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Department of Anatomy and Cell Biology (A.-J.S., R.S.), College of Physicians and Surgeons, Columbia University, New York, New York 10032; Department of Psychology (R.S.), Barnard College, New York, New York 10027; and Department of Psychology (M.W., B.K., R.S.), Columbia University, New York, New York 10027
Address all correspondence and requests for reprints to: Dr. Rae Silver, Columbia University, Mail Code 5501, 1190 Amsterdam Avenue, New York, New York 10027. E-mail: qr{at}columbia.edu
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Abstract |
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Introduction |
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The presence of MC in the central nervous system (CNS) of many species has been described (4). It has been suggested that in rats over 80% of the histamine in discrete brain regions, particularly the thalamus, is derived from MC rather than from neurons (5). In adult rodents, the number of MC is highly variable but they are most prominent around the medium size blood vessels of the thalamus and in regions of the hippocampus (6). These cells can secrete following IgE coupling and in response to other stimuli including neurotransmitters (7) (for review, see Ref. 8). MC contain a wide range of biologically active molecules, including biogenic amines, heparin (or heparan) sulfated proteoglycans, neutral proteases, and neuropeptides. They also make a large number of additional factors when stimulated (9). Given these characteristics, it is clear that even a small number of such potent unicellular glands have a significant effect on CNS blood vessel permeability (10) and on neuronal activation (or suppression) (11).
MC display a secretory cycle in which they alternate between resting and active states. Following activation, they can replenish their specific products. MC have two forms of secretion, termed anaphylactic and piecemeal degranulation. The former is characterized by compound exocytosis (12), whereas in the latter, vesicles bud off from the parent granule, leaving the granule with an electron lucent core (13). In cells undergoing compound exocytosis, fusion chambers are formed with one granule fusing with the plasma membrane while other granules fuse with the first in a chain reaction (12). The opening of the granule to the extracellular space results in the influx of water and the resultant hydration of the proteoglycans. This form of secretion results in the emptying of the majority of the granule’s contents (14). Following secretion, MC recover by increased protein synthesis and the production of new granules and/or the refilling of old ones (15, 16).
In ring doves, courtship reproducibly results in a highly significant increase in the number of MC in the medial habenula within 2 h of pairing; such elevations are easily detectable both qualitatively (17) and quantitatively (18, 19). Courtship represents the initiation of a cascade of events leading to reproductive readiness, mating, and parental behavior in this species (20). The first phase of this reproductive cycle is characterized by an increase in circulating gonadal steroids in both sexes.
In the present study, we have asked several questions concerning the phenomenon of MC appearance in the dove CNS including: 1) Can sex-specific gonadal steroids induce an elevation in the number of detectable MC in the medial habenula? 2) Are there any sex differences? 3) Is there evidence for degranulation (activation) of these cells? We have used histochemical and quantitative ultrastructural methods to obtain answers to these questions.
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Materials and Methods |
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For light microscopic studies, animals were given SILASTIC brand implants (Dow Corning, Midland, MI) of one of the following hormones: cholesterol (n = 5 females and n = 8 males), testosterone (T, n = 7 males), dihydrotestosterone (DHT, n = 9 males); 17ß estradiol (E, n = 8 females). For electron microscopy, the following groups were studied: control animals included males in visual isolation (n = 2) and those given SILASTIC capsules containing cholesterol (n = 3 males, n = 3 females). Experimental animals included females given E (n = 3); males treated either with T (n = 3) or DHT (n = 3), and males that had been courting for 2 h (n = 3). The latter animals were placed in cages measuring 80 x 45 x 35 cm with a female.
Steroid capsule preparation and implantation
SILASTIC capsules (id = 0.058, od = 0.07, l = 1 cm, with a
0.25 cm plug at each end) were used to administer the steroids at
approximately physiological levels (21). Capsules were implanted sc for
7 days, which is the approximate duration of courtship in this species
(22). For capsule implantation, animals were anesthetized with 0.3 ml
of chloropent (4.25% chloral hydrate; 2.12% magnesium sulfate; 0.88%
phenobarbital, 14.25% ethanol and 33% propylene glycol injected
im).
