Physiology&Behavior,Vol.52, pp. 1009-1013, 1992

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Cerebral Glucose Utilization During Conditioned Sexual Arousal F. H. DE J O N G E , *~ J. A. D. M. T O N N A E R , I " H. VAN L E E U W E , * A. J. P. C. TIELEMANS,I" A. L. L O U W E R S E * A N D N. E. VAN DE POLL*

*Netherlands Institute for Brain Research, Meibergdreef 33, l l05AZ Amsterdam ZO, The Netherlands and ~'Organon International BV, Department of Neuropharmacology, Postbox 20, 5340BH, Oss, The Netherlands Received 30 March 1992 DE JONGE, F. H., J. A. D. M. TONNAER, H. VAN LEEUWE, A. J. P. C. TIELEMANS, A. L. LOUWERSE AND N. E. VAN DE POLL. Cerebralglucose utilization duringconditioned sexual arousal. PHYSIOL BEHAV 52(5) 1009- l013, 1992.--Local cerebral glucose utilization was investigated in male rats during conditioned sexual arousal. Increased glucose utilization was found in three amygdaloid nuclei after exposure to a stimulus associated with exposure to a sexually active female. No changes were observed in areas known to be of crucial importance for the expression of consummatory aspects of sexual behavior. These results corroborate and extend previous results showing a dissociation between the expression of appetitive and consummatory aspects of sexual behavior at a neural level. Cerebral glucose uptake

Sexualarousal

Conditioning

THERE is good knowledge concerning the neural circuitry involved in the expression of male sexual behavior, that is to say, of male copulatory responses (l 0). It remains to be established, however, which brain areas are involved in sexual arousal or the expression of sexually motivated behavior. For the expression of male copulatory responses, the hypothalamus, and in particular the medial preoptic area (mPOA), is most conspicuously involved. Lesions in this region impair copulatory behavior in a great variety of species (3,8,10,23), without eliminating sexual drive (8,23). Thus, mPOA-lesioned male rats and rhesus monkeys continue to press a lever in order to get access to an estrous female, but do not copulate when the female becomes available. Apparently the neural control of copulation, at least at the preoptic level, has to be dissociated from that of sexually motivated behavior. The need for a conceptual differentiation between appetitive and consummatory aspects of sexual behavior, both at a behavioral and at a neural level, was already recognized by Beach in 1956 (2). Despite recently advanced behavioral techniques (4,8), however, the neural control of appetitive sexual behavior is still poorly understood. As a first approach to the question how the brain controls appetitive sexual behavior, we investigated which brain areas are activated during sexual arousal. We therefore studied the effects of conditioned sexual arousal on local cerebral

Males

Rats

glucose utilization (LCGU) as a direct measure of neuronal activity. The procedure used to study cerebral glucose utilization is described in detail by Room (18), who adapted the method from Sokoloff's original publication (24), for investigations in freely moving, unstressed animals. It is essentially based on the characteristics of the glucose analogue [14C]2-deoxyglucose that is metabolized in competition with the natural substrate through one or more steps of the metabolic pathway until it is eventually converted to [t4C]2-deoxyglucose-phosphate, which cannot be further metabolized and accumulates in the tissue. To induce conditioned sexual arousal, we followed a procedure developed by Zamble et al. (27), in which a formerly neutral stimulus (CS, conditioned stimulus) gains salience by its association with sexual-arousing stimuli (US, unconditioned stimulus) during five association trials. By presentation of the CS alone on the final trial, cerebral glucose utilization could be studied in sexually aroused male rats without actually presenting the primary cue (sexually active female). Control rats received the same number of neutral and sexual-arousing stimuli, but in this group, presentation of the CS was always separated from presentation of the US by a delay of 8 h, to avoid direct CS-US association. Thus, effects of conditioned sexual arousal on cerebral glucose utilization could be studied without any con-

Requests for reprints should be addressed to F. H. de Jonge at her present address: Agricultural University, Department of Animal Husbandry, Postbox 338, 6700AH Wageningen, The Netherlands.

