Brain &search
Bullerin, Vol. 2, pp. 307-312,
All rights of reproduction
1971. Copyright 0 ANKHO ~nt~rnationa1 Inc. in any form reserved. Printed in the U.S.A.
Modifications in Gonadotropin Control and Reproductive Behavior in the Female Rat by Hypothalamic and Preoptic Lesions’ DWIGHT M. NANCE, LARRY W. CHRISTENSEN, JAMES E. SHRYNE AND ROGER A. GORSKI
Department of Anatomy and Brain Research Institute, UCLA School ofMedicine, Los Angeles, CA 90024 (Received 3 I December 1976) NANCE, D. M., L. W. CHRISTENSEN, J. E. SHRYNE AND R. A. GORSKI. Modifications in gonadotropin controland reproductive behavior in the female rat by hypothalamic and preoptic lesions. BRAIN RES. BULL. 2(4) 307-312, 19’77. - Effects of smatl hypothalamic and preoptic area lesions on vaginal cyclicity, ovulation and ovarian weights of female rats were examined. Animals were then gonadectom~ed and tested for lordosis behavior following injections of estrogen alone and estrogen plus progesterone. Male sex behavior was also measured during daily treatment with testosterone. Relative to sham operated rats, lesions in the dorsal preoptic area produced a significant increase in lordosis behavior, virtual elimination of male sex behavior, and only marginal effects on ovarian function. Animals with lesions in the ventral preoptic area showed constant vaginal cornification, lack of ovulation and si~ifi~~y smaller ovaries than the other groups. These rats also tended to show more female sex behavior and less maie sex behavior than sham operated rats. Animals with lesions in the anterior hypothalamus and dorsomedial hypothalamus showed normal ovarian function and levels of female and male sex behavior comparable to the ventral preoptic lesioned rats. Animals with Iesions in the ventromedi~ hypothalamus tended to show lower levels of lordosis behavior than sham animals, but displayed a dramatic and significant increase in male copulatory behavior relative to the other groups. These data indicate a clear dissociation between the neural control of cyclic gonadotropin activity and sex specific reproductive behavior. Vaginal cyclicity Sexual behavior
Ovulation
Preoptic area
Ventromedial
AS A RESULT of the presence or absence of androgens during the neonatal critical period, a female cyclic pattern of gonadotropin regulation is generally associated with a potential to dipslay high levels of lordotic behavior, whereas a tonic, male pattern of gonadotropin control is frequently associated with a heightened capacity to display male copulatory behavior [I I]. Yet, these correlated phenomena may be mediated by relatively separate and distinct neural systems. Administration of different doses of systemic or intracranial testosterone at different ages during early development can produce various degrees of dissociation in these reproductive processes [ 131, data which strongly support the notion of differential neural control of reproductive function. The present experiment, which examines the effects of small brain lesions on ovulation, lordotic behavior, and male sex behavior, further supports the hypothesis that sexually dimorphic patterns of reproduction in the rat are mediated by relatively discrete neural systems f I 11.
were maintained in groups of five to eight per cage with ad lib access to Purina rat chow and water in a reverse light room. Lights were on from 1O:OO p.m. to 1 I:00 a.m. Vaginal smears were collected daily for 17 days, and animals showing three regular 4-5 day estrous cycles were selected for the experiment. Animals were then randomly divided into lesion and sham operated groups. Small bilateral electrolytic lesions were made in the dorsal preoptic area (D-POA), ventral preoptic area (V-POA), anterior hypothalamus (A-HYP), or ventromedial hypothalamus (VMH) by passing anodal current (1 mA/ 10 set) through a 00 stainless steel insect pin, insulated except for 0.5 mm at the tip. Sham operated rats, included with each lesion group, were treated the same as the lesion animals, except that no current was passed through the electrode. With the skull level between the incisors and intraaural canal, modified stereotaxic coordinates from de Groot 181 for the various lesions were as follows: D-POA, A-P = bregma, lateral = 0.35 mm and 6.6 mm below the dura; V-PO& A-P = bregma, lateral = 0.3 mm and 7.4 mm below the dura; A-HYP, A-P = 1.O mm posterior to bregma, lateral - 0.5 mm and 6.8 mm below the dura; and VMH, A-F = 2.2 mm posterior to bregma, lateral
METHOr> Sixty-day-old
female
rats
(Simonsten
hypothalamus
Sprague-Dawiey)
’ Research supported by USPHS Grants HD-01182, AM-I 8254 and the Ford Foundation. 307
NANCE ET AL.
