Depression by estrogen of electrical activity in the hypothalamic ventromedial nucleus of female cats

Psychoneuroendocrinology, Vol. 5, pp. 13 to 24.

0306-453018010101-0013502.00/0

© Pergamon Press Ltd. 1980. Printed in Great Britain.

DEPRESSION BY ESTROGEN OF ELECTRICAL ACTIVITY IN THE HYPOTHALAMIC VENTROMEDIAL NUCLEUS OF FEMALE CATS SUSAN E. HOLBROOKE* and RICHARD P. M1CHAEL'~ *Department of Psychiatry, Institute of Psychiatry, University of London, England tDepartment of Psychiatry, Emory University School of Medicine, Atlanta, GA 30322, U.S.A., and Georgia Mental Health Institute (Received 28 November 1978)

SUMMARY (1) Hypothalamic and cortical EEG activities were telemetered from freely-moving ovariectomized female cats during mating tests with a vigorous male conducted over periods of many weeks. (2) Records were analyzed for long-term energy changes in 3 frequency bands: 4--8, 8-16 and 16-32 Hz. In Hvm, energy levels (Voltage Indices) were higher in the higher frequency bands while the reverse trend was observed in anterior and posterior hypothalamus. (3) Estrogen treatment had widespread effects on energy levels in the brain, but consistently depressed energy levels in Hvm, particularly in the highest frequency band. (4) Estrogen treatment did not consistently exert this effect in anterior and posterior hypothalamus or in cortical sites. (5) Behavioral factors also contributed to the effects observed. Although these were difficult to interpret, the particular depression observed in Hvm during mating activity did not occur elsewhere. (6) There is evidence that these localized changes in EEG activity relate to the regulation of estrous behavior. Key Words--Hypothalamus; telemetry; EEG energy analysis; estrogen.

INTRODUCTION HYPOTHALAMIC implants of estrogen induce sexual receptivity in ovariectomized cats by a local and direct action on the brain (Harris & Michael, 1964; Harris, Michael & Scott, 1958; Michael, 1962, 1965), and the hypothalamus and related areas of the limbic system selectively accumulate radioactively-labelled estrogen from the systemic circulation (Glascock & Michael, 1962; Michael, 1965). Since the hypothalamus of the female cat appears to possess receptors for estrogens and these hormones induce behavioral estrus (Michael, 1961), it was of interest to determine if their administration would result in any changes in hypothalamic electrical activity: the presumption being that biochemical changes in the brain antecede changes in neural activity, and that the latter would then be responsible for the altered behavioral patterns that characterize the estrous state. Earlier studies in the female cat have been concerned principally with the effects of estrogen on neural responses to sensory, particularly genital, stimulation (Alcaraz, GuzmanFlores, Salas & Beyer, 1969; Beyer, Almanza, de la Torre & Guzman-Flores, 1971; Michael, 1973; Porter, Cavanaugh, Critchlow & Sawyer, 1957; Sutin & Michael, 1970), but neither Porter et aL (1957), nor Sutin & Michael (1970) were able to detect, by visual inspection, any differences between the basic characteristics of the hypothalamic E E G of 13

14

SUSANE. HOLBROOKEand RICHARDP. MICHAEL

ovariectomized cats and those treated with estrogen. The present study c o m p a r e d the longterm effects o f estrogen on the electrical activity o f the h y p o t h a l a m i c ventromedial nucleus ( H v m ) with its effects at o t h e r neural sites recorded via chronically i m p l an t ed m a c r o electrodes. T o this end, a m e t h o d for telemetering and analyzing the E E G o f freely-moving animals was developed (Michael, H o l b r o o k e & Weller, 1977; Michael, Weller & Wolff, 1965a, b). Th e radio-transmitters were implanted beneath the skin and therefore did not impede the free m o v e m e n t o f females during mating tests with vigorous males. The m e t h o d o f energy analysis o f the E E G was particularly suitable for correlating changes in electrical activity with changes in behavior. This analyzed c o n t i n u o u s changes in energy within wide frequency bands rather than changes in individual frequencies occurring within epochs. These techniques m a d e possible l o n g - t e r m studies on changes in b e h a v i o r and electrical activity following h o r m o n e treatment, and also facilitated the c o m p a r i s o n o f activity at different neural sites in the same animal.

