0013-7227/78/0102-0001$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 1 Printed in U.S.A.
The Arcuate Nucleus and the Control of Gonadotropin and Prolactin Secretion in the Female Rhesus Monkey (Macaca
mulatto)* T. M. PLANT,f L. C. KREY4 J. MOOSSY, J. T. McCORMACK,§ D. L. HESS.fif AND E. KNOBIL** Department of Physiology and Division of Neuropathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 ABSTRACT. Attempts were made to destroy selectively the arcuate nucleus with radiofrequency current in adult female rhesus monkeys as a first step in identifying the areas of the mediobasal hypothalamus (MBH) that are responsible for the neural control of gonadotropin secretion in this species. Extensive or complete destruction of the arcuate region was produced in three animals and in two of these the lesion was confined primarily to the arcuate region and the dorsal aspect of the posterior median eminence. These lesions resulted in the cessation of LH and FSH secretion and blocked the positive feedback action of estradiol on gonadotropin release but did not appear to influence grossly basal thyroid and adrenocortical function, or to abolish GH discharge in response to insulin hypoglycemia. Adenohypophysial infarcts were not observed and exogenous LHRH and TRH induced marked discharges of the appropriate anterior pituitary hormones. In two additional animals with large hypothalamic lesions, destruc-
tion of the arcuate region was incomplete. In this group only partial inhibition of gonadotropin secretion was observed. LH and FSH secretion did not appear to be influenced in one animal bearing a large MBH lesion that entirely spared the arcuate region. Although serum prolactin remained at pre-lesion control levels after placement of the two relatively discrete lesions confined to the arcuate region, unambiguous increases in the secretion of this hormone were observed when the area of destruction encompassed tissue anterior and/or dorsal to the arcuate region. These observations suggest that the arcuate region is the primary structure mediating the hypothalamic control of gonadotropin secretion in the rhesus monkey. They also suggest that, in this species, the regions of the MBH involved with the regulation of gonadotropin release and those which control prolactin secretion are anatomically distinct. (Endocrinology 102: 52, 1978)
P
brain does not appear to interfere with either tonic gonadotropin secretion or with the positive feedback action of estradiol on LH and FSH release (1). Furthermore, estrogen-induced gonadotropin surges can be elicited in acutely decerebrated rhesus monkeys in which Received April 4, 1977. * This research was supported by Grants HD 03968 all neural tissue dorsal and anterior to the and HD 08610 from the NIH and by a Grant from the optic chiasm has been removed (2). These Ford Foundation. A preliminary report of this investigaobservations have led us to the conclusion tion was presented at the 58th Annual Meeting of the Endocrine Society, San Francisco, California, 1976 (Abthat, in the rhesus monkey, the central comstract 202). ponents of the control systems that govern t Postdoctoral Research Fellow of the NICHHD, NIH. both the tonic and preovulatory modes of ^ Present address: The Rockefeller University, York gonadotropin secretion are resident within the Avenue and 66th Streets, New York, New York 10021. § Postdoctoral Research Fellow of the Ford Founda- MBH-hypophyseal apparatus. That the neural tion. Present address: Department of Anatomy, Louisiana State University School of Medicine, New Orleans, Louis- portion of this apparatus plays a major role in the control of gonadotropin secretion in the iana 70112. H Present address: California Primate Research Center, monkey, as in other species (3), is underlined Davis, California 95616. * * To whom requests for reprints should be addressed. by the findings that passive immunization
REVIOUS studies from this laboratory have shown that, in the rhesus monkey, surgical disconnection of the medial basal hypothalamus (MBH) from the remainder of the
52
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ARCUATE NUCLEUS AND LH AND FSH SECRETION
53
with antisera to LHRH inhibits tonic LH and FSH secretion (4) and that administration of exogenous LHRH induces a release of these hormones (5, 6). The purpose of the present study was to identify the structures within the MBH that are instrumental in this regard. As a first step toward this end, attempts were made to destroy the arcuate nucleus with radiofrequency (RF) current and to determine the effects of the resulting lesions on LH and FSH secretion. Materials and Methods One intact adult female rhesus monkey (679, 4.8 kg BW) and six adult ovariectomized monkeys (5.4-6.9 kg BW) were studied. The MBH of one additional adult female (556, 6.0 kg BW) with a lesion in the preoptic area, was used as a control for histological purposes. The housing and general maintenance of the animals has been described in detail elsewhere (1). RF lesions were produced with monopolar coagulating electrodes with 0.5 mm tips, made from 30-gauge hypodermic stainless steel tubing, and an indifferent electrode made from a 1.5 inch, 23-gauge hypodermic stainless steel needle. A set