- Email: [email protected]
Possible role of anterior inputs to the medial basal hypothalamus (MBH) in regulating prolactin release during pseudopregnancy in the rat
Brain Research, 83 (1975) 51-58 © Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands
51
POSSIBLE ROLE OF ANTERIOR INPUTS TO THE MEDIAL BASAL HYPOTHALAMUS (MBH) IN REGULATING PROLACTIN RELEASE DURING PSEUDOPREGNANCY IN THE RAT*
Y. A R A I Arid K. Y A M A N O U C H I
Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113 (Japan) (Accepted September 3rd, 1974)
SUMMARY
The effects of transection of fiber connections with the medial basal hypothalamus (MBH) on induction or maintenance of pseudopregnancy (PSP) were studied. PSP was induced 'pharmacologically' by injecting reserpine (1 mg/kg) on the day of diestrus I in 4-day cycling rats or the corresponding day after induction of ovulation in persistent estrous animals. Postero-bilateral deafferentation (PBLD) which cuts dorsal, lateral and posterior connections to the MBH failed to influence the induction of PSP. All rats resulted in PSP following reserpine injection. Anterior deafferentation (AD) which interrupts the preoptic-hypothalamic connections induced anovulatory persistent vaginal estrus. In these AD rats, reserpine could not activate new corpora lutea which had been formed by a pretreatment with 10 I.U. HCG. However, replacement therapy with 20 I.U. prolactin was successful in activating these corpora lutea. In spite of the AD, all rats thereafter remained in diestrus, massive deciduomata being formed following uterine trauma. In the second series of experiments, the AD or roof deafferentation (RD) was performed 3 days after reserpine injection. The AD effectively interrupted reserpine-induced PSP. However, this effect of the AD was reversed by the replacement with prolactin from the day of operation. PSP was also not interrupted by the AD when reserpine was given supplementarily from the day of the AD through the day before autopsy, but a single dose of reserpine only on the day of the AD was not effective in maintaining PSP in AD rats. The RD which removes dorsal afferents to the preoptic area (POA) and hypothalamus did not interfere with PSP; it continued in all rats of the group. These results suggest that neural influence originating from the POA or more rostral regions is necessary to maintain prolonged release of prolactin during PSP. Since successive * A part of this studywas presentedat the Fourth InternationalCongressof Psychoneuroendocrinology, Berkeley, 1973.
52
Y. ARAI AND K. YAMANOUCHI
injections of reserpine were needed to keep the AD rats pseudopregnant, reserpine sensitive neural structures which may locate behind the surgical cut seem to be unable to support independently prolonged release of prolactin during PSP.
INTRODUCTION
It is generally believed that prolactin secretion is chronically inhibited by the central nervous system (CNS), since the removal of neural influences on the anterior pituitary causes a rapid increase in prolactin secretion. Many stimuli can act through the CNS to induce prolactin release. Mechanical stimulation of the uterine cervix activates the corpora lutea of the rat and results in pseudopregnancy (PSP) as effectively as does mating with a vasectomized male 6. A number of drugs which depress the CNS (such as reserpine) have been found capable of stimulating prolactin secretiona,9,13. Several investigators have suggested the existence of possible sites which are positively involved in the induction or maintenance of PSP. Everett and Quinn 7 reported that electrical stimulation of the dorsomedial nucleus and related areas of the medial hypothalamus was effective in evoking PSP but not in inducing ovulation. This finding strongly suggests the specific contribution of these neural substrates to the inductive process of PSP. Recently we1 have pointed out the possible participation of the preoptic projection to the medial basal hypothalamus (MBH) in induction of PSP, because the interruption of fiber connections between the preoptic area (POA) and MBH effectively prevented the development of PSP following the cervical stimulation. Carrer and Taleisnik5 found that a similar transection not only inhibited the induction of PSP but also interrupted an established one. The functional role of the POA-hypothalamic connections is well documented in triggering ovulatory discharge of gonadotropins, but is less known in controlling prolactin secretion. In the present study, 3 types of hypothalamic deafferentations were performed to elucidate the nature of the preoptic influence on prolactin secretion during PSP. MATERIALS AND METHODS
Virgin female Wistar rats (200--250 g) were housed in an air-conditioned room with controlled lighting (24 ± 1.4°C, 14/10h light/dark illumination). Vaginal smears were taken daily and only animals with 4-day cycles were selected for the experiments. Three types of hypothalamic deafferentations were performed under ether anesthesia according to a modified technique described by Halhsz and GorskP °, Anterior deafferentation (AD). A half-dome cut (Fig. 1) was made at the anterior or posterior end of the suprachiasmatic nucleus with the use of the Hal~sz knife having a 2.0 mm lateral × 2.0 mm vertical blade. This type of the deafferentation interrupted anterior connections to the MBH, but other connections were left intact. Postero-bilateral deafferentation (PBLD). The dorsal, lateral and posterior
PREOPTICAREAAND PROLACTINSECRETION
53
OC
RD,~~.*FX,
AD
y
.
