Brain Research, 101 (1976) 47-66 47 ~, Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

SENSORY REQUIREMENTS FOR THE LORDOSIS REFLEX IN FEMALE RATS

LEE-M1NG KOW AND DONALD W. PFAFF

The Rockefeller University, New York, N.Y. 10021 (U.S.A.)

(Accepted June 30th, 1975)

SU MMARY

(1) Visual, auditory and olfactory input were not necessary for the performance of the lordosis reflex by female rats. Since taste is not involved in this behavior, somatosensory input must be sufficient for triggering the lordosis reflex.

(2) Surgical cutaneous denervation markedly reduced the frequency and strength of lordosis. The most effective operation denervated the perineum, tailbase, posterior rump and ventral flanks. This pattern of results agrees with film observa- tions and manual stimulation of lordosis.

(3) Effects of surgical cutaneous denervation were confirmed in experiments with local anesthesia produced by subcutaneous procaine injections.

(4) It is deduced that cutaneous mechanoreceptors in specific regions of hairy skin of the female rat play a crucial role in triggering the lordosis reflex.

INTRODUCTION

An estrous female rat responds to the male rat with a display of sexual behavior which includes the lordosis reflex. Lordosis occurs rapidly following mounting by the male, which involves contact between the male and specific portions of the skin of the female rat 20,zl. In the absence of the male rat, the lordosis reflex can be triggered by stimulation on specific portions of the skin of the female, exerted by the experi- menter's hand s,')t. Thus, somatosensory stimulation appears to be sufficient for the prompt initiation of lordosis in rats. Although the possible roles of other modalities have not been studied comprehensively, some information is available. Female rabbits rendered blind, deaf and anosmic were found to be sexually active 4. In female rats, removal of olfactory input did not reduce the performance of lordosis 9,1s. A more inclusive test of the necessity for non-somatosensory information is one object of the present studies (experiment I).

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The choice of skin regions tbr cutaneous desensitization operations in the present series of experiments on the female rat has been guided by previous observations of rat mating behavior. Film analyses show that the male rat's forepaws and forearnls touch portions of the female's flanks, and that parts of the male rat's lower abdolnen, rear legs and pelvic region touch the female's rump, tailbase and perineum z°,zl. Manual pressure exerted by the experimenter on those same skin regions of the estrous female rat can trigger lordosis 8. Electrophysiological recording from the pudendal nerve has demonstrated input from rapidly adapting cutaneous receptors which can be influenced by estrogen 14,~5. Cutaneous information importanl for lordosis appears to enter the spinal cord through dorsal roots L1 through $1 ~7 and ascend the spinal cord in the anterolateral columns (manuscript in preparation)l~L Somatosensory stimulation from inside the vagina apparently is not necessary for lordosis, since removal", anesthetization 4 or denervation 3 of the vagina in various species does not prevent the performance of female mating behavior. Based on these various observations a series of experiments was designed to test the necessity and sufficiency of somatosensory input for stimulating lordosis in female rats, and further to determine those precise regions of skin from which cutaneous input is necessary for lordosis.

The first experiment was done to see if female rats would pertbrm lordosis with no afferent input other than somatosensory, and when it was found that they could, the effect of subsequent cutaneous denervation was investigated. Then, the effects on lordosis of desensitization of increasingly wider areas of skin were studied using surgical cutaneous denervation (experiments I1 and Ili). Conclusions from these experiments were confirmed using a different method of cutaneous desensitization, subcutaneous injections of local anesthetic (experiment IV).

EXPERIMENT I " LORDOSIS IN BLINDED, DEAFENED, ANOSMIC FEMALE RATS

To rule out the necessity of visual, auditory and olfactory inputs, lordosis reflex tests can be conducted with animals which have been surgically blinded, deafened and rendered anosmic. Since taste cannot play a role in this behavior (female rats do not regularly lick the males), the somatosenses provide the only afferent input. In female rats thus deprived of all distance senses, we then initiated the study of the effects of surgical cutaneous denervation on lordosis.

Methods

Adult ovariectomized female rats were obtained from Hormone Assay Labora- tories weighing between 210 and 330 g. Experiments were begun no less than two weeks following ovariectomy. Females were daily given 5/~g estradiol benzoate per day, in- jected subcutaneously in sesame oil during the period of behavioral testing and for at least two weeks before. Progesteione was not used, primarily because it was considered inadvisable for experiments in which daily testing often was required ~.

Females were tested for the occurrence and strength of the lordosis reflex on at

49

least 3 different days before operations for cutaneous denervation. Each test began with 5 attempts to elicit lordosis through manual stimulation by the experimenter. Each of the 5 times the experimenter rapidly scratched the flank regions of the female rat bilaterally, and then briefly touched the rump and tailbase areas of the female while assuming a 'forked' position of the hand around the rear end of the female rat: in this position the thumb is pressing against one flank of the rat, the ring finger against the other flank, while the index and third fingers are forked posteriorly around either side of the tailbase of the female such that the tips of the fingers can press up- wards against the perineal region. Following the rapid application of manual pressure in this forked position, the experimeter rated the lordosis as absent, fair, good or excellent (scoring 0, 1, 2, or 3, respectively). The female's score for lordosis reflex response to manual stimulation in each test was the sum of scores from the 5 stimulus applications. Following manual stimulation testing, females were tested for lordosis in response to 15 mounts by vigorous 'stud' male rats, in testing cages (23 in. × 7 in. × 15 in.) with glass fronts. The female"s reflex response to each mount was scored as lordosis absent, weak, or strong (quantitative scores 0, 1, or 2 respectively), on the basis of the extent of dorsiflexion of the vertebral column, indicated by head elevation, thorax lowering, and rump and tailbase elevation. The female's lordosis quotient for each test is defined as the percentage of male mounts on which she performed lordosis (whether the reflex was weak or strong). Testing was carried out shortly after the be- ginning of the dark phase of a reversed light-dark cycle. All tests were done without knowledge of the experimental treatment of the animals.

On the basis of averaged lordosis responses for all preoperative testing, matched pairs of animals were constructed. In each matched pair of females, one received an operation for cutaneous denervation, while the other received a sham operation. Ex- periments were usually conducted with squads of 3 or 4 such matched pairs.

