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Inverse correlation between Synaptic Э ribbon number and the density of adrenergic nerve endings in the pineal gland of various mammals.

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THE ANATOMICAL RECORD 205:93-99 (1983)
Inverse Correlation Between “Synaptic” Ribbon Number
and the Density of Adrenergic Nerve Endings in the
Pineal Gland of Various Mammals
Department of Anatomy, The University of Texas Health Science Center at Sun Antonio,
J.B., J.T.H.,
L.J.P.,R.J.R.) and Labomtory of
San Antonio, TX 78284 ( M X . , T.S.K.,
Electron Microscopy, Department of Pathological Anatomy, Institute of Pathology,
Medical School, Look, Poland ( M X . )
The number of “synaptic” ribbons was inversely correlated with
the density of the adrenergic nerve endings of the pineal gland compared among
a diverse group of species including the fox, cat, rat, cotton rat, white-footed
mouse, Djungarian hamster, ground squirrel, and chipmunk. The concentration
of norepinephrine paralleled the number of adrenergic nerve terminals in the
pineal glands of the cotton rat, rat, and ground squirrel, the only species in which
norepinephrine concentrations were measured. The number of ribbon fields paralleled numbers of “synaptic” ribbons in all species examined. Adrenergic nerve
endings were observed primarily within the perivascular spaces, although some
endings also were found among parenchymal cells. Adrenergic nerve endings
forming synaptic junctions with pinealocytes were not observed in any of these
species, nor was there any physical association between these nerve endings and
“synaptic” ribbons.
The functionally enigmatic pineal “synaptic” ribbon (SR) has been described in every
mammalian species studied thus far (for review, see Pevet, 1979). A circadian rhythm in
SR numbers, high at night and low during the
daytime, has been reported in many of these
species (guinea-pig: Vollrath, 1973; rat: Kurumado and Mori, 1977; King and Dougherty,
1980; hamster: Hewing, 1979; baboon: Theron
et al., 1979). Because the gland’s adrenergic
innervation regulates most, if not all pineal
circadian rhythms (for review, see Vollrath,
1981), the rhythms in SR formation may similarly be regulated by the adrenergic innervation. This hypothesis has been examined with
a variety of experimentally altered conditions
including acute (Vollrath and Howe, 1976)and
chronic (Vollrath and HUSS,1973; Lues, 1971;
Hewing, 1980; King and Dougherty, 1982a;
Vollrath, 1975)changes in the 1ight:dark cycle,
blinding (Kurumado and Mori, 1980), pineal
sympathectomy (Romijn, 1975,1976; King and
Dougherty, 1982b) as well as pharmacological
manipulation of the adrenergic function (Karasek, 1974; Vollrath and Howe, 1976; Romijn
and Gelsema, 1976; King and Dougherty,
1982a,b). Common to each of these studies is
0003-276W83/2051-0093$02.500 1983 ALAN R. LISS, IN(:
the conclusion that the adrenergic innervation
of the pineal gland influences the level of SR
A comparison between previously reported
qualitative descriptions of the density of pineal
adrenergic nerve endings (e.g. Wolfe, 1965; Ito
and Matsushima, 1967; Matsushima and Reiter, 1977; Matsushima et al., 1979; Karasek
and Hansen, 1982) and quantitative analyses
of SR numbers suggest a potential relationship
between these two pineal components. This relationship is apparent in experimental studies
concerning SR formation following the destruction of the adrenergic innervation to the
pineal gland (e.g., Romijn, 1975; Karasek, 1976;
Romijn and Gelsema, 1976; King and Dougherty, 1982b). Synaptic ribbon numbers increase in the absence of adrenergic nerve fibers
in the pineal glands. In order to examine this
potential relationship under nonexperimental
natural conditions, we attempted to correlate
morphometrically the number of adrenergic
nerve endings with the density of “synaptic”
ribbons in a diverse number of mammalian
species. Norepinephrine concentrations also
Received June 1,1982;accepted September 20, 1982.
