Physiology & Behavior, Vol. 19, pp. 223-237. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.

Effects of Medial Hypothalamic Lesions on the Lordosis Response and Other Behaviors in Female Golden Hamsters CHARLES W. M A L S B U R Y , * t LEE-MING KOWt AND DONALD W. P F A F F #

*Departments o f Psychiatry and Psychology, University o f Pittsburgh School o f Medicine, Pittsburgh, PA 15261 and t The Rockefeller University, New York, N Y 10021

(Received 9 March 1977) MALSBURY, C. W., L.-M. KOW AND D. W. PFAFF. Effects of medial hypothalamic lesions on the lordosis response and other behaviors in female golden hamsters. PHYSIOL. BEHAV. 19(2) 223-237, 1977.- The lordosis response in ovariectomized, hormone-primed females was measured in repeated weekly tests before and after bilateral lesions of various medial hypothalamic regions. Changes in body weight, frequency of vaginal marking, and agonistic behavior were also recorded. Forty-two females were divided into five groups based on lesion location: (1) medial preoptic (MPO), (2) medial anterior hypothalamus (MAH), (3) dorsomedial nucleus-dorsal premammillary nucleus (DM-DPM), (4) dorsal ventromedial nucleus (Dorsal VMN), (5) medial basal hypothalamus (MBH). Unoperated control females were tested in an identical manner. Only lesions in the region of the ventromedial nucleus eliminated lordosis. Lesions which disrupted lordosis also increased agonistic behavior in male-female pairs. The frequency of vaginal marking was reduced by lesions in the MPO, MAH, and Dorsal VMN groups. Rapid weight increases and obesity were also seen following certain medial hypothalamic lesions, however no significant correlations were found between degree of weight change and degree of lordosis deficit. A quantitative analysis of VMN damage revealed a relatively low, but statistically significant positive correlation between degree of VMN damage and lordosis deficits. There were no significant correlations between degree of VMN damage and weight change. Copulatory behavior Ventromedial nucleus

Lordosis Medialhypothalamus Sex hormones Agonistic behaviour Scent marking Hamster

LITTLE INFORMATION is available concerning the neural control of female copulatory behavior in hamsters. In a number of other species medial hypothalamic tissue is important for sexual receptivity, and several studies indicate this may be the case in the hamster as well. Early work [69] suggested that medial hypothalamic lesions can eliminate lordosis in the hamster, but methodological problems make the results difficult to interpret. More recently, the locations of estradiol-concentrating neurons in the medial hypothalamus have been described in the hamster [20,38], and Ciaccio and Lisk [9] have found that estrogen implanted directly into the medial hypothalamus can facilitate lordosis in the hamster. It is likely that progress in analyzing neural mechanisms of female hamster copulatory behavior will depend on knowledge of the critical brain regions for this behavior, areas where restricted lesions will interrupt pathways necessary for the behavior. The present experiment was carried out in order

Weightregulation

Lesions

to identify important medial preoptic or hypothalamic regions. Lesions of the medial hypothalamus have completely eliminated female mating behavior in the following mammalian species: golden hamster [69], rat [5, 10, 17, 24, 29, 33, 34, 40, 63], guinea pig [4, 16, 23], rabbit [60], cat [1, 61, 62] and ewe [11,19]. Although the lordosis response of the rat has been most extensively studied, even in this species the identity of the hypothalamic substrate for lordosis is not well defined. One set of studies finds that lesions in the medial anterior hypothalamus (MAH) can abolish or disrupt lordosis [29, 40, 63]. Other studies have reported that lesions in the medial basal hypothalamus (MBH) including the area of the ventromedial nucleus (VMN) can disrupt lordosis [5, 17, 33, 34]. Partial lordosis deficits with recovery have also been reported following lesions in this area [39]. Recently, VMN lesions were reported to nearly eliminate lordosis in

Supported by: Department of Psychiatry, University of Pittsburgh School of Medicine and NIH Grant HD-05751 to Dr. Donald W, Pfaff. 2The authors thank Michael Montgomery, Annette Malsbury, Heidi Phillips, Joanne Daood and Mary Lou Sedloek for assistance with these experiments and in preparation of the manuscript. 223