Surgery Two weeks before the beginning of behavioral testing all females were blinded

(by enucleation), deafened (by puncturing the ear drum and damaging the auditory ossicles), and rendered anosmic (by suction removal of the olfactory bulbs). These 3 operations were performed under halothane (Fluothane) anesthesia, and were done bilaterally. Completeness of removal of at least the anterior half of the olfactory bulbs bilaterally was confirmed after the experiment by visual inspection through a dissect- ing microscope following cardiac perfusion, and also by histologic verification with serial frozen sections stained with cresyl violet. All animals included in the report of this experiment had a complete interruption of olfactory input.

After pretesting, operations for cutaneous denervations began by anesthetiza- tion with halothane (Fluothane). After the hair was shaved, an incision was made in the middle of the area to be denervated. Throughout the area to be denervated the skin was separated from the muscle by spreading hemostats. Subcutaneous layers of fascia between muscles and skin were torn or cut to interrupt nerves travelling toward the skin and to expose the largest segments of the cutaneous nerves, lying on top of the muscle. All visible nerves in the area above the muscles were cut (and, if they ran with

50

large blood vessels, tied illst) usually near the point where they emerged from between muscles. Layers of fat were also cut thoroughly so that nerves running through them or next to them would be interrupted, in the perineal region, fat and fascia were cut extensively, and were interrupted as close to the walls of the internal vaginal and rectal passages as possible without damaging those structures, The incision was sutured and

Aureomycin powder rubbed onto the closed incision. The cutaneous denervation was repeated on the second side, and in all cases operations were intended to be bilaterally symmetrical. Just before the animal emerged from anesthesia, a stiffrubber collar, like a ruff, was fitted around its neck, the tightness adjusted with a small piece of wire so that the animal could breathe but could not remove the collar. The diameter of the collar (3.5 in.) prevented cutaneous desensitized animals from ripping open their incisions. These collars, modeled after those used by Roth and Rosenblatt 2~ were teft in place until sacrifice, including the period of postoperative lordosis reflex testing.

Sham-operated animals were treated in all respects like the cutaneous denervated animals except that as soon as the incision was made it was sutured. The skin was not separated from the muscles, nor any nerves, fascia or fat intentionally cut. Sham- operated animals were also fitted with collars. Postoperative health was checked daily. When necessary, wounds were repaired by cleaning and resuturing.

Lordosis testing procedures after cutaneous operations were identical to those preoperative, and began within 24 h of denervation and sham operations. Because of steady changes in lordosis pertbrmance with increasing duration ofestradiol treatment, and because of the possibility of nerve regeneration with increasing postoperative time, we decided that the most reliable comparison was between lordosis scores in the last test before and the first test after a sham or denervation operation. These were separated by less than 48 I1 and were run in the same way for denervated and sham- operated animals. Results illustrated are based on this comparison.

Since each animal contributed matched pairs of preoperative and postoperative results, statistical evaluations were made using the non-parametric Walsh test ''a tbr two related samples, where n ~-- 6 or larger, and are presented in the figures. Where n < 6, statistical comparisons of data from all preoperative tests to all postoperative tests were made for each animal, using the non-parametric Mann-Whitney U-test":', and are summarized in the text.

Results

Deaf, blind, anosmic female rats performed strong lordosis reflexes on a high proportion of mounts by the male rat, and also responded with lordosis to manual stimulation ( 'pre ' in Fig. I A and B) before cutaneous operations. Extensive cutane- ous denervation of the flanks, back, rump and perineum caused a large decrease in lordosis quotient and abolished the lordosis response to manual stimulation (P < 0.01, 'post ' in Fig. IA). The amount of reduction of lordosis quotient (about 85 ~ reduction) was comparable to that seen in animals with normal sight, hearing and olfactory input (see Fig. 2C). Cutaneous denervation on just the dorsal and lateral surfaces (Fig. 1B) also gave a significant reduction (P <: 0.05) in lordosis responses to the male rat or to

51

Skin area d e n e r v a t e d Side v iew V e n t r a l v iew

(done bilaterally)

Lordosis response

to male rat

Lordosis response

to manual stimulation

I00

8O

6O

4O

20 ..

o N pre post

(n=6)

d_ nerv sham I

rv

pre post pre post

(n=6) (n=6)

sham

V7m pre post

(n=6)

I00[ denerv sham 15[

40 6 denerv

2 3

pre post pre post pre post

(n=4) (n=4) (n=4)

sham

pre post

(n=4)

Fig. 1. Lordosis reflexes in female rats blinded, deafened and rendered anosmic two weeks before the cutaneous denervation experiment. Results, to the right, show mean lordosis responses before ('pre') and after ('post') cutaneous denervation of selected areas of the female rat's skin. Lordosis reflexes were tested in response to mounting by the male zat (lordosis quotient, 0-100) or by the female's response to manual stimulation by the experimenter (0-15). Shading in each rat picture shows the region of female's skin in which all visible cutaneous nerves were cut and the fascia inter- rupted. Sites of incisions are shown by dark lines in the region of denervation. All operations were bilaterally symmetrical. Statistical comparisons of postoperative to preoperative lordosis performance are shown where N = 6 or larger: **P < 0.01.

manual stimulation (Fig. 1B), a somewhat larger reduction than that seen after equivalent operations in animals with normal sight, hearing and olfactory input (see Fig. 2D). Additional analyses of responses taking into account the strength of the lordosis reflex, as well as its occurrence, gave these same results.

EXPERIMENT II" SECTIONING OF PUDENDAL NERVES

The results of experiment I showed that afferent information other than somatosensory input is not necessary for lordosis in the female rat. Since cutaneous denervation did reduce the ability of females to perform lordosis, a more extensive series of studies was indicated to define the precise skin regions contributing to afferent input for the lordosis reflex. In previous investigations 15,17 we found that in female rats the skin of the perineal region is innervated exclusively by the pudendal

52

nerve. The present experiment was performed to see the effect of pudendal nerve inter- ruption on the lordosis reflex. The experiment included two different hormonal con- ditions, to look for interactions between estrogen levels and the effects of perincal desensitization by pudendal nerve section.

