye molecules is not
necessary for fluorescence, as has been widely
assumed, but may actually be detrimental; e.g.
several triarylmethane dyes emitted much more
intense fluorescence than anthraquinone and
dioxazine dyes. Within groups of closely
related dyes, IR fluorescence was determined by
t h e nature and position of substituents, and by
t h e extent of n-electron orbitals. Insulating
groups tended t o enhance IR fluorescence; large
molecular orbitals favored dissipation of
energy via radiationless transitions. Picric
and phosphomolybdic acid a r e regarded as
fluorescence quenchers. In this study, their
effect varied widely from dye to dye and ranged
from enhancement t o abolition of IR
fluorescence. Often, the two reagents affected
the same dye differently. Chelation with
chromium or copper has long been known to
abolish fluorescence of dyes; however, a few
metal complex dyes showed strong 1R
fluorescence when bound t o human tissues.
These d a t a indicate that IR fluorescence
is a function of the electronic configuration
of dye molecules and is affected by alterations
of electron orbitals.
Supported in part by Biomedical Research
Support Grant # 2 SO7 RR05365-20.
Mong, F.S.F., M . C . Farach, and M. MartinezCarion, Depts. of Anatomy and Biochemistry,
Medical College of V i r g i n i a , Richmond, VA.
Acetylcholine Receptor (AChR) a c t i v i t y i n f r e e
muscle g r a f t s of r a t s .
Postsynaptic a c e t y l c h o l i n e r e c e p t o r (AChR)
a c t i v i t y i s an i n d i c a t o r of f u n c t i o n a l c a p a c i t y
of s k e l e t a l muscle. For example, p a t i e n t s w i t h
myathenia g r a v i s have very l o w AChR a c t i v i t y .
C o n t r a c t i l e s t u d i e s showed t h a t f r e e muscle
g r a f t s a r e f u n c t i o n a l . However, AChR a c t i v i t y
i n t h e g r a f t s has never been s t u d i e d . Previous
i n v e s t i g a t i o n s show t h a t , i n muscle g r a f t s , presynaptic c h o l i n e a c e t y l f e r a s e (CAT, an e s s e n t i a l
enzyme f o r t r a n s m i t t e r AChR s y n t h e s i s i n axon
t e r m i n a l s ) w a s only 60% of t h a t of normal muscle.
The purpose of t h e p r e s e n t i n v e s t i g a t i o n i s t o
study postsynaptic AChR of muscle g r a f t s and
compare them t o t h a t of normal muscles. The
extensor digitorum longus (EDL) muscle of rats
w a s removed and r e t u r n e d , i n a r e v e r s e manner,
t o t h e o r i g i n a l s i t e and i t s two ends were sut u r e d t o t h e tendon stumps l e f t i n s i t u . The
muscle g r a f t s were removed from 1 t o 105 days
a f t e r surgery. For comparison, normal and denervated ( f o r 1 4 days) muscle were a l s o taken from
d i f f e r e n t animals. For measurement of AChR act i v i t y , t h e muscles or g r a f t s y e r e r i n s e d i n 50
mM phosphate b u f f e r (pH 7.4/10 M EDTA) a t 4OC
and then minced. They were then c e n t r i f u g e d a t
100,OOOg f o r 30 min., e x t r a c t e d i n t h e same
medium containing 1 . 5 % t r i t o n X100, and incubated for 1 5 hours a t 4OC. Afterward, t h e y were
c e n t r i f u g e d again a s above and t h e supernatant
w a s use 25mmediately f o r AChR a s s a y by binding
with I
-' 'a-bungaratoxin a t 4OC overnight by a
modification of t h e method of Schmidt and
Raftery (Anal. Biochem. 52:349, 1973). The
r e s u l t s showed t h a t t h e r e c e p t o r a c t i v i t y of t h e
g r a f t was i n i t i a l l y low. They gradually increased t o t h e peak a c t i v i t y around 1 4 days
p o s t o p e r a t i v e l y (15.63 2 3.59 n mole/g p r o t e i n ) ,
which i s s i g n i f i c a n t l y hiEher than t h a t of
denervated ( f o r 1 4 days) muscle (3.3 n mole/g
p r o t e i n ) . Due t o t h e a c t i o n of r e g e n e r a t i n g
nerves, t h e e x t r a - j u n c t i o n a l AChR of muscle
f i b e r s i n t h e g r a f t s was g r a d u a l l y reduced, so
t h a t , by l o 5 days, t h e t o t a l a c t i v i t y o f t h e
g r a f t (0.478 2 0.03 n mole/g p r o t e i n ) was ins i g n i f i c a n t l y d i f f e r e n t from t h a t Of normal musc l e s (0.375 n molelg p r o t e i n ) . It i s c o n c d d e d ,
t h e r e f o r e , t h a t AChR a c t i v i t y Of EDL-muscle
g r a f t s resemble c l o s e l y t h a t o f normal muscle.
149
outer scale surface a s well as the inner
surface and hinge region. In f a c t , a l l s i x
a-keratin polypeptides found in the whole
scale epidermis are also present in epidermis
taken exclusively from the outer scale surface. Three of these polypeptides demonstrate
a specific enrichment when compared t o the
polypeptides of the e n t i r e scale epidermis.