of three electrodes, spaced 1.5 mm apart in an electrode carrier, was implanted on each side of midline in FIG. 1. Lateral roentgenogram showing final placement the anteroposterior (AP) plane. Using procedures of the bilateral electrode array above the sella turcica previously described (1), stereotaxic coordinates for the production of a RF lesion. Note that the posterior were derived for the placement of the tips of the electrodes are positioned immediately above the dorsal posterior electrodes of the bilateral array 0.8-1.7 aspect of the posterior clinoid. Three electrodes were mm above the dorsal surface of the posterior clinoid placed on either side of midline in an attempt to effect complete bilateral destruction of the elongated arcuate process and 0.6-1.0 mm from the midline, at 6-8° nucleus. from the vertical. Each animal was anesthetized with sodium pen- under these conditions ranged from 20-30 mA rms. tobarbital (30 mg/kg BW, iv), placed in a stereoThe electrodes were then removed from the taxic frame equipped with steel offset ear bars and brain. Gelfoam discs were placed on the dura, and riser block (Kopf 1404, 865) and given an im injec- the subcutaneous fascia and skin were closed. The tion of 4-8 mg dexamethasone to reduce brain entire procedure was completed in 3 or 4 h and edema. The dura was exposed through a trephined the animals were usually sitting on their perches opening 1.0 cm in diameter, and the bilateral elec- within 3 h after removal from the stereotaxic introde array was positioned immediately above the strument. Each animal received daily im injections dura. A lateral roentgenogram of the head was then of Terramycin (50 mg) as well as penicillin (300,000 taken to verify the derived AP coordinate. The two U) every other day for 5 days after placement of sets of electrodes were then lowered simultaneously the lesion. and their descent monitored by repeated lateral These RF lesions resulted in the development roentgenograms until final placement was achieved of polydipsia and polyuria in five animals (475, 537, (Fig. 1). The indifferent electrode was placed deep 542, 568, and 562) but not in one other (477). This into the temporalis muscle and a 40-V root mean diabetes insipidus was less severe than that obsquare (rms) RF current (2.25 MHz), generated by served following complete surgical disconnection of a Grass lesion maker (LM3), was applied to each the MBH (1). electrode in turn for 30 sec. The current achieved Blood samples were taken daily or every other
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54
PLANT ET AL.
day by femoral venipuncture, usually between 0900 and 1100 h, for at least 3 days before placement of lesions and for 30-107 days thereafter. One female (679), however, was studied for only 5 days after placement of the lesion. Blood samples were allowed to clot at 4 C overnight and were centrifuged; the serum was stored at — 20 C until required for assay. RIAs previously described were used to determine serum LH (7), FSH (8, heterologous FSH RIA), prolactin (9), GH (10), estrogen (11), cortisol (10), and thyroxine (12) concentrations. The negative and positive feedback actions of estradiol on gonadotropin secretion were assessed by the sc administration of estradiol benzoate (EB, 42 jug/kg BW) in oil (13), which resulted in peak concentrations of serum estrogen of 360-840 pg/ml 12-24 h after injection. Blood samples were taken at 12-h intervals for 4 days following EB injection. In order to test pituitary responsiveness to exogenous hypothalamic releasing factors, animals were placed in restraining chairs and fitted with indwelling cephalic or saphenous vein catheters. The next day, blood samples were drawn at 20-min intervals into heparinized syringes for 1 h before administration of the releasing factor and for 4 h thereafter. Red blood cells were returned to the animals upon completion of the test. LHRH (LRF, K-18-5-2, and K-136-06 Guillemin) and TRH (Guillemin) were administered as single iv injections of 20 and 25 [ig, respectively. Two animals (477 and 568) received a continuous infusion of LHRH (6.8 jtxg/h) for 2 days and during this time blood samples were taken at 12-h intervals. The competence of the pituitary to discharge GH in response to insulin (0.2 U/kg BW, Squibb) hypoglycemia was determined while animals, fitted with a saphenous or cephalic vein catheter, were restrained in primate chairs and fasted overnight as described previously (10, 14). At the termination of the experiments, the animals were killed and their brains fixed, in situ, with 10% formalin as previously described (1). The brain was removed from the skull and trimmed to a block of tissue containing the hypothalamus. Hypodermic tubing (18 gauge) was driven through the hypothalamus in the AP plane on either side of midline at the level of the anterior commissure to provide reference holes for the subsequent alignment of coronal sections during reconstruction of the lesion. The hypothalamus was embedded in paraffin, and serial coronal sections (10 and 20 ju,m) were made through the MBH and stained with cresyl violet. The pituitary was removed from the sella turcica, embedded in paraffin, sectioned at 6 or 10 jiim, and stained with cresyl violet.