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ovariectomized, estradiol benzoate (EB) treated females, but surprisingly, these deficits disappeared when progesterone was given in addition to EB [ 4 6 ] . In s u m m a r y , it appears that both the MAH and MBH may be part of the h y p o t h a l a m i c substrate necessary for sexual receptivity in the rat. In the guinea pig also, lesions in both the MAH [4] and MBH [23] have disrupted female copulatory behavior. A similar situation exists with observations made following intracerebral estrogen implants. Implantation of estrogen into the medial preoptic area and MAH [2,421 and MBH [2,18] has been reported to induce receptivity in ovariectomized rats. In the guinea pig, implants in both areas are effective, with the MBH more sensitive to the facilitating action of estrogen [ 5 0 ] . In the rabbit, the MBH is the most effective site [ 5 4 ] . Lesions more rostral to either of these areas in the medial preoptic area have p r o d u c e d either no deficit [53,63] or a facilitation of lordosis in rats [ 4 0 , 5 7 ] . The lack of conclusive information on the identity of the crucial medial h y p o t h a l a m i c region or regions in the above studies arises from several factors. The first is a lack of comparison of the effects of restricted lesions in various parts of the medial h y p o t h a l a m u s in the same study under standard conditions. There is also a general lack of adequate description of the size and locations of lesions making comparisons between studies difficult. The case for an i m p o r t a n t role for the MBH in female copulatory behavior is particularly weakened because of methodological problems. Female c o p u l a t o r y behavior was directly observed in only seven studies using the lesion technique to investigate this area (hamster: [ 6 9 ] ; rat: [17, 39, 46] ;guinea pig: [23] ; ewe: [ 11,19] ). In the others, the presence of sperm or a vaginal plug was relied on as indirect evidence of female receptivity following cohabitation with males. With such a procedure, the a m o u n t of stimulation received from males is not controlled, and the response of the female is not quantified. Even in studies where female behavior was directly observed, detailed measures are rarely reported. Only Goy and Phoenix [ 2 3 ] , LaVaque and Rodgers [ 3 9 ] , and Mathews and Edwards [461 report detailed behavioral effects of MBH lesions. Also, most of these studies were done in females with ovaries intact, the vaginal smear indicating h o r m o n e levels adequate to support female copulatory behavior. The clear demonstration of a direct MBH lesion effect on behavioral systems, rather than an indirect effect via a possible subtle postoperative change in pattern or a m o u n t of gonadal secretion, requires control of the appropriate h o r m o n e s through injection. Only Kennedy [33] and Mathews and Edwards [46] using the rat, and Goy and Phoenix [23] using the guinea pig, have followed this procedure. The previous study on the hamster [69] suffers both from inadequate behavioral measures and lack of control over gonadal h o r m o n e levels. We have tried to avoid these methodological problems in the present study. In order to identify the critical medial h y p o t h a l a m i c region or regions involved in the control of copulatory behavior in the female hamster, the lordosis response in ovariectomized, h o r m o n e - p r i m e d females was measured in repeated weekly tests before and after bilateral lesions were made at various levels from just anterior to the anterior commissure back through the level of the ventromedial nucleus. Changes in body weight, frequency of vaginal marking, and agonistic behavior are also reported. A preliminary report of the results has appeared in abstract form [361.

METHOD

Animals Data are presented from 49 adult, female golden hamsters (Mesocricetus auratus) obtained from Lakeview Hamster Colony, Newfield, NJ (outbred strain, L V G : L A K ) . All subjects were housed individually in 7 in. × 10 in. x 7 in. stainless steel, solid b o t t o m cages with Sanicel corn cob bedding and paper tissue nesting material. R a t chow, guinea pig chow, sunflower seeds and fresh cabbage were supplied. At least one week after arrival, all were ovarie c t o m i z e d and then allowed at least one week recovery. Following this recovery and adaptation period a weekly h o r m o n e replacement t r e a t m e n t was begun. Each female was given 1 0 u g estradiol benzoate (EB) on Day 1 and 500 ug progesterone (P) on Day 3 (test day). When it was noticed that some of the lesioned females rapidly gained weight postoperatively, h o r m o n e s were administered according to individual body weight in subsequent animals. These 26 females were given EB at 61.4 ~g/kg and P at 3 . 0 m g / k g . The h o r m o n e s were administered by subcutaneous injection in a p p r o x i m a t e l y 0.1 ml sesame oil. The first weekly h o r m o n e t r e a t m e n t was intended to sensitize the female, and hence no data were collected following the first t r e a t m e n t [3 ].

Behavior Tests Testing was done in the animal rooms which were kept on a 12/12 or 14/10 light-dark cycle with lights off at 10 a.m.. Weekly on Day 3, beginning with the second week of h o r m o n e treatment, all females were given two behavior test sessions, a morning EB only and an a f t e r n o o n EB + P test. The EB only test was carried out one-half to two hr following lights off. At this time the females had been primed with estrogen for abour 42 hr, but no P had yet been injected. Immediately after the first test session each female was given P, and five hours later tested again in an identical m a n n e r under the EB + P condition. Little lordosis is seen in the a.m. tests following EB alone, but it is almost always maximal under the EB + P condition in the p.m. tests. The test schedule described above was designed to reveal either facilitations or disruptions of lordosis that might occur postlesion. To begin a test each animal was removed from its cage and placed on the test surface, a 14 in. diameter circular platform. This surface could be rotated, enabling the e x p e r i m e n t e r to conveniently apply manual stimulation to the animal. After an adaptation period of at least 30 sec, the female was exposed to a sexually active male hamster for 2 rain. During this period instances of vaginal marking, fighting and lordosis were recorded. Vaginal marking consists of lowering the hindquarters, touching the floor with the perineal area and dorsiflexion of the tail. The male was allowed to m o u n t the female, but intromissions, which may cause a decline in receptivity [6] were prevented. This was done using the eraser end of a pencil to gently push the male's hindquarters away from the female's perineum when intromission seemed possible. The two-rain observation was followed by a series of tests to evaluate the responsiveness of the animal to various lordosis-eliciting stimulations and her ability to maintain the lordosis posture once elicited. Each of the tests was begun when the female was not in lordosis. Her responsiveness was tested with three kinds of stimulation in the following order: fingering, cupped hand and placing a male. The fingering was done by lightly