AD
--PBLO
OC'
~
A
B
Fig. 1. Schematic representation of the surgical procedures. AD, anterior deafferentation; PBLD, postero-bilateral deafferentation; RD, roof deafferentation; AC, anterior commissure; AH, anterior hypothalamic nucleus; AL, anterior lobe of pituitary; ARC, arcuate nucleus; DM, dorsomedial nucleus; FX, fornix; MB, mammillary body; OC, optic chiasma; PH, posterior hypothalamic nucleus; PL, posterior lobe of pituitary; POA, preoptic area; PV, paraventricular nucleus; RE, nucleus reuniens; SC, suprachiasmatic nucleus; VM, ventromedial nucleus.
connections to the MBH were severed with a knife having a 1.4 mm lateral 5< 2.0 mm vertical blade. The MBH was left in neural contact only with the anterior hypothalamic area (Fig. 1). Roof deafferentation (RD). A horizontal cut was made at the level of the anterior commissure with an L-shaped knife having a horizontal blade of 2.5 mm (Fig. 1). This cut interrupted the dorsal connections of the POA and hypothalamus. Sham deafferentation (SD). The knife was lowered through the sagittal sinus to the base of the brain and removed without rotation. In the first series of experiments, the effect of hypothalamic deafferentation on the induction of PSP was studied. For induction of PSP, reserpine was injected instead of applying the cervical stimulation, because the vaginal stimulation has been found to be simply replaced by the treatment with neuroleptic drugsa,9, la. Thirty days after the deafferentation, vaginal smears were taken daily. Since the SD and PBLD do not interfere with ovulatory mechanisms 10, following completion of 4 normal cycles, a single injection of 1 mg/kg of reserpine (Serpasil, CIBA) was given subcutaneously on the day of diestrus I in SD and PBLD groups (groups 2 and 3). Three days later, a thin thread was placed in one of the uterine horns to test for a deciduoma reaction. Animals were killed 4 days after the uterine trauma. In agreement with earlier findings t°, the rats with the AD showed persistent estrus. They failed to ovulate. To obtain ovulation in these rats, 10 I.U. of H C G (Teikoku Hormone Mfg., Co., Ltd., Tokyo) was injected subcutaneously for 2 successive days. Reserpine injection was given on the first day of diestrus. Three days after the reserpine injection uterine traumatization with a thread was made and autopsy was performed 4 days later (group 4). A small number of rats with the AD were injected with 20 I.U. of ovine
54
Y. ARAI A N D K. Y A M A N O U C H 1
prolactin (NIH-prolactin-SI0) daily from the day of reserpine injection to the day before autopsy (group 5). In the second series of experiments, influence of the AD or RD on the maintenance of PSP was examined. A single subcutaneous injection o f reserpine (1 mg/kg) was administered on the day of diestrus I to develop PSP. Three days after reserpine, rats were given A D (group 8), R D (group 9) or SD (group 7), and uterine trauma. Animals were sacrificed 4 days later. To study the effect of hormonal replacement a number of rats with A D were injected with 20 I.U. of prolactin daily from the day of the operation to the day before they were killed (group 10). In addition, supplemental reserpine treatment was given to the A D rats from the day of the operation to the day before autopsy (group 12). Dosage of reserpine was 1 mg/kg on the day of the AD, and then reduced to 0.5 mg/kg from the next day. A number of rats received a supplemental injection o f reserpine (1 mg/kg) only on the day of the AD, no additional treatment being given (group 11). At the termination of the various experiments, body and organ weights were recorded for each rat. A segment of the deciduomata or uterine tissues (with a thread) and ovaries were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with H - E . Each brain was removed and the precise localization of each hypothalamic deafferentation was determined histologically. RESULTS
Effect o f hypothalamic deafferentation on induction o f P S P
Results are summarized in Table I. When a single injection of reserpine (1 mg/kg) was given to the rats receiving the PBLD (group 3) or SD (group 2) on the day of diestrus I, all rats treated continued in diestrus. At autopsy, massive deciduoma TABLE
1
EFFECT OF HYPOTHALAMIC DEAFFERENTAT1ON ON INDUCTION OF PSEUDOPREGNANCY*
Group Treatment
No. of rats
Deciduomaresponse Deciduomata --
Control uterine horn
Traumatized uterine horn
(Mean _+_S.E., mg/lOOg body weight)
1 2 3
Intact control Sham deafferentation (SD) Postero-bilateral deafferentation (PBLD) Anterior deafferentation (AD) AD -}-20 I.U. prolactin***
4 5
5 7
5 7
0 0
100~ 100
62± 7 58 ± 16
308 zk 40 352 :~ 35
7
7
0
100
61 L-- 14
408 ~ 28
7 5
0 5
7 0
0 100 K
(116 =k 7)** 85~- 6
(126 zk 13)** 416jz 10
* Pseudopregnancy was induced by injecting reserpine (1 mg/kg) on the day of diestrus I. The uterine weight of the animals showing negative deciduoma response is given in parentheses. *** Prolactin was injected from the day of reserpine injection to the day before autopsy. **
55
PREOPTIC AREA AND PROLACTIN SECRETION TABLE 1I EFFECT OF H Y P O T H A L A M I C DEAFFERENTATION ON M A I N T E N A N C E OF P S E U D O P R E G N A N C Y *
Group Treatment
Deciduoma response (no. positive/no, treated)
Control uterine horn
Traumatized uterine horn
(Mean -4-S.E., mg/ lO0 g body weight) 6
Intact control
7/8
87.5~ + --
7
SD
7/7
100.0~o + --
8
AD
4/16
25.0~o + --
9
Roofdeafferentation(RD) 6/6 AD + prolactin***
7/7
100.0~ + --
ll
12
AD + reserpine on the dayofAD*** AD -4- reserpine from the day of AD through the day before autopsy* * *
3/9 6/7
(1)**
(7)
33.3~o + -85.7 ~ -4--
59 ± 3 89 69 ± 8
(0)
--
(4)
58 ± 5
(12)
100.0~ -4- (6) --
10
(7)**
(0)
108
-4- 9
61 -4- 4
337 ! 36 94 445 ± 53 3 1 4 i 59 1 2 9 -4- 10 218 -4- 58
--
(7)
63 -4- 5
(0)
--
(3) (6) (6) (1)
72 90-4- 11 76 -4- 4 95
218 i 67 242 1094- 9 180 ~: 30 103
* Pseudopregnancy was induced by injecting reserpine on the day of diestrus I. ** The number of rats which showed positive or negative response is given in parentheses. *** For dosage, see text. f o r m a t i o n was seen. I n c o n t r a s t , the rats with the A D § in which o v u l a t i o n h a d been i n d u c e d b y 2 s u b c u t a n e o u s injections o f 10 I.U. H C G on 2 consecutive d a y s showed d i e s t r o u s smears f o r a while after reserpine injection o n the first d a y o f diestrus, b u t corn±fled vaginal smears a p p e a r e d a g a i n until the d a y o f a u t o p s y ( g r o u p 4). T h e p l a c e m e n t o f a t h r e a d in one o f the uterine h o r n s failed to induce d e c i d u o m a response in all 7 rats o f this group. A n o t h e r 5 rats with A D received t r e a t m e n t w i t h 20 I.U. p r o l a c t i n ( g r o u p 5). P r o l a c t i n injections were c o n t i n u e d f r o m the d a y o f reserpine injection to the d a y before a u t o p s y . A l l rats t r e a t e d w i t h p r o l a c t i n r e m a i n e d in diestrus. Massive d e c i d u o m a t a were f o u n d at a u t o p s y . T h e m e a n weight o f t r a u m a t i z e d h o r n s was a l m o s t c o m p a r a b l e with those o f P B L D , S D a n d i n t a c t c o n t r o l g r o u p s (Table I).
