Tuberoinfundibular dopaminergic neurons and lactotropes in young and old female rats

NeurobiologytfAging, Vol. 13, pp. 275-281, 1992

0197-4580/92 $5.00 + .00 Copyright(©1992 PergamonPress Ltd.

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Tuberoinfundibular Dopaminergic Neurons and Lactotropes in Young and Old Female Rats G I O V A N N I L. R O S S I , ~G I L B E R T O E. B E S T E T T 1 A N D M A R I A N N E J. R E Y M O N D *

Division of Experimental Pathology, Institute of Animal Pathology, University of Berne, Berne, and *Division of Endocrinology, Department of lnternal Medicine, University Hospital, Lausanne, Switzerland R e c e i v e d 6 N o v e m b e r 1990; A c c e p t e d 20 S e p t e m b e r 1991 ROSSI G. L., G. E. BESTETTI, AND M. J. REYMOND. Tuberoin[undibulardoparninergicneuronsand lactotropes in young and oldfi,male rats. NEU ROBIOL AGING 13(2) 275-281, 1992.--Aging in female rats is accompanied by several endocrine dysfunctions, such as reproductive decline associated with characteristic hyperprolactinemia, lactotrope hyperplasia, and functional impairment of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. The aim of this morphometrical, immunocytochemical, and densitometrical study was to gain a better anatomical knowledge of TIDA neurons and axons as well as of lactotropes in old female rats with (A) or without (NA) pituitary adenomas, compared with young animals. At the hypothalamic level, we found that tyrosine hydroxylase (TH)-labeled neurons in the arcuate nucleus were comparable in young and old NA yet their size and TH-content were increased in A animals. Also the TH-labeled median eminence axons did not differ significantly between young and old NA but were more numerous in the old A rats. Independently from adenomas, both number of prolactin (PRL)-labeled structures and content ofimmunoreactive PRL were increased in pituitaries of old rats, the plasma PRL levels, however, were high only in A. Our findings support the documented lactotrope hypertrophy and hyperplasia in old female rats and suggest that TlDA-neuron changes only occur in hyperprolactinemicanimals carrier ofadenomas. Rat Female Arcuate nucleus

Aging Prolactin axis Tuberoinfundibular dopaminergic neurons Median eminence Prolactinoma Morphology Densitometry

Tyrosine hydroxylase

A reduced number of DA-fluorescent varicosities on supraoptic perikarya and LHRH fibers was found in the ME of old male rats (37). Yet, the number ofTH-positive perikarya in the arcuate nucleus (AN) and circumventricular regions of old female rats was unchanged (27). Finally, damaged TIDA neurons in the AN and reduced DA-fluorescent terminals in the ME were seen in old rats bearing prolactinomas (33). Although the pathogenesis of the reduced TIDA-neuron activity is not yet clear, damaging effects of long-lasting elevated plasma estrogen and/or PRL levels have been suggested (2). Morphological studies have demonstrated harmful estrogen effects in young adult female rats, i.e., lactotrope hypertrophy, followed by microadenomas, macroadenomas, and neuronal degeneration as well as increased astrocytic and microglial activity in the AN (6). These changes are comparable to those seen in old animals (36). Furthermore, a possible neurotoxic PRL activity on TIDA neurons has been suggested by some (33-35) but denied by others (21). Although many questions regarding the origin of these agerelated central and peripheral neuroendocrine dysfunctions remain unsolved, the mediobasal hypothalamus (MBH) appeared to play a crucial role, at least in animal models. To further define the role of the MBH in the pathogenesis of the age-related disturbed PRL secretion, we measured by morphometry and immunocytochemical densitometry TH-la-