Imnunofluorescent examination shows t h a t
the a-keratins present in the outer scale
surface a r e restricted t o the Stratum Basale
and Stratum Intermedium w i t h no detectable
antigen in the Stratum Corneum. The Bkeratins of the outer scale surface a r e
localized in the Stratum Intermedium and
Stratum Corneum with no detectable fluorescense in the Stratum Basale.
Supported by NSF Grant #PCM 8011745.
ODOR, D. Louise and Richard J . BLANDAU, Department of Anatomy, School of Medicine, University
of S o u t h Carolina, Columbia, South Carolina and
Department of Biological Structure, School of
Medicine, University of Washington, Seattle,
Washington. Solitary Cilium of Secretory Cells
of Oviductal Epithelium.
The presence of a single cilium on a variety
of cell types has been reported. I t arises from
one of the diplosomal centrioles. Due t o their
r a r i t y in TEM sections, information about them
i s scanty. I t has been speculated that they are
immotile. I n our TEM and SEM studies on oviductal epithelium of the rabbit, pig-tailed
monkey, baboon and human we noted a single
cilium on some of the nonciliated c e l l s . In the
rabbit they occurred most frequently in ovariectomized and ovariectomized-progesterone treated
and less often in estrous, ovulatory and ovariectomized-estrogen treated animals. I n tissue
cultures of rabbit oviductal epithelium a
solitary cilium occurred on many c e l l s . Many
such c i l i a exhibited a n unusual vortical motili t y , beating 11-28 beats per second. The
single cilium was shorter than c i l i a of c i l i a t e d
c e l l s . Interestingly, in some TEM pictures two
basal f e e t lay on opposite sides of the basal
body. In a study in Mollusca, Gibbons reported
(JBBC =179,'61)
t h a t the effective stroke of
the c i l i a r y beat was toward the basal foot. We
propose t h a t the vortical motility i s related
in some way t o the presence of two basal feet.
A normal axonemal complex was visible. During
the primate menstrual cycle (pig-tailed monkey,
baboon) a single cilium was observed more often
during the l a t e luteal and early f o l l i c u l a r
phases t h a n during midcycle. I n imnature
(baboon), anovulatory (baboon) and ovariectomized (monkey) primates such c i l i a were
encountered more often than a t any time during
the cycle. I n specimens from menopausal women
a s o l i t a r y cilium was noted also. I n the monkey,
baboon and human, as in the rabbit, two basal
f e e t were often observed. (Supported by
USPHS g r a n t HD-03752 and contacts HD-3-2788
and 70-2141 and 70-2142).
PEPPLER, R.D. and J. CANALE*. Departments of
Anatomy and Obstetrics and Gynecology, QuillenDishner College of Medicine, East Tennessee
State University, Johnson City, Tennessee and
Louisiana State University Medical Center, New
Orleans, Louisiana. Follicular development 4
rats bearing a copper or plastic intrauterine
device.
Copper IUDs are effective but conclusive evidence concerning their safety is lacking. Rats
with a copper IUD show a decrease in the number
of eggs ovulated from the ipsilateral ovary after one estrous cycle (Contraception 12: 327,
1975) and progesterone secretion is reduced (J.
Reprod. Fertil. 33: 519, 1973). Copper from an
IUD is distributed systemically (Anat. Rec. 196:
247, 1980), but does not affect the release of
219, 1980).
gonadotropins (IRCS Med.
Collectively, these data suggest the contraceptive action of copper is at the ovarian level.
The present study examined the effect of a copper or plastic IUD on follicular development.
Nulliparous, female, Holtzman rats (180 g) were
maintained under fluorescent illumination (05.00
to 19.00 h). After three consecutive estrous
cycles, a segment of pure copper wire or plastic
tubing was inserted into alternate left or right
uterine horns at metestrus. A l l animals were
killed at proestrus after one cycle; one month;
three, six or 1 2 months. Ovaries were removed,
sectioned serially (10 vm) and stained with H&E.
A l l follicles > 295 vm were counted in both ovaries. Differences in the number of follicles
were not apparent after one cycle or one month
among the three groups. However, at three and
six months, the ovary on the IUD (copper) side
contained 46% and 36% less follicles respectively than the contralateral ovary. This difference was not caused by only the ovulatable follicles ( > 448 vm) being affected. In rats with
a copper IUD at one year post-insertion. the IUD
side ovary contained 61% less follicles (6.3 i
2.3) than the non-IUD side ovary (16.3 f 0.9)
and 47% less than the control ovary ( 1 1 . 8 t 1 . 6 ) .
Differences in follicular development between
the control rat and those containing plastic
IUDs (both IUD and non-IUD sides) were not apparent at the other time periods (three, six and
12 months after insertion). The results demonstrate a disturbance in the normal pattern of
follicular development by the copper IUD.
Supported by E.G. Schlieder Educational Foundation and NIH Biomedical Research Grant to ETSU.
s.
a:
O ' G U I N , M.W.*and SAWYER, R.H.:
Department of
Biology, University of South Carolina,
Columbia, S.C. 29208 (Introduced by Dr. W.L.
Poteat). Determination of a and B-type
keratin distribution in avian scutate scales
by biochemical and imnunohistochemical
analysis.