Endo • 1978 Vol 102 • No 1
The lesions were reconstructed by projecting every 10th or 20th section on graph paper and the distance, in the dorsoventral (DV) plane, from the reference holes to the dorsal and ventral aspects of the structures of interest were measured. The shape of the lesions, as seen in coronal section, usually resembled that of a dumbbell (Fig. 2) with greatest destruction in the DV plane occurring lateral to midline. The DV dimension of the lesion at midline, however, was used to reconstruct the area of destruction which was then superimposed on a diagrammatic parasagittal section of the monkey hypothalamus. The lesions were characterized histologically by varying degrees of cavitation and coagulative necrosis with destruction of virtually all neuronal perikarya. Lipid-containing phagocytes in varying numbers were present within the lesions and at their margins. An astroglial response was also evident at the margins of the lesions adjacent to and intermixed with zones of granulation tissue and fibrosis. With the exception of a minimal reaction by phagocytes along the electrode tracts, tissue damage such as infarcts or hemorrhages was not detected in the neural parenchyma beyond the limits of the RF lesions. The leptomeningial and parenchymal vessels outside the lesions appeared to be morphologically normal.
Results The precise anterior and posterior limits of the arcuate nucleus could not be determined consistently by examination of serial coronal sections of the hypothalamus of control animals and of lesioned animals in which this region of the MBH was spared. A distinct aggregate of ventral periventricular perikarya was observed in some animals immediately posterior to the optic chiasm whereas, in others, this group of neurons became apparent only more caudally. In either case, this nucleus continued posteriorly, without interruption, to the ventral premammillary region. Premammillary nuclei could not be distinguished from presumptive arcuate neurons. For these reasons, the term arcuate region is used to describe the most ventral periventricular area between the optic chiasm and mammillary bodies. In one intact animal (679), a large lesion, which spared the arcuate nucleus, was produced in the MBH extending in the AP plane
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55
ARCUATE NUCLEUS AND LH AND FSH SECRETION from the caudal aspect of the optic chiasm to a position posterior to the midtuberal level (Figs. 2 and 3). This lesion did not grossly influence basal serum gonadotropin concentrations, nor did it interfere with the feedback actions of estradiol on LH and FSH secretion (Fig. 3). Incomplete destruction (approximately 10-60%) of the arcuate region was produced in a group of three ovariectomized monkeys. One of these animals (542) bore a massive lesion which, although extending along the entire AP plane of the MBH from the caudal boundary of the optic chiasm to the mammillary bodies, spared the ventral aspect of the arcuate region (Fig. 4). Photomicrographs of coronal sections taken at the retrochiasmatic, midtuberal, and premammillary levels of the MBH of this animal are shown in Fig. 5. The retrochiasmatic area of the MBH was largely destroyed as was the ventromedial nucleus and the medial premammillary region. The median eminence remained intact. While this lesion resulted in an abrupt decline in LH and FSH concentrations, gonadotropin levels remained well above the limit of detectability (LH: 2 ng/ml; FSH: 10 ng/ml). The administration of EB to this monkey resulted in the expected reduction in serum LH and FSH levels which was interrupted 36 h after the injection by a characteristic surge of these hormones (Fig. 4). Although qualitatively similar, the magnitude of this discharge was
0
Z
FIG. 3. Time courses of serum LH, FSH, and prolactin concentrations in an intact animal (679) before and after placement of a RF lesion (on day 0) dorsal to the arcuate region. The reconstruction of the lesion from serial coronal sections of the brain is superimposed upon a diagrammatic parasagittal section of the hypothalamus. Note that this large lesion (see also Fig. 2) did not appear to interfere with either tonic gonadotropin secretion or with the positive feedback action of estradiol. ARC: arcuate nucleus; MM: mammillary body; SC: suprachiasmatic nucleus; VMN: ventromedial nucleus; AC: anterior commissure; LT: lamina terminalis; OCH: optic chiasm. The arrow indicates the sc administration of EB.
22
FIG. 2. Coronal section of the hypothalamus at the midtuberal level of monkey 679 (see also Fig. 3).
FIG. 4. Time courses of serum LH, FSH, and prolactin concentrations in an ovariectomized animal (542) before and after placement of a large RF lesion that spared the ventral aspect of the arcuate region (see also Fig. 5). The reconstruction of the lesion from serial coronal sections of the hypothalamus is shown as in Fig. 3. Note that gonadotropin secretion is only partially suppressed.
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PLANT ET AL.