MATERIALS AND METHODS

Animals Seventeen mature, female cats of mixed breed were used for electrode implantations. They were maintained together in a pen and received a diet of proprietary cat foods and vitamin supplements. Three intact male cats were employed for behavioral testing. They were housed separately, each in a large cage, and received the same diet as the females. An ovariectomized female cat kept in continuous estrus by estrogen administration was used to train the males to mate and to maintain their sexual activity (see below). Surgical procedures All surgical procedures were carried out under pentobarbitone sodium anesthesia (Nembutal, Abbott Laboratories Ltd., 36 mg/kg body wt, i.p.). (1) Ovariectomy. Both ovaries and a small portion of the upper ends of the uterine horns were excised via bilateral flank incisions. Examination of the ovaries and uteri established that all animals were post-pubertal. Ovariectomies were carried out 7-73 weeks prior to implantation of electrodes and transmitters. (2) Implantation of electrodes. One or two pairs of electrodes were implanted in each animal with the stereotaxic machine, using the atlas of Jasper & Ajmone-Marsan (1954). Stainless steel screws were used as extra-dural electrodes, and enamel-insulated, stainless steel wires (0.25 mm dia) with 0.5 mm bare tips were used as deep electrodes. (3) Implantation of transmitters. These were implanted subcutaneously on the cat's head between the ears and were anchored to the skull by nylon screws and dental cement. The transmitter and electrode leads were embedded in an insulating layer of impermeable wax. Over this, a thin layer of colloidal graphite was applied to minimize stray capacitance, and then a final layer of dental cement provided mechanical protection before closing the skin. The procedure has been described fully (Michael et al., 1977). (4) Exchange of electrode leads. Because transmitters were single channel, it was necessary to connect to another pair of electrodes to obtain recordings from a different site in the same animal. The procedure was similar to that employed for connecting leads during implantation; a heat shunt was used to prevent brain damage when connections were re-soldered. Transmitters The single channel sub-miniature transmitters were of the type described by Michael, et al. (1965a, b), modified by the inclusion of a remotely-actuated solid-state switch (Weller, 1966) to prolong the life of the cell. The biological signals directly modulated the capacity of a varactor diode, which gave corresponding deviations in the carrier fiequency of the oscillator. The transmitters had an input resistance of 1 Mffl, a carrier frequency of 20-22 MHz and current consumption of 150-200 laA. They were 21 x 11 x 9 mm, weighed 3 g and were totally implantable. The transmitter could be switched on and off by means of a radio pulse directed through the intact skin of the cat's head. Full details of the transmitter, a circuit diagram and the technique for remote switching have been published (Michael et al., 1977).

ESTROGEN AND THE HYPOTHALAMUS

15

Receiving and recording system Transmitters do not radiate uniformly in all directions and so the signal strength will change with changes in orientation of the animal's head. To overcome this effect, three orthogonally placed aerials were wound round the outside of the behavioral testing cage, and an automatic aerial selection system was employed on the receiver. When the signal strength on a given aerial fell below a threshold level, logic circuits ensured the selection of another aerial that was receiving an adequate signal. None of the variance of the Voltage Index was due to changes in the orientation of the cat. A general coverage receiver was used in conjunction with a limiter and Forster-Seely discriminator to detect the narrow band F M transmissions and to provide particularly rigorous rejection of the amplitude modulation which resulted from movements of the animals. The receiver output was fed in parallel to a pen recorder and an F M tape-recorder. The majority of experiments was conducted within a screened room to prevent interference from other radio sources. Hormone administration Ovariectomized cats were given 100 lag stilbestrol dipropionate (Organon Laboratories Ltd.) s.c. in 0.5 ml ethyl oleate for 3 days or until mating occurred. Receptivity was maintained by twice weekly doses of either 50 or 100 lag. Mating tests (1) Behavior testing cage. Mating tests were conducted in a suitable pen 1.53 m long × 0.77 m wide × 0.90 m high. It was constructed of perspex and wood and the three orthogonal aerials were wound round it. The pen was divided into two compartments by a removable partition. (2) Male cats. Training the male cats to mount females, and maintaining males in a sexually active condition was an important part of the study. This was done by repeatedly presenting them with receptive females in a familiar environment where males had established territory rights. (3) Testing procedure. Tests were generally of 30 min duration and followed the general lines described earlier (Michael, 1961). Tests were divided into five behavioral situations: (A) the female alone in one compartment of the testing pen and the male in a different room; (B) male and female together in testing pen, but separated by partition; (C) male and female together (partition removed); (D) male and female again separated by partition; (E) the female again alone and the male in a different room. During each situation A-E, the behavioral interactions were observed and recorded on tape, and EEG records were obtained. Generally, a sequence of 7 mating tests was conducted while records were obtained from each electrode site in all females before and during treatment with estrogen; this could usually be accomplished in 8-10 weeks for a given female. (4) Control tests. These were the same as those described above, but a female partner was substituted for the test male. EEG analysis The method of continuous EEG analysis of Byford (1964) was adapted to the present study. The EEG was filtered into 3 frequency bands (4--8, 8-16 and 16-32 Hz), full-wave rectified and integrated. The signals were displayed as gradients proportional to the amplitude-time integrals of the EEG on a 3-pen recorder (Michael et aL, 1977). EEG records were analyzed at the lowest integration rate of the analyzer and the slowest paper speed (22.5 mm/min) of the 3-pen recorder. Thus, a record of 30 min duration was condensed on to a chart of 675 mm. The variable input gain of the analyzer facilitated optimal display of the gradients about 45 ° and different input gain settings were required for different animals. It was essential to establish the relationship between the input voltage at the center frequency of each band and the gradients displayed. A calibration procedure was employed for each input gain setting of the analyzer which enabled data from all animals to be displayed on a single scale (Voltage Index). Numerical treatment of results The tangents of the gradients in the write-out of the 3-pen recorder were determined for each behavioral situation (A-E) of each test and termed "Energy Indices" of the EEG. An Overall Mean Energy Index for a hormone condition was determined. This consisted of all behavioral situations for all tests recorded during that hormone condition from each electrode site. By means of the calibration procedure, the equivalent input voltage at the center frequency of the band was determined for each Overall Mean Energy Index. This value was termed a "Voltage Index", and its use enabled group data from all cats to be represented on one scale. Voltage Indices recorded from different animals were grouped acenrding to neurological site, and any

