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Electrochemically stimulated release of luteinizing hormone and ovulation after surgical interruption of lateral hypothalamic connections in the rat
Brain Research, 131 (1977) 335-344 © Elsevier/North-Holland Biomedical Press
335
E L E C T R O C H E M I C A L L Y S T I M U L A T E D R E L E A S E OF L U T E I N I Z I N G H O R M O N E A N D O V U L A T I O N A F T E R S U R G I C A L I N T E R R U P T I O N OF L A T E R A L H Y P O T H A L A M I C C O N N E C T I O N S I N T H E RAT*
CHRISTOPHER P. PHELPS** and CHARLES H. SAWYER Department of Anatomy and Brain Research Institute, School of Medicine, University of California, Los Angeles, Calif., 90024 (U.S.A.)
(Accepted November 1lth, 1976)
SUMMARY Plasma L H and FSH were studied in adult female rats following bilateral electrochemical stimulation (ECS) of the medial preoptic area (MPOA). By stereotaxic surgery frontal (FC) and frontal-lateral (LFC) retrochiasmatic "deafferenting" cuts were made with a Hal~sz knife (1.5 m m radius). At 3 and 10 weeks after surgery rats were given pentobarbital (32 mg/kg, i.p.) at 13:30 h and stimulated at 15:00 h with anodal direct current (20/zA for 60 sec) via concentric bipolar steel electrodes placed bilaterally 0.9 m m f r o m the midline. Stimulation at 3 weeks after FC increased plasma LH from a prestimulation level of 95 4- 22 ng/ml to 227 ± 51 ng/ml 80 min after ECS, from which it fell to 111 ± 29 ng/ml at 160 min, with 2 of 7 animals ovulating the next day. L F C females had similar pre-ECS plasma LH levels 3 weeks after surgery (71 ± 10 ng/ml), but LH concentrations at 80 min (104 :k 21 ng/ml) and 160 min post-ECS were significantly lower than those of FC rats, and 0 of 5 rats ovulated. Following a similar protocol 10 weeks after surgery, stimulating the M P O A resulted in comparable elevations in plasma LH and 4 of 10 FC animals ovulated; however, the L F C group still retained a significant blocking effect on ovulation (only 1 of 12 ovulated) when compared with controls the next day. Plasma FSH concentrations were not significantly altered by M P O A stimulation at the parameters employed, either before or after deafferentation. However, L F C resulted in reduced ovarian and uterine weights when compared with controls at both 3 and 10 weeks, whereas FC exerted no observable effect on these organs at these intervals of study. The results of these studies suggest that lateral input to the medial basal hypothalamus contributes to M P O A mediated release of LH and ovulation as well as to tonic maintenance of ovarian and uterine function.
* Supported by grants from NIH (NS01162) and the Ford Foundation. ** Postdoctoral Fellow of NIAMDD (AM03298). Present address: Department of Anatomy, College of Medicine, University of South Florida, Tampa, Fla., 33620, U.S.A.
336 INTRODUCTION Surgical interruption of neural pathways leading to and from the medial basal hypothalamus (MBH) has provided a useful approach to the study of brain pathways involved in endocrine regulationg, 1~. We have recently combined the techniques of anterior hypothalamic deafferentation and electrochemical stimulation (ECS) of the preoptic area 19 for the purpose of evaluating the extent of anterior hypothalamic input required to induce a surge of LH release in the rat 17. During the course of this investigation an increase in plasma LH was detected 1 h following bilateral ECS of the medial preoptic area (MPOA) rostral to the interruption of preoptico-tuberal pathways considered essential for ovulation in the rat 2,19. Serial reconstructions of electrode and frontal cut (FC) locations in each rat revealed that at least one stimulation site extended lateral to FC locations, suggesting a possible extension of ECS to fibers en passage through the lateral preoptic area coursing towards the MBH s,14,16. We have now inve3tigated these lateral connections to the MBH for their contribution to gonadotropin release from the anterior pituitary following ECS. Included is a comparative study of the variable capacities for response to ECS which occur with time after surgical interruption of anterior and anterolateral inputs to the MBH. MATERIALS AND METHODS Female rats (200-250 g) were selected for study after they had shown at least two consecutive 4-day estrous cycles. All animals were maintained on Purina lab chow and tap water ad libitum in a controlled environment with lights on from 5:00 to 19:00 h.
Anterior deafferentation Animals selected for FC were deafferented according to the method of Hal~tsz and Pupp 11 using a knife with a retractable blade 17. Briefly the procedure consisted of positioning the tip of the blade 1.6 mm behind bregma, drawing the blade up into the carrier and then lowering the carrier 5.5 mm through the superior sagittal sinus. The knife blade was then extruded in the median sagittal plane with the tip of the blade resting at the posterior border of the optic chiasm. FC was produced by rotating the knife 10-15 times 180° through the periosteum of the sphenoid bone, returning the blade to the sagittal plane and retracting it into the carrier. Sham FC surgeries consisted of extrusion and retraction of the blade in the median sagittal plane only. The knife blade had a turning radius of 1.5 mm for FC.