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DE JONGE ET AL.

founding effects from increased locomotor or olfactory activity. Prior to this experiment, we confirmed that presentation of the CS induces the same endocrine and behavioral changes in the male rat as those obtained after presentation of the unconditioned stimulus (i.e., estrous female behind a wire mesh) (9,11 ). In particular, we confirmed that presentation of the CS induces a 200% increase in plasma LH levels within 10 min, and a reduction in ejaculation latency during a subsequent sexual interaction test (5,6,25). Local cerebral glucose utilization was measured in 22 different areas selected for their putative role in the expression of aspects of sexual behavior (see Table 1). These areas are the primary olfactory areas, including the main and accessory olfactory bulbs that transmit information on olfactory cues (26) to the amygdaloid areas, which act in concert to integrate prior social experience, higher order cognitive information, and incoming sensory information (7,13). Only the corticomedial amygdala is of known relevance for the expression of sexual (i.e., ejaculatory) behavior in males (6,13), but a special role with respect to the integration of stimulus reward associations has been dedicated to other parts of the amygdala (i.e., the basolateral nucleus) by a number of authors (1,8). The preoptic/hypothalamic areas, and in particular the mPOA and the sexually dimorphic nucleus of the mPOA, which receive projections from the amygdala, are most conspicuously involved in copulatory behavior and were selected for that reason (3,10). The limbic and frontal cortical areas were selected since recent studies, showing dopamine release from the nucleus accumbens in response to sexually related cues ( 14,17), suggest a specific role for dopaminergically innervated areas in relation to the presentation of sexually related cues. Limbic and frontal cortical areas with high (nucleus accumbens, medial prefrontal cortex minus area 2), medium (frontal cortex area 2, orbital prefrontal cortex, lateral septum), and low (frontal cortex area 1, parietal cortex) dopaminergic innervation were therefore selected. In addition to the areas that were selected on the basis of their presumed function in the control of sexually related processes, white and gray matter (viz. the dorsal part of the caudate putamen, which is explicitly involved in the control of locomotor activity) were selected as reference areas.

the cannulas were slid into two bent steel tubes and attached to the skull of the head with polycarboxylate cement. Surgical procedures were exactly according to those previously described in Room et al. (18).

Conditioning Procedure Since the IV injection with the radioactive glucose analogue [~4C]2-deoxyglucose forms an essential element in the procedure of the study of cerebral glucose utilization, sham injections were chosen as the CS during conditioning trials. Zamble et al. (27) showed that estrous females behind a wire mesh (unconsummated sexual arousal) are the most effective stimuli to produce conditioned sexual arousal. Therefore, exposure to estrous females behind a wire mesh was presented as the US. The exact procedure of CS and US presentation was as follows: the animals were left to recover for 1 week after surgery, before five daily conditioning trials started. During the conditioning trials, male rats of the sexually aroused group were transported in their home cage to an experimental room, and were left to adapt for 10 min before receiving a sham injection (CS). The CS was then followed by presentation of the US (female behind a wire mesh) with a varying interval of 3, 6, 9, 6, and 3 min, respectively. Males were then returned to their room. Control rats went through exactly the same procedure but received the US (female behind a wire mesh) with a delay of 8 h. All stimuli were presented during the dark phase of the circadian cycle, and experimental rooms were carefully cleaned in between association trials.

Experimental Test Procedure Following the five daily conditioning trials, the male rats went through the same procedure on the final experimental day, except that a) no estrous females were presented and b) all rats received an IV injection of [~4C]2-deoxyglucose (150 uCi/kg, specific activity 58 mCi/mmol) instead of a sham injection as the conditioned stimulus. Fourteen timed arterial blood samples (100 ul each) were drawn at 0, 0.25, 0.5, 0.75, 1, 2, 3, 5, 7.5, 10, 15, 25, 35, and 45 min following the IV injection with [~4C]2-deoxyglucose. Plasma glucose and plasma 14C concentrations were determined in these samples. Forty-five min after injection with

METHOD

Animals and Housing Conditions Two groups (n = 7) of sexually experienced, adult male albino Wistar rats obtained from the central animal supply house of T.N.O. (Zeist, The Netherlands) were individually housed under a 12:12 h reversed clark:light cycle. The two groups of males (group 1: sexually aroused group, and group 2: control group) were housed in separate but identical rooms. Ovariectomized stimulus females (n = 16) were artificially kept in heat by subcutaneously implanted 0.5 cm long silastic tubes (0.8 mm i.d., 1.4 mm o.d., Talas, Ommen, The Netherlands) containing estradiol (Diosynth, Oss). The stimulus females were maintained in groups of four per cage and were housed in the same rooms as the males. All animals were handled daily, and food and water was always available ad lib.