16

SUSANE. HOLBROOKEand RICHARDP. M[CHAEL

differences before and during estrogen administration were assessed using Wilcoxon matched-pairs signedranks tests (2-tailed) (Siegel, 1956). In an individualanimal the significance of the effects of hormone treatment and of the behavioral situation on the Energy Indices at a given recording site was tested by a split-plot analysis of variance. This was modified in the following way for use on data derived from individual subjects: all terms were tested against the residual (within-test) variation, the between-test variation being treated as a separate term, and the degrees of freedom were reduced by Box's correction (Box, 1954; Greenhouse & Geisser, 1959), a conservative procedure. To assess further the significance of differences established by the analysis of variance, an adaptation of the Student t-test incorporating the error variance given in the analysis of variance table was used.

Brain histology Brains were perfused with 10~o formal saline via each carotid artery with the electrodes in situ. Blocks containing the hypothalamus or other deep recording sites were isolated and, after embedding in celloidin, serial sections were cut at 25 lam. Alternate sections were stained by the method of Kluver and Barrera and by the silver method of Bodian to locate electrode tips accurately. RESULTS E E G records were telemetered satisfactorily over periods of several weeks from 16 of the 17 animals, a n d from 28 of the 30 b r a i n sites. D a t a were o b t a i n e d b o t h before a n d during estrogen a d m i n i s t r a t i o n from 14 cats a n d 21 sites. Recordings were made during a total of 378 tests, a n d of these, 357 were sufficiently complete for quantitative treatment. Only data from positive m a t i n g tests were included in the estrogenized condition a n d 27 tests were excluded from the statistical calculations on these grounds.

Effect o f estrogen on activity in hypothalamic ventromedial nucleus Figure 1 shows two illustrative examples of raw E E G records together with their corresponding Voltage Index measurements. The Voltage Indices of the 3 frequency b a n d s of the E E G recorded from 9 hypothalamic ventromedial nuclei (8 cats) are shown in Fig. 2. Data both before a n d during estrogen a d m i n i s t r a t i o n were obtained from 7 cats a n d were

Anterior Hypothalamus L7

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J sec. FIG. 1. Two illustrative examples of raw EEG records together with their corresponding Voltage Indices. Upper trace: anterior hypothalarnus, 4-8 Hz, Voltage Index = 307 laV; 8-16 Hz, Voltage Index = 223 I~V; 16-32 Hz, Voltage Index = 196 laV. Lower trace: Hvm, 4-8 Hz, Voltage Index = 257 pV; 8-16 Hz, Voltage Index = 221 laV; 16-32 Hz, Voltage Index = 409 ~tV.