MEDIAL HYPOTHALAMIC LESIONS AND LORDOSIS r u b b i n g t h e lateral flank o f the f e m a l e b e t w e e n t h e t h o r a c i c cage a n d p e r i n e u m w i t h t h e t h u m b o n o n e a n d m i d d l e o r i n d e x finger o n t h e o t h e r side. The s e c o n d t y p e of s t i m u l a t i o n was d o n e b y c u p p i n g t h e h a n d over t h e w h o l e dorsal a n d lateral b o d y surface c a u d a l t o t h e f r o n t s h o u l d e r s a n d g e n t l y r u b b i n g b o t h sides o f t h e b o d y , i n c l u d i n g t h e h i n d q u a r t e r s . In a p r e v i o u s e x p e r i m e n t [ 3 7 ] we f o u n d t h a t b r u s h i n g the h i n d q u a r t e r s will readily elicit lordosis f r o m o v a r i e c t o m i z e d , EB + P i n j e c t e d females. Since t h e c u p p e d h a n d s t i m u l a t i o n covers a skin area o n the h i n d q u a r t e r s i n c l u d i n g a n d m u c h larger t h a n t h a t stimu l a t e d by fingering, the c u p p e d h a n d s t i m u l a t i o n is m o r e effective. T h e t h i r d t y p e of s t i m u l a t i o n was p r o d u c e d b y the e x p e r i m e n t e r h o l d i n g a male b y the loose skin o f the u p p e r b a c k a n d placing h i m o n the h i n d q u a r t e r s of the female. R e p e a t e d placing was used in a t t e m p t s to trigger lordosis. Lordosis was d e f i n e d as t h e s i m u l t a n e o u s occurrence a n d m a i n t e n a n c e for at least 5 sec of b o d y imm o b i l i t y with lack of large sideways e x p l o r a t o r y h e a d m o v e m e n t s , the tail raised at least h o r i z o n t a l , and the surface of t h e b a c k e i t h e r f l a t t e n e d or concave. During this p o s t u r e t h e rear legs were e x t e n d e d to various degrees a n d t h e r e was a l m o s t always e i t h e r slowing or c o m p l e t e lack o f vibrissae m o v e m e n t s . A s t i m u l a t i o n was d e f i n e d as effective in eliciting lordosis if it c o u l d elicit a n d m a i n t a i n the p o s t u r e for at least 5 sec. The time limits for eliciting lordosis were 15 sec for fingering a n d c u p p e d h a n d stimu l a t i o n a n d 30 sec for placing a male. To test the ability of the female to h o l d lordosis, the p o s t u r e , if it c o u l d be elicited, was elicited a n d m a i n t a i n e d for 5 sec b y placing a male on h e r b a c k a n d t h e n a t t e m p t i n g t o m a i n t a i n the p o s t u r e by fingering for as long as possible u p to a limit o f 3 0 0 sec f o l l o w i n g r e m o v a l of the male. The d u r a t i o n of u n i n t e r r u p t e d lordosis a n d t h e q u a l i t y of t h e lordosis p o s t u r e were r e c o r d e d . T h e q u a l i t y was r a t e d as e x c e l l e n t , good, fair, or p o o r . Criteria for t h e t e r m i n a t i o n o f lordosis i n c l u d e d m o v i n g b o t h rear feet, or one rear f o o t twice, or the tail t o u c h i n g the g r o u n d . Measuring lordosis in r e s p o n s e to m a n u a l s t i m u l a t i o n h a s t h e advantage t h a t s t i m u l a t i o n can be p r e s e n t e d in a u n i f o r m m a n n e r a n d is n o t a f f e c t e d b y the f e m a l e ' s behavior. H o w e v e r , it s e e m e d i m p o r t a n t to d e t e r m i n e if a l e s i o n - p r o d u c e d lordosis deficit seen in m a n u a l s t i m u l a t i o n tests w o u l d also a p p e a r in tests w i t h an active male. T h u s , d u r i n g the course of t h e e x p e r i m e n t it was d e c i d e d to include a d d i t i o n a l tests for lordosis and agonistic b e h a v i o r in the h o m e cage of an active male. These tests were carried o u t in 15 females. I m m e d i a t e l y f o l l o w i n g each a f t e r n o o n m a n u a l s t i m u l a t i o n test u n d e r the EB + P c o n d i t i o n , each female was r e m o v e d f r o m the test p l a t f o r m a n d placed in t h e 7 in. × 10 in. × 7 i n . h o m e cage o f a s e x u a l l y active male. T h e pair was left t o g e t h e r for 3 0 0 sec while lordosis l a t e n c y a n d t o t a l lordosis d u r a t i o n for t h e female, a n d the n u m b e r of o n - b a c k p o s t u r e s d i s p l a y e d b y each a n i m a l were r e c o r d e d . T h e o n - b a c k p o s t u r e in w h i c h an a n i m a l is e i t h e r lying o n its b a c k or side is indicative o f a m o d e r a t e level o f aggressive b e h a v i o r in t h e pair [ 2 0 ] . This i n d e x of agonistic b e h a v i o r was c h o s e n because it is easy to recognize a n d can be scored w i t h o u t l e t t i n g a full-blown fight w i t h severe b i t i n g develop. The d e v e l o p m e n t of vigorous fights was p r e v e n t e d by s e p a r a t i n g the animals w i t h a gloved h a n d f o r a p p r o x i m a t e l y 3 0 sec a n d t h e n p e r m i t t i n g c o n t a c t again. If r e p e a t e d bites o c c u r r e d , t h e session was e n d e d before 3 0 0 sec t o p r e v e n t w o u n d i n g of t h e animals.

