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Southern society of anatomists. Twenty-third annual meeting October 5 У8 1983

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SOUTHERN SOCIETY OF ANATOMISTS
Twenty-Third Annual Meeting
October 5-8, 1983
Atlantic Beach, North Carolina
HOST
EAST CAROLINA UNIVERSITY
461
SOUTHERN SOCIETY OF ANATOMISTS
OFFfCERS 1982-83
President
CARL R. MORGAN
(East Carolina University-Greenville, NC)
President-E lect
MARILYN L. ZIMNY
(Louisiana State University-New Orleans)
Past-President
RICHARD G. SKALKO
(East Tennessee State University-Johnson
City)
Secretary- Treasurer
GEORGE E. GOODE
(Eastern Virginia Medical School-Norfolk)
Councilors
RICHARD D. PEPPLER
(East Tennessee State University-Johnson City)
FRANK N. LOW
(Louisiana State University-New Orleans)
W. KEITH O’STEEN
(Bowman Gray Medical School-Winston-Salem)
HUBERT W, BURDEN
(East Carolina University-Greenville, NC)
462
The Southern Society of Anatomists thanks the following firms for their
support of this convention:
CAROLINA
BIOLOGICAL
COMPANY
EASTERNMICROSCOPE
COMPANY/ZEISS
ELECTRON
MICROSCOPY
SCIENCES
FISHERSCIENTIFIC
COMPANY
HEALTHSCIENCES
CONSORTIUM
JEOL
LIPSHAW,INC.
METTLERINSTRUMENT
COMPANY
OPELCO/OLYMPUS
PREISER
SCIENTIFIC/NIKON
SOUTHERN
MICROINSTRUMENTS
(SMI)
VASHAWSCIENTIFIC~LEITZ
463
OPENING SESSION
Carl R. Morgan, Professor and Chairman
Department of Anatomy, East Carolina University
School of Medicine
President, Southern Society of Anatomists
SESSION I
“Reproductive Biology”
(Chairperson: Richard G. Skalko)
SESSION I1
‘‘Cell Biology 1”
(Chairperson: Jack E. Brinn)
SESSION I11
“Neurobiology 1”
(Chairperson: James D. Fix)
SESSION IV
“Neurobiology 2”
(Chairperson: James R. Augustine)
SESSION V
“Methods”
(Chairperson: Carl R. Morgan)
SESSION VI
“Cell Biology 2”
(Chairperson: Marilyn L. Zimny)
Abstracts of papers from platform and poster presentations can be found
alphabetically on pages 465-479.
464
ALLEN, E.R. Department of Anatomy, LSU Medical
Center, 1100 F l o r i d a Avenue, New Orleans, LA
70119. THE MUSCULUS COMPLEXUS OF NORMAL AND
DYSTROPHIC CHICKEN EMBRYOS
Female r a t s were e i t h e r vagotomized ( V x ) o r
sham
vagotomized (SVx) on the morning of
p r o e s t r u s . A t h i r d group underwent SVx and was
p a i r fed (PF) w i t h Vx r a t s . Estrous c y c l e s ,
food i n t a k e and body weight were monitored
daily.
Rats
were
b l e d by o r b i t a l sinus
puncture each a f t e r n o o n a t 1600 h. Seven days
a f t e r surgery, r a t s were d e c a p i t a t e d between
0900 and 1200 h.
Vx d i d n o t a l t e r t h e p r o e s t r u s LH surge on
t h e afternoon f o l l o w i n g surgery. Rats i n a l l 3
groups entered e s t r u s on the day f o l l o w i n g
surgery. Throughout t h e 7 day period, Vx r a t s
SVx
rats
and
weighed
ate
less
then
s i g n i f i c a n t l y l e s s than SVx r a t s a t t h e end o f
PF r a t s a l s o l o s t
t h e experiment (P<0.05).
weight a t a r a t e s i m i l a r t o t h a t o f Vx r a t s .
F o l l o w i n g the i n i t i a l e s t r o u s period, 9 o f 10
Vx r a t s were a c y c l i c f o r 7 days. A l l SVx r a t s
entered a second e s t r u s 4-5 days a f t e r t h e
i n i t i a l e s t r u s . F i v e o f seven PF r a t s a l s o
e n t e r e d e s t r u s a t 5-6 days a f t e r t h e i n i t i a l
e s t r u s . U t e r i n e weights o f Vx and PF r a t s were
lower
than
uterine
weights o f SVx r a t s
(P<0.05),
but
ovarian
weights
were n o t
d i f f e r e n t (P>0.05). These data c o n f i r m the
observation t h a t estrous c y c l i c i t y i s a l t e r e d
i n vagotomized r a t s . We have a l s o shown t h a t
when i n t a c t r a t s are p a i r f e d w i t h vagotomized
rats,
body
weight decreases, b u t e s t r o u s
cyclicity
continues.
These
observations
support t h e hypothesis t h a t t h e vagus nerve
may p l a y a r o l e as modulator o f r e p r o d u c t i v e
function.
(Supported by NICHHD g r a n t s 16364 and 06899)
Abnormalities have p r e v i o u s l y been reported
i n t h e p e c t o r a l muscle of embryos and young
chicks from a pure s t r a i n of New Hampshire Red
chickens homozygous f o r i n h e r i t e d muscular
dystrophy. Fine s t r u c t u r a l s t u d i e s of t h e
musculus complexus i n normal and d y s t r o p h i c
embryos w e r e undertaken because of a sharp
decrease i n hatching by t h e diseased b i r d s .
The complexus was removed from s e r i a l - a g e d ,
perfused embryos and young c h i c k s , c u t i n t o
s m a l l e r p i e c e s , p o s t f i x e d and prepared f o r
transmission e l e c t r o n microscopy.
Thick
(approx. lpm) s e c t i o n s were mounted on g l a s s
s l i d e s and s t a i n e d w i t h a 0.1% s o l u t i o n of
t o l u i d i n e b l u e i n o r d e r t o i n s u r e proper cont e n t and o r i e n t a t i o n before o b t a i n i n g t h i n
ones. E l e c t r o n micrographs were obtained with
a P h i l i p s 300 e l e c t r o n microscope.
Swollen
and d i s t o r t e d o r g a n e l l e s , a s observed i n t h e
p e c t o r a l muscle, were a l s o seen i n t h e
musculus complexus. A l a c k of t h e presence of
l a r g e polyribosomes (myosin s y n t h e s i s ) and t h e
formation of pseudostraps during d i f f e r e n t i a t i o n i n t h e myopathic hatching muscle w e r e
u l t r a s t r u c t u r a l d i f f e r e n c e s found between
normal and d y s t r o p h i c embryos. The musculus
complexus i s indeed a f f e c t e d by i n h e r i t e d
muscular dystrophy.
ALLEN,*L.G.,CAWRENCE,I.E.,Jr.,BURDEN,H.W.
and
HOOSON,*C.A.
Depts. o f Anatomy and O b s t e t r i c s
and Gynecology,East C a r o l i n a U n i v e r s i t y School
o f Medicine, Greenville,N.C. 27834. Analysis
o f t h e e f f e c t o f vagotomy on e s t r o u s c y c l i c i t y
i n the r a t .
Previous s t u d i e s have shown t h a t abdominal
vagotomy
a t p r o e s t r u s or e s t r u s causes a
prolonged p e r i o d o f a c y c l i c i t y i n t h e r a t . I n
t h i s study, we made a f u r t h e r a n a l y s i s o f
t h i s phenomenon i n c l u d i n g determinations o f
cyclicity,
food
intake, body weight,
and
l u t e i n i z i n g hormone l e v e l s f o l l o w i n g vagotomy.
AUGUSTINE, James R. and James F. WHITE*, Departments o f Anatomy and Surgery, U n i v e r s i t y o f
South C a r o l i n a School o f Medicine, Columbia,
South C a r o l i n a . F u n c t i o n a l o r g a n i z a t i o n o f t h e
accessory nucleus i n the savanna baboon, Papio
cynocephal us.
The o r q a n i z a t i o n o f t h e accessory nerve nucl e u s o r accessory nucleus (Nomina Anatomica,
1977) i n t h e savanna baboon, Papio cynocephalus,
i s c u r r e n t l y being i n v e s t i g a t e d u s i n g horseradi s h peroxidase neurohistochemistry.
In a series
465
t i s s u e s . The muscle was s t r e t c h e d b r i s k l y a t
a frequency of 5Hz under loads between 1 and
10 grams. Animals were i n j e c t e d w i t h 25yCi of
14C,ZDG. Anesthesia was maintained and
stimulation continued f o r 45 minutes a f t e r t h e
t r a c e r i n j e c t i o n . Control animals were sham
operated and i n j e c t e d w i t h t r a c e r , but were
not stimulated. Sections of quick-frozen
brains were c o l l e c t e d s e r i a l l y and autoradiographed f o r 10-14 days. Results were
evaluated qua1 i t a t i v e l y . Autoradiographs
showed t r a c e r concentrated above control
l e v e l s b i l a t e r a l l y i n t h e following areas: 1)
accessory cuneate nuclei; 2 ) i n f e r i o r o l i v a r y
n u c l e i ; 3) deep c e r e b e l l a r nuclei; 4) red
n u c l e i ; 5) v e n t r o l a t e r a l thalamic nuclei; 6 )
corpus s t r i a t u m ; 7 ) motor cortex. Labelling
was r e s t r i c t e d t o small areas and o t h e r
portions of t h e same a r e a s were not labeled
above control l e v e l s . No s i g n i f i c a n t l a b e l l i n g
was seen i n nuclei of the dorsal column-medial
lemniscus pathway. These r e s u l t s suggest t h a t
cuneocerebellar and cerebellothalamic pathways
a r e primarily responsible f o r muscle s t r e t c h
relays t o motor cortex.
of baboons, e i t h e r the sternocleidomastoid o r
the t r a p e z i u s muscle was s u r g i c a l l y exposed and
the appropriate branch of the spinal portion of
the accessory nerve (XI) supplying t h a t muscle
was i d e n t i f i e d and e l e c t r i c a l l y stimulated.
This branch was then transected close t o i t s
point of e n t r y i n t o t h e muscle and a 2 cm piece
of s t e r i l e Argyle tubing placed over the d i s t a l
1 cm of the nerve. The proximal end of the
tubing was secured t o the nerve with the a i d of
surgical adhesive (alpha cyanoacrylate). Several m i c r o l i t e r s o f a 25% solution of HRP i n
d i s t i l l e d water were i n j e c t e d i n t o t h e d i s t a l
end of the tubing thus bathing the c u t end of
the nerve i n the HRP s o l u t i o n . The d i s t a l end
of the tubing was then sealed w i t h a small piece
of muscle t h a t was glued i n place. After a 48hour survival time t h e animals were s a c r i f i c e d
via vascular perfusion-fixation, t h e brains d i s sected, and frozen sections (40 microns) c u t
through the brain stem and cervical spinal cord.
The r e s u l t i n g s e c t i o n s were t r e a t e d according t o
the tetramethyl benzidine (TMB)-HRP method of
Mesulam (1978). Our i n i t i a l observations indic a t e t h a t the r o s t r a l p a r t of the accessory n u cleus i n the baboon, extending from lower medull a r y l e v e l s through C 1 and i n t o C 2 , supplies
the sternocleidomastoid muscle. The caudal portion of the accessory nucleus i n t h e baboon, on
the o t h e r hand, extending from C2 cord l e v e l s ,
through C3 and C4 l e v e l s and i n t o the r o s t r a l
p a r t of C5, was found t o supply the trapezius
muscle. Although labeled neurons of the accessory nucleus were confined t o Rexed's lamina IX
throughout t h e i r r o s t r a l -caudal e x t e n t and on
the same side a s the i n j e c t i o n , t h e more r o s t r a l
portion of t h i s nucleus occupies a medial position whereas i t s caudal portion occupies a more
l a t e r a l position i n the ventral horn. The rel a t i o n s h i p of the accessory nucleus t o c e r t a i n
brain stem motor nuclei w i l l be discussed.
BISHOP*,A.L., 0.F7. HENSON, J R . , M.M. HENSON
and D.B. JENKINS, Departments of Anatomy and
S u r g e r y , D i v i s i o n of O t o l a r y n g o l o g y , U n i v e r s i t y
of North C a r o l i n a , Chapel B i l l , North C a r o l i n a ,
(Sponsored by 0.5'. Henson, J r . ) The e f f e r e n t
a u d i t o r y s y s t e m of t h e mustache b a t .
Mustache b a t s , P t e r o n o t u s p a r n e l l i i , have
s p e c i a l h e a r i n g c a p a c i t i e s which e n a b l e them t o
r e s o l v e s m a l l changes i n f r e q u e n c y and u t i l i z e
a "Doppler s h i f t compensation" t y p e o f b i o s o n a r .
One p a r t of t h e i r a u d i t o r y system which seems
w e l l developed compared t o o t h e r Doppler s h i f t
compensating b a t s i s t h e system of e f f e r e n t
n e r v e f i b e r s t r a v e l i n g from t h e b r a i n s t e m t o
t h e h a i r c e l l s of t h e c o c h l e a . T h i s s t u d y w a s
u n d e r t a k e n as t h e f i r s t p a r t of a combined beh a v i o r a l , p h y s i o l o g i c a l and a n a t o m i c a l s t u d y
of t h e e f f e r e n t system i n P t e r o n o t u s .
The
c o u r s e , d i s t r i b u t i o n and m o r p h o l o g i c a l c h a r a c t e r i s t i c s of t h e system were s t u d i e d w i t h HRP
t r a c i n g techniques, acetylcholinesterase hist o c h e m i s t r y , z i n c iodide/osmium ( M a i l l e t ) s t a i n i n g , and by t r a n s m i s s i o n and s c a n n i n g e l e c t r o n
microscopy. H W s t u d i e s showed t h a t many of
t h e crossed e f f e r e n t f i b e r s have t h e i r c e l l
b o d i e s i n a d i s c r e t e n u c l e a r group l o c a t e d medi a l t o t h e LSO w h i l e u n c r o s s e d f i b e r s h a v e
t h e i r c e l l b o d i e s i n a w e l l d e f i n e d group medi a l t o t h e MSO.
Staining f o r acetylcholinesterase showed t h a t t h e c r o s s i n g f i b e r s t r a v e r s e
t h e b r a i n s t e m 2 mm r o s t r a l t o t h e obex. Here
t h e y l i e d i r e c t l y b e n e a t h t h e f l o o r of t h e
f o u r t h v e n t r i c l e and form a r i b b o n - l i k e band
a p p r o x i m a t e l y 300 um wide. The f i b e r s e n t e r
t h e c o c h l e a and w i t h i n t h e o s s e o u s s p i r a l l a m i n a t h e y e x i s t as a d i s c r e t e i n t r a g a n g l i o n i c
s p i r a l b u n d l e . M a i l l e t - s t a i n e d whole mounts of
t h e c o c h l e a showed t h a t t h e e f f e r e n t f i b e r s
pass through t h e habenula p e r f o r a t a t o enter
a w e l l developed i n n e r s p i r a l b u n d l e b e n e a t h
t h e i n n e r h a i r c e l l s . From h e r e f i b e r s p a s s
between p i l l a r c e l l s , f r e q u e n t l y i n b u n d l e s of
BATTON, ROBERT R. Department of Anatomy,
Marshall University School o f Medicine,
Huntington, West Virginia. CNS representation
of r a t forelimb d i g i t a l f l e x o r muscles traced
w i t h 14C, 2-deoxyglucose.