Effect o f hypothalamic deafferentation on maintenance o f P S P T a b l e II presents the effects o f h y p o t h a l a m i c deafferentations on reserpinei n d u c e d PSP. I n t a c t a n d S D rats (groups 6 a n d 7) s h o w e d d i e s t r o u s smears c o n t i n u o u s ly after reserpine injection on the d a y o f diestrus I. A t a u t o p s y , a massive d e c i d u o m a response was o b t a i n e d in 7 o u t o f 8 rats in g r o u p 6 a n d in all 7 rats in g r o u p 7, A m o n g rats receiving AD§§, 4 o u t o f 16 s h o w e d persistent diestrus a n d gave d e c i d u o m a § In all rats of groups 4 and 5, the AD was made at the posterior end of the suprachiasmatic nucleus (Fig. 1). §§ The AD was performed at the level oftbe anterior or posterior end of the suprachiasmatic nucleus in groups 8, 10, 11 and 12, but results were not significantly different between the two types of cut.
56
Y. ARAI A N D K. Y A M A N O U C H I
response. The other rats with AD exhibited cornified smears 2-4 days after AD and remained in estrus until sacrificed (group 8). At autopsy, uteri were markedly distended with fluid and no deciduoma formation was found in these rats. This macroscopic finding was confirmed by histological evidence that showed the absence of decidual cells in the endometrial tissue close to a thread. There was no significant difference in weight between intact and traumatized uterine horns, but both were significantly heavier than intact horns of other groups, because of uterine stimulation by endogenous estrogen (Table II). The POA-roof transection (RD) permitted the deciduoma response; all rats had massive deciduomata at autopsy (group 9). Twenty I.U. of prolactin were injected daily into the rats with AD for 4 days beginning the day of the operation (group 10). Prolactin effectively reversed the interruptive effect of the AD on the maintenance of PSP. The animals remained in diestrus and, at autopsy, sizes of deciduomata and weights of traumatized uterine horns were almost comparable to those of intact and SD controls or RD rats. In the groups of rats with AD receiving supplemental injections of reserpine, successive injections for 4 days from the day of surgery were effective in overcoming the effect of the AD (group 12). In 6 out of 7 rats, deciduoma response was obtained, although the size of deciduomata was considerably smaller. In rats receiving a single injection of reserpine on the day of the AD without further treatment (group 11 ), small uterine swellings at the site of traumatization were observed in 6 out of 9 rats, but histological examinations of these uterine tissues showed that deciduomata were highly degenerative in 3 out of 6 rats. DISCUSSION
The finding that the PBLD failed to inhibit the induction of PSP in the present study indicates that fiber connections with the MBH interrupted by the PBLD were not directly involved in reserpine-induced PSP. This is consistent with a previous report that a similar PBLD did not prevent the induction of PSP by vaginal-cervical stimulation1. Since i t has been reported that the transection of the POA-hypothalam ic connections inhibited establishment of PSP following vaginal-cervical stimulation1,~, the sensory pathways related to the induction of PSP seem to ascend the brain stem dorsal or lateral to the MBH and converge on the POA before giving the information to the MBH. This conjecture is well supported by electrophysiological evidence indicating that impulses of the cervical origin can be recognized in the anterior, lateral or dorsal hypothalamus, POA and thalamus 3,4,12,~5,1~. In the present study as previously reported by Carrer and Taleisnik5, the interruption of POA-hypothalamic connections (AD) also interfered with reserpine-induced PSP; however, it is still questionable whether these sensory neural substrates are involved in 'pharmacological' induction of PSP. It has been suggested that the MBH is a possible focus for reserpine action to cause prolactin release, based on the results of implantation experiments of reserpine crystal into the brain11,17. The results shown in Table I might be interpreted to mean that the preoptic projection to the MBH is primarily involved in initiating PSP
PREOPTIC AREAAND PROLACT1NSECRETION
57