Endo • 1978 Vol 102 • No 1
FIG. 5. Coronal sections of the hypothalamus of monkey 542 bearing a lesion in the MBH (right) and of a control animal (556) at the retrochiasmatic (top), midtuberal (middle) and premammillary levels (bottom).
smaller than that observed in response to EB administration preoperatively. After placement of this lesion, serum prolactin levels showed a dramatic rise, increasing from less than 20 ng/ml to values of approximately 120 ng/ml (Fig. 4). In another animal (475) the lesion was restricted primarily to the retrochiasmatic region and destroyed only small portions of the anterior aspects of the arcuate
region and of the median eminence (Fig. 6). The endocrinological sequelae of this lesion were similar to those seen in animal 542, namely, a partial reduction of serum LH and FSH concentrations, a decline in circulating gonadotropin levels followed by a discharge of these hormones in response to EB injection, and a sustained rise in serum prolactin levels. The lesion in the third animal of this group
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ARCUATE NUCLEUS AND LH AND FSH SECRETION LESION
% 120 — 80
400 300 200 100 12
4 0 2
6
10 DAYS
14
18
22
26
FlG. 6. Time courses of serum LH, FSH, and prolactin concentrations in an ovariectomized animal before and after placement of a RF lesion that encroached on the anterior aspects of the arcuate region and the median eminence.
(562) was confined primarily to the right side of the third ventricle where it involved the median eminence, the arcuate region, and more dorsal areas of the MBH. On the other side of midline, however, the arcuate region and median eminence remained largely intact. This lesion resulted in a progressive rise in serum concentrations of prolactin, which plateaued at approximately 150 ng/ml 8 days later and remained at this elevated level for the duration of the experiment (102 days). During this prolonged period of hyperprolactinemia serum levels of LH and FSH were maintained at concentrations only slightly lower than prelesion control values and administration of EB on two separate occasions induced characteristic gonadotropin surges (Fig. 7). The time courses of these discharges were similar to those observed prior to placement of the lesion but their magnitude was somewhat reduced. A lesion which included the entire arcuate region (537), reduced serum LH and FSH to undetectable levels (Fig. 8). In this animal, the lesion extended from above the arcuate region to the base of the MBH resulting in extensive destruction of the median eminence. All direct neural inputs to the median eminence appeared to be interrupted with the
57
exception of those entering through a small undamaged medial area near the anterior boundary of the lesion at the posterior aspect of the optic chiasm. Laterally, the lesion extended along the base of the MBH toward the optic tracts and impinged on the mammillary bodies caudally. After placement of this lesion, serum LH concentrations declined to 2 ng/ml or less 5 days later and remained at undetectable levels for the duration of the 34day period of postoperative study (Fig. 8). The time course of the decline of serum FSH levels was similar to that of LH. The administration of EB failed to induce a discharge in either of the gonadotropic hormones. A 3-fold increase in prolactin levels was observed within the first 3 days after the operation. Serum prolactin concentrations remained relatively constant thereafter (Fig. 8). In two animals (477 and 568), lesions were produced which were limited largely to the arcuate region. In monkey 568 the lesion extended in the AP plane from the rostral boundary of the mammillary bodies along the
12
100
FIG. 7. Time courses of serum LH and prolactin concentrations in an ovariectomized animal before and after placement of a RF lesion. Note that this lesion, which produced unilateral damage to the arcuate region and median eminence, markedly elevated serum prolactin concentrations without dramatically influencing LH secretion. The time course of FSH (not shown) was similar to that of LH.
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PLANT ET AL.
1
120
4
0
2
6
10
18
22
DAYS
FIG. 8. Reduction in serum LH and FSH concentrations to undetectable levels and abolition of the positive feedback action of estradiol in an ovariectomized animal by a lesion which completely destroyed the arcuate region and encroached on the median eminence. The corresponding time course of serum prolactin concentrations is shown on the upper panel.
base of the third ventricle to the anterior aspect of the arcuate region (Fig. 9). The anterior infundibulum and the retrochiasmatic area remained intact but, more posteriorly, the lesion encroached upon the dorsal aspect of the median eminence. This lesion was restricted to the arcuate region on the left side of the third ventricle, but on the right side the destruction extended laterally along the base of the MBH, toward the medial aspect of the optic tract. Although the lesion in animal 477 (Fig. 10) was similar to that in monkey 568, a greater part of the rostral aspect of the arcuate region was left intact, destruction of the median eminence was less obvious and, on the left side of the third ventricle, the lesion encroached onto the basal aspect of the ventromedial nucleus. Photomicrographs of coronal sections taken at the retrochiasmatic, midtuberal, and premammillary levels of the MBH of these monkeys are shown in Fig. 11. In both animals, there was a rapid and dramatic fall in circulating LH and FSH levels following placement of the lesions (Figs. 9 and 10). Within 72 h, serum LH levels had decreased to less than 10% of
Endo • 1978 Vol 102 • No 1
prelesion control values. In one of these monkeys (568) LH levels continued to fall, and from the ninth postoperative day until the time of sacrifice LH in daily serum samples was detected only infrequently. In animal 477, however, the decline of serum LH levels was less complete with concentrations of this hormone ranging from
Vol. 102, No. 1 Printed in U.S.A.