ESTROGEN AND THE HYPOTHALAMUS HYPOTHALAMIC

VENTROMEDIAL

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NUCLEUS

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600

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7 Hvm

NUMBERS

OVARIECTOMIZED OVARIECTOMIZEO. ESTROGEN-TREATED

~= 4 - 8 Hz

"=8-)6 Hz

~]= 16-32 HZ

FIG. 2. Voltage Indices of the 3 frequency bands of the EEG recorded by telemetry from 9 hypothalamic ventromedial nuclei in female cats. Highest Voltage Indices were in the highest frequency band, and during estrogen treatment Voltage Indices were consistently depressed (Wileoxon 2-tailed, p <0.01). c o m p a r e d u s i n g t h e W i l c o x o n m a t c h e d - p a i r s s i g n e d - r a n k s test (2-tailed). E s t r o g e n a d m i n i s t r a t i o n r e s u l t e d in a significant d e p r e s s i o n o f e n e r g y levels ( V o l t a g e I n d i c e s ) in t h e t w o h i g h e r f r e q u e n c y b a n d s ( 8 - 1 6 H z : N = 7, T = 2, p < 0 . 0 5 . 16-32 H z : N = 7, T = 0, TABLE I. EFFECTS OF ESTROGENADMrrasrRATION ON THE OVERALLMEANENERGY INDICES OF THE EEG IN FREQUENCY BANDS I ( 4 - 8 H z ) , II (8-16Hz) and III (16-32Hz) OBTAINED FROM THE HYPOTHALAMIC VENTROMEDIAL NUCLEI OF 70VARIECTOMIZED FEMALE CATS

Cat No. Site 1 2 3 4 5 6 7

Hvm Hvm Hvm Hvm Hvm Hvm Hvm

Significances of differences between Energy Indices before and during estrogen treatment I II III n.s. n.s. - p <0.001 n.s. - p <0.001 - p <0.001 - p <0"01 - p <0"01 - p <0.001 n.s. - p <0.001 - p <0.001 - p <0.001 + p <0.01 --p <0.001 n.s: - p <0.01 - p <0.01 - p <0.001 - p <0.001 --p <0.001

F Ratios and degrees of freedom after Box (1954), from split-plot analyses of variance I II Ill 2"69 (I, 13.3) 0"52 (1, 11.7) 96.39 (1, 15.4) 3.64 (1, 17-8) 31-14 (1, 18"0) 20"64 (1, 12.9) 9.56 (1, 29.1) 9-24 (1, 36.9) 199.9 (1, 28-6) 0-59 (1, 31"3) 72.77 (1, 23.9) 445.4 (I, 36.8) 138-4 (1, 27.7) 12.89 (1, 35.4) 59-38 (1, 30"5) 0.66 (1, 11.5) 11.69 (1, 9-48) 12.27 (1, 11.8) 15.94 (1, 33-4) 239.1 (1, 27-9) 464.8 (1, 28.2)

This table gives significance levels for the differences illustrated in Fig. 1. A ' - ' sign indicates a decrease and a ' + ' sign an increase in Energy Indices.

18

SUSAN E, HOLBROOKEand RICHARD P. MICHAEL

p <0.02), but not in the lowest frequency band (4-8 Hz: N ---- 7, T ---- 3, n.s.). When data for all 3 frequency bands for all 7 cats were analyzed together, estrogen administration was again seen significantly to depress the Voltage Indices (N = 21, T -----9, p <0.01). Table I shows the significance of differences in energy levels before and during estrogen administration in each frequency band for each of the 7 H v m sites where data were available, and this table may be studied in conjunction with Fig. 2. The split-plot analyses of variance after applying Box's conservative correction showed highly significant (p <0.001) decreases in energy levels during estrogenization in 6 of 7, 3 of 7 and 2 of 7 cases in the 16-32, 8-16 and 4-8 Hz bands, respectively.

Effect of estrogen on activity in anterior and posterior hypothalamus Figures 3 and 4 show the Voltage Indices of the 3 frequency bands of the E E G recorded from 13 anterior and posterior hypothalamic sites (10 cats). Data both before and during estrogen administration were obtained from 4 anterior and 4 posterior hypothalamic sites (7 cats). When all 3 frequency bands were grouped together, Wilcoxon tests showed no significant effects of estrogen on energy levels in either the 4 anterior hypothalamic sites (N = 11, T ---- 17, n.s.), or the 4 posterior hypothalamic sites (N ---- 12, T ---- 32, n.s.). When data from all 8 anterior and posterior hypothalamic sites were combined, no significant effect of estrogen could be demonstrated (4-8 Hz: N ---- 8, T = 15, n.s. 8-16 Hz: N ---- 7, T ---- 17, n.s. 16-32 Hz: N = 8, T ~ 20, n.s. All bands: N ~ 23, T ----- 164, n.s.). The split-plot analyses of variance showed highly significant (p <0.001) changes in energy ANTERIOR

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NUMBERS

OVARIECTOM~ZEO OVARIECTOMIZEO~ ESTROGEN-TREATED

~=4-8Hz

1T=8-16 HZ ]~=16-:52Hz

FIo. 3. Voltage Indices of the 3 frequency bands of the EEG from 8 anterior hypothalamic sites in female cats. During estrogen treatment Voltage Indices both increased and decreased and changes were not consistent in direction 0Nileoxon 2-tailed, n.s.).