in response to reserpine. However, it is also probable that the AD interfered with PSP after it had been established. This is at least true for the results presented in Table I I, where the AD effectively interrupted the PSP which had already been initiated. Since administration of prolactin was successful in reversing the inhibitory effect of the AD on PSP, anterior inputs removed by the AD seem to play an important role in supporting prolactin release during PSP. A similar situation has been reported for the interruption of pregnancy in rats receiving the POA-hypothalamic transection 2. Furthermore, the preoptic roof section which cuts dorsal afferents to the POA, did not interfere with maintenance of PSP in the present study. This suggests that the neural influence supporting prolactin release during PSP seems to originate from the POA itself or some structures anterior to the POA. Supplemental treatment of the AD rats with reserpine could maintain the PSP, although sizes of deciduomata and weights of traumatized uterine horns were markedly reduced, compared with control groups. This indicates that a neural structure capable of responding to reserpine may locate behind the surgical cut, presumably in the MBH, as suggested by Kanematsu and Sawyer11 and Van Maanen and Smelik17. However, it seems unlikely that this structure can independently permit secretion of a sufficient amount of prolactin required for the maintenance of PSP, because histological examinations showed that deciduomata were degenerative in the AD rats receiving a single injection of reserpine on the day of the operation alone. To support prolonged release of prolactin, either successive injections of reserpine or neural influence from the POA appeared to be necessary in our rats. Recently a nocturnal prolactin surge occurring each night during PSP has been reported by Freeman and Neill8. Since the levels of prolactin in the pituitary and blood were not measured in the present study, it is not clear whether the situation of 'pharmacologically' induced PSP is the same with the PSP induced by vaginal-cervical stimulation in which the stimuli are thought to be memorized in the CNS 8. It is highly possible that the AD interfered with the mechanisms for a nocturnal surge and thereby inhibited the onset or maintenance of PSP in our rats. In this connection, it is of interest to note the findings of Neil114 that the surge of prolactin secretion following estrogen treatment was inhibited by a similar AD. However, it still remains to be solved whether anterior inputs inhibit hypothalamic PIF activity or increase hypothalamic PRF activity under these conditions. ACKNOWLEDGEMENTS The authors wish to express their thanks to Mrs. K. Serisawa for her excellent assistance. We are also grateful to Dr. R. W. Bates, NIAMD, Bethesda, U.S.A., for supplying prolactin preparations. This study was supported by grants from the Ministry of Education of Japan.
REFERENCES 1 ARAI, Y., Effectof hypothalamic de-afferentationon inductionof pseudopregnancyby vaginalcervical stimulationin the rat, J. Reprod. Fertil., 19 (1969) 573-575.
58
Y. ARAI AND K. YAMANOUCHI
2 ARAI, Y., Possible participation of central nervous system (CNS) in blastocyst implantation in the rat, Brain Research, 66 (1974) 393-400. 3 BARRACLOUGH,C. A., AND SAWYER,C. H., Induction of pseudopregnancy in the rat by reserpine and chlorpromazine, Endocrinology, 65 (1959) 563-571. 4 BARRACLOUGH,C. A., ANn CROSS, B A., Unit activity in the hypothalamus of the cyclic female rat: effect of genital stimuli and progesterone, J. Endocrinol., 26 (1963) 339-359. 5 CARRER, H. F., AND TALEISNIK,S., Induction and maintenance of pseudopregnancy after interruption of preoptic hypothalamic connections, Endocrinology, 86 (1970) 231-236. 6 EVERETT, J.W., Central neural control of reproductive functions of the adenohypophysis, Physiol. Rev., 44 (1954) 373-431. 7 EVERETT, J. W., AND QU~NN, D. L., Differential hypothalamic mechanisms inciting ovulation and pseudopregnancy, Endocrinology, 78 (1966) 141-150. 8 FREEMAN,M. E., AND NEILL, J. D., The pattern of prolactin secretion during pseudopregnancy in the rat: a daily nocturnal surge, Endocr&ology, 90 (1972) 1292-1294. 9 GOLD, E. M., AND GANONG, W. F., Effects of drugs on neuroendocrine processes. In L. MARTIM AND W. F. GANONG(Eds.), Neuroendocrinology 11, Academic Press, New York, 1967, pp. 377-437. 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 KANEMATSU, S., AND SAWYER, C . H . , Effects of intrahypothalamic implants of reserpine on lactation and pituitary content of prolactin in the rabbit, Proc. Soc. exp. Biol. (N. Y.), 113 (1963) 967-969. 12 KAWAKAMI, M., AND SAITO, H., Unit activity in the hypothalamus of the cat: effect of genital stimuli, luteinizing hormone and oxytocin, Jap. J. Physiol., 17 (1967) 466-486. 13 MEITES,J., Pharmacological control of prolactin secretion and lactation. In R. GUILLEMIN(Ed.), Proc. First Int. Pharmacol. Meet. I, Pergamon Press, Oxford, 1962, pp. 151-181. 14 NEILL, J. D., Sexual differences in the hypothalamic regulation of prolactin secretion, Endocrinology, 90 (1972) 1154-1159. 15 PORTER, R. W., CAVANOUGH,E. B., CRITCHLOW, B. V., AND SAWYER, C. H., Localized changes in electrical activity of the hypothalamus in estrous cats following vaginal stimulation, Amer. J. Physiol., 189 (1957) 145-151. 16 RAMIREZ,V. D., KOMISARUK,B. R., WH1TMOYER,O. I., AND SAWYER,C. H., Effects of hormones and vaginal stimulation on the EEG and hypothalamic units in rats, Amer. J. Physiol., 212 (1967) 1376-1384. 17 VAN MAANEN, J. H., AND SMELIK, P. G., Induction of pseudopregnancy in rats following local depletion of monoamines in the median eminence of the hypothalamus, Neuroendocrinology, 3 (1968) 177-186.
