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Copulation and eating during electrical stimulation of the rat hypothalamus
Physiology and Behavior. Vol. 7, pp. 587-593. Pergamon Press, 1971. Printed in Great Britain
Copulation and Eating During Electrical Stimulation of the Rat Hypothalamus' FRIEDRICH
K. S T E P H A N , E L L I O T S. V A L E N S T E I N ~ A N D I R V I N G Z U C K E R
Department of Psychology, University of California, Berkeley, U.S.A. (Received 19 N o v e m b e r 1970) STEPHAN, F. K., E. S. VALENSTEINAND I. ZUCKEg. Copulationand eating during electrical stimulation of the rat hypothalamus. PHYSIOL.BEHAV.7 (4) 587-593, 1971.--Copulatory behavior of male rats was recorded during tests in which
hypothalamic stimulation was provided in altemating 3 rain-on, 3 rain-off sequences. The animals were also tested separately for stimulation-bound eating. Hypothalamic stimulation shortened the post-ejaculatory refractory period and decreased the interval between successive intromissions. The number of intromissions per ejaculation and the number of mounts per intromission was either unchanged or increased by electrical stimulation. Seven of 14 rats displayed stimulation-bound eating; post-stimulation inhibition of eating was observed in 3 of these animals and in 4 rats that were not stimulation-bound eaters. Our results suggest that the phenomenon of stimulation-bound copulation may be in part a reflection of post-stimulation depression in behavior rather than a specific enhancement of sexual behavior. Hypothalamic electrode sites associated with the greatest augmentations in copulatory behavior could not be distinguished anatomically from sites yielding stimulation-bound eating; the conclusion that posterior hypothalamic stimulation is more likely to elicit copulation than eating was thus not supported by our findings. Some disadvantages of the on-off brain stimulation paradigm for the analysis of copulatory behavior are discussed. Eating
Copulation
Sexual arousal
Stimulation-bound behavior
Hypothalamus
Self-stimulation
example, that non-specific stimuli such as handling and electrical shock applied to the male rat's skin can facilitate sexual behavior [1, 5, 13]. A number of investigators have attempted to induce copulation with electrical stimulation of various limbichypothalamic sites [14, 16], but with the exception of the striking increase in sex behavior shown by some male rats stimulated in the preoptic area [10, 19], electrical activation of sex behavior has fallen far short of that demonstrated for many other behaviors. In the cases of preoptic stimulation, the animals may exhibit a decrease in the number of intromissions preceding an ejaculation and in some instances a very striking increase in the number of ejaculations has been reported [10, 19]. The influence of more posterior hypothalamic stimulation on male sexual performance has been of a different nature [3, 4, 6]. Animals with posterior hypothalamic electrodes have been tested with estrous females during intermittent periods of stimulation with the result that the frequency of copulation during stimulation-on periods often greatly exceeded that during stimulation-off intervals. The elicitation of copulation with a high degree of control could be of great value in analyzing interactions among hormonal, environmental and neural factors involved in male sexual behavior. However, the sudden depression in
A NUMBER OF consummatory behaviors, including eating, drinking, gnawing, hoarding and attack, can be reliably elicited by electrical stimulation of the hypothalamus [17, 18]. In most instances the observed behavior can readily be attributed to electrical stimulation of the brain (ESB); for example, eating, which is reliably elicited in sated rats during electrical stimulation of the hypothalamus, only rarely occurs in the absence of such stimulation. Although it has been claimed that ESB can also elicit copulatory behavior in the male rat [3, 6], the demonstrations are generally not comparable. Animals usually are not tested when their sexual appetites have been completely sated and, as a result, sexual responses directed toward an estrous female are observed in the absence of central stimulation. Previous studies have often emphasized that, when intermittent ESB is presented, the male may display a preponderance of sexual activity during the stimulation periods. While these demonstrations, together with those involving peripheral stimulation [1, 5, 13], provide evidence of modification of the temporal patterning of copulatory behavior, it has not always been clear whether a concomitant increase in sexual motivation or behavior has been achieved. Furthermore, facilitation of copulatory activities may reflect an activation of the substrate specifically involved in sexual behavior, but it might also be a manifestation of more general arousal. It has been shown, for
1Supported by an NIMH predoctoral fellowship to F.K.S., NIMH grants M--4529, Career Scientist Award MH-4947 and research grant NsG-437 from NASA to E.S.V., Grant HD-02982 and Career Development Award K4 HD-42413 from NICHD, NIH Biomedical Science Support Grant FR-7006 and the Committee on Research, University of California, to I.Z. We are grateful to Sharon Robinson, Pat Salber and Barbara Case for technical assistance. 2Now at Department of Psychology and Neuroscience Laboratory, University of Michigan, Ann Arbor, Mich., U.S.A. 587
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STEPHAN, VALENSTEIN AND ZUCKER
copulation coincident with termination of ESB suggests that hypothalamic stimulation may cause a post-stimulation depression of sex behavior [4], and that it is only by comparison with this lowered performance that sex behavior seems augmented. This alternative hypothesis is consistent with the demonstration that many animals exhibit a poststimulation depression of eating even when tested following a food deprivation period [7]. In the present study we attempted to induce stimulationbound copulation; in addition, we sought to evaluate the evidence for sexual facilitation by hypothalamic ESB through a comparison with normative data and by determining the consistency among a broader spectrum of behavioral measures. We also planned to establish whether stimulationbound sexual behavior is an artifact of a generalized poststimulation depression in behavior or a true activation of specific sexual mechanisms. Furthermore, since one of us has recently emphasized that different behaviors may be elicited from the same anatomical stimulation [16], we determined whether brain stimulation which augments or inhibits copulation exerts similar effects on non-sexual behavior. METHOD
Animals These experiments were performed on 10 Sprague-Dawley and 4 Long-Evans adult male rats. The animals were housed in individual cages with free access to food and water at all times, except as indicated below. A 12-hr light-dark cycle was in effect throughout the experiments. Ovariectomized rats used in the mating tests were brought into behavioral estrus by a subcutaneous injection of 30 t~g estradiol benzoate followed by 0.4 mg progesterone approximately 36hr later. Estradiol benzoate (Progynon) and progesterone (Proluton) were generously supplied by the Schering Corporation, Bloomfield, N.J. Prior to each test the female's receptivity to a non-experimental indicator male was established. Only females displaying high levels of receptivity were used in the mating tests.
Surgery Two bipolar stainless-steel electrodes (Plastic Products, Roanoke, Virginia, MS-303-0.018) insulated except for the tips were implanted under methoxyflurane (Metofane) anesthesia in males that had previously been selected for vigorous copulatory behavior. With the dorsal surface of the skull level between bregma and lambda, stereotaxic coordinates for the lateral hypothalamic area (LHA) were 3-3.5 mm posterior to bregma, 1.8 mm lateral to the midline and 8.8 mm below the top of the skull. The corresponding coordinates for a second electrode aimed at the posterior hypothalamic area were posterior 4-4.5 mrn, lateral 1.2 mm and vertical 9 ram.
Apparatus and Behavioral Categories The electrical stimulus was a sine wave passed through a 1.0 M f~ resistor in series with the animal to keep current levels approximately constant. Current levels were monitored and calibrated on an oscilloscope and peak values ranged from 10-60 o~A. Mating tests were conducted in a semi-circular arena with a Plexiglas front to permit observations (radius 18 in., height 16 in.). W o o d shavings covered the floor of the arena
during copulation tests; only food and water were available during eating-drinking tests. Illumination of the arena was provided by a dim white overhead light; mating tests were conducted 3-5 hr after the onset of the dark phase of the illumination cycle. Males were habituated to the arena for about 1 2 m in before the female was introduced. During control tests, males were connected to the stimulation wires but no current was passed through the electrodes. The male's sexual behavior was recorded in terms of mounts, intromissions, and ejaculations. In brief, a mount was scored when the male placed his forepaws on the female's hindquarters while oriented posteriorly to her; mounts were often, but not always, accompanied by pelvic thrusting. Intromissions were similar to mounts and included penetration into the vagina. Ejaculations were distinguished from intromissions by the greater depth of the final thrust and by the longer duration of the ejaculatory penetration. An EsterlineAngus multiple channel event recorder was used to record these 3 separate components of sexual behavior. Several derived measures were also used in the behavioral analyses: the post-ejaculatory interval (PEI), defined as the time in see from ejaculation to the next intromission; the number of intromissions per ejaculation (I/E); and the mean interval in see between successive intromissions (III). In addition, as a measure of the efficiency of the sexual behavior, the number of mounts per intromission (M/I) was tabulated for each animal. The tests for self-stimulation of the brain were performed in a small Skinner box with a grid floor and a lever mounted on one side. Rats were first tested for self-stimulation and current levels which supported stable rates without producing motor side-effects were recorded. All animals were retested for self-stimulation at the end of the experiment. Only rats with electrodes that supported self-stimulation behavior on both tests were included in the present experiment. None of the rats self-stimulated at both electrode sites.