Lateral-frontal deafferentation In addition to the FC surgery other groups of animals were subjected to lateral frontal cuts (LFC) by extending the lateral edges of the FC 2.5 mm posteriorly towards the mammillary bodies. The lateral portion of this surgery was executed according to the method of Halfisz and Pupp 11 with some modifications. Briefly it consisted of rotating the 1.5 mm radius blade 90 ° from the median sagittal plane towards the side
337 of intended interruption. With the knife blade in this position the carrier was moved 2.5 mm posteriorly, then 2.5 mm anteriorly and the blade returned to the sagittal plane. All lateral cuts were made bilateral by repeating this procedure on the opposite lateral hypothalamus. Sham LFC consisted of a sham FC in addition to a 2.5 mm midline posterior-anterior course for the carrier with the blade retracted.
Experimental design Animals were subjected to FC, LFC or appropriate sham surgery on a morning of vaginal estrus. The rats were caged in pairs and vaginal smears taken daily during the course of experiments. On an afternoon of vaginal proestrus at approximately 3 or 10 weeks following hypothalamic surgery each animal was given pentobarbital (Pb-Diabutal, 32 mg/kg, i.p.) at 13:30. At 15:00 h it was bled (0.7 ml) by jugular venipuncture, immediately placed in a stereotaxic apparatus and a bipolar concentric electrode was lowered into the medial preoptic area (MPOA) according to the atlas of De Groot 7. The MPOA was stimulated bilaterally by passing 20/~A DC for 60 sec on each side, and the electrode was later checked for its ability to deposit iron in agar gel containing ferro-ferricyanide~0. The rat was bled at 40 min intervals (40, 80, 120, 160 min after stimulation) and the plasma was separated and stored for future L H and FSH assay. On the following morning both ovaries were removed, and the oviduct was examined microscopically for ova. Under deep ether anesthesia the animal was then perfused with saline followed by a saline-5 ~ potassium ferro-ferricyanide solution and finally 10 ~ formalin. The brains were removed and prepared for histological examination which included localization of Prussian Blue spots relative to FC and LFC according to the atlas of Albe-Fessard et al. 1. Ovarian, uterine and anterior pituitary weights were taken at autopsy. Plasma LH and FSH were measured by radioimmunoassay using the N I A M D D kits (A. F. Parlow) with the appropriate rat pituitary standards as reference preparations. Changes in organ weights and plasma gonadotrophin concentrations were analyzed with Student's t test, whereas ovulation data were compared by the Fisher Exact Probability Test. RESULTS
Estrous cycles Female rats subjected to FC alone showed variable vaginal cytology. The predominant finding was a fluctuating number of cornified cells present daily for periods up to 10 days with sporadic interruptions in this cytology by leukocytic-epithelial composites. When LFC surgerie3 were performed, vaginal smears remained predominantly leukocytic for most of the experimental period. During the last half of the 70-80 day period of observation in L F C rats a small proportion of nucleated and cornified epithelial cells appeared with the leukocytes every 5-7 days.
Electrochemically stimulated ovulation Ovulation following MPOA stimulation was significantly reduced (P = 0.025)
338 TABLE I Ovulation and organ weights after frontal deafferentation Brain surgery
Number o f days between brain surgery and stimulation
Ovulation by Ovarian weight Uterine weight Anterior POA stimula- (rag) (rag) pituitary weight tion in pento( mg) barbital-blocked rats
Sham FC FC
17-21 17-21
7/7 2/7*
84.9 i 4.4 74.0 ± 7.9
438.3 ± 20.7 432.5 ± 28.3
12.4 ± 0.5 12.4 ± 0.9
Sham FC FC
70-80 70-80
8/10 4/10
79.4 ± 3.7 72.8 ~ 10.1
489.9 -I- 36.9 500.5 24-29.4
12.9 ± 0.7 14.4 ± 1.1
* P - 0.025 when compared with shams.
17-2 i days after F C when compared with sham-operated controls, whereas c o m p a r i s o n with controls at the 70-80 day interval revealed statistically similar percentages of a n i m als with C> 5) tubal ova (Table I). Females receiving L F C registered a significant reduction in ovulation at 17-21 days (P < 0.01) which persisted through the 70-80 day (P < 0.01) period o f study (Table II). A c c o m p a n y i n g this reduction in o v u l a t i o n after M P O A s t i m u l a t i o n was a loss in ovarian weights when compared with controls or when L F C rats were compared across the two intervals of study (Table II). L F C also resulted in a loss in uterine weights at 17-21 days, which persisted t h r o u g h the remainder of the study (Table II). Plasma LH and FSH
H y p o t h a l a m i c deafferentation ( F C or LFC) resulted in a reduction of the capacity for M P O A stimulation to elevate plasma L H c o n c e n t r a t i o n at either interval TABLE II Ovulation and organ weights after frontaLlateral deafferentation Brain surgery
Number o/days between brain surgery and stimulation
Ovulation by Ovarian weight Uterine weight Anterior POA stimula- (mg) (mg) pituitary weight tion in pento(mg) barbital-blocked rats
Sham L F C * * * LFC
17-21 17-21
7/7 0/5*
90.0 i 6.7 63.8 5- 5.2*
Sham LFC*** LFC
70-80 70-80
11/12 1/12"
83.2 ± 4. I 529.3 :~ 24.0 36.8i3.1",** 246.1 ~ 37.9"
444.6 ± 17.8 10.9 ~- 0.7 296.9 ± 45.2* 10.0 ± 1.2 12,7 ± 0.3 12.2±0.8
* P <- 0.01 when compared with shams. ** P < 0.01 when compared with LFC 17-21 days after surgery. *** Rats 377 and 441 (see text) were removed from LFC groups and added to their respective sham LFC groups because of incompleteness of deafferentation revealed by histology.