Tissue preparation for light microscopy
On day 8 after implantation, a lethal dose of chloropent (1.0
ml) was administered (im) followed 5–10 min later by 0.33 ml heparin
(300 U, Sigma, St. Louis, MO). The bird was
perfused via the carotid artery using a peristaltic pump (Harvard
apparatus) at 8 ml/min. An initial flush with 50–100 ml of 0.1
M phosphate buffer (PB), pH 7.4 was followed by 150 ml of
4% paraformaldehyde containing 0.1% glutaraldehyde (vol/vol) in PB.
After perfusion, the brain was removed from the cranium and placed in
the same fixative at room temperature for 2 h. Tissue blocks were
then cryoprotected with 20% sucrose and embedded in 12% gelatin
(Fisher Scientific International, Inc., Springfield,
NJ), which facilitated preparation of brain sections. The
gelatin is hardened by refrigeration and then fixed in a solution of
4% paraformaldehyde and 20% sucrose.
Sections were cut on a freezing microtome at 40 µm in the sagittal plane and collected sequentially. For acidic toluidine blue staining, sections were mounted on double subbed slides (subbing solution: 0.75% gelatin with 0.5% chromium potassium sulfate) and air dried.
Acidic toluidine blue histochemistry
This aniline dye at acidic pH, reacts with the sulfated
proteoglycans in the MC granules producing metachromasia. Toluidine
blue (Sigma) was dissolved in 60% ethanol and acidified
to pH 2.0 with HCl. Sections were rinsed in water, and processed
through 60% ethanol before being treated for 5 min with the dye.
Following staining the sections were cleared in acetone, cleared in
Hemo-De (both from Fisher Scientific International, Inc.)
and coverslipped using Permount.
Analysis of sections for LM
For the light microscopic histochemical procedure, alternate
sections through the entire medial habenula for each experimental
subject were stained for (acidic) toluidine blue metachromasia and the
numbers of MC were counted.
Because alternate sections were used for each marker, cells can appear in more than one section. Based on the fact that mast cells are essentially round, the Abercrombie factor (23) was applied to correct overestimation of cell number. The correction factor was calculated as follows: N = 2n (T/T + D), where N is the corrected cell number, n is the uncorrected cell number multiplied by 2 (because of using alternate sections), T is the section thickness, and D is the mean cell diameter calculated from mean cell surface area (24).
Electron microscopy
The animals were administered the same anesthetic as above and
perfused through the carotid artery with 2% paraformaldehyde (made
fresh from powder, pH 7.3) containing 2.5% EM grade glutaraldehyde
(Sigma). Tissue blocks containing the habenula were
postfixed at room temperature for at least 4 h and were stored in
this fixative for up to 4 days. If brains could not be processed
immediately they were cryoprotected with 20% sucrose followed by
"anti-freeze" (25) and stored at -20 C. This latter procedure
preserves cytoarchitecture. Tissue blocks were embedded in gelatin
(vide supra) and 50 µm sections were cut on a vibratome
(DSK Microslicer, Ted Pella, Inc., Redding, CA). The medial
habenula was identified under the dissecting microscope and dissected
using a scalpel blade. These small pieces were osmicated [2%
OsO4 in 0.9% NaCl containing 1.5%
K4 Fe(CN)6] for 2 h, room temperature;
dehydrated in ascending alcohol series, cleared in propylene oxide and
embedded in EPON (Electron Microscopy Sciences, Fort Washington,
PA). Ultrathin sections were cut at 70 nm, mounted on
Formvar-coated slot hole grids (Ted Pella, Inc.) and viewed with
a JEOL (Peabody, MA) 1200EX electron microscope.
Cells (21–51/bird), cut through the plane of the nucleus, were identified in 1-µm sections using basic toluidine blue (borate buffer, pH 11) for each bird and then photographed at 6–8,000x; final printing was at 18–20,000x. The aim was to determine if there was evidence for changes in state of activation of MC under the conditions tested. Two methods were used to estimate cellular activity. The first method was point-counting stereology to determine the volume fraction of different granular subtypes within MC of the different groups (26). This approach proved problematic as organelles are not distributed evenly through a MC (e.g. its Golgi is highly polarized). A more serious difficulty arose from the fact that many of cells in the steroid-treated animals were highly degranulated (see Results), and it was impossible to score the finely particulate material as within a granule or as free in the cytoplasm.