Histology At the completion of the experiment, animals were anesthetized and perfused with saline and formalin. Frozen brain sections were stained with cresylecht violet and examined for localization of electrode tips.
Pre-test Procedures Twelve of the 14 rats served as subjects in a preliminary experiment in which parameters of brain stimulation were varied during sex behavior tests. All males were also tested for stimulation-bound eating and drinking with only food and water present in the mating arena. During these tests the current was on for 0.5 min and off for 1 min in alternating sequences. Animals received 4 such tests, each 1 hr in duration, and one 8-hr test in which current was on for 0.5 rain and off for 4.5 min in alternating sequences. If stimulation-bound behayior was not apparent at the end of these tests, the rat was designated a nonstimulation-bound eater and drinker. At least two weeks intervened between pro-test and test procedures.
Test Procedures Sexual behavior. During mating tests ESB was delivered automatically in the form of a continuous sine wave in alternating 3 rain-on, 3 min-off sequences. Half of the males received 2 current-on periods and the other half 2 current-on plus one current-off period prior to introduction
COPULATION, EATING AND BRAIN STIMULATION of the estrous female into the arena. This procedure was chosen because most animals were somewhat hyper-reactive during the initial ESB period and this could have interfered with copulation or increased the variability of the results. In addition, introduction of the female at different times ensured that half of the sex tests began with a current-off period and the remainder with a current-on period. Sex tests were terminated when the male completed an intromission following the fourth ejaculation. Each animal was given two control sex tests and one sex test with intermittent brain stimulation. Eating. During these tests animals received 20 trials of 0.5 min continuous sine wave stimulation followed by 1 rain of no stimulation. Prior to the first formal test for stimulationbound eating and drinking (Test 3, Table 1) animals were maintained on an ad lib food and water schedule. The highest current levels were determined at which eating was elicited in these sated rats without producing motor side-effects or excessive activation. These current levels were considerably lower than those necessary to support stable self-stimulation behavior. Current levels for animals that did not display stimulation-bound behavior were selected by observing these rats until searching and exploratory behavior was elicited by ESB. These current levels were used in subsequent tests (Tests 4 and 5, Table 1). Prior to Test 5, rats were deprived of food but not water for 24 hr in order to assess post-stimulation effects on eating. All animals were allowed to eat for 2 rain before the first ESB trial. As in Test 3, rats were given 20 trials of 0.5 rain ESB followed by 1 min of no stimulation. After the last of the ESB trials, rats were observed for an additional 5 rain to determine whether they would eat. The logic underlying this procedure is that in the absence of inhibitory effects produced by the stimulation, eating should occur in fooddeprived rats during both stimulation-on and stimulation-off periods. This paradigm is analogous to that for sexually rested male rats who, in the absence of inhibitory effects, could be expected to copulate during current-on and currentoff periods. The sequence of 7 tests given all experimental males is summarized in Table 1. A constant interval (2 days) between ESB tests was used to minimize variability produced by
TABLE 1 TESTINO SEQUENCEFOR MALE RATS
Tests 1
Inter-Test Interval
Self-Stimulation Variable
2 Control Se~ Test 6 days 3 Stimulus-Bound Eating (Sated) 2 days 4 Sex Test with ESB 2 days 5 Stimulus-Bound Eating (24 hr Deprivation) 6 days 6 Control Sex Test Variable 7 Self-Stimulation
589 possible threshold changes at the electrode tip as a function of time [20]; sex tests were separated by 8 days to minimize possible sexual exhaustion effects [2]. The intervals between Tests 1 and 2 and between Tests 6 and 7 were variable.
RESULTS
Copulatory Behavior Correlation with stimulation.
The effects of ESB on copulatory behavior were not as pronounced as those reported previously [4], but in general the frequency of sexual activity during stimulation-on periods exceeded that during stimulation-off periods. Indeed, one rat (No. 70) displayed all of its sexual activity during the stimulation-on periods. Twelve of 14 rats had on-to-off mount ratios exceeding 1.0, 10 had ratios above 2.0, and 6 of these animals produced ratios above 3.0. However, only 1 of the 14 rats had an on-to-off intromission ratio greater than 3.0; on/off ratios for ejaculation were also much less striking in the present study. Mounts/intromissions. The number of mounts per intromission (M/I), which is one measure of reproductive efficiency, was analyzed for current-on and current-off periods and compared to values for the control tests. The M/I measure was considered to be significantly affected by ESB only if current-on and current-off values were more than twice or less than half those for the control periods. Eight of 14 rats showed a significant increase in the number of mounts per intromission during current-on periods; a comparable increase during current-off periods was observed for 2 of these rats. M/I ratios were decreased in only 1 rat during current-on periods and for 6 animals during current-off periods (Table 2). Inter-intromission interval. The mean interval between successive intromissions (IlI) during current-on periods (III-on) was less than the HI during the first control test (Test 2) for 13 of the 14 rats (p < 0.01). The reduction exceeded 30 per cent for 9 of the rats. When animals were retested in a second control test (Test 6), the mean IIIs of 7 rats were similar to those for their initial control test (:k15 per cent); for 2 rats the mean l~IIs increased by 35 per cent. The IIIs of the remaining 5 rats, however, were reduced during the second control test to levels comparable to those for the current-on periods of Test 4. Whether the reduction in IlIs for these animals represents a long-term effect of ESB cannot be assessed on the basis of the present data, but the results do illustrate the necessity of adequate control procedures in assessing the effects of ESB on sexual behavior. The post-stimulation effects of ESB were assessed by comparing the mean I I I during the stimulation-on, stimulation-off, and control periods. Post-stimulation effects were considered to have occurred only if the stimulation-on and -off scores differed from each other and each of these differed from the control period scores. The results were not consistent for all animals. We inferred that rats (Nos. 10, 209, 175, 70) exhibiting HI-on < III-C < III-off were demonstrating post-stimulation inhibition of sexual behavior. This inference is indirectly supported by the substantial increase in the standard deviations of the HI during the off period for these rats. F o r 4 rats (Nos. 0, 205, 6, 72) we inferred post-stimulation facilitation as IIt-off < III-on < III-C. We did not assess post-stimulation effects in terms of on-off ratios since comparison to control values is difficult and not necessarily meaningful for this measure. Intromissions/ejaculation. The number of intromissions
590
STEPHAN, VALENSTEIN AND ZUCKER TABLE 2 SEXUALPERFORMANCE(MOUNTS,INTROMIgSION$,EJACULATIONS)AND EATINGDURINGCONTROLAND STIMULATION(ESB) TESTS
On/Off Ratios1" Mounts Intro. Ejac. Rat No. I0" 209* 175" 70 0* 205 6 72 226* 204* 136* 186 163 201
12/5 20•4 12/3 26/0 17/11 3/14 14/3 12/3 30/10 17/4 8/3 6/1 9[7 12/15
13/8 10/8 5/7 8/0 3/10 7/16 13/11 6/6 12/15 8/9 14/7 11/4 12/8 12/10
3/1 3/1 2/2 4/0 2/2 2/2 2/2 4/0 3/I 2/2 3/1 3/1 0/4 2/2
Inter-Intro. Interval Control ESB Test 2 Test 6 Test 4 On Off M SD M SD M SD M SD 30 57 52 106 51 57 40 86 90 26 66 60 79 58
16 35 23 48 19 32 25 120 59 10 26 31 31 33
27 77 49 102 22 52 34 27 53 36 37 68 84 38
12 24 17 61 7 21 21 17 22 23 25 23 32 13
23 47 19 48 34 50 21 48 45 26 30 17 38 42
21 26 4 31 20 30 14 22 18 13 23 6 15 10
43 64 60 -29 34 15 35 68 28 42 51 35 47
34 46 32 -17 17 5 18 56 5 31 25 20 28
Mounts per Intro. Eating ( # out of 20):~ Control ESB After Deprivation Tests 2+6 Test 4 on/off Stim.-on Stim.-off
0.4 2.8 1.1 0.5 1.5 1.4 0.4 1.2 2.4 0.3 1.0 0.8 0.3 0.5
0.9/0.6 2.0/0.5 2.4/0.4 3.3/-5.7/1.1 0.4/0.9 1.1/0.3 2.0/0.5 2.5/0.7 2.1/0.4 0.6/0.4 0.6/0.3 0.8/0.9 1.0/1.5
20 20 14 0 20 0 1 0 18 20 20 0 12 7
0 1 15 3 1 2 17 7 19 18 11 4 16 15
*Stimulation-boundeaters (Validated on Test 3). 1"Data summed for all stimulation-on and off periods (Test 4). ,Animals were deprived of food for 24 hr prior to Test 5 and were administered twenty 30-sec stimulation trials and twenty l-rain interstimulus trials in the presence of food. M--Mean. SD--Standard deviation of the mean.