308
= 0.5 mm and 8.0 mm below the dura. Sodium methohexital (Brevital) was used as anesthesia. Beginning three weeks after surgery, vaginal cycles were monitored for 2-6 weeks in order to assess the effects of the lesions on vaginal cyclicity. Using ether anesthesia, ovulation was determined by visually examining the ovary on the morning of estrus following a regular 4-5 day estrous cycle for the presence of dilated ampullae. If the ampullae were dilated, the ovaries were removed, the oviduct dissected out, and the number of ova counted under a microscope. Ovarian weights were also recorded. Behavior tests were begun 3-8 weeks after ovariectomy. Female sexual behavior was tested initially on the fourth day following three daiiy injections of 0.5 fig estradiol benzoate (EB). Behavior tests were begun at 1:00 p.m. and consisted of placing the test animals in a Plexiglas arena with 2-3 sexually vigorous Long-Evans male rats. Following 20 mounts, the animals were removed and a lordosis quotient (LQ) computed for each animal by dividing the number of lordotic responses by the number of mounts [ 201 and multiplying by 100. Immediately following the behavior test. animals were injected with 0.S mg progesterone and a second test for female sexual behavior conducted 4-6 hr later (15 mount test). Four to eight weeks following the female behavior tests, all animals were injected daily with 150 pg testosterone propionate (TP) and tested for masculine sexual behavior on injection Days 5, 9, 13, and 17. Tests for masculine sexual behavior were 1.5 min in length. Test animals were adapted to the test arena for 3 min prior to the introduction of a stimulus female (EB-progesterone primed spayed rat), and the stimulus females were changed halfway through the 15 min test. On each of the four tests the following measures were made: mount frequency (MF), total number of mounts in the 15 min test; mount latency (ML), latency to the first mount; intromission frequency (IF), total number of intromission patterns exhibited in the entire test; and intromission latency (IL), time from the start of test until the occurrence of the first intromission pattern. After completion of all tests, the animals were killed, perfused with 10% Formatin and their brains removed for histological examination.
damage in the more ventral aspect of the VMN-arcuate complex, with most of the damage in the posterior half of the VMN. A second group of animals had comparable lesions in the same coronal plane, but differed from the VMH lesion group in that the lesions were 0.5-1.0 mm more dorsal, and included the dorsomedial hypothalamus (DMN) and the most dorsal aspect of the VMN. The data from the experimental animals were then grouped and analyzed by t-test and analysis of variance [34] in these five lesioned and one sham operated groups. Results referred to as significant were at a probability of 0.05 or less. Representative microphotographs for the five lesion sites are shown in Fig. I. Ovarian Function Results of the various lesions on ovarian function are summarized in Table 1. Ovulation was completely disrupted by the V-POA lesions, and S/9 rats showed an absence of corpora lutea and all displayed constant vaginal cornification. The small ovarian weights reflect the ste’lity induced by V-POA lesions (Table I ). Although 3/9 the D-POA lesioned animals showed abberrant estrous cycles and a failure to ovulate, these three animals had the most ventrally placed preoptic lesions in the D-POA group. However, all animals in the D-POA group had corpora lutea. The remaining six D-POA animals that did ovulate showed normal 4-5 day estrous cycles and ovulated a normal complement of ova. Although ovarian weights of the D-POA lesioned animals were significantly lower than the sham animals, their ovaries were significantly heavier than the V-POA animals. The other three lesion groups, A-HYP, VMH and DMH, all showed regular estrous cycles and full ovulation. Ovarian weights of the A-HYP animais were not significantly different from the sham operated rats (Table I), but the VMH and DMH groups tended to have smaller ovaries than the sham group. TABLE EFFECTS
1
OF PREOPTIC AND HYPOTHALAMIC LESIONS OVULATION AND OVARIAN WEIGHT
-._____
~. 9%
RESULTS
Group
N
Since the sham operated animals included with each lesion group were comparable in terms of both ovarian function and sexual behavior, data for these animals were pooled. A total of five separate lesion groups were derived from histological examination of the brains. D-POA lesions were located at the coronal level of the suprachiasmatic nucleus (SCN) and consisted of bilateral lesions in the dorsai 1/Z to Z/3 of the preoptic area, ventral and generally just posterior to the midline junction of the anterior commissure. V-POA lesions were in the same A-P plane as the D-POA lesions, but more ventrically located. These lesions generally damaged or destroyed the SCN and the ventral third of the preoptic area. A-HYP lesions tended to be located in the more dorsal and posterior aspect of the anterior hypothalamus at a coronal level, generally anterior or continuous with the anterior pole of the ventromedial nucleus (VMN) of the hypothalamus. Animals in the VMH lesion group fell into two distinct groups based upon histological differences. VMH lesioned animals had bilateral
SHAMS D-POA V-POA A-HYP VMH DMH
20 9 9 6 5 6
Ovulation
--
Mean No. Ova/ Ovulation _-~ -
95 67 0
8.6 + 0.5 8.3 + 1.3 0
100
8.0 rl: 1.5
100 100
8.6 ir 0.9 9.8 5 0.7
ON
Mean Ovarian Weight (mg)
80.1 68.0 44.2 89.1
tI + ir
2.8 2.6 4.5
4.2 69.6 t 5.2 68.1 I 3.6
-
I_--
Female Sexual Behavior Effects of the brain lesions on lordosis behavior are plotted in Fig. 2. Relative to sham animals, D-POA rats showed a significantly higher mean LQ when tested after priming with EB alone. Although there was a tendency for V-POA and A-HYP lesioned rats to be behaviorally more responsive to EB than sham animals, this effect was not statistically reliable. Likewise, the decreased LQ in response
BRAIN
FIG.
LESIONS
AND REPRODUCTIVE
1. Representative
FUNCTION
microphotographs of lesions (D), dorsomedial
hypothalamus
in the dorsal preoptic area (A and B), ventral preoptic hypothalamus (E) and ventromedial h~pothal~us (F).
to EB only produced by VMH lesions, relative to control rats, was not statistically significant. All groups showed a significant increase in mean LQ following progesterone with the D-POA and A-HYP lesioned administration, animals showing a significantly higher level of lordosis behavior than the sham operated rats. Although the VMH lesioned rats showed a lower mean LQ than did the sham animals following progesterone administration, this difference was not significant.