Surgery After

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225 tests, bilateral lesions were m a d e in t h e m e d i a l p r e o p t i c a n d h y p o t h a l a m i c regions u n d e r s o d i u m p e n t o b a r b i t a l a n e s t h e s i a ( 8 0 mg/kg). Surviving o p e r a t e d females weighed 1 3 4 - 1 9 9 g w i t h a m e a n weight of 1 6 7 g at surgery. Surgery was p e r f o r m e d d u r i n g t h e earlier half o f the interval b e t w e e n t w o successive weekly tests. T h e r e f o r e , weekly test intervals were n o t i n t e r r u p t e d . Only females with c o n s i s t e n t high levels of sexual r e c e p t i v i t y d u r i n g the first 3 or 4 weekly tests c o n t i n u e d in the e x p e r i m e n t e i t h e r as u n o p e r a t e d c o n t r o l s or as lesioned animals. B o t h e l e c t r o l y t i c (N = 25) and r a d i o - f r e q u e n c y c u r r e n t (N = 38) lesions were m a d e using a 0 . 0 0 7 in. d i a m e t e r , tefloni n s u l a t e d p l a t i n u m - i r i d i u m wire. F o r the e l e c t r o l y t i c ( E L ) lesions o n l y the tip cross section was bared, while for r a d i o - f r e q u e n c y ( R F ) lesions 0.8 m m o f tip was bared. EL lesions were m a d e with a direct c u r r e n t of 2.0 m A passed for 10 sec. The e l e c t r o d e tip was the a n o d e and a rectal p r o b e the c a t h o d e . In m o s t cases these lesions were m a d e at t w o d e p t h s , 0.2 m m apart, on each side of the brain. R F lesions were m a d e w i t h a Grass LM4 l e s i o n - m a k e r using 9 - 1 0 m A passed for 60 sec b e t w e e n the e l e c t r o d e tip a n d a rectal p r o b e . P o s t o p e r a t i v e m o r t a l i t y was h i g h o n l y for females with lesions a i m e d for the MPO. W h e n all females with a t t e m p t e d bilateral lesions were divided i n t o those w i t h e l e c t r o d e s aimed for the MPO and t h o s e aimed for o t h e r medial h y p o t h a l a m i c areas, the m o r t a l i t y in the first p o s t o p e r a t i v e week came to 6 / 2 0 or 30% for the MPO, a n d 3 / 4 3 or 7% for all o t h e r p l a c e m e n t s . Lesions were a i m e d for the medial p r e o p t i c area (MPO), MAH, d o r s o m e d i a l h y p o t h a l a m u s ( D M H ) and MBH in a c c o r d a n c e w i t h a skull-flat s t e r e o t a x i c atlas d e v e l o p e d for the h a m s t e r (Malsbury, u n p u b l i s h e d m a n u s c r i p t ) . Using this system, the skull was a d j u s t e d in the s t e r e o t a x i c i n s t r u m e n t so t h a t a line t h r o u g h the skull s u t u r e s b r e g m a and l a m b d a w o u l d be h o r i z o n t a l . Bregma was used as the anteriorp o s t e r i o r (AP) zero p o i n t , w h i l e d e p t h ( D ) w a s d e t e r m i n e d f r o m the cortical surface. All m e a s u r e s are in millimeters. The c o o r d i n a t e s were: MPO; A P = + 1.8 to + 1.9, lateral (L) = 0.5 to 0.7, D = 6.1 to 6.5, M A H ; AP = + 1.0, L = 0.5 to 0.6, D = 6.5 to 6.9, DMH; AP = 0.0 to - 0 . 4 , L = 0.5 to 0.6, D = 6.8 to 7.2, MBH; AP = 0.0 to - 0 . 4 , L = 0.5 to 0.6, D = 7.3 to 7.9. Usual s t e r e o t a x i c operative p r o c e d u r e s w e r e followed. T h e h a m s t e r skull differs f r o m t h e rat's in t h a t significant b l e e d i n g occurs f r o m the skull itself as it is drilled. This bleeding was c o n t r o l l e d with b o n e wax. Since t h e elect r o d e s were l o w e r e d close to the m i d l i n e , the skull was drilled t h i n over the m i d l i n e and the last layer of b o n e carefully r e m o v e d w i t h fine forceps. The s u p e r i o r sagittal sinus was rarely d a m a g e d , and this was used as the m i d l i n e l a n d m a r k . It was s o m e t i m e s necessary to g e n t l y push the sinus clear of the e l e c t r o d e tip as it was l o w e r e d . A dissecting m i c r o s c o p e was a great help w i t h this p r o c e d u r e . A f t e r l o w e r i n g the tip to the desired d e p t h , t h e lesion was m a d e following a waiting p e r i o d of at least 5 m i n to allow t h e b r a i n to recover f r o m the c o m p r e s s i o n caused b y e l e c t r o d e insertion. It was felt t h a t this gave a m o r e accurate d e p t h m e a s u r e m e n t .