Methods

Eleven adult, ovariectomized Sprague-Dawley rats (Hormone Assay Lab., Inc.) were used. Their body weights ranged from 299 to 339 g at the beginning of the experi- ment. The subjects, housed individually under a reversed light-dark cycle, were randomly divided into experimental (n := 7) and control (n = 4) groups.

The experiment was divided into 4 stages: sensitization, preoperative, recovery and postoperative. Hormones were injected subcutaneously in 0.1 ml sesame oil. During the first stage each rat was given two weekly 'sensitization' treatments 1° of 10 #g estradiol benzoate (EB)/rat and then 500/~g progesterone (P)/rat two days later. The rats showed strong lordosis following the second sensitization treatment. The lordosis-inducing effect of the sensitization treatment was allowed to fade out before the beginning of the preoperative stage, during an interval of more than two weeks in which no hormone was given. Both the pre- and postoperative stages, separated by the two-week recovery stage, used the same hormone treatment schedule. In one week, each rat was given 10/zg EB, followed two days later by 500/~g P. In the last week of both stages 10 #g EB/rat was given daily for 7 days and on the last day 500/~g P/rat was also given.

Approximately 6 h after the P injection, each female rat was put into an observa- tion cage (9 in. x 17 in. × 7 in.) occupied by a stud male rat and was observed for 15 mounts by the male or 15 min, whichever came first. The lordosis quotient (LQ), defined as (number of lordoses/nurnber of mounts by the male) × 100, was calculated from the observations. Postoperatively, behavioral tests were carried out 'blind', i.e., the observer had no knowledge about the type of operation performed on each subject. All the behavioral tests were carried out several hours after the beginning of the dark phase of the light cycle.

Surgery A surgical operation was performed on every female at the completion of the

preoperative tests. The rats were anesthetized with Equi-Thesin (Jensen-Salsberg Lab., 0.3 ml/100 g body weight, i.p.), and were given atropine (0.12 mg/rat, i.p.) to prevent excessive saliva secretion and Longicil (Fort Dodge Lab., 0.15 ml/rat, i.m.) to prevent infection. After the hair was clipped, a midline incision was made on the skin near the dorsal tail base. Both pudendal nerves, one on each side, were exposed by cutting the muscles attached to the caudal vertebral columns. For the control (sham operation) group the pudendal nerves on both sides were exposed but no injury was made. For the experimental group, the pudendal nerves were physically interrupted bilaterally by removing a segment of a few millimeters from each of them. The muscles and the skin were then sutured, and the females allowed to recover for two weeks.

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T A B L E I

EFFECTS OF SECTIONING OF PUDENDAL NERVES ON THE LORDOSIS REFLEX UNDER TWO HORMONAL CONDI- TIONS

Lordosis response to male rat (mean LQ ± S.E.M.)*

Estrogen priming o f female 10 llg EB tO llg EB x 7

Denervated (n = 7)

Sham (n - 4)

Pre-op 88 ± 4 96 ~ 2 Pos t -op 24 ± 11 90 ± 5

Decrease 73 6 P** < 0.01 N.S. Pre-op 67 ± 23 95 -: 3 Pos t -op 42 ± 23 82 - 14 ~o Decrease 37 14 P** N.S. N.S.

* Mean lordosis quot ient ± s t andard error of mean. ** P value, two-tailed, t-test. The non-paramet r i c Walsh test gave the same pat tern of stat ist ical results.

At the end of the experiment the pudendal nerves of all females were examined. In the control group no physical interruption of the pudendal nerve was seen in any subject. To make sure that the sham operation did not cause significant functional damage to the nerves, electrophysiological responses of the pudendal nerves to mechanical stimulation (brushing) on perineal skin were recorded at a site central to the segment exposed in the first operation. All the pudendal nerves of the subjects in the control group responded to the skin stimulation, indicating that the sham opera- tion did not functionally interrupt the pudendal nerves. In the experimental group, the pudendal nerve was also examined bilaterally. In most of the cases the cut ends could be found. In the cases in which the cut ends could not be located, electrophysio- logical recording was used, and the interruption of the pudendal nerves was verified.

For each estradiol dose, every rat contributed both preoperative and post- operative lordosis results. Differences were analyzed statistically using the t-test for matched pairs and non-parametric Walsh test.

Results

Sectioning of the pudendal nerves led to a large and significant decrease of lordosis when the lower estrogen dosage was applied but not when estrogen levels were raised by 7 daily injections (Table I). The sham operation did not lead to a significant effect on lordosis under either hormonal condition. Thus, cutaneous input from the perineal skin of the female rat does contribute to lordosis, but elevated es- trogen levels can override the effects of loss of this input.

EXPERIMENT I l l ; CUTANEOUS DENERVATION

Since desensitizing perineal skin by pudendal nerve section did not eliminate

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Skin areo denervafed Side view Ventral view

(done bilorerolly)

Lordosis Lordosis response response

1o male rat to manual stimulation

pre pOST pre post pre post pre post

(n =12) (n=12) (n=12} (n=12)

(n=8) (n=8) (n=a] (n=8)

I00[ denerv 15[ denerv

80[ sham ,2 t

sham 40 6

2 5

pre pos% pre posl p~ posl r~ s

(n=8( (n:8) (n=8) (n=8)

Skin aree denerveted Side view Ventral view

Cdone bilQlerall~)

Lordosis Lordosis response response

to male rut :lo monu81 stimulation

1

I00 r denerv sham 15[denerv sham

re s re os re os pre post p pot p p I p p t {n~) In=31 (n=3) In=3(

,oo I ,5[ denervdenerv 80 / sham 12 / sham

60

40

20

C pre post we ~os~ pre post l~e post

(n=~] (n~5) (n=~) in=3)

Fig. 2. Effects of surgical cutaneous denervations of different regions of the female rat's skin on the lordosis reflex. Lordosis was measured in response to mounting by the male rat (lordosis quotient, 0-100) and by scoring the lordosis response to manual stimulation by the experimenter (0-15). In each rat picture, shading shows the area of the female's skin in which all visible cutaneous nerves were cut and the fascia interrupted. Sites of incisions are shown by dark lines in the areas of opera- tion. All operations were bilaterally symmetrical. Results, to the right of each set of operation pictures, show mean lordosis responses before ('pre') and after ('post') either denervation or sham operations. Statistical comparisons of postoperative to preoperative results are shown when N = 6 or larger: *P < 0.01; **P < 0.004.