Effects
of the brain
lesions
on TM and ML during
the
area (C), anterior
four behavior tests are plotted in Figs. 3 and 4, respectively. Across all four tests, VMH lesioned rats showed significantly more mounts than all other groups (Fig. 3). Both the D-POA and DMH lesioned rats showed significantly fewer total mounts than did sham operated rats. A-HYP and V-POA lesioned animals were comparable to sham animals, whereas the V-POA, A-HYP and DMH lesioned rats were not significantly different from the D-POA lesioned animals. in terms of latency to mount (Fig. 4), VMH lesioned rats showed a significantly shorter ML than all other groups. D-POA lesioned animals showed a significantly greater ML than did sham animals. There was a tendency for the
310
100.
NANCE ET AL.
n
SHAM
a
O-WA
q
V-WA
g
A-HYP
0
VYH
6
1
t 0.5
ALONE
m6 PROGESTERONE
0.5,1&y EB X 3 DAYS
DAYS- 5 FIG. 2. Mean lordosis quotient (t Standard Error) of sham operated, dorsal preoptic lesioned (D-POA), ventral preoptic lesioned (V-POA), anterior hypothalamus lesioned (A-HYP), ventromedial hypothalamus lesioned (VMH) and dorsomedial hypothalamus Iesioned (DMH) female rats. AII animals were tested following three daily injections of OS pg estradiol benzoate (EB)/day (EB alone) and retested four to six hr later following 0.5 mg progesterone. Numbers at the base of the bar graphs the number of animals per group.
SHAM(19)
h +
D-POA (71
I---
VMH (5)
c-
A-HYP(6)
x-
VPOA
o----
DMH (6)
I
X-w,
--w_
refer to
1’
DAYS-
5
:
I3
9 150 ~9
TPI
I7
FIG. 4. Mean latency to first mount shown by sham, preoptic, and lesioned rats (same groups as described in Fig. 1) tested during daily injections of 150 pg testosterone propionate (TP)/day. Animals were tested on injection Days 5, 9, 13 and 17. Number of animals/group is shown in parentheses.
hypothalamic
DISCUSSION
1’
I
I3
J(
1’
i
9 TP/ 5A’f
V-POA lesioned rats to show shorter ML than D-POA animals, although this difference was not significant. Mount latencies for DMH, A-HYP and V-POA lesioned groups were intermediate between sham and D-POA but not reliably different from either group. There were no statistically significant group differences in terms of the IF and IL data, which are not illustrated.
: (6)
l50r0
17
DAY
FIG. 3. Mean total number of mounts shown by sham, preoptic and hypothalamic lesioned rats (same groups as described in Fig. l), tested during daily injections of 150 pg testosterone propionate (TP)/day. Animals were tested on injection Days 5, 9, 13 and 17. Number of animals/group is shown in parentheses.
The present data indicate a clear dissociation between the neural control of cyclic gonadotropin activity and sex specific male and female reproductive behavior in that each one can be modified by a specific brain lesion independent of the other parameters of reproductive function. Consistent with prior experiments, the ventral aspect of the preoptic area appears critically related to the maintenance of ovarian cycles and ovulation in the rat (2,4] in that lesions in this ventral region produce an acyclic pattern of gonadotropin release. As shown by Barraclough, et al. [ 21, and demonstrated in this experiment, lesions in the dorsal aspect of the medial preoptic area do not disrupt ovarian function. Lesions which tend to overlap these dorsal and ventral aspects of the preoptic area appear to produce mixed effects, as shown by the three animals mentioned earlier in the D-POA lesioned group. However, small lesions in the A-HYP. VMH and DMH have minimal effects on ovarian function. Normal estrous cycles following VMH lesions have been observed by others [ 20,2 11, The marked increase in lordosis behavior following estrogen priming produced by D-POA lesions is consistent with an earlier report by Powers and Valenstein 1301, and is strikingly similar to the effect of septal lesions on lordosis behavior of female rats [ 261 The slight increase in lordosis behavior found with A-HYP lesions is somewhat surprising, considering reports that A-HYP lesions may actually block
BRAIN
LESIONS
AND REPRODUCTIVE
311
FUNCTION
or attenuate female sex behavior [ 17,3 11. Besides possible differences in lesion sites, differences in the lesion size may critically determine subsequent responsiveness to hormones [25]. This latter point will be considered below. The small decrement in female sexual behavior reported here for VMH lesions has also been noted by others [ 20,211. The virtual elimination of male copulatory behavior following preoptic lesions (Figs. 3 and 4) is consistent with numerous studies suggesting a critical role for this brain area in the mediation of male sex behavior [ 10,141. The present data further suggest that lesions in the dorsal aspects of the preoptic area are more effective for attenuating male sexual behavior than lesions in the V-POA. The dramatic effect of small VMH lesions on male sex behavior was quite unexpected. Although D6rner, et al. [ 91 reported increased male sexual behavior following lesions in the central hypothalamus, their methods of behavioral assessment are subject to criticism [ 111 and difficult to interpret. However, the present results, based upon a more standardized index of male sexual behavior, provides positive support for their conclusions. Also, a degree of localization is suggested in the present experiment. Lesions placed 0.5-t .O mm more dorsal in the medial hypothalamus do not potentiate male sexual behavior, but rather may actually produce a slight decrement in male copulatory behavior. Thus, the neural control of gonadotropins, iordotic behavior and male copulatory behavior appear to be mediated by relatively discrete neural systems with a considerable degree of independence. A relatively consistent body of literature suggests a primary involvement of the preoptic area in male sex behavior. Stimulation of the preoptic area can potentiate