ESTR~3ENANDTHEH

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19

POSTERIOR HYPOTHALAMUS

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ESTROGEN - TREATEO I~

1 6 - 3 2 HZ

FIo. 4. Voltage Indices of the 3 frequency bands of the EEG from 5 posterior hypothalamic sites in female cats. During estrogen treatment, changes in Voltage Indices were not consistent in direction and results resembled those in anterior hypothalamns (Wilcoxon 2-tailed, n.s.). levels during estrogenization in 8 of 8, 6 of 8 and 5 of 8 cases in the 16-32, 8-16 and 4-8 Hz bands, respectively, but the direction of the differences was not consistent. Estrogen resulted in increases in energy levels in 3 sites and decreases in 5 sites, and the increases were of greater magnitude than the decreases.

Effect of estrogen on activity in extra-hypothalamic sites (cortex and thalamus) Figure 5 shows the Voltage Indices of the 3 frequency bands of the E E G recorded from 6 extra-hypothalamic sites (6 cats). Data before and during estrogen administration were obtained from one thalamic and 4 cortical sites. When all 3 frequency bands of the 4 cortical sites were grouped together, Wilcoxon tests showed no significant effect of estrogen on energy levels (N = 11, T = 24.5, n.s.). The split-plot analyses o f variance showed highly significant (p <0.001) changes in energy levels during estrogenization in 2 of 5 cases in each of the 3 frequency bands: however, these changes were not consistent in direction.

Control tests with a female partner A total of 23 control tests was conducted when a female cat was substituted for the male, 6 before and 17 during estrogenization, and data were obtained from a total of 6 electrode sites in 4 cats. Data recorded during a given hormone condition were more similar to each other than those recorded during the opposite hormone condition, independently of the sex of the testing partner. There was no clear indication, therefore, that the sex of the partner was substantially influencing results, although no data during mounting or intromission could be obtained.

Relation between the energy characteristics of the EEG and the response to estrogen The cortical EEG is well known in the cat and need not be described again here. The Hvm E E G contained predominantly high frequency activity and had a spiky appearance

20

SUSANE. HOLBROOKEand RICHARDP. MICHAEL CAUOATE NUCLEUS ~

THALAMUS

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CAT NUMBERS I l l - - Ill OVARIECTOMIZED X - - - X OVARIECTOMIZED~ ESTROC-~N-TREATED ~" 4-SHz

~[= 8-16 Hz

TIT= 16- 32 NZ

FIG. 5. Voltage Indices of the 3 frequency bands of the EEG from 6 extra-hypothalamic sites in female cats. Changes during estrogen treatment were not consistent in direction (Wilcoxon 2-tailed, n.s.). while those from anterior and posterior hypothalamus contained predominantly lower frequency activity and had a rather flat appearance. Correspondingly, as will be apparent from Figs. 2-5, energy levels in Hvm had a characteristic pattern that was quite different from those in other sites. In Hvm, the Voltage Index was, in general, considerably higher in the highest frequency band, whereas this trend occurred in only one of the other sites, and the reverse trend was apparent in anterior and posterior hypothalamus (Fig. 1). Table II gives the significance of differences between Voltage Indices in the 3 frequency bands for different neural sites, and only in Hvm, where energy levels were higher in the higher frequency bands, were energy levels consistently depressed by estrogen (see also Table III). Influence of behavioral situation on hormone-induced d(fferences in EEG energy levels This proved to be very difficult. We asked the question: did the behavioral situation in which the female cat was placed influence the differences in energy levels induced by estrogen treatment? To answer this, we examined the 'between situations' terms and the 'hormone × situations' interaction terms of the split-plot analyses of variance for statistical significance. In 18 of the 20 sites and in 37 of the 60 cases (3 frequency bands per site) there were significant differences between situations. The 'hormones x situations' interaction term was statistically significant in 14 of the 20 sites and in 25 of the 60 cases. Hvm appeared to differ both from other hypothalamic sites and from the cerebral cortex, and detailed