Sensory receptors i n muscles convey
information needed f o r automatic and voluntary
motor c o n t r o l . Perturbations of ongoing
movement o r s t a t i c muscle tone a r e accompanied
by feedback e s p e c i a l l y through Group I
a f f e r e n t s . Feedback helps r e s t o r e motor
performance t o p a t t e r n s e s t a b l i s h e d by c e n t r a l
mechanisms. Experiments i n r a t s were designed
t o s t i m u l a t e predominately muscle spindles i n
an e f f o r t t o c o r r e l a t e t h e i r anatomical
representation and their functional c o n t r i bution t o motor control. Although both
long- and short-latency pathways a r e
postulated f o r musculosensory feedback t o t h e
motor cortex, their s e p a r a t e courses have not
been described. Q u a l i t a t i v e t r a c i n g w i t h 14C,
2-deoxyglucose (2DG) can provide information
simultaneously about nervous system s t r u c t u r a l
and functional organization. Anesthetized r a t s
were prepared f o r the study by d i s s e c t i n g t h e
s u p e r f i c i a l d i g i t a l f l e x o r f r e e of confining
466
two, as upper tunnel fibers. Most fibers lie
in a deep sulcus of a Deiters' cell, and they
terminate as large vesiculated endings which
often indent the hair cell base. Mailletstained whole mounts show efferent endings on
hair cells throughout the cochlea and the distribution to the three outer rows appears
uniform.
Supported by grants from the National Institutes of Health, NS 19031; NS 12445
the primary input of nerve terminals which
transmit sensations of pain. There are two
types of glomerular terminals; fluoride resistant acid phosphatase (FFAP) positive terminals
and terminals which contain substance P. The
terminal fields of these two types occupy the
ventral and dorsal portions of substantia
gelatinosa respectively. Capsaicin administration has been shown to reduce substance P
and FRAF' activity, and, in adult rats, causes
degeneration of central synaptic glomerular
terminals of substantia gelatinosa. Animals
treated with capsaicin exhibit some analegsia
to thermal or chemical stimulation. A surprising result of experiments in which neonatal rats were treated with capsaicin was that,
as young adults, they possess 27% more of the
central terminals than their vehicle injected
littermates. What is the origin of these
terminals? Since unmyelinated afferent fibers
and terminals ending in rat substantia gelatinosa are damaged or functionally compromised by capsaicin, the results reported here
suggest a compensatory afferentation by the
formation of additional replacement terminals,
or by the retention of more glomerular terminals in response to neonatal capsaicin treatment. Recent evidence suggests that A6 fiber
terminals may be involved in this "compensation".
BJORKMAN*, O.R. and G.W. KALMUS* (sponsored by
M.E. MCNEILL), Department o f Biology, East
C a r o l i n a U n i v e r s i t y , Greenvi 11e, North
Carolina. C y c l i c AMP d i s t r i b u t i o n as a
determinant o f neural t u b e morpholog
The r e l a t i v e c o n c e n t r a t i o n and d i t t r i b u t i o n
o f cAMP were determined by i m u n o f l u o r e s c e n c e
a1 ong t h e developing p o s t e r i o r neural tube o f
t h e e a r l y (Stage 10) c h i c k embryo. The normal
d i s t r i b u t i o n o f cAMP i n t h e neural tube was
recorded p h o t o g r a p h i c a l l y i n f r o z e n t r a n s v e r s e
s e c t i o n s o f embryos s t a i n e d immunohistochemically w i t h a f l u o r e s c e i n l a b e l e d antibody
t o CAMP. A l l photographic v a r i a b l e s were h e l d
constant.
R e l a t i v e cAMP c o n c e n t r a t i o n s were
c a l c u l a t e d f o r each s e c t i o n from t r a n s m i s s i o n
densitometer scans across t h e photographic
images o f t h e neural t i s s u e . Stage 10 c h i c k
embryos c o n t a i n a l l l e v e l s o f n e u r u l a t i o n :
neural p l a t e , neural groove, neural f o l d and
closed neural tube. R e l a t i v e CAMP
concentration i s highest i n the p o s t e r i o r
neural p l a t e and decreases a n t e r i o r l y through
t h e neural f o l d t o a minimum i n t h e closed
neural tube. C y c l i c AMP i s l o c a l i z e d along
membranes, a t t h e l a t e r a l l e a d i n g edges o f t h e
neural f o l d , and along t h e presumptive lumenal
s u r f a c e o f t h e tube. Neural tube c l o s u r e can
be i n h i b i t e d by concanavalian A (Con A).
A p p l i c a t i o n o f 2-100 u g h 1 Con A t o embryos i n
c u l t u r e i n h i b i t e d neural tube c l o s u r e i n 60 t o
Immunofluorescence
90% o f t h e embryos t r e a t e d .
f o r cAMP i n Con A t r e a t e d embryos revealed a
change i n cAMP d i s t r i b u t i o n but no s i g n i f i c a n t
change i n r e l a t i v e cAMP concentration.
I n the
t r e a t e d embryos, cAMP f a i l e d t o concentrate i n
t h e l e a d i n g edges o f t h e neural f o l d o r along
t h e lumenal surface o f t h e developing neural
tube.
P e r t u r b a t i o n o f t h e cAMP d i s t r i b u t i o n
w i t h o u t change i n c o n c e n t r a t i o n may be
s u f f i c i e n t t o i n h i b i t CAMP-dependent
developmental processes p o s s i b l y by
interference with the microfilament
c o n s t r i c t i o n known t o be r e q u i r e d t o t r a n s f o r m
t h e neural p l a t e i n t o a tube.
.
Campbell*, S.E. and D.R. Hilbelink, Department
of Anatomv. Universitv of South Florida. Collene
of Medicine, Tampa, FL. Organ blood flow determinations using radioactive microspheres in the
golden hamster.
The radioactive microsphere "reference sample" technique is routinely used in determining
regional or organ blood flows in species ranging
in size from rats to sheep. This technique involves the administration of microspheres into
the left ventricle by either direct cardiac
puncture or by the placement of a cannula into
the left ventricle by way of the common carotid
artery. Both of these approaches present major
problems in species smaller than the rat. The
cardiac puncture technique, when employed in
the smaller laboratory animals, results in a
high percentage of discarded animals because of
the difficulty of accurately entering and maintaining the tip of the injection needle in the
left ventricle. Ventricular cannulation in the
smaller species results in major obstruction of
normal cardiac output due to blockage of the
outflow vessel (aorta) of the left ventricle by
the cannula.
Modifications of the cannulation technique
were employed in this study to determine the
normal blood flow to several organs in the golden hamster. The right common carotid artery was
cannulared with the tip of the cannula positioned at the junction of the right brachiocephalic
artery and the arch of the aorta. This positioning of the cannula, coupled with a slower
infusion rate for the microspheres, allowed for
the adequate mixing of the microspheres with
the blood without obstructing cardiac outflow.
Statisically reliable data was obtained for
normal organ blood flow values (absolute and
L
BROWN, H . Keith, Robert J. LAIRD, Nick N.
PALERMO* and Donn L. SMITH*, Departments of
Anatomy and Pharmacology, University of South
Florida, College of Medicine, Tampa, Florida.
Synaptic reorganization following capsaicin
administration.
It is well established that the substantia
gelatinosa of spinal cord dorsal horn receives
467
.
r e l a t i v e ) and percent c a r d i a c output t o 11
s p e c i f i c organs i n t h e hamster.
By u t i l i z a t i o n
of t h i s modified cannulation technique, i t
should now be p o s s i b l e t o a c c u r a t e l y determine
r e g i o n a l blood flow i n even t h e s m a l l e s t of
l a b o r a t o r y mammals. D e t a i l s of t h e r e g i o n a l
blood flow f o r t h e hamster, as determined by
t h e above described technique, w i l l be presented.
CARSON, Keith A . , WANGQ, Vera D . , and Anne M.
PINKERTONQ, Department of B i o l o g i c a l Sciences,
Old Dominion University and the Department of
Anatomy, Eastern V i r g i n i a Medical School,
Biochemical, p h y s i o l o g i c a l , and pharmacol o g i c a l evidence supports the hypothesis t h a t
c h o l i n e r g i c mechanisms a r e a c t i v e i n the cont r o l of oxytocin and vasopressin ' s e c r e t i o n by
neurosecretory neurons i n the hypothalamic
p a r a v e n t r i c u l a r (PVN) and s u p r a o p t i c (SON)
n u c l e i . However, t h e r e has been no d i r e c t anatomical demonstration of t h i s suspected cholinergicpathway. These experiments were designed
t o i n v e s t i g a t e the u l t r a s t r u c t u r a l f e a t u r e s of
the i n n e r v a t i o n of PVN and SON neurons using
AChE as a marker f o r c h o l i n e r g i c axons and
s y n a p t i c t e r m i n a l s . In t h e s e s t u d i e s , r a t s and
mice were a n e s t h e t i z e d , perfused i n t r a c a r d i a l l y
with f i x a t i v e and had the b r a i n s removed. The
hypothalamus was s e c t i o n e d using a vibratome
and the s e c t i o n s were washed i n b u f f e r and
r e a c t e d f o r the demonstration of AChE a c t i v i t y
using a modification o f the L e w i s and Shute
copper t h i o c h o l i n e method. The s e c t i o n s were
osmicated, dehydrated, and embedded i n wafers
between Teflon-coated c o v e r g l a s s e s . The AChE
s t a i n i n g technique produced abundant r e a c t i o n
product i n t h e s e c t i o n s . By l i g h t microscopy,
the neurons of the PVN and SON were moderately
s t a i n e d . The n e u r o p i l i n t h e s e n u c l e i contained
l i t t l e r e a c t i o n product. Under the e l e c t r o n
microscope t h e r e a c t i o n product was observed i n
the n u c l e a r envelope, endoplasmic reticulum,
and Golgi apparatus of PVN and SON neurons,
The n e u r o p i l contained very l i t t l e AChE r e a c t i o n
product. There w e r e no AChE-positive synapses
c o n t a c t i n g PVN o r SON neurons. The t y p i c a l
p a t t e r n o f AChE r e a c t i o n product l o c a l i z a t i o n
observed i n a r e a s r e c e i v i n g c h o l i n e r g i c innervat i o n w a s n o t observed i n the PVN o r SON. The
r e s u l t s of t h e s e u l t r a s t r u c t u r a l cytochemical
s t u d i e s do n o t support the presence of a d i r e c t
c h o l i n e r g i c p r o j e c t i o n t o t h e PVN o r SON.
and t h e changes induced by o l f a c t o r y
bulbectomy (Obx) were c o r r e l a t e d !ith
locomotor behavior and a t y p i c a l , r e f l e x i v e "
b i t i n g behavior a c t i v i t i e s i n male mice.
O l f a c t o r y - b x was performed i n a d u l t , male CD-1
mice housed 8/caye.
Sham operated animals, i n
which t h e o l f a c t o r y bulbs were exposed but n o t
manipulated, served as c o n t r o l s . Locomotor
a c t i v i t y was monitored w i t h actophotometers
and b i t i n g behavior was q u a l i f i e d by s c o r i n g
t h e head and t a i l l e s i o n s r e s u l t i n g from
i n t r a s p e c i e s f i g h t i n g . Between 2 and 35 days
post-Obx, an increase i n t h e number and
f l u o r e s c e n t - i n t e n s i t y o f indolaminec o n t a i n i n g neurons was i d e n t i f i e d i n the
o l f a c t o r y tubercle, l a t e r a l o l f a c t o r y t r a c t
(bed neurons) and p i r i f o r m cortex. Neurons i n
t h e nucleus accumbens and a n t e r i o r o l f a c t o r y
nucleus a l s o demonstrated increased
h i s t o f l u o r e s c e n c e by Day 35 post-0bx as
compared w i t h c o n t r o l s , b u t t o a l e s s e r e x t e n t
than t h a t observed i n t h e o t h e r o l f a c t o r y
l o c i . The increase i n s e r o t o n i n c o n c e n t r a t i o n s was i n v e r s e l y r e l a t e d t o
locomotor a c t i v i t y and d i r e c t l y c o r r e l a t e d
w i t h b i t i n g behavior i n Obx mice. No change
i n e i t h e r locomotor a c t i v i t y o r b i t i n g
behavior was noted i n c o n t r o l s throughout t h e
study. These r e s u l t s demonstrate t h a t t h e
development o f t h e depressed locomotor
behavior was temporally c o r r e l a t e d w i t h an
enhanced s e r o t o n i n h i s t o f l u o r e s c e n c e i n
s p e c i f i c f o r e b r a i n , o l f a c t o r y regions i n Obx
mice.
Clark, George and R.C. Pennington, Veterans
A d m i n i s t r a t i o n Medical Center; Departments o f
Pathology and Anatomy, Medical U n i v e r s i t y o f
South Carolina, Charleston, South Carolina.
The Musto S t a i n f o r E l a s t i c Tissue.
The Verhoeff s t a i n f o r e l a s t i c t i s s u e has
been used f o r s e v e n t y - f i v e years. There i s
no question t h a t e l a s t i c t i s s u e i s s t a i n e d
b u t o t h e r s t r u c t u r e s are a l s o stained. The
need f o r d i f f e r e n t i a t i o n has always l e n t a
s u b j e c t i v e tone t o t h e questionable s p e c i f i c i t y o f t h e s t a i n . Musto (1981) has r e c e n t l y
published a v a r i a n t which she says does not
r e q u i r e d i f f e r e n t i a t i o n . Our attempts t o use
t h i s s t a i n proved i n t e r e s t i n g but t h e r e was
always overstaining. We have developed a
s i m p l i f i e d v a r i a n t which i s c o n s i s t e n t and
which appears valuable e s p e c i a l l y f o r t h e
study o f diseased blood vessels. A l s o
e l a s t i c f i b e r s i n t h e t r a b u c l e a and capsule
o f t h e spleen and t h e l o n g i t u d i n a l e l a s t i c
f i b e r s i n t h e esophagus are d i s t i n c t l y
stained. A f t e r removal from t h e hematoxylin
s o l u t i o n excess s t a i n i s removed i n t h e count e r s t a i n i n g s o l u t i o n . The procedure developed i s : (1) Oewax and hydrate, ( 2 ) S t a i n
f o r 45 minutes i n 15 m l o f 2% hematoxylin i n
95% ethanol, 10 m l o f 2.5% aqueous
FeC13.6H20, and 25 m l o f 50% ethanol, ( 3 )
Wash thoroughly i n water, ( 4 ) S t a i n 5 minutes
i n 1%woodstain s c a r l e t i n 4% phosphotungstic
acid, ( 5 ) Rinse t h r e e times i n d i s t i l l e d
water, (6) Three changes o f n - b u t y l alcohol,
( 7 ) Clear and mount. E l a s t i c f i b e r s i n blood
CHAMBERLAIN: J.K., O.R. G a r r i s and J.P.