preceding ejaculation (ejaculation threshold) has frequently been used as an index of sexual efficiency [2]. The on-off paradigm used in the present and previous studies [4] complicates data analysis with respect to this measure since the ejaculation latency almost always exceeds 3 rain and therefore overlaps stimulation-on and stimulation-off periods. It is possible, for example, that a reduction in ejaculation threshold induced by ESB might be off-set by post-stimulation inhibitory effects. Nevertheless, the results of the present study suggest that ESB in no case produced a dramatic change in the number of intromissions required to produce ejaculation. Post-ejaculation interval For 9 of the 14 rats the mean duration of post-ejaculatory refractory periods was reduced by ESB even though some portion of the post-ejaculatory period may have overlapped a current-off interval. Males were frequently observed pursuing females very soon after an ejaculation that occurred during a current-on period or immediately after the onset of ESB when ejaculation occurred during current-off intervals. In the absence of brain stimulation the males adopted the post-ejaculatory resting posture (e.g. [9]). The mean interval between ejaculation and the next intromission (PEI) was substantially reduced in 6 of the 14 rats as compared to the average PEI for the two control tests (range of decrease, 14-36 per cen0. The mean latency between ejaculation and the first post-ejaculatory mount was even more substantially reduced for 9 of the rats (range 14-64 per cen0.
Eating. Seven of the 14 rats were stimulation-bound eaters (SBE). When tested while sated, six of these rats ate on at least 19 of the 20 current-on trials and on no more than one of the current-off trials (Test 3). One rat (No. 175) ate on 12 current-on trials and on one current-off trial. One animal (No. 204) displayed stimulation-bound drinking on a preliminary test but had switched to stimulation-bound eating by the present test. This rat occasionally drank during ESB periods; however, only eating responses were scored for all rats. When food deprived (Test 5), three of these animals (Nos. 10, 209, 0) showed strong post-stimulation inhibition of eating; they ate during all 20 current-on trials but on not more than one current-off trial, even though they had been without food for a 24-hr period prior to testing. Four of the 7 non-SBE (70, 205, 72, 186) showed inhibition of eating during both current-on and current-off periods, while rat No. 6 exhibited inhibition of eating during current-on periods only. All animals in which post-stimulation inhibition of eating occurred resumed eating 3-5 min after the last stimulation period and were observed eating continuously for several minutes. It should be noted that all rats had also been observed eating for 2 min prior to the first stimulation period. Four of the SBE rats and 3 non-SBE rats did not display post-stimulation depression of eating. Self-stimulation. As all rats used in the present experiment were selected on the basis of positive self-stimulation performance it was not possible to determine whether stimulusbound behavior could be observed in non-self-stimulating
COPULATION, EATING AND BRAIN STIMULATION
animals. Current values for self-stimulation were always higher than those used during the remaining tests.
Comparison Between Measures Copulation. An analysis of the data summarized in Table 2 reveals the absence of consistent relations among the different measures of sexual performance. For example, rats showing the greatest decrease in III frequently required more intromissions per ejaculation than during control tests while the post-ejaculatory interval was either decreased or unchanged. High on/off ratios for mounts occurred without a substantial reduction in the HI (Nos. 204, 209). Rat 163, on the other hand, had a very low on/off ratio (all ejaculations occurred during stimulation-off periods) but showed a 50 per cent reduction in the III. In short, While ESB generally decreased the ItI and PEI, and resulted in high on/off ratios for mounts, in many animals these changes were not correlated.
Comparison of post-stimulation effects on eating and copulation. Three of 4 animals showing post-stimulation depression of copulatory behavior (Nos. 10, 209, 70, the exception 175) as inferred by an increase in III also displayed inhibition of eating during post-stimulation periods. A similar relation was observed for 5 of the 6 rats (Nos. 10, 209, 70, 72, 186, the exception 136) exhibiting the largest on-off sex ratios after combining scores for mounts, intromissions and ejaculations. During the twenty l-rain current-off periods these rats occasionally groomed themselves; but in spite of the 24-hr food deprivation period, the food pellets tended to be ignored. In the case of rats 70, 72 and 186 stimulation also interferred with eating during the stimulation periods. Thus, with some hypothalamic electrodes there may be a general post-stimulation inhibition of consummatory behavior, but during stimulation different behaviors may not be affected equally. In the sex tests, which had a longer currentoff period (3 min), some copulatory activity was observed (except for No. 70), but the inter-intromission intervals were longer and irregularly spaced, giving the impression of a general disruption of copulation. There was no tendency for the converse to be true, however, as animals did exhibit post-stimulation inhibition of eating without any comparable inhibition of copulation. It would appear, therefore, that post-stimulation inhibition of eating and copulatory behavior is not congruent; but there may be a tendency for stimulation, which produces an inhibitory after-effect in a sex test, to also produce inhibition in an eating test. Anatomical analysis. An extensive analysis of the hypothalamic sites capable of eliciting copulatory behavior and eating is not possible with a sample of only 14 electrodes. In spite of this limitation some anatomical observations may be helpful. There has been a tendency to view posterior hypothalamic sites as more likely to elicit copulatory behavior [3, 4, 6], and more rostral sites, as, for example, the perifornical area, as having a greater probability of eliciting eating. No support for this view can be found in our sample (Fig. 1). Of the 6 animals exhibiting the highest on/off ratios in sexual behavior, four electrodes were located at A-P planes containing the ventromedial and/or arcuate nucleus; only two were located more posteriorly. Rat 70, for example, had the highest on/off ratio, but one of the most anterior placements in our sample (cf. Fig. 1). Four of the 8 animals displaying stimulus-bound eating had posterior hypothalamic electrodes located at planes in which the mammillary bodies were evident. No pattern could be discerned for the electrode locations producing post-stimulation inhibition of behavior or specific effects on other copulatory measures.