The second method, results of which are presented here, sorted MC into
five categories, according to morphological appearance of their
granules during secretory and recovery phases (Fig. 1
). The categories are based on detailed
observations of the appearance of MC in doves that were sexually active
(27), developing (24), or treated with Compound 48/80 (a polyamine
degranulating agent) (10); as well as work on other avian species (28, 29) and mammalian MC studies (30). These categories are shown
schematically in Fig. 1, and are defined as follows: (I) resting; three
phases of activation namely (II) initial activation; (III) fully
degranulated; (IV) piecemeal degranulation; and (V) resynthesizing.
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Scoring of MC
Micrographs (n = 564) were placed in random order and
scored into the five categories described above by two observers (M.W.
and A.-J.S.), who were blind to the experimental groups of the animals
from which the micrographs derived. Interobserver reliability was
98.6%, representing agreement on 558/564 cells. The statistical
outcome of the experiment was identical whether or not the eight cells
for which the raters disagreed were included in the analysis. As there
were no differences between isolated animals (n = 2) and those
treated with cholesterol (n = 6) across the five categories used,
we merged these groups for the data analysis.
Statistical analysis
Overall differences among groups were determined using a
factorial ANOVA and Fisher’s post hoc test (P <
0.05), unless otherwise specified.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (ANOVA F = 4.9, df = 3,33,
P = 0.007). Significant differences were found between
control animals and each of the steroid-treated groups (Fisher’s post
hoc, C vs. E, P = 0.04 increase of 230%; C
vs. T, P = 0.002 increase of 319%; C
vs. DHT, P = 0.007 increase of 273%). There was
no statistically significant difference in MC numbers among animals
treated with E, T, or DHT.
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). In others, the granules were more
dispersed within the cell, with paler zones of granule-free cytoplasm
or which contained dispersed granules (Fig. 3B). This variability
suggested the occurrence of both activated and resting MC in the medial
habenula which was confirmed in the EM material (below).
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summarizes these
data and shows the percentage of MC in each category for each
experimental group.
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, A–E. These are
characterized in the following paragraphs. The resting cell (I) is
filled with electron dense secretory granules of varying subgranular
architecture. Some granules are homogeneously dense and have a
semilunar lucent cap. Others have scroll or lattice-like interiors or
show densely particulate patterns. As with all MC, the resting cell has
a heterochromatic nucleus and filamentous processes or filopodia on
their external surface. A representative resting MC, taken from a male
bird that had been kept in visual isolation from conspecifics, is shown
in Fig. 4A.
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, B–D; all micrographs are taken from estrogen-treated
animals. At the initiation of secretion (II), the MC have a few fusion
chambers, but these do not predominate in the cytoplasm. Other granule
subtypes appear swollen and less electron dense than in resting cells.
At higher magnification (Fig. 4B), stacks of granules (incipient fusion
chambers) are apparent, and those granules closest to the plasma
membrane have the most dispersed interiors. For the cell shown in Fig. 4B1 a single extruded granule is present in the expanded extracellular
space, created in part by the filopodia of the MC.
Fully degranulated (III) MC have few dense granules. The cytoplasm is
dominated by widely expanded fusion chambers filled with finely
particulate material (Fig. 4C). At higher magnification (Fig. 4C1), the
hydration of the proteoglycan core of the fusion chamber has stretched
such that the granular membrane loses its electron density and cannot
be resolved.
When cells undergo piecemeal degranulation (IV), many of the secretory
granules are electron lucent. Some electron dense granules, such as
those found in the resting state, are also present (Fig. 4D). Closer
examination (Fig. 4D1) of the lucent granules shows that there are
focal deposits of dense material at the inside of the granules membrane
and particulate material in the core. Piecemeal degranulation and
compound exocytosis can occur in the same cell.
Resynthesizing cells (V) have relatively few granules, most of
which are electron dense. These cells are filled with the organelles
associated with protein synthesis and packaging, have a prominent Golgi
apparatus (Fig. 4E). Abundant elements of the smooth endoplasmic
reticulum are also present.