lo rdos i s in f ema le rats , and the size o f lo rdos i s r e d u c t i o n was d e p e n d e n t u p o n e s t rogen

levels ( e x p e r i m e n t II) , m o r e ex tens ive c u t a n e o u s d e n e r v a t i o n s s eemed ind ica ted . I n

th is e x p e r i m e n t an inc reas ing ly w ide r a rea o f surg ica l c u t a n e o u s d e n e r v a t i o n was

tes ted, a n d t h e n an o p t i m u m p a t t e r n o f d e n e r v a t i o n (grea te r r e d u c t i o n o f lo rdos i s wi th

less sk in a rea d e n e r v a t e d ) was de t e rmined .

Methods

This e x p e r i m e n t was c o n d u c t e d in a m a n n e r iden t ica l to t h a t o f e x p e r i m e n t I in

all respects , excep t t ha t in the p r e sen t e x p e r i m e n t all ra ts h a d n o r m a l sight, hea r i ng

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and olfaction. Also, in the present experiment, more and different skin regions were tested for effects of cutaneous denervation, compared to experiment I. Otherwise, source and condition of animals, hormone treatments, methods of testing for lordosis, schedules of testing and construction of matched pairs of animals, statistical evalua- tions, and methods of cutaneous denervation and sham operation were all identical to experiment 1.

Results

Cutaneous denervation of the posterior rump, tailbase and perineum caused a 51 ~o decrease in the frequency of lordosis in response to mounts by the male rat, and a 60 ~o decrease in the lordosis score in manual stimulation tests (P < 0.01 ) (Fig. 2A). The effects produced here by surgical denervation of the posterior rump, tailbase and perineum are comparable to those seen after local anesthesia (see experiment IV, and Fig. 4A).

Enlarging the denervated area of the rump and flanks to include the skin on the anterior surface of the rear legs and just in front of the rear legs led to a larger denerva- tion effect on lordosis (lordosis quotient decreased by 80~o; P < 0.004) (Fig. 2B). Extending the flank and back denervation even further anterior (Fig. 2C) did not proportionally increase the magnitude of effect on lordosis. Extensive cutaneous denervation on the dorsal side of the body, including the rump, flanks and back, while leaving the perineum untouched, reduced lordosis quotient by 55 ~o, and abolished lordosis responses to manual stimulation (P < 0.05) (Fig. 2D). On the other hand, extensive denervation on the ventral side of the body, including the perineum, the inner surface of the legs, and the ventral flank region where the male's paws palpate the female's skin, virtually abolished lordosis responses both to the male rat and to manual stimulation (P < 0.05) (Fig. 2E).

From a consideration of all these denervation results it appeared that the most critical skin areas for lordosis might include the perineum, tailbase, most posterior rump (Fig. 2A and B), and the most ventral part of the flanks at the front of the rear legs where the male's paws palpate the female's skin (Fig. 2B and E). An experiment designed to denervate just those skin regions (Fig. 2F) gave a very large (84~) denervation effect on lordosis response to the male rat and abolished response to manual stimulation (P < 0.05). This appeared to be an optimal pattern of denerva- tion, since a large decrement in lordosis was obtained with a relatively small area of skin desensitized.

Denervation of the dorsal aspect of the thorax, as an operative control, gave no significant effect on lordosis.

Supplementary response measures, taking into account different strengths of lordosis reflex response to male rat mounting, gave the same results as those reported above for simple lordosis quotients.

The time course of recovery of the lordosis reflex from the effect of cutaneous denervation presumably reflects the course of peripheral nerve regeneration. However, the shapes of recovery curves varied from experiment to experiment, and may also

56

have been influenced by the development of paraesthesias (see Discussion), infection, and by the overall scope and site of denervation.

The experiment shown in Fig. 2A, with averages of 51 ~'o and 60% decreases in lordosis responses to male rat and manual stimulation respectively, was run in 3 sepa- rate squads, each with several matched pairs of sham-operated and denervated animals. One squad, weighing an average of 300 g, had a preoperative lordosis quotient of 55 and preoperative manual stimulation score of 8.6. This squad showed the largest effects of cutaneous denervation: a 70 ~,,~ decrease in lordosis quotient and 80,,%o decrease in manual stimulation lordosis score. In contrast, another squad, weighing an average of 220 g, had preoperative lordosis quotients averaging 84 and manual stimulation score, 12.0. This squad showed the smallest effect of cutaneous denervation : a 26 ?,/, decrease in lordosis quotient and a 40°/~o decrease in manual stimulation lordosis score.

Thus, when the dose of estrogen per body weight was high, lordosis deticits produced by cutaneous denervation were reduced. In the experiments illustrated in Fig. 2B and C, also, rats with higher initial lordosis quotientsin response to preopera- tive estrogen doses showed less severe effects of cutaneous denervation. Here, as in the experiment on pudendal nerve section (experiment II, Table I), higher estrogen dose or greater responsiveness to a given dose can partially compensate for sensory loss in the control of lordosis,

E X P E R I M E N T 1V " L O C A L C U T A N E O U S A N E S T H E S I A

Another method of achieving cutaneous desensitization, aside from surgical denervation, is subcutaneous injection of local anesthetic. To confirm the conclusions based on denervation experiments, we investigated the effects of subcutaneous injec- tions of procaine on lordosis in female rats.

Methods

Adult ovariectomized female rats (N -- 18) were obtained from Hormone Assay Laboratories, weighing between 280 and 340 g. Experiments were begun no less than 3 weeks following ovariectomy. All hormone injections were given subcutaneously, in sesame oil. The first squad of animals (n ~- 6, see Fig. 3) received 6 #g EB 48 h before each behavioral test. The second squad of females (n ~- 12, see Fig. 4) received, for the first 4 tests, 2 k~g EB 48 h before each test, and then received, for the last 4 tests, daily injections of 10 ktg EB/day. For all females, P (0.5 mg) was given 4--6 h before each behavioral test.