591 DISCUSSION
The reports [3, 4, 6] that electrical stimulation could activate a copulation reward site have given rise to the hope that it might be possible to identify and to study the properties of the neural structures controlling sexual motivation and/or performance. One of the major lines of support for this conclusion is derived from the finding that male rats receiving intermittent posterior hypothalamic stimulation exhibit most of their sexual activity during stimulation rather than nonstimulation periods. We have attempted to elaborate on this phenomenon by extending the range of observations to the standardized measures of sexual behavior that have been well studied in the past. The lack of agreement among different measures of male sexuality has to be regarded as disappointing. Although 10 of the 14 rats exhibited twice the number of mounts when the stimulation was on, compared to when it was off, there was no comparable effect on intromissions or ejaculations. Hypothalamic stimulation also increased or left unchanged the number of intromissions per ejaculation in 11 of the 14 rats. This finding argues against a general facilitation of all components of copulatory behavior. General increases have been reported [10, 19] from electrical stimulation of the preoptic area and following large electrolytic lesions which destroyed portions of the posterior hypothalamus, posterior medial thalamic nuclei, and the rostral part of the central grey matter in the mesencephalon [11]. In contrast to the present findings, lesioned male rats ejaculated after relatively few intromissions and with a shorter latency, and were sexually refractory for shorter intervals following ejaculation [11]. The increase in the number of mounts per intromission recorded during stimulation periods in the present study suggests that instead of augmenting sexual performance, ESB may have interfered with the efficiency of copulation, perhaps by excessively arousing the animals. The measures that seemed most influenced by hypothalamic stimulation were the inter-intromission interval and the post-ejaculatory interval. In general, the inter-intromission interval was decreased by stimulation in most rats. This seems to be true even though in some instances a second control test given after the stimulation test suggests either a lasting effect of stimulation or a change in performance over time (which would have exaggerated the facilitating influence of stimulation). However, the fact that there were a number of animals that exhibited a decrease in III during stimulation periods that did not also show a significant "stimulus-bound behavior" effect (as reflected in the on/off ratio), suggests that the relation between these measures is not strong. Overall, the results suggest that hypothalamic stimulation tended to facilitate the rate of sexual activity, to shorten the time of inactivity during post-ejaeulatory refractory periods, and to modify the temporal patterning of the behavior, although not all aspects of sexual behavior were equally affected. In a recent study [14] involving lateral preoptic area stimulation it was reported that there was general agreement among measures of sexuality, but the behavior was primarily analyzed in terms of its temporal course. Response rate measures also predominated in other studies [4]. With the two exceptions noted [10, 19] involving the preoptic area, the amount of copulatory behavior displayed by male rats has not been altered by brain stimulation. Since the variables which are affected by hypothalamic stimulation (PEI, Ill) are also changed by a number of types of nonspecific stimulation [1, 5, 13], investigators have attempted
592
STEPHAN, VALENSTEIN AND ZUCKER f
(
•
t "! . .;,,.,'> t ,
~-:.-..' ~; 136
7O
204
0
2O9 72 186
163
205 6
I0
226
FIG. 1. Histological identification of hypothalamic sites at which electrical stimulation affected eating and copulation of male rats. Brain sections are modified from Konig and Klippel [12]; only those loci designated by triangles yielded stimulation-bound eating. All sites supported selfstimulation behavior.
to provide other evidence that brain stimulation was specifically affecting sexual behavior. To date this evidence, consisting of data indicating that stimulated animals will press a lever to obtain ~ s to an estrous female [4, 6] or that self-stimulation rates can be influenced by steroid hormone manipulations [4, 15], has been preliminary, and in our opinion far from conclusive in demonstrating a correlation either with a discrete hypothalamic area or the facilitation of sexual behavior. Since only 1 rat in our sample of 14 exhibited convincing stimulation-bound copulation, we cannot claim to have shown that this phenomenon is a general consequence of post-stimulation depression in sexual behavior. We do, however, consider this to be the case. The existence of animals such as Rat 70, which did not exhibit any sexual behavior during the inter-stimulus intervals, makes it abundantly clear that an inhibitory process may be triggered with the offset of the stimulation. This inhibitory process does not affect all behaviors equally, as tbe blocking of eating and copulatory activity during the inter-stimulus intervals was not always consistent. Again Rat No. 70 served as an illuminating example as this animal, which displayed all its sexual behavior during the stimulation periods, did not eat at all when stimulated in Test 5 even though it had been food-deprived. It should be noted that several animals which did not show stimulation-bound eating nevertheless
provided strong evidence for post-stimulation inhibition of eating (Nos. 70, 205, 72, 186)• O~ber animals (e.g., No. 0) displayed post-stimulation inhibition in the eating test, but very little evidence of such inhibition in the sex test. Some of this discrepancy might be attributed to differences in sexual and hunger motivational levels or the unequal compellingness of the goal objects (receptive female and food) for an individual male, but it could not be argued, convincingly, that if these variables could be controlled, perfect congruence would h a w resulted. In general, the results suggested that the estrous female was the more compelling stimulus, and if post-stimulation inhibition was evident during the sex test, it was likely, but not inevitably, to be present during the food test. In a recent summary [8] of the neural sites at which electrodes can elicit eating, drinking and gnawing, the conclusion was drawn that those areas are widespread throughout the rat hypothalamus. Although there were some hypothalamic sites that did not appear to be capable of ¢liciting the behaviors studied, it was noted that throughout the positive areas there did not seem to be distinct regions that were more likely to activate one behavior than another. The anatomical data obtained from this study indicate that within the hypothalamic areas explored (preoptie, anterior and medial hypothalamic areas were not in our sample) sites that are capable of producing changes in
COPULATION, EATING AND BRAIN STIMULATION
593
copulatory behavior are not easily distinguishable from stimulation-bound eating sites. None of the arguments advanced above should be inferred to mean that we are suggesting either that all hypothalamic stimulation produces the same general effect or that a distinct neural system subserving sexual motivation and performance may not exist. Clearly, all hypothalamic stimulation does not have the same effect as there exist too many cases in the literature of different behavioral results being produced from different sites in the same animal. Furthermore, there are many cases that have been reported (including the present account) of stimulation from the same electrode having different consequences for different behaviors. It may be that some central stimulation shares with some peripheral stimulation (handling, shock to tail or back) the capacity to elevate the response rate and alter the temporal pattern of male sexual behavior without specifically involving the neural
elements that normally participate in sexual activity. One conclusion forced upon us by the present study is that the technique of presenting intermittent periods of stimulation during observations of sexual behavior has some serious shortcomings. Unlike the situations demonstrating stimulus-bound eating or drinking, the operant level of male sexual behavior in the presence of a receptive female is not close to zero. The abnormally low amount of sexual behavior often displayed during the alternating, non-stimulation periods probably results from an active inhibitory process. Consequently, comparisons between stimulation and nonstimulation periods may be misleading. Furthermore, in contrast to most other behaviors studied, sexual behavior involves a longer and more serially dependent sequence of responses, that cannot be arbitrarily broken up into stimulation and non-stimulation periods without producing interactive effects that may be difficult to interpret.
REFERENCES 1. Barfield, R. J, and B. D. Sachs. Sexual behavior: stimulation by painful electric shock to the skin of male rats. Science 161 : 392-394, 1968. 2. Beach, F. A. and L. Jordan. Sexual exhaustion and recovery in the male rat. Q. Jl exp. Psychol. 8: 121-133, 1956. 3. Caggiula, A. R. Specificity of copulation-reward systems in the posterior hypothalamus. Proc. Am. Psychol. Ass. 2: 125-126, 1967. 4. Caggiula, A. R. Analysis of the copulation-reward properties of posterior hypothalamic stimulation in male rats. J. comp. physiol. Psychoi. 70: 399-412, 1970. 5. Caggiula, A. R. and R. Eibergen. Copulation of virgin male rats evoked by painful peripheral stimulation. J. comp. physiol. Psychoi. 69: 414-419, 1969. 6. Caggiula, A. R. and B. G. Hocbel. "Copulation-reward site" in the posterior hypothalamus. Science 153: 1284-1285, 1966. 7. Cox, V. C., J. W. Kakolewski and E. S. Valenstein. Inhibition of eating and drinking following hypothalamic stimulation in the rat. J. comp. physiol. Psychol. 68: 530-535, 1969. 8. Cox, V. C. and E. S. Valenstein. Distribution of hypothalamic sites yielding stimulus-bound behavior. Brain Behav. Evolut. 2: 359-376, 1969. 9. Dewshury, D. A. A quantitative description of the behavior of rats during copulation. Behaviour 29: 154-178, 1967. 10. Dis, H. van and K. Larsson. Induction of sexual arousal in the castrated male rat by intracranial stimulation. Physiol. Behav. 6: 85-86, 1971. 11. Heimer, L. and K. Larsson. Drastic changes in the mating behaviour of male rats following lesions in the junction of diencephalon and mesencephalon. Experientia 20: 460-463, 1964.