Alpha and B-keratins a r e the products of
d i s t i n c t , developmentally regulated, structural genes. I t has been generally accepted
from histological, f i n e structural and X-ray
diffraction studies of the normal overlapping
(scutate) scale of the chick t h a t only 8-type
keratins are present in the outer epidermal
surface of the scale while a-keratins are
found exclusively in the epidermis of the
inner scale surface and hinge region. We
have reexamined the distribution of aand B-keratins in scutate scales by sodium
dodecyl sul fate-polyacrylamide gel electrophoresis and indirect imnunofluorescence
with non-cross reacting antisera which we
have produced t o both a- and @-keratins.
Contrary t o previous studies, we find t h a t
a-keratins are definitely present in the
150
POTEAT, William L. and Miller 3 . SULLIVAN J r . ,
Department of Anatomy U.S.C. ,School of Medicine,
Columbia, S.C. The Effect of Clomiphene Citrate,
Estradiol, and Proqesterone on Glycogen Phosphorylase and Lipid Accumulation in the Luminal
Epithelium of the Ovariectomized Rat.
Previous work in t h i s laboratory has demons t r a t e d stimulatory e f f e c t of clomiphene ci t r a t e
on glycogen depostiion in the uterine luminal
epithelium of the r a t , an effect inhibited by
progesterone and n o t mimicked by estradiol.Lipid
and glycogen phosphorylase have now been studied
i n these c e l l s by histochemical methods. Ovariectomized r a t s were treated with various combinations of colmiphene c i t r a t e (0.25 mg/kg), estradiol dipropionate ( 1 . 0 ~ 9 )and progesterone (5.0
mg). The r a t s received three consecutive daily
dosages and were killed 24 hours a f t e r the l a s t
dose. Pieces of tissue were removed from the
animal and imnediately frozen in isopentane
chilled in liquid nitrogen. The tissue was
transferred t o a cryostat where sections (12pm
and 20pm) were cut and mounted on glass slides.
Some of these were processed for glycogen demons t r a t i o n by the glycogen phosphorylase technique
u s i n g glucose-1-phosphate a s a substrate. Others
were stained with o i l red 0 t o demonstrate lipid.
The glycogen phosphorylase enzyme appeared i n
greatest concentration i n the same treatment
groups in which glycogen was in the greatest
concentration (estradiol, clomiphene, and clomiphene + estradiol).Progesterone did not s i g n i f i cantly increase phosphorylase concentration nor
was there any apparent increase in phosphorylase
a c t i v i t y in the progesterone combination groups.
Apparently progesterone inhibits clomipheneinduced glycogenesis by a mechanism other than
stimulation of phosphorylase activity. Lipid
accumulation in the luminal epithelium was
greatly increased i n the progesterone-treated
r a t s versus the controls. L i t t l e o r no l i p i d
was observed in these c e l l s a f t e r treatment with
e i t h e r clomiphene o r estradiol. However, a
significant amount of l i p i d was present a f t e r
treatment w i t h progesterone and clomi phene.
Therefore, progesterone does inhibit clomiphene's pronounced increase i n epithelial glycogen, but the drug appears t o have no e f f e c t on
the hormone's stimulation of l i p i d deposition in
the c e l l s .
After six days the tissue fragments are
obviously rounded but remain connected to the
filter by a foot process. Microscopically
the cultured cyst wall fragment is composed of
a central core of connective tissue which is
covered by a simple squamous to columnar
epithelium. The epithelium cells resemble
those of uncultured cysts, are bound by apical
junctions and rest on a basal lamina. The
foot process is a continuation of the
epithelium which spreads over the filter. This
epithelium is squamous and devoid of ciliated
cells.
The hilar region of the ovaries of immature
animals, also has been cultured. After 14 days
of culture this tissue was implanted subcutaneously in the animal from which the
tissue originated. Fifty days after its
implantation this tissue contains cystic
dilations similar to those of the in situ
ovary.
These preliminary studies indicate that the
differentiated cyst wall can be maintained in
culture for at least 15 days and that the
precystic tissue can be cultured and then
implanted to become vascularized and cystic.
These findings are part of an ongoing Study
to determine the effect of certain steroids
on the growth of the serous cystadenoma.
(Supported by NIH Grant 00652)
Department of
SCHWEISTHAL, Michael R . ,
Anatomy, Oral Roberts University, Tulsa,
Oklahoma.
The use of pseudoisocyanin to
stain delta cells.
Pancreas and duodenum were removed en
bloc from adult Sprague-Dawley rats and
fixed in Bouin's fluid. Following routine
histological procedures, the tissues were
embedded in paraplast and sectioned at 4 pm.
Tissue sections were stained with pseudoisocyanin (PCYN) after acid hydrolysis (Solcia
et al., Stain Tech., 43:257, 1968), photographed, destained by being placed in 0.01 N
HC1 for 20 minutes and restained with the
indirect immunofluorescent technique for
somatostatin (Coons et al., J. Exp. Ned.,
102:49, 1955) o r the unlabeled antibody
peroxidase-antiperoxidase
technique
for
somatostatin (Sternberger et al., J. Histochem. Cytochem., 18:315, 1970). A comparison of positive staining PCYN cells and
positive
immunologically
stained
cells
revealed that in every case of pancreatic
tissue they matched. These results confirm
the use of PCYN for staining pancreatic
delta cells in the rat.
However, the
results obtained with staining the duodenum
o f the rat were inconclusive.