A G I N G is associated with several functional endocrine changes, of which the impaired reproductive function in the old female is the most evident. Functional and structural lesions of the hypothalamo-pituitary axis and gonads were well documented in old female rats (4,29,36) that show irregular estrous cycles, followed by constant estrus, recurrent pseudopregnancy, or acyclicity (for reviews see 4,19). Other important changes were hyperprolactinemia (17,19) and increased occurrence of prolactin (PRL)secreting pituitary adenomas ( 15,18). The dysfunction of lactotropes in old rats is most probably related to the impaired activity of tuberoinfundibular dopaminergic (TIDA) neurons (28,29). In vivo and in vitro studies on the pathogenesis of the age-related increased PRL secretion in old compared with young rats have indeed shown in the former reduced secretory activity of TIDA neurons (for review see 29), mainly ascribed to decreased biosynthetic activity (2,9,29) and to loss of neuronal response to PRL, in particular (30). Age-related alterations in the Ca ++ channel or in tyrosine hydroxylase (TH) phosphorylation (23,38) or changes in the amount of TH may be involved in the impaired dopamine (DA) biosynthesis in TIDA neurons, but the amount of TH in the median eminence (ME) of aged female rats, either diestrous or constant estrous animals was greater than in cycling estrous rats (24). Morphological data on TIDA neurons in old rats are scarce.

JRequests for reprints should be addressed to G. L. Rossi, P.O. Box 2735, CH-3001, Berne, Switzerland. 275

276

ROSSI ET AL.

beled neurons of AN and axon cross-sections of the ME as well as plasma PRL levels and PRL-labeled lactotropes of the anterior pituitary gland in young and old female rats with or without pituitary prolactinomas. MATERIALSAND METHOD

Genera/ Virgin female Long-Evans rats (Centre d'01evage R. Janvier, Le Genest-St-lsle, France), aged 10 ( 11 rats) or 38 ( 19 rats) weeks, were housed in animal quarters of the Centre Hopitalier Universitaire Vaudois (Lausanne, Switzerland) under controlled conditions of temperature (22°C), humidity, and light (on at 6:00 a.m., off at 6:00 p.m.) and had free access to tap water and standard rat diet. Cytological examinations of vaginal smears began at age 11 weeks or 22 months. The estrous cycles of the old animals were irregular, with long diestrus periods interspaced by one or two days of proestrus or estrus, thus compatible with recurrent pseudopregnancy (4,12). The young animals were killed on diestrus, the old rats with repeated pseudopregnancy after five days or more ofdiestrus. To minimize the effects of circadian rhythms, all animals were killed between 8:00 a.m. and I0:00 a.m. At autopsy animals were aged 3 and 23-24 months old. Under i.p. ketamine hydrochloride anesthesia, whole body perfusion fixation was performed as earlier described (32). Briefly, alter taking a blood sample from the jugular vein for PRL determination, the abdominal aorta was cannulated and the blood vessels were rinsed with Ringer solution during 20 s. Fixation was achieved by 10 rain perfusion with phosphate buffered (pH 7.4) 2% glutaraldehyde-1.5% paraformaldehyde solution, at a pressure of 130 mm Hg. After opening the skull, the rats were left in the fixative solution for at least additional 4 h to complete fixation. A complete autopsy was performed and principal organs were examined to discard animals with gross pathological changes.

Tissue Processing The brain was then removed from the skull, weighed (Table 1), and dissected under a stereomicroscope to obtain the MBH specimens as previously described (5,32). Briefly, each MBH was cut free hand into a series of six 0.3-0,4 mm thick rostrocaudal frontal sections. From each of these sections (levels I to VI) semithin sections were obtained. By recognition of fixed anatomical reference points and/or characteristics (i.e., shape of the third ventricle and of its lateral recesses, shape of the median eminence, presence, and shape of the pituitary pars tuberalis, etc.) we chose three comparable serial frontal sections in each animal, spaced about 100 urn, from MBH level Ili (Fig.

FIG, 1. Mediobasal hypothalamus: a) TH-labeled neurons in the arcuate nucleus and b) TH-labeled axon cross-sections in the median eminence. Semithin sections, anti-TH peroxidase-anti-peroxidaseimm unocytochemistry (250X).

1). At this level, the profile of the 3rd ventricle recesses has an angle of 70-90 °, the outer profile of the ME is roughly parallel to the base of the 3rd ventricle or slightly protruding and it is laterally delimited by a thin portion of the pituitary pars tuberalis. This level comprehends a substantial portion of the antero-basal AN (5,32). After extraction from the basal meninges, the pituitary gland was examined for the presence of macroadenomas, weighed (Table 1), cut longitudinally into two halves along the sagittal axis, and a 0.5 m m thick frontal slice was cut from one

TABLE 1 GENERALAND RIA DATA Group

Body Weight(g)

Brain (g)

PituitaryGland (mg)

Ovaries(rag)