Histology Analysis A f t e r c o m p l e t i n g 3 to 8 weekly p o s t o p e r a t i v e tests the l o c a t i o n a n d e x t e n t of the lesions in each female were e x a m i n e d histologically. Tissue was p r e p a r e d in t h e f o l l o w i n g way. A n i m a l s were sacrificed with an overdose o f s o d i u m p e n t o b a r b i t a l followed by cardiac p e r f u s i o n w i t h 0.9% saline a n d t h e n 10% F o r m a l i n . Brains were r e m o v e d

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and after soaking in 10% F o r m a l i n for a week, followed by a p p r o x i m a t e l y 2 days in a 30% sucrose-10% Formalin solution, frozen 50 u sections were cut through the area of damage. These coronal sections were alternately m o u n t e d and stained with either the cresyl violet or Weil m e t h o d . Each lesion was outlined on coronal plane atlas plates spaced at 0.2 m m intervals. When lesions are made close to a ventricle, ventricular pressure will gradually produce some degree of collapse of normal tissue into the lesion cavity resulting in expansion of the ventricle. In this study because the animals were sacrificed at least four weeks following surgery, the third ventricle was typically enlarged at the level of the lesion resulting in considerable tissue distortion. With the aid of the m i c r o p r o j e c t o r the area of missing tissue was outlined by using the remaining normal tissue landmarks. Because of the lordosis deficits seen following lesions in the region of the VMN, a quantitative analysis of the a m o u n t of damage to the VMN was done for each female sustaining VMN damage (N = 17). The p r o m i n e n c e of the VMN in cresyl-violet stained coronal sections made this possible. The area of VMN tissue remaining was calculated at three particular AP levels from the anterior, middle and posterior VMN. Distinctive anatomical landmarks near the base of the brain were used to identify these levels in b o t h unoperated controls and lesioned females.

Method o f determining levels o f VMN for quantitative analysis o f lesion damage from cresyl-stained 50 ~ sections. Level, A, Anterior Pole o f VMN. The first section on which prominent, darkly-staining neurons are seen at the ventral surface of MBH bilaterally serves as a landmark section. Anterior-most VMN cells also appear about here. Then move posterior two cresyl sections (200 u). This is level A. Level A is about equal to AP level 0.0 in the skull flat coordinate system used here. A well-developed VMN with p r o m i n e n t pars lateralis is present. The arcuate nucleus is also first clearly identifiable at this level. Level B, Mid-VMN. Move posterior 3 cresyl sections (300 u) from Level A. This is Level B with large VMN, pars lateralis less prominent, little arcuate left directly below third ventricle, but arcuate still present lateral to ventral part of third ventricle. Prominent darkly-staining neurons at ventral surface of MBH have nearly disappeared by this level. This is the anterior border of median eminence. Level C, Posterior Pole o f VMN. Move posterior 3 cresyl sections ( 3 0 0 u ) from Level B. This is Level C. VMN is smaller, less distinct. Its lateral border is s o m e w h a t difficult to define here. The median eminence is well-developed. Darkly-staining cells at ventral surface of brain are completely absent. DPM is usually clearly visible dorsal to VMN here. Each of the three levels was separated from the n e x t by a p p r o x i m a t e l y 300 u. The area of VMN remaining in each hemisphere was traced o n t o drawing paper using a microprojector. The outlines were then traced o n t o finely squared graph paper. F o r each female the mean VMN area remaining for the two hemispheres was calculated at each level. To determine the percentage of VMN lesioned, these areas were c o m p a r e d to the mean VMN areas calculated at the same three levels from 5 unoperated control females. RESULTS