51
POSSIBLE ROLE OF ANTERIOR INPUTS TO THE MEDIAL BASAL HYPOTHALAMUS (MBH) IN REGULATING PROLACTIN RELEASE DURING PSEUDOPREGNANCY IN THE RAT*
Y. A R A I Arid K. Y A M A N O U C H I
Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113 (Japan) (Accepted September 3rd, 1974)
SUMMARY
The effects of transection of fiber connections with the medial basal hypothalamus (MBH) on induction or maintenance of pseudopregnancy (PSP) were studied. PSP was induced 'pharmacologically' by injecting reserpine (1 mg/kg) on the day of diestrus I in 4-day cycling rats or the corresponding day after induction of ovulation in persistent estrous animals. Postero-bilateral deafferentation (PBLD) which cuts dorsal, lateral and posterior connections to the MBH failed to influence the induction of PSP. All rats resulted in PSP following reserpine injection. Anterior deafferentation (AD) which interrupts the preoptic-hypothalamic connections induced anovulatory persistent vaginal estrus. In these AD rats, reserpine could not activate new corpora lutea which had been formed by a pretreatment with 10 I.U. HCG. However, replacement therapy with 20 I.U. prolactin was successful in activating these corpora lutea. In spite of the AD, all rats thereafter remained in diestrus, massive deciduomata being formed following uterine trauma. In the second series of experiments, the AD or roof deafferentation (RD) was performed 3 days after reserpine injection. The AD effectively interrupted reserpine-induced PSP. However, this effect of the AD was reversed by the replacement with prolactin from the day of operation. PSP was also not interrupted by the AD when reserpine was given supplementarily from the day of the AD through the day before autopsy, but a single dose of reserpine only on the day of the AD was not effective in maintaining PSP in AD rats. The RD which removes dorsal afferents to the preoptic area (POA) and hypothalamus did not interfere with PSP; it continued in all rats of the group. These results suggest that neural influence originating from the POA or more rostral regions is necessary to maintain prolonged release of prolactin during PSP. Since successive * A part of this studywas presentedat the Fourth InternationalCongressof Psychoneuroendocrinology, Berkeley, 1973.
52
Y. ARAI AND K. YAMANOUCHI
injections of reserpine were needed to keep the AD rats pseudopregnant, reserpine sensitive neural structures which may locate behind the surgical cut seem to be unable to support independently prolonged release of prolactin during PSP.
INTRODUCTION
It is generally believed that prolactin secretion is chronically inhibited by the central nervous system (CNS), since the removal of neural influences on the anterior pituitary causes a rapid increase in prolactin secretion. Many stimuli can act through the CNS to induce prolactin release. Mechanical stimulation of the uterine cervix activates the corpora lutea of the rat and results in pseudopregnancy (PSP) as effectively as does mating with a vasectomized male 6. A number of drugs which depress the CNS (such as reserpine) have been found capable of stimulating prolactin secretiona,9,13. Several investigators have suggested the existence of possible sites which are positively involved in the induction or maintenance of PSP. Everett and Quinn 7 reported that electrical stimulation of the dorsomedial nucleus and related areas of the medial hypothalamus was effective in evoking PSP but not in inducing ovulation. This finding strongly suggests the specific contribution of these neural substrates to the inductive process of PSP. Recently we1 have pointed out the possible participation of the preoptic projection to the medial basal hypothalamus (MBH) in induction of PSP, because the interruption of fiber connections between the preoptic area (POA) and MBH effectively prevented the development of PSP following the cervical stimulation. Carrer and Taleisnik5 found that a similar transection not only inhibited the induction of PSP but also interrupted an established one. The functional role of the POA-hypothalamic connections is well documented in triggering ovulatory discharge of gonadotropins, but is less known in controlling prolactin secretion. In the present study, 3 types of hypothalamic deafferentations were performed to elucidate the nature of the preoptic influence on prolactin secretion during PSP. MATERIALS AND METHODS
Virgin female Wistar rats (200--250 g) were housed in an air-conditioned room with controlled lighting (24 ± 1.4°C, 14/10h light/dark illumination). Vaginal smears were taken daily and only animals with 4-day cycles were selected for the experiments. Three types of hypothalamic deafferentations were performed under ether anesthesia according to a modified technique described by Halhsz and GorskP °, Anterior deafferentation (AD). A half-dome cut (Fig. 1) was made at the anterior or posterior end of the suprachiasmatic nucleus with the use of the Hal~sz knife having a 2.0 mm lateral × 2.0 mm vertical blade. This type of the deafferentation interrupted anterior connections to the MBH, but other connections were left intact. Postero-bilateral deafferentation (PBLD). The dorsal, lateral and posterior
PREOPTICAREAAND PROLACTINSECRETION
53
OC
RD,~~.*FX,
AD
y
.