Females were tested for the presence of the lordosis reflex in response to mount- ing by the male rat. Each test was 8 min long, and was conducted in a cage (23 in. × 7 in. x 15 in.) with a glass front. Tests were conducted in the early part of the dark phase of a reversed dark-light cycle. To vary the area and strength of somatosensory stimulation by the male, two different types of stud (sexually experienced) male rats were used. Large normal males, weighing more than 500 g, were used for the first

57

half of each test. To give a weaker, smaller stimulus, hypophysectomized males, weighing about 300 g and equipped with subcutaneous pellets of testosterone, were used for the second half of each test, since it is known that hypophysectomized male rats can respond to testosterone with mating behavior 19. In all major respects, results were the same regardless of the size of the stud male rat, and therefore in all data presented, the results are combined. The presence of the lordosis reflex in the female was determined by observing the dorsiflexion of the vertebral column (elevation of rump, tailbase and head, while the thorax is lowered) in response to mounts by the male. The lordosis quotient was defined as in the previous experiments. Among all mounts by the male, 3 different types were distinguished. The simple mount involves merely the approach by the male to the rear of the female, and bilaterally symmetrical grasping of the female's flanks. The mount with thrust involves all the movements of the simple mount plus pelvic hindquarters thrusting by the male against the rear end of the female. The intromission involves all the movements of the mount with thrust plus a final vigorous thrust terminated by a rapid springing dismount, which is well established as the correlate of penile insertion into the vagina. In preliminary tests with these animals, not included in the Results, the correlation of rapid dismount with penile insertion was confirmed by filming mating encounters from below a glass- bottomed enclosure. The fintromission quotient' was defined in order to measure the success of the male in obtaining penile insertion, and is the number of intromissions divided by the total number of mounts, with the ratio multiplied by 100: that is, (intromissions/simple mounts + mounts with thrusts ÷ intromissions) × 100.

Local anesthesia was achieved by subcutaneous injections of 0.25 ml procaine solution at each site on the female marked in Figs. 3 and 4. We used a 2~o solution of procaine hydrochloride in isotonic saline (Abbott Laboratories), with adrenaline hydrochloride (1 mg/60 ml) added to constrict local blood vessels and prolong local anesthetic action. Pilot testing showed that subcutaneous injections, but not topical applications, produced reliable local anesthesia. During these informal tests of re- sponses to pain it appeared that peak anesthetic action occurred between 5 and 20 min after subcutaneous procaine injections. Therefore, lordosis reflex tests (8 rain in duration) were begun 6-9 min after procaine injection. Control tests were run with 0.25 ml isotonic saline injected at the sites marked in Figs. 3 and 4.

Tests for lordosis were separated by 7 days, and in all cases each female served as her own control. For the first squad of females (n 6, Fig. 3) 6 tests were given for each female with control injections. Then, two 'procaine' tests were conducted. Finally, a postprocaine control test was run for each female. Statistics were used to compare 'preprocaine' mean values with mean values in the procaine tests. For the second squad of females (n -- 12, Fig. 4) each female was used in all 4 conditions - - pelvic region procaine or saline control (Fig. 4A) and flank-back procaine or saline control (Fig. 4B) - - and the experiment was counterbalanced against order effects.

Statistical evaluations of differences between procaine-injection and control- injection tests were made using the non-parametric Walsh and Sign tests 25.

This experiment has been reported in preliminary form 21.

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Injection Side view

(done bilaterally)

sites Ventral view

Lordosis quotient

(L°rd°ses x ~ I00)

° t ¼ Subcutaneous .~/~j Injections: Saline control 35 ( pre - procaine)

x o x Procaine 07 '1' Saline control 4 7 (poet- procaine)

"lntromission quotient"

Intromissions x I00)

19

02" 54

~p<.02 Fig. 3. Effect of subcutaneous injections of procaine in the flank, back and perineal regions of the female rat on the performance of lordosis by the female (mean lordosis quotient) in response to mounts by the male, and on the achievement of penile insertion by the male (mean intromission quotient). At each site marked by x, procaine hydrochloride in 0.25 ml saline, or the vehicle control, was injected. Treatments were bilaterally symmetrical.

Results

Injection sites were chosen to anesthetize regions of the skin of the female rat touched by the male during mounting and pelvic thrusting, as determined by film analysis 2°,zl. Anesthetization of the female's flanks and back (touched by the male's forepaws and forearms during mounting) in addition to anesthetization of the female's perineum (touched by the male during pelvic thrusting) led to a significant decrease in lordosis quotient (Fig. 3). Moreover, local anesthesia of the female's skin caused a significant decrease in the male's intromission quotient, when the female received pro- caine treatment (Fig. 3).

The area of female's skin anesthetized as shown in Fig. 3 is quite large. Further experiments in a second squad of females were designed to fractionate this large area. Anesthetization of skin on the female's perineum and tailbase resulted in a large de- crease in lordosis performance (Fig. 4A). For this effect, it is important that skin on and near the midline, including the immediate perivaginal and perianal regions, be anesthetized thoroughly. As in the previous experiment, cutaneous anesthesia of the female significantly reduced the performance of intromission by the anale rat (Fig. 4A). Procaine injections restricted to the region touched by the male's paws and forearms gave less striking effects on lordosis than procaine injections in the perineal and tail- base region. The only significant effect on lordosis occurred when the females were given high estrogen levels (Fig. 4B), and no effect of flank and back procaine was seen on the performance of intromission by the male (Fig. 4B).

Figs. 3 and 4 show two ways in which changed conditions of females during lordosis-mount encounters alter the opportunity of the male to achieve an intro- mission. First, cutaneous desensitization of the female, interfering with lordosis, secondarily interferes with the opportunity of the male to achieve intromission (re- duced intromission quotient). Second, increased estrogen levels in the female (10 fig/

59

Inject on sites Side view Ventra l view

(done bitulerG I!y]

L I

f,.