8

The male rats were provided with two polyethylene cannulas in the left aorta dorsalis and vena cava in order to enable injection of[ 14C]2-deoxyglucose through the vena cava, and to take timed arterial plasma samples from the aorta dorsalis, in the freely moving, unstressed rat (18). For that purpose, the free ends of

*

• ** * * *

* *

.

7

-56 E

4

Surgical Procedures

*

i

0.25

i

1

i

L

5 15 minutes ( log scale )

i

45

FIG. 1. Plasmaglucoseconcentrationsin sexuallyaroused(0) and control (O) male rats in 14 timed arterial blood samples (time in log scale). The arrow indicates injection with [~4C]2-deoxyglucose(T = 0). Data are expressed as mean (+_SEM);*p < 0.05; **p < 0.01; ***p < 0.001.

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the radioactive ligand, a n i m a l s were sacrificed by a bolus injection o f 1 ml (60 mg) s o d i u m pentobarbital a n d the brains were r e m o v e d a n d processed for cryostat sectioning (20 ~tm).

Histological Procedures Sections o f the brain, together with '4C plastic calibration standards ( A m e r s h a m , PDA-504), were exposed d u r i n g 4 days to A m e r s h a m Hyper-film (~-max, RPN.9), a n d were subsequently c o u n t e r s t a i n e d with t h i o n i n . '4C c o n c e n t r a t i o n s in the a u t o r a d i o g r a m s were d e t e r m i n e d with the aid o f a c o m p u t e r i z e d image analysis system (IBAS; K o n t r o n , M u n i c h ) t h a t superimposed the a u t o r a d i o g r a m s a n d the thionin-stained sections, in which anatomically defined structures were d e m a r c a t e d according to the b o u n d a r i e s given in Paxinos a n d W a t s o n (16). '4C concentrations (nCi/g) were linearly transformed into L C G U ( a m o l / 1 0 0 g tissue/minute) by use of the Sokoloff equation, adapted for varying plasma glucose levels by Savaki et al. (21). All areas t h a t were m e a s u r e d are presented in Table 1. RESULTS Plasma glucose levels for sexually aroused a n d control rats are presented in Fig. 1; glucose levels were analysed by A N O V A for repeated m e a s u r e m e n t s including a total o f seven rats in

each t r e a t m e n t group. Post hoc contrasts were r u n by univariate F-tests a n d a p < 0.05 with F ( I , 12) > 5.5 was considered significant. T h e analysis revealed a significant interaction effect between the factors group a n d t i m e (p < 0.001). Post hoc contrasts showed t h a t sexually aroused subjects had significantly higher glucose levels during the first 7.5 m i n o f testing (see Fig. 1). T h e L C G U o f the reference areas were first calculated in a preliminary analysis, a n d did not differ between the groups (ttest, p > 0.48). Although L C G U calculation included a correction for varying plasma glucose levels (2 I), L C G U ratios were subsequently calculated (as a % of reference areas) in order to avoid a n y r e m a i n i n g systematic bias related to group differences in plasma glucose levels. T h e L C G U ratios were analysed by a univariate two-way A N O V A including a total of seven rats in each t r e a t m e n t group. A p < 0.05 with F(1, 12) > 8.7 was considered significant. T h e analysis indicated that L C G U ratios were significantly increased in 1) the basolateral amygdaloid nucleus (BLA), 2) the bed nucleus of the accessory olfactory tract (BAOT), a n d 3) the central amygdala (CEA) b o t h w h e n analyzed relative to white a n d to gray m a t t e r (for p values, see Table 1). O t h e r areas, including those that are explicitly k n o w n to be involved in the expression o f copulatory responses (viz. the medial preoptic area, the sex-

TABLE 1 LOCAL CEREBRAL GLUCOSE UTILIZATION IN SEXUALLY AROUSED AND CONTROL RATS Percent of White Matter Control Primary olfactory areas Olfactory bulb Accessory olfactory bulb Amygdaloid areas Bed n. acc. olfactory tract Corticomedial amygdala Basolateral amygdala Central amygdala Preoptic/hypothalamic areas Lateral preoptic area Medial preoptic area Sexual dimorphic n. Suprachiasmatic nucleus Lateral hypothalamic area Anterior hypothalamic area Ventromedial n. Limbic & frontal cortical areas Lateral septum N. accumbens Medial prefrontal cortex (-FR2) Frontal cortex area 2 Orbital prefrontal cortex Frontal cortex area 1 Parietal cortex area 1 Reference areas Caudate putamen White matter