339
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Fig. 1. Changes in plasma LH concentrations following electrochemical stimulation (20/~A DC for 60 sec) of the medial preoptic area in rats 17-21 days and 70-80 days after anterior (FC) and anterolateral (LFC) deafferentation of the hypothalamus. In the 17-21 day experiment LH levels were significantly higher at 80 and 160 min in the FC group than in the LFC group (* P < 0.02; ** P < 0.04). Such differences were not apparent at 70-80 days after surgery. N values are given in the tables.
(3 or 10 weeks) o f study (Figs. 1 and 2). However, at 17-21 days after brain surgery there were statistically significant differences in plasma L H at 80 (P < 0.02) and 160 (P < 0.04) min following M P O A stimulation when c o m p a r i n g the F C and L F C groups, L F C values being lower (Fig. 1). These differences were not apparent at the 70-80 day interval o f study, wherein the F C and L F C groups presented comparable increases in L H concentration during the 160 min sampling period. M P O A stimulation at the parameters employed did not induce a rise in plasma F S H levels in even the sham-operated groups, so there were no significant differences in plasma F S H concentrations when either F C or L F C groups were c o m p a r e d with their respective controls at the same times o f sampling following M P O A stimulation (Fig. 2).
Brain histology Composite reconstructions of F C and L F C locations relative to the centers o f bilateral anodal stimulation sites (Prussian Blue reaction) reflected a general uniformity in the placement o f both types o f cuts at the two intervals o f study (Fig. 3). All o f the F C animals had visible ventral scars posterior to the optic chiasm indicating the completeness o f surgery at that location. In the case o f the L F C surgeries, all
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Fig. 2. Failure of electrochemical stimulation of MPOA to induce a significant rise in plasma FSH either in sham-deafferented rats or in animals with anterior (FC) or anterolateral (LFC) deafferentation of the hypothalamus. N values are given in the tables.
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Fig. 3. Composite horizontal reconstructions of anterior (FC) and anterolateral (LFC) deafferentatlon cuts and electrochemical stimulation sites (dots) in the rat hypothalamus and medial preoptic area. The plus mark ( ÷ ) lies in the midsagittal plane 7 mm rostral to the vertical interaural plane. The scales at the left measure in mm rostral to the interaural plane.
341 animals exhibited complete cuts posterior to the chiasm; however, the bilateral posterior 2.5 m m extension of the knife was incomplete in 2 cases (Nos. 377 and 441). Because of the different nature of these incomplete cuts they will be described separately below. Animal No. 377 presented an L F C at 75 days that was incomplete on the left side of the brain throughout the last (most posterior) millimeter of the lateral cut. The release pattern for plasma L H in this rat was also markedly different from the remainder of the animals: 40 min following ECS, L H rose from a prestimulation level of 74 ng/ml to 511 ng/ml. At 80 rain after ECS, plasma L H had risen further to 1295 ng/ml, falling to 537 ng/ml at 120 min and 275 ng/ml at 160 min post-ECS. At autopsy this animal had ovulated 7 ova with an ovarian weight comparable (41.4 mg) to the group mean; however, the uterine weight (520.6 mg) was more than twice that of the group mean (Table II). Plasma F S H concentrations were not different from long-term L F C group means (Fig. 2). In another L F C rat (No. 441) autopsied 18 days following deafferentation, the lateral portion of the cut was incomplete bilaterally in that there was an 0.2 m m ventral gap along both of the 2.5 m m lateral portions of the cut (Fig. 4). Plasma L H concentrations rose from a pre-ECS level of 67 ng/ml to 648 ng/ml at 80 min after stimulation and remained elevated at 120 min (572 ng/ml) and 160 min (314 ng/ml) when compared with L F C group means (Fig. 1). Ovarian weights (47.2 mg) in rat No. 441 were similar to those of complete L F C rats at the interval of study; however, No. 441 was the only animal in the experimental group that had tubal ova (7) at autopsy
Fig. 4. Photomicrographs of frontal sections of the "LFC" deafferented hypothalamus and median eminence in which the knife blade reached the base of the brain (A) but not in the brain of rat 441 (B), in which arrows point to the sites of continuity between lateral and medial hypothalamus.