AD
--PBLO
OC'
~
A
B
Fig. 1. Schematic representation of the surgical procedures. AD, anterior deafferentation; PBLD, postero-bilateral deafferentation; RD, roof deafferentation; AC, anterior commissure; AH, anterior hypothalamic nucleus; AL, anterior lobe of pituitary; ARC, arcuate nucleus; DM, dorsomedial nucleus; FX, fornix; MB, mammillary body; OC, optic chiasma; PH, posterior hypothalamic nucleus; PL, posterior lobe of pituitary; POA, preoptic area; PV, paraventricular nucleus; RE, nucleus reuniens; SC, suprachiasmatic nucleus; VM, ventromedial nucleus.
connections to the MBH were severed with a knife having a 1.4 mm lateral 5< 2.0 mm vertical blade. The MBH was left in neural contact only with the anterior hypothalamic area (Fig. 1). Roof deafferentation (RD). A horizontal cut was made at the level of the anterior commissure with an L-shaped knife having a horizontal blade of 2.5 mm (Fig. 1). This cut interrupted the dorsal connections of the POA and hypothalamus. Sham deafferentation (SD). The knife was lowered through the sagittal sinus to the base of the brain and removed without rotation. In the first series of experiments, the effect of hypothalamic deafferentation on the induction of PSP was studied. For induction of PSP, reserpine was injected instead of applying the cervical stimulation, because the vaginal stimulation has been found to be simply replaced by the treatment with neuroleptic drugsa,9, la. Thirty days after the deafferentation, vaginal smears were taken daily. Since the SD and PBLD do not interfere with ovulatory mechanisms 10, following completion of 4 normal cycles, a single injection of 1 mg/kg of reserpine (Serpasil, CIBA) was given subcutaneously on the day of diestrus I in SD and PBLD groups (groups 2 and 3). Three days later, a thin thread was placed in one of the uterine horns to test for a deciduoma reaction. Animals were killed 4 days after the uterine trauma. In agreement with earlier findings t°, the rats with the AD showed persistent estrus. They failed to ovulate. To obtain ovulation in these rats, 10 I.U. of H C G (Teikoku Hormone Mfg., Co., Ltd., Tokyo) was injected subcutaneously for 2 successive days. Reserpine injection was given on the first day of diestrus. Three days after the reserpine injection uterine traumatization with a thread was made and autopsy was performed 4 days later (group 4). A small number of rats with the AD were injected with 20 I.U. of ovine
54
Y. ARAI A N D K. Y A M A N O U C H 1
prolactin (NIH-prolactin-SI0) daily from the day of reserpine injection to the day before autopsy (group 5). In the second series of experiments, influence of the AD or RD on the maintenance of PSP was examined. A single subcutaneous injection o f reserpine (1 mg/kg) was administered on the day of diestrus I to develop PSP. Three days after reserpine, rats were given A D (group 8), R D (group 9) or SD (group 7), and uterine trauma. Animals were sacrificed 4 days later. To study the effect of hormonal replacement a number of rats with A D were injected with 20 I.U. of prolactin daily from the day of the operation to the day before they were killed (group 10). In addition, supplemental reserpine treatment was given to the A D rats from the day of the operation to the day before autopsy (group 12). Dosage of reserpine was 1 mg/kg on the day of the AD, and then reduced to 0.5 mg/kg from the next day. A number of rats received a supplemental injection o f reserpine (1 mg/kg) only on the day of the AD, no additional treatment being given (group 11). At the termination of the various experiments, body and organ weights were recorded for each rat. A segment of the deciduomata or uterine tissues (with a thread) and ovaries were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with H - E . Each brain was removed and the precise localization of each hypothalamic deafferentation was determined histologically. RESULTS
Effect o f hypothalamic deafferentation on induction o f P S P
Results are summarized in Table I. When a single injection of reserpine (1 mg/kg) was given to the rats receiving the PBLD (group 3) or SD (group 2) on the day of diestrus I, all rats treated continued in diestrus. At autopsy, massive deciduoma TABLE
1
EFFECT OF HYPOTHALAMIC DEAFFERENTAT1ON ON INDUCTION OF PSEUDOPREGNANCY*
Group Treatment
No. of rats
Deciduomaresponse Deciduomata --
Control uterine horn
Traumatized uterine horn
(Mean _+_S.E., mg/lOOg body weight)
1 2 3
Intact control Sham deafferentation (SD) Postero-bilateral deafferentation (PBLD) Anterior deafferentation (AD) AD -}-20 I.U. prolactin***
4 5
5 7
5 7
0 0
100~ 100
62± 7 58 ± 16
308 zk 40 352 :~ 35
7
7
0
100
61 L-- 14
408 ~ 28
7 5
0 5
7 0
0 100 K
(116 =k 7)** 85~- 6
(126 zk 13)** 416jz 10
* Pseudopregnancy was induced by injecting reserpine (1 mg/kg) on the day of diestrus I. The uterine weight of the animals showing negative deciduoma response is given in parentheses. *** Prolactin was injected from the day of reserpine injection to the day before autopsy. **
55
PREOPTIC AREA AND PROLACTIN SECRETION TABLE 1I EFFECT OF H Y P O T H A L A M I C DEAFFERENTATION ON M A I N T E N A N C E OF P S E U D O P R E G N A N C Y *
Group Treatment
Deciduoma response (no. positive/no, treated)
Control uterine horn
Traumatized uterine horn
(Mean -4-S.E., mg/ lO0 g body weight) 6
Intact control
7/8
87.5~ + --
7
SD
7/7
100.0~o + --
8
AD
4/16
25.0~o + --
9
Roofdeafferentation(RD) 6/6 AD + prolactin***
7/7
100.0~ + --
ll
12
AD + reserpine on the dayofAD*** AD -4- reserpine from the day of AD through the day before autopsy* * *
3/9 6/7
(1)**
(7)
33.3~o + -85.7 ~ -4--
59 ± 3 89 69 ± 8
(0)
--
(4)
58 ± 5
(12)
100.0~ -4- (6) --
10
(7)**
(0)
108
-4- 9
61 -4- 4
337 ! 36 94 445 ± 53 3 1 4 i 59 1 2 9 -4- 10 218 -4- 58
--
(7)
63 -4- 5
(0)
--
(3) (6) (6) (1)
72 90-4- 11 76 -4- 4 95
218 i 67 242 1094- 9 180 ~: 30 103
* Pseudopregnancy was induced by injecting reserpine on the day of diestrus I. ** The number of rats which showed positive or negative response is given in parentheses. *** For dosage, see text. f o r m a t i o n was seen. I n c o n t r a s t , the rats with the A D § in which o v u l a t i o n h a d been i n d u c e d b y 2 s u b c u t a n e o u s injections o f 10 I.U. H C G on 2 consecutive d a y s showed d i e s t r o u s smears f o r a while after reserpine injection o n the first d a y o f diestrus, b u t corn±fled vaginal smears a p p e a r e d a g a i n until the d a y o f a u t o p s y ( g r o u p 4). T h e p l a c e m e n t o f a t h r e a d in one o f the uterine h o r n s failed to induce d e c i d u o m a response in all 7 rats o f this group. A n o t h e r 5 rats with A D received t r e a t m e n t w i t h 20 I.U. p r o l a c t i n ( g r o u p 5). P r o l a c t i n injections were c o n t i n u e d f r o m the d a y o f reserpine injection to the d a y before a u t o p s y . A l l rats t r e a t e d w i t h p r o l a c t i n r e m a i n e d in diestrus. Massive d e c i d u o m a t a were f o u n d at a u t o p s y . T h e m e a n weight o f t r a u m a t i z e d h o r n s was a l m o s t c o m p a r a b l e with those o f P B L D , S D a n d i n t a c t c o n t r o l g r o u p s (Table I).