and even induce male sex behavior. [ 18,22], whereas lesions in this brain area attenuate male sexual behavior ([ 10,141 ; and Fig. 3). Sex steroids implanted in the preoptic area of adult rats can induce male copulatory behavior [ 6,7], and finally, steroid implants into the preoptic area of neonatal rats potentiates the capacity to display male sex behavior when the animals are primed with hormones in adulthood [ 131. To the extent that the preoptic area is a focal point for the regulation of male sex behavior, the medial forebrain bundle (MFB) appears to comprise at least one essential pathway for its expression 15, 15, 191, and may, in fact, exert some inhibitory control over lordotic behavior [ 23 ] The ability of VMH lesions to potentiate male sex behavior is also compatible with the probable involvement of the MFB in male sex behavior. As shown for self-stimulation [ 161, for example, the VMH appears to exert a tonic restraint on the more lateral aspects of the hypothalamus. Thus, increases in male sex behavior after a VMH lesion may represent a release of lateral hypothalamic influence, via the preoptic area, on male sex behavior. Although the present results of preoptic area lesions on female sexual behavior suggest that this brain region may exert inhibitory control over lordotic behavior, such a conclusion appears premature considering major inconsistencies in the literature. For example, lesions in the preoptic-anterior hypothalamic area of female rats are reported to attenuate female sex behavior [ 17,3 I ] , whereas both inhibitor 124,291 and facilitatory (Negoro and Gorski, unpublished data) effects of electrical stimulation in the preoptic area have been observed on lordotic behavior. Also, mixed behavioral effects have been reported
following direct implants of estrogen into the preoptic region, in that increases in running behavior have been reported without any changes in lordosis behavior [33]. Yet others have found that the preoptic area is a highly effective implant site for the induction of female sex behavior by hormones [ 121. Also, implants of protein synthesis inhibitors into the preoptic area as well as the VMH can block the priming effect of systemic estrogen on lordosis behavior [ 111. Differential behavioral effects have also been reported for estrogen implants in the VMH [ 1,331. Thus, the regulatory role of the preoptic area in the mediation of female sex behavior cannot be characterized clearly at the present time. It is possible that differences in animal strain, testing procedures, location of implantation or lesion sites, etc. may eventually account for these major inconsistencies, but if we contrast these studies concerned with female sex behavior with the more orderly and convergent results based upon different strains and reported from different laboratories for the neural control of male sex behavior, these possible explanations seem less hopeful. These divergent experimental results concerning the neural control of lordotic behavior may not be resolved without some modification in experimental approach or interpretation. As an initial approach to this latter possibility, the actual size of various brain lesions in relation to subsequent behavioral responsiveness to estrogen may be considered a critical variable. In the case of feeding behavior, another hormone responsive system, complete destruction of the VMH-arcuate region can attenuate the depressive effects of estrogen on food intake and body weight, whereas unilateral or incomplete lesions in this region not only leave the response to estrogen intact, but may even produce a heightened responsiveness to estrogen ([ZS] and unpublished data). The potential importance of lesion size on the attenuation of the effects of estrogen on feeding and body weight has also been noted by Wade [ 321 and Beatty, et al. [3]. It has been hypothesized that incomplete destruction of estrogen sensitive neurons in the VMH-arcuate area may result in a type of denervation hypersensitivity in the remaining estrogen sensitive neurons [25] _ A similar hypothesis has also been proposed to account for the marked increase in lordosis behavior induced by estrogen following septal lesions in female rats
[281,
Thus, the failure to see decrements in lordotic behavior reported here for preoptic and A-HYP lesions may reflect the relatively small lesions compared to those produced by Singer [ 3 I ] and Herndon and Neil1 [ 171. Although Powers and Valenstein [ 301, who reported (as found in the present experiment) an increase in estrogen sensitivity with preoptic lesions, did not indicate the size or extent of their lesions, it seems likely that their use of a platinum electrode for making lesions would have resulted in relatively small lesions. Thus, the effects of preoptic-A-HYP lesions on lordosis behavior may be partially clarified by a parametric analysis of various size lesions on subsequent lordotic behavior. However, an additional and perhaps important variable is pointed out by the work of La Vaque and Rodgers [ 211. Whereas they noted, as had Kennedy and Mitra 1201, a marked decrement in mating behavior in female rats following VMH lesions, La Vaque and Rodgers observed a gradual recovery of lordotic behavior to control levels when the animals were tested across several sessions. Whether this reflects an effect of repeated testing or a time dependent change in the effects of a brain lesion on
312
NANCE /DAL.
behavior focus
behavioral had
has yet
to be determined. Although beyond the experiment, it is possible that different would have been observed if the animals
of the present been
effects tested
in closer
proximity
to the time
ACKNOWLEDGEMENT
The authors gratefully Mrs. Erna Freiberg.
acknowledge
the histological
assistance
of
of lesioning.
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2.
3.