342 the morning after ECS. The uterus was larger (472.0 mg) in this female, wereas plasma FSH concentrations were similar to mean concentration for the group (Fig. 2). DISCUSSION
A functional role for lateral input to the M B H has been suggested by studies wherein lateral deafferentation reduced norepinephrine content of the hypothalamus 6, and posterior-lateral deafferentation reduced the incidence of ovulation in the rat TM. There is considerable anatomical and physiological evidence to suggest a relationship between adrenergic transmitters and gonadotropin release factors3A~, ~8. In the present study when retrochiasmatic deafferentations were extended 1.5 mm laterally from the midline and 2.5 m m posteriorly as far as the mammillary bodies (LFC), there was a reduction in both ovarian and uterine weights when compared with those in sham controls 3 weeks after LFC. This change did not occur in animals after FC alone. Prior to autopsy, these differences in organ mass might have been predicted by vaginal cytology. Females with L F C were characterized by periods of predominantly leukocytic smears over the 10 week period of study, whereas the principal cell type in FC rats was the cornified epithelial cell. There was also a further decline in ovarian weights progressing from 3 to 10 weeks in the L F C rats when compared with controls, whereas no change occurred in FC animals over a similar period. Changes in plasma LH concentration following electrochemical stimulation (ECS) of the M P O A rostral to cuts also revealed differences between LFC and FC rats. Three weeks after hypothalamic surgery stimulation rostral to FC resulted in an increase in plasma LH over the 2.5 h sampling period, although the levels obtained were much less than in sham FC rats. Females 3 weeks after FC did, however, reveal higher plasma L H levels at the 80 and 160 min sampling times than those following LFC. Such differences between FC and LFC L H curves were not significant at 10 weeks after surgery (Fig. 1). The LFC results suggest that the passage of time improves LH response to brain ECS, but not the ovulatory endpoint. In fact, there is a decrease in ovarian weight following LFC, progressing from 3 to 10 weeks with a continued reduction in uterine mass. The results with L F C females can be contrasted with the responses of FC rats to the same ECS and recovery periods : similar ovarian and uterine weights at both intervals with an improvement at 10 weeks in ovulatory response to changes in LH concentration essentially the same as those occurring after ECS at 3 weeks. These findings suggest a role for lateral hypothalamic input to the MBHS,14,16 in both the LH response to ECS of the M P O A (3 week study) and apparent increase in gonad sensitivity to LH, evidenced by the restoration of ovulation in FC rats after ECS at 10 weeks. This conclusion is supported by the findings in animals 377 and 441, in which accidentally incomplete L F C permitted the retention of quantitatively and qualitatively different connections at the lateral hypothalamus with the remainder of the brain, i.e., connections which were associated with dramatic increases in LH levels after ECS as well as with ovulatory and uterine parameters. Our inability to detect changes in plasma FSH concentrations between experimental and control groups following MPOA stimulation is in good agreement with
343 the works of K a l r a et al. 12, who reported that ECS (100 # A × 60 sec) of the M P O A does n o t result in a n increase in p l a s m a F S H c o n c e n t r a t i o n on the a f t e r n o o n o f proestrus at 0.5, 1 a n d 3 h after stimulation. Other laboratories 4,5 report elevations in p l a s m a F S H following electrochemical s t i m u l a t i o n of the M P O A ; however, these increases occur after quantitatively larger ECS t h a n that employed in the present study, which m a y have obscured or delayed a n y possible difference 4 between control a n d experimental groups in our results. ACKNOWLEDGEMENTS The authors wish to t h a n k Miss K a t h e r i n e Bangs a n d Mr. D a v i d W h i t m o y e r for technical assistance, Ms. Lois fels for secretarial aid, Ms. Arlene K o i t h a n for histological p r e p a r a t i o n s a n d Mr. Bob McAllister for the drawings.
REFERENCES 1 Albe-Fessard, D., Stutinsky, F. et Libouban, S., Atlas Stdrdotaxique du Diencdphale da Rat Blanc, Centre Nationale de la Recherche Scientifique, Paris, 1966, pp. 1-58. 2 Barraclough, C. A., Sex steroid regulation of reproductive neuroendocrine processes. In R. O. Greep (Ed.), Handbook of Physiology, Section 7, Endocrinology, Vol. II, Part I, Williams and Wilkins, Baltimore, Md., 1974, pp. 29-56. 3 Brownstein, M. J., Palkovits, M., Saavedra, J. M. and Kizer, J. S., Distribution of hypothalamic hormones and neurotransmitters within the diencephalon. In L. Martini and W. F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. IV, Raven Press, New York, 1976, pp. 1-23. 4 Chappel, S. C. and Barraclough, C.A., Hypothalamic regulation of pituitary FSH secretion, Endocrinology, 98 (1976) 927-935. 5 Clemens, J. H., Shaar, C. J., Kleber, J. W. and Tandy, W. A., Areas of the brain stimulatory to LH and FSH secretion, Endocrinology, 88 (1971) 180-184. 6 Cuello, A. C., Weiner, R. I. and Ganong, W. F., Effect of lateral deafferentation on the morphology and catecholamine content of the mediobasal hypothalamus, Brain Research, 59 (1973) 191300. 7 De Groot, J., The rat forebrain in stereotaxic coordinates, Trans. roy. Neth. Aead. Sci., 52 (1959) 1~40. 8 Hagino, N., Kosaras, B. and Flerk6, B., Neuroendocrinological approach to investigation of the septo-tuberal tract in the rat, Anat. Rec., 181 (1975) 369 (abstract). 9 Hal~sz, B., The endocrine effects of isolation of the hypothalamus from the rest of the brain. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology 1969, Oxford Univ. Press, London, 1969, pp. 307-342. 10 Hal~sz, B. and Gorski, R. A., Gonadotrophic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus, Endocrinology, 80 (1967) 608-622. 11 Hal~sz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotropic area, Endocrinology, 77 (1965) 553-562. 12 Kalra, S. P., Ajika, K., Krulich, L., Fawcett, C. P., Quijada, M. and McCann, S. M., Effects of hypothalamic and preoptic electrochemical stimulation on gonadotropin and prolactin release in proestrous rats, Endocrinology, 88 (1971) 1150-1158. 13 Krey, L. C., Butler, W. R. and Knobil, E., Surgical disconnection of the medial basal hypothalamus and pituitary function in the Rhesus monkey. I. Gonadotropin secretion, Endocrinology, 96 (1975) 1073-1087. 14 Krieg, W. S., The hypothalamus of the albino rat, J. comp. NeuroL, 55 (1932) 19-89. 15 McCann, S. M., Regulation of secretion of follicle-stimulating hormone and luteinizinghormone. In R. P. Greep (Ed.), Handbook of Physiology, Section 7, Endocrinology, Vol. IV, Part 2, Williams and Wilkins, Baltimore, Md., 1974, pp. 489-517.