Prolactin(ng/ml)

Young(n = l l) OId-NAC(n = 12) OId-AC(n = 17)

232_+ 3.8 352 + 5.9* 331 _+ 9.8*

1.79___ 0,02 2.08 _+ 0.03* 2.02 _+ 0.02*

15.1 _+ 0.51 20.3 _+ 0.56* 28.6 _+ 5.36"t

124 + 3.7 141 _+ 8.7 144 _+ 13.4

19.1 _+ 3.52 13.4 _+ 1.39 69.9 _+ 10.24"t

Values are mean _+ SE. Body, brain, pituitary and ovary weight, and plasma prolactin of young and old female rats not carrier (NAC) or carrier (AC) of pituitary adenoma. *p < 0.01 old versus young (Mann-Whitney U-Test). tP < 0.01 old-AC versus old-NAC (Mann-Whitney U-Test).

TIDA NEURONS AND LACTOTROPES half at the level of the maximal pituitary height to yield one standard tissue block per animal (22). Sections of each pituitary gland were stained with Toluidine blue for light microscopical identification of microadenomas. The old animals were divided into two groups depending on the presence or not of pituitary, adenomas. Tissue blocks for immunocytochemistry were not postfixed and after dehydration, infiltration, and embedding in Spurr's low viscosity medium, semithin sections were cut. The immunocytochemical labelling of the hormones was performed by the PAP method (31 ). The antibodies were rabbit A-bovine TH (Eugene Tech International Inc., Allendale, NJ') and rabbit A-human PRL (Dako Corporation, Santa Barbara, CA). AntiTH was used at the original dilution and anti-PRL diluted 1:200. Both commercially available antibodies are known to have negligible cross-reactivity for other hormones. To assay the PRL immunoreaction specificity, we either omitted the primary antiserum or preabsorbed it with purified rat PRL (4 ~g/ 100 ~1 PBS: UCB-Bioproducts, Brussels, Belgium)according to the protocol of Lloyd and Childs (16). Both procedures completely abolished the immunocytochemical reaction. The specificity of the TH immunoreaction was confirmed by omission of the primary antiserum in our laboratory and by Western blot and immunocytochemistry by the producer (Eugene Tech International Inc.).

Densitometr.v The apparatus was a video-based, computer-linked system programmed to identify and measure optical densities in the selected images. Screen displays showing the amount of the immunoreactive substance in gray tones as well as the automatic measuring and evaluation of particles whose gray tone distinguished them clearly from the background were the main functions of the program. Furthermore, the interactive editing of images allowed us to eliminate possible artifacts. We used a personal computer (AT OPC-286, Oriental Precision Co. Ltd., Seoul, Korea) with a 20 Mb hard drive and monochrome data monitor, a digitizing tablet (SummaSketch Plus, Summagraphics Co., Fairfield, CT), a digitizing card (FG- 100 AT, Imaging Technology Inc., Woburn, MA), a high resolution display-screen (Sword Rapier ll, Cotron Electronics Ltd., Coventry, Warwick, England), a chip black and white television camera (CF6, Kappa Messtechnik, Gleichen, FRG), a microscope equipped with light stabilizer (Axioplan, Carl Zeiss AG Schweiz, Zurich, Switzerland), and a luxmeter (Illuminance Meter T - 1M, Minolta Camera Co., Osaka, Japan). The software applied was a morphometrical, densitometrical, and statistical package (Diasys I, Datalab, Thrrigen, Switzerland). The immunoreactive material in the semithin sections was quantified (7), with some personal modifications. Briefly, the measuring procedure was as follows: The densitometer was calibrated daily to grant constant conditions throughout the experiment. The light setting was constant for each measuring series (TH 650, PRL 240 lux). To improve the contrast a dark green and a gray (Zeiss 0.5) filter were used. The MBH images were taken by a 40X, the pituitary ones by a 25X objective. The black and white television camera transferred the microscopical image to a screen on which a digitized cursor was electronically generated. The instrument was initially calibrated in the gray level analysis mode (128 gray levels) by determining the gray level (density) of labeled and unlabeled regions. The recording of the image after careful focusing induced the au-