Barfield, R. .I., J. J. Chen and P. G. McDonald. Activation of estrous behavior by intracerebral implants of estradiol benzoate (EB) in ovariectomized rats. Eastern Conference on Reproductive Behavior, Nags Head, NC, 1975 (Abstr.). Barraclough, C. A., S. Yrarrazaval and R. Hatton. A possible hypothalamic site of action of progesterone in the facilitation of ovulation in the rat. Endocrinology 75: 838-845. 1964. Beatty, W. W., D. A. O’Briant and T. R. Vilberg. Effects of ovariectomy a.nd estradiol injections on food intake and body weight in rats with ventromedial hypothalamic lesions.
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Bullerin, Vol. 2, pp. 307-312,
All rights of reproduction
1971. Copyright 0 ANKHO ~nt~rnationa1 Inc. in any form reserved. Printed in the U.S.A.
Modifications in Gonadotropin Control and Reproductive Behavior in the Female Rat by Hypothalamic and Preoptic Lesions’ DWIGHT M. NANCE, LARRY W. CHRISTENSEN, JAMES E. SHRYNE AND ROGER A. GORSKI
Department of Anatomy and Brain Research Institute, UCLA School ofMedicine, Los Angeles, CA 90024 (Received 3 I December 1976) NANCE, D. M., L. W. CHRISTENSEN, J. E. SHRYNE AND R. A. GORSKI. Modifications in gonadotropin controland reproductive behavior in the female rat by hypothalamic and preoptic lesions. BRAIN RES. BULL. 2(4) 307-312, 19’77. - Effects of smatl hypothalamic and preoptic area lesions on vaginal cyclicity, ovulation and ovarian weights of female rats were examined. Animals were then gonadectom~ed and tested for lordosis behavior following injections of estrogen alone and estrogen plus progesterone. Male sex behavior was also measured during daily treatment with testosterone. Relative to sham operated rats, lesions in the dorsal preoptic area produced a significant increase in lordosis behavior, virtual elimination of male sex behavior, and only marginal effects on ovarian function. Animals with lesions in the ventral preoptic area showed constant vaginal cornification, lack of ovulation and si~ifi~~y smaller ovaries than the other groups. These rats also tended to show more female sex behavior and less maie sex behavior than sham operated rats. Animals with lesions in the anterior hypothalamus and dorsomedial hypothalamus showed normal ovarian function and levels of female and male sex behavior comparable to the ventral preoptic lesioned rats. Animals with Iesions in the ventromedi~ hypothalamus tended to show lower levels of lordosis behavior than sham animals, but displayed a dramatic and significant increase in male copulatory behavior relative to the other groups. These data indicate a clear dissociation between the neural control of cyclic gonadotropin activity and sex specific reproductive behavior. Vaginal cyclicity Sexual behavior
Ovulation
Preoptic area
Ventromedial
AS A RESULT of the presence or absence of androgens during the neonatal critical period, a female cyclic pattern of gonadotropin regulation is generally associated with a potential to dipslay high levels of lordotic behavior, whereas a tonic, male pattern of gonadotropin control is frequently associated with a heightened capacity to display male copulatory behavior [I I]. Yet, these correlated phenomena may be mediated by relatively separate and distinct neural systems. Administration of different doses of systemic or intracranial testosterone at different ages during early development can produce various degrees of dissociation in these reproductive processes [ 131, data which strongly support the notion of differential neural control of reproductive function. The present experiment, which examines the effects of small brain lesions on ovulation, lordotic behavior, and male sex behavior, further supports the hypothesis that sexually dimorphic patterns of reproduction in the rat are mediated by relatively discrete neural systems f I 11.
were maintained in groups of five to eight per cage with ad lib access to Purina rat chow and water in a reverse light room. Lights were on from 1O:OO p.m. to 1 I:00 a.m. Vaginal smears were collected daily for 17 days, and animals showing three regular 4-5 day estrous cycles were selected for the experiment. Animals were then randomly divided into lesion and sham operated groups. Small bilateral electrolytic lesions were made in the dorsal preoptic area (D-POA), ventral preoptic area (V-POA), anterior hypothalamus (A-HYP), or ventromedial hypothalamus (VMH) by passing anodal current (1 mA/ 10 set) through a 00 stainless steel insect pin, insulated except for 0.5 mm at the tip. Sham operated rats, included with each lesion group, were treated the same as the lesion animals, except that no current was passed through the electrode. With the skull level between the incisors and intraaural canal, modified stereotaxic coordinates from de Groot 181 for the various lesions were as follows: D-POA, A-P = bregma, lateral = 0.35 mm and 6.6 mm below the dura; V-PO& A-P = bregma, lateral = 0.3 mm and 7.4 mm below the dura; A-HYP, A-P = 1.O mm posterior to bregma, lateral - 0.5 mm and 6.8 mm below the dura; and VMH, A-F = 2.2 mm posterior to bregma, lateral
METHOr> Sixty-day-old
female
rats
(Simonsten
hypothalamus
Sprague-Dawiey)
’ Research supported by USPHS Grants HD-01182, AM-I 8254 and the Ford Foundation. 307
NANCE ET AL.