Although
there were matches of positive PCYN and
antisomatostatin cells, there were too often
no matches for positive PCYN cells. Some
possible explanations will be explored and
our studies are being continued.
Quattropani, Steven L. Department of Anatomy,
Medical College of Virginia, Richmond, Va.
23298. Culture of Ovarian Serous Cysts.
The rete ovarii of the guinea pig regularly
forms serous cysts as the animal ages.
Because these cysts develop spontaneously and
are similar in appearance to serous cysts which
develop in humans, the nature of their development has been subject to investigation.
Fraqents of ovarian cyst wall approximately
1 mm have been removed and cultured up to 15
days. Cyst fragments were placed on a Millipore filter supported by a stainless steel
grid in Waymouth's MB752/1 medium supplemented
with 10% calf serum and containing penicillin
and streptomycin. Medium level was adjusted
to the level of the filter allowing wetting of
the tissue fragment. Cultures were maintained
at 37.5' in a humidified atmosphere containing
5% C02. Medium was changed at three day
intervals.
SINGH, B . B . , J.L. BOSHELL, D.E. STEFLIK, J.R.
SMITH AN0 R . V . MCKINNEY. Department of Oral
Biology, Medical College of Georgia, Augusta and
Riverbanks Zoological P a r k , Columbia, South
151
o f t h e same d i e t w i t h s u c r o s e r e p l a c i n g t h e
e t h a n o l . A d m i n i s t r a t i o n o f t h e d i e t s began on
g e s t a t i o n a l day 1 2 and c o n t i n u e d u n t i l p o s t n a t a l day 7 ; a t which t i m e t h e e t h a n o l and sucr o s e d i e t s were d i s c o n t i n u e d and t h e g r o u p s
r e t u r n e d t o chow and water d i e t s . L i t t e r s were
s a c r i f i c e d on p o s t n a t a l d a y s 7 , 1 4 and 2 1 f o r
m o r p h o l o g i c a l e v a l u a t i o n . The t i s s u e w a s proc e s s e d e i t h e r w i t h r a p i d G o l g i O K Golgi-Cox
t e c h n i q u e s and s e c t i o n e d in t h e p a r a s a g g i t a l
p l a n e . As a r e s u l t o f p e r i n a t a l e x p o s u r e t o
e t h a n o l , t h e P u r k i n j e c e l l s p r e s e n t a morphoNormally,
l o g i c a l p i c t u r e of r e t a r d e d growth.
t h e murine P u r k i n j e c e l l d i s p l a y s p e r i s o m a t i c
s p i n e s a t 7 days and has a rudimentary a p i c a l
In t h e e t h a n o l - t r e a t e d group, t h e
dendrite.
s p i n e s a r e p r e s e n t , b u t t h e r e i s no e v i d e n c e of
a n a p i c a l d e n d r i t e . By 1 4 d a y s , t h e e t h a n o l t r e a t e d group d i s p l a y s a n a r b o r t h a t i s more
t r u n c a t e d and d i s o r i e n t e d t h a n e i t h e r o f t h e
In a d d i t i o n , some o f t h e neurc o n t r o l groups.
ones have g r o t e s q u e s i l h o u e t t e s .
By 2 1 d a y s ,
t h e r e i s less d i s c e r n i b l e d i f f e r e n c e which l e a d s
one t o s u g g e s t t h a t t h e r e i s some r e c o v e r y from
this alcoholic insult.
shown v a r i a b l e t i n c t o r i a l p r o p e r t i e s of keratohyalin granules (KHG) from f i l i f o r m p a p i l l a e of
d i f f e r e n t species (Anat. Record 199:305, 1981).
These KHG were e i t h e r basophilic (BKHG) o r
eosinophilic (EKHG) i n nature; however, some
granules s t a i n e d both with hematoxylin andeosik,
Therefore, the o b j e c t i v e of t h i s study was t o
c h a r a c t e r i z e the type of KHG i n various primates
Specimens from t h e dorsal s u r f a c e of tongues
from human, rhesus monkey, s p e c i o s i s monkey,
black howler monkey, and lemur were fixed in105
neutral buffered formalin o r 80% methanol f o r
l i g h t microscopy, and 3% gultaraldehyde and 1%
osmic acid f o r e l e c t r o n microscopy. For histochemical s t u d i e s consecutive 5um p a r a f f i n sections were stained a s follows: hematoxylinandlor
eosin f o r t i n c t o r i a l c h a r a c t e r i z a t i o n , Pauly's
reagent f o r proteins r i c h i n h i s t i d i n e and the
Sakaguchi oxine reaction f o r p r o t e i n s r i c h i n
arginine. Light microscopic observations revealed t h a t rhesus and s p e c i o s i s monkeys contained a l a r g e number of E K H G w i t h only an occasional BKHG being observed i n s e r i a l sections.
The human p a p i l l a e s t i l l contained a prominant
EKHG population; however, more BKHG were observed than the two previous s p e c i e s . Theblack
howler monkey and lemur apparently contained
moderate amounts OT both types of granules.