277 tomatic digitizing function which comprised: calculation of a mean image from three subsequently digitized images, subtraction of a previously set background image, gray calibration, and image inversion. Specific areas were then selected by a cursor-controlled draw-measure function (hypothalamus) or by setting windows (pituitary gland). The number of pixels activated by the label in each structure was read and calculated by the computer microdensitometer. Threshold values for the detection of the label were separately preset for each field so that the computer would read only the label present in the selected structures. As to the MBH, in one standard semithin section from level Ill (5,32), we measured the TH-labeled neuronal perikarya and axon cross-sections in the AN as well as the TH-labeled axon cross-sections in the ME (Fig. 1). The draw-measure function allowed the discrimination of TH-labeled structures from unlabeled tissue. We considered all TH-labeled structures present in the AN during the first measuring series, i.e., all neuronal perikarya (also those from neurons without a visible nucleus) and axons. In a second series of measurements performed on three standard semithin sections, cut serially from level Ill of each rat, we considered only the TH-labeled neuronal perikarya from neurons that contained a clearly visible nucleus. The following measurements were calculated: a) number of anatomical structures, b) total area of the immunoreactive substance, and c) number of pixels per gray level. Subsequently, we then calculated the total number of pixels in each gray level and the sum of the products of number of pixels times the corresponding gray level and termed this sum total immunoreactivity. The latter is considered a relative quantitative evaluation of the antigen content ( I ). We also noted the immunoreactivity per unit area and per structure. To consider only technically perfect tissue, a few rats per group were excluded. As to the pituitary gland, in one standard frontal hemisection we measured the PRL-labeled cytoplasmic portions oflactotropes (Fig. 2). We obtained the following measurements: a) number of PRL-labeled anatomical structures, b) total area of the immunoreactive substance, and c) number of pixels per gray level. We then calculated the total immunoreactivity (see

FIG. 2. Pituitary lactotropes: a) young rat and b) old rat not carrier of adenoma. Semithin sections, anti-PRL anti-phosphatase immunocytochemistry (100X).

278

ROSSI ET AL. TABLE 2 EFFECT OF AGE AND PITUITARY ADENOMASON TH-IMMUNOREACTIVITYOF ARCUATE NUCLEUS STRUCTURES Group

Total TH-Labeled Area (um2)

Total TH-lmmunoreactivity (X 10 3)

Young(n = 9) OId-NAC(n = 8) OId-AC(n = 7)

1335 _+ 169.0 1032 + 198.0 1448 _+ 221.6

834 + 117.6 676 _+ 140.4 970 + 141.4

Values are mean _+ SE. Total area and immunoreactivity of TH-labeled structures (perikaryal cytoplasms and axon cross-sections) in the arcuate nucleus of young and old female rats not carrier (NAC) or carrier (AC) of pituitary adenoma.

MBH). F o u r windows were measured. We considered only technically perfect tissue so a few rats per group were excluded.

RIA The P R L concentration was determined in the plasma by RIA using the reagents generously supplied by the N I D D K and National H o r m o n e and Pituitary Program (University of Maryland, School o f Medicine, Baltimore, MD). The antigen used for iodination was N I D D K r P R L - I - 5 , the antiserum was a n t i - r P R L - S - 9 (1:12000). Results were expressed in term of N I D D K r P R L - R P - 3 and all determinations were done in duplicates.

Statistics G r o u p means and standard errors (SE) were calculated. The significance o f differences between groups was tested by the M a n n - W h i t n e y - W i l c o x o n U-test after checking equality of distribution (Kolmogoroff-Smirnoff 2-sample test). For the measurements on neuronal perikarya from AN neurons comparisons between groups were performed using the Kruskall-Wallis H-test. RESULTS

General Data Body, brain, and pituitary weights were higher in old than in young animals: ovarian weight was comparable (Table 1) and no obesity was observed.

R/A Plasma P R L levels were comparable in young and old rats without a d e n o m a s although greatly increased in old rats with a d e n o m a s (Table 1).

ltistoh~gy By histological examination of the pituitary gland, adenomas were detected in 7 older rats. Twelve old and all (11 rats) young animals were without adenomas.