DaVanzo: East 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.
I d e n t i f i c a t i o n o f indol ami ne-concentrati n i
o l f a c t o r y neurons associated w i t h m o d i f i e
locomotor behavior i n bulbectomized mice.
The h i s t o f l uorescent i d e n t i f i c a t i o n o f
i n d o l amine ( s e r o t o n i n ) - c o n c e n t r a t i n g neurons
468
was t h e n made and b o t h c a r o t i d a r t e r i e s were
exposed. S i l k s u t u r e was t h e n p l a c e d under
each c a r o t i d b u t n o t t i e d o f f . The s u t u r e
m a t e r i a l was l e f t i n s i d e and t h e i n c i s i o n was
c l o s e d . On t h e n e x t day t h e wound c l i p s were
removed and b o t h c a r o t i d a r t e r i e s were
o c c l u d e d f o r 30 m i n u t e s . Animals s u b j e c t e d t o
t h e e n t i r e p r o c e d u r e i n v a r i a b l y showed
d e f i n i t e signs o f b r a i n asphyxia (stroke).
However, 76% (16 o u t o f 21) o f t h e l e s s t h a n
one week o l d animals e x h i b i t e d s t r o k e s i g n s
prior t o carotid ligations. O f the eight
a d u l t s s u b j e c t e d t o t h e procedure, none
e x h i b i t e d signs o f stroke p r i o r t o c a r o t i d
l i g a t i o n . D u r i n g t h e i n i t i a l placement o f t h e
c a r o t i d l i g a t u r e s , we observed a marked
d i f f e r e n c e i n t h e development o f t h e c a r o t i d
a r t e r i e s between newborn and a d u l t a n i m a l s .
I n t h e l e s s t h a n one week o l d guinea p i g s , t h e
c a r o t i d a r t e r i e s appeared t o be small and
p o o r l y p e r f u s e d when compared t o a d u l t s . T h i s
r e l a t i v e change i n c a r o t i d s i z e may be a t
l e a s t a p a r t i a l e x p l a n a t i o n f o r t h e observed
d i f f e r e n c e s i n response t o f o u r - v e s s e l
o c c l u s i o n between a d u l t and newborn q u i n e a
pigs. This study p o i n t s t o p r e v i o u s l y
unsuspected d i f f e r e n c e s i n n e o n a t a l and a d u l t
c i r c u l a t i o n i n t h i s species.
vessels, spleen, esophahgus and t r a c h e a a r e
s t a i n e d b l a c k (mouse) o r b l u e (human). There
i s some s t a i n i n g o f f a t c e l l s , and t h e
c y t o p l a s i a o f l i v e r and p a n c r e a t i c a c i n a r
c e l l s a r e h e a v i l y s t a i n e d . Muscle i s red,
c o l l a g e n i s c o l o r l e s s t o red.
DAVENPORT, W.D. AND E.R. ALLEN, Departments
of Oral Pathology and Anatomy, Louisiana
State University Medical Center, 1100 Florida
Avenue, New Orleans, LA 70119.
TEM
OBSERVATIONS ON THE PERMEABILITY OF THE
MICROVASCULATURE IN THE BURSA OF FABRICIUS OF
'GALLUS DOMESTICUS.'
30
years
the
Within
the
past
significance of the thymus and the bursa of
Fabricius in terms of development and
function of the immune system has been
realized.
Considerable information is
available on both organs from a functional
standpoint and considerable morphological
information is available on the thymus. Very
little correlation has been done with respect
to structure-function relationships for the
bursa.
In
this
preliminary
study
the
permeability of the bursa microvasculature
was investigated at the ultrastructural level
using horseradish peroxidase (HRP) as a
tracer. HRP was injected intravascularly via
the wing veins of young chickens and allowed
to circulate for 5, 15 and 30 minutes. After
the appropriate time interval the chicken was
decapitated, exsanquinated and the bursa
removed.
Conventional techniques were used
for development of HRP activity and tissue
processing for electron microscopy.
Careful examination of the microvascular
bed revealed that these vessels prevented
leakage
of
tracer material
into
the
extracellular space suggesting that a bloodbursa1 barrier may exist similar to that
found in the thymic cortex.
As suggested
with the thymus, this selective barrier to
intravascularly circulating macromolecules
may play an important role in the development
and maintenance of the imune system.
DAVIS*, D.G.S.,
R . SALDANHA*, T.M. LOUIS and
A.E. KOPELMAN*, Departments o f P e d i a t r i c s and
Anatomy, East C a r o l i n a U n i v e r s i t y School of
Medicine, G r e e n v i l l e , N o r t h C a r o l i n a . S t r o k e
a f t e r e x p e r i m e n t a l b r a i n i s c h e m i a i n neGJborn
and a d u l t g u i n e a p i g s .
I n an a t t e m p t t o develop an animal model
f o r p e r i n a t a l b r a i n asphyxia, we used a f o u r v e s s e l o c c l u s i o n procedure on newborn and
a d u l t guinea p i g s . Under Metofane"
anaesthesia, a m i d - d o r s a l i n c i s i o n was made i n
t h e neck. A f t e r s e p a r a t i n g t h e s u r r o u n d i n g
muscles, b o t h o f t h e v e r t e b r a l a r t e r i e s were
cauterized. A mid-ventral c e r v i c a l i n c i s i o n
DUDEK,* Ronald Walter, Thomas CHARLES* and
A l a n F. BOYNE, *Department o f Anatomy, E a s t
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 , NC; Department
o f Pharmacology, U n i v e r s i t y o f Maryland,
B a l t i m o r e , MD. (Sponsored b y Hubert Burden)
C C I
I.,.
I n e b e t a c e l l s e c r e t o r y g r a n u l e has been
c l a s s i c a l l v d e s c r i b e d as c o n t a i n i n a an
e l e c t r o n - d e n s e c o r e surrounded b y Zn
e l e c t r o n - l u c e n t h a l o u s i n g aqueous f i x a t i o n
methodoloqy. Our s t u d i e s us1ng q u i c k - f r e e z e
f i x a t i o n methodology show new m o r p h o l o g i c a l
aspects o f t h e s e c r e t o r y g r a n u l e . We c u l t u r e d
a d u l t r a t i s l e t s f o r seven d a s i n R P M I 1640
c o n t a i n i n g 11.1 mM g l u c o s e . z u b s e q u e n t l y , t h e
i s l e t s were q u i c k - f r o z e n a a i n s t a copper b a r
a t l i q u i d n i t r o g e n tempera7ure f o l l o w e d b y
freeze-dryin or freeze-substitution
acetone
c o n t a i n i n g 18 osmium t e t r o x i d e and 0.65% u r a n y l
a c e t a t e ) . The i s l e t s were embedded i n A r a l d i t e
502, sectioned, and c o u n t e r s t a i n e d w i t h l e a d
c i t r a t e and u r a n y l a c e t a t e . Some s e c t i o n s were
s t a i n e d immunocytochemical l y f o r i n s u l i n u s i n g
P r o t e i n A-gold.
We observed t h a t i n q u i c k frozen beta cells:
1) t h e s e c r e t o r y g r a n u l e s
c o n t a i n e d a cob-web, f i l a m e n t o u s m a t e r i a l thaf;
f i l l e d the e n t i r e granule i n c l u d i n g t h e "halo ,
2) t h e f i l a m e n t o u s m a t e r i a l demonstrated
i n s u l i n i m m u n o r e a c t i v i t y , and 3) i n f r e e z e substituted i s l e t s , the granules contained t h e
f i l a m e n t o u s m a t e r i a l and t h e c o r e demonstrated
a p r o m i n e n t r i n g around i t s p e r i m e t e r . We
suggest t h a t t h i s f i l a m e n t o u s m a t e r i a l
demonstratin i n s u l i n immunoreactivity i s
p r e f e r e n t i a l l y extracted during t r a d i t i o n a l
aqueous f i x a t i . o n methodology. Consequently,
t h i s has l e d i n v e s t i g a t o r s t o b e l i e v e t h a t
i n s u l i n i s l o c a l i z e d s o l e l y over the electrondense c o r e o f t h e a r a n u l e . Our r e s u l t s would
i n d i c a t e t h a t i n s u r i n i s present i n the granule
as a f i l a m e n t o u s m a t e r i a l f i l l i n g i t s e n t i r e
c o n t e n t s and t h a t t h e c o r e may be a c o m b i n a t i o n
469
o f i o n s and i n s u l i n b i n d i n g p r o t e i n . I f t h i s
i s true, then conventional t h i n k i n g o f , t h e
e x o c y t o t i c e v e n t which r e q u i r e s e x p u l s i o n o f
t h e e n t i r e c o r e t h r o u g h a l a r g e opening i n t h e
c e l l membrane may r e q u i r e r e - e x a m i n a t i o n . I n
l i g h t o f o u r f i n d i n g , j,t c o u l d be p o s t u l a t e d
t h a t o n l y s m a l l "pores o r i n c r e a s e d membrane
p e r m e a b i l i t y a r e needed f o r t h e f i l a m e n t o u s
m a t e r i a l ( i n s u l i n ) t o be r e l e a s e d f r o m t h e b e t a
cell.
vironment in the limited spawning areas of this
anadromous species, and because of the expanding importance of the species in landlocked
sport fisheries. Preliminary to studies offreshwater-saltwater adaptation, we have conducted a
study of the surface ultrastructure of the gill
arch using routine scanning electron microscopic procedures. Each of the four gill arches
supports two rows of filaments extending posterolaterally and two rows of rakers extending anteromedially. Rows of secondary lamellae cover
the lateral surfaces of each filament, and
numerous spines stud the pharangeal surface of
each raker. All surfaces of the gill arch are
covered by a mozaic of ridged eipithelial cells,
although the ridges are less prominent on the
secondary lamellae. The cells measure 7.5? 2 . 1
pm in diameter, and the ridges measure 0.2 f0.03
Um in width. Apices of chloride cells are present along adjacent borders of ridged cells only
on the apposing surfaces of filaments and at the
bases of secondary lamellae. These apices often
appear as depressions in the epithelium, measure
3.16f 1 . 0 5 pm in diameter, and exhibit numerous
cellular extensions. The lateral row of rakers
on the first gill arch consists of long, thin
appendages. The medial row of rakers on the
first gill arch and all rakers on the other
arches consist only of short, spine-studded,
raised areas on the pharangeal surface of the
arch. There are nine pairs of rakers on each
arch. Taste buds, usually on raised areas ofthe
epithelium are present in a single row on the
pharangeal surfaces of the gill arch itselfand
on the long rakers, and are distributed randomly between the spines on the short rakers.
Taste bud surfaces measure 3.06 +1.1 um in
diameter and exhibit large and small microvilli,
numbering 9 t 4 and 163i56, respectively, per
taste bud.
Donation of fish by the Tennessee Wildlife
Agency is acknowledged and appreciated.
GARRIS, D.R.,
M.S. Dar*and M.M. McConnaughey:
East 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.
E f f e c t s o f - o v a r i a n s t e r o i d s on u t e r i n e
v a s c u l a r r e s p o n s i v i t y t o catecholamines.
U t e r i n e b l o o d f l o w IUBF). o v a r i a n s t e r o i d
hormones and catecholamines ( i A ) a r e
r e c o g n i z e d m o d u l a t o r s o f u t e r i n e metabolism,
g r o w t h and d i f f e r e n t i a t i o n . The i n t e r a c t i o n
and e f f e c t s o f t h e s e parameters on c y c l i c
a l t e r a t i o n s i n t h e guinea p i g u t e r u s were
examined.
UBF was measured and compared t o
c y c l i c v a r i a t i o n s i n u t e r i n e norepinephrine
(NE), a l p h a and b e t a r e c e p t o r c o n t e n t on Days
0,2,6,8 and 12 (Day 0 = o v u l a t i o n ) o f t h e
e s t r o u s c y c l e . The m o d u l a t i o n o f t h e s e
parameters by o v a r i a n s t e r o i d hormones was
assessed i n o v a r i e c t o m i z e d animals f o l l o w i n g 3
days o f o i l , e s t r a d i o l (E: l u g ) , p r o g e s t e r o n e
(P: 2 mg) o r combined PE t r e a t m e n t . U t e r i n e
weight, UBF, alpha and b e t a r e c e p t o r s were
e l e v a t e d on Day 0 when t i s s u e NE was a t b a s a l
l e v e l s . UBF and t i s s u e NE demonstrated a
reciprocal r e l a t i o n s h i p throughout t h e estrous
c y c l e , w h i l e b o t h a l p h a and b e t a r e c e p t o r
l e v e l s remained r e l a t i v e l y c o n s t a n t . P and
E-treatments induced a s i g n i f i c a n t depression,
w h i l e PE-treatment i n c r e a s e d t i s s u e NE l e v e l s .
PE and E - t r e a t m e n t s i n d u c e d a marked i n c r e a s e
i n UBF, a l p h a and b e t a r e c e p t o r l e v e l s as
compared t o o i l - t r e a t e d c o n t r o l s .
UBF i n
E - t r e a t e d animals was u n a f f e c t e d by t o p i c a l
exposure t o e p i n e p h r i n e o r 5-HT, b u t
demonstrated a marked i n c r e a s e f o l l o w i n g
combined ACH- NE t r e a t m e n t . P-treatment
p r e v e n t e d t h e s e CA-induced responses i n UBF.
The p r e s e n t r e s u l t s i n d i c a t e t h a t UBF i s
i n c r e a s e d by E-treatment w h i l e NE l e v e l s a r e
depressed.
P a l o n e had minimal e f f e c t s on any
o f t h e s e u t e r i n e parameters.
It i s suggested
t h a t E-induced u t e r i n e hyperemia i s mediated
v i a d i r e c t e f f e c t s on t h e u t e r i n e v a s c u l a t u r e
and by i n d i r e c t s u p p r e s s i o n o f t i s s u e NE
l e v e l s i n t h e guinea p i g .
HENSON, M.M., D.B. JENKINS, A.L. BISHOP,* and
O.W. HENSON, JR., Departments of Anatomy and
Surgery, Division of Otolaryngology, The
University of North Carolina, Chapel Hill,
North Carolina. A new type of sustentacular
cell in the organ of Corti.