308
= 0.5 mm and 8.0 mm below the dura. Sodium methohexital (Brevital) was used as anesthesia. Beginning three weeks after surgery, vaginal cycles were monitored for 2-6 weeks in order to assess the effects of the lesions on vaginal cyclicity. Using ether anesthesia, ovulation was determined by visually examining the ovary on the morning of estrus following a regular 4-5 day estrous cycle for the presence of dilated ampullae. If the ampullae were dilated, the ovaries were removed, the oviduct dissected out, and the number of ova counted under a microscope. Ovarian weights were also recorded. Behavior tests were begun 3-8 weeks after ovariectomy. Female sexual behavior was tested initially on the fourth day following three daiiy injections of 0.5 fig estradiol benzoate (EB). Behavior tests were begun at 1:00 p.m. and consisted of placing the test animals in a Plexiglas arena with 2-3 sexually vigorous Long-Evans male rats. Following 20 mounts, the animals were removed and a lordosis quotient (LQ) computed for each animal by dividing the number of lordotic responses by the number of mounts [ 201 and multiplying by 100. Immediately following the behavior test. animals were injected with 0.S mg progesterone and a second test for female sexual behavior conducted 4-6 hr later (15 mount test). Four to eight weeks following the female behavior tests, all animals were injected daily with 150 pg testosterone propionate (TP) and tested for masculine sexual behavior on injection Days 5, 9, 13, and 17. Tests for masculine sexual behavior were 1.5 min in length. Test animals were adapted to the test arena for 3 min prior to the introduction of a stimulus female (EB-progesterone primed spayed rat), and the stimulus females were changed halfway through the 15 min test. On each of the four tests the following measures were made: mount frequency (MF), total number of mounts in the 15 min test; mount latency (ML), latency to the first mount; intromission frequency (IF), total number of intromission patterns exhibited in the entire test; and intromission latency (IL), time from the start of test until the occurrence of the first intromission pattern. After completion of all tests, the animals were killed, perfused with 10% Formatin and their brains removed for histological examination.
damage in the more ventral aspect of the VMN-arcuate complex, with most of the damage in the posterior half of the VMN. A second group of animals had comparable lesions in the same coronal plane, but differed from the VMH lesion group in that the lesions were 0.5-1.0 mm more dorsal, and included the dorsomedial hypothalamus (DMN) and the most dorsal aspect of the VMN. The data from the experimental animals were then grouped and analyzed by t-test and analysis of variance [34] in these five lesioned and one sham operated groups. Results referred to as significant were at a probability of 0.05 or less. Representative microphotographs for the five lesion sites are shown in Fig. I. Ovarian Function Results of the various lesions on ovarian function are summarized in Table 1. Ovulation was completely disrupted by the V-POA lesions, and S/9 rats showed an absence of corpora lutea and all displayed constant vaginal cornification. The small ovarian weights reflect the ste’lity induced by V-POA lesions (Table I ). Although 3/9 the D-POA lesioned animals showed abberrant estrous cycles and a failure to ovulate, these three animals had the most ventrally placed preoptic lesions in the D-POA group. However, all animals in the D-POA group had corpora lutea. The remaining six D-POA animals that did ovulate showed normal 4-5 day estrous cycles and ovulated a normal complement of ova. Although ovarian weights of the D-POA lesioned animals were significantly lower than the sham animals, their ovaries were significantly heavier than the V-POA animals. The other three lesion groups, A-HYP, VMH and DMH, all showed regular estrous cycles and full ovulation. Ovarian weights of the A-HYP animais were not significantly different from the sham operated rats (Table I), but the VMH and DMH groups tended to have smaller ovaries than the sham group. TABLE EFFECTS
1
OF PREOPTIC AND HYPOTHALAMIC LESIONS OVULATION AND OVARIAN WEIGHT
-._____
~. 9%
RESULTS
Group
N
Since the sham operated animals included with each lesion group were comparable in terms of both ovarian function and sexual behavior, data for these animals were pooled. A total of five separate lesion groups were derived from histological examination of the brains. D-POA lesions were located at the coronal level of the suprachiasmatic nucleus (SCN) and consisted of bilateral lesions in the dorsai 1/Z to Z/3 of the preoptic area, ventral and generally just posterior to the midline junction of the anterior commissure. V-POA lesions were in the same A-P plane as the D-POA lesions, but more ventrically located. These lesions generally damaged or destroyed the SCN and the ventral third of the preoptic area. A-HYP lesions tended to be located in the more dorsal and posterior aspect of the anterior hypothalamus at a coronal level, generally anterior or continuous with the anterior pole of the ventromedial nucleus (VMN) of the hypothalamus. Animals in the VMH lesion group fell into two distinct groups based upon histological differences. VMH lesioned animals had bilateral
SHAMS D-POA V-POA A-HYP VMH DMH
20 9 9 6 5 6
Ovulation
--
Mean No. Ova/ Ovulation _-~ -
95 67 0
8.6 + 0.5 8.3 + 1.3 0
100
8.0 rl: 1.5
100 100
8.6 ir 0.9 9.8 5 0.7
ON
Mean Ovarian Weight (mg)
80.1 68.0 44.2 89.1
tI + ir
2.8 2.6 4.5
4.2 69.6 t 5.2 68.1 I 3.6
-
I_--
Female Sexual Behavior Effects of the brain lesions on lordosis behavior are plotted in Fig. 2. Relative to sham animals, D-POA rats showed a significantly higher mean LQ when tested after priming with EB alone. Although there was a tendency for V-POA and A-HYP lesioned rats to be behaviorally more responsive to EB than sham animals, this effect was not statistically reliable. Likewise, the decreased LQ in response
BRAIN
FIG.