12. Konig, J. F. R. and R. A. Klippel. The Rat Brain: d Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams and Wilkins, 1963. 13. Larsson, K. Non-specific stimulation and sexual behavior in the male rat. Behaviour 20:110-114, 1963. 14. Madlafousek, J., K. Freund and I. Grofovfi. Variables determining the effect of electrostimulation in the lateral preoptic area on the sexual behavior of male rats. J. comp. physioL Psychol. 72: 28--44, 1970. 15. Olds, J. Effects of hunger and male sex hormone on selfstimulation of the brain. J. comp. physiol. Psychoi. 51 • 320-324, 1958. 16. Roberts, W. W., M. I. Steinberg and L. W. Means. Hypothalamic mechanisms for sexual, aggressive and other motivational behaviors in the opossum. J. comp. physiol. Psychol. 64: 1-15, 1967. 17. Valenstein, E. S., V. C. Cox and J. W. Kakolewski. Reexamination of the role of the hypothalamus in motivation. Psychol. Rev. 77" 16-31, 1970. 18. Valenstein, E. S. Behavior elicited by hypothalamic stimulation: A prepotency hypothesis. Brain Behav. Evolut. 2: 296-316, 1969. 19. Vaughan, E. and A. E. Fisher. Male sexual behavior induced by intracranial electrical stimulation. Science 137: 758-760, 1962. 20. Wise, R. A. Hypothalamic motivational systems: Fixed or plastic neural circuits. Science 162: 377-379, 1968.
Copulation and Eating During Electrical Stimulation of the Rat Hypothalamus' FRIEDRICH
K. S T E P H A N , E L L I O T S. V A L E N S T E I N ~ A N D I R V I N G Z U C K E R
Department of Psychology, University of California, Berkeley, U.S.A. (Received 19 N o v e m b e r 1970) STEPHAN, F. K., E. S. VALENSTEINAND I. ZUCKEg. Copulationand eating during electrical stimulation of the rat hypothalamus. PHYSIOL.BEHAV.7 (4) 587-593, 1971.--Copulatory behavior of male rats was recorded during tests in which
hypothalamic stimulation was provided in altemating 3 rain-on, 3 rain-off sequences. The animals were also tested separately for stimulation-bound eating. Hypothalamic stimulation shortened the post-ejaculatory refractory period and decreased the interval between successive intromissions. The number of intromissions per ejaculation and the number of mounts per intromission was either unchanged or increased by electrical stimulation. Seven of 14 rats displayed stimulation-bound eating; post-stimulation inhibition of eating was observed in 3 of these animals and in 4 rats that were not stimulation-bound eaters. Our results suggest that the phenomenon of stimulation-bound copulation may be in part a reflection of post-stimulation depression in behavior rather than a specific enhancement of sexual behavior. Hypothalamic electrode sites associated with the greatest augmentations in copulatory behavior could not be distinguished anatomically from sites yielding stimulation-bound eating; the conclusion that posterior hypothalamic stimulation is more likely to elicit copulation than eating was thus not supported by our findings. Some disadvantages of the on-off brain stimulation paradigm for the analysis of copulatory behavior are discussed. Eating
Copulation
Sexual arousal
Stimulation-bound behavior
Hypothalamus
Self-stimulation
example, that non-specific stimuli such as handling and electrical shock applied to the male rat's skin can facilitate sexual behavior [1, 5, 13]. A number of investigators have attempted to induce copulation with electrical stimulation of various limbichypothalamic sites [14, 16], but with the exception of the striking increase in sex behavior shown by some male rats stimulated in the preoptic area [10, 19], electrical activation of sex behavior has fallen far short of that demonstrated for many other behaviors. In the cases of preoptic stimulation, the animals may exhibit a decrease in the number of intromissions preceding an ejaculation and in some instances a very striking increase in the number of ejaculations has been reported [10, 19]. The influence of more posterior hypothalamic stimulation on male sexual performance has been of a different nature [3, 4, 6]. Animals with posterior hypothalamic electrodes have been tested with estrous females during intermittent periods of stimulation with the result that the frequency of copulation during stimulation-on periods often greatly exceeded that during stimulation-off intervals. The elicitation of copulation with a high degree of control could be of great value in analyzing interactions among hormonal, environmental and neural factors involved in male sexual behavior. However, the sudden depression in
A NUMBER OF consummatory behaviors, including eating, drinking, gnawing, hoarding and attack, can be reliably elicited by electrical stimulation of the hypothalamus [17, 18]. In most instances the observed behavior can readily be attributed to electrical stimulation of the brain (ESB); for example, eating, which is reliably elicited in sated rats during electrical stimulation of the hypothalamus, only rarely occurs in the absence of such stimulation. Although it has been claimed that ESB can also elicit copulatory behavior in the male rat [3, 6], the demonstrations are generally not comparable. Animals usually are not tested when their sexual appetites have been completely sated and, as a result, sexual responses directed toward an estrous female are observed in the absence of central stimulation. Previous studies have often emphasized that, when intermittent ESB is presented, the male may display a preponderance of sexual activity during the stimulation periods. While these demonstrations, together with those involving peripheral stimulation [1, 5, 13], provide evidence of modification of the temporal patterning of copulatory behavior, it has not always been clear whether a concomitant increase in sexual motivation or behavior has been achieved. Furthermore, facilitation of copulatory activities may reflect an activation of the substrate specifically involved in sexual behavior, but it might also be a manifestation of more general arousal. It has been shown, for
1Supported by an NIMH predoctoral fellowship to F.K.S., NIMH grants M--4529, Career Scientist Award MH-4947 and research grant NsG-437 from NASA to E.S.V., Grant HD-02982 and Career Development Award K4 HD-42413 from NICHD, NIH Biomedical Science Support Grant FR-7006 and the Committee on Research, University of California, to I.Z. We are grateful to Sharon Robinson, Pat Salber and Barbara Case for technical assistance. 2Now at Department of Psychology and Neuroscience Laboratory, University of Michigan, Ann Arbor, Mich., U.S.A. 587
588
STEPHAN, VALENSTEIN AND ZUCKER
copulation coincident with termination of ESB suggests that hypothalamic stimulation may cause a post-stimulation depression of sex behavior [4], and that it is only by comparison with this lowered performance that sex behavior seems augmented. This alternative hypothesis is consistent with the demonstration that many animals exhibit a poststimulation depression of eating even when tested following a food deprivation period [7]. In the present study we attempted to induce stimulationbound copulation; in addition, we sought to evaluate the evidence for sexual facilitation by hypothalamic ESB through a comparison with normative data and by determining the consistency among a broader spectrum of behavioral measures. We also planned to establish whether stimulationbound sexual behavior is an artifact of a generalized poststimulation depression in behavior or a true activation of specific sexual mechanisms. Furthermore, since one of us has recently emphasized that different behaviors may be elicited from the same anatomical stimulation [16], we determined whether brain stimulation which augments or inhibits copulation exerts similar effects on non-sexual behavior. METHOD
Animals These experiments were performed on 10 Sprague-Dawley and 4 Long-Evans adult male rats. The animals were housed in individual cages with free access to food and water at all times, except as indicated below. A 12-hr light-dark cycle was in effect throughout the experiments. Ovariectomized rats used in the mating tests were brought into behavioral estrus by a subcutaneous injection of 30 t~g estradiol benzoate followed by 0.4 mg progesterone approximately 36hr later. Estradiol benzoate (Progynon) and progesterone (Proluton) were generously supplied by the Schering Corporation, Bloomfield, N.J. Prior to each test the female's receptivity to a non-experimental indicator male was established. Only females displaying high levels of receptivity were used in the mating tests.