The outer tunnel of the organ of Corti is
surrounded by a number of different types of
supporting cells. Most of these are well known,
e.x., the cells of Boettcher, Deiters and
Henson, but others have only recently been identified (Henson %&., Hearing Res. 10: 153,
1983). A cell type which has not been fully
described is the one that we have designated
the basal cell of the outer tunnel. This cell
is characteristically present in the organ of
Corti of Microchiroptera. TEM, LM and SEM
studies on the mustache bat, Pteronotus E. parnellii have revealed that these basal cells
exist in a single row between the third row of
Deiters' cells and the cells of Boettcher. A
distinctive feature of these cells is a highly
distorted nucleus which can be easily seen in
whole mount preparations. This distortion is
the result of intra- and extracellular accumulations of "empty" vacuoles and of several
different types of granular material. The in-
(Supported by The R o c k e f e l l e r Foundation)
*HARPOLE, Jody, and Fred E. HOSSLER. Department of Anatomy, Quillen-Dishner College of
Medicine, East Tennessee State University,
Johnson City, Tennessee. Surface ultrastructure
of the gill arch of the striped bass, Morone
saxitalis.
Interest in the biology of the striped bass,
Morone saxitalis, has increased recently because of acute awareness of damage to the en-
470
t r a c e l l u l a r m a t e r i a l a p p e a r s t o be w i t h i n w e l l
d e f i n e d compartments which c a n be v i s u a l i z e d i n
TEM m i c r o g r a p h s and by s p e c i a l SEM t e c h n i q u e s .
Some o f t h e e x t r a c e l l u l a r m a t e r i a l i s c o n t i n u o u s w i t h a n o s m i o p h i l i c l a y e r of t h e b a s i l a r
membrane; i r r e g u l a r clumps c h a r a c t e r i s t i c a l l y
occupy compartments wedged between t h e b a s a l
c e l l s and t h e c e l l s o f B o e t t c h e r . The c e l l
s u r f a c e f a c i n g t h e o u t e r t u n n e l i s covered w i t h
a sparse l a y e r of m i c r o v i l l i . A l l of the o r g a p
e l l e s of t h e c e l l are crowded i n t o a s m a l l area
b e n e a t h t h e n u c l e u s and a d j a c e n t t o t h e b a s i l a r
membrane. Some l i t e r a t u r e s u g g e s t s t h a t t h e s e
c e l l s may b e p r e s e n t i n o t h e r mammals b u t t h e y
a p p e a r t o b e h i g h l y modified in b a t s . The
f u n c t i o n of t h e s e c e l l s remains t o be d e t e r mined.
Supported by g r a n t s from t h e N a t i o n a l
I n s t i t u t e s o f H e a l t h , NS 12445; NS 19031.
The c o c h l e a of t h e mustache b a t , P t e r o n o t u s
p. p a r n e l l i i h a s many s p e c i a l i z e d f e a t u r e s and
h a s been t h e s u b j e c t of numerous i n v e s t i g a t i o n s .
L i g h t m i c r o s c o p i c and s c a n n i n g and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c s t u d i e s have
shown t h a t t h e s p i r a l l i m b u s i s b a s i c a l l y s i m i l a r t o t h a t of o t h e r m a m m a l s i n b e i n g composed
of a d e n s e c o n n e c t i v e t i s s u e framework and i n t e r d e n t a l cells. Unlike d e s c r i p t i o n s a v a i l a b l e
f o r t h e s p i r a l l i m b u s of o t h e r m a m m a l s , howe v e r , t h e i n t e r d e n t a l c e l l s of P t e r o n o t u s are
a r r a n g e d i n t i g h t l y packed, r a d i a l l y o r i e n t e d
c o r d s which l i e w i t h i n f u r r o w s between d e n s e
c o n n e c t i v e t i s s u e r i d g e s ( a u d i t o r y t e e t h of
Huschke).
Each of t h e c e l l s h a s a n e n l a r g e d ,
i r r e g u l a r l y shaped b a s a l p a r t . a s t a l k - l i k e
c e n t r a l portion, and a t h i n , f l a t a p i c a l p a r t
which expands t o c o v e r t h e a d j a c e n t areas o f
t h e s u r f a c e of t h e limbus.
Several adjacent
c e l l s may p a r t i c i p a t e i n f o r m a t i o n of " s t a l k s " .
and c y t o p l a s m i c c h a n n e l s as d e s c r i b e d by L i m
f o r t h e limbus of t h e g u i n e a p i g ( J . L a r y n g o l .
O t o l . , 84: 1241, 1970) a r e n o t e v i d e n t . The
expanded b a s a l p o r t i o n s of t h e i n t e r d e n t a l
c e l l s a r e t i g h t l y molded t o n e i g h b o r i n g c e l l s
and t h e c o r d s of c e l l s e x t e n d from t h e a t t a c h ment p o i n t o f R e i s n e r ' s membrane t o t h e i n n e r
s u l c u s ; i n t h e l a t t e r area t h e c e l l s u n i t e
w i t h a c o n t i n u o u s , s p i r a l l y a r r a n g e d group o f
c e l l s t h a t rest on t h e u n d e r s u r f a c e o f t h e
v e s t i b u l a r l i p of t h e l i m b u s . Only a s m a l l
p e r c e n t a g e o f t h e c e l l s seem t o be a s s o c i a t e d
w i t h t h e t e c t o r i a l membrane. I t h a s been widel y advocated t h a t t h e i n t e r d e n t a l cells are
a s s o c i a t e d w i t h t h e f o r m a t i o n and p e r h a p s maint e n a n c e o f t h e t e c t o r i a l membrane. Our anat o m i c a l f i n d i n g s and t h e s t u d y of s t r u c t u r a l
i n t e r r e l a t i o n s h i p s r a i s e questions about t h i s
interpretation.
Supported by g r a n t s from t h e N a t i o n a l
I n s t i t u t e s of H e a l t h , NS 12445; NS 19031.
HENSON, O . W . , J R . , M.M. HENSON, A.L. BISHOP,*
Departments of Anatomy and
and D.B. JENKINS.
S u r g e r y , D i v i s i o n of O t o l a r y n g o l o g y , The
U n i v e r s i t y of N o r t h C a r o l i n a , Chapel H i l l ,
N o r t h C a r o l i n a . O b s e r v a t i o n s on t h e a f f e r e n t
f i b e r s of t h e c o c h l e a i n t h e b a t , P t e r o n o t u s
p. p a r n e l l i i .
The c o c h l e a of t h e mustache b a t , P t e r o n o t u s
p a r n e l l i i , h a s a n enormous b a s a l t u r n w i t h a
prominent, e a s i l y v i s u a l i z e d a r r a y o f s p i r a l
g a n g l i o n c e l l s . A combination o f TEM, SEM and
whole mount s t a i n i n g t e c h n i q u e s h a s a l l o w e d us
t o t r a c e t h e c o u r s e , d i s t r i b u t i o n and terminat i o n o f many f i b e r s i n more d e t a i l t h a n h a s
p r e v i o u s l y been p o s s i b l e .
Groups of a f f e r e n t
f i b e r s l e a v e t h e o s s e o u s s p i r a l lamina v i a t h e
habenula p e r f o r a t a and immediately p a s s t o t h e
b a s e s o f t h e i n n e r h a i r c e l l s . These unbranched f i b e r s s u r r o u n d t h e b a s e of e a c h h a i r c e l l .
The few a f f e r e n t f i b e r s t h a t do n o t s y n a p s e
with t h e inner h a i r c e l l s course obliquely
a c r o s s t h e f l o o r of t h e t u n n e l o f C o r t i and
f i r s t s p a c e of Nuel. Here t h e y are b u r i e d i n
s u l c i of t h e c e l l membranes o f t h e p i l l a r c e l l s
but they surface a t regular i n t e r v a l s .
The
p o i n t s a t which t h e y s u r f a c e c o r r e s p o n d t o t h e
j u n c t i o n s between a d j a c e n t p i l l a r c e l l s . A t
these p o i n t s t h e diameter of each f i b e r nearly
doubles. After crossing the f i r s t space of
Nuel t h e f i b e r s c o n t i n u e t o r u n i n a p a r a l l e l
a r r a y on t h e i n n e r s u r f a c e of t h e f i r s t row of
D e i t e r s ' c e l l s ; o n l y h e r e does b r a n c h i n g o c c u r
and a t e a c h branch p o i n t t h e f i b e r s are
s w o l l e n . The f i b e r s r e a c h e a c h h a i r c e l l by
p a s s i n g t h r o u g h a s i n g l e o p e n i n g i n t h e cupshaped p o r t i o n o f t h e Deiters' c e l l . Within
t h i s opening t h e individual f i b e r s l i e i n isol a t e d grooves. Five t o seven f i b e r s t y p i c a l l y
innervate each o u t e r h a i r cell.
Supported by g r a n t s from t h e N a t i o n a l
I n s t i t u t e s o f H e a l t h , NS 12445; NS 19031.
LEONARD,* M . J . , C.P. SMITH*, C.A. HODSON*, T.M.
LOUIS, 1.E. LAWRENCE, JR., L.G. ALLEN* AND H.Y.
BURDEN, Departments o f Anatomy and O b s t e t r i c s
and
Gynecology,
East
Carolina University,
Greenville,
North Carolina.
The e f f e c t o f
vagotomy on o v a r i a n f o l l i c u l a r development
U n i l a t e r a l ovariectomy (ULO) i n r a t s doubles
the
number
o f ovulating f o l l i c l e s i n the
remaining
ovary
as a r e s u l t o f increased
f o l l i c u l a r recruitment.
Increased weight o f
t h e ovary i . e . compensatory o v a r i a n hypertrophy
(COH) thus r e s u l t s from t h e increased number o f
corpora l u t e a . Abdominal vagotomy ( V x ) i n h i b i t s
COH.
H i s t o l o g i c a l a n a l y s i s o f these o v a r i e s
suggests
increased
interstitial
g l a n d and
d i m i n u t i v e f o l l i c u l a r development. Since t h e
i n t e r s t i t i a l gland i s d e r i v e d p r i m a r i l y from
atretic
follicles,
the
c u r r e n t s t u d y was
conducted t o q u a n t i t a t e t h e number o f h e a l t h y
and a t r e t i c f o l l i c l e s a t v a r i o u s times a f t e r V x
o r sham surgery ( s V X ) .
%rum t e s t o s t e r o n e and
basal LH l e v e l s were a l s o determined i n t h e two
groups.
Vx o r sVx was performed on t h e morning
of p r o e s t r u s ( 0 h, day 0) between 0900 and 1100
h
under
ether
anesthesia.
Rats
were
subsequently d e c a p i t a t e d a t 24 h, 48 h, 96 h,
o r 8 days.
Trunk blood was c o l l e c t e d and t h e
serum was separated and s t o r e d a t -60°C u n t i l
radioimmunoassay
for
LH and t e s t o s t e r o n e .
.
JENKINS, D.B., A.L. BISHOP,* O.W. HENSON, J R .
and M.M. HENSON. The Departments o f Anatomy
and S u r g e r y , D i v i s i o n of O t o l a r y n g o l o g y , The
U n i v e r s i t y o f North C a r o l i n a , Chapel H i l l ,
North C a r o l i n a . The s t r u c t u r e and c e l l u l a r
o r g a n i z a t i o n o f t h e s p i r a l limbus of t h e
cochlea.
471
O v a r i e s were s e r i a l l y s e c t i o n e d and t h e t o t a l
number o f h e a l t h y and a t r e t i c a n t r a l f o l l i c l e s
were counted.
Vx ( 1 ) i n c r e a s e d (p<0.05) t h e
number
o f h e a l t h y f o l l i c l e s 151-394 um i n
diameter
at
24 and 48 hs, ( 2 ) i n c r e a s e d
(p<0.05)
the
number
of a t r e t i c f o l l i c l e s
151-394 um a t 96 h and 8 days, ( 3 ) decreased
(p<0.05) t h e number o f l a r g e h e a l t h y a n t r a l
f o l l i c l e s (395-570 urn and > 570 urn) a t 24 h,
( 4 ) i n c r e a s e d (p<0.05) a t r e s i a i n t h e 395-570
um f o l l i c l e s a t 48 h . There was no d i f f e r e n c e
i n t h e number o f l a r g e f o l l i c l e s a t 96 h b u t Vx
caused a decrease (p<0.05) i n t h e number o f
l a r g e h e a l t h y f o l l i c l e s >570 um a t 8 days. Vx
had no e f f e c t on serum t e s t o s t e r o n e l e v e l s a t
any t i m e s t u d i e d .
However, LH l e v e l s were
depressed a t 48 h (p<0.05), 96 h (p<0.005) and
8 days (p' 0.001).
C o l l e c t i v e l y these r e s u l t s
suggest t h a t t h e vagus. n e r v e p a r t i c i p a t e s i n
the
control
of
LH
secretion, f o l l i c u l a r
recruitment,
a t r e s i a and i n t e r s t i t i a l g l a n d
formation i n the r a t .
(Supported b y NICHHD Grant HDD6889).
McGinty, J.F.* Dept. o f Anatomy, East 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.
qt.rain d i f f e r s n r s s i n t h e d i s t r i b u t i o n o f o p i o i d p e p t i d e immunoreactive neurons i n t h e
c e r e b r a l c o r t e x and hippocampus o f r a t s .
A w i d e s w e a d o o o u l a t i o n o f neurons
c o n t a i n i n g ' proenkephal in-A and/or
p r o e n k e p h a l i n - B i m m u n o r e a c t i v i t y has been
detected i n the cerebral cortex o f r a t s
(McGinty, Van d e r Kooy, and Bloom, i n p r e s s ) .
Noted h e r e a r e d i f f e r e n c e s i n t h e d i s t r i b u t i o n
and i n t e n s i t y o f s t a i n i n g o f t h e s e o p i o i d
p e p t i d e - c o n t a i n i n g neurons i n two w h i t e r a t
s t r a i n s , Sprague Dawley and F i s h e r 344, and
one brown r a t s t r a i n , B D I X . A n t i s e r a t o
e n k e p h a l i n and b o v i n e adrenal m e d u l l a r y
p e p t i d e (BAM), fragments o f proenkephalin-A,
and an a n t i s e r u m t o dynorphin-A, a fragment o f
proenkephalin-B, s t a i n e d neurons i n l a y e r s
11-111 and V - V I o f n e o c o r t e x as w e l l as i n
l a y e r s 11-111 o f o l f a c t o r y c o r t e x . I N E D I X
r a t s , enkephalin-immunoreactive neurons were
f a r more numerous and i n t e n s e l y s t a i n e d i n
these r e q i o n s and i n t h e s u h i c u l a r and
hippocampal f i e l d s t h a n i n Spraque Dawley r a t s
of t h e same sex and age. I n F i s h e r r a t s ,
dynorphin-immunoreactive neurons were inore
numerous i n t h e o l f a c t o r y c o r t e x and n e o c o r t e x
t h a n i n Sprague Dawley r a t s . Mossy f i b e r s i n
t h e hippocampus c o n t a i n e d a h i g h c o n c e n t r a t i o n
of d y n o r p h i n i n b o t h w h i t e r a t s t r a i n s .