Morphometo' and Densitometry In the hypothalamus, the n u m b e r of TH-labeled nucleated AN neurons were comparable in the three groups (young: 24.1 _+ 2.80, old without adenoma: 27.0 _+ 2.81, and old with adenoma: 23.3 _+ 2.61). Because of high individual variations, the total area and immunoreactivity of TH-labeled AN structures did not differ significantly a m o n g the three groups yet values of old rats with a d e n o m a s were higher than those of young animals (Table 2). The perikaryal cytoplasm area of TH-labeled nucleated A N neurons as well as the T H - i m m u n o r e a c t i v i t y per neuron of old rats without a d e n o m a s were comparable with those of young rats (Table 3). These measurements were higher in the carriers of a d e n o m a s than in both young rats and old rats without ade n o m a s (Table 3). The T H - i m m u n o r e a c t i v i t y per um 2 perikaryal cytoplasm area was unchanged in adenoma-ffee,

TABLE 3 EFFECT OF AGE AND PITUITARY ADENOMASON TH-IMMUNOREACTIVITYOF ARCUATE NUCLEUS NEURONS TH-Immunoreactivity Group

Cytoplasm Area (#m 2)

Per ~tm2 Cytoplasm

Per Neuron (× 10 3)

Young(n = 10) OId-NAC(n = 8) OId-AC (n = 7)

20.42 _+ 0.47 22.10 _+ 0.70 24.41 _+ 0.63**tt

870.73 _+ 3.01 871.44 _+ 3.24 882.22 _+ 4.06*

17.75 + 0.41 19.36 _+ 0.66 21.64 _+ 0.60**it

Values are mean _+ SE. Area ofperikaryal cytoplasm of TH-labeled nucleated neurons and total immunoreactivity per cytoplasm unit area and per neuron in three cross-sections of the arcuate nucleus of young and old female rats NAC or AC of pituitary adenoma (242 cells from young, 217 cells from old-NAC, and 163 from old-AC rats were considered). *p < 0.05. **p < 0.01 old versus young (Kruskall-Wallis//-test). t t p < 0.01 old-AC versus old-NAC (Kruskall-Wallis H-test).

TIDA NEURONS AND LACTOTROPES

279

TABLE 4 EFFECTOF AGE AND PITUITARYADENOMASON TH-IMMUNOREACTIVITYOF MEDIAN EMINENCEAXONCROSS-SECTIONS TH-Immunoreactivity Group

Number

Total Area(urn2)

Total(× 10 3)

Per Axon

Young(n = 8) OId-NAC(n = 8) OId-AC(n = 7)

833 ± 130.8 728 ± 114.6 1176 ± 171.3tt

1190 ± 161.5 975 ± 209.7 1779 + 323.5

806 ± 113.3 668 + 145.2 1236 ± 236.3

1007 + 141.7 960 + 137.3 1132 ± 111.8

Values are mean ± SE. Number, total area, and immunoreactivity ofTH-labeled axon crosssections in the median eminence of young and old female rats of young and old female rats NAC or AC of pituitary adenoma. t t p < 0.05 old-AC versus old-NAC (Mann-Whitney U-Test).

slightly higher in old adenoma-carrier compared with young rats (Table 3). In the ME, the number, total area, and total immunoreactivity of TH-labeled axon cross-sections did not differ significantly in the young and old rats adenoma-free (Table 4). The number of TH-labeled axon cross-sections was higher in old adenoma-carrier than in young rats (Table 4). Also, the total area and total immunoreactivity ofTH-labeled axon cross-sections showed the same tendency (Table 4). In the anterior pituitary gland, the number, total area, and total immunoreactivity of PRL-labeled structures were higher in old rats with or without adenomas than in young animals (Table 5).

DISCUSSION The aim of this study was to gain a better knowledge of the structure of the TIDA neurons in the AN and of their axons in the ME as well as of the lactotropes in the anterior pituitary gland in young and old female rats with or without pituitary prolactinomas. It is commonly accepted that with increasing age the TIDA neuron secretory activity is reduced, thus leading to hyperprolactinemia (see 29), due both to increased lactotrope number and to greater PRL release per lactotrope (8). Yet, studies leading to this conclusion generally did not specifically exclude animals that carried microprolactinomas (see 25). These tumors, however, were frequent in old female rats (15,18). Recent investigations suggested that elevated plasma PRL levels were due to the presence of prolactinomas (38). Furthermore, the