344 16 Millhouse, O. E., A Golgi study of the descending medial forebrain bundle, Brain Research, 15 (1969) 341-363. 17 Phelps, C. P., Krieg, R.J. and Sawyer, C. H., Spontaneous and electrochemically stimulated changes in plasma LH in the female rat following hypothalamic deafferentation, Brain Research, 101 (1976) 239-249. 18 Sawyer, C. H., First Geoffrey Harris Memorial Lecture: some recent developments in brainpituitary-ovarian physiology, Neuroendocrinology, 17 (1975) 97-124. 19 Tejasen, T. and Everett, J. W., Surgical analysis of the preoptico-tuberal pathway controlling ovulatory release of gonadotropins in the rat, Endocrinology, 81 (1967) 1387-1396. 20 Terasawa, E. and Sawyer, C. H., Electrical and electrochemical stimulation of the hypothalamoadenohypophysial system with stainless steel electrodes, Endocrinology, 84 (1969) 918-925.
335
E L E C T R O C H E M I C A L L Y S T I M U L A T E D R E L E A S E OF L U T E I N I Z I N G H O R M O N E A N D O V U L A T I O N A F T E R S U R G I C A L I N T E R R U P T I O N OF L A T E R A L H Y P O T H A L A M I C C O N N E C T I O N S I N T H E RAT*
CHRISTOPHER P. PHELPS** and CHARLES H. SAWYER Department of Anatomy and Brain Research Institute, School of Medicine, University of California, Los Angeles, Calif., 90024 (U.S.A.)
(Accepted November 1lth, 1976)
SUMMARY Plasma L H and FSH were studied in adult female rats following bilateral electrochemical stimulation (ECS) of the medial preoptic area (MPOA). By stereotaxic surgery frontal (FC) and frontal-lateral (LFC) retrochiasmatic "deafferenting" cuts were made with a Hal~sz knife (1.5 m m radius). At 3 and 10 weeks after surgery rats were given pentobarbital (32 mg/kg, i.p.) at 13:30 h and stimulated at 15:00 h with anodal direct current (20/zA for 60 sec) via concentric bipolar steel electrodes placed bilaterally 0.9 m m f r o m the midline. Stimulation at 3 weeks after FC increased plasma LH from a prestimulation level of 95 4- 22 ng/ml to 227 ± 51 ng/ml 80 min after ECS, from which it fell to 111 ± 29 ng/ml at 160 min, with 2 of 7 animals ovulating the next day. L F C females had similar pre-ECS plasma LH levels 3 weeks after surgery (71 ± 10 ng/ml), but LH concentrations at 80 min (104 :k 21 ng/ml) and 160 min post-ECS were significantly lower than those of FC rats, and 0 of 5 rats ovulated. Following a similar protocol 10 weeks after surgery, stimulating the M P O A resulted in comparable elevations in plasma LH and 4 of 10 FC animals ovulated; however, the L F C group still retained a significant blocking effect on ovulation (only 1 of 12 ovulated) when compared with controls the next day. Plasma FSH concentrations were not significantly altered by M P O A stimulation at the parameters employed, either before or after deafferentation. However, L F C resulted in reduced ovarian and uterine weights when compared with controls at both 3 and 10 weeks, whereas FC exerted no observable effect on these organs at these intervals of study. The results of these studies suggest that lateral input to the medial basal hypothalamus contributes to M P O A mediated release of LH and ovulation as well as to tonic maintenance of ovarian and uterine function.
* Supported by grants from NIH (NS01162) and the Ford Foundation. ** Postdoctoral Fellow of NIAMDD (AM03298). Present address: Department of Anatomy, College of Medicine, University of South Florida, Tampa, Fla., 33620, U.S.A.
336 INTRODUCTION Surgical interruption of neural pathways leading to and from the medial basal hypothalamus (MBH) has provided a useful approach to the study of brain pathways involved in endocrine regulationg, 1~. We have recently combined the techniques of anterior hypothalamic deafferentation and electrochemical stimulation (ECS) of the preoptic area 19 for the purpose of evaluating the extent of anterior hypothalamic input required to induce a surge of LH release in the rat 17. During the course of this investigation an increase in plasma LH was detected 1 h following bilateral ECS of the medial preoptic area (MPOA) rostral to the interruption of preoptico-tuberal pathways considered essential for ovulation in the rat 2,19. Serial reconstructions of electrode and frontal cut (FC) locations in each rat revealed that at least one stimulation site extended lateral to FC locations, suggesting a possible extension of ECS to fibers en passage through the lateral preoptic area coursing towards the MBH s,14,16. We have now inve3tigated these lateral connections to the MBH for their contribution to gonadotropin release from the anterior pituitary following ECS. Included is a comparative study of the variable capacities for response to ECS which occur with time after surgical interruption of anterior and anterolateral inputs to the MBH. MATERIALS AND METHODS Female rats (200-250 g) were selected for study after they had shown at least two consecutive 4-day estrous cycles. All animals were maintained on Purina lab chow and tap water ad libitum in a controlled environment with lights on from 5:00 to 19:00 h.