The results indicate significant differences among groups in the
state of their MC. Control animals (those treated with cholesterol and
those housed in isolation) had significantly more cells in the resting
state than did animals in any of the gonadal steroid-treated groups
(overall ANOVA F = 3.6, df = 4,15, P = 0.03;
Fisher’s post hoc test C vs. T, P = 0.03; C
vs. E, P = 0.04; C vs. DHT,
P = 0.02). Similarly, animals that had courted for
2 h also had significantly more resting cells that did the
steroid-treated animals (Fisher’s posthoc 2 h vs. T
P = 0.04; 2 h vs. DHT P = 0.03,
2 h vs. E, P = 0.06) (Fig. 5). Conversely, the hormone-treated
animals had more MC in the secretory cycle (described further
below).
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). A more detailed analysis indicates a
significant difference among hormone-treated groups in the initial
activation (II) of MC secretion (ANOVA F = 4.23, df = 3,13,
P = 0.03). Estrogen treatment results in fewer cells in this
category than does DHT (Fisher’s post-hoc test, P = 0.004),
and this difference almost reaches significance in the comparison
between E and T (P = 0.059).
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Resynthesizing (V) cells were few in number in all of the groups, and there were no differences among groups.
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Discussion |
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Increases in mast cell number
In previous work we demonstrated that the size of the MC
population is increased within 2 h of the initiation of sexual
behavior (19). It is known that circulating levels of T and DHT are
elevated in males within 4 h of pairing (the earliest time point
tested) and remain elevated until the time eggs are laid, approximately
7 days (22). The present data show that this increase in the MC
population can be triggered by circulating steroids. The numbers of MC
in the medial habenula of DHT- and T-treated males and E-treated
females are within the range found in animals that had courted for
2 h (19). We conclude first, that courtship behavior itself is not
necessary for an increase in the population of habenular MC. Second,
the sexes respond similarly to homotypical gonadal steroids. Third, T
and DHT were equally potent in increasing MC numbers and stimulating
their activation, indicating that aromatization to 17ß estradiol is
not necessary. Finally, the number of detectable MC in the medial
habenula of cholesterol-treated birds was similar to that seen in males
kept in visual isolation (19), indicating that the stress of anesthesia
and capsule implantation is not a factor in the current phenomenon,
though it has been reported that stress alters MC populations (33). The
hypothesis that follows from these studies is that the increase in MC
number in the medial habenula, a relay station between the limbic
forebrain and mid-brain involved in motivational states (34), plays a
role in reproductive physiology and behavior.
There have been numerous reports of correlations between MC numbers and hormonal state. For example, newly parturient rats have significantly more MC in the thalamus than do virgin controls (35, 36). This may be related to the stress of pup removal, and stress is known to alter the numbers of thalamic mast cells in rats (33). That the increases in MC number observed following pup removal are due to MC migration is based on our demonstration that MC labeled ex vivo with vital dyes can be found within the CNS within an hour of intravascular injection of the labeled cells (35, 36).
Gonadal steroids could influence MC activation and/or migration by acting directly on the MC, or indirectly by stimulating neuronal elements that subsequently release a chemotactic molecule. Nuclear estrogen receptors have been reported in MC of the rat peritoneum (37) and bladder (38). Although direct experimental tests of MC migration in the presence of gonadal steroids have not been performed, substantial in vivo evidence exists that there is appreciable reorganization of the location of MC in reproductive organs during the estrous or menstrual cycle (39, 40, 41).
It is known that the medial habenula (in contrast to the lateral habenula) has no steroid receptors. However, there are afferents to the medial habenula that arise from steroid containing areas such as the septal region (see above) and the raphe (34, 42). The only molecular candidate known to be released in the medial habenula that is chemotactic for MC is ATP (43). This originates from the nerve terminals of triangular and septo-fimbrial neurons which terminated in the medial habenula and release ATP (44). These septal regions also contain neurons with estrogen and/or androgen receptors (45). The released ATP could act as a chemotactic factor on MC resident in the pia. ATP could also act as a fast neurotransmitter (46) inducing medial habenula neurons to release a chemoattractant.
Mast cell activation
Based on the quantitative electron microscopic results, it is
clear that elevation in circulating sex-specific gonadal steroids
results in a shift out of the resting state (I) into actively secretory
ones (II, III, IV). The control animals had more resting MC than did
any of the hormone-treated groups (Fig. 5), and conversely,
hormone-treated animals had more cells in the remaining, activated
categories (Fig. 6). There were few group differences within any
specific activated categories, presumably reflecting the rapidity of
the secretory events and the transition from one active category to
another. Such rapid response occurs in human skin where degranulation
following IgE coupling by allergen begins within 15 sec upon exposure
to allergen and complete degranulation is found in 95% of MC within 5
min (47).