plasma PRL levels of prolactinoma-free female rats older than age 30 months were similar to those of young control animals (25). We found that the perikaryal cytoplasm area, the TH-immunoreactivity per neuron, and per unit cytoplasmic area of the TH-positive neurons in the AN were comparable in young and old adenoma-free animals. In old adenoma-carrier rats, on the contrary, all these measurements were increased which contrasts the reduced TH mRNA found in AN neurons of old prolactinoma-carrier rats (38), yet the neurons of the latter were damaged by the growth of tumors (38). Our findings also illustrate that alterations of TIDA perikarya in the AN of old female rats only occur when aging is associated with presence of prolactinomas. Our data also confirmed that aging is not parallelled by loss of TIDA neurons, as previously reported (27) but contrasted the overall decline in AN neurons earlier described by others (13). The TH-positive neuron changes as well as the increased number of TH-labeled axon cross-sections found in our old adenoma-carrier rats may reflect the effect of the long-term elevated plasma PRL concentration (10) on the autoregulatory PRL feedback on TIDA neurons (11,20). In the adenohypophysis, the increased number as well as total area and immunoreactivity of PRL-labeled pituitary structures were found in our old adenoma-free compared with young rats supported lactotrope hyperplasia (8) but not enhanced PRL release (8). Plasma PRL levels of our old adenoma-free rats, in fact, were comparable to those of young animals. Our results parallelled the finding of increased number of round PRL secretory granules without substantial change in serum PRL levels in old male rats (25). This might suggest a

TABLE 5 EFFECTOF AGE AND PITUITARYADENOMASON PRL-IMMUNOREACTIVITYOF PITUITARYLACTOTROPES Group Young(n = 11) OId-NAC(n = 11) OId-AC(n = 6)

Number 7408 ± 385.3 11361 ± 262.6* 11104 ± 685.3*

TotalArea(um 2)

TolalPRL-lmmuno~activ~ty (X 10 3)

24371 ± 2546.9 42634 ± 2940.3* 42506 + 4972.7*

2677 + 291.4 4714 ± 363.2* 4611 ± 544.7*

Values are mean _+SE. Number, total area, and immunoreactivity of PRL-labeled structures in the anterior pituitary gland of young and old female rats NAC or AC of pituitary adenoma, *p < 0.01 old versus young (Mann-Whitney U-Test).

280

ROSSI ET AL.

disturbed balance o f synthesis and release, leading to increased storage of P R L granules, Despite the high plasma P R L levels of the old adenoma-carrier animals, the n u m b e r as well as total area and i m m u n o r e activity of the PRL-labeled pituitary structures were not different from those of old animals without adenomas. It is well known that a d e n o m a t o u s cells synthesize and immediately secrete large a m o u n t s of P R L without storage into granules (15,18), In conclusion, our data on the pituitary gland of old female rats further substantiated the PRL-cell disorders, those on M B H suggest that T I D A - n e u r o n changes only occur in hyperprolactinemic old female rats carrier of adenomas.

ACKNOWLEDGEMENTS We thank Mrs. U. Forster, Mr. S. Grimm, Mr. G. DiLullo, and Miss W. Benotto for their skillful technical assistance. We are also indebted to Mrs. E. Rossi for her helpful criticism and assistance in the preparation of the article. This work was supported in part by the Swiss National Science Foundation Grant 3.659-0.87, 32-28273.90 (to GLR and GEB) 3.394-0.86 (to M JR), and by the Bern University Foundation for Research Promotion (Hochschulstifiung) Grants 11.6.1985, 25.3.1986, 26.6,1987, 15.6.1988, and 13.6.1989 (to GEB). The material used in the assay of PRL was generously provided by the NIDDK and the National Hormone and Pituitary Program, University of Maryland, School of Medicine, Baltimore, MD.