Histology Analysis As m e n t i o n e d above, bilateral lesions were m a d e in a

total of 63 females. Nine died, and two were used to test new lesion electrodes, or practice operative techniques, thus no behavioral data were collected. This left 52 surviving lesioned females with behavioral data. Ten of these females had lesions that proved to be very small or asymmetrical or placed outside the h y p o t h a l a m i c areas of interest. The c o p u l a t o r y behavior of these ten females will be discussed briefly below. However, since the type of damage p r o d u c e d in these animals was not equivalent to that seen in the other 42, they are not included in group analyses intended to reveal the importance of particular medial h y p o t h a l a m i c regions. The remaining 42 animals had symmetrical lesions which in most cases were confined to the medial preoptic and medial h y p o t h a l a m i c regions within 1.3 mm of the midline. When the individual lesions drawings (see M E T H O D ) were complete, these 42 animals were divided into five groups based on lesion location. Lesions centered between the anterior boundary of the third ventricle and the posterior edge of the anterior commissure were put in the MPO group. The MPO appears at AP levels + 2.2 to + 1.6 (Fig. 1). The medial anterior h y p o t h a l a m u s (MAH), AP levels + 1.4 to + 0.2, is directly posterior and c o n t i n u o u s with the MPO, b o u n d e d anteriorly by the anterior commissure and posteriorly by the anterior tip of the ventromedial nucleus. Lesions centered in this region were placed in the MAH group. Lesions at the level of the ventromedial nucleus lAP levels 0.0 to 0.6) were divided into three groups based on the dorsal-ventral location of the lesion. Those which damaged the area dorsal to the ventromedial nucleus w i t h o u t encroaching on the nucleus itself, were put in the DM-DPM group. This group sustained damage to the dorsomedial nucleus of the h y p o t h a l a m u s (DM) and the dorsal premammillary nucleus (DPM). Animals with lesions centered in the dorsal part of the ventromedial nucleus, or border b e t w e e n the ventromedial nucleus and dorsomedial nucleus directly above, were placed in the Dorsal VMN group. Lesions damaging the ventromedial nucleus and extending ventrally toward the base of the brain were placed in the medial basal h y p o t h a l a m i c (MBH) group. Three of the four lesions in this last group p r o d u c e d bilateral damage to the arcuate nucleus. Unavoidably there is some overlap of damage between the groups. Group composites of overlapping lesion outlines were prepared to indicate the area of lesion damage included in each of the five groups. Each individual lesion was included at every composite level where it produced bilateral damage. Thus all regions where any individual female had bilateral damage are included in the group lesion outlines. These group drawings are shown in Fig. 1.

Lordosis." EB + P Condition The data clearly demonstrate functional differences between medial h y p o t h a l a m i c regions in the control of lordosis. Table 1 shows the mean percent of weekly postoperative tests in which experimenter-applied stimuli elicited lordosis from the females in each group. Only lesions in the region of the ventromedial nucleus interfered with the elicitation of lordosis. F r o m examining the ranges in this table it can be seen that the only failures to elicit lordosis in at least two of the three or four postoperative tests were with females sustaining damage to the VMN region (Dorsal VMN and MBH groups). Also, in some of the females with lesions in the VMN region, lordosis was never elicited postoperatively. The total N for the Dorsal VMN

M E D I A L H Y P O T H A L A M I C L E S I O N S AND L O R D O S I S

227

1'I';;14

FIG. 1. Overlapping lesion outlines indicate the area of damage included in each of the five groups. Each individual lesion was included at every composite level where it produced bilateral damage. The VMN appears at levels 0.0 to -0.6. A lateral scale (in mm) is presented below each group's drawings.

228

MALSBURY,

TABLE

KOW AND PFAFF

l

POSTOPERATIVE ELICITATION OF LORDOSIS BY EXPERIMENTER-APPLIED STIMULI: MEAN PERCENT OF WEEKLY TESTS IN WHICH LORDOSIS WAS ELICITED FROM THE FEMALES IN EACH GROUP

G r o u p (NI

T y p e of Stimulation* B

A X% 93 96 96 95 49~ 56

Control(7) MPO(7) M A H (13) DM-DPM(5) Dorsal V M N (13) M B H (4)

(ranget (75-100) (75-100) (50-100) (75-100) (0-100) (0-100)

X% 89 100 96 95 67~ 75

(range) (50-1001 (100) (50-100) (75-100) t0-100) (0-100)

C X% 100 100 98 95 61+ 69

(range) (100) (100) (75-100) (75-100) (0-100) (0-100)

*A = Fingers, B = Placing Male, C = Cupped Hand, See Methods for further description. +The n u m b e r of females showing a postoperative decrease is significant at p < 0 . 0 0 5 , Sign test, one-tailed.