LESIONS
AND REPRODUCTIVE
1. Representative
FUNCTION
microphotographs of lesions (D), dorsomedial
hypothalamus
in the dorsal preoptic area (A and B), ventral preoptic hypothalamus (E) and ventromedial h~pothal~us (F).
to EB only produced by VMH lesions, relative to control rats, was not statistically significant. All groups showed a significant increase in mean LQ following progesterone with the D-POA and A-HYP lesioned administration, animals showing a significantly higher level of lordosis behavior than the sham operated rats. Although the VMH lesioned rats showed a lower mean LQ than did the sham animals following progesterone administration, this difference was not significant.
Effects
of the brain
lesions
on TM and ML during
the
area (C), anterior
four behavior tests are plotted in Figs. 3 and 4, respectively. Across all four tests, VMH lesioned rats showed significantly more mounts than all other groups (Fig. 3). Both the D-POA and DMH lesioned rats showed significantly fewer total mounts than did sham operated rats. A-HYP and V-POA lesioned animals were comparable to sham animals, whereas the V-POA, A-HYP and DMH lesioned rats were not significantly different from the D-POA lesioned animals. in terms of latency to mount (Fig. 4), VMH lesioned rats showed a significantly shorter ML than all other groups. D-POA lesioned animals showed a significantly greater ML than did sham animals. There was a tendency for the
310
100.
NANCE ET AL.
n
SHAM
a
O-WA
q
V-WA
g
A-HYP
0
VYH
6
1
t 0.5
ALONE
m6 PROGESTERONE
0.5,1&y EB X 3 DAYS
DAYS- 5 FIG. 2. Mean lordosis quotient (t Standard Error) of sham operated, dorsal preoptic lesioned (D-POA), ventral preoptic lesioned (V-POA), anterior hypothalamus lesioned (A-HYP), ventromedial hypothalamus lesioned (VMH) and dorsomedial hypothalamus Iesioned (DMH) female rats. AII animals were tested following three daily injections of OS pg estradiol benzoate (EB)/day (EB alone) and retested four to six hr later following 0.5 mg progesterone. Numbers at the base of the bar graphs the number of animals per group.
SHAM(19)
h +
D-POA (71
I---
VMH (5)
c-
A-HYP(6)
x-
VPOA
o----
DMH (6)
I
X-w,
--w_
refer to
1’
DAYS-
5
:
I3
9 150 ~9
TPI
I7
FIG. 4. Mean latency to first mount shown by sham, preoptic, and lesioned rats (same groups as described in Fig. 1) tested during daily injections of 150 pg testosterone propionate (TP)/day. Animals were tested on injection Days 5, 9, 13 and 17. Number of animals/group is shown in parentheses.
hypothalamic
DISCUSSION
1’
I
I3
J(
1’
i
9 TP/ 5A’f
V-POA lesioned rats to show shorter ML than D-POA animals, although this difference was not significant. Mount latencies for DMH, A-HYP and V-POA lesioned groups were intermediate between sham and D-POA but not reliably different from either group. There were no statistically significant group differences in terms of the IF and IL data, which are not illustrated.
: (6)
l50r0
17
DAY
FIG. 3. Mean total number of mounts shown by sham, preoptic and hypothalamic lesioned rats (same groups as described in Fig. l), tested during daily injections of 150 pg testosterone propionate (TP)/day. Animals were tested on injection Days 5, 9, 13 and 17. Number of animals/group is shown in parentheses.
The present data indicate a clear dissociation between the neural control of cyclic gonadotropin activity and sex specific male and female reproductive behavior in that each one can be modified by a specific brain lesion independent of the other parameters of reproductive function. Consistent with prior experiments, the ventral aspect of the preoptic area appears critically related to the maintenance of ovarian cycles and ovulation in the rat (2,4] in that lesions in this ventral region produce an acyclic pattern of gonadotropin release. As shown by Barraclough, et al. [ 21, and demonstrated in this experiment, lesions in the dorsal aspect of the medial preoptic area do not disrupt ovarian function. Lesions which tend to overlap these dorsal and ventral aspects of the preoptic area appear to produce mixed effects, as shown by the three animals mentioned earlier in the D-POA lesioned group. However, small lesions in the A-HYP. VMH and DMH have minimal effects on ovarian function. Normal estrous cycles following VMH lesions have been observed by others [ 20,2 11, The marked increase in lordosis behavior following estrogen priming produced by D-POA lesions is consistent with an earlier report by Powers and Valenstein 1301, and is strikingly similar to the effect of septal lesions on lordosis behavior of female rats [ 261 The slight increase in lordosis behavior found with A-HYP lesions is somewhat surprising, considering reports that A-HYP lesions may actually block
BRAIN
LESIONS
AND REPRODUCTIVE
311
FUNCTION
or attenuate female sex behavior [ 17,3 11. Besides possible differences in lesion sites, differences in the lesion size may critically determine subsequent responsiveness to hormones [25]. This latter point will be considered below. The small decrement in female sexual behavior reported here for VMH lesions has also been noted by others [ 20,211. The virtual elimination of male copulatory behavior following preoptic lesions (Figs. 3 and 4) is consistent with numerous studies suggesting a critical role for this brain area in the mediation of male sex behavior [ 10,141. The present data further suggest that lesions in the dorsal aspects of the preoptic area are more effective for attenuating male sexual behavior than lesions in the V-POA. The dramatic effect of small VMH lesions on male sex behavior was quite unexpected. Although D6rner, et al. [ 91 reported increased male sexual behavior following lesions in the central hypothalamus, their methods of behavioral assessment are subject to criticism [ 111 and difficult to interpret. However, the present results, based upon a more standardized index of male sexual behavior, provides positive support for their conclusions. Also, a degree of localization is suggested in the present experiment. Lesions placed 0.5-t .O mm more dorsal in the medial hypothalamus do not potentiate male sexual behavior, but rather may actually produce a slight decrement in male copulatory behavior. Thus, the