B i \;

Lordosis ' rntromission quotient quotient"

\{ L°rd°ses Xmounts ,OO) \(Inff°missi°ns x ~ [OO)

EstradiOllnjections~SUbcutaneous 2 ~g IOffg/day 2/zg IO/zg/day

Procaine 16 ~ 4 9 " * 6 * t 2 5 " *

SQline Control 41 8"2 28 / 52

Procaine vs. Conlrol, • p< .05

" ' p < .005

Lordosis "lnlromiss[on quotient quotient"

Lordoses x [00) /lntromissions I00) mounts ] ~ x

EstrodiOllnjections=SubcutGneous2 p.g IO/zg/day 2.p.g IO/zg/doy

Procdine ~7 56 ~ 25 ~5

Snlir~e coalro[ 45 82 27 45

Procaine vs. ControE, ~p< .O5

Fig. 4. Effects of subcutaneous injections of procaine in the pelvic region (A) or flank and back region (B) of the female rat on performance of lordosis by the female (mean lordosis quotient) in response to mounts by the male, and on achievement of penile insertion by the male (mean intro- mission quotient). The results of each series of procaine injections are shown in tables to the right. Females were given either single injections of 2 #g estradiol benzoate, or daily injections of 10 #g. At each site marked ×, 0.25 ml of 2 % procaine hydrochloride solution or the saline control was injected. All treatments were bilaterally symmetrical.

day), leading to stronger lordosis performance, also led in turn to more frequent intro- missions by the male (Fig. 4).

I f contact by the male's forepaws and forearms on the female's flanks, and con-

tact by the male hindquarters on the female's rump and perineum are considered as two 'categories' o cutaneous stimulation by the male, a third category is intravaginal

stimulation by penile insertion. The importance of this kind of stimulation for lordosis was tested by preventing intromission with local anesthesia of the penis 1,6,~4. Sub- cutaneous injections, 0.25 ml of 2 % procaine hydrochloride at each site, were given to sexually experienced male rats (n = 8) to anesthetize the dorsal nerve of the penis.

Two injections were given on each side of the base of the penis. Tests 8 rain in duration were begun 6-9 min following treatment of the male. Ovariectomized females (n = 12)

were primed two days before testing with 2 fig EB ,and 0.5 mg P was injected 4 h before lordosis tests. Under these conditions, local anesthesia of the male's penis abolished penile insertion (intromission quotient decreased to 0) but the lordosis quotient did not decrease (Table II). Thus, intravaginal stimulation from penile in-

sertion by the male rat is not a critical element in the stimulation of lordosis.

Under normal conditions, the performance of lordosis by the estrous female rat is significantly correlated to the performance of intromission by the sexually ex-

60

TABLE I1

EFFECTS OF MALE'S PENILE ANESTHESIA WITH PROCAINE ON LORDOSIS BY FEMALE AND ON Pt:NILE INSt R IION

( 'INTROMISSION') BY MALE (MEAN S.E.M.)

Lordosis quotient lntromission quotient

Mounts \ Mounts .

Treatment o f male: Penile procaine 56 ~ 7 0* Control (no injections) 34 ~ 8 15 ~: 3

Procaine vs. control, *P < 0.01

perienced male rat (see Fig. 8 in ref. 21). This could not be due to intromission causing lordosis, because prevention of intromission does not prevent lordosis (Table II), and because of other reasons based on the timing of lordosis and on the response of the female to simple mounts with thrusts (see pp. 270-271 of ref. 21). On the other hand, since treatments of the female which affect the performance of lordosis - - namely, desensitization of the tailbase and perineal area, which reduces lordosis, and increased estrogen dosage, which increases lordosis-- also affect performance of intromission by the male, it seems likely that the reason for the correlation between the occurrences of lordosis and intromission is that lordosis allows intromission.

DISCUSSION

Location of cutaneous receptors important for lordosis To trigger the lordosis reflex of the female rat, the male rat first touches the

female's flanks (with his forearms and forepaws) and sometimes touches her back with his nose or chin. Then, by stepping forward with his rear legs, he contacts her tailbase and rump areas and begins pelvic thrusting against her tailbase and perineum 2°;2t. Finally, after the female is in the lordosis posture, he achieves penile insertion, thereby providing intravaginal stimulation.

Among the regions of cutaneous stimulation provided by the male rat, posterior rump, tailbase and perineal stimuli are the most important for lordosis. Within that region, removal of a small field of receptors, by cutting the pudendal nerve, has an effect on lordosis under certain hormonal conditions (Table I). Desensitization of the perineum, tailbase and posterior rump regions, by surgical denervation or by procaine injection, leads to a decrease of about 50 ~ in lordosis quotient in response to the male rat (Figs. 2A and 4A). Enlarging the field of surgical cutaneous denervation by extending the operation forward on the dorsal side of the body (Figs. IA and 2BIC) increases the deficit in the lordosis reflex. The small skin region whose desensitization most effectively extended the deficit produced by perineum-tailbase-posterior rump denervation was the vertical band of skin on the lateral aspects of the flank and abdomen (touched by the male rat's forearms and forepaws during palpation).

61

Desensitization of this region added to perineum-tailbase-posterior rump desensitiza- tion (Figs. 2F and 3) leads to large deficits in the lordosis reflex, considering the rela- tively small area of skin treated.

In contrast, desensitization of skin on the dorsal thorax by nerve section (ex- periment II1) or by skin separation from muscle (Kow and Pfaff, unpublished observa- tions) did not affect lordosis. Neither did prevention of intravaginal stimulation reduce lordosis (Table ll). Since the thorax denervation was large, and yet did not affect lordosis, denervation effects elsewhere can be attributed to the specific contributions of those skin regions rather than to the mere fact of surgery.

Visual, auditory and olfactory stimuli coming from the male rat apparently are not essential for triggering the lordosis reflex. Before cutaneous denervation, blinded, deafened, and anosmic female rats performed lordosis frequently, and the effects of cutaneous denervation in these animals were roughly comparable to effects in females which had normal sight, hearing and smell (Fig. IA and B).

The two methods of cutaneous desensitization used in these experiments gave comparable results. That is, desensitization of the perineal, tailbase and posterior rump region by procaine injection (Fig. 4A) or by surgical cutaneous denervation (Fig. 2A) both gave about a 50 ~ decrease in lordosis quotient. Adding to this region of desensitization similar treatment of the flank and lateral abdomen region contacted by the male's forearms and forepaws increased the deficit in lordosis quotient to about 80~o, whether the desensitization was achieved by procaine (Fig. 3) or surgical de- nervation (Fig. 2F).