Histochemically the EKGH reacted s t r o n g l y w i t h
the Sakaguchi oxine reaction and the BKHGstrongl y w i t h Pauly's reagent. T h i s confirms our previous observations of o t h e r mammals. Ultras t r u c t u r a l l y both EKHG and BKHG exhibited variat i o n s in morphology and e l e c t r o n d e n s i t y and
were associated w i t h ribosomes and/or tonofilaments. No limiting membrane was noticed cons t r a i n i n g the KHG of any species. EKHG appeared g e n e r a l l y l a r c e r t h a n BKHG, and some components of EKHG were l e s s e l e c t r o n dense i n comparison t o BKHG. T h i s study suggests t h a t
primates e x h i b i t v a r i a t i o n s i n t h e amount and
proportion of EKHG and BKHG. Based upon t h e i r
biological composition this may r e f l e c t species
s p e c i f i c i t y of primate tongue KHG.
This i n v e s t i g a t i o n w a s s u p p o r t e d by a g r a n t
from t h e D i s t i l l e d S p i r i t s Council o f t h e
United S t a t e s , I n c .
SPANGLER*, Kevin M . , Craig K. HENKEL, I n g l i s
J . M I L L E R , JR., Department of Anatomy, Bowman
Gray School of Medicine, Winston-Salem, North
Carolina. (Sponsored by W . J . Bo) LocaZizat i o n of the motor neurons inneruating the r a t
stapedius and tensor tympani muscles: A s t u d y
usinq HRP.
The stapedius (ST) and tensor tympani (TT)
muscles c o n t r a c t i n response t o high i n t e n s i t y
sound s t i m u l a t i o n , i n d i c a t i n g some influence
of brainstem auditory pathways onto t h e motor
neurons f o r these muscles. The l o c a l i z a t i o n
of the neurons innervating t h e ST and TT was
studied i n t h e r a t , using the method of r e t r o grade axonal t r a n s p o r t of horseradish peroxidase (HRP). Unilateral i n j e c t i o n s of a 30%
solution of HRP were made i n t o e i t h e r ST o r TT.
Following a 24 t o 48 hour survival time,
s e r i a l frozen s e c t i o n s through the brainstem
were processed f o r HRP histochemistry. In the
TT cases reaction product was localized i n a
s t r i n g of 25-60 small neurons (12-20 11)
ventral t o t h e motor trigeminal nucleus,
p a r t i c u l a r l y i n i t s r o s t r a 1 extent. These
c e l l s bordered on and were occasionally found
i n t h e l a t e r a l lemniscus. Preliminary r e s u l t s
a f t e r ST i n j e c t i o n s showed small oval and
fusiform neurons f i l l e d w i t h reaction product,
j u s t ventral and medial t o t h e f a c i a l motor
nucleus. These c e l l s were found along the
rostral-caudal e x t e n t of the nucleus. All TT
and ST neurons were found t o l i e o u t s i d e the
trigeminal and f a c i a l motor n u c l e i , respect i v e l y . However, they appear t o be i d e n t i f i a b l e subpopulations associated w i t h t h e s e
nuclei. The position of t h e neurons suggests
possible connections t o auditory s t r u c t u r e s
which could provide pathways mediating the
acoustic middle e a r r e f l e x e s .
SMITH", Diane E . , and David L. D a v i e s , Department o f Anatomy, L o u i s i a n a S t a t e U n i v e r s i t y
Medical C e n t e r , New O r l e a n s , L o u i s i a n a .
(Spons o r e d by M a r i l y n L. Zimny). A l t e r a t i o n s i n t h e
p o s t n a t a l development of t h e c e r e b e l l a r P u r k i n j e
cell following p e r i n a t a l ethanol administration.
The c e r e b e l l u m undergoes a s i g n i f i c a n t port i o n o f i t s development d u r i n g t h e p e r i n a t a l
period.
The s e n s i t i v i t y o f i t s n e u r o n s d u r i n g
t h i s growth s p u r t make them a n e x c e l l e n t model
f o r the study of environmental manipulation.
In o r d e r t o examine t h e m o r p h o l o g i c a l changes
i n t h e Purkinje c e l l a f t e r exposure t o ethanol,
a series of e x p e r i m e n t s were i n i t i a t e d u s i n g a
s t r a i n of mice known t o h a v e a h i g h p r e f e r e n c e
f o r and b e h a v i o r a l t o l e r a n c e t o e t h a n o l (C57BL/
6 3 . Time-pregnant mice w e r e d i v i d e d i n t o t h r e e
groups: c o n t r o l s f e d mouse chow and w a t e r
libitum; experimentals f e d a chocolate-flavored
l i q u i d (Nutrament) f o r m u l a t e d t o c o n t a i n 25% o f
i t s c a l o r i c v a l u e as e t h a n o l (5.4% v / v ) ; and
n u t r i t i o n a l c o n t r o l s p a i r - f e d i s o c a l o r i c amounts
Supported in p a r t by Grant No. BNS 7918832
from the National Science Foundation.
152
SPENCER*, M., H. PUCHTLER and S. N.
MELOAN, Department of Pathology, Medical
College of Georgia. Application -of molecular
orbital theories to biological stains.
The color of biological stains is usually
ascribed to quinonoid rings, as suggested by
Nietzki in 1888. This hypothesis was
discarded by chemists in 1905. Different
quinonoid forms of a dye have been regarded
as resonance structures. However, the
resonance theory does not assume shifts of
charges and rearrangement of bonds from
limiting structure to limiting structure. In
fact, the latter have no real existence, but
indicate that the true configuration of a dye
cannot be shown by either formula. During the
last decade, the resonance theory has been
superseded by mlecular orbital theories. We
applied these concepts to biological stains.