Anterior deafferentation Animals selected for FC were deafferented according to the method of Hal~tsz and Pupp 11 using a knife with a retractable blade 17. Briefly the procedure consisted of positioning the tip of the blade 1.6 mm behind bregma, drawing the blade up into the carrier and then lowering the carrier 5.5 mm through the superior sagittal sinus. The knife blade was then extruded in the median sagittal plane with the tip of the blade resting at the posterior border of the optic chiasm. FC was produced by rotating the knife 10-15 times 180° through the periosteum of the sphenoid bone, returning the blade to the sagittal plane and retracting it into the carrier. Sham FC surgeries consisted of extrusion and retraction of the blade in the median sagittal plane only. The knife blade had a turning radius of 1.5 mm for FC.
Lateral-frontal deafferentation In addition to the FC surgery other groups of animals were subjected to lateral frontal cuts (LFC) by extending the lateral edges of the FC 2.5 mm posteriorly towards the mammillary bodies. The lateral portion of this surgery was executed according to the method of Halfisz and Pupp 11 with some modifications. Briefly it consisted of rotating the 1.5 mm radius blade 90 ° from the median sagittal plane towards the side
337 of intended interruption. With the knife blade in this position the carrier was moved 2.5 mm posteriorly, then 2.5 mm anteriorly and the blade returned to the sagittal plane. All lateral cuts were made bilateral by repeating this procedure on the opposite lateral hypothalamus. Sham LFC consisted of a sham FC in addition to a 2.5 mm midline posterior-anterior course for the carrier with the blade retracted.
Experimental design Animals were subjected to FC, LFC or appropriate sham surgery on a morning of vaginal estrus. The rats were caged in pairs and vaginal smears taken daily during the course of experiments. On an afternoon of vaginal proestrus at approximately 3 or 10 weeks following hypothalamic surgery each animal was given pentobarbital (Pb-Diabutal, 32 mg/kg, i.p.) at 13:30. At 15:00 h it was bled (0.7 ml) by jugular venipuncture, immediately placed in a stereotaxic apparatus and a bipolar concentric electrode was lowered into the medial preoptic area (MPOA) according to the atlas of De Groot 7. The MPOA was stimulated bilaterally by passing 20/~A DC for 60 sec on each side, and the electrode was later checked for its ability to deposit iron in agar gel containing ferro-ferricyanide~0. The rat was bled at 40 min intervals (40, 80, 120, 160 min after stimulation) and the plasma was separated and stored for future L H and FSH assay. On the following morning both ovaries were removed, and the oviduct was examined microscopically for ova. Under deep ether anesthesia the animal was then perfused with saline followed by a saline-5 ~ potassium ferro-ferricyanide solution and finally 10 ~ formalin. The brains were removed and prepared for histological examination which included localization of Prussian Blue spots relative to FC and LFC according to the atlas of Albe-Fessard et al. 1. Ovarian, uterine and anterior pituitary weights were taken at autopsy. Plasma LH and FSH were measured by radioimmunoassay using the N I A M D D kits (A. F. Parlow) with the appropriate rat pituitary standards as reference preparations. Changes in organ weights and plasma gonadotrophin concentrations were analyzed with Student's t test, whereas ovulation data were compared by the Fisher Exact Probability Test. RESULTS
Estrous cycles Female rats subjected to FC alone showed variable vaginal cytology. The predominant finding was a fluctuating number of cornified cells present daily for periods up to 10 days with sporadic interruptions in this cytology by leukocytic-epithelial composites. When LFC surgerie3 were performed, vaginal smears remained predominantly leukocytic for most of the experimental period. During the last half of the 70-80 day period of observation in L F C rats a small proportion of nucleated and cornified epithelial cells appeared with the leukocytes every 5-7 days.
Electrochemically stimulated ovulation Ovulation following MPOA stimulation was significantly reduced (P = 0.025)
338 TABLE I Ovulation and organ weights after frontal deafferentation Brain surgery
Number o f days between brain surgery and stimulation
Ovulation by Ovarian weight Uterine weight Anterior POA stimula- (rag) (rag) pituitary weight tion in pento( mg) barbital-blocked rats
Sham FC FC
17-21 17-21
7/7 2/7*
84.9 i 4.4 74.0 ± 7.9
438.3 ± 20.7 432.5 ± 28.3
12.4 ± 0.5 12.4 ± 0.9
Sham FC FC
70-80 70-80
8/10 4/10
79.4 ± 3.7 72.8 ~ 10.1
489.9 -I- 36.9 500.5 24-29.4
12.9 ± 0.7 14.4 ± 1.1
* P - 0.025 when compared with shams.