21

ESTROGEN AND THE HYPOTHALAMUS

TABLE II. SIGNIFICANCE OF DIFFERENCE BETWEEN VOLTAGE INDICES (I.IV) OF THE 3 FREQUENCY BANDS OF THE

EEG (aOTH HOgMO~Ecor~rnoNs) IN 3 ~URAL aFXnor~s Neural region Hvm

N 16

Anterior and Posterior Hypothalamus Cortex

21 8

I

vs n.s. T=39"5

Comparisons between frequency bands II I vs III II p <0-01 T=5

p <0-01 T = 11

p <0.01 T=23-5

vs

III p <0-01 T=3.5

p <0-05 T=58.5

p <0.02

p <0.01 T=0

T=I

n.s. T=15

The p value appears under the frequency band with the higher Voltage Index. I = 4 - 8 Hz, II=8--16 Hz, I I I = 16-32 Hz. (Wilcoxon matched-pairs signed-ranks test, 2-tailed. N=number of matched pairs, minus the number with no difference between Voltage Indices. T=smaller sum of like-signed ranks.) TABLE III. SHOWS THE CONSISTENCY WITH WHICH E E G ENERGY LEVELS DECREASED IN H v m IN THE 1 6 - 3 2 H z BAIRD DURING THE MATING SITUATION ( C )

Behavioural situation Direction of change Ovariectomy Ovariectomy and estrogen

4-8 8-16 16-32 4-8 8-16 16-32

Hz Hz Hz Hz Hz Hz

A-B + 0 1 1 0 3 0 0 1 0 0 2 1

+ 0 0 2 1 0 0

B--C 1 1 0 0 0 6

C--D + 0 0 0 0 0 2 1 0 2 0 2 1

D--E + 2 0 1 0 0 0 0 0 0 0 0 0

( + ) Increases and ( - ) decreases in energy levels in the second situation (e.g. 13) with respect to the first (e.g. A) as determined by t-tests on the split-plot analysis of variance. The Table shows the incidence, out of 7 cases, of significant differences in energy levels between behavioral situations.

statistical e x a m i n a t i o n s o f these changes were c a r r i e d out. Results were complex, with considerable a n i m a l to a n i m a l variation, a n d these d e t a i l e d findings will n o t be given here. The m o s t consistent finding, however, was in H v m in the 16--32 H z b a n d , where a m a r k e d decrease in energy o c c u r r e d in situation C (mating behavior) in the estrogenized c o n d i t i o n (Table III). T w o examples are illustrated in Fig. 6. In contrast, in a n t e r i o r a n d p o s t e r i o r h y p o t h a l a m u s (Fig. 6), even t h o u g h there was a highly significant difference (p <0.001) in overall m e a n E n e r g y Indices before a n d d u r i n g estrogen, there was no significant decrease in energy in situation C (mating behavior). I n general, the results p o i n t e d to a consistent interaction between the b e h a v i o r a l situation a n d h o r m o n e c o n d i t i o n in H v m (6 o f 7 cases) t h a t was n o t o b s e r v e d in either a n t e r i o r o r p o s t e r i o r h y p o t h a l a m u s (I o f 8 cases). DISCUSSION This study used telemetry to d e m o n s t r a t e the l o n g - t e r m (weeks) changes in electrical activity in the h y p o t h a l a m u s o f female cats induced b y estrogen a d m i n i s t r a t i o n . R e c o r d i n g s were m a d e successfully for p e r i o d s o f weeks o r m o n t h s f r o m h e a l t h y females free to interact d u r i n g m a t i n g tests with a sexually vigorous male. D a t a were subjected to a f o r m o f energy analysis t h a t gave results which c o u l d be interpreted in terms o f electrode location, h o r m o n a l

22

SUSANE. HOt~ROOXEand RICHAaDP. MICHAEL HYPOTHALAMIC VENTROMEDIAL NUCLEUS

I-2

HYPOTHALAMIC VENTROMEDIAL NUCLEUS

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P
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Overall Mean

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FIG. 6. Examples of Mean Energy Indices of the 16-32 band from hypothalamic sites in each of 4 female cats plotted to show the influence of the behavioral situation in which the female was placed (situations A--E). Estrogenization significantly depressed the overall mean energy levels in all 4 cases. However, significant depressions of Energy Indices were observed during the mating situation (C) in the estrogen-treated condition only in Hvm. ' - ' denotes a decrease in energy, ' + ' denotes an increase in energy. status and behavioral situation. The most consistent finding was a depression of the energy of the EEG in Hvm during estrogen administration in all females, particularly marked in the 16-32 Hz band. Although significant changes in energy levels were also observed in individual animals in other sites, their direction was variable. Consequently, when the grouped data were considered, the only statistically significant depressions in energy occurring during estrogen treatment were in Hvm. Depressions of energy were most marked in the highest frequency band which seemed to be specially influenced by the hormone. It is difficult, in the present state of knowledge, to correlate these effects with changes in neuronal activity. However, the interaction between the behavioral situation in which the female was placed and her hormonal condition supported the view that the changes in Hvm had physiological significance in relation to the neural control of estrous behavior: such changes have not previously been described for any species. In this context the shortterm suppressions of the amplitude of the EEG that are co-terminous with intromission