Aromatic rings do not have alternating
single and double bonds. Instead, each carbon
atom contributes a electron to the molecular
orbitals. Heteroatoms can donate one or two
electron pairs. Frequently, electron orbitals
extend over the whole dye molecule, e.g. in
Pararosaniline, Thionine. The positive charge
is not associated with certain groups, but is
distributed oyer the whole dye molecule, hence
t h e term delocalized cationic dyes.
To facilitate application of mlecular
orbital theories to light and fluorescence
microscopy, we redesigned structural dye
formulas in accordance with chemical data by
Hilckel, Wizinger and others. Molecular models
confirmed the validity of these structures and
visualized steric effects of bridge formation
by heteroatoms, e.g. in the transformation of
Malachite Green to Rhodamines. These data
also elucidated concomitant alterations of
fluorescence properties.
These pilot studies indicate that dye
formulas based on molecular orbital theories
are more informative and easier to interpret
than traditional quinonoid formulas.
Supported in part by Biomedical Research
Support Grant // 2 SO7 RR05365-20.
STEFLIK, O.E., B.B. SINGH, J.R. SMITH AND R.V.
Department of Oral Pathology, Medical
College of Georgia, Augusta, and Riverbanks
Zoological Park, Columbia, South Carolina.
A Histological and Electron Microscopic Investigation of Wallaby Tongue Filiform Papillae.
For the past few years our laboratories have
been involved i n studying epithelial cell differentiation of f i l i f o m papillae of various
species i n order t o elucidate the c e l l u l a r basis
underlying t h e i r variable morphology. The obj e c t i v e of t h i s investigation was to examine the
filiform papillae of a marsupial, the DANA
WALLABY of the genus Macropus. For t h i s study,
samples were taken from the dorsal surface of
the tongues of two adult wallabies and processed
f o r l i g h t microscopy as well as scanning and
transmission electron microscopy. SEM displayed
a complex structure of the f i l i f o m papillae
consisting of a thick principle projection,
surrounded by many, long spine-like secondary
projections . Apparently, a1 1 projections originated from a cormnon "bulb-like'' structure. Histologically, the principle projections consisted of
MCKINNEY.
a t l e a s t two major cell l i n e s , d i s t i n c t from a
third interpapillary cell l i n e . The dominent
s o f t keratin papillary cell l i n e differentiated
i n the presence of keratohyalin granules (KHG)
which were eosinophilic or basophilic i n nature.
The t h i n , hard keratin papillary c e l l l i n e lacked keratohyal in granules. Ultrastructurally,
the KHG exhibited varied morphology and electron
density and were associated w i t h ribosomes and
tonofilaments. The granular cell l i n e differentiated into clearer c e l l s containing primarily
f i n e filamentous material. The differentiated
nongranular c e l l s were packed w i t h coarse bundles of tonofilaments rendering these cellsmore
electron dense than the differentiated keratinocytes of the s o f t keratin line. Also, the
secondary projections displayed circular prof i l e s of slender epithelial c e l l s arranged in a
concentric pattern. This study suggests t h a t
the wallaby tongue f i l i f o m papillae iscomposed
of multiple projections eminating from a common
"bulb-like" structure. The projections include
a thicker principle papillae, comprised of s o f t
and hard keratin cell l i n e s , surrounded by mult i p l e spinous projections consisting of concent r i c rings of epithelium. Further, the filiform
papillae of the Dana Wallaby displays similari t i e s with ungulates, specifically the pig.
TERRACIO, L. and DOUGLAS, W.H.J., Department of
Anatomy, University of South Carolina, Columbia,
S.C. and Tufts University, Boston, MA. Organotypic Culture of Prostatic Epithelial Cells.
Monolayer cultures permit the study of a
single parameter on prostatic epithelial c e l l s
without the complicating influences of the
stroma, however, these cultures f a i l t o maintain
the histological organization of the epithelial
c e l l s . This report describes a procedure for
the establishment of organotypic cultures of r a t
ventral prostate (RVP) and canine prostate ( C P )
epithelial c e l l s thar retain a histological
organization similar t o prostatic acini. Aggregates of epithelial c e l l s were isolated from
minced RVP and CP using a collagenase digestion
and selective attachment procedure (Anat. Rec.
197:239, 1980, I n Vitro 16:213, 1980). The
epithelial aggregates were innoculated a t h i g h
density onto a three-dimensional collagen sponge
matrix (Gelfoam; UPJOHN). The cultures were fed
with F12K tissue culture medium supplemented
with 10%horse serum (HS) and maintained in a
humidified atmosphere of 5% CO2 in a i r . The
F12K-10% HS was changed every other day and the
cultures were maintained for 3 weeks in v i t r o .
A t weekly intervals selected cultures were
stained with 2,3,5 triphenyl tetrazoliun chloride ( I n Vitro 16:306, 1980), fixed with 2.5%
glutaraldehyde and 2.0% paraformaldehyde in
0.1M cacodylate buffer, dehydrated in ethanol
and embedded in Epon. Some cultures were fixed
with Bouin's f l u i d , processed for paraffin histology and stained for acid phosphatase immunofluorescence (Fed. Proc. 39:414, 1980). A t a l l
3 time periods, the cultures consisted of cuboidal t o columnar epithelial c e l l s organized
into acini with l i t t l e or no stromal c e l l s present. A t one week in v i t r o the c e l l s contained
what appeared t o be a few scattered secretory
granules. After 2 weeks, the number of granul e s had increased giving the cultures an appear-
153
ance s i m i l a r t o t h e i n t a c t gland. The organot y p i c c u l t u r e s were p o s i t i v e f o r a c i d phosphat a s e immunofluorescence a t a l l 3 time periods.