17-2 i days after F C when compared with sham-operated controls, whereas c o m p a r i s o n with controls at the 70-80 day interval revealed statistically similar percentages of a n i m als with C> 5) tubal ova (Table I). Females receiving L F C registered a significant reduction in ovulation at 17-21 days (P < 0.01) which persisted through the 70-80 day (P < 0.01) period o f study (Table II). A c c o m p a n y i n g this reduction in o v u l a t i o n after M P O A s t i m u l a t i o n was a loss in ovarian weights when compared with controls or when L F C rats were compared across the two intervals of study (Table II). L F C also resulted in a loss in uterine weights at 17-21 days, which persisted t h r o u g h the remainder of the study (Table II). Plasma LH and FSH
H y p o t h a l a m i c deafferentation ( F C or LFC) resulted in a reduction of the capacity for M P O A stimulation to elevate plasma L H c o n c e n t r a t i o n at either interval TABLE II Ovulation and organ weights after frontaLlateral deafferentation Brain surgery
Number o/days between brain surgery and stimulation
Ovulation by Ovarian weight Uterine weight Anterior POA stimula- (mg) (mg) pituitary weight tion in pento(mg) barbital-blocked rats
Sham L F C * * * LFC
17-21 17-21
7/7 0/5*
90.0 i 6.7 63.8 5- 5.2*
Sham LFC*** LFC
70-80 70-80
11/12 1/12"
83.2 ± 4. I 529.3 :~ 24.0 36.8i3.1",** 246.1 ~ 37.9"
444.6 ± 17.8 10.9 ~- 0.7 296.9 ± 45.2* 10.0 ± 1.2 12,7 ± 0.3 12.2±0.8
* P <- 0.01 when compared with shams. ** P < 0.01 when compared with LFC 17-21 days after surgery. *** Rats 377 and 441 (see text) were removed from LFC groups and added to their respective sham LFC groups because of incompleteness of deafferentation revealed by histology.
339
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Fig. 1. Changes in plasma LH concentrations following electrochemical stimulation (20/~A DC for 60 sec) of the medial preoptic area in rats 17-21 days and 70-80 days after anterior (FC) and anterolateral (LFC) deafferentation of the hypothalamus. In the 17-21 day experiment LH levels were significantly higher at 80 and 160 min in the FC group than in the LFC group (* P < 0.02; ** P < 0.04). Such differences were not apparent at 70-80 days after surgery. N values are given in the tables.
(3 or 10 weeks) o f study (Figs. 1 and 2). However, at 17-21 days after brain surgery there were statistically significant differences in plasma L H at 80 (P < 0.02) and 160 (P < 0.04) min following M P O A stimulation when c o m p a r i n g the F C and L F C groups, L F C values being lower (Fig. 1). These differences were not apparent at the 70-80 day interval o f study, wherein the F C and L F C groups presented comparable increases in L H concentration during the 160 min sampling period. M P O A stimulation at the parameters employed did not induce a rise in plasma F S H levels in even the sham-operated groups, so there were no significant differences in plasma F S H concentrations when either F C or L F C groups were c o m p a r e d with their respective controls at the same times o f sampling following M P O A stimulation (Fig. 2).
Brain histology Composite reconstructions of F C and L F C locations relative to the centers o f bilateral anodal stimulation sites (Prussian Blue reaction) reflected a general uniformity in the placement o f both types o f cuts at the two intervals o f study (Fig. 3). All o f the F C animals had visible ventral scars posterior to the optic chiasm indicating the completeness o f surgery at that location. In the case o f the L F C surgeries, all
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Fig. 2. Failure of electrochemical stimulation of MPOA to induce a significant rise in plasma FSH either in sham-deafferented rats or in animals with anterior (FC) or anterolateral (LFC) deafferentation of the hypothalamus. N values are given in the tables.
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Fig. 3. Composite horizontal reconstructions of anterior (FC) and anterolateral (LFC) deafferentatlon cuts and electrochemical stimulation sites (dots) in the rat hypothalamus and medial preoptic area. The plus mark ( ÷ ) lies in the midsagittal plane 7 mm rostral to the vertical interaural plane. The scales at the left measure in mm rostral to the interaural plane.
341 animals exhibited complete cuts posterior to the chiasm; however, the bilateral posterior 2.5 m m extension of the knife was incomplete in 2 cases (Nos. 377 and 441). Because of the different nature of these incomplete cuts they will be described separately below. Animal No. 377 presented an L F C at 75 days that was incomplete on the left side of the brain throughout the last (most posterior) millimeter of the lateral cut. The release pattern for plasma L H in this rat was also markedly different from the remainder of the animals: 40 min following ECS, L H rose from a prestimulation level of 74 ng/ml to 511 ng/ml. At 80 rain after ECS, plasma L H had risen further to 1295 ng/ml, falling to 537 ng/ml at 120 min and 275 ng/ml at 160 min post-ECS. At autopsy this animal had ovulated 7 ova with an ovarian weight comparable (41.4 mg) to the group mean; however, the uterine weight (520.6 mg) was more than twice that of the group mean (Table II). Plasma F S H concentrations were not different from long-term L F C group means (Fig. 2). In another L F C rat (No. 441) autopsied 18 days following deafferentation, the lateral portion of the cut was incomplete bilaterally in that there was an 0.2 m m ventral gap along both of the 2.5 m m lateral portions of the cut (Fig. 4). Plasma L H concentrations rose from a pre-ECS level of 67 ng/ml to 648 ng/ml at 80 min after stimulation and remained elevated at 120 min (572 ng/ml) and 160 min (314 ng/ml) when compared with L F C group means (Fig. 1). Ovarian weights (47.2 mg) in rat No. 441 were similar to those of complete L F C rats at the interval of study; however, No. 441 was the only animal in the experimental group that had tubal ova (7) at autopsy
Fig. 4. Photomicrographs of frontal sections of the "LFC" deafferented hypothalamus and median eminence in which the knife blade reached the base of the brain (A) but not in the brain of rat 441 (B), in which arrows point to the sites of continuity between lateral and medial hypothalamus.