ESTROGE~ANDTHEH ~ M U S

23

were also localized to Hvm (Michael, 1973; Michael et al., 1977). Many diverse functions have been attributed to Hvm and changes in its electrical activity may also have relevance for other behavioral changes that accompany estrus, such as the decline in food intake. Our observations on the characteristics of the E E G in different hypothalamic regions were consistent with those of Brooks (1959) in unanesthetized, unrestrained cats. Previous studies on the chronic effects of estrogen on E E G activity have been mainly concerned with the durations of slow wave and paradoxical sleep in the female rat and guinea pig (Malven & Sawyer, 1966). Changes in EEG activity in five frequency bands between 2 and 30 Hz were reported to accompany the induction of estrus in the rabbit (Kawakami, Terasawa, Tsuchihashi & Yamanaka, 1966) but the combinations of changes occurring in low and high frequency bands differed from those in the present study. Lincoln (1967) reported th'.t: the spontaneous firing rates of large populations of neurons in preoptic, anterior hyp~,thalamic and septal regions of urethane-anesthetized rats were higher in ovariectomized females than in estrogen-treated females and females in persistent estrus; the reverse tendency was observed in the lateral hypothalamus. Cross & Dyer (1971) and Dyer, Pritchet & Cross (1972) also observed changes in anterior hypothalamic-preoptic unit activity during the estrous cycle of rats. Estrogen also affects spontaneous unit activity in acute cats (Kawakami & Saito, 1969) and rabbits (Duly, Vincent, Bensch, Aumonier & Faure, 1969). It is apparent that estrogens may affect neural activity in a variety of ways in different species. Nevertheless, there are obvious advantages to using telemetry in conscious, freely-moving animals when attempts are being made to correlate changes in brain activity with changes in ongoing behavior, and it was clear that the estrogen-induced changes in the hypothalamus were not simply the result of changes in cortical activity and the level of arousal. Although this study involved extensive observations on the behavior of cats during more than 300 mating tests, many more electrode placements, particularly in limbic system sites, need investigation before any unique properties are ascribed to the changes observed in Hvm. Nevertheless, the hypothalamus possesses hormone sensors, and hypothalamic implants cause behavioral changes by a direct action on an estrogen-receptor system. One might speculate, therefore, that the altered level of electrical activity in these brain regions during estrogenization has a gating effect which results in a change in the perception of afferent stimuli so that the male's coital activities, which are noxious and aversive to ovariectomized females, become pleasant and rewarding during estrus. We thank the Foundations' Fund for Research in Psychiatry, the Medical Research Council (U.K.), and N.I.M.H. (grant MH19506) for supporting this work. Dr. G. H. Byford, R.A.F. Institute of Aviation Medicine, advised us on the EEG analyses and grateful acknowledgement is given. These studies formed part of Miss S. E. Holbrooke's Ph.D. thesis, University of London. REFERENCES ALCAP.AZ,M., GUZMAN-FLORES,C., SALAS,M. & BEY~R,C. (1969) Effect of estrogen on the responsivity of hypothalamic and mesencephalic neurons in the female cat. Brain Research 15, 439--446. BEYER, C., ALMANZA, J., DE LA TORRE, L. & GUZMAN-FLORES, C. (1971) Effect of genital stimulation on the brain stem multi-unit activity of anestrous and estrous cats. Brain Research 32, 143-150.

Box, G. E. P. (1954)Some theorems on quadratic forms applied in the study of analysis of variance problems. II. Effects of inequality of variance and of correlation between errors in the two-way classification. Ann. math. Statist. 25, 484--498.