Effect o f hypothalamic deafferentation on maintenance o f P S P T a b l e II presents the effects o f h y p o t h a l a m i c deafferentations on reserpinei n d u c e d PSP. I n t a c t a n d S D rats (groups 6 a n d 7) s h o w e d d i e s t r o u s smears c o n t i n u o u s ly after reserpine injection on the d a y o f diestrus I. A t a u t o p s y , a massive d e c i d u o m a response was o b t a i n e d in 7 o u t o f 8 rats in g r o u p 6 a n d in all 7 rats in g r o u p 7, A m o n g rats receiving AD§§, 4 o u t o f 16 s h o w e d persistent diestrus a n d gave d e c i d u o m a § In all rats of groups 4 and 5, the AD was made at the posterior end of the suprachiasmatic nucleus (Fig. 1). §§ The AD was performed at the level oftbe anterior or posterior end of the suprachiasmatic nucleus in groups 8, 10, 11 and 12, but results were not significantly different between the two types of cut.
56
Y. ARAI A N D K. Y A M A N O U C H I
response. The other rats with AD exhibited cornified smears 2-4 days after AD and remained in estrus until sacrificed (group 8). At autopsy, uteri were markedly distended with fluid and no deciduoma formation was found in these rats. This macroscopic finding was confirmed by histological evidence that showed the absence of decidual cells in the endometrial tissue close to a thread. There was no significant difference in weight between intact and traumatized uterine horns, but both were significantly heavier than intact horns of other groups, because of uterine stimulation by endogenous estrogen (Table II). The POA-roof transection (RD) permitted the deciduoma response; all rats had massive deciduomata at autopsy (group 9). Twenty I.U. of prolactin were injected daily into the rats with AD for 4 days beginning the day of the operation (group 10). Prolactin effectively reversed the interruptive effect of the AD on the maintenance of PSP. The animals remained in diestrus and, at autopsy, sizes of deciduomata and weights of traumatized uterine horns were almost comparable to those of intact and SD controls or RD rats. In the groups of rats with AD receiving supplemental injections of reserpine, successive injections for 4 days from the day of surgery were effective in overcoming the effect of the AD (group 12). In 6 out of 7 rats, deciduoma response was obtained, although the size of deciduomata was considerably smaller. In rats receiving a single injection of reserpine on the day of the AD without further treatment (group 11 ), small uterine swellings at the site of traumatization were observed in 6 out of 9 rats, but histological examinations of these uterine tissues showed that deciduomata were highly degenerative in 3 out of 6 rats. DISCUSSION
The finding that the PBLD failed to inhibit the induction of PSP in the present study indicates that fiber connections with the MBH interrupted by the PBLD were not directly involved in reserpine-induced PSP. This is consistent with a previous report that a similar PBLD did not prevent the induction of PSP by vaginal-cervical stimulation1. Since i t has been reported that the transection of the POA-hypothalam ic connections inhibited establishment of PSP following vaginal-cervical stimulation1,~, the sensory pathways related to the induction of PSP seem to ascend the brain stem dorsal or lateral to the MBH and converge on the POA before giving the information to the MBH. This conjecture is well supported by electrophysiological evidence indicating that impulses of the cervical origin can be recognized in the anterior, lateral or dorsal hypothalamus, POA and thalamus 3,4,12,~5,1~. In the present study as previously reported by Carrer and Taleisnik5, the interruption of POA-hypothalamic connections (AD) also interfered with reserpine-induced PSP; however, it is still questionable whether these sensory neural substrates are involved in 'pharmacological' induction of PSP. It has been suggested that the MBH is a possible focus for reserpine action to cause prolactin release, based on the results of implantation experiments of reserpine crystal into the brain11,17. The results shown in Table I might be interpreted to mean that the preoptic projection to the MBH is primarily involved in initiating PSP
PREOPTIC AREAAND PROLACT1NSECRETION
57