Sexually Aroused

Percent of Caudate Putamen Control

102.2 _+ 3.6 50.4 _+ 2.9

367.6 _+ 11.8 180.9 + 8.1

362.6 + 11.7 178.2 + 8.8

188.2 174.6 247.0 167.0

+ 3.9 _+ 6.4 _+ 1.8 + 4.7

213.1 182.8 282.6 185.8

+ 7.0t + 4.1 _+ 8.3 ~t +_ 5.4*

52.0 48.1 68.1 46.0

+ 2.3 -z-_2.0 + 2.2 _+ 1.5

59.8 51.4 79.8 52.2

_+ 0.8t + 0.8 + 3.3t + l.lt

263.8 188.8 174.8 166.6 234.4 219.7 182.6

+ 9.1 + 5.6 _+ 4.8 + 6.5 + 8.1 _+ 8.2 +_ 6.5

266.8 191.4 171.6 176.3 239.8 225.8 184.7

+_ 7.9 _+ 5.6 + 3.2 _+ 9.3 + 4.6 + 9.5 + 10.8

72.4 51.8 48.0 46.1 64.4 60.5 50.2

_+ 1.9 +_ 0.8 _+ 0.8 _+ 2.6 _+ 2.3 + 2.4 + 1.8

75.0 53.9 48.3 49.5 67.5 64.0 51.8

+ 1.7 _+ 1.6 + 0.9 + 2.2 + 1.4 _+ 3.1 _+ 2.4

199.8 357.8 375.4 360.0 354.3 367.1 367.9

_+ 8.0 _+ 12.8 + 11.6 + 11.8 + 14.7 _ 17.5 + 20.6

196.2 347.6 369.5 364.6 347.0 367.8 382.1

+ 7.1 + 5.3 + 9.6 _+ 11.3 + 11.4 + 14.0 +_ 13.4

54.8 98.2 103.1 98.8 97.0 100.6 100.6

+ 1.3 _+ 2.7 + 2.6 + 2.4 + 2.4 _ 3.5 _+ 3.2

55.2 98.1 104.2 102.6 97.9 106.2 107.7

_+ 1.6 _+ 2.9 _+ 3.5 + 3.1 + 4.0 _+ 4.3 ___4.1

357.4 + 13.3 100.0 + 0.0

353.4 + 8.3 100.0 -+ 0.0

100.9 _+ 2.1 49.7 + 2.0

Sexually Aroused

100.0 _+ 0.0 27.6 _+ 0.9

100.0 _+ 0.0 28.2 +- 0.7

Data are presented relative to white and gray matter (dorsal part of the caudate putamen) and are expressed as mean + SEM. * p < 0.05. t p < O.Ol. p < 0.001.

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DE JONGE ET AL.

ually dimorphic nucleus of the medial preoptic area, the primary olfactory areas, and the corticomedial amygdala), did not respond during sexual arousal (Table 1 and Fig. 2).

n.s.

300

~ ~

= ~

~ r ~

DISCUSSION

Behavioral Specfwity This is the first report to describe changes in cerebral glucose metabolism during sexual arousal in male rats. This was achieved using a conditioning procedure that prevents sensory and locomotor activation associated with presentation of the unconditioned stimulus (sexually active female). The fact that LCGU in the caudate putamen was not increased during sexual arousal further corroborates our impression that sexually aroused and control rats did not differ in locomotor activity after presentation of the CS. Yet, in the absence of associated behavioral changes, how do we know that sexual arousal has actually been produced? Different plasma glucose levels between experimental and control subjects reflect differences in vigilance state, but they do not prove that these differences were induced by sexual arousal. Prior to this experiment, we therefore validated the procedure, by showing endocrine and behavioral changes after conditioned sexual arousal that were similar to those reported after presentation of an estrous female, but not a male incentive (6,9,11). In particular, we confirmed that presentation of the CS induces a 200% increase in plasma LH levels and a reduction in ejaculation latency during a subsequent sexual interaction test (5,6,25). The criticism may be made that frustration rather than sexual arousal could underly the metabolic changes since males of the sexually aroused group expect, but do not receive, an attractive female on the final trial. Therefore, by introducing unpredictable intervals between CS and US during the preceding association trials, we also prevented possible frustration effects. There is no doubt, therefore, that it is indeed conditioned sexual arousal that induced the underlying metabolic changes in three amygdaloid nuclei. It should be kept in mind, however, that this does not exclude the possibility that stimuli conditioned to emotionally charged conditions other than sexual arousal might also cause such a pattern of metabolic changes.