(The
B D I X s t r a i n was n o t t e s t e d w i t h d y n o r p h i n
a n t i s e r u m ) . However, BAM-immunoreactivity was
o n l y e v i d e n t i n mossy f i b e r s o f t h e F i s h e r
r a t s and u n d e t e c t a b l e i n t h o s e o f t h e Sprapiie
Dawley r a t s . These d a t a may i n d i c a t e
prominent d i f f e r e n c e s among- r a t s t r a i n s i n t h e
e x p r e s s i o n o f o p i o i d p e p t i d e r g i c genes and
s h o u l d serve as a caveat f o r p e p t i d e a n a l y z e r s
who t h i n k a r a t i s a r a t i s a r a t .
Supported by NS 20451.
MASTRIANNI: J.A., A.J. 1ngenito"and D.R.
G a r r i s , East 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.
Localization o f beta-endorphin concentrating
neurons i n t h e m e d u l l a o f t h e spontaneously
hypertensive r a t : association with
c a t e c h o l a m i n e - c o n t a i n i n g neurons.
The d i s t r i b u t i o n o f b e t a - e n d o r p h i n (BE)c o n c e n t r a t i n g , and catecholamine (CA)c o n t a i n i n g , neurons w i t h i n t h e m e d u l l a o f
c o n t r o l and spontaneoulsy h y p e r t e n s i v e (SHR)
r a t s was determined u s i n g b o t h i n v i v o and i n
v i t r o a u t o r a d i o g r a p h y and h i s t o f l u o r e s c e n c e 7
P u r i f i e d 125[I]-8E was a d m i n i s t e r e d b y
e i t h e r i n t r a v e n t r i c u l a r i n j e c t i o n o r was
i n c u b a t e d on f r o z e n b r a i n s e c t i o n s . A l t e r n a t e
s e c t i o n s were prepared f o r CA l o c a l i z a t i o n .
Neuronal b i n d i n g o f 125[I]-BE was b o t h
s p e c i f i c and s a t u r a b l e , and was found t o
c o n c e n t r a t e i n t h e brainstem, e s p e c i a l l y ,
w i t h i n the p e r i v e n t r i c u l a r medullary n u c l e i .
Both BE and CA neurons e x h i b i t e d a s i m i l a r
d i s t r i b u t i o n p a t t e r n i n the medulla.
B E - c o n c e n t r a t i n g c e l l s were l o c a t e d i n t h e
n u c l e u s t r a c t u s s o l i t a r i u s (NTS), r e t i c u l a r
f o r m a t i o n (magnocel l u l a r RF-M), accessory
cuneate n u c l e u s (CUN), and nucleus t r a c t u s
s p i n o - t r i g e m i n a l (NTST), i n c o n t r o l and SHR
r a t s , b u t were decreased i n number i n SHR
animals. C A - c o n t a i n i n g neurons were l o c a l i z e d
i n t h e same n u c l e i as w e l l as i n t h e n u c l e u s
ambiguus (NA). The number o f CA neurons was
e l e v a t e d i n SHR animals as compared w i t h
controls.
These r e s u l t s i n d i c a t e t h a t BE- and
CA-neurons have a s i m i l a r d i s t r i b u t i o n p a t t e r n
w i t h i n the medullary n u c l e i recognized t o
modulate CNS i n f l u e n c e s on t h e c a r d i o v a s c u l a r
system. The decreased b i n d i n g o f BE and
i n t e n s e CA h i s t o f l u o r e s c e n c e i n SHR animals as
compared w i t h c o n t r o l s suggests t h a t a causal
i n t e r a c t i o n e x i s t s between t h e s e n e u r o n a l
systems i n t h e r e g u l a t i o n o f c a r d i o v a s c u l a r
dynamics.
D.I..,
D . J . FLETCHER,* R.4. DUDEK,*
McNEILL,*
BRINN,*
Department o f Anatomy, E a s t
and -1.E.
Carolina
University,
Greenville,
North
Burden)
An
Caroliqa.
(Sponsored b y H.W.
immunocytochemical
s t u d y o f the p a n c i e a t z
i s l e t system o f t h e channel c a t f i s h , I c t a l u r u s
ounctatas.
The
endocrine
pancreas o f t h e channel
c a t f i s h i s segregated i n t o a l a r g e p r i m a r y
i s l e t and numerous s m a l l e r secondary i s l e t s .
Several
earlier
studies
have
reported
approximations o f the various endocrine c e l l
populations i n t h e p r i n c i p a l i s l e t . b u t t o
d a t e no q u a n t i f i c a t i o n s have been r e p o r t e d on
t h e e n t i r e i s l e t system.
I n view o f i s l e t
cell
d i s t r i b u t i o n d i f f e r e n c e s i n mammalian
i s l e t s o f v e n t r a l and d o r s a l p r i m o r d i a , we
decided
to
d e t e r m i n e i f t h e p r i m a r y and
secondary i s l e t s i n t h e c a t f i s h had s i m i l a r
differences.
Sections
of
primary
and
secondary
islets
were immunocytochemically
stained using a n t i s e r a t o a n g l e r f i s h i n s u l i n
and
porcine
glucagon.
The
appropriate
a b s o r p t i o n c o n t r o l s were a p p l i e d i n each case.
Cell
types
were
quantified
on
light
m i c r o g r a p h s b y p o i n t - c o u n t i n g p r o c e d u r e s . The
r e l a t i v e percentages o f i n s u l i n - r e a c t i v e c e l l s
(Supported b y a g r a n t f r o m The American H e a r t
Association: North Carolina A f f i l i a t e )
472
were
31.Rt1.2
and
32.4~1.6 i n the
p r i m a r y a d secondary i s l e t s , r e s p e c t i v e l y .
Similarly,
the
glucagon-reactive
cells
amounted t o 23.2+1.8% and 24.2+1.1% i n
the
principal
and
secondTry
islets,
respectively.
Thus,
the
glucagon
cell
p o p u l a t i o n does n o t r e f l e c t any d i f f e r e n c e s
between t h e two groups o f i s l e t s as m i g h t be
expected
if
the
secondary
islets
were
homologous
with
the
mammalian
ventral
It i s i n t e r e s t i n g t o
pancreatic d e r i v a t i v e .
note t h a t t h e i n s u l i n c e l l p o p u l a t i o n i s l e s s
than
has
been r e p o r t e d e a r l i e r f o r t h i s
species and i s about h a l f o f t h a t r e p o r t e d f o r
mamalian i s l e t s .
This f u r t h e r supports t h e
suggestion t h a t f i s h are n o t as dependent as
mamnals on i n s u l i n as a r e g u l a t o r y agent.
Q u a n t i f i c a t i o n s o f somatostatin and p a n c r e a t i c
polypeptide
cells
are i n progress as an
additional clue t o the possible s i m i l a r i t i e s
between c a t f i s h i s l e t s and t h e two d e r i v a t i v e s
of t h e mammalian pancreas.
E.C. SIMPSON*, J.T. BRAY*,
MCNEILL, M.E.,
CRULE*, P.C. YOUNT* and C.R. MORGAN,
Departments o f Anatomy, Biology and Surgery,
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 , North
Carolina. Whole body z i n c analyses o f d i a b e t i c
and c o n t r o l r a t s .
-changes
i n zinc (zn) l e v e l s i n
selected tissues i n i n s u l i n d e f i c i e n t r a t s
suggests t h a t Zn metabolism i s a l t e r e d i n
experimental diabetes.
P r e l i m i n a r y data show
decreased Zn i n some t i s s u e s and elevated
l e v e l s i n o t h e r t i s s u e s from male r a t s w i t h
a l l o x a n - d i a b e t e s o f 10 months d u r a t i o n (McNeill
et. a l . , 1981). A b a s i c question i s whether
t h e t o t a l amount o f Zn i n d i a b e t i c animals i s
elevated, depressed, o r normal. This study
compares t h e whole body c o n c e n t r a t i o n o f Zn i n
chronic alloxan-diabetic r a t s t o t h a t o f
c o n t r o l animals.
E i g h t male Sprague-Dawley r a t s made i n s u l i n
d e f i c i e n t w i t h an i.v. i n j e c t i o n o f a l l o x a n (40
mg/kg) were p a i r e d w i t h age-matched c o n t r o l s .
Terminal body weight and blood glucose l e v e l s
were determined a t s a c r i f i c e 10 months l a t e r .
Each animal was weighed a f t e r d i s p o s i n g of t h e
contents o f t h e d i g e s t i v e t r a c t and u r i n a r y
b l a d d e r and was heated i n an autoclave f o r one
hour. A whole r a t homogenate was prepared.
Three r e p l i c a t e s from each animal were placed
i n acid-washed tubes, freeze d r i e d and weighed.
Zn c o n c e n t r a t i o n was determined by atomic
a b s o r p t i o n spectrophotometry.
The mean c o n c e n t r a t i o n i n t h e c h r o n i c
d i a b e t i c animals was 76 t 2 ug Zn/g d r y t i s s u e
compared to 31 t 1 i n t h g c o n t r o l s ( p < 0.01).
The values were-19 5 1 pg Zn/g carcass i n t h e
d i a b e t i c r a t s and 16 t 1 i n t h e c o n t r o l s
( p < 0.01).
The cause(s) o f t h e increased Zn
c o n c e n t r a t i o n i n i n s u l i n d e f i c i e n t r a t s and i t s
consequences remain( s ) unknown.
M.D.
(Supported i n p a r t by U n i t e d Way Grant
0- 58026. )
Mehl, Robert E., and James D. F i x ,
Department o f Anatomy, Marshall U n i v e r s i t y
School o f Medicine, Huntington, West V i r g i n i a .
neuronal populations based on N i s s l s t u d i e s
(Olszewski and B a x t e r 1954). However, more
r e c e n t r e p o r t s have i n d i c a t e d more than two
types o f neurons e x i s t i n t h e L.C. o f some
l a b o r a t o r y animals (Swanson 1976, C i n t r a e t .
a l . 1982). I n t h i s r e p o r t we d e s c r i b e t h r e e
d i f f e r e n t neuron p o p u l a t i o n s based on G o l g i
impregnations o f several human brainstems.
B r a i n s were perfused w i t h i n 24 hours
postmortem w i t h h e p a r i n i z e d s a l i n e , and f i x e d
i n f o r m a l i n f o r l e s s than 48 hours. This was
f o l l o w e d by c l a s s i c a l r a p i d Golgi f i x a t i o n
w i t h potassium dichromate and impregnation
w i t h s i l v e r n i t r a t e . Transverse s e c t i o n s
o f 120 lm were c u t on a vibratome and
examined under t h e 1i g h t microscope. Neurons
were grouped i n t o t h r e e major types based on
c y t o a r c h i t e c t u r a l data.
Large m u l t i p o l a r c e l l s which c h a r a c t e r i s t i c a l l y accumulate melanin have e l l i p t i c a l
somata t h a t d i s p l a y spines and l o n g e r t h i n
processes. Each soma g i v e s r i s e t o f o u r t o
seven 1arge primary d e n d r i t e s . One d e n d r i t e
usually t r a v e l s ventro-medially, o f t e n t o
t h e Raphe n u c l e i . Primary d e n d r i t e s
t y p i c a l l y branch i n t o secondary o r t e r t i a r y
d e n d r i t e s which show an i n t i m a t e r e l a t i o n s h i p
t o vasculature.
I n t e r m e d i a t e s i z e d b i p o l a r neurons were
observed i n a dorso-ventral o r i e n t a t i o n .
The somata o f these c e l l s a r e f u s i f o r m i n
shape and do n o t e x h i b i t spines o r o t h e r
l o n g e r processes seen on melaninated neurons.
Each g i v e s r i s e t o two primary d e n d r i t e s ,
which were observed t o branch i n t o several
secondary d e n d r i t e s t r a v e l ing up t o
200 microns.
A t h i r d t y p e o f c e l l observed i s m u l t i p o l a r and c h a r a c t e r i z e d by a t r i a n g u l a r
soma. Each p o l e g i v e s r i s e t o a primary
d e n d r i t e t h a t branches dichotomously.
These c e l l s do n o t e x h i b i t somatic spines.
NITZ? A.J., and D.H. MATULIONIS, Department of
Anatomy, University of Kentucky Medical Center,
Lexington, Kentucky. Electrophysiologic and
motor end-plate response to pneumatic tourniquet compression.
Tissue ischemia during limb surgery is routinely produced by the pneumatic tourniquet.
This often leads to functional and electromyographic (EMG) abnormalities. The present study
evaluates EMG and histologic (motor end-plate)
changes in rat legs following tourniquet application. The right or left hindlimb of 90 rats
(Sprague-Dawley, male, 300-3508) was compressed
by a tourniquet with contralateral limb serving
as control. The animals were equally divided
(n=5) into groups based on 1)tourniquet pres-
sure(ZO0, 300, o r 400 mmHg), 2 ) d u r a t i o n of comp r e s s i o n ( 1 , 2, o r 3 h r s ) , and 3)tim e of s a c r i f i c e ( l . 3 , o r 6 wks post-compression).
Prior
t o s a c r i f i c e l e g muscles were examined f o r f i b r i l l a t i o n p o t e n t i a l s and t h e s c i a t i c n e r v e
s t i m u l a t e d t o document compound a c t i o n potent i a l s . Subsequently, t i b i a l i s a n t e r i o r muscles
were removed and s t a i n e d f o r motor e nd-pla te s
( z i n c iodide-osmium t e t r o x i d e ) . End-plates
were a s s e s s e d by dete rm ining e nd-pla te l e n g t h ,
p e r cent age of end- p la te s w i t h t e r m i n a l s p r o u t s ,
and l e n g t h of t e r m i n a l s p r o u t s . EMG e v a l u a t i o n
re v eal ed f i b r i l l a t i o n p o t e n t i a l s i n a l l animal
groups. These w e r e owst pronounced a t s e ve n
days and decr eas ed p r o g r e s s i v e l y by t h r e e and
s i x weeks f ol l ow i ng compression. Compound act i o n p o t e n t i a l s were a l t e r e d s i m i l a r l y , i.e.
a l l animals w e r e a d v e r s e l y a f f e c t e d a t one week
(d e cr eas ed a r e a under t h e c urve ) w i t h progre s s i v e improvement no te d a t t h r e e and s i x weeks.