Surgery Two bipolar stainless-steel electrodes (Plastic Products, Roanoke, Virginia, MS-303-0.018) insulated except for the tips were implanted under methoxyflurane (Metofane) anesthesia in males that had previously been selected for vigorous copulatory behavior. With the dorsal surface of the skull level between bregma and lambda, stereotaxic coordinates for the lateral hypothalamic area (LHA) were 3-3.5 mm posterior to bregma, 1.8 mm lateral to the midline and 8.8 mm below the top of the skull. The corresponding coordinates for a second electrode aimed at the posterior hypothalamic area were posterior 4-4.5 mrn, lateral 1.2 mm and vertical 9 ram.
Apparatus and Behavioral Categories The electrical stimulus was a sine wave passed through a 1.0 M f~ resistor in series with the animal to keep current levels approximately constant. Current levels were monitored and calibrated on an oscilloscope and peak values ranged from 10-60 o~A. Mating tests were conducted in a semi-circular arena with a Plexiglas front to permit observations (radius 18 in., height 16 in.). W o o d shavings covered the floor of the arena
during copulation tests; only food and water were available during eating-drinking tests. Illumination of the arena was provided by a dim white overhead light; mating tests were conducted 3-5 hr after the onset of the dark phase of the illumination cycle. Males were habituated to the arena for about 1 2 m in before the female was introduced. During control tests, males were connected to the stimulation wires but no current was passed through the electrodes. The male's sexual behavior was recorded in terms of mounts, intromissions, and ejaculations. In brief, a mount was scored when the male placed his forepaws on the female's hindquarters while oriented posteriorly to her; mounts were often, but not always, accompanied by pelvic thrusting. Intromissions were similar to mounts and included penetration into the vagina. Ejaculations were distinguished from intromissions by the greater depth of the final thrust and by the longer duration of the ejaculatory penetration. An EsterlineAngus multiple channel event recorder was used to record these 3 separate components of sexual behavior. Several derived measures were also used in the behavioral analyses: the post-ejaculatory interval (PEI), defined as the time in see from ejaculation to the next intromission; the number of intromissions per ejaculation (I/E); and the mean interval in see between successive intromissions (III). In addition, as a measure of the efficiency of the sexual behavior, the number of mounts per intromission (M/I) was tabulated for each animal. The tests for self-stimulation of the brain were performed in a small Skinner box with a grid floor and a lever mounted on one side. Rats were first tested for self-stimulation and current levels which supported stable rates without producing motor side-effects were recorded. All animals were retested for self-stimulation at the end of the experiment. Only rats with electrodes that supported self-stimulation behavior on both tests were included in the present experiment. None of the rats self-stimulated at both electrode sites.
Histology At the completion of the experiment, animals were anesthetized and perfused with saline and formalin. Frozen brain sections were stained with cresylecht violet and examined for localization of electrode tips.
Pre-test Procedures Twelve of the 14 rats served as subjects in a preliminary experiment in which parameters of brain stimulation were varied during sex behavior tests. All males were also tested for stimulation-bound eating and drinking with only food and water present in the mating arena. During these tests the current was on for 0.5 min and off for 1 min in alternating sequences. Animals received 4 such tests, each 1 hr in duration, and one 8-hr test in which current was on for 0.5 rain and off for 4.5 min in alternating sequences. If stimulation-bound behayior was not apparent at the end of these tests, the rat was designated a nonstimulation-bound eater and drinker. At least two weeks intervened between pro-test and test procedures.
Test Procedures Sexual behavior. During mating tests ESB was delivered automatically in the form of a continuous sine wave in alternating 3 rain-on, 3 min-off sequences. Half of the males received 2 current-on periods and the other half 2 current-on plus one current-off period prior to introduction
COPULATION, EATING AND BRAIN STIMULATION of the estrous female into the arena. This procedure was chosen because most animals were somewhat hyper-reactive during the initial ESB period and this could have interfered with copulation or increased the variability of the results. In addition, introduction of the female at different times ensured that half of the sex tests began with a current-off period and the remainder with a current-on period. Sex tests were terminated when the male completed an intromission following the fourth ejaculation. Each animal was given two control sex tests and one sex test with intermittent brain stimulation. Eating. During these tests animals received 20 trials of 0.5 min continuous sine wave stimulation followed by 1 rain of no stimulation. Prior to the first formal test for stimulationbound eating and drinking (Test 3, Table 1) animals were maintained on an ad lib food and water schedule. The highest current levels were determined at which eating was elicited in these sated rats without producing motor side-effects or excessive activation. These current levels were considerably lower than those necessary to support stable self-stimulation behavior. Current levels for animals that did not display stimulation-bound behavior were selected by observing these rats until searching and exploratory behavior was elicited by ESB. These current levels were used in subsequent tests (Tests 4 and 5, Table 1). Prior to Test 5, rats were deprived of food but not water for 24 hr in order to assess post-stimulation effects on eating. All animals were allowed to eat for 2 rain before the first ESB trial. As in Test 3, rats were given 20 trials of 0.5 rain ESB followed by 1 min of no stimulation. After the last of the ESB trials, rats were observed for an additional 5 rain to determine whether they would eat. The logic underlying this procedure is that in the absence of inhibitory effects produced by the stimulation, eating should occur in fooddeprived rats during both stimulation-on and stimulation-off periods. This paradigm is analogous to that for sexually rested male rats who, in the absence of inhibitory effects, could be expected to copulate during current-on and currentoff periods. The sequence of 7 tests given all experimental males is summarized in Table 1. A constant interval (2 days) between ESB tests was used to minimize variability produced by
TABLE 1 TESTINO SEQUENCEFOR MALE RATS
Tests 1
Inter-Test Interval
Self-Stimulation Variable
2 Control Se~ Test 6 days 3 Stimulus-Bound Eating (Sated) 2 days 4 Sex Test with ESB 2 days 5 Stimulus-Bound Eating (24 hr Deprivation) 6 days 6 Control Sex Test Variable 7 Self-Stimulation
589 possible threshold changes at the electrode tip as a function of time [20]; sex tests were separated by 8 days to minimize possible sexual exhaustion effects [2]. The intervals between Tests 1 and 2 and between Tests 6 and 7 were variable.
RESULTS
Copulatory Behavior Correlation with stimulation.