The Arcuate Nucleus and the Control of Gonadotropin and Prolactin Secretion in the Female Rhesus Monkey (Macaca
mulatto)* T. M. PLANT,f L. C. KREY4 J. MOOSSY, J. T. McCORMACK,§ D. L. HESS.fif AND E. KNOBIL** Department of Physiology and Division of Neuropathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 ABSTRACT. Attempts were made to destroy selectively the arcuate nucleus with radiofrequency current in adult female rhesus monkeys as a first step in identifying the areas of the mediobasal hypothalamus (MBH) that are responsible for the neural control of gonadotropin secretion in this species. Extensive or complete destruction of the arcuate region was produced in three animals and in two of these the lesion was confined primarily to the arcuate region and the dorsal aspect of the posterior median eminence. These lesions resulted in the cessation of LH and FSH secretion and blocked the positive feedback action of estradiol on gonadotropin release but did not appear to influence grossly basal thyroid and adrenocortical function, or to abolish GH discharge in response to insulin hypoglycemia. Adenohypophysial infarcts were not observed and exogenous LHRH and TRH induced marked discharges of the appropriate anterior pituitary hormones. In two additional animals with large hypothalamic lesions, destruc-
tion of the arcuate region was incomplete. In this group only partial inhibition of gonadotropin secretion was observed. LH and FSH secretion did not appear to be influenced in one animal bearing a large MBH lesion that entirely spared the arcuate region. Although serum prolactin remained at pre-lesion control levels after placement of the two relatively discrete lesions confined to the arcuate region, unambiguous increases in the secretion of this hormone were observed when the area of destruction encompassed tissue anterior and/or dorsal to the arcuate region. These observations suggest that the arcuate region is the primary structure mediating the hypothalamic control of gonadotropin secretion in the rhesus monkey. They also suggest that, in this species, the regions of the MBH involved with the regulation of gonadotropin release and those which control prolactin secretion are anatomically distinct. (Endocrinology 102: 52, 1978)
P
brain does not appear to interfere with either tonic gonadotropin secretion or with the positive feedback action of estradiol on LH and FSH release (1). Furthermore, estrogen-induced gonadotropin surges can be elicited in acutely decerebrated rhesus monkeys in which Received April 4, 1977. * This research was supported by Grants HD 03968 all neural tissue dorsal and anterior to the and HD 08610 from the NIH and by a Grant from the optic chiasm has been removed (2). These Ford Foundation. A preliminary report of this investigaobservations have led us to the conclusion tion was presented at the 58th Annual Meeting of the Endocrine Society, San Francisco, California, 1976 (Abthat, in the rhesus monkey, the central comstract 202). ponents of the control systems that govern t Postdoctoral Research Fellow of the NICHHD, NIH. both the tonic and preovulatory modes of ^ Present address: The Rockefeller University, York gonadotropin secretion are resident within the Avenue and 66th Streets, New York, New York 10021. § Postdoctoral Research Fellow of the Ford Founda- MBH-hypophyseal apparatus. That the neural tion. Present address: Department of Anatomy, Louisiana State University School of Medicine, New Orleans, Louis- portion of this apparatus plays a major role in the control of gonadotropin secretion in the iana 70112. H Present address: California Primate Research Center, monkey, as in other species (3), is underlined Davis, California 95616. * * To whom requests for reprints should be addressed. by the findings that passive immunization
REVIOUS studies from this laboratory have shown that, in the rhesus monkey, surgical disconnection of the medial basal hypothalamus (MBH) from the remainder of the
52
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ARCUATE NUCLEUS AND LH AND FSH SECRETION
53
with antisera to LHRH inhibits tonic LH and FSH secretion (4) and that administration of exogenous LHRH induces a release of these hormones (5, 6). The purpose of the present study was to identify the structures within the MBH that are instrumental in this regard. As a first step toward this end, attempts were made to destroy the arcuate nucleus with radiofrequency (RF) current and to determine the effects of the resulting lesions on LH and FSH secretion. Materials and Methods One intact adult female rhesus monkey (679, 4.8 kg BW) and six adult ovariectomized monkeys (5.4-6.9 kg BW) were studied. The MBH of one additional adult female (556, 6.0 kg BW) with a lesion in the preoptic area, was used as a control for histological purposes. The housing and general maintenance of the animals has been described in detail elsewhere (1). RF lesions were produced with monopolar coagulating electrodes with 0.5 mm tips, made from 30-gauge hypodermic stainless steel tubing, and an indifferent electrode made from a 1.5 inch, 23-gauge hypodermic stainless steel needle. A set of three electrodes, spaced 1.5 mm apart in an