in response to reserpine. However, it is also probable that the AD interfered with PSP after it had been established. This is at least true for the results presented in Table I I, where the AD effectively interrupted the PSP which had already been initiated. Since administration of prolactin was successful in reversing the inhibitory effect of the AD on PSP, anterior inputs removed by the AD seem to play an important role in supporting prolactin release during PSP. A similar situation has been reported for the interruption of pregnancy in rats receiving the POA-hypothalamic transection 2. Furthermore, the preoptic roof section which cuts dorsal afferents to the POA, did not interfere with maintenance of PSP in the present study. This suggests that the neural influence supporting prolactin release during PSP seems to originate from the POA itself or some structures anterior to the POA. Supplemental treatment of the AD rats with reserpine could maintain the PSP, although sizes of deciduomata and weights of traumatized uterine horns were markedly reduced, compared with control groups. This indicates that a neural structure capable of responding to reserpine may locate behind the surgical cut, presumably in the MBH, as suggested by Kanematsu and Sawyer11 and Van Maanen and Smelik17. However, it seems unlikely that this structure can independently permit secretion of a sufficient amount of prolactin required for the maintenance of PSP, because histological examinations showed that deciduomata were degenerative in the AD rats receiving a single injection of reserpine on the day of the operation alone. To support prolonged release of prolactin, either successive injections of reserpine or neural influence from the POA appeared to be necessary in our rats. Recently a nocturnal prolactin surge occurring each night during PSP has been reported by Freeman and Neill8. Since the levels of prolactin in the pituitary and blood were not measured in the present study, it is not clear whether the situation of 'pharmacologically' induced PSP is the same with the PSP induced by vaginal-cervical stimulation in which the stimuli are thought to be memorized in the CNS 8. It is highly possible that the AD interfered with the mechanisms for a nocturnal surge and thereby inhibited the onset or maintenance of PSP in our rats. In this connection, it is of interest to note the findings of Neil114 that the surge of prolactin secretion following estrogen treatment was inhibited by a similar AD. However, it still remains to be solved whether anterior inputs inhibit hypothalamic PIF activity or increase hypothalamic PRF activity under these conditions. ACKNOWLEDGEMENTS The authors wish to express their thanks to Mrs. K. Serisawa for her excellent assistance. We are also grateful to Dr. R. W. Bates, NIAMD, Bethesda, U.S.A., for supplying prolactin preparations. This study was supported by grants from the Ministry of Education of Japan.
REFERENCES 1 ARAI, Y., Effectof hypothalamic de-afferentationon inductionof pseudopregnancyby vaginalcervical stimulationin the rat, J. Reprod. Fertil., 19 (1969) 573-575.
58
Y. ARAI AND K. YAMANOUCHI
2 ARAI, Y., Possible participation of central nervous system (CNS) in blastocyst implantation in the rat, Brain Research, 66 (1974) 393-400. 3 BARRACLOUGH,C. A., AND SAWYER,C. H., Induction of pseudopregnancy in the rat by reserpine and chlorpromazine, Endocrinology, 65 (1959) 563-571. 4 BARRACLOUGH,C. A., ANn CROSS, B A., Unit activity in the hypothalamus of the cyclic female rat: effect of genital stimuli and progesterone, J. Endocrinol., 26 (1963) 339-359. 5 CARRER, H. F., AND TALEISNIK,S., Induction and maintenance of pseudopregnancy after interruption of preoptic hypothalamic connections, Endocrinology, 86 (1970) 231-236. 6 EVERETT, J.W., Central neural control of reproductive functions of the adenohypophysis, Physiol. Rev., 44 (1954) 373-431. 7 EVERETT, J. W., AND QU~NN, D. L., Differential hypothalamic mechanisms inciting ovulation and pseudopregnancy, Endocrinology, 78 (1966) 141-150. 8 FREEMAN,M. E., AND NEILL, J. D., The pattern of prolactin secretion during pseudopregnancy in the rat: a daily nocturnal surge, Endocr&ology, 90 (1972) 1292-1294. 9 GOLD, E. M., AND GANONG, W. F., Effects of drugs on neuroendocrine processes. In L. MARTIM AND W. F. GANONG(Eds.), Neuroendocrinology 11, Academic Press, New York, 1967, pp. 377-437. 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 KANEMATSU, S., AND SAWYER, C . H . , Effects of intrahypothalamic implants of reserpine on lactation and pituitary content of prolactin in the rabbit, Proc. Soc. exp. Biol. (N. Y.), 113 (1963) 967-969. 12 KAWAKAMI, M., AND SAITO, H., Unit activity in the hypothalamus of the cat: effect of genital stimuli, luteinizing hormone and oxytocin, Jap. J. Physiol., 17 (1967) 466-486. 13 MEITES,J., Pharmacological control of prolactin secretion and lactation. In R. GUILLEMIN(Ed.), Proc. First Int. Pharmacol. Meet. I, Pergamon Press, Oxford, 1962, pp. 151-181. 14 NEILL, J. D., Sexual differences in the hypothalamic regulation of prolactin secretion, Endocrinology, 90 (1972) 1154-1159. 15 PORTER, R. W., CAVANOUGH,E. B., CRITCHLOW, B. V., AND SAWYER, C. H., Localized changes in electrical activity of the hypothalamus in estrous cats following vaginal stimulation, Amer. J. Physiol., 189 (1957) 145-151. 16 RAMIREZ,V. D., KOMISARUK,B. R., WH1TMOYER,O. I., AND SAWYER,C. H., Effects of hormones and vaginal stimulation on the EEG and hypothalamic units in rats, Amer. J. Physiol., 212 (1967) 1376-1384. 17 VAN MAANEN, J. H., AND SMELIK, P. G., Induction of pseudopregnancy in rats following local depletion of monoamines in the median eminence of the hypothalamus, Neuroendocrinology, 3 (1968) 177-186.