REFERENCES 1. Agnati, L. F.; Fuxe, K.: Zoli, M.; Zini, I.: H~irfstrand, A.; Toflhno, G.: Goldstein, M. Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase and neuropeptide Y-immunoreactive neurons in the rostral medulla oblongala of the adult and old male rat. Neuroscience 26:461-478; 1988, 2. Arita, J.: Kimura, F. Effect of aging on in vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices. Brain Res. 369:391-394:1986. 3. Arita, J.; Kimura, F. Direct inhibitory effect of long term estradiol treatment on dopamine synthesis in tuberoinfundibular dopaminergic neurons: In vitro studies using hypothalamic slices. Endocrinology 121:692-698; 1987. 4. Aschheim, P. Aging in the hypothalamic-hypophyseal ovarian axis in the rat. In: Everitt, A. V.; Burgess, J. A. eds. Hypothalamus, pituitary and aging, Springfield, 1L: Charles C. Thomas. 1976:376-418. 5. Bestetti, G.; Rossi, G. L. Hypothalamic lesions in rats with longterm streptozotocin-induced diabetes mellitus. Acta Neuropathol. (Berl) 52:119-127: 1980. 6. Casanueva, F.; Cocchi, D.: Locatelli, V.; Flauto, C.; Zambotti, F.: Bestetti, G.; Rossi, G. L.; Mfiller, E. Defective central nervous system dopaminergic function in rats with estrogen-induced pituitary tumors, as assessed by plasma prolactin concentrations. Endocrinology 110:590-599:1982. 7. Childs, G. V.; Lloyd, J. M.; Unabia, G.; Gharib, S, D.: Wierman, M. E.; Chin, W. W. Detection ofluteinizing hormone fl-messenger ribonucleic acid (RNA) in individual gonadotropes after castration: Use of a new in situ hybridization method with a photobiotinylated complementary RNA probe. Mol. Endocrinol. 1:926932; 1987. 8. Chuknyiska, R. S.; Blackman, M. R.; Hymer, W. C.: Roth, G. S. Age-related alterations in the number and function of pituitary lactotropic cells from intact and ovariectomized rats. Endocrinology 118:1856-1862; 1986. 9. Demarest, K. T.: Moore, K. E.: Riegle, G. D. Dopaminergic neuronal function, anterior pituitary dopamine content, and serum concentrations of prolactin, luteinizing hormone and progesterone in the aged female rat. Brain Res. 247:347-354; 1982. 10. Demarest, K. T.; Riegle, G. D.; Moore, K. E. Prolactin-induced activation of tuberoinfundibular dopaminergic neurons: Evidence for both a rapid "'tonic" and a delayed k'induction" component. Neuroendocrinology 38:467-475: 1984. 11. Demarest, K. T.: Moore, K. E.; Riegle, G. D. Responsiveness of tuberoinfundibular dopamine neurons in the aged female rat to the stimulatory actions ofprolactin. Neuroendocrinology 45:227232; 1987. 12. Fayein, N. A.: Aschheim, P. Age-related temporal changes of levels of circulating progesterone in repeatedly pseudopregnant rats. Biol. Reprod. 23:616-620; 1980. 13. Hsu, H. K.; Peng, M. T. Hypothalamic neuron number of old female rats. Gerontology 25:19-22; 1978. 14. Huang, H. H,; Marshall, S.; Meites, J. Capacity of old v e r s u s

15.

16.

17.

18,

19. 20. 21.

22. 23. 24.

25.

26. 27.

28. 29.