The effects of ESB on copulatory behavior were not as pronounced as those reported previously [4], but in general the frequency of sexual activity during stimulation-on periods exceeded that during stimulation-off periods. Indeed, one rat (No. 70) displayed all of its sexual activity during the stimulation-on periods. Twelve of 14 rats had on-to-off mount ratios exceeding 1.0, 10 had ratios above 2.0, and 6 of these animals produced ratios above 3.0. However, only 1 of the 14 rats had an on-to-off intromission ratio greater than 3.0; on/off ratios for ejaculation were also much less striking in the present study. Mounts/intromissions. The number of mounts per intromission (M/I), which is one measure of reproductive efficiency, was analyzed for current-on and current-off periods and compared to values for the control tests. The M/I measure was considered to be significantly affected by ESB only if current-on and current-off values were more than twice or less than half those for the control periods. Eight of 14 rats showed a significant increase in the number of mounts per intromission during current-on periods; a comparable increase during current-off periods was observed for 2 of these rats. M/I ratios were decreased in only 1 rat during current-on periods and for 6 animals during current-off periods (Table 2). Inter-intromission interval. The mean interval between successive intromissions (IlI) during current-on periods (III-on) was less than the HI during the first control test (Test 2) for 13 of the 14 rats (p < 0.01). The reduction exceeded 30 per cent for 9 of the rats. When animals were retested in a second control test (Test 6), the mean IIIs of 7 rats were similar to those for their initial control test (:k15 per cent); for 2 rats the mean l~IIs increased by 35 per cent. The IIIs of the remaining 5 rats, however, were reduced during the second control test to levels comparable to those for the current-on periods of Test 4. Whether the reduction in IlIs for these animals represents a long-term effect of ESB cannot be assessed on the basis of the present data, but the results do illustrate the necessity of adequate control procedures in assessing the effects of ESB on sexual behavior. The post-stimulation effects of ESB were assessed by comparing the mean I I I during the stimulation-on, stimulation-off, and control periods. Post-stimulation effects were considered to have occurred only if the stimulation-on and -off scores differed from each other and each of these differed from the control period scores. The results were not consistent for all animals. We inferred that rats (Nos. 10, 209, 175, 70) exhibiting HI-on < III-C < III-off were demonstrating post-stimulation inhibition of sexual behavior. This inference is indirectly supported by the substantial increase in the standard deviations of the HI during the off period for these rats. F o r 4 rats (Nos. 0, 205, 6, 72) we inferred post-stimulation facilitation as IIt-off < III-on < III-C. We did not assess post-stimulation effects in terms of on-off ratios since comparison to control values is difficult and not necessarily meaningful for this measure. Intromissions/ejaculation. The number of intromissions
590
STEPHAN, VALENSTEIN AND ZUCKER TABLE 2 SEXUALPERFORMANCE(MOUNTS,INTROMIgSION$,EJACULATIONS)AND EATINGDURINGCONTROLAND STIMULATION(ESB) TESTS
On/Off Ratios1" Mounts Intro. Ejac. Rat No. I0" 209* 175" 70 0* 205 6 72 226* 204* 136* 186 163 201
12/5 20•4 12/3 26/0 17/11 3/14 14/3 12/3 30/10 17/4 8/3 6/1 9[7 12/15
13/8 10/8 5/7 8/0 3/10 7/16 13/11 6/6 12/15 8/9 14/7 11/4 12/8 12/10
3/1 3/1 2/2 4/0 2/2 2/2 2/2 4/0 3/I 2/2 3/1 3/1 0/4 2/2
Inter-Intro. Interval Control ESB Test 2 Test 6 Test 4 On Off M SD M SD M SD M SD 30 57 52 106 51 57 40 86 90 26 66 60 79 58
16 35 23 48 19 32 25 120 59 10 26 31 31 33
27 77 49 102 22 52 34 27 53 36 37 68 84 38
12 24 17 61 7 21 21 17 22 23 25 23 32 13
23 47 19 48 34 50 21 48 45 26 30 17 38 42
21 26 4 31 20 30 14 22 18 13 23 6 15 10
43 64 60 -29 34 15 35 68 28 42 51 35 47
34 46 32 -17 17 5 18 56 5 31 25 20 28
Mounts per Intro. Eating ( # out of 20):~ Control ESB After Deprivation Tests 2+6 Test 4 on/off Stim.-on Stim.-off
0.4 2.8 1.1 0.5 1.5 1.4 0.4 1.2 2.4 0.3 1.0 0.8 0.3 0.5
0.9/0.6 2.0/0.5 2.4/0.4 3.3/-5.7/1.1 0.4/0.9 1.1/0.3 2.0/0.5 2.5/0.7 2.1/0.4 0.6/0.4 0.6/0.3 0.8/0.9 1.0/1.5
20 20 14 0 20 0 1 0 18 20 20 0 12 7
0 1 15 3 1 2 17 7 19 18 11 4 16 15
*Stimulation-boundeaters (Validated on Test 3). 1"Data summed for all stimulation-on and off periods (Test 4). ,Animals were deprived of food for 24 hr prior to Test 5 and were administered twenty 30-sec stimulation trials and twenty l-rain interstimulus trials in the presence of food. M--Mean. SD--Standard deviation of the mean.
preceding ejaculation (ejaculation threshold) has frequently been used as an index of sexual efficiency [2]. The on-off paradigm used in the present and previous studies [4] complicates data analysis with respect to this measure since the ejaculation latency almost always exceeds 3 rain and therefore overlaps stimulation-on and stimulation-off periods. It is possible, for example, that a reduction in ejaculation threshold induced by ESB might be off-set by post-stimulation inhibitory effects. Nevertheless, the results of the present study suggest that ESB in no case produced a dramatic change in the number of intromissions required to produce ejaculation. Post-ejaculation interval For 9 of the 14 rats the mean duration of post-ejaculatory refractory periods was reduced by ESB even though some portion of the post-ejaculatory period may have overlapped a current-off interval. Males were frequently observed pursuing females very soon after an ejaculation that occurred during a current-on period or immediately after the onset of ESB when ejaculation occurred during current-off intervals. In the absence of brain stimulation the males adopted the post-ejaculatory resting posture (e.g. [9]). The mean interval between ejaculation and the next intromission (PEI) was substantially reduced in 6 of the 14 rats as compared to the average PEI for the two control tests (range of decrease, 14-36 per cen0. The mean latency between ejaculation and the first post-ejaculatory mount was even more substantially reduced for 9 of the rats (range 14-64 per cen0.
Eating. Seven of the 14 rats were stimulation-bound eaters (SBE). When tested while sated, six of these rats ate on at least 19 of the 20 current-on trials and on no more than one of the current-off trials (Test 3). One rat (No. 175) ate on 12 current-on trials and on one current-off trial. One animal (No. 204) displayed stimulation-bound drinking on a preliminary test but had switched to stimulation-bound eating by the present test. This rat occasionally drank during ESB periods; however, only eating responses were scored for all rats. When food deprived (Test 5), three of these animals (Nos. 10, 209, 0) showed strong post-stimulation inhibition of eating; they ate during all 20 current-on trials but on not more than one current-off trial, even though they had been without food for a 24-hr period prior to testing. Four of the 7 non-SBE (70, 205, 72, 186) showed inhibition of eating during both current-on and current-off periods, while rat No. 6 exhibited inhibition of eating during current-on periods only. All animals in which post-stimulation inhibition of eating occurred resumed eating 3-5 min after the last stimulation period and were observed eating continuously for several minutes. It should be noted that all rats had also been observed eating for 2 min prior to the first stimulation period. Four of the SBE rats and 3 non-SBE rats did not display post-stimulation depression of eating. Self-stimulation. As all rats used in the present experiment were selected on the basis of positive self-stimulation performance it was not possible to determine whether stimulusbound behavior could be observed in non-self-stimulating
COPULATION, EATING AND BRAIN STIMULATION
animals. Current values for self-stimulation were always higher than those used during the remaining tests.
Comparison Between Measures Copulation. An analysis of the data summarized in Table 2 reveals the absence of consistent relations among the different measures of sexual performance. For example, rats showing the greatest decrease in III frequently required more intromissions per ejaculation than during control tests while the post-ejaculatory interval was either decreased or unchanged. High on/off ratios for mounts occurred without a substantial reduction in the HI (Nos. 204, 209). Rat 163, on the other hand, had a very low on/off ratio (all ejaculations occurred during stimulation-off periods) but showed a 50 per cent reduction in the III. In short, While ESB generally decreased the ItI and PEI, and resulted in high on/off ratios for mounts, in many animals these changes were not correlated.
Comparison of post-stimulation effects on eating and copulation. Three of 4 animals showing post-stimulation depression of copulatory behavior (Nos. 10, 209, 70, the exception 175) as inferred by an increase in III also displayed inhibition of eating during post-stimulation periods. A similar relation was observed for 5 of the 6 rats (Nos. 10, 209, 70, 72, 186, the exception 136) exhibiting the largest on-off sex ratios after combining scores for mounts, intromissions and ejaculations. During the twenty l-rain current-off periods these rats occasionally groomed themselves; but in spite of the 24-hr food deprivation period, the food pellets tended to be ignored. In the case of rats 70, 72 and 186 stimulation also interferred with eating during the stimulation periods. Thus, with some hypothalamic electrodes there may be a general post-stimulation inhibition of consummatory behavior, but during stimulation different behaviors may not be affected equally. In the sex tests, which had a longer currentoff period (3 min), some copulatory activity was observed (except for No. 70), but the inter-intromission intervals were longer and irregularly spaced, giving the impression of a general disruption of copulation. There was no tendency for the converse to be true, however, as animals did exhibit post-stimulation inhibition of eating without any comparable inhibition of copulation. It would appear, therefore, that post-stimulation inhibition of eating and copulatory behavior is not congruent; but there may be a tendency for stimulation, which produces an inhibitory after-effect in a sex test, to also produce inhibition in an eating test. Anatomical analysis. An extensive analysis of the hypothalamic sites capable of eliciting copulatory behavior and eating is not possible with a sample of only 14 electrodes. In spite of this limitation some anatomical observations may be helpful. There has been a tendency to view posterior hypothalamic sites as more likely to elicit copulatory behavior [3, 4, 6], and more rostral sites, as, for example, the perifornical area, as having a greater probability of eliciting eating. No support for this view can be found in our sample (Fig. 1). Of the 6 animals exhibiting the highest on/off ratios in sexual behavior, four electrodes were located at A-P planes containing the ventromedial and/or arcuate nucleus; only two were located more posteriorly. Rat 70, for example, had the highest on/off ratio, but one of the most anterior placements in our sample (cf. Fig. 1). Four of the 8 animals displaying stimulus-bound eating had posterior hypothalamic electrodes located at planes in which the mammillary bodies were evident. No pattern could be discerned for the electrode locations producing post-stimulation inhibition of behavior or specific effects on other copulatory measures.