O([actory Stimulation One previous study investigated the effects ofestrous female odors on cerebral glucose metabolization in male rats (15). Regretfully, these authors limited their observations in the amygdala to the corticomedial amygdala, which revealed, similar to our observations, no metabolic changes. These authors did find, however, increased cerebral glucose utilization in the lateral hypothalamus (LHA) after presentation of estrous female odors. Since we did not observe similar changes in cerebral metabolization during conditioned sexual arousal while female odors were absent, the increased metabolization in the LHA in the study ofOrsini et al. should, most likely, be attributed to olfactory stimulation, and not to sexual arousal.

Plasma Glucose Levels" Sexually aroused subjects had significantly higher plasma glucose levels during the first 7.5 min of testing, which may reflect a physiological anticipation to the metabolic demands of appetitive and consummatory sexual behavior. Since an estimated 85% of the radioactive ligand is trapped in the tissue within the first 10 min after injection (24), the elevated plasma glucose levels during the first 7.5 min also indicate that sexual arousal is induced during the period that is crucial for the

(13)

200

e.i1)

a.

100

MEA

BLA

l/ CEA

BAOT

FIG. 2. Local cerebral glucose uptake in amygdaloid areas of sexually aroused (hatched bars) and control rats (gray bars), expressed as a percentage of white matter (mean _+SEM). MEA: corticomedial amygdala; BLA: basolateral amygdala; CEA: central amygdala; BAOT: bed n. of the accessory olfactory tract. NS: not significant;*p < 0.05; **p < 0.01 ; ***p < 0.001.

expression of neuronal functional activity by the deoxyglucose method.

Quantification of Glucose Metabolism Plasma glucose levels obviously affect relative ~4C uptake rate in cerebral tissue. Semiquantitative analysis of optical density ratios circumvents this problem (22), but may introduce sources of error (12) that are eliminated in the present experiment by calculating LCGU ratios rather than optical density ratios. White matter is most often taken as a reference area, but may be less sensitive than gray matter to changes in plasma glucose levels. Therefore, LCGU ratios were calculated relative to white and to gray matter. Since both analyses yielded the same results, it is concluded that increased LCGU ratios in the amygdaloid nuclei are not secondary to group differences in plasma glucose levels.

The Amygdala in Relation to Conditioned Sexual Arousal Changes in LCGU ratios were observed in three amygdaloid areas (BLA, CEA, and BAOT), but not in regions that are known to be explicitly involved in the expression of copulatory behavior such as the MEA, mPOA, the n. accumbens, or the primary olfactory areas (3,8,10). Our results therefore confirm that different neural mechanisms underly appetitive and consummatory aspects of sexual behavior, and are consistent with the hypothesis that it is primarily the amygdala that integrates sensory information with higher order cognitive processes for the expression of emotional behavior (1). It is well known that amygdaloid nuclei are anatomically and functionally differentiated, each one contributing in a specific way to the integration of emotional stimuli. Yet, how do the BLA, BAOT, and CEA act in concert during conditioned sexual arousal? Although further investigations are needed to answer this question, it can already be stated that the BLA is involved in stimulus-reward associations (1,8). However, the BLA does not functionally discriminate between sexual and, for instance, food reward (8). Similarly, the CEA translates conditioned stimuli into appropriate autonomic output, but fails to discriminate between stimuli with different associational affective value

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(19,20). The BAOT, therefore, seems to be the most likely candidate as a center that relates specific sexual associative information to the stimulus-reward association, which is then further integrated by BLA and CEA. Such a hypothetical role is suggested, since specifically the B A O T receives input from the vomeronasal system that is involved in reproductive behavior (7,25). Although it remains a challenge to investigate how different amygdaloid nuclei collaborate as a "sensory gateway to e m o t i o n s " (1), the present results suggest that the integrated action o f BLA, CEA, and B A O T forms the essential gateway

to the behavioral and endocrine c o n c o m i t a n t s of sexual arousal. ACKNOWLEDGEMENTS We wish to thank C. W. Pool, J. Wester, and A. Klop for implementation of computer programs for automated image analysis; G. van der Meulen, H. Stoffels, W. T. P. Verweij, and T. Eikelboom for preparing tables and figures; and F. H. Lopes da Silva, J. P. C. de Bruin, M. A. Corner, and J. L. Slangen for valuable comments on earlier drafts of the manuscript. In particular we wish to thank P. Room for theoretical advice on the deoxyglucose method.

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