neural control of gonadotropins, iordotic behavior and male copulatory behavior appear to be mediated by relatively discrete neural systems with a considerable degree of independence. A relatively consistent body of literature suggests a primary involvement of the preoptic area in male sex behavior. Stimulation of the preoptic area can potentiate and even induce male sex behavior. [ 18,22], whereas lesions in this brain area attenuate male sexual behavior ([ 10,141 ; and Fig. 3). Sex steroids implanted in the preoptic area of adult rats can induce male copulatory behavior [ 6,7], and finally, steroid implants into the preoptic area of neonatal rats potentiates the capacity to display male sex behavior when the animals are primed with hormones in adulthood [ 131. To the extent that the preoptic area is a focal point for the regulation of male sex behavior, the medial forebrain bundle (MFB) appears to comprise at least one essential pathway for its expression 15, 15, 191, and may, in fact, exert some inhibitory control over lordotic behavior [ 23 ] The ability of VMH lesions to potentiate male sex behavior is also compatible with the probable involvement of the MFB in male sex behavior. As shown for self-stimulation [ 161, for example, the VMH appears to exert a tonic restraint on the more lateral aspects of the hypothalamus. Thus, increases in male sex behavior after a VMH lesion may represent a release of lateral hypothalamic influence, via the preoptic area, on male sex behavior. Although the present results of preoptic area lesions on female sexual behavior suggest that this brain region may exert inhibitory control over lordotic behavior, such a conclusion appears premature considering major inconsistencies in the literature. For example, lesions in the preoptic-anterior hypothalamic area of female rats are reported to attenuate female sex behavior [ 17,3 I ] , whereas both inhibitor 124,291 and facilitatory (Negoro and Gorski, unpublished data) effects of electrical stimulation in the preoptic area have been observed on lordotic behavior. Also, mixed behavioral effects have been reported
following direct implants of estrogen into the preoptic region, in that increases in running behavior have been reported without any changes in lordosis behavior [33]. Yet others have found that the preoptic area is a highly effective implant site for the induction of female sex behavior by hormones [ 121. Also, implants of protein synthesis inhibitors into the preoptic area as well as the VMH can block the priming effect of systemic estrogen on lordosis behavior [ 111. Differential behavioral effects have also been reported for estrogen implants in the VMH [ 1,331. Thus, the regulatory role of the preoptic area in the mediation of female sex behavior cannot be characterized clearly at the present time. It is possible that differences in animal strain, testing procedures, location of implantation or lesion sites, etc. may eventually account for these major inconsistencies, but if we contrast these studies concerned with female sex behavior with the more orderly and convergent results based upon different strains and reported from different laboratories for the neural control of male sex behavior, these possible explanations seem less hopeful. These divergent experimental results concerning the neural control of lordotic behavior may not be resolved without some modification in experimental approach or interpretation. As an initial approach to this latter possibility, the actual size of various brain lesions in relation to subsequent behavioral responsiveness to estrogen may be considered a critical variable. In the case of feeding behavior, another hormone responsive system, complete destruction of the VMH-arcuate region can attenuate the depressive effects of estrogen on food intake and body weight, whereas unilateral or incomplete lesions in this region not only leave the response to estrogen intact, but may even produce a heightened responsiveness to estrogen ([ZS] and unpublished data). The potential importance of lesion size on the attenuation of the effects of estrogen on feeding and body weight has also been noted by Wade [ 321 and Beatty, et al. [3]. It has been hypothesized that incomplete destruction of estrogen sensitive neurons in the VMH-arcuate area may result in a type of denervation hypersensitivity in the remaining estrogen sensitive neurons [25] _ A similar hypothesis has also been proposed to account for the marked increase in lordosis behavior induced by estrogen following septal lesions in female rats
[281,
Thus, the failure to see decrements in lordotic behavior reported here for preoptic and A-HYP lesions may reflect the relatively small lesions compared to those produced by Singer [ 3 I ] and Herndon and Neil1 [ 171. Although Powers and Valenstein [ 301, who reported (as found in the present experiment) an increase in estrogen sensitivity with preoptic lesions, did not indicate the size or extent of their lesions, it seems likely that their use of a platinum electrode for making lesions would have resulted in relatively small lesions. Thus, the effects of preoptic-A-HYP lesions on lordosis behavior may be partially clarified by a parametric analysis of various size lesions on subsequent lordotic behavior. However, an additional and perhaps important variable is pointed out by the work of La Vaque and Rodgers [ 211. Whereas they noted, as had Kennedy and Mitra 1201, a marked decrement in mating behavior in female rats following VMH lesions, La Vaque and Rodgers observed a gradual recovery of lordotic behavior to control levels when the animals were tested across several sessions. Whether this reflects an effect of repeated testing or a time dependent change in the effects of a brain lesion on
312
NANCE /DAL.
behavior focus
behavioral had
has yet
to be determined. Although beyond the experiment, it is possible that different would have been observed if the animals
of the present been
effects tested
in closer
proximity
to the time
ACKNOWLEDGEMENT
The authors gratefully Mrs. Erna Freiberg.
acknowledge
the histological
assistance
of
of lesioning.
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