young female rats to secrete LH, FSH and prolactin. Biol. Reprod. 14:538-543: 1976. Kovacs, K.: Horvath, E.: llse, R. G.; Ezrin, C.: llse, D. Spontaneous pituitary adenomas in aging rats. A light microscopic, immunocytological and fine structural study. Beitr. Pathol. 161:116: 1977. Lloyd, J. M.: Childs, G. V. Differential storage and release oflutel nizi ng hormone and follicle-releasing hormone from individual gonadotropes separated by centrifugal elutriation. Endocrinology 122:1282-1290: 1988. Lu, K. H.: Hopper, B. R.: Vargo, T. M.: Yen, S. S. C. Chronological changes in sex steroid, gonadotropin and prolactin secretion in aging female rats displaying different reproductive states. Biol. Reprod. 21:193-203:1979. McComb, D. J.: Kovacs, K.; Beri. J.: Zak, F. Pituitary adenomas in old Sprague-Dawley rats: A histologic, ultrastructural, and immunocytochemical study. J. Nat. Cancer Inst. 73:1143-1166: 1984. Meites, J. Changes in neuroendocrine control of anterior pituitary function during aging. Neuroendocrinology 34:151-156; 1982. Moore, K. E.: Annunziato, L.: Gudelsky, G. A. Studies on tuberoinfundibular dopamine neurons. Adv. Biochem. Psychopharm. 19:193-204: 1978. Morgan, W. W.; Bartke, A.; Herbert, D. C. The effect of long-term diethylstilbestrol treatment or hyperprolactinemia on the response of the tuberoinfundibular dopamine neurons to elevated prolactin. Brain Res. 335:330-333: 1985, Pitton, 1.: Bestetti, G. E.: Rossi, G. L. The changes in the hypothalamo-pituitary-gonadal axis of streptozotocin-treated male rats depend from age at diabetes onset. Andrologia 19:464-473; 1987. Porter, J. C. In situ activity and phosphorylation of tyrosine hydroxylase in the median eminence. Mol. Cell. Endocrinol. 46:21 27: 1986. Porter, J. C. Relationship of age, sex, and reproductive status to the quantity oftyrosine hydroxylase in the median eminence and superior cervical ganglion of the rat. Endocrinology 118:14261432: 1986. Putten van, L, J, A,: Zwieten van, M. L; Mattheij, J. A. M.; Kemenade van, J. A. M. Studies on prolactin-secreting cells in aging rats of different strains. I. Alterations in pituitary histology and serum prolactin levels as related to aging. Mech. Ageing Dev. 42:75-90: 1988. Reymond, M. J.; Porter, J. C. Secretion of hypothalamic dopamine into pituitary stalk blood of aged female rats. Brain Res. Bull, 7:69-73; 1981. Reymond, M. J,; Arita, J.; Dudley, C. A.; Moss, R. L.: Porter, J. C. Dopaminergic neurons in the mediobasal hypothalamus of old rats. Evidence for decreased affinity of tyrosine hydroxylase for substrate and cofactor. Brain Res, 304:215-223; 1984. Reymond, M. J.; Porter, J. C. Involvement of hypothalamic dopamine in the regulation of prolactin secretion. Horm. Res. 22:142-152: 1985. Reymond, M. J,; Donda, A.; Lemarchand-Brraud, Th. Neuro-

TIDA NEURONS AND LACTOTROPES

30. 31.

32.

33.

endocrine aspects of aging: Experimental data. Horm. Res. 31:3238: 1989. Reymond, M. J. Age-related loss of the responsiveness of the tuberoinfundibular dopaminergic neurons to prolactin in the female rate. Neuroendocrinology 52:490-496; 1990. Rossi, G. L4 Bestetti, G. Morphological changes in the hypothalamic-hypophyseal-gonadal axis of male rats after twelve months of streptozotocin-induced diabetes. Diabetologia 21:476-481; 1981. Rossi, G. L.; Bestetti, G. Technical aspects in the study of pathologic lesions in the hypothalamus of the rat. In: Jones, T. C.; Mohr, U.: Hunt, R. D. eds. Monographs on pathology of laboratory animals, endocrine system. New York: Springer Verlag; Berlin: Heidelberg; 1983:311-316. Sarkar, D. K.: Gottschall, P. E.; Meites, J. Damage to hypothalamic dopaminergic neurons is associated with development of prolactin-secreting pituitary, tumors, Science 218:684-686; 1982.

281 34. Sarkar, D. K.; Gottschall, P. E.; Meites, J. Decline oftuberoinfundibular dopaminergic function resulting from chronic hyperprolactinemia in rats. Endocrinology 115:1269-1274; 1984. 35. Sarkar, D. K.; Gottschall, P. E.; Xie, Q. w.; Meites, J. Reduced tuberoinfundibular dopaminergic neuronal function in rats with in situ prolactin-secreting pituitary tumors. Neuroendocrinology 38:498-503: 1984. 36. Schipper, H.: Brawer, J. R.: Nelson, J. F.; Felicio, L. S.; Finch, C. E. Role of the gonads in the histologic aging of the hypothalamic arcuate nucleus. Biol. Reprod. 25:413-419; 1981. 37. Sladek, J. R. Jr.; Khachaturian, H.; Hoffman, G. E.; Sch61er, J. Aging of central endocrine neurons and their aminergic afferents. Peptides 1:141-157: 1980. 38. Voogt, J. L4 Arbogast, L. A.: Quadri, S. K.; Andrews, G. Tyrosine hydroxylase messenger RNA in the hypothalamus, substatia nigra and adrenal medulla of old female rats. Mol. Brain Res. 8:55-62: 1990.