TABLE

2

EFFECT OF MEDIAL HYPOTHALAMIC LESIONS ON LORDOSIS UNDER EB+P CONDITION AND ON WEIGHT GAIN U nop. Control

MPO

MA H

DMDPM

Dorsal VMN

MBH

Lordosis PLP* (N, S.D.)

86 (7, 19)

66 (7, 34)

89 (13, 24)

94 (5, ll)

26t (13, 28t

38 (4, 45)

Weight Gain, g (N, S.D.)

5 (7, 5)

4 (6, 15)

22 (9, 311

7 (4, 5)

20~(8, 14)

54+ (4, 22)

*Post-lesion p e r f o r m a n c e (PLP) for each female was scored as: ( m e a n p o s t l e s i o n lordosis d u r a t i o n / m e a n p r e l e s i o n lordosis d u r a t i o n ) x 100. L o r d o s i s w a s elicited and m a i n t a i n e d by experimenter-applied stimulation. +Significant difference from unoperated control group ( M a n n - W h i t n e y U test, p < 0 . 0 5 , two-tailed).

TABLE

3

MEAN TOTAL LORDOSIS DURATIONS (SEC) UNDER EB ONLY CONDITION Unop. Control

M PO

MA H

DMDPM

Dorsal VMN

MBH

(7)

(7)

(13)

(5)

(13)

(4)

Preop. Mean (S.D.I

47.6 (83.6)

0.0 (0.0)

2.4 t4.5)

4.0 (7.2t

0.9 (3.1)

8.2 (14.8)

Postop. Mean ( S. D. I

34.3 (67. I I

26. I (50.61

38.5 (81. I )

0.0 (0.01

0.2 (11.6)

5.4 (111.8)

(N)

M E D I A L H Y P O T H A L A M I C LESIONS AND L O R D O S I S

,oo

j.

~ 200

o

__o

,50

2--

g

229

/

MAH(~3)

DM-DPM(,5) +I D°rs°l VMN(I3) • MBH (4)

{\

/\ ~ ///

.

/

///~~

•

•

~00

o

•

50

Preoperative t / P o s t o p e r o l i v e I

I

I

I

2

5

I

I 5

4

I 6

I 7

Weeks

FIG. 2. Pre- and postoperative weekly lordosis performance expressed as mean total lordosis duration for unoperated controls and five lesion groups. Lordosis was elicited and maintained by experimenter-applied stimulation. For abbreviations, see text. The numbers in parentheses are the N's for each group.

60

/" 50 -- Controls(7) 40 cn o

30--

0

MPO (6) MAH (9) DM- DPM (4) DORSAL VMN (8)

[] + • •

MBH (4)

II

/ / /

•

/ •

E °

20-

._~

÷ 1 $

O

i 4

,

i 3

,

,

i

,

,~

i

FIG. 3. Overlapping lesion outlines from the five females with the greatest lordosis deficits (PLP range, 0 - 3 ) . All sustained damage in the region of the VMN (levels 0.0 to -0.6). -I0 Preoperative I I

I 2

/ I ~ 1 5

Postoperative

4

I 5

I 6

I 7

Weeks

FIG. 4. Pre- and postoperative weekly weight changes in grams calculated from the last preoperative measure for the unoperated controls and five lesion groups. For abbreviations, see text. The numbers in parentheses are the N's for each group.

230

M A L S B U R Y , KOW A N D P F A F F

FIG. 5. Overlapping lesion outlines from the six females with the greatest postoperative weight gains: (A) two similar lesions from the MAH group with no damage to the VMN, gains by the fourth postoperative week, 66 and 75 g; (B) four lesions in the region of the VMN. The gains in these females ranged from 46 to 81 g by the fourth postoperative week.

and MBH g r o u p s c o m b i n e d is 17. In 8 of these females lordosis c o u l d n o t be elicited b y any of the m a n u a l stimuli in at least o n e weekly test p o s t l e s i o n . In 3 of these 8, lordosis was n e v e r elicited b y any stimuli p o s t o p e r a t i v e l y for at least 4 c o n s e c u t i v e weekly tests. B o t h EL and R F lesions can e l i m i n t e lordosis if placed in this region. Lesion effects o n lordosis were n o t all or n o n e . In those females w h i c h displayed lordosis following damage in the area of the V M N , varying degrees of deficit were observed. This was expressed in r e d u c t i o n s of lordosis d u r a t i o n s in response to 3 0 0 sec of c o n t i n u o u s m a n u a l s t i m u l a t i o n . The weekly lordosis p e r f o r m a n c e for each group o n this measure is s h o w n in Fig. 2. The greatest deficits are seen in the dorsal VMN a n d MBH groups. Postlesion p e r f o r m a n c e (PLP) for each female was scored as: ( m e a n p o s t l e s i o n lordosis d u r a t i o n / m e a n prelesion lordosis d u r a t i o n ) x 100. T h e m e a n lordosis PLP scores for each group are s h o w n in the u p p e r h a l f of Table 2. C o m p a r i n g each lesion g r o u p with the u n o p e r a t e d c o n t r o l s revealed significantly l o w e r PLP scores in the Dorsal V M N g r o u p only ( M a n n - W h i t n e y U test, p < 0 . 0 5 , two-tailed).