were determined in three of these species, each
having either high, medium, or low numbers
of adrenergic nerve endings. The norepinephrine content then was correlated with the density of the adrenergic innervation in the pineal
The pineal gland of rats (four males), foxes
(two males and two females), cats (four females), Djungarian hamsters (two males and
two females), ground squirrels (two males and
two females), chipmunks (two males and two
females), cotton rats (two males and two females), and white-footed mice (two males and
two females) were used for ultrastructural
analysis. Rats and Djungarian hamsters were
kept in a 1ight:dark cycle of 14:lO h (lights on
at 0600 h), whereas the other species were kept
under natural lighting conditions. All animals
were sacrificed by decapitation between 1200
h and 1400 h. The pineal glands were immediately removed and immersion-fixed in 3.5%
glutaraldehyde-2% formaldehyde in 0.067 M
cacodylate buffer (pH 7.2 at 4°C). All glands
were post-fixed in 1% osmium tetroxide in 0.1
M cacodylate buffer, dehydrated in a graded
series of acetones and embedded in Spurr’s
(1969) low vicosity epoxy resin. One thin section from a series of thin sections exhibiting
light gold interference colors (70-90 nm) was
taken from the largest transverse diameter of
each gland, stained with uranyl acetate and
lead citrate and examined with a Siemens 1A
electron microscope. Five grid squares (200
mesh; total area of 45,125 p.m2)containing pineal parenchyma were chosen without prior
examination and at random for analysis. The
numbers of “synaptic” ribbon (SR),ribbons fields
(RF), and adrenergic nerve terminals (NT) were
counted by using a consecutive series of parallel scans across each grid square at 12,000
to 14,000 x magnification. Ribbon fields were
defined as one or more spatially related SR
(Vollrath and Huss, 1973; King and Dougherty, 1982a,b). Data was expressed as the number of SR, RF, or NT & SEM per 20,000 p.m2.
For biochemical estimation of norepinephrine (NE) the pineal glands of rats (six males),
cotton rats (three males and three females),
and ground squirrels (four males and two females) were used. Norepinephrine in the pineal gland was assayed by high performance
liquid chromatography with electrochemical
detection (LCEC) as previously described
(Hansen and Christie, 1981). Essentially, this
procedure involved the extraction of pineal NE
with 0.1 M perchloric acid, absorption onto acidwashed alumina and elution from the alumina
with 0.1 M perchloric acid. The internal stan-
dard dihydroxybenzylamine (DHBA) (2 ng/20
p1) was added to each sample before processing. Aliquots (20 pl) of the alumina eluate were
valve loaded (Altex) onto a reverse phase U1trasphere ODS 5 pm column (Beckman).ARer
elution from the column, the sample passed
through a thin-layer flow cell containing a
glassy carbon electrode (Bioanalytical Systems) at a preset potential of + 0.72 V. The
oxidation current, which was directly proportional to the concentration of NE passing by
the electrode, was monitored on a strip chart
recorder (Houston Omniscribe). For calibration purposes, a sample consisting of proper
strength (2 ng/20 p.1) synthetic NE and DHBA
was eluted along with the pineal samples. To
calculate NE concentration, the peak height
ratios relative to the internal standard DHBA
for unknown pineal samples was compared to
that of the synthetic standard whose concentration was known. Protein determinationswere
performed by the method of Lowry et al. (1951)
as adapted for use with a Technicon Autoanalyzer. Results were expressed as ng of NE per
mg protein.
Data were analyzed first by a one-way analysis of variance. Significant differences between means were determined using the Newman-Keuls multiple range test.
“Synaptic” ribbons in the pineal glands of
all species examined were morphologically
similar. The structure of the SR core varied in
appearance from a trilaminar (Fig. 1)to platelike (Fig. 2) configuration with a variety of
intermediate forms (Figs. 2, 31, depending on
the plane of section. There were significant differences between the numbers of both SR and
RF among the species examined (Fig. 6). Low
numbers of SR and RF were observed in the
cotton rat, white-footed mouse, fox, cat, and
rat. In contrast, significantly higher numbers
(p < 0.001 vs other species) of SR and RF were
present in the chipmunk (Fig. 4) and ground
squirrel, whereas the pineal gland of DjunFigs. 1,Z. Rat. Typical appearance of “synaptic”ribbons
relative to the plane of section from trilaminar (TIto platelike (P) with an intermediate form (I). Figure 1, x 47,000;
Figure 2, x 36,000.
Fig. 3. Rat. Plate-like and intermediate form of “synaptic”ribbons adjacent to plasmalemma. NT, nerve ending.
x 11,000.
Fig. 4. Chipmunk. Numerous ribbon fields farrows). Pi,
pinealocyte;G, glial cell. x 16,000.
Fig. 5. Chipmunk. Large ribbon field. x 49,000.
8 200-
Ribbon Fmlda
“Synaptic” Ribbona
Nerve Terminala
Fig. 6. Inverse correlation between numbers of “synaptic” ribbons (ribbon fields) and adrenergic nerve terminals in
eight mammalian species. Half brackets refer to SEM.
TABLE 1 . Relation of ribbon fields (RF) and distribution
Vulpes uulpes domesticus
B. Sigmodon hispidw
(Cotton rat)
C. Rattus mttus
(Sprague-Dawley rat)
D. Felix domesticus
E. Perornyscus leucopus
(White-footed mouse)
F. Phoabpus sungorus
(Djungarian hamster)
G. Spernwphilus richardsonii
(Ground squirrel)
H. T a m h striatus
RF adjacent
to cell membrane
* 4.3b
NT in
perivascular spaces
NT in
4.4 i 2.8
39.5 t 2.8C
83.0 t 0.4h
2.4 t 1.4
88.8 t 3.1’
78.2 t 3.3k
93.8 t 2.2
nerve endings (NT) in the pineal parenchyma
92.4 t 0.8
94.1 t 0.6
92.9 t 0.8
94.4 t 1.2
Data are percents and represent means f SEM.Letters indicate statistically significant differences(SD) at level p < 0.05 vs species noted.
a: SD vs A, E, F; b: SD vs A-H; c: SD vs B-H; d: SD vs G , H; e: SD vs G , H; E SD vs C, G , H; g: SD vs B-H h SD vs G , H,i: SD vs E; j:
SD vs G , H, k SD vs G , H.
garian hamsters exhibited intermediate numbers of SR and RF (p < 0.001 vs other species).
Most RF in the species studied were associated
with the plasmalemma (Fig. 3), with the exception of those in the cat (Table 1). In the
chipmunk and ground squirrel, RF frequently
consisted of large numbers of SR (Fig. 5). “Synaptic” ribbons did not appear to have any consistant relationship to nerve fibers or their
endings, to endothelial cells, to glial cells or to
other pinealocytes. So-called “paired’ RF, i.e.,
RF lying opposite one another in adjacent pinealocytes, constituted only a small percent of
the total number of RF in all species studied
(Table 1).
The adrenergic nerve terminals in all species
studied were typical morphologically of previously described adrenergic nerve terminals
(Fig. 7) (see Vollrath, ’81).The number of NT
varied significantly among the species studied
(Fig. 6). The highest numbers (p < 0.001 vs
other species) of NT were observed in the cot-
Fig. 7. Fox. High density of nerve endings (asterisks)
found in the pineal parenchyma. Pi, pinealocyte: GP, glial
process. x 16,000.Inset, Morphologically typical adrenergic
nerve ending. x 48,000.
ton rat and fox, whereas in the chipmunk and
ground squirrel they were lowest (p < 0.005
vs other species). The majority of NT in all
species studied were located in the perivascular spaces (Table 1).A lower number of NT
was present in the parenchyma of the pineal
glands of the chipmunk and ground squirrel
when compared to other species examined (Table 1).An inverse correlation was observed between the numbers of either SR or RF and the
numbers of NT (r = -0.6247 and -0.6942, respectively; p < 0.05).
Norepinephrine concentrations were measured in three species, one having low (ground
squirrel), another intermediate (rat), and a
third, high (cotton rat) NT numbers. Concentrations of NE were highest in the cotton rat
10.0 ng/mg protein), intermediate in
the rat (21.5 2 5.1 ng/mg protein), and lowest
in the ground squirrel (9.1 5 1.0 ng/mg protein). Concentration of NE in the pineal gland
of the cotton rat was significantly greater (p <
0.001) than that in the rat and ground squirrel.
Although the concentrationof NE in the ground
squirrel was 137%lower than that in the rat,
the difference was not statistically significant
because of variation between animals.
gan culture of the rat pineal gland which
essentially can be considered a form of denervation, likewise results in an increase in SR
numbers (Karasek, 1976; Romijn and Gelsema, 1976). Our results suggest a similar relationship between SR formation and the density of adrenergic innervation of the pineal
gland, The pineal glands of those species having few nerve endings exhibit the greatest populations of SR. In turn, those species where
pineal glands are richly innervated by adrenergic nerves contain the fewest number of SR.
Despite the relationship between a high density of adrenergic innervation and low SR
numbers, the addition of norepinephrine to
cultured rat pineal glands (Karasek, 1974) or
the administration of a synthetic adrenergic
agonist (isoproterenol)to rats which are acutely
sympathectomized by superior cervical ganglionectomy (King and Dougherty, 1982b)leads
to an increase in SR formation. King and
Dougherty (1982a) have offered a working hypothesis which may explain this apparent paradox. They found that SR formation decreases
during continuous light but can be increased
with the administration of isoproterenol. The
density of beta-adrenergic receptors increases
during continuous light in apparent compenDISCUSSION
sation for reduced levels of norepinephrine
Although a relationship between the adre- (Cantor et al., 1981). On the other hand, SR
nergic innervation of the pineal gland and SR formation increases during continuous darkformation has been hypothesized (Vollrath, ness when the density of these receptors is low.
1973;Vollrath and Howe, 1976; Karasek, 1976; Thus, King and Dougherty (1982a)suggest that
King and Dougherty, 1980),the nature of this the function of SR formation is related to a
relationship has been speculative. Our study decrease in the density of these receptors along
demonstrates an inverse correlation between the plasmalemma of the pinealocyte, presumthe density of adrenergic nerve endings and ably by membrane turnover associated with
SR or RF numbers in the pineal gland of a SR vesicles. The potential association between
diverse number of mammalian species. The membrane turnover and SR vesicle function
concentration of NE paralleled the density of has been proposed by various investigators
NT in three species (cotton rat, rat, and ground (Spadaroet al., 1978;King and Dougherty, 1979,
squirrel-the only species in which NE con- 1980; McNulty, 1980).
The inverse relationship between adrenergic
centration have been measured). These results
indicate that SR numbers are inversely pro- nerve endings and SR numbers observed among
portional not only to NT numbers but also to diverse mammalian species may offer a collecNE concentrations. These results represent the tive model system for further study of the regfirst evidence for such a relationship under ulation and function of SR formation in the
normal conditions. However, this relationship mammalian pinealocyte. Assuming that SR
has been suggested by various other investi- formation is related to changes in the density
gators using experimentally altered conditions of adrenergic receptors along the plasmato affect SR formation in the pineal gland of lemma of pinealocytes, then the inverse correlation between fewer adrenergic nerve endrodent species.
Denervation of the mammalian pineal gland ings and higher numbers of SR in the pineal
by superior cervical ganglionectomy (Romijn, gland of some mammalian species may be as1975; Romijn and Gelsema, 1976; King and sociated with a compensatory increase in the
Dougherty, 1981b) or by chemical sympathec- density of beta-adrenergic receptors in these
tomy (6-hydroxydopamine:Romijn, 1976) pro- species. Our study may present further eviduce consistently elevated numbers of SR. Or- dence for the working hypothesis that the for-
mation of pineal SR serves to regulate the density of beta-adrenergic receptors along the
plasmalemma and, therefore, adrenergic control of pinealocyte metabolism in general.
The authors wish to thank Dr. William W.
Morgan for his help with the present work, Ms
Gwynne Duke for her excellent technical assistance, and Mrs. Nancy Elms for typing the
manuscript. This study was supported by NSF
grant #PCM 8003441to R. J. R. and by a grant
from the Polish Academy of Sciences, within
project 10.4 to M.K. T.S.K. is a postdoctoral
fellow in the Center for Training in Reproductive Biology HD 07139. J.T.H.
is the recipient
of NIH RCDA KO4 HL-00680.
This paper was presented in part at Ninety
Fifth Annual Session of the American Association of Anatomists, April 4-8, 1982 (abstract published in Anatomical Record 1982,
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mammal, synaptic, ending, nerve, adrenergic, ribbon, density, various, correlation, gland, pineal, number, inverse
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