End-plate l e n g t h , p e rc e nta ge of t e r m i n a l
s p r o u t s , and l e n g t h o f s p r o u t s i n c r e a s e d s i g n i f i c a n t l y f ol l ow i n g compression w i t h t h e
g r e a t e s t i n c r e a s e observed a t t h r e e weeks. By
s i x weeks post-compression t h e above pa ra m e te rs
p ro gr es s ed toward normalcy, but were s t i l l elev a t e d when compared t o c o n t r o l v a l u e s . These
a l t e r a t i o n s are con s ide re d t o r e p r e s e n t a rep a i r r es pons e t o n e r v e i n j u r y . The r e s u l t s re p o r t ed i n d i c a t e t h a t t o u r n i q u e t p r e s s u r e s and
d u r a t i o n of compression used c l i n i c a l l y both
p lay a r o l e i n producing n e r v e i n j u r y and sugg e s t t h a t t o u r n i q u e t u s e during limb s u r g e r y
should be r e- eval uate d.
this study provide t h e possible anatomical substrate
for t h e explanation of t h e phenomenon of referred
pain within t h e orofacial area; however, t h e r e may b e
other central nervous system connections t h a t
contribute t o referred pain.
ODOR, D. Louise, Department of Anatomy, School
of Medicine,
University of South Carolina,
Columbia, S.C. Cytologic changes in smooth muscle
cells of the rabbit mesoturbarium in relation t o
estradiol levels.
Preliminary observations on t h e
smooth muscle of t h e mesoturbarium superius, one
of t h e mesenteries a t t a c h e d t o t h e oviduct, a r e
centered on whether these muscle cells respond in
a similar way t o estradiol a s those of t h e
myometrium.
Tissues
from
ovulatory,
ovariectomized and ovariectornized-estradiol (6 or
10 pg/day) t r e a t e d rabbits were examined by light
and electron microscopy. A f t e r long-termed ovariectomy t h e cells become narrower, have more
heterochromatin, a smaller nucleolus and fewer
cytoplasmic organelles than cells from ovulatory
rabbits. After estradiol t r e a t m e n t nuclear changes
include an increase in overall size of t h e nucleus,
in size of the nucleolus and in amount of
euchromatin. After 3 days of t r e a t m e n t t h e
nucleolus is larger than t h a t of ovariectomized
rabbits and by day 6, larger than t h a t of ovulatory
animals. The entire cell hypertrophies and t h e
number of organelles increases.
Mitochondria
elongate and a r e more abundant a f t e r 4 days of
t r e a t m e n t than in ovariectomized animals. By day
3 of t r e a t m e n t more polysomes and GER cisternae
a r e present. By day 6 t h e GER cisternae have
elongated, become branched and a r e especially
numerous in t h e extensive sarcoplasm at t h e ends
of t h e nucleus.
The Golgi complex enlarges
somewhat. The most striking change is the
appearance a s early a s day 1 of t r e a t m e n t of clumps
of glycogen particles in t h e perinuclear and subplasmalemmal regions. The size and number of
particle aggregates increase t o a maximum by day
6 and remain at t h a t level through day 10 of
treatment. Usually the glycogen is associated with
long AER cisternae. Thus, t h e smooth muscle of
this mesentery does undergo cytologic changes
similar t o those of t h e uterus under estrogenic
stimulation.
(USPHS grants
HD-03752
and
RRO-0166 and contracts 70-2141 and 70-2142.)
NllJ*, Shi-Lin and Richard M. DOM. Department of
Anatomy, Medical University of South Carolina,
Charleston, South Carolina.
An Anatomical
Explanation for Referred Pain.
Recent advancements in retrograde labelling of
neurons with fluorescent compounds allows for one
neuron t o be labelled with more than one compound.
For instance, a neuron projecting t o more than one
site could be labelled with two or more fluorescent
compounds by injecting different fluorescent
substances into different ta rge t areas. The multiple
fluorescent labeling technique has been utilized in
this investigation of the neurons of the trigeminal
ganglion of t h e LEH r a t t o determine if one neuron,
by axonal branching, supplies more than a single
anatomical locus. To investigate this possibility, a
saturated solution of True Blue was injected into the
subcutaneous tissue overlying one temporomandibular
joint and powdered Nuclear Yellow was placed into
the pulp cavity of t he ipsilateral mandibular incisor.
After the appropriate survival time, t h e animals were
perfused with 10% cacodylate buffered formalin, the
trigeminal ganglia sectioned a t 20 microns on an
lnternational Co. cryostat, the sections mounted
directly on slides and studied using a fluorescent
microscope equipped for ultraviolet excitation.
The majority of fluorescing trigeminal ganglion
cells was labelled with either True Blue or Nuclear
Yellow.
However, some neurons were double
labelled, having True Blue in the cytoplasm and
Nuclear Yellow in t he nucleus. Thus, one neuron has
processes supplying t h e incisor as well as the
ipsilateral temporomandibular joint. The results of
PETRALI, J . P . , K.R. MILLS, D.M. MAXWELL and
GREEN, Comparative Pa t h o l o g y , B a s i c and
Applied Pharmacology, USAMRICD, Aberdeen Proving
Ground, Maryland. Soman-Induced Myopathy.
Soman, a powerful i r r e v e r s i b l e i n h i b i t o r of
acetylcholinesterase was studied f o r its acute
and c h r o n i c t o x i c i t y i n r a t diaphragm. Young
a d u l t male r a t s s u r v i v i n g e i t h e r an a c u t e s i n g l e
subcutaneous LD50 dose (140 microgramsfkilogram)
o r a c h r o n i c 0 . 4 LD50 dose d a i l y , f o r 2 2 d a y s ,
p r e s e n t e d moderate t o s e v e r e u l t r a s t r u c t u r a l
a b n o r m a l i t i e s o f t h e diaphragm a t 27 h o u r s
M.D.
474
SCHENCK, M.H.* and D.J. ALLEN, Department o f
Anatomy, Medical C o l l e g e o f Ohio, Toledo, Ohio.
S t r u c t u r e o f n a t i v e human, b o v i n e and p o r c i n e
p a r i e t a l pericardium.
T h i s i n v e s t i g a t i o n used TEM and SEM t o
s t u d y t h e u l t r a s t r u c t u r e of human, b o v i n e and
porcine p a r i e t a l pericardium. A f t e r being f i x e d
i n 3% b u f f e r e d g l u t a r a l d e h y d e , washed i n 0.144N
c a c o d y l a t e b u f f e r and p o s t o s m i c a t e d (2%), t h e
t i s s u e specimens were r o u t i n e l y processed f o r
e l e c t r o n microscopy. T i s s u e samples were a l s o
q u a l i t a t i v e l y analyzed u s i n g h i s t o c h e m i c a l t e c h niques t o survey f o r polysaccharides, proteins
and enzymes i n t h e p e r i c a r d i u m . The p r i m a r y
purpose o f t h i s s t u d y was t o compare t h e h i s t o c h e m i s t r y and morphology o f t h e s e t i s s u e s t o
o b t a i n a d a t a base f o r t h e e v a l u a t i o n o f p r e and p o s t i m p l a n t a t i o n changes i n c a r d i a c v a l v e s
made o f mammalian p e r i c a r d i u m .
The most apparent m o r p h o l o g i c a l d i f f e r e n c e
among t h e s p e c i e s was t h e shape and t e n a c i t y of
t h e m e s o t h e l i a l c e l l s . I n b o t h b o v i n e and p o r c i n e p e r i c a r d i u m , m e s o t h e l i a l c e l l s were f l a t
o r squamous i n shape and e a s i l y removed from t h e
s e r o s a l s u r f a c e , whereas t h e human s e r o s a l c e l l s
were l o w - c u b o i d a l and much more a d h e r e n t t o t h e
u n d e r l y i n g c o n n e c t i v e t i s s u e l a y e r . The a b i l i t y
o f t h e s e human c e l l s t o w i t h s t a n d more i n s u l t
t h a n e i t h e r t h e b o v i n e o r p o r c i n e may be due t o
numerous i n t e r d i g i t a t i o n s o f t h e b a s a l l a m i n a
i n t o t h e submesothel i a l c o n n e c t i v e t i s s u e l a y e r .
The t h r e e s p e c i e s had s u b m e s o t h e l i a l c o l l a g e n
bundles p a r a l l e l i n g t h e s e r o s a l s u r f a c e , b u t
notable v a r i a t i o n s o f t h i s l a y e r included a
l a y e r o f f i b r o b l a s t s i n human t i s s u e and t h i c k
e l a s t i c f i b e r s i n p o r c i n e p e r i c a r d i u m . In each
o f t h e s e , t h e f i b r o s a c o n s i s t e d o f c o l l a g e n and
e l a s t i c f i b e r s p e n e t r a t e d by v a s c u l a r and n e u r a l
elements and c o n n e c t i v e t i s s u e c e l l s . E l a s t i c
f i b e r s o f p a r i e t a l p e r i c a r d i u m were s i m i l a r t o
t h o s e o f dura mater, h a v i n g s e v e r a l l a y e r s o f
m i c r o f i b r i l s r a t h e r t h a n t h e s i n g l e l a y e r seen
i n most e l a s t i c t i s s u e . The o r i e n t a t i o n o f
e l a s t i n w i t h i n t h e f i b r o s a was r e v e a l e d w i t h SEM
a f t e r d i g e s t i o n o f t h e n o n - e l a s t i c components
w i t h 0.5N NaOH. The e p i p e r i c a r d i u m c o n t a i n e d a
network o f f i n e e l a s t i c f i b e r s o f v a r i a b l e
d e n s i t y o v e r l y i n g t h i c k c o l l a g e n bundles and
appeared smoothest i n human p e r i c a r d i u m due t o
t h e presence o f fewer e l a s t i c f i b e r s .
(Supported by g r a n t s from t h e M e d i c a l C o l l e g e
o f Ohio and t h e F.M. Douglass F o u n d a t i o n )
(acute group) apparently continuing to 22 days
(acute and chronic group) the last time period
observed. Ultrastructural changes followed a
typical progression of severe and persistent
contraction, widening of sarcoplasmic spaces,
focal and highly specific Z band disruption,
loss of banding with sarcomeric streaming, loss
of myofilaments, vesiculation of sarcoplasmic
reticulum, and a proliferation of intermyofibrillar myelin figures associated with the
sarcoplasmic reticulum. Curiously, mitochondria
were only minimally involved or appeared normal.
In the chronic group, acetylcholinesterase
activity of whole blood and diaphragm was
depressed to 6% and 49% respectively of control
values, while calcium levels of diaphragm, were
elevated to 2X that of control values. These
observations are reminiscent of the delayed
toxicities reported for some, but not all, of
the organophosphorous inhibitors of acetylcholinesterase.
Rowley, W.H. * , W.D. Kornegay 111, and M.C.
Poole, Department o f Anatomy, School o f
p r o e s t r o u s surges o f p i t u i t a r y g o n a d o t r o p i n s
(LH and FSH) when a d m i n i s t e r e d d u r i n g d i e s t r u s
2. The p r e s e n t s t u d y a s c e r t a i n s t h e
gonadotrope p o p u l a t i o n and m o r p h o l o g i c a l
changes a s s o c i a t e d w i t h t h i s suppression.
C y c l i n g 60 d a y - o l d female r a t s ( C h a r l e s R i v e r
Sprague-Dawley) were i n j e c t e d w i t h p r o g e s t e r o n e
( 1 mg/100 g body w t . , s . c . ) a t 1000 d i e s t r u s 2,
and a t 1000 p r o e s t r u s . An a d d i t i o n a l c o n t r o l
group r e c e i v e d i n j e c t i o n s o f c o r n o i l . The
animals were s a c r i f i c e d b y d e c a p i t a t i o n a t
1600, 1800, and 2000 p r o e s t r u s . A t s a c r i f i c e ,
t h e a n t e r i o r p i t u i t a r i e s were removed and
processed f o r e l e c t r o n microscopy, and t h e
b l o o d was c o l l e c t e d f o r LH and FSH
radioimmunoassay. The assays showed t h a t LH
and FSH were suppressed d u r i n g p r o e s t r u s i n
t h o s e animals r e c e i v i n g p r o g e s t e r o n e on 1000
d i e s t r u s 2, however, t h e r e was no s u p p r e s s i o n
i n t h o s e animals r e c e i v i n g p r o g e s t e r o n e a t 1000
p r o e s t r u s . A p r e l i m i n a r y morphometric a n a l y s i s
r e v e a l e d t h a t t h e r e was no d i f f e r e n c e i n t h e
c e l l volumes o f t h e Type I and I 1 gonadotropes,
and s u r p r i s i n g l y t h e r e was no change i n t h e
gonadotrope p o p u l a t i o n s f o l l o w i n g p r o g e s t e r o n e
suppression.
A q u a l i t a t i v e analysis revealed t h a t the
rough endoplasmic r e t i c u l u m was s u b s t a n t i a l l y
i n c r e a s e d i n b o t h t h e progesterone-suppressed
Type I gonadotropes and i n t h e c o n t r o l s .
However, t h e G o l g i complex was g r e a t e r i n t h e
p r o g e s t e r o n e suppressed Type I gonadotropes
than i n t h e proestrous controls. Also t h e
number o f s e c r e t o r y g r a n u l e s was reduced i n t h e
p r o g e s t e r o n e suppressed gonadotropes. The
u l t r a s t r u c t u r e o f t h e Type I 1 gonadotropes was
n o t a f f e c t e d by t h e progesterone. These
o b s e r v a t i o n s suggest t h a t p r o g e s t e r o n e can
suppress g o n a d o t r o p i n s e c r e t i o n b y r e d u c i n g
s e c r e t o r y g r a n u l e packaging a t t h e G o l g i
comp 1e x .
(Supported by N I H HD 15259)
SINGH, B.B., SCHUSTER, G.S., ERBLANO, J.F.,
SCHAFFNER, D.L. and STEFLIK, D.E. Department o f
O r a l B i o l o g y , Medical C o l l e g e o f Georgia,
Augusta. E f f e c t s o f 12-0-Tetradecanoyl-phorbol1 3 - a c e t a t e on Hamster Cheek Pouch Mucosa.
A s u r v e y o f t h e l i t e r a t u r e on e x p e r i m e n t a l
o r a l c a r c i n o g e n e s i s shows a l a c k o f s t u d i e s on
t h e e f f e c t s o f 12-0-Tetradecanoyl-phorbol-13a c e t a t e (TPA), t h e most p o w e r f u l p r o m o t i n g agent, on o r a l e p i t h e l i u m . The a i m o f t h i s i n v e s t i g a t i o n was t o d e t e r m i n e t h e e f f e c t s o f TPA on
hamster cheek pouch mucosa. Male S y r i a n golden
hamsters were d i v i d e d i n t o f i v e groups. Each
group was t r e a t e d t h r e e t i m e s d u r i n g a s i n g l e
e i g h t h o u r p e r i o d as f o l l o w s : Group 1, 5119 TPA
i n 0.25 m l acetone; Group 2 , 1Opg f l u o c i n o l o n e
475
SOBOL*, T.L.,
G.W. KALMUS* and M.E. MCNEILL,
Departments o f Biology and Anatomy, 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 , North
Comparative study o f t h e normal and
Carolina.
d i a b e t i c Chinese hamster Harderian gland.
T e C inese amster C r i c e t u us r i s e u s )
soan:ane!usl
v d k e l o ~ s abetes' a n d h e n
used t o studjl t h e pineal-gonadal axis.