These results show maximum lordosis deficits from treatment of skin areas not only which are touched by the male rat during mating 20, but also which must be touched during manual stimulation by the experimenter to elicit the lordosis reflex: the flanks, the posterior rump, the 'notch' by the tailbase between the vertebral column and the rear leg, and the perineumS, 21. Taken together, film observations of the lordo- sis reflex, observations from manual stimulation, and the present desensitization ex- periments generate a model of sensory control over the lordosis reflex zl,2z in which the earliest responses by the female rat to the first cutaneous stimuli from the male allow the male to apply subsequent stimuli. The resulting chain of stimuli and responses by the male and female are not only important for the female's reflex progression to the lordosis posture, but also are important for allowing the male to achieve penile in- sertion.

Comments on methodology

The operations used to produce cutaneous anesthesia can be described precisely but the skin region actually desensitized may vary somewhat from what was intended. With surgical nerve section, if a nerve passing through a corner of the region treated is cut on its way to another region of innervation, then the desensitization would be wider than intended. If, on the other hand, nerves enter the skin outside the region of surgical treatment and enter the treated area in the skin itself, the region of desen- sitization would be smaller than intended. With injections of procaine,uncertainty about the exact extent of desensitization comes from our lack of knowledge about exactly

62

how far the procaine diffused beneath the skin. One concern about the procaine treat- ment, that it might directly affect muscles involved in lordosis rather than having a purely sensory effect, turns out not to be justified, since the effects of procaine ireat- ment on lordosis agreed with the effects of surgical cutaneous denervation. Finally. in preliminary experiments (Kow and Pfaff, unpublished observations) we found that less effective means of cutaneous desensitization, such as by merely lifting the skin off the muscle, lead to correspondingly smaller deficits of lordosis quotient, compared to the effects of actual cutaneous nerve section. Just lifting the skin off the muscle fails to remove extensive innervation coming from around the side of the operation through the skin 17.

While the manipulations used would ideally produce a total loss of sensation in the skin regions treated, this ideal situation may not always be the case. In the experi- ments of HeadV~,~zL following cutaneous nerve section in man there would be a center region which was totally anesthetized, but this region was surrounded by skin on which light stimuli could give poorly localized, diffuse, disagreeable sensations ('proto- pathic sensibility'). In our present experiments, data on the differential localization of critical regions of skin would still be valid, even if these paresthesias did play some part. It would appear that the main importance of abnormal sensations during nerve regeneration would be to affect the shape and meaning of recovery curves. This was one reason that we chose, tbr the most valid comparison, to present results l¥om the last day before surgical denervation and the first day after surgical denervation.

It is interesting that definition of the sensory requirements for lordosis as precise as given by the experiments above depends to some extent on the doses of hormones used to prime female rats, and on factors influencing the hormone sensitivity of those females. When the dose or effectiveness of hormone was markedly increased, sensory requirements for lordosis were to some extent decreased. With cutaneous denervation of the posterior rump, tailbase and perineum (Fig. 2A), smaller animals with higher estrogen doses suffered a less severe loss of lordosis from cutaneous desensitization. A similar point could be made with the results of pudendal nerve section (Table I). During informal, preliminary experiments, also, it was noticed that high estrogen doses given to small ovariectomized female rats would reduce the effectiveness of cutaneous desensitization accomplished by surgical denervation or by procaine in- jections. Finally, in a recent experiment using manual stimulation of lordosis in ani- mals given different estrogen and progesterone doses, we observed that increasing hormone doses would compensate ['or decreased strength of stimulation s. The mechan- isms of these 'trading relations' between sensory input and hormone level for trigger- ing lordosis are as yet unknown. They could in part be peripheral, with higher estrogen dosage increasing the sensitivity of cutaneous receptors ~a, but it is also likely that central integrative mechanisms play an important role.

Nature oJ'receptors Jor lordosis reflex in J~,male rat Since removal of the distance receptors for sight, hearing and olfaction did not

interfere with the ability of the female rat to perform lordosis (see preoperative data in Fig. 1 ) and since taste is presumed not to be important, somatosensory stimuli must

63

be of primary importance. Film observations and the sufficiency of somatosensory manual stimuli for lordosis support this point 2°,21. With the present results we have shown that stimuli from the male rat primarily on the posterior rump, tailbase and perineum of the female, facilitated by stimuli on a vertical band of flank and lateral abdomen, are necessary for the normal and frequent occurrence of lordosis. All of these regions are composed of hairy skin. Since cutaneous manipulations can cause very large reductions in the performance of lordosis (80-100 ~o reductions in response to the male rat or manual stimulation; see Figs. 1, 2E, 2F and 3), it appears that skin receptors themselves must be crucial for lordosis, and conversely that deep receptors, particularly those in and between muscles, are not sufficient for the normal occurrence of lordosis in a large percentage of cases. These considerations indicate that cutaneous mechanoreceptors in the hairy skin on the posterior rump, tailbase and perineum, and possibly the flanks, play a crucial role in triggering the lordosis reflex in the female rat.

Regarding the temporal characteristics of cutaneous receptors important for lordosis, some receptor types can be ruled out by considering response latency and stimulus frequency. Female rats start to raise their rump with a mean latency of 161 msec after the male's paws grasp their flanks 20. Receptors associated with C fibers require strong stimulation for at least 150-200 msec for response s. Since time must be allowed for impulse conduction, spinal neuronal integration, and development of the motor response to a point where it is visible, it seems unlikely that mechanoreceptors associated with C fibers are crucial for the initial triggering of lordosis.