The lesions f r o m the 5 females w i t h the greatest lordosis deficits (PLP range, 0 - 3 ) are s h o w n in Fig. 3. F o u r of these lesions were from the Dorsal V M N g r o u p a n d one f r o m the MBH group. T h r e e were electrolytic lesions and t w o were m a d e with r a d i o - f r e q u e n c y c u r r e n t . The data f r o m the ten females e x c l u d e d f r o m g r o u p analyses because o f small or a s y m m e t r i c a l lesions are in a g r e e m e n t w i t h the data discussed above c o n c e r n i n g the i m p o r t a n c e of the VMN region. These females can be classified i n t o t h r e e categories: (1) females w i t h lesions a n t e r i o r to a n d n o t e n c r o a c h i n g on VMN, N = 4, PLP X = 95, range 8 0 - 1 0 0 ; (2) females w i t h lesions e n c r o a c h i n g on VMN at least unilaterally, N = 4, PLP X = 44, range 27 58; (3) females with lesions p o s t e r i o r to and n o t e n c r o a c h i n g o n VMN, N = 2, PLP X = 72, range 4 4 - 1 0 0 . T h e histology of females in the second c a t e g o r y above indicates t h a t in c o m b i n a t i o n with damage to the dorsomedial nucleus, even slight damage to the dorsal b o u n d a r i e s of the VMN can p r o d u c e a decrease in lordosis. N o n e of the females in the s t u d y were particularly h a r d to h a n d l e p o s t o p e r a t i v e l y , including the 5 females with the greatest lordosis deficits. These 5 females did n o t try to

MEDIAL HYPOTHALAMIC LESIONS AND LORDOSIS

231

TABLE 4

I00

CORRELATION COEFFICIENTS BETWEEN PERCENTAGE OF VMN LESIONED AT THREE LEVELS SHOWN lN THE ACCOMPANYING DIAGRAM AND POSTOPERATIVE LORDOSIS DEFICITS OR WEIGHT CHANGES Lordosis % Decrease N = 17 r~

VMN Level A B C A+B+C

+ + + +

Weight Change N = 12 rs

.48* .51" .27 .52*

+ + +

.36 .17 .29 .30

¢,.) + o3 + 80 < "O ~D cO

m

rs = + . 5 2

B

p


40

+

0 0

c

0

VMN in Sagittal Section

Anterior Posterior A

B

C

a t t a c k the e x p e r i m e n t e r in m a n u a l s t i m u l a t i o n tests, b u t simply i g n o r e d the stimuli in m o s t cases a n d c o n t i n u e d t o e x p l o r e t h e test p l a t f o r m . No o b v i o u s m o t o r difficulties were seen in any of the o p e r a t e d females. A p r e v i o u s s t u d y of t h e effects of V M N area lesions in male h a m s t e r s also r e p o r t e d n o change in resistance to c a p t u r e or r e a c t i v i t y to h a n d l i n g p o s t o p e r a t i v e l y [45 ].

Lordosis: EB Only Condition O v a r i e c t o m i z e d female h a m s t e r s usually show little or n o lordosis w h e n given EB alone. The m e a n t o t a l lordosis d u r a t i o n s for t h r e e p r e o p e r a t i v e a n d f o u r p o s t o p e r a t i v e tests in this c o n d i t i o n are s h o w n for each g r o u p in Table 3. T h e r e were n o significant d i f f e r e n c e s b e t w e e n p r e - a n d p o s t o p e r a t i v e scores for a n y g r o u p (Walsh test, all p > 0 . 0 5 , two-tailed). T h e r e was great variability b e t w e e n animals in each group, a n d this is r e f l e c t e d in the large s t a n d a r d d e v i a t i o n s in T a b l e 3. In a d d i t i o n , in t h o s e females w h i c h displayed lordosis in this c o n d i t i o n t h e r e was great variability f r o m o n e week to t h e n e x t b o t h pre- a n d postoperatively. This c o n t r a s t s sharply w i t h the s t a b i l i t y of the same a n i m a l s ' lordosis p e r f o r m a n c e s in t h e EB + P c o n d i t i o n f r o m week to w e e k p r e o p e r a t i v e l y .

o*

B

z

~9

0 0

cl

Dorsal

0

+#,

20 *Significant positive correlation. (p