Infert i l i t y i s a complication o f t h e diabetic s t a t e
i n t h i s animal.
I n t h e d i a b e t i c Chinese
hamster a l t e r a t i o n s have been observed i n t h e
It has
f i n e s t r u c t u r e o f t h e Harderian gland.
been suggested t h a t t h e Harderian gland which
i s l o c a t e d i n t h e o r b i t o f t h e eye has both
endocrine and e x o c r i n e f u n c t i o n s . Among these
suggestions are: (1) t h a t t h e Iiarderian gland
serves as a source o f l u b r i c a t i o n f o r t h e eye
through l i p i d s e c r e t i o n ; ( 2 ) t h a t i t may
i n f l u e n c e p i n e a l and/or r e p r o d u c t i v e f u n c t i o n
through a c t i o n s as an e x t r a r e t i n a l photoreceptor; and, ( 3 ) t h a t i t may serve a
communication f u n c t i o n through pheromone
s e c r e t i o n . I n t h e present study, t h e d i a b e t i c
Chinese hamster Harderian gland weight was
decreased i n comparison t o c o n t r o l s .
Furthermore, t h e Harderian glands o f d i a b e t i c
animals were found t o have a decrease i n
a l v e o l a r size, t h e number o f c e l l s p e r
a l v e o l u s , t h e c e l l r a d i u s and t h e number o f
Type I 1 c e l l s per alveolus. No Type I 1 c e l l s
were observed i n t h r e e d i a b e t i c animals.
S t r u c t u r a l changes seen i n t h e Harderian glands
o f d i a b e t i c animals may i n d i c a t e p o s s i b l e
disturbances i n s e c r e t o r y s t a t e . A t t h e TEM
l e v e l c o n c e n t r i c l a m e l l a e were observed which
v a r i e d i n e x t e n t and c o n f i g u r a t i o n as well as
l o c a t i o n and a s s o c i a t i o n w i t h o t h e r o r g a n e l l e s
w i t h i n t h e s e c r e t o r y c e l l s o f t h e Harderian
gland. D i a b e t i c animals were observed t o have
increased r e l a t i v e c o n c e n t r i c l a m e l l a e area t o
t o t a l cytoplasm as compared w i t h c o n t r o l s .
C e l l s w i t h c l e f t s t r u c t u r e s were observed i n
t h e Harderian glands o f t h e d i a b e t i c animals t o
a f a r g r e a t e r e x t e n t than c o n t r o l s . C l e f t
s t r u c t u r e s appear t o be r e l a t e d t o t h e
f o r m a t i o n o f vacuoles w i t h i n t h e cytoplasm o f
Type I 1 c e l l s .
acetonide (FA), a s t e r o i d a l a n t i - i n f l a m m a t o r y
agent, i n 0.25 m l acetone; Group 3, FA f o l l o w e d
by TPA; Group 4, 0.25 m l acetone; and Group 5,
u n t r e a t e d animals. The animals were s a c r i f i c e d
72 hours a f t e r treatment f o l l o w i n g i n j e c t i o n
w i t h v i n b l a s t i n e s u l f a t e o r c o l c h i c i n e . The t i s sues were processed by h i s t o l o g i c techniques and
s t a i n e d w i t h hematoxylin and eosin, P a u l y ' s
Reagent f o r h i s t i d i n e - r i c h p r o t e i n ( s ) , and
methyl-green p y r o n i n (MGP) f o r RNA. TPA induced
marked inflammation and s i g n i f i c a n t l y enhanced
e p i t h e l i a l m i t o t i c a c t i v i t y i n basal and parabasal l a y e r s . The e p i t h e l ium e x h i b i t e d hyperp l a s i a , the t o t a l e p i t h e l i a l thickness i n c r e a s i n g 1.5 t o 2 times as compared w i t h t h e c o n t r o l s .
V a r i a b i l i t y o f c e l l s i z e and shape i n t h e lower
s t r a t a was a l s o observed. Many e p i t h e l i a l
c e l l s s t a i n e d prominantly w i t h MGP i n d i c a t i v e
o f enhanced RNA synthesis, and a prominent
g r a n u l a r c e l l l a y e r showing abundant k e r a t o h y a l i n granules r e a c t e d p o s i t i v e l y w i t h P a u l y ' s
Reagent i n d i c a t i v e o f d i f f e r e n t i a t i o n . FA
markedly i n h i b i t e d the inflammatory process as
w e l l as the h y p e r p l a s t i c s t i m u l a t i o n induced by
TPA. Previous s t u d i e s i n o u r l a b o r a t o r i e s have
shown t h a t TPA enhances m i t o t i c a c t i v i t y and
produces a1t e r a t i o n s i n e p i t h e l i a l c e l l morphology o f c u l t u r e d hamster pouch e x p l a n t s as determined by h i s t o l o g i c and scanning e l e c t r o n
microscopic observations. T h i s study suggests
t h a t TPA i s a l s o a p o t e n t agent s t i m u l a t i n g
hyperplasia o f hamster cheek pouch e p i t h e l i u m
in v i v o under the c o n d i t i o n s described.
h
Supported p a r t l y by g r a n t DE 6239
SLEMMONS*, K.D., M.E. MCNEILL, and E.C.
SIMPSON*, Department o f Biology, East C a r o l i n a
U n i v e r s i t y , and Department 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 , North Carolina.
Zinc
c o n c e n t r a t i o n s i n s e l e c t e d ti=
from acute
a l l o x a n - d i a b e t i c male r a t s .
Current evidence suaaests t h a t t h e d i a b e t i c
c o n d i t i o n may be accompanied by a l t e r a t i o n s i n
z i n c metabolism. The purpose o f t h i s study was
t o compare t h e z i n c concentrations i n s e l e c t e d
t i s s u e s from acute a l l o x a n - d i a b e t i c and normal
male r a t s . O f p a r t i c u l a r i n t e r e s t were t i s s u e s
associated w i t h t h e complications o f diabetes
me11 i t u s .
Experimental diabetes was induced i n male
Sprague-Dawl ey r a t s by intravenous a1 loxan
i n j e c t i o n ( 4 0 mg/kg body weight). Four weeks
a f t e r alloxan administration, t i s s u e zinc
l e v e l s were determined by atomic absorption
spectrophotometry. Zinc c o n c e n t r a t i o n s were
s i g n i f i c a n t l y h i g h e r i n t h e adenohypophysis
( p < 0.03), b r a i n ( p < 0.03), and p r o s t a t e
( p < 0.01) o f t h e d i a b e t i c group. Zinc
concentrations i n t h e lens, r e t i n a , p i n e a l ,
neurohypophysis, hypothalamus, aorta, l i v e r ,
kidney, and pancreas were s i m i l a r between t h e
two groups.
These r e s u l t s , along w i t h t h e r e s u l t s o f
o t h e r s t u d i e s ( F a i l l a and K i s e r , 1981; McNeill
e t . a1 , 1981), suggest t h a t experimental
diabetes r e s u l t s i n t h e b u i l d u p o f z i n c i n
p a r t i cul a r tissues.
STEFLIK, O . E . , MCKINNEY, R.V., JR., SINGH, B.B.
and KOTH, D.L. Department o f Oral Pathology,
Medical College o f Georgia, Augusta and Department o f Fixed P r o s t h e t i c s , UNC a t Chapel H i l l .
A l t e r n a t i v e U l t r a s t r u c t u r a l M o d a l i t i e s t o Examine the Tissue Response t o Oral Implants.
Our l a b o r a t o r i e s have been i n v e s t i g a t i n g
the response o f the o r a l t i s s u e s t o t h e s i n g l e
c r y s t a l sapphire (Al,O,)
endosseous d e n t a l i m p l a n t . Our previous experimental animal s t u d i e s
i n v o l v i n g t r a d i t i o n a l TEM, SEM and h i s t o l o g i c a l
analyses demonstrated t h a t v i a b l e e p i t h e l i u m
e x i s t e d adjacent t o t h i s i m p l a n t as w e l l as
showing a c l o s e j u x t a p o s i t i o n i n g o f bone. F o l lowing these s t u d i e s we decided t o r e - e v a l u a t e
the h i s t o l o g i c a l d a t a by r e t r i e v i n g t h e s p e c i mens f o r a l t e r n a t i v e u l t r a s t r u c t u r a l a n a l y s i s .
The purpose o f t h i s paper i s t o present two
a l t e r n a t i v e m o d a l i t i e s which permi t t r u e c o r r e l a t i v e histological/ultrastructural s t u d i e s .
H i s t o l o g i c a l specimens were perfused w i t h formal i n and embedded i n polymethyl m e t h a c r y l a t e
.
(Supported i n p a r t by U n i t e d Way Grant
0-58026- )
476
(PMMA). These specimens were sect i oned a t llOu,
s t a i n e d and h i s t o l o g i c a l l y recorded. For SEM,
th e se specimens were then surface-etched w i t h
oxygen plasma, w h i c h exposed s u r f a c e topography
and allowed SEM a n a l y s i s . For TEM, s i m i l a r s p e c imens were c r y o f r a ct ured by consecutive i m mersion i n t o b o il in g water and l i q u i d ni t rogen.
This c r e a t e d a f r a c t u r e plane along t h e bioniater i a l - e p i t h e l i a l i n t e r f a c e ; t h u s permitting t he
reembedding of the gingiva i n t o fresh PMMA f o r
subsequent TEM study. Results have allowed us
t o d e f i n i t i v e l y c o r r e l a t e l i g h t and SEM observ at io n s which has f a c i l i t a t e d t he judging of
c e l l u l a r o r i e n t a t i o n t o the implant, which was
o cc a si o n a ll y a problem i n our t r a d i t i o n a l E.M.
s t u d i e s . SEM displayed e x c e l l e n t gingival ret e n t i o n and provided a unique means t o examine
maturation p a t t e r n s of t h e epithelium. TEM demonstrated e x c e l l e n t r e t e n t i o n of t h e u l t r a s t r u c t u r a l morphology of t hese h i s t o l o g i c a l l y
prepared specimens, p a r t i c u l a r l y a t the basal
c e l l regions w i t h e x c e l l e n t preservat i on of hemidesniosonies Since the reenibedded specimens were
alr e a d y s t a i n e d , o r i e n t a t i o n was f a r e a s i e r than
i n previous s t u d i e s . This study has shown t h a t
properly prepared h i s t o l o g i c a l specimens can be
r e t r i e v e d f o r E.N. a n a l y s i s and t h a t p r e c i s e
c o r r e l a t i v e s t u d i e s can be accomplished. Further, when o r i e n t a t i o n of samples i s c r i t i c a l ,
th e se procedures can be extremely useful .
conversion ratios for 5a -reduced C1902-steroids
/4-androstene-3,17-dione t o be 3.8 (EC: 0.85 mg P)
and 0.53 (F: 0.75 mg P) a t 300 nM and 39.0 (EC: 0.85
mg P) and 0.89 (F: 0.75 mg P) a t 3 nM substrate.
For specimen I1 ratios were 2.3 (EC: 0.30 rng Pf and
0.57 (F: 0.57 mg P). In both EC and F cultures,
androsterone and Sa -dihydrotestosterone were
respectively the predominant and minor constituents
of the 5a -C1902-rnetabolites owing to extensive
17-oxosteroid formation. Contrary t o previous
reports based on mechanical epithelial-stromal
separation, w e surmise that EC and not F a re the
predominant source of testosterone 5a-reductase
in human BPH tissue. Supported by CA 29513 and
a USC American Cancer Society Grant.
.
TERRACIO, L., REIMAN, J.A., and B U G G Y , J . ,
D e p a r t m e n t s o f Anatomy a nd P h y s i o l o g y ,
U n i v e r s i t y of South C a r o l i n a School of
M e d i c i n e , C o l u m b i a , SC 2 9 2 0 8 .
Immunohi stochemi c a l -Local i z a t i on o f a S o d i um
Exc re ting Hormone Regulating Renal ATPase.
When s a l t i n t a k e i s h i q h . r e n a l sodium
e xc re tion is increased by a himoral agent which
a c t s by i n h i b i t i n g sodium-potassiun ATPase and
the associated a c t i v e menbrane ion t r a n s p o r t .
C a r d i a c g l y c o s i d e s such as digoxin or ouabain
compete w i t h this n a t r i u r e t i c hormone ( N H ) a t
t h e same r e c e p t o r s i t e , suggesting t h a t NH is
the endogenous 1 iga nd r e g u l a t i n g r e n a l Na-K
ATPase.
Other i n v e s t i g a t o r s have shown t h a t
some a n t i b o d i e s r a i s e d a g a i n s t d i g o x i n a l s o
r e c o g n i z e NH, presunably s i n e they share sane
common s t r u c t u r e t h a t i n t e r a c t s w i t h t h e
r e g u l a t o r y s i t e on Na-K ATPase. In this s tudy,
t h e renal s i t e o f a c t i o n of NH was l o c a l i z e d
u s i n g a d i g o x i n a n t i b o d y (w hic h c r o s s - r e a c t s
w i t h NH) i n a peroxidase-antiperoxidase (PAP)
immunohistochmical procedure. To induce high
l e v e l s of n a t r i u r e t i c hormone, an animal model
c o m b i n i n g h i g h d i e t a r y sodium i n t a k e w i t h
reduced renal e xc re tory f u n c t i o n was s t u d i e d .
In a d u l t male Sprague-Dawley r a t s , 75% of t o t a l
renal mass was s u r g i c a l l y removed. The h i g h
s a l t ( a n d t h u s high NH) group of r a t s had only
1%NaCl to drink whereas the low s a l t (and t h u s
low N H ) c o n t r o l group dra nk d e i o n i z e d water
w i t h a low sodium feed. A fte r 4 weeks on these
d i e t s , t h e a n i m a l s were s a c r i f i c e d ,
exsanguinated w i t h a s a l i n e f l u s h , t h e k i d n e y s
removed f o r f i x a t i o n i n Bouin's f l u i d , embedded
i n p a r a f f i n , s e c t i o n e d a t 5 m i c r o n s , and
m o u n t e d on g l a s s s l i d e s . A f t e r removal o f
p a r a f f i n , s l i d e s were p r o c e s s e d f o r s t a n d a r d
PAP imnunohistochemistry. As c o n t r o l s , primary
o r secondary a n t i b o d y o r PAP were o m i t t e d o r
prima ry a n t i b o d y preabsorbed w i t h digoxin was
substituted.
NH-like immunoreactiv i t y was
1 o c a l i z e d i n t h i c k a s c e n d i n g 1 imbs, d i s t a l
t u b u l e s , and c o l l e c t i n g t u b u l e s o f r e n a l
NH-like
m e d u l l a o f r a t s on h i g h s a l t .
i m m u n o r e a c t i v i t y was n o t e v i d e n t i n o t h e r
kidney r e g i o n s o r i n m e d u l l a of r a t s on low
s a l t . T h i s local i z a t i o n corresponds t o the
r e g i o n a l d i s t r i b u t i o n of renal Na-K ATPase and
suggests t h a t immunore a c tive NH was bound t o
re c e ptors on ATPase.