The triggers for the MC degranulation observed in this study are not known, although they may be similar to those listed above. The numbers and distribution across categories of activated MC in the birds that had courted for 2 h were similar to that of control animals. These findings indicate that the signals triggering increased numbers of MC in the medial habenula (19) are separable from the steroid-related signal(s) that stimulates degranulation.
In vitro treatment with estrogen alters the release characteristics of the MC in response to neurotransmitters such as substance P and carbachol, a stable agonist of nicotinic acetylcholine receptor; the steroid augments the secretion of histamine or 5HT following exposure to these substances (48). In our experiment, both T and DHT also resulted in a large number of activated cells. In contrast, mammalian connective tissue MC show a decreased responsiveness to substance P in the presence of testosterone in vitro (48). Clearly, either the phenotype of the MC or the microenvironment of the medial habenula differs from the connective tissue cells and/or in vitro conditions.
MC activation occurs in other reproductive-related conditions. For example, in the primate endometrium there is a dramatic increase in MC activation immediately before and during menstruation (49). MC degranulation releases mediators that can up-regulate the secretion of metalloproteases and activate latent ones (50). Such events result in the degradation of the basal lamina of the endometrial glands, the eventual breakdown of the endometrium, and the bleeding at menstruation. It appears that the MC is the central effector cell leading to the onset of endometrial failure and bleeding following the withdrawal of estrogen and progesterone (50).
In summary, treatment of adult ring doves with sex-specific gonadal steroids results in an increase in mast cell number in the medial habenula. This hyperplasia is accompanied by degranulation of the MC and release of MC mediators. These mediators have the potential of altering the blood brain barrier (10), neuronal properties (6, 11), or synaptic arrangements (51). Further studies are needed to understand hormone-brain-mast cell interactions.
Gordon Research Conference, Reproductive Tract Biology Connecticut College, New London, CT, July 1–6, 2000
The topics will include the following: female reproductive tract: novel mechanisms of growth control in the ovary; male reproductive tract: factors that regulate development of the sex cells and reproductive organs; grand rounds in reproductive tract biology; signaling mechanisms that mediate critical transitions in the mammary gland; genes that regulate placental development; uterine responses that are critical to implantation and placentation; biology of the reproductive tract of large animals; nuclear receptors as master regulators of hormone actions. There will also be a panel discussion entitled "Reproduction has a past, but does it have a future?"
The invited speakers and panel participants include: Donna Baird, Fuller Bazer, Richard Behringer, Henning M. Beier, Elizabeth A. Bonney, David Brigstock, James Cross, Anne Croy, Rodney D. Geisert, Nava Dekel, Beatrice Desvergne, S. K. Dey, Jeane Djiane, Patricia Donahoe, Dean Edwards, Denise Faustman, Asgerally Fazleabas, Birgit Gellersen, Vincent Giguere, Stanley R. Glasser, Thomas "Tod" Hansen, Lothar Henninghausen, Holly Ingraham, Hiroaki Kiyokawa, Kenneth Korach, Ronald Magness, Manuel Mark, John McLachlan, Sergio Ojeda, Ok-Kyong Park-Sarge, Renee Reijo Pera, JoAnne Richard, Michael Roberts, G. Shyamala, Thomas E. Spencer, Colin Stewart, Hugh Taylor, Salli Tazuke, Corey Teuscher, Axel Thomson, Zena Werb (Keynote Speaker), Deborah Wolgemuth, and Koji Yoshinaga.
Connecticut College is a new site for this conference. The college is located near the Thames River and is a short distance from Ocean Beach Park on Long Island Sound.
For more information, please write, fax, or e-mail: Susan Fisher, University of California San Francisco, 513 Parnassus Avenue, HSW-604, San Francisco, California, USA 94143-0512. Fax: 415 502-7338; e-mail: sfisher@cgl.ucsf.edu.
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Acknowledgments |
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Footnotes |
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Received June 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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"-dihydrotestosterone levels in
the peripheral plasma of male and female ring doves (Streptopelia
risoria) during the reproductive cycle. Biol Reprod 16:666–677[Abstract]
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