All stimuli from the male on the skin regions of the female important for lordosis appear to be moving stimuli. Frame-by-frame analysis of movie films (54 frames/sec) of 132 mating encounters between 8 receptive female rats and vigorous males (Kow and Pfaff, unpublished observations) have revealed dominant frequencies of stimula- tion by the male's pelvic thrusting (against the female's posterior rump, tailbase and perineum) about 15-16/sec ( m e a n - 15.7/sec) and by the male's forepaws (on the female's ventral flanks) about 12-13/sec (mean = 12.3/sec). While these 'frequency analyses' have several limitations, they quantify what the observer sees with the un- aided eye and suggest that dominant frequencies of the male rat's stimulation on the female's skin lie between 10 and 20/sec. In view of this, it seems unlikely that Pacinian corpuscles or hair receptors of the G1 type found in cat are very important for female rat lordosis. These are very difficult to stimulate at frequencies below 40-60/sec, and indeed appear to be ' tuned' to frequencies between 100 and 400/sec (ref. 5).

Cutaneous mechanoreceptors which are not ruled out from consideration for lordosis control on the basis of frequency response or latency include hair receptors associated with Aa or A5 fibers, Iggo Type II receptors and 'field' receptors 5. It is interesting to note that recording from the pudendal nerve of the female rat has shown the existence of very sensitive fast-adapting mechanoreceptors in the skin of the perineum whose sensitivity is affected by estrogen 15.

Implications for organization of lordosis reflex With knowledge of the sensory (cutaneous) requirements for lordosis, and with

observations on the motor topography of lordosis z°,21, initial questions about lordosis

64

reflex organization can be raised. Do facts about lordosis presently available force the postulation of new spinal reflex types? Insofar as we can conclude that lordosis properties do fit with known spinal reflex patterns, this would not deny the importance of descending supraspinal, hormone-dependent influences. We assume 2~,zz that such influences, especially from the hypothalamus, are important for facilitating appropri- ate spinal reflex patterns composing lordosis, and for adding the feature of hormone- dependence.

Reflexes to ipsilateral cutaneous stimuli have been described most carefully in the decerebrate cat 7. Most commonly observed are withdrawal reflexes, which in the case of a limb usually means flexion reflexes. One exception to the rule of ipsilateral hind limb flexion reflexes is that mechanical stimulation on the upper thigh and perineum can cause bilateral extension of the hind limbs (p. 72 of tell 7). In female rats, also, we have observed that light cutaneous stimulation of the medial surface of the thigh and/or the perineum and tailbase can caused marked bilateral hind limb extension and tailbase elevation (Pfaff and Lewis, unpublished observations) ~. A second important exception to the rule of ipsilateral extension is that cutaneous stimulation directly over an extensor muscle can cause excitation of that extensor and inhibition of the antagonist flexor muscles II. In addition, light massage of an extensor muscle itself can set up a reflex contraction presumably due to the muscle's stretch receptor reflex mechanism (p. 72 of ref. 7). Given these established spinal reflex patterns and the delineation of somatosensory receptors important for lordosis as discussed above, what can be deduced about the possible nature of sensory control over the lordosis reflex ?

The possible reflex role of cutaneous stimulation on the perineum is most clear. Observations in decerebrate cats (p. 72 of ref. 7) and in female rats (Pfaff and Lewis, unpublished observations) s show that light cutaneous perineal stimuli cause bilateral rear leg extension and tailbase elevation. In fact, the rump and tailbase elevation characteristic of lordosis following stimulation of perineum by the male, fits the pattern for a 'withdrawal reflex' (a polysynaptic reflex upon cutaneous stimulation, not necessarily flexion ; vis-a-vis a monosynaptic stretch reflex) since elevation of the perineum withdraws the skin surface from the direction of stimulation.

Stimulation on the female's tailbase and the most posterior part of the rump also is associated with movements by the female which might constitute a withdrawal reflex, since the 'notch' next to the tailbase, moving upward and finally bending slight- ly forward in the dorsiflexed posture, is moving the tailbase away from the main focus of stimulation. In addition, deep stimulation in this region by pressure from the male may stretch muscles or tendons of the deep back muscle system which executes the vertebral dorsiflexion of lordosis, thus triggering stretch reflexes in that system (Pfaff e t al. 21, and Brink, Morrell, Modianos and Pfaff, unpublished observations).

The possible reflex involvement of stimuli and ~esponses involving the female's flanks appear to be the most complicated. Pressure from the male's forearms may deform muscles, triggering stretch reflexes in the deep back longissimus dorsi system which are important for dorsiflexion of the vertebral column and thus could partici- pate in lordosis (Pfaff e t al. el and Brink, Morrell, Modianos and Pfaff, unpublished

65

observations). Second, stimulation on the flanks themselves or on the lateral abdomen might cause enough skin deformation on the anterior thigh to initiate bilateral rear leg extension as can be observed in spinal animals (p. 72 of ref. 7), or, since this vertical band of cutaneous stimulation shares dorsal root field L2 with the anterior thigh 17, reflex responses typical of thigh stimulation might thus be triggered. In any case, we have observed that rapid scratching of the flanks by an experimenter can cause slight rear leg extension or leg abduction in female rats (Pfaff and Lewis, un- published observations) s. Finally, palpation by the male's forepaws on the lateral abdomen probably stimulates the field of the genitofemoral nerve, and could help to initiate rump elevation or bilateral rear leg extension by the same types of mechanisms

used by the pudendal nerve (for the perineal region) or the nerves from the anterior thigh (p. 72 of ref. 7). Since rear leg extension and rump elevation move the posterior abdomen away from the direction of stimulation, this segment of lordosis could have the character of a withdrawal reflex.

Descriptions of lordosis reflex organization based primarily on film analysis (Fig. l0 of ref. 21) have made it clear that the lordosis posture of the female rat is the

result of a sequence of component reflexes. From the new considerations above it seems possible that lordosis is a concatenation of spinal reflex mechanisms whose existence has already been established individually in physiological studies of the cat spinal cord. According to this notion, supraspinal influences on lordosis could work by simply increasing activity in the designated spinal reflex circuits. If the types of reflex patterns suggested here do participate as component lordosis mechanisms, then there exists a good match between cutaneous stimuli from the male and spinal reflexes available in the female rat.

ACKNOWLEDGEMENTS

Supported by N I H Grant HD-05751 and by an institutional grant from the Rockefeller Foundation for the Study of Reproductive Biology.

We thank Catherine Lewis for excellent technical assistance and Gabrielle Zummer for help in preparation of the manuscript.

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