These r e s u l t s i n d i c a t e t h a t p r o s t a t i c e p i t h e l i a l c e l l s can be maintained i n organotypic cult u r e and t h a t t h e c u l t u r e s maintain much of the
d i f f e r e n t i a t e d morphology seen i n t h e i n t a c t
gland.
Supported by NIH Grant CA26063 and USC American
Cancer S o c i e t y I n s t i t u t i o n a l Award.
located a t the p o s t e r i o r a s p e c t of t h e t h i r d
v e n t r i c l e between t h e habenular and p o s t e r i o r
comnissures. A c o r r e l a t i v e scanning (SEM) and
transmission (TEM) e l e c t r o n microscopic study
was undertaken t o determine t h e r e l a t i o n s h i p
o f t h e deep pineal t o the pineal r e c e s s a s well
a s t h e p o s s i b i l i t y of ependymal s p e c i a l i z a t i o n
w i t h i n t h e pineal r e c e s s a s i s proposed f o r
o t h e r circumventricular organs. Adult male
Mongolian g e r b i l s were used which had been maintained i n a c o n s t a n t l i g h t environment
(LD 14:lO). The tissue was f i x e d by perfusion
w i t h 3% glutaraldehyde, 1%paraformaldehyde i n
0.1W phosphate b u f f e r . The region o f t h e deep
pineal and pineal r e c e s s were d i s s e c t e d o u t and
f u r t h e r processed f o r SEM. The tissue was
c r i t i c a l p o i n t d r i e d , coated w i t h gold and viewed w i t h a JEOL JSM-35. The blocks of t i s s u e
were then embedded i n S p u r r ' s epoxy r e s i n and
sectioned f o r c o r r e l a t i v e TEM. Employing SEM,
t h e pineal r e c e s s could be divided i n t o 3 zones:
c e n t r a l , paracentral and p e r i p h e r a l . The surf a c e c e l l s of the c e n t r a l zone show p r i m a r i l y
m i c r o v i l l i and a v a r i a b l e number o f supraependyma1 c e l l s . The c e l l s of the p a r a c e n t r a l zone
possess m i c r o v i l l i and t u f t s of c i l i a while
those of t h e peripheral zone a r e c h a r a c t e r i z e d
by dense c i l i a t i o n . TEM revealed a deep pineal
gland c o n s i s t i n g o f p i n e a l o c y t e s and g l i a l c e l l s
w i t h t h e pinealocytes predominating. Most s i g n i f i c a n t was t h e appearance of spaces between
t h e a d j a c e n t pinealocytes t h a t contained prof i l e s of numerous c i l i a . The ependymal c e l l s
of t h e c e n t r a l zone a r e f l a t t e n e d although t h e r e
were c a s e s i n which no ependymal c e l l s could
be d e t e c t e d . T h u s t h e morphology o f t h e deep
pineal gland, t h e ependyma and the supraependyma1 s t r u c t u r e s imply t h a t the pineal r e c e s s i s
s p e c i a l i z e d f o r communication between the deep
pineal and t h e cerebrospinal f l u i d .
WALDROP, F. S., H. PUCHTLER and 5. N.
MELOAN, Department of Pathology, Medical
College of Georgia. Infrared fluorescence
patterns of stained muscle in various diseases.
Previous studies of stained myofibrils demonstrated decrease or loss of infrared (IR)
fluorescence in diseases of muscle, e.g., in
Duchenne-type muscular dystrophy, and in myocardial and arterial lesions. W e therefore
extended these studies to muscle samples from
patients with a variety of diseases.
Experiments were carried out on human
autopsy material. Methacarn-fixed paraffin
sections w e r e stained with Alphazurin A and
photographed with Ektachrome infrared f i l m as
described (Histochemistry g:211-230, 1980).
When the same areas were photographed w i t h
conventional and with infrared color f i l m , IR
fluorescence often showed alterations which
were not identifiable by direct light microscopy. Comparative studies of sections from
patients with Duchenne-type, pseudo-hypertrophic and Werdnig-Hofmann i n f a n t i l e muscular
dystrophy demonstrated distinct differences in
fluorescence patterns. Changes of IR fluorescence in spastic quadriplegia, hemiparesis due
to malformation of brain, and Parkinson's disease w e r e again different and easily distinguishable from muscular dystrophies and from
lesions associated w i t h toxic conditions, e.g.
eclampsia, or metabolic disturbances, e.g.
anorexia nervosa. Various pathological conditions not usually regarded as diseases of
muscle, e.g. hepatic cirrhosis, were associated
with striking anomalies of IR fluorescence
patterns and partial loss of fluorescence.
These pilot studies indicate that IR
fluorescence of stained muscle is a sensitive
indicator of alterations of myofibrils. Owing
to the heterogeneity of our test material, t h e
potential diagnostic significance of various IR
fluoresence patterns could not yet be
determined.