TERRACIO, L., OFNER, P. and VENA, R.L.,
Department of Anatomy, University of South
Carolina, Columbia, SC and Department of Urology,
Tufts University School of Medicine, Boston, MA.
Radiotestosterone Metabolism by Human Prostatic
Epithelial Cells and Fibroblasts.
The
role
of
orostatic
stromal-eoithelial
interactions in the metabolic disposition of
androgens is important to the understanding of
prostatic function in health and disease. We
investigated the ability of epithelial cells (EC) and
fibroblasts (F) derived from open-prostatectomy
BPH specimens to metabolize radiotestosterone &
vitro. EC and F were established in culture using
a modification of our published procedure for the
canine (J. Androl 3:269-280, 1982). Both cell types
were characterized by light (LM) and electron
microscopy (EM) and histochemistry (HC). EC
exhibited a polygonal shape in confluent monolayers
(LM). Employing EM, adjacent EC were joined by
tight junctions and desmosomes to form lumen-like
structures.
The EC contained immunoreactive
prostatic acid phosphatase (PAP). F had an
elongated spindle shape by L M and were not
attached by junctions but did possess dilated profiles
of RER by EM. The F were negative for PAP but
positive for collagen production by HC. Primary
cultures were grown to semiconfluency in RPMI
1640 with antibiotics and 1 0 % horse serum and the
rinsed cells were then incubated with [3Hl
testosterone a t 37OC in serum-free medium. In this
preliminary comparison of reductive and oxidative
pathways, cultures containing 0.30-0.85 mg of cell
protein (PI metabolized 83-99% of 3 nM and 300 nM
substrate in 24 hr. For specimen I, we found the
477
/
VIDIC, Branislav and Peter H. BURRI, Departments
of h a t o m y , Georgetown University, Washington,
D.C. and University of Rerne, Rerne, Switzerland.
Quantitative cellular and subcellular organization of
the postnatal r a t pulmonary epithelium.
In the r a t the t o t a l pulmonary volume increases
from 0.57 t o 2.25 cm3 durinq the f i r s t three weeks of
postnatal development. The volumetric proportion
between the parenchymal and non-parenchymal
divisions, however, remains relatively constant.
While, during the same time, the airspace and blood
vessel volumes become proportionately increased t o
the
total
parenchymal
volume,
the
tissue
subcompartment diminishes f r o m 28.2 t o 19.4% o f
the t o t a l Darenchymal volume. This i s mostly due t o a
reduction in the volumetric density o f the cellular
population in the interstitium. These structiiral
chanqes r e f l e c t the postnatal differentiation o f the
organ from the immature, neonatal t o the adult form:
I. formation and capillarisation of the secondary
septum; and 2. establishment of gas-exchanging
oarenchyma. In the course o f this pulmonary
rnorohogenesis (day 4 t o 21) the alveolar suface area
expands f r o m 442.9 t o 2159 cm2 and the eoithelial
volume f r o m 27.7 to 93.2 mm3. The volumetric r a t i o
between the type 1 and I1 c e l l populations decreases
significantly i n spite of a decrease in the individual
type 11 c e l l and the intracellular organellar size. The
t o t a l type I1 c e l l mass increases from 10.3 t o 6 1 and
that of lamellar bodies from 0.97 t o 5.05 mm3. These
results surgest a parallel growth pattern hetween the
t o t a l volume o f lamellar hodies and the t o t a l alveolar
surface are, showing a value of 0.022 um on the day 4
and 0.023 um on the day 21 o f postnatal development.
ments and s y n a p t i c c o n f i g u r a t i o n s i n p e d i c l e s
o f r o d and cone c e l l s were discerned. The
i n n e r p l e x i f o r m l a y e r , presumbly a s y n a p t i c
zone where c e n t r i f u g a l t e r m i n a l s i n n e r v a t e t h e
amacrine c e l l s , d i d n o t c o n t a i n any myelinated
axons. Numerous s y n a p t i c b u t t o n s c o n t a i n i n g
s y n a p t i c v e s i c l e s o f v a r i o u s s i z e and d e n s i t i e s
were observed i n t h i s l a y e r . Myelinated axons
were n o t observed i n t h e o p t i c f i b r o u s l a y e r .
The o p t i c nerve (through which t h e c e n t r i f u g a l
f i b e r s p r o j e c t t o t h e r e t i n a ) d i d c o n t a i n some
non-myelinated axons. ' However, most o p t i c
axons were myelinated.
We speculate t h a t c e n t r i f u g a l f i b e r s t o t h e
r e t i n a o f t h e g a r t e r snake are non-myelinated
axons. Our t r a c i n g o f t h e W a l l e r i a n degene r a t i o n i n t h e r e t i n a and H3-macromolecule
l a b e l i n g o f t h e c e n t r i f u g a l system w i l l f u r t h e r
l o c a l i z e the e f f e r e n t terminals i n the r e t i n a .
I t should a i d i n the i d e n t i f i c a t i o n o f those
non-myelinated axons. (Supported by a BRSG fund
from Marshall U n i v e r s i t y School of Medicine and
AMA Educational Resources Fund)
WHITEHEAD: D.S.,
D.R. G a r r i s and C.R. Morgan,
East 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.
E f f e c t s o f alloxan-induced diabetes on u t e r o o v a r i a n s t r u c t u r e and f u n c t i o n i n t h e
pseudopregnant r a t .
The e f f e c t s o f a l l o x a n (40 mg/kg)-induced
diabetes on t h e s t r u c t u r e and f u n c t i o n o f t h e
u t e r o - o v a r i a n a x i s were examined i n
pseudopregnant (PSP: Day 0 = o v u l a t i o n ) r a t s .
A l l o x a n was administered e i t h e r p r i o r (PA) t o
Day 0 o r on Day 1 (D-1A) o f PSP and t h e
e f f e c t s on serum progesterone (P) and glucose
(G) l e v e l s monitored and compared w i t h s a l i n e t r e a t e d c o n t r o l s (C). I n C r a t s , serum P
l e v e l s were elevated, and G l e v e l s were
normal (100 mg/dl ), throughout PSP.
In
c o n t r a s t , a l l o x a n - a d m i n i s t r a t i o n p r i o r t o Day
0 completely prevented corpus luteum f u n c t i o n ,
as i n d i c a t e d by t h e depressed serum P l e v e l s ,
when compared w i t h c o n t r o l s ( p < 0.01).
Alloxan-treatment on Day 1 o f PTP induced
l u t e o l y s i s by Day 6 as compared w i t h c o n t r o l
(P i 0.05) P l e v e l s . Both PA and D-1A
treatments induced s i g n i f i c a n t ( p i 0.001)
increases i n serum G l e v e l s as compared w i t h
controls.
Both o v a r i a n and u t e r i n e weights
were depressed i n PA and D-1A groups as
compared w i t h c o n t r o l s .
D a i l y treatment o f
D-1A r a t s w i t h 6 I U o f bovine i n s u l i n r e s t o r e d
o v a r i a n and u t e r i n e weights t o c o n t r o l l e v e l s
and elevated serum P l e v e l s above ( p < 0.05)
t h e D-1A group, b u t not t o c o n t r o l l e v e l s .
Associated w i t h t h e diabetes induced
depression o f l u t e a l f u n c t i o n i n D-1A and PA
r a t s was a l o s s o f endometrial s t r u c t u r a l
i n t e g r i t y , c h a r a c t e r i z e d by a l o s s o f columnar
e p i t h e l i a l c e l l s and a t h i c k e n e d basement
membrane. These r e s u l t s i n d i c a t e t h a t
diabetes-associated r e p r o d u c t i v e d y s f u n c t i o n
r e s u l t s from t h e depression o f
insulin-dependent, o v a r i a n steroidogenesis i n
t h e r a t . Endometrial d i s r u p t i o n i s probably a
secondary r e s u l t o f t h e diabetes-induced,
o v a r i a n impai rment.
Supported hy Grants F06-TW00124-01AI from the
Fogarty International Center USPHS and 3.293.78
f r o m t he Swiss National Science Foundation.
WANG, R.T. and J . CAPITO*, Department o f
Anatomy, Marshall U n i v e r s i t y School o f
Medicine, Huntington, WV. C e n t r i f u g a l f i b e r s t o
the r e t i n a o f g a r t e r snakes may be nonmyel i n a t e d
The nucleus o f t h e v e n t r a l s u p r a o p t i c decuss a t i o n i n t h e g a r t e r snake's diencephalon has
been i d e n t i f i e d as t h e source o f c e n t r i f u g a l
f i b e r s t o t h e c o n t r a l a t e r a l r e t i n a (Halpern,
Wang and Colman, 1976). I n t h i s study an
attempt was made t o i d e n t i f y the s y n a p t i c
connection o f t h e c e n t r i f u g a l f i b e r s i n t h e
retina.
Normal r e t i n a e and o p t i c nerves from g a r t e r
snakes (Thamnophis s i r t a l i s ) were i s o l a t e d from
animals f o l l o w i n g c a r d i a c p e r f u s i o n w i t h a Na
cacodylate b u f f e r (0.18M, pH 7.2) and EM
f i x a t i v e s (2% paraformaldehyde and 3% g l u t a r aldehyde i n 0.10M Na cacodylate b u f f e r , pH
7.2). Tissues were osmicated and s t a i n e d w i t h
u r a n y l a c e t a t e p r i o r t o dehydration and embedding. U l t r a t h i n s e c t i o n s o f t i s s u e were
p o s t - s t a i n e d w i t h u r a n y l a c e t a t e and l e a d
c i t r a t e . A H i t a c h i HU-11C TEM operated a t
75Kv was used f o r t i s s u e examinations.
The r e t i n a o f t h e g a r t e r snake resembled
other vertebrate r e t i n a e i n containing a threeneuron-chain organized i n t o 10 p a r a l l e l laminae.
T y p i c a l membranous f o l d i n g s i n t h e o u t e r seg-
-.
478
WILLIAMS: S., C. Smith-West: L. West*and D.R.
Garris, East Carolina University School of
Medicine, Greenville, NC 27834.
diabetes and endometkial structure was
examined in genetically-diabetic Chinese
hamsters. Uterine samples were collected from
animals exhibiting pre-diabetic to overt
diabetic conditions and from matched control
animals. In controls (with blood glucose
levels < 145 mg/dl) the endometrium was
typifiea by an intact luminal epithelium, a
thin underl-ving basement membrane and a well
organized stroma layer. In contrast, mildly
diabetic (150-250mg/dl) animals exhibited a
compressed luminal epithelium which was
embedded in a thickened basement membrane
infiltrated with phagocytic blood elements.
The stromal layer contained several irreqular
cells which were characterized by cytoplasmic
vacuolization and collagen fiber separation by
an amyloid-like, intercellular ground
substance. In overt-diabetic animals (2 300500 rng/dl), the luminal epithelial cells were
located over a greatly thickened basement
membrane which was infiltrated by phagocytic
blood elements and degenerating stromal cell
membranes. The lumina of the glands were
closed and the stromal cells were separated by
an increased intercellular space occupied by
an amyloid-like ground substance. Many
stromal cells exhibited cytoplasmic
vacuolization. These studies demonstrate that
the uterine involution associated with
diabetes occurs in a sequential manner and
that the structural changes are temporally
related to progressive elevations in blood
glucose levels.
l i m b i c c o r t e x of t h e r a b b i t have been described
previously (J. Rose & Woolsey,
Comp.
89:279, 1948; Berger e t a l . , Brain Res., 201:411
1980), t h e s e r e p o r t s e i t h e r d e t a i l d e s c r i p t i o n s
l i m i t e d t o thalamic p r o j e c t i o n p a t t e r n s based on
the e f f e c t s of c o r t i c a l a b l a t i o n (J. Rose &
Woolsey) o r , i f c u r r e n t enzyme mapping technique
i s employed, only t h e r e c i p r o c a l c o r t i c o - c o r t i c a l connections between a n t e r i o r and p o s t e r i o r
limbic a r e a s are discussed (Berger e t a l . ) . The
p r e s e n t study i s p a r t o € a s e r i e s of experiments
designed t o c h a r a c t e r i z e completely, using mode r n t r a c i n g procedures, t h e a f f e r e n t and e€f e r e n t connections of t h e c i n g u l a t e c o r t e x of
the rabbit.
Horseradish peroxidase was i n j e c t e d i n t o vari o u s p o r t i o n s of the p o s t e r i o r limbic c o r t e x i n
8 r a b b i t s . Approximately 48 h r s a f t e r i n j e c t i o n
t h e b r a i n s were removed, sectioned a t 50 pm, and
t h e s e c t i o n s processed following t h e c o b a l t
c h l o r i d e modification of Adams (Neurosci., 2 : 1 4 1
1 9 7 7 ) . Microscopic examination revealed t h a t ,
with t h e exception of l a b e l i n t h e c o n t r a l a t e r a l
c o r t i c a l a r e a s homotypic t o t h e i n j e c t i o n s i t e s ,
a l l l a b e l l e d neuronal soma were found only i p s i l a t e r a l t o the i n j e c t i o n . Cortical l a b e l l i n g
included a n t e r i o r l i m b i c and s u b i c u l a r r e g i o n s ,
but even following i n j e c t i o n s i n t o d o r s a l r e t r o s p l e n i a l c o r t e x ( a r e a 29d) l a b e l l e d c e l l s were
not apparent i n sensory o r motor c o r t i c e s . This
observation s t a n d s i n c o n t r a d i c t i o n t o r e c e n t
o b s e r v a t i o n s by Vogt and M i l l e r (J.
Neurol., 216:192, 1983). Thalamic c e l l s cont a i n i n g HW r e a c t i o n product were contained
almost e x c l u s i v e l y w i t h i n t h e a n t e r o v e n t r a l and
a n t e r o d o r s a l n u c l e i , w i t h some l a b e l l e d c e l l s
appearing i n t h e l a t e r a l d o r s a l nucleus i f t h e
i n j e c t i o n f e l l i n t h e most p o s t e r i o r p a r t of
t h e r e t r o s p l e n i a l a r e a . Only a very few c e l l s
were observed i n t h e midline o r i n t r a l a m i n a r
thalamic n u c l e i a f t e r p o s t e r i o r limbic c o r t e x
i n j e c t i o n s . Labelled neuronal samae were a l s o
found i n the medial s e p t a l - d i a g o n a l band n u c l e i
and t h e tegmentum of t h e brainstem.
L.
a.,
m.
WOODRUFF, Michael L. and Ronald H. BAISDEN, D e partment of Anatomy, Quillen-Dishner College of
Medicine, East Tennessee S t a t e U n i v e r s i t y , John-
put t o t h e p o s t e r i o r p o r t i o n of t h e midline
479
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meeting, october, 1983, annual, society, anatomists, southern, twenty, third
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