342 the morning after ECS. The uterus was larger (472.0 mg) in this female, wereas plasma FSH concentrations were similar to mean concentration for the group (Fig. 2). DISCUSSION
A functional role for lateral input to the M B H has been suggested by studies wherein lateral deafferentation reduced norepinephrine content of the hypothalamus 6, and posterior-lateral deafferentation reduced the incidence of ovulation in the rat TM. There is considerable anatomical and physiological evidence to suggest a relationship between adrenergic transmitters and gonadotropin release factors3A~, ~8. In the present study when retrochiasmatic deafferentations were extended 1.5 mm laterally from the midline and 2.5 m m posteriorly as far as the mammillary bodies (LFC), there was a reduction in both ovarian and uterine weights when compared with those in sham controls 3 weeks after LFC. This change did not occur in animals after FC alone. Prior to autopsy, these differences in organ mass might have been predicted by vaginal cytology. Females with L F C were characterized by periods of predominantly leukocytic smears over the 10 week period of study, whereas the principal cell type in FC rats was the cornified epithelial cell. There was also a further decline in ovarian weights progressing from 3 to 10 weeks in the L F C rats when compared with controls, whereas no change occurred in FC animals over a similar period. Changes in plasma LH concentration following electrochemical stimulation (ECS) of the M P O A rostral to cuts also revealed differences between LFC and FC rats. Three weeks after hypothalamic surgery stimulation rostral to FC resulted in an increase in plasma LH over the 2.5 h sampling period, although the levels obtained were much less than in sham FC rats. Females 3 weeks after FC did, however, reveal higher plasma L H levels at the 80 and 160 min sampling times than those following LFC. Such differences between FC and LFC L H curves were not significant at 10 weeks after surgery (Fig. 1). The LFC results suggest that the passage of time improves LH response to brain ECS, but not the ovulatory endpoint. In fact, there is a decrease in ovarian weight following LFC, progressing from 3 to 10 weeks with a continued reduction in uterine mass. The results with L F C females can be contrasted with the responses of FC rats to the same ECS and recovery periods : similar ovarian and uterine weights at both intervals with an improvement at 10 weeks in ovulatory response to changes in LH concentration essentially the same as those occurring after ECS at 3 weeks. These findings suggest a role for lateral hypothalamic input to the MBHS,14,16 in both the LH response to ECS of the M P O A (3 week study) and apparent increase in gonad sensitivity to LH, evidenced by the restoration of ovulation in FC rats after ECS at 10 weeks. This conclusion is supported by the findings in animals 377 and 441, in which accidentally incomplete L F C permitted the retention of quantitatively and qualitatively different connections at the lateral hypothalamus with the remainder of the brain, i.e., connections which were associated with dramatic increases in LH levels after ECS as well as with ovulatory and uterine parameters. Our inability to detect changes in plasma FSH concentrations between experimental and control groups following MPOA stimulation is in good agreement with
343 the works of K a l r a et al. 12, who reported that ECS (100 # A × 60 sec) of the M P O A does n o t result in a n increase in p l a s m a F S H c o n c e n t r a t i o n on the a f t e r n o o n o f proestrus at 0.5, 1 a n d 3 h after stimulation. Other laboratories 4,5 report elevations in p l a s m a F S H following electrochemical s t i m u l a t i o n of the M P O A ; however, these increases occur after quantitatively larger ECS t h a n that employed in the present study, which m a y have obscured or delayed a n y possible difference 4 between control a n d experimental groups in our results. ACKNOWLEDGEMENTS The authors wish to t h a n k Miss K a t h e r i n e Bangs a n d Mr. D a v i d W h i t m o y e r for technical assistance, Ms. Lois fels for secretarial aid, Ms. Arlene K o i t h a n for histological p r e p a r a t i o n s a n d Mr. Bob McAllister for the drawings.
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344 16 Millhouse, O. E., A Golgi study of the descending medial forebrain bundle, Brain Research, 15 (1969) 341-363. 17 Phelps, C. P., Krieg, R.J. and Sawyer, C. H., Spontaneous and electrochemically stimulated changes in plasma LH in the female rat following hypothalamic deafferentation, Brain Research, 101 (1976) 239-249. 18 Sawyer, C. H., First Geoffrey Harris Memorial Lecture: some recent developments in brainpituitary-ovarian physiology, Neuroendocrinology, 17 (1975) 97-124. 19 Tejasen, T. and Everett, J. W., Surgical analysis of the preoptico-tuberal pathway controlling ovulatory release of gonadotropins in the rat, Endocrinology, 81 (1967) 1387-1396. 20 Terasawa, E. and Sawyer, C. H., Electrical and electrochemical stimulation of the hypothalamoadenohypophysial system with stainless steel electrodes, Endocrinology, 84 (1969) 918-925.