electrode carrier, was implanted on each side of midline in FIG. 1. Lateral roentgenogram showing final placement the anteroposterior (AP) plane. Using procedures of the bilateral electrode array above the sella turcica previously described (1), stereotaxic coordinates for the production of a RF lesion. Note that the posterior were derived for the placement of the tips of the electrodes are positioned immediately above the dorsal posterior electrodes of the bilateral array 0.8-1.7 aspect of the posterior clinoid. Three electrodes were mm above the dorsal surface of the posterior clinoid placed on either side of midline in an attempt to effect complete bilateral destruction of the elongated arcuate process and 0.6-1.0 mm from the midline, at 6-8° nucleus. from the vertical. Each animal was anesthetized with sodium pen- under these conditions ranged from 20-30 mA rms. tobarbital (30 mg/kg BW, iv), placed in a stereoThe electrodes were then removed from the taxic frame equipped with steel offset ear bars and brain. Gelfoam discs were placed on the dura, and riser block (Kopf 1404, 865) and given an im injec- the subcutaneous fascia and skin were closed. The tion of 4-8 mg dexamethasone to reduce brain entire procedure was completed in 3 or 4 h and edema. The dura was exposed through a trephined the animals were usually sitting on their perches opening 1.0 cm in diameter, and the bilateral elec- within 3 h after removal from the stereotaxic introde array was positioned immediately above the strument. Each animal received daily im injections dura. A lateral roentgenogram of the head was then of Terramycin (50 mg) as well as penicillin (300,000 taken to verify the derived AP coordinate. The two U) every other day for 5 days after placement of sets of electrodes were then lowered simultaneously the lesion. and their descent monitored by repeated lateral These RF lesions resulted in the development roentgenograms until final placement was achieved of polydipsia and polyuria in five animals (475, 537, (Fig. 1). The indifferent electrode was placed deep 542, 568, and 562) but not in one other (477). This into the temporalis muscle and a 40-V root mean diabetes insipidus was less severe than that obsquare (rms) RF current (2.25 MHz), generated by served following complete surgical disconnection of a Grass lesion maker (LM3), was applied to each the MBH (1). electrode in turn for 30 sec. The current achieved Blood samples were taken daily or every other
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54
PLANT ET AL.
day by femoral venipuncture, usually between 0900 and 1100 h, for at least 3 days before placement of lesions and for 30-107 days thereafter. One female (679), however, was studied for only 5 days after placement of the lesion. Blood samples were allowed to clot at 4 C overnight and were centrifuged; the serum was stored at — 20 C until required for assay. RIAs previously described were used to determine serum LH (7), FSH (8, heterologous FSH RIA), prolactin (9), GH (10), estrogen (11), cortisol (10), and thyroxine (12) concentrations. The negative and positive feedback actions of estradiol on gonadotropin secretion were assessed by the sc administration of estradiol benzoate (EB, 42 jug/kg BW) in oil (13), which resulted in peak concentrations of serum estrogen of 360-840 pg/ml 12-24 h after injection. Blood samples were taken at 12-h intervals for 4 days following EB injection. In order to test pituitary responsiveness to exogenous hypothalamic releasing factors, animals were placed in restraining chairs and fitted with indwelling cephalic or saphenous vein catheters. The next day, blood samples were drawn at 20-min intervals into heparinized syringes for 1 h before administration of the releasing factor and for 4 h thereafter. Red blood cells were returned to the animals upon completion of the test. LHRH (LRF, K-18-5-2, and K-136-06 Guillemin) and TRH (Guillemin) were administered as single iv injections of 20 and 25 [ig, respectively. Two animals (477 and 568) received a continuous infusion of LHRH (6.8 jtxg/h) for 2 days and during this time blood samples were taken at 12-h intervals. The competence of the pituitary to discharge GH in response to insulin (0.2 U/kg BW, Squibb) hypoglycemia was determined while animals, fitted with a saphenous or cephalic vein catheter, were restrained in primate chairs and fasted overnight as described previously (10, 14). At the termination of the experiments, the animals were killed and their brains fixed, in situ, with 10% formalin as previously described (1). The brain was removed from the skull and trimmed to a block of tissue containing the hypothalamus. Hypodermic tubing (18 gauge) was driven through the hypothalamus in the AP plane on either side of midline at the level of the anterior commissure to provide reference holes for the subsequent alignment of coronal sections during reconstruction of the lesion. The hypothalamus was embedded in paraffin, and serial coronal sections (10 and 20 ju,m) were made through the MBH and stained with cresyl violet. The pituitary was removed from the sella turcica, embedded in paraffin, sectioned at 6 or 10 jiim, and stained with cresyl violet.