591 DISCUSSION
The reports [3, 4, 6] that electrical stimulation could activate a copulation reward site have given rise to the hope that it might be possible to identify and to study the properties of the neural structures controlling sexual motivation and/or performance. One of the major lines of support for this conclusion is derived from the finding that male rats receiving intermittent posterior hypothalamic stimulation exhibit most of their sexual activity during stimulation rather than nonstimulation periods. We have attempted to elaborate on this phenomenon by extending the range of observations to the standardized measures of sexual behavior that have been well studied in the past. The lack of agreement among different measures of male sexuality has to be regarded as disappointing. Although 10 of the 14 rats exhibited twice the number of mounts when the stimulation was on, compared to when it was off, there was no comparable effect on intromissions or ejaculations. Hypothalamic stimulation also increased or left unchanged the number of intromissions per ejaculation in 11 of the 14 rats. This finding argues against a general facilitation of all components of copulatory behavior. General increases have been reported [10, 19] from electrical stimulation of the preoptic area and following large electrolytic lesions which destroyed portions of the posterior hypothalamus, posterior medial thalamic nuclei, and the rostral part of the central grey matter in the mesencephalon [11]. In contrast to the present findings, lesioned male rats ejaculated after relatively few intromissions and with a shorter latency, and were sexually refractory for shorter intervals following ejaculation [11]. The increase in the number of mounts per intromission recorded during stimulation periods in the present study suggests that instead of augmenting sexual performance, ESB may have interfered with the efficiency of copulation, perhaps by excessively arousing the animals. The measures that seemed most influenced by hypothalamic stimulation were the inter-intromission interval and the post-ejaculatory interval. In general, the inter-intromission interval was decreased by stimulation in most rats. This seems to be true even though in some instances a second control test given after the stimulation test suggests either a lasting effect of stimulation or a change in performance over time (which would have exaggerated the facilitating influence of stimulation). However, the fact that there were a number of animals that exhibited a decrease in III during stimulation periods that did not also show a significant "stimulus-bound behavior" effect (as reflected in the on/off ratio), suggests that the relation between these measures is not strong. Overall, the results suggest that hypothalamic stimulation tended to facilitate the rate of sexual activity, to shorten the time of inactivity during post-ejaeulatory refractory periods, and to modify the temporal patterning of the behavior, although not all aspects of sexual behavior were equally affected. In a recent study [14] involving lateral preoptic area stimulation it was reported that there was general agreement among measures of sexuality, but the behavior was primarily analyzed in terms of its temporal course. Response rate measures also predominated in other studies [4]. With the two exceptions noted [10, 19] involving the preoptic area, the amount of copulatory behavior displayed by male rats has not been altered by brain stimulation. Since the variables which are affected by hypothalamic stimulation (PEI, Ill) are also changed by a number of types of nonspecific stimulation [1, 5, 13], investigators have attempted
592
STEPHAN, VALENSTEIN AND ZUCKER f
(
•
t "! . .;,,.,'> t ,
~-:.-..' ~; 136
7O
204
0
2O9 72 186
163
205 6
I0
226
FIG. 1. Histological identification of hypothalamic sites at which electrical stimulation affected eating and copulation of male rats. Brain sections are modified from Konig and Klippel [12]; only those loci designated by triangles yielded stimulation-bound eating. All sites supported selfstimulation behavior.
to provide other evidence that brain stimulation was specifically affecting sexual behavior. To date this evidence, consisting of data indicating that stimulated animals will press a lever to obtain ~ s to an estrous female [4, 6] or that self-stimulation rates can be influenced by steroid hormone manipulations [4, 15], has been preliminary, and in our opinion far from conclusive in demonstrating a correlation either with a discrete hypothalamic area or the facilitation of sexual behavior. Since only 1 rat in our sample of 14 exhibited convincing stimulation-bound copulation, we cannot claim to have shown that this phenomenon is a general consequence of post-stimulation depression in sexual behavior. We do, however, consider this to be the case. The existence of animals such as Rat 70, which did not exhibit any sexual behavior during the inter-stimulus intervals, makes it abundantly clear that an inhibitory process may be triggered with the offset of the stimulation. This inhibitory process does not affect all behaviors equally, as tbe blocking of eating and copulatory activity during the inter-stimulus intervals was not always consistent. Again Rat No. 70 served as an illuminating example as this animal, which displayed all its sexual behavior during the stimulation periods, did not eat at all when stimulated in Test 5 even though it had been food-deprived. It should be noted that several animals which did not show stimulation-bound eating nevertheless
provided strong evidence for post-stimulation inhibition of eating (Nos. 70, 205, 72, 186)• O~ber animals (e.g., No. 0) displayed post-stimulation inhibition in the eating test, but very little evidence of such inhibition in the sex test. Some of this discrepancy might be attributed to differences in sexual and hunger motivational levels or the unequal compellingness of the goal objects (receptive female and food) for an individual male, but it could not be argued, convincingly, that if these variables could be controlled, perfect congruence would h a w resulted. In general, the results suggested that the estrous female was the more compelling stimulus, and if post-stimulation inhibition was evident during the sex test, it was likely, but not inevitably, to be present during the food test. In a recent summary [8] of the neural sites at which electrodes can elicit eating, drinking and gnawing, the conclusion was drawn that those areas are widespread throughout the rat hypothalamus. Although there were some hypothalamic sites that did not appear to be capable of ¢liciting the behaviors studied, it was noted that throughout the positive areas there did not seem to be distinct regions that were more likely to activate one behavior than another. The anatomical data obtained from this study indicate that within the hypothalamic areas explored (preoptie, anterior and medial hypothalamic areas were not in our sample) sites that are capable of producing changes in
COPULATION, EATING AND BRAIN STIMULATION
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copulatory behavior are not easily distinguishable from stimulation-bound eating sites. None of the arguments advanced above should be inferred to mean that we are suggesting either that all hypothalamic stimulation produces the same general effect or that a distinct neural system subserving sexual motivation and performance may not exist. Clearly, all hypothalamic stimulation does not have the same effect as there exist too many cases in the literature of different behavioral results being produced from different sites in the same animal. Furthermore, there are many cases that have been reported (including the present account) of stimulation from the same electrode having different consequences for different behaviors. It may be that some central stimulation shares with some peripheral stimulation (handling, shock to tail or back) the capacity to elevate the response rate and alter the temporal pattern of male sexual behavior without specifically involving the neural
elements that normally participate in sexual activity. One conclusion forced upon us by the present study is that the technique of presenting intermittent periods of stimulation during observations of sexual behavior has some serious shortcomings. Unlike the situations demonstrating stimulus-bound eating or drinking, the operant level of male sexual behavior in the presence of a receptive female is not close to zero. The abnormally low amount of sexual behavior often displayed during the alternating, non-stimulation periods probably results from an active inhibitory process. Consequently, comparisons between stimulation and nonstimulation periods may be misleading. Furthermore, in contrast to most other behaviors studied, sexual behavior involves a longer and more serially dependent sequence of responses, that cannot be arbitrarily broken up into stimulation and non-stimulation periods without producing interactive effects that may be difficult to interpret.
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