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SUSANE. HOLBROOKEand RICHARDP. MICHAEL

BROOKS, D. C. (1959) Electrical activity of the ventromedial nucleus of the hypothalamus. Am. J. Physiol. 197, 829-834. BYFORD, G. H. (1964) Signal variance and its application to continuous measurements of LEG activity. Proc. R. Soc. B 161, 421-437. CROSS,B. A. & DYER, R. G. (1971) Cyclic changes in neurons of the anterior hypothalamus during the rat estrous cycle and the effect of anesthesia. In Steroid Hormones and Brain Function,UCLA Forum Med. Sci. No. 15, C. H. Sawyer and R. Gorski (Eds.), pp. 95-102. University of California Press, Los Angeles. DutY, B., VINCENT,J. D., BENSCH,C., AUMONIER,M. & FAURE,J. M. A. (1969) Activities unitaires hypothalamique chez la lapine chronique: influence des oestrogenes. J. PhysioL (Paris) 61 Suppl. 2, 279. DYER, R. G., PRrrcn~Tr, C. J. & CROSS,B. A. (1972) Unit activity in the diencephalon of female rats during the oestrous cycle. J. Endocr. 53, 151-160. GLASCOCK,R. F. & MICHAEL,R. P. (1962) The localization of oestrogen in a neurological system in the brain of the female cat. J. Physiol. (Lond.) 163, 38-39. GREENHOUSE,S. W. & GEISSER,S. (1959) On methods of analysis of profile data. Psychometrika 24, 95-112. HARRIS, G. W. & MICHAEL,R. P. (1964) The activation of sexual behavior by hypothalamic implants of oestrogen. J. PhysioL (Lond.) 171, 275-301. HARRIS,G. W., MICHAEL,R. P. & SCOTT,P. P. (1958) Neurological site of action of stilboestrol in eliciting sexual behaviour. In Ciba Foundation Symposium on the Neurological Basis of Behaviour, G. E. W. Wolstenholme and C. M. O'Connor (Eds.), pp. 236-254. Churchill, London. JASPER,H. H. & AJMONE-MARSAN,C. (1954) A Stereotaxic Atlas of the Diencephalon of the Cat. The National Research Council of Canada, Ottawa. KAWAKAMI,M. d~ SAITO, H. (1969) The analysis of inter-spike interval fluctuation of hypothalamic unit activity in response to luteinizing hormone and oxytocin. Jap. J. Physiol. 19, 243-259. KAWAKAMI,M., TERASAWA,E., TSUCmHASHI,S. & YAMANAKA,K. (1966) Differential control by sex hormones of brain activity in the rabbit and its physiological significance. In Steroid Dynamics, G. Pincus, K. Nakao and J. F. Tait (Eds.), pp. 237-302. Academic Press, New York. LINCOLN,D. W. (1967) Unit activity in the hypothalamus, septum and preoptic area of the rat: characteristics of spontaneous activity and the effect of oestrogen. J. Endocr. 37, 177-189. MALVEN, P. V. & SAWYER,C. H. (1966) Sleeping patterns in female guinea pigs: effects of sex hormones. ExpL Neurol. 15, 229-239. MICHAEL, R. P. (1961) Observations upon the sexual behaviour of the domestic cat (Felis cattus, L.)under laboratory conditions. Behaviour 18, 1-24. MICHAEL,R. P. (1962) Estrogen-sensitive neurons and sexual behaviour in female cats.Science 136, 322-323. MICHAEL,R. P. (1965) Oestrogens in the central nervous system. Br. reed. Bull. 21, 87-90. MICHAEL, R. P. (1973) The effects of hormones on sexual behaviour in female cat and rhesus monkey. In Handbook of Physiology, Section 7, Endocrinology, VoL II, Reproductive System - - Female, R. O. Greep and E. B. Astwood (Eds.), pp. 187-221. American Physiological Society, Washington, D.C. MICHAEL,R. P., HOLBROOKE,S. E. & WELLER,C. (1977) Telemetry and continuous energy analysis of hypothalamic LEG changes in female cats during intromission by the male. Psychoneuroendocrinology 2, 287-301. MICHAEL, R. P., WELLER,C. ~£ WOLFF,H. S. (1965a) Telemetry, brain activity and animal behaviour. In Digest of the 6th International Conference on Medical Electronics and Biological Engineering, Tokyo, pp. 201-202. MICHAEL,R. P., WELLER,C. d~ WOLFF,H. S. (1965b) A totally implantable transmitter for telemetering the electrical activity of the brain in cats and monkeys. J. PhysioL (Lond.) 180, 3-5. PORTER,R. W., CAVANAUGH,E. B., CRITCHLOW,B. V. • SAWYER,C. H. (1957) Localized changes in electrical activity of the hypothalamus in estrous cats following vaginal stimulation. Am. J. Physiol. 189, 145-151. SIEOEL,S. (1956) Nonparametric Statistics for the Behavioral Sciences, pp. 75-83. McGraw-Hill, New York. StrrlN, J. & MICHAEL,R. P. (1970) Changes in brain electrical activity following vaginal stimulation in estrous and anestrous cats. PhysioL Behav. 5, 1043-1051. WELLER, C. (1966) Remotely actuated solid-state switch. Electron. Lett. 2, 191-192.