Endo • 1978 Vol 102 • No 1
The lesions were reconstructed by projecting every 10th or 20th section on graph paper and the distance, in the dorsoventral (DV) plane, from the reference holes to the dorsal and ventral aspects of the structures of interest were measured. The shape of the lesions, as seen in coronal section, usually resembled that of a dumbbell (Fig. 2) with greatest destruction in the DV plane occurring lateral to midline. The DV dimension of the lesion at midline, however, was used to reconstruct the area of destruction which was then superimposed on a diagrammatic parasagittal section of the monkey hypothalamus. The lesions were characterized histologically by varying degrees of cavitation and coagulative necrosis with destruction of virtually all neuronal perikarya. Lipid-containing phagocytes in varying numbers were present within the lesions and at their margins. An astroglial response was also evident at the margins of the lesions adjacent to and intermixed with zones of granulation tissue and fibrosis. With the exception of a minimal reaction by phagocytes along the electrode tracts, tissue damage such as infarcts or hemorrhages was not detected in the neural parenchyma beyond the limits of the RF lesions. The leptomeningial and parenchymal vessels outside the lesions appeared to be morphologically normal.
Results The precise anterior and posterior limits of the arcuate nucleus could not be determined consistently by examination of serial coronal sections of the hypothalamus of control animals and of lesioned animals in which this region of the MBH was spared. A distinct aggregate of ventral periventricular perikarya was observed in some animals immediately posterior to the optic chiasm whereas, in others, this group of neurons became apparent only more caudally. In either case, this nucleus continued posteriorly, without interruption, to the ventral premammillary region. Premammillary nuclei could not be distinguished from presumptive arcuate neurons. For these reasons, the term arcuate region is used to describe the most ventral periventricular area between the optic chiasm and mammillary bodies. In one intact animal (679), a large lesion, which spared the arcuate nucleus, was produced in the MBH extending in the AP plane
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ARCUATE NUCLEUS AND LH AND FSH SECRETION from the caudal aspect of the optic chiasm to a position posterior to the midtuberal level (Figs. 2 and 3). This lesion did not grossly influence basal serum gonadotropin concentrations, nor did it interfere with the feedback actions of estradiol on LH and FSH secretion (Fig. 3). Incomplete destruction (approximately 10-60%) of the arcuate region was produced in a group of three ovariectomized monkeys. One of these animals (542) bore a massive lesion which, although extending along the entire AP plane of the MBH from the caudal boundary of the optic chiasm to the mammillary bodies, spared the ventral aspect of the arcuate region (Fig. 4). Photomicrographs of coronal sections taken at the retrochiasmatic, midtuberal, and premammillary levels of the MBH of this animal are shown in Fig. 5. The retrochiasmatic area of the MBH was largely destroyed as was the ventromedial nucleus and the medial premammillary region. The median eminence remained intact. While this lesion resulted in an abrupt decline in LH and FSH concentrations, gonadotropin levels remained well above the limit of detectability (LH: 2 ng/ml; FSH: 10 ng/ml). The administration of EB to this monkey resulted in the expected reduction in serum LH and FSH levels which was interrupted 36 h after the injection by a characteristic surge of these hormones (Fig. 4). Although qualitatively similar, the magnitude of this discharge was
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FIG. 3. Time courses of serum LH, FSH, and prolactin concentrations in an intact animal (679) before and after placement of a RF lesion (on day 0) dorsal to the arcuate region. The reconstruction of the lesion from serial coronal sections of the brain is superimposed upon a diagrammatic parasagittal section of the hypothalamus. Note that this large lesion (see also Fig. 2) did not appear to interfere with either tonic gonadotropin secretion or with the positive feedback action of estradiol. ARC: arcuate nucleus; MM: mammillary body; SC: suprachiasmatic nucleus; VMN: ventromedial nucleus; AC: anterior commissure; LT: lamina terminalis; OCH: optic chiasm. The arrow indicates the sc administration of EB.
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FIG. 2. Coronal section of the hypothalamus at the midtuberal level of monkey 679 (see also Fig. 3).
FIG. 4. Time courses of serum LH, FSH, and prolactin concentrations in an ovariectomized animal (542) before and after placement of a large RF lesion that spared the ventral aspect of the arcuate region (see also Fig. 5). The reconstruction of the lesion from serial coronal sections of the hypothalamus is shown as in Fig. 3. Note that gonadotropin secretion is only partially suppressed.
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FIG. 5. Coronal sections of the hypothalamus of monkey 542 bearing a lesion in the MBH (right) and of a control animal (556) at the retrochiasmatic (top), midtuberal (middle) and premammillary levels (bottom).
smaller than that observed in response to EB administration preoperatively. After placement of this lesion, serum prolactin levels showed a dramatic rise, increasing from less than 20 ng/ml to values of approximately 120 ng/ml (Fig. 4). In another animal (475) the lesion was restricted primarily to the retrochiasmatic region and destroyed only small portions of the anterior aspects of the arcuate
region and of the median eminence (Fig. 6). The endocrinological sequelae of this lesion were similar to those seen in animal 542, namely, a partial reduction of serum LH and FSH concentrations, a decline in circulating gonadotropin levels followed by a discharge of these hormones in response to EB injection, and a sustained rise in serum prolactin levels. The lesion in the third animal of this group
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ARCUATE NUCLEUS AND LH AND FSH SECRETION LESION
% 120 — 80
400 300 200 100 12
4 0 2
6
10 DAYS
14
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FlG. 6. Time courses of serum LH, FSH, and prolactin concentrations in an ovariectomized animal before and after placement of a RF lesion that encroached on the anterior aspects of the arcuate region and the median eminence.
(562) was confined primarily to the right side of the third ventricle where it involved the median eminence, the arcuate region, and more dorsal areas of the MBH. On the other side of midline, however, the arcuate region and median eminence remained largely intact. This lesion resulted in a progressive rise in serum concentrations of prolactin, which plateaued at approximately 150 ng/ml 8 days later and remained at this elevated level for the duration of the experiment (102 days). During this prolonged period of hyperprolactinemia serum levels of LH and FSH were maintained at concentrations only slightly lower than prelesion control values and administration of EB on two separate occasions induced characteristic gonadotropin surges (Fig. 7). The time courses of these discharges were similar to those observed prior to placement of the lesion but their magnitude was somewhat reduced. A lesion which included the entire arcuate region (537), reduced serum LH and FSH to undetectable levels (Fig. 8). In this animal, the lesion extended from above the arcuate region to the base of the MBH resulting in extensive destruction of the median eminence. All direct neural inputs to the median eminence appeared to be interrupted with the
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exception of those entering through a small undamaged medial area near the anterior boundary of the lesion at the posterior aspect of the optic chiasm. Laterally, the lesion extended along the base of the MBH toward the optic tracts and impinged on the mammillary bodies caudally. After placement of this lesion, serum LH concentrations declined to 2 ng/ml or less 5 days later and remained at undetectable levels for the duration of the 34day period of postoperative study (Fig. 8). The time course of the decline of serum FSH levels was similar to that of LH. The administration of EB failed to induce a discharge in either of the gonadotropic hormones. A 3-fold increase in prolactin levels was observed within the first 3 days after the operation. Serum prolactin concentrations remained relatively constant thereafter (Fig. 8). In two animals (477 and 568), lesions were produced which were limited largely to the arcuate region. In monkey 568 the lesion extended in the AP plane from the rostral boundary of the mammillary bodies along the
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100
FIG. 7. Time courses of serum LH and prolactin concentrations in an ovariectomized animal before and after placement of a RF lesion. Note that this lesion, which produced unilateral damage to the arcuate region and median eminence, markedly elevated serum prolactin concentrations without dramatically influencing LH secretion. The time course of FSH (not shown) was similar to that of LH.
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PLANT ET AL.
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FIG. 8. Reduction in serum LH and FSH concentrations to undetectable levels and abolition of the positive feedback action of estradiol in an ovariectomized animal by a lesion which completely destroyed the arcuate region and encroached on the median eminence. The corresponding time course of serum prolactin concentrations is shown on the upper panel.
base of the third ventricle to the anterior aspect of the arcuate region (Fig. 9). The anterior infundibulum and the retrochiasmatic area remained intact but, more posteriorly, the lesion encroached upon the dorsal aspect of the median eminence. This lesion was restricted to the arcuate region on the left side of the third ventricle, but on the right side the destruction extended laterally along the base of the MBH, toward the medial aspect of the optic tract. Although the lesion in animal 477 (Fig. 10) was similar to that in monkey 568, a greater part of the rostral aspect of the arcuate region was left intact, destruction of the median eminence was less obvious and, on the left side of the third ventricle, the lesion encroached onto the basal aspect of the ventromedial nucleus. Photomicrographs of coronal sections taken at the retrochiasmatic, midtuberal, and premammillary levels of the MBH of these monkeys are shown in Fig. 11. In both animals, there was a rapid and dramatic fall in circulating LH and FSH levels following placement of the lesions (Figs. 9 and 10). Within 72 h, serum LH levels had decreased to less than 10% of
Endo • 1978 Vol 102 • No 1
prelesion control values. In one of these monkeys (568) LH levels continued to fall, and from the ninth postoperative day until the time of sacrifice LH in daily serum samples was detected only infrequently. In animal 477, however, the decline of serum LH levels was less complete with concentrations of this hormone ranging from