Supported in part by Biomedical Research
Support Grant 2 SO7 RR05365-20.
Woodruff, M. L. Baisden, R. H., Department
of Anatany, Quillen-Dishner C o l l e g e of
Medicine, East Tennessee State U n i v e r s i t y ,
Johnson C i t y , Tennessee 37614. The R o l e of
e Fornix
-t h_
_ _ _i n Mcdulation o f P l a E XTRLevels.
The h i e a m p s is thought to be
involved i n r e g u l a t i o n of p i t u i t a r y - a d r e n a l
f u n c t i o n , p a r t i c u l a r l y following perids of
" p y c h o g e n i c stress". T y p i c a l l y plasma
c u r t i c o s t e r o n e l e v e l s have keen measured
a f t e r s t r e s s f u l e v e n t s i n i n t a c t animals and
animals with damage to t h e h i p a m p a l
system. A t p r e s e n t there are m e x t a n t
remrts of t h e e f f e c t of such damage on
plasma a d r e n m r t i c o t r o p i n ( X C H ) l e v e l s
a f t e r stress. I n t h i s experiment rats with
f o r n i x l e s i o n s , sham-operated rats, and
unoperated rats were t r a i n e d i n a shock
avoidance task. Blood w a s drawn a f t e r l i g h t
e t h e r i z a t i o n v i a c a r d i a c p l n c t u r e f r a n rats
i n each s u r g i c a l group either imnediately
a f t e r t r a i n i n g , 1 h r a f t e r t r a i n i n g , or 24 h r s
a f t e r t r a i n i n g . Other rats, matched to the
s u r g i c a l groups t r a i n e d i n shock avoidance,
were e t h e r i z e d and had blood witbdrawn to
provide c o n t r o l plasma samples f o r t h e
e f f e c t s of the blood withdrawal procedure.
Plasma w a s obtained f r a n all blood samples
WELSH, MARCIA G . , Department of Anatomy, School
of Medicine. Universitv of South Carolina.
Columbia, SC 29208. IIntroduced by Dr. R.J.
Weymouth). C o r r e l a t i v e SEM/TEM I n v e s t i g a t i o n of
t h e Pineal Recess of the Mongolian G e r b i l .
The pineal system o f t h e Mongolian g e r b i l
(Meriones unguiculatus) c o n s i s t s of a s u p e r f i c i a l pineal gland t h a t i s connected by a s t a l k
t o a deep pineal gland. The deep pineal i s
154
For the ultrastructural studies a small
piece of tissue was processed routinely f o r TEH.
For the biochemical studies tissue was washed,
minced, lyophilized and weighed. A small portion
of the sample underwent hydroxyproline and
hexosamine assays. The remaining sample was
analyzed for authentic type I and type 111
collagens.
Ultrastructurally, three d i s t i n c t cell
types were noted. There was a population of
relatively typical fibroblasts with thin prof i l e s of rough endoplasmic reticulum containing
4nm microfilaments. The second type was a
myofibroblast-like c e l l , exhibiting a basal
lamina, pinocytotic vesicles, microfilament
bundles, and attachment plaques f o r intercell u l a r connections. The third was mast c e l l s .
Biochemically, the hydroxyproline content o f
the clubfoot tissue was not significantly different from t h a t o f our control, while the
hexosamine content decreased slightly. The
analyses of collagens revealed t h a t about 1015%of collagens in posterior capsule and
talonvicular j o i n t capsule of clubfeet i s of
type 111.
The presence of type 111 collagen was evident in the clubfeet medial connective tissue
a s opposed t o t h a t of the control. There
appears t o be a relationship between the presence of type 111 collagen a n d the presence of
the myofibroblast-like c e l l s . I t has been
postulated t h a t mast c e l l s stimulate the contraction of myofibroblast-like c e l l s . This
observation i s similar t o t h a t seen in
by centrifugation and was subsequently
assayed for ACPH in duplicate using human
MTH standard, ACIW 1-125, and rabbit
anti-ACPH serum in a pfccedure described
by C m k et al. (Ehdocrinolcw,93, 1973).
Statistical analysis of the results of the
assay indicated that none of the ether stress
only groups differed fran each other in
plasma concentrations of ACPH, nor did the
ACl'H concentrations differ among the groups
having blccd drawn 24 hrs after avoidance
training. However, the rats with fornix
transections had significantly higher plasma
concentrations bth imnediately after,
and 1 hr after, avoidance training. These
results are interpreted to indicate that the
hippocamps inhibits ACPH release in rats
with intact brains subjected to severe, but
not rrpderate, environmental stress.
Supported by NIH BRDG #l-SOB-RR
09171-02.
H. SHOJI, R . D'AMBROSIA and 5 .
WILLIG. Ultrastructural and biochemical studies
of s o f t tissue chanqes in clubfeet. Louisiana
S t a t e University Medical Center, New Orleans,
Louisiana .
Although abnormalities in genetics, neuromuscular system and osseous development have
been implicated in clubfoot, few studies have
focused on the s o f t tissues. I n the present
study the ultrastructural and biochemical changes
in ten clubfoot cases ranging in age from s i x
months t o seven years were assessed. The
connective tissues studied were taken from the
medial aspect a t t h e junction of the t i b i o t a l a r navicular j o i n t .
ZIElNY, M. L.,
155
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