close

Вход

Забыли?

вход по аккаунту

?

Fine structure of the dorsal lingual epithelium of Trachemys scripta elegans (Chelonia Emydidae)

код для вставкиСкачать
THE ANATOMICAL RECORD 250:80–94 (1998)
Quantitative Light Microscopic Study on the Heterogeneity
in the Superficial Pineal Gland of the Rat
YOSHIKI HIRA, YUKO SAKAI, AND SHOJI MATSUSHIMA*
Department of Anatomy, Asahikawa Medical College, Nishikagura, Asahikawa 078, Japan
ABSTRACT
Background: The previous results regarding regional and
day-night differences in pinealocyte size in rats are conflicting. The
relationships between these differences and the vascularity and sympathetic innervation have scarcely been investigated.
Methods: Wistar-King rats, kept under light/dark 12:12, were killed at
midday or midnight in October. The nuclear density of pinealocytes in the
superficial pineal was measured on the dorsoperipheral, dorsocentral,
ventroperipheral, and ventrocentral regions at distal, middle, and proximal levels at daytime and nighttime. The total area of blood vessels per
unit area at daytime and nighttime and total length of tyrosine hydroxylase (TH)-immunoreactive fibers per unit area at daytime were determined on the same regions at the same levels.
Results: Pinealocyte size was larger toward the distal levels and in the
periphery than in the center at any level. The area of blood vessels and
length of TH fibers were also larger toward the distal levels; the former in
the ventral region and the latter in the dorsal and ventral regions were
larger in the periphery than in the center. Ventral pinealocytes, but not
dorsal ones, showed day-night changes in size. Prominent day-night
rhythms in area of blood vessels occurred in the ventral region, where TH
fibers were more abundant than in the dorsal region.
Conclusions: Pinealocyte size shows the distal to proximal and peripheral to central gradients, which may be related to the differential distribution of blood vessels and sympathetic fibers. Since pinealocytes and blood
vessels, showing prominent day-night changes in size, are localized in the
more richly innervated regions, sympathetic fibers may play an important
role in controlling these rhythms. Anat. Rec. 250:80–94, 1998.
r 1998 Wiley-Liss, Inc.
Key words: pineal gland; regional difference; circadian rhythm; vascurarity; sympathetic innervation
The unequivocal morphological evidence for a circadian rhythmicity in the pineal gland of mammals was
first described by Quay and Renzoni (1966), who demonstrated that the nuclear and nucleolar sizes of the
pinealocytes of the rat show similar rhythms, with
maximums at midday and low values during the nighttime, and that the nuclear and nucleolar sizes are
similar between peripheral (cortical) and central (medullary) regions. Thereafter, determinations of the size
of the pinealocytes in the peripheral and central regions
at various time points during a 24-h period in rats have
been repeated by many investigators (Diehl, 1981;
Becker and Vollrath, 1983; Diehl et al., 1984; Lew et al.,
1984; Karasek et al., 1990). However, the original
findings by Quay and Renzoni (1966) have not always
been reproducible. It is therefore an open question whether the size of pinealocytes differs in different regions
and whether 24-h rhythms in the size of pinealocytes
exist in different regions in the pineal gland of the rat.
r 1998 WILEY-LISS, INC.
Our previous studies revealed that the size of pinealocytes of mice (Matsushima et al., 1989) and Chinese
hamsters (Matsushima et al., 1983; Sakai et al., 1986;
Hira et al., 1989) exhibited a 24-h rhythm similar to
that described by Quay and Renzoni (1966) in rats.
Thus, if the problem of the regional differences in the
size of pinealocytes is to be settled, it is expected that
24-h rhythms in the size of pinealocytes will be demonstrable also in rats. In a previous study concerning the
effect of a magnetic field on the pinealocytes of the rat,
we found, using transverse sections, that in shamexposed rats killed in April and October, the peripheral
pinealocytes were larger than the central pinealocytes
at the middle level of the proximodistal extent of the
superficial pineal gland, and distal pinealocytes were
*Correspondence to: Shoji Matsushima, Department of Anatomy,
Asahikawa Medical College, Nishikagura, Asahikawa 078, Japan
Received 12 May 1997; Accepted 29 July 1997
HETEROGENEITY OF RAT PINEAL
81
Fig. 1. For nuclear area measurements, transverse profiles of the superficial pineal gland were divided
into dorsal or ventral, outer peripheral (DOP or VOP), inner peripheral (DIP or VIP), outer central (DOC
or VOC) and inner central (DIC or VIC) regions.
larger than proximal pinealocytes, and also that distal
and proximal pinealocytes in rats killed in October, but
not those in animals killed in April, showed apparent
day-night changes in size (Matsushima et al., 1993).
Thus, in the present study, more detailed comparisons
of size of pinealocytes in different regions (peripheral
and central; dorsal and ventral) at different levels
(distal, middle, and proximal) were made in transverse
sections of the superficial pineal gland of normal rats
killed in October. Subsequently, whether or not the size
of pinealocytes in these regions exhibits day-night
rhythms was examined.
Although cell size is generally interpreted as reflecting cellular activity, the nature of the changes in the
size of pinealocytes is not exactly known. The present
investigation further aimed to elucidate the significance of the regional differences in pinealocyte size and
its 24-h rhythms from the morphological aspect. Thus,
we determined the area of blood vessels and length of
sympathetic fibers in the same regions as used for the
measurement of pinealocyte size, and investigated the
relationships between the distribution patterns of blood
vessels and sympathetic fibers and regional differences
in the size of pinealocytes in the rat. In addition,
whether or not the area of blood vessels in these regions
shows day-night changes was examined, and the area
of blood vessels and the size of pinealocytes at daytime
and nighttime were compared.
MATERIALS AND METHODS
Twenty-six male Wistar-King rats were used when
20–25 weeks old (330–425 g body weight). The animals
were kept under conditions of controlled lighting (LD
12:12; lights on at 0600 h) and temperature (21 6 2°C).
Light was provided by cool-white fluorescent lamps,
with the intensity at the level of the cages being
approximately 20 lux. The animals were housed two
per clear plastic cage and were provided food and water
ad libitum.
For the measurements of nuclear density and nuclear
size, each group of five animals was killed by decapitation at 1200 or 2400 h, i.e., the middle of the light or
dark period, respectively, on October 16. In a previous
study dealing with the effects of magnetic field exposure on the size of pinealocytes in the superficial pineal
gland of the rat, the size of pinealocytes in certain
regions was found to show day-night rhythms in October, but not in April, in sham-exposed rats (Matsushima et al., 1993). Thus, October was selected as the
month of sacrifice in the present study. A small portion
of the brain, 2- to 3-mm-thick, containing the superficial pineal gland and the distal part of the pineal
82
Y. HIRA ET AL.
Figs. 2,3. Transverse sections of an ink-injected, superficial pineal gland (P) at distal (Fig. 2) and
proximal (Fig. 3) levels. Slightly stained with hematoxylin. The upper portion of the pineal profiles is
directed dorsally. G, great cerebral vein. 3160.
parenchymal stalk, was rapidly removed, fixed for 20 h
in Bouin’s fluid, dehydrated in a graded series of
ethanol, cleared in benzene, and embedded in paraffin.
The embedded brain tissues were oriented so that the
pineal gland could be cut transversely from the distal
end. Serial sections were prepared at a thickness of 8
µm, and were stained with hematoxylin and eosin.
The size of pinealocytes was expressed as nuclear
density as previously described (Matsushima et al.,
1993). Nuclear density was determined by counting the
HETEROGENEITY OF RAT PINEAL
83
Fig. 4. Profile area of the superficial pineal gland at daytime (A) and nighttime (B) in every tenth
transverse section.
number of pinealocyte nuclei per unit area of 0.01 mm2;
10 unit areas from each region were used for each
animal. We have shown, using transverse sections, that
differences in nuclear density of pinealocytes exist
between peripheral and central regions of the middle
portion of the superficial pineal gland of the rat (Matsushima et al., 1993). In the present study, determinations of nuclear density in peripheral and central
regions were performed on the distal, middle, and
proximal levels. In addition, transverse profiles of the
superficial pineal gland at each level were divided into
dorsal and ventral halves. Thus, nuclear density was
estimated in the four regions (dorsoperipheral, dorsocentral, ventroperipheral, and ventrocentral) at the three
different levels. The most distal or proximal section,
large enough to cover at least more than one unit area
in each region, was selected for nuclear density determinations in the distal or proximal portions, respectively.
The section at the middle level between the distal and
proximal sections was used for nuclear density in the
middle portion. Adjacent sections were also examined
until the number of unit areas in each region attained
10. Pineal profile areas were determined from distal to
proximal levels as previously described (Sakai et al.,
1996). Serial transverse sections used for nuclear density determinations were examined at a magnification
of 3200, camera lucida drawings of pineal profiles were
made, and areas of the profiles were measured using an
image analyzer (Videoplan, Kontron, Munich, Germany).
As mentioned, nuclear density of pinealocytes differed between peripheral and central regions of the
middle portion of the superficial pineal gland of the rat
(Matsushima et al., 1993). In the present study, we
attempted to examine if the size of pinealocytes exhibits gradual changes from peripheral to central regions.
Thus, transverse profiles of the superficial pineal gland
in a section at the middle level were divided into four
concentric regions from peripheral to central (Fig. 1).
Since the area of each region was too small for nuclear
density determinations, nuclear profile areas of pinealocytes were measured.
84
Y. HIRA ET AL.
Fig. 5. Nuclear density of pinealocytes at 1200 (open circle) and 2400 h (solid circle) in the
dorsoperipheral (DP), dorsocentral (DC), ventroperipheral (VP), and ventrocentral (VC) regions at the
distal (D), middle (M) and proximal (P) levels.
Nuclear profile areas of pinealocytes were obtained in
the middle portion of the superficial pineal gland of
animals, which were killed at 1200 h and used for
nuclear density determinations. First, the number of
nuclei sufficient to obtain accurate values for the nuclear
profile areas was determined. Five alternate sections
from the middle portion of an animal were examined in
an Olympus microscope equipped with a drawing tube,
and outline drawings of pinealocyte nuclei were made
at the magnification of 31,000. Nuclear areas were
measured using an image analyzer (Videoplan, Kontron, Munich, Germany). One hundred nuclei were
randomly selected from each section. The differences
between the mean nuclear profile areas obtained from
each of the five sets of 100 nuclei and from the 500
nuclei were less than 5%. Thus, in this study, the mean
nuclear profile areas were obtained from 100 nuclei in
each region at the middle level of each animal. The
entire profile of the superficial pineal gland on the
section used for the nuclear density determinations in
the middle portion was photographed at 3200 and
enlarged to 31,000 to make montage micrographs.
Transverse profiles of the superficial pineal gland were
roughly oval in shape, with the long axis lying horizontally (Fig. 1). The middle portion of the dorsal surface
was usually invaginated by the great cerebral vein.
Three concentric figures, similar to the profile, were
depicted on the micrograph so that the intersections
with the long and short diameters of the profile divide
each diameter into eight equal parts. Midline and
horizontal line segments, which are intercepted by the
profile and cross at their midpoints, were regarded as
short and long diameters, respectively. Thus, the pineal
profile was divided into four concentric areas from
central to peripheral. These areas were then subdivided
into dorsal and ventral halves by horizontal line segments. Profile areas of 100 pinealocyte nuclei in the
above eight regions were measured. The pineal glands,
the profiles of which were not symmetric with respect to
the midline because of the deviation of the great
cerebral vein, were discarded.
Ten animals were used to examine the distribution
and area of blood vessels in the superficial pineal gland.
Each group of five animals was anesthetized with
sodium pentobarbital (80 mg/kg, i.p.) at 1200 or 2400 h
in October, and perfused transcardially with 50 ml
phosphate-buffered saline containing heparin (4,000
IU/kg) for 2 min, followed by 75 ml undiluted Indian ink
(Rotring, Germany) for 3 min. Animals killed at 2400 h
were anesthetized under dim red light and their heads
were covered by black cloth during perfusion. The
superficial pineal glands with the surrounding tissue
were removed and fixed in Bouin’s fluid for 20 h,
dehydrated in a graded series of ethanol, and embedded
in paraffin in vacuo. Serial transverse sections were
prepared at a thickness of 8 µm, and were stained
85
HETEROGENEITY OF RAT PINEAL
TABLE 1. ANOVA of nuclear density of pinealocytes in
peripheral and central regions, and at distal, middle and
proximal levels, in dorsal (A, C) or ventral (B, D) regions
at daytime (A, B) or at nighttime (C, D)
(A)
(B)
(C)
(D)
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
209.83
369.68
5.03
463.10
1
2
2
24
209.83
184.84
2.51
19.30
Total
1047.64
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
349.80
509.50
11.60
491.86
1
2
2
24
349.80
254.75
5.80
20.49
Total
1362.76
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
262.85
646.39
33.81
371.12
1
2
2
24
262.85
323.19
16.90
15.46
Total
1314.17
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
353.63
1257.54
1.42
308.24
1
2
2
24
353.63
628.77
0.71
12.84
Total
F
p
10.87
9.58
0.13
,0.01
,0.01
.0.10
F
p
17.07
12.43
0.28
,0.01
,0.01
.0.10
F
p
16.99
20.90
1.09
,0.01
,0.01
.0.10
F
p
27.53
48.96
0.06
,0.01
,0.01
.0.10
TABLE 2. ANOVA of nuclear density of pinealocytes at
daytime and nighttime, and at distal, middle and
proximal levels, in peripheral (A, C) or central (B, D)
regions in dorsal (A, B) or ventral (C, D) regions
(A)
(B)
(C)
(D)
29
slightly with hematoxylin. In addition to the most
distal or proximal section, or the section at the middle
level between them, nine sections adjacent to each of
the above three sections were used for the observation
of blood vessels. The most distal and proximal sections
were determined in the same way as that used for
nuclear density computations. Camera lucida drawings
of all the blood vessels in these sections were made at a
magnification of 3400, and the total profile areas of
blood vessels per unit area of 1 mm2 in the four regions
at distal, middle and proximal levels were determined
using a digitizer (KD4610A, Graphitec, Yokohama,
Japan) and computer (NIH, Image 1.59).
The distribution of sympathetic nerve fibers in the
superficial pineal gland was examined in five animals.
In addition, one animal, which was subjected to superior cervical ganglionectomy 1 week prior to sacrifice,
was used to examine the effect of ganglionectomy on
sympathetic nerve fibers. These animals were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) at 1200
h in October, and perfused through the left cardiac
ventricle with phosphate-buffered saline containing
heparin (4,000 IU/kg), followed by ice-cold Bouin’s
fixative without acetic acid. The superficial pineal
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
3.70
380.17
5.84
400.33
1
2
2
24
3.70
190.09
2.92
16.68
Total
790.05
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
13.33
632.69
36.20
433.90
1
2
2
24
13.33
316.34
18.10
18.08
Total
1116.12
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
1043.59
779.97
45.72
448.80
1
2
2
24
1043.59
389.98
22.86
18.70
Total
2318.08
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
1050.21
896.13
58.25
351.31
1
2
2
24
1050.21
448.07
29.13
14.64
Total
2355.90
29
F
p
0.22
11.40
0.18
.0.10
,0.01
.0.10
F
p
0.73
17.50
1.00
.0.10
,0.01
.0.10
F
p
55.81
20.85
1.22
,0.01
,0.01
.0.10
F
p
71.75
30.61
1.99
,0.01
,0.01
.0.10
TABLE 3. ANOVA of nuclear area of pinealocytes in
dorsal and ventral regions (R1), and from peripheral to
central regions (R2)
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
R1
R2
R1 3 R2
Error
0.14
7.22
0.19
2.35
1
3
3
32
0.14
2.41
0.06
0.07
Total
9.89
39
F
p
1.93
32.81
0.84
.0.10
,0.01
.0.10
gland together with the surrounding tissue was removed and fixed in the same fixative for 20 h at 4°C,
dehydrated in a graded series of ethanol, and embedded
in paraffin in vacuo. Serial transverse sections were
prepared at a thickness of 8 µm.
Sympathetic nerve fibers were stained with the antisera against tyrosine hydroxylase (TH; Chemicon International, Temecula, CA; Code No. AB152) using Vectastain Elite ABC kit (Vector, Burlingame, CA). Sections
were pretreated in 3% H2O2, preincubated in 1.5%
normal goat serum for 30 min at room temperature,
86
Y. HIRA ET AL.
TABLE 4. ANOVA of nuclear density of pinealocytes (A),
total area of blood vessels (B) and total length of TH-fibers
(C) at daytime in dorsal and ventral regions, and at distal,
middle and proximal levels
Source
of
variation
Sum
of
squares
(A) Region (R) 431.48
Level (L)
859.01
R3L
20.18
Error
1531.22
Total
Source
of
variation
(B) Region (R)
Level (L)
R3L
Error
Total
Source
of
variation
2841.89
Sum
of
squares
0.0121
0.0200
0.0021
0.0334
0.0676
Sum
of
squares
Degrees
of
freedom
1
2
2
54
Mean
of
squares
431.48
429.51
10.09
28.36
F
p
15.22 ,0.01
15.15 ,0.01
0.36 .0.10
59
Degrees
of
freedom
1
2
2
54
Mean
of
squares
F
p
0.0121 19.64 ,0.01
0.0100 16.18 ,0.01
0.0010 1.69 .0.10
0.0006
59
Degrees
of
freedom
(C) Region (R)
Level (L)
R3L
Error
0.0033
0.0216
0.0012
0.0349
1
2
2
54
Total
0.0610
59
Mean
of
squares
F
p
0.0033 5.17 ,0.01
0.0108 16.76 ,0.01
0.0006 0.91 .0.10
0.0006
and then incubated at room temperature overnight in
the antisera against TH (diluted 1:100). Sections were
incubated with biotinylated goat anti-rabbit IgG (diluted 1:200) for 1 h at 32°C. Finally, sections were
reacted with 0.01% diaminobenzidine tetrahydrochloride and 0.01% H2O2 in phosphate-buffered saline for 2
min at 37°C.
The density of sympathetic nerve fibers was expressed as the total length of TH-immunoreactive
fibers per unit area of various regions at the distal,
middle, and proximal levels of the superficial pineal
gland. Camera lucida drawings of TH fibers were made
at a magnification of 31,000, and the length of all TH
fibers per unit area of 0.0064 mm2 was measured using
an image analyzer ( Videoplan, Kontron, Munich, Germany). In one animal, the estimation of the total length
of these fibers per unit area was made on 200 unit
areas, which were randomly selected from a section of
the middle portion. The differences between the values
obtained from each of the five sets of 40 unit areas and
those from the 200 unit areas were less than 10%. Thus,
in this study, the total length of TH fibers per unit area
was expressed as the average of the values from 40 unit
areas. Sections from the distal, middle, or proximal
levels were selected in the same way as that used for
nuclear density determinations; transverse pineal profiles on the selected sections were divided into the four
regions, and the total length of TH fibers in each region
was estimated.
Data were expressed as the mean 6 the standard
error of the mean. Statistical analysis of data was
performed by a two-way analysis of variance (ANOVA).
The total area of blood vessels was analyzed after the
inverse sine transformation.
RESULTS
The great cerebral vein usually ran in the median
plane and just over the entire length of the superficial
pineal gland (Figs. 2, 3). Occasionally, it was displaced
laterally to a variable extent. The great cerebral vein,
large enough to cover a considerable part of the dorsal
surface of the distal portion of the gland (Fig. 2),
decreased in diameter toward the proximal level (Fig.
3). The dorsal surface was flat or slightly depressed at
the distal level (Fig. 2). The depressions deepened
toward the proximal level (Fig. 3). Profile areas of the
superficial pineal gland decreased toward distal or
proximal levels (Fig. 4). From the shape and size of the
profiles, it appears that the superficial pineal gland of
the rat usually has the form of a spindle, flattened or
depressed dorsally.
Nuclear density of pinealocytes increased significantly toward proximal levels (Fig. 5; Tables 1, 2, 4). If
comparisons of nuclear density between central and
peripheral regions were made on the dorsal or ventral
regions separately, nuclear density was significantly
larger in the central region than in the peripheral
region (Fig. 5; Table 1). The nuclear area of pinealocytes
showed a significant decrease from the outer peripheral
to inner central regions in both the dorsal and ventral
regions (Fig. 6; Table 3). Significant differences in the
nuclear area were not detected between dorsal and
ventral pinealocytes (Table 3). There were no day-night
differences in nuclear density in the dorsal region,
whereas in the ventral region, nuclear density exhibited significant day-night changes, decreasing at daytime and increasing at nighttime (Fig. 5; Table 2). At
daytime, nuclear density was significantly larger in the
dorsal region than in the ventral region (Fig. 5; Table 4).
The blood vessels in the superficial pineal gland were
almost completely filled with the Indian ink. The
profiles of the blood vessels were variable in size (Figs.
2, 3). At the distal level, the profiles appeared to be
larger in number and size in the ventroperipheral
region than elsewhere (Fig. 2). The profiles became less
numerous toward the proximal level (Fig. 3). Examination of the sections containing the pineal gland showed
that the density of blood vessels was apparently smaller
in the gland than in the cerebral hemispheres and the
midbrain. The total area of blood vessels decreased
significantly toward proximal levels (Fig. 7; Tables 4, 5,
6). The total area of blood vessels in the ventral region,
but not in the dorsal region, was significantly larger in
the peripheral region than in the central region (Fig. 7;
Table 5). Significant day-night differences existed in the
total area of blood vessels with its increase and decrease occurring at daytime and nighttime, respectively
(Fig. 7; Table 6); the differences were particularly
prominent in the ventral region. In daytime, the total
area of blood vessels was significantly larger in the
ventral region than in the dorsal region (Fig. 7; Table 4).
TH-immunoreactive fibers appeared as relatively
thick bundles in the pineal capsule and the surface of
the pineal parenchyma; the bundles split into a network of thinner bundles or single fibers toward the
interior of the gland (Fig. 8). The network was more
dense in the ventral region than in the dorsal region
HETEROGENEITY OF RAT PINEAL
87
Fig. 6. Nuclear area of pinealocytes at 1200 h in the dorsal or ventral, outer peripheral (DOP or VOP),
inner peripheral (DIP or VIP), outer central (DOC or VOC) and inner central (DIC or VIC) regions at the
middle level.
(Fig. 8). The total length of TH-immunoreactive fibers
was significantly larger toward the distal levels (Fig. 9;
Tables 4, 7); it was significantly larger in the peripheral
region than in the central region (Fig. 9; Table 7)s well
as in the ventral region than in the dorsal region (Table
4). TH-immunoreactive fibers disappeared completely
from the superficial pineal gland following bilateral
removal of the superior cervical ganglia.
DISCUSSION
Regional Difference in the Size of Pinealocytes
Previous results concerning the differences in the
size of pinealocytes between peripheral and central
regions in the rat are inconsistent. In earlier literature,
no apparent differences have been described in sizes of
pinealocyte nuclei and pinealocytes between the two
regions (Quay and Renzoni, 1966; Blumfield and Tapp,
1970), whereas more recent studies have shown that
the nuclear volumes of pinealocytes are significantly
larger in the periphery than in the center (Diehl, 1981;
Becker and Vollrath, 1983). The present results are
compatible with the findings of Diehl (1981) and Becker
and Vollrath (1983). We demonstrate, examining transverse sections, that pinealocyte size, expressed as
nuclear density, is larger in the peripheral region than
in the central region at any level from distal to proximal
and, in addition, the nuclear size of pinealocytes increases gradually toward the periphery. An inverse
relationship exists between nuclear size and nuclear
density of pinealocytes (Blumfield and Tapp, 1970;
Matsushima et al., 1990). It seems therefore that the
pinealocyte size in the rat is larger toward the periphery in transverse planes. Thus, the conception that the
parenchyma of the superficial pineal gland of the rat is
composed of two subdivisions, peripheral and central,
should be modified.
We were also able to demonstrate, using transverse
sections, that pinealocytes increased in size toward the
distal levels. This observation is compatible with our
previous results (Matsushima et al., 1993), although
the experimental conditions were not identical in both
studies. The present data indicate that the use of
transverse sections is essential for the study of the
regional differences in pinealocyte size in the rat. In
view of the presence of the distal to proximal and
peripheral to central gradients in pinealocyte size, it is
evident that the structure of the superficial pineal
gland of the rat is even more complex than previously
thought.
Taking into account the present results, it is postulated that pinealocytes varying in size are arranged in a
very regular fashion in the superficial pineal gland of
the rat, as displayed schematically in Figure 10. The
pineal parenchyma is composed of thin, dome-shaped
laminae of pinealocytes, which are superimposed in a
proximodistal direction (Fig. 10A). Pinealocytes are of
the same size in each lamina, increasing progressively
in size in more distal laminae. It is obvious that, in this
88
Y. HIRA ET AL.
Fig. 7. Total area of blood vessels at 1200 (open circle) and 2400 h (solid circle). Abbreviations are the
same as used in Figure 5.
model, pinealocytes are larger in the more peripheral
regions in a transverse plane (Fig. 10B).
The significance of the regional differences in the size
of pinealocytes of the rat is not known at present.
Relationships between the regional differences in the
size of pinealocytes and the distribution of blood vessels
and sympathetic fibers will be discussed later. Brief
considerations of the developmental aspects of the
regional differences in the size of pinealocytes of the rat
are given here. The embryonic development of the
superficial pineal gland of the rat was examined by
Calvo and Boya (1981), who found that the transformation of the pineal gland from a saccular structure into a
compact organ proceeds from distal to proximal levels.
This indicates that the development progresses faster
in the distal region than in the proximal region. Thus, it
is possible to assume that the differentiation process of
pinealocytes is different in different regions, and that
the regional difference in the size of pinealocytes in a
proximodistal direction is related to their developmental histories. However, nothing has been found in the
literature concerning the differentiation of larger (distal or peripheral) and smaller (proximal or central)
pinealocytes of the rat.
Distribution and Area of Blood Vessels, and Day-Night
Changes in Area of Blood Vessels
Studies by means of the India ink injection method
have scarcely been made on the intrapineal distribution
of blood vessels (von Bartheld and Moll, 1954; Hodde,
1979), and no quantitative data have so far been
obtained on this subject. The recent cast preparation
technique with the scanning electron microscope contributes greatly to our knowledge of the pineal vascularization (Hodde, 1979; Hodde and Veltman, 1979; Murakami et al., 1988; Chunhabundit and Somana, 1991).
This technique is quite useful for examining blood
vessels supplying or draining the pineal gland. However, a classical light microscopic study using serial
histological sections of India ink-injected specimens
may be more suitable for the demonstration of the
intrapineal distribution of blood vessels. Scanning electron microscopic studies of corrosion casts indicate that
intrapineal blood vessels consist mostly of capillaries
and form a homogeneous network in Wistar rats (Hodde,
1979; Murakami et al., 1988). In the present study,
however, the total area of blood vessels per unit area,
which may represent the number and size of blood
89
HETEROGENEITY OF RAT PINEAL
TABLE 5. ANOVA of total area of blood vessels in
peripheral and central regions, and at distal, middle and
proximal levels, in dorsal (A, C) or ventral (B, D) regions
at daytime (A, B) or at nighttime (C, D)
(A)
(B)
(C)
(D)
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
0.00030
0.00561
0.00060
0.00458
1
2
2
24
0.00030
0.00281
0.00030
0.00019
Total
0.01109
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
0.01513
0.01649
0.00384
0.00895
1
2
2
24
0.01513
0.00824
0.00192
0.00037
Total
0.04440
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
0.00001
0.00273
0.00033
0.00855
1
2
2
24
0.00001
0.00137
0.00016
0.00036
Total
0.01162
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
0.00801
0.01450
0.00527
0.02089
1
2
2
24
0.00801
0.00725
0.00264
0.00087
Total
0.04868
29
F
p
1.55
14.70
1.56
.0.10
,0.01
.0.10
F
p
40.57
22.10
5.14
,0.01
,0.01
.0.05
F
p
0.03
3.83
0.45
.0.10
,0.05
.0.10
F
p
9.20
8.32
3.03
,0.01
,0.01
.0.05
vessels, was found to show regional differences in
Wistar-King rats. Interestingly, the regional differences
in the area of blood vessels are almost identical with
those in the size of pinealocytes; pinealocytes are larger
in size in more richly vascularized regions. Thus, the
size of pinealocytes may be proportional to the degree of
vascularization.
One of the most interesting observations in the
present study is the demonstration of day-night changes
in the total area of blood vessels throughout the superficial pineal gland of the rat. The results suggest that
pineal capillaries dilate at daytime and constrict at
nighttime. The present data showing larger areas of
blood vessels at midday as compared to those at midnight are not consistent with the well-known nocturnal
enhancement of pineal metabolic activity related to
melatonin production in many mammalian species
including the rat. Our previous study revealed that the
areas of blood vessels in the pineal gland of mice
showed a 24-h rhythm similar to that described in the
present study in rats (Matsushima et al., 1989). Pineal
glands of many inbred strains of mice are reported to be
unable to synthesize significant amounts of melatonin
(Ebihara et al., 1986; Goto et al., 1989). Thus, it seems
TABLE 6. ANOVA of total area of blood vessels at
daytime and nighttime, and at distal, middle and
proximal levels, in peripheral (A, C) or central (B, D)
regions in dorsal (A, B) or ventral (C, D) regions
(A)
(B)
(C)
(D)
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
0.00229
0.00288
0.00037
0.00616
1
2
2
24
0.00229
0.00144
0.00018
0.00026
Total
0.01170
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
0.00116
0.00521
0.00081
0.00698
1
2
2
24
0.00116
0.00260
0.00041
0.00029
Total
0.01416
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
0.00942
0.03573
0.00051
0.02184
1
2
2
24
0.00942
0.01786
0.00025
0.00091
Total
0.06749
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Time (T)
Level (L)
T3L
Error
0.00404
0.00366
0.00020
0.00801
1
2
2
24
0.00404
0.00183
0.00010
0.00033
Total
0.01590
29
F
p
8.93
5.62
0.71
,0.01
,0.01
.0.10
F
p
3.99
8.95
1.39
,0.05
,0.01
.0.10
F
p
10.35
19.63
0.27
,0.01
,0.01
.0.10
F
p
12.10
5.49
0.29
,0.01
,0.01
.0.10
difficult to correlate the rhythm in the area of pineal
blood vessels with melatonin formation. Although the
presence of a 24-h rhythm in the size of intrapineal
blood vessels has not been reported in the rat, the
number of erythrocytes per unit volume of the pineal
gland of rats has been found to decrease from the end of
the dark period to the beginning of the light period
(Quay, 1972). Our data may not correspond to the
results obtained by Quay (1972), although the total
area of blood vessels and the number of erythrocytes
were not determined at the identical times. Measurements of the area of blood vessels at more time points
over a 24-h period are needed to explain the discrepancy between the two studies.
It is known that the blood flow to the pineal gland is
as high as that to some endocrine organs and the
choroid plexus in rats (Goldman and Wurtman, 1964;
Nikitovitch-Winer and Goldman, 1986) and pigs (Madsen et al., 1990). The pineal blood flow in sheep was
found to decrease from the end of the dark period to the
beginning of the light period (Rollag et al., 1978). This
observation may be compatible with the above-mentioned data on the pineal blood content in rats (Quay,
1972). Determinations of the pineal blood flow at vari-
90
Y. HIRA ET AL.
Fig. 8. TH-immunoreactive fibers in a transverse section of the left two-thirds of the middle region of the
superficial pineal gland. The upper portion of the pineal profile is directed dorsally. 3225.
ous time points and comparisons of the data with the
area of blood vessels in rats seem important to understand the significance of the day-night changes in the
area of the blood vessels.
Distribution of TH-Immunoreactive Fibers
A glyoxylic acid-induced fluorescence study (Schröder, 1987) and dopamine-b-hydroxylase immunohistochemistry (Schröder and Vollrath, 1985) have shown
that the sympathetic fibers are homogeneously distributed in the superficial pineal gland of the rat. In
addition, the sympathetic fibers in the pineal gland of
the rat have been repeatedly studied by means of
formaldehyde-induced fluorescence method (Møller and
van Veen, 1981; Vollrath, 1981) or tyrosine hydroxylase- or neuropeptide Y immunohistochemistry (Schon
et al., 1985; Zhang et al., 1991) but no mention has been
made of their distribution in relation to the regions of
the organ. The present quantitative observations have
clearly shown, for the first time, the regional difference
in the distribution of sympathetic fibers in the superfi-
cial pineal gland of the rat. In the rat, sympathetic
fibers enter the gland at its distal portion by way of the
nervi conarii and from its surface via the perivascular
spaces of pial vessels (Kappers, 1960, 1965; Bowers et
al., 1984). The differences in the density of sympathetic
fibers between distal and proximal regions and between
peripheral and central regions may be due to the
distance from the entrance of these fibers. Since the
density of sympathetic fibers is higher in the ventral
region than in the dorsal region, the nervi conarii may
ramify more extensively in the ventral region.
The similarities of the regional differences among the
distribution of the sympathetic fibers, the area of the
blood vessels, and the size of the pinealocytes indicate a
close correlation among them. The regional differences
in the size of the pinealocytes and the area of the blood
vessels may be caused by the uneven distribution of
sympathetic fibers. Sympathetic fibers and their endings are mainly located in perivascular spaces in the
superficial pineal gland of the rat (Matsushima and
Reiter, 1977). The present observation that a 24-h
HETEROGENEITY OF RAT PINEAL
91
Fig. 9. Total length of TH-immunoreactive fibers at 1200 h. Abbreviations are the same as used in
Figure 5.
TABLE 7. ANOVA of total length of TH-fibers in
peripheral and central regions, and at distal, middle and
proximal levels, in dorsal (A) or ventral (B) regions
(A)
(B)
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
0.00246
0.01445
0.00071
0.01338
1
2
2
24
0.00246
0.00723
0.00035
0.00056
Total
0.03101
29
Source
of
variation
Sum
of
squares
Degrees
of
freedom
Mean
of
squares
Region (R)
Level (L)
R3L
Error
0.01076
0.00837
0.00068
0.00686
1
2
2
24
0.01076
0.00418
0.00034
0.00029
Total
0.02667
29
F
p
4.42
12.96
0.63
,0.05
,0.01
.0.10
F
p
37.63
14.63
1.19
,0.01
,0.01
.0.10
rhythm in the area of the blood vessels is more prominent in the more richly innervated regions suggests
that sympathetic innervation plays an important role
in the regulation of the rhythm.
Day-Night Changes in Size of Pinealocytes
Day-night rhythms in the size of pinealocytes of the
rat, since the original description by Quay and Renzoni
(1966), have been studied by many subsequent investigators, and there is much confusion in the results thus
far obtained. The nuclear area or volume, or the
cytoplasmic area of pinealocytes was reported to be
larger at day than at night (Quay and Renzoni, 1966;
Lew et al., 1984), whereas a converse relationship
between daytime and nighttime values was also found
(Diehl et al., 1984; Karasek et al., 1990). In addition,
the pattern of the rhythm varied depending on the
regions in the gland or the day examined (Diehl, 1981;
Becker and Vollrath, 1983). Thus, it is generally accepted that the estimation of the size of pinealocytes is
not a reliable method for studying the day-night rhythm
of these cells (Diehl et al., 1984).
The present study is the first report to demonstrate
that there are two populations of pinealocytes: one
showing day-night changes in size and the other without such a rhythm, in the superficial pineal gland of the
rat, and that the former and latter pinealocytes are
localized in the ventral and dorsal regions, respectively.
Thus, it is evident that the size of pinealocytes can be
used as a reliable indicator of functional activity of
these cells, and that the pinealocytes are composed of
92
Y. HIRA ET AL.
Fig. 10. Diagrams showing an arrangement of pinealocytes in a
horizontal plane through the longitudinal axis of the superficial pineal
gland (A) and in a transverse plane at its middle level (B). Laminae of
pinealocytes are demarcated by dotted lines, and pinealocytes in each
lamina are expressed as those arranged in a single row. Bars in A
indicate the level of transection. Pinealocytes and their size are
expressed as solid circles of various size.
two distinct types, which probably differ in function.
The present finding substantiates the hypothesis proposed by Becker and Vollrath (1983) that the inconsistent karyometric results obtained from rats may be due
to the structural and functional complexity of the
pineal gland of this animal. It is clear that the distal to
proximal and peripheral to central gradients of pinealocyte size and the subdivisions into dorsal and ventral
regions according to the presence or absence of its
day-night changes are responsible for the previous
contradictory data concerning the 24-h rhythm in the
size of pinealocytes of the rat.
The discrepancy among the previous data obtained
from several species casts some doubt upon the importance of the measurement of pinealocyte size. Daynight rhythms, similar to those shown here in the rat,
were obtained in mice (Matsushima et al., 1989) and
Chinese hamsters (Matsushima et al., 1983; Sakai et
al., 1986; Hira et al., 1989), whereas nuclear or cytoplasmic volumes of pinealocytes over a 24-h period in golden
hamsters (Dombrowski and McNulty, 1984) and gerbils
(Welsh et al., 1979) were different from those in the
above animals. The reason for this discrepancy remains
unknown. Further studies are needed to examine
whether or not factors other than species variation are
responsible for the differing results.
The present finding that the total area of blood
vessels in the ventral region and the size of ventral
pinealocytes exhibit prominent day-night changes suggests that both rhythms are closely related. In this
context, our quantitative electron microscopic observations obtained from mice (Matsushima et al., 1989) are
worthy of mention. In mice, nuclear and cytoplasmic
areas of pinealocytes and area of capillary lumen were
HETEROGENEITY OF RAT PINEAL
also larger during daytime than during nighttime. In
addition, there was a marked similarity between the
24-h rhythms of pericapillary spaces in mice and rats.
Quay (1974) demonstrated a diurnal increase and a
nocturnal decrease in the width of pineal canaliculi of
rats, which correspond to pericapillary and wide intercellular spaces. The area of pericapillary spaces and
their extensions in the pineal gland of mice also showed
similar changes (Matsushima et al., 1989). These observations suggest that 24-h rhythms in areas of blood
vessels and perivascular and intercellular spaces, and
the size of pinealocytes in both animals are generated
by similar mechanisms. The size of pinealocytes of the
rat exhibits day-night rhythms in the particular region
where sympathetic fibers are abundantly distributed
and where prominent day-night rhythms in the size of
the blood vessels are observed. Thus, sympathetic
innervation may be important for the control of all of
these rhythms in the rat. The influence of superior
cervical ganglionectomy on the rhythms of areas of
blood vessels and perivascular spaces has not hitherto
been investigated. Peschke et al. (1989) have reported
that ganglionectomy induces changes in the pattern of
24-h rhythms in the nuclear size of pinealocytes of the
rat. In this study, however, regions of the organ are not
considered. The effects of ganglionectomy on the rhythm
of pinealocyte size should be reexamined.
The significance of day-night changes in the size of
pinealocytes is obscure. Electrophysiological investigations have demonstrated the presence of two types of
pinealocytes of the rat; one shows a 24-h rhythm of
spontaneous electrical activity and the other does not
(Reuss, 1987). However, no studies have been made to
correlate the rhythm in the size of pinealocytes with
that in the electrical activity of these cells. The daynight rhythm in pinealocyte size may have no relation
to its regional differences, because the rhythm occurs
only in the ventral region and throughout this region.
Immunohistochemical studies have revealed distal to
proximal gradients in the intensity of staining for
neuron-specific enolase (McClure et al., 1986) or Santigen (Korf et al., 1985) in the superficial pineal
gland of the rat. Attempts to examine the relationships
between these electrophysiological and immunohistochemical data and the present findings, i.e., the regional differences in the size of pinealocytes, and the
differential localization of sympathetic fibers and pinealocytes showing day-night rhythms in their size, seem
essential for a better understanding of the heterogeneity of the superficial pineal gland of the rat.
LITERATURE CITED
Becker, U.G., and L. Vollrath 1983 24-hour-variation of pineal gland
volume, pinealocyte nuclear volume and mitotic activity in male
Sprague-Dawley rats. J. Neural Transm., 56:211–221.
Bowers, C.W., L.M. Dahm, and R.E. Zigmond 1984 The number and
distribution of sympathetic neurons that innervate the rat pineal
gland. Neuroscience, 13:87–96.
Blumfield, M., and E. Tapp 1970 Measurements of pineal parenchymal
cells and their nuclei in the albino rat at different ages. Acta
Morphol. Neerl. Scand., 8:1–8.
Calvo, J., and J. Boya 1981 Embryonic development of the rat pineal
gland. Anat. Rec., 200:491–500.
Chunhabundit, P., and R. Somana 1991 Scanning electron microscopic
study on pineal vascularization of the common tree shrew (Tupaia
glis). J. Pineal Res., 10:59–64.
Diehl, B.J.M. 1981 Time-related changes in size of nuclei of pinealocytes in rats. Cell Tissue Res., 218:427–438.
93
Diehl, B.J.M., U. Heidbüchel, H.A. Welker, and L. Vollrath 1984
Day/night changes of pineal gland volume and pinealocyte nuclear
size assessed over 10 consecutive days. J. Neural Transm.,
60:19–29.
Dombrowski, T.A., and J.A. McNulty 1984 Morphometric analysis of
the pineal complex of the golden hamster over a 24-hour light:
dark cycle: I. The superficial pineal in untreated and optically
enucleated animals. Am. J. Anat., 171:359–368.
Ebihara, S., T. Marks, D.J. Hudson, and M. Menaker 1986 Genetic
control of melatonin synthesis in the pineal gland of the mouse.
Science, 231:491–493.
Goldman, H., and R.J. Wurtman 1964 Flow of blood to the pineal body
of the rat. Nature, 203:87–88.
Goto, M., I. Oshima, T. Tomita, and S. Ebihara 1989 Melatonin content
of the pineal gland in different mouse strains. J. Pineal Res.,
7:195–204.
Hira, Y., Y. Sakai, and S. Matsushima 1989 Comparisons of sizes of
pinealocyte nuclei and pinealocytes in young and adult Chinese
hamsters (Cricetulus griseus) under different photoperiod conditions. J. Pineal Res., 7:411–418.
Hodde, K.C. 1979 The vascularization of the rat pineal organ. Prog.
Brain Res., 52:39–44.
Hodde, K.C., and W.A.M. Veltman 1979 The vascularization of the
pineal gland (epiphysis cerebri) of the rat. Scan. Electr. Microsc.,
III:369–374.
Kappers, J.A. 1960 The development, topographical relations and
innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch., 52:163–215.
Kappers, J.A. 1965 Survey of the innervation of the epiphysis cerebri
and the accessory pineal organs of vertebrates. Prog. Brain Res.,
10:87–153.
Karasek, M., B. Stankov, V. Lucini, F. Scaglione, G. Esposti, M.
Mariani, and F. Fraschini 1990 Comparison of the rat pinealocyte
ultrastructure with melatonin concentrations during daytime and
at night. J. Pineal Res., 9:251–257.
Korf, H.-W., M. Møller, I. Gery, J.S. Zigler, and D.C. Klein 1985
Immunocytochemical demonstration of retinal S-antigen in the
pineal organ of four mammalian species. Cell Tissue Res., 239:81–
85.
Lew, G.M., K. Washko, and W.B. Quay 1984 Quantitation of ultrastructural twenty-four-hour changes in pineal nuclear dimensions. J.
Pineal Res., 1:61–68.
Madsen, F.F., F.T. Jensen, M. Væth, and J. Ch. Djurhuus 1990
Regional cerebral blood flow in pigs estimated by microspheres.
Acta Neurochir., 103:139–147.
Matsushima, S., and R.J. Reiter 1977 Fine structural features of
adrenergic nerve fibers and endings in the pineal gland of the rat,
ground squirrel and chinchilla. Am. J. Anat., 148:463–478.
Matsushima, S., Y. Morisawa, I. Aida, and K. Abe 1983 Circadian
variations in pinealocytes of the Chinese hamster, Cricetulus
griseus. A quantitative electron-microscopic study. Cell Tissue
Res., 228:231–244.
Matsushima, S., Y. Sakai, and Y. Hira 1989 Twenty-four-hour changes
in pinealocytes, capillary endothelial cells and pericapillary and
intercellular spaces in the pineal gland of the mouse. Semiquantitative electron-microscopic observations. Cell Tissue Res., 255:323–
332.
Matsushima, S., Y. Sakai, and Y. Hira 1990 Effect of photoperiod on
pineal gland volume and pinealocyte size in the Chinese hamster,
Cricetulus griseus. Am. J. Anat., 187:32–38.
Matsushima, S., Y. Sakai, Y. Hira, M. Kato, T. Shigemitsu, and Y.
Shiga 1993 Effect of magnetic field on pineal gland volume and
pinealocyte size in the rat. J. Pineal Res., 14:145–150.
McClure, C.D., P.J. McMillan, and A. Miranda 1986 Demonstration of
differential immunohistochemical localization of the neuronspecific enolase antigen in rat pinealocytes. Am. J. Anat., 176:461–
467.
Møller, M., and Th. vanVeen 1981 Fluorescence histochemistry of the
pineal gland. In: The Pineal Gland, Vol. I, Anatomy and Biochemistry. R.J. Reiter, ed. CRC Press, Boca Raton, pp. 69–93.
Murakami, T., A. Kikuta, T. Taguchi, and A. Ohtsuka 1988 The blood
vascular architecture of the rat pineal gland: A scanning electron
microscopic study of corrosion casts. Arch. Histol. Cytol., 51:61–
69.
Nikitovitch-Winer, M.B., and H. Goldman 1986 Effect of hypothalamic
deafferentation on hypophysial and other endocrine gland blood
flows. Endocrinology, 118:1166–1170.
Peschke, E., M. Schön, S. Tertsch, D. Peschke, and J. Peil 1989
Morphometric investigations of the pineal gland after ganglionectomy and thyroidectomy under the aspect of circadian and
seasonal variations. J. Hirnforsch., 30:399–407.
94
Y. HIRA ET AL.
Quay, W.B. 1972 Twenty-four-hour rhythmicity in carbonic anhydrase
activities of choroid plexuses and pineal gland. Anat. Rec., 174:279–
287.
Quay, W.B. 1974 Pineal canaliculi: Demonstration, twenty-four-hour
rhythmicity and experimental modification. Am. J. Anat., 139:81–
93.
Quay, W.B., and A. Renzoni 1966 Twenty-four-hour rhythms in pineal
mitotic activity and nuclear and nucleolar dimensions. Growth,
30:315–324.
Reuss, S. 1987 Electrical activity of the mammalian pineal gland.
Pineal Res. Rev., 5:153–189.
Rollag, M.D., P.L. O’Callaghan, and G.D. Niswender 1978 Dynamics of
photo-induced alterations in pineal blood flow. J. Endocrinol.,
76:547–548.
Sakai, Y., I. Aida, and S. Matsushima 1986 Effect of continuous
darkness on circadian morphological rhythms in pinealocytes of
the Chinese hamster, Cricetulus griseus. Cell Tissue Res., 245:127–
134.
Sakai, Y., Y. Hira, and S. Matsushima 1996 Regional differences in the
pineal gland of the cotton rat, Sigmodon hispidus: Light microscopic, electron microscopic, and immunohistochemical observations. J. Pineal Res., 20:125–137.
Schon, F., J.M. Allen, J.C. Yeats, Y.S. Allen, J. Ballesta, J.M. Polak, J.S.
Kelly, and S.R. Bloom 1985 Neuropeptide Y innervation of the
rodent pineal gland and cerebral blood vessels. Neurosci. Lett.,
57:65–71.
Schröder, H. 1987 Aminergic innervation pattern of the rodent pineal
gland: No apparent influence of time of day. Acta Anat., 129:22–
26.
Schröder, H., and L. Vollrath 1985 Distribution of dopamine-betahydroxylase-like immunoreactivity in the rat pineal organ. Histochemistry, 83:375–380.
Vollrath, L. 1981 The pineal organ. In: Handbuch der Mikroskopischen Anatomie des Menschen, VI/7, A. Oksche and L. Vollrath,
eds. Springer, Berlin-Heidelberg-New York, pp. 186–193.
von Bartheld, F., and J. Moll 1954 The vascular system of the mouse
epiphysis with remarks on the comparative anatomy of the
venous trunks in the epiphyseal area. Acta. Anat., 22:227–235.
Welsh, M.G., I.L. Cameron, and R.J. Reiter 1979 The pineal gland of
the gerbil, Meriones unguiculatus. II. Morphometric analysis over
a 24-hour period. Cell Tissue Res., 204:95–109.
Zhang, E.-T., J.D. Mikkelsen, and M. Møller 1991 Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fibers in the
pineal complex of untreated rats and rats following removal of the
superior cervical ganglia. Cell Tissue Res., 265:63–71.
Документ
Категория
Без категории
Просмотров
3
Размер файла
513 Кб
Теги
structure, epithelium, dorsal, trachemys, elegans, linguam, emydidae, chelonia, fine, script
1/--страниц